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ANNUAL  REPORT 
OF 
PROGRAM  ACTIVITIES 
lA  <?       NATIONAL  HEART  AND  LUNG  INSTITUTE 
Fiscal  Year  1975 


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NATIONAL  INSTITUTES  OF  HEALTH 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

Annual  Report 

July  1,  1974  -  June  30,  1975 

Office  of  the  Director 


The  mission  of  the  National  Heart  and  Lung  Institute  is  1)  to  conduct  and 
support  research  on  the  heart,  blood  vessels,  blood,  and  lungs  and  on  their 
diseases;  2)  to  develop  and  evaluate  means  of  prevention,  diagnosis,  and 
treatment  and  to  encourage  application  of  proven  techniques  by  the  medical 
community;  and  3)  to  provide  support  for  the  training  of  new  research 
workers,  clinical  scientists,  and  teachers  in  the  cardiovascular  and 
pulmonary  disease  fields.  Some  of  the  Institute's  activities  in  key 
program  areas  during  fiscal  year  1975  are  briefly  highlighted  below. 

Heart  and  Vascular  Diseases 

A  major  concern  of  the  Institute  has  been  the  support  of  clinical  trials 
to  determine  whether  and  to  what  extent  illness  and  death  from  coronary  heart 
disease  and  other  cardiovascular  diseases  can  be  reduced  by  timely  interven- 
tions against  major  risk  factors.   Studies  recently  completed  or  in  progress 
include : 

— The  Coronary  Drug  Project,  begun  some  8  years  ago  and  completed 
this  fiscal  year,  reveals  that  lip id- lowering  drugs  are  not  of 
value  in  improving  long-term  survival  among  patients  who  have 
already  sustained  one  or  more  heart  attacks. 

~^The  Multiple  Risk  Factor  Intervention  Trial  (MRFIT)  will  test 
the  hypothesis  that  reducing  blood  cholesterol  levels,  reducing 
elevated  blood  pressure,  and  reducing  or  eliminating  cigarette 
smoking  may  reduce  morbidity  and  mortality  from  coronary  heart 
disease  among  men  at  increased  risk  because  of  the  presence  of 
some  combination  of  these  factors.  Recruitment  of  the  12,000 
volunteers  needed  for  the  study  is  well  along  at  the  20  parti- 
cipating centers  and  should  be  completed  by  January,  1976. 

— The  Hypertension  Detection  and  Followup  Program  involves  14 
participating  centers  and  more  than  11,000  patients  with  high 
blood  pressure  in  a  5-year  study  to  assess  the  effectiveness 
of  adequate  blood-pressure  control  in  reducing  morbidity  and 
mortality  from  cardiovascular  diseases. 

— The  Coronary  Primary  Prevention  Trial,  underway  at  12  Lipid 
Research  Clinics,  will  evaluate  the  preventive  value  of  lipid- 
lowering  diets  and  drugs  in  4,000  men  who  have  elevated  blood 
lipids,  but  have  not  experienced  a  heart  attack. 


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— Recruitment  of  patients  began  in  May  for  the  Aspirin-Myocardial 
Infarction  Study  (AMIS)  to  determine  whether  aspirin,  which 
inhibits  the  aggregation  of  blood  platelets,  will  reduce  the 
threat  of  recurrent  heart  attacks  and  heart  attack  deaths 
among  persons  who  have  had  at  least  one  such  attack.  The 
study  will  involve  30  participating  centers,  4,000  subjects, 
and  will  run  for  three  years. 

Another  clinical  trial,  scheduled  to  begin  in  the  near  future  at  12  clinical 
centers,  will  compare  coronary  artery  bypass  graft  surgery  with  non-surgical 
medical  management  of  coronary  artery  disease  to  determine  the  clinical 
conditions  under  which  such  surgery  is  appropriate  and  most  likely  to  yield 
good  results . 

Other  activities  in  the  heart  and  vascular  disease  fields  included  1) 
establishment  of  9  Specialized  Centers  of  Research  for  basic  and  clinical 
studies  on  acute  and  chronic  ischemic  heart  disease;  2)  continued  support  of 
research  on  means  of  protecting  ischemic  heart  muscle,  minimizing  permanent 
heart  damage  resulting  from  acute  heart  attacks,  and  accurately  assessing 
the  extent  of  that  damage;  3)  continued  development  of  non-invasive  instrumen- 
tation for  the  detection  and  evaluation  of  cardiovascular  diseases  in 
symptomatic  and  asymptomatic  patients;  and  4)  research  concerned  with  the 
causes,  prevention,  and  emergency  treatment  of  sudden  cardiac  death. 

Lung  Diseases 

The  lung  contains  approximately  40  different  types  of  cells,  each  with  some 
specific  function  that  may  be  altered  in  disease.  Recent  developments  in 
cell-culture  and  separation  techniques  show  promise  of  permitting  detailed 
in  vitro  studies  of  various  cell  types,  including  their  response  to  various 
agents  and  insults  that  may  operate  in  the  development  of  lung  disorders . 
Receiving  special  emphasis  has  been  research  on  the  cells  of  lung  connective 
tissue  and  their  synthesis  of  the  structural  proteins  collagen  and  elastin, 
and  research  on  the  Type  II  cell,  which  plays  a  role  in  lung  repair  and  also 
secretes  the  surfactant  needed  to  prevent  collapse  of  the  air-sacs,  or 
alveoli,  of  the  lung.   Other  basic  studies  planned  or  in  progress  are 
concerned  with  respiratory  mucin,  animal  models  of  pulmonary  fibrosis,  and 
the  development  of  markers  for  various  types  of  lung  cells . 

Hyaline  membrane  disease,  or  neonatal  respiratory  distress  syndrome,  is  a 
major  cause  of  death  in  the  newborn,  especially  among  premature  infants. 
Mortality  from  this  disorder  may  be  dramatically  reduced  as  a  result  of  two 
recent  developments:   one  is  a  test  involving  sampling  of  the  amnionic  fluid 
that  can  identify  the  high-risk  infant  before  birth;  the  other  is  a  safe, 
effective  method  of  applying  continuous  positive  airway  pressure  to  inflate 
infants'  collapsed  lungs  and  preserve  adequate  respiratory  function. 

Support  was  continued  during  fiscal  1975  for  clinical  trials  of  the  extra- 
corporeal membrane  oxygenator  in  the  management  of  acute  respiratory  failure 
at  9  participating  institutions.  Acute  respiratory  failure  carries  a 


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mortality  rate  of  about  40  percent.  It  is  believed  that  this  rate  can  be 
substantially  reduced  by  using  oxygenators  to  provide  temporary  respiratory 
support  until  the  patients'  lungs  can  recover  sufficiently  to  resume  their 
respiratory  duties . 

The  Division  of  Lung  Diseases  has  completed  the  first  phase  of  its  National 
Pulmonary  Faculty  Training  Program,  designed  to  strengthen  pulmonary  faculties 
at  schools  of  medicine  or  osteopathy  that  have  not  yet  developed  adequate 
programs  in  respiratory  diseases .  Applications  will  soon  be  invited  from 
these  schools  nominating  junior  faculty  members  for  extensive  pulmonary 
training  at  medical  centers  selected  for  their  strong  academic  programs  in 
these  fields. 

The  Institute  also  sponsored  a  number  of  workshops  in  pulmonary-disease 
subject  areas.   The  proceedings  of  some  of  these  were  published  for  distribu- 
tion to  the  scientific  and  medical  communities.   Published  reports  included 
Hematological  Analysis  of  Extracorporeal  Circulation;  Lung  Metabolism;  and 
Lung  Cell  Separation,  Identification,  and  Culture. 

Blood  Diseases  and  Blood  Resources 

Hemophilia  remains  the  prime  target  of  most  Institute-supported  research  on 
hemorrhagic  diseases .  Recent  research  has  indicated  that  most  hemophiliacs 
have  an  abnormal  form  of  antihemophilic  factor  (AHF)  rather  than  an  absolute 
deficiency  of  this  clotting  factor.  The  abnormal  AHF  does  not  permit  adequate 
blood  clotting,  however,  so  that  normal  AHF  must  be  provided,  usually  as  a 
concentrate,  to  prevent  or  control  bleeding  episodes.   But  data  from  research 
on  this  aspect  of  hemophilia  also  hold  promise  of  yielding  more  reliable 
means  for  detecting  carriers  of  hemophilia  before  they  give  proof  of  it  by 
bearing  hemophilic  sons.   One  promising  diagnostic  scheme  has  been  developed 
and  is  currently  being  evaluated. 

At  the  other  pole  from  hemorrhagic  disorders  are  clotting  complications  of 
heart  and  blood  vessel  diseases  that  are  often  responsible  for  their 
disabling  or  lethal  manifestations.  Noninvasive  techniques  have  been 
developed  for  diagnosis  of  thrombotic  lesions  early  in  their  development. 
These  have  been  used  effectively  in  recent  clinical  trials  evaluating  the 
effectiveness  of  anticoagulants  and  inhibitors  of  platelet  aggregation  in  the 
prevention  of  deep  venous  thrombosis.  Highly  effective  in  some  patients, 
these  agents,  unfortunately,  appeared  to  work  least  well  in  patients  undergoing 
orthopedic  surgery,  who  are  particularly  susceptible  to  clotting  complications 
during  the  postoperative  period. 

Institute-supported  research  on  sickle-cell  anemia  has  included  clinical 
trials  of  anti-sickling  agents  for  the  prevention  or  relief  of  sickle  cell 
crises .  A  clinical  trial  of  oral  cyanate  was  discontinued  when  the  drug  was 
found  associated  with  peripheral  neural  disturbances  and  the  development  of 
abnormal  reflexes  in  some  patients . 


OD-3 


Research  in  Cooley's  anemia  ranges  from  studies  of  the  faulty  hemoglobin- 
synthesizing  machinery  of  red  cells  from  patients  suffering  from  the  disease 
to  the  quest  for  effective  chelating  agents  to  treat  the  iron  overload  that 
often  develops  as  a  consequence  of  repeated  transfusions  required  by  these 
patients . 

Other  studies  are  seeking  inexpensive  and  effective  methods  for  identifying 
abnormal  hemoglobins  during  the  prenatal  period,  at  birth,  or  during  later 
life.  One  recently  developed  method  for  identifying  hemoglobin  A„  may  also 
be  of  value  for  detecting  a  carrier  state  of  Cooley's  anemia. 

Intramural  Research 

Among  the  findings  reported  by  the  Division  of  Intramural  Research  during 
FY  1975  were  the  following: 

— The  extent  and  severity  of  heart-muscle  damage  resulting  from 
acute  heart  attack  may  be  a  critical  factor  affecting  survival 
and  also  the  amount  of  residual  disability  after  recovery. 
NHLI  scientists  have  demonstrated  in  animals  that  nitroglycerin, 
given  in  combination  with  methoxamine  or  phenylephrine,  reduced 
the  extent  of  heart-muscle  damage  resulting  from  induced  heart 
attacks  and  reduced  the  threat  of  heart-rhythm  disturbances 
that  are  a  frequent  and  sometimes  lethal  consequence  of  such 
attacks.   This  drug  regimen  is  currently  being  clinically 
evaluated  at  NHLI. 

— NHLI  surgeons  report  that  xenograft  heart  valves  (from  pigs) , 
mounted  on  prosthetic  frames  for  ease  of  insertion,  have  out 
performed  artificial  disc  valves  for  mitral  or  tricuspid  valve 
replacement .  Recipients  of  xenograft  valves  had  a  much  lower 
mortality  rate  during  the  first  six  months  after  surgery  than 
did  disc-valve  recipients.  Moreover,  during  this  period  the 
xenograft  recipients,  though  receiving  no  anticoagulants 
postoperatively,  developed  no  clotting  complications,  whereas 
30  percent  of  the  disc-valve  recipients  developed  such 
complications  despite  maintenance  on  anticoagulant  drugs. 

— NHLI  scientists  have  developed  methods  for  casting  very  thin 
membranes  from  silicone  rubber  that  are  free  of  the  pinhole 
defects  that  have  been  the  chief  cause  of  membrane  failure 
in  blood  oxygenators.   The  membranes,  incorporated  into  a 
spiral  coil  membrane  oxygenator  developed  at  NHLI,  have  shown 
excellent  gas-exchange  and  blood-compatibility  characteristics 
during  prolonged  periods  of  blood  oxygenation  in  experimental 
animals  (sheep) . 

— In  hemoglobin  synthesis,  two  protein  chains — the  alpha  chain 
and  the  beta  chain — are  combined  to  form  the  finished 
molecule.   In  Cooley's  anemia,  however,  beta  chain  synthesis 


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is  abnormally  slow;  excess  alpha  chains  pile  up,  then 
precipitate  in  the  red  blood  cells,  causing  them  to  be 
destroyed.  NHLI  studies  have  shown  that  the  problen  in 
Cooley's  anemia  is  a  scarcity  of  the  mRNA  that  directs  the 
assembly  of  the  beta  chain.  The  betaglobin  mRNA  is  normal, 
as  are  the  beta  chains  it  produces;  but  the  red  cells  from 
victims  of  Cooley's  anemia  contain  too  little  of  it  to 
keep  pace  with  the  normal  rate  of  alpha-chain  synthesis. 

National  Research  and  Demonstration  Centers  Program 

During  fiscal  1975,  the  Institute  awarded  funds  for  the  establishment  and 
support  of  three  National  Research  and  Demonstration  Centers  under  a 
provision  of  the  National  Heart,  Blood  Vessel,  Lung  and  Blood  Act  of  1972. 
The  Act  authorizes  the  eventual  establishment  and  support  of  up  to  30  such 
centers  to  1)  carry  out  basic  and  clinical  research  on  heart  and  blood  vessel 
diseases,  lung  diseases,  blood  diseases  or  blood  resources;  2)  provide 
demonstrations  of  advanced  methods  of  prevention,  diagnosis  and  treatment; 
3)  provide  a  training  resource  for  scientists  and  physicians  concerned  with 
these  diseases ;  and  4)  conduct  information  and  education  programs  for  health 
professionals  and  the  general  public. 

Of  the  three  centers  established  thus  far,  the  program  of  the  Baylor  Center 
will  focus  on  heart  and  blood  vessel  diseases,  particularly  arteriosclerosis 
and  its  complications.   The  center  established  at  the  University  of  Vermont 
will  concentrate  on  lung  diseases,  with  special  emphasis  on  occupational 
pulmonary  disorders  resulting  from  prolonged  exposure  to  harmful  dusts  and 
fumes  in  various  industries  and  occupations.  And  the  center  established 
at  the  King  County  Central  Blood  Bank  in  Seattle  will  be  concerned  mainly 
with  improvement  of  procedures  for  the  acquisition,  processing,  storage, 
distribution,  and  clinical  use  of  blood  and  blood  products. 

Other  Institute  Activities 

In  March,  1975,  the  Institute  completed  and  forwarded  to  the  President  for 
transmittal  to  the  Congress  the  second  in  a  series  of  annual  leports 
required  under  the  National  Heart,  Blood  Vessel,  Lung,  and  Blood  Act  of  1972. 
The  report  highlights  activities,  progress,  and  accomplishments  during  1974 
and  outlines  plans  for  the  National  Heart,  Blood  Vessel,  Lung,  and  Blood 
Program  over  the  next  five  years .  A  second  annual  report  was  also  prepared 
and  submitted  by  the  Institute's  principal  advisory  body:   the  National 
Heart  and  Lung  Advisory  Council. 

The  Institute  also  continued  its  participation  in  the  National  High  Blood 
Pressure  Education  Program  (NHBPEP) ,  which  was  initiated  in  1972  and  became 
fully  operational  in  1973.  NHLI  was  designated  the  lead  coordinating  agency 
for  the  program,  but  it  is  a  joint  effort  involving  some  15  federal  agencies 
and  over  four  hundred  other  participating  groups — professional  and  private, 


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national  and  local.  Its  program,  aimed  at  alerting  health  professionals  and 
the  general  public  to  the  dangers  of  untreated  high  blood  pressure  and  the 
beneficial  effects  of  therapy,  encompasses  a  wide  range  of  activities  in 
education,  research,  planning,  and  community  services. 

The  activities  of  NHBPEP  continue  throughout  the  year,  but  usually  peak  in 
May,  which  has  been  designated  National  High  Blood  Pressure  Month  for  two 
successive  years.   In  conjunction  with  the  National  High  Blood  Pressure  Month, 
1975,  the  Institute's  High  Blood  Pressure  Information  Center  distributed  to 
organizations  desiring  to  participate  more  than  55,000  kits  containing 
education,  detection,  and  treatment  guidelines.   In  the  average  year,  the 
HBP  Information  Center  receives  some  113,000  requests  for  information  and 
mails  out  more  than  1.9  million  publications. 

As  an  indication  of  the  program's  effectiveness,  the  number  of  patient  office 
visits  for  high  blood  pressure  remained  abbut  the  same  during  the  8  years 
prior  to  1972;  this  number  increased  by  15  percent  in  1972,  20  percent  in 
1973,  and  30  percent  in  1974. 

The  Institute's  Public  Inquiries  and  Reports  Branch  has  worked  closely  with 
the  HBP  Information  Center  in  such  matters  as  the  design,  production,  and 
staffing  of  HBP  exhibits,  production  of  publications,  and  also  in  setting  up 
the  highly-successful  HBP  screening  program  carried  out  as  a  service  to  NIH 
employees  during  the  past  year.   Providing  information  on  high  blood  pressure 
is  only  one  function  of  this  Branch,  which  during  FY  1975  handled  more  than 
32,000  inquiries;  mailed  out  more  than  1  million  publications;  oversaw  the 
production  and  distribution  of  59  new  publications  issued  by  the  Branch  or 
other  NHLI  divisions,  and  issued  27  news  releases  on  a  variety  of  program  or 
research  topics.   In  addition,  the  PIRB  has  analyzed  the  trends  of  incoming    ' 
public  and  professional  inquiries  in  order  to  identify  areas  requiring 
additional  affirmative  action. 

The  NHLI  portion  of  the  U.S .-U.S .S.R.  Health  Exchange  Program  made  significant 
progress  during  fiscal  1975.  There  was  a  continued  exchange  of  Soviet  and 
American  health  researchers  as  well  as  productive  planning  sessions  which 
were  convened  here  or  in  the  Soviet  Union.   In  several  areas  of  the  cardio- 
vascular disease  program,  joint  research  has  moved  from  the  plannning  stage 
to  ongoing  basic  or  clinical. studies.  Areas  which  have  seen  particularly 
successful  collaboration  include  the  pathogenesis  of  arteriosclerosis, 
myocardial  metabolism,  and  congenital  heart  disease.   In  addition,  the 
separate  agreement  on  artificial  heart  research  and  development  has  resulted 
in  an  expanding  scientific  and  technological  exchange. 


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ANNUAL  REPORT  -  DIVISION  OF  HEART  AND  VASCULAR  DISEASES,  NHLI 
July  1,  1974  -  June  30,  1975 


Overview 

The  Division  of  Heart  and  Vascular  Diseases  (DHVD)  is  responsible  for  planning 
and  directing  the  National  Heart  and  Lung  Institute's  research  grant,  contract 
and  training  programs  in  heart  and  vascular  diseases.  These  programs  encompass 
basic  research;  targeted  research;  clinical  trials;  and  education,  demonstration 
and  control  activities.  The  Division  maintains  surveillance  over  developments 
in  its  program  areas  and  assesses  the  national  need  for  research  in  the  causes , 
prevention,  diagnosis  and  treatment  of  cardiovascular  diseases  and  for  manpower 
training  in  these  disease  areas.  Despite  some  slight  downturn  in  recent  years 
of  the  death  rate  in  coronary  heart  disease,  cardiovascular  disease  continues 
to  be  the  number  one  killer  in  the  United  States.  A  major  focus  of  the 
Division  is  on  arteriosclerosis  and  hypertension  which  together  account  for 
over  1,000,000  deaths  annually.  The  programs  of  the  Division  are  broad-based, 
employing  all  available  funding  mechanisms  in  an  attempt  to  responsibly  take 
three  kinds  of  action:  support  of  new  basic  research;  clinical  evaluation  of 
existing  basic  research  findings  and  concepts  through  clinical  trials;  and 
Education,  Demonstration  and  Control  Programs  such  as  the  National  High 
Blood  Pressure  Education  Program  translating  research  concepts  to  practical 
patient  care. 

Administrative  Highlights 

In  September  advisory  committees  for  four  program  areas  of  the  Division 
were  established  with  new  or  revised  charters.  They  are:  the  Cardiac  Advisory 
Committee,  the  Clinical  Applications  and  Prevention  Advisory  Committee,  the 
Lipid  Metabolism  Advisory  Committee  and  the  Arteriosclerosis  and  Hypertension 
Advisory  Committee.  Each  Committee's  purpose  is  to  advise  the  Director  of  DHVD 
on  planning,  executing  and  evaluating  the  Division's  programs  in  the  specified 
area.  Three  of  the  Committees  held  meetings  this  fiscal  year.  These  committees 
have  been  very  helpful  to  the  Program  Directors  and  to  the  Director,  DHVD  in 
their  discussions  and  recommendations.  The  fourth  committee,  the  Arteriosclerosis 
and  Hypertension  Advisory  Committee  will  hold  its  first  meeting  early  in  the  Fall. 

On  July  1,  1974  responsibility  for  the  National  High  Blood  Pressure  Education 
Program  (NHBPEP)  was  transferred  to  the  Division  from  the  Office  of  the  Director, 
NHLI.  This  organizational  transfer,  accompanied  by  a  physical  transfer  of  the 
NHBPEP  to  the  same  building  housing  most  other  programs  of  the  Division, 
has  resulted  in  better  and  closer  coordination  between  all  the  components  of 
the  Division's  hypertension  program. 


Two  Branches  in  the  Cardiology  Area  have  been  renamed.  The  name  of  the 
Clinical  Cardiac  Diseases  Branch  was  changed  to  Cardiac  Diseases  Branch. 
The  former  Cardiovascular  Devices  Branch  is  now  the  Devices  and  Technology 
Branch.  Concurrent  with  its  change  in  name,  the  Devices  and  Technology 
Branch  became  responsible  for  grants  activities  in  its  field,  in  addition 
to  its  previous  responsibilities  for  the  research  contracts  in  the  Division's 
Circulatory  Assistance  program. 

Research  Highlights 

Following  are  but  a  few  abbreviated  examples  of  the  research  progress  made 
during  the  past  year: 

o  The  cellular  basis  of  familial  hypercholesterolemia  (Type  II  Hyperlipo- 
porteinemia)  has  been  demonstrated.  The  finding  that  some  cells  at 
least  (fibroblasts)  in  tissue  culture  have  specific  receptors  for  the 
binding  and  removal  of  circulating  low  density  lipoproteins  and  that 
these  receptors  are  deficient  or  lacking  in  the  disorder  has  opened  up  a 
major  new  area  for  research  and  study. 

°  Progress  is  being  made  on  developing  promising  techniques  for  protecting 
ischemic  myocardium,  thus  diminishing  the  amount  of  heart  muscle  lost 
from  myocardial  infarction.  The  techniques  are  in  part  built  upon 
improved  understanding  of  the  basic  biochemical  metabolism  of  heart 
muscle  and  its  derangement  when  the  myocardium  becomes  ischemic. 
Experimental  therapy  includes  methods  for  providing  chemical  substrates 
and  agents  to  enhance  their  entry  into  cells ,  methods  to  improve  blood 
flow  in  the  coronary  arteries,  and  methods  to  diminish  the  workload 
and  thus  the  energy  needs  of  the  heart. 

o  Further  progress  has  been  achieved  in  the  development  of  phonoangio- 
graphy  as  a  functional  non- invasive  technique  for  the  detection  of 
arteriosclerosis.  In  a  clinical  trial  involving  35  patients  with 
carotid  artery  stenosis,  a  comparison  of  the  stenotic  diameters 
(0.5mm  to  5.0mm)  predicted  by  the  non- invasive  methods  with  the 
diameters  estimated  by  x-ray  showed  agreement  within  +_  0.46mm  (S.D.). 

°  Computer  methods  to  achieve  image  enhancement  and  quantification  of 
arterial  disease  on  conventional  femoral  arteriograms  have  also  been 
developed  which  1)  display  detected  vessel  edges;  2)  estimate  the 
vessel  mid- line  and  non-diseased  lumen;  and  3)  provide  a  numerical 
measure  of  edge  irregularity.  A  clinical  prototype  for  a  stand- 
alone interactive  computer  image -processing  system  is  expected  to 
be  operational  July  1975. 


-2 


°  In  the  area  of  hypertension  the  preliminary  finding  that  patients  with 
low  renin  hypertension  excrete  an  excess  of  a  novel  16 -OH  sterol  has 
stimulated  several  investigators  to  examine  this  discovery. 

°  As  one  of  the  few  studies  providing  epidemiological  data  on  women,  an 
analysis  of  changes  in  risk  factors  among  Framingham  women  passing  through 
the  menopause  has  been  completed.  The  most  significant  finding  was  that 
serum  cholesterol  increases  abruptly  after  menopause.  However,  this  does 
not  account  for  the  3-4  fold  increase  in  the  rate  of  CHD  in  the  immediate 
post -menopausal  years,  which  still  remains  to  be  "explained". 

o  The  level  of  alpha- lipoprotein  cholesterol  in  the  blood  has  emerged  as  a 
strikingly  significant  independent  discriminant  of  risk  of  myocardial 
infarction  within  the  collaborative  studies  in  Framingham,  Albany, 
Honolulu,  Claxton  and  Israel.  Although  this  association  has  been  reported 
earlier,  its  strength  of  relationship  even  at  ages  65-74  provides  a 
potentially  important  further  refinement  of  risk  assessment  both  for 
predictive  purposes  and  for  monitoring  intervention  effects.  Prospective 
data  from  these  studies  will  become  a\railable  to  confirm  this  initial  cross 
sectional  finding. 

°  Direct  correlation  of  risk  factor  levels  during  life  with  subsequent 

severity  of  atherosclerotic  lesions  in  the  coronary  arteries  at  autopsy  is 
being  found  in  the  special  protocol  autopsy  studies  which  are  part  of  the 
Puerto  Rico  and  Honolulu  prospective  epidemiological  studies.  Significant 
associations  of  the  average  percent  of  intimal  surface  involved  with 
raised  atherosclerotic  lesions  have  been  found  with  preceding  levels  of 
serum  cholesterol,  blood  glucose,  systolic  and  diastolic  blood  pressure. 
These  important  findings  are  beginning  to  provide  a  more  direct  evaluation 
of  risk  factor  relationships  to  specific  lesions  of  atherosclerosis  as 
endpoints  whereas  previous  epidemiological  data  have  only  related  risk 
factors  to  clinical  events  of  coronary  heart  disease. 

Ongoing  Research  Programs  and  New  Initiatives 

0  Specialized  Centers  of  Research  in  Ischemic  Heart  Disease 

Nine  grants  have  been  awarded  to  investigators  in  this  new  network 
of  specialized  centers ,  replacing  the  Myocardial  Infarction  Research 
Units  (MIRU's)  which  pioneered  in  developing  innovative  and  effective 
techniques  in  basic  and  clinical  research  and  treatment  of  heart  attacks. 
These  new  specialized  centers  have  a  broader  mandate  than  the  MIRU's  and 
are  conducting  basic  and  clinical  research  in  both  acute  and  chronic 
ischemic  heart  disease. 


-  3  - 


d  Specialized  Centers  of  Research  in  Hypertension 

Five  Hypertension  SCOR's  were  established  five  years  ago  and  have 
been  productive  in  the  study  of  the  etiology  and  pathogenesis  of 
hypertension  and  in  the  development  and  application  of  new  knowledge 
essential  for  the  improved  diagnosis  and  management  of  hypertension. 
This  year  a  new  competition  was  held  for  Hypertension  SCOR's.  Fifteen 
applications  were  received  and  are  under  review  and  consideration  for 
i  funding  in  FY  1976. 

5  Specialized  Centers  of  Research  in  Arteriosclerosis 

The  thirteen  current  Arteriosclerosis  SCOR  grants  are  due  to  expire  next  fiscal 
year.  An  evaluation  of  the  current  program  and  activities,  by  NHL I  staff, 
advisors  and  SCOR  participants ,  indicated  that  good  progress  had  been  made  in 
the  integration  of  basic  and  clinical  research  efforts  and  in  establishing 
effective  multidisciplinary  bases  for  the  research  programs.  Based  on  this 
determination  that  the  purposes  and  approach  of  this  program  were  valid,  an 
announcement  for  new  open  competition  for  the  continuation  of  this  program  was 
issued  this  fiscal  year  for  funding  in  FY  1977. 

3  Protecting  Ischemic  Myocardium  and  Minimizing  Infarct  Size 

A  program  focussed  upon  protecting  ischemic  myocardium  was  established  in  1971. 
The  central  importance  of  the  problems  in  this  area  and  the  evidence  that  promising 
)  techniques  and  approaches  could  be  further  rapidly  developed,  led  to  the 
release  of  a  competitive  RFP  last  December;  of  the  57  proposals  received,  13 
were  selected,  after  review  by  outside  experts-  and  the  DHVD  staff,  for  award 
of  contract. 

5  Quantifying  the  Size  of  Infarcted  Myocardium 

Techniques  to  quantify  infarct  size  have  great  usefullness  in  prognosis  as  well 
as  in  assessing  the  efficacy  of  various  proposed  interventions  designed  to  reduce 
or  limit  the  size  of  ischemia,  infarction  or  scarring  of  the  myocardium.  A 
contract  program  in  this  area  has  been  conducted  since  1971.  Review  of  the  needs 
and  progress  in  this  important  area  led  to  the  decision  to  continue  the  program 
and  an  RFP  was  issued  last  December.  Of  the  56  proposals  received  and  reviewed 
by  outside  experts  and  the  staff  of  the  Division,  9  were  selected  for  award 
of  contract. 

■>   Sudden  Cardiac  Death 

J  A  contract  program  in  this  area  has  been  in  existence  since  1970  and  has  been  very 
productive.  Research  in  this  program  is  directed  toward  a  better  understanding 
and  prevention  of  sudden  death  and  includes  in  its  scope  prophylaxis  and  early 


• 


-  4  - 


therapy,  evaluation  of  pathophysiological  mechanisms,  precipitating  factors 
and  recognition  of  risk  factors.  Upon  careful  review  of  the  breadth 
and  scope  of  the  program  it  was  judged  more  appropriate  to  move  the 
continuation  and  expansion  of  this  program  to  the  grant  ledger  rather 
than  to  continue  it  in  the  contract  area.  Applications  for  this  program 
are  currently  under  review. 

o  Development  of  Non- Invasive  Diagnostic  Instrumentation 

In  recognition  of  the  pressing  need  to  improve  the  diagnosis  and 
evaluation  of  asymptomatic  as  well  as  symptomatic  patients  and  to 
enhance  the  effective  evaluation  of  current  treatment  modalities  the 
Division  issued  an  RFP  for  the  development  of  noninvasive  diagnostic 
instrumentation.  Of  the  34  proposals  received  in  response  to  the 
solicitation,  8  have  been  selected  for  funding  this  year  and  a  ninth 
is  under  consideration  for  funding  next  fiscal  year. 

o  Non-Human  Primate  Models  of  Arteriosclerosis,  Hypertension  and 
Dys  1  ipoprot  e  memia 

A  need  of  high  priority  has  been  the  development  of  resources  of 
appropriate  models  in  nonhuman  primates  to  facilitate  research  into 
the  chronic  development  and  regression  of  lesions  and  into  the 
pathophysiology  of  arteriosclerosis,  hypertension  and  dislipopro- 
teinemia. To  meet  that  need  the  Division  issued  an  RFP  for  the 
development  and  supply  of  suitable  animals.  Of  the  sixteen  proposals 
received,  six  have  been  selected  for  funding  this  fiscal  year. 

o  National  Cardiovascular  Research  and  Demonstration  Center  in  Houston 

The  National  Research  and  Demonstration  Center  for  Cardiovascular 
Diseases  was  formally  dedicated  at  Baylor  University  in  Houston,  Texas 
in  March.  This,  the  first  such  Center  in  Cardiovascular  Diseases 
authorized  under  the  Heart  and  Lung  Act  of  1972,  has  a  well  rounded 
and  integrated  program  of  basic  and  clinical  research,  education  and 
demonstration  projects.  This  new  Center  holds  great  promise  of  being 
in  itself  a  model  and  demonstration  of  the  validity  of  the  concept 
that  both  basic  research  and  application  of  research  results  ave 
something  to  gain  from  being  in  close  proximity,  and  that  the  gap 
between  the  bench  and  the  bedside  can  be  narrowed.  An  important  and 
innovative  ingredient  built  into  the  program  of  the  NRDC  is  a  comprehensive 
evaluation  plan  under  which  all  activities  of  the  Center  will  undergo 
periodic  and  searching  evaluation,  by  the  Center  itself,  by  the  NHL I 
and  by  the  scientific  and  medical  communities. 


-  5 


0  National  High  Blood  Pressure  Education  Research  Program 

The  purpose  of  this  program,  now  in  its  second  year,  is  to  foster 
efforts  that  will  result  in  a  better  understanding  of  the  characteristics 
of  an  individual  that  may  relate  to  adherence  behavior  and  the  education 
interventions  that  will  enhance  a  patient's  initial  and  long-term 
adherence  to  hypertensive  therapy.  Six  grants  were  awarded  last  year, 
and  this  year,  in  response  to  the  second  solicitation,  an  additional 
five  grants  were  awarded. 

o  Nutrition  Research  and  Education 

Good  progress  continues  to  be  made  in  the  area  of  nutrition  research  and 
education.  Among  the  activities  of  this  fiscal  year  was  a  Nutrition - 
Behavior  Conference  which  brought  together  the  investigators  involved  in 
nutrition  studies  from  the  Lipid  Research  Clinics,  Multiple  Risk  Factor 
Intervention  Trial,  Specialized  Centers  of  Research  Program  and  the  Cardio- 
vascular Research  and  Demonstration  Center  to  share  the  experience  and  results 
of  these  studies.  The  nutrition  teaching  materials  which  have  been  produced 
within  these  programs  are  expected  to  have  wide-spread  general  value.  The 
common  LRC-MRFIT  system  for  processing  diet  information  and  calculating 
nutrient  values  has  been  placed  in  operation  at  the  Nutrition  Coding  Center 
at  the  University  of  Minnesota. 

o  Education  and  Pemonstration  Programs 

Several  components  of  the  Division's  program  in  education  and  demonstration 
are  described  elsewhere  in  this  report,  namely  the  National  Research  and 
Demonstration  Center  and  the  High  Blood  Pressure  Education  Research  Program. 
A  report  on  the  opportunities  and  needs  in  cardiac  rehabilitation  was  prepared 
and  issued  by  a  Task  Force  established  by  the  Division.  As  a  beginning 
step  in  implementation  of  that  report  the  Division  is  planning,  with  the 
help  of  expert  consultants  a  baseline  survey  of  knowledge,  attitudes  and 
behavior  on  the  part  of  physicians  and  their  patients  vis  a  vis  cardiac 
rehabilitation.  The  National  High  Blood  Pressure  Education  Program  continues 
to  make  solid  progress.  Over  100  National,  State  and  local  organizations, 
including  voluntary  groups,  are  involved  in  this  activity,  under  the  leadership 
of  the  Division.  Public  awareness  is  increasing  about  the  importance  of 
regular  blood  pressure  checks  and  of  regular  taking  of  medicine  by  persons 
found  to  be  hypertensive.  Many  communities  have  launched  high  blood  pressure 
screening  and  follow-up  programs.  Over  300  communities  have  received  assistance 
and  materials  from  the  Division's  High  Blood  Pressure  Information  Center. 
In  addition  the  Center  has  responded  to  well  over  a  half  million  requests 
for  information  and  has  developed  and  is  operating  an  ongoing  mass  media 
education  campaign  which  has  presented  education  messages  to  an  estimated 
75  percent  of  the  Nation's  population.  Much  remains  to  be  done  in  this  area, 
however,  particularly  with  respect  to  increasing  adherence  to  available 
treatment  on  the  part  of  persons  with  high  blood  pressure. 


Clinical  Trials 

This  year  one  of  the  Division's  Clinical  Trials  came  to  its  scheduled  end  and 
another  began,  bringing  to  five  the  number  of  large  scale  Clinical  Trials  the 
Institute  is  currently  supporting.  Brief  descriptions  of  their  purpose  and  status 
follow. 

The  Coronary  Drug  Project  ended  this  year  in  accordance  with  its  planned  schedule. 
This  study,  begun  over  eight  years  ago,  evaluated  the  efficacy  of  several  lipid 
lowering  drugs  in  patients  who  had  had  at  least  one  heart  attack.  Two  years  ago 
three  of  the  drugs  were  dropped  from  the  study  because  they  had  adverse  effects 
without  compensating  benefits.  Neither  of  the  two  drugs  that  were  continued  for  the 
entire  duration  of  the  study,  clofibrate  and  nicotinic  acid,  had  any  effect  on 
mortality.  Clofibrate,  in  fact  was  associated  with  a  high  degree  of  cardiovascular 
morbidity  and  while  nicotinic  acid  decreased  angina  and  new  heart  attacks  it 
was  associated  with  frequent  side  effects.  These  results  have  been  widely  cir- 
culated in  the  medical  literature.   It  should  be  emphasized  that  these  negative 
findings  refer  only  to  patients  who  have  had  previous  heart  attacks,  and  do  not 
indicate  whether  the  two  drugs  are  useful  for  persons  who  have  not  had  any 
heart  attack.  Despite  the  disappointing  negative  results,  the  study  was  extremely 
worthwhile  in  two  respects :  first ,  it  should  result  in  lower  costs  to  patients 
who  will  not  have  these  drugs  prescribed,  and  second,  the  information  obtained  on 
the  natural  history  of  myocardial  disease  is  very  useful. 

The  Hypertension  Detection  and  Follow-Up  Program  is  a  multicenter  cooperative 
effort  designed  to  determine  the  effectiveness  of  systematic  antihypertensive 
therapy  in  reducing  clinical  morbidity  and  mortality  in  persons  with  elevated 
blood  pressure  in  fourteen  community-based  populations.  Participants  include 
men  and  women  between  the  ages  of  30-69,  many  with  mild  hypertension  as  well  as 
those  with  moderate  and  severe  disease,  in  a  cross -section  of  major  ethnic  and 
racial  groups  and  socio-economic  strata.  More  than  11,300  hypertensive  partici- 
pants have  been  randomized  to  either  stepped  care  or  referred  care.  "Stepped 
care"  is  a  series  of  carefully  controlled  drug  regimens  for  persons  who  are 
assigned  to  HDFP  clinics  for  their  hypertension  medication.  Persons  not  selected 
for  "stepped  care"  are  referred  to  their  own  physicians  for  treatment,  and  are 
in  the  "referred  care"  group.  At  12  months  after  baseline,  approximately  761  of 
the  stepped  care  participants  are  maintaining  active  participation  and  mean 
diastolic  pressure  reduction  is  13.8  mm  Hg.  About  501  of  the  referred  care 
participants  are  also  under  therapy  and  the  mean  reduction  of  diastolic  blood 
pressure  is  7.3  mm.  More  energetic  efforts  will  be  needed  to  increase  the  pro- 
portion of  the  stepped  care  group  who  reach  goal  blood  pressure  because  of  the 
greater  than  expected  treatment  effect  in  the  referred  care  group. 

The  Multiple  Risk  Factor  Intervention  Trial  is  designed  to  study  whether  the 
reduction  of  serum  cholesterol ,  reduction  of  elevated  blood  pressure  and 
elimination  or  reduction  of  cigarette  smoking  will  produce  a  significant 
reduction  in  morbidity  and  mortality  from  coronary  heart  disease.  Twenty 


-  7  - 


clinical  centers  are  each  screening  125000  to  20,000  men  aged  35-57  to  identify 
and  enroll  a  total  of  12,000  men  who  are  at  increased  risk  of  developing  coronary 
heart  disease  because  of  combination  of  these  three  major  risk  factors.  Over 
7,000  of  the  12,000  participants  needed  for  the  trial  have  been  enrolled  so  far 
and  the  primary  recruitment  phase  of  the  study  is  expected  to  be  completed  by 
January  1,  1976.  The  study  period  for  each  person  in  the  Trial  is  to  be  six 
years  from  enrollment. 


Th 


e  Lipid  Research  Clinics  Coronary  Primary  Prevention  Trial: is  designed  to 
test  the  hypothesis  that  lowering  the  levels  of  lipids  in  the  blood  (by  diet 
and  drugs)  will  decrease  the  incidence  of  heart  attacks  and  heart  attack  deaths. 
Twelve  clinics  are  conducting  this  study,  which  will  involve  4000  male  subjects 
who  have  high  blood  levels  of  lipids  but  who  have  not  had  a  heart  attack. 
Over  1200  subjects  have  already  been  randomized  into  the  study,  and  it  is  expected 
that  recruitment  will  be  completed  by  January,  1976.  The  study  is  scheduled 
to  run  for  7  years. 

The  Coronary  Arteiy  Surgery  Trial  has  been  in  the  planning  and  protocol  prepara- 
tion  phase  for  the  past  two  years  and  will  be  entering  its  full  operational  phase 
late  this  fiscal  year.  Twelve  clinical  centers  will  evaluate  coronaiy  artery 
bypass  graft  surgery  by  a  series  of  randomized  studies  and  a  patient  registry. 
Over  25,000  such  operations  are  being  performed  each  year.  This  study  hopes 
to  provide  careful  assessment  of  the  clinical  conditions  in  which  such  surgery 
is  appropriate  and  compare  the  results  of  surgery  with  non-surgical  medical 
\ treatment,  both  in  terms  of  morbidity  and  mortality. 

The  Aspirin -Myocardial  Infarction  Study  was  initiated  this  year.  It  has  been 
observed  in  a  number  of  retrospective  studies  that  regular  ingestion  of  aspirin 
was  associated  with  lower  incidence  of  heart  attacks.  This  study  is  a  carefully 
designed  prospective  study  of  the  possible  efficacy  of  aspirin  as  a  preventive 
for  recurrent  heart  attacks  and  heart  attack  death  among  persons  who  have  had 
at  least  one  such  attack.  Thirty  clinical  centers  are  participating  in  this 
study,  which  will  involve  4000  men  and  women,  each  of  whom  will  be  followed 
for  at  least  3  years.  ." 

Evaluation  Studies  in  Clinical  Trials 

The  size  and  importance  of  the  Division's  clinical  trials  make  it  imperative 
that  the  Division  ensure  that  they  are  being  conducted  in  as  effective  and 
efficient  manner  as  possible,  from  both  the  technical,  scientific  and  the 
administrative  points  of  view.  Accordingly  the  Division  proposed  and  received 
authorization  from  HEW  to  conduct  evaluation  studies  of  two  aspects  of  the 
'trials:  the  central  laboratories  that  perform  the  laboratory  analysis  of  blood, 
urine,  lipids,  etc.,  and  the  central  coordinating  centers  that  play  a  key  role 
in  the  accumulation,  processing  and  analysis  of  the  large  amount  of  data  that 
are  generated  in  the  studies.  The  evaluation  of  the  central  laboratories  was 
started  in  the  Spring.  Plans  for  the  evaluation  of  the  coordinating  centers 
are  in  process  and  it  is  expected  that  an  RFP  for  the  conduct  of  the  study 


will  be  issued  shortly.  These  evaluation  studies,  in  which  outside  experts 
not  directly  involved  in  the  operations  of  the  centers  will  provide  advice 
and  recommendations  to  the  Division,  will  be  valuable  not  only  with  respect 
to  the  current  trials  but  also  with  respect  to  future  trials  in  this  Division 
or  elsewhere. 

Manpower  Programs 

)Since  its  inception  last  year,  the  Manpower  Branch  has  been  concerned  with  the 
Weinberger  Postdoctoral  Fellowship  Program,  Impoundment  Restoration  Order, 
Stipend  Equalization,  National  Research  Service  Award  Programs  and,  finally,  the 
recent  administration  moratorium  on  FY  76  Institutional  Fellowship  Awards.  The 
Division  plans  to  make  84  new  Individual  Fellowship  Awards,  25  new  Institutional 
Fellowship  Awards  and  25  new  Career  Development  Awards  in  Fiscal  Year  1975.  The 
cardiovascular  community,  and  of  even  more  importance  the  young  potential  cardio- 
vascular research  scientists,  are  being  confused  if  not  demoralized,  by  the  ever 
changing  Federal  plans  and  regulations  on  fellowship^ support.  It  is  hoped  that 
some  order  and  stability  can  be  brought  into  this  area  in  the  next  year. 


-  9 


ANNUAL  REPORT 

OF 

DIVISION  OF  BLOOD  DISEASES  AND  RESOURCES,  NHLI 

July  1,  1974  through  June  30,  1975 


The  programs  of  the  Division  of  Blood  Diseases  and  Resources  seek  to 
improve  the  diagnosis,  prevention,  treatment,  and  cure  of  diseases  of 
the  blood  and  related  disorders,  and  to  improve  utilization  of  the 
nation's  blood  resources.   These  programs  encompass  basic  research,  targeted 
applied  research,  clinical  trials  and  demonstrations  and  applications  of 
these  research  findings  in  four  programmatic  areas:   bleeding  and  clotting 
disorders,  sickle  cell  disease  and  related  disorders,  blood  resources, 
and  biomaterials  research  and  development.   The  Division  continually 
assesses  the  national  needs  for  research  in  these  areas  and  develops  and 
supports  programs  to  address  those  needs  through  the  research  grants  and 
contracts.   Some  highlights  of  accomplishments  of  the  past  year  are 
briefly  mentioned  below. 

The  Division  continues  to  maintain  interest  in  hemophilia  as  a 
major  portion  of  its  research  in  bleeding  and  clotting  disorders.   The 
understanding  of  the  molecular  nature  of  Factor  VIII  continues  to  increase. 
It  is  now  appreciated  that  most  hemophiliacs  have  an  abnormal  Factor  VIII 
molecule  which  does  not  permit  adequate  blood  clotting.   Data  derived 
from  research  on  this  aspect  of  hemophilia  has  allowed  the  design  of  a 
scheme  to  diagnose  hemophilia  carriers.   A  special  workshop  was  coordinated 
and  sponsored  by  the  Blood  Division  and  the  National  Hemophilia  Foundation   . 
to  study  the  adequacy  of  this  method.   Hemophilia  B,  or  Factor  IX  deficiency, 
must  be  treated  with  a  plasma  fraction  different  from  that  used  for 
Hemophilia  A,  or  Factor  VIII  deficiency.   The  Factor  IX  concentrates  have 
been  associated  with  a  high  risk  of  thrombotic  complications.   Recent 
studies  have  helped  clarify  the  nature  of  this  problem  and  led  to  methods 
designed  to  overcome  them.   A  major  problem  in  the  care  of  the  Factor  VIII 
deficient  hemophiliac  has  been  the  spontaneous  appearance  of  inhibitors 
to  Factor  VIII.   A  study  of  the  incidence  and  clinical  importance  of  these 
inhibitors  has  been  designed  by  the  E.lood  Division  and  will  be  implemented 
early  in  fiscal  year  1976.   It  is  hoped  that  the  information  obtained  will 
improve  treatment  of  patients  with  inhibitors  and  reduce  the  strain  on  the 
blood  resource  created  by  these  patients. 

Studies  on  the  mechanism  of  thrombosis  and  means  for  its  prevention 
and  control  are  being  pursued  at  many  levels.   There  is  reason  to  hope  that 
blood  tests  will  be  developed  to  identify  patients  with  a  high  risk  of 
thrombotic  problems.   Non-invasive  diagnostic  methods,  utilizing  physical 
and  chemical  means,  have  been  developed  to  diagnose  thrombotic  lesions  in 
their  early  stages  of  development.   These  methods  have  been  utilized  to 
perform  clinical  trials  which  have  demonstrated  that  certain  anticoagulant 
and  antiplatelet  agents  can  prevent  the  formation  of  deep  venous  thrombosis 
and  perhaps  pulmonary  embolism.   The  status  of  this  field  was  recently 
summarized  at  a  workshop  sponsored  by  the  Blood  Division  and  the  American 
Heart  Association.   The  information  issuing  from  that  workshop,  which  will 


be  published  and  available  to  the  community,  will  help  in  the  formulation 
of  future  research  plans  and  development  of  educational  programs  for  the 
biomedical  community. 

Patients  undergoing  orthopedic  surgery  are  particularly  susceptible  to 
postoperative  thromboembolic  complications.   These  patients  have  shown  the 
least  promising  results  with  anticoagulant  and  antiplatelet  agents.   The 
Blood  Division  is  supporting  four  clinical  trials  utilizing  such  agents 
alone  and  in  combination  to  clarify  the  status  of  this  problem.   These 
studies  are  underway  and  should  be  completed  within  one  year. 

The  Division's  interest  in  Cooley's  Anemia  (thalassemia)  has  resulted 
in  the  funding  of  a  clinic  to  test  and  evaluate  new  techniques  of  screening 
and  genetic  counseling.   Fundamental  research  is  also  being  funded  from 
which  abnormal  sub-cellular  mechanisms  are  elucidated  in  the  red  cells  of 
thalassemia  patients.   The  problem  of  iron  overload,  which  develops  as  a 
result  of  chronic  transfusions  of  thalassemia  patients,  has  necessitated 
a  search  for  iron  chelating  agents.   Presently,  a  clinical  trial  of  the 
iron  chelator,  Desf errioxamine,  is  under  consideration. 

As  part  of  the  Division  research  emphasis  on  sickle  cell  disease  and 
related  disorders,  studies  of  the  ant i-sickling  agent  cyanate  indicated 
tuat  this  agent,  when  given  by  the  oral  or  by  the  parenteral  route,  is 
associated  with  peripheral  neuropathy,  characterized  by  an  alteration  of 
the  nerve  conduction  time  and  the  development  of  abnormal  reflexes.   These 
findings  have  resulted  in  discontinuance  of  clinical  trials  with  oral 
cyanate.   In  the  meantime,  extracorporeal  studies  utilizing  cyanate  are 
being  continued.   A  number  of  other  anti-sickling  agents  are  also  being 
investigated.   These  studies  are  in  the  early  developmental  stage  and  it  is 
too  early  to  determine  possible  success  of  these  agents. 

Efforts  continue  to  develop  more  effective  and  economical  procedures 
to  identify  abnormal  hemoglobins  during  the  prenatal  period,  at  birth  and 
at  later  times  in  life.   Procedures  to  identify  hemoglobin  A,  S  and  F  during 
the  prenatal  period  have  been  developed  and  are  now  being  refined.   A 
micro-column  chromatography  procedure,  which  was  developed  a  year  ago  and 
is  presently  being  field  tested,  will  allow  for  the  identification  of 
hemoglobin  An  which  will  perhaps  make  a  procedure  available  to  identify 
a  carrier  state  of  Cooley's  Anemia. 

Recent  accomplishments  in  the  development  of  blood  compatible  bio- 
materials  include  the  expanded  and  improved  capability  for  biological 
testing  and  evaluation  of  candidate  materials.   Also,  a  workshop  on  extra- 
corporeal membrane  oxygenators  was  jointly  sponsored  and  coordinated  with 
the  Division  of  Lung  Diseases  which  reviewed  the  current  state  of  the  art 
and  formulated  clinical  and  research  problems  on  the  relation  of  blood 
elements  to  membrane  oxygenators. 

In  the  area  of  blood  resources,  the  Division's  efforts  have  continued 
to  play  a  catalytic  role  vis-a-vis  the  American  Blood  Commission,  initially 


in  its  formation,  and  presently  in  the  support  of  its  task  groups,  which 
will  have  a  major  role  in  providing  the  data  upon  which  the  Commission  will 
implement  the  goals  of  the  National  Blood  Policy.   Other  activities  include 
clinical  trials  of  hepatitis  B  immune  globulin  as  passive  immunization  for 
populations  at  high  risk  of  contracting  hepatitis  B.   These  trials  are 
nearing  completion;  results  will  be  reported  early  in  FY'76.   A  prospective 
epidemiological  study  of  post-transfusion  hepatitis  has  been  started  to 
assess,  on  an  ongoing  basis,  the  nature  and  extent  of  this  problem  as  we 
move  towards  the  implementation  of  an  all-volunteer  blood  donation  system. 
During  this  year  the  Division  has  supported  the  development  of  prototype 
stei'ile  connecting  devices  between  plastic  containers.   Such  devices  have 
many  applications  in  blood  banking  practice,  one  of  which  is  the  extension 
of  the  outdating  period  for  frozen-thawed  red  cells. 

The  Division,  as  a  major  focus  for  research  and  clinical  training  in 
hematology  and  related  areas  including  blood  banking  sciences,  currently 
supports  over  180  trainees.   Two  coordinated  studies  are  being  initiated  to 
assess  the  further  national  training  needs  in  these  areas. 


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DIVISION  OF  LUNG  DISEASES 
ANNUAL  REPORT 
July  1,  1974  through  June  30,  1975 

The  Division  of  Lung  Diseases  plans,  directs  and  evaluates  extramural  pro- 
grams addressed  to  pulmonary  diseases  and  respiratory  disorders.   Through 
four  Branches  (Etiology,  Pathophysiology,  Special  Programs  and  Resources, 
and  Centers  and  Control  Programs)  responsible  for  both  grants  and  contracts, 
the  Division  sponsors  programs  for  (1)  Research  and  Development,  (2)  Man- 
power, (3)  Research  and  Demonstration  Centers  and  (4)  Prevention,  Control  and 
Education.   These  activities  are  supported  through  investigator-initiated  re- 
search and  program  project  grants,  goal-oriented  center  grants,  manpower  de- 
velopment awards  and  targeted  research  contracts.   Every  effort  is  made  to 
achieve  a  coordinated  program  and  all  goal-oriented  and  targeted  programs  are 
evaluated  at  regular  intervals  by  both  the  Division's  staff  and  the  Pulmonary 
Diseases  Advisory  Committee,  frequently  with  the  advice  of  ad  hoc  consultants. 
Program  evaluations  are  presented  to  the  National  Heart  and  Lung  Advisory 
Council  and  Institute  staff. 

Tangible  accomplishments  are  evident  in  the  Division's  effort  to  interest  and 
involve  investigators  from  basic  disciplines,  and  to  foster  fundamental  stud- 
ies1 of  the  lung  in  health  and  disease.   Interdisciplinary  research  with  a  ma- 
jor emphasis  on  basic  problems  is  now  supported  through  17  program  project 
grants,  an  increase  of  42  percent  since  fiscal  1974.   The  Young  Investigator 
Pulmonary  Research  Grant  has  attracted  new  investigators  to  the  pulmonary  field 
and.it  is  gratifying  to  note  that  this  year,  as  was  the  case  in  1974,  approx- 
imately half  of  the  new  grants  are  addressed  to  lung  structure  or  function. 
National  Research  Service  Institutional  Grants  and  Postdoctoral  Fellowship 
Awards  approved  this  year  are  also  largely  concerned  with  fundamental  problems. 
Contract  programs  addressed  to  various  aspects  of  lung  structure  and  function 
(for  example,  studies  of  lung  elastin,  and  separation .and  culturing  of  lung 
cells)  have  stimulated  interest  in  fundamental  approaches  that  are  now  being 
extended,  through  issuance  of  requests  for  contract  proposals,  to  studies  of 
respiratory  mucin,  pulmonary  fibrosis  in  animal  models,  and  the  development 
of  markers  for  various  types  of  individual  lung  cells.   In  addition,  the  Divi- 
sion continues  to  support  fundamental  as  well  as  clinical  research  through  its 
Specialized  Centers  of  Research,  and  the  investigator-initiated  research  grant 
continues  to  be  addressed  in  large  part  to  basic  investigations. 

As  Specialized  Centers  of  Research  (SCOR)  grants  are  approaching  the  time  for 
competitive  renewal,  the  Division  has  announced  a  new  Pulmonary  SCOR  competi- 
tion that  invites  new  .applicants  as  well  as  renewal  of  present  active  grants. 
On  the  basis  of  four  years  of  experience  with  this  supportive  mechanism  and 
consonant  with  the  objectives  of  the  Division's  National  Plan,  this  competi- 
tion requires  each  SCOR  to  be  limited  to  one  of  four  major  disease  categories; 
namely,  Chronic  Airways  Diseases,  Pediatric  Pulmonary  Diseases,  Fibrotic  and 
Immunologic  Pulmonary  Diseases,  and  Pulmonary  Vascular  Diseases.   It  also  re- 
quires a  clinical  emphasis  but  strongly  encourages  fundamental  investigations 
relevant  to  the  SCOR  goals.   The  Division  believes  that  the  SCOR  mechanism 

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should  not  duplicate  opportunities  available  through  other  supportive  mecha- 
nisms, such  as  the  program  project  grant.   Moreover,  institutions  with  strong  "~ 
programs  in  more  than  one  disease  category  are  encouraged  to  submit  more  than 
one  SCOR  application. 

The  Division's  Prevention,  Control  and  Education  Program  has  been  initiated 
with  two  groups  of  contracts  for  educational  programs  addressed  to  recogni- 
tion and  early  treatment  of  acute  respiratory  insufficiency  in  adults   and 
neonatal  respiratory  distress  syndrome.   Other  demonstration  and  education 
activities  are  supported  through  the  Vermont  Research  and  Demonstration  Lung 
Center. 

Because  pulmonary  academic  manpower  is  still  insufficient  to  meet  national 
needs,  the  Division  initiated  a  new  program — the  National  Pulmonary  Faculty 
Training  Program — designed  to  enable  institutions  with  inadequate  pulmonary 
academic  staffs  to  capitalize  on  resources  for  excellent  training  at  other 
institutions.   This  is  a  two-phase  program.   The  first  phase  has  been  com- 
pleted with  the  review  of  applications  from  institutions  wishing  to  provide 
pulmonary  training.   The  Division  anticipates  (by  August  1,  1975)  inviting 
applications  from  medical  schools  that  wish  to  nominate  junior  faculty  mem- 
bers who  will  receive  training  at  one  of  the  approved  Training  Centers. 

The  Division  is  strongly  committed  to  continuing  evaluation  of  its  goal- 
oriented  and  targeted  programs.   During  the  year  it  has  drawn  upon  ad  hoc 
consultants  as  well  as  the  Pulmonary  Diseases  Advisory  Committee  to  assess 
the  Pulmonary  SCOR,  Pulmonary  Academic  Award,  Contract  and  Epidemiology 
Programs  as  well  as  a  completed  group  of  contracts  on  Oxygen  Toxicity.   Re- 
view of  the  Epidemiology  Contracts  and  of  the  Pulmonary  Academic  Award  Pro- 
gram involved  on-site  visits.   In  addition,  the  Pulmonary  Diseases  Advisory 
Committee  visited  the  Vermont  Research  and  Demonstration  Center  in  association 
with  the  regular  Committee  "meeting. 

To  coordinate  the  different  facets  of  its  pr'ogram  the  Division  sponsors  small 
workshops,  which  involve  SCOR  investigators,  contractors,  grantees  and  expert 
consultants,  for  discussions  of  issues  that  are  timely  and  important  relative 
to  the  Division's  National  Program.   The  Division  also  works  with  national 
societies  in  arranging  symposia  at  their  annual  meetings.   Reports,  already 
issued  or  in  preparation,  on  workshops  held  since  the  last  Annual  Report  are 
these:  Hematological  Analysis  of  Extracorporeal  Membrane  Oxygenation,    Lung 
Metabolism,    and  Lung  Cell   Separation,   Identification   and  Culture.      A  sympo- 
sium sponsored  jointly  with  the  American  College  of  Chest  Physicians  was  ad- 
dressed to  the  use  of  extracorporeal  membrane  oxygenation  in  acute  respira- 
tory failure. 

For  the  information  of  the  biomedical  community,  the  Division  distributed 
reports  on  its  Contract  Program    (updated) ,  Current  Pulmonary  Research  Pro- 
grams   (updated) ,  and  Bioeng ineer ing  Program;    as  well  as  Procedures   for 
Standardized  Measurements   of  Lung  Mechanics   and  Principles   of  Body   Plethys- 
mography,   and  Report  of  Conference  on  Scientific  Basis  of  Respiratory   Therapy 


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Finally,  the  Division  held  meetings  of  two.  steering  groups  to  obtain  expert 
consultation  in  developing  its  plans  for  two  small  task  groups  to  be  initiated 
in  the  Fall.   One,  addressed  to  Epidemiology  of  Respiratory  Diseases,  the  other 
to  Prevention,  Control  and  Education  in  Pulmonary  Diseases,  will  advise  the 
Division  on  what  is  known,  what  is  being  done,  what  needs  to  be  done,  and  how 
the  Division  can  best  contribute  to  these  two  important  areas. 

The  Division's  major  problem  is  the  same  as  last  year;  namely,  to  acquire  a 
professional  staff  of  appropriate  background  and  adequate  size  to  monitor 
responsibly  its  complex  and  growing  programs. 


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NATIONAL  HEART  AND  LUNG  INSTITUTE 

DIVISION  OF  EXTRAMURAL  AFFAIRS 

Annual  Report 

July  1,  1974  -  June  30,  1975 


The  Division  of  Extramural  Affairs  is  responsible  for  advising  the  Director, 
NHLI,  on  research  contract,  grant,  and  training  program  policy.   It  also 
represents  the  Institute  on  overall  NIH  extramural  and  collaborative  program 
policy  committees,  coordinates  such  policy  within  NHLI,  and  coordinates  the 
Institute's  research  grant  and  training  programs  with  the  National  Heart  and 
Lung  Advisory  Council.   Other  major  services  provided  by  the  Division 
include:   (a)  grant  and  contract  management  and  processing  services  for  the 
Institute's  other  divisions,  (b)  reports  and  statistics  related  to  the 
Institute's  grant  and  contract  programs,  and  (c)  initial  scientific  merit 
review  of  project  grants  and  research  contracts  for  the  Institute. 

Increased  emphasis  was  placed  upon  improved  implementation  of  the  Freedom  of 
Information  Act  and  the  recently  enacted  Privacy  Act  amendments  scheduled  to 
become  effective  later  this  year.  It  therefore  became  essential  that  the 
Institute  centralize  these  activities.  The  Acting  Director  of  the  Institute 
appointed  a  Freedom  of  Information  Officer  in  the  Division  of  Extramural 
Affairs  to  accomplish  this.  An  index  of  Institute  documents  affecting  grant 
and  contract  programs  was  developed,  and  inquiries  for  technical  information 
are  now  channeled  through  this  officer. 

The  Division  has  continued  to  serve  as  the  primary  liaison  to  the  National 
Heart  and  Lung  Advisory  Council.   In  collaboration  with  the  Council,  several 
mechanisms  were  developed  which  should  help  the  Division  to  more  efficiently 
carry  out  its  responsibilities.   One  of  these  are  criteria  that  define  the 
Program  Project  Grant  as  conceptualized  and  utilized  by  the  NHLI.   Guide- 
lines have  also  been  implemented  for  the  individual  review  of  Program  Proj- 
ects by  the  Council.   The  Council  has  asked  that  criteria  for  the  support  of 
conferences  also  be  developed  for  their  use. 

The  Division  continues  to  provide  broad  services  to  the  rest  of  the  Insti- 
tute.  These  include: 

1.  Management  requirements  for  grants  and  contracts. 

2.  Central  storage  and  maintenance  of  official  files  for  all 
grant  programs. 

3.  Preparation  of  review  materials  for  Council. 

4.  Preparation  of  official  and  summary  minutes  of  Council  actions. 

5.  Follow-up  and  close-out  for  all  terminated  grants. 

6.  Operation  of  the  program  Policy  and  Procedures  Office. 

7.  Committee  management  functions. 

The  initial  technical  merit  review  of  research  grant  applications  and 
research  contract  proposals  has  continued  to  result  in  markedly  increased 
responsibilities.   The  types  of  reviews  in  the  grant  program  included: 


Clinical  Trials 

Conference  Grants 

High  Blood  Pressure  Education  Research  Program 

Hypertension  SCOR  Grants 

Institutional  Fellowship  Grants 

Ischemic  Heart  Disease  SCOR  Grants 

National  Pulmonary  Faculty  Training  Program 

Program  Project  Grants 

Pulmonary  Academic  Awards 

Sudden  Cardiac  Death  Research  Grants 

Thrombosis  SCOR  Grants 

Young  Pulmonary  Investigator  Program 

In  addition,  sixteen  contract  reviews  were  held  directly  related  to  new  RFPs 
issued  by  the  Institute,  and  nine  renewal  contract  reviews  were  held.   Thus, 
a  total  of  25  review  committees  were  convened  to  review  contracts  during 
Fiscal  Year  1975.   Furthermore,  nine  mail  reviews  related  to  unsolicited  or 
sole  source  proposals  were  conducted  by  the  Review  Branch. 

In  the  Reports  and  Evaluations  Branch,  the  basic  NHLI  Information  System 
became  operational.   It  consists  of  several  components  which  may  be  linked 
to  provide  (a)  data  relating  to  the  management  of  grants  and  contracts, 
(b)  data  related  to  research  and  development  activities  supported  by  con- 
tracts and  grants,  as  well  as  (c)  data  about  the  major  programs  and  program 
areas  in  each  division.   It  is  expected  that  the  basic  system,  which  is 
currently  on  tape,  will  soon  be  converted  to  an  on-line  disc.   This  will 
shorten  response  (turn  around)  time.   Overall  development  of  the  data  system 
continues  with  the  incorporation  of  specific  items  based  on  the  definition 
of  needs  by  program  managers  and  administrative  staff  of  the  Institute. 
These  changes  have  broadened  the  capability  of  the  system  and  have  resulted 
in  increased  use  by  program  and  administrative  staff.   With  basic  operations 
in  the  Reports  and  Evaluations  Branch  finally  underway,  increased  attention 
will  be  placed  on  analytical  and  evaluative  functions  in  relation  to  the 
Institute's  overall  programs. 

The  morale  of  the  Division  staff  has  been  adversely  affected  by  increased 
individual  workloads  coupled  with  strict  hiring  limitations.   In  addition, 
lack  of  adequate  working  space  in  the  Westwood  Building  has  aggravated  the 
morale  situation.   The  lack  of  sufficient  space  has  resulted  in  (a)  dis- 
placement of  one  supervisor  from  her  office  in  order  that  it  could  be  used 
for  temporary  storage,  (b)  conversion  of  office  space  for  a  professional 
staff  member  into  a  storage  and  processing  room  during  peak  workload  periods, 
and  (c)  continued,  and  valid,  complaints  from  the  Montgomery  County  Fire 
Marshal,  as  well  as  neighboring  organizations,  because  of  cluttered  hallways. 

The  increase  in  the  Institute's  programs  and  the  Division's  workload  has 
also  brought  about  a  significant  increase  in  grant  and  contract  reviews, 
management  procedures,  and  coordinating  activities.   Although  the  Division 
has  continued  to  meet  its  deadlines,  unless  additional  positions  are  made 
available  and  our  physical  space  allocation  is  increased,  the  real 
possibility  exists  that  future  deadlines  may  not  be  met  and  the  morale  of 
the  staff  will  continue  to  deteriorate. 


It  is  anticipated  that  next  year  will  bring  an  increase  both  in  the  workload 
and  responsibilities  of  the  Division.   We  are  therefore  studying  possible 
internal  reorganizations  which  may  increase  the  efficiency  of  our  use  of 
existing  space  and  personnel.   However,  without  an  increase  in  our  current 
personnel  ceiling  and  space  allocation,  it  is  doubtful  that  we  can  continue 
to  carry  out  our  responsibilities  in  the  highly  professional  manner  that  has 
been  characteristic  of  this  Division. 


INTRAMURAL  RESEARCH 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

July  1,  1974  -  June  30,  1975 


INTRAMURAL  RESEARCH 
Project  Reports 

Laboratory  of  Biochemical  Genetics 

Summary 1 

Acetylcholine  receptors 5 

Morphine  receptors  as  regulators  of  adenylate  cyclase 7 

Regulation  of  Receptor  Activity 9 

Studies  of  action  potential  and  receptor  ionophores 10 

The  biosynthesis  of  neurotransmitters  in  cell  hybrids 12 

Ultrastructure  of  neuroblastoma  somatic  cell  hybrids 14 

Ornithine  decarboxylase  induction  in  neural  cells 16 

Protein  phosphorylation  in  neuroblastoma  cells 17 

Gene  expression  for  neural  properties 18 

Naturally  occurring  anti-tumor  antibodies 20 

Glutamic  acid  decarboxylase  in  developing  chick  retina 22 

Muscarinic  acetylcholine  receptors  in  cultured  cell  lines 23 

Developmental  regulation  of  excitability 25 

Genetics  of  cyclic-AMP  metabolism 28 

Expression  of  type  C  virus  in  human-mouse  cell  hybrids 29 

Chromosome  segregation  in  hybrid  cells 31 

Genetic  analysis  of  differentiation  using  cell  hybrids 33 

Genetic  analysis  of  hyperlipidemias  using  cell  hybrids 34 

Bacteriophage  resistance  in  transformed  Bacillus  subtilis 35 

Type  C  virus  particles  in  normal  and  diseased  humans 37 

The  biology  of  cyclic  nucleotides  in  E^  coli 39 

Mechanism  in  protein  synthesis 41 

Laboratory  of  Biochemistry 

Section  on  Enzymes 

.  Summary 43 

Metabolism  of  the  branched-chain  amino  acids 47 

Kinetics  and  mechanisms  of  biochemical  reactions 50 

Cellular  regulation  of  enxyme  levels 58 

Regulation  of  the  cascade  control  of  E_^  coli  glutamine  synthetase 61 

Regulation  of  glutamine  synthetase 64 

Biochemical  genetics  of  NH3-assimilatory  enzymes  in  E^_  coli  K^2 66 

Metabolite  regulation  of  coupled  covalent  modification  cascase  systems-  69 

Enzyme  degradation  in  Klebsiella  aerogenes 73 

Section  on  Intermediary  Metabolism  and  Bioenergetics 

Summary 75 

Role  of  selenium  in  anaerobic  electron  transport.   CH4  biosynthesis 77 

Stereochemical  studies  of  enzymatic  reactions 81 

Characterization  of  a  bacterial  selenoprotein 84 

Electron  transport  system  associated  with  proline  metabolism 86 

Menadione-dependent  p-nitrophenylphosphatase  of  Clostridium  sticklandii  87 

Section  on  Protein  Chemistry 

Summary 89 

Protein  structure:  Enzyme  action  and  control 91 


Laboratory  of  Cell  Biology 

Summary 

Electron  transport  in  E^   coli  and  rat  liver  mitochondria 103 

DNA  synthesis  in  E\_  coli 106 

Differential  scanning  calorimetry  of  fibrinogen 110 

Proteolytic  fragmentation  of  fibrinogen HI 

Circular  dichroism  studies  on  reduced  alkylated  lysozyme 114 

Tritium  labeling  of  binding  site  residues 116 

Interaction  of  SHj-blocked  myosin  with  actin  and  ATP 119 

Actin-myosin  interaction:  Control  by  native  tropomyosin 121 

The  interaction  of  actin  and  myosin 124 

Mechanism  of  myosin  and  actomyosin-Mg-ATPase 127 

Acanthamoeba  actin:   Isolation  and  role  in  cell  motility 131 

Acanthamoeba  myosin  cof actor:   Isolation  and  function 134 

Plasma  membrane  and  phagosome  membranes  of  Acanthamoeba 136 

Cytology  of  Acanthamoeba 140 

Structure,  assembly  and  function  of  microtubules 145 

Laboratory  of  Cellular  Metabolism 

Summary 151 

Regulation  of  lipid  synthesis  in  mammalian  cells 157 

Histamine  release  from  mast  cells;  immediate  hypersensitivity 160 

Regulation  of  rat  liver  phosphodiesterases 163 

Inhibition  of  histamine  metabolism  by  salicylates 165 

Cyclic  nucleotide  metabolism  in  human  umbilical  artery 167 

Cyclic  nucleotide  metabolism  in  cultured  cells 170 

Cyclic  nucleotide  metabolism  in  human  leukocytes 172 

Regulation  of  cyclic  AMP  phosphodiesterase  activity 175 

Regulation  of  hormone-sensitive  lipase  activity 178 

Laboratory  of  Chemical  Pharmacology 

Summary 181 

Metabolic  activation  of  a-methyldopa 187 

Effect  of  spironolactone  analogues  on  testicular  P-450 189 

The  role  of  cytochrome  b5  in  cytochrome  P-450  systems 192 

Role  of  guanine  nucleotides  in  vision 195 

Studies  on  the  covalent  binding  of  chloramphenicol 198 

Role  of  intestinal  flora  on  nitrobenzene-induced  toxicities 201 

Role  of  lung  cyclic  nucleotides  in  paraquat  toxicity 207 

Effects  of  Ca-H-  and  carbachol  on  cGMP  in  lung  cells 209 

Role  of  cyclic  nucleotides  in  hormone  action 212 

Paraquat  toxicity  in  rat  lung 217 

Ethanol,  isopropanol,  and  CCl4-induced  liver  toxicity 221 

Hydrazines  1.   Human  isoniazid  acetylation  and  metabolism 223 

Hydrazines  2.   Chemical  synthesis  of  labeled  compounds 229 

Hydrazines  3.   Studies  of  reactive  metabolites  in  vitro 233 

Hydrazines  4.   Studies  of  reactive  metabolites  in  rats 236 

Phenacetin- induced  toxicity:   N-oxidation  of  phenacetin 244 

Laboratory  of  Chemistry 

Summary 247 

Electrochemical  methods  of  analysis  and  synthesis 253 

Nuclear  magnetic  resonance  of  natural  products 254 


Structure  of  natural  products  using  Instrumental  methods 257 

Isolation  and  characterization  of  natural  products 259 

X-ray  structural  R&D  for  physiologically  important  molecules 261 

Application  of  mass  spectrometry  to  problems  in  biochemistry 266 

Use  of  digital  computing  in  problems  in  biochemistry 268 

Laboratory  of  Kidney  and  Electrolyte  Metabolism 

Summary 271 

Study  of  the  effect  of  cholera  toxin  on  toad  urinary  bladder 277 

Control  of  protein  phosphorylation  in  toad  urinary  bladder 280 

Separation  by  morphologic  type  and  study  of  responsiveness  to 

vasopressin  of  toad  bladder  epithelial  cells » 283 

Effect  of  parathyroid  hormone  on  protein  phosphorylation  in  rabbit 

renal  cortical  tubules « 286 

Regulation  of  cation  permeability  in  duck  erythrocytes 288 

Urinary  acidification  by  proximal  straight  tubules 291 

Glucose  transport  in  the  proximal  convoluted  tubule — 294 

Mechanism  of  salt  and  water  transport  by  proximal  renal  tubules 296 

Ion  transport  in  the  cortical  collecting  tubule 300 

Mechanism  of  salt  transport  by  isolated  segments  of  amphibian 

distal  nephron 302 

Anion  transport  across  individual  amphibian  erythrocytes 304 

Volume  regulation  in  nucleated  erythrocytes 307 

Cardioglobulin  B-s  of  human  serum 311 

Laboratory  of  Technical  Development 

Summary 315 

Angle  rotor  councercurrent  chromatography 321 

New  flow-through  centrifuge  without  rotating  seals 325 

Eye  motion  measurement  by  ultrasound 328 

Membrane  lung  systems  for  long  term  support 330 

Analysis  of  microcirculation  by  coherent  light  scattering 338 

Fluorescent  complexes  of  proteins 341 

Applications  of  fluorescence  in  biochemistry 343 

Methodology  in  fluorescence  measurements 345 

An  automated  method  for  rapid  bacterial  and  mammalian  cell  growth 

and  assay 348 

Blood  gas  monitoring  for  extended  periods 351 

Blood  flow  measurement  using  nuclear  magnetic  resonance  techniques —  354 

Discrete  cell  temperature  measurement  study 357 

Instrumentation  for  the  study  of  pre-steady  state  enzyme  kinetics 359 

Development  of  microcalorimeters  for  clinical  chemistry 363 

Italy-U.S.  cooperative  science  program  -  blood  gas  instruments  - 

Project  78 366 

On-line  cardiac  output  measurement  during  extracorporeal  membrane 

oxygenation 370 

Cardiology  Branch 

Summary ■ 373 

Effects  of  altered  autonomic  innervation  of  the  heart  on  ventricular 

electrical  stability  in  chronic  heart  failure 381 

Enhanced  survival  during  acute  myocardial  infarction  in  reserpine 

treated  dogs 384 

3  ar 


Cholinergic  enhancement  of  ventricular  electrical  stability: 

Adrenergic  dependency  or  primary  action? 386 

Clinical  characteristics  of  asymmetric  septal  hypertrophy 388 

Comparison  of  two-dimensional  echocardiographic  systems 389 

Echocardiographic  findings  in  patients  with  hypereosinophilia 390 

Congenital  heart  disease  associated  with  ASH 392 

Measurement  of  mitral  orifice  area  by  two-dimensional  echocardiography  394 

Mechanism  of  beneficial  action  of  TNG-methoxamine  in  AMI 396 

Factors  affecting  the  operative  mortality  in  aortic  valvular  disease —  398 
Differential  diagnosis  of  great  artery  anomalies  by  two-dimensional 

echocardiography 400 

Long-term  effects  of  operation  on  obstruction  and  LV  hypertrophy  in 

IHSS 402 

Distribution  of  the  cardiomyopathy  in  IHSS 404 

Pathophysiology  and  prediction  of  onset  of  atrial  fibrillation 406 

A  real  time  system  for  two-dimensional  echocardiography 408 

Effects  of  space  flight  on  cardiac  function 409 

Mitral  valve  position  in  patients  with  ASH 411 

Aortic  regurgitation:   Cardiac  function  and  operative  result 412 

Identification  of  cyanotic  heart  disease  in  infants  by  two-dimensional 

echocardiography 414 

Intramitochondrial  glycogen  deposits  in  cardiac  muscle 415 

Phosphorylation  of  cardiac  muscle  proteins 416 

Sudden  infant  death  syndrome:   Potential  cardiac  mechanisms 417 

Asymmetric  septal  hypertrophy  in  childhood 419 

Nitroglycerin,  nitroprusside  and  myocardial  ischemia 421 

Nitroglycerin  therapy  for  acute  myocardial  infarction  in  man 423 

Effects  of  vasodilators  on  coronary  collateral  flow 425 

Determinants  of  ventricular  septal  motion 427 

Isolation  and  characterization  of  myosin  from  patients  with  asymmetric 

septal  hypertrophy 429 

Indices  of  reversibility  in  heart  failure 431 

Echocardiographic  characteristics  of  infiltrative  cardiomyopathy 433 

Growth  of  aortic  smooth  muscle  cells  in  vitro 434 

Physical  factors  determining  cardiac  motion 435 

Growth  of  cells  in  tissue  culture  from  patients  with  ASH 436 

Dynamic  EKG-gated  scintiangiography 437 

Scintigraphy  in  the  assessment  of  coronary  artery  disease 439 

Stress  myocardial  imaging  in  the  evaluation  of  cardiac  disease 441 

Pre-  and  postoperative  exercise  performance  in  patients  with  ASH 443 

Left  ventricular  function  using  roentgen  videodensitometry 444 

Effect  of  TNG  during  exercise  in  valvular  heart  disease 446 

Persistent  paradoxic  septal  motion  following  ASD  closure 447 

Scintigraphic  detection  of  asymmetric  septal  hypertrophy 449 

The  interventricular  septum  in  ASH 451 

Limitations  of  the  electrocardiographic  response  to  exercise  in 

predicting  coronary  artery  disease; 452 

Phosphorylation  of  myosin  from  muscle  and  non-muscle  sources : 454 

Contractile  proteins  from  adult,  embryonic  and  malignant  cells 456 

Hypertension-Endocrine  Branch 

Summary 459 

4  Z* 


Outpatient  hypertension  diagnostic  screening  program 467 

Adrenal  steroid  secretion  in  hypertension 469 

Aminoglutethimide  in  low  renin  essential  hypertension 471 

Effects  of  spironolactone 475 

Adrenergic  nervous  system  function  in  hypertension 478 

Relation  of  K  to  vascular  response  of  BP 480 

Studies  in  Bartter's  syndrome 481 

Control  of  renin:   the  role  of  the  vagus  nerves 483 

Renal  prostaglandins,  sodium  and  blood  pressure 485 

Spironolactone  on  plasma  renin  and  dog  renal  histology 488 

Metabolism  of  albumin  on  patients  with  idiopathic  edema 490 

Coronary  artery  disease  and  urinary  steroids 492 

Nephrogenous  cyclic  AMP  as  a  parathyroid  function  test 493 

Aminoglycoside  effects  on  urinary  calcium 496 

Vitamin  D  metabolism  in  renal  stone  disease 497 

Chemical  regulators  of  parathyroid  gland  secretion 498 

Evaluation  of  sodium  cellulose  phosphate  (S.C.P.) 499 

Binding  and  metabolic  effects  of  neurotoxic  drugs 501 

Electron  transport  in  bovine  adrenal  medullary  vesicles 503 

Release  of  norepinephrine  from  neuronal  vesicles 506 

Receptors  participating  in  the  induction  of  tyrosine  hydroxylase 509 

Regulation  of  tyrosine  hydroxylase  by  steroid  receptors 511 

Regulation  of  tyrosine  hydroxylase  in  carotid  body 513 

Role  of  second  messengers  in  tyrosine  hydroxylase  induction 515 

A  radioimmunoassay  for  dopamine-B-hydroxylase 517 

Synthesis  of  dopamine-B-hydroxylase  in  a  cell  free  system 519 

Biochemistry  of  the  spontaneously  hypertensive  rats 521 

Regulation  of  catecholamine  synthesis 523 

Regulation  of  hydroxy  indole  pathway  in  the  pineal  gland 525 

Studies  on  the  properties  of  iron-sulfur  proteins 527 

Characterization  of  human  dopamine- B-hydroxylase 529 

Catalogue  of  isohormones  of  human  growth  hormone 531 

Control  of  growth  hormone  secretion 533 

Growth  hormone  secretion  and  structure  in  cultured  cells 535 

Juvenile  diabetes  mellitus 537 

Characterization  of  the  active  site(s)  of  dopamine-g-hydroxylase 539 

Mitochondrial  monoamine  oxidase 542 

P-chloromethamphetamine  and  tryptophan  hydroxylase 544 

Prostaglandins  in  renal  and  vascular  physiology 547 

Kinins  in  urine 551 

Clinical  investigations  of  vasoactive  systems 553 

Plasma  prekallikrein  in  hypertension 556 

Renal  kallikrein  and  plasma  kininogen  in  hypertension 559 

Urinary  kallikrein  and  kinin 561 

Urokinase  excretion  and  function 565 

Studies  on  the  structure  of  villikinin 567 

Clinical  biochemistry  of  the  kallikrein-kinin  system 569 

Peptide  biochemistry 571 

Biochemistry  of  the  kallikrein-kininogen-kinin  system 573 

Histamine,  prostaglandins  and  esterase  from  human  lung 578 

Purification  of  SRS-A  from  human  lung 581 

Biosynthesis  of  SRS-A  in  monkey  lung 584 


The  role  of  prostaglandins  in  the  vascular  system 586    d 

Amino  acid  sequence  determination  of  polypeptides 590 

Fibrinolytic  inhibitors  and  vascular  endothelium 593 

Angiotensin  converting  enzymes  (ACE)  in  endothelial  cells 595 

Taste  and  olfaction 599 

Trace  metal  metabolism 618 

Hormonal  effects  on  sensory  and  neural  function 629 

Molecular  Disease  Branch  / 

Summary 635 

The  lipoproteins  of  Tangier  Disease 639 

Characterization  of  the  human  plasma  apolipoproteins 643 

Rat  plasma  lipoproteins  and  apolipoproteins 648 

NHLI  Type  II  coronary  intervention  study 652 

The  biochemistry  and  metabolism  of  plasma  lipoproteins 657 

Lipid  constituents  of  human  tissues 660 

Tissue  lipidoses  and  hyperlipoproteinemias 663 

Microscopic  studies  in  tissue  lipid  storage  diseases 667 

Structure  and  function  of  parathyroid  hormone 670 

Structure  and  function  of  the  plasma  lipoproteins 676 

Molecular  Hematology  Branch 

Summary 685 

Mechanism  of  hemoglobin  biosynthesis  in  cell-free  systems 689 

Evolution  of  the  protein  synthesizing  machinery 692 

Mechanism  of  action  of  the  enzyme  RNA-directed  DNA  polymerase 694 

Mechanism  of  globin  messenger  RNA  transcription  in  bone  marrow  cells —  696    i 

Globin  gene  expression  in  somatic  cell  hybrids 698 

The  mechanisms  of  regulation  of  the  respiratory  function  of  blood 700 

Evaluation  of  alteration  in  blood  oxygen  affinity  as  a  basis  for  the 

treatment  of  sickle  cell  anemia 702 

Regulation  of  hemoglobin  chain  synthesis  in  beta-thalassemia— 704 

The  mechanism  of  hemoglobin  switching  in  sheep  and  goats 706 

Iron  chelation  therapy  in  transf usional  hemosiderosis 708 

Cardiac  hemolytic  anemia 710 

Pulmonary  Branch 

Section  on  Pulmonary  Biochemistry 

Summary 713 

Models  of  lung  growth 719 

Control  of  collagen  synthesis  in  lung  cell-free  systems 723 

Collagen  synthesis  in  cultured  lung  cells 725 

The  accumulation  of  collagen  in  human  lung 727 

Experimental  models  of  the  interstitial  lung  disorders 729 

Heterogeneity  of  lung  collagen 731    ( 

Studies  of  patients  with  fibrotic  lung  disease 733 

Section  on  Molecular  Pharmacology 

Summary 737 

Influence  of  age,  infection  and  drugs  on  lung  amines 741 

Influence  of  aspirin  on  amine  and  purine  metabolism  in  tumor  cells 744 

Disorders  of  amine  metabolism  in  human  disease 7A7 

i 

6  izz; 


Sensitive  assay  for  serotonin  in  tissues 751 

Interaction  of  adrenochrome  with  red  cell  ghost  membranes 753 

Spin  trapping  studies  of  the  microsomal  metabolism  of  CC^Br 756 

Acetylcholinesterase  from  Torpedo  californica 759 

Membrane  fluidity  in  contact  inhibited  and  transformed  cells 762 

Acridine  spin  labels  as  probes  for  nucleic  acids 765 

Spin  label  studies  of  Halobacterium  halobium  purple  membranes 769 

Physicochemical  studies  of  lung  surfactant  lipoprotein 772 

Further  spin  label  studies  of  egg  white  avidin 774 

Purification  and  characterization  of  Na+  +  IC*"-ATPase 777 

Chemical  characterization  of  pharmacological  receptors 779 

Biochemical  effects  of  paraquat  on  the  lung 782 

Information  retrieval  in  pharmacology 784 

Clinic  of  Surgery 

Summary 787 

Cardiac  valve  replacement  with  the  Hancock  porcine:   A  five  year 

clinical  experience 791 

Cardiac  patient  data  profile 792 

Surgical  management  of  acquired  tricuspid  valve  disease 793 

Phonocardiographic  detection  of  ball  variance 794 

NU-5  atomic  powered  pacemaker  -  experimental  and  clinical  evaluation —  796 

Silastic  ball  variance  detection 797 

Topical  hypothermia  system ■ 798 

Symposium  on  intraoperative  protection  of  the  myocardium 799 

The  effect  of  dipyridamole  and  methylprednisolone  on  intimal  pro- 
liferation in  venous  autografts  used  for  arterial  bypass 800 

Protection  of  the  myocardium  during  anoxic  arrest 801 

The  contribution  of  atrial  contraction  to  right  heart  function  before 

and  after  right  ventriculotomy:  Experimental  and  clinical  evaluation  803 

A  mechanical  device  for  sutureless  aortosaphenous  vein  anastomosis 804 

Subendocardial  ischemia  during  partial  coronary  occlusion  in  dogs: 
The  significance  of  S-T  segment  elevation  in  subendocardial 

electrograms 805 

Subepicardial  and  subendocardial  ischemia  following  coronary  occlusion  807 
Alterations  in  regional  ischemia  following  partial  coronary  occlusion: 
The  effects  of  changes  in  blood  pressure,  heart  rate,  and  cardiac 

output 808 

Temporal  changes  in  regional  coronary  flow  after  partial  coronary 

occlusion 809 

Extended  storage  of  the  canine  heart  for  transplantation 811 

Anatomic  correction  of  transposition  of  the  great  vessels 812 

Study  of  the  rate  of  development  and  severity  of  arterial  atheromas 

in  high  and  normal  flow  states  of  hypercholesterolemic  animals 813 

The  effects  of  isoproterenol  and  dopamine  on  regional  coronary  flow 

distribution  in  dogs  with  partial  coronary  occlusion 814 

Distribution  of  blood  flow  before  and  after  repair  of  coarctation 

of  aorta 815 

Study  of  arterial  wall  permeability  as  a  function  of  flow  rate 816 


mr 


Office  of  the  Director,  Division  of  Intramural  Research 

Section  on  Experimental  Atherosclerosis 

Summary 817 

The  arterial  intimal  tissue  responses  following  endothelial  injury  —  823 

The  relationship  of  arterial  intimal  Evans  blue  dye  accumulation  to 

surface  reflectance  and  light  absorption 825 

The  study  of  arterial  transport  processes  in  an  in  vitro  life  support 

system 828 

The  arterial  ultrastructure  in  areas  of  increased  and  decreased 

transvascular  transport  830 

The  mechanics  of  albumin  transport  into  the  arterial  intimal  medial 

space 832 

Blood  velocity  profiles  and  hemodynamic  stresses  in  the  aorta  and  its 

major  branches  835 

Vascular  mechanics:   arterial  wall  properties  837 

Trial  of  psychophysiologic  techniques  for  the  amelioration  of 

hypertension  840 

Quantitation  of  the  apolipoproteins  in  plasma  by  two-dimensional 

Immunoelectrophoresis  842 

Hyperlipoproteinemia  and  atherosclerosis:   changes  in  plasma  lipo- 
proteins and  apolipoproteins  induced  by  cholesterol  feeding  in 
dogs,  swine,  rats,  rabbits,  and  Patas  monkeys  844 

Aortic  metabolism  of  plasma  lipoproteins  846 

Animal  models  for  study  of  atherosclerosis  848 

Tissue  culture  studies  of  aortic  smooth  muscle  cells  and  skin  fibro- 
blasts: cell  growth  and  metabolism  in  response  to  incubation  with 
various  lipoprotein  classes  850 

Section  of  Pathology 

Summary 853 

The  coronary  arteries  in  ischemic  heart  disease  859 

The  coronary  arteries  in  fatal  coronary  events  860 

The  coronary  arteries  in  coronary  heart  disease  861 

The  coronary  arteries  in  ischemic  heart  disease  862 

Coronary  thrombosis  in  myocardial  infarction  863 

Thrombosis,  atherosclerosis  and  ischemic  heart  disease  864 

Acute  myocardial  infarction  and  angiographically  normal  coronary 

arteries  865 

Myocardial  embolus  to  coronary  artery  866 

Tuberous  xanthoma  in  Type  II  hyperlipoproteinemia  867 

The  pathology  of  Tangier  disease  868 

The  hypertensive  diseases.   The  extent  of  hypertension  as  a  risk 

factor 869 

Cardiac  pathology  after  valve  replacement  using  disc  prostheses  871 

Observations  after  insertion  of  Hufnagel  prostheses  in  descending 

aorta 873 

Aortic  valve  atresia:   A  necropsy  study  of  73  cases  874 

Endocardial  structure  in  carcinoid  heart  disease  876 

The  unruptured  sinus  of  valsalva  aneurysm  877 

Endocardial  papillary  elastofibromas  878 

Ultrastructural  features  of  endocardial  fibroelastosis  879 

Morphologic  evaluation  of  myocardial  protection  -  °™ 

Cardiac  morphologic  changes  produced  by  ethanol 

8  SZZ&- 


Massive  myocardial  hemosiderosis 882 

Cardiac  ultrastructure  in  the  cardiomyopathies 883 

Nuclear  membranes  in  hypertrophied  human  myocardium 886 

Intranuclear  glycogen  in  myocardium 887 

Cardiac  lesions  in  bone  marrow  transplantation 888 

The  structural  basis  of  abnormal  cardiac  function 890 

Section  on  Theoretical  Biophysics 

Summary 891 

Mathematical  theory  of  renal  function 895 

Computer  simulation  of  renal  function 899 


i£- 


ANNUAL  REPORT  OF  THE 

LABORATORY  OF  BIOCHEMICAL  GENETICS 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

July  1,  1974  through  June  30,  1975 

Studies  in  the  Laboratory  of  Biochemical  Genetics  focus  on  defining 
mechanisms  which  enable  cells  to  communicate  with  one  another  and  to  respond 
to  hormones  and  other  external  influences. 

During  the  past  few  years,  numerous  neuroblastoma  cell  lines  were  ob- 
tained and  characterized  with  respect  to  neuronal  properties  such  as  trans- 
mitter synthesis,  storage,  and  catabolism,  receptors,  and  effects  of  receptor 
activation. 

Additional  cell  lines  with  new  neural  phenotypes  were  generated  and 
questions  of  dominance  of  gene  expression  and  complementation  were  explored 
by  fusing  neuroblastoma  with  other  cells  and  obtaining  hybrid  cell  lines. 
The  expression  of  genes  for  neural  properties  was  found  to  be  dominant  with 
most  matings.   Some  hybrid  clones  expressed  the  neural  phenotype  of  the 
neuroblastoma  parent  50  or  more  cell  generations  after  fusion;  many  others 
had  specific  defects  in  transmitter  synthesis,  storage,  and  catabolism; 
response  to  neurotransmitters;  action  potential  reactions;  and  so  forth. 
Another  class  of  hybrids  had  acquired  new  neural  properties  which  were  not 
detected  with  parental  neuroblastoma  cells.   For  example,  we  previously 
showed  that  fusion  of  mouse  neuroblastoma  cells  with  rat  glioma  cells,  both 
lacking  choline  acetyltransf erase,  yields  hybrid  cell  lines  with  high  choline 
acetyltransferase  activity  which  store  acetylcholine  and  have  clear  vesicles 
identical  in  appearance  to  those  found  at  synaptic  junctions.   Additional 
studies  now  show  that  fusion  of  mouse  neuroblastoma  cells  which  lack  tyrosine 
hydroxylase  activity  with  cells  from  normal  sympathetic  ganglia  from  mouse 
embryos  yields  hybrid  cells  with  high  tyrosine  hydroxylase  activity  that 
synthesize  dopamine,  possess  muscarinic  excitatory  acetylcholine  receptors, 
and  have  both  small  and  large  dense-core  vesicles.   Fusion  of  neuroblastoma 
cells  with  cells  from  normal  retina  yielded  some  cell  lines  that  synthesize 
catecholamines  and  another  that  synthesizes  acetylcholine.   These  results 
show  that  fusion  of  neuroblastoma  cells  with  cells  from  the  normal  nervous 
system  generates  hybrid  cells  with  new  neural  properties  which  have  not  been 
detected  with  the  parental  neuroblastoma  cells.   The  new  neural  phenotypes 
are  inherited  and  thus  far  have  been  perpetuated  in  a  fairly  stable  fashion 
for  more  than  100  cell  generations.   This  approach  would  appear  to  be  a 
general  one  that  can  be  used  to  obtain  cell  lines  with  other  differentiated 
properties  that  can  be  used  to  elucidate  reactions  that  are  required  for  cell 
communication . 

Cyclic  GMP  levels  of  some  neuroblastoma  and  hybrid  cell  lines  were  found 
to  increase  up  to  200-fold  upon  activation  of  muscarinic  acetylcnoline  recep- 
tors, resulting  in  intracellular  cGMP  concentrations  greater  than  600  pmoles 
per  mg  protein.   Both  sensitive  and  insensitive  cell  lines  were  found.   The 
cells  also  have  receptors  for  PGE..  and  adenosine  which,  upon  activation, 
result  in  rapid  elevations  of  cAMF  levels.   Thus,  the  effects  of  activating 
pairs  of  receptors  which  are  functionally  coupled  to  cGMP  or  cAMP  cell  responses 
were  studied.   The  results  reveal  considerable  complexity  in  the  regulation 


of  receptor  mediated  events.   Activation  of  the  muscarinic  acetylcholine 
receptor  elicits  both  an  elevation  in  cGMP  and  a  decrease  in  cAMP  levels. 
Conversely,  activation  of  adenosine  receptors  elevates  cAMP  and  depresses 
cGMP  levels.   Unexpectedly,  PGE  was  found  to  increase  the  concentrations  of 
both  cGMP  and  cAMP.   The  results  suggest  that  one  species  of  PGE   receptor 
affects  cAMP  levels  and  another  receptor,  cGMP  levels.   Carbamylcholine  and 
PGE  dependent  increases  in  cGMP  are  additive;  whereas,  PGE  and  adenosine 
dependent  increases  in  cAMP  levels  are  not  additive.   These  results  show  that 
informational  molecules  impinging  upon  a  cell  regulate  in  at  least  4  ways 
cell  responses  to  other  species  of  informational  molecules.   Current  studies 
focus  on  defining  the  mechanisms  which  underlie  the  observed  phenomena,  for 
similar  events  may  well  occur  at  synapses.   The  results  also  show  that  genes 
determining  receptor  species  for  putative  neurotransmitters  can  be  expressed 
in  dividing  cells,  that  the  parental  programs  of  gene  expression  are  inherited, 
and  that  dividing  cells  can  be  programmed  with  respect  to  their  ability  to 
receive  information  from  different  kinds  of  neurons. 

In  collaboration  with  Dr.  Werner  Klee,  various  neuroblastoma  and  hybrid 
cell  lines  were  assayed  for  morphine  receptors .   Cell  lines  with  and  without 
stereospecif ic,  high  affinity,  morphine  receptors  were  found.   Such  receptors 
are  particularly  abundant  in  a  neuroblastoma  x  glioma  hybrid  cell  line,  for 
the  average  hybrid  cell  possesses  approximately  3  x  10  narcotic  receptors. 
The  neuroblastoma  parent  possesses  relatively  few  narcotic  receptors  and  the 
glioma  parent  lacks  narcotic  receptors.   The  results  suggest  that  gene  expres- 
sion for  opiate  receptors  may  be  dominant  in  the  hybrid  cell  lines  studied. 

Further  studies  revealed  that  morphine  inhibits  adenylate  cyclase  activity 
of  cells  with  morphine  receptors  but  does  not  affect  adenylate  cyclase  activity 
of  cells  without  these  receptors.   Thus,  two  forms  of  adenylate  cyclase  were 
distinguished;  one  form  sensitive,  the  other  insensitive,  to  narcotics. 
Questions  pertaining  to  the  mechanism  of  narcotic  addiction  and  dependence 
currently  are  being  explored  with  this  system. 

12  5 
[   IJ-Labeled-a-bungarotoxin  has  been  used  as  a  specific  probe  for 

acetylcholine  receptors.   A  highly  sensitive  histochemical  technique  for 

localization  of  acetylcholine  receptors  was  devised  which  is  based  on  the 

binding  of  bungarotoxin  to  the  receptor  followed  by  complex  formation  with 

rabbit  antibody  against  the  bungarotoxin.   A  double  antibody  step  follows 

involving  complex  formation  with  horseradish  peroxidase  conjugated  antibody 

against  rabbit  antiserum.   Using  this  procedure,  it  has  been  possible  to  show 

that  mouse  diaphragm  endplates  have  a  limited  distribution  of  acetylcholine 

receptors,  that  denervated  muscle  shows  spreading  of  receptors  and  that 

myasthenia  gravis  is  characterized  by  the  presence  of  a  serum  factor  that 

interferes  with  the  binding  of  toxin  to  normal  muscle  endplates. 

Some  properties  of  the  Na   influx  system  associated  with  the  nicotinic 
acetylcholine  receptor  ionophore  were  studied.   Na  uptake  behaves  cooperatively 
upon  stimulation  by  cholinergic  agonists  and  activation  of  Na   transport  ex- 
hibits a  different  temperature  profile  than  the  transport  process.   Activation 
of  the  Na   ionophore  by  alkaloid  neurotoxins  is  completely  inhibited  by  diva- 
lent cations.   Other  studies  indicate  that  the  ionophore  is  regulated  coopera- 
tively by  another  component  that  interacts  specifically  with  polypeptide 
toxins. 


A  variety  of  cell  types  maintained  in  tissue  culture  respond  to  environ- 
mental or  developmental  changes  with  respect  to  their  complement  of  ionophores . 
Inhibition  of  the  growth  of  neuroblastoma  cells  by  butyric  acid  or  dibutyryl- 
cAMP  results  in  increased  ionophore  activity.   Embryonic  chick  skeletal 
muscle  in  culture  develops  Na  ionophore  activity  only  after  fusion  of  the 
cells  into  myotubes.   In  contrast,  stable  cultures  of  rat  skeletal  muscle 
have  ionophore  activity  before  fusion  which  is  only  increased  approximately 
two-fold  after  fusion.   Adult  heart  cells  possess  3  or  more  types  of  ionophores, 
one  for  Na  ,  one  for  Ca   and  another  for  K  .   Use  of  the  inhibitor  D-600  has 
shown  that  embryonic  chick  heart  has  a  population  of  ionophores  that  changes 
with  age. 

Induced  enzyme  synthesis  in  Ej_   coli  requires  cAMP.   The  blockade  of  such 
induced  enzyme  synthesis  by  glucose  or  other  catabolizable  sugars  is  due  to 
their  effects  in  lowering  cAMP  levels.   We  have  been  exploring  the  mechanism 
by  which  glucose  decreases  cAMP  levels.   We  have  shown  that,  while  glucose 
does  not  affect  adenylate  cyclase  in  vitro,  it  effectively  inhibits  the  enzyme 
in  intact  cells.   The  substitution  of  a-methyl  glucoside,  a  compound  that  can 
penetrate  cells,  but  not  be  metabolized  further  than  the  phosphorylated  form 
for  glucose  as  an  inhibitor  of  adenylate  cyclase  indicates  that  extensive 
metabolism  of  glucose  is  not  required  for  adenylate  cyclase  inhibition. 

The  spectrum  of  sugars  that  inhibit  E.  coli  adenylate  cyclase  varies 
with  the  conditions  under  which  the  cells  have  been  cultured.   A  variety  of 
studies  indicate  that  the  presence  of  transport  systems  for  sugars  endows  E. 
coli  with  the  capacity  to  have  its  adenylate  cyclase  inhibited  by  the  sugars. 
These  studies  suggest  that  adenylate  cyclase  in  E^  coli  may  be  regulated  in 
an  inhibitory  sense  by  a  mechanism  similar  to  that  by  which  mammalian  cell 
adenylate  cyclases  are  regulated  positively  by  many  hormones. 

Friend-virus  transformed  mouse  erythroleukemia  cells  produce  hemoglobin 
on  exposure  to  dimethylsulf oxide  and  have  high  levels  of  acetylcholinesterase. 
Hybrids  formed  between  these  cells  and  human  or  mouse  fibroblasts  were  found 
not  to  express  these  differentiated  functions  and  not  to  produce  hemoglobin 
messenger  RNA  which  suggests  that  hemoglobin  gene  transcription  is  repressed 
in  the  hybrid  cells. 

Fusion  of  a  transformed  tissue  culture  cell  line  to  normal  cells  which 
have  not  been  cultured  results  in  hybrid  lines  in  which  chromosomes  from  the 
normal  parent  are  preferentially  lost.   This  property  has  been  used  for  gene 
mapping  by  tracing  the  segregation  of  chromosomes  and  loss  of  certain  enzymes. 
We  have  found  that  at  least  15  isozymes  are  asyntenic  in  the  mouse.   In  addition, 
we  observed  that  genes  which  are  linked  in  man  are  on  different  chromosomes 
in  the  mouse,   and  that  some  genes  that  are  on  the  same  chromosome  arm  in  the 
human  are  linked  in  the  mouse. 

Hybrids  between  human  and  mouse  cells  have  been  analyzed  for  the  expres- 
sion of  oncornavirus.  Our  results  indicate  that  human  genes  can  regulate  the 
production  of  the  viral  RNA-dependent  DNA  polymerase  but  do  not  affect  the 
expression  of  the  viral  structural  proteins.  Human  chromosomes  14,  21  and 
possibly  12  appear  to  be  responsible  for  this  regulation.  We  also  found  that 
human  genes  can  block  the  induction  of  type  C  viral  genes  from  mouse  integra- 
tion sites  and  can  alter  the  host  range  of  mouse  virus. 


Project  No.  z01  HL  00001-04  LBG 

1.  Biochemical  Genetics 

2 .  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Acetylcholine  Receptors 

Previous  Serial  Number :   NHLI-305 

Principal  Investigators:   Mathew  Daniels,  Ph.D.  and  Zvi  Vogel,  Ph.D. 

Other  Investigators:   S.  P.  Ringel,  M.D.  (MNB) ,  A.  N.  Bender,  M.D.  (MNB) , 

B.  W.  Festoff,  M.D.  (MNB),  W.  K.  Engel,  M.D.  (MNB), 
Tom  Reese,  M.D.  (LNNS) ,  M.  DuBois,  M.D.  (IDB) 

Cooperating  Units:   NINDS 

Project  Description: 

125 
Objectives:   Investigators  in  this  laboratory  and  others  have  utilized    I 

labelled  a -bungarotoxin  (aBT)  as  a  label  for  nicotinic  acetylcholine  receptors 
in  intact  and  cultured  skeletal  muscle,  and  in  embryonic  and  mature  retina. 
The  objectives  of  this  study  were  to  devise  a  his tochemical  technique  of 
greater  sensitivity  and  resolution  for  localizing  bound  aBT  and  to  apply  this 
technique  to  studying  the  ultras tructural  distribution  of  acetylcholine  recep- 
tors in  the  peripheral  and  central  nervous  system  during  development,  in 
culture,  and  in  the  mature  state. 

Methods  Employed:  We  have  employed  indirect  immunoperoxidase  staining  of 
cryostat  sectioned,  teased,  or  monolayer  cultured  materials  to  which  aBT  has 
been  bound.  These  materials  are  subsequently  examined  by  light  and  electron 
microscopy. 

Major  Findings:   We  devised  a  technique  of  greater  sensitivity  and  resolu- 
tion utilizing  (rabbit)  antibody  against  aBT  and  horseradish  peroxidase- 
conjugated  antibody  against  rabbit.   Using  this  double  immunotechnique,  we 
observed  the  limited  ultrastructural  distribution  of  acetylcholine  receptor 
within  mouse  diaphragm  endplates . 

The  technique  has  now  found  application  in  three  other  studies  involving 
acetylcholine  receptor  distribution  on  muscle.   (1)  In  human  muscle  disease 
involving  denervation,  the  method  has  been  used  to  detect  denervated  fibers, 
in  which  there  is  spreading  of  the  receptors.   (2)  In  myasthenia  gravis,  a 
muscle  weakness  disease,  the  method  has  been  used  to  detect  a  serum  factor 
which  blocks  aBT  binding  at  normal  muscle  endplates.   (3)  In  cultured  embryonic 


Project  No.   ZQ1  HL  Q0Q01-O4  LBG 
muscle  the  method  is  being  used  to  characterize  the  ultrastructure  of  the 
regions  of  the  muscle  membrane  containing  a  high  concentration  of  receptors. 

Significance  to  Biomedical  Research:   Knowledge  of  the  ultrastructural 
distribution  of  acetylcholine  receptor  is  of  clear  importance  in  any  attempt 
to  understand  the  role  of  neurotransmitters  and  their  receptors  in  the  function 
and  development  of  the  nervous  system.   The  a-bungarotoxin-immunoperoxidase 
technique  already  has  shown  promise  for  the  diagnosis  and  analysis  of  mechanisms 
in  human  neuromuscular  disorders . 

Proposed  Course:   We  plan  to  complete  the  study  on  cultural  skeletal  muscle 
and  continue  our  collaboration  in  characterizing  the  myasthenia  gravis  serum 
factor.   We  are  also  continuing  to  modify  the  technique  for  attempts  at  ultra- 
structural  visualization  of  acetylcholine  receptors  in  retina. 

Publications: 

1.  Daniels,  M.  P.  and  Vogel,  Z.:  Immunoperoxidase  staining  of  a-bungarotoxin 
bound  to  acetylcholine  receptors  in  mouse  motor  endplates.  J.  Cell  Biol. , 
63,  76a,  1974. 

2.  Daniels,  M.  P.  and  Vogel,  Z.:   Immunoperoxidase  staining  of  a-bungarotoxin 
binding  sites  in  muscle  endplates  shows  distribution  of  acetylcholine 
receptors.   Nature,  253,  339-341,  1975. 

3.  Bender,  A.  N. ,  Ringel,  S.  P.,  Engel,  W.  K. ,  Daniels,  M.  P.  and  Vogel,  Z.: 
Myasthenia  Gravis:   A  serum  factor  blocking  acetylcholine  receptors  of  the 
human  neuromuscular  junction.   Lancet,  March  15,  607-609,  1975. 

4.  Bender,  A.  N. ,  Ringel,  S.  P.,  Festoff,  B.  W.  ,  Engel,  W.  K. ,  Vogel,  Z.  and 
Daniels,  M.  P.:   Denervated  skeletal  muscle  fibers  identified  by  immuno- 
peroxidase localization  of  extrajunctional  alpha-bungarotoxin  binding. 
Nature,  In  press. 


Project  No.  Z01  HL  00002-02  LBG 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Morphine  Receptors  as  Regulators  of  Adenylate 
Cyclase 

Previous  Serial  Number:   NHLI-300 

Principal  Investigators:   Marshall  Nirenberg,  Ph.D.  and  Shail  Sharma, 

Ph.D.  Guest  Worker  (F.I.C.) 

Other  Investigators:   Werner  Klee,  Ph.D.  (LGCB) 

Cooperating  Units:   NIMH 

Project  Description: 

Objectives:   The  objectives  are  to  elucidate  molecular  mechanisms  of  narcotic 
dependence  and  tolerance,  and  the  effects  of  narcotics  upon  the  sensitivities 
of  other  receptors  - 

In  collaboration  with  Dr.  Werner  Klee,  various  neuroblastoma  and  hybrid 
cell  lines  were  assayed  for  morphine  receptors.   Cell  lines  with  and  without 
stereospecif ic,  high  affinity,  morphine  receptors  were  found.   Such  receptors 
are  particularly  abundant  in  a  neuroblastoma  x  glioma  hybrid  cell  line,  for 
the  average  hybrid  cell  possesses  approximately  3  x  10  narcotic  receptors. 
The  neuroblastoma  parent  possesses  relatively  few  narcotic  receptors  and  the 
glioma  parent  lacks  narcotic  receptors.   The  results  suggest  that  gene  expres- 
sion for  opiate  receptors  may  be  dominant  in  the  hybrid  cell  lines  studied. 

Further  studies  revealed  that  morphine  inhibits  adenylate  cyclase  activity 
of  cells  with  morphine  receptors  but  does  not  affect  adenylate  cyclase  activity 
of  cells  without  these  receptors.   Thus,  two  forms  of  adenylate  cyclase  were 
distinguished;  one  form  sensitive,  the  other  insensitive  to  narcotics.   The 
interactions  between  (narcotic  receptor)  moieties  and  the  adenylate  cyclase 
complex  exhibit  positive  cooperativity ;  whereas  the  interactions  between  nar- 
cotic and  receptor  are  not  cooperative. 

Significance  to  Biomedical  Research:   A  molecular  mechanism  for  narcotic 
dependence  was  proposed  wherein  the  number  of  adenylate  cyclase  molecules  per 
cell  increases  over  a  period  of  days  when  cells  are  cultured  in  the  presence 
of  narcotics.   Cyclic  AMP  levels  then  are  normal  in  the  presence  of  a  narcotic 


Project  No.  Z01  HL  00002-02  LBG 

inhibitor  of  adenylate  cyclase  but  are  abnormally  high  in  the  absence  of  the 
narcotic. 

Honors  and  Awards :   None 

Publications : 

1.  Klee,  W.  A.  and  Nirenberg,  M. :   A  neuroblastoma  x  glioma  hybrid  cell  line 
with  morphine  receptors.   Proc.  Nat.  Acad.  Sci. ,  USA,  71,  3474-3477,  1974. 

2.  Sharma,  S.  K. ,  Nirenberg,  M. ,  and  Klee,  W.  A.:   Morphine  receptors  as 
regulators  of  adenylate  cyclase  activity.   Proc.  Nat.  Acad.  Sci. ,  USA,  72, 
590-594,  1975. 

3.  Klee,  W.  A.,  Sharma,  S.  K. ,  and  Nirenberg,  M. :   Opiate  receptors  as  regu- 
lators of  adenylate  cyclase.   Life  Sciences,  In  press. 


Project  No.  Z01  HL  00003-02  LBG 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Regulation  of  Receptor  Activity 

Previous  Serial  Number:   NHLI-306 

Principal  Investigators:   Hiroshi  Matsuzawa,  Ph.D.  and  Marshall  Nirenberg, 

Ph.D. 

Other  Investigators :   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   The  objective  is  to  define  synaptic  events  using  clonal 
cells  as  model  systems. 

Cyclic  GMP  levels  of  some  neuroblastoma  and  hybrid  cell  lines  were  found 
to  increase  up  to  200-fold  upon  activation  of  muscarinic  acetylcholine 
receptors,  resulting  in  intracellular  cGMP  concentrations  greater  than  600 
pmoles  per  mg  protein.   Both  sensitive  and  insensitive  cell  lines  were 
found.   The  cells  also  have  receptors  for  PGE  and  adenosine  which,  upon 
activation,  result  in  rapid  elevations  of  cAMP  levels.   Thus,  the  effects  of 
activating  one  species  of  receptor  upon  cell  responses  mediated  by  another 
species  of  receptor  were  studied.   The  results  reveal  considerable  complexity 
in  the  regulations  of  receptor  mediated  events.   Activation  of  the  muscarinic 
acetylcholine  receptor  elicits  both  an  elevation  in  cGMP  and  a  decrease  in 
cAMP  levels.   Conversely,  activation  of  adenosine  receptors  elevates  cAMP 
and  depresses  cGMP  levels.   Unexpectedly,  PGE  was  found  to  increase  the 
concentrations  of  both  cGMP  and  cAMP.   The  results  suggest  that  one  species 
of  PGE..  receptor  affects  cAMP  levels  and  another  receptor,  cGMP  levels. 
Carbamylcholine  and  PGE  dependent  increases  in  cGMP  are  additive;  whereas, 
PGE  and  adenosine  dependent  increases  in  cAMP  levels  are  not  additive. 
These  results  show  that  informational  molecules  impinging  upon  a  cell  regulate 
in  at  least  4  ways  cell  responses  to  other  species  of  informational  molecules. 
The  results  also  show  that  genes  determining  receptor  species  for  putative 
neurotransmitters  can  be  expressed  in  dividing  cells,  that  the  parental 
programs  of  gene  expression  are  inherited,  and  that  dividing  cells  can  be 
programmed  with  respect  to  their  ability  to  receive  information  from  different 
kinds  of  neurons.   Current  studies  focus  on  defining  the  mechanisms  which 
underlie  the  observed  phenomena,  for  similar  events  may  well  occur  at  synapses. 

Publications:   None 


Project  No.  Z01  HL  00004-02  LBG 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Studies  of  Action  Potential  and  Receptor  Ionophores 

Previous  Serial  Number:   NHLI-307 

Principal  Investigator:   William  A.  Catterall,  Ph.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   The  objectives  of  this  project  are  (1)  to  develop  biochemical 
methods  for  study  of  action  potential  and  receptor  ionophores,  (2)  to  use 
these  methods  to  study  the  mechanism  of  action  of  receptor  and  action  poten- 
tial ionophores,  and  (3)  to  use  these  methods  to  study  the  regulation  and 
genetic  expression  of  action  potential  and  receptor  ionophores  in  cultured 
cells. 

Methods  Employed:   Biochemical  assays  which  measure  changes  in  passive  Na 
influx  were  used  to  study  the  acetylcholine  receptor  ionophore  and  action 
potential  Na   ionophores. 

Major  Findings:   (1)   Studies  of  Na   transport  by  the  acetylcholine  recep- 
tor Na   ionophore  in  cultured  muscle  cells  led  to  the  following  conclusions: 
(a)  Activation  of  the  nicotinic  acetylcholine  receptor  by  cholinergic  agonists 
is  cooperative  whereas  inhibition  by  antagonists  is  not.   (b)  The  processes 
of  activation  and  desensitization  are  temperature  sensitive  while  the  process 
of  ion  transport  is  not.   (c)   The  ionophore  functions  as  an  ion  channel 
rather  than  as  an  ion  carrier.   (d)   The  channel  is  saturable  with  K  for  Na 
of  150  mM  at  0°  and  a  turnover  number  of  2-3  x  10   ions /min/ channel. 


(2)   Studies  of  activation  of  the  action  potential  Na   ionophore  by  neuro- 
toxins led  to  the  following  conclusions:   (a)   the  alkaloid  neurotoxins 
veratridine,  batrachotoxin,  and  aconitine  activate  the  ionophore  by  reversible 
interaction  with  a  single  class  of  sites;   (b)   divalent  cations  are  competitive 
inhibitors  of  the  activation  by  alkaloid  neurotoxins;   (c)   the  polypeptide 
toxins  of  scorpion  venom  activate  the  ionophore  by  interaction  with  a  different 
class  of  sites  from  the  alkaloid  toxins;   (d)   the  sites  of  action  of  the 
alkaloid  toxins  and  scorpion  toxins  are  allosterically  coupled  in  a  highly 


/a 


Project  No.  ZQ1  HL  00004-02  LBG 
cooperative  manner;   and  (e)   tetrodotoxin  is  a  noncompetitive  inhibitor  (K 
=  8  nM)  of  activation  by  neurotoxins.   Experiments  from  other  labs  suggest 
that  tetrodotoxin  acts  at  the  ion  transport  site  for  Na  .   These  results 
suggest  that  the  activity  of  the  action  potential  Na  ionophore  is  modulated 
by  two  regulatory  components  which  bind  activating  neurotoxins  and  interact 
cooperatively  in  controlling  the  activity  of  an  ion  transport  component  which 
binds  tetrodotoxin. 

Significance  to  Biomedical  Research:   The  results  provide  new  insights 
into  the  mechanism  of  action  and  regulation  of  membrane  macromolecules  involved 
in  information  transfer  and  processing  in  the  nervous  system  and  in  maintenance 
of  normal  beating  in  heart . 

Proposed  Course:   Planned  investigations  include  (1)  completing  the  kinetic 
analysis  of  ion  transport  by  the  nicotinic  acetylcholine  receptor  of  cultured 
muscle  cells  (2)  initiating  studies  of  ion  transport  changes  associated  with 
activation  of  muscarinic  acetylcholine  receptors  of  neuroblastoma  and  heart 
cells,  and  (3)  purifying  the  active  components  of  the  scorpion  toxin  mixture 
used  in  these  studies,  radioactively  labelling,  and  studying  binding  by  nerve 
cells  and  heart  cells . 

Honors  and  Awards :   None 

Publications : 

1.  Catterall,  W.  A.:   Sodium  transport  by  the  acetylcholine  receptor  of 
cultured  muscle  cells.   J.  Biol.  Chem. ,  250,  1776,  1975. 

2.  Catterall,  W.  A.:   Activation  of  the  action  potential  Na  ionophore  of 
cultured  neuroblastoma  cells  by  veratridine  and  batrachotoxin.   J_.  Biol. 
Chem. ,  In  press. 

3.  Catterall,  W.  A.:   Cooperative  activation  of  the  action  potential  Na 
ionophore  by  neurotoxins.   Proc.  Nat.  Acad.  Sci . ,  USA,  In  press. 


// 


Project  No.  Z01  HL  00005-02  LBG 

1.  Biochemical  Genetics 

2 .  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   The  Biosynthesis  of  Neurotransmitters  in  Cell  Hybrids 

Previous  Serial  Number:   NHLI-303 

Principal  Investigator:   Eliahu  Heldman,  Ph.D. 

Other  Investigators:   None 

Cooperating  Units :   None 

Project  Description: 

Major  Findings:   Established  cell  lines  that  are  able  to  form  synapses  in 
tissue  culture  may  be  very  useful  as  a  model  for  investigating  the  mechanism 
of  cellular  communication  and  synaptic  interactions .   Cell  hybrids  between 
neuroblastoma  and  normal  neuronal  tissue  may  provide  such  a  system  since 
normal  neuronal  properties  that  are  carried  by  the  hybrids  may  be  potentially 
important  in  cell  recognition  and  in  their  ability  to  form  synapses.   The 
ability  to  synthesize  neurotransmitters  and  to  store  them  like  normal  neuronal 
cells  is  one  of  the  requirements  in  this  context.   Cell  hybrids  between  the 
established  line  of  neuroblastoma  -  N18TG2G  and  rat  or  mouse  retina  were 
tested  for  their  ability  to  synthesize  neurotransmitters.   The  cells  were 
incubated  with  radioactive  precursors  for  potential  neurotransmitters  and  the 
products  were  extracted,  separated  by  high  voltage  electrophoresis  and  identi- 
fied.  Four  classes  of  cell  hybrids  were  observed:   (1)  Those  that  accumulated 
catecholamines.   (2)  Those  that  accumulated  acetylcholine.   (3)  Those  that 
accumulated  both  catecholamines  and  acetylcholine.   (4)  Those  that  did  not 
accumulate  any  of  the  possible  neurotransmitters  tested  (catecholamines, 
acetylcholine,  serotonin  and  GABA) .   Some  of  the  hybrids  were  tested  for 
their  GABA  content  and  they  did  show  significant  amount  of  that  substance. 
The  precursor  for  the  GABA  was  not  glutamic  acid  but  putrescine. 

Those  cells  that  were  capable  of  accumulating  both  catecholamines  and 
acetylcholine  were  recloned  under  various  conditions.   Three  classes  of  clones 
were  observed:   (1)  Those  that  retained  newly  synthesized  acetylcholine.   (2) 
Those  that  retained  both,  newly  synthesized  catecholamines  and  newly  synthe- 
sized acetylcholine.   (3)  Those  that  did  not  accumulate  either  of  them.   Not 
a  single  clone  with  the  ability  to  accumulate  only  newly  synthesized  catechol- 
amines was  isolated.   The  cell  hybrids  were  also  tested  for  their  choline 
acetyltransf erase  (CAT)  and  tyrosine  hydroxylase  activities  (TH) .   Lines  able 


/5- 


Project  No.  Z01  HL  00005-02  LBG 
to  accumulate  catecholamines  had  high  TH  activity  (70-300  pmole/mg  protein/ 
min) .   Among  the  lines  accumulating  acetylcholine  only  N18RE101  had  significant 
CAT  activity  (30  pmole/mg  protein/min) .   The  other  lines  had  low  activity 
indicating  that  these  lines  are  able  to  retain  well,  slowly  synthesized  acetyl- 
choline. 

The  catecholamines  were  identified  by  three  different  chromatographic 
systems.   In  N18RE103  dopamine  (DA)  was  found  to  be  the  major  product.   Small 
quantities  of  NE  were  also  found.   DOPA  was  not  accumulated  and  apparently 
was  converted  immediately  to  DA.   In  N18RE1200  the  only  product  was  DA.   In 
N18ME1  and  N18ME3  DA  was  the  major  product,  small  quantities  of  NE  were  also 
found  and  DOPA  was  also  accumulated  to  some  extent . 

Significance  to  Biomedical  Research:   Knowledge  of  the  biochemistry  of 
neuroblastoma  and  cell  hybrids  in  culture  provide  us  with  understanding  of 
neuronal  mechanism  and  may  explain  certain  disorders  in  neuronal  communication 
and  synaptic  function  in  vivo. 

Honors  and  Awards :   None 

Publications :   None 


/3 


Project  No.  Z01  HL  00006-02  LBG 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Ultrastructure  of  Neuroblastoma  Somatic  Cell  Hybrids 

Previous  Serial  Number:   NHLI-308 

Principal  Investigator:   Mathew  Daniels,  Ph.D. 

Other  Investigators:   John  Minna,  M.D.,  Mr.  Doyle  Mullinax  (M.  A.),  Zvi  Vogel, 

Ph.D.,  Marshall  Nirenberg,  Ph.D. 

Cooperating  Units:   Microbiological  Associates 

Project  Description: 

Objectives:   Previous  and  ongoing  work  in  this  laboratory  has  shown  that 
some  somatic  cell  hybrids  between  neuroblastoma  and  other  cell  types  can 
express  neuronal  characteristics  to  varying  degrees,  in  some  cases  to  a  greater 
degree  than  the  parent  cells.   The  objective  of  the  present  project  was  to 
extend  these  observations  to  the  ultras true tural  level  by  means  of  electron 
microscopy  of  the  intact  cell  cultures  of  hybrid  lines  derived  by  crosses 
between  neuroblastoma  and  glioma,  L-cells,  human  fibroblasts,  or  embryonic 
nerve  cells. 

Methods  Employed:   We  are  applying  standard  transmission  electron  micro- 
scopic techniques  to  monolayer  and  rotation-mediated  aggregate  cell  cultures 
fixed  and  embedded  without  any  dislocation. 

Major  Findings:   We  have  studied  the  ultrastructure  of  aggregates  of  several 
lines  of  neuroblastoma  x  Chinese  hamster  retina  (NCE)  and  neuroblastoma  x  rat 
retina  (NRE)  somatic  cell  hybrids.   In  the  NCE  hybrid  lines  there  was  a  wide 
variation  in  the  ability  to  aggregate.   This  ability  was  generally  correlated 
with  the  frequency  of  specialized  intracellular  junctions.   Further,  there 
appeared  to  be  at  least  2  classes  of  junction-forming  cells,  those  with  only 
small,  "macula  adherens"  (MA)  type  junctions  and  those  with  both  MA  junctions 
and  larger  (with  a  narrower  gap),  "zonula  adherens"  (ZA)  type  junctions. 
These  specialized  junctions  were  also  observed  in  aggregates  of  the  NRE  hybrids. 
In  addition,  two  of  these  hybrid  lines  showed  extensive  neurite  formation  in 
aggregates,  a  feature  not  observed  in  the  NCE  lines. 

Significance  to  Biomedical  Research:   This  investigation  may  yield  informa- 
tion as  to  the  pattern  of  inheritance  of  neuronal  characteristics  in  the 
somatic  cell  hybrids  as  well  as  the  appropriateness  of  these  cells  for  use  as 

1  ltf. 


Project  No.  Z01  HL  0Q006-O2  LBG 

neuronal  models.   This  type  of  information  is  ultimately  important  in  the 
attempt  of  this  laboratory  to  understand  the  biochemical  and  genetic  basis 
for  nervous  system  function  and  development. 

Proposed  Course:   We  plan  to  describe  the  intercellular  junctions  in  more 
detail  and  compare  them  to  those  of  retinal  and  neuroblastoma  cells.   In 
addition,  it  should  be  of  interest  to  co-aggregate  with  retinal  cells  the  NRE 
lines  which  form  neurites,  since  these  cells  may  have  more  tendency  for  inter- 
action. 

Honors  and  Awards :   None 

Publications : 

1.   Daniels,  M.  P.  and  Hamprecht,  B. :   The  ultrastructure  of  neuroblastoma 
glioma  somatic  cell  hybrids.   Expression  of  neuronal  characteristics 
stimulated  by  dibutyryl  adenosine  3', 5'  cyclic  monophosphate.   J_.  Cell 
Biol.,  63,  691,  1974. 


/S~ 


Project  No.  Z01  HL  00007-01  LBG 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Ornithine  Decarboxylase  Induction  in  Neural  Cells 

Previous  Serial  Number:   None 

Principal  Investigator:   Uriel  Bachrach,  Ph.  D. 

Other  Investigators:   None 

Cooperating  Units  :   None 

Project  Description: 

Major  Findings:   The  naturally  occurring  polyamine,  spermine,  spermidine 
and  putrescine  are  wide-spread  in  biological  material,  including  the  nervous 
system.   Cellular  polyamine  levels  fluctuate  during  the  growth  cycle  and  cor- 
relate well  with  cellular  RNA  concentrations.   The  rate-limiting  step  in  poly- 
amine synthesis  is  ornithine  decarboxylase,  which  catalyzes  the  conversion  of 
ornithine  to  putrescine.   We  were  able  to  show  that  ornithine  decarboxylase 
activity  of  neuroblastoma  N115  and  Glioma  C6-Bu-1  cells  was  high  in  prolifer- 
ating cells  and  declined  when  they  reached  confluency.   When  confluent  cells 
were  fed  with  fresh  medium,  ornithine  decarboxylase  activity  (ODC)  increased 
precipitiously  after  a  lag  of  2  hours  and  was  1000-fold  higher  than  the  basal 
activity,  4  hours  later  these  changes  in  ODC  were  accompanied  by  changes  in 
cellular  putrescine  levels.   This  unique  increase  in  ornithine  decarboxylase 
activity  was  accompanied  by  accumulation  of  polyamines,  and  by  resumption  of 
RNA  synthesis.   The  induction  of  ODC,  by  fresh  medium,  could  be  prevented  by 
Actinomycin  D  and  by  cycloheximide  and  was  also  accomplished  by  the  addition 
of  dibutyrvl  cAMP.  isoproterenol,  norepinephrine  or  PGE^  (prostaglandin)  to 
confluent  neuroblastoma  and  glioma  cells,  respectively.   Phosphodiesterase 
inhibitors,  such  as,  theophylline,  Ro  20-1274  [4-(3-butoxy-4-methoxybenzyl)- 
2-imidazolidinone]  and  IBMX  (3-isobutyl-l-methylxanthine) ,  also  caused  the 
induction  of  ODC  when  added  to  confluent  cells.   Since  all  these  agents  are 
known  to  bring  about  the  accumulation  of  cAMP ,  it  has  been  suggested  that  ODC 
induction  is  mediated  by  cAMP,  which  probably  operates  on  the  level  of  gene 
transcription. 

Significance  to  Biomedical  Research:   The  activity  of  ornithine  decarboxyl- 
ase (ODC),  the  initial  enzyme  in  polyamine  biosynthetic  pathwa-",  fluctuates 
during  the  growth  cycle  of  neuroblastoma  and  glioma  cells.   The  activity  of 
the-  enzyme  is  1000  times  higher  in  proliferating  cells  compared  with  stationary 
ones.   This  study  indicates  that  cAMP  mediates  the  induction  of  ODC.   Growth 
of  neural  cells  may  thus  be  regulated  by  cAMP  by  modulating  cellular  ODC  and 
polyamine  levels. 

1  U 


Project  No.  Z01  HL-00008-01  LBG 

1.  Biochemical  Genetics 

2 .  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-IIH 

Individual  Project  Report 

July  1,  197^  through  June  30,  1975 


Project  Title:   Protein  Phosphorylation  in  Neuroblastoma  Cells 

Previous  Serial  Number:   None 

Principal  Investigators:   Steven  Sabol,  M.D.  ,  Ph.D.  and  Marshall  Nirenberg, 

Ph.  D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Major  Findings:   C-1300  neuroblastoma  cells  have  been  shown  to  exhibit  a 
variety  of  electrophysiological  responses  to  putative  neurotransmitters. 
Clone  N115  cells  respond  to  iontophoretic  application  of  cholinergic  agents 
by  transient  hyperpolarization  of  the  plasma  membrane.  Dr.  H.  Matsuzawa 
demonstrated  that  cholinergic  agents  also  cause  a  large  transient  increase 
in  the  content  of  guanosine  3'5f  monophosphate  (cyclic  GMP)  of  these  cells. 
Both  responses  follow  similar  time  courses  (5-60  seconds)  and  are  mediated 
by  muscarinic  cholinergic  receptors.   Because  of  the  existence  in  eukaryotic 
cells  of  protein  kinases  activated  by  cyclic  nucleotides,  it  was  hypothesized 
that  membrane  potential  changes  in  nerve  or  neuroblastom  cells  exposed  to 
muscarinic  cholinergic  agents  may  be  the  result  of  cyclic  GMP-dependent 
phosphorylation  of  membrane  proteins  concerned  with  ion  transport.   Pre- 
liminary studies  demonstrated  the  existence  in  N115  cells  of  histone  kinase 
activity  which  was  stimulated  by  physiological  concentrations  of  cyclic  GMP 
and  which  could  be  chromatographically  resolved  from  some  of  the  adenosine 
3 ' 5 '  monophosphate  (cyclic  AMP)  -  dependent  protein  kinase  activity.   In 
a  search  for  endogenous  substrates  for  protein  kinases  in  N115  cells,  gel 
electrophoresis  and  autoradiography  were  employed  to  identify  several  soluble 
proteins  which  were  phosphorylated  in  a  manner  dependent  on  cyclic  AMP  and 
to  equal  or  lesser  extent  on  cyclic  GMP.   No  strictly  cyclic  GMP-dependent 
kinase  substrates  were  found.   Electrophoretic  analysis  of  proteins  from 
whole  cells  treated  for  various  times  with  carbamyl choline  revealed  no 
obvious  changes  in  phosphoproteins  which  could  be  ascribed  to  the  elevation 
of  cyclic  GMP  concentration.   However,  more  highly  resolving  electrophoretic 
methods  are  currently  being  applied  to  this  problem. 

Significance  to  Biomedical  Research:   Through  this  work,  an  attempt  is  made 
to  understand  the  mechanism  of  generation  of  slow  postsynaptic  potentials 
which  occur  in  some  neurons  in  response  to  muscarinic  cholinergic  stimulation. 
Such  potentials  are  thought  to  be  important  in  the  control  of  nerve  excitation. 

i  /r 


Project  No.  Z01  HL-00009-01  LBG 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  197^  through  June  30,  1975 

Project  Title:   Gene  Expression  for  Neural  Properties 

Previous  Serial  Number  :   None 

Principal  Investigators:   Eliahu  Heldman ,  Ph.  D.  and  Marshall  Nirenberg,  Ph.D. 

Other  Investigators:   John  Minna,  M.D.  and  Hayden  Coon,  M.D.  (NCl). 

Cooperating  Units:   National  Cancer  Institute,  LCB 

Project  Description: 

Major  Findings:   During  the  past  few  years,  numerous  neuroblastoma  cell 
lines  were  obtained  and  characterized  with  respect  to  neuronal  properties  such 
as  transmitter  synthesis,  storage,  catabolism,  receptors  and  effects  of 
receptor  activation. 

Additional  cell  lines  with  new  neural  phenotypes  were  generated  and  questions 
of  dominance  of  gene  expression  and  complementation  were  explored  by  fusing 
neuroblastoma  with  other  cells  and  obtaining  hybrid  cell  lines.   The  expres- 
sion of  genes  for  neural  properties  was  found  to  be  dominant  with  most 
matings.   Some  hybrid  clones  expressed  the  neural  phenotypes  of  the  neuro- 
blastoma parent  50  or  more  cell  generations  after  fusion  ;  many  others  had 
specific  defects  in  transmitter  synthesis,  storage,  and  catabolism;  response 
to  neurotransmitters,  action  potential  reactions;  and  so  forth.   Another  class 
of  hybrids  had  acquired  new  neural  properties  which  were  not  detected  with 
parental  neuroblastoma  cells.   For  example,  we  previously  showed  that  fusion 
of  mouse  neuroblastoma  cells  with  rat  glioma  cells ,  both  lacking  choline 
acetyltransferase  activity  which  store  acetylcholine  and  have  clear  vesicles 
identical  in  appearance  to  those  found  at  synaptic  junctions.   Additional 
studies  now  show  that  fusion  of  mouse  neuroblastoma  cells  which  lack  tyrosine 
hydroxylase  activity  with  cells  from  normal  sympathetic  ganglia  from  mouse 
embryos  yields  hybrid  cells  with  high  tyrosine  hydroxylase  activity  that 
synthesize  dopamine,  possess  muscarinic  excitatory  acetylcholine  receptors, 
and  have  both  small  and  large  dense-core  vesicles.   Fusion  of  neuroblastoma 
cells  with  cells  from  normal  retina  yielded  some  cell  lines  that  synthesize 
catecholamines  and  another  that  synthesizes  acetylcholine.   These  results 
show  that  fusion  of  neuroblastoma  cells  with  cells  from  the  normal  nervous 
system  generates  hybrid  cells  with  new  neural  properties  which  have  not  been 
detected  with  the  parental  neuroblastoma  cells.   The  new  neural  phenotypes 


H 


Project  No.   Z01  HL  00009-01  LBG 


are  inherited  and  thus  far  have  "been  perpetuated  in  a  fairly  stable  fashion 
for  more  than  100  cell  generations.   This  approach  would  appear  to  be  a 
general  one  that  can  be  used  to  obtain  cell  lines  with  other  differentiated 
properties  that  can  be  used  to  elucidate  reactions  that  are  required  for  cell 
communication. 

Publications  : 

1.  Breakfield,  X.  0.  and  Nirenberg,  M.  W.:   Selection  for  neuroblastoma  cells 
that  synthesize  certain  transmitters.   Proc.  Nat.  Acad.  Sci.,  USA,  71: 
2530-2533,  19lh. 

2.  Giller,  E.  L. ,  Breakfield,  X.  0.,  Christian,  C.  N.,  Neale,  E.  A.  and 
Nelson,  P.  G. :   Expression  of  neuronal  characteristics  in  culture:  some 
pros  and  cons  of  primary  cultures  and  continuous  cell  lines.   In: 
Santini,  M.  (Ed.)  Golgi  Centennial  Symposium:   Perspectives  in  Neuro- 
biology. New  York,  Raven  Press,  pp.  603-623,  1975. 

3.  Breakfield,  X.  0.:   Reserpine  sensitivity  of  catecholamine  metabolism 
in  murine  neuroblastoma  clone  NIE-115+.   J.  Neurochem.,  197*+,  In  press. 


f* 


Project  No.  Z01  HL  00010-01  LBG 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Naturally  Occurring  Anti-Tumor  Antibodies 

Previous  Serial  Number :   None 

Principal  Investigator:   Sue  Ellen  Martin 

Other  Investigators:   William  J.  Martin,  M.D.,  (NCI) 

Cooperating  Units:   National  Cancer  Institute,  VLL 

Project  Description: 

Objectives:   Detection  and  characterization  of  tumor  related  cell  surface 

antigens. 

Methods  Employed:   In  vitro  cultivation  of  tumor  cells  of  both  mesodermal 
and  non-mesodermal  origin.   Antigenic  analysis  of  the  tumor  cell  lines  using 
both  immume  and  naturally  occurring  antibodies  in  the  complement  dependent, 
antibody-mediated  cytotoxicity  assay. 

Major  Findings:   Normal  mouse  sera  were  shown  to  contain  a  wide  variety  of 
naturally  occurring  antibodies  (NOA)  reactive  with  both  recently   derived 
and  long  term  transplantable  tumor  cell  lines.   The  occurrence  of  NOA  in  sera 
of  congenitally  athymic  (nude)  mice  indicated  that  the  production  of  these 
antibodies  was  thymus  independent.   Absorption  studies  revealed  that  the 
various  tumor  cell  lines  expressed  both  individually  distinct  and  shared 
antigens.   An  antigen  recognized  by  NOA  on  a  murine  cell  line  derived  from 
a  neuroblastoma  adrenal  metastasis  of  a  spontaneous  murine  ovarian  teratoma 
was  found  to  be  present  on  normal  brain  tissue  of  species  as  diverse  as  man 
and  chicken.   Many  of  the  tumor  antigens  recognized  by  NOA  could  not,  however, 
be  detected  on  normal  tissues  and  appeared  to  be  tumor  specific.   A  strain 
of  mouse  was  identified  which  had  a  genetically  determined  defect  in  the 
production  of  tumor  reactive  NOA.   These  mice  did  not  have  an  unusually  high 
incidence  of  spontaneous  tumors. 

Significance  to  Biomedical  Research:   While  it  has  generally  been  assumed 
that  the  immume  system  plays  an  important  role  in  the  detection  and  elimi- 
nation of  nascent  tumors,  little  evidence  in  support  of  this  hypothesis  has 
been  forthcoming.   The  recent  demonstration  that  nude  (congenitally  athymic) 
mice  do  not  have  an  increased  incidence  of  spontaneous  tumors  has  seriously 


0o 


Project  No.  Z01  HL  00010-01  LBG 

challenged  the  concept  that  T  cell  immunity  plays  a  crucial  role  in  immune 
surveillance  against  tumors. 

The  demonstration  that  some  NOA  are  directed  against  normal  tissue  antigens 
may  indicate  that  naturally  occurring  antibodies  serve  some  function  other 
than  immune  surveillance.   Such  anti-self  antibodies  may  play  an  important 
role  in  the  prevention  of  tissue  destructive  autoimmunity. 

Naturally  occurring  antibodies  provide  a  very  sensitive  method  with  which 
to  detect  both  tumor-associated  and  normal  tissue  antigens  and  a  powerful 
tool  for  the  identification  and  isolation  of  these  components. 

Proposed  Course:   Naturally  occurring  antibodies  will  be  used  for  the 
identification  and  isolation  of  cell  surface  components. 

Publications : 

1.  Martin,  S.  E.  and  Martin,  W.  J.:  Anti-tumor  antibodies  in  normal  mouse 
sera.   Int.  J.  Cancer,  In  press. 

2.  Martin,  S.  E.  and  Martin,  W.  J.:   Interspecies  brain  antigen  detected 
by  naturally  occurring  mouse  anti-brain  autoantibody.   Proc.  Nat.  Acad. 
Sci. ,  USA,  In  press. 


M 


Project  No.  z01  HL  00011-01  LBG 

1.  Biochemical  Genetics 

2 .  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Glutamic  Acid  Decarboxylase  in  Developing  Chick  Retina. 

Previous  Serial  Number:   None 

Principal  Investigator:   Fernando  DeMello ,  M.  D. 

Other  Investigators:   Marshall  Nirenberg,  Ph.  D. 

Cooperating  Units:   None 

Project  Description: 

Major  Findings:   The  objective  is  to  follow  the  appearance  of  glutamic 
acid  decarboxylase  activity  during  the  course  of  retina  differentiation 
and  attempt  to  correlate  it  with  synaptogenesis .   Glutamic  acid  decarboxylase 
(GAD)  activity  increases  and  shows  a  remarkable  elevation  which  levels  off 
after  hatching.  The  level  of  gamma  amino  butyric  acid  in  the  retina  increases 
with  the  increase  of  GAD  activity. 

Significance  to  Biomedical  Research:   The  role  neurotransmitters  play  in 
synaptogenesis  is  of  clear  importance  in  any  attempt  to  understand  the 
function  and  development  of  the  nervous  system. 

Proposed  Course:  We  are  now  attempting  to  study  the  regulation  processes 
involved  in  the  development  of  glutamic  acid  decarboxylase  in  chick  retina. 
Emphasis  is  to  be  made  on  the  role  of  cyclic  nucleotides  in  this  process. 

Publications:   None 


$3- 


Project  No.  Z01  HL  00012-01  LBG 

1.  Biochemical  Genetics 

2 .  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Muscarinic  Acetylcholine  Receptors  in  Cultured  Cell  Lines 

Previous  Serial  Number:   None 

Principal  Investigators:   Marshall  Nirenberg,  Ph.D.  and  William  Klein, 

Ph.D. 

Other  Investigators :   None 

Cooperating  Units:   None 

Project  Description: 

Previous  experiments  have  indicated  that  a  number  of  cell  lines  isolated 
in  the  laboratory  possess  active  acetylcholine  receptors.   (Unpublished 
results,  Nirenberg,  Matsuzawa,  Sharma,  Catterall,  and  Gibbons).   As  clonal 
lines  present  a  number  of  advantages  for  studying  neural  phenomena,  the 
presence  of  such  receptors  on  these  lines  may  be  particularly  useful  in 
studying  receptor  action.   The  cells  afford  an  opportunity  to  study  the 
molecular  basis  of  receptor  function  as  well  as  such  important  aspects  as 
short-term  and  long-term  regulation  of  receptor  activity.   Investigating  the 
nature  of  receptor  function  and  regulation  is  the  long-range  focus  for  this 
line  of  experimentation.   Initial  results  are  described  below. 

Direct  binding  studies  have  been  employed  to  confirm  and  quantitate  the 
occurence  of  acetylcholine  receptors  in  several  cell  lines.   Radioactive 
quinuclidinyl  benzilate  (QNB) ,  a  highly  sensitive  and  specific  muscarinic 
antagonist,  was  prepared  and  purified  and  used  to  assay  receptor  concentra- 
tions in  intact  cells  under  physiological  conditions.   Cell  lines  with 
hyperpolarizing  responses  to  acetylcholine  and  others  with  depolarizing 
responses  have  been  measured  to  have  50-100  fmoles  of  muscarinic  receptor 
per  mg  protein.   Control  cells  with  no  measurable  levels  of  receptor  have 
been  found.   The  assay  is  sensitive  to  less  than  5  fmoles  per  mg  protein. 
Evidence  that  QNB  binds  to  muscarinic  receptors  as  expected  is  seen  in  the 
100-fold  greater  sensitivity  of  binding  to  oxotremorine  than  to  tubocurarine. 
Oxotremorine  and  tubocurarine  are  drugs  relatively  specific  for  muscarinic 
receptors  and  nicotinic  receptors,  respectively.   The  dissociation  constant 
for  (  H)-QNB  is  approximately  10   M.   Competition  experiments  done  with 
labeled  atropine  and  various  muscarinic  agents  are  in  agreement  with  results 
obtained  using  QNB. 


c23 


Project  No.  Z01  HL  00012-01  LBG 
Significance  to  Biomedical  Research:   A  variability  in  receptor  levels 
seen  from  day  to  day  suggests  that  the  amount  of  receptor  per  cell  may  be 
regulated.   Preliminary  experiments  suggest  that  receptor  concentration  may 
increase  with  the  age  of  culture. 

Culturing  cells  in  the  presence  of  muscarinic  agonists  appears  to  lower  the 
level  of  specific  QNB  binding.   Also,  a  shorter  pulse  of  agonist  appears  to 
desensitize  the  receptor,  lowering  QNB  binding  for  a  period  lasting  several 
hours.   Removal  of  oxygen  for  30  minutes  lowers  receptor  levels  about  50%  and 
starvation  of  cultures  lowers  receptor  levels  considerably  more. 

+       +2 
Removal  of  Na  and  Ca   from  the  assay  medium  has  no  effect  on  oxotremorine- 

sensitive  QNB  binding.   However,  a  large  oxotremorine-insensitive  component 

seen  in  complete  medium  is  greatly  reduced.   This  oxotremorine-insensitive 

binding  is  also  lost  after  cell  homogenization,  but  specific  binding  is  also 

lower  than  can  be  accounted  for  by  randomization  of  membrane  sidedness. 

Honors  and  Awards :   None 

Publications:   None 


&¥ 


Project  No.  Z01  HL  00013-01  LBG 

1.  Biochemical  Genetics 

2.  Molecular  Biology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Developmental  Regulation  of  Excitability 

Previous  Serial  Number:   None 

Principal  Investigator:   Jonas  B.  Galper,  M.D.  and  William  A. 

Catterall,  Ph.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   The  objectives  of  this  project  are  (1)  to  devise  biochemically 
manipulable  cell  culture  systems  for  studying  the  development  of  action  poten- 
tial generation  and  receptor  function  in  excitable  cells,  (2)  to  use  these 
cell  culture  systems  to  document  the  changes  in  action  potential  generation 
and  receptor  function  occurring  during  development,  and  (3)  to  understand  the 
role  of  hormonal  stimuli,  cell-cell  interaction,  and  synapse  formation  in 
these  developmental  changes. 

Methods  Employed:   Cultures  of  clonal  lines  of  mouse  neuroblastoma  and  rat 
skeletal  muscle  and  cultures  of  primary  embryonic  chick  muscle  were  prepared 
by  standard  procedures.   Primary  cultures  of  chick  embryonic  heart  cells  were 
prepared  either  as  monolayers  of  individual  beating  cells  or  as  suspension 
cultures  of  aggregates  of  a  few  hundred  synchronously  beating  cells.   The  ion 
transport  activity  of  action  potential  and  receptor  ionophores  was  studied 
using  measurements  of   Na  influx  as  described  previously  (Project  NHLI-307) . 

Major  Findings:   (1)  Neuroblastoma  cells  logarithmically  growing  in  either 
monolayer  or  suspension  culture  exhibit  significant  levels  of  action  potential 
Na  ionophore  activity  indicating  that  this  differentiated  property  is 
expressed  in  the  dividing  cell.   Inhibition  of  cell  growth  with  either  butyric 
acid  or  dibutyryl  cyclic  AMP  causes  a  marked  increase  in  ionophore  activity. 
(2)  Primary  cultures  of  embryonic  chick  skeletal  muscle  have  undetectable 
levels  of  action  potential  Na  ionophore  activity  until  at  least  one  day  after 
fusion  of  the  cells  into  multinucleate  myotubes.   The  observed  activity  is 
always  sensitive  to  tetrodotoxin  (K  =  1-3  x  10   M) .   Cultures  of  clonal 
lines  of  rat  skeletal  muscle  have  significant  levels  of  action  potential  Na 
ionophore  activity  before  fusion  and  exhibit  only  a  2-3  fold  increase  in 
activity  concomitant  with  fusion.   The  observed  activity  is  always  relatively 

i  s.r 


Project  No.  Z01  HL  00013-01  LBG 
insensitive  to  tetrodotoxin  (K  =  1-3  x  10   M) .   Thus  the  developmental 
regulation  of  the  action  potential  Na  ionophore  in  avian  and  mammalian  muscle 
appears  different.   (3)  At  least  three  distinct  ionophores  are  involved  in 
the  action  potential  in  normal  adult  heart:   a  nerve-like  action  potential  Na 
ionophore,  an  action  potential  Ca   ionophore  which  also  transports  Na  ,  and 
a  K  ionophore  required  for  repolarization.   During  development  in  ovo,  beating 
of  embryonic  chick  hearts  becomes  increasingly  more  sensitive  to  inhibition 
by  tetrodotoxin,  an  inhibitor  of  the  nerve-like  action  potential  Na  ionophore. 
At  the  same  time,  beating  becomes  less  sensitive  to  inhibition  by  compound 
D-600  which  is  thought  to  be  an  inhibitor  of  the  action  potential  Ca   /Na 
ionophore.   Beating  of  embryonic  heart  cells  in  monolayer  culture  is  insensitive 
to  tetrodotoxin  regardless  of  the  age  of  the  embryo  from  which  the  cells  were 
obtained  but  the  sensitivity  of  monolayer  cells  to  D-600  decreases  with 
embryonic  age  as  jLn  ovo.   Despite  the  tetrodotoxin  insensitivity  of  beating 
of  monolayer  heart  cells,  these  cells  have  significant  levels  of  neurotoxin 
stimulated   Na  uptake  indicating  significant  levels  of  action  potential  Na 
ionophore  activity  and„this  Na  transport  activity  has  -normal  sensitivity  to 
tetrodotoxin  (K  =10   M) .   Thus  these  cells  have  substantial  tetrodotoxin- 
sensitive  action  potential  Na  ionophore  activity  which  is  not  required  for 
beating.   This  transport  activity  is  also  inhibited  by  compound  D-600.   The 
inhibition  is  competitive  with  respect  to  the  activating_neurotoxins._,The  K 
for  inhibition  of  uptake  by  D-600  increases  from  5  x  10   M  to  1  x  10   M 
with  increasing  embryonic  age  as  does  the  K  for  inhibition  of  beating.   Thus 
D-600  can  inhibit  both  action  potential  Na   ionophore  activity  and  beating 
which  is  dependent  on  Ca   /Na   ionophore  activity  by  a  common  mechanism  whose 
sensitivity  changes  with  embryonic  age.   (4)   Aggregate  cultures  of  embryonic 
chick  heart  cells  beat  synchronously  in  culture.   The  beating  becomes  increas- 
ingly sensitive  to  tetrodotoxin  with  increasing  age  of  the  embryo  from  which 
the  cells  were  derived.   Aggregates  whose  beating  is  inhibited  by  tetrodotoxin 
reactivate  over  a  period  of  2  to  3  hours  in  culture  in  a  process  that  appears 
to  require  protein  synthesis.   Reactivation  occurs  only  in  cultures  prepared 
from  "transitional"  hearts,  those  whose  beating  is  partially  sensitive  to 
tetrodotoxin.   It  is  highly  dependent  on  the  choice  of  medium  and  serum. 
Reactivation  in  the  presence  of  tetrodotoxin  may  represent  a  rapid  response 
of  the  cells  to  inhibition  of  rhythmic  activity  which  involves  modification 
of  components  of  tetrodotoxin-sensitive  action  potential  ionophores.   (5)   A 
fraction  of  the  aggregates  of  chick  embryonic  heart  cells  formed  by  our  methods 
exhibit  asynchronous  beating  superficially  resembling  some  clinically  described 
arrhythmias.   The  fraction  of  aggregates  exhibiting  arrhythmias  varies  with 
culture  conditions  and  age  in  ovo. 

Significance  to  Biomedical  Research:   The  results  provide  new  insight  into 
the  developmental  regulation  of  membrane  macromolecules  involved  in  information 
transfer  and  processing  in  the  nervous  system  and  in  maintenance  of  normal 
beating  in  heart. 

Proposed  Course:   Planned  investigations  include  (1)  studying  further  the 
influence  of  cell  surface  interactions  and  cell  growth  rate  on  action  potential 
Na   ionophore  activity  of  neuroblastoma  cells;  (2)  extending  the  study  of 
developmental  regulation  in  muscle  to  include  other  mammalian  systems  and 
assessing  the  influence  of  nerve  on  the  development  of  muscle  action  potential 


tit 


Project  No.   ZQ1  HL  00013-01  LBG 
Na  ionophore;  (3)  studying  in  more  detail  the  differences  between  the  action 
potential  Na  ionophore  in  heart  cells  that  require  its  activity  for  beating 
and  those  that  do  not:  (4)  documenting  the  changes  in  activity  of  the  action 
potential  Na  and  Ca  /Na  ionophores  during  reactivation  of  heart  cell  aggre- 
gates made  quiescent  by  tetrodotoxin;  (5)  studying  the  effects  of  nerve  on 
the  action  potential  ionophore  activity  of  heart  cell  aggregates;  and  (6) 
developing  rational  procedures  for  inducing  arrhythmias  in  heart  cell  aggregates 
and  for  pharmacologic  treatment  of  such  arrhythmias. 

Honors  and  Awards :   None 

Publications:   None 


^r 


Project  No.   Z01  HL  00076-05  LBG 

1.  Biochemical  Genetics 

2.  Somatic  Cell  Genetics 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Genetics  of  Cyclic-AMP  Metabolism 

Previous  Serial  Number:   NHLI  308 

Principal  Investigator:   John  D.  Minna,  M.D. 

Other  Investigators:   Stuart  Brown,  Ph.  D.,  Thomas  Marshall,  Ph.  D. , 

Richard  Lemons,  Ph.  D.  and  Alfred  Gilman,  M.D. ,  Ph.D. 
(UVA) . 

Cooperating  Unit:   University  of  Virginia,  Department  of  Pharmacology. 

Project  Description: 

Major  Findings:   Clonal  mammalian  cells  replicating  in  vitro  have  been 
phenotyped  for  enzymes  and  receptors  relevant  to  cAMP  metabolism.   Cell  fusion 
studies  were  then  performed  and  hybrid  cells  characterized  for  their  phenotype 
for  these  properties.   To  date  prostaglandin  E^,  g  adrenergic  responses , 
phosphodiesterase ,  and  adenylate  cyclase  (basal  and  levels  following  hormone 
stimulation),  as  well  as  binding  of  labeled  prostaglandins  and  catecholamines 
are  being  studied.   We  are  concentrating  at  present  on  the  dominantly 
inherited  PGE   receptor-adenylate  cyclase  system  in  human  mouse  hybrid 
segregating  either  human  or  mouse  chromosomes. 

Significance  to  Biomedical  Research:   By  phenotyping  the  hybrids  for  their 
PGE-,  responsiveness  and  then  determining  their  chromosome  composition,  as- 
signment of  human  and  mouse  genes  for  these  metabolically  important  functions 
can  be  achieved. 

Honors  and  Awards :   None 

Publications:   Maguire ,  M.E. ,  Sturgill,  T.  W. ,  Anderson,  H.  J.,  Minna,  J.D. 
Gilman,  A.  G. :   Hormonal  control  of  cyclic  AMP  metabolism  in 
parental  and  hybrid  somatic  cells.   Advances  in  Cyclic 
Nucleotide  Research,  Vol.  5,  1977. 


A6 


Project  No.  Z01  HL  00077-03  LBG 

1.  Biochemical  Genetics 

2.  Somatic  Cell  Genetics 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Expression  of  Type  C  virus  in  Human-Mouse  Cell  Hybrids 

Previous  Serial  Number:   NHLI-311 

Principal  Investigators:   John  D.  Minna,  M.D.,  Thomas  H.  Marshall,  Ph.D., 

Richard  Lemons,  Ph.D.,  Ronald  Yasbin,  Ph.D.,  and 
Stuart  Brown,  Ph.D. 

Other  Investigators:   S.  H.  Wilson,  Ph.D.  and  A.  Gazdar,  M.D. 

Cooperating  Units:   NCI 

Project  Description: 

Human  x  rodent  hybrid  cell  lines  segregating  either  human  or  rodent  chromo- 
somes have  been  analyzed  for  the  expression  of  type  C  particle  markers  (oncor- 
navirus) using  RNA  dependent  DNA  polymerase  (RDDP)  assays,  specific  antisera 
against  viral  proteins,  electron  microscopy,  and  viral  biologic  activity. 
The  hybrid  cell  experiments  have  explored  three  general  systems:   1)  the  regu- 
lation of  characterized  rodent  virus  by  human  genes;  2)  the  ability  of  the 
hybrid  cells  to  support  the  replication  of  exogenously  applied  virus;  3)  the 
de  novo  (spontaneous  or  induced)  production  of  new  virus  by  the  hybrid  cells. 
In  order  to  undertake  these  studies  with  clinically  available  material  techniques 
were  established  that  allow  the  generation  of  hybrid  cells  after  fusion  to 
small  samples  of  normal  or  malignant  tissue  taken  directly  from  patients. 
The  following  results  have  been  obtained.   We  can  reproducibly  generate  large 
numbers  of  mouse  x  human  hybrids  with  all  leukemia  cell  types .   We  have  demon- 
strated that  human  genes  can  regulate  the  production  of  viral  RDDP  but  have 
no  apparent  effect  on  expression  of  the  main  viral  structural  protein. 

By  analyzing  hybrids  segregating  human  chromosomes  it  is  possible  to  assign 
these  regulatory  genes  to  human  chromosomes  14,  21  and  possibly  12.  Mouse 
xenotropic  virus  able  to  replicate  in  human  but  not  mouse  cells  has  a  complex 
genetic  control  pattern  when  tested  in  human  x  mouse  hybrid  cells.   In  addition, 
we  find  human  genes  can  block  the  induction  of  type  C  viral  genes  from  mouse 
genetic  integration  sites,  and  can  alter  the  host  range  of  mouse  virus.   Thus 
xeno  and  ecotropic  control  involves  surface  receptors,  intracellular  preinte- 
gration  regulation,  post  integration  control,  and  host  range  modification. 
By  applying  viruses  known  to  cause  neoplasia  in  primates  (woolly  monkey, 
gibbon  ape  lymphoma  virus)  to  hybrid  cells  segregating  human  chromosomes  we 


Af 


Project  No.   Z01  HL  00077-03  LBG 
are  systematically  looking  for  the  human  genes  required  for  their  replication 
and  integration.   Hamster-mouse  hybrids  losing  mouse  chromosomes  are  also 
being  studied  to  determine  which  mouse  genes  are  required  for  viral  gene 
replication.   Clones  able  and  clones  unable  to  support  murine  leukemia  viral 
replication  have  been  isolated.   This  should  widely  extend  the  previous  mouse 
genetic  studies. 

Significance  to  Biomedical  Research:   These  studies  will  allow  a  genetic 
analysis  of  human  and  mouse  genes  important  in  the  regulation,  production, 
replication  and  structure  of  virus  particles  known  to  play  an  important  role 
in  growth  regulation,  neoplasia  and  possible  differentiation.   In  addition, 
they  demonstrate  the  highly  evolved  mechanisms  for  oncogenic  virus  control  in 
humans . 

Publications: 

1.   Gazdar,  A.  F.,  Russell,  E.  K. ,  and  Minna,  J.:   Biologic  properties  of  a 
type  C  virus  isolated  from  a  human  x  mouse  hybrid  cell  line.   Proc.  Soc. 
Exp.  Med.  Biol. ,  In  press. 


3o 


Project  No.  Z01  HL  00078-03  LBG 

1.  Biochemical  Genetics 

2.  Somatic  Cell  Genetics 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Chromosome  Segregation  in  Hybrid  Cells 

Previous  Serial  Number:   NHLI-313 

Principal  Investigator:   John  D.  Minna,  M.D. 

Other  Investigators:   Thomas  Marshall,  Ph.D.,  Stuart  Brown,  Ph.D.,  Richard 

Lemons,  Ph.D.,  Ronald  Yasbin,  Ph.D.,  and  Hayden 
Coon,  M.D.  (NCI) 

Cooperating  Units:   Laboratory  of  Cell  Biology,  NCI 

Project  Description: 

We  have  found  that  chromosome  segregation  patterns  can  be  varied  by  proper 
selection  of  the  parent  cells  introduced  into  a  cell  fusion  reaction.   The 
fundamental  principal  we  have  discovered  is  that  fusion  of  a  transformed  tissue 
culture  line  to  fresh  normal  cells  from  a  laboratory  animal  results  in  the 
segregation  of  the  chromosomes  of  the  normal  cell  parent.   This  allows  gene 
mapping  by  testing  of  the  hybrid  lines  as  they  segregate  chromosomes.   The 
phenomonon  itself  is  of  fundamental  biologic  importance  with  respect  to  control 
of  foreign  genetic  information.   The  linkage  studies  will  allow  among  other 
things  the  development  of  a  genetic  evolutionary  map  for  the  mammalian  chromo- 
somes.  At  present  we  have  assayed  21  different  isozymes  in  mouse  x  hamster, 
mouse  x  human,  and  Chinese  hamster  x  human  hybrid  cells  losing  various  patterns 
of  chromosomes.   This  has  enabled  us  to  derive  new  linkage  data  for  the  mouse. 
Of  importance:  1)  we  have  demonstrated  that  at  least  15  different  isozymes 
are  asyntenic  in  the  mouse;  2)  that  genes  linked  in  the  human  that  are  on 
different  chromosome  arms  are  unlinked  in  the  mouse;  and  3)  that  at  least 
some  genes  on  the  same  chromosome  arm  in  the  human  are  linked  in  the  mouse. 

In  addition,  human  x  mouse  hybrid  cells  have  been  generated  by  mating  mouse 
tissue  culture  lines  to  cells  taken  from  human  patients  with  dominantly  inheri- 
ted genetic  diseases  and  malignant  leukemic  cells.   These  are  being  analyzed 
for  their  pattern  of  chromosome  segregation.   To  date  we  have  found  that  segre- 
gation of  human  chromosomes  in  such  hybrids  is  non-random  with  preferential 
elimination  of  some  human  chromosomes  while  others  are  selectively  retained. 

Significance  to  Biomedical  Research:   These  observations  are  of  fundamental 
biologic  importance  and  will  allow  the  construction  of  a  mammalian  evolutionary 


3t 


Project  No.  Z01  HL  00078-03  LBG 
chromosome  map.   This  genetic  approach  to  malignancy  and  other  genetic  disorders 
is  novel  and  should  uncover  human  genes  important  for  growth  and  virus  regula- 
tion. 

Honors  and  Awards :   None 

Publications : 

1.   Minna,  J.  D.  and  Coon,  H.  G. :   Human  x  mouse  hybrid  cells  segregating  mouse 
chromosomes  and  isozymes.   Nature,  252,  401-404,  1974. 


3% 


Project  No.      Z01  HL  00079-02   LBG 

1.  Biochemical  Genetics 

2.  Somatic  Cell  Genetics 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Genetic  Analysis  of  Differentiation  Using  Cell  Hybrids 

Previous  Serial  Number:   NHLI-305 

Principal  Investigators:   John  D.  Minna,  M.D.  and  Richard  Lemons,  Ph.D. 

Other  Investigators:   A.  Deisseroth,  M.D.,  A.  Neinhuis,  M.D.,  and  F. 

Anderson,  M.D. 

Cooperating  Units:   Molecular  Hematology  Branch,  NHLI 

Project  Description: 

Genetic  analysis  of  differentiated  functions  related  to  the  erythropoetic 
system  was  carried  out  using  somatic  cell  hybridization  techniques.   Friend 
virus  transformed  murine  erythroleukemia  cells  replicate  as  clonal  lines  in 
vitro  and  express  many  differentiated  functions  of  red  cells.   A  6-thioguanine 
resistant  mutant,  deficient  in  hypoxan thine  phosphoribosyl  transferase,  was 
isolated  and  shown  to  be  unable  to  replicate  in  selective  HAT  medium,  produced 
hemoglobin  after  induction  with  dimethylsulf oxide ,  and  exhibited  high  levels 
of  acetylcholinesterase .   These  cells  were  mated  to  mouse  and  human  fibroblasts 
not  expressing  these  differentiated  functions  and  the  resultant  hybrids  isolated 
and  characterized.   The  hybrids  before  chromosome  segregation  (with  retention 
of  chromosome  bearing  hemoglobin  structural  genes)  were  found  to  have 
extinguished  these  differentiated  functions.   The  specific  level  of  gene  regu- 
lation was  determined  by  molecular  hybridization  experiments.   The  hybrids 
were  found  to  produce  no  hemoglobin  messenger  RNA. 

Significance  to  Biomedical  Research:  This  represents  the  first  determination 
of  the  level  of  gene  regulation  following  extinction  of  differentiated  functions 
in  hybrid  cells. 

Honors  and  Awards :   None 

Publications:   None 


33 


Project  No.  Z01  HL  00080-02  LBG 

1.  Biochemical  Genetics 

2.  Somatic  Cell  Genetics 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Genetic  Analysis  of  Hyperlipidemias  Using  Cell  Hybrids. 

Previous  Serial  Number:   NHLI  312 

Principal  Investigator:   John  D.  Minna,  M.  D. 

Other  Investigators:   Thomas  Marshall,  Ph.  D. ,  Stuart  Brown,  Ph.  D. , 

Richard  Lemons,  Ph.  D.  and  Howard  Sloan,  M.D.  (MDB) 

Cooperating  Units:   NHLI,  Molecular  Diseases  Branch 

Project  Description: 

Major  Findings:   Fibroblasts  from  patients  with  homozygous  type  II  hyper- 
cholesterolemia bearing  the  defect  in  regulation  of  3-hydroxy-3-methyl- 
glutaryl  coenzyme  A  reductase  (EC  1.1.1.34,  HmGCoA)  were  fused  to  mouse  cells 
not  demonstrating  this  defect  and  hybrid  clones  isolated.   Hybrid  lines 
segregating  human  chromosomes  will  be  phenotyped  for  the  defect  and  for  their 
human  chromosome  content.   We  are  analyzing  the  lines  for  their  human  gene 
content.   To  date  21  different  human  isozymes  assigned  to  15  different  human 
chromosomes  have  been  tested  in  15  independently  isolated  hybrid  clones . 

Significance  to  Biomedical  Research:   Analysis  of  the  chromosome  and 
HmGCoA  phenotype  should  allow  a  systematic  genetic  analysis  of  human  genes 
involved  in  the  production  and  regulation  of  lipoproteins  and  chromosome 
assignment  of  the  gene  for  this  clinically  important  disorder. 

Honors  and  Awards :   None 

Publications :   None 


3* 


Project  No.  Z01  HL  00081-01  LBG 

1.  Biochemical  Genetics 

2.  Somatic  Cell  Genetics 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Bacteriophage  Resistance  in  Transformed  Bacillus 
subtilis 

Previous  Serial  Number:   None 

Principal  Investigator:   Ronald  E.  Yasbin,  Ph.D. 

Other  Investigators:   Frank  E.  Young,  M.D.,  Ph.D. 

Cooperating  Units:   University  of  Rochester,  Department  of 
Microbiology 

Project  Description: 

B_.  subtilis  W23,  unlike  B_.  subtilis  168,  is  resistant  to  bacteriophages 
SPP1,  SP02,  tf>105  and  <j>29.   This  resistance  has  been  attributed  to  the  inability 
of  these  bacteriophages  to  absorb  the  cell  wall  of  strain  W23.   On  the  other 
hand,  bacteriophages  4>e,  <J>25,  SP01,  and  SP82  plaque  with  an  efficiency  of  10 
to  10   on  strain  W23  as  compared  to  strain  168.   However,  these  four  bacterio- 
phages absorb  equally  well  to  the  cell  walls  of  B_.  subtilis  168  and  ^•_fi 
subtilis  W23.   Strain  168  was  transformed,  at  a  very  low  frequency  (10   ), 
to  SPP1/SP02  resistance  by  DNA  isolated  from  strain  W23.   Characterization 
of  four  isolated  transformants  revealed  that  all  were  completely  resistant 
to  bacteriophages  cj>105  and  <j)29.   One  of  the  strains  (RUB824)  was  completely 
resistant  to  both  bacteriophages  SPP1  and  SP02  while  RUB823  was  completely 
resistant  to  bacteriophage  SPP1  and  only  partially  resistant  to  bacteriophage 
SP02.   In  addition,  the  remaining  two  transformed  strains  (RUB821  and  RUB822) 
were  only  partially  resistant  to  both  bacteriophages  SPP1  and  SP02.   Strain 
RUB824  was  completely  resistant  to  bacteriophage  SP82  while  partially  resis- 
tant to  bacteriophage  SP01.   On  the  other  hand,  strains  RUB821,  822,  and  823 
were  completely  resistant  to  bacteriophage  SP01  while  being  only  partially 
resistant  to  bacteriophage  SP82.   The  successful  absorption  of  bacteriophage 
to  these  transformed  strains  does  not  necessarily  result  in  a  successful 
infection.   These  results  indicate  the  complex  nature  of  bacteriophage 
resistance  in  B_.  subtilis.   Additionally,  utilizing  the  processes  of  transfor- 
mation and  transfection,  it  appears  that  bacteriophage  DNA  is  discriminated 
against  in  these  four  strains.   These  results  suggest  the  acquisition  and/or 
activation  of  nucleases  in  the  transformed  strains  which  were  not  present  in 
the  parent  168  strain.   The  action  of  these  nucleases  is  presently  under 
inves  t  igat  ion . 


3T 


Project  No.  ZQ1  HL  Q0Q81-01  LBG 

Significance  to  Biomedical  Research:   This  research  is  designed  to 

elucidate  the  types  of  molecules  used  as  viral  receptors  on  cell  surfaces. 

Publications :   None 


Zl 


Project  No.   Z01  HL  00082-01  LBG 

1.  Biochemical  Genetics 

2.  Somatic  Cell  Genetics 

3.  Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Type  C  Virus  Particles  in  Normal  and  Diseased  Humans. 

Previous  Serial  Number:   None 

Principal  Investigators:   Stuart  Brown,  Ph.  D. ,  Richard  Lemons,  Ph.  D.  and 

John  D.  Minna,  M.D. 

Other  Investigators:   Dr.  R.  Young  (NCI)  and  Dr.  P.  Schein  (GUH) 

Cooperating  Units:   National  Cancer  Institute,  Medicine  Branch  and 
Georgetown  University  Hospital,  Department  of 
Oncology 

Project  Description: 

Major  Findings:   Type  C  virus  particles  have  been  implicated  in  neoplastic 
disease,  growth  regulation,  and  intracellular  information  transfer.   Certain 
biochemical,  biologic  and  immunologic  characteristics  of  these  particles 
allow  for  their  identification.   To  be  clinically  useful,  assay  for  such 
particles  must  be  performed  using  small  samples  of  easily  obtained  patient 
material,  collected  under  conditions  readily  available,  followed  by  a  minimum 
of  processing.   We  have  established  these  conditions  as  well  as  the  assay  for 
one  such  marker,  RNA  dependent  DNA  polymerases  in  small  samples  of  peripheral 
blood  body  fluid,  or  tissue  and  then  assayed  a  large  number  of  normal  in- 
dividuals.  Of  interest  we  find  in  plasma,  serum  and  platelets  a  particle 
sedimenting  at  40,000  x  g  but  not  10,000  x  g  which  contains  a  DNA  polymerase 
activity.   By  a  series  of  tests  we  have  established  that  the  assay  for  this 
activity  is  reproducible,  sensitive,  and  can  be  quantitatively  performed  on 
crude  biologic  preparations.   Partial  characterization  of  the  activity  shows 
it  to  be  similar  to  a  class  of  cytoplasmic  DNA  polymerases  (type  III,  or 
gamma),  that  are  related  but  not  identical  to  type  C  virus  DNA  polymerase. 
The  source  of  the  particle  is  at  present  unknown  but  could  be  released  from 
platelets  during  hemostasis.   Its  biologic  role  is  unknown  but,  because  of 
its  transmission  during  transfusion  therapy,  and  because  of  its  ability  to 
circulate  around  the  body,  could  play  an  important  physiologic  as  well  as 
pathologic  role.   A  large  number  of  samples  have  been  collected  from  patients 
with  neoplastic  and  non-neoplastic  diseases  of  possible  type  C  viral  origin 
(eg.  systemic  lupus  erythematosis)  and  are  being  assayed  using  the  above 
described  techniques.   By  fusing  human  cells  to  mouse  cells  carrying  an 
integrated  sarcoma  virus  genome,  a  hybrid  cell  can  be  made  that  will  be  of 
potential  use  for  biologic  detection  of  virus  particles.  When  the  hybrid 
cell  is  infected  with  a  replicating  type  C  virus  (leukemia  virus)  the  sarcoma 

1  37 


Project  Number  Z01  HL  00082-01  LBG 

virus  can  be  "rescued"  and  transforms  the  cells'  morphology  yielding  a 
scorable  "focus  or  plaque".   Because  of  the  human  genes  present  human 
derived  virus  may  be  introduced  into  such  a  hybrid,  and  this  cell  can  be 
used  for  focus  formation  assay. 

Honors  and  Awards:   None 

Publications:   None 


n 


Project  No.  Z01  HL  00151-05  LBG 

1.  Biochemical  Genetics 

2.  Macromolecules 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   The  Biology  of  Cyclic  Nucleotides  in  Ej_   coli 

Previous  Serial  Number:   NHLI-309 

Principal  Investigator:   Alan  Peterkofsky,  Ph.D.,  James  Harwood,  Ph.D.,  and 

Jose  Gonzales,  Ph.D. 

Other  Investigators:   Mrs.  Celia  Gazdar 

Cooperating  Units:   None 

Project  Description: 

Major  Findings:   Cyclic  AMP  (cAMP)  plays  a  key  role  in  metabolic  control 
in  15.  coli.   It  is  required  for  transcription  of  the  genes  for  many  induced 
enzymes.   Glucose  is  effective  in  regulating  cAMP  levels.   Previous  studies 
showed  an  inverse  correlation  between  the  presence  of  glucose  in  culture 
medium  and  the  accumulation  of  cAMP.   This  observation  could  not  be  explained 
by  the  action  of  glucose  as  a  repressor  of  adenylate  cyclase  synthesis,  as 
a  stabilizer  of  cAMP  phosphodiesterase ,  or  as  a  direct  inhibitor  of  adenylate 
cyclase  activity  in  cell-free  preparations.   Our  recent  development  of  an 
in  vivo  assay  for  adenylate  cyclase  provided  a  basis  for  further  exploring 
the  inhibitory  action  of  glucose  in  intact  cells.   With  this  assay  it  was 
possible  to  show  that,  while  glucose  does  not  affect  adenylate  cyclase  in 
vitro,  it  rapidly  inhibits  the  enzyme  activity  in  intact  cells.   Extensive 
metabolism  of  glucose  is  not  required  since  a-methyl  glucoside  also  inhibits 
adenylate  cyclase  in  vivo.   Dose-response  studies  indicate  that  low 
concentrations  of  glucose  lead  to  essentially  complete  inhibition  of  adenylate 
cyclase  activity  while  only  moderately  decreasing  intracellular  cAMP  levels. 
We  therefore  concluded  that  the  decreased  cellular  cAMP  levels  resulting 
from  glucose  addition  to  intact  cells  can  be  accounted  for  by  inhibition  of 
adenylate  cyclase  without  any  significant  effect  on  cAMP  phosphodiesterase 
or  the  transport  of  cAMP  from  the  cells  into  the  medium. 

When  E_.  coli  B  is  grown  in  glucose-supplemented  medium,  it  possesses 
adenylate  cyclase  activity  which  can  be  inhibited  by  glucose  but  relatively 
few  other  compounds.   When  the  bacteria  are  grown  on  a  variety  of  other 
compounds  such  as  fructose,  mannitol,  or  lactose,  the  organisms  contain 
adenylate  cyclase  activity  which  is  inhibited  by  that  carbon  source,  while 
maintaining  the  capacity  of  the  enzyme  to  be  inhibited  by  glucose.   Those 
compounds  which  are  effective  as  inhibitors  of  adenylate  cyclase  in  bacteria 

1  39 


grown  under  various  conditions  are  also  effective  in  controlling  cellular 
cAMP  levels.   A  comparison  of  the  kinetics  of  induction  of  the  transport 
system  for  mannitol  and  the  acquisition  of  mannitol-sensitivity  of  adenylate 
cyclase  suggested  a  relationship  of  the  two  processes.   Other  studies 
indicated  that  utilization  of  substrate  through  a  transport  system  is  required 
for  adenylate  cyclase  inhibition. 

Significance  to  Biomedical  Research:   We  have  established  that  adenylate 
cyclase  in  broken  cell  preparations  shows  no  regulation  by  effectors;  however, 
in  intact  cells,  glucose  effectively  inhibits  adenylate  cyclase.   Sugars 
other  than  glucose  will  inhibit  adenylate  cyclase  provided  their  transport 
systems  are  present.   Our  currently  available  data  suggest  that  the  membrane- 
bound  adenylate  cyclase  of  E_.  coli  represents  a  powerful  model  system  for 
the  regulation  by  effectors  of  adenylate  cyclase.   Further  studies  in  this 
system  may  provide  insight  into  the  mechanism  by  which  receptor-bound  hormones 
influence  adenylate  cyclase. 

Publications: 

1.  Peterkofsky,  A.  and  Gazdar,  C. :   Glucose  inhibition  of  adenylate  cyclase 
in  intact  cells  of  Escherichia  coli  B.   Proc.  Nat.  Acad.  Sci. ,  USA,  71, 
2324-2328,  1974. 

2.  Peterkofsky,  A.,  Harwood,  J.,  and  Gazdar,  C:  Inducibility  of  sugar 
sensitivity  of  adenylate  cyclase  of  E_.  coli  B.  J_.  Cyclic  Nucleotide 
Res.,  1,  11-20,  1975. 


& 


Project  No.  Z01  HL  00152-02  LBG 

1.  Biochemical  Genetics 

2 .  Macromolecules 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  197^  through  June  30,  1975 


Project  Title:   Mechanism  in  Protein  Synthesis 

Previous  Serial  Number:   NHLI-310 

Principal  Investigators:   Chandan  Prasad,  Ph.  D.  and  Alan  Peterkofsky,  Ph.  D, 

Other  Investigators :  None 

Cooperating  Units:   None 

Project  Description: 

Major  Findings:   Pyroglutamic  acid  (pGlu)  occurs  at  the  amino-terminus  of 
peptides  and  proteins  including  immunoglobulin  chains.  While  studies  on 
the  initiation  of  protein  synthesis  in  eucaryotic  cells  have  shown  that 
methionine  is  the  initiating  amino  acid,  the  initiation  of  synthesis  of 
proteins  with  blocked  N-terminal  amino  acids  was  not  well  understood.   We 
have  now  been  able  to  show  that  methionine  transiently  labels  the  amino 
terminus  of  an  immunoglobulin  light  chain  which  contains  amino-terminal 
pGlu.   We  have  therefore  concluded  that  methionine  is  the  initiator  amino 
acid  for  the  synthesis  of  mouse  plasmacytoma  light  chain  containing  N- 
terminal  pGlu. 

Significance  to  Biomedical  Research:   Methionine  is  the  initiator  amino 
acid  for  the  synthesis  of  an  immunoglobulin  light  chain  which  contains 
amino-terminal  pyroglutamic  acid. 

Proposed  Course:  We  will  continue  to  explore  the  mechanism  of  synthesis 
of  pyroglutamic  acid  in  proteins  and  peptides. 

Publications: 

1.   Prasad,  C.  and  Peterkofsky,  A.:   Initiation  by  methionine  of  mouse 

immunoglobulin  light  chain  containing  NH2~terminal  pyroglutamic  acid. 
J.  Biol.  Chem.  250:   171-171+ ,  1975- 


ff 


ANNUAL  REPORT  OF  THE 

LABORATORY  OF  BIOCHEMISTRY 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

July  1,  1974  through  June  30,  1975 

SECTION  ON  ENZYMES 

Research  in  the  Section  on  Enzymes  is  concerned  with  studies  on  biochemi- 
cal mechanisms  of  cellular  regulation  and  mechanisms  of  enzyme  action. 

Biochemical  Mechanisms  of  Cellular  Regulation. 

(A)  Glutamine  Synthetase.   Regulation  of  glutamine  synthetase  in  E.  coli 

is  mediated  by  a  "closed"  bicyclic  nucleotidylylation  cascade.   One  cycle 

involves  the  uridylylation  and  deuridylylation  of  the  regulatory  protein,  ?xi' 

which  is  catalyzed  by  uridylyltransferase  (UT)  and  uridylyl  removing  (UR) 

enzyme  respectively.   The  other  cycle  involves  adenylylation  and  deadenylyl- 

ation  of  glutamine  synthetase  (GS)  both  of  which  are  catalyzed  by  adenylyl- 

transferase  (ATase) .   Coupling  of  the  two  cycles  derives  from  the  fact  that 

the  unmodified  form  of  Pjj  (^iia)  stimulates  adenylylation  activity  of  ATase, 

whereas  the  uridylylated  Pj-j-  (Pjid)  stimulates  the  deadenylylation  activity  of 

ATase.   A  steady  state  analysis  of  this  unique  bicyclic  cascade  showed  that 

for  any  given  metabolic  state  the  level  of  glutamine  synthetase  adenylylation 

is  determined  by  the  relationship  [Eq  (1)1, 

12  k1k3UR 
(1)  GS-  =  -!— - — - — — - — ■ — - — ,  in  which  n  =  the  average  number  of  covalently 

K^lC/  Uti  t  iCi  '^'3  Up 

bound  adenylyl  groups  per  mole  of  GS;  k-^ ,  k2,  ko,  and  k,  are  the  specific  rate 
constants  for  the  deuridylylation,  uridylylation,  adenylylation  and  deadenylyl- 
ation reactions  respective,  and  Uj.  and  UR  denote  the  uridylyltransferase  and 
uridylyl  removing  enzyme  activities,  respectively.   Bearing  in  mind  that  the 
■activity  of  GS  is  inversely  proportional  to  the  state  of  adenylylation,  this 
equation  illustrates  the  enormous  allosteric  control  potential  of  this  cascade; 
variations  in  any  one  or  all  of  the  6  parameters  in  response  of  fluctuations 
in  the  concentrations  of  multiple  allosteric  effectors  will  lead  to  different 
state  levels  of  adenylylation. 

With  the  separation  and  purification  of  GS ,  ATase,  Pjj,  and  the  UTase-UR 
enzyme  complex  it  has  become  possible  to  examine  the  effects  of  various  meta- 
bolites on  each  step  in  the  cascade. 

The  PTTA  supported  adenylylation  of  GS  by  ATase  (k^  in  equation  (1))  is 
inhibited  synergistically  by  Ptid  and  CC-ketoglutarate,  and  is  stimulated  by 
glutamine,  methionine  and  tryptophan.   The  P^jp  stimulated  deadenylylation 
(k/  of  equation  (1))  requires  the  presence  of  a-ketoglutarate  and  ATP,  and  is 
inhibited  by  glutamine,  UTP,  P-enolpyruvate,  and  3-phosphoglycerate.   The 
uridylylation  of  P-r-r  (k£  of  equation  (1))  is  stimulated  by  a-ketoglutarate, 
ATP,  and  is  inhibited  by  glutamine  and  orthophosphate;  whereas  the  deuridylyl- 
ation of  Ptxd  (ki  of  equation  1)  is  stimulated  by  Mn  +  and  glutamine  and  is 
inhibited  by  CMP,  UMP,  and  CoA.   A  high  molecular  weight  endogeneous  inhibitor 
of  UR  activity  was  partially  purified  and  characterized  as  a  relatively 


43 


specific  CMP  and  UMP  binding  compound.   Activity  of  this  binding  substance  is 
resistant  to  treatment  with  proteases,  nucleases,  phospholipases,  and  lysozyme. 
The  demonstration  that  there  are  reciprocal  effects  of  glutamine  and  a-keto- 
glutarate  at  almost  every  step  in  the  cascade,  emphasizes  the  key  role  of  these 
substances  in  regulating  GS  activity. 

Studies  on  the  mechanism  of  cumulative  feedback  inhibition  of  unadenylyl- 
ated  glutamine  synthetase  were  continued.   From   detailed  kinetic  analyses, 
inhibition  constants  were  obtained  for  ATP,  CTP,  AMP,  histidine,  tryptophan, 
and  alanine.   Determinations  of  the  coefficients,  OC,   which  reflects  the  degree 
of  interaction  between  two  inhibitors  that  bind  to  separate  sites  on  the 
enzyme,  disclosed  that  ATP  and  CTP  react  at  a  common  site  whereas  AMP,  histi- 
dine, and  tryptophan  each  react  at  different  sites  on  the  enzyme.   Furthermore, 
it  was  shown  that  at  infinite  concentrations,  AMP,  alanine,  and  glycine  produce 
complete  inhibition,  but  inhibition  by  histidine  is  only  507c.   In  general,  the 
results  support  earlier  conclusions  that  there  are  separate  binding  sites  on 
the  enzyme  for  most  inhibitors,  but  in  addition  they  show  that  there  is  con- 
siderable interaction  between  most  binding  sites. 

Earlier  work  from  this  laboratory  determined  that  the  genes  designating 
glutamine  synthetase  (GS)  glutamate  synthase  (GAT) ,  and  glutamate  dehydrogenase 
(GDH)  do  not  constitute  a  contiguous  operon  in  E.  coli  and  may  be  located  at 
approximate  map  positions  77',  50',  and  21'  respectively.   Enzymic  analysis  of 
a  group  of  revertants  from  gin-  to  gln+  revealed  a  thermolabile  GS  in  each 
case  thereby  substantiating  that  the  gene  locus  at  77',  gin  A,  is  the  struc- 
tural gene  for  GS .   Two  of  the  revertants  with  thermolabile  GS  exhibited  poor 
derepression  on  limiting  NH3  although  the  adenylylation  values  were  low  as 
expected.   To  reconcile  these  data  with  the  autogenous  regulation  scheme  pro- 
posed by  Magasanik  et  a_l.  the  scheme  should  be  expanded  to  include  a  positive 
activation  or  GS  as  well  as  repression  by  adenylylated  GS .   Results  of  pre- 
liminary investigations  or  the  relationship  between  methionine  sulfoxamide 
resistance  and  GS  derepression  suggest  that  if  the  structural  gene  for  GS  is 
autogenously  regulated,  it  functions  in  cooperation  with  some  other  element  - 
perhaps  a  component  of  GAT. 


Significance  of  Cascade  Control  Processes. 

The  glutamine  synthetase  bicyclic  cascade  system  is  unique  since  the  two 
interconvertible  forms  of  Pj-j-  are  oppositional  in  their  capacities  to  stimu- 
late ATase  to  catalyze  adenylylation  and  deadenylylation  of  GS .   More  con- 
ventional cascade  systems  are  of  the  type  involved  in  the  activation  of 
muscle  phosphorylase.   Here  regulation  is  mediated  by  cyclic  covalent  modifi- 
cation reactions  in  which  only  the  active  form  of  an  enzyme  in  one  cycle  is  a 
catalyst  for  the  covalent  modification  of  an  enzyme  in  the  next.   Steady  state 
analysis  of  such  systems  shows  that  when  the  cascade  involves  (n-1)  successive 
cyclic  covalent  modification  reactions  and  an  allosteric  activation  of  the 
first  enzyme  in  the  series,  then  under  steady  state  equilibrium 
conditions,  the  fraction  of  the  modified  form  of  the  target  enzyme  (Ena)  is 
described  by  Eq.  (2), 


4* 


Jna 


En  "  'M+1^Y"L  <^r2  +     ^T~  (2) 

with  the  assumptions  that:   (a)  the  catalytic  constants  for  the  forward  step, 

kf  =  klf  El  =  k2fE2  =  •'•  k(n-l)fE(n-l)>  where  klf>  k2f>  '"  k(n-l)  are 
specific  rate  constants  for  successive  forward  steps  in  the  cycles  and  Ei, 
E2>    ' ' "  En  are  total  concentrations  of  enzymes  undergoing  activation  at  the 
first  step,  first  cycle  and  (n-1)  cycle  respectively;  (b)  the  catalytic  con- 
stants for  the  regeneration  of  the  unmodified  forms,  k1  =  ki   Ri  =  k   R  = 

k(n-l)rRn-l'  where  klr>  k2r>  ""  k(n-l)r  are  specific  rate  constants  for 
the  successive  regeneration  steps  and  Rj_ ,  R2,  -••  R(n_]\  are  concentrations  of 
enzymes  catalyzing  the  regeneration  steps.   Equation  (2)  demonstrates  the 
tremendous  amplification  potential  of  such  cascade  systems.   It  follows  that 
the  concentration  of  effector,  e,  required  to  produce  50%  conversion  of  En  to 
Ena,  decreases  in  such  a  manner  that  log  e0-5  is  inversely  proportional  to  the 
number  of  cycles  in  the  cascade.   Thus,  when  Kd  =  1  mM  and  k^/kf  =  0.1,  eQ  5 
is  approximately  equal  to  1,  0.1,  0.01,  and  0.001  mM  for  a  0,  1,  2,  and  3 
cycle  cascade,  respectively.   In  addition  to  this  amplification  capacity,  such 
systems  exhibit  an  enormous  capacity  for  allosteric  control.   It  is  evident 
from  Eq.  (2)  that  positive  or  negative  allosteric  interactions  with  any  one  or 
all  of  the  several  enzymes  in  the  cascade  will  affect  the  catalytic  constants 
of  these  enzymes  and  thereby  regulate  the  over-all  ratio  of  k^/kt,  which  in 
turn  determines  the  degree  of  amplication. 

(B)  Regulation  of  Enzyme  Levels.   Among  the  more  important  mechanisms  of 
metabolic  regulation  are  those  concerned  with  the  regulation  of  enzyme  levels. 
The  concentration  of  any  particular  enzyme  in  the  cell  reflects  balance  between 
its  de  novo  synthesis  and  its  degradation.   In  a  continuing  effort  to  develop 
a  convenient  model  system  for  studying  the  regulation  of  specific  enzyme 
degradation,  the  mechanisms  that  underlie  the  disappearance  of  some  key  enzymes 
in  nitrogen  metabolism  is  being  investigated  in  resting  cultures  of  E.  coli  and 
Klebsiella  aerogenes  subjected  to  conditions  of  nitrogen  starvation. 

a.  Inactivation  of  aspartokinases  in  E.  coli.   Studies  with  suspensions 
of  nitrogen  starved,  permeabilized  (toluene  treated)  E.  coli  cells  have  shown 
that  the  selective  inactivation  of  both  the  lysine-sensitive  and  the  threonine- 
sensitive  aspartokinase  isozymes  is  dependent  upon  a  carbon  source  and  is 
inhibited  by  anaerobiosis,  EDTA,  HCN,  and  chloramphenicol.   A  soluble  enzyme 
system  that  catalyzes  inactivation  of  the  threonine-sensitive  but  not  the 
lysine-sensitive  aspartokinase  has  been  partially  purified  from  cell  free 
extracts.   A  heat  stable  factor  that  is  essential  for  this  enzyme  catalyzed 
inactivation  was  isolated  from  boiled  extracts  and  was  identified  as  glycerol. 
The  nature  of  the  inactivation  process  in  under  investigation. 

b.  Inactivation  of  the  lysine-sensitive  aspartokinase  and  glutamine 
synthetase  in  K.  aerogenes.   Inactivation  of  the  lysine-sensitive  asparto- 
kinase  and  glutamine  synthetase  activities  in  K.  aerogenes  is  induced  by 
nitrogen  starvation.   The  loss  of  either  enzyme  activity  is  dependent  upon  the 
presence  of  an  energy  source,  is  inhibited  by  dinitrophenol  and  is  greatly 


*r 


stimulated  by  concentrations  of  chloroamphenicol  that  inhibit  growth  and 
protein  synthesis.   The  loss  in  glutamine  synthetase  activity  is  associated 
with  a  loss  of  cross  reactivity  with  glutamine-specif ic  antibodies,  suggesting 
that  the  loss  in  activity  could  be  due  to  protein  degradation.   The  results 
suggest  further  that  the  chloramphenicol  induced  enzyme  degradation  is  due  to 
inhibition  of  the  de  novo  synthesis  of  these  enzymes,  thereby  upsetting  the 
balance  between  synthesis  and  degradation. 

(C)  The  Mechanism  of  Enzyme  Action. 

a.  Glutamine  synthetase.   The  mechanism  of  glutamine  synthetase  catalysis 
has  been  a  subject  of  considerable  controversy.   Meister  has  proposed  that  the 
reaction  proceeds  by  a  sequential  mechanism  in  which  ATP  and  glutamate  react 
first  to  produce  an  enzyme  bound  glutamyl-P  intermediate  which  then  reacts 
with  ammonia  to  produce  glutamine  and  Pi,  whereas  Boyer  concludes  that  the 
reaction  occurs  by  a  concerted  mechanism  in  which  all  substrates  (ATP,  gluta- 
mate and  ammonia)  react  simultaneously  on  the  enzyme  to  produce  a  transition 
state  intermediate  which  then  decomposes  to  yield  the  products.   With  the 
application  of  fast  reaction  techniques  and  other  physical  and  chemical  methods 
the  ability  of  glutamine  synthesis  to  catalyze  5  different  reactions  has  been 
studied  in  detail.   From  the  experimental  data  all  5  reactions  can  be  explained 
by  an  integrated  mechanism  in  which  all  reactions  occur  at  the  same  catalytic 
site.   By  following  the  changes  in  intrinsic  fluorescence  due  to  substrate 
binding  and  the  overall  biosynthetic  reaction,  the  kcat  and  individual  rate 
constants  of  the  reaction  catalyzed  by  the  Mg^+  activated  enzyme  could  be 
determined.   From  the  biphasic  nature  of  the  kinetic  data  obtained  in  the 
absence  of  ammonia  it  is  obvious  that  two  different  intermediates  are  formed. 
However,  in  the  presence  of  limiting  ammonia  only  the  fast  forming  intermediate 
is  observed.   From  the  time  required  for  consumption  of  ammonia  and  the 
kinetics  obtained  when  ammonia  is  added  to  the  enzyme-Mg-ATP-glutamate  complex, 
it  is  deduced  that  ammonia  reacts  only  with  the  second  intermediate.   Although 
these  and  the  other  results  could  be  explained  by  either  the  Meister  or  the 
Boyer  hypothesis,  they  are  more  compatible  with  the  postulate  that  glutamyl-P 
is  an  intermediate. 

b.  Role  of  vitamin  B]^2  coenzyme  in  conversion  of  CU-leucine  to  p-leucine. 

The  catabolism  of  the  branched  chain  amino  acids,  leucine,  isoleucine,  and 
valine,  is  incompletely  undsrstood.   Certain  inborn  errors  of  metabolism  have 
been  described  which  implicate  faulty  catabolism  of  these  amino  acids;  chief 
among  these  are  maple  syrup  disease  and  isovaleric  acidemia.   In  an  effort  to 
investigate  the  mechanism  of  catabolism  of  these  amino  acids  strains  of 
Clostridia  have  been  isolated  that  can  utilize  these  amino  acids  as  a  sole 
source  of  carbon,  nitrogen  and  energy  for  growth.   Evidence  was  obtained 
supporting  the  conclusion  that  leucine  degradation  in  one  of  these  organisms, 
Clostridium  sporogenes,  strain  EC-9,  occurs  by  the  following  mechanism: 
a-leucine  -.  f}-leucine  -.  p-ketoisocaproate  ->  acetate  +  isobutyrate.   The  mutase 
catalyzing  the  conversion  of  ct-leucine  to  p-leucine  has  been  partially  purified 
and  has  been  shown  to  require  B12-coenzyme  for  activity.   This  is  the  first 
time  that  Bj^-coenzyme  has  been  implicated  in  an  a   _  p  mutase  reaction.   The 
mutase  reaction  has  been  detected  in  livers  of  rats,  sheep  and  the  Rhesus 
monkey,  and  in  rat  kidney.   It  could  not  be  detected  in  either  chicken  or  dog 
livers . 


<M 


Project  No.  Z01   HL   00201-04   LB 
Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Metabolism  of  the  Branched-Chain  Amino  Acids 

Previous  Serial  Number:   NHLI-4 

Principal  Investigator:   J.  M.  Poston 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   The  catabolism  of  the  branched-chain  amino  acids,  leucine, 
isoleucine,  and  valine,  is  incompletely  understood.   Catabolic  pathways 
have  been  outlined  in  bovine  and  rat  liver  and  one  or  two  enzymes  have 
been  partially  purified  and  studied.   Certain  inborn  errors  of  metabolism 
have  been  described  which  implicate  faulty  catabolism  of  these  amino  acids, 
chief  among  these  are  maple  syrup  disease  and  isovaleric  acidemia.   These 
amino  acids  have  been  shown  to  participate  in  the  Stickland  reaction  and 
many  organisms  are  capable  of  fermenting  these  compounds  in  Stickland  pairs. 
Until  recently,  a  fermentation  in  which  one  of  these  amino  acids  serves  both 
as  the  carbon  and  energy  source  had  not  been  described.   Several  strains  of 
organisms  have  been  isolated  which  grow  on  leucine  in  a  single  amino  acid 
fermentation.   The  objectives  of  this  project  are  to  establish  the  fermenta- 
tion pathways  of  leucine  and  the  other  branched-chain  amino  acids  in  these 
organisms  and  to  examine  the  enzymes  responsible  for  the  various  metabolic 
steps  in  these  fermentations. 

Major  Findings: 

As  reported  previously,  when  cells  or  extracts  of  cells  of  Clostridium 
sporogenes  strain  EC-9  (one  of  the  strains  that  ferment  leucine)  are  incu- 
bated with  L-leucine,  several  metabolic  products  are  formed  that  are  consis- 
tent with  the  pathway  reported  in  mammals.   The  production  of  isobutyrate , 
however,  could  not  be  explained  on  the  basis  of  the  mammalian  pathway,  for 
it  does  not  arise  in  any  direct  way  from  leucine.   It  was  postulated  that  the 
pathway 

a-leucine  >  g-leucine  — -  3-ketoisocaproate '-->   acetate  +  isobutyrate 

might  be  operating  in  C^.  sporogenes.   When  evidence  was  sought  to  support  the 
postulated  mechanism,  it  was  found,  among  others,  that  incubation  of  extracts 
with  labelled  leucine  gave  rise  to  labelled  g-ketoisocaproate,  incubation  of 
extracts  with  increasing  amounts  of  B-ketoisocaproate  gave  rise  to  increasing 

1  47 


Project  No.  Z01   HL   00201-04   LB 

amounts  of  acetate,  and  incubation  of  extracts  with  g-leucine  gave  rise  to 
substrate-dependent  production  of  a-leucine. 

Direct  conversion  of  a-leucine  to  g-leucine  was  demonstrated  by  the 
formation  of  labelled  g-leucine  when  radioactive  a-leucine  was  incubated 
with  extracts  of  C.  sporogenes.   Because  g-leucine  is  very  difficult  to 
measure  directly,  most  investigations  of  the  enzyme  carrying  out  the  inter- 
conversion  of  a-  and  g-leucine  have  used  the  reverse  reaction  in  which  g- 
leucine  was  the  substrate  and  the  a-leucine  formed  was  measured  by  reaction 
with  nihnydrin.   In  general,  total  ninhydrin  reaction  has  been  measured,  but 
the  actual  formation  of  a-leucine  has  been  demonstrated  to  parallel  the  total 
ninhydrin  reaction.   This  was  done  using  the  automatic  amino  acid  analyzer. 

Initial  experiments  reported  last  year  have  been  extended  to  explore 
the  involvement  of  cobalamin  with  the  mutase  reaction.   Both  the  forward  and 
reverse  mutase  reactions  are  inhibited  by  intrinsic  factor.   When  coenzyme- 
B  _  is  added  to  the  incubations  there  is  a  marked  stimulation  of  the  reverse 
reaction.   Iron  does  not  seem  to  be  involved  in  this  mutation  nor  is  there 
any  effect  upon  addition  of  S-adenosylmethionine.   Whether  pyridoxal  phos- 
phate is  involved  in  the  mutase  reaction  is  not  yet  certain.   This  is  the 
first  known  example  of  an  a-g  mutation  that  is  B  -dependent.   Other  similar 
mutations  are  stimulated  by  iron  and  pyridoxal  phosphate  alone,  and  are  un- 
affected by  the  presence  or  absence  of  any  B-.  ?  coenzyme. 

The  leucine  mutase  activity  can  be  fractionated  with  ammonium  sulfate 
and  recovered  on  gel  filtration  but,  when  it  has  been  treated  with  DEAE- 
cellulose,  the  activity  recovered  has  not  been  stable  to  freezing  or  storage. 
Activity  levels  in  extracts  prepared  from  various  batches  of  cells  vary 
widely.   It  is  not  yet  clear  why,  but  both  the  stage  of  the  culture  (i.e., 
whether  or  not  sporulation  has  begun  to  occur)  and  the  nutrition  of  the  cells 
seem  to  influence  the  activity  levels. 

Other  organisms  have  been  surveyed  for  leucine  mutase  activity  and 
Clostridium  lentoputrescens  was  found  to  be  a  good  source.   Several  other 
Clostridia  including  C^.  kluyveri  and  a  choline-fermenting  Clostridium  had 
low  levels  of  the  activity.   C.  sticklandii  and  C.  propionicum  had  no  demon- 
strable activity. 

In  view  of  the  importance  of  the  liver  in  catabolizing  leucine  in  humans, 
several  species  were  examined  for  leucine  mutase  activity.   Rat,  sheep,  and 
Rhesus  monkey  livers  had  the  activity,  but  chicken  and  dog  livers  did  not. 
Rat  kidney  also  has  appreciable  activity. 

Proposed  Course  of  Action: 

The  leucine  mutase  will  be  purified  and  characterized.   To  this  end, 
the  conditions  which  yield  maximum  activity  in  cell  cultures  will  be  estab- 
lished.  The  nature  of  the  B-.  „  involvement  will  be  established.   The  g- 
leucine  deaminase  will  be  examined  and  the  fate  of  the  nitrogen  will  be 
determined.   The  g-ketoisocaproate  cleavage  enzyme  will  be  examined  and  its 

2  ¥B 


Project  No.  Z01   HL  00201-04   LB 

cof actors  established.   The  distribution  of  this  pathway  in  nature  will  be 
explored  and  it  will  be  determined  if  it  plays  any  part  in  human  metabolism. 

Relevance  to  Biomedical  Research: 

This  study  impinges  on  at  least  two  areas  of  medical  concern,   1)  the 
mode  of  action  of  yitamin  B  „  in  its  metabolic  roles,  and   2)  the  means  by 
which  organisms  catabolize  food  materials.   This  second  area  is  directly 
concerned  with  several  inborn  errors  of  metabolism  that  have  been  shown  to 
be  devastating  to  the  well-being  of  humans,  especially  in  the  case  of  maple 
syrup  urine  disease,  isovaleric  acidemia,  and  disorders  of  the  catabolism  of 
short-chain  acids.   The  mode  of  action  of  B  „  is  imperfectly  understood,  but 
its  importance  in  hematopoiesis  and  in  the  maintenance  of  proper  neurological 
function  is  exemplified  in  the  disease  of  its  metabolic  deficiency,  pernicious 
anemia . 

Keyword  Descriptors: 

Branched-chain  Amino  Acids,  a-Leucine,  g-Leucine,  Clostridium  sporogenes , 
Clostridium  lentoputrescens ,  Cobalamin,  Coenzyme-B   ,  Leucine  Mutase. 

Honors  and  Awards:   None 

Publications : 

1.   J.  Michael  Poston  and  Thressa  C.  Stadtman:   Cobamides  as  Cofactors: 
Methylcobamides  and  the  Synthesis  of  Methionine,  Methane,  and  Acetate.   In 
Babior,  Bernard  M.  (Ed.):   Cobalamin:   Biochemistry  and  Pathophysiology. 
New  York,  John  Wiley  and  Sons,  Inc.  1975,  pp.  111-139. 


<& 


Project  No.  Z01   HL   00202-04   LB 
Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS -NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Title:   Kinetics  and  Mechanisms  of  Biochemical  Reactions 

Previous  Serial  Number:   NHLI-10 

Principal  Investigators:   P.  B.  Chock 

Sue  Goo  Rhee 

Other  Investigators:   None 

Cooperating  Units:   M.  Greifner,  Biomedical  Engineering  &  Instrumentation 
Branch,  Division  of  Research  Service,  NIH 
S.  Chock  and  E.  Einsenberg,  Lab  of  Cell  Biology,  National 
Heart  &  Lung  Institute,  NIH 

Project  Description: 

Objectives:   1)  To  set  up  a  laboratory  for  the  study  of  fast  kinetics  of 
reactions,  particularly  for  studying  individual  steps  of  enzymic  reactions 
and  protein-ligand  interactions.   2)  With  this  fast  kinetic  technique  and 
other  physical  and  chemical  methods,  to  elucidate  the  biochemical  action  of 
glutamine  synthetase  from  Escherichia  coli.   3)  To  study  the  kinetics  and 
mechanism  of  DNA-repressor  interaction  utilizing  the  fluorescence  technique. 

Major  Development: 

1.  A  stopped-flow  cell  with  a  dead  time  of  500  u  sec  have  been  designed 
and  built.   The  short  dead  time  is  accomplished  under  mild  conditions,  such 
as  60  psi  of  driving  pressure.   In  addition,  the  signal  output  is  directly 
processed  by  a  PDP  11  computer  and  displayed  on  a  Tektronix  4010-1  computer 
display  terminal.   The  latter  set  up  has  decreased  enormously  the  number  of 
man-hours  required  to  analyze  the  data  obtained. 

2.  High  voltage  discharge  temperature-jump  machine  is  in  operation  with 
an  improved  sensitivity  for  the  optical  density  and  fluorescent  detectors. 

Major  Findings: 

1.   We  have  shown  experimentally  that  an  integrated  mechanism  can  be 
used  to  explain  reactions  (1)  to  (5)  catalyzed  by  the  unadenylylated  glutamine 
synthetase  from  E.  coli. 

Me2+^ 
L-Glutamate  +  ATP  +  NH3  r; L-Glutamine  +  ADP  +  Pt  (1) 


£b 


Project  No.  Z01   HL   00202-04   LB 

Me2+ 

L-Glutamine  +  NH90H  _  7-glutamylhydroxamate  +  NHo     (2) 

z    s ADP,  Pi  or  Asi 

Me2"1"  ^ 
L-Glutamate  +  ATP  ^     —  pyrrolidone  carboxylate  +  ADP  +  P-^         (3) 

2+ 

L-Glutamine  +  H90   ,  ^e >  L-Glutamate  +  NHo  (4) 

1       ADP,  Asi  J 

Me2+,  Pi  or  Asi 
N]TP  +  N2DP  ^  N2TP  +  NiDP  (5) 

Glutamate,  Glutamine 

The  abbreviation  Me2+,  Asi,  Pi,  N-i  and  N„  represent  divalent  metal  ions, 
arsenate,  orthophosphate,  nucleoside  1  and  nucleoside  2,  respectively.   The 
enzymic  activities  which  catalyze  the  various  reactions  are  referred  to  as 
follows:   reaction  (1),  biosynthetic  activity,  reaction  (2),  transferase; 
reaction  (3),  ATPase;  reaction  (4),  arsenate  dependent  glutaminase;  and 
reaction  (5),  transphosphorylase. 

The  proposed  mechanistic  scheme  is:  (The  parenthesis  indicates  enzyme 
bound  complex.) 


S? 


Project  No.  ZQ1   HL  00202-04   LB 


+• 
t 

(  *-T- 


0_ 

+■ 

-f- 


r«» 


Project  No.  Z01  HL  00202-04   LB 

Evidence  for  supporting  the  above  mechanistic  scheme  are:   (i)  Glutamate 
for  reactions  (1)  and  (3),  and  glutamine  for  reactions  (2)  and  (4)  occupy  the 
same  binding  site.   (ii)  ATP  for  reactions  (1)  and  (3),  and  ADP  for  reactions 
(2)  and  (4)  occupy  the  same  site.   (iii)  Orthophosphate  and  arsenate  bind  at 
a  site  which  overlaps  the  /-phosphate  site  of  nucleoside  triphosphate, 
(iv)  Both  Mg2+  and  Mn^+  support  the  formation  of  reaction  intermediate 
observed  in  reactions  (1)  and  (3).   In  addition  they  both  support  the  reverse 
biosynthetic  reaction.   (v)  Observed  transphosphorylation  between  N^TP  and 
NoDP  is  consistent  with  the  coupling  of  both  forward  and  reverse  biosynthetic 
reactions.   (vi)18o  transfer  is  reversible  between  orthophosphate  and  7-acyl 
group  of  glutamate.   (vii)  Arsenate,  a  better  nucleophile  than  P^  functions 
as  a  more  effective  substrate  for  the  transferase  reaction.   However,  ATP 
derived  from  P^  and  ADP  is  more  stable  than  the  corresponding  arsenate 
derivative,  therefore  P^  is  a  better  substrate  than  arsenate  for  the  reverse 
biosynthetic  reaction. 

In  the  proposed  scheme,  two  possible  mechanisms  are  included.   One 
involves  the  formation  of  /-glutamyl  phosphate  (or  arsenate)  (V)  as  a  required 
intermediate  for  the  biosynthetic  reaction,  and  the  other  involves  partial 
bond  breaking  and  forming  transition  state  intermediates  such  as  I,  II,  and 
III.   Both  mechanisms  require  the  interaction  of  P^  (or  As^)  oxygen  with  the 
5-carbon  of  glutamine  for  initiating  the  reverse  biosynthetic  and  transfer 
reactions;  and  the  interaction  of  7-acyl  oxygen  of  glutamate  with  the 
/-phosphorus  of  nucleoside  triphosphate  for  forward  biosynthetic  and  glutamate 
dependent  ATPase  reactions.   The  mechanism  which  requires  /-glutamyl  phosphate 
as  an  intermediate  implies  that  in  the  reverse  biosynthetic  and  transfer 
reactions,  ADP  does  not  interact  directly  with  Pj^  (or  arsenate)  to  form  ATP; 
instead,  ADP  is  utilized  for  inducing  (or  stabilizing)  the  protein  conformation 
needed,  and  provides  free  energy  for  the  formation  of  /-glutamyl  phosphate. 
A  direct  ADP-P-^  interaction  in  the  reverse  biosynthetic  reaction  would  proceed 
by  III  -•  II  ->  V  ->  I  which  means  that  the  forward  biosynthetic  reaction  will 
proceed  by  I  -»  V  ->  II  -»  III.   In  other  words,  the  NH3  addition  step  would  be 
II  -»  III,  which  is  inconsistent  with  the  assumption  that  NH3  attacks 
/-glutamyl  phosphate.   In  the  second  mechanism,  /-glutamyl  phosphate  is  formed 
only  when  NH3  and  NH2OH  are  not  present.   With  this  mechanism,  the  biosynthetic 
reaction  will  proceed  from  glutamate,  ATP  j!  I  ^  II  S  III  ^  glutamine,  ADP  and 
Pi,  while  the  transfer  reaction  pathway  is  glutamine  ->  III  -»  II  -»  IV  -» 
/-glutamyl  hydroxamate. 

2.   The  rates  of  ADP  and  P^  binding  to  the  unadenylylated  glutamine 
synthetase  from  E.  coli  were  studies  by  the  stopped-flow  technique. 

The  following  six  reactions  were  studied. 

(1)  ADP  +  EMn 

(2)  ADP  +  EMn  ?± 

(3)  V±     +   EMn 

(4)  Pi  +  EMn  ADP 

(5)  ADP  +  EMg 

(6)  ADP  +  EMg  Pi 


r3 


Project  No.  Z01   HL   00202-04   LB 

Experimental  results  obtained  at  various  substrate  concentrations  suggest  that 
there  are  two  steps  involved;  the  first  is  formation  of  a  binary  complex  (ES) , 
followed  by  a  relatively  slower  conformational  change  to  ES ' 

E  +  S  vk_L    ES  ^ ES'  (6) 

Under  the  psuedo  first  order  conditions,  k^g  obtained  from  the  analysis  of 
the  rates  of  the  fluorescence  change  can  be  described  by  equation  (7). 

—  +  k_2  (7) 


"obs 

1  + 


kTTsT 


where  Ki  = 


Utilizing  a  curve  fitting  computer  program,  the  following  values  were 
obtained  at  15°. 

Kl  k2  k-2         Kdiss 

Reaction  1  3.9  x  104  M"1       90  sec"1        9.5sec_1  3   x  10"6 

Reaction  2  4.9  x  104  220  0.5      3.5  x  10"8 

Reaction  4        230  216  1.8      3.7  x  10"5 

Kdiss  ls  calculated  from  the  equation  Kd^ss  =  k_2/Kik2- 

Reaction  (3),  (5),  and  (6)  are  too  fast  and  their  Kdiss  are  too  high  to 
permit  accurate  measurement  of  the  rate  constant  such  that  a  concentration 
dependent  rate  profile  can  be  established.   However  maximum  values  of  kobs 
which  is  independent  of  substrate  concentration,  can  be  estimated  by  using 
our  home-made  fast  mixing  cell.   The  values  of  kobs  are 

kobs 

Reaction  3         1000  sec"1 
Reaction  5         1000 
Reaction  6  310 

The  values  of  Kdiss  for  reaction  (1)  -  (6)  were  also  measured  independently 
by  fluorescence  titration  method.  These  results  are  in  good  agreement  with 
the  Kdiss  calculated  from  the  kinetic  data. 

Kdiss 


Reaction  1 

4.6 

X 

10_b 

Reaction  2 

5 

X 

10"8 

Reaction  3 

1.4 

x 

10"3 

Reaction  4 

3 

X 

10-5 

Reaction  5 

4 

X 

10-5 

Reaction  6 

3 

X 

10-5 

M 


S* 


Project  No.  Z01  HL   00202-04   LB 

The  rate  of  ADP  release  from  the  Mn^"1"  activated  enzyme  with  the  presence  of 
P-l  is  very  slow  (k_2  =0.5  sec"'-).   This  slow  rate  is  responsible  for  the 
observed  inactivation  of  biosynthetic  reaction  by  Mn^+  with  a  conventional 
assay  method  for  ADP  or  P^  detection. 

3.  With  the  unadenylylated  enzyme  in  the  presence  of  Mg^+3  L-glutamine 
binds  to  the  enzyme  and  enhances  the  tryptophan  fluorescence  of  the  protein. 
The  reaction  rate  is  very  fast.   Due  to  relatively  high  rate  constants  and 
high  dissociation  constant  (K^gg  =  7  mM,  obtained  from  the  amplitude),  one 
cannot  obtain  accurately  the  glutamine  concentration  dependent  rate.   But  the 
concentration  independent  rate  is  120  sec~l,  which  is  corresponding  to  the 
sum  of  the  k2  and  k_2  (see  equation  7).   In  the  case  of  Mn2+  enzyme,  the 
L-glutamine  binding  is  enhanced  by  the  presence  of  ADP.   The  enhancement 
factor  is  equal  to  2  as  indicated  by  fluorescence  titration  of  ADP  to  the 
enzyme  and  enzyme-glutamine  complex. 

4.  By  following  the  protein  fluorescence  changes,  due  to  substrate 
binding  and  overall  biosynthetic  reaction,  we  can  evaluate  the  kcat  and 
individual  rate  constants  of  the  reaction  process  catalyzed  by  the  Mg^+ 
activated  enzyme.   The  data  indicate  that  two  different  intermediates  are 
formed  during  the  course  of  the  reaction.   In  the  absence  of  NH3,  the  two 
intermediates,  whose  rates  of  formation  are  different,  are  revealed  by  the 
biphasic  nature  of  the  kinetic  data.   However,  in  the  presence  of  limiting 
amount  of  NH3,  only  the  fast  forming  intermediate  is  observed.   From  the  time 
required  for  the  consumption  of  ammonia,  one  can  calculate  the  k  at  which  is 
in  good  agreement  with  that  obtained  from  steady-state  data.   As  soon  as  NH3 
is  consumed,  the  second  intermediate  is  reformed.   The  fact  that  a  fluores- 
cence signal  corresponding  to  the  second  intermediate  was  not  observed  in  the 
presence  of  NH3,  is  due  to  the  rapid  reaction  rate  of  NH3  with  the  second 
intermediate,  as  indicated  by  the  kinetic  studies  of  the  addition  of  NH3  to 

MgEATP-Glut. 

5.  Utilizing  stopped-flow  spectrometer,  a  technique  was  introduced  for 
determining  ATP  concentration  in  either  cell  crude  extract  or  purified  system 
with  luciferase-luciferin  reaction.   Due  to  the  fact  that  light 

produced  from  luciferase-luciferin-02_ATP  reaction  decays  rapidly,  and  the 
decay  rate  is  shown  to  be  ATP  concentration  dependent.   In  addition,  if  the 
sample  contains  ADP  and  other  nucleoside  triphosphates,  it  is  observed  that 
nucleoside  diphosphokinase  in  the  firefly  lantern  will  catalyze  the  conversion 
of  ADP  to  ATP  in  a  relatively  slower  rate  than  the  luciferin-luciferase 
reaction.   Therefore  it  is  necessary  to  capture  the  initial  rise  of  light 
intensity  produced  by  the  luciferase  catalyzed  reaction.   Stopped-flow 
spectrometer  is  used  to  record  the  initial  light  intensity.   This  technique 
is  shown  to  be  a  more  quantative  method  for  ATP  assay  when  luciferin- 
luciferase  system  is  used;  and  with  the  addition  of  luciferin,  which  will 
increase  light  intensity  by  a  factor  of  50  to  100,  one  can  measure  accurately 
picomole  amounts  of  ATP.   In  addition,  the  interference  from  other  nucleotides 
can  be  detected  and  differentiated  by  the  stopped-flow  technique. 


rr 


Project  No.  Z01   HL   00202-04   LB 

It  is  interesting  to  point  out  that  first  order  rate  is  followed  for  at 
least  877o  of  the  light  producing  reaction.   This  is  the  oxidation  of 
luciferase-luciferin-AMP  complex  by  oxygen  to  form  oxyluciferin,  AMP,  and 
CO2.   The  observed  rate  constant  is  2.3+0.2  sec"l  at  25°.   The  observed 
rate  is  independent  of  ATP  concentration  from  0.5  uM  to  0.1  mM  range.   This 
suggests  that  (a)  oxygen  (ca  *>  1  mM)  is  not  limited  since  the  amplitude  is 
directly  proportional  to  ATP  concentration  and  (b)  the  rate  determining  step 
is  a  unimolecular  reaction  of  luciferase-luciferin-AMP-02  complex  to  form 
oxyluciferin. 

6.   Fast  reaction  kinetic  studies  of  the  interaction  of  actin, 
subfractment-1  of  myosin  and  ATP  were  carried  out  with  the  collaboration  of 
Drs.  Stephen  Chock  and  Evan  Eisenberg.   With  light  scattering  and  fluores- 
cence methods  to  detect  the  interaction  of  myosin  with  actin  and  actomyosin 
with  ATP  respectively,  it  is  possible  to  follow  the  whole  cycle  for  ATP 
hydrolysis  by  actomyosin.   The  results  suggest  that  ATP  first  binds  to 
actomyosin  to  initiate  the  dissociation  of  actomyosin  to  actin  and  ATP-myosin. 
The  ATP-myosin  complex  then  undergoes  conformational  changes  which  accompanied 
by  partial  hydrolysis  of  ATP.   Complete  ATP  hydrolysis  was  accomplished  when 
actin  rebind  to  the  myosin. 

Significance  to  Bio-Medical  Research: 

The  aim  is  to  gain  better  understanding  of  how  enzymes  function  with 
respect  to  its  catalytic  and  regulatory  properties. 

Proposed  Course  of  Research: 

1)  To  further  improve  the  existent  machines  with  respect  to  their 
sensitivity  and  shortening  the  dead-time  of  the  stopped-flow  machine.   In 
addition,  to  build  a  laser  heating  temperature-jump  machine  and  a  slow  heat 
exchange  temperature-jump  machine  to  cover  the  nanosecond  and  second  range 
respectively. 

2)  To  further  explore  the  physical  and  chemical  properties  of  the 
unadenylylated  and  adenylylated  glutamine  synthetase  with  respect  to  their 
catalytic  and  regulatory  functions.   In  particular,   we  will  utilize  the 
stopped-flow  technique  to  study  the  kinetics  of  inhibition  and  catalysis  at 
high  enzyme  level  such  as  that  present  in  vivo. 

3)  To  study  the  kinetic  and  mechanism  of  DNA-repressor  interaction 
utilizing  the  tryptophan  fluorescence  of  the  repressor. 

Keyword  Descriptors: 

Glutamine  synthetase,  steady-state  kinetics,  fast  reaction  kinetics, 
transient  kinetics,  mechanistic  study,  luciferin-luciferase,  actin, 
actomyosin. 

Honors  and  Awards:   None 


$& 


Project  Zo.  Z01  HL  00202-04   LB 
Publications: 

1.  R.  B.  Tiramons,  S.  G.  Rhee ,  D.  L.  Luterman,  and  P.  B.  Chock:   Mechanistic 
Studies  of  Glutamine  Synthetase  from  E.  coli.   I.  Fluorometric  Identification 
of  a  Reactive  Intermediate  in  the  Biosynthetic  Reaction.   Biochem.  13: 
4479-4485,  1974. 

2.  P.  Hambright  and  P.  B.  Chock:   Metal-Porphyrin  Interaction.   III.  A 
Dissociative-Interchange  Mechanism  for  Metal  Ion  Incorporation  into  Porphyrin 
Molecules.   J.  Am.  Chem.  Soc.  96:   3123-3131,  1974. 

3.  J.  R.  Sutter,  P.  Hambright,  P.  B.  Chock,  and  M.  Krishnamurthy :  Temperature- 
Jump  Kinetic  Study  of  a  Ferric  Porphyrin  Monomer-Dimer  Equilibrium  in  Aqueous 
Solution.   Inorg.  Chem.  13:   2764-2765,  1974. 

4.  P.  Hambright  and  P.  B.  Chock:   Metal-Porphyrin  Interactions.   IV.  Electron- 
Transfer  Kinetics  between  Dithionite  and  Manganese  (III)  and  Cobalt  (III) 
Porphyrins.   Inorg.  Chem.  13:   3029-3031,  1974. 

5.  R.  B.  Timmons,  C.  Y.  Huang,  E.  R.  Stadtman,  and  P.  B.  Chock:   Fluorescence 
Studies  of  Glutamine  Synthetase  from  E.  coli:  €-Adenylylated  and  Unadenylylated 
Enzymes.   In  Fisher,  E.  H. ,  Krebs,  E.  G. ,  Neurath,  H. ,  and  Stadtman,  E.  R. 

(Eds.):  Third  International  Symposium  on  Metabolic  Interconversion  of  Enzymes. 
New  York,  Springer-Verlag,  1974,  pp.  209-220. 

6.  P.  Hambright,  M.  Krishnamurthy,  and  P.  B.  Chock:   Metal-Porphyrin  Inter- 
actions.  V.  Kinetics  of  Cyanide  Addition  to  a  Water  Soluble  Iron  Porphyrin 
Dimer.   J.  Inorg.  Nucl.  Chem.  37:   557-561,  1975. 

7.  S.  G.  Rhee,  M.  I.  Greifner,  and  P.  B.  Chock:   Determination  of  Adenosine 
5 ' -Triphosphate  by  the  Luciferin-Luciferase  System  with  a  Stopped-Flow 
Spectrometer.   Anal.  Biochem.  64:   1975. 


£7 


Project  No.  ZQ1  EL   QQ2Q3-02  LB 
Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  ^Maryland 

PHS-NIK 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Cellular  Regulation  of  Enzyme  Levels 

Previous  Serial  Number:   NHLI-2 

Principal  Investigator:   Donald  M.  Pehlke 

Other  Investigators:   E.  R.  Stadtman 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Control  of  cellular  enzyme  concentration  is  an  important  mech- 
anism of  metabolic  regulation.   The  level  of  any  functional  protein  reflects 
the  balance  between  its  de  novo  synthesis  and  its  degradation.   While  inves- 
tigations of  the  process  by  which  proteins  are  synthesized  have  yielded  im- 
portant understandings  directly  relevant  to  biomedica]  applications  (e.g., 
mechanisms  of  hormone  action,  antibiotics) ,  comparatively  little  is  known 
about  the  process  by  which  proteins  are  degraded.   Since  metabolic  energy, 
RNA  synthesis,  and  protein  synthesis  are  required  for  protein  degradation, 
the  probability  that  the  process  plays  an  important  metabolic  role  is  high. 

In  procaryotes,  the  transition  from  the  logarithmic  phase  of  growth  to 
stationary  phase  provokes  the  rapid  loss  of  certain  enzymes  and  an  increase 
in  protein  degradation  as  measured  by  the  release  of  radio-labelled  amino  acids 
from  pre-labelled  proteins.   To  date,  this  process  has  required  the  intact 
cell  in  E_.  coli.   Studies  from  this  laboratory  of  alterations  in  fifteen  dif- 
ferent enzymes  in  E_.  coli  led  to  the  observation  that  significant  losses  of 
enzymatic  activity  occurred  with  threonine-sensitive  aspartokinase,  lysine- 
sensitive  aspartokinase,  and  homoserine  dehydrogenase.   These  are  key  enzymes 
involved  in  the  synthesis  of  the  aspartate  family  of  amino  acids.   The  loss 
of  these  activities,  therefore,  represents  a  significant  and  rational  metabolic 
adaptation  to  nitrogen  starvation.   These  enzymes  were  selected  for  further 
detailed  study  of  the  biochemical  mechanisms  underlying  their  degradation  as 
a  possible  model  with  which  to  investigate  the  regulation  of  the  protein  break- 
down system. 

Major  Findings: 

I.   Whole  cell  studies:   A  system  was  developed  to  assess  the  above  en- 
zyme activities  in  intact  cells  of  E_.  coli  W  using  frozen-thawed  cells  briefly 
exposed  to  toluene  which  renders  the  cell  membrane  permeable  to  small  mole- 


SS 


Project  No.  Z01  HL  Q02Q3-Q2  LB 

cules.   Inactiyation  of  the  lysine  sensitiye  aspartokinase  (Lys  AK)  is  not 
affected  by  2,4  dinitrophenol  but  is  blocked  by  anaerobic  conditions  or  cy- 
anide under  standard  conditions  of  nitrogen  starvation.   A  carbon  source 
(glucose  or  glycerol)  is  required.   No  inactivation  is  obseryed  in  the  absence 
of  diyalent  cations.   Loss  of  Lys  AK  activity  is  blocked  by  inhibition  of 
protein  synthesis  with  chloramphenicol.   Phenylmethanesulf onylf luoride  (PMSF), 
an  inhibitor  of  serine  proteases,  has  no  effect.   Amino  acid  end  products  of 
the  aspartate  pathway  did  not  affect  Lys  AK  inactivation  in  the  intact  cell. 

Threonine  sensitive  aspartokinase  (Thr  AK)  inactivation  in  responce  to 
nitrogen  starvation  was  also  studied  with  the  aboye  effectors.   2,4  DNP  has 
no  effect  while  O2  deprivation  and  exposure  to  KCN  block  inactivation.   EDTA 
and  chloramphenicol  prevent  activity  loss;  PMSF  is  not  effective.   A  carbon 
source  seems  to  be  required.   Threonine  partially  blocks  Thr  AK  inactivation 
while  lysine  increases  the  rate  of  loss. 

Observations  on  the  homoserine  dehydrogenase  activity  (HSDH)  qualitatively 
paralleled  the  Thr  AK  findings.   Since  both  enzymatic  activities  are  present 
on  the  same  polypeptide  in  E_.  coli ,  this  is  not  surprising.   However,  quanti- 
tative differences  between  the  two  activity  losses  were  noted  which  suggest 
interesting  regulatory  possibilities. 

II.   Cell  free  studies:   A  cell  free  extract  capable  of  inactivating  par- 
tially purified  Thr  AK  and  HSDH  but  not  Lys  AK  has  been  discovered  in  station- 
ary phase  cells.   This  inactivating  activity  is  enzymatic  in  nature  and  re- 
quires an  activator  or  co-factor  present  in  boiled  cell  extracts.   The  activa- 
tor has  been  purified  and  identified  from  boiled  cell  extracts.   Purification 
of  the  protein  responsible  for  inactivating  activity  is  in  progress. 

Relevance  to  biomedical  research: 

While  it  is  difficult  to  estimate  the  impact  of  the  elucidation  of  a  basic 
cellular  process,  an  understanding  of  the  mechanisms  and  regulation  of  protein 
degradation  would  have  broad  application  in  biochemistry,  bacteriology,  and 
medicine.   In  bacteria,  the  probability  that  protein  degradation  is  necessary 
for  successful  adaptation  to  new  cellular  environments  might  be  exploited  in 
the  design  of  new  antibiotics. 

Studies  of  mammalian  muscle  have  demonstrated  that  protein  degradation  is 
central  to  the  negative  nitrogen  balance  and  muscle  atrophy  associated  with 
disuse.   Similar  questions  must  be  asked  about  the  role  of  protein  breakdown 
in  situations  such  as  wound  healing,  anoxic  myocardial  injury,  and  diabetes. 
Better  understanding  of  the  proteolytic  enzymes  of  the  coagulation,  comple- 
ment, and  kinin  systems  may  arise  from  the  study  of  protein  degradation. 

Nutritional  disorders  and  aging  are  more  directly  related  to  the  subject 
of  this  report.   Evidence  is  accumulating  that  there  is  an  increase  in  func- 
tionally abnormal  proteins  with  age.   The  fate  of  these  proteins,  the  metabolic 
significance  of  their  presence,  and  the  question  of  whether  their  presence 

2  £*? 


Project  No.  Z01  HL  00203-02  LB 


reflects,  an  altered  degradation  system  relate  to  a  basic  understanding  of 
the  aging  process.   Similarly,  the  study  of  the  nitrogen  starved  system  dis- 
cussed in  this  report  may  lead  to  new  insights  about  the  processes  involved 
in  protein  malnutrition  in  man. 

Finally,  while  there  are  no  presently  known  genetic  disorders  thought  to 
be  due  to  defective  protein  degradation,  there  are  -many  examples  of  cross 
reacting  material  (CKM)  negative  enzyme  deficiency  states.   These  have  been 
interpreted  as  deficiencies  of  de  novo  synthesis.   Many  could  alternatively 
be  interpreted  as  defects  in  control  of  the  degradative  process  with  in- 
creased loss  of  the  protein  of  interest. 

Proposed  Course  of  Action: 

Purification  of  the  Thr  AK/HSDH  inactivating  protein  will  continue  with 
the  hope  of  attaining  a  homogeneous  preparation  with  which  to  study  the 
mechanism  by  which  AK  activity  is  lost.   It  will  be  necessary  to  determine 
whether  the  loss  of  activity  is  due  to  enzyme  modification  or  proteolytic 
cleavage,  whether  the  reaction  is  specific  for  Thr  AK  or  is  generalized,  how 
the  reaction  is  controlled,  and  how  it  relates  to  the  regulation  of  cell 
metabolism.   In  addition  to  enzymologic  and  physical  approaches,  antibodies 
will  be  raised  to  both  the  Thr  AK/HSDH  and  to  the  inactivating  protein  in 
order  to  study  their  interaction  immuno-chemically.   Efforts  at  isolating  a 
genetic  mutant  lacking  the  inactivating  activity  will  be  made. 

Keyword  Descriptors : 

E.  coli,  threonine  sensitive  aspartokinase,  lysine  sensitive  asparto- 
kinase ,  homoserine  dehydrogenase,  protein  degradation. 

Honors  and  Awards:   None. 

Publications:   None. 


60 


Project  No.  Z01   HL  00210-02   LB 

1.  Laboratory  of  Biochemistry 

2.  Section  on  Enzymes 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Regulation  of  the  Cascade  Control  of  E_.  coli  Glutamine 
Synthetase 

Previous  Serial  Number:   NHLI-1 

Principal  Investigator:   Edgar  G.  Engleman 

Sharron  H.  Francis 

Other  Investigator:   Earl  R.  Stadtman 

Cooperating  Units:   None 

Project  Description: 

Objectives:   In  addition  to  regulation  by  cumulative  feedback  inhibition, 
glutamine  synthetase  from  _E.  coli  W  is  regulated  by  the  covalent  attachment 
of  an  AMP  moiety  to  a  tyrosyl  residue  in  each  of  the  enzyme  subunits.   The 
attachment  and  removal  of  the  AMP  is  catalyzed  by  an  enzyme  known  as 
adenylyltransf erase  (ATase).   The  activity  of  ATase  is  modulated,  however, 
by  a  second  protein,  Pjt,  which  exists  in  two  forms.   Pjia  and  PlID  promote 
adenylylating  activity  and  deadenylylating  activity  of  the  ATase,  respectively. 
PIIA  ^-s   converted  to  Pun  by  the  covalent  attachment  of  a  UMP  group  to  a 
tyrosine  in  each  of  its  subunits.   The  interconversion  of  Pjja  and  PILD  is 
catalyzed  by  the  uridylyltransferase  and  uridylyl-removing  enzyme(s). 

Major  Findings: 

In  our  studies  we  have  examined  the  effects  of  a  number  of  metabolites 
on  the  respective  enzymatic  steps  in  the  cascade  control  of  E_.  coli  glutamine 
synthetase.   The  ATase,  Pjia  and  PIID  and  the  uridylyl  transferase-uridylyl 
removing  enzyme Cs)  were  isolated  for  these  studies  and  the  role  of  effectors 
in  each  step  examined. 

The  complex  control  of  the  cascade  is  evidenced  by  the  large  number  of 
substances  which  exert  potent  inhibitory  or  stimulatory  effects  at  physiolo- 
gical levels.   The  reciprocal  effects  of  glutamine  and  a-ketoglutarate  are 
seen  at  every  step,  suggesting  not  only  an  important  regulatory  function  for 
these  two  compounds,  but  also  a  specific  binding  site  for  these  ligands  on 
the  respective  proteins. 

The  Pixa  supported  adenylylation  of  glutamine  synthetase  by  the  adenylyl 
transferase  is  inhibited  by  the  addition  of  either  Pjxd  or  a-ketoglutarate. 
However,  a-ketoglutarate  and  Pxjn  in  combination  act  synergystically  to  in- 

1 


Project  No.  Z01   HL  00210-02   LB 


hibit  this  activity.   Of  the  other  substances  tested,  only  methionine  and 
tryptophan  stimulated  adenylylation  at  physiological  levels,  and  this  stimu- 
lation appears  to  be  mediated  by  a  direct  effect  on  ATase  and  is  not  depen- 
dent on  the  Pj-jA-ATase  complex.   At  high  (12mM)  levels  coenzyme  A  and  3- 
phosphoglycerate  inhibit  adenylylation. 

The  PxiD  supported  deadenylation  requires  the  presence  of  a-ketoglutarate 
and  ATP  and  the  level  of  a-ketoglutarate  required  for  activity  is  closely 
related  to  the  level  of  ATP.   At  0.03  mM  a-ketoglutarate  and  2.5  mM  ATP, 
PI1D  supported  deadenylation  is  80%  of  the  maximal  activity.   Under  these 
conditions  glutamine  inhibits  deadenylation  but  Pjia  has  little  or  no  effect. 
However,  the  combination  of  Ptta  and  glutamine  produce  a  greater  inhibition 
than  glutamine  alone,  suggesting  synergism  analogous  to  Pj-jp-a-ketoglutarate 
inhibition  of  adenylylation.   At  high  concentrations  (10  mM)  a  variety  of 
metabolites  inhibit  the  deadenylation,  i.e.,  CoA,  UTP ,  3-phosphoglycerate, 
phosphoenol  pyruvate,  fructose  1,6-diphosphate,  and  tryptophan.   However, 
at  1  mM  concentrations  only  UTP,  PEP,  and  3-PGA  show  any  effects. 

A  search  for  endogenous  inhibitors  of  the  UR  enzyme  seemed  warranted  by 
the  observation  that  this  activity  could  not  be  assayed  in  the  crude  homo- 
genate  from  E_.  coli.   Furthermore,  addition  of  E.  coli  crude  extract  to 
partially  purified  UR  effectively  inhibited  its  activity.   Two  inhibitors, 
CMP  and  UMP,  were  identified  from  these  E_.  coli  extracts  in  the  following 
manner.   Passage  of  a  streptomycin  supernatant  over  Sephadex  G-200  yielded 
a  large  inhibitor  complex  which  when  heated  at  60°  C  for  30  minutes  dissocia- 
ted into  an  active  dialyzable  fraction  that  was  adsorbed  to  charcoal  and 
further  separated  over  a  Dowex  50  column.   Thin-layer  chromatography  yielded 
two  active  moieties,  which  were  identified  as  CMP  and  UMP  on  the  basis  of 
Kf  values  and  UV  spectra. 

The  existence  of  a  large  relatively  specific  binding  substance  for  CMP 
and  UMP  is  demonstrated  by  the  isolation  of  the  inhibitors  from  the  void 
fraction  from  a  G-200  column.   Radioactively  labeled  CMP  and  UMP  readily 
exchange  into  this  fraction  but  TMP ,  IMP,  and  AMP  are  bound  to  a  lesser  de- 
gree.  Further  characterization  of  this  binding  substance  has  been  difficult 
because  it  is  resistant  to  proteases,  nucleases,  phospholipases  and  lysozyme. 

Although  all  nucleotides  directly  tested  inhibited  the  Mn  supported  UR 
activity  the  monophosphates  were  at  least  one  order  of  magnitude  greater  in 
potency  than  the  diphosphates  which  were  more  potent  than  the  triphosphates. 
Of  the  other  substances  tested  coenzyme  A  was  the  most  potent  inhibitor  of 
UR  activity.   None  of  these  inhibitors  of  Mn-supported  UR  had  any  effect  on 
the  UR  activity  supported  by  Mg-ATP-a-KG  UR-activity,  suggesting  that  the 
two  UR  activities  may  involve  different  mechanisms.   Nonetheless,  both  activi- 
ties were  stimulated  by  glutamine. 


££L 


Project  No.  ZQ1  HL  00210-02  LB 


Unlike  UR  which  is  unaffected  by  a-ketoglutarate,  the  "UTase  requires 
a-ketoglutarate  for  activity;  Q.l  onM  a-ketoglutarate  supports  65%  of  the 
maximal  activity  and  the  UTase  is,  potently  inhibited  by  glutamine.   For 
example,  at  O.loriM  a-ketoglutarate  and  0.1  onM  glutamine  the  "UTase  activity 
is  approximately  60%  inhibited.   Like  the  Mg'ATP* a-ketoglutarate  UR-actiyity, 
UTase  was  unaffected  by  nucleotide  monophosphates ,  coenzyme  A  and  other  meta- 
bolites tested,  i.e.,  phosphoenol  pyruvate,  fructose  1,6  diphosphate,  3-phos- 
phoglycerate,  tryptophan,  glycine,  histidine,  DPN,  TPN. 

Thus,  all  steps  in  the  cascade  control  are  sensitive  to  the  metabolites 
primarily  involved  in  the  glutamine  synthetase  reaction;  i.e.  glutamine  and 
a-ketoglutarate.   In  addition,  a  number  of  other  metabolites  affect  the 
various  steps  in  the  cascade  but  the  physiological  significance  of  these 
effects  are  not  fully  understood. 

Proposed  Course  of  Project: 

1.  Further  attempts  to  purify  UR-UTase  using  techniques  such  as 
CMP/UMP  affinity  chromatography,  and  if  successful,  physical  characterization 
of  the  enzyme. 

2.  Characterization  of  the  CMP/UMP  binding  substance  and  studies  regard- 
ing its  possible  interactions  with  the  UR  enzyme. 

Keyword  Descriptors:   Glutamine  Synthetase,  Regulation,  Cascade  Control. 

Honors  and  Awards:   None 

Publications:   None 


43 


Project  No.  Z01  HL  00211-02  LB 
Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS -NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Title:   Regulation  of  Glutamine  Synthetase 

Previous  Serial  Number:   NHLI-3 

Principal  Investigators:   Robert  Park 

Pauline  Smyrniotis 

Other  Investigators:   E.  R.  Stadtman 

Cooperating  Units:   None 

Project  Description: 

Objectives:   1)  Using  unadenylylated  glutamine  synthetase  in  the  presence  of 
Mg"1-1",  to  examine  the  effect  of  substrate  and  effector  concentrations  upon  the 
catalytic  constants  of  each  of  the  substrates  in  the  /-glutamyl  transfer 
reaction.   2)  Through  a  kinetic  analysis,  to  determine  if  certain  end  products 
of  glutamine  metabolism  achieve  inhibition  by  reacting  at  a  common  site  or  at 
multiple  sites  on  glutamine  synthetase. 

Major  Findings: 

1 .  Effect  of  substrate  concentration  on  catalytic  constants. 

ADP  Arsenate  Ms 
Glutamine  +  NH20H  5 7-glutamylhydroxymate       (1) 

The  apparent  Michaelis  constants  (app.  Y^)    for  glutamine,  ADP,  and  arsenate  in 
the  transfer  reaction  (Reaction  1)  are  highly  dependent  upon  the  binding  of 
each  of  the  other  reactants.   If  the  arsenate  concentration  increases  from 
5  mM  to  20  mM,  the  app.  K^,  decreases  from  14  mM  to  10  mM  for  glutamine,  and 
from  175  uM  to  50  |iM  for  ADP.   Likewise,  if  ADP  is  gradually  saturated,  the 
app.  K  decreases  from  69  mM  to  6  mM  for  arsenate,  and  from  18  mM  to  10  mM  for 
glutamine.   As  glutamine  is  saturated,  the  app.  1^  for  arsenate  decreases  from 
11  mM  to  7  mM.   This  pattern  of  decreasing  app.  Km  as  a  result  of  gradual 
substrate  saturation  suggests  that  the  substrate  binding  of  the  transfer 
reaction  is  random.   Further  kinetic  studies  with  respect  to  hydroxylamine  and 
Mg"1-*"  are  in  progress  to  resolve  this  issue. 

2.  Mechanism  of  cumulative  feedback  inhibition.   We  have  continued  to 
explore  the  mechanism  of  feedback  inhibition  of  glutamine  synthetase  by 
alanine,  AMP,  histidine,  and  tryptophan  by  utilizing  graphical  procedures  of 
Cleland  (1)  and  Yagi  and  Ozawa  (2).   Inhibition  constants  were  obtained  for 

1  l* 


Project  No.  Z01   HL   00211-02  LB 

ATP,  CTP,  AMP,  histidine,  tryptophan,  and  alanine.   Thereafter,  by  following 
enzyme  activity  and  varying  one  inhibitor  (1^)  at  fixed  levels  of  a  second 
inhibitor  (I2) >    an  interaction  coefficient,  a,  can  be  obtained  from  the 
graphical  plots  which  reflects  whether  Ij_  and  Io  compete  for  the  same  site  or 
occupy  separate  sites  on  the  enzyme.   Using  this  approach,  it  was  found  that 
ATP  and  CTP  have  a  common  site,  and  AMP,  histidine,  and  tryptophan  each  have 
separate  sites.   These  findings  were  further  supported  by  results  of  the 
graphical  method  of  Yagi  and  Ozawa  (2)  in  which  plots  of  1/V  versus  increasing 
concentration  of  2  inhibitors  varied  at  constant  molar  ratio  yield  straight 
lines  for  2  inhibitors  occupying  the  same  site.   Data  obtained  for  alanine 
reveal  conflicting  results  from  the  two  different  graphical  methods,  and  this 
is  currently  being  resolved.   Fractional  inhibition  plots  for  AMP  and  alanine 
show  that  they  are  complete  inhibitors,  whereas  histidine  achieves  only  about 
507o  inhibition  at  infinite  concentrations. 

Significance  to  Bio-Medical  Research: 

The  mechanism  of  enzyme  regulation  through  feedback  inhibition  is  the 
basis  of  certain  metabolic  diseases  such  as  hypercholesterolemia,  and  together 
with  other  mechanisms  to  regulate  enzyme  activity,  constitutes  the  means  by 
which  a  multitude  of  metabolic  pathways  are  controlled.   Because  of  its  key 
role  in  bacterial  nitrogen  metabolism,  glutamine  synthetase  serves  as  a  unique 
model  to  elucidate  principles  of  metabolic  regulation. 

Proposed  Course  of  Research: 

Studies  will  be  completed  on  the  effect  of  substrate  binding  upon  the 
app.  Kjp  for  the  transfer  reaction  of  glutamine  synthetase.   In  addition, 
kinetic  studies  will  be  continued  to  further  elucidate  the  mechanism  of  feed- 
back inhibition  and  explore  the  relationship  between  binding  sites  of  each  of 
the  inhibitors  of  glutamine  synthetase. 

Keyword  Descriptors: 

Feedback  inhibition,  glutamine  synthetase,  kinetics,  catalytic  constants, 
inhibitors. 

Honors  and  Awards:   None 

Publications: 

1.   J.  B.  Hunt,  P.  Z.  Smyrniotis,  A.  Ginsburg,  and  E.  R.  Stadtman:   Metal  Ion 
Requirement  by  Glutamine  Synthetase  of  Escherichia  coli  in  Catalysis  of 
/-Glutamyl  Transfer.   Arch.  Biochem.  Biophys.  166:   102-124,  1975. 


4S~ 


Project  No.  Z01   HL   00212-04   LB 
Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Biochemical  Genetics  of  NH3-Assimilatory  Enzymes  in  E.  coli 
Ki2 

Previous  Serial  Number:   NHLI-5 

Principal  Investigator:   Mary  Anne  Berberich 

Other  Investigators:   None 

Cooperating  Units:   Dr.  J.  Phillips,  Section  on  Laboratory  Animal  Medicine 
and  Surgery,  National  Heart  and  Lung  Institute,  NIH 

Project  Description: 

Objectives:   1)  To  isolate  and  characterize  biochemically,  mutants  in  which 
the  activity  of  glutamine  synthetase,  glutamate  synthase  or  glutamate  dehy- 
drogenase is  affected  and  to  conduct  genetic  experiments  for  the  purpose  of 
locating  these  mutations  on  the  E.  coli  chromosomal  map.   2)  To  develop 
methods  to  study  the  physiological  inter-relationship  of  the  NH^-assimilatory 
enzymes  in  E.  coli. 

Major  Findings: 

Earlier  work  from  this  laboratory  determined  that  the  genes  designating 
glutamine  synthetase  (GS) ,  glutamate  synthase  (GAT) ,  and  glutamate  dehydro- 
genase (GDH)  do  not  constitute  a  contiguous  operon  in  E.  coli  and  may  be 
located  at  approximate  map  positions  771 ,  50' ,  and  20' ,  respectively.   Enzymic 
analysis  of  a  group  of  revertants  from  gin"  to  gln+  revealed  a  thermolabile 
GS  in  each  case  thereby  substantiating  that  the  gene  locus  at  77',  gin  A,  is 
the  structural  gene  for  GS.   Continued  investigation  suggested  that  these 
enzymes  might  be  involved  in  a  more  subtle  scheme  relating  the  regulation  of 
amino  acid  metabolism  and  NH3  assimilation. 

Two  of  the  revertants  with  thermolabile  GS  exhibited  poor  derepression 
on  limiting  NH3  although  the  adenylylation  values  for  the  GS  were  low  as 
expected.   In  order  to  reconcile  these  data  with  the  autogenous  regulation 
scheme  proposed  by  Magasanik  ej:  al_.  the  scheme  should  be  expanded  to  include 
a  positive  activation  by  GS  as  well  as  repression  by  adenylylated  GS . 
Derepression  on  limiting-NH.3  and  derepression  on  glutamate  are  probably 
related  to  an  increase  in  intracellular  concentration  of  a-ketoglutarate.   One 
of  the  two  revertants  described  above  was  also  unable  to  derepress  when  grown 
on  glutamate  whereas  the  other  derepressed  to  approximately  307»  wild-type 
extent.   These  results  may  indicate  involvement  of  an  additional  component  in 

1  4>6> 


Project  No.  Z01  HL  00212-04  LB 

derepression  which  can  only  be  discerned  from  the  pattern  of  regulation  of  the 
altered  revertant  enzyme.   This  is  being  examined  further. 

Preliminary  evidence  from  another  laboratory  suggests  that  constitutivity 
of  GS  can  result  from  mutations  affecting  enzymes  involved  in  the  chain  of 
biochemical  events  leading  to  the  modification  of  GS  by  adenylylation  (Abstr. 
of  Ann.  Meeting  of  ASM,  New  York,  1975) .   It  is  interesting  that  the  genes 
gin  B  and  gin  D,  apparently  designating  p^j  regulatory  protein  and  uridylating 
enzyme  have  been  tentatively  assigned  map  positions  approximating  that  for 
GAT  (501).   Another  locus,  gin  E,  the  apparent  determinant  for  uridylyl 
removing  enzyme  is  designated  in  the  general  map  region  of  GDH  (^20').   This 
genetic  juxtaposition  may  be  significant  in  the  scheme  of  regulation  of  NH3 
assimilation  and  is  being  examined  further. 

Growth  in  a  minimal  salts  medium  containing  glucose  at  33  mm  and  glutamate 
at  60  mm  gives  optimal  derepression  of  GS .   This  is  supplemented  where  neces- 
sary with  the  amino  acids  threonine,  leucine,  histidine  and  arginine  at  100 
ug/ml  which  collectively  show  no  repressive  effects,  e.g.  via  NH3  contribution, 
in  wild- type.   Under  these  conditions,  (with  or  without  amino  acid  supplement) 
the  activity  of  GAT  is  almost  completely  repressed  and  there  is  a  marked 
elevation  in  the  level  of  GDH  activity.   The  extent  of  derepression  appears 
independent  of  the  state  of  adenylylation  of  GS .   Also,  there  is  no  corrobo- 
ration in  E.  coli  of  the  observation  reported  for  Klebiella  that  a  reciprocal 
relationship  exists  between  GS  and  GDH.   According  to  Brenchley  (personal 
communication)  studies  with  S_.  typhimurium  agree  with  the  E.  coli  findings. 
Furthermore,  there  is  no  evidence  in  either  coli  or  typhimurium  for  NH3 
induction  of  GDH.   In  wild-type  E.  coli,  the  activities  of  both  GDH  and  GAT 
may  be  decreased  to  ~  507o  original  value  by  increasing  NHo  concentration  to 
100  mM.   These  differences  may  reflect  the  adaptive  changes  dictated  by  the 
milieu  of    soil-Living   vs.  enteric  organisms. 

Some  recent  findings  with  a  temperature-sensitive  glutamate  t-rna 
synthetase  mutant  indicate  that,  at  the  restrictive  temperature,  the  activity 
of  GAT  is  elevated  as  is  that  of  GS  (La  Pointe,  J.,  personal  communication). 
The  interpretation  offered  is  that  charged  glutamyl-t-rna  is  a  co-repressor 
for  both  GS  and  GAT.   However  the  observations  of  repressed  GAT  with  dere- 
pression of  GS  on  high  glutamate  reported  here  do  not  agree  with  the  idea  of 
unity  in  control  for  these  two  enzymes.   The  relationship  between  GAT  and  GS 
is  currently  being  investigated  from  another  point  of  view  (see  last  section 
below) . 

Attempts  to  correlate  the  phenomenon  of  MS0  resistance  with  the  mechanism 
of  GS  derepression  are  in  progress  and  early  work  suggests  that,  if  the 
structural  gene  for  GS  is  autogenously  regulated,  it  functions  via  a 
cooperation  with  some  other  element  -  perhaps  a  component  of  GAT.   In  this 
regard,  it  is  interesting  that,  in  the  case  of  Klebsiella,  the  gin  C" 
regulatory  type  mutations  which  are  now  assigned  positions  within  the  gin  A 
structural  gene  locus,  were  originally  derived  from  a  GAT"  parent.   The 
observation  that  methionine  potentiates  MSO  resistance  is  being  investigated 
in  view  of  the  fact  that  methionine  is  a  potent  inhibitor  of  GAT.   Methionine 


17 


Project  No.  Z01   HL  00212-04  LB 

has  also  been  observed  to  reduce  the  growth  rate  of  wild-type  cells  in 
unsupplemented  minimal  medium. 

Antibody  has  been  prepared  in  the  rabbit  against  purified  GAT.   Although 
no  cross-reactivity  could  be  observed  between  Ptj  A  or  D;  GS  n^  or  7  and 
anti-GAT  antiserum  via  a  micro  precipitin  test,  approximately  357o  neutraliza- 
tion of  enzyme  activity  could  be  observed  when  anti-GAT  serum  was  added  to 
GStt— 7  prior  to  transferase  assay.   At  this  antiserum  concentration  the 
activity  of  the  homologous  antigen  (GAT)  was  687,  inhibited.   Control  serum  did 
not  inhibit  and  GS  h  1.0  appears  unaffected.   Some  inhibition  of  p-^  activity 
(/W157»)  was  observed  using  very  small  amounts  of  antiserum  however  with  larger 
amounts  the  control  serum  showed  inhibition.   These  results  are  preliminary 
and  the  antiserum  will  be  fractionated  in  an  attempt  to  eliminate  non-specific 
inhibition.   Sub-unit  sharing  among  the  NH3  assimilatory  enzymes  may  represent 
an  additional  regulatory  opportunity. 

Proposed  Course  of  Project: 

Genetic  studies  to  refine  the  chromosomal  regions  containing  the  struc- 
tural genes  for  glutamine  synthetase,  glutamate  dehydrogenase,  glutamate 
synthase  will  continue  with  an  emphasis  on  isolation  of  regulatory  mutants. 
Enzymic  analysis  of  revertants  of  structural  gene  mutants  will  continue. 
Physiological  and  biochemical  studies  on  the  interrelationships  of  NH3- 
assimilatory  enzymes  in  mutant  strains  will  continue. 

Keyword  Descriptors: 

Genetics  of  glutamine  synthetase,  regulation  of  NH3  metabolism,  drug 
resistance  and  derepression  of  glutamine  synthetase. 

Honors  and  Awards:   None 

Publications:   None 


*8 


Project  No.  Z01   HL  00213-01   LB 
Laboratory  of  Biochemistry 
Section  on  Enzymes 
Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Title:   Metabolite  Regulation  of  Coupled  Covalent  Modification  Cascade 
Systems 

Previous  Serial  Number:   NHLI-3 

Principal  Investigator:   E.  R.  Stadtman 

Other  Investigators:   P.  B.  Chock 
S.  P.  Adler 

Cooperating  Units:   None 

Project  Description: 

Objectives:   To  make  a  theoretical  analysis  of  the  steady  state  functions 
involved  in  the  allosteric  regulation  of  key  enzymes  in  metabolism  by  cascades 
of  cyclic  covalent  modification  reactions. 

Major  Findings: 

Regulation  of  several  key  enzymes  in  metabolism  is  mediated  by  cyclic 
covalent  modification  reactions  in  which  the  active  form  of  an  enzyme  in  one 
cycle  is  a  catalyst  for  the  covalent  modification  of  an  enzyme  in  the  next. 
When  the  cascade  involves  (n-1)  successive  cyclic  covalent  modification 
reactions  and  an  allosteric  activation  of  the  first  enzyme  in  the  series,  it 
can  be  shown  that  under  steady  state  equilibrium  conditions,  the  fraction  of 
the  modified  form  of  the  target  enzyme  (Ena)  is  described  by  Eq.  (1), 


5*  +1 


k^n-1 

kT 


v'\n-2 
Kr> 


k' 
kT 


+  1 


(1) 


with  the  assumptions  that:   (1)  the  catalytic  constants  for  the  forward  step, 

kf  =  klf  El  =  k2fE2  =  *"  k(n-l)fE(n-l)'  where  klf'  k2f>  •••k(n-l)f  are 
specific  rate  constants  for  successive  forward  steps  in  the  cyclas  and  E]^, 

E2,  • • •  En  are  total  concentrations  of  enzymes  undergoing  activation  at  the 
first  step,  first  cycle  and  (n-1)  cycle  respectively;  (2)  the  catalytic  con- 
stants for  the  regeneration  of  the  unmodified  forms,  k^.  =  k^rR^  =  k~  Ro  = 
*'■  k(n-l)rRn-l'  where  k^r,  k2r,  '■■  k(n_i)r  are  specific  rate  constants  for 
the  successive  regeneration  steps  and  R]^,  R£,  ■••  R(n-1)  are  concentrations  of 
enzymes  catalyzing  the  regeneration  steps.   Equation  (1)  demonstrates  the 
tremendous  amplification  potential  of  such  cascade  systems.   It  follows  that 


if 


Project  No.  Z01   HL   00213-01   LB 

the  concentration  of  effector,  e,  required  to  produce  50%  conversion  of  En  to 
Ena,  decreases  in  such  a  manner  that  log  eg  5  is  inversely  proportional  to 
the  number  of  cycles  in  the  cascade.   Thus,  when  Kd  =  1  mM  and  kr/kf  =0.1, 
eQ  5  is  approximately  equal  to  1,  0.1,  0.01  and  0.001  mM  for  a  0,  1,  2,  and  3 
cycle  cascade,  respectively.   In  addition  to  this  amplification  capacity,  such 
systems  exhibit  an  enormous  capacity  for  allosteric  control.   It  is  evident 
from  Eq.  (1)  that  positive  or  negative  allosteric  interactions  with  any  one  or 
all  of  the  several  enzymes  in  the  cascade  will  affect  the  catalytic  constants 
of  these  enzymes  and  thereby  regulate  the  over-all  ratio  of  kr/kf,  which  in 
turn  determines  the  degree  of  amplification. 

The  foregoing  cascade  model  is  patterned  after  the  system  that  regulates 
the  activities  of  phosphorylase  or  glycogen  synthetase  in  which  a  series  of 
protein  kinases  and  phosphatases  catalyze  the  phosphorylation  and  dephos- 
phorylation  of  the  target  enzymes.   A  somewhat  different  kind  of  cascade 
regulates  the  activity  of  glutamine  synthetase  in  E.  coli.   Here,  regulation  is 
mediated  by  the  coupling  of  two  cyclic  nucleotidylylation  processes.   The 
first  cycle  consists  of  uridylylation  and  deuridylylation  of  the  Pj;j  regulatory 
protein.   This  is  catalyzed  by  a  protein  complex  exhibiting  both  uridylyl- 
transferase  (UTase)  and  uridylyl  removing  (UR)  activities,  and  involves  the 
attachment  and  removal  of  a  uridylyl  group  to  and  from  a  tyrosyl  hydroxyl 
group  in  each  of  four  identical  subunits  of  the  regulatory  protein.   A  second 
cycle,  involves  adenylylation  and  deadenylylation  of  a  tyrosyl  hydroxyl  group 
in  each  of  the  12  subunits  of  glutamine  synthetase  (GS) ,  and  is  catalyzed  by 
adenylyltransferase  (ATase) .   Coupling  of  the  two  cycles  is  obtained  by  almost 
complete  dependence  of  GS-adenylylation  on  the  unmodified  form  (Pjt^)  and  of 
GS-deadenylylation  on  the  uridylylated  form  (Pud)  °f  the   regulatory  protein. 
Because  the  two  interconvertable  forms  of  P^^  are  oppositional  in  their 
capacities  to  stimulate  the  Atase  catalyzed  adenylylation  and  deadenylylation 
reactions,  the  steady  state  level  of  adenylylated  GS  is  given  by  the 
expression, 

12  k!k3UR 

GOri  — 


k2k4Ux  +  k^k3ljR 


where  n  =  the  average  number  of  covalently  bound  adenylyl  groups  per  mole  of 
GS ;  k-^ ,  ko,  ko,  and  k,  are  the  specific  rate  constants  for  the  deuridylylation, 
uridylylation,  adenylylation  and  deadenylylation  reactions,  respectively;  and, 
Uj  and  Ud  are  the  concentrations  of  UTase  and  UR  removing  activities,  respec- 
tively.  Variations  in  any  one  or  all  of  these  parameters,  in  response  to 
fluctuations  in  the  relative  concentrations  of  allosteric  effectors  (viz,  UTP, 
ATP,  CMP,  UMP,  glutamine,  CU-ketoglutarate,  Pi,  Mg2+  and  Mn2+)  lead  to  the 
establishment  of  different  steady  state  levels  of  GS  adenylylation  and  hence 
its  catalytic  activity.   In  the  above  derivations  it  is  assumed  that  the 
affinities  of  the  various  modifying  enzymes  for  their  protein  substrates  is 
relatively  small;  i.e.,  that  protein-protein  interactions  do  not  affect 
significantly  the  concentrations  of  the  various  enzyme  forms.   If  the 
affinities  are  high,  then  preferential  binding  of  one  catalyst  to  the  modified 
or  unmodified  forms  of  its  substrate  could  result  in  deviations  from  the 
theoretical  relationship  described.   Clearly  in  the  above  cascade  systems,  the 


To 


Project  No.  Z01  HL  00213-01   LB 

coupling  of  nucleoside  triphosphate  dependent  covalent  modification  reactions 
with  regeneration  of  the  unmodified  enzyme  forms,  results  in  the  net  decom- 
position of  ATP  and  UTP.   It  is  assumed  that  the  concentrations  of  ATP  and  UTP 
are  constant  for  a  given  metabolic  state.   Nevertheless,  the  apparent  loss  in 
ATP  energy  accompanying  the  cyclic  processes  is  not  wasted,  this  energy  is 
used  to  provide  the  driving  force  that  is  needed  to  maintain  the  modified  and 
unmodified  forms  of  the  various  enzymes  at  metabolite  specified  steady  state 
levels  that  are  away  from  true  thermodynamic  equal ibrium  values.   The  con- 
sumption of  ATP  energy  is  therefore  the  price  that  must  be  paid  to  support  the 
elegant  cascade  type  of  cellular  regulation.   In  the  last  analysis  cascade 
systems  represent  physiological  computers,  the  circuitry  of  which  consists  of 
a  series  of  interconnected  terminals  in  the  form  of  interconvertable  enzymes. 
By  means  of  allosteric  and  active  site  interactions,  these  interconvertable 
enzymes  are  programmed  to  sense  fluctuations  in  the  concentrations  of  a 
multiplicity  of  metabolites;  this  leads  to  automatic  adjustments  in  the 
specific  activities  and  rate  constants  of  the  several  cascade  enzymes.   Through 
this  system  the  multiple  inputs  are  integrated  and  registered  as  a  single  out- 
put; i.e.,  the  fractional  modification  of  the  target  enzyme  which  ultimately 
determines  its  catalytic  activity. 

Proposed  Course  of  Research: 

The  theoretical  steady  state  analysis  of  cascade  systems  will  be  extended 
to  include  a  consideration  of  the  effects  of  protein-protein  interactions  and 
also  to  evaluate  the  expenditure  of  ATP  energy  needed  to  maintain  steady  states 
away  from  thermodynamic  levels.   An  attempt  will  be  made  to  determine  the 
specific  rate  constants  for  the  reaction  involved  in  the  glutamine  synthetase 
cascade  in  cider  to  test  the  validity  of  the  theoretical  steady  state  analysis. 

Keyword  Descriptors: 

Cascade  regulation,  metabolic  regulation,  covalent  modification, 
glutamine  synthetase  system,  adenylylation-deadenylylation,  and  uridylylation- 
deuridylylation. 

Honors  and  Awards:  None 

Publications: 

1.  S.  P.  Adler  and  E.  R.  Stadtman:   Cascade  Control  of  E.  coli  Glutamine 
Synthetase.   In  Richter,  D.  (Ed.):  Lipmann  Symposium.   Energy,  Regulation  and 
Biosynthesis  in  Molecular  Biology.   Walter  de  Gruyter,  Berlin-New  York,  1974, 
pp.  28-39. 

2.  E.  R.  Stadtman,  J.  E.  Ciardi,  P.  Z.  Smyrniotis,  A.  Segal,  A.  Ginsburg,  and 
S.  P.  Adler:   Role  of  Adenylylated  Glutamine  Synthetase  Enzymes  and  Uridylylated 
Regulatory  Protein  Enzymes  in  the  Regulation  of  Glutamine  Synthetase  Activity 
in  Escherichia  coli.   In  Market,  C.  L.  (Ed.):  Isoenzymes  II:  Physiological 
Function.   New  York,  Academic  Press,  1975,  pp.  715-732. 


Tf 


Project  No.  Z01   HL  00213-01   LB 

3.  R.  E.  Miller,  E.  Shelton,  and  E.  R.  Stadtman:   Zinc  Induced  Paracrystalline 
Aggregation  of  Glutamine  Synthetase.   Arch.  Biochem.  Biophys.  163:  155-171, 
1974. 

4.  A.  Ginsburg  and  E.  R.  Stadtman:   Glutamine  Synthetase  of  Escherichia  coli: 
Structure  and  Regulation.   In  Ebner,  K.  E.  (Ed.):  Subunit  Enzymes:  Biochemistry 
and  Function.   New  York,  M.  Dekker,  Inc.,  1975  (in  press). 


7A 


Project  No.  Z01   HL  00214-01   LB 

1.  Laboratory  of  Biochemistry 

2.  Section  on  Enzymes 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Enzyme  Degradation  in  Klebsiella  aerogenes 

Principal  Investigator:   Richard  M.  Fulks 

Other  Investigators:   Earl  R.  Stadtman 

Project  Description: 

The  biochemical  mechanisms  involved  in  protein  breakdown  and  its 
regulation,  and  the  role  protein  breakdown  plays  in  controlling  the  amount 
of  a  protein  present  within  a  cell. 

Major  Findings: 

Klebsiella  aerogenes ,  an  organism  closely  related  to  E.  coli,  and  one 
that  has  proved  useful  in  many  biochemical  and  genetic  studies  was  selected 
as  experimental  material.   Preliminary  experiments  showed  that  the  asparto- 
kinase  activity  of  this  organism  began  to  fall  within  minutes  of  onset  of 
stationary  phase  due  to  nitrogen  limitation.   Within  three  hours,  over  half 
of  the  activity  present  at  the  beginning  of  stationary  phase  was  lost.   At 
the  same  time  glutamine  synthetase  activity  increased  in  these  nitrogen- 
limited  cells  and  this  showed  that  the  inactivation  process  was  specific  in 
nature.   Aspartokinase  inactivation  also  occurred  in  cell  suspensions  and 
this  meant  that  large  volumes  of  cells  could  be  grown  at  once  and  stored 
frozen,  then  small  portions  of  cells  thawed  and  used  for  individual  experi- 
ments as  needed. 

Addition  of  glucose  promoted  the  inactivation  process  and  dinitrophenol 
caused  reduction  in  the  rate  of  inactivation.   These  findings  are  consistent 
with  an  energy-dependent  inactivation  process,  as  has  been  proposed  for  most 
instances  of  intracellular  protein  degradation.   Chloramphenicol,  an  inhibi- 
tor of  protein  synthesis,  caused  a  more  rapid  loss  of  aspartokinase  activity. 
This  result  suggested  that  in  the  absence  of  inhibitor,  the  cells  were  syn- 
thesizing new  enzyme,  but  they  were  inactivating  it  at  a  rate  that  exceeded 
synthesis.   As  was  true  in  the  absence  of  chloramphenicol,  dinitrophenol 
inhibited  inactivation  of  the  enzyme  when  chloramphenicol  was  present. 

Glutamine  synthetase  activity  also  declined  when  chloramphenicol  was 
present,  in  contrast  to  the  rise  in  activity  that  occurred  when  these  nitro- 
gen-limited cells  were  incubated  in  the  absence  of  chloramphenicol.   As  was 
true  for  aspartokinase,  the  inactivation  of  glutamine  synthetase  in  the  pre- 
sence of  chloramphenicol  was  inhibited  by  dinitrophenol.   When  dinitrophenol 


73 


Project  No.  Z01   HL   00214-01   LB 

alone  was  added  to  the  medium,  the  level  of  glutamine  synthetase  remained 
nearly  constant  throughout  5  hours  of  incubation. 

Study  of  the  inactivation  of  glutamine  synthetase  offered  several  advan- 
tages over  study  of  aspartokinase  inactivation.   One  important  advantage  was 
that  antibody  to  the  E.  coli  glutamine  synthetase  had  already  been  prepared 
in  this  laboratory  by  Tronick  e_t  al.  and  this  antibody  cross-reacted  with  the 
Klebsiella  enzyme.   Incubation  of  Klebsiella  cells  in  the  presence  of  chlor- 
amphenicol resulted  in  a  loss  of  antigenic  material  which  probably  reflects 
intracellular  degradation  of  glutamine  synthetase.   Control  experiments 
showed  that  chloramphenicol  did  not  simply  interact  directly  with  the  enzyme 
to  denature  it.   These  findings  are  consistent  with  the  interpretation  that 
in  the  presence  of  chloramphenicol,  the  amount  of  glutamine  synthetase  present 
declined  because  the  cells  degraded  the  enzyme  but  were  unable  to  synthesize 
new  enzyme. 

Inactivation  of  glutamine  synthetase  by  covalent  attachment  of  adenylyl 
groups  to  the  enzyme  affords  one  important  way  for  controlling  glutamine 
synthetase  activity  in  these  cells.   However,  adenylylation  was  not  responsible 
for  the  changes  observed  in  the  present  experiments,  because  the  assays  used 
to  measure  catalytic  activity  were  equally  sensitive  to  adenylylated  or  un- 
adenylyla ted  enzyme,  and  the  antibody  used  reacts  with  both  forms  of  the  en- 
zyme.  Although  adenylylation  by  itself  is  unable  to  explain  the  inactivation 
and  decomposition  of  glutamine  synthetase  in  these  experiments,  adenylylation 
may  nevertheless  play  a  role.   For  example,  modification  of  glutamine  synthe- 
tase appears  to  facilitate  its  degradation  in  cultured  liver  cells. 

Proposed  Research: 

Experiments  to  further  characterize  the  chloremphenicol-induced  degrada- 
tion of  glutamine  synthetase  will  be  carried  out.   Also  effects  of  other  in- 
hibitors such  as  puromycin  will  be  examined.   These  studies  should  lay  the 
groundwork  for  experiments  with  cell-free  preparations,  which  can  provide  more 
precise  information  about  the  steps  involved  in  protein  degradation  and  its 
regulation.   Genetic  approaches  may  also  be  fruitful  in  the  study  of  protein 
breakdown  in  these  cells. 

Keyword  Descriptors: 

Protein  degradation,  enzyme  regulation,  glutamine  synthetase,  lysine- 
sensitive  aspartokinase,  Klebsiella  aerogenes,  stationary  phase,  antibody, 
immunodiffusion. 

Honors  and  Awards:   None 
Publications:   None: 


7t 


Annual  Report  of  the 

Section  on  Intermediary  Metabolism 

and  Bioenergetics 

Laboratory  of  Biochemistry 

National  Heart  and  Lung  Institute 

July  1,  1974  through  June  30,  1975 

Research  in  the  Section  on  Intermediary  Metabolism  and  Bioenergetics  has 
been  concerned  with  (1)  the  anaerobic  metabolism  of  amino  acids  and  other 
nitrogen-containing  compounds  with  particular  reference  to  the  identification 
and  characterization  of  the  components  of  the  electron  transport  systems 
linked  to  glycine  reductase  and  to  proline  reductase,  to  the  roles  of  selenium, 
iron,  sulfur  and  flavins  in  the  amino  acid  reductase  reactions,  and  to  the 
mechanism  of  the  Bi o-coenzyme  dependent  Cf-methyleneglutarate  mutase  reaction 
(an  intermediate  step  in  the  anaerobic  decomposition  of  nicotinic  acid)  and 
(2)  the  metabolism  of  formate  and  other  one-carbon  compounds  by  methane- 
producing  bacteria  and  the  roles  of  selenium,  molybdenum  and  tungsten  in  the 
methane  fermentation. 

Selenium  Biochemistry  and  Anaerobic  Qxido-Reduction  Reactions: 

In  Clostridium  sticklandii ,  Clostridium  lentoputrescens  and  related  amino 
acid- fermenting  bacteria  that  utilize  glycine  as  a  terminal  electron  acceptor 
an  essential  component  of  the  reductase  system  has  been  shown  to  be  a  low- 
molecular  weight,  acidic  selenoprotein.   The  unusual  ultraviolet  spectrum 
characteristic  of  the  oxidized  form  of  this  protein  is  explained  by  the  absence 
of  tryptophan  and  the  presence  of  5  residues  of  phenylalanine  and  1  residue  of 
tyrosine  in  the  polypeptide  chain.   The  protein  contains  cysteine  and  methio- 
nine residues  and  as  yet  unidentified  selenium-containing  organic  compound  in 
covalent  linkage.   Reduction  of  the  protein  in  neutral  solution  with  borohy- 
dride  causes  an  instantaneous  increase  in  absorbancy  in  the  low  ultraviolet 
(maximum  about  238  nm)  which  is  similar  to  the  spectral  changes  observed  when 
certain  diselenides  are  reduced  to  selenols  under  the  same  conditions.   Rapid 
reoxidation  of  the  protein  and  the  model  compounds  is  indicated  by  immediate 
reversal  of  the  spectral  changes  upon  exposure  to  air.   These  studies,  together 
with  properties  of  the  reduced  and  carboxymethylated  protein,  suggest  that  the 
selenium  moiety  is  converted  to  a  selenol  (-SeH)  upon  reduction  and  the  ease  of 
reversibility  of  this  process  might  explain  its  biological  role. 

In  addition  to  greater  chemical  stability  of  the  selenium  moiety  of  the 
protein  which  is  observed  upon  alkylation  of  the  reduced  form  of  the  seleno- 
protein, both  the  biological  activity  and  the  antigenic  specificity  of  the 
protein  are  lost.   Specific  antibodies  prepared  to  the  pure  native  seleno- 
protein fail  to  detect  any  cross-reacting  precursor  protein  in  extracts  of 
selenium-deficient  bacteria  but  do  appear  to  react  with  a  selenium-containing 
tryptic  peptide  of  the  native  protein.   Hence  the  sensitive  immunologic 
approach  may  aid  in  characterization  of  the  selenium  moiety  of  the  protein  and 
its  mode  of  biosynthesis. 

The  formate  dehydrogenase  of  the  methane-producing  organism,  Methanococcus 
vannielii,  also  is  a  selenoprotein  and  procedures  for  the  partial  purification 
of  this  enzyme  have  been  developed.   This  complex,  oxygen-sensitive  enzyme  is 


7r 


of  interest  for  studies  as  to  the  precise  nature  of  oxygen  sensitivity  of  this 
class  of  enzymes,  and  the  role  of  its  selenium,  molybdenum  and  iron  components. 
Failure  of  the  complex  form  of  the  enzyme  from  the  methane  organism  to  react 
with  antibodies  to  the  glycine  reductase  selenoprotein  may  indicate  that  the 
chemical  form  of  selenium  in  the  two  proteins  is  different  but  smaller  molec- 
ular weight  forms  of  the  selenium-containing  polypeptide  will  also  be  examined. 

Proline  Reductase: 

The  proline  reductase  of  C.  sticklandii,  C.  lentoputrescens  and  related 
bacteria  transfers  reducing  equivalents  to  proline  which,  like  glycine,  also 
can  serve  as  a  terminal  electron  acceptor  for  these  organisms.   Proline 
reductase  has  been  solubilized  from  the  membrane  of  the  cell  by  the  use  of 
detergents  and  purified  to  homogeniety  by  standard  enzymological  techniques. 
The  pure  reductase,  molecular  weight  298,000,  consists  of  subunits  of  61,500 
and  50,500  and  is  a  f lavoprotein.   Marked  sensitivity  of  the  enzyme  to  boro- 
hydride,  hydroxyiamine  and  other  carbonyl  reagents  was  earlier  shown  by  Abeles 
et  al .  to  be  the  result  of  modification  of  an  essential  pyruvate  moiety  cova- 
lently  bound  to  the  enzyme.   The  pure  reductase  is  unable  to  react  directly 
with  the  normal  electron  donor,  a  reduced  pyridine  nucleotide,  and  one  or  more 
low  molecular  weight  carriers  must  be  added  to  reconstitute  the  natural 
electron  transport  chain.   Preliminary  data  showing  copurif ication  of  proline 
reductase  activity  and  radioactive  selenium  from  75se-labeled  extracts  suggest 
that  a  selenium-containing  catalyst  may  be  part  of  the  electron  transport 
chain. 

Quinone-Dependent  Phosphatase  of  C.  sticklandii: 

Continued  studies  on  the  unusual  mercaptan  and  quinone-dependent 
p-nitrophenylphosphatase  of  clostridial  origin  have  resulted  in  a  greatly 
improved  method  of  isolation  of  the  pure  enzyme  in  good  yield  and  established 
additional  chemical  properties  of  the  protein  structure.   The  functional  form 
of  the  enzyme  presumably  is  a  quinone  adduct  of  one  or  more  of  the  four 
sulfhydryl  groups  that  can  be  titrated  on  the  protein.   The  possible  function 
of  this  enzyme  as  a  phosphorylated  intermediate  in  an  energy  conservation 
process  or  in  a  transport  process  is  under  investigation. 

q-Methylene  Glutarate  Mutase: 

As  part  of  a  continuing  investigation  on  the  precise  mechanism  of  the 
chemical  rearrangements  catalyzed  by  various  Bj^  coenzyme  dependent  enzymes, 
the  stereochemistry  of  the  interconversion  of  a-methyleneglutarate  and 
methylitaconate  by  a-methyleneglutarate  mutase  of  the  nicotinate  fermenting 
organism,  Clostridium  barkeri,  is  under  study.   The  necessary  substrates, 
labeled  with  tritium  and  deuterium  for  the  determination  of  the  stereochemical 
course  of  the  reaction  and  with  ^C  to  serve  as  a  marker  of  the  extent  of 
interconversion,  have  been  synthesized  by  improved  procedures  developed 
especially  for  the  problem.   The  necessary  enzymes  were  prepared  and  better 
methods  for  assay  of  the  reaction  were  developed  in  order  that  the  critical 
experiments  with  the  doubly  labeled  substrates  can  be  carried  out  with 
precision.   This  type  of  careful  study  is  one  of  the  valid  approaches  to  the 
elucidation  of  the  exact  mechanism  of  this  group  of  poorly  understood  B]^ 
dependent  reactions. 


76 


Project  No.  Z01   HL   00205-20   LB 

1.  Laboratory  of  Biochemistry 

2.  Section  on  Intermediary 

Metabolism  &  Bioenergetics 
3-  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Role  of  selenium  in  anaerobic  electron  transport. 
CH  biosynthesis. 

Previous  Serial  Number:   NHLI-13 

Principal  Investigator:   T.  C.  Stadtman 

Other  Investigators:   Belinda  Seto  (Staff  Fellow;  see  individual  report) 
Joyce  Cone  (Staff  Fellow;  see  individual  report) 
Raphael  Martin  (Guest  Scientist  from  Spain;  Spanish 

Fellowship;  January  1975  starting  date.) 
Jay  Jones  (Technical  Assistant  and  Predoctoral  Student 

at  George  Washington  U. ) 
Joe  Nathan  Davis  (Research  Assistant;  anaerobic  labora- 
tory operator  and  advisor  to  new  fellows  regarding 
culture  of  microorganisms,  operation  of  amino  acid 
analyzers,  disc  gel  electrophoresis  equipment,  etc.) 
See  separate  research  report. 

Project  Description: 

1)   Anaerobic  metabolism  of  certain  amino  acids  with  special  reference  to  the 
role  of  selenium,  quinones ,  flavins  and  non-heme  iron  proteins  in  the  electron 
transfer  and  phosphorylation  reactions  involved.   a)   Structure  and  function 
of  the  selenoprotein  component  of  glycine  reductase  and  its  interreaction 
with  the  other  protein  components  of  glycine  reductase.   Nature  of  the  elec- 
tron transfer  and  phosphorylation  reactions  linked  to  glycine  reduction  by 
Clostridium  sticklandii  and  related  amino  acid  fermenting  bacteria.   b)  Isola- 
tion and  characterization  of  proline  reductase  of  C_.  sticklandii  and  identifi- 
cation of  proteins  and  cofactors  required  for  electron  transport  between  re- 
duced pyridine  nucleotides  (e.g.,  DPNH)  and  proline,  the  terminal  electron 
acceptor.   This  project  investigated  primarily  by  Dr.  Belinda  Seto.   2)   Mech- 
anism of  formate  oxidation  to  carbon  dioxide  by  Me thano coccus  vanielii  and 
roles  of  selenium,  molybdenum  and  vitamin  B   in  methane  biosynthesis  from 
formate.   Studies  to  determine  whether  stimulation  of  growth  of  M.  vannielii 
by  tungstate  is  the  result  of  substitution  of  tungsten  for  molybdenum  in 
formate  dehydrogenase.   Parallel  studies  on  formate  dehydrogenase  of  C_.  stick- 
landii to  determine  the  nature  of  the  selenium-containing  moiety  of  this  en- 
zyme, and  its  biochemical  role.   Isolation  and  characterization  of  the  selenium- 
dependent  formate  dehydrogenase  of  M.  vannielii  is  research  project  of  Jay  B. 
Jones.   3)   Characterization  of  the  quinone-dependent  p-nitrophenylphosphatase 


77 


Project  No.  Z01   HL   00205-20   LB 

of  Clostridium  sticklandii  (see  report  of  J.  N.  Davis). 
Major  Findings: 

la.   The  chemical  and  physical  properties  of  the  selenoprotein  (Protein 
A)  of  clostridial  glycine  reductase  have  been  further  characterized.   Unlike 
one  other  pure  selenoprotein,  glutathione  peroxidase,  under  investigation  in 
other  laboratories  the  glycine  reductase  selenoprotein  is  remarkably  stable 
to  a  variety  of  chemical  procedures.   Only  when  the  native  selenoprotein  is 
oxidized  with  peroxide  or  with  iodine  is  the  selenium  quantitatively  cleaved 
from  the  protein  and  lost  as  selenite.   The  protein  exhibits  an  abnormal  ultra- 
violet absorption  spectrum;  the  marked  fine  structure  in  the  region  of  250  to 
270  nm  is  explained  by  the  presence  in  the  protein  of  5  phenylalanine  residues 
and  1  tyrosine  residue  and  the  complete  absence  of  tryptophane.   Two  cysteine 
residues  are  present  in  the  protein  as  determined  by  amino  acid  analyses  of 
the  hydrolyzed  carboxymethylated  protein  (2  carboxymethylcysteine  residues) 
and  by  analyses  of  hydrolysates  after  performic  acid  oxidation  (2  cysteic 
acid  residues).   Two  methionine  residues  have  been  found  in  a  number  of  hy- 
drolyzed samples  of  the  protein.   The  selenium  containing  residue  in  the  pro- 
tein is  clearly  distinguishable  from  selenomethionine  and  from  Se-methyl- 
selenocysteine.   Although  the   Se-labeled  compound  isolated  from  acid  hydroly- 
sates of  the  carboxymethylated  selenoprotein  cochromatographs  in  thin  layer 
systems  and  on  the  amino  acid  analyzer  with  Se-carboxymethyl  selenocysteine , 
it  appears  to  be  more  stable  than  the  authentic  reference  compound  and  thus 
its  precise  identity  is  still  in  doubt.   The  marked  increase  in  absorbancy  at 
238  nm  observed  when  the  protein  is  reduced  with  borohydride  at  neutral  pH 
may  be  attributed  to  the  conversion  of  the  selenium  moiety  to  a  selenol  (-SeH) . 
Model  diselenides  exhibit  such  absorbancy  when  reduced  at  neutral  pH.   Con- 
tinued attempts  to  obtain  a  derivative  of  the  selenocompound  suitable  for  mass 
spectral  analysis  are  in  progress. 

Investigations  carried  out  by  Dr.  Raphael  Martin  to  determine  the  amino 
acid  composition  of  the  amino  and  carboxyl  ends  of  the  selenoprotein  molecule 
are  in  progress.   The  selenium  containing  moiety  is  located  internally  and  is 
not  among  the  amino  acid  residues  liberated  from  the  amino  terminus  by  leucine 
amino  peptidase  nor  from  tha  carboxy  terminus  by  carboxypeptidase. 

Specific  antibodies  to  the  native  selenoprotein  were  produced  in  rabbits 
and  purified  by  Dr.  Belinda  Seto.   Extracts  and  enzyme  preparations  that  con- 
tain biologically  active  selenoprotein  exhibit  strong  precipitin  tests  with 
the  antibodies  but  no  cross-reacting  material  was  detected  in  selenium- 
deficient  extracts  that  lack  the  selenoprotein  activity.   This  suggests  that 
either  the  selenium-moiety  is  a  very  important  immunological  determinant  or 
synthesis  of  the  protein  ceases  when  selenium  is  unavailable  to  the  cell. 
Current  studies  with  chemically  modified  selenoprotein  and  with  tryptic  pep- 
tides containing  the  selenium  moiety  are  in  progress  to  aid  in  characteriza- 
tion of  the  selenoprotein  by  immunological  methods. 

lb.   The  D-proline  reductase  of  C.    sticklandii  which  was  partially  puri- 
fied and  studied  in  1954-56,  and  further  characterized  as  regards  electron 
transport  properties  by  Dr.  Arnold  Schwartz  and  T.  C.  Stadtman  (1955-58)  has 

2  73 


Project  No.  Z01   HL   00205   LB 

now  been  obtained  in  homogenous  form  by  Dr.  Belinda  Seto.   The  pure  reductase, 
a  flavoprotein  containing  covalently  bound  pyruvate,  is  completely  resolved 
of  the  normal  electron  carriers  that  transport  reducing  equivalents  from 
reduced  pyridine  nucleotides  (e.g.,   DPNH) . 

A  large  molecular  weight  complex  of  proline  reductase  that  had  been  pre- 
pared earlier  by  Schwartz  and  stored  at  -80°  still  exhibited  activity  with 
DPNH  and  served  as  a  source  of  components  needed  to  couple  the  completely 
resolved  reductase  to  the  natural  electron  donor.   The  availability  of  a  pure 
resolved  terminal  electron  acceptor  (the  proline  reductase)  now  will  allow 
isolation  and  characterization  of  the  components  of  this  interesting  anaerobic 
electron  transport  system.   Earlier  indications  that  ferredoxin  and  a  labile 
protein  component  plus  catalytic  levels  of  acetyl-CoA  were  required  to  re- 
constitute the  electron  transport  chain  now  can  be  reexamined. 

2.   The  formate  dehydrogenase  of  M.  vannielii  was  purified  75-fold  from 
crude  extracts  by  a  series  of  steps  involving  heat  denaturation,  ammonium 
sulfate  precipitation  and  chromatography  on  DEAE-cellulose.   All  of  these  pro- 
cedures were  carried  out  in  the  absence  of  oxygen  in  the  anaerobic  laboratory 
to  prevent  destruction  of  the  very  oxygen-labile  enzyme.   The  75-fold  purified 
enzyme  preparation  separated  into  two  catalytically  active  bands  when  subjected 
to  electrophoresis  on  slabs  of  polyacrylamide  gel.   Two  different  forms  of  the 
enzyme  also  were  suggested  by  the  double  optimal  pH  profile  (pH  7.5  and  pH  8.5) 
exhibited  by  the  purified  material.   Some  stimulation  of  ability  of  the  puri- 
fied enzyme  to  oxidize  formate  with  triphenyltetrazolxum  as  electron  acceptor 
by  a  low  molecular  weight  cofactor  preparation  suggests  the  gradual  separation 
of  the  enzyme  from  a  cofactor  as  purification  progresses. 

As  reported  last  year,  evidence  from  growth  experiments  and  from   Se- 
labeling  experiments  indicates  that  the  M.  vannielii  formate  dehydrogenase  is 
a  selenoprotein.   There  is  some  evidence  that  the  enzyme  from  other  sources 
also  may  contain  molybdenum  and  iron.   The  two  forms  of  the  enzyme  from  M. 
vannielii  will  be  useful  to  compare  analytically  in  view  of  possible  diffe- 
rences in  cofactor  and  electron  carrier  composition.   Of  particular  importance 
is  the  identification  of  the  oxygen-labile  moiety  of  the  protein. 

The  antibody  to  the  clostridial  selenoprotein  prepared  by  Dr.  Belinda 
Seto  failed  to  exhibit  any  cross-reactivity  with  either  the  crude  or  the  most 
purified  form  of  the  M.  vannielii  formate  dehydrogenase.   Further  experiments 
using   Se-labeled  peptide  fragments  from  the  methane  bacterial  protein  will 
also  be  made  to  test  for  possible  homology. 

Keyword  Descriptors: 

Anaerobic  electron  transport  and  phosphorylation,  selenoprotein  (selen- 
ium-containing protein),  glycine  reductase  from  anaerobic  bacteria,  proline 
reductase  from  anaerobic  bacteria,  non-heme  iron  proteins  (ferredoxins) , 
molybdenum  and  tungsten  in  proteins,  methane  biosynthesis,   formate  dehydro- 
genase, acidic,  heat-stable  proteins,  amino  acid  composition  of  selenoprotein, 
atomic  absorption  spectrometry. 

7f 


Project  No.  Z01   HL  00205-20   LB 


Publications : 

1.  M.  Tanaka,  M.  Haniu,  K.  T.  Yasunobu,  J.  B.  Jones  and  T.  C.  Stadtman: 
Amino  Acid  Sequence  Determination  of  Clostridium  M-E  Ferredoxin  and  a  Comment 
on  the  Role  of  the  Aromatic  Residues  in  the  Clostridial  Ferredoxin. 
Biochemistry  13:   pp.  5284-5289,  1974. 

2.  J.  M.  Poston  and  T.  C.  Stadtman:   Cobamides  as  Cof actors :   Methylcobamides 
and  the  Synthesis  of  Methionine,  Methane,  and  Acetate.   In  Babior,  Bernard  M. 
(Ed.):   Cobalamin:   Biochemistry  and  Pathophysiology.   New  York,  John  Wiley 
and  Sons,  Inc.  1975,  pp.  111-139. 

3.  J.  Retey,  F.  Kunz,  T.  C.  Stadtman  and  D.  Arigoni:   Zur  Kenntnis  der  g- 
Lysin-Mutase  Reaktion:   Mechanismus  und  sterischer  Verlauf.   Manuscript  sub- 
mitted to  Helvetica. 

4.  F.  Kunz,  J.  Retey,  D.  Arigoni,  L.  Tsai  and  T.  C.  Stadtman:  Zur  Kenntnis 
der  g-Lysin-Mutase  Reaktion:  Die  absolute  Konf iguration  der  3 ,5-Diaminohex- 
ansSure.   Manuscript  submitted  to  Helvetica. 


eo 


Project  No.  Z01   HL  00206-16   LB 
Laboratory  of  Biochemistry 
Section  on  Intermediary 

Metabolism  &  Bioenergetics 
Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Stereochemical  Studies  of  Enzymatic  Reactions 

Previous  Serial  Number:   NHLI-11 

Principal  Investigator:   Lin  Tsai 

Other  Investigator:   E.  Caveney 

Cooperating  Units:   None 

Project  Description: 

Objective  1:   For  the  study  of  the  steric  course  of  the  rearrangement  cataly- 
zed by  aMG-mutase,  it  is  necessary  to  establish  various  methodologies. 

Major  Findings: 

14 
1.   A   C-labelled  substrate  of  fairly  high  specific  radioactivity  is 

needed  as  a  marker  for  the  study  of  the  rearrangement  reaction.   A  procedure 

to  render  optimal  incorporation  of    C-atom  to  the  methylene  group  of  a- 

methyleneglutaric  acid  was  developed  according  the  following  sequence  of 

reactions : 

CO,CH,     „,  .-£•£> ,~  K+ 

CM      CH  «>%KOH-      .  CHX  CH 

CHidC     2sch,:    ^Co1ch3        ch,oh *    C-Hi^c"     "o£    ^CO^CH^ 


— >  ok       cx  >  ,c*^s     ycs 

CHjO^C    "CH,.   COjCH3  ho^C     CH,.   CovH 

After  numerous  experimentation,  the  optimum  condition  for  the  Mannich  reaction 
was  found  to  be  the  use  of  50%  excess  of  the  trimethyl  ester  with  respect  to 

C-formaldehyde.   Under  this  condition,  a  55%  of  radioactive  yield  was  ob- 
tained. 

2.   The  combined  enzymatic  activities  of  aMG-mutase  and  KIT-isomerase 
were  determined  by  a  colorimetric  assay  of  2 ,4-dinitrophenylhydrazine  deriva- 
tive of  dimethylmaleic  anhydride.   It  was  noted  that  the  published  procedure 
did  not  give  consistent  results;  therefore,  a  different  procedure  was  worked 


tl 


Project  No.  Z01   HL   00206-16  LB 

out  involving  extraction  of  the  DNP -derivative  into  known  volume  of  methylene 
chloride,  from  which  the  chromophoric  material  was  extracted  into  known  volume 
of  2.5  N  NaOH.   Thus,  a  linear  standard  curve  could  be  constructed  for  0.1  - 
0.4  ymole  of  dimethylmaleic  anhydride. 

3.   Crude  extract  having  aMG-mutase  and  MIT-isomerase  activities  was  ob- 
tained in  the  35  -  65%  ammonium  sulfate  fraction.   This  usually  gave  a  7  -  10% 
conversion  of  a-methyleneglutarate  to  dime  thy  lmaleate.   Using  -^C-substrate, 
this  extract  did  yield  l^C-dimethy lmaleate  of  about  the  same  specific  activity, 
although  some   C  was  found  in  other  volatile  acid,  probably  acetic  acid. 
The  quantitative  aspect  of  -*-  C-substrate  to  product  is  still  under  study. 

Proposed  Course  of  Action: 

To  work  out  a  procedure  for  degradation  of  dime thy lmaleate  to  acetate  so 
as  to  apply  it  to  study  the  enzymatic  reaction  with  doubly  labelled  substrate. 

Objective  2:   Since  2-amino-5-hydroxyhexanoic  acid  is  an  unusual  amino  acid 
isolated  from  plant,  it  would  be  of  interest  to  determine  which  one  of  the 
diastereomers  from  the  synthetic  compound  correspond  to  the  natural  product. 

Major  Findings: 

An  improved  method  for  the  preparation  of  2-amino-5-hydroxyhexanoic  acid 
was  accomplished  as  outlined  below: 

CH3CO  CHaP^  c  (coxC«H&  iO  vo%  koh-c^SO*    CH3COCH2CH2CH  CO3.C1W5- 
MHCOCH3     Co>  HC*c-ioo°  WHC0CH3 


No^HA     .    CH3CHCHj.CH3.CKCOiC.Hs  in  HCI    CH1CHCV^CHrCHC03_H 
OH       NHCQCU3  OH       NH^ 


This  method  gave  consistently  better  results  than  the  previous  approach  which 
required  strongly  acidic  condition  for  the  hydrolysis  of  the  acetamidomalonate 
derivative.   Numerous  attempts  were  made  to  separate  the  diastereomeric  mix- 
ture with  only  minor  success.   After  tedious  column  chromatograph  of  the 
acetamidolactone  derivative,  only  a  small  amount  of  one"  of  the  isomers  could 
be  obtained.   The  main  difficulty  in  the  problem  is  the  lack  of  distinction 
between  these  diastereomers  in  their  physical  and  chemical  properties.   For 
instance,  the  synthetic  product,  which  must  be  a  mixture  of  isomers,  showed 
the  same  chromatographic  behaviors  as  the  natural  product  in  TLC,  amino  acid 
analyser,  as  well  as  GLC  of  derivatives. 


6*- 


Project  No.  Z01  HL  00206-16  LB 

The  evidence  that  the  synthetic  product  was  indeed  a  mixture  of  diaster- 
omers  came  only  after  careful  examination  of  the  proton  (PMR)  and  carbon 
(CMR)  magnetic  resonance  spectra.   In  the  PMR  of  the  amino  acid  in  D2O,  only 
the  H  at  C2  showed  a  small  difference  in  chemical  shift,  0.014  ppm.   Similarly, 
in  the  CMR  spectrum,  the  l^C  resonance  of  the  two  diastereomers  differed  at 
C5  and  C5  by  0.10  and  0.13  ppm  respectively. 

Proposed  Course  of  Action: 

To  continue  to  search  for  methods  of  separation  of  the  isomers  of  syn- 
thetic product  so  as  to  correlate  it  with  the  natural  product. 

Keyword  Descriptors: 

Stereochemistry  of  enzyme  reaction,  a-methyleneglutarate  mutase, 
coenzyme-B-i  o  • 

Honors  and  Awards :   None 
Publications:   None: 


83 


Project  No.  Z01   HL  00207-01   LB 
Laboratory  of  Biochemistry 
Section  on  Intermediary 

Metabolism  &  Bioenergetics 
Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Characterization  of  a  bacterial  selenoprotein 

Previous  Serial  Number:   NHLI-15 

Principal  Investigator:   Joyce  E.  Cone 

Other  Investigators:   Thressa  C.  Stadtman 
Raphael  Martin 
Belinda  Seto 

Cooperating  Units:   Joe  Nathan  Davis  (_amino  acid  analyses) 

Project  Description: 

Objectives:   Chemical  characterization  of  the  selenium  protein  required  for 
glycine  reduction  in  Clostridium  sticklandii;  purification  of  additional 
enzyme  and  electron  transport  components  required  for  the  overall  reaction. 

Major  Findings: 

The  fundamental  molecular  properties  of  the  selenium  protein  have  been 
determined  as  a  result  of  large  scale  purification  procedures  and  a  simplified 
extraction  assay  for  glycine  reductase  activity.   To  date,  studies  on  the 
identity  of  the  selenium  moiety  of  the  protein  appear  to  rule  out  seleno- 
methionine although  certain  biological  properties  indicate  the  chemical  form 
of  selenium  may  be  an  ether.   Although  the  precise  nature  of  the  selenium 
moiety  is  still  unknown,  sufficient  amounts  of  protein  and  chemical  procedures 
are  at  hand  to  pursue  structural  studies. 

Proposed  Course  of  Research: 

Structural  studies  on  the  selenium  moiety  of  protein  A  will  be  continued 
in  conjunction  with  the  effects  of  chemical  modification  on  the  biological 
activity  of  the  native  protein.   Alternatively  to  its  presumptive  function  as 
an  electron  carrier,  the  selenium  component  may  serve  as  a  group  carrier 
during  the  reductive  deamination  of  glycine  to  yield  acetic  acid. 

It  is  also  proposed  to  study  the  effects  of  inhibitors  of  protein  synthe- 
sis on  the  production  of    Se-labeled  protein  in  order  to  obtain  information 
on  whether  selenium  is  incorporated  into  the  protein  during  ribosomal  protein 
synthesis  or  whether  selenium  is  introduced  into  inactive  (but  pre-existing) 
protein  as  a  "post-translational"  modification. 


& 


Project  No.  Z01   HL  00207-01   LB 


Keyword  Descriptors; 

Glycine  reduction,  Clostridium  sticklandii,  selenium  protein,  chemical 
and  physical  characterization. 

Honors  and  Awards:   None 

Publications:   None 


S£T 


Project  No.  ZQ1   HL   00208-02   LB 

1.  Laboratory  of  Biochemistry 

2.  Section  on  Intermediary 

Metabolism  &  Bioenergetics 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Electron  Transport  System  Associated  with  Proline  Metabolism. 

Previous  Serial  Number:   NHLI-14 

Principal  Investigator:   Belinda  Seto 

Other  Investigators:   t.  C.  Stadtman 

Project  Description: 

Objectives:  (1)  To  purify  and  characterize  proline  reductase  in  Clostridium 
sticklandii.  (2)  To  determine  the  nature  of  the  electron  transfer  processes 
involved  in  the  reduction  of  proline. 

Major  Findings: 

(1)  Proline  reductase  has  been  purified  to  homogeneity  on  the  basis  of 
ultracentrifugation  and  gel  electrophoresis.   It  has  a  molecular  weight  of 
298,000  and  consists  of  subunits  of  61,500  and  50,500.   Spectral  studies  indi- 
cate that  it  contains  a  flavin  coenzyme.   Preliminary  data  also  suggested 
the  incorporation  of  Se-'-'  into  the  protein. 

(2)  NADH  can  be  used  as  an  electron  donor  for  crude  preparations  of 
proline  reductase.   However,  electrons  cannot  be  transferred  directly  from 
NADH  to  purified  proline  reductase.   Presently,  experiments  are  performed 
to  identify  the  electron  carrier (s)  involved  in  proline  reduction. 

(3)  As  a  cooperating  project  with  Thressa  C.  Stadtman,  antiserum  against 
purified  selenoprotein  (protein  A)  of  glycine  reductase  was  prepared.   The 
antigenic  specificity  of  the  antiserum  was  determined.   It  failed  to  cross 
react  with  native  proline  reductase  or  with  formate  dehydrogenase  (Methano- 
coccus  yannielii)  which  is  also  a  selenium-containing  protein.   The  anti- 
serum will  be  used  specifically  to  study  the  electron  transport  component 
(selenoprotein)  of  glycine  reductase. 

Keyword  Descriptors: 

Proline  reductase,  anaerobic  electron  transport,  f lavoprotein,  seleno- 
protein, reduced  pyridine  nucleotides,  specific  antisera. 

Honors  and  Awards:   None. 

Publications:   None. 

66 


Project  No.  Z01   HL   002Q9-Q5   LB 

1.  Laboratory  of  Biochemistry 

2.  Section  on  Intermediary 

Metabolism  &  Bioenergetics 

3.  Bethesda,  Maryland 

PHS-N1E 
individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Menadione-Dependent  p-nitrophenylphosphatase  of  Clostridium 
sticklandii 

Previous  Serial  Number:   NHL1-12 

Principal  Investigator:   J.  N.  Davis 

Other  Investigator:   T.  C.  Stadtman 

Cooperating  Units:   None 

Project  Description: 

Objectives:   1)   To  identify  the  natural  substrate  of  the  phosphatase. 

2)   To  determine  the  composition  and  some  physical  properties  of  the  enzyme 

protein. 

Major  Findings: 

1.  Titration  of  the  reduced  protein  with  the  sulphydryl  group  reagent 
5,5 '-dithiobis  (2-nitrobenzoic  acid)  indicated  that  four  sulphydryl  groups 
are  present  on  the  enzyme  protein. 

2.  A  simple  and  greatly  improved  method  of  isolation  of  the  menadione 
dependent  phosphatase  in  high  yield  was  devised.   This  made  use  of  the  obser- 
vation that  the  enzyme  forms  a  tight  complex  with  a  dye  known  as  Cibacron 
blue  F3GA  which  has  a  high  affinity  for  a  number  of  enzymes  involved  in  phos- 
phate metabolism.   When  dye  coupled  to  DEAE-cellulose  was  used  virtually 
pure  phosphatase  could  be  eluted  selectively  in  a  single  step  procedure  by 
the  appropriate  concentration  of  ammonium  sulfate. 

3.  Although  the  fluorescense  absorption  and  emission  spectra  of  the 
pure  protein  indicated  the  presence  of  tryptophan,  preliminary  results  on 
the  amino  acid  composition  following  hydrolysis  with  p-toluenesulfonic  acid 
(under  conditions  that  do  not  destroy  tryptophan)  failed  to  detect  this 
amino  acid. 


8T 


Project  No.  ZQ1   HL   Q02Q9-05   LB 


Proposed  Course  of  Action; 

1)  Additional  determinations  of  the  amino  acid  composition  of  the  phos- 
phatase will  be  made   (a)   following  carboxymethylation  and  hydrolysis  to 
determine  the  total  cysteine  content  and   (h)   following  hydrolysis  with 
methane  sulfonic  acid  to  establish  the  presence  or  absence  of  tryptophan. 

2)  Attempts  will  be  continued  to  identify  the  natural  phosphate  ester 
substrate  of  the  enzyme  by   (a)   preparation  of  a  naphthoquionol  monophos- 
phate ester  and   (b)   by  preparation  of  -^P-labeled  extracts  of  C_.  sticklandii 
which  can  be  tested  as  substrates.   The  possibility  that  the  phosphorylated 
intermediate  formed  in  the  glycine  reductase  system  may  serve  as  substrate 
will  be  tested.   The  selenoprotein  component  of  glycine  reductase  will  be 
phosphorylated  chemically  and  tested.   It  is  of  considerable  interest  to  know 
whether  this  quinone-dependent  phosphatase  normally  participates  in  a  transfer 
process  that  eventually  leads  to  ATP  synthesis,  whether  it  is  involved  in  some 
energy-dependent  membrane  transport   system  that  involves  a  quinone  catalyst 
or  is  a  component  of  a  regulatory  system. 

Keyword  Descriptors: 

Menadione,  quinone-dependent  phosphatase,  sulphydryl  enzyme,  p-nitrophenyl 
phosphatase,  Clostridium  sticklandii ,  Cibacron  blue-DEAE  cellulose  chroma- 
tography. 

Honors  and  Awards:   None. 

Publications:   None. 


Annual  Report  of  the 

Section  on  Protein  Chemistry 

Laboratory  of  Biochemistry 

National  Heart  and  Lung  Institute 

July  1,  1974  through  June  30,  1975 

Research  in  the  Section  on  Protein  Chemistry  consists  of  studies  on  the 
physical  and  chemical  properties  of  macromolecules  of  biological  interest  and 
on  the  roles  of  ligand  binding  and  protein-protein  interactions  in  enzyme 
catalysis  and  regulation. 

The  metal  ion  and  substrate  binding  properties  of  glutamine  synthetase 
from  E.  coli  are  being  studied  further.   A  calorimetric  investigation  of  the 
binding  of  L-glutamine  to  the  unadenylylated  Mn-enzyme  has  given: 
dH°  d  -10  kcal/mole  and  Kp  i  7  x  10-3  M  (in  the  absence  of  ADP)  and 
AH°  d  -6  kcal/mole  and  Kj,  ^  2  x  10"3  M  (in  the  presence  of  saturating  ADP). 
Microcalorimetric  measurements  are  being  used  to  obtain  information  on  protein 
binding  sites,  on  the  separateness  of  binding  sites  for  allosteric  effectors 
and  substrates,  on  proton  release  and  uptake,  and  on  kinetic  intermediates. 

The  ATP: glutamine  synthetase  adenylyltransf erase,  an  enzyme  involved  in 
the  adenylylation  and  deadenylylation  of  glutamine  synthetase  in  E.  coli,  has 
been  purified  2300-fold  and  partially  characterized.   Although  this  enzyme  is 
difficult  to  purify,  it  was  found  that  its  activity  could  be  stabilized  con- 
siderably with  phosphate-MgCl2  buffers.   Recent  studies  have  shown  that  the 
adenylyltransferase  is  a  single  polypeptide  chain  of  115,000  molecular  weight 
with  s2~   =  5.6  S.   Circular  dichroism  measurements  indicate  that  the  enzyme 
has  an  CH-helical  content  of  *°  267».   Amino  acid  analyses  show  116  arginine  + 
lysine,  248  glutamic  +  aspartic  acids,  8  cysteine  (no  disulfides),  15  trypto- 
phan, and  22  tyrosine  residues  per  mole.   The  intrinsic  tryptophanyl  residue 
fluorescence  of  adenylyltransferase  is  2-fold  greater  than  that  of  free  tryp- 
tophan; this  property  has  been  used  to  monitor  ligand-induced  conformational 
changes  in  the  enzyme.   Activators  of  the  adenylylation  reaction  (ATP, 
L-glutamine,  or  the  regulatory  Pjj  protein)  produced  an  enhancement  of 
fluorescence;  a-ketoglutarate ,  an  inhibitor  of  adenylylation  and  an  activator 
of  deadenylylation,  caused  a  net  fluorescence  decrease.   Studies  of  the  inter- 
action between  glutamine  synthetase,  adenylyltransferase,  and  the  regulatory 
Pjj  protein  are  in  progress. 

Studies  on  the  glutamyl-tRNA  synthetase  of  E.  coli  have  indicated  that 
this  enzyme  is  a  single  polypeptide  chain  of  ~"  63,000  molecular  weight.   The 
existence  of  a  reported  complex  between  the  catalytic  unit  and  a  regulatory 
protein  in  crude  extracts  could  not  be  demonstrated.   Nevertheless,  bovine 
serum  albumin  or  the  regulatory  P-r-r  protein  activate  the  enzyme  and  decrease 
the  K^  value  for  glutamate  2-fold.   These  effects  could  be  related  to  a  regu- 
lation of  glutamyl-tRNA  synthetase  activity  in  the  cell  through  a  loosely 
associated  complex  between  this  enzyme  and  another  protein  or  between  this 
enzyme  and  a  membrane  component. 


ef 


Project  No.  ZQ1   HL   00204-08   LB 
Laboratory  of  Biochemistry 
Section  on  Protein  Chemistry 
Bethesda,  Maryland 

PHS -NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Protein  Structure:  Enzyme  Action  and  Control 

Previous  Serial  Number:   NHLI-7 

Principal  Investigator:   Ann  Ginsburg 

Other  Investigators:   Carlos  E.  Caban 
Donald  M.  Powers 
Andrew  Shrake 

Cooperating  Units:   D.  Zoph,  National  Institute  of  Arthritis,  Metabolism  and 
Digestive  Diseases,  NIH 

Project  Description: 

Objectives:   1)  To  study  the  physical  and  chemical  properties  of  glutamine 
synthetase  from  Escherichia  coli,  particularly  with  respect  to  the  correlation 
of  the  structure  and  catalytic  function  of  this  enzyme.   2)  To  study  confor- 
mation and  stabilization  changes  of  a  protein  macromolecule  effected  through 
the  specific  binding  of  small  molecules,  and  the  relationship  of  such  effects 
to  enzyme  catalysis  and  regulation.   3)  Ultracentrifugation  studies  to  deter- 
mine macromolecular  properties  of  biologically  important  proteins.   4)  To 
purify  and  study  the  ATP-glutamine  synthetase  adenylyltransferase  from 
E.  coli,  with  emphasis  on  the  mechanism  of  action  and  the  physical  structure 
of  this  enzyme.   5)  To  isolate  the  glutamyl -tRNA  synthetase  from  E.  coli  in 
order  to  investigate  the  structure  and  regulation  of  this  enzyme. 

Major  Findings: 

1.   Mutual  interactions  of  divalent  cations  and  other  effectors  with 
glutamine  synthetase  from  E.  coli.   (Investigators:   A.  Shrake,  D.  M.  Powers, 
and  A.  Ginsburg).   This  study  is  an  extension  of  previous  investigations  on 
the  role  of  various  effectors  in  enzyme  catalysis.   A  newly  calibrated  LKB 
batch-type  microcalorimeter  has  been  used  to  obtain  the  enthalpy  and  free 
energy  for  the  binding  of  L-glutamine  to  unadenylylated  glutamine  synthetase 
in  the  presence  of  Mn^+  at  pH  7.2  (30°)  from  Scatchard  plots  of  the  heats  of 
binding  at  different  glutamine  concentrations.   With  and  without  10"^  M  ADP 
present,  respectively,  ZlH°  ^-6  and  ^-10  kcal/mole  whereas  KD  &2   and  ^7  mM. 
By  using  two  buffer  systems  with  quite  different  heats  of  protonation  in 
these  studies,  little,  if  any,  proton  uptake  was  found  to  occur  upon  the 
binding  of  glutamine  to  the  native  enzyme.   This  is  in  contrast  to  previous 
results  obtained  with  freshly  tightened  glutamine  synthetase,  (i.e.  enzyme 
from  which  Mn^+  had  been  removed  and  readded) .   A  conversion  of  the  tightened 

i  *r 


Project  No.  Z01   HL   00204-08   LB 

form  to  the  native  conformation  may  occur  slowly  and  can  be  investigated  by 
calorimetry . 

2.  Ultracentrifugation  studies.   (Investigator:   A.  Ginsburg.)   A  small 
amount  of  the  regulatory  Pjj  protein  (involved  in  adenylylation-deadenylyla- 
tion  of  glutamine  synthetase)  was  purified  by  Dr.  S.  P.  Adler  from  E.  coli. 
Weight  average  molecular  weights  of  41,700  t   2300  for  the  native  protein  and 
of  13,400  +  2000  for  the  protein  in  6.0  M  guanidine-HCl  were  determined. 
Preliminary  results  indicated  that  there  was  about  87o  dissociation  of  the 
tetramer  to  the  dimer  in  the  studies  with  the  native  protein  in  M/10  K-PO^ 
buffer  at  pH  7.1. 

Goat  Anti-LND-I  antibody  was  purified  by  Dr.  D.  Zoph  (NIAMDD)  to  immuno- 
logical and  electrophoretic  homogeneity  by  affinity  chromatography,  and 
dialyzed  vs  0.02  M  Na-PO^  -  0.1  M  NaCl  buffer  at  pH  7.4.   Sedimentation 
velocity  studies  gave  sSq  w  =  6.76  S  T  0.02S,  with  no  significant  concentra- 
tion dependence  from  3.2  -  0.8  mg/ml  protein  concentrations.   Sedimentation 
equilibrium  studies  indicated  a  weight  average  molecular  weight  (M^)  of 
158,000  t  6000  for  this  goat  /-globulin.   A  diffusion  coefficient  (D^q  w)  of 
3.8  x  10~7  cm2/sec  was  calculated.   The  purified  goat  Anti-LNF-II  antibody,  in 
contrast  to  the  LND-I  antibody,  shows  some  cooperativity  in  binding  hapten 
which  may  be  explored  further  in  ultracentrifugal  studies. 

3.  Studies  on  the  ATP: glutamine  synthetase  adenylyltrans ferase  from 
E.  coli.   (Investigators:   C.  E.  Caban  and  A.  Ginsburg.)   Regulation  of 
glutamine  synthetase  in  E.  coli  is  mediated  by  adenylylation  and  deadenylyla- 
tion,  a  covalent  modification  of  glutamine  synthetase  catalyzed  by  adenylyl- 
transferase  and  involving  also  a  small  regulatory  protein.   Heretofore, 
purified  adenylyltransferase  has  been  unstable  and  consequently  difficult  to 
characterize. 

A  new  purification  procedure  (with  Mg^+  included  in  all  chromatographic 
steps)  has  resulted  in  a  relatively  stable  enzyme  form.   In  K-PO4  (10  - 
100  mM,  pH  7.8)  containing  1  mM  MgCl0,  the  enzyme  was  stable  for  months  when 
stored  at  -80°  or  for  days  at  0-4°  at  concentrations  above  1  mg/ml.   At  very 
low  concentration  (8  ug/ml) ,  the  half-life  of  the  enzyme  was  192  hrs  at  0°  or 
46  hrs  at  25°  in  this  buffer.   The  enzyme  is  considerably  less  stable  in  Tris 
or  imidazole  buffers  with  or  without  added  MgC^.   In  studies  reported  last 
year,  the  homogeneity  of  the  enzyme  preparation  was  established  (s^q  w  =  5.6S; 
M^,  =  115,000  T  5000)  as  was  the  fact  that  the  enzyme  consists  of  a  single 
polypeptide  chain.   Circular  dichroism  studies  indicate  that  the  adenylyl- 
transferase has  an  a-helical  content  of  *>  267»  and  an  estimated  277o  p-pleated 
sheet  structure.   Amino  acid  analyses  show  a  high  arginine  and  lysine  content 
(116  residues/mole)  and  even  higher  levels  of  glutamic  and  aspartic  acids 
(248  residues/mole),  which  are  responsible  for  the  acidic  nature  of  the 
protein  (pi  =  4.98).   In  addition,  the  enzyme  contains  8  cysteine  (no  disul- 
fides), 15  tryptophan,  and  22  tyrosine  residues  per  mole.   The  intrinsic 
tryptophanyl  residue  fluorescence  of  adenylyltransferase,  which  is  2-fold 
greater  than  that  of  free  tryptophan,  was  used  to  monitor  ligand-induced 
conformational  changes  in  the  enzyme.   Activators  of  the  adenylylation 

2  ?5- 


Project  No.  Z01   HL  00204-08   LB 

reaction  (glutamine,  ATP,  or  the  regulatory  Pjj  protein,  which  itself  has  a 
very  low  fluorescence  yield)   produced  an  enhancement  of  fluorescence.   A  net 
fluorescence  decrease  was  caused  by  a-ketoglutarate,  which  is  an  inhibitor  of 
the  adenylylation  reaction  and  an  activator  of  deadenylylation. 

An  analog  of  L-glutamine,  2-chloroketone,  will  be  tested  for  a  possible 
covalent  modification  of  the  allosteric  activating  site  for  glutamine. 
Determination  of  the  stability  constant  and  stoichiometry  of  the  interaction 
between  adenylyltransferase  and  the  regulatory  Ptt  protein  will  be  attempted. 
From  previous  results,  we  suspect  that  a  limited  proteolysis  may  produce  an 
active,  low  molecular  weight  form  (^70,000  mol.  wt.)  with  a  loss  in  its 
capacity  to  be  stimulated  by  the  regulatory  protein.   This  and  other  pro- 
perties of  the  enzyme  currently  are  being  investigated  also. 

4.   Glutamyl-tRNA  synthetase  from  E.  coli.   (Principal  investigator: 
D.  M.  Powers.)   Glutamyl-tRNA  synthetase  (GluRS)  of  E.  coli  has  been  reported 
by  J.  Lapointe  and  D.  Soil  (J.  Biol.  Chem.  247:  4966,  1972)  to  exist  as  a 
102,000  MW  complex  consisting  of  a  catalytic  subunit  (56,000  MW)  and  a  regu- 
latory component  (46,000  MW) .   After  finding  that  purification  of  GluRS  led 
to  a  single  polypeptide  chain  of  **  63,000  mol.  wt.,  cell  extracts  were 
examined  directly  for  the  presence  of  a  regulatory  component.   For  this  pur- 
pose, two  techniques  were  used:   (a)  The  size  of  GluRS  was  determined  by 
electrophoresis  on  gels  containing  different  polyacrylamide  concentrations 
(5-107o)  by  comparison  with  appropriate  protein  standards.   (b)  Kinetic 
measurements  of  K^  values  for  glutamate  were  made,  since  the  presence  of  a 
regulatory  component  is  reported  to  decrease  this  K^  value  20-fold  to  *>  5  [M. 
The  following  strains  of  E.  coli  were  examined:   E.  coli  B,  K-12,  W,  and  K-12 
(CA-244),  a  gift  from  D.  Soil.   Growth  conditions  were  varied  from  a  defined 
medium  (glycerol  or  glucose  +  ammonia)  to  the  enriched  medium  of  Soil  (yeast 
extract  +  tryptophan) ,  and  cells  were  harvested  at  different  stages  of  growth. 
Cell  extracts  were  prepared  with  a  French  press  in  a  buffer  of  107o  glycerol, 
10  mM  Tris-HCl  (pH  8),  10  mM  MgCl2,  10  mM  NH^Cl,  and  20  mM  2-mercaptoethanol. 
Ribosomes  and  other  large  components  were  removed  by  high  speed  centrifugation 
or  by  partitioning  in  a  two  phase  aqueous  system.   When  cells  were  harvested 
at  different  stages  of  growth,  GluRS  activity  was  1.7-fold  lower  in  stationary 
than  in  exponential  growth  phase,  but  the  Kl  value  for  glutamate  remained 
constant  at  ^  50  uM.   In  all  cases  tested  on  polyacrylamide  gels,  only  one 
peak  of  GluRS  activity  was  found;  this  corresponded  to  a  size  of  ^63,000  mol. 
wt.   The  addition  of  divalent  cations  to  the  cell  extract  (Mg2+,  Mn^+,  Ca^+, 
or  Zn^+) ,  an  omission  of  glycerol  or  2-mercaptoethanol  from  the  ceil  beakage 
buffer,  or  an  addition  of  a  0.5  M  NH^Cl  ribosome  wash  had  no  effect  on  either 
the  GluRS  molecular  weight  or  the  Kjl  value  for  glutamate. 

GluRS  was  purified  to  homogeniety  from  E.  coli  B  grown  into  exponential 
growth  phase.   A  single  species  of  62,500  MW  was  observed  on  polyacrylamide 
gels  in  sodium  dodecyl  sulfate.   A  potential  interrelationship  between  GluRS 
and  the  glutamine  synthetase  regulatory  system  was  investigated.   The 
adenylyltransferase  from  E.  coli  and  regulatory  (P;q)  protein  from  E.  coli 
and  from  P.  putida  were  found  to  stimulate  GluRS  activity  2-fold  and  to 
decrease  the  K^  for  glutamate  2- fold.   However,  bovine  serum  albumin  at 

3  93 


Project  No.  Z01   HL   00204-08   LB 

equivalent  concentrations  had  a  similar  effect;  at  1  mg/ml  serum  albumin, 
GluRS  activity  was  stimulated  nearly  6-fold.   Since  all  three  proteins  have 
approximately  the  same  acidic  isoelectric  points,  this  effect  appears  to  be 
non-specific.   The  effects  of  these  acidic  proteins  on  GluRS  suggest,  however, 
that  the  conformation  of  the  enzyme  may  be  affected  by  protein-protein  or 
possibly  by  protein-membrane  interactions.   We  are  currently  isolating 
sufficient  quantities  of  GluRS  to  study  its  physical  and  chemical  properties 
and  to  explore  further  the  regulation  of  GluRS  through  protein-protein 
interactions . 

Significance  to  Bio-Medical  Research: 

The  regulation  and  control  of  enzymic  activities  rn  vivo  is  of  funda- 
mental importance  in  cellular  metabolism.   Through  studies  _in  vitro,  these 
processes  can  be  understood  more  fully.   The  study  of  structural  changes  that 
can  be  induced  in  a  protein  macromolecule  are  important  in  understanding 
cellular  processes  on  a  molecular  basis. 

Proposed  Course  of  Project: 

1)  To  study  conformational  and  stabilization  changes  of  a  protein 
macromolecule  effected  through  the  specific  binding  of  small  molecules,  and 
the  relationship  of  such  effects  to  enzyme  catalysis  and  regulation.   Ultra- 
centrifugation,  microcalorimetry,  spectral,  fluorescence,  equilibrium  binding, 
and  kinetic  techniques  will  be  used.   In  addition,  a  gel  method  of  zone 
transport  will  be  standardized  for  measuring  affinity  constants  of  interacting 
molecules . 

2)  To  study  mutual  interactions  of  divalent  cations  and  other  effectors 
with  glutamine  synthetase  from  E.  coli. 

3)  Physical  and  chemical  studies  of  the  E.  coli  ATP: glutamine  synthetase 
adenylyltransferase  will  be  continued. 

4)  To  purify  and  characterize  the  E.  coli  glutamyl -tRNA  synthetase; 
possible  mechanisms  for  regulating  this  activity  in  E.  coli  will  be  explored. 

Keyword  Descriptors: 

Protein  structure,  enzyme  catalysis  and  regulation,  microcalorimetry, 
ultracentrifugation,  conformation  and  stabilization  changes  in  proteins, 
protein-protein  interactions,  E.  coli  glutamine  synthetase,  E.  coli  ATP: 
glutamine  synthetase  adenylyltransferase,  E.  coli  glutamyl-tRNA  synthetase. 

Honors  and  Awards:   None 

Publications: 

1.   E.  R.  Stadtman  and  A.  Ginsburg:   The  Glutamine  Synthetase  of  Escherichia 
coli:  Structure  and  Control.   In  Boyer,  P.  D.  (Ed.):  The  Enzymes.   3rd  ed. 

4  I* 


Project  No.  Z01   HL   00204-08   LB 
New  York,  Academic  Press,  1974,  Vol.  X,  pp.  755-807. 

2.  A.  Ginsburg  and  E.  R.  Stadtman:   Glutamine  Synthetase  of  Escherichia  coli: 
Structure  and  Regulation.   In  Ebner,  K.  E.  (Ed.):  Subunit  Enzymes : 
Biochemistry  and  Function.   New  York,  M.  Dekker,  Inc.,  1975  (in  press). 

3.  J.  B.  Hunt,  P.  Z.  Smyrniotis,  A.  Ginsburg,  and  E.  R.  Stadtman:   Metal  Ion 
Requirement  by  Glutamine  Synthetase  of  Escherichia  coli  in  Catalysis  of 
/-Glutamyl  Transfer.   Arch.  Biochem.  Biophys.  166:   102-124,  1975. 


?r 


Annual  Report  of  the 
Laboratory  of  Cell  Biology 
National  Heart  and  Lung  Institute 
July  1,  1974  through  June  30,  1975 

The  Laboratory  of  Cell  Biology  was  formed  in  November,  1974  mostly  from  the 
Sections  on  Cellular  Physiology  and  Cellular  Biochemistry  and  Ultrastructure, 
Laboratory  of  Biochemistry.   Two  new  sections  were  formed:   Section  on  Memb- 
rane Biochemistry  and  Section  on  Organelle  Biochemistry.   The  research  of 
the  Laboratory  of  Cell  Biology  includes  the  biochemistry  of  muscle  contraction; 
the  chemistry  and  ultrastructure  of  cell  motility;  the  structure,  assembly 
and  function  of  microtubules;  the  structural  and  functional  interrelationships 
among  the  plasma  membrane  and  intracellular  membrane  systems  during  endo- 
cytosis;  the  mechanisms  of  electron  transport  and  energy  trandsuction;  multi- 
enzyme  complexes  involved  in  DNA  synthesis;  the  structure  and  conformation 
of  proteins. 

Muscle  Biochemistry:  The  repeating  contractile  unit  of  skeletal  muscle  cons- 
ists of  thin  actin  filaments  attached  to  two  Z-lines,  that  define  the  sar- 
comere, and  thick  myosin  filaments  that  lie  between  the  actin  filaments.  The 
cyclical  interaction  of  the  actin  and  myosin  activates  the  Mg++-ATPase  acti- 
vity of  the  myosin.   In  the  presence  of  ATP,  the  myosin  undergoes  a  conforma- 
tional change  pulling  the  actin  filaments  and  attached  Z-lines  towards  each 
other  (contraction)  as  the  myosin  cyclically  binds  to  and  releases  from  the 
actin.   During  active  contraction,  only  a  small  portion  of  the  myosin  molecu- 
les are  attached  to  the  actin  filaments.   Four  other  proteins,  troponin  I, 
C  and  T  and  tropomyosin  are  associated  with  the  actin  filaments  and  regulate 
the  system  by  making  it  dependent  on  the  presence  of  Ca   ,  in  addition  to 
Mg   .   The  detailed  molecular  events  of  this  morphological  contractile  cycle 
are  still  incompletely  understood.   They  cannot  be  studied  in  intact  muscle 
or  with  pure  actin  and  myosin  because  of  the  insolubility  of  myosin  and 
actomyosin.   In  the  last  few  years  considerable  progress  has  been  made  in 
this  Laboratory  by  studying  the  model  system  in  which  myosin  is  replaced  by 
its  proteolytic  fragments  heavy  meromyosin  (HMM)  or  subfragment-1  (S-l), 
soluble  derivatives  with  full  enzymatic  activity. 

It  was  previously  found  by  analytical  ultracentrifugation  that  under  condi- 
tions of  excess  actin  and  ATP,  and  therefore  maximum  ATPase  activity,  most 
of  the  HMM  was  not  bound  to  the  actin.   These  paradoxical  data  were  best  ex- 
plained by  hypothesizing  a  refractory  state  of  myosin  that  could  not  bind  to 
actin  in  the  presence  of  ATP  and  a  slow,  rate-determining  step  for  the  conver- 
sion of  refractory  myosin  to  non-refractory  myosin  that  could  bind  to  actin 
in  the  presence  of  ATP. 

In  the  last  year  the  existence  of  two  states  of  HMM  were  confirmed  and  exten- 
ded to  S-l  by  laser-light  scattering,  turbidity  and  fluorescence  measurements 
of  the  interaction  of  HMM  and  S-l  with  actin  in  the  presence  and  absence  of 
ATP.   It  has  also  been  shown  that  refractory  HMM  and  S-l  are  normal  molecules, 
only  transiently  in  the  refractory  state,  by  re-isolating  them  and  showing 
they  have  normal  EDTA  and  act  in- activated  Mg"*-1"- ATPase.   These  experiments 
under  steady  state  conditions  have  been  supplemented  by  studying  the  inter- 
action of  actin,  S-l  and  ATP  by  measuring  light  scattering  changes  in  a  stop- 


<?7 


flow  apparatus  during  a  single  catalytic  cycle  in  which  one  molecule  of  ATP 
is  hydrolyzed  per  molecule  of  S-l.   The  rate  of  re-binding  of  S-l  to  actin 
is  found  to  be  equal  to  the  steady  state  ATPase  rate  (measured  with  excess 
ATP),  to  be  10  times  slower  than  the  rate  of  dissociation  of  S-l  from  actin 
and  to  become  constant  at  high  levels  of  actin.   These  data  confirm  the 
existence  of  a  slow,  rate-determining  conformational  change  of  S-l  (refract- 
ory to  non-refractory  state)  required  for  its  binding  to  actin  in  the  pre- 
sence of  ATP. 

When  one  sulfhydryl  group  of  HMM  is  blocked  with  N-ethylmaleimide  its  ATPase 
activity  is  activated  only  3-fold  by  actin,  instead  of  the  usual  200-fold. 
Since  under  conditions  of  maximal  ATPase  activity  only  a  small  portion  of 
the  NEM-HMM  is  bound  to  actin  (as  previously  found  for  HMM) ,  it  was  proposed 
that  NEM-HMM  also  underwent  a  conversion  from  refractory  to  non-refractory 
state.   This  hypothesis  has  now  been  supported  by  demonstrating  that  the  NEM- 
HMM  that  is  not  bound  to  actin  is  indistinguishable  from  the  actin-bound  NEM- 
HMM  and  that  the  formation  of  the  non-refractory  NEM-HMM  is  the  rate-deter- 
mining step  in  the  hydrolysis  of  ATP  by  actin-activated  NEM-HMM. 

Further  details  of  the  catalytic  cycle  have  been  revealed  by  comparing  the 
events  occurring  when  only  molecule  of  ATP  is  added  per  molecule  of  HMM  in 
a  stop-flow  apparatus  (pre-steady  state  kinetics)  to  the  usual  events  when 
unlimited  ATP  is  present  (steady  state  kinetics) .   It  had  been  found  last 
year  that  ATP  is  bound  essentially  irreversibly  to  the  active  site  of  HMM 
with  the  rapid  exponential  release  of  0.4  IT  per  bound  ATP  and  an  induced 
conformational  change  in  HMM  that  could  be  measured  by  a  change  in  intrinsic 
tryptophan  fluorescence.   There  follows  a  slower  exponential  release  of 
0.6H+  per  ATP  which  equals  the  catalytic  rate  of  hydrolysis  under  steady 
state  conditions.   The  following  scheme  was  proposed: 

kj^            k2       j,     Kcat 
myosin  +  ATP     N  myosin-ATP ^myosin" -ATP ^myosin  +  ADP  +  0.6H 

k  -,  + 

"i  0.4H 

The  postulated  two  step  interaction  between  myosin  and  ATP  has  now  been  sup- 
ported by  showing  that  lowering  the  ionic  strength  decreases  the  equilibrium 
constant  for  binding  of  myosin  and  ATP  and  that  lowering  the  temperature 
decreases  the  rate  of  conformational  change  (k2)  and  increases  the  associa- 
tion of  myosin  and  ATP.   When  actin  is  present,  ATP  dissociates  actin-HMM 
(measured  by  turbidity  decrease)  more  rapidly  that  the  induced  change  in 
fluorescence  caused  by  ATP  binding  to  HMM.   The  interaction  of  ATP  and  HMM 
is  facilitated  by  the  presence  of  actin  and  the  rate  of  decay  of  the  HMM-ATP 
complex  (fluorescence  decay)  equals  the  rate  of  formation  of  actin-HMM 
(turbidity  increase) . 

Thus,  evidence  has  been  obtained  for  the  occurrence  of  molecular  events  in 
vitro  that  are  counterparts  of  the  mechanical  events  _in  vivo.   The  dissocia- 
tion of  actin-HMM  by  ATP  is  coincident  with  the  formation  of  an  HMM-ATP  com- 
plex which  is  associated  with  a  conformational  change  in  the  HMM  protein. 
Hydrolysis  of  the  ATP  allows  reformation  of  the  actin-HMM  complex. 


n 


Cytoplasmic  Actin  and  Myosin:   Many  types  of  cell  motility  are  based  on  cyto- 
plasmic actins  and  myosins,  proteins  very  similar  to,  but  different  from, 
their  muscle  counterparts.   Several  years  ago  we  characterized  these  proteins 
in  Acanthamoeba  castellanii  and  now  we  are  re- investigating  their  properties 
in  detail.   Previous  efforts  were  hampered  by  the  very  poor  yields  of  cyto- 
plasmic actin  from  all  systems.   New  procedures  have  been  developed  for  the 
rapid  isolation  of  Acanthamoeba  actin  in  high  yield  and  purity  so  that  it 
should  now  be  possible  to  characterize  the  protein  fully. 

Acanthamoeba  myosin  is  unique  among  all  known  myosins  in  having  a  much  lower 
molecular  weight  (180,000  vs  about  420,000)  and  in  its  requirement  for  a  co- 
factor  protein  for  the  actin-activation  of  its  Mg++-ATPase.   Cof actor  and 
myosin  are  present  in  only  very  small  amounts  in  Acanthamoeba  and  their  puri- 
fication in  quantities  sufficient  for  the  desired  studies  is  difficult. 
Recent  experiments  indicate  that  cofactor  and  Acanthamoeba  myosin  can  be  sep- 
arated from  each  other,  and  from  actin,  on  ATP-agarose  columns.   A  preparative 
procedure  may  be  developed  based  on  these  observations. 

Microfilaments  and  Endocytosis:   One  motility  system  thought  to  involve  cyto- 
plasmic actin  and  myosin  is  phagocytosis.  Scanning  electron  micrographs  show 
that  particles  to  be  phagocytosed  by  Acanthamoeba  initially  bind  to  the 
acanthopods  (small  filopodia  containing  bundles  of  actin  filaments)  and  time- 
lapse  motion  pictures  show  that  phagocytosis  occurs  within  about  60  seconds 
of  initial  contact  of  the  particle  with  the  cell.   Electron  microscopy  of 
thin-sections  shows  a  marked  accumulation  of  cytoplasmic  actin  filaments 
perpendicular  to  the  limited  regions  of  plasma  membrane  in  contact  with  the 
particle  to  be  ingested.   As  phagocytosis  continues  the  filaments  form  a 
thick  rim,  lying  parallel  to  the  membrane,  around  the  forming  phagosome. There 
are  no  actin  filaments  associated  with  the  internalized  phagosome  membrane. 
This  rapid  dissociation  of  filamentous  actin  is  one  of  the  apparent  differ- 
ences between  cytoplasmic  actin  and  muscle  actin  that  needs  to  be  studied 
with  pure  cytoplasmic  actin.  Within  the  cell  the  movement  of  phagosomes 
seems  to  be  randomly  controlled  by  cytoplasmic  streaming. 

Composition  of  Acanthamoeba  plasma  membrane  and  phagosome  membrane:   Previous 
work  in  this  Laboratory  had  shown  that  the  Acanthamoeba  plasma  membrane  con- 
sists of  about  one-third  each  of  protein,  lipid  (phospholipid  +  sterol)  and 
a  novel  polymeric  glycosphingolipid,  lipophosphonoglycan.   The  proteins  were 
shown  to  consist  mostly  of  a  15,000  dalton  polypeptide.  Work  this  year  has 
suggested  that  the  15,000  dalton  polypeptide  may  be  associated  specifically 
with  plasma  membranes  isolated  from  stationary  phase  or  encysting  cells  and 
may  not  be  a  major  component  of  the  membranes  of  rapidly  growing  amoebae. 
Phagosome  membranes  are,  as  discussed  above,  derived  from  the  plasma  membrane 
but  then  fuse  with  the  membranes  of  intracellular  vesicles.   Isolated  phago- 
some membranes  have  now  been  found  to  have  protein/phospholipid  ratio  about 
four  times  greater  than  the  ratio  for  plasma  membranes.   This  suggests  that 
there  are  significant  changes  in  the  plasma  membrane  after  it  is  internalized 
as  a  phagosome  membrane.   Dodecyl  sulfate  gel  electrophoresis  confirms  the 
absence  of  actin  in  the  phagosome  membrane  preparations  (in  contrast  to  its 
co-isolation  with  plasma  membranes)  and  little,  if  any,  of  the  15,000  dalton 
polypeptide  is  present. 


*f 


Membrane  Fusion:   During  normal  growth  the  cell  surface  of  Acanthamoeba  is 
internalized  about  10-50  times/hour  in  the  process  of  pinocytosis.   Morpho- 
metric  measurements  of  cells  ingesting  yeast  support  the  hypothesis  that 
intracellular  membranes  move  to  the  surface  at  the  same  rate.   The  surface 
area  of  the  cell  remains  constant  during  active  phagocytosis  but  the  surface 
area  of  large  cytoplasmic  vacuoles  decreases  in  an  amount  equivalent  to  the 
plasma  membrane  internalized  as  phagosome  membranes.   It  seems  probable  that 
the  vacuole  membranes  lost  from  the  cell's  interior  fuse  with  the  plasma 
membrane.   Analysis  is  complicated  by  the  many  fusions  that  occur  between 
phagosomes  and  intracellular  vesicles  and  by  the  probable  changes  in  membrane 
proteins  discussed  above. 

Despite  the  fact  that  the  plasma  membrane  contains  many  enzymes  of  phospho- 
lipid metabolism,  evidence  has  not  been  found  for  their  function  in  the  mol- 
ecular events  of  membrane  fusion.   We  have  now  shown  that  phospholipid  bi- 
layer  vesicles  fuse  with  the  plasma  membrane  of  viable  Acanthamoeba  under 
conditions  where  enzymatic  catalysis  is  unlikely  to  be  involved.   When  such 
fusion  occurs  the  contents  of  the  internal  aqueous  space  of  the  phospholipid 
vesicle  are  introduced  into  the  cytoplasm  of  the  cell.   These  experiments 
therefore  provide  a  basis  for  introducing  otherwise  impermeable  molecules 
into  the  cell's  cytoplasm.   Endocytosis  of  phospholipid  vesicles  can  also 
occur,  under  other  conditions,  as  an  alternate  mechanism  of  uptake  and,  in 
this  case,  the  vesicle  and  its  contents  are  introduced  into  the  lysosomal 
system  of  the  cell. 

Microtubule  Assembly  and  Function:  Cytoplasmic,  flagellar  and  ciliary  micro- 
tubules are  the  basis  for  different  types  of  cell  motility.   Just  as  cyto- 
plasmic microfilaments  are  a  polymeric  form  of  globular  actin,  so  micro- 
tubules are  cylinders  consisting  of  13  protofilaments  each  of  which  is  formed 
by  the  polymerization  of <Xand  0  -tubulin  subunits  (55,000  daltons) .   Poly- 
merization and  depolymerization  of  cytoplasmic  microtubules  is  regulated  by 
unknown  control  mechanisms.   Depolymerization  can  be  induced  in  vitro  by  low 
temperature,  high  ionic  strength,  colchicine  or  high  Ca++,  none  of  which  can 
reasonably  be  invoked  for  the  jLn  vivo  phenomenon.   Research  in  this  Labora- 
tory is  focussed  on  three  possible  regulatory  mechanisms  (1)  a  cyclic  AMP- 
stimulated  phosphorylation  of  a  single  serine  residue  in  ^-tubulin,  (2)  the 
binding  of  guanine  nucleotides,  (3)  the  specific  enzymatic  addition  of  tyro- 
sine to  the  COOH-terminus  of  o^.- tubulin. 

Partially  purified  tubulin  from  brain  is  tyrosylated  by  free  tyrosine  in  the 
presence  of  ATP,  Mg++,  and  KC1.   Highly  purified  tubulin  is  a  receptor  for 
tyrosyl  groups  but  only  in  the  presence  of  a  partially  purified  enzyme  from 
bovine  brain.   Preliminary  results  suggest  that  tubulin  need  not  be  fully 
tyrosylated  to  polymerize  in  vitro. 

The  apparent  requirement  for  bound  guanosine  nucleotides  for  polymerization 
of  tubulin  may  be  more  complex  than  previously  thought.   Ca    (ImM)  inhibits 
tubulin  polymerization  but  not  binding  of  nucleotides  or  transphosphorylation 
of  phosphate  from  free  to  bound  nucleotides.   Contrary  to  what  was  previously 
believed,  ATP  cannot  be  used  to  study  the  transfer  of  phosphate  from  free  to 
bound  nucleotides  because  ATP  also  phosphorylates  tubulin  serine  residues. 


fta 


GTP,  however,  specifically  undergoes  transphosphorylation  and  does  not  phos- 
phorylate  serine  residues.   Recent  studies  indicate  that  only  half  of  the 
tubulin  preparations  that  polymerize  are  substrates  for  the  GTP- transphos- 
phorylation reaction.   It  is  not  clear,  therefore,  whether,  as  previously 
supposed,  phosphorylation  of  tubulin-bound  GDP  to  bound  GTP  is  a  requisite 
for  polymerization. 

Flagellar  microtubules  are  organized  into  a  structure  consisting  of  9  outer 
doublet  microtubules  surrounding  a  central  pair  of  single  microtubules.  The 
9  outer  doublets  are  connected  to  each  other  by  "arms"  and  to  the  central 
pair  by  radial  spokes.   Sliding  of  the  filaments  is  thought  to  be  induced 
by  an  ATPase,  dynein,  that  is  a  component  of  the  arms.   In  addition  to  dynein, 
previous  work  in  this  Laboratory  has  led  to  the  discovery  of  a  low  molecular 
weight  Ca-ATPase  in  Chlamydomonas  flagellae.   The  function  of  this  enzyme, 
of  dynein-ATPase  and  of  other  flagellar  enzymes  are  being  investigated. 
Results  to  date  are  as  follows.   (1)  A  mutant  has  been  isolated  which  results 
in  paralyzed  flagellae  in  Chlamydomonas  and  in  which  the  low  molecular  weight 
Ca++-ATPase  and  the  central  pair  of  microtubules  are  missing.   (2)  Evidence 
has  been  found  that  certain  preparations  of  ATP  contain  a  specific  inhibitor 
of  dynein  ATPase  since  with  one  commercial  preparation  of  ATP  the  Ca++-ATPase 
of  dynein  is  normal  but  its  Mg+^-ATPase  is  907»  inhibited.  The  nature  of  this 
inhibition  is  under  study.  (3)  In  addition  to  the  dynein-ATPase  and  the  low 
molecular  weight  Ca++-ATPase,  Chlamydomanas  flagellae  have  been  shown  to  con- 
tain an  adenylate  kinase  and  nucleoside  diphosphokinase  of  unknown  function 
in  flagellar  movement. 

Electron  Transport  in  E.  coli  and  Mitochondria: Energy  transduction  occurs  in 
the  inner  mitochondrial  membrane  of  eukaryotic  cells  and  in  the  plasma  membr- 
ane of  bacteria.   In  general  terms,  electrons  from  NADH  are  transferred  through 
a  series  of  membrane -bound  intermediates  to  O2,  the  terminal  acceptor,  which 
is  reduced  to  water.  Each  of  the  intermediates  has  a  characteristic  oxidation- 
reduction  potential  (midpoint  redox  potential)  where  507o  of  the  molecules  are 
reduced  and  50%  are  oxidized.  The  oxidation  of  each  intermediate  can  be  follo- 
wed by  measuring  the  increase  in  absorption  at  a  wavelength  characteristic  of 
that  intermediate.  By  these  titration  curves  of  potential  versus  absorption 
spectra,  the  number  of  intermediates  in  the  chain  and  their  sequence  can  be 
determined.  At  several  points  in  the  passage  of  electrons  down  this  electro- 
potential  gradient  the  energy  released  is  converted  to  a  form  used  by  the  cell, 
usually,   if  not  always,  ATP. 

Data  from  the  laboratory  of  Britten  Chance  suggested  the  presence  in  non-ener- 
gized mitochondria  of  two  forms  of  cytochrome  b  with  redox  potentials  of  -30 
and  +65  mV.   When  mitochondria  were  energized  by  ATP  two  forms  were  detected 
with  redox  potentials  of  +260  and  +65  mV.   It  was  suggested  that  the  conver- 
sion of  a  cytochrome  b  from  a  molecule  with  redox  potential  of  -30  mV  to  a 
potential  of  +260  mV  might  be  the  long- sought  high  energy  intermediate. 

Similar  experiments  in  this  Laboratory  with  E.  coli  membranes  (non-energized) 
revealed  three  forms  of  cytochrome  b-^  (redox  potentials  of  -50,  +110  and  +220 
mV)  but  none  of  the  mid-point  potentials  changes  when  the  membranes  are  ener- 
gized by  ATP.  This  observation  led  to  a  re-investigation  of  the  mitochondria 
system.  It  was  found  that  in  non-energized  mitochondria,  in  the  range  of 
+110  to  +350  mV,  there  may  be  an  anomalous  change  in  absorption  of  cytochrome 

5  tOl 


b  in  a  direction  opposite   to  that  to  be  expected.   The  titration  data  are, 
therefore,  subject  to  the  re-interpretation,  by  computer  modeling,  that  in 
mitochondria,  as  in  E.  coli,  there  are  three,  not  two,  forms  of  cytochrome  b. 
Because  of  the  optical  anomaly  in  non-energized  mitochondria  one  of  these  may 
be  undetected.   Upon  energizing  the  system  with  ATP  the  undetected  cytochrome 
b  may  undergo  a  spectral  shift,  rather  than  the  previously  proposed  change  in 
redox  potential,  and  is  then  detected.   Whether  this  proposed  ATP-induced 
spectral  shift  represents  a  "high-energy"  intermediate  remains  to  be  determined. 

DNA  Synthesis  in  E.  coli:  Several  lines  of  evidence  suggest  that  DNA  synthesis 
may  occur  in  membrane-associated  enzyme  complexes.  A  search  for  such  a  system 
has  led  to  the  partial  purification  of  an  enzyme  complex  (not  membrane-assoc- 
iated), with  an  apparent  "molecular"  weight  of  390,000,  that  synthesizes  DNA, 
is  stimulated  by  ATP  and  prefers  native  to  heat-denatured  DNA  as  primer.  Alth- 
ough the  complex  contains  polymerase  I  (Kornberg's  enzyme)  it  differs  in  its 
activity  from  pure  polymerase  I  in  its  ATP-stimulation  and  its  preference  for 
native  DNA.  The  complex  also  contains  the  recBC  enzyme  known  to  have  both  ATP- 
dependent  DNase  activity  and  DNA-dependent  ATPase  activity.  Complexes  isolated 
from  recBC  mutants  that  lack  the  DNase  activity  but  retain  the  ATPase  activity 
still  show  ATP- stimulated  DNA  synthesis  in  70  mM  KC1.  Thus,  it  seems  that  the 
ATPase  activity  but  not  the  DNase  activity  of  the  recBC  enzyme  may  be  required. 
Complexes  from  these  mutants  differ  from  complexes  from  wild-type  cells,  how- 
ever, in  being  inhibited  by  ATP  in  150  mM  KC1,  under  which  conditions  the  wild- 
type  complex  still  shows  ATP-stimulation  suggesting  that  the  apparent  complex 
between  recBC  and  polymerase  I  may  be  less  stable  in  the  mutant  than  in  the 
wild-type  cells. 

Structure  of  Fibrinogen;   Fibrinogen  is  the  circulating  protein  in  the  blood 
plasma  that  is  converted  to  fibrin  by  selective  removal  of  a  few  amino  acids 
by  the  specific  protease  thrombin.  Fibrinogen  (MW=335,000)  consists  of  six 
polypeptide  chains,  two  each  of  chains  designated  o^^and  ^T  Electron  micros- 
copy and  thermodynamic  data  suggested  that  these  polypeptides  were  arranged 
in  two  sets  ofo^ftandj^chains  in  a  symmetrical  molecule  consisting  of  a  central 
globular  region  (E) ,  containing  all  six  chains,  and  two  identical  "satellite" 
globular  regions  (D) ,  each  containing  one  set  of  three  chains  branching  from 
the  central  region.   This  model  has  now  been  given  strong  support  by  quantita- 
tive kinetic  analysis  of  the  fragments  produced  by  controlled  trypsin  digestion 
of  native  fibrinogen.  To  construct  the  proper  model  it  was  necessary  to  sep- 
arate the  fragments  produced  at  different  stages  of  proteolysis  by  Sephadex 
chromatography  or  sodium  dodecyl  sulfate  electrophoresis  and  determine  their 
absolute  yields  and  molecular  weights  in  order  to  account  for  all  of  the  fibr- 
inogen molecule  in  the  products.   In  the  earliest  stages  of  trypsin  digestion 
a  major  fragment  X  is  formed  containing  the  major  globular  regions  of  D  and  E 
still  intact  but  with  a  loss  of  a  number  of  small  peptides  from  those  portions 
of  the  six  polypeptide  chains  that  extend  beyond  the  globular  regions  of  D. 
Further  digestion  produces  two  major  fragments:  one  D  (MW=85,000)  and  Y  (MW= 
134,000)  which  consists  of  a  second  D  still  linked  to  E.  Continued  proteolysis 
splits  Y  into  D  and  E  (MW=^7,000)  so  that,  finally,  two  D  and  one  E  are  formed. 
This,  2D+E=1 70, 000+47, 000=21 7, 000  with  the  small  peptide  fragments  accounting 
for  the  remainder  of  the  original  mass  of  fibrinogen  (335 ,  000) .  This  structure 
is  supported  by  calorimetry.  The  thermal  transitions  at  61°  and  100°  of  separ- 
ated D  and  E  are  the  same  as  those  of  fibrinogen  indicating  physical  independ- 
ence of  the  covalently-linked  subunits  in  the  native  molecule. 


fta. 


Project  No.  Z01  HL  00401-09  LCB 

1.  Laboratory  of  Cell  Biology 

2 .  Membrane  Enzymology 

3.  Bethesda,  Md.  20014 


Project  Title: 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Electron  transport  in  E.  coli  and  rat  liver  mitochon- 
dria. 


Previous  Serial  No.:  NHLI-16 

Principal  Investigator:  Richard  W.  Hendler 

Other  Investigators:  None 
Cooperating  Units: 


Electrical  and  Electronic  Engineering  Section  of  the 
Biomedical  Engineering  and  Instrumentation  Branch 
in  the  Division  of  Research  Services. 


Project  Description: 

A.   Redox  potentials  of  b-type  cytochromes  of  E.  coli  and  rat  liver  mito- 
chondria. 

We  have  recently  shown  that  E.  coli  possesses  3  species  of  cyt  b^  having 
midpoint  reduction  potentials  about  -50,  +110,  and  +220mV.   Because  it  has 
been  proposed  that  b-type  cytochromes  of  mammalian  systems  can  participate 
in  energy  transduction  and  ATP  formation,  we  tested  whether  the  redox 
properties  of  any  of  the  three  was  changed  by  the  addition  of  ATP  or  the 
uncoupler  dinitrophenol.   Because  reactions  that  phosphorylate  ADP  are 
generally  sensitive  to  (ATP)/ (ADP)  (P)  concentration  ratios,  parallel  experi- 
ments were  performed  in  phosphate-containing  and  phosphate- free  buffers.   In 
no  case,  was  any  evidence  found  for  energy-dependent  changes  of  redox 
potential.   Having  failed  to  confirm  an  energy-dependent  character  for  redox 
potentials  of  b-type  cytochromes  in  E.  coli,  we  decided  to  study  a  mammalian 
system  more  closely.   We  found  that  the  apparent  energy-dependent  change  of 
redox  potential  of  one  of  the  cytochromes  b  in  rat  liver  mitochondria 
depended  on  the  assumption  that  only  two  species  of  this  cytochrome  were 
present.   The  data,  however,  were  much  better  fit  to  a  three  component  system. 
In  this  case  there  was  no  longer  any  evidence  for  the  change  of  a  low  redox 
potential  species  to  a  high  potential  form.   The  same  two  species  present 
in  non-energized  mitochondria  (i.e.  -30  and  +65mV)  were  present  in  energized 
mitochondria.   The  energized  mitochondria  had  a  third  species  with  a 
potential  of  about  +260mV.   The  non-energized  mitochondria  displayed 
anomalous  optical  behavior  in  the  voltage  ran  gs  where  the  high  potential 
species  would  be  revealed  (i.e.  +110  to  +350mV) .   The  extent  of  cytochrome 
reduction  is  monitored  by  the  difference  in  optical  absorption  between  a 
peak  and  a  reference  wavelength.   This  ^.O.D.  normally  increases  upon  chemical 
reduction  (i.e.  a  lowering  of  solution  potential).   The  anomalous  response 


A>3 


Project  No.  Z01  HL  00401-09  LCB 

was  a  decrease  in  AO.D.  accompanying  a  lowering  of  solution  potential. 
Therefore,  instead  of  finding  an  energy-dependent  change  in  redox  potential, 
we  observed  an  energy  dependent  optical  anomaly.   We  believe  that  the 
phenomenon  may  be  due  to  a  shift  in  absorption  spectrum  so  that  the  original 
peak  wavelength,  subsequently  represents  a  lower  optical  density  relative 
to  the  reference  wavelength  optical  density.   Experiments  to  test  this 
explanation  are  further  discussed  below  (parts  C  and  D) . 

B.  Determination  of  whether  three  chromatographically  separated  E.  coli 
fractions  containing  cytochrome  b-^, represent  three  different  redox  potential 
forms  of  cytochrome  bi. 

We  have  previously  separated  the  E.  coli  respiratory  chain  into  various 
fractions  containing  different  dehydrogenases  and/or  cytochromes.   Three  of 
these  contain  cytochrome  b]_;  one  complexed  with  succinate  dehydrogenase 
(D.E.  succ)  ,  one  uncomplexed  (DE-Fe-2)  and  one  associated  with  cytochrome 
oxidase  (CO.).   It  was  found  that  although  both  "DE  succ"  and"DE-Fe2"  were 
markedly  enriched  with  the  lowest  potential  species,  relative  to  the  other 
two,  all  three  species  were  present  and  there  was  no  clear-cut  distinction 
between  the  two  fractions.   "CO."  contained  all  three  species  of  cytbi 
with  a  relatively  high  amount  of  the  highest  potential  species. 

C.  Redox  characteristics  of  E.  coli  cytochrome  oxidase  (cyt  d) . 

Cytochrome  d  shows  a  very  pronounced  optical  anomaly  in  the  voltage  region 
from  50  to  200mV.   Just  as  was  seen  with  rat  liver  mitochondrial  cytochrome 
b,  the  ^O.D.  decreased  with  lower  solution  potentials.   A  voltage-  (or 
energy-?)  dependent  shift  in  absorption  spectrum  could  be  responsible  for 
the  phenomenon.   To  test  this  idea,  a  series  of  absolute  spectra  were  taken 
at  different  voltages.   It  was  found  that  the  absorption  peak  used  for 
cytochrome  d  did  shift  as  a  function  of  the  oxidizing  potential.   The  nature 
of  the  shift  was  such  that  it  did  qualitatively  account  for  the  observed 
optical  anomaly.   A  problem  preventing  the  obtaining  of  accurate  quantita- 
tive data  is  that  the  oxidizing  potential  of  the  cuvette-contents  continually 
changes  so  that  optical  scans  at  fixed  voltages  cannot  be  obtained.   In 
principal  this  kind  of  analysis  could  be  applied  to  the  energy-dependent 
optical  anomaly  of  the  rat  liver  mitochondria  system.   However,  the 
spontaneous  voltage  drift  in  that  system  is  too  great.   Another  limitation 
found  in  the  potentiometric  analysis  of  cytochrome  d  was  that  the  highest 
oxidizing  potentials  that  we  have  been  able  to  obtain  with  chemical  oxidants 
(K3Fe(CN)5  and  KMn04)  are  less  than  that  normally  maintained  in  air  saturated 
solutions.   Because  of  this,  we  have  not  been  able  to  analyze  an  apparent 
very  high  potential  component  of  cyt  d. 

D.  Development  of  automatic  and  controlled  voltage  potentiometry. 

Because  of  1)  The  inability  to  obtain  spectral  scans  at  fixed  voltages. 
2)  The  inability  to  obtain  and  hold  oxidation  potentials  above  600mV. 


/c4 


Project  No.  Z01  HL  00401-09  LCB 

and  3)  because  of  the  cumbersome  and  laborious  nature  of  manually  performed 
potentiometric  titrations,  efforts  were  undertaken  to  improve  the  experimental 
techniques.   Initial  steps  were  directed  towards  developing  a  system  using 
electrically  controlled  digital  burette  delivery  systems  to  introduce 
chemical  oxidants  and  reductants  at  rates  designed  to  hold  a  fixed  potential 
or  to  maintain  a  fixed  rate  of  change.   Recently,  a  radically  new  approach 
was  thought  of  and  adopted.   Instead  of  introducing  chemical  oxidants  and 
reductants,  these  agents  are  generated  in  situ  electrically  from  a  second 
set  of  electrodes.   The  generating  electrode  is  in  direct  contact  with  the 
suspension  of  respiratory  components, and  the  reaction  products  of  its 
auxiliary  electrode  are  isolated  from  the  vessel  by  virtue  of  their 
insolubility  (i.e.  Ag  or  AgCl)  and  by  a  sleeve  of  KCl-AgCl-Agar.   We  have 
found  that  such  a  system  can  generate  oxidizing  potentials  as  high  and  as 
low  as  desired.   Preliminary  experiments  have  shown  that  fixed  solution 
voltages  can  be  attained  and  maintained  as  well  as  programmed  rates  of 
change  of  voltage.   Problems  that  have  not  yet  been  resolved  deal  with 
fluctuations  of  voltage  above  and  below  the  pre-set  values  due  to  cycling 
of  the  feed  back  control  system  and  the  limitations  imposed  by  mixing  times 
required  to  disperse  the  rapidly  generated  electrode  products  throughout 
the  solution. 

E.   Proposed  course. 

Work  will  continue  towards  the  development  of  the  combined  coulometric 
potentiometric  system.   Using  this  system,  it  should  be  easy  to  complete  the 
potentiometric  analysis  for  f lavoproteins  and  cytochromes  of  intact  E.  coli, 
isolated  components  and  of  6? her  respiratory  systems.   A  new  kind  of  overall 
analysis  of  respiratory  chains  may  be  possible.   A  series  of  complete  spectral 
scans  at  fixed  voltages  could  be  used  to  generate  a  series  of  difference 
spectra  as  a  function  of  voltage.   These  difference  spectra  can  be  mathame- 
tically  resolved  into  individual  absorption  peaks  which  can  be  assigned  to 
components  of  the  chain  based  on  the  observed  redox  potentials.   This  approach 
applied  to  energized  and  non-energized  respiratory  chains  should  also  reveal 
energy-dependent  spectral  shifts. 

A  somewhat  different  line  of  approach  in  this  overall  project  will  also  be 
pursued  later  this  year  when  a  new  visiting  fellow  arrives.   We  will  try  to 
reconstitute  a  functionally  integrated  respiratory  chain  from  the  isolated 
subunits  we  have  been  able  to  obtain. 

Keywork  Descriptors:   Respiration,  cytochromes,  redox  potentials,  potentio- 
metric titration,  membrane  function,  bioenergetics,  energy  transduction. 

Publications: 

Hendler,  R.W. ,  Towne,  D.W.  and  Shrager,  R.I. :  Redox  properties  of  b-type 
cytochromes  in  Escherichia  coli  and  rat  liver  mitochondria  and  techniques 
for  their  analysis.   Biochim.  Biophys.  Acta.  376:  42-64  (1975) . 


/*r 


Project  No.  Z01  HL  00402-03  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Section  on  Membrane  Enzymology 

3.  Bethesda,  Md.  20014 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  DNA  synthesis  in  E.  coli 

Previous  Serial  No. :     NHLI-17 

Principal  Investigators : Richard  W.  Hendler 

Raymond  Scharff 
Clark  Springgate 

Project  Description: 

A.  Functional  differentiation  between  free  and  complexed  DNA  polymerase  I. 

At  150mM  KC1,  free  polymerase  was  inhibited  44%  by  added  ATP  whereas  a 
particulate  fraction,  P3,  was  stimulated  by  21%.   Free  polymerase  was  added 
to  P3  and  the  combined  system  was  27%  inhibited  by  ATP.   From  the  amounts 
of  activity  due  to  the  added  polymerase  and  that  of  the  polymerase  in  P3 
it  was  calculated  that  the  27%  inhibition  was  what  would  be  expected  from 
the  arithmetic  sum  of  21%  stimulation  of  the  endogenous  polymerase  and  51% 
inhibition  of  the  added  enzyme.   The  same  experiment  performed  at  70mM 
KC1  showed  that  the  free  enzyme  was  not  affected  by  ATP  while  P3  was 
stimulated  90%.   The  combined  system  was  stimulated  by  34%  which  was  the 
amount  predicted  from  the  summation  of  the  two  independent  responses.   There- 
fore, free  DNA  polymerase  responds  differently  to  ATP  than  does  the  DNA 
polymerase  present  in  the  cell  system. 

B.  Ability  of  deoxynucleoside  triphosphate  (dNTP)  to  serve  as  rATP. 

At  70mM  KC1,  ATP  causes  a  doubling  of  the  DNA  synthesis  rate.   If  ATP  is 
needed  for  synthesis,  why  isn't  the  system  more  ATP-dependent?   There  are 
two  obvious  possibilities  to  explain  the  high  background  activity.   One  is 
that  there  may  be  enough  endogenous  non-ATP-dependent  DNase  activity  to 
provide  short  gaps  for  a  polymerase  to  copy.   Further  purification  of  the 
complex  may  then  lead  to  a  reduction  of  this  activity.   The  other  possibility 
is  that  dNTP,  present  to  sustain  DNA  synthesis,  may  be  used  as  ATP.   To  test 
this  possibility,  individual  dNTP's  or  ATP  was  added  in  small  aliquots  to 
incorporating  systems  containing  all  4  dNTP's.   It  was  found  that  dATP  was 
at  least  as  effective  as  rATP,  but  the  other  dNTP's  could  not  substitute  for 
ATP.   Therefore,  even  in  the  absence  of  added  rATP,  a  background  level  of 
usable  triphosphate  bond  energy  is  available  to  the  system. 

C.  Effect  of  recBC  mutation  on  properties  of  the  complex. 


foU 


Project  No.  Z01  HL  00402-03  LCB 

At  15QmM  KC1,  DNA  synthesis  in  an  extract  from  wild  type  cells  was  stimulated 
about  25%  by  added  ATP,  whereas  an  extract  from  recB~  cells  was  inhibited 
by  7%.   However,  at  70mM  KC1  the  mutant  extract  showed  about  80%  of  the  ATP 
stimulation  of  the  wild  type  extract.   Similar  results  were  obtained  with 
the  isolated  complexes  from  rec+  and  rec~  cells.   ATP  stimulation  as  a 
function  of  KCl  concentration  showed  that  the  mutant  complex  was  more 
sensitive  to  salt  than  the  wild  type  complex.   The  recBC  enzyme,  in 
addition  to  being  an  ATP-dependent  DNase  is  also  a  DNA-dependent  ATPase. 
The  two  activities  are  independent  and  although  recBC  mutants  are  known 
to  be  deficient  in  nuclease  activity,  their  ATPase  activity  may  be  unimpaired. 
The  recBC  mutation  we  are  studying,  affected  the  protein  so  that  its 
nuclease  activity  was  lost  and  its  affinity  for  polymerase  I  was  altered. 
However,  the  ability  to  demonstrate  an  active  complex  in  the  mutant  at  low 
salt  concentration  suggests  that  it  is  not  the  nuclease,  but  rather  the 
ATPase  that  is  required  in  the  complex. 

The  role  of  the  ATPase  has  not  been  established,  but  it  could  be  involved  in 
unwinding  the  duplex  in  preparation  for  copying  by  polymerase. 

D.   Kinetics  of  DNA- synthesizing  system. 

The  rate  of  DNA  syntheses  by  the  system  under  study  is  m  kedly  concentration- 
dependent.   Time  course  experiments  over  a  wide  range  of  concentrations  show 
that  the  shape  of  the  incorporation  curve  goes  through  a  continuous  series 
of  changes.   The  curve  is  sigmoidal  at  very  low  concentrations,  becomes 
hyperbolic  and  then  linear  with  increasing  concentration,  and  starts  to 
slope  off  with  still  higher  concentrations,  to  produce  curves  with  plateaus 
or  peaks  of  incorporation  in  the  middle  of  the  incubation  period.   The 
Y-intercept  obtained  by  extrapolation  from  two  fixed  time  points  (i.e.  30 
and  60  mins.)  is  positive  at  very  low  concentrations,  goes  through  zero  and 
becomes  negative  as  concentration  is  increased  through  the  hyperbolic  phase, 
increases  to  zero  when  the  linear  incorporation  range  is  reached,  and 
becomes  increasingly  positive  as  the  incorporation  curve  slopes  off.   A  very 
useful  function  has  been  developed  as  an  indicator  of  the  kind  of  incor- 
poration curve  in  operation.   The  Y-intercept  divided  by  the  incorporation 
rate  from  30  to  60  minutes  (Y/S)  allows  comparisons  to  be  made  with 
preparations  having  widely  different  activities.   The  Y/S  value  is  "+"  in 
the  sigmoidal  range  "o"  and  "-"  through  the  hyperbolic  range,  '"g"    again  at 
linearity,  "+"  during  early  stages  of  sloping  off,  "*0"  when  the  30  and 
60  min.  points  lie  on  a  plateau  and  "-"  when  the  60  min.  incorporation  point 
is  lower  than  that  at  30  mins.   The  specific  activity  of  a  given  preparation 
increases  with  concentration  to  a  maximum  just  before  the  linear  incorporation 
rate  is  attained  and  then  decreases,  even  to  the  extent  of  becoming  negative 
at  high  concentrations.   A  plot  of  the  percent  of  the  linear  incorporation 
rate  vs.  Y/S  yields  a  curve  which  enables  one  to  correct  the  observed 
specific  activity  at  any  concentration  to  the  specific  activity  at  linearity 
(i.e.  Y/S=0) .   The  kind  of  behavior  just  described  is  most  unusual  for 
enzymes.   From  some  of  the  known  characteristics  of  the  incorporation  system, 
however,  a  model  has  been  developed  which  may  account  for  the  unusual 


for 


Project  No.  Z01  HL  00402-03  LCB 
properties.   The  model  is  based  on  the  following  considerations: 

1.  Duplex  DNA  must  be  "prepared"  for  copying  by  the  polymerase. 

2.  The  polymer ase-recBC  complex  has  a  dissociation  constant,  Kpp. 

3.  Polymerase  activity  is  markedly  enhanced  when  the  polymerase  is  complexed 
with  recBC. 

4.  The  complex  has  DNase  activity  associated  with  both  of  its  components. 

Let  A  represent  native  duplex  DNA 

B  represent  partially  hydrolyzed  DNA 

C  represent  DNA  containing  incorporated  nucleotides 

D  represent  partially  hydrolyzed  DNA 

k-[_  represent  rate  of  conversion  of  A  to  B 

k2  represent  rate  of  conversion  of  B  to  C 

k3  represent  rate  of  conversion  of  C  to  D 

a     kl   ^B >S2 -)   C    k^  y  n 

At  low  enzyme  concentrations  where  much  of  the  complex  is  dissociated, 
polymerase  activity  is  low  relative  to  recBC  activity  (i.e.  ki/k2  is  high). 
Therefore,  B  will  tend  to  accumulate.   As  B  accumulates,  the  rate  of  forma- 
tion of  C  will  increase.   This  can  explain  the  lag  seen  at  low  concentrations. 
With  increasing  concentration,  more  complex  is  formed,  which  increases  poly- 
merase activity  (decreases  ki/k2)  and  eliminates  the  lag.   At  still  higher 
concentrations  the  nuclease  activity  of  the  complex  begins  to  prevail. 
During  the  incubation,  partially  digested  DNA  becomes  more  easily  hydrolyzed 
leading  to  a  decrease  in  the  level  of  radioactive  DNA.   This  general  model 
has  been  discussed  with  an  enzyme  kinecicist,  John  Hearon,  and  a 
mathematician  experienced  with  kinetic  problems,  Richard  Shrager.   Both  believe 
that  it  may  account  for  the  observations  and  are  willing  to  collaborate  in 
evaluating  the  model. 

E.   Dissociation  and  reconstitution  of  DNA  synthesizing  complex. 

The  ATP  stimulation  of  DNA  synthesis  in  crude  systems  is  lost  at  280m  KC1 
but  is  completely  regained  by  diluting  the  KC1  concentration  to  70mM.   Isola- 
ted complex  when  re-chromatographed  on  Bio  Gel  A1.5M  in  280mM  KC1  no  longer 
migrates  as  a  3  90,000  molecular  weight  entity.   Instead  non-ATP  stimulated 
polymerase  activity  is  seen  in  the  elution  volume  for  DNA  polymerase  I, 
indicating  the  dissociation  of  the  complex.   Mixing  this  polymer ase  with  a 
portion  of  the  column  eluate  from  the  270, 000M. W.  region  (i.e.  where  free 
recBC  enzyme  should  be  located)  leads  to  a  marked  enhancement  of  polymerase 
activity  plus  the  reappearance  of  an  ATP  stimulation.   The  ratio  of  the  two 
enzymes  appears  to  be  critical  in  order  to  achieve  an  ATP  stimulation. 
Re- chromatography  of  the  mixture  on  Bio  Gel  A1.5m,  however,  did  not  lead 
to  the  isolation  of  reconstituted  complex.   Considerations  which  may  be 
pertinent  to  the  above  findings  are:  1)  The  necessity  of  re-constituting 

3  'oQ 


Project  No.  Zol  HL  00402-03  LCB 

in  the  presence  of  DNA.   2)  Another  factor  present  in  crude  preparations 
may  be  required.   3)  The  two  enzymes  may  be  able  to  complement  each  other 
without  forming  a  stable  complex  in  a  manner  similar  to  that  of  unjoined 
fragments  of  ribonuclease. 

Proposed  Course  of  Research: 

After  learning  of  the  concentration  dependence  of  the  DNA  synthesizing  system, 
it  was  possible  to  convert  observed  activities  to  uniform  linear  rate  incor- 
poration values.   This  revealed  that  a  major  part  of  the  system  was  being 
lost  to  the  debris  fraction  of  the  initial  20000g  centrifugation.   Efforts  to 
release  and  retrieve  this  activity  will  be  made. 

The  recBC  enzyme  will  be  purified  and  the  formation  of  complex  from  pure 
recBC  and  polymerase  I  enzymes  will  be  attempted.   If  necessary,  other  cell 
fractions  will  be  sought  to  effect  the  formation  of  a  stable  complex. 
Attempts  will  be  made  to  purify  further  the  endogenous  complex  and  to 
unequivocably  identify  recBC  as  a  constituent.   Kinetics  and  the  concentration 
dependence  of  the  purified  complex  will  be  studied.   The  DNA  product  of  the 
purified  and  crude  systems  will  be  more  fully  characterized. 

Keyword  Descriptors:   DNA  synthesis,  recombination  enzymes,  enzyme  complex, 
DNA  polymerase  complex,  recBC. 

Publications : 

Hendler,  R.W. ,  Pereira,  M. ,  and  Scharff,  R. :  DNA  synthesis  involving  a 
complexed  form  of  DNA  polymerase  I  in  E.  coli  extracts.   Proc.  Nat.  Acad. 
Sci.  U.S.A.  71:  (1975)  (in  press) . 


tof 


Project  No.  Z01  HL  00403-01  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Cellular  Physiology 

3.  Bethesda,  Md.  20014 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Differential  scanning  calorimetry  of  fibrinogen. 

Principal  Investigator:   Elemer  Mihalyi 

Cooperating  Units:       Western  Regional  Research  Center,  Agricultural 

Research  Service,  U.S.  Dept.  of  Agriculture, 
Berkeley,  California  94710  Dr.  John  W.  Donovan 

Objectives:   To  investigate  the  possibility  of  independent  thermal  unfolding 
of  subunits  in  the  fibrinogen  molecule  and  to  correlate  these  subunits 
with  the  large  fragments  obtained  by  proteolysis.   Further  to  investigate 
the  effect  of  clotting  on  unfolding  of  the  subunits. 

Major  Findings: 

Solutions  of  fibrinogen  show  two  endothermal  (denaturing)  transitions  at 
61°  and  at  100°,  when  heated  in  a  differential  scanning  calorimeter. 
Similar  transitions  are  observed  for  a  mixture  of  the  fragments  D  and  E 
obtained  by  limited  proteolysis  of  fibrinogen.   Isolated  fragment  E  shows 
only  a  single  transition,  at  97^.   The  independent  thermal  denaturation  of 
these  portions  of  fibrinogen  supports  the  three-nodular  model  proposed  for 
fibrinogen.   The  D  and  E  subunits  retain  their  characteristic  denaturation 
behavior  when  fibrinogen  is  clotted  by  thrombin  addition,  but  over  a  period 
of  about  one  hundred  clotting  times,     the  denaturation  temperature  of  the 
D  subunit  increases  by  9°  and  its  enthalpy  of  denaturation  by  one-third. 
Since  this  change  takes  place  in  the  absence  of  Factor  XIII  activity,  and 
its  rate  is  proportional  to  thrombin  concentration,  it  is  presumed  to  be 
mediated  by  a  proteolytic   cleavage  distinct  from  those  which  liberate  the 
A  and  B  peptide. 

Methods  Employed:   Differential  scanning  calorimetry. 

Project:  This  phase  of  the  project  completed.  Further  investigation  will  be 
directed  toward  elucidation  of  the  mechanism  of  the  slow  action  of  thrombin. 
Publication: 

Donavan,  J.W.  and  Mihalyi,  E.   Conformation  of  fibrinogen:  Calorimetric 
evidence  for  a  three-nodular  structure.   Proc.  Nat.  Aca.  Sci.  ,  U.S.A.  71; 
4125-4128  (1974). 

Keyword  Descriptors:  fibrin,  blood  clotting,  differential  scanning,  calori- 
metry, protein,  subunits,  thermal  denaturation. 


/fO 


Project  No.  Z01  HL  00404-16  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Cellular  Physioloey 

3.  Bethesda,  Md.  20014 

NIH-PHS 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title  Proteolytic  fragmentation  of  fibrinogen. 

Principal  Investigator:  Elemer  Mihalyi 

Other  Investigator:  David  Towne 

Previous  Serial  No. :  NHLI-153 

Cooperating  Units:       Division  of  Computer  Research  and  Technology, 

Laboratory  of  statistical  and  Mathematical  Methodol- 
ogy, Richard  Shrager. 

Objectives:   The  purpose  of  the  work  performed  during  the  last  5  years  was  to 
provide  sufficiently  accurate  data  for  a  complete  kinetic  analysis  of  the 
proteolytic  degradation  of  fibrinogen.   For  this  it  was  necessary  to  estimate 
the  fraction  of  optical  density  in  each  of  the  reaction  products  along  the 
reaction  path.   Further,  the  specific  optical  densities  were  needed  to  convert 
optical  density  distribution  into  mass  distribution.   It  had  to  be  proved 
that  the  mass  distribution  obeyed  the  law  of  mass  conservation.   The  mass 
distribution  and  the  independently  determined  molecular  weights  of  the  fragments 
could  be  used  to  determine  the  number  of  the  fragments  derived  from  one  native 
molecule  of  fibrinogen.   With  all  these  data  the  kinetics  could  be  worked  out 
for  the  whole  process,  accounting  for  all  the  fragments  formed. 

Methods  Employed:  Sodium  dodecyl  sulfate-polyacrylamide  gel  electrophoresis 
with  UV-scanning,  Sephadex  C-200  chromatography,  equilibrium  ultracentrifuga- 
tion,  amino  acid  analysis,  peptide  mapping,  kinetic  modeling  by  the  computer. 

Major  findings:   The  bovine  fibrinogen-trypsin  system  was  worked  out  in  more 
detail.   The  optical  density  conversion  factors  of  the  fragments  were  found 
as  follows : 

Table  1 

Material  Specific  Optical  Density 

Fibrinogen   14.78    Fragment  D    20.04 

Fragment  X   16.62    Fragment  E     8.97 

Fragment  Y   15.61    Fragment  PI    21.73 

Fragment  P2    4.60 

With  these,  the  sum  of  the  calculated  masses  of  the  fragments  remained  constant 
and  equdl  to  that  of  native  fibrinogen  through  the  whole  reaction.   The  mass 
distribution,  at  the  stage  where  fragments  D,  E,  PI  and  P2  reach  their 

1  AY 


Project  No.  Z01  HL  00404-16  LCB 

maximum  aboundance,  was  used  to  calculate  the  molecular  weights  of  these 
components.   The  data  are  listed  in  table  2  together  with  the  molecular 
weights  determined  by  other  methods. 

The  fragments  were  isolated  by  recycling  on  the  Sephadex  G230   column  and  were 
homogeneous  in  sodium  dodecylsulfate-polyacrylamide  gel  electrophoresis.   In 
high  speed  sedimentation  equilibrium  runs  these  gave  the  molecular  weights 
listed  also  in  table  2. 

In  serial  digests,  as  judged  from  sodium  dodecylsulfate  polyacrylamide  gel 
electrophoresis,  the  molecular  weights  of  the  components  remained  nearly 
constant  throughout  the  entire  digestion  phase  investigated  here.   The 
average  of  the  molecular  weights  obtained  are  also  given  below: 

Table  2 

Molecular  Weights  Obtained  by  Various  Methods 


Material 

Sedimentation 
equilibrium 

SDS-elect 

Fibrinogen 

335,000 



X 

230,000 

210,000 

Y 

133,800 

132,000 

D 

85,000 

85,000 

E 

47,000 

42,000 

PI 

16,500 

16,000 

Mass  Distribution 


82,300 
51,400 
17,400 


The  computer  analysis  showed  that  the  peptide  fractions,  PI  and  P2,  are  not 
connected  with  the  fragmentation  into  the  D  and  E  fragments .   The  early 
phase  was  not  resolved  sufficiently  in  our  studies,  however,  it  is  clear 
that  the  final  fragment  X  is  derived  from  native  fibrinogen  by  removing  all 
the  PI  and  P2.   Because  of  this  low  degree  of  resolution  the  first  phase,  i.e. 
F  -«-X  +  PI  +  P2,  could  be  approximated  as  a  single  step  and  described  by  a 
single  rate  constant.   Strictly  speaking,  this  means  that  the  whole  PI  +  P2 
segment  was  removed  in  one  piece.   This  is  probably  not  far  from  truth,  because 
other  workers  f round  the  appearance  of  a  40,000  molecular  weight  piece  in  the 
early  digests  and  proved  that  this  is  derived  from  the  C-terminal  portion  of 
the  A iX -chains.   We  have  also  demonstrated  by  finger  printing  that  P2  almost 
entirely  originated  from  the  same  segment. 

The  following  sequence  of  events:   X  -*Y  +D  and  Y-*D  +E,  could  be  described 
by  a  single  rate  constant,  that  was  close  to  the  average  rate  of  cleavage 
of  the  peptide  bonds  in  the  slow  reaction,  as  determined  in  the  pH  stat. 
However,  for  the  curve  fitting  it  was  necessary  to  assume,  either  that  there 
are  3-chains  on  either  side  of  the  D-E-D  structure,  that  all  have  to  be 
cleaved  in  order  to  separate  the  fragments,  or  that  fragment  X  is  an 
obligatory  intermediate.   The  latter  case  would  mean  that  PI  and  P2  somehow 
protect  the  linkage  between  D  and  E,  and  this  linkage  cannot  be  cleaved 
until  the  protection  is  removed.   With  the  protected  model  a  single  cleavage 


ttl- 


Project  No.  Zol  HL  00404-16  LCB 

i.e.  one  connecting  chain,  adequately  describes  the  process.   Since  the 
chemical  data  show  that  there  are  3  chains  between  the  subunits,  we  prefer 
the  first  model.   The  data  also  suggest  that  in  each  chain  there  is  only  one 
critical  bond  that  is  cleaved.   This  agrees  with  the  observation  that 
peptide  release  does  not  seem  to  be  associated  with  the  fragmentation  into 
D  and  E  fragments.   The  whole  analysis  is  remarkable  for  the  fact  that 
such  a  complicated  process  could  be  described  with  only  two  rate  constants. 
This  is  the  first  case  when  a  complete  analysis  of  a  proteolytic  fragmenta- 
tion was  possible.   It  is  axiomatic  that  kinetics  seldom  prove  anything. 
However,  in  this  case  all  the  intermediates  were  isolated  and  characterized 
and  the  mechanism  was  suggested  by  the  chemical  data  of  the  structure  of  the 
molecule.   These  facts  restricted  the  modeling  to  such  a  degree  that  the 
results  cannot  be  far  from  reality. 

The  data  obtained  for  the  plasmin  digestion  of  bovine  fibrinogen  (NHLI-153 
report  1970-1971)  were  not  accurate  for  the  present  purposes.   These 
experiments  were  redone  under  the  improved  conditions  and  supplemented  with 
data  on  the  digestion  of  human  fibrinogen  by  the  same  enzyme.   Also,  the 
data  obtained  on  human  fibrinogen  in  the  previous  year  (NHLI  report  1973-74) 
were  utilized  in  the  computer  modeling.   All  four  systems,  bovine  fibrinogen 
and  human  fibrinogen  cleaved  by  plasmin  and  by  trypsin,  were  remarkably  similar 
both  with  respect  to  fragments  produced  and  the  kinetics  of  the  process.   The 
main  differences  were  with  respect  to  the  F  -»X  step,  and  this  is  undoubtedly 
due  to  the  sequence  variability  of  the  AOt-chain  segment  removed.   The 
liberation  of  the  D  and  E  fragments  followed  an  identical  course  with  all 
four  cases,  only  with  rate  differences  between  them.   Human  fibrinogen 
appears  to  be  fragmented  by  trypsin  with  about  half  the  rate  observed  with 
bovine  fibrinogen.   A  similar  rate  difference,  although  musch  less  accentuated, 
seems  to  hold  for  the  plasmin  digestion  of  the  two  proteins. 

Project:   This  phase  of  the  project  completed.   Other  aspects  of  the 
proteolytic  fragmentation  of  fibrinogen  will  be  continued. 

Publications:   None 

Keyword  Descriptors:   Blood  coagulation,  fibrinogen,  fibrinolysis,  proteolytic 
degradation  products,  kinetics. 


t(3 


Project  No.  Z01  HL  00405-01  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Cellular  Phvsiologv 

3.  Bethesda,  Md.  20014 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Circular  dichroism  studies  on  reduced  alkylated 

lysozyme 

Principal  Investigator:   F.  H.  White,  Jr. 

Other  Investigator:      A.  G.  Wright,  Jr.  (Technical) 

Project  Description: 

Objectives: 

To  explore  the  possible  appearance  of  conformational  structure  in  a  fully 
reduced,  alkylated  protein. 

Methods : 

1.  Previously  established  methods  (F.H.  White,  Methods  in  Enzymology, 
Vol.  25,  p.  387  (1972))  were  employed  for  the  reduction  of  lysozyme  with 
3-mercaptoetbanol  in  the  presence  of  urea,  followed  by  alkylation  with 
iodoacetate  or  iodoacetamide. 

2.  Methods  recently  developed  in  this  lab  were  used  for  the  selective 
alkylation  of  SH  groups  in  reduced  lysozyme  with  triphenylvinylphosphonium 
bromide  (TVP) .   This  reagent  was  originally  developed  by  J.  Swan  and  S.Wright 
Aus.  J.  Chem.  24,  777  (1971)  for  alkylation  of  amino  groups  in  lysine 
residues. 

3.  Circular  dichroism  studies  were  conducted  on  a  Cary  Model  60  spectrophoto- 
meter with  a  Model  #6001  CD  attachment.   The  data  were  treated  by  the 
procedure  of  N.  Greenfield  and  G.  Fashman  (Biochem.  8_,  4108  (1969)  )  . 

4.  Phosphorus  assays  to  measure  the  incorporation  of  triphenylethyl- 
phosphonium  (TEP)  groups,  resulting  from  alkylation  with  TVP,  were  conducted 
by  the  procedure  of  L.  Lazarus  and  S.  Chou,  Anal.  Biochem.  4_5,  557  (1972)  . 

5.  Amino  acid  analysis  was  carried  out  by  the  procedure  of  S.  Moore  and 
W.  Stein,  Anal.  Biochem.  3JD,  1190  (1958). 

Major  Findings: 

1.   The  use  of  TVP  has  been  studied  extensively  in  this  laboratory  and  two 
findings  have  been  made. 

r<4 


Project  No.  Z01  HL  00405-01  LCB 

a.  It  has  a  high  selectivity  for  SH  groups  between  pH7  and  8. 

b.  Reduced  TEP  lysozyme  is  soluble  up  to  pH6  in  0.07  5M  sodium  phosphate, 
whereas  the  carboxymethyl  and  carboxamidomethyl  derivatives  are  insoluble 
above  pH4.   Hence  the  use  of  reduced  TEP  lysozyme  made  possible  a  study  of 
pH  effects  on  structure  over  a  wider  range. 

2.  Reduced  lysozyme  samples  after  alkylation  with  iodoacetate,  iodoacetamide , 
or  TVP,  were  examined  by  circular  dichroism.   Evidence  of  ordered  structure 
was  found  in  all  samples  when  dissolved  in  either  dilute  phosphate  or 

dilute  HC1.   The  observed  structures  were  0-8%  ahelix,  approximately  30% 
8  structure,  and  approximately  60%  random  coil. 

3.  Circular  dichroism  in  8M  urea  or  6M  guanidine  showed  no  evidence  of 
structure.   Digestion  of  reduced  alkylated  lysozyme  samples  with  pepsin  also 
destroyed  the  conformational  structure. 

Significance: 

It  has  long  been  established  that  development  of  conformational  structure 
is  dependent  on  amino  acid  sequence,  but  the  exact  relationship,  despite 
extensive  empirical  and  theoretical  studies,  has  never  been  elaborated. 

The  present  results  suggest  a  new  approach  to  this  problem,  since  it  has 
never  been  firmly  established  that  a  protein  chain,  in  the  absence  of 
disulfide  bonds,  could  develop  conformational  structure  to  a  measureable 
extent.   Further  investigation  should  shed  some  light  on  the  amino  acid 
sequences  responsible  for  the  various  structures  observed  by  circular 
dichroism. 

Proposed  Course : 

It  is  proposed  that  this  project  should  be  continued  in  two  directions: 

a.  To  examine  other  proteins,  after  reduction  and  alkylation,  for  the 
presence  of  conformational  structure. 

b.  To  employ  degradative  procedures  on  reduced  alkylated  proteins  for  the 
purpose  of  identifying  the  smallest  conformationally  functional  unit  of 
amino  acid  sequence. 

Publications : 
None 

Keyword  Descriptors:  Conformational  structure,  circular  dichroism,  reduced 
alkylated  lysozyme,  a-helix,  8-structure,  random  coil. 


//r 


Project  No.  Z01  HL  00406-03  LCB 

1.  Laboratory  of  cell  biology 

2.  Cellular  Physiolo?»v 

3.  Bethesda,  Md.  20014 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Tritium  labeling  of  binding  site  residues 

Previous  Serial  No.  NHLI-23 

Principal  Investigator:  F.  H.  White,  Jr. 

Other  Investigator:  A.  G.  Wright,  Jr.  (Technical) 

Project  Description: 

Objectives:  To  investigate  the  mechanism  of  a  reaction,  whereby  the  binding 
site  residues  of  alpha-chymotrypsin  become  preferentially  labeled  with  tritium. 

Methods : 

1.  Chymotrypsin  was  labeled  with  tritiated  diisopropylf luorophosphate 
(T-DFP)  (Cohn  et  al. ,  Methods  in  Enzymol.  XI,  688  (1967))  to  attach  the 
tritiated  diisopropylphosphoryl  (T-DIP)  group  to  the  serine  residue  of 
Position  195.   The  T-DFP  had  been  labeled  by  a  commercial  source  to  a  specific 
activity  of  3. 3Ci/mmole,  the  highest  available. 

2.  The  labeled  protein  was  exposed  to  tritiated  hydrogen  sulfide  (HST) 
(White, et  al. ,  Radiation  Res.  32,  744  (1967)),  which  labels  the  carbon  free- 
radicals  that  develop  from  exposure  to  ionizing  radiation. 

3.  Amino  acid  analysis  with  scintillation  flow  counting  was  employed  for 
analysis  of  the  labeled  protein  hydrolysate  as  describe!  by  F.H.  White  and 
C.R.  Mencken,  Anal.  Biochem.  34,  470  (1968. 

Major  Findings: 

Earlier  (Ann.  Rep.  for  1974  (#23)  and  F.H.  White,  J.  Labelled  Compounds, 
(in  press) )  it  was  observed  that  the  reaction  of  T-DIP  chymotrypsin  with 
tritiated  hydrogen  sulfide  (HST)  effected  a  transfer  of  tritium  onto  residues 
close  to  the  binding  site. 

The  following  reactions  constitute  an  hypothesis  to  account  for  this  transfer, 
and  the  hypothesis  was  then  tested  as  described  below: 

self 

T-DIP-chymotrypsin   _ -^      T-DIP-chymotrypsin (C- )  (1) 

radiolysis 


//c 


Project  No.  Z01  HL  00406-03  LCB 
HST  -y  T-DIP-chymotrypsin  (C-T)  +  HS'  (2) 

HS,+T-DIP-chymotrypsin--^HST+DIP(C-  ).-chymotrypsin  (3) 

A  radical  migration  to  residues  close  to  the  DIP  group  would  be  followed  by: 
amino  acid  (C- ) +HST ->amino  acid  (C-T)  +HS"  (4) 


Reaction  (1) ,  whereby  carbon  free-radicals  form  on  amino  acid  residues,  ensues 
as  a  result  of  self-radiolysis,  as  shown  earlier  (F.H.  White  and  G.  Wright, 
Abstract  Vth  Intern.  Cong,  of  Rad.  Res.,  Seattle  (1974)). 

It  is  well  established  that  HST  reacts  with  the  resulting  free  radical  as 

in  reaction  (2)  (White,  et  al. ,  Radiation  Res.  47,  8  (1971)),  to  liberate  the 

HS-  radical. 

It  is  then  hypothesized  that  this  radical  is  capable  of  abstracting  tritium 
from  the  T-DIP  group,  to  leave  a  radical  (C-)  on  the  latter  group,  as  in 
reaction  (3) .   There  is  abundant  evidence  to  support  radical  migration 
(e.g.  see  J.H.  Miller,  et  al. ,  Photochem.  and  Photobiol.  14,  577  (1971)), 
which  would  result  in  appearance  of  radicals  on  nearby  residues.   These 
radicals  would  react  with  HST  as  in  reaction  (4) . 

This  hypothesis  has  been  tested  as  follows,  to  determine  whether  or  not 
abstraction  of  tritium  by  HS*  proceeds  under  the  reaction  conditions 
employed. 

Samples  of  tritiated  lysozyme,  prepared  as  by  F.  H.  White  et  al.  (Anal.  Biochem. 
30  295  (1969)),  were  exposed  either  to  gamma-radiation  or  electrical  discharge 
to  create  a  content  of  carbon  free-radicals  approximating  that  produced  by 
self-radiolysis  as  in  reaction  (1)  . 

Subsequent  exposure  to  H2S  resulted  in  tritium  removal  from  the  carbon- 
tritium  bond  to  a  maximum  of  20-30%. 

Conclusions: 

These  results  support  the  hypothesis  that  HS"  abstracts  tritium  from  the 
carbon- tritium  bond,  and  therefore  also  support  the  proposed  reaction 
mechanism. 

Significance: 

The  tritium-labeling  of  binding  site  residues  suggests  applications  to  the 
study  of  protein  binding  sites.   First,  however,  it  is  necessary  to  understand 
the  reaction  mechanism, and  the  present  results  shed  light  on  this  subject. 
Moreover,  the  abstraction  of  tritium  from  carbon  by  the  SH  radical  appears 
not  to  have  been  demonstrated  previously. 

til 


Project  No.   Z01  HL  00406-03  LCB 

Proposed  Course  of  Research: 

With  emphasis  on  the  possible  use  of  this  reaction  in  binding  site  studies, 
it  is  planned  to  continue  by  seeking  other  model  protein-ligand  complexes 
to  obtain  information  as  to  the  general  applicability  of  the  labeling 
reaction.   Such  information  is  deemed  necessary  prior  to  serious  application 
to  proteins  whose  structure  is  less  well  understood. 

Publication: 

White,  F.  H. ,  Preferential  tritium  labelling  of  binding  site  residues  in 
alpha  chymotrypsin  by  exposure  of  the  1,3-^H-diisopropylphosphoryl  derivative 
to  tritiated  hydrogen  sulfide.   J.  Labelled  Compounds  (in  press) . 

Keyword  Descriptors:   Alpha-chymotrypsin,  tritium- labeling,  carbon  free- 
radicals,  radical  migration,  protein-ligand  complex. 


//6 


Project:   Z01  IIL  00407-02  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Cellular  Physiology 

3.  Bethesda,  Md.  20014 

NIH-PHS 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Interaction  of  SH^-blocked  myosin  with 

actin  and  ATP. 


Principal  Investigators:  Sally  Mulhern 

Evan  Eisenberg 
W.  Wayne  Kielley 

Project  Description: 

Objectives:   The  interaction  of  myosin  and  actin  in  the  presence   of  ATP 
is  the  central  reaction  involved  in  muscle  contraction.   In  order  to  under- 
stand this  reaction,  experiments  were  performed  on  actin  and  HMM  which  is 
a  proteolytic  digestion  product  of  myosin.   It  was  demonstrated  that  the 
actin  can  activate  the  HMM  ATPase  200-fold  while  a  large  fraction  of  HMM 
remains  not  bound  to  actin.   Since  it  was  demonstrated  in  this  laboratory 
that  SHi-blocked  HMM  (NEM-HMM)  was  only  3-fold  activated  by  actin  experiments 
were  performed  to  find  which  step  in  the  actin  activation  of  NEM-HMM  ATPase 
was  blocked.   In  previous  studies  we  demonstrated  that  as  with  unmodified 
HMM  very  little  NEM-HMM  is  bound  to  actin  during  ATP  hydrolysis  under 
conditions  of  maximum  actin  activation.   From  this  it  was  suggested  that 
during  a  cycle  of  interaction  with  actin  and  ATP,  NEM-HMM  underwent  a  rate 
limiting  conversion  from  a  refractory  state  which  is  unable  to  bind  to 
actin  to  a  non-refractory  state  which  can  bind  to  actin.   This  model 
predicts  that  as  with  unmodified  HMM  the  ATP  turnover  rate  per  mole  of  actin 
at  high  JNEM-HMMJ  would  be  much  higher  than  the  ATP  turnover  rate  per  mole 
of  NEM-HMM  at  high   £actinj  .   This  conclusion  depends  on  there  being  one 
species  of  modified  HMM  present,  i.e.  it  must  be  demonstrated  that  NEM-HMM 
unbound  to  actin  during  ATP  hydrolysis  has  the  same  activity  as  the  original 
NEM-HMM.   In  the  present  study  we  determined  both  the  ATP  turnover  rate 
per  mole  of  NEM-HMM  and  actin  at  high  (actinj  and  high  ^NEM-HMM/  respectively. 
We  also  employed  an  analytical  ultracentrifuge  equipped  with  a  separation 
cell  to  determine  if  the  NEM-HMM  which  remains  unbound  to  actin  in  the 
absence  of  salt  and  maximal  actin  activation  has  the  same  ATPase  activity 
as  the  original  NEM-HMM. 

Methods  Employed  and  Major  Findings: 

Double-reciprocal  plots  of  the  ATPase  rate  at  high  /NEM-HMM] in  the  presence 
of  2yM  actin  and  double  reciprocal  plots  of  the  ATPase  rate  at  high  £actin/ 
in  the  presence  of  5yM  NEM-HMM  were  compared.   Results  demonstrated  that 
the  ATP  turnover  rate  per  mole  of  actin  was  more  than  10-fold  higher  than 
the  ATP  turnover  rate  per  mole  of  NEM-HMM  both  in  the  presence  and  absence 


't? 


Project  No.  Z01  HL  00407-02  LCB 

of  salt.   The  analytical  ultracentrifuge  equipped  with  a  separation  cell  was 
used  to  isolate  the  HMM  which  remained  unbound  to  actin  during  ATP  hydrolysis 
under  conditions  of  nearly  maximum  actin  activation  in  the  presence  and 
absence  of  salt.   It  was  shown  that  the  unbound  HMM  had  the  same  3-fold 
maximum  actin  activation  just  as  the  original  NEM-HMM.   These  results  demon- 
strate that  NEM-HMM  consists  of  one  species  which  binds  to  actin  and  shows 
actin  activation.   This  NEM-HMM  undergoes  a  rate  limiting  transition  from  the 
refractory  to  the  non-refractory  state  during  interaction  with  actin  and  ATP. 
Since  the  actin  activation  of  NEM-HMM  is  lower  than  that  of  normal  HMM,  this 
transition  may  be  slower  for  NEM-HMM  than  for  normal  HMM. 

Proposed  Course  of  Research: 

In  order  to  determine  if  these  findings  apply  to  subfragment-1  which  has 
only  a  single  head  in  contrast  to  HMM,  we  propose  to  use  the  analytical 
ultracentrifuge  to  investigate  the  binding  of  NEM-subfragment-1  to  actin  both 
in  the  presence  and  absence  of  salt.   We  also  plan  to  use  the  analytical 
ultracentrifuge  to  investigate  the  binding  of  actin  to  HMM  which  has  been 
modified  with  NEM  both  at  the  SHj  and  SH2  sites. 

Publication: 

Mulhern,  S.,  Eisenberg,  E. ,  and  Kielley,  W.W.   The  interaction  of  actin  with 
SHj-blocked  heavy  meromyosin  in  the  presence  and  absence  of  actin. 
Biochemistry  (in  press) . 

Keyword  descriptors:   Muscle,  myosin,  SH^-blocked  HMM,  actin. 


{}& 


Project  No.  Z01  HL  00408-03  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Cellular  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Actin-myosin  interaction:  Control  by  native 

tropomyosin. 

Previous  Serial  No.      NHLI-26 

Principal  Investigator;   Evan  Eisenberg 

Other  Investigator:      Louis  Dobkin 

Collaborating  Investigators:  David  Kominz  and  Barbra  Eaton,  NIAMD,  NIH. 

Project  Description: 

Objectives : 

It  is  now  clear  that  relaxation  of  skeletal  muscle  is  caused  by  removal  of 
Ca2+  from  the  sarcoplasm  by  the  sarcoplasmic  reticulum  and  that  contraction 
is  triggered  by  the  release  of  the  Ca2+.   It  is  now  also  clear  that  this 
effect  of  Ca2+  is  mediated  by  a  complex  of  proteins  called  "native  tropomyosin' 
which  binds  to  the  actin  filament,  and  prevents  the  myosin  bridges  from 
binding  to  actin  in  the  absence  of  Ca2+.   in  previous  work  we  investigated 
the  activity  of  the  three  troponin  components ,  troponin  I ,  T  and  C  with 
and  without  tropomyosin  present.   We  found  that  all  three  troponin 
components  plus  tropomyosin  had  to  be  present  to  confer  Ca-sensitivity  on 
the  interaction  of  actin  and  HMM.   But  we  also  found  that  troponin  I  and  T 
alone  could  inhibit  the  actin-HMM  interaction  even  without  tropomyosin  being 
present  suggesting  that  tropomyosin  may  not  be  necessary  for  inhibition  of 
the  actin-HMM  interaction.   This  result  is  not  consistent  with  a  recent 
model  of  troponin-tropomyosin  action  which  suggests  that  tropomyosin  alone 
blocks  the  binding  of  HMM  to  actin  filaments  with  the  role  of  the  troponin 
being  to  simply  orient  the  tropomyosin  on  the  actin  filament  so  as  to  make 
the  blocking  effect  of  the  tropomyosin  Ca-sensitive.   In  this  model,  the 
tropomyosin  is  thought  to  be  able  to  occupy  only  two  positions  on  the  actin 
filamnet  —  one  position  which  blocks  the  actin-HMM  interaction  in  the 
absence  of  Ca  and  one  which  accentuates  it  in  the  presence  of  Ca.   To  further 
investigate  this  question  we  studied  the  binding  of  tropomyosin  to  actin  both 
in  the  presence  and  absence  Of  the  troponin  components  and  correlated  this 
binding  with  inhibition  of  the  acto-HMM  ATPase. 

Methods  Employed  and  Major  Findings: 

Direct  binding  studies  of  tropomyosin  to  actin  using  il25  labeled  tropo- 
myosin showed  that  at  KC1  concentrations  below  0.1M,  tropomyosin  only  binds 

1  «/ 


Project  No.  Z01  HL  00408-03  LCB 

to  actin  at  Mg  concentrations  greater  than  lmM.   Furthermore  under  conditions 
where  tropomyosin  binds  it  always  causes  60%  inhibition  of  the  actin- 
activated  HMM  ATPase.   This  result  is  not  compatible  with  a  model  for 
tropomyosin  action  which  allows  the  tropomyosin  to  occupy  only  2  positions 
on  the  actin  filament  —  an  on  or  off  position.   Further  evidence  against 
this  simple  model  comes  from  evidence  that  troponin  I  induces  tropomyosin 
to  bind  to  actin  under  conditions  where  the  tropomyosin  itself  does  not 
bind.   Since  troponin  I  is  not  thought  to  directly  interact  with  tropomyosin, 
these  data  suggest  that  troponin  I  may  have  a  direct  effect  on  the  actin 
filament  which  in  turn  induces  the  tropomyosin  to  bind,  a  result  which 
would  not  be  compatible  with  the  simple  model  of  troponin-tropomyosin  action 
given  above.   Further  evidence  for  the  complex  interaction  of  these  proteins 
comes  from  data  showing  that  even  under  conditions  where  the  effect  of 
troponin  I  on  the  actin- HMM  interaction  is  reversed  by  troponin  C,  the 
troponin  I  is  still  able  to  induce  the  tropomyosin  to  bind  to  actin,  a 
result  which  suggests  a  dual  role  for  troponin  I.   Finally  our  recent 
results  indicate  ahat  although  in  the  presence  of  ATP  tropomyosin  inhibits 
the  actin-HMM  interaction,  at  low  ATP  concentration  the  tropomyosin  increases 
the  HMM  binding  in  a  cooperative  manner  as  originally  suggested  by  A.M.  Weber 
and  her  collaborators.   Analogously  we  have  found  that  in  the  absence  of  ATP, 
HMM  can  induce  tropomyosin  binding  under  conditions  where  the  tropomyosin 
alone  does  not  bind.   However  it  is  still  not  clear  whether  the  cooperative 
effect  of  tropomyosin  occurs  only  at  low  ATP  concentration  and  also  how  the 
presence  of  troponin  affects  this  cooperativity.   Further  work  will  be 
necessary  in  this  area  but  a  report  of  much  of  the  above  data  is  presently 
in  press  in  Biochemistry. 

Proposed  Course  of  Research: 

First  the  inhibition  of  Lhe  acto-HMM  ATPase  by  tropomyosin  alone  will  be 
investigated  at  high  ATP  concentration  to  determine  whether  the  inhibition 
can  be  reversed  by  increasing  the  HMM  concentration  —  a  result  which  would 
suggest  that  cooperative  interaction  of  actin,  HMM  and  tropomyosin  occurs 
even  at  high  ATP  concentration.   Second  the  activating  effect  which  the 
troponin-tropomyosin  complex  has  on  the  acto-HMM  ATPase  in  the  presence  of 
Ca  will  be  investigated  to  determine  exactly  which  troponin  components  are 
necessary  for  this  effect.   We  will  also  investigate  whether  this  activating 
effect  depends  cooperatively  on  the  HMM  concentration  both  at  high  and  low 
ATP  concentration.   Third  we  will  continue  our  earlier  studies  in  collaboration 
with  Dr.  E.  Korn's  group  on  the  interaction  of  tropomyosin  and  troponin  with 
the  actin  and  myosin  presently  being  isolated  from  Acanthameba  —  an 
investigation  which  might  reveal  both  how  the  troponin  and  tropomyosin 
effects  depend  on  specific  configurations  of  the  actin  and  myosin  and  might 
also  reveal  how  cellular  actin  differs  from  the  actin  found  in  organized 
skeletal  muscle  myofibrils. 

Publications: 

Eaton,  B.L.,  Kominz,  D.R.  and  Eisenberg,  E.   Correlation  between  the  inhibi- 
tion of  the  acto-heavy  meromyosin  ATPase  and  the  binding  of  tropomyosin  to 
F-actin:   Effects  of  Mg++,  KC1,  troponin  I,  and  troponin  C.   Biochemistry 

2  ft* 


Project  No.  Z01  HL  00408-03  LCB 
(in  press) . 
Keyword  Descriptors:   Troponin,  tropomyosin,  muscle  relaxation,  actomyosin. 


f*3 


Serial  No.  Z01  HL  00409-05  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Cellular  Physiology 

3.  Bethesda,  Md.  20014 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  The  interaction  of  actin  and  myosin 

Previous  Serial  No. :     NHLI-25 

Principal  Investigators:  Evan  Eisenberg 

W.  Wayne  Kielley 

Collaborating  Investigators  for  Theoretical  work:  Terrell  Hill  and 

Richard  Podolsky,  NIAMD 

Collaborating  Investigators  for  laser  light  scattering:  Allan  Fraser 
and  Francis  D.  Carlson,  Johns  Hopkins  University 

Collaborating  Investigators  for  stop  flow  studies:  S.  Chock  and 

P.B.  Chock 

Other  Investigators:      Louis  Dobkin 

Project  Description: 

Objectives:   It  is  now  generally  recognized  that  contraction  of  muscle  involves 
the  interaction  of  the  two  proteins  actin  and  myosin  with  ATP.   It  is  there- 
fore of  considerable  importance  to  determine  the  nature  of  the  interaction 
which  occurs  between  actin  and  myosin  in  vitro  as  ATP  is  hydrolyzed.   In 
particular  it  is  of  importance  to  elucidate  the  steps  occurring  during 
the  hydrolytic  cycle,  since  these  steps  may  correspond  to  the  steps  of  the 
contractile  cycle  in  vivo.   This  is  difficult  to  accomplish  with  myosin 
because  it  occurs  as  insoluble  filaments  at  low  ionic  strength.   However, 
heavy  meromyosin  (HMM)  and  subfragment-1  (S-l) ,  double  and  single  headed 
proteolytic  digestion  of  myosin  respectively,  are  soluble  at  low  ionic 
strength  and  therefore  their  interaction  with  actin  can  be  more  easily 
studied.   In  the  present  study  we  continued  our  investigation  of  the 
refractory  state  of  HMM  and  S-l,  a  state  which  we  discovered  occurs  during 
the  cycle  of  interaction  of  myosin  with  actin  and  ATP  during  whijh  the 
myosin  head  is  unable  to  bind  to  actin.   Only  when  the  refractory  state 
transforms  to  the  non-refractory  state  is  the  myosin  able  to  interact 
with  actin  and  the  transition  from  the  refractory  state  to  the  non- 
refractory  state  seems  to  be  one  of  the  major  rate  limiting  steps  in 
the  cyclic  interaction  of  myosin  with  actin  and  ATP.   The  occurrence  of  a 
refractory  state  has  considerable  implications  for  the  actin-myosin  cycle 
which  occurs  in  vivo  and  therefore  in  the  present  study  we  have  put  consid- 
erable effort  into  proving  its  existence  in  vitro  as  well  as  beginning 


Project  No.  Z01  HL  00409-05  LCB 

a  study  of  the  implication  of  such  a  state  for  in  vivo  muscle  models. 

Methods  Employed  and  Major  Findings: 

The  original  finding  which  led  us  to  postulate  the  exsitence  of  a  refractory 
state  is  that  considerable  dissociation  of  the  acto-HMM  occurs  even  under 
conditions  where  the  actin  maximally  activates  the  HMM  or  S-l  ATPase,  as 
shown  by  ultracentrifuge  and  ATPase  studies.   We  have  now  completed  studies 
using  laser-light  scattering,  turbidity  and  viscosity  to  measure  the  inter- 
action of  HMM  and  S-l  with  actin.   In  the  absence  of  ATP,  all  of  these 
techniques  suggest  that  marked  interaction  is  occurring  between  the  actin 
and  HMM  or  S-l.   On  the  other  hand,  in  the  presence  of  ATP, when  the  ATPase 
is  nearly  maximally  activated  by  actin  all  three  of  the  techniques  suggest 
that  less  than  10%  of  the  HMM  or  S-l  is  interacting  with  the  actin.   There- 
fore these  measurements  also  indicate  that  a  large  fraction  of  both  the  HMM 
and  S-l  are  in  a  refractory  state  during  their  interaction  with  actin  and 
ATP.   This  work  is  presently  in  press  in  Biochemistry.   In  addition  to  this 
study  we  have  completed  a  detailed  analytical  ultracentrifuge  investigation 
of  the  binding  of  HMM&S-l  to  actin  under  conditions  of  maximal  actin- 
activation  of  the  HMM  and  S-l  ATPase.   Using  a  special  separation  cell  and 
ATP32  assay  we  have  been  able  to  isolate  the  HMM  and  S-l  which  do  not  bind 
to  actin,  i.e.,  are  in  the  refractory  state,  and  show  that  they  have 
essentially  normal  EDTA  and  actin- activated  ATPase  activity.   Therefore,  the 
HMM  and  S-l  which  remain  unbound  to  actin  are  the  same  as  normal  protein  — 
they  are  simply  transiently  in  the  refractory  state  during  their  inter- 
action with  actin  and  ATP.   In  the  ultracentrifuge  study  we  have  also  been 
able  to  demonstrate  that  only  half  as  much  S-l  as  HMM  binds  to  actin  under 
conditions  of  maximal  actin  activation  of  the  ATPase,  possibly  because  S-l 
has  only  one  head  while  HMM  has  two  heads  and  one  bound  HMM  head  can  carry 
the  other  head  down  with  the  actin.   Finally,  in  this  ultracentrifuge  study 
we  have  found  that,  as  expected,  as  the  KC1  concentration  is  increased 
and  the  actin-activated  ATPase  decreases  the  amount  of  bound  HMM  at  a 
given  actin  concentration  also  decreases.   We  have  also  been  able  to 
demonstrate  that  a  4-fold  change  in  g  has  no  effect  on  the  amount  of  HMM 
bound,  suggesting  that  the  sedimenting  process  itself  has  no  effect  on  the 
ratio  of  free  and  bound  HMM.   In  summary  the,  this  detailed  analytical  ultra- 
centrifuge study  confirms  our  original  evidence  for  the  refractory  state. 
It  is  presently  being  written  up  for  publication. 

In  addition  to  this  ultracentrifuge  study  we  have  begun  a  stopped -flow 
light  scattering  study  of  the  interaction  of  actin,  S-l  and  ATP.   By  adding 
a  stoichiometric  amount  of  ATP  to  the  acto-S-1  complex,  we  are  able  to  observe 
a  single  cycle  of  interaction  of  the  S-l  with  actin  and  ATP.   Oar  results 
show  that  in  this  cycle,  first  a  rapid  dissociation  of  the  acto-S-1  complex 
occurs,  followed  by  a  10- fold  slower  rebinding  of  the  S-l  to  the  actin. 
Furthermore,  the  rate  of  rebinding  of  the  S-l  to  actin  is  always  equal 
to  the  steady-state  ATPase  rate  and  levels  off  at  high  actin  concentration 
just  as  the  steady  rate  does.   The  finding  that  the  rate  of  rebinding 
does  not  increase   linearly  with  actin  concentration,  but  rather  reaches 
a  maximum  value  strongly  implies  that  the  S-l  undergoes  a  conformational 


Project  No.  Z01  HL  00409-05  LCB 

change  prior  to  its  rebinding  to  actin  and  at  high  actin  concentration  this 
conformational  change  becomes  rate  limiting.   This  conformational  change  is 
apparently  what  we  have  previously  described  as  the  transition  from  the 
refractory  to  the  non-refractory   state.   The  ability  to  observe  a  single 
cycle  of  interaction  of  S-l,  actin  and  ATP  should  be  a  powerful  technique 
for  clarifying  the  cycle  of  interaction  of  the  myosin  bridge  with  actin 
myosin  and  ATP  which  occurs  in  vivo. 

In  this  regard,  in  addition  to  these  experimental  studies,  we  are  involved 
in  theoretical  studies  on  the  mechanism  of  muscle  contraction  in  vivo.   One 
paper  has  already  been  published  in  Biophys.  J.  based  on  this  work  and  we 
are  presently  working  with  Terrell  Hill  on  a  model  which  icnludes  a 
refractory  state  as  part  of  the  mechanism  of  cross-bridge  interaction  in  vivo. 

Proposed  Course  of  Research: 

We  plan  to  continue  our  stopped- flow  experiments  extending  them  to  higher 
temperatures  and  also  using  HMM  as  well  as  S-l.   We  also  plan  to  perform 
these  stopped  flow  measurements  using  a  3-syringe  stopped-flow  apparatus 
which  will  permit  us  to  mix  the  S-l  and  ATP  and  at  varying  times  thereafter 
add  the  actin.   This  will  allow  us  to  determine  rate  constants  in  the  cycle 
which  cannot  be  determined  using  the  2-syring  apparatus.   We  also  plan  to 
continue  our  collaboration  with  Terrell  Hill  on  the  theoretical  implication 
of  the  refractory  state  for  models  of  muscle  contraction. 

Significance  to  Bio-Medical  Research: 

This  work  is  aimed  at  gaining  a  better  understanding  of  the  basic  mechanism 
of  muscle  motility  and  the  control  of  this  motility,  phenomena  which  occur 
not  only  in  skeletal  muscle,  but  also  in  such  diverse  systems  as  cardiac 
muscle,  arterial  smooth  muscle,  platelets,  and  perhaps  within  all  cells 
where  protoplasmic  streaming  occurs. 

Publications : 

Hill,  T.L. ,  Eisenberg,  E. ,  Chen,  Y-D. ,  and  Podolsky,  R.J.   Some  self- 
consistent  two-state  sliding  filament  models  of  muscle  contraction. 
Biophys.  J.  Vol.  15:  335-372  (1975)  . 

Fraser,  A.B-,  Eisenberg,  E. ,  Kielley,  W.W. ,  and  Carlson,  F.D.   The  inter- 
action of  heavy  meromyosin  and  subfragment-1  with  actin:  Physical  measurements 
in  the  presence  and  absence  of  ATP.   Biochemistry  (in  press) . 

Keyword  Descriptors:   Actin,  myosin,  muscle  biochemistry,  ATPase. 


rU> 


Project  No.  Z01  HL  00410-02  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Cellular  Physiology 

3.  Bethesda,  Md.  20014 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Mechanism  of  myosin  and  actomyosin-Mg-ATPase 

Previous  Serial  No.      NHLI-19 

Principal  Investigators:  Stephen  P.  Chock 

Evan  Eisenberg 

Collaborating  Investigator:  P.  B.  Chock 

Project  Description: 

Actin  and  myosin  appear  to  be  ubiquitous  proteins  that  not  only  provide  the 
basis  of  motility  in  higher  animals  and  protozoa,  but  also  may  be  intimately 
involved  in  cellular  development  as  well.   In  muscle,  they  constitute  the 
main  proteins  involved  in  the  cyclic  process  of  myosin-actin  crossbridge 
formation  which  provides  the  basis  of  muscle  contraction  and  thus  motility 
itself.   The  important  roles  of  actin  and  myosin  in  most  living  systems  makes 
it  necessary  for  us  to  understand  the  mechanism  of  their  function. 

Objectives: 

Since  it  is  evident  that  muscle  contraction  constitutes  the  main  aspect  of 
animal  motility,  the  understanding  of  how  muscle  works  will  no  doubt  provide 
basic  information  on  the  topic  of  motility  as  a  whole.   In  muscle,  the 
hydrolysis  of  ATP  by  myosin  in  the  presence  of  Mg++  provides  the  energy 
source  for  the  contractile  process.   It  is  therefore  logical  to  approach 
the  problem  of  muscle  contraction  by  first  studying  the  mechanism  of  myosin- 
ATP  interaction.   With  the  information  gained  from  the  above  studies  one 
can  then  approach  the  more  complex  system  of  actin-myosin-ATP  interaction 
which  is  the  key  event  in  muscle  contraction  itself. 

In  spite  of  the  fact  that  the  myosin  and  actomyosin  Mg-ATPase  has  been 
studied  for  decades  the  basic  information  of  how  myosin  and  actomyosin  inter- 
act with  ATP  in  the  presence  of  Mg++  is  still  lacking.   This  information 
is  important  because  it  might  explain  how  the  basic  biochemical  steps  relate 
to  the  physiological  mechanism  of  contractile  process. 

Methods  Employed  and  Major  Findings: 

Since  steady  state  kinetics  alone  cannot  provide  sufficient  information  on 
the  elementary  steps  of  the  interaction  of  myosin  and  actomyosin  with  ATP, 
the  use  of  presteady  state  kinetics  becomes  necessary.   With  the  accessibility 
of  an  excellent  stopflow  system  designed  and  built  by  Dr.  P.B.  Chock  here 


Project  No.  Z01  HL  00410-02  LCB 

at  NIH,  significant  information  concerning  the  nature  of  the  interaction  of 
myosin  and  actomyosin  with  ATP  has  been  elucidated. 

Pre-steady  state  and  steady  state  H+  release 

By  simultaneously  monitoring  both  the  prestt-ady  and  steady  state  time  course 
of  H+  release  following  the  addition  of  ATP  to  heavy  meromyosin  (HMM,  a 
proteolytic  fragment  of  myosin)  under  conditions  of  excess  Mg++,  0.5M  KC1 
pH8,  250C,  the  following  information  concerning  the  interaction  of  myosin 
active  site  with  ATP  has  been  obtained:  (1)  The  binding  of  ATP  to  the 
myosin  active  site  is  essentially  irreversible  and  both  of  the  HMM  active 
sites  bind  ATP  equally  well  with  no  observable  head  to  head  interaction 
during  ATP  binding;  (2)  Under  the  single  turnover  condition,  that  is,  when 
the  concentration  of  HMM  active  sites  is  less  than  or  equal  to  the  concen- 
tration of  ATP  added,  the  release  of  H+  following  the  addition  of  ATP 
proceeds  in  two  exponential  phases  with  two  distinct  rate  constants  —  a 
very  fast  initial  phase  with  an  apparent  second  order  rate  constant  of 
7X10^M-lg-l  and  a  slow  exponential  phase  with  a  first  order  rate  constant, 
kcat,  of  0.016S-!;  (3)  Under  steady  state  conditions,  that  is,  when  the 
concentration  of  ATP  added  is  much  greater  than  the  concentration  of  HMM 
active  sites,  the  two  exponential  phases  of  H+  release  are  interspaced  with 
a  linear  steady  state.   The  fact  that  this  linear  steady  state  rate  is  equal 
to  kcat  when  calculated  on  the  basis  of  the  molecular  weight  of  each  active 
HMM  head,  signifies  that  in  the  steady  state  both  myosin  active  sites 
hydrolyze  ATP  independently  and  at  equal  rate;  (4)  the  magnitude  of  the 
initial  proton  release  equals  0.4H+  released  per  ATP  bound,  and  the  magnitude 
of  the  H+  released  in  the  slow  exponential  phase  equals  0.6H+  released  per 
ATP  bound.   Together,  they  total  1  H+  released  per  ATP  hydrolyzed  as  expected 
for  the  hydrolysis  of  ATP  at  pH8.   (5)  The  fact  that  the  characteristic  of 
the  initial  H+  burst  is  sufficiently  different  from  the  initial  phosphate 
burst  as  reported  by  Taylor  et  al. ,  suggests  that  the  H+  burst  does  not 
represent  the  phosphate  burst.   In  other  words,  if  Taylor's  data  on  the 
rate  of  the  phosphate  burst  was  correct  then  our  result  suggests  that  the 
fast  initial  H+  burst  preceeds  the  cleavage  of  ATP  and  that  no  additional 
H+  is  released  in  the  cleavage  step  represented  by  the  phosphate  burst 
until  the  rate  limiting  step  controlled  by  kcat-   This  in  turn  suggests 
that  the  H+  burst  is  related  to  ATP  binding  rather  than  to  the  cleavage 
step  as  described  by  Taylor  et  al. 

Pre-steady  and  steady  state  fluorescence  changes 

By  measuring  the  intrinsic  tryptophan  fluorescence  change  following  the 
addition  o  f  ATP  to  HMM  using  the  stopf low  technique  the  following  events 
are  observed:  (1)  There  is  a  very  fast  fluorescence  change  following  the 
binding  of  ATP  with  an  apparent  second  order  rate  constant  equal  to  that 
observed  for  the  H+  burst;  (2)  under  the  condition  of  a  single  turnover  of 
ATP  the  fast  initial  fluorescence  change  is  followed  by  a  slow  exponential 


A3  8 


Project  No.  Z01  HL  00410-02  LCB 

fluorescence  decay  with  a  rate  constant  equal  to  kcat;  (3)  Under  the 
steady  state  condition,  the  fluorescence  level  remains  constant  for  a  length 
of  time  equal  to  the  time  that  the  steady  state  ATPase  occurs  before  the 
ATP  is  depleted;  (4)  The  amplitude  of  the  presteady  state  fluorescence 
change  is  proportional  to  the  concentration  of  ATP  added  and  reaches  a 
maximum  when  the  concentration  of  ATP  added  equals  the  concentration  of  HMM 
active  sites.   This  therefore  represents  a  method  for  myosin  active-site 
titration.   Based  on  the  above  observations  the  following  conclusions  are 
drawn:  (1)  Both  the  preteady-state  fluorescence  change  and  the  H+  burst 
represent  a  conformational  change  in  the  myosin  active  site  following  the 
binding  of  ATP.   (2)  The  0.4H+  released  in  the  burst  might  well  represent 
an  ionization  of  H+  from  the  protein  due  to  a  conformational  change 
following  the  binding  of  ATP.   It  probably  does  not  represent  the  hydrolysis 
of  ATP  per  se  because  at  pH8  1  H+  would  be  released  per  ATP  hydrolyzed; 
unless  the  pk  of  bound  phosphate  were  quite  different  from  that  of  the 
normal  phosphate.  (3)  Since  the  2nd  order  rate  constant  for  ATP  binding  is 
only  7X10^  which  is  much  slower  than  a  diffusion  limited  rate  constant, the 
ATP  probably  binds  in  a  2-step  process  involving  first  the  rapid 
formation  of  a  collison  intermediate  followed  by  a  slower  conformational 
change.   The  simplest  scheme  that  can  be  drawn  for  the  interaction  of  myosin 
with  ATP  in  the  presence  of  Mg++  is  therefore  as  follows: 

kl       k2              kcat 
M+T  v    -  MT  >  MT*  >  M+Product 

k_x  +0.4H+  +0.6H+ 

where  M  is  myosin  active  site  and  T  is  ATP  and  MT*  represents  the  fluorescence 
intermediate.   The  above  scheme  predicts  that  at  high  IT],  the  rate  of  the 
fluorescence  burst  will  plateau  at  k2-   In  recent  work  this  has  indeed 
been  observed  at  low  ionic  strangth  and  at  temperatures  below  20°C. 

Effect  of  temperature  and  ionic  strength 

The  effect  of  temperature  and  ionic  strength  on  the  binding  of  ATP  has 
also  been  studied  using  the  fluorescence  technique.   The  conclusions  are: 
(1)  Lowering  the  KC1  concentration  from  0.5M  to  0.1M,  increases  the 
magnitude  of  the  equilibrium  constant (*i. )  by  about  6-fold,  while  it  decreases 

k-1 
the  rate  of  the  conformational  change  (k2)  by  about  3-fold.   (2)  Lowering 
the  temperature  from  20OC  to  10oc  decreases  k2  by  about  5-fold  while  it 
increases  the  value  of  k-|   by  about  3-fold.   The  effect  of  ionic  strength 

k-l 
is  therefore  primarily  on  the  equilibrium  step  while  the  effect  of  temperature 
is  primarily  on  the  step  involving  the  conformational  change.   This  in 
turn  suggests  that  the  conformational  change  involves  a  large  energy  of 
activation  which  implies  that  it  might  involve  a  large  cooperative 
structural  change  in  the  myosin  following  the  binding  of  ATP. 

Actomyosin-ATP  interaction 

By  employing  both  light  scattering  and  fluorescence  techniques  using  the 
stopflow  apparatus,  the  mechanism  of  the  actin-myosin-ATP  interaction  has 
been  explored.   Since  the  association  and  dissociation  of  the  actomyosin 

3  'tf 


Project  No.  Z01  HL  00410-02  LCB 

complex  can  be  measured  by  light  scattering, the  rate  of  the  dissociation 
and  reassociation  of  the  actomyosin  complex  can  be  correlated  with  a 
decrease  and  increase  in  turbidity.   In  preliminary  experiments  we  found 
that  the  rate  of  the  turbidity  decrease  i.e.  the  rate  of  actomyosin 
dissociation  in  the  presence  of  ATP,  is  faster  than  the  rate  of  the  fluores- 
cence change;  and  that  the  maximum  rate  of  the  fluorescence  change  is  the 
same  whether  or  not  actin  is  present.   This  implies  that:  (1)  ATP  dissociates 
actomyosin  prior  to  the  formation  of  the   [mt*j  intermediate  and  the  conforma- 
tional change  occurs  after  the  dissociation  of  the  myosin  from  the  actin. 
(2)  The  fact  that  Vmax  for  the  formation  of  MT*  is  attained  at  lower  ATP 
concentration  in  the  presence  of  actin  than  in  the  absence  of  actin,  suggests 
that  actin  enhances  the  accessiblity  of  myosin  ATP-binding  sites  through  a 
specific  spatial  orientation  of  the  myosin  head.   In  other  words  ATP  binds 
better  to  actomyosin  than  to  myosin  alone.   (3)  The  rate  of  decay  of  the 
C  MT*]  intermediate  equals  the  rate  of  the  turbidity  rerise  which  in  turn 
equals  the  steady  state  actomyosin  ATPase.   This  suggests  that  not  only  is 
[MT*J  an  intermediate  in  the  mechanism  of  the  actomyosin  ATPase  but  its 
disappearance  is  intimately  involved  with  the  rate  limiting  step  in  the 
cyclic  interaction  of  myosin  with  actin  in  the  presence  of  ATP,  i.e.  with 
the  transition  from  the  refractory  to  the  non-refractory  state.   In  summary, 
the  above  observations  suggest  that  for  the  first  time  one  can  observe  a 
cyclic  process  of  actin-myosin-ATP  interaction  in  vitro  which  might  be 
analogous  to  the  mechanistic  events  taking  place  in  vivo. 

Proposed  course  of  research 

More  detailed  experiments  are  still  needed  to  clarify  the  elementary  steps 
of  the  catalytic  mechanism  of  both  the  myosin  and  actomyosin  ATP  interaction. 
Furthermore,  it  is  still  unclear  whether  the  two  active  sites  of  myosin  are 
kinetically  identical.   Our  preliminary  experiments  suggest  that  they  are 
the  same,  but  more  detailed  studies  are  required  before  a  firm  conclusion 
can  be  drawn.   Finally,  in  collaboration  with  Dr.  Sally  Mulhern  we  are  in 
the  process  of  studying  the  mechanism  of  SH^-blocked  myosin.   This  is  of 
interest  because  SHi-blocked  myosin  binds  ATP  almost  as  well  as  normal  myosin, 
but  its  ATPase  activation  by  actin  is  much  decreased  and  we  hope  to  determine 
which  steps  in  the  cycle  are  altered  by  blocking  SH^. 

Publications : 

Chock,  S.P.  and  Eisenberg,  E. ,  Heavy  meromyosin  Mg-ATPase:  Pre-steady  state 
and  steady  state  H+  release.   Proc.Nat.  Acad.  Sci.  71,  4915  (1974). 

Keyword  descriptors:   Kinetics,  myosin,  actomyosin. 


tlo 


Project  No.  Z01  HL  00501-01  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Cellular  Biochemistry  and 
Ultrastructure 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Acanthamoeba  Actin:   Isolation  and  Role  in  Cell 

Motility 

Previous  Serial  Number:    None 

Principal  Investigators:   Edward  D.  Korn 

David  J  .  Gordon 

Project  Description: 

Objectives:   In  the  years  1968-1972,  this  Laboratory  provided  some  of  the 
earliest  and  most  definitive  evidence  for  the  presence  of  actin  in  cells.   An 
isolation  procedure  was  developed  based  on  the  known  properties  of  skeletal 
muscle  actin  and  the  pure  Acanthamoeba  actin  that  was  prepared  was  shown  to 
be  very  similar,  but  not  identical,  to  muscle  actin  in  amino  acid  composition, 
polymerization  to  F-actin  and  ability  to  activate  the  Mg   -ATPase  of  muscle 
myosin.   However,  the  yield  of  actin  was  very  low,  perhaps  17»  of  the  total 
cytoplasmic  actin.  and  there  were  apparently  significant  differences  noted 
between  the  Acanthamoeba  and  muscle  actins  . 

This  research  has  been  re-initiated  with  the  long  range  goal  of  understanding 
the  role  of  cytoplasmic  actomyosin  in  cell  movement  in  Acanthamoeba  castell- 
anii  and,  by  extrapolation,  in  all  cells  .   Toward  this  end,  the  following 
lines  of  research  have  been  initiated:   1)  the  isolation  of  pure  native 
Acanthamoeba  actin  in  sufficient  yield  for  careful  physical  and  kinetic  stu- 
dies .   2)  Complete  characterization  of  its  physical  and  enzymatic  properties, 
including  viscosity,  bound  nucleotide,  the  depolymerization-polymerization 
cycle,  binding  and  activation  of  muscle  myosin  subfragments,  and  interaction 
with  the  skeletal  muscle  regulatory  proteins.   Particular  attention  will  be 
paid  to  those  differences  in  properties  between  Acanthamoeba  and  muscle  actin 
which  might  account  for  the  widely  differing  nature  of  the  contractile  proces- 
ses in  these  cell  types.   3)  Reconstruction  of  the  Acanthamoeba  actomyosin 
system  from  its  components  and  characterization  of  its  enzymatic  properties. 
This  system  is  of  particular  interest  because  of  the  requirement  for  a  co- 
factor  (as  yet  poorly  characterized)  for  activation  of  Acanthamoeba  myosin 
by  actin.   4)  Investigation  of  how  the  energy  of  the  Acanthamoeba  actomyosin 
ATPase  system  is  transduced  into  cell  movement.   In  particular,  attention  has 
been  focused  on  how  actin  microfilaments  might  be  anchored  to  the  plasma  mem- 
brane, with  which  they  appear  closely  associated  in  intact  cells  and  in 
isolated  plasma  membranes . 


/*/ 


Project  No.  Z01  HL  00501-01  LCB 

Methods  Employed:   The  purification  methods  which  have  been  tried  individually 
and  in  combination  to  isolate  actin  from  whole  amoebae  or  from  ethanol  or  ace- 
tone powders  of  amoebae  include  gel  filtration,  ion  exchange  chroma tography, 
ammonium  sulfate  fractionation  and  sedimentation  of  actin  filaments  under 
appropriate  ionic  conditions .   Sodium  dodecyl  sulfate-gels,  stimulation  of 
Mg  ATPase  activity  of  muscle  myosin  subfragments,  and  gross  changes  in  vis- 
cosity, flow  birefringence,  and  sedimentation  velocity  have  been  used  to 
monitor  the  purity  and  state  of  Acanthamoeba  actin  preparations. 

Preliminary  Results:   The  major  problems  in  isolating  Acanthamoeba  actin  have 
been  to  circumvent  the  high  proteolytic  activity  of  the  amoeba  homogenate  and 
to  separate  actin  from  myosin  and  other  associated  proteins  by  methods  suffi- 
ciently mild  to  avoid  denaturation .   The  most  promising  method  to  date  involves 
extraction  of  fresh  amoebae  in  a  low  salt,  Ca   -ATP  buffer,  adsorption  of  the 
proteins  to  DEAE-cellulose  by  a  KCl-gradient  .   This  procedure  gives  a  high 
yield  of  80-907o-pure  actin  which  shows  flow  birefringence  under  polymerizing 
conditions  and  can  activate  muscle  myosin  subfragment-1  with«~»157c,  of  the 
specific  activity  of  skeletal  muscle  actin.   This  activity  is  only  slightly 
less  than  that  observed  by  Weihing  and  Korn  (this  Laboratory,  1971)  for  puri- 
fied actin  obtained  in  much  lower  yield  by  an  arduous  procedure. 

In  addition  to  lower  myosin-activation,  it  also  appears  that  Acanthamoeba 
actin  has  different  polymerization  properties  than  muscle  actin.   In  the  pre- 
sence of  Ca   -ATP  and  0 . 1M  KC1,  muscle  actin  polymerizes  rapidly  (within 
seconds)  at  0°C;  Acanthamoeba  actin  remains  almost  totally  depolymerized 
under  these  conditions,  even  after  36  hrs  at  0°.   If  Mg"1-1"  is  added  or  if  the 
mixture  is  warmed  to  25°,  polymerization  of  Acanthamoeba  actin  is  promoted, 
though  it  is  never  as  complete  as  for  muscle  actin.   Furthermore,  under  con- 
ditions where  muscle  actin  depolymerizes ,  some  Acanthamoeba  actin  remains  in 
filaments .   An  example  of  this  is  in  preparation  of  plasma  membranes  with 
associated  actin  filaments  which  remain  polymerized  in  lOmM  Tris-HCl  buffer. 
Finally,  there  is  a  tendency  for  Acanthamoeba  actin,  isolated  under  some 
conditions,  to  form  precipitates  rather  than  filaments  when  KC1  is  added. 
Further  work  is  needed  to  decide  which  of  these  differences  from  muscle  actin 
are  trivial  artifacts  of  the  purification  methods  employed  and  which  reflect 
physiologically  important  differences  in  the  properties  of  cytoplasmic  actin 
or  of  cytoplasmic  actin  in  association  with  other  proteins  in  the  cytoplasmic 
contractile  system. 

Proposed  Course  of  Research:   Since  it  appears  that  we  are  close  to  accomp- 
lishing the  first  goal,  the  purification  in  high  yield  of  Acanthamoeba  actin, 
work  next  year  will  probably  be  largely  concerned  with  fully  characterizing 
the  Acanthamoeba  actin  as  listed  in  Objective  2.   Should  Acanthamoeba  myosin 
and  cofactor  be  available  (see  Project  No.  Z01  HL  00502-01  LCB) their  inter- 
action with  Acanthamoeba  actin  will  also  be  studied  (Objective  3)  . 

Keyword  Descriptors  : 

Acanthamoeba  castellanii,  amoeba,  actin,  microfilaments,  cytoplasmic  acto- 


/32 


Project  No.  Z01  HL  00501-01  LCB 
myosin,  contractile  proteins,  cell  motility,  membrane-associated  proteins. 
Honors  and  Awards :   None 
Publications:   None 


H3 


Project  No.  Z01  HL  00502-01  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Cellular  Biochemistry  and 
Ultrastructure 

3.  Bethesda,  Maryland 
PHS-NIH 

Individual  Project  Report 
July  I,    1974  through  June  30,  1975 

Project  Title:  Acanthamoeba  Myosin  Cofactor:   Isolation  and 

Function 

Previous  Serial  Number:    None 

Principal  Investigators:   Edward  D.  Korn 

Hiroshi  Maruta 

Project  Description: 

Objectives:   In  the  years  1969-1972,  this  Laboratory  provided  some  of  the 
earliest  and  most  definitive  evidence  for  the  presence  of  a  myosin  ATPase  in 
the  cytoplasm  of  cells  .   This  molecule,  together  with  cytoplasmic  actin,  is 
undoubtedly  responsible  for  many  types  of  cell  motile  processes  in  Acanth- 
amoeba .   Acanthamoeba  myosin  is  unique  among  known  myosins  in  having  a  much 
smaller  molecular  weight,  about  180,000  vs  420,000,  and  in  requiring  a  co- 
factor  protein  for  actin-activation  of  the  Acanthamoeba  myosin  Mg   -ATPase  . 
Acanthamoeba  cofactor  also  increases  the  activity  of  muscle  actomyosin. 
Although  the  Acanthamoeba  myosin  was  previously  obtained  in  a  highly  purified 
form,  Acanthamoeba  cofactor  was  obtained  only  as  a  relatively  crude  fraction 
separated  from  the  myosin  by  hydroxy lapatite,  the  last  step  in  the  purifica- 
tion of  Acanthamoeba  myosin.   We  have  now  renewed  this  research  with  the 
specific  purpose  of  purifying  the  Acanthamoeba  myosin  cofactor  and  studying 
the  nature  of  its  interaction  with  Acanthamoeba  actin  and  myosin  and  with 
skeletal  muscle  actin  and  myosin  and  the  muscle  regulatory  proteins  .   This 
problem  has  wide  implications  because  of  the  universality  of  cytoplasmic 
actin  and  myosin  and  recent  evidence  that  some  of  the  mammalian  cytoplasmic 
systems  may  also  have  a  cofactor  protein. 

Methods  and  Preliminary  Findings : 

1 .   Attempt  to  purify  rapidly  Acanthamoeba  myosin  free  of  actin  and  cofactor  . 
This  is  necessary  in  order  to  have  a  myosin  fraction  suitable  for  the  assay 
of  cofactor  activity.   Crude  Acanthamoeba  extract  can  be  applied  to  an  ATP- 
agarose  column  in  0.01M  imidazole,  pH  7,  -0 .05M  KC1  and  cofactor  eluted  by 
raising  the  KC1  concentration  to  0.1  M  in  the  presence  of  2  mM  EDTA .   Myosin 
is  eluted  only  at  0.5M  KC1.   Prepared  in  this  way,  cofactor  is  free  of  myosin 
and  actin  and  myosin  is  free  of  cofactor  and  actin.   Unfortunately,  the  cap- 
acity of  the  ATP-agarose  column  is  very  low  and  since  the  cofactor  and  myosin 
each  comprise  less  than  1%  of  the  total  protein  this  procedure  is  not  suitable 
for  large  scale  isolations.   It  may  be  adequate,  however,  for  providing 
sufficient  cofactor-free,  actin-free  myosin  for  assay  purposes.   Cofactor  is 
unstable  unless  kept  at  -80°  in  the  presence  of  albumin  (lmg/ml) . 


/5^ 


Project  No.  Z01  HL  00502-01  LCB 

2.  Attempt  to  purify  Acanthamoeba  cof actor  on  DEAE-cellulose  and  by 
ammonium  sulfate  fractionation.   The  cofactor  fraction  from  the  ATP-agarose 
column  can  be  adsorbed  to  DEAE-cellulose  from  0.01M  Tris-HCl,  pH  8.0,  -0.01M 
KC1  and  eluted  with  0.045M  KC1 .   Further  purification  was  not  possible  because 
of  rapid  loss  of  activity.   When  crude  Acanthamoeba  extracts  were  applied  to 
the  DEAE-cellulose,  cofactor  activity  was  eluted  only  together  with  the  myosin 
in  0.09-0.13M  KC1  and  precipitated  with  the  myosin  between  1.1  and  1  .6M 
ammonium  sulfate  suggesting  that  all  of  the  Acanthamoeba  cofactor  exists  as 

a  myosin  complex. 

3.  Attempt  to  purify  Acanthamoeba  cofactor  by  gel  filtration.  Cofactor  pro- 
tein and  myosin  partially  dissociate  in  0.2MKC1  and  cofactor  elutes  slightly 
after  myosin  on  Sephadex  G-200  and  Biogel  P-300  columns . 

Proposed  Course  of  Research:   It  is  apparent  that  isolation  of  Acanthamoeba 
cofactor  will  not  be  easy  because  of  its  instability  when  separated  from 
myosin,  its  low  concentration  in  the  Acanthamoeba  extracts  and  the  low 
capacity  of  the  most  reliable  method  for  separating  cofactor  and  myosin, 
ATP-agarose  columns.   Two  alternatives  will  be  pursued.   First,  the  lengthy 
procedure  previously  developed  for  the  purification  of  Acanthamoeba  myosin 
will  be  carried  out  until  the  last  step  for  which  ATP-agarose  separation  will 
be  substituted.   In  this  way,  by  purifying  the  complex  of  Acanthamoeba  myosin 
and  cofactor,  we  can  maintain  activity  of  the  cofactor  and  perhaps  obtain 
both  purified  myosin  and  cofactor.   Second,  attempts  will  be  made  to  purify 
myosin  and  cofactor  by  complexing  them  to  polystyrene  beads  to  which  F-actin 
has  been  previously  adsorbed.   It  has  been  shown  by  others  that  polystyrene 
will  bind  muscle  F-actin  and  that  muscle  myosin  will  bind  to  the  actin  that 
is  bound  to  the  polystyrene.   It  may  then  be  possible  to  recover  Acanthamoeba 
myosin-cofactor  from  crude  Acanthamoeba  extracts  or  from  partially  purified 
fractions  freed  of  actin. 

Keyword  Descriptors  : 

Acanthamoeba  castellanii ,  amoeba,  myosin,  myosin  cofactor,  cell  motility, 
cytoplasmic  actomyosin,  cytoplasmic  myosin. 

Honors  and  Awards:   None 

Publications:   None 


/3S" 


Project  No.  Z01  HL  00504-10  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Cellular  Biochemistry  and 
Ultrastructure 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual   Project   Report 

July    1,    1974   through   June    30,    1975 

Project   Title:  Plasma   Membrane   and   Phagosome   Membranes   of  Acanth- 

amoeba 

Previous  Serial  Number:   NHLI-26,  NHLI-27 

Principal  Investigator:    Edward  D.  Korn 

Other  Investigators:       Sharron  Smith 

Shmuel  Batzri 

Project  Description: 

Objectives:   It  is  the  purpose  of  this  research  ultimately  to  determine  the 
complete  chemical  composition  and  macromolecular  organization  of  the  amoeba 
plasma  membrane  and  related  intracellular  membranes  as  well  as  the  chemical 
mechanism  of  their  fusions  and  interconversions .   In  past  years  we  have  found 
that  the  plasma  membrane  consists  of  approximately  one-third  each  by  mass  of 
protein,  lipid  (phospholipids+sterols)  and  lipophosphonoglycan,  a  novel  poly- 
meric glycosphingolipid .   In  addition  cytoplasmic  actin  filaments  seem  to  be 
intimately  associated  with  the  plasma  membrane.   Phagosome  membranes,  which 
are  derived  from  the  plasma  membrane  but  undergo  subsequent  fusions  with 
intracellular  membranes,  have  been  shown  to  have  a  lipid  composition  similar 
to  the  plasma  membrane  .   The  plasma  membrane  also  contains  many  of  the  enzy- 
mes of  phospholipid  hydrolysis  and  re-synthesis  but  is  not  capable  of  total 
phospholipid  synthesis  de  novo.  Work  this  year  has  largely  been  concerned 
with  a  more  detailed  comparison  of  the  protein  composition  of  plasma  memb- 
ranes and  phagosome  membranes  and  studies  on  the  mechanism  of  membrane  fusion 
with  a  model  system  of  phospholipid  vesicles . 

Methods  and  Findings: 

Composition  of  Plasma  Membranes  and  Phagosome  Membranes:   Isolated  plasma 
membranes  had  previously   been  found  to  contain  two  major  polypeptides:  actin 
and  a  polypeptide  of  about  15,000  daltons  .   Actin  is  not  a  true  membrane 
component  and  can  be  removed  by  any  of  several  procedures  that  depolymerize 
actin  filaments .   We  have  now  found  that  membranes  from  which  actin  has  been 
removed  by  6M  KI  show  a  fairly  constant  ratio  of  about  0.5  mg  protein/umole 
of  phospholipid  (about  0.65  mg  protein/mg  phospholipid),  although  there  may 
be  more  protein  in  the  plasma  membranes  of  cells  at  late  stationary  phase  of 
growth.   The  15,000  dalton  polypeptide  increases  from  a  low  of  about  107»  of 
the  actin-free  plasma  membrane  protein  to  about  507»  as  the  cells  progress 
from  early  log  growth  to  late  stationary  phase.   It  appears  likely,  therefore, 


/36 


Project  No.  Z01  HL  00504-10  LCB 
that  this  protein  is  a  component  of  the  membranes  of  late  stationary  or 
encysting  cells,  some  of  which  are  present  in  all  cultures.   At  all  stages  of 
cell  growth  the  ratio  of  lipophosphonoglycan  to  phospholipid  in  the  plasma 
membrane  seems  to  be  relatively  constant. 

Phagosomes  can  be  readily  isolated  from  cells  that  have  ingested  polystyrene 
beads  and  the  phagosome  membranes  can  be  isolated  after  mild  sonication.   The 
phagosome  membranes  contain  about  the  same  ratio  of  lipophosphonoglycan/phos- 
pholipid  as  the  plasma  membranes.   But  data  thus  far  collected  indicate  that 
the  protein/phospholipid  ratio  is  about  4  times  higher  in  phagosome  membranes 
than  in  plasma  membranes,  about  2  mg  protein/fimole  phospholipid.   This  inc- 
rease in  protein  content  is  compatible  with  some  recent  freeze-cleavage 
electron  microscopic  images  obtained  by  Dr.  Blair  Bowers,  in  this  Laboratory, 
which  show  at  least  10  times  more  intramembranous  particles  (presumably 
protein)  in  phagosome  membranes  than  in  the  plasma  membranes .   Since  the  mem- 
branes of  the  vesicles  with  which  the  phagosomes  fuse  do  not  have  a  high 
frequency  of  intramembranous  particles  it  seems  possible  that  their  high  con- 
centration in  the  phagosome  membranes  arises  by  loss  of  phospholipid  and 
lipophosphonoglycan  rather  than  acquisition  of  protein. 

Structure  of  Lipophosphonoglycan:   Studies  on  the  chemical  structure  of  this 
molecule  have  recently  been  resumed.   The  compound  consists  of  fatty  acids, 
neutral  sugars  (glucose,  mannose,  galactose,  xylose),  amino  sugars,  phyto- 
sphingosine,  glycerol,  inositol,  aminophosphonic  acids  and  phosphate.   From 
compositional  analysis  of  the  products  of  partial  acid  and  alkaline  hydrolysis 
we  have  now  developed  the  following  working  hypothesis  for  the  general  struc- 
ture of  the  molecule: 


4  glucose 
2  mannose 
1  galactose 


4  mannose 
1  xylose 


5  galactosamine,  13  phosphate,  30  aminophosphonate ,  20  glycerol,  6  inositol! 


12   Ceramides 
Sodium  dodecyl  sulfate  gel  electrophoresis  separates  lipophosphonoglycan  into 
two  bands  .   Preliminary  analysis  of  the  two  bands  separated  by  preparative 
gel  electrophoresis  indicates  that  one  of  the  bands  contains  xylose,  mannose 
and  a  small  amount  of  glucose  but  no  galactose  while  the  other  band  contains 
mannose,  galactose  and  glucose  but  no  xylose.   These  data  are  qualitatively 
in  agreement  with  the  data  from  the  partial  hydrolyses  and  suggest  that  the 
two  postulated  oligosaccharide  chains  may  be  on  separable  molecules . 

Membrane  Fusion:  Studies  on  the  interaction  of  single  bilayer  phospholipid 
vesicles  with  Acanthamoeba  have  been  continued  this  year.   Vesicles  are  made 
from  either  egg  phosphatidyl  choline  or  dipalmitoylphosphatidyl  choline  with 
the  bilayer  labeled  with  radioactive  phospholipid  and  the  internal  aqueous 
space  labeled  with  either  radioactive  glucose,  radioactive  inulin  or  amylase. 
Uptake  by  Acanthamoeba  of  both  preparations  of  phospholipid  vesicles  is  very 
rapid.   Uptake  of  the  egg  phosphatidyl  choline  vesicles  seems  to  be  by  endo- 


f3? 


Project  No.  Z01  HL  00504-10  LCB 
cytosis  since  uptake  is  inhibited  by  dini trophenol  and  incubation  at  4° 
(inhibitors  of  endocytosis) ,    uptake  of  markers  of  the  internal  aqueous  space 
is  exactly  equivalent  to  uptake  of  the  phospholipid  bilayer  and  the  phospho- 
lipid vesicle  membranes  can  be  visualized  by  electron  microscopy  within  endo- 
cytic  vesicles.   Uptake  of  the  dipalmitoyl  phosphatidyl  choline  vesicles  does 
not  seem  to  be  by  endocytosis  because  it  is  not  inhibited  by  dinitrophenol  or 
4°  and  occurs  even  when  the  cells  are  previously  fixed  with  glutaraldehyde  . 
Exchange  of  phospholipid  has  been  ruled  out  by  showing  that  the  net  uptake  of 
phospholipid  equals  the  uptake  of  radioactive  phospholipid.   Adsorption  of 
the  vesicles  to  the  cells  is  eliminated  by  showing  equal  uptake  of  vesicles 
irrespective  of  their  charge,  by  the  difference  between  the  dipalmitoyl  phos- 
phatidyl choline  vesicles  and  the  egg  phosphatidyl  choline  vesicles  whose 
surface  properties  are  similar  and  by  the  uptake  kinetics  .   The  most  likely 
explanation  is  fusion  of  the  dipalmi toylphospha tidyl  choline  vesicle  bilayer 
with  the  amoeba  plasma  membrane.   This  interpretation  is  supported  by  experi- 
ments with  multilamellar  vesicles  in  which  it  can  be  shown  by  electron  micro- 
scopy that  the  internal  vesicle  membranes  are  introduced  into  the  cytoplasm 
of  the  cell  but  are  not  within  an  endocytic  vesicle.   During  the  fusion 
process  about  40%  of  the  contents  of  unilamellar  vesicles  enter  the  Acanth- 
amoeba .   This  system  then  provides  a  model  for  a  fusion  process  that  should 
allow  the  introduction  of  otherwise  impermeable  substances  into  the  cytoplasm 
of  cells.   At  the  same  time  it  argues  that  metabolic  events,  such  as  hydrolysis 
and  re-synthesis  of  phospholipids,  are  not  obligatory  for  membrane  fusion  to 
occur  . 

Proposed  Course  of  Research: 

1.  The  separated  chains  of  lipophosphonoglycan  will  be  isolated  and  their 
chemical  composition  determined  and  compared  to  the  original  material  and  to 
each  other  .   If  possible  structural  studies  on  the  isolated  chains  will  be 
commenced . 

2.  We  will  continue  to  compare  the  composition  of  the  amoeba  plasma  membrane 
and  phagosome  membrane  at  different  stages  of  growth  and  attempt  to  correlate 
the  chemical  data  with  the  electron  microscopic  images  obtained  by  Dr.  Blair 
Bowers . 

Keyword  Descriptors : 

Acanthamoeba  cas tellanii ,  plasma  membranes,  phagosome  membranes,  membrane 
fusion,  lipophosphonoglycan. 

Honors  and  Awards :   None 

Publications : 

Korn,  E  .D  .  :   Preface.   In  Korn,  E  .D .  (Ed.):   Methods  in  Membrane  Biology. 
New  York,  Plenum  Press,  1974,  Vol.  1,  pp.  vii-x. 

Korn,  E.D.:   Preface.   In  Korn,  E  .D .  (Ed.):   Methods  in  Membrane  Biology, 
New  York,  Plenum  Press,  1974,  Vol.  2,  pp.  vii-viii . 


rt6 


Project  No.  Z01  HL  00504-10  LCB 

Korn,  E  .D .  :  Biochemistry  of  the  Plasma  Membrane.  In  Zweifach,  B.W.,  Grant, 
L.  and  McClusky,  R.T.  (Eds.):  The  Inflammatory  Process.  New  York,  Academic 
Press,  1974,  Vol.  I,  pp.  51-113. 

Korn,  E  .D . :   The  Isolation  of  the  Amoeba  Plasma  Membrane  and  the  Use  of  Latex 
Beads  for  the  Isolation  of  Phagocytic  Vacuole  (Phagosome)  Membranes  from 
Amoebae  Including  the  Culture  Techniques  for  Amoebae.   In  Fleischer,  S.  and 
Packer,  L.  (Eds.):   Methods  in  Enzymology,  New  York,  Academic  Press,  1974, 
pp.  686-698. 

Victoria,  E.J.  and  Korn,  E  .D . :   Enzymes  of  Phospholipid  Metabolism  in  the 
Plasma  Membrane  of  Acanthamoeba  castellanii  .   J .  Lipid  Res  .   16:  54-60,  1975. 

Korn,  E  .D  . :  Preface.  In  Korn,  E  .D  .  (Ed.):  Methods  in  Membrane  Biology, New 
York,  Plenum  Press,  1974,  Vol.  3,  pp.  IX-XI . 

Korn,  E  .D  .  :  Biochemistry  of  Endocytosis  .  In  Fox,  C .F .  and  Strominger,  J. 
(Eds  .) :  The  Biochemistry  of  Cell  Walls  and  Membranes,  London,  Butterworth 
&  Co.,  1975,  Vol.  2,  pp.  1-26. 

Editor:  Korn,  E .D .  (Ed.):  Methods  in  Membrane  Biology.  New  York,  Plenum 
Press,  1975,  Vol.  2,  263  pp.       

Editor:  Korn,  E .D .  (Ed.):  Methods  in  Membrane  Biology.  New  York,  Plenum 
Press,  1975,  Vol.  3,  246  pp. 


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Project  No.  Z01  HL  00505-09  LCB 
1. Lab. Cell  Biology 
PHS-NIH  2.Cell.Biochem.& 

Individual  Project  Report  Ultrastructure 

July  1,  1974  through  June  30,  1975      3.Bethesda,Marvland 

Project  Title:  Cytology  of  Acanthamoeba 

Previous  Serial  Number:    NHLI-28 

Principal  Investigator:    Blair  Bowers 

Other  Investigators:       Edward  D.  Korn 

Antoinette  Ryter 

Project  Description: 

Objectives:   To  elucidate  the  structural  basis  of  biochemical  and  physiological 
events  in  the  soil  ameba ,  Acanthamoeba  castellanii  .   Our  current  emphasis  is 
on  understanding  more  about  the  interrelationships  and  possible  interconver- 
sions  of  internal  membrane  systems  with  those  of  the  plasma  membrane  through 
biochemical  and  morphological  studies  of  endocytosis  and  through  morphological 
studies  of  the  membranes  with  freeze- fracture  replication  and  enzyme  cyto- 
chemistry . 

Methods  Employed: 

1.  Transmission  electron  microscopy  is  being  used  for  the  study  of  fixed  and 
embedded  cells  and  for  evaluation  of  isolated  cell  fractions  . 

2.  The  light  microscope  with  phase  contrast  and  Nomarski  interference  optics 
and  time-lapse  cinematography  are  being  used  for  study  of  living  cells. 

3.  The  technique  of  freeze-f racture  replication  (also  dependent  on  trans- 
mission electron  microscopy)  is  being  used  for  the  study  of  surface  and  inter- 
nal membrane  morphology. 

4.  Routine  and  special  biochemical  and  cytochemical  procedures  are  carried 
out  as  necessary  to  prepare  specimens  for  observation  with  the  microscopic 
procedures  and  to  supplement  morphological  data  . 

5.  Stereological  procedures  are  being  used  to  quantitate  and  interpret  elec- 
tron micrographs  . 

Major  Findings  : 

1.   Acanthamoeba  is  a  cell  with  a  very  high  endocytic  rate.   This  property 
implies  a  rapid  turnover  of  surface  membrane  because  each  endocytic  event 
carries  surface  membrane  into  the  cell.   It  is  probable  that  tne  large  number 
of  vacuoles  and  vesicles  visible  in  the  cytoplasm  of  Acanthamoeba  are  interre- 
lated with  the  surface  membrane,  possibly  as  part  of  a  system  for  recircula- 
tion of  surface  membrane.   Although  pinocytosis  appears  to  account  for  the 
major  portion  of  membrane  turnover  in  cells  cultured  on  soluble  medium,  a 


/>& 


Project  No.  Z01  HL  00505-09  LCB 

better  system  for  study  is  that  of  phagocytosis  since  the  ingested  particles 
provide  convenient  markers  for  newly  internalized  membranes.  The  problem  is 
being  approached  by  Dr.  A.  Ryter,  guest  worker,  in  two  ways:  one  by  cyto- 
chemical  methods  at  the  level  of  the  light  and  electron  microscopes,  and  the 
other,  by  using  a  morphometric  method  that  determines  relative  surface  areas 
of  membranous  structures  seen  in  the  electron  micrographs  . 

a)  The  cytochemical  study  is  attempting  to  localize  acid  phosphatase,  one 
of  the  characteristic  lysosomal  enzymes,  which  is  present  in  high  amount  in 
Acanthamoeba .   The  first  step  has  been  a  technical  study  on  the  conditions  of 
fixation,  washing,  cytochemical  incubations  and  physiological  states  of  the 
cells  .   All  of  these  factors  play  a  role  in  the  membrane  permeability  to  sub- 
strates used  in  the  cytochemical  localizations.   In  spite  of  many  difficul- 
ties due  to  a  low  and  variable  permeability  of  the  cells  some  quantitative 
results  concerning  the  behavior  of  digestive  vacuoles  during  and  after  phago- 
cytosis have  been  obtained  .   Cells  which  have  not  phagocytosed  particles  con- 
tain on  the  order  of  sixteen  vacuoles  per  cell.   Half  of  these  vacuoles  are 
acid  phosphatase  positive  (assessed  from  thin  sections)  .   After  allowing  cells 
to  phagocytose  yeast  until  an  average  uptake  of  8  yeast  per  cell  was  obtained, 
about  half  of  the  vacuoles  containing  acid  phosphatase  disappear  whereas 
about  half  of  the  phagosomes  containing  ingested  yeasts  become  acid  phospha- 
tase positive.   It  is  still  unknown  whether  the  apparent  fusion  of  acid  phos- 
phatase-positive  vacuoles  with  phagosomes  occurs  by  direct  fusion  or  after 
progressive  fragmentation  of  the  vacuoles  into  smaller  vesicles. 

b)  The  morphometric  study  is  in  its  preliminary  stages,  but  seems  to  be 
very  fruitful.   It  has  already  been  shown  that  during  phagocytosis  of  yeasts, 
the  surface  area  of  the  plasma  membrane  remains  constant  in  spite  of  the  high 
amount  of  internalized  membrane.   By  contrast,  the  surface  area  of  the  inter- 
nal vacuolar  membranes  decreases  and  this  decrease  corresponds  exactly  to  the 
surface  area  of  plasma  membrane  internalized  as  phagosomes  .   These  observa- 
tions suggest  that  the  renewal  of  the  plasma  membrane  may  be  made  by  the 
fusion  of  the  vacuoles  with  the  plasma  membrane . 

2.   Phagocytosis  is  one  expression  of  cell  motility.   Korn,  Weihing  and 
Pollard  (in  this  laboratory)  have  previously  identified  and  characterized  the 
major  proteins  (actin,  myosin  and  cofactor)  responsible  for  the  motility  of 
Acanthamoeba ,  but  the  way  in  which  these  proteins  function  is  totally  unknown. 
Phagocytosis  is  an  aspect  of  cell  motility  that  is  limited  in  time  and  space 
and  therefore  is  amenable  to  morphological  analysis.   This  morphological 
analysis  may  prove  useful  in  correlating  biochemical  studies  of  motility  with 
cell  behavior.   The  observations  in  this  study  are  relevant  to  our  interest 
in  plasma  membrane  recirculation  as  well.   Since  microfilaments  (shown  by 
Pollard  and  Korn  to  contain  actin)  are  presumably  a  marker  for  areas  of  the 
cytoplasm  that  are  actively  participating  in  cellular  movements,  particular 
attention  has  been  paid  to  their  distribution. 

Fine  structural  observations  are  necessarily  of  stopped  events,  therefore  we 
have  also  made  time-lapse  movies  of  living  cells  to  determine  time  periods 


/*/ 


Project  No.  Z01  HL  00505-09  LCB 

involved  in  the  uptake  event.   In  addition,  observations  of  phagocytosing 
cells  have  been  made  with  a  scanning  electron  microscope  (SEM)  to  determine 
if  the  phagocytic  process  is  limited  to  certain  areas  of  the  cell  and  to 
attempt  to  assess  the  role  of  acanthopods  in  particle  capture. 

The  movies  show  that  contact  and  binding  of  a  particle  do  not  necessarily 
result  in  ingestion.   Amebas  were  observed  to  crawl  over  or  under  particles 
(yeast  or  latex  beads)  or  drag  bound  particles  along  with  them  for  some  time 
without  ingestion.   In  one  case  food  cup  formation  was  observed  adjacent  to 
an  adhering  but  non-phagocytosed  particle.   SEM  images  show  clearly  that 
acanthopods  bind  particles  and  therefore  probably  play  an  important  role  in 
particle  capture.   Both  the  movies  and  SEM  observations  show  no  preferential 
uptake  by  particular  regions  of  the  surface  (e.g.  the  uropod) .   When  the 
"decision"  is  made  by  the  ameba  to  ingest,  engulfment  takes  place  rapidly  and 
with  little  hesitation.   Measurements  of  eight  engulfment  events  from  time- 
lapse  movies  shows  the  time  elapsed  between  particle  contact  and  discernible 
motion  of  the  particle  in  cytoplasmic  streaming  within  the  cell  varied  between 
33  and  81  seconds,  most  being  around  60  seconds. 

We  have  examined  the  process  of  uptake  in  the  transmission  electron  microscope 
(TEM)  with  three  kinds  of  particles:   bacteria,  latex  beads  of  various  sizes 
and  lipid-extracted  yeasts  .   The  uptake  of  all  three  appear  to  be  similar  but 
the  most  informative  images  have  been  made  from  yeast  uptake  studies  .   The 
TEM  images  show  that  limited  regions  of  membrane  in  contact  with  the  particle 
have  a  marked  accumulation  of  microfilaments  associated  with  the  membrane. 
The  orientation  of  these  filaments  appears  to  be  primarily  perpendicular  to 
the  membrane.   As  the  particle  is  enveloped  by  the  plasma  membrane  the  fila- 
ments may  fo^m  a  thick  rim  extending  all  around  the  particle  and  the  pre- 
dominant orientation  becomes  parallel  to  the  membrane.   Intermediate  stages 
between  almost  complete  closure  of  the  plasma  membrane  around  the  particle 
and  the  appearance  of  the  particle  within  the  cytoplasm  without  its  cortex 
of  microfilaments  are  rare,  presumably  because  this  phase  of  engulfment  is 
especially  rapid.   The  channel  of  entry  into  the  cytoplasm  is  marked  for  a 
period  of  time  by  the  presence  of  a  convoluted  tubule  about  45  nm  in  diameter 
that  is  surrounded  by  a  compact  net  of  microfilaments .   The  images  suggest 
that  the  final  fusion  event  that  separates  the  phagosome  membrane  from  the 
surface  membrane  is  the  vesiculation  of  a  narrow  tube  that  literally  may  be 
squeezed  together  by  a  contractile  event .   Once  inside  the  cell  the  phago- 
some membrane  no  longer  shows  the  association  of  microfilaments .   The  phago- 
some then  becomes  accessible  to  other  vesicular  components  of  the  cytoplasm 
and  a  number  of  fusions  take  place.   These  observations  point  to  an  important 
role  for  microfilaments  in  the  engulfment  of  large  particles  .   The  apparent 
failure  of  binding  of  a  particle  to  the  surface  of  the  ameba  to  entrain  in- 
gestion and  the  generally  capricious  nature  of  uptake  are  surprising.   The 
implication  is  that  engulfment  may  be  a  random  process  that  depends  upon  some 
precondition  of  the  cytoplasm  or  plasma  membrane . 

Acanthamoeba  pinocytoses  continuously  and  one  of  the  first  fusion  events  of 
phagosomes  is  with  pinosomes .   (Pinosomes  can  be  readily  labeled  with  exo- 
genous horse-radish  peroxidase .)   Pinocytosis  appears  to  occur  mainly  by 


rf> 


Project  No.  Z01  HL  00505-09  LCB 

very  small  vesiculations  of  the  surface  membrane  and  an  association  of  micro- 
filaments with  this  process  has  not  been  discerned .   Subsequently  hydrolytic 
enzymes  gain  access  to  the  phagosome,  but  their  route  of  entry  has  not  yet 
been  satisfactorily  determined,  since  we  have  so  far  been  unable  to  get 
wholly  reliable  cytochemical  reactions  for  any  of  these  enzymes . 

Time-lapse  cinematography  shows  that  once  the  phagosome  is  completely  severed 
from  the  plasma  membrane  it  appears  to  be  moved  passively  by  cytoplasmic 
streaming . 

3.   Last  year  we  provided  a  description  of  the  structure  of  the  plasma  mem- 
brane of  Acanthamoeba  as  shown  by  freeze-fracture  replication.   This  techni- 
que reveals  some  aspects  of  the  micromorphology  of  membranes,  especially  the 
distribution  of  polypeptides  which  are  visualized  as  particles  of  various 
sizes  and  shapes  .   During  the  past  year  we  have  begun  experimental  manipula- 
tion of  the  membranes  to  gain  insight  into  the  nature  and  identity  of  the 
morphological  features  of  the  membrane. 

Experiments  with  cationized  ferritin  show  that  both  sides  of  the  membrane 
are  uniformly  covered  with  negatively  charged  groups  at  least  as  close  to- 
gether as  12  nm  (diameter  of  one  ferritin  molecule)  and  that  the  distribution 
of  these  charges  is  unrelated  to  the  intramembranous  particles.   These  obser- 
vations confirm  previous  evidence  that  most  of  the  charged  groups  on  the 
membrane  are  associated  with  lipophosphonoglycan  and  provide  additional 
information  about  surface  charge  density. 

The  outer  surface  of  Acanthamoeba  plasma  membrane  has  many  particles  that 
presumably  represent  polypeptides  extending  from  the  cell  surface.  However, 
repeated  attempts  to  remove  the  particles  by  exposure  of  intact  cells  to 
proteases  in  solution  did  not  appear  to  alter  the  morphology  of  the  outer 
surface  of  the  plasma  membrane .  We  subsequently  attempted  to  assess  the 
degree  and  specificity  of  digestion  by  monitoring  the  results  with  sodium 
dodecyl  sulfate  gel  electrophoresis  of  isolated  plasma  membranes.   Three 
enzymes  were  chosen  for  initial  experiments,  two  broad-spectrum  proteases, 
pronase  and  papain,  and  one  more  bond-specific  protease,  trypsin.   The  enzy- 
matic digestions  were  carried  out  either  on  intact  cells  or  on  isolated 
plasma  membranes.   In  the  case  of  intact  cells,  the  membranes  were  subsequen- 
tly isolated  for  gel  electrophoresis. 

Not  unexpectedly  we  observed  that  isolated  membranes  were  much  more  suscept- 
ible to  enzymatic  attack  by  proteases  than  intact  membranes  .   Both  papain 
and  pronase  digestion  of  isolated  membranes  caused  decreases  in  all  poly- 
peptide bands.   Papain-digested  intact  cells  showed  no  interpretable  changes 
in  band  patterns .   Trypsin  digestion  of  either  isolated  membranes  or  intact 
cells  was  much  more  selective  and  appears  to  alter  at  least  two  bands  .  These 
limited  results  indicate  that  this  experimental  approach  may  be  feasible  but 
considerable  further  effort  must  be  put  into  the  basic  procedures  to  determine 
why  separate  membrane  isolations  often  show  considerable  variation  in  the  gel 
pattern.   Also  for  this  project  it  will  be  desirable  to  find  a  gel  system 
that  provides  higher  resolution  of  the  membrane  polypeptides  . 

4  fift 


Project  No.  Z01  HL  00505-09  LCB 

Our  interpretation  of  this  data,  taken  together  with  freeze-fracture  observa- 
tions, is  that  the  outer  surface  of  Acanthamoeba  probably  does  contain  exposed 
polypeptides,  but  the  failure  of  some  proteases  to  attack  intact  cells  is 
probably  due  to  some  protective  function  of  the  lipophosphonoglycan . 

Significance  to  Bio-medical  Research:   Phagocytosis  is  a  major  mechanism  of 
defense  against  infection  and  pinocytosis  promises  a  means  of  therapy  in 
certain  storage  diseases  in  which  lysosomal  enzymes  are  defective  .   Both  of 
these  processes  take  place  at  high  rates  in  the  ameba  and  can  easily  be  stud- 
ied in  Acanthamoeba . 

Changes  in  cell  surface  properties  appear  to  play  a  major  role  in  proliferation 
of  cancerous  cells  .   Acanthamoeba  is  in  some  senses  an  analog  of  transformed 
cells  and  thus  information  on  the  organization  of  its  surface  membrane  may 
prove  useful  in  understanding  malignant  cells. 

Proposed  Course  of  Research:   The  interrelationships  of  surface  membrane  and 
internal  membrane  systems  in  Acanthamoeba  cas tellanii  will  be  studied  by  cyto- 
chemical  and  stereological  techniques  and  with  freeze-fracture  replication. 

Keyword  Descriptors : 

Membrane  morphology,  endocytosis,  Acanthamoeba  castellanii ,  electron  micro- 
scopic, freeze-fracture,  plasma  membrane 

Honors  and  Awards :   None 

Publications : 

Bowers,  B.  and  Korn,  E  .D . :   Localization  of  lipophosphonoglycan  on  both  sides 
of  Acanthamoeba  plasma  membrane.   J .  Cell  Biol  .  62:  533-540,  1974. 

Bowers,  B.,  Levin,  G.  and  Cabib,  E.:   In  vivo  effect  of  polyoxin  D  on  chitin 
synthesis  and  septum  formation  in  Saccharomyces  cerevisiae.   J.  Bacteriol. 
119:  564-575,  1974. 

Cabib,  E.,  Ulane,  R.E.  and  Bowers,  B.:   A  Molecular  Model  for  Morphogenesis: 
The  Primary  Septum  of  Yeast.   In  Horecker,  B  .L .  and  Stadtman,  E.R.  (Eds.): 
Current  Topics  in  Cellular  Regulation.   New  York,  Academic  Press,  1974,  pp. 
1-32. 

Korn,  E.D.,  Bowers,  B.,  Batzri,  S.,  Simmons,  S.R.,  and  Victoria,  E .J . :  Phos- 
pholipids and  Membrane  Fusion  in  Endocytosis  and  Exocytosis  .   J.  Supramolecular 
Structure.   2:  517-528,  1974. 


/¥¥ 


Project  No.  Z01  HL  00503-03  LCB 

1.  Laboratory  of  Cell  Biology 

2.  Section  on  Organelle  Bio- 
chemistry 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Structure,  Assembly  and  Function  of  Microtubules 

Previous  Serial  Number:  NHLI-6 

Principal  Investigators:  Martin  Flavin 

Yoko  Nagata 
Daniel  Raybin 
Keith  Summers 

Project  Description: 

Objectives:   Microtubules,  found  in  the  cytoplasm  and  dividing  nuclei   of  all 
eukaryotic  cells,  are  cylinders  composed  of  13  parallel  protofilaments,  for- 
med by  the  polymerization  of  a  dimeric  precursor  consisting  of  2  very  similar 
55,000  dalton  subunits,  CC   and  (3  tubulin.   Polymerization  is  induced  by  warm- 
ing suitable  brain  extracts,  and  we  measure  it  by  light  scattering,  low-speed 
centrifugation  of  polymer,  electron  microscopy  or,  recently,  by  darkfield 
light  microscopy.   Polymerization  is  reversed  by  cooling  and  tubulin  can  be 
purified  by  repeated  cycles  of  warming  and  cooling.   This  rn  vitro  polymeri- 
zation appears  to  be  nucleated  by  open  rings  of  short  protofilaments  which 
grow  into  sheets  prior  to  closure  to  cylinders  .  Whether  this  sequence  pre- 
cisely matches  _in  vivo  assembly  is  not  known.   Polymers  consisting  only  of 
tubulin  have  been  reported  to  form  slowly  under  special  circumstances,  but 
usually  small  amounts  of  certain  other  proteins  copolymerize .   The  most  con- 
spicuous of  these  MAPS  (microtubule  associated  proteins)  bands  as  2  high 
molecular  weight  proteins  in  sodium  dodecyl  sulfate  gel  electrophoresis, 
appears  to  form  a  filamentous  coating  on  the  outer  microtubule  surface,  and 
may  be  related  to  flagellar  dynein.   The  status  of  other  MAPS  is  still  obscure, 
but  they  probably  include  a  protein  kinase  and  a  tubulin-GDP  transphosphory- 
lase . 

Microtubules  make  up  the  mitotic  spindle  responsible  for  chromosome  movements, 
and  are  essential  for  intracellular  transport  of  material  and  for  flagellar 
and  ciliary  motility.   They  are  also  implicated  in  the  development  and  main- 
tenance of  anisometric  cell  form,  and  thus  may  lead  towards  a  nulecular  under- 
standing of  cellular  morphogenesis .   In  living  cells  microtubules  may  form 
and  dissolve  quite  rapidly,  for  example  in  the  mitotic  spindle  and  in  relation 
to  the  movement  of  pigment  granules,  at  a  time  when  the  total  cell  content  of 
tubulin  appears  constant.   Although  all  tubulins  studied  appear  to  be  structur- 
ally very  similar,  it  is  possible  in  some  cases  that  there  may  be  multiple 
tubulin  genes  coding  proteins  destined  only  for  one  or  another  specific 
organelle.   More  generally  it  seems  that  transcriptional  and  translational 

i  //r 


Project  No.  Z01  HL  00503-03  LCB 

controls  could  not  be  sufficient  to  regulate  microtubule  assembly  in  vivo . 
Moreover,  none  of  the  conditions  currently  used  to  depolymerize  microtubules 
in  vitro  (cold,  high  ionic  strength,  colchicine,  mM  Ca   )  is  likely  to  have  a 
regulatory  function  in  vivo .   Our  research  is  now  focussed  on  certain  post- 
translation  modifications  of  tubulin  which  might  modulate  the  assembly,  or 
functions,  of  microtubules.   One  of  these  is  a  cAMP  stimulated  phosphoryla- 
tion of  a  single  serine  residue  in  g-tubulin  (several  residues  in  a  high 
molecular  weight  MAPS  are  also  phosphorylated)  .   Second,  tubulin  dimer  binds 
guanine  nucleotides  at  2  sites,  one  exchangeable  (E)  and  one  not  (N) ,  and 
some  evidence  has  suggested  that  polymerization  involves  the  transformations 
indicated  by: 

GTP  ATP 

E-dimer-N-GDP-»  GTP-E-dimer-N-GDP  ->  GTP-E-dimer-N-GTP-^GDP-E-polymer-N-GDP 

(or  GTP) 
Although  an  enzyme  catalyzing  transphosphorylation  of  the  N-GDP  has  not  been 
identified,  and  there  is  conflicting  evidence  as  to  whether  the  polymer  con- 
tains only  GDP,  nucleotide  binding  and  transphosphorylation  constitute  tubulin 
modifications  which  might  control  assembly  or  function.   A  third  post- transla- 
tional  modification  which  we  have  recently  identified  is  the  specific  addition 
of  a  tyrosine  residue  to  Qt-tubulin,  apparently  by  peptide  linkage  at  the  C- 
terminal  end  of  the  polypeptide  chain. 

Flagellar  microtubules  consist  of  an  array  of  9  parallel  outer  doublet  micro- 
tubules surrounding  a  central  pair  of  single  microtubules  .   Paired  arms 
extend  from  one  tubule  of  each  outer  doublet  towards  the  adjacent  doublet, 
and  radial  spokes  extend  inward  from  each  doublet,  terminating  in  enlarged 
spoke  heads  at  a  central  sheath  which  surrounds  the  central  microtubules  . 
ATP  is  utilized  for  motility  by  energy- transducing  protein(s)  called  dynein, 
which  are  located  in  the  outer  doublet  arms,  and  are  believed  to  induce  sliding 
of  doublets  past  each  other  by  translocation  through  binding  sites  on  the 
adjacent  doublet.   Dynein  can  be  solubilized  as  a  heterogeneous,  high  molecular 
weight  ATPase,  activated  by  either  Mg  ^  or  Ca   .  We  have  previously  reported 
that  Chlamydomonas  flagella  contain,  in  addition  to  dynein,  a  distinct  low 
molecular  weight,  Ca   -specific  ATPase.   Our  objective  is  to  determine  the 
function  of  this  enzyme,  and  we  consider  two  principal  possibilities  .   There 
is  evidence  that  during  flagellar  bending  there  must  be  translocation  of  spoke 
heads  along  the  central  sheath,  and  the  enzyme  might  be  derived  from  a  spoke 
head  energy-transducing  system.   Secondly,  the  enzyme  might  function  in  steer- 
ing or  tactic  responses,  in  which  Ca"1"  ,  and  possibly  ATP  as  well,  have  been 
implicated . 

Major  Findings : 

1 .   Post-translational  modifications  of  tubulin  and  microtubule  assembly . 

la  .   Tyrosylation  of  a- tubulin  (D .  Raybin) :   It  was  recently  shown  that 
rat  brain  extracts  could  incorporate  a  tyrosine  residue  into  a  specific  pro- 
tein having  many  of  the  characteristics  of  tubulin.   The  incorporation  appeared 
to  be  through  a  peptide  bond  at  the  C-terminal  end  of  the  protein.   The  react- 
ion utilized  free  tyrosine  rather  than  tyrosyl-tRNA,  required  only  ATP,  Mg+2 
and  KC1,  and  appears  to  be  unprecedented.   We  have  confirmed  these  results  and 


f& 


Project  No.  Z01  HL  00503-03  LCB 

shown  by  high  resolution  SDS  gel  electrophoresis  that  a-tubulin  is  the  only 
protein  in  brain  extracts  which  incorporates  radioactive  tyrosine.   Tubulin 
purified  by  ion-exchange  chromatography  retains  the  over-all  reaction,  but 
tubulin  purified  by  3  cycles  of  polymerization  does  not.   Using  the  latter  as 
substrate,  we  have  been  able  to  establish  an  assay  for  the  tyrosylating 
enzyme,  and  to  partially  purify  it  from  bovine  brain  and  separate  it  from 
tubulin.   Tubulin  purified  by  3  cycles  of  polymerization  has  the  capacity  to 
accept  at  least  0.25  moles  of  tyrosine  per  mole  of  a-tubulin.   At  the  same 
time,  the  radioactivity  incorporated  by  a  crude  extract  can  copolymerize 
through  several  cycles  with  added  purified  tubulin.   These  preliminary  results 
suggest  at  least  that  tubulin  can  polymerize  _in  vitro  without  being  exclusive- 
ly in  the  tyrosylated  or  detyrosylated  state. 

lb.   Tubulin  bound  nucleotides  (Y .  Nagata,  D.  Raybin) :   The  assay  for  GTP 
binding  to  the  exchangeable  (E)  site  and  for  transphosphorylation  of  GDP  at 
the  non-exchangeable  (N)  site  is  based  on  incubating  tubulin  in  the  presence 
of  colchicine  with  H3,  y  -P32-labeled  GTP  or  ATP.   Free  nucleotides  are  rapid- 
ly separated  from  tubulin  dimer ;  the  H3in  the  latter  indicates  the  extent  of 
binding  at  the  E  site,  and  the  excess  of  P   over  H3  indicates  the  extent  of 
transphosphorylation.   Since  the  assay  is  based  on  isotope  ratios,  we  first 
ascertained  that  tubulin  did  not  form  any  guanosine  polyphosphates  ;  specifi- 
cally no  ppGpp  (or  cGMP)  was  formed  even  in  the  presence  of  trapping  pools  . 
Concentrations  of  Ca   which  inhibit  polymerization  (1  mM)  did  not  inhibit 
binding  or  transphosphorylation.   A  closer  examination  of  the  inhibition  of 
polymerization  by  Ca   was  also  consistent  with  this  result:   by  varying  the 

concentration  of  free  Ca"^2  in  the  presence  of  2  fixed  concentrations  of  Ca- 

4-9 
GTP  chelate  it  was  shown  that  free  Ca   was  the  active  inhibitory  species. 

Contrary  to  previous  results  we  found  that  ATP  could  not  be  used  to  study 
transphosphorylation:   instead  of  a  maximum  of  1  mole  of  "excess"  P   bound 
per  tubulin  dimer,  2  or,  in  the  presence  of  cAMP,  3  moles  were  bound,  much  of 
which  was  covalently  linked,  presumably  to  serine  residues .   This  phenomenon 
was  not  observed  with  GTP.  With  GTP  a  high  extent  (0.5  -  1.0  moles  excess 
P   /mole  tubulin)  of  transphosphorylation  has  been  observed  with  only  half  of 
our  tubulin  preparations  which  are  competent  to  polymerize;  either  transphos- 
phorylation is  not  prerequisite  to  polymerization  or  the  tubulin  is  already 
charged  with  GTP  on  the  N  site  and  cannot  accept  additional  P   .   However  the 
tubulin  was  obtained  by  cold-depolymerization  after  3  cycles  of  polymerization 
and,  according  to  the  results  of  others,  should  contain  only  GDP.   Therefore 
we  are  now  trying  to  identify  directly  the  nucleotides  bound  in  the  polymer; 
the  problem  is  to  remove  unbound  nucleotides  completely  without  causing  de- 
polymerization  . 

lc  .   Direct  observation  of  microtubule  polymerization  and  depolymerization 
by  darkfield  light  microscopy  (K .  Summers  and  D.  Raybin) .   Previous  electron 
microscopic  observations  of  polymerization  have  indicated  that  some  form  of 
nucleation  may  be  required  and  that  open  sheets  of  protof ilaments  are  inter- 
mediates .   Drawbacks  are  that  observation  can  only  be  made  at  arbitrary 
intervals  and  artifacts  may  be  produced  by  fixation  and  drying  the  material 
on  grids  for  negative  staining.   Although  the  diameter  of  a  microtubule  (25 
nanometers)  is  only  1/10  of  the  resolving  power  of  the  light  microscope,  we 

3  '*n 


Project  No.  Z01  HL  00503-03  LCB 

have  found  that  they  can  be  clearly  visualized  by  darkfield  illumination. 
Microtubules  can  be  observed  directly  and  continuously  under  _in  vitro  poly- 
merization and  depolymerization  conditions  .   Length  distributions  can  easily 
be  determined,  and  we  find  that  completed  microtubules  maintain  a  straight 
configuration  even  after  achieving  a  length  1000  times  their  diameter.  Inter- 
mediate stages,  which  may  correspond  to  long  open  sheets,  are  more  flexible 
and  less  stable.   Depolymerization  by  Ca    seems  to  involve  destabilization 
along  the  entire  length  of  the  microtubule,  whereas  colchicine  induces  depoly- 
merization from  one  end .   A  curling  up  is  sometimes  observed  in  the  absence 
of  GTP. 

2.   Flagellar  Motility. 

+  2 
2a  .   The  function  of  the  low  molecular  weight  Ca   -activated  ATPase  in 

Chlamydomonas  flagella  (K .  Summers)  .   The  localization  of  this  enzyme  in  the 

flagella  should  give  a  clue  to  its  function,  and  one  approach  to  localization 

is  to  see  if  it  is  altered  in  quality  or  quantity  in  mutants  with  specific 

flagellar  structural  defects  .   Four  genes  have  been  identified  mutations  in 

which  result  in  paralyzed  flagella  lacking  the  central  pair  of  microtubules. 

We  have  examined  3  of  these  and  found  that  the  Ca-ATPase  is  similar  to  that 

of  wild  type  in  2,  but  is  probably  totally  absent  from  the  third,  pf-15. 

Further  work  is  required  to  rule  out  the  possibility  that  this  result  might 

be  due  to  increased  leakiness  or  fragility  of  the  pf-15  flagella. 

2b  .   Specific  inhibitors  or  activators  of  dynein  (Y .  Nagata  and 
T.  Watanabe)  .   Solubilized  dynein  has  an  ATPase  activity  activated  almost 

-4-9        -4-9 

equally  well  by  Ca  '  or  Mg   .   We  have  one  commercial  preparation  of  ATP  with 
which  the  Ca  ^-ATPase  activity  is  normal,  but  the  Mg   -ATPase  is  reduced  by 
907o .   Either  traces  of  an  inhibitor  are  present  in  it,  or  traces  of  an  acti- 
vator in  all  other  preparations.   The  inhibitory  preparation  has  lower  amounts 
of  many  metals  including  calcium;  Ca    supplementation  does  not  reverse  the 
inhibition.   It  has  a  higher  concentration  of  iron. 

2c  .   Flagellar  adenylate  kinase  and  nucleoside  diphosphokinase 
(T  .  Watanabe)  .   Last  year's  report  described  the  properties  of  these  enzymes 
and  their  possible  significance  for  flagellar  regeneration  and  motility. 
Although  not  currently  being  pursued,  further  work  showed  the  presence  in 
Chlamydomonas  of  at  least  3  molecular  species  of  each  enzyme.   For  each 
enzyme  one  species  appeared  to  be  uniquely  flagellar  and  one  to  be  shared 
with  cell  bodies.   Another  project  of  last  year's  report  not  currently  being 
pursued  is  the  study  of  mutants  resistant  to  drugs  which  inhibit  microtubule 
assembly  or  function. 

Proposed  Course  of  Research:   We  plan  to  purify  the  tubulin  tyrojylating 
enzyme,  to  characterize  the  reaction,  and  determine  whether  tyrosine  removal 
is  catalyzed  by  the  same  enzyme.   The  reaction  seems  quite  specific  for 
tyrosine  (only  phenylalanine  has  been  shown  to  replace  it,  with  a  Km   100  times 
higher)  ;  however  it  is  possible  that  free  tyrosine  is  not  the  actual  substrate 
For  example  the  enzyme  might  function  in  the  incorporation  of  tubulin  into 
membranes,  and  it  may  be  informative  to  determine  the  cellular  localization 
of  the  enzyme.   We  will  determine  whether  flagella  contain  the  enzyme  and 


f¥* 


Project  No.  Z01  HL  00503-03  LCB 

whether  flagellar  doublet  microtubules  can  be  tyrosylated.   We  hope  to  obtain 
a-tubulin  in  completely  tyrosylated  and  detyrosylated  states,  and  then  to 
assess  the  role  of  this  modification  in  the  conversion  of  monomer  to  dimer, 
dimer  to  polymer,  or  in  microtubule  function.   If  both  forms  can  polymerize 
in  vitro  we  will  analyze  the  polymers  by  microscopy,  compare  the  kinetics  of 
polymerization,  and  determine  which  MAPS  copolymerize  by  gel  electrophoresis. 
Eventually  we  will  examine  cells  in  which  microtubules  undergo  rapid  rever- 
sible assembly  to  see  whether  these  changes  correlate  with  extent  of  tyro- 
sylation . 

With  regard  to  tubulin  nucleotides,  we  will  try  various  procedures  to  deter- 
mine what  nucleotides  are  present  in  the  polymer  and  whether  hydrolysis  of 
both  GTPs  is  essential  for  polymerization.   We  will  determine  whether  the 
transphosphorylation  reaction  is  catalyzed  by  tubulin  itself  or  by  a  separable 
enzyme.   In  the  latter  case  we  will  purify  the  enzyme  in  the  expectation  that 
it  is  likely  to  be  a  regulatory  protein. 

If  we  confirm  that  the  absence  of  the  Ca-ATPase  from  pf-15  flagella  reflects 
its  association  with  central  structures  of  the  flagella,  we  will  characterize 
these  mutant  flagella  further  by  electron  microscopy  and  SDS  gel  electro- 
phoresis. We  may  be  interested  to  determine  enzyme  patterns  in  phototaxis  as 
well  as  other  paralyzed  mutants,  and  in  other  species;  we  know  that  the  Ca- 
ATPase  is  absent  from  extracts  of  Tetrahymena  cilia,  but  a  wider  survey  is 
needed  to  ascertain  whether  there  is  a  significant  correlation  with  the  differ- 
ent motility  systems  of  ciliates,  bif lagellates  and  monof lagellates  .   Apart 

from  the  Ca-ATPase,  we  are  interested  in  the  broader  problem  of  the  involve- 

+  2 
ment  of  Ca    in  cell  steering.   For  this  purpose  we  would  like  to  work  with 

reactivated  flagella,  i.e.  detached  flagella  which  have  been  partially 

demembranated  and  become  motile  only  when  ATP  is  added,  and  to  see  at  first 

whether  Ca   affects  the  asymetry  of  flagellar  beating. 

Keyword  Descriptors : 

Cilia,  microtubules,  motility 

Honors  and  Awards:   None. 

Publications : 

Flavin,  M.:   Methionine  Biosynthesis.   In  Greenberg,  D.M.  (Ed.):  Metabolism 
of  Sulfur  Compounds .   New  York,  Academic  Press,  1975,  pp.  457-503. 

Summers,  K.:   The  role  of  flagellar  structures  in  motility.   Biochim.Biophys  . 
Acta  416:  in  press,  1975. 


/¥f 


ANNUAL  REPORT  OF  THE 
LABORATORY  OF  CELLULAR  METABOLISM 
NATIONAL  HEART  AND  LUNG  INSTITUTE 
July  1,  1974  through  June  30,  1975 


Although  there  have  been  some  changes  in  emphasis  as  well  as  in  the 
direction  and  scope  of  specific  projects  during  the  past  year,  the  research 
program  of  the  Laboratory  of  Cellular  Metabolism  continues  to  be  concen- 
trated in  five  main  areas.   These  are  (1)  the  control  of  cyclic  nucleotide 
phosphodiesterase  and  adenylate  cyclase  activities,  (2)  the  metabolism  and 
functions  of  cyclic  nucleotides  in  lung,  leukocytes  and  arterial  smooth 
muscle,  (3)  the  regulation  of  hormone-sensitive  lipase  activity  in  adipose 
tissue,  (4)  the  mechanisms  by  which  histamine  release  from  mast  cells  is 
initiated  and  terminated,  (5)  the  control  of  lipid  synthesis,  particularly 
cholesterol  synthesis  in  mammalian  cells.   Some  of  the  major  findings  are 
outlined  below. 

1.   Control  of  Cyclic  Nucleotide  Phosphodiesterase  and  Adenylate  Cyclase 
Activities. 

We  have  had  a  long  standing  interest  in  the  mechanisms  by  which  cyclic 
nucleotide  degradation  is  regulated  and  were  among  the  first  to  show  that 
cyclic  AMP  phosphodiesterase  activity  can  be  (1)  rapidly  increased  in  fat 
cells  by  insulin  and  cyclic  AMP,  (2)  "induced"  in  cultured  fibroblasts  by 
maintaining  intracellular  cyclic  AMP  at  an  elevated  level  for  many  hours 
and  (3)  decreased  in  hepatoma  cells  by  glucocorticoids.   We  have  reported 
that  in  fat  cells  it  is  a  particulate  high  affinity  phosphodiesterase  whose 
activity  is  altered  by  insulin,  dexamethasone  and  cyclic  AMP.   Numerous 
attempts  over  the  past  two  years  to  solubilize  this  enzyme  (or  enzymes) 
for  further  study  have  met  with  little  success.   Recently,  however,  we  have 
discovered  a  method  for  reproducibly  solubilizing  the  membrane-bound 
phosphodiesterase  activity  in  essentially  100%  yield  and  hope  that  purifi- 
cation by  standard  techniques  of  ion  exchange,  affinity  and  gel  chromato- 
graphy will  now  be  possible. 

Another  phosphodiesterase  with  particularly  interesting  regulatory 
properties  has  been  partially  purified  from  the  soluble  fraction  of  rat 
liver.   The  kinetics  of  cyclic  AMP  hydrolysis  with  this  enzyme  are  consis- 
tent with  those  of  an  allosteric  enzyme  displaying  positive  cooperativity 
between  catalytic  sites.   We  have  now  completed  detailed  kinetic  studies  of 
the  effects  of  (1)  cyclic  GMP,  cyclic  IMP  and  cyclic  XMP  on  cyclic  AMP 
hydrolysis,  (2)  cyclic  IMP,  cyclic  AMP  and  cyclic  XMP  on  cyclic  GMP 
hydrolysis,  and  (3)  cyclic  GMP,  cyclic  AMP  and  cyclic  XMP  on  cyclic  IMP 
hydrolysis.   It  appears  that  this  phosphodiesterase  favors  cyclic  GMP  both 
as  a  substrate  and  as  an  effector.   We  are  at  present  working  to  complete 
purification  of  the  enzyme  as  a  prerequisite  to  characterization  of  its 
structural  and  regulatory  properties. 

Work  on  adenylate  cyclases  this  year  has  been  relatively  more  limited 
than  in  the  recent  past.   The  effects  of  choleragen  on  the  enzyme  in  rat 


AT/ 


fat  cells  and  in  cultured  fibroblasts  have  been  investigated.   The  latter 
cells  seem  likely  to  be  especially  useful  for  study  of  the  nature  of  the 
choleragen  receptor.   The  fibroblasts  lack  the  capacity  to  synthesize  GM-^ 
ganglioside  which  has  been  postulated  by  several  workers  to  be  the  receptor 
or  a  critical  part  of  it.   When  grown  in  the  usual  manner,  however,  they 
take  up  and  incorporate  gangliosides  from  the  serum  contained  in  the  medium. 
Using  a  serum-free  medium  we  have  now  grown  cells  that  are  deficient  in  GM^ 
ganglioside  and  in  which  the  adenylate  cyclase  does  not  respond  to  cholera- 
gen.  By  restoration  of  the  ganglioside  in  current  experiments,  we  expect 
to  learn  more  about  the  nature  of  the  choleragen  receptor  and  its  relation 
to  adenylate  cyclase. 

2.   Metabolism  of  Cyclic  Nucleotides  in  Lung,  Leukocytes  and  Arterial 
Smooth  Muscle. 

When  these  studies  were  initiated  several  years  ago,  virtually 
nothing  was  known  about  the  factors  that  influence  cyclic  GMP  metabolism 
or  the  functions  subserved  by  this  nucleotide  in  mammalian  cells.   Our 
experiments  with  lung  slices  provided  evidence  that  bradykinin  as  well  as 
acetylcholine  (which  was  shown  elsewhere  to  act  in  other  tissues)  could 
cause  accumulation  of  cyclic  GMP  and  led  us  to  suggest  that  cyclic  GMP 
might  play  a  role  in  the  regulation  of  prostaglandin  synthesis.   In  recent 
work  we  have  used  two  other  systems  that  provide  relatively  more  homogeneous 
populations  of  cells  and  a  better  opportunity  for  correlation  of  changes 
in  cyclic  nucleotide  content  with  modifications  of  cellular  function. 

The  availability  of  a  large  body  of  physiological  data  on  the  human 
umbilical  artery  suggested  that  it  would  be  useful  for  study  as  an  example 
of  vascular  smooth  muscle.   This  proved  to  be  the  case.   It  was  found  that 
acetylcholine,  bradykinin,  histamine,  serotonin  and  K"*"  ions,  all  of  which 
produce  contraction  of  the  artery,  also  cause  a  rapid  accumulation  of  cyclic 
GMP  in  this  tissue.   These  agents  do  not  alter  the  cyclic  AMP  content  of 
the  artery  which  we  have  found  to  be  elevated  only  be  prostaglandin  E^ 
(PGE-^) ,  an  agonist  that  induces  relaxation  of  the  artery.   In  many  tissues 
calcium  (Ca"1-1")  plays  a  critical  role  in  cyclic  nucleotide  metabolism.   The 
effects  of  acetylcholine,  bradykinin,  histamine  and  K+  were  abolished  in 
the  Ca++-depleted  artery  and  restored  after  return  of  Ca^  to  the  incuba- 
tion  medium.   Two  ionophores  (drugs  that  facilitate  the  movement  of  Ca 
through  membranes)  mimicked  the  effects  of  these  Ca   -dependent  agonists 
on  cyclic  GMP.   Accumulation  of  cyclic  GMP  induced  by  serotonin,  on  the 
other  hand,  was  not  diminished  in  Ca++-depleted  arteries  and,  in  fact, 
seemed  to  be  inhibited  by  Ca"*-1"  at  the  concentration  usually  present  in 
the  medium.   These  studies  demonstrated  for  the  first  time  the  existence 
of  two  different  mechanisms  for  control  of  cyclic  GMP  accumulation.   One, 
Ca  -dependent s  has  been  observed  in  several  other  tissues.   The  other,  not 
requiring  exogenous  Ca"1-1"  has  thus  far,  to  our  knowledge,  been  found  only 
in  the  umbilical  artery  and  in  human  leukocytes  (see  below). 

Oxygen  (O2)  acts  in  two  (or  more)  separate  ways  to  initiate  closure 
of  the  umbilical  artery  at  birth.   It,  apparently  directly,  causes  contrac- 
tion and  further  plays  a  "permissive"  role  in  the  action  of  other  chemical 
agents  that  cause  contraction.   We  have  found  that  O2  rapidly  raises  the 

2  fS-X- 


cyclic  GMP  content  of  the  artery  in  a  Ca  -dependent  manner  without  affect- 
ing the  cyclic  AMP  content  or  the  effect  of  PGE^  on  it.   It  is  striking 
that  the  effect  of  O2  on  cyclic  GMP  is  not  prevented  by  inhibitors  of 
oxidative  phosphorylation.   O2  was  required  for  demonstration  of  the  Ca"*-1"- 
dependent  accumulation  of  cyclic  GMP  in  response  to  bradykinin,  histamine 
and  ionophore.   In  contrast,  serotonin,  and  also  methylene  blue  and  ascor- 
bate  like  those  of  serotonin  were  inhibited  by  Ca   as  well  as  by  Oo.   These 
studies  have  shown  that  there  exist  in  the  umbilical  artery,  and  presumably 
in  other  tissues  also,  at  least  two  separate  systems  for  control  of  cyclic 
GMP  synthesis  that  are  influenced  differently  by  Ca"*-1"-  and  C^-linked 
processes.   Elucidation  of  the  mechanisms  through  which  neurohumoral  agents 
and  O2  modify  cyclic  GMP  synthesis  and  influence  the  contractility  of 
arterial  smooth  muscle  should  aid  in  understanding  the  physiological  control 
of  perfusion  in  localized  vascular  beds  and  the  pathogenesis  of  certain 
circulatory  disorders. 

In  studies  with  human  leukocytes  we  found  last  year  that  serotonin, 
melatonin  and  related  derivatives  of  tryptamine  caused  accumulation  of 
cyclic  GMP  and  the  effect  of  these  amines  was  predominantly  if  not  solely 
on  the  monocytes.   While  searching  for  a  functional  correlate  we  have  now 
found  in  collaboration  with  the  Laboratory  of  Clinical  Investigation,  NIAID, 
that  serotonin  enhances  the  responsiveness  of  these  cells  to  a  chemotactic 
stimulus.   Ascorbic  acid  and  carbamylcholine  also  stimulate  monocyte  chemo- 
taxis  and  we  have  shown  that  they  too  increase  the  cyclic  GMP  content  of 
human  monocytes.   All  of  our  findings  are  consistent  with  a  role  for  cyclic 
GMP  in  modulation  of  chemo taxis  and/ or  cell  movement. 

In  monocytes  (as  in  the  umbilical  artery)  the  effects  of  serotonin 
and  ascorbic  acid  on  cyclic  GMP  were  unimpaired  by  the  absence  of  exogenous 
Ca   .   The  ionophore  A23187  which  causes  Ca  -dependent  accumulation  of 
cyclic  GMP  in  the  artery  (and  other  tissues)  did  not  increase  cyclic  GMP 
but  caused  significant  rises  in  cyclic  AMP  in  monocytes  and  polymorpho- 
nuclear leukocytes.   The  effect  of  the  ionophore  was  not  dependent  upon 
the  presence  of  Ca"1-1"  or  Mg"*-1"  in  the  incubation  medium.   We  are  at  present 
evaluating  the  relationship  of  the  changes  in  cyclic  AMP  content  to  effects 
of  the  ionophore  on  chemotaxis.   In  any  case,  it  appears  that  this  drug  can 
influence  cyclic  nucleotide  metabolism  through  more  than  one  mechanism  and 
not  all  of  its  effects  are  necessarily  attributable  to  alterations  in  Ca 
movement . 

Cyclic  nucleotides  have  now  been  implicated  in  several  aspects  of 
leukocyte  function  relative  to  inflammatory  and  immunological  responses. 
In  collaboration  with  the  Laboratory  of  Clinical  Investigation,  NIAID,  we 
have  studied  the  effects  of  human  transfer  factor  on  cyclic  GMP  and  cyclic 
AMP  in  human  leukocytes.   These  preparations  transfer  cell-mediated  immune 
responsiveness  and  apparently  have  both  antigen-specific  and  antigen- 
independent  effects.   When  mononuclear  cells  were  incubated  with  dialyzable 
transfer  factor  from  human  mononuclear  cells,  their  cyclic  GMP  content  was 
rapidly  and  dramatically  increased  with  no  significant  change  in  cyclic  AMP. 
Studies  with  purified  populations  of  leukocytes  established  that  the 
response  occurred  chiefly  if  not  solely  in  monocytes  and  neutrophil  cyclic 
GMP  was  unaffected.   The  transfer  factor  preparations  contained  serotonin 

3  /S? 


and  ascorbic  acid  in  concentrations  previously  shown  to  raise  cyclic  GMP 
in  monocytes.   Four  fractions  separated  from  dialyzable  transfer  factor 
preparations  by  gel  chromatography  caused  elevation  of  cyclic  GMP  in  mono- 
cytes.  The  first  two  contained  ascorbate.   The  others,  one  of  which  was 
the  fraction  that  contained  the  transfer  factor  activity,  contained  no 
ascorbate  or  serotonin.   Thus  it  appears  that  preparations  of  human  transfer 
factor  contain,  in  addition  to  ascorbic  acid  and  serotonin,  another  substance 
or  substances  capable  of  causing  accumulation  of  cyclic  GMP  in  human  mono- 
cytes.  Any  or  all  of  these  may  contribute  to  their  clinical  effects 
perhaps  by  amplifying  subthreshold  cellular  inflammatory  or  immune  responses. 

We  have  now  amassed  a  number  of  clues  to  the  ways  in  which  cyclic  GMP 
metabolism  may  be  modulated  in  intact  cells  and  the  types  of  cellular 
processes  in  which  it  may  play  a  regulatory  role.   In  the  next  phase  of 
investigation,  attention  will  be  focussed  on  the  enzymes  that  synthesize 
and  degrade  cyclic  GMP,  particularly  the  guanylate  cyclases. 

3.  Regulation  of  Hormone-Sensitive  Lipase  Activity  in  Fat  Cells. 

This  enzyme  which  catalyzes  the  rate-limiting  step  in  triglyceride 
breakdown  continues  to  resist  all  attempts  in  our  laboratory  and  elsewhere 
at  extensive  purification.   By  taking  advantage  of  its  substrate  affinity 
we  have  obtained  20  to  25  fold  purification  at  an  early  stage  but  the 
instability  of  the  enzyme  following  this  procedure  has  limited  its  useful- 
ness.  In  most  preparations  the  lipase  tends  to  be  associated  with  lipids 
and  other  proteins  in  large  aggregates.   We  found  last  year  that  by  using 
gel  chromatography  in  the  presence  of  1  M  NaCl  the  lipase  from  rat  fat  could 
be  obtained  in  a  form  with  an  apparent  molecular  weight  of  <100,000.   In 
this  state  it  is  relatively  stable.   When  subjected  to  further  fractiona- 
tion, however,  by  a  variety  of  means,  large  losses  of  activity  have 
invariably  resulted.   Considering  that  the  lipase  from  adipose  tissue  of 
another  species  might  be  more  amenable  to  study  we  carried  out  exploratory 
experiments  with  fat  from  other  rodents  and  from  the  chicken.   None  of  the 
sources  tested,  however,  appeared  to  offer  any  advantages  over  the  rat  tissue. 

As  we  reported  a  few  years  ago,  the  hormone-sensitive  lipase  from 
the  rat  is  inactivated  by  incubation  with  ATP,  Mg"1-1"  and  ascorbic  acid.   The 
requirements  for  Mg   and  ascorbic  acid  are  highly  specific  but  we  have 
recently  found  that  the  nucleotide  requirement  is  relatively  nonspecific. 
Thus  ADP,  GTP,  GDP,  CTP  or  CDP  can  replace  ATP  and  CTP  is  effective  at 
lower  concentrations  than  are  any  of  the  other  nucleotides.   The  related 
nucleoside  monophosphates  and  cyclic  monophosphates  are  inactive.   Until 
more  purified  preparations  of  the  lipase  are  available  the  significance  of 
the  apparent  preference  for  CTP  in  this  reaction  remains  unclear. 

4.  Release  of  Histamine  from  Mast  Cells. 

The  release  of  histamine  and  other  vasoactive  compounds  from  mast 
cells  probably  plays  a  role  in  the  pathogenesis  of  many  allergic  and  inflam- 
matory processes.   Dextran  causes  release  of  histamine  in  a  genetically 
determined  reaction  that  in  many  ways  resembles  anaphylactic  release.   It 


/*Y 


was  used  in  our  earlier  work  which  established  that  cell  desensitization 
limits  the  duration  of  histamine  release  and  is  therefore  a  major  determi- 
nant of  the  magnitude  of  release.   Studies  now  completed  confirmed  the 
preliminary  findings  reported  last  year  that  the  rate  of  cell  desensitiza- 
tion is  also  a  critical  determinant  of  the  amount  of  histamine  that  is 
released  as  a  result  of  an  antigen-antibody  reaction. 

The  systemic  reaction  of  rats  to  the  administration  of  dextran  has 
been  described  as  resembling  anaphylaxis  and  termed  "anaphylactoid" . 
Extensive  studies  begun  last  year  have,  however,  failed  to  demonstrate 
either  precipitating  antibodies  to  dextran  or  antibodies  of  the  IgE  type 
in  the  serum  of  dextran-reactive  rats.   We  have  concluded,  therefore,  that 
the  dextran  reaction  is  due  to  the  existence  of  natural  dextran  receptors 
on  mast  cells  and  not  to  the  presence  of  cytotropic  antibodies.   A  careful 
comparison  of  the  anaphylactoid  reaction  to  dextran  and  the  reaction  to 
antigen  in  rats  immunized  with  ovalbumin  yielded  findings  consonant  with 
this  view.   It  seems  clear  that  the  anaphylactoid  reaction  to  dextran  is 
not  identical  with  anaphylaxis  and  its  mechanism  remains  to  be  elucidated. 

Another  project  related  to  histamine  metabolism  was  carried  out  this 
year  in  collaboration  with  members  of  the  Pulmonary  Branch,  NHLI,  who  had 
found  that  administration  of  aspirin  or  sodium  salicylate  to  animals  of 
several  species  alters  the  metabolism  of  histamine  such  that  the  formation 
of  5' -phosphor ibosylimidazoleacetate,  normally  an  excretory  product,  is 
largely  prevented.   In  an  attempt  to  determine  the  mechanism  of  this  effect 
of  salicylates,  the  enzyme  responsible  for  the  conversion  of  imidazoleacetate 
to  its  phosphoribosyi  derivative  was  prepared  from  rat  liver.   The  purified 
imidazoleacetate  phosphoribosyi  transferase  was  inhibited  by  those  salicylate 
derivatives  that  are  active  jin  vivo  in  concentrations  that  would  be  achieved 
in  tissues.   Other  anti-inflammatory  agents  that  do  not  alter  the  excretion 
of  phosphor ibosylimidazoleacetate  were  not  inhibitory. 

5.   Regulation  of  Lipid  Synthesis  in  Mammalian  Cells. 

Work  on  the  hormonal  control  of  lipid  metabolism  has  been  largely 
suspended  during  the  past  year  while  efforts  were  concentrated  on  studies 
of  the  nature  of  the  metabolic  defect  in  Type  II  hyperlipoproteinemia  as 
outlined  below.   In  addition,  we  have  investigated  the  effects  of  cholesterol 
feeding  on  hepatic  sterol  synthesis  in  the  rat  and  have  found  that  although 
it  undergoes  a  rapid  decline  when  cholesterol  is  added  to  the  diet  (as  is 
well  known)  the  depression  is  not  sustained,  i.e.,  with  prolonged  cholesterol 
intake  the  rate  of  synthesis  rises  again.   This  intriguing  observation  when 
explained  could  shed  some  light  on  the  effects  of  dietary  manipulation  on 
cholesterol  metabolism  in  man. 

We  found  several  years  ago  that  cholesterol  synthesis  in  normal  human 
fibroblasts  was  low  when  whole  serum  was  present  in  the  medium  and  increased 
over  a  period  of  hours  when  the  serum  was  removed  or  was  replaced  with  lipid- 
free  serum.   The  rate-limiting  step  in  cholesterol  synthesis  in  many  tissues 
is  catalyzed  by  hydroxymethylglutaryl  coenzyme  A  (HMGCoA)  reductase  and  it 
was  shown  that  the  activity  of  this  enzyme  in  the  normal  fibroblasts  was 


/ST 


altered  in  parallel  with  cholesterol  synthesis.   Last  year  we  reported 
that  cultured  fibroblasts  from  three  patients'  homozygous  for  Type  II 
hyperlipoproteinemia  (familial  hypercholesterolemia)  exhibited  an  impair- 
ment in  this  negative  feedback  regulation  of  HMGCoA  reductase  activity 
in  response  to  serum  lipoproteins.   Cells  from  one  more  patient  have 
since  been  studied.   In  all  of  these  cell  lines  (obtained  from  the 
Molecular  Disease  Branch,  NHLI)  HMGCoA  reductase  activity  was  depressed 
somewhat  by  whole  serum  albeit  much  less  than  was  observed  with  normal 
fibroblasts. 

Goldstein  and  Brown,  however,  had  reported  a  complete  lack  of  effect 
of  serum  on  HMGCoA  reductase  activity  in  fibroblasts  from  several  other 
patients  with  familial  hypercholesterolemia  and  it  was  unclear  whether  the 
differences  between  their  findings  and  ours  were  due  to  differences  in 
experimental  conditions  or  were  an  indication  of  heterogeneity  in  the 
population  pheno typically  designated  as  Type  II  hyperlipoproteinemia.   We 
therefore  obtained  fibroblasts  from  one  of  the  patients  originally  studied 
by  Goldstein  and  Brown  and  from  another  apparently  similar  patient.   The 
changes  in  HMGCoA  reductase  activity  in  response  to  serum  in  those  cells 
and  in  the  cells  that  we  had  originally  studied  were  compared.   It  was 
found  that  the  defect  in  feedback  regulation  in  the  cells  from  the  NIH 
patients,  although  very  real,  is  much  less  severe  than  that  in  the  other 
two  cell  lines.   Thus,  it  appears  that  the  syndrome  clinically  described 
as  Type  II  hyperlipoproteinemia  is  not  the  result  of  a  single  genetic 
abnormality.   A  similar  conclusion  was  reached  by  Goldstein  and  coworkers 
who  have  recently  studied  fibroblasts  from  three  patients  that  behave 
very  much  like  those  from  the  NIH  patients.   They  have  related  the  metabolic 
abnormalities  in  different  Type  II  fibroblasts  to  defects  in  the  cell 
surface  receptors  for  low  density  lipoproteins.   In  this  regard,  we  have 
found  that  the  rate  of  uptake  of  radio-labeled  triglycerides  from  low 
density  and  very  low  density  lipoproteins  is  only  about  10%  of  normal  with 
cells  from  the  Type  II  patient  that  are  designated  "receptor  negative"  by 
Goldstein  et  al.  and  about  50%  of  normal  with  the  apparently  less  severely 
defective  cells  from  the  NIH  patients.   These  studies  which  are  still  in 
the  initial  stages  are  part  of  a  continuing  investigation  of  lipid  trans- 
port and  synthesis  in  human  fibroblasts  with  the  goal  of  defining  in 
detail  the  nature  of  the  abnormalities  in  hypercholesterolemia  and  other 
disorders  of  lipid  metabolism. 


(% 


Project  No.  Z01  HL  00601-06  LCM 

1.   Laboratory  of  Cellular  Metabolism 
3.   Bethesda,  Md. 


PHS  -  NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 


Project  Title:  Regulation  of  Lipid  Synthesis  in  Mammalian  Cells 

Previous  Serial  No.:  NHLI-29 

Principal  Investigator:  Joel  Avigan,  Ph.D. 

Other  Investigator:  Marta  E.  Schreiner,  B.S. 

Cooperating  Units:  Molecular  Disease  Branch,  NHLI 

Project  Description: 

The  objective  of  this  project  was  to  study  hormonal  and  feedback 
control  of  sterol  and  fatty  acid  synthesis  in  cells  grown  in  culture  and 
in  tissues  of  laboratory  animals.   Cultures  of  human  fibroblasts  that  were 
originally  obtained  from  investigators  in  the  Molecular  Disease  Branch  of 
NHLI  as  well  as  other  cell  lines  received  from  the  NIGMS  Genetic  Mutant 
Cell  Repository  were  grown  in  monolayers  to  conf luency  and  then  incubated 
in  minimal  essential  medium  with  additions  as  indicated;  e.g. ,  hormones, 
serum,  or  fractions  thereof.   Fatty  acid  and  sterol  synthesis  were 
determined  by  incubating  cell  cultures  with  radioactive  acetate.   The 
activity  of  hydroxymethylglutaryl  coenzyme  A  (HMGCoA)  reductase  was 
assayed  in  cell  homogenates  incubated  with  HMGCoA  followed  by  isolation  of 
the  radioactive  mevalonic  acid  by  a  method  that  was  previously  modified  in 
this  laboratory  (NHLI-29,  1974).   The  uptake  of  triglycerides  by  fibroblasts 
from  serum  lipoproteins  was  measured  following  incubation  of  the  cells 
with  medium  containing  lipoproteins  labeled  in  vitro  with  radioactive 
tripalmitin.   Rat  hepatic  microsomal  HMGCoA  reductase  was  determined  in 
preparations  obtained  from  animals  maintained  for  0-3  weeks  on  diets 
containing  cholesterol  and  sacrificed  at  the  time  of  peak  activity  of  the 
enzyme  in  the  diurnal  cycle. 

Effects  of  glucocorticoids.   In  our  previously  reported  studies 
(NHLI-276,  1973)  it  was  observed  that  dexamethasone  stimulates  fatty  acid 
and  nonsaponifiable  lipid  synthesis  in  several  diploid  cell  lines  and  that 
no  such  effect  occurred  in  some  permanent  lines.   It  was  subsequently  shown 
that  sterol  synthesis  in  an  organized  tissue  (rabbit  aortic  media  and 
intima)  was  also  stimulated  by  50%  following  incubation  for  2  days  in  vitro 
with  dexamethasone,  0.1  or  1  uM.   Four  compounds  with  glucocorticoid 
activity  in  vivo  (dexamethasone,  hydrocortisone,  fluocinolone  acetonide 
and  prednisolone)  were  studied  with  respect  to  their  effects  on  lipid 
synthesis  in  human  skin  fibroblasts.   At  concentrations  of  0.1  or  1  uM 

1  /T7 


Project  No.  Z01  HL  00601-06  LCM 


all  stimulated  this  process  but  only  prednisolone  was  active  at  0.01  yM. 
Regulation  of  sterol  synthesis  is  believed  to  occur  mostly  through 
changes  in  the  activity  of  HMGCoA  reductase  -  a  key  enzyme  on  the 
biosynthetic  pathway.   Dexamethasone,  0.1  or  1  yM,  did  not  consistently 
affect  reductase  activity  in  human  fibroblasts  but  100  yM  increased  it 
greatly.   On  the  other  hand,  in  a  transformed  cell  line  (L-cells)  the 
HMGCoA  reductase  was  not  induced  by  dexamethasone  even  at  100  yM,  which 
was  consistent  with  the  previously  observed  lack  of  stimulation  of  lipid 
synthesis  in  these  cells. 

Feedback  control  of  cholesterol  synthesis  in  human  fibroblasts  derived 
from  Type  II  hyperlipemic  homozygous  and  from  normal  donors.   The  previous 
study  was  extended  to  a  cell  line  described  by  Goldstein  and  Brown  (Nat. 
Acad.  Sci.  USA  70,  2809,  1973)  as  feedback  receptor  negative.   Following 
incubations  under  standard  conditions  in  the  presence  and  in  the  absence 
of  low  density  serum  lipoproteins,  we  confirmed  that  in  this  cell  line 
there  is  a  total  absence  of  effect  of  LDL  on  HMGCoA  reductase  activity. 
On  the  other  hand,  in  cells  from  the  four  type  II  homozygous  patients 
studied  at  the  NIH,  LDL  decreased  HMGCoA  reductase  activity  although  to 
a  lesser  degree  than  in  control  cells.   The  basis  for  the  difference  in 
responsiveness  of  these  cell  lines  is  not  clear  at  the  present  time, 
but  the  abnormal  condition  may  be  polygenic  and  caused  by  more  than  one 
genetic  mutation. 

Triglyceride  uptake  by  normal  and  type  II  homozygous  fibroblasts. 
It  was  shown  that  the  type  II  cells  take  up  labeled  triglycerides  complexed 
with  low  density  or  very  low  density  serum  lipoproteins  at  a  slower  rate 
than  do  normal  cells.   Further  studies  concerning  the  deficiencies  in 
lipid  transport  are  now  in  progress. 

The  effect  of  feeding  cholesterol  on  rat  hepatic  HMGCoA  reductase. 
It  has  been  repeatedly  reported  in  literature  that  feeding  cholesterol  to 
rats  causes  a  rapid  decline  in  the  level  of  hepatic  cholesterol  synthesis 
and  of  HMGCoA  reductase  activity.   There  was  a  valid  interest  in  exploring 
the  effect  of  sustained  dietary  intake  of  cholesterol  on  the  activity  of 
the  enzyme  as  compared  with  that  of  a  short-term  feeding.   It  was  shown 
that  contrary  to  expectations  the  activity  of  hepatic  microsomal  HMGCoA 
reductase  was  higher  following  a  2-week  period  of  feeding  a  diet  containing 
5%  cholesterol  than  after  a  period  of  3  days.   The  physiological  signifi- 
cance of  the  rebound  in  enzyme  activity  on  extended  cholesterol  feeding 
is  presently  unknown. 

It  is  proposed  to  study  further  the  nature  of  the  abnormality  in 
lipoprotein  transport  and  in  the  regulatory  system  affecting  cholesterol 
synthesis  in  fibroblasts  grown  from  patients  with  type  II  hyperlipoprotein- 
emia and  also  to  investigate  the  transport  and  metabolism  of  triglycerides 
in  cells  derived  from  individuals  with  other  types  of  hyper lipidemia. 


sse 


Project  No.  Z01  HL  00601-06  LCM 


Publications: 


Avigan,  J.,  Bhathena,  S.  J.,  and  Schreiner,  M.  E. :   Control  of 
sterol  synthesis  and  of  hydroxymethylglutaryl  CoA  reductase  in 
skin  fibroblasts  grown  from  patients  with  homozygous  type  II 
hyperlipoproteinemia.   J.  Lipid  Res.  16:  151-154,  1975. 


Keywords : 


Cholesterol  synthesis  -  HMGCoA  reductase  -  tissue  culture  - 
human  skin  fibroblasts  -  glucocorticoids  -  type  II  hyperlipo- 
proteinemia -  triglyceride  uptake  -  feedback  control  -  arterial 
tissue  -  hepatic  microsomes. 


/ST 


Project  No.  Z01  HL  00602-05  LCM 

1.   Laboratory  of  Cellular  Metabolism 
3.   Bethesda,  Md. 


PHS  -  NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 


Project  Title:  Histamine  Release  from  Mast  Cells;  Immediate 

Hypersensitivity 

Previous  Serial  No.:  NHLI-30 

Principal  Investigator:  James  H.  Baxter,  M.D. 

Other  Investigator:  Ronald  Adamik,  B.S. 

Project  Description: 

Objective:  to  define  the  mechanisms  that  control  release  of 
mediators  from  mast  cells,  and  that  regulate  other  cellular  functions 
in  the  immediate  hypersensitivity  reaction.   Rat  peritoneal  mast  cells 
are  used  for  the  in  vitro  studies.   Prior  to  harvesting  the  cells,  the 
donor  rats  may  be  immunized  with  an  antigen  (egg  albumin)  and  pertussis 
vaccine  in  order  to  sensitize  the  cells  with  cytotropic  antibody.   The 
release  of  histamine  (and  other  mediators)  by  the  antigen,  and  by 
dextran  and  other  releasing  agents  is  then  studied  under  various 
conditions.   Studies  are  also  made  on  the  systemic  reactions  of  rats 
to  antigen  and  to  dextran. 

Role  of  cell  desensitization  in  controlling  histamine  release  by 
antigen.   Continuing  the  studies  on  mast  cell  desensitization  by  dextran 
described  last  year,  histamine  release  from  immunized  rat  mast  cells  by 
antigen  was  studied  in  terms  of  rate  of  release  and  duration  of  release, 
which  together  determine  the  total  amount  released.   With  the  use  of 
various  antigen  concentrations  and  incubation  temperatures,  release 
always  stopped  at  about  the  same  time  that  the  cells  became  desensitized 
to  the  antigen.   It  appeared,  therefore,  that  the  rate  of  desensitization 
(under  the  influence  of  environmental  factors)  determined  the  duration  of 
release,  and  thereby  was  an  important  determinant  of  the  total  amount  of 
histamine  released. 

Calcium  and  phosphatidyl  serine  (PS)  effects  in  mast  cell  histamine 
release  by  dextran.   Histamine  release  from  rat  mast  cells  by  dextran 
(together  with  7  ug/ml  PS)  required  greater  than  0.1  mM  Ca   ,  and  maximal 
release  (at  pH  7)  about  1  mM  Ca   .   Cell  desensitization  by  dextran 
likewise  required  Ca"1-'".   Spontaneous  leakage  of  histamine  from  the  cells 
was  decreased  by  Ca"1-1".   Cells  suspended  for  15  min  in  Ca   -free  medium 
had  to  be  preincubated  with  Ca4"1"  for  10  min  (at  25°)  before  their  respon- 
siveness to  dextran  was  maximally  restored;  responsiveness  never  returned 

1  fto 


Project  No.  Z01  HL  00602-05  LCM 


to  the  original  level.   Histamine  release  could  be  stopped  short  of 
completion  by  adding  EDTA  or  glucose,  or  by  diluting  the  cells  (and 
dextran) .   Na+  and  K  were  not  required  for  histamine  release,  and  ouabain 
was  without  effect  on  the  reaction.   St"1""*",  Ba"1-1"  and  Mg44"  were  ineffective 
in  replacing  Ca"*-^,  but  did  decrease  histamine  leakage.   The  studies 
described  above  were  made  with  PS  in  the  medium.   In  the  presence  of  Ca"H", 
PS  greatly  enhanced  release  by  dextran,  by  increasing  the  rate  of  release 
without  affecting  the  rate  of  cell  desensitization.   Some  evidence  of 
an  interaction  between  Ca44"  and  ps  (in  their  effects  on  histamine  release) 
was  demonstrated,  in  that  the  two  agents  were  strongly  synergistic,  and 
when  PS  was  present  a  greater  concentration  of  Ca   was  required  for 
maximal  release  by  dextran. 

Anaphylactoid  reaction  in  rat:  non-antibody  dependence  and  non- 
identity  with  anaphylaxis.   Systemic  reactions  of  some  nonimmunized 
animals  to  injections  of  certain  substances  which  do  not  harm  most  animals 
are  well  known.   These  reactions  are  often  species  (or  strain)  specific; 
they  resemble  anaphylaxis,  and  have  been  suspected  of  being  due  to  anti- 
bodies.  We  have  investigated  the  basis  of  one  such  reaction,  the  "ana- 
phylactoid" reaction  of  the  rat  to  dextran.   Not  only  did  we  fail  to 
demonstrate  precipitating  antibodies  against  dextran  in  the  serum  of 
dextran-reactive  (Sprague-Dawley)  rats,  but  also  the  serum  of  such  rats 
failed  to  prepare  the  skin  of  non-dextran-reactive  (Wistar/Furth)  rats  for 
passive  cutaneous  anaphylaxis  or  their  peritoneal  mast  cells  for  histamine 
release  by  dextran.   The  negative  results  with  dextran  were  obtained  in 
parallel  with  positive  control  results  with  egg  albumin  after  use  of 
serum  from  rats  that  had  been  immunized  with  egg  albumin  and  pertussis 
vaccine.   Therefore,  we  conclude  that  the  dextran  reaction  is  a  result 
of  natural  dextran  receptors  on  the  mast  cells,  and  not  of  the  presence 
of  cytotropic  antibodies. 

A  comparison  of  the  anaphylactoid  reaction  to  dextran  and  the  reaction 
to  antigen  (egg  albumin)  in  Sprague-Dawley  rats  that  had  been  immunized 
with  the  antigen  and  pertussis  vaccine,  indicated  that  the  two  reactions 
were  different,  and  therefore  may  involve  different  mechanisms.   When 
compared  at  approximately  equal  levels  of  mortality,  the  dextran  reaction 
was  characterized  by  severe  acral  edema,  whereas  the  reaction  to  antigen 
exhibited  intestinal  edema  and  more  severe  respiratory  disturbances. 

We  plan  to  study:  (1)  release  of  serotonin  and  heparin,  as  well  as 
histamine,  from  mast  cells;  (2)  interaction  of  lipids  and  other  substances 
with  mast  cell  membranes;  (3)  basis  of  differences  in  the  rat  reactions 
to  dextran  and  antigen. 


/a 


Project  No.  ZOl  HL  0602-05  LCM 


Significance  to  heart  and  lung  research:   Histamine  and  other 
physiologically  active  substances  which  are  released  from  mast  cells 
produce  important  effects  on  the  small  blood  vessels  and  on  bronchial 
smooth  muscle. 


Publications: 

Baxter,  J.  H.  and  Adamik,  R. :   Control  of  histamine  release: 
effects  of  various  conditions  on  rate  of  release  and  rate 
of  cell  desensitization.   J.  Immunol.  114:  1034-1041,  1975. 


Keywords : 


Mast  cells,  histamine  release,  cell  desensitization, 
dextran,  antigen,  cytotropic  antibody,  calcium,  phosphatidyl 
serine,  anaphylaxis,  anaphylactoid  reaction,  passive  cutaneous 
anaphylaxis. 


ft* 


Project  No.  Z01  HL  00603-01  LCM 

1.   Laboratory  of  Cellular  Metabolism 
3.   Bethesda,  Md. 

PHS  -  NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Regulation  of  Rat  Liver  Phosphodiesterases 

Previous  Serial  No. :      None 

Principal  Investigators:   Joel  Moss,  M.D.,  Ph.D. 

Martha  Vaughan,  M.D. 

Other  Investigators:      Vincent  C.  Manganiello,  M.D. ,  Ph.D. 

Sally  Stanley,  B.S. 

Project  Description: 

Objectives:   To  define  the  structural  and  kinetic  characteristics 
of  phosphodiesterases  responsible  for  cyclic  nucleotide  hydrolysis  in 
rat  liver. 

Methods:   Phosphodiesterase  activity  is  measured  by  methods 
previously  developed  in  this  laboratory.   The  enzyme  will  be  purified 
by  affinicy  chromatography. 

Major  Findings:   A  previously  identified  guanosine  cyclic 
3' ,5'-monophosphate-stimulated  adenosine  cyclic  3' ,5'-monophosphate 
phosphodiesterase  was  partially  purified  from  the  100,000  g  supernatant 
fraction  of  rat  liver.   The  kinetics  of  cyclic  AMP  hydrolysis  were 
consistent  with  those  of  an  allosteric  enzyme  displaying  positive 
cooperativity  between  catalytic  sites.   In  the  presence  of  ymolar 
cyclic  GMP,  hydrolysis  of  ymolar  cyclic  AMP  was  stimulated  10-fold. 
The  marked  sigmoidicity  of  the  curve  relating  rate  of  cyclic  AMP 
hydrolysis  to  cyclic  AMP  concentration  was  not  evident  when  cyclic  GMP 
was  present.   In  addition  to  cyclic  GMP,  cyclic  IMP  and  cyclic  XMP 
stimulated  cyclic  AMP  hydrolysis.   The  Ka's  for  cyclic  IMP  and  cyclic  XMP 
were  approximately  one  and  three  orders  of  magnitude  higher  than  that  for 
cyclic  GMP. 

The  purified  phosphodiesterase  also  catalyzed  the  hydrolysis  of 
cyclic  GMP,  the  K^   being  approximately  half  that  noted  for  cyclic  AMP. 
Cyclic  IMP,  cyclic  AMP  and  cyclic  XMP  accelerated  the  hydrolysis  of 
cyclic  GMP  when  the  latter  was  present  at  nmolar  concentrations.   Cyclic 
IMP  proved  to  be  the  most  potent  effector  (Ka=l-2  ymolar)  followed  by 
cyclic  AMP  (Ka=2-4  ymolar)  and  cyclic  XMP  (Ka=400  ymolar) .   Hydrolysis 
of  nmolar  cyclic  IMP  was  stimulated  by  cyclic  GMP,  cyclic  AMP  and 
cyclic  XMP  in  order  of  increasing  K  .   The  kinetic  data  suggest  that 

1  /4S 


Project  No.  Z01  HL  00603-01  LCM 


this  phosphodiesterase  favors  cyclic  GMP  both  as  a  substrate  and  an 
effector.   The  physiological  significance  of  this  is  unclear. 

Significance  to  Heart  and  Lung  Research:   Control  of  cyclic  GMP 
and  cyclic  AMP  hydrolysis  is  important  in  the  action  of  hormones  on 
the  cardiovascular  system  and  in  the  contraction  of  smooth  muscle  in 
the  lung . 

Proposed  Course:   The  phosphodiesterase  will  be  further  purified 
and  its  kinetic  and  structural  characteristics  studied. 

Publications:   None 

Keywords: 

Phosphodiesterase,  cyclic  nucleotides,  adenosine  3 ',5'- 
monophosphate,  guanosine  3' ,5' -monophosphate. 


tit- 


Project  No.  Z01  HL  00604-01  LCM 

1.   Laboratory  of  Cellular  Metabolism 
3.   Bethesda,  Md. 

PHS  -  NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Inhibition  of  Histamine  Metabolism  by 

Salicylates 

Previous  Serial  No. :       None 

Principal  Investigators:   Joel  Moss,  M.D.,  Ph.D. 

Maria  C.  de  Mello,  Ph.D. 
Martha  Vaughan,  M.D. 
Michael  A.  Beaven,  Ph.D. 

Cooperating  Units:         Pulmonary  Branch,  NHLI 

Dr.  de  Mello  is  supported  by  the  Brazilian 
National  Research  Council 

Project  Description: 

Objectives:   To  define  the  mechanism  for  the  in  vivo  inhibition 
by  salicylates  of  the  formation  of  the  histamine  metabolite,  5'-phospho- 
ribosy  li  m  idazoleacetate. 

Methods:   Imidazoleacetate  phosphoribosyl  transferase  was  isolated 
by  standard  chromatographic  and  ultracentrifugal  procedures.   Product 
identification  was  based  on  methods  developed  by  Beaven,  et  al.  (1). 

Major  Findings:   Beaven  and  coworkers  (1)  previously  demonstrated 
that  the  administration  of  sodium  salicylate  or  aspirin  alters  the 
metabolism  of  histamine  in  several  animal  species.   The  formation  of 
the  histamine  metabolite,  5'-phosphoribosylimidazoleacetate,  was 
inhibited  by  these  salicylates,  but  was  not  affected  by  other  anti- 
inflammatory agents.   In  an  attempt  to  define  the  mechanism  of  this 
salicylate  effect,  the  enzyme  responsible  for  the  conversion  of  imidazole- 
acetate to  its  phosphoribosyl  derivative  was  purified  from  rat  liver  by 
ultracentrifugation  and  DEAE-cellulose  chromatography.   The  purified 
transferase  preparation  was  inhibited  by  those  salicylate  derivates 
active  in  vivo  at  concentrations  that  would  be  achieved  in  tissues. 

Significance  to  Heart  and  Lung  Research:   Histamine  exerts  a 
profound  effect  on  the  smooth  muscle  of  the  lung.   Inhibition  of  one  of 
the  pathways  for  histamine  degradation  by  salicylates  may  thus  be 
relevant  in  the  pathogenesis  of  certain  disease  states. 


/&r 


Project  No.  Z01  HL  00604-01  LCM 


Proposed  Course:   The  effects  of  salicylate  metabolites  and 
anti-inflammatory  agents  on  the  transferase  reaction  will  be  evaluated. 

Publications: 

(1)   Beaven,  M.A.,  Horakova,  Z.,  and  Keiser,  H. :   Inhibition 

by  aspirin  of  ribose  conjugation  in  the  metabolism  of  histamine. 

European  J.  Pharm.  29:  138-146,  1974. 


Keywords: 


Salicylates,  aspirin,  anti-inflammatory  agents, 

5 ' -phosphoribosylimidazoleacetate ,  imidazoleacetate 

phosphoribosyl  transferase. 


*6 


Project  No.  Z01  HL  00605-02  LCM 

1.   Laboratory  of  Cellular  Metabolism 
3.   Bethesda,  Md. 


PHS  -  NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 


Project  Title:  Cyclic  Nucleotide  Metabolism  in  Human 

Umbilical  Artery 

Previous  Serial  No.:        NHLI-36 

Principal  Investigators:    Ronald  Clyman,  M.  D. 

Vincent  Manganiello,  M.D.  ,  Ph.D. 
Martha  Vaughan,  M.  D. 

Other  Investigator:         Adam  Blacksin 

Project  Description: 

Objectives:   To  determine  the  effects  of  divalent  cations  and 
oxygen  on  cyclic  nucleotide  metabolism  in  the  human  umbilical  artery. 

Methods:   Umbilical  cords  from  term  (gestational)  pregnancies 
were  obtained  within  30  min  of  delivery.   Umbilical  artery  segments 
were  prepared  as  described  by  Clyman  et  al.  (1).   Variations  in  incubation 
medium  Ca-r-content  and  02-content  were  made  as  described  previously. 
cGMP  and  cAMP  were  measured  as  described  in  Ref.  (2). 

Major  Findings:   We  have  previously  demonstrated  that  in  term 
gestational  human  umbilical  artery  segments  incubated  in  room  air  at  37°C, 
histamine,  acetylcholine,  bradykinin,  K  and  serotonin  (agonists  that 
cause  contraction)  cause  accumulation  of  cGMP  without  altering  the  content 
of  cAMP;  prostaglandin  E-^  (PGEi ) ,  which  relaxes  the  artery,  caused  cAMP 
accumulation  without  affecting  the  cGMP  content. 

Calcium  (Ca  )  appears  to  be  Important  in  the  regulation  of  cyclic 
nucleotide  content  in  several  tissues.   In  the  umbilical  artery  the  control 
of  cAMP  content  by  PGE-i  was  independent  of  Ca   .   After  incubation  in 
Ca+"^-free  medium,  the  cGMP  content  of  the  artery  segments  was  decreased 
by  50%  and  was  unaffected  by  histamine,  acetylcholine,  bradykinin  and  K  . 
Readdition  of  Ca"1-1"  (2.7  mM)  or  Sr4^"  (3.6  mM)  to  the  medium  partially 
restored  the  basal  cGMP  content  and  the  agonist  effects  on  the  cGMP  content. 
However,  Sr   was  not  as  effective  as  Ca   in  this  regard.   Ionophores 
A23187  and  X537A  (agents  that  facilitate  Ca   movement  through  membranes) 
mimicked  the  effects  of  these  Ca^-dependent  agonists  on  cGMP  content. 
Incubation  with  the  phosphodiesterase  inhibitor  3-isobutyl-l-methyl 
xanthine  (0.1  mM)  increased  both  the  basal  content  of  cGMP  and  the  histamine 


'67 


Project  No.  Z01  HI.  00605-02  LCM 


induced  accumulation  3-fold.   This  effect  was  dependent  on  the  presence 
of  Ca   also.   Accumulation  of  cGMP  induced  by  serotonin,  on  the  other 
hand,  was  not  diminished  in  Ca   -depleted  arteries  and,  in  fact,  seemed 
to  be  inhibited  by  2.7  idM  Ca   .   Thus  agonists  controlling  cGMP  accumu- 
lation appear  to  act  through  two  different  mechanisms:  one  Ca   -dependent, 
the  other  Ca4-1"- inhibited. 

O2  acts  in  at  least  two  separate  ways  to  initiate  closure  of  the 
umbilical  artery  at  birth.   O2,  itself,  apparently  directly  induces 
constriction;  it  plays  further  a  "permissive"  role  in  the  action  of 
other  chemical  agents  that  cause  contraction.   We  found  that  Oo  increased 
the  cGMP  content  of  the  artery  in  a  Ca   -dependent  manner  without  affecting 
cAMP  content.   Inhibitors  of  oxidative  phosphorylation  (oligomycin  and 
2,4-dinitrophenol)  did  not  inhibit  this  effect  of  C^.   O2  was  required  for 
demonstration  of  the  Ca  -dependent  accumulation  of  cGMP  in  response  to 
bradykinin,  histamine,  and  ionophore  A23187.   The  effect  of  isobutyl 
methyl  xanthine  on  basal  content  and  on  the  bradykinin-induced  accumulation 
was  also  dependent  on  the  presence  of  02-   Methylene  blue  and  sodium 
ascorbate  caused  cGMP  accumulation  in  C^-deprived  arteries.   Their  effects 
were  not  diminished  in  Ca  -depleted  arteries  and,  in  fact,  seemed  to  be 
inhibited  when  2.7  mM  Ca"*-1"  was  present  in  the  medium.   The  effects  of 
these  agents  and  of  serotonin  on  cGMP,  which  were  inhibited  by  Ca   ,  were 
also  inhibited  by  C^-   These  non-Ca  -,  non-02~dependent  agonists 
(methylene  blue,  ascorbate,  and  serotonin)  did  not,  however,  permit 
demonstration  of  the  effects  of  the  Ca  -  and  C^-dependent  agonists 
on  02~deprived  arteries.   It  appears  that  there  are  in  the  umbilical 
artery  at  least  two  separate  mechanisms  for  control  of  cGMP  synthesis  that 
are  influenced  differently  by  Ca"1-1"-  and  C^-linked  processes. 

Significance  to  Heart  and  Lung  Research:   Elucidation  of  the 
mechanisms  by  which  neurohumoral  agents  and  O2  influence  the  contractility 
of  arterial  smooth  muscle  should  aid  in  understanding  the  physiological 
control  of  perfusion  in  localized  vascular  beds  and  the  pathogenesis  of 
certain  circulatory  disorders. 

Proposed  Course:   Studies  with  the  human  umbilical  artery  will  be 
terminated.   The  control  of  cyclic  GMP  metabolism  and  particularly  of 
guanylate  cyclase  activity  will  be  further  investigated  using  cultured 
arterial  smooth  muscle  cells. 

Publications: 

(1)   Clyman,  R.I.,  Sandler,  J. A.,  Manganiello,  V.C. ,  and 

Vaughan,  M.   Guanosine  3' ,5' -monophosphate  and  adenosine 

3' ,5 '-monophosphate  content  of  human  umbilical  artery. 
J.  Clin.  Invest.  55:  120-125,  1975. 


/*8 


Project  No.  Z01  HL  00605-02  LCM 


(2)   Clyman,  R.I.,  Blacksin,  A.S.,  Sandler,  J. A.,  Manganiello, 
V.C.,  and  Vaughan,  M.   Role  of  Ca"*""1"  in  the  regulation  of  cyclic 
nucleotide  content  in  human  umbilical  artery.   J.  Biol.  Chem. 
1975,  in  press. 


Keywords: 


Umbilical  artery,  calcium,  cyclic  nucleotides,  oxygen, 
ascorbate,  methylene  blue,  serotonin,  bradykinin, 
histamine. 


Mf 


Project  No.  Z01  HL  00606-04  LCM 

1.   Laboratory  of  Cellular  Metabolism 
3.   Bethesda,  Md. 

PHS  -  NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Cyclic  Nucleotide  Metabolism  in  Cultured  Cells 

Previous  Serial  No.:        NHLI-S1  and  32 

Principal  Investigators:    Vincent  C.  Manganiello,  M.D.,  Ph.D. 

Joel  Moss,  M.D.,  Ph.D. 
Martha  Vaughan,  M.D. 

Other  Investigators:        Betty  Horn,  B.S. 

Sally  Stanley,  B.S. 

Cooperating  Units:  P.  Fishman,  Developmental  and  Metabolic 

Neurology  Branch,  NINDS 

Project  Description: 

Objectives:   To  study  control  of  cAMP  and  cGMP  metabolism  in 
cultured  cells.   In  the  past  year  human  fibroblasts  have  been  used  to 
investigate  the  mechanism  of  action  of  choleragen  or  adenylate  cyclase 
and  the  part  played  by  gangliosides  in  the  cell  surface  receptor  for 
choleragen. 

Methods:   Measurement  of  cAMP  by  method  of  Gilman  (Proc.  Nat. 
Acad.  Sci.  67:  305,  1970);  adenylate  cyclase  by  the  method  described 
by  us  (manuscripts  in  preparation) ;  phosphodiesterase  by  our  published 
methods  (Proc.  Nat.  Acad.  Sci.  69:  269,  1972;  70:  3830,  1973). 

Major  Findings: 

Effects  of  choleragen  on  adenylate  cyclase  activity.   Within  30  min 
after  addition  of  choleragen  cAMP  content  of  cultured  human  fibroblasts  was 
increased.   Coincident  with  the  increase  in  cAMP  content  was  an  apparent 
alteration  in  response  to  isoproterenol  and  PGE^.   At  all  concentrations 
of  isoproterenol,  the  increment  in  cAMP  produced  during  a  10  min  incubation 
with  isoproterenol  was  enhanced  in  the  toxin-treated  cells.   In  the 
presence  of  maximally  effective  concentrations  of  PGE^,  the  increment  in 
cAMP  produced  by  PGE-i  was  either  similar  in  both  control  cells  or  toxin- 
treated  cells  or  actually  lower  in  the  toxin-treated  fibroblasts.   At 
lower  concentrations  of  PGE-.  ,  accumulation  of  cAMP  was  enhanced  in  the 
toxin-treated  cells. 

After  incubation  with  cholera  toxin,  although  basal  adenylate 
cyclase  activity  of  fibroblast  homogenates  was  increased,  no  enhancement 

1  f70 


Project  No.  Z01  HL  00606-04  LCM 

of  the  response  to  isoproterenol  or  PGEi  was  observed.   To  elucidate 
mechanism  of  interaction  of  cholera  toxin  with  adenylate  cyclase,  calls 
were  incubated  with  I    cholera  toxin;  membrane  fractions  were  prepared 
and  solubilized  with  the  nonionic  detergent  Lubrol  PX.   Although  both 
cyclase  activity  and  I    toxin  were  found  to  co-chromatograph,  such 
studies  did  not  establish  any  definite  association  between  toxin  and 
adenylate  cyclase. 

GM-]  ganglioside  and  the  action  of  choleragen.   Human  fibroblasts 
lack  capacity  to  synthesize  GM-^  ganglioside.   Since  GM-^  ganglioside 
is  thought  to  serve  as  the  cell  surface  receptor  for  cholera  toxin,  and 
since  the  fetal  calf  serum  used  in  our  growth  medium  contains  high 
concentrations  of  GM^,  we  have  assumed  that  fibroblasts  incorporate 
exogenous  GMi  into  their  cell  membranes,  and  this  binding  accounts  for 
the  responsiveness  of  these  cells  to  cholera  toxin.   We  have  now  shown 
that  cells  grown  in  chemically  defined  medium,  in  the  absence  of  serum, 
do  not  respond  to  cholera  toxin.   In  current  studies  with  replacement  of 
GM-^  ganglioside  we  expect  to  learn  more  about  the  nature  of  the  choleragen 
receptor  and  its  relation  to  adenylate  cyclase. 

Significance  to  Heart  and  Lung  Research:   Studies  of  the  regulation 
of  cAMP  and  cGMP  metabolism  in  homogeneous  populations  of  cultured  cells 
should  aid  in  understanding  the  nature  of  cellular  regulatory  processes 
through  which  hormones,  prostoglandins  and  other  humoral  agents  act 
on  the  lung  and  cardiovascular  system. 

Proposed  Course:   The  mechanism  of  action  of  choleragen  will  be 
investigated  in  human  fibroblasts  and  other  types  of  cells,  especially 
those  that  can  be  grown  in  chemically  defined,  serum-free  medium.   We 
also  plan  to  study  the  interrelationships  between  cAMP  and  cGMP  metabolism 
in  cultured  cells,  especially  cells  of  smooth  muscle  origin. 

Publications: 

Vaughan,  M. :   Effects  of  choleragen  and  fluoride  on  adenylate 
cyclase.   In  Dumont,  J.E.,  Brown,  B. ,  and  Marshall,  N.  (Eds.): 
Regulation  of  Function  and  Growth  of  Eukaryotic  Cells  by 
Intracellular  Cyclic  Nucleotides,  New  York,  Plenum  Publ.  Corp., 
1975,  in  press. 


Keywords: 


Tissue  culture,  cAMP,  cGMP,  choleragen,  GM]^  ganglioside, 
adenylate  cyclase,  receptor  isoproterenol,  PGEls  L-2071  cells. 


f7f 


Project  No.  Z01  HL00607-02  LCM 

1.   Laboratory  of  Cellular  Metabolism 
3.   Bethesda,  Md. 


PHS  -  NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 


Project  Title:  Cyclic  Nucleotide  Metabolism  in  Human  Leukocytes 

Previous  Serial  No.:       NHLI-35 

Principal  Investigators:   Jeffrey  A.  Sandler,  M.  D. 

Martha  Vaughan,  M.  D. 

Other  Investigators:       Vincent  C.  Manganiello,  M.D. ,  Ph.D. 

Ronald  I.  Clyman,  M.  D. 

Cooperating  Unit:  Dr.  John  I.  Gallin 

Dr.  Charles  H.  Kirkpatrick 


Laboratory  of  Clinical  Investigation,  NIAID 


Project  Description: 


Objectives:   To  assess  (1)  the  relationship  between  chemotaxis 
and  cyclic  nucleotide  content  in  human  polymorphonuclear  and  mononuclear 
leukocytes,  (2)  the  effects  of  dialyzable  transfer  factor  on  cyclic  GMP 
content  of  leukocytes,  (3)  the  relationship  between  cyclic  AMP  and 
cyclic  GMP  content  in  human  monocytes,  and  (4)  the  role  of  divalent 
cations  in  cyclic  nucleotide  generation  in  leukocytes. 

Methods:   Cells  were  separated  and  incubated  and  cyclic  nucleotides 
extracted,  purified  and  assayed  by  standard  methods.   Chemotaxis  was 
evaluated  by  a  modification  of  the  Boyd en  chamber  procedure. 

Major  Findings: 

1.  Chemotaxis  and  cyclic  nucleotides:  Serotonin,  ascorbic  acid 
and  carbamylcholine  enhanced  the  responsiveness  of  human  monocytes  to  a 
chemotactic  stimulus  (endotoxin- treated  serum).   These  agents  caused 
significant  accumulation  of  cyclic  GMP  in  monocytes  providing  further 
evidence  for  a  relationship  between  intracellular  cyclic  GMP  and  monocyte 
movement.   PMN  leukocyte  chemotaxis  was  also  enhanced  by  these  agents 
although  significant  increases  in  cyclic  GMP  were  not  demonstrated. 

2.  Effect  of  dialyzable  transfer  factor  on  cyclic  GMP  in  human 
monocytes: 

Incubation  of  human  leukocytes  for  5  min  with  dialysates  of 
leukocyte  lysates  that  contained  transfer  factor  or  with  leukocyte 
dialysates  devoid  of  transfer  factor  caused  a  rise  in  their  cGMP  content 

1  tri. 


Project  No.  Z01  HL 00607-02  LCM 

with  little  change  in  cAMP.   The  accumulation  of  cGMP  occurred  pre- 
dominately if  not  exclusively  in  monocytes.   Substances  that  increased 
monocyte  cGMP  could  be  obtained  from  several  cell  populations  including 
mononuclear  cells  from  Hypaque-Ficoll  gradients,  plastic-adherent 
monocytes,  non-adherent  lymphocytes  and  neutrophils,  but  were  not  present 
in  dialysates  of  leukemic  lymphocytes  from  patients  with  the  Sezary 
syndrome. 

Dialysates  of  lysed  mononuclear  cells  contained  serotonin, 
ascorbate  and  an  unidentified  cholinergic  activity  as  well  as  transfer 
factor.   Passage  of  these  dialysates  through  a  column  of  Sephadex  G-25 
yielded  four  fractions  that  increased  leukocyte  cGMP.   Two  of  these 
fractions  contained  ascorbate;  two  other  active  fractions,  including  the 
one  that  caused  conversion  of  delayed  skin  tests,  did  not  contain 
detectable  ascorbate  or  serotonin.   When  a  dialysate  of  lysed  neutrophils 
which  contained  no  transfer  activity  was  passed  over  the  same  column, 
only  the  fractions  that  contained  ascorbate  caused  accumulation  of 
cyclic  GMP  in  mononuclear  cells. 

These  observations  are  consistent  with  the  possibility  that  some 
aspects  of  transfer  factor  activity  may  be  effected  through  cyclic  GMP 
dependent  processes. 

3.  Relationship  between  cAMP  and  cGMP  in  human  monocytes:   When 
the  cGMP  content  of  monocytes  was  increased  by  serotonin  or  ascorbic  acid 

and  the  cAMP  content  was  elevated  by  PGE, ,  polystyrene  beads  or  the 
ionophore  A23187,  there  was  no  change  in  basal  cGMP  content  but 
the  increment  produced  by  serotonin  or  ascorbic  acid  was  markedly 
reduced.   Serotonin  did  not  interfere  with  the  effects  of  PGE,  on  cAMP. 
We  have  not  yet  defined  the  mechanism  by  which  agents  that  raise  cAMP 
interfere  with  the  accumulation  of  cGMP  in  monocytes. 

4.  Role  of  Ca   and  Mg   in  cyclic  nucleotide  metabolism.   The 
effects  of  serotonin  and  ascorbic  acid  on  accumulation  of  cyclic  GMP 

in  human  monocytes  are  apparently  independent  of  extracellular  Ca"1-*"  and 
Mg   .   This  is  in  contrast  to  observations  with  other  tissues  in  which 
accumulation  of  cGMP  in  response  to  several  agents  does  not  occur  in  the 
absence  of  exogenous  Ca 

Ionophore  A23187,  an  agent  reported  to  enhance  calcium  movement 
across  biologic  membranes  (and  to  cause  accumulation  of  cGMP  Iv   other 
tissues)  did  not  increase  cGMP  but  caused  a  significant  accumulation  of 
cAMP  in  both  monocytes  and  polymorphonuclear  leukocytes.   This  effect 
of  the  ionophore  did  not  require  the  presence  of  extracellular  calcium 
or  magnesium. 


m 


Project  No.  Z01  HL  00607-02  LCM 


Significance  to  Heart  and  Lung  Research:   Phagocytosis  is  a 
fundamental  cellular  function  relevant  to  host  defense  and  the  pathogenesis 
of  inflammatory  and  degenerative  processes  in  all  tissues  including  heart, 
lung  and  blood  vessels.   Information  concerning  cyclic  GMP,  the  factors 
that  control  its  metabolism  and  the  role  that  it  plays  in  cell  physiology 
is  at  present  fragmentary.   Available  data  suggest,  however,  that  it  may 
be  of  especial  importance  in  the  latter  tissues. 

Proposed  Course:  The  guanylate  cyclase  and  phosphodiesterases 
of  monocytes  will  be  assayed  and  the  effects  of  cAMP  on  these  enzymes 
investigated. 

Publications: 

Sandler,  J.  A.,  Clyman,  R.  I.,  Manganiello,  V.  C,  and 
Vaughan,  M. :   The  effect  of  serotonin  (5-hydroxytryptamine) 
and  derivatives  on  guanosine  3' ,5' -monophosphate  in 
human  monocytes.   J.  Clin.  Invest.  55:  431-435,  1975. 

Vaughan,  M. :   Metabolism  of  3' ,5 '-guanosine  monophos- 
phate in  vascular  smooth  muscle,  leukocytes,  and  lung. 
In  Dumont,  J.E.,  Brown,  B. ,  and  Marshall,  N.  (Eds.): 
Regulation  of  Function  and  Growth  of  Eukaryotic  Cells 
by  Intracellular  Cyclic  Nucleotides,  New  York,  Plenum 
Publ.  Corp.,  1975,  in  press. 


Keywords: 


Leukocytes,  polymorphonuclear  leukocytes,  monocytes,  cGMP, 
cAMP,  dialyzable  transfer  factor,  calcium  ionophore, 
serotonin,  ascorbic  acid,  chemotoxis. 


m 


Project  No.  Z01  HL  00608-02  LCM 

1.   Laboratory  of  Cellular  Metabolism 
3.   Bethesda,  Md. 


PHS  -  NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 


Project  Title:  Regulation  of  Cyclic  AMP  Phosphodiesterase 

Activity 

Previous  Serial  No.:  NHLI-33 

Principal  Investigators:       C.  J.  Lovell-Smith,  M.D.,  Ph.D. 

Martha  Vaughan,  M.  D. 

Other  Investigators:  V.  C.  Manganiello,  M.D.,  Ph.D. 

Ferol  Lieberman,  M.S. 

Project  Description: 

In  order  to  clarify  the  complex  mechanisms  through  which  cyclic 
nucleotide  phosphodiesterase  activity  is  regulated,  we  have  attempted  to: 
(1)  reproduce  the  in  vivo  effects  of  triiodothyronine  (T3)  on  fat  cell 
phosphodiesterase  in  an  in  vitro  system;  (2)  solubilize  and  purify  the 
membrane-bound  fat  cell  phosphodiesterase(s)  that  is  subject  to  control 
by  insulin,  glucocorticoids,  cyclic  AMP  and  To;  and  (3)  use  choleragen 
to  modify  phosphodiesterase  in  fat  cells.   Phosphodiesterase,  adenylate 
cyclase  and  cyclic  AMP  were  assayed  by  standard  methods. 

1.   Effects  of  Tn  on  fat  cells.   In  preliminary  studies  last  year 
it  appeared  that  these  might  be  related  to  a  decrease  in  adenylate  cyclase 
activity  but  further  work  has  shown  that  it  is  probably  secondary  to 
the  increased  activity  of  a  high,  affinity  particulate  phosphodiesterase 
in  the  fat  cells  from  thyroidectomized  rats.   Treatment  of  these  animals 
for  4  days  with  T3  reversed  the  changes  in  phosphodiesterase  and  restored 
responsiveness  of  the  fat  cells  to  isoproterenol.   It  is  probably  fortuitous 
that  these  effects  of  T3  closely  resemble  those  produced  by  the  addition  of 
T3  to  fat  cells  in  vitro.   The  latter  apparently  pharmacological  effects 
of  T3  are  immediate  and  rapidly  reversible  and  the  concentrations  required 
are  several  orders  of  magnitude  greater  than  those  found  in  blood. 

In  order  to  prove  that  the  results  of  To  treatment  in  vivo  are  due 
to  a  direct  effect  on  fat  cells  (not  secondary  to  some  other  hormonal 
changes  resulting  from  T3  administration)  it  will  be  necessary  to  demon- 
strate this  in  vitro.   As  this  presumably  physiological  effect  of  T3  is 
evident  only  after  many  hours  we  carried  out  studies  with  fat  cells  (or 
fragments  of  tissue)  incubated  for  one  or  more  days  under  several  different 
conditions.   In  all  instances,  unfortunately,  the  fat  cells  failed  to 
retain  normal  metabolic  responsiveness  and  no  effects  of  T3  were  demonstrated. 

1  //r 


Project  No.  Z01  HL  00608-02  LCM 


Currently,  we  are  attempting  to  study  the  effect  of  low  concentra- 
tions of  T3  in  cultured  cells  particularly  those  that  will  grow  in  the 
absence  of  serum,  or  in  the  presence  of  serum  from  hypothyroid  animals. 
As  yet,  there  is  no  evidence  that  To  under  these  circumstances  affects 
the  phosphodiesterases  (soluble  or  particulate)  of  the  lines  under  study. 

2.  Solubilization  of  phosphodiesterase  from  fat  cells.   A  large 
number  of  detergents  and  other  compounds  that  were  tested  failed  to 
solubilize  the  enzyme  and /or  caused  extensive  losses  of  activity.   We 
have  recently  found  that  the  high  affinity  particulate  phosphodiesterase 
activity  can  be  solubilized  in  essentially  100%  yield  using  a  combination 
of  BRIJ  30,  0.1%,  and  1  M  NaCl.   It  is  hoped  that  the  enzyme  or  enzymes 
will  now  be  susceptible  to  purification  by  standard  techniques  of  ion- 
exchange,  affinity  and  gel  chromatography. 

3.  Effects  of  choleragen  on  fat  cells.   As  first  demonstrated 
in  this  laboratory  several  years  ago,  choleragen  increases  lipolysis 

in  fat  cells  after  a  delay  of  about  one  hour.   Measurements  of  fat  cell 
cyclic  AMP  content  in  similar  experiments  revealed  that  it  was  signifi- 
cantly elevated  only  after  4  hr  of  exposure  to  choleragen;  i.e.,  well 
after  lipolysis  was  stimulated.   However,  by  carrying  out  incubations  in 
the  presence  of  theophylline  to  inhibit  cyclic  AMP  degradation,  effects 
of  choleragen  on  fat  cell  cyclic  AMP  were  demonstrable  as  early  as  one 
hour.   The  conclusion  that  adenylate  cyclase  was  activated  by  choleragen 
within  one  hour  was  confirmed  by  direct  assay  of  the  enzyme  in  particulate 
fractions  and  cyclase  activity  continued  to  rise  during  the  second  and 
third  hours  of  exposure  to  choleragen.   The  particulate  phosphodiesterase 
activity  was  also  increased  by  choleragen  consistent  with  our  earlier 
observations  that  when  the  fat  cell  cyclic  AMP  content  is  elevated 
(whether  by  increasing  its  rate  of  synthesis  or  decreasing  degradation) 
phosphodiesterase  activity  is  enhanced. 

In  relation  to  the  mechanism  of  action  of  choleragen  it  is  notable 
that  the  magnitude  of  isoproterenol  stimulation  is  the  same  with  the 
choleragen-activated  adenylate  cyclase  as  it  is  with  the  enzyme  from 
control  cells. 

Significance  to  biomedical  research:   It  is  likely  that  the 
phosphodiesterase  of  many  tissues  is  under  regulation  by  several  factors, 
including  hormones.   This  will  probably  play  an  increasingly  important 
role  in  studies  of  the  cyclic  AMP  system.   The  study  of  thyroid  hormones 
contributes  to  the  relatively  little  that  is  known  of  the  mode  of  action 
of  these  hormones. 

Proposed  course:   (a)  to  define  the  effect  of  T3  on  phospho- 
diesterase activity  in  an  in  vitro  system,   and  (b)  to  purify  and 
■characterise  the  low  K_  particulate  phosphodiesterase  of  rat  adipocytes. 


'TC 


Project  No.  Z01  HL  00608-02  LCM 


Publications: 


Vaughan,  M. :   Regulation  of  3' ,5' -adenosine  monophosphate 

phosphodiesterase  activity.   In  Dumont,  J.E.,  Brown,  B. , 

and  Marshall,  N.   (Eds.):   Regulation  of  Function  and 

Growth  of  Eukaryotic  Cells  by  Intracellular  Cyclic  Nucleotides, 

New  York,  Plenum  Publ.  Corp.,  1975,  in  press. 


Keywords: 


Phosphodiesterase,  isolated  fat  cells,  triiodothyronine, 
cyclic  AMP,  cholera  toxin  (choleragen) ,  adenylate 
cyclase,  enzyme  solubilization. 


(77 


Project  No.  Z01  HL  00609-07  LCM 

1.   Laboratory  of  Cellular  Metabolism 
3.   Bethesda,  Md . 

PHS  -  NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Regulation  of  Hormone-Sensitive  Lipase  Activity 

Previous  Serial  No.:         NHLI-34 

Principal  Investigators:      Su-Chen  Tsai,  Ph.D. 

Martha  Vaughan,  M.  D. 

Other  Investigators:         None 

Project  Description: 

Objectives:   (1)   To  purify  the  fat  cell  hormone-sensitive  lipase  and 
the  enzymes  that  regulate  its  activity.   (2)   To  elucidate  the  mechanisms 
of  lipase  activation  and  inactivation. 

3 
Methods:   Assay  of  lipase  activity  using  H-glyceryl  trioleate  as 

substrate,  precipitation  of  the  unhydrolyzed  substrate  with  5%  trichloro- 
acetic acid  and  radioassay  of  H-glycerol  in  the  supernatant  fluid.   Frac- 
tionation and  purification  of  hormone-sensitive  lipase  using  ammonium 
sulfate  precipitation,  gel  chromatography  and  disc  gel  electrophoresis. 

Major  Findings: 

1.   Purification  of  the  lipase  from  rat  adipose  tissue.   The  ammonium 
sulfate-precipitated  lipase  was  incubated  for  5  min  at  30°  \vTith  an  emulsion 
of  triolein  and  mixed  phospholipids.   After  centrifugation  about  60%  of  the 
lipase  activity  and  3  to  5%  of  the  protein  was  associated  with  the  floating 
lipid  layer.   When  emulsions  were  made  with  pure  triolein  or  with  phospho- 
lipids alone  <10%  of  the  lipase  was  bound  to  them.   The  mixed  emulsions 
of  phospholipids  and  triolein  similar  to  that  used  as  a  substrate  for  the 
enzyme  were  most  efficient  for  separating  the  lipase  from  other  proteins 
in  the  solution  of  the  ammonium  sulfate  fraction.   Lipase  activity  could 
be  recovered  from  the  emulsion  after  removal  of  lipids  with  acetone-ether 
extraction  but  yields  were  low.   Dissociation  of  the  lipase  by  incubation 
of  the  emulsion  in  buffer  containing  1  M  NaCl  was  more  effective  but 
unfortunately  the  preparations  obtained  were  relatively  unstable  in  subse- 
quent purification  steps.   It  was  possible  to  show  that  most  of  the  lipase 
activity  in  these  preparations  behaved  as  a  molecule  of  <100,000  when  chroma- 
tographed  in  the  presence  of  1  M  NaCl.   We  had  previously  found  that  in  the 
presence  of  1  M  NaCl  most  of  the  ammonium  sulfate-precipitated  lipase 
was  dissociated  to  that  size  whereas  gel  chromatography  in  the  absence  of 
NaCl  yields  lipase  activity  distributed  through  a  heterogenous  family  of 
very  large  molecules  or  aggregates. 

i  ns 


Project  No.  Z01  HL  00609-07  LCM 

2.  The  lipase  in  adipose  tissue  from  chickens.   We  considered  that 
the  lipase  from  adipose  tissue  of  species  other  than  the  rat  might  be  more 
amenable  to  purification  and  study.   There  have  been  a  few  reports  concerning 
the  hormone  sensitive  lipase  in  chicken  fat  and  this  tissue  would  offer 
obvious  practical  advantages.   We  found,  however,  in  exploratory  experiments 
that  the  specific  activity  of  the  ammonium  sulf ate-precipitated  lipase  from 
chicken  fat  was  only  20%  of  that  of  preparations  from  rat  fat  and  the  chicken 
enzyme  appeared  to  offer  no  particular  advantages.   In  particular,  inactiva- 
tion  of  the  chicken  lipase  with  ATP,  Mg"1-1"  and  ascorbate  was  not  demonstrable. 

3.  Cholesteryl  esterase  activity  in  lipase  preparations.   We  had 
found  that  the  ammonium  sulf ate-precipitated  lipase  hydrolyzed  cholesteryl 
esters  as  well  as  triglycerides.   In  an  attempt  to  determine  whether  the 
same  enzyme  acted  on  both  substrates  activities  against  triolein  and 
cholesteryl  oleate  were  compared  under  a  variety  of  conditions  known  to 
activate  or  inactivate  the  lipase.   The  effects  inhibitors  were  also  compared. 
These  studies  were  complicated  by  the  fact  that  the  apparent  cholesterol 
esterase  activity  could  be  varied  widely  by  very  minor  changes  in  the  method 
of  substrate  preparation  or  in  time  elapsed  between  ints  preparation  and 

use.   All  of  the  data,  however,  were  consistent  with  the  conclusion  that 
two  different  enzymes  act  on  the  two  substrates. 

4.  Inactivation  of  the  rat  lipase.   As  we  reported  a  few  years  ago, 
the  hormone-sensitive  lipase  from  the  rat  is  inactivated  by  incubation  with 
ATP,  Mg   and  ascorbic  acid.   The  requirements  for  Mg   and  ascorbic  acid 
are  highly  specific  but  we  have  recently  found  that  the  nucleotide  require- 
ment is  relatively  nonspecific.   Thus  ADP,  GTP,  GDP,  CTP  or  CDP  can  replace 
ATP  and  CTP  is  effective  at  lower  concentrations  than  are  any  of  the  other 
nucleotides.   The  related  nucleoside  monophosphates  and  cyclic  monophos- 
phates are  inactive.   Until  more  purified  preparations  of  the  lipase  are 
available  the  significance  of  the  apparent  preference  for  CTP  in  this 
reaction  remains  unclear. 

Significance  to  Heart  and  Lung  Research:   Availability  of  plasma  FFA, 
an  important  energy  substrate  for  heart,  is  regulated  via  the  hormone 
sensitive  lipase  activity  of  adipose  tissue.   Purification  of  the  lipase 
and  the  inactivating  enzyme  are  essential  before  the  mechanisms  through 
which  its  activity  is  regulated  can  be  elucidated. 

Proposed  Course:   Purification  of  the  hormone  sensitive  lipase  from 
adipose  tissue  will  be  continued  along  with  studies  of  the  ATP,  Mg   , 
ascorbate-dependent  inactivation  of  the  enzyme. 

Publications:   None 

Keywords:   cholesterol  esterase;  lipase,  hormone-sensitive;  lipase  inactiva- 
tion; enzyme  regulation. 


(79 


Annual  Report  of  the 
LABORATORY  OF  CHEMICAL  PHARMACOLOGY 
National  Heart  and  Lung  Institute 
July  1,  197U  through  June  30,  1975 

DRUG  METABOLISM  AS  A  CAUSE  OF  DRUG  TOXICITY 

Many  organic  compounds  are  transformed  in  the  body  to  potent  alkylating  and 
arylating  metabolites  that  combine  covalently  with  various  tissue  components, 
including  proteins,  lipids  and  nucleic  acids.   During  the  past  few  years  this 
laboratory  has  been  developing  an  integrated  approach  for  determining  l) 
whether  these  chemically  reactive  metabolites  mediate  the  different  toxici- 
ties caused  by  their  parent  substances,  2)  whether  there  is  a  dose  threshold 
for  the  toxicity  and  the  covalent  binding  of  the  chemically  reactive  metabo- 
lites, 3)  the  mechanisms  of  formation  and  inactivation  of  the  reactive  metabo- 
lites, h)   whether  enzymes  present  in  a  given  tissue  account  for  the  formation 
of  the  metabolite  that  becomes  covalently  bound  in  that  tissue,  5)  the  mech- 
anisms by  which  various  treatments  alter  the  formation  and  elimination  of  the 
reactive  metabolites  and  thereby  change  the  incidence  and  severity  of  the 
toxicity  and  6)  the  conditions  under  which  extrapolation  of  data  obtained 
in  vitro  to  living  animals  will  be  reasonably  valid  and  when  it  won't. 

To  evaluate  the  approach  the  laboratory  searches  for  drugs  and  other  foreign 
compounds  that  cause  tissue  lesions  and  determines  whether  radiolabeled  tox- 
icants are  covalently  bound  to  macromolecules  in  target  tissues.   It  then 
studies  whether  the  amount  of  covalently  bound  metabolite  in  the  target 
tissue  is  approximately  proportional  to  the  dose  of  the  toxicant  and  whether 
various  treatments  that  alter  the  activity  of  various  enzymes  that  are  known 
to  metabolize  foreign  compounds  cause  parallel  changes  in  the  amount  of  co- 
valently bound  metabolite  in  the  tissues  and  in  the  incidence  and  severity 
of  the  tissue  lesion.   If  the  changes  in  covalent  binding  and  the  tissue 
lesion  do  not  parallel  each  other,  the  covalent  binding  of  double -labeled 
derivatives  of  the  foreign  compound  and  its  conjugates  is  studied  to  deter- 
mine whether  only  a  part  of  the  molecule  of  the  toxicant  or  its  conjugate 
becomes  covalently  bound.   Concurrent  studies  in_  vitro  aid  in  elucidating  the 
enzyme  that  catalyzes  the  formation  of  the  reactive  metabolite,  the  part  of 
the  toxicant's  molecule  that  is  activated  and  whether  enzymes  in  the  target 
tissue  can  account  for  the  amount  of  reactive  metabolite  that  becomes  co- 
valently bound  in  the  target  tissues.   Concurrent  studies  on  the  pharmaco- 
kinetics of  the  toxicant  in_  vivo  and  on  the  distribution  of  its  urinary  met- 
abolites aid  in  elucidating  possible  mechanisms  for  dose  thresholds  for  co- 
valent binding  and  in  resolving  apparent  discrepancies  between  in  vitro  and 
in  vivo  results.   The  laboratory  is  also  testing  the  validity  of  a  simple 
mathematical  model,  based  on  irreversible  kinetics,  as  an  aid  in  resolving 
possible  mechanisms  by  which  treatments  may  alter  covalent  binding  and  tox- 
icity. 

With  this  integrated  approach  the  laboratory  has  discovered  that  the  liver 
necrosis  caused  by  halogenated  benzenes,  acetaminophen  and  furosemide  in 
animals  occurs  only  after  threshold  doses  are  exceeded  and  that  these  dose 
thresholds  are  due  to  dose  dependent  changes  in  the  metabolism  of  the  tox- 
icants.  It  has  also  discovered  that  the  incidence  of  liver  damage  caused  by 

1  /*/ 


other  drugs  and  foreign  compounds,  including  carbon  tetrachloride,  isoniazid 
and  iproniazid,  does  not  depend  on  a  threshold  dose  but  is  approximately 
proportional  to  the  dose  of  the  toxicant.   In  addition,  the  laboratory  has 
discovered  why  a  given  treatment  may  increase  the  incidence  of  toxicity 
caused  by  a  given  toxicant  at  one  dose  but  decreases  it  at  another  or  in- 
creases it  in  one  animal  species  but  decreases  it  in  another.   We  are  thus 
gaining  a  better  understanding  of  the  mechanisms  by  which  organic  compounds 
cause  tissue  lesions  and  of  the  effects  of  various  treatments  and  different 
dosage  schedules  on  drug-induced  toxicities. 

Isoniazid  and  related  drugs 

Programs  in  which  isoniazid  was  used  prophylatically  to  prevent  tuberculosis 
were  stopped  because  about  1%   of  the  patients  manifested  a  hepatitis-like 
syndrome  which  in  some  cases  resulted  in  death.   Last  year,  we  reported  evi- 
dence that  the  hepatitis  caused  by  isoniazid  occurred  predominately  in  pa- 
tients that  acetylated  the  drug  rapidly  and  suggested  that  the  hepatitis  may 
be  caused  by  a  chemically  reactive  metabolite  formed  by  the  following  se- 
quence of  events:   l)  Isoniazid  is  first  acetylated  to  form  acetylisoniazid 
which  in  turn  is  hydrolyzed  by  an  amidase  to  isonicotinic  acid  and  acetyl 
hydrazine.   2)  The  acetyl  hydrazine  then  is  hydroxylated  to  form  a  chemically 
reactive  metabolite  by  a  cytochrome  P-U50  enzyme  in  liver  microsomes.   In 
accord  with  this  view,  considerably  more  isoniazid  is  excreted  as  isonico- 
tinic acid  in  fast  acetylators  of  the  drug  than  in  slow  acetylators .   But 
there  was  no  difference  between  fast  and  slow  acetylators  in  the  proportion 
of  the  dose  of  acetylisoniazid  excreted  into  urine  as  isonicotinic  acid. 
Thus,  the  increase  in  acetylisoniazid  formation  by  fast  acetylators  accounts 
for  the  increase  in  the  formation  of  isonicotinic  acid  and  acetyl  hydrazine. 

It  also  seems  likely  that  the  liver  necrosis  observed  in  patients  receiving 
iproniazid  may  be  caused  by  a  similar  sequence  of  events,  that  is:   l)  ipro- 
niazid is  hydrolyzed  to  isonicotinic  acid  and  isopropyl  hydrazine  and  2)  the 
isopropyl  hydrazine  in  turn  is  converted  to  a  chemically  reactive  metabolite 
by  a  cytochrome  P-U50  enzyme  in  liver  endoplasmic  reticulum. 

The  view  that  the  liver  damage  caused  by  isoniazid  and  iproniazid  is  medi- 
ated by  acetyl  hydrazine  and  isopropyl  hydrazine  rather  than  isonicotinic 
acid  is  supported  by  the  following  facts:   l)  The  doses  required  to  produce 
liver  necrosis  in  rats  are  much  smaller  with  acetyl  hydrazine  or  isopropyl 
hydrazine  than  with  acetyliproniazid  or  iproniazid.   2)  The  acetyl  group  of 
acetylisoniazid  is  covalently  bound  in  rat  liver  to  a  greater  extent  than  is 
the  isonicotinic  acid  group  after  administration  of  doubly  labeled  acetyl- 
isoniazid.  3)  Changes  in  the  amount  of  the  acetyl  group  of  acetylisoniazid 
and  acetyl  hydrazine  and  in  the  amount  of  the  isopropyl  group  of  isopropyl 
hydrazine  that  become  covalently  bound  to  liver  protein  in_  vivo  parallel 
changes  in  the  severity  of  the  liver  necrosis  caused  by  the  pretreatment  of 
animals  with  phenobarbital  or  cobaltous  chloride. 

The  identities  of  the  reactive  metabolites  of  acetyl  hydrazine  and  isopropyl 
hydrazine  remain  to  be  determined.   In_  vitro  experiments  have  confirmed  the 
view  that  both  acetyl  hydrazine  and  isopropyl  hydrazine  are  converted  to 
their  reactive  metabolites  by  a  cytochrome  P-^50  enzyme  in  liver  endoplasmic 


ftJL 


reticulum.   Experiments  with  double -labeled  acetyl  hydrazine  have  revealed 
that  the  acetyl  group  of  the  reactive  metabolite  probably  becomes  covalently 
bound  intact  and  that  most  of  it  is  converted  to  acetate.   Thus,  the  reactive 
metabolite  is  probably  an  N-hydroxyl  derivative  of  acetyl  hydrazine.   In  vivo 
the  acetate  is  converted  to  carbon  dioxide.   Indeed,  changes  in  the  formation 
of  radiolabeled  carbon  dioxide  in  vivo  after  the  administration  of  acetyl- 
labeled  acetylisoniazid  or  acetyl  hydrazine  parallel  changes  in  the  co- 
valent  binding  of  the  acetyl  group  to  liver  proteins .   The  formation  of 
radiolabeled  carbon  dioxide  may  thus  be  used  as  an  indirect  measure  of  the 
formation  of  the  reactive  metabolite  of  acetyl  hydrazine.   Experiments  with 
l^C-1,  3ft_2_i sop ropy 1  hydrazine  indicate  that  the  isopropyl  group  of  its 
reactive  metabolite  is  also  covalently  bound  to  liver  proteins  intact  and 
thus  the  reactive  metabolite  is  probably  an  N-hydroxyl  derivative  of  iso- 
propyl hydrazine.   Some  of  the  reactive  metabolite  is  converted  to  propane. 
But  studies  in_  vivo  have  revealed  that  phenobarbital  pretreatment  decreases 
the  formation  of  propane  from  isopropyl  hydrazine  even  though  it  increases 
covalent  binding  of  the  reactive  metabolite.   The  relationships  between  co- 
valent  binding  of  the  reactive  metabolite  and  propane  formation  therefore 
need  to  be  clarified. 

OTHER  STUDIES  RELATED  TO  THE  METABOLISM  AND 
TOXICITY  OF  DRUGS 

Chloramphenicol  ( d- ( - ) -threo-1- (p-nitrophenyl )  -2- ( di chlcroacet amide ) -1 , 3- 
propanediol)  -  Previous  studies  have  shown  that  chloramphenicol  in_  vivo  is 
covalently  bound  predominately  to  proteins  in  liver,  bone  marrow  and  plasma, 
but  the  mechanism  of  activation  was  unclear.   During  the  past  year,  chloram- 
phenicol was  labeled  with  3h  in  the  1-position  of  the  propanediol  and  with 
l^C  in  the  dichloroacetyl  group.   In  vitro  studies  revealed  that  about  20% 
more  of  the  -^H-labeled  derivative  was  covalently  bound  to  liver  microsomes 
than  was  the  -^C-labeled  derivative.   By  contrast,  in  vivo  studies  revealed 
that  5-10  times  more  of  the   C-label  was  covalently  bound  to  tissue  proteins 
than  was  the  3n-label,  suggesting  that  the  chloramphenicol  was  cleaved  either 
before  or  after  it  became  covalently  bound.   In  either  case,  most  of  the  co- 
valent binding  occurring  in_  vivo  does  not  appear  to  be  mediated  by  the  re- 
duction of  the  nitro  group.   In  this  regard,  it  may  be  important  that   C- 
dichloroacetic  acid  is  also  covalently  bound  extensively  to  tissue  proteins 
in  vivo. 

Nitrobenzene  and  other  nitrobenzenes  -  Nitrobenzenes  are  known  to  cause  met- 
hemoglobinemia presumably  through  their  reduction  to  nitroso  or  hydroxylamine 
derivatives.   Although  nitro  compounds  may  be  reduced  by  several  different 
mammalian  enzymes  including  NADPH  cytochrome  c_  reductase,  cytochrome  P-^50, 
xanthine  oxidase  and  aldehyde  oxidase,  they  also  may  be  reduced  by  intestinal 
flora.   Indeed,  the  finding  that  nitrobenzene  given  either  intraperitoneally 
or  orally  does  not  cause  methemoglobinemia  in  germ-free  rats  or  in  those 
treated  with  antibiotics  suggests  that  the  reduction  of  nitrobenzene  in_  vivo 
is  predominately  by  intestinal  bacteria.   Surprisingly,  in  rats  kept  under 
ordinary  laboratory  conditions ,  the  methemoglobinemia  caused  by  nitrobenzene 
is  greater  when  it  is  administered  intraperitoneally  than  when  it  is  given 
orally.   Thus,  intraperitoneal  administration  of  drugs  does  not  preclude  the 
possibility  that  they  might  be  metabolized  by  intestinal  bacteria  even  when 

3  /9S 


the  drugs  are  not  excreted  in  "bile. 

Role  of  cytochrome  be  in  the  formation  of  superoxide  by  cytochrome  P-l+50 
systems  -  btuaies  on  tne  relative  rates  01  oxidation  or  i\IAuh  ana  imADFH  have 
revealed  that  at  least  2/3  of  the  electrons  required  for  the  reduction  of 
oxygenated-cytochrome  P-^O-substrate  complexes  in  liver  microsomes  are 
mediated  by  cytochrome  b^.   The  finding  that  an  anti-cytochrome  be  antibody 
preparation  does  not  inhibit  NADPH  oxidation  as  much  as  it  inhibits  drug 
metabolism,  however,  suggests  that  the  oxygenated  cytochrome  P-U50-substrate 
complexes  may  decompose  when  the  rate  of  reduction  of  the  complexes  to 
"active  oxygen"  cytochrome  P-i+50-substrate  complexes  is  the  rate-controlling 
step  in  drug  metabolism.   In  accord  with  this  view,  superoxide  is  formed  by 
the  cytochrome  P-U50  system  in  liver  microsomes  and  its  rate  of  formation  is 
increased  by  the  anti-cytochrome  be,  antibody. 

Pretreatment  of  male  rats  with  either  spironolactone  or  pregnenolone-l6a- 
carbonitrile  (PCN)  also  leads  to  the  formation  of  cytochrome  P-U50  systems  in 
which  the  rate-limiting  step  is  apparently  the  reduction  of  the  oxygenated- 
cytochrome  P-U50  complex.   After  the  addition  of  substrates  to  liver  micro- 
somes from  rats  pretreated  with  these  substances ,  the  rate  of  substrate-de- 
pendent NADPH  oxidation  is  greater  than  the  rate  of  drug  metabolism.   This 
extra  NADPH  oxidation  is  apparently  due  to  the  formation  of  superoxide,  be- 
cause more  superoxide  is  formed  by  these  liver  microsomes  than  is  formed  by 
microsomes  from  phenobarbital  pretreated  rats.   Moreover,  studies  on  the  rate 
of  reduction  of  cytochrome  be  by  NADH  or  NADPH  indicate  that  these  reactions 
are  slower  in  liver  microsomes  from  PCN  treated  rats  than  in  those  from 
phenobarbital  pretreated  rats. 

q-Methyl  dopa  -  Large  doses  of  a-methyl  dopa  (>  250  mg/kg)  in  rats  cause  a 
mild  hepatic  injury  characterized  by  diffuse  acidophilic  bodies,  without  de- 
pletion of  glutathione  or  increases  in  diene  conjugation  of  phospholipids. 
In  vitro  studies  have  shown  that  a-methyl  dopa  is  converted  to  a  chemically 
reactive  metabolite  by  enzyme  systems,  such  as  cytochrome  P-U50  in  liver 
microsomes  and  xanthine  oxidase.   Since  superoxide  dismutase  and  various 
catechols  inhibit  the  covalent  binding  of  radiolabeled  a-methyl  dopa,  in  the 
presence  of  either  enzyme,  it  seems  probable  that  the  reactive  metabolite  is 
formed  indirectly  by  the  superoxide  produced  by  these  enzymes  rather  than  by 
a  direct  action  of  these  enzymes  on  a-methyl  dopa.   Whether  the  formation  of 
chemically  reactive  metabolites  of  a-methyl  dopa  i_n  vivo  are  mediated  by 
superoxide,  however,  remains  to  be  determined. 

Paraquat  -  This  herbicide  causes  edema  and  necrosis  in  pulmonary  alveoli 
followed  by  interstitial  fibrosis  and  death.   Although  it  is  commonly 
believed  that  the  toxicity  is  mediated  by  superoxide  formed  during  the 
autoxidation  of  the  reduced  form  of  the  herbicide,  we  have  not  been  able  to 
demonstrate  that  lipid  peroxidation,  which  is  caused  by  superoxide,  occurs 
either  in  living  animals  or  in  lung  slices.   Last  year,  we  reported  that  the 
toxicity  might  be  affected  by  altering  8-adrenergic  responses  in  lung  because 
the  lethal  effects  of  the  herbicide  are  potentiated  by  isoproterenol  and  are 
decreased  by  propranolol.   However,  the  relationships  between  the  3-adre- 
negric  system  in  lung  and  paraquat  toxicity  are  obscure  because  paraquat 
causes  a  decrease  rather  than  an  increase  in  cyclic  AMP.   Moreover,  the  pro- 

k  /ft 


tective  effects  of  propranolol  and  similar  3-adrenergic  blocking  agents  may 
be  due  largely  to  the  inhibition  of  the  paraquat  active  transport  system  in 
lung  slices  discovered  by  Rose  et_  al_ .  (Nature  252:31^,  197*0  even  though 
propranolol  did  not  appear  to  decrease  appreciably  the  uptake  of  paraquat 
into  rat  lung. 

PHYSIOLOGICAL  CONTROL  MECHANISMS 

Cyclic  nucleotide  formation  -  Last  year,  we  reported  that  the  guanylate 
cyclase  in  lung  supernatant  requires  divalent  ions  but  was  not  activated  by 
carbamyl  choline.   During  the  past  year,  it  was  found  that  2.5  mM  Ca   in- 
creases the  accumulation  of  cyclic-GMP  70-fold  and  increases  that  of  cyclic- 
AMP  30-fold  in  lung  cells.   Moreover,  a  combination  of  carbamyl  choline  and 
2.5  mM  Ca+  increases  the  accumulation  of  cyclic-AMP  70-fold,  but  partially 
inhibits  the  accumulation  of  cyclic-GMP.   These  findings  are  thus  consistent 
with  the  view  that  carbamyl  choline  exerts  its  effects  on  cyclic  nucleotide 
formation  in  part  by  modifying  intracellular  Ca++  concentrations. 

We  have  confirmed  that  succinylation  of  cyclic-GMP  and  cyclic-AMP  increases 
the  sensitivity  of  the  immunoassay  methods  for  these  substances  by  100-fold. 
With  these  sensitive  methods,  we  have  shown  that  cyclic-AMP  in  tracheal 
smooth  muscle  is  increased  by  epinephrine  and  a  vasoactive  intestinal  poly- 
peptide and  that  theophylline  potentiates  these  effects.   We  have  also  shown 
that  cyclic-GMP  in  isolated  pancreatic  acinar  cells  is  increased  by  choli- 
nergic stimulants  and  that  atropine  blocks  these  effects. 


/ssr 


Project  No.  Z01  HL  00801-01  LCP 

1.  Chemical  Pharmacology 

2 .  Enzyme-Drug  Interaction 

3 .  Bethesda  ,  Md . 


PHS  -NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Metabolic  activation  of  CH-methyldopa 

Previous  Serial  Number:   None 


Principal  Investigators: 


Dr.  E.  Dybing 

Dr  .  J .  R.  Mitchell 

Dr.  S  .  D.  Nelson 

Dr.  J.  R.  Gillette 


Other  Investigators: 


Mr.  Kenneth  Greene 
Mr  .  John  George 

Dr.  Dybing  is  a  Fogarty  International  Fellow, 


Cooperating  Unit: 

Project  Description: 

Objectives :   Renewed  interest  in  the  hepatic  injury  produced  by  methyl- 
dopa  (MD)  has  been  stimulated  by  recent  reports  that  the  antihypertensive 
drug  may  initiate  chronic  active  liver  disease,  occasionally  with  a  fatal 
outcome.   The  hepatic  damage  has  been  attributed  to  hypersensitivity  rather 
than  to  direct  toxicity,  but  careful  review  of  the  literature  reveals  that 
the  syndrome  is  similar  to  that  produced  by  isoniazid.  Most  individuals 
fail  to  show  constitutional  features  indicative  of  an  allergic  response  but 
usually  demonstrate  hepatic  injury  upon  rechallenge  only  after  lengthy  re- 
exposure  to  MD .   Moreover,  MD  produces  mild,  clinically  covert,  hepatic  in- 
jury in  157o  of  recipients  when  liver  function  tests  are  monitored  and  thus 
the  injury  is  not  restricted  to  rare,  idiosyncratic  individuals.   We  have 
been  interested  in  elucidating  the  role  of  the  liver  microsomal  cytochrome 
P-450  system  in  a  possible  metabolic  activation  of  MD . 

Methods  Employed:   To  assess  the  direct  hepatotoxicity  of  MD ,  large 
doses  (100-400  mg/kg)  were  administered  i .p .  or  i.v.  to  animals.   3h-MD  was 
incubated  with  rat  or  mice  microsomal  protein  in  the  presence  of  a  NADPH- 
generating  system,  and  the  covalent  binding  of  reactive  intermediates  to 
microsomal  proteins  was  determined  at  various  substrate  concentrations  and 
under  varying  incubation  conditions  according  to  conventional  methods. 

Major  Findings:   MD  produced  mild  hepatic  injury  with  diffuse  acido- 
philic bodies  in  male  Fisher  rats  (min.  toxic  dose  250  mg/kg),  but  no  in- 
crease in  lipid  diene  conjugation  nor  depletion  of  glutathione  were  found. 
A  large  amount  of  covalent  binding  occurred  when  -%-MD  was  incubated  with  rat 
or  mice  microsomal  protein  in  the  presence  of  NADPH  and  O2  (Vmax  0.5  nmoles/ 
mg/min  in  rats,  0.4  nmoles/mg/min  in  mice,  K^  50  microM) .   The  binding  was 


M 


Project  No.  Z01HL  00801-01  LCP 


inhibited  by  a  CO:02  atmosphere  (9:1),  indicating  the  involvement  of  cyto- 
chrome P-450.   Moreover,  antibody  prepared  against  NADPH  cytochrome  £  re- 
ductase inhibited  binding  by  49% .   However,  MD  did  not  show  P-450  binding 
spectra  (Type  I,  II,  or  III)  and  its  covalent  binding  was  inhibited  by  super- 
oxide dismutase,  ascorbic  acid  (1  mM) ,  ethy lenediamine  (20  mM)  and  gluta- 
thione (1  mM)  ,  indicating  that  activation  by  superoxide  anion  probably  to  the 
semiquinone  radical  was  occurring.   The  covalent  binding  was  inhibited  by 
analogs  such  as  1-dopa,  dopamine,  epinephrine,  norepinephrine,  catechol  and 
resorcinol  but  not  by  3-methoxy-CU-methyl-tyrosine  (3-O-methyldopa)  .   These 
analogs,  but  not  3-O-methyldopa,  also  were  found  to  covalently  bind  after 
microsomal  activation.   Additional  studies  with  MD  demonstrated  that  the  rat 
microsomal  system  could  be  replaced  by  human  hepatic  microsomes  or  by  a 
xanthine  oxidase  system  and  the  binding  again  was  inhibited  by  superoxide 
dismutase.   Metabolic  activation  by  superoxide  anion  may  play  a  role  in  the 
toxicity  of  many  catechols  and  catechol-like  compounds. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
The  results  demonstrate  that  MD  can  be  converted  by  hepatic  cytochrome  P-450 
to  a  potent  arylating  agent.   This  reaction  mechanism  may  be  responsible  for 
the  hepatotoxicity  of  MD  in  patients. 

Proposed  Course  of  Project:   Nearing  completion;  manuscripts  are  in 
preparation . 

Keyword  Description: 

methy  idopa 

superoxide  anion  radicals 
superoxide  dismutase 
xanthine  oxidase 


Honors  and  Awards:  None 
Publications:   None 


Project  No.  Z01  HL  00802-03  LCP 


1.  Chemical  Pharmacology 

2.  Enzyme-Drug  Interaction 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  197)+  through  June  30,  1975 

Project  Title:   Effect  of  Spironolactone  Analogues  on  Testicular  P-^50 

Previous  Serial  No.  NHLI-39,  NHLI  191 

Principal  Investigators:   Dr.  H.  Borner 

Dr.  J.R.  Gillette 

Other  Investigator:       Mr.  John  George 

Cooperating  Units :        None 

Project  Description: 

Objectives :   In  this  laboratory  it  was  shown  that  treatment  with 
spironolactone  causes  a  specific  breakdown  of  testicular  cytochrome  P-U50 
in  vitro.   In  order  to  elucidate  the  destruction  mechanism,  in  vivo  and  in 
vitro  studies  with  spironolactone  and  structural  analogues  were  carried  out. 

Methods  Employed:   Standard  biochemical  techniques  were  used. 

Major  Findings:   In  vitro  studies  with  spironolactone  revealed  that 
spironolactone  causes  a  breakdown  of  testicular  cytochrome  P-i+50  only  when 
NADPH  is  present  and  the  incubation  is  carried  out  at  37°C.   The  testicular 
cytochrome  P-^50  of  the  guinea  pig  was  the  most  sensitive  to  spironolactone 
among  rats,  mice,  rabbits  and  dogs. 

Therefore,  the  effect  of  various  analogues  on  the  testicular  cytochrome 
P-i+50  of  guinea  pig  was  checked  in  the  presence  of  NADPH  at  37°C. 

Final  concentrations  of  lk   to  5^0  uM  were  used.   Here  the  limiting  factor 
is  the  low  solubility  of  the  compounds.   Propylene  glycol  was  used  to  facili- 
tate solution  of  spironolactone  and  analogues.   However,  propylene  glycol 
caused  destruction  of  cytochrome  P-^50  at  concentrations  higher  than  300  ul/ 
3  ml.   Spironolactone  dissolved  in  aqueous  propylene  glycol  (100  yl/ml) 
causes  a  50%   destruction  on  testicular  P-U50  within  30  minutes  "In  the 
presence  of  NADPH  but  not  in  the  absence  of  NADPH.   But  all  the  other  com- 
pounds decreased  the  P-i+50  by  less  than  10%.   It  seems  likely  that  the 
initial  hydrolysis  of  the  thioacetyl-ester  group  which  is  assumed  to  be 
necessary  before  the  formation  of  the  reactive  metabolite  is  hindered. 

The  studied  compounds  were: 


/8f 


Project  No.  Z01  HL  00802-03  LCP 


oS0 


_r  =  S-C-CHo  =  Spironolactone 
f, 
=   __S-C-C(-CH3)3  =   Sc    27825 


'/ 


=   __ S-C-Cl^-CHg-/- 1  =  Sc   27937 
=  __S-C  fS>  ~      Sc   27996 


C02-CH3 


Sc    25152 


£U 


Sc   23133 


Dtiiroxazone 


Emdabol 


C?0 


Project  No.  Z01  HL  00802-03  LCP 

Significance  to  Biomedical  Research  and  to  the  Program  of  the  Institute: 
The  elucidation  of  the  mechanism  by  which  spironolactone  causes  a  breakdown 
of  cytochrome  P-l+50  may  give  information  which  could  be  helpful  for  the 
future  design  of  diuretic  steroids  without  side  effects. 

Proposed  Course  of  Project:   In  vivo  studies  should  be  carried  out  for 
comparison. 

Keyword  Description:   Spironolactone,  Cytochrome  P-U50  and  Testicular 

Honors  and  Awards :   None 

Publications :   None 


/*/ 


Project  No.  Z01  HL  OO8CR-O6  LCP 

1.  Chemical  Pharmacology 

2.  Enzyme-Drug  Interaction 

3.  Bethesda,  Md . 

PHS  -NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  role  of  cytochrome  b^  in  cytochrome  P-450  systems 

Previous  Serial  Number:   NHLI  41 

Principal  Investigators:   Dr.  Henry  A.  Sasame 

Dr.  James  R.  Gillette 

Other  Investigators:      None 

Cooperating  Unit:         None 

Project  Description: 

Objectives :   Last  year  we  reported  the  results  of  immunochemical  studies 
which  demonstrated  unequivocally  that  the  synergistic  effect  of  NADH  on 
NADPH-dependent  metabolism  of  various  compounds  by  cytochrome  P-450  enzymes 
in  liver  microsomes  is  mediated  by  cytochrome  b^ .   In  addition,  we  also 
demonstrated  that  cytochrome  b^  also  plays  a  role  in  the  NADPH-dependent 
metabolism  of  various  compounds  even  in  the  absence  of  NADH.   However,  the 
anti-cytochrome  b5  antibody  inhibited  drug  metabolism  more  than  it  inhibited 
NADPH  oxidation.   The  objective  of  this  project  was  therefore  to  determine 
the  reason  for  this  discrepancy. 

Methods  Employed:   An  anti-cytochrome  be  antibody  fraction  was  prepared 
from  antiserum  of  sheep  immunized  against  cytochrome  br  purified  from  rat 
liver  microsomes  as  reported  in  last  year's  report.   The  rate  of  superoxide 
anion  formation  by  cytochrome  P-450  enzymes  in  rat  liver  microsomes  was 
assayed  spectrophotometrically  by  following  the  rate  of  adrenochrome  forma- 
tion from  epinephrine.   The  reduction  rate  of  cytochrome  br  in  rat  liver 
microsomes  was  measured  in  a  stop-flow  apparatus  attached  to  Aminco  DW-2 
spectrophotometer.   Standard  biochemical  procedures  were  used  to  measure 
other  functional  components  of  the  microsomal  cytochrome  P-450  system. 

Major  Findings:   1)   We  have  discovered  that  after  the  anti-cytochrome 
be;  antibody  blocks  the  transfer  of  electrons  from  cytochrome  b^  to  the  oxy- 
genated cytochrome  P-450  substrate  complex,  the  concentration  of  the  complex 
increases  thereby  increasing  its  rate  of  dissociation  to  oxidized  cytochrome 
P-450  and  superoxide  anion  (O^-)  •   The  latter  was  detected  by  measuring  the 
cherry-red  colored  adrenochrome  formed  from  epinephrine  in  the  incubation 
media.   As  expected,  the  difference  in  the  rate  of  adrenochrome  formation  in 
the  presence  and  absence  of  the  antibody  gamma  globin  was  greater  when  both 
NADH  and  NADPH  were  present  than  when  only  NADPH  was  present  in  the  incuba- 
tion media.   In  the  absence  of  the  antibody,  NADH  increases  the  rate  of  re- 

1  ft* 


Project  No.  Z01  HL  00803-06  LCF 

duction  of  the  complex  formed  in  the  presence  of  NADPH  thereby  decreasing  the 
rate  of  formation  of  superoxide,  whereas  in  the  presence  of  the  antibody, 
NADH  scarcely  alters  the  rate  of  superoxide  formation.   Since  reduction  of 
the  oxygenated  cytochrome  P-450  complex  is  required  for  drug  metabolism,  the 
formation  of  superoxide  accounts  for  the  inhibition  of  drug  metabolism  without 
inhibition  of  NADPH  oxidation. 

2)   Further  evidence  supporting  the  role  of  cytochrome  be  as  a  source  of 
second  electron  in  NADPH-mediated  cytochrome  P-450  system  in  rat  liver  micro- 
somes was  unveiled  by  studies  with  liver  microsomes  isolated  from  rats 
pretreated  with  pregnenolone  carbonitrile  (PCN)  .   Like  the  induction  caused 
by  phenobarbital,  the  induction  by  PCN  increases  both  NADPH  cytochrome  £ 
reductase  and  cytochrome  P-450  levels  in  rat  liver  microsomes.   However  PCN 
markedly  decreases  the  rate  of  cytochrome  be,  reduction  and  increases  in  the 
uncoupling  between  drug  metabolism  and  NADPH  oxidation.   These  conclusions 
were  based  on  the  following  facts:   i)   The  rates  of  cytochrome  bc_  reduction 
by  either  NADH  or  NADPH  were  slower  in  liver  microsomes  from  PCN  treated  rats 
than  in  those  from  phenobarbital  treated  rats.   ii)   The  addition  of  ethyl- 
morphine  increased  the  rate  of  NADPH-mediated  adrenochrome  formation  by  liver 
microsomes  to  a  greater  extent  after  the  PCN  treatment  than  after  the  pheno- 
barbital treatment.   iii)   The  decrease  in  the  steady  state  level  of  cyto- 
chrome br  caused  by  the  addition  of  ethylmorphine  was  much  greater  in  micro- 
somes from  PCN  treated  rats  than  in  those  from  phenobarbital  treated  animals. 
The  presence  of  the  anticytochrome  br  antibody  decreased  the  steady-state 
level  of  reduced  cytochrome  be  in  liver  microsomes  from  PCN  treated  rats  and 
prevented  the  decrease  caused  by  ethylmorphine.  Moreover,  the  addition  of 
ethylmorphine  lowered  the  NADH  dependent  steady  state  level  of  reduced  cyto- 
chrome b^  in  liver  microsomes  from  PCN  treated  animals  but  had  no  effect  on 
the  steady  state  level  in  microsomes  from  phenobarbital  treated  rats.   iv) 
The  anti -cytochrome  b5  antibody  inhibited  the  NADPH  dependent  ethylmorphine 
metabolism  in  microsomes  from  PCN  treated  rats  by  as  much  as  50%,  suggesting 
that  PCN  treatment  also  slows  the  reduction  of  the  oxygenated  cytochrome 
P-450  substrate  complex  by  NADPH  cytochrome  £  reductase  as  well  as  that  by 
cytochrome  b^ . 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
Since  it  has  been  suggested  that  superoxide  may  cause  cytotoxic  effects,  it 
is  important  to  delineate  the  role  of  cytochrome  b^  in  decreasing  superoxide 
formation . 

Proposed  Course  of  Project:   We  shall  study  the  effects  of  the  anti- 
cytochrome b<j  antibody  and  PCN  treatment  on  the  in  vitro  metabolism  of  various 
drugs  and  steroids  that  cause  toxicity  in  animal  models. 

Keyword  Description: 

cytochrome  b5 
cytochrome  P-450 
superoxide 


f9t 


Project  Wo.   Z01  HL  00803-06  LCP 


Honors  and  Awards:   None 
Publications : 


Sasame,  H.A.,  Thorgeirsson,  S.  S.,  Mitchell,  J.R.  and  Gillette,  J.R.: 
The  role  of  cytochrome  be  in  cytochrome  P-450  enzymes.   In  the 
Proceedings  of  the  Second  Philadelphia  Conference  on  Heme  Protein  P-450 
New  York,  Plenum  Press,  in  press. 


t9* 


Project  No.  Z01  HL  0082S-01  LCF 

1.  Chemical  Pharmacology 

2.  Drug-Tissue  Interaction 

3.  Bethesda,  Md . 

PHS  -NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Role  of  guanine  nucleotides  in  vision 

Previous  Serial  Number:   None 

Principal  Investigator:   Dr.  G.  Krishna 

Other  Investigators:     Dr.  N.  Krishnan 

Dr.  G.  Chader  (NEI) 

Cooperating  Unit:        Laboratory  of  Vision,  NEI 

Project  Description: 

Objectives:   Retinal  rod  outer  segment  which  is  the  photo  receptor  unit 
of  the  neuro  retina  contains  extraordinarily  high  enzyme  activities  for  the 
synthesis  and  degradation  of  cyclic  GMP .   Moreover,  it  contains  very  high 
protein  kinase  activity  which  specifically  phosphorylates  opsin.   The  role 
of  these  enzymes  in  vision  is  not  clearly  understood.   The  main  objective  of 
this  study  is  to  examine  the  effect  of  light  exposure  of  the  rod  outer  seg- 
ments on  the  activities  of  these  enzyme  systems  and  to  indicate  the  possible 
role  of  cyclic  GMP  in  vision. 

Methods  Employed:   Dark  adapted  bovine  retinal  rod  outer  segments  were 
prepared  by  a  method  which  did  not  involve  homogenization .   The  rod  outer 
segments  were  purified  by  sucrose  gradient  and  were  suspended  in  Tris 
hydrochloride  buffer  pH  7.6  containing  5  mM  Mgc^  (1  mg  protein/ml).   The 
segments  were  exposed  to  light  (100  ft  candles)  for  specific  periods  of  time, 
The  assays  of  enzyme  activities  were  carried  out  in  the  dark  under  diffused 
red  light . 

Guanylate  cyclase  and  cyclic  GMP  phosphodiesterase  were  assayed 
according  to  the  methods  described  in  last  year's  report.   GTP -opsin  kinase 
or  ATP -opsin  kinase  was  measured  using  either  100  p.M  ?-32p  GTP  or  7-32p  ATP. 
The  phosphorylated  protein  were  separated  on  a  millipore  filter.   In  some 
experiments  the  phosphorylated  opsin  was  isolated  on  sepharose  columns. 
Cyclic  GMP  was  assayed  according  to  the  methods  described  in  one  of  this 
year's  reports  (Frandsen  and  Krishna). 

Major  Findings:   Bovine  retinal  rod  outer  segments  contain  the  highest 
guanylate  cyclase  activity  of  any  tissues  thus  far  examined  (5  nmoles  of 
cyclic  GMP  formed  per  mg  protein  per  minute).   This  enzyme  undergoes  light- 
induced  inhibition  (30%)  within  seconds.   At  the  same  time,  the  enzyme 


/*r 


Project  NO...ZQ1  HL  .0082S-01  LHP 

responsible  for  degradation  of  cyclic  GMP  undergoes  light-induced  activation 

(10-fold)  which  requires  the  presence  of  ATP  or  GTP .   Other  nucleotides, 

ITP  or  UTP ,  have  a  smaller  effect  on  the  enzyme  system  while  CTP  has  no  effect. 

Cyclic  GMP  is  present  in  very  high  concentration  in  the  rod  outer 
segments  (500  pmoles  per  mg  protein)  which  is  200-300  times  higher  than  any 
other  tissues.   The  identity  of  cyclic  GMP  has  been  verified  by  three  inde- 
pendent procedures  including  high  pressure  liquid  chromatography.   This 
nucleotide  appears  to  be  sequestered  in  the  rod  outer  segments  and  does  not 
undergo  rapid  degradation  by  the  enzyme  system  present  in  the  rod  outer 
segments.   The  cyclic  GMP  also  undergoes  light-induced  changes  to  a  very 
small  extent  (207»)  . 

Cyclic  AMP  is  also  present  but  only  to  the  extent  of  5  pmoles/mg  protein. 

The  specific  protein  kinase  present  in  the  rod  outer  segments  undergoes 
rapid  activation  (within  one  second)  by  light  exposure  of  rod  outer  segments. 
This  light-induced  activation  is  mainly  due  to  the  conversion  of  rhodopsin 
to  opsin  by  the  light,  and  the  enzyme  phosphory lates  only  opsin  as  shown  by 
chromatography  on  sepharose  columns.   The  light  activated  opsin  phosphory- 
lation is  effected  by  GTP  or  ATP.   GTP  appears  to  be  more  efficient  and  the 
phosphorylation  by  GTP  is  markedly  inhibited  by  cyclic  AMP  and  other  adenine 
nucleotides.   Inorganic  phosphate  also  maredly  inhibits  GTP  opsin  kinase. 
The  ATP  opsin  kinase  is  not  inhibited  to  the  same  extent  by  adenine  nucleo- 
tides and  is  maredly  activated  by  phosphate.   These  differential  effects  as 
well  as  direct  experimentation  indicate  that  GTP  opsin  phosphorylation  is  not 
mediated  through  ATP. 

Calcium,  which  is  known  to  mimic  light  in  its  ability  to  hyperpolarize 
the  membrane  rod  outer  segments,  markedly  inhibits  light -induced  GTP  opsin 
kinase.   This  indicates  the  effect  of  calcium  in  mimicking  light  may  occur 
at  the  step  beyond  light-induced  changes  in  enzyme  activities. 

Preliminary  experiments  indicate  that  the  phosphorylated  opsin  molecule 
can  transfer  phosphate  to  ADP .   The  exact  mechanism  of  ATP  formation  within 
the  disc  membrane  is  not  clear,  but  the  possibility  of  the  involvement  of 
phosphorylation  of  opsin  in  the  transfer  of  phosphate  from  outside  to  inside 
the  disc  membrane  is  indicated,  and  this  may  result  in  the  efflux  of  calcium 
from  the  disc  involving  calcium  ATPase.   The  increase  in  calcium  will  block 
effectively  the  sodium  channels  resulting  in  hypopolarization  and  chus 
resulting  in  the  conversion  of  light  to  electrical  energy. 

The  above  experiments  strongly  indicate  the  phosphorylation  of  opsin  by 
GTP  and  modulation  by  cyclic  AMP  and  other  adenine  nucleotides  may  play  an 
important  role  in  vision. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
The  finding  that  rod  outer  segments  contain  very  high  concentrations  of  cyclic 
GMP  and  the  enzyme  system  for  degradation  and  synthesis  indicate  that  the 
photo  receptor  unit  of  the  retina  may  represent  a  unique  system  where  cyclic 

2  fn 


Project  No.  Z01  HL  00825-01  LCP 

GMP  and  guanine  nucleotides  play  a  more  important  role  than  the  adenine 
nucleotides.   This  study  will  enable  us  to  understand  the  role  of  cyclic  GMP 
and  GTP  in  other  systems  including  the  heart  and  lung  in  modulating  choliner- 
gic functions. 

Proposed  Course  of  Project:   The  role  of  GTP -opsin  kinase  in  the  transfer 
of  phosphate  from  outside  of  the  disc  membrane  to  inside  and  the  role  of 
calcium  in  mimicking  light  will  be  investigated  in  detail. 

Keyword  Description: 


cyclic  GMP 

guanine  nucleotides 

opsin  phosphorylation 


retinal  rod  outer  segments 
vision 


Honors  and  Awards:   None 


Publications : 


Chader,  Gerald  J.,  Fletcher,  R.T.,  and  Krishna  G.:   Light-induced 
phosphorylation  of  rod  outer  segments  by  guanosine  triphosphate. 
Biochem.  &  Biophys.  Research  Comm.,  in  press. 


irr 


Project  No.  Z01  HL  00826-02  LCP 

1.  Chemical  Pharmacology 

2.  Drug-Tissue  Interaction 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  197^  through  June  30,  1975 

Project  Title:   Studies  on  the  Covalent  Binding  of  Chloramphenicol 

Previous  Serial  No.  NHLI-52 

Principal  Investigators:   Dr.  Lance  R.  Pohl 

Dr.  B.G.  Reddy 
Dr.  Gopal  Krishna 

Other  Investigator:       Ms.  Ethel  Boykins 

Cooperating  Unit :         None 

Project  Description: 

Objectives :   Chloramphenicol  (-*-  C)  has  previously  been  shown  to  be 
covalently  bound  in_  vivo  predominantly  to  proteins  of  the  liver,  bone 
marrow,  and  plasma.   The  present  research  is  being  conducted  in  order 
to  determine  the  mechanism(s)  by  which  chloramphenicol  is  activated  to 
metabolites  that  rc-act  with  tissue  macromolecules .   This  study  will  hope- 
fully lead  to  a  better  general  understanding  of  the  mechanism(s)  involved 
in  chloramphenicol-induced  bone  marrow  damage.   In  addition,  it  may  also 
lead  to  an  intimate  understanding  of  the  chemical  reactions  between  re- 
active metabolites  and  biological  molecules.   Such  interactions  appear  to 
be  involved  in  the  hepatotoxicity ,  and  carcinogenicity  induced  by  several 
chemical  agents.   Similar  interactions  may  also  be  involved  in  the  develop- 
ment of  blood  dyscrasias ,  such  as  chloramphenicol-induced  asplastic  anemia. 

Methods  Employed:   l)  ^-Chloramphenicol  (Fig.l)  was  synthesized  by 
reaction  of  the  keto-derivative  of  chloramphenicol  with  ^H-calcium  boro- 
hydride.   The  keto  derivative  of  chloramphenicol  was  prepared  by  oxidation 
of  chloramphenicol  with  N-bromosuccinamide. 

2)  Covalent  binding  of  chloramphenicol  was  studied  in  vitro  utilizing 
rat  liver  microsomes  and  NADPH  as  described  in  earlier  reports.   For  these 
studies  double-labeled  (l^C  and  3h)  chloramphenicol  was  employed. 

3)  In_  vivo  covalent  binding  to  bone  marrow  and  other  tissues  of  the  rat 
was  studied  by  injecting  doiible-labeled  (1^C  and  -%)  chloramphenicol  (Fig.l) 
(30  mg/kg,  po)  to  phenobarbital-pretreated  rats.   Various  tissues  were 
removed  at  the  end  of  2h   hours  and  covalent  binding  to  various  tissues  was 
studied  as  reported  earlier. 


/ffl 


Project  No.  Z01  HL  00826-02  LCP 

Major  Findings:   l)  Covalent  Binding  in  vitro.   Both  -%  and   C  chlor- 
amphenicol bind  to  rat  liver  microsomes  at  the  rate  of  200  pmoles/mg  protein. 
The  rate  of  the  reaction  appears  to  slow  after  h   minutes  of  incubation. 
After  10  minutes  about  20%  more  H  than   C  chloramphenicol  is  bound  co- 
valently  to  rat  liver  microsomes.   These  results  indicate  that  the  majority 
of  the  covalently  bound  molecules  contain  the  entire  molecule  of  chloram- 
phenicol.  The  most  likely  bioactivation  of  chloramphenicol  that  could  lead 
to  covalent  binding  appears  to  be  arene  oxide  formation,  hydroxylation  and/or 
free  radical  formation  at  the  dichloroacetamide  group. 

2)  Covalent  Binding  in  vivo.   When  double  labeled  chloramphenicol  was 
administered  to  rats  at  a  dose  of  30  mg/kg  po ,  both  H  and  -1-  C  appear  to 
be  bound  covalently  to  various  tissue  macromolecules .   The  covalent  binding 
of   C  chloramphenicol  appears  to  be  similar  to  that  obtained  in  last  year's 
studies.   However,  the  binding  cf  ^H  chloramphenicol  is  markedly  different  in 
various  tissues.   Liver  contains  about  30-^0%  of  3h  chloramphenicol  bound  co- 
valently as  compared  to  ■*■  C  chloramphenicol  while  bipod  and  bone  marrow  con- 
tain only  10%  of  ^h  chloramphenicol  as  compared  to   C  chloramphenicol.   Thus, 
it  appears  that  in  vivo  chloramphenicol  may  undergo  cleavage  of  the  dichloro- 
acetamide group  either  before  or  after  binding  to  macromolecules  resulting  in 
differential  binding  of  -^C  and  -%  chloramphenicol.   It  is  also  conceivable 
that  bioactivation  may  involve  an  oxidation  of  chloramphenicol  keto  compound 
resulting  in  the  loss  of  -%  before  covalent  binding. 

Significance  cf  Biomedical  Research  and  the  Program  of  the  Institute:   The 
finding  that  the  covalent  binding  of  -^C  and  ^H  chloramphenicol  is  markedly 
different  in  vivo  in  comparison  to  in  vitro  indicates  a  more  complex  nature 
of  bioactivation  of  drug  molecules  in_  vivo  which  may  be  responsible  for  the 
drug-Induced  tissue  damage.   Thus  it  is  not  possible  to  predict  any  mechanism 
of  drug  activation  from  studies  utilizing  liver  microsomes  in_  vitro  alone. 

Proposed  Course  of  Project:   Various  tissue  macromolecules  containing 
covalently  bound  ^H  and  14C  chloramphenicol  will  be  hydrolyzed  with  pronase 
in  order  to  isolate  the  amino  acids  containing  the  label  from  the  chloram- 
phenicol molecules.   The  analysis  of  the  material  would  enable  us  to  arrive 
at  a  mechanism  of  activation  and  covalent  binding  of  chloramphenicol. 

We  also  propose  to  study  the  suceptability  of  various  animal  species  to 
chlorampheni col-induced  bone  marrow  damage.   Since  a  number  of  possible 
metabolites  of  chloramphenicol,  such  as  dichloracetic  acid,  chloramphenicol 
base  and  keto  derivative  of  chloramphenicol,  are  available  in  sufficient 
amounts  we  propose  to  study  whether  these  metabolites  are  covalently  bound 
and  are  eapable  of  producing  bone  marrow  damage  in  various  animal  species. 

Since  numerous  attempts  in  this  laboratory  and  others  have  failed  to 
induce  aplastic  anemia  in  experimental  animals  with  chloramphenicol,  we 
propose  to  determine  whether  chloramphenicol  causes  aplastic  anemia  in  animals 
whose  hemopoietic  system  is  markedly  stimulated  by  pretreatment  with  phenyl- 
hydrazine  . 

2  /*? 


Project  No.     Z01  HL  00826-02  LCP 

Keyword  Description:   Covalent  Binding  and  Chloramphenicol 

Honors  and  Awards:   None 

Publications : 

Krishna,  G.  and  Bonanomi ,  L. :   Covalent  Binding  of  Chloramphenicol 
as  a  Biochemical  Basis  for  Chloramphenicol-induced  Bone  Marrow 
Damage.  In  Morselli ,  P.L.,  Garattini ,  S.  and  Cohen,  S.N.  (Eds.): 
Drug  Interactions.   New  York,  Raven  Press,  197*+,  pp.  173-180. 

Krishna,  G. :   Covalent  binding  of  drugs  to  tissue  macromolecules  as 
a  biochemical  mechanism  of  drug  toxicities  with  special  emphasis  on 
chloramphenicol  and  thiamphenicol.   Postgraduate  Medical  Journal  50: 
73-77,  1971*. 

Rao,  G.S.,  Krishna,  G.  and  Gillette,  J.R.:   The  enzymatic  formation^ of 
chemically  reactive  metabolites  of  N-nitroso-monomethyl  tripelennamine 
by  a  mechanism  other  than  N-dealkylation.   Biochemical  Pharmacology, 
in  press . 

Asghar,  K. ,  Reddy,  B.G.  and  Krishna,  G. :   Hi sto chemical  localization 
of  glutathione  in  tissues.   The  Journal  of  Histochemistry  and 
Cytochemistry,  in  press. 

Docks,  E.L.  and  Krishna,  G. :   Covalent  binding  of  trans-stilbene  to 
rat  liver  microsomes.   Biochemical  Pharmacology,  in  press. 


HO-C-H™ 

H-C-NH  CO  CHC12 
CH20H 

*l*+c      **% 

Fig.  1   CHLORAMPHENICOL 


$jCO 


Project  Wo.  Z01  HL  00827-01  LCP 


1.  Chemical  Pharmacology 

2.  Drug-Tissue  Interaction 
3  .   Bethesda ,  Md . 

PHS  -NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Role  of  intestinal  flora  on  nitrobenzene-induced  toxicities. 

Previous  Serial  Number:  None 

Principal  Investigators:   Dr.  B.  G.  Reddy 

Dr.  Lance  R.  Pohl 
Dr.  Gopal  Krishna 

Other  Investigators:      None 

Cooperating  Unit:  None 

Project  Description: 

Objectives :   Nitrobenzene  has  been  known  to  have  a  toxic  effect  upon 
the  hemopoetic  system.   One  of  the  major  pathological  changes  observed  with 
nitrobenzene  administration  in  acute  studies  is  methemoglobinemia.   In  order 
to  understand  the  mechanism  of  formation  of  methemoglobin ,  we  have  studied 
the  relationship  of  biotransformation  of  nitrobenzene  to  the  toxicity.   This 
investigation  will  further  serve  as  a  model  study  for  more  complex  molecules. 
In  particular,  these  studies  may  serve  as  an  important  model  for  studying 
various  toxicities  produced  by  the  antibiotic  chloramphenicol  which  contains 
a  p-nitropheny 1  group. 

Methods  Employed:   Male,  Sprague  Dawley  rats,  180-220  g,  from  Hormone 
Assay  Labs  were  used  in  this  study.   Nitrobenzene  (200  mg/kg)  and  m-dinitro- 
benzene  (20  mg/kg)  were  administered  intraperitoneally  to  rats,  and  the  rate 
of  formation  and  disappearance  of  methemoglobin  was  measured.   In  order  to 
understand  the  role,  if  any,  of  the  cytochrome  P-450  systems  in  these  re- 
actions, the  experiments  have  also  been  conducted  with  phenobarbita 1  and 
piperonyl  butoxide  pretreated  rats  since  these  two  agents  are  known  to  induce 
and  inhibit,  respectively,  these  systems.   Similar  experiments  have  also  been 
conducted  in  rats  whose  intestinal  flora  was  destroyed  by  antibiotic  treat- 
ment and  in  germ -free  rats.   These  studies  were  run  in  order  to  assess  the 
involvement  of  the  intestinal  flora  in  the  biotransformation  and  toxicity 
of  nitrobenzene . 

Major  Findings:   As  shown  in  Table  1,  route  of  administration  had  a 
significant  (P  <  .01)  effect  on  the  level  of  methemoglobin  formation  by  both 
nitrobenzene  and  meta -dinitrobenzene  .   The  effect  on  methemoglobin  formation 
was  more  pronounced  when  nitrobenzene  was  administered  intraperitoneally  in 
comparison  to  the  oral  route.   When  nitrobenzene  was  administered  to  rats  by 
oral  route,  the  methemoglobin  levels  never  reached  above  6%  of  total 


2.0f 


Project  No.  Z01  HL  00827-01  LCP 


hemoglobin  during  the  8  hr  period  whereas,  administration  of  nitrobenzene  by 
intraperitoneal  route,  the  methemoglobin  levels  reached  a  peak  within  2  hr 
and  then  declined.   It  is  evident  from  the  table,  the  route  of  administration 
had  a  small  but  significant  effect  (P  <  .01)  on  the  methemoglobin  formation 
induced  by   meta-dinitrobenzene .   Based  on  these  observations,  the  intra- 
peritoneal route  was  chosen  for  further  studies.   The  highest  levels  of 
methemoglobin  in  both  i.p.  and  oral  route  was  found  to  be  between  1  and  2  hr 
after  administration  of  these  compounds. 

The  effects  of  phenobarbital  and  piperonyl  butoxide  pretreatments  on 
methemoglobin  formation  are  shown  in  Table  2.   Table  2(a)  shows  that  pheno- 
barbital increased  significantly  (P  <  .05)  methemoglobin  formation  by  nitro- 
benzene compared  to  normal  rats.   This  suggests  that  the  liver  may  have  a 
role  in  the  bioactivation  of  nitrobenzenes  to  a  metabolite  which  is  respon- 
sible for  methemoglobin  formation.   Piperonyl  butoxide  pretreatment  did  not 
significantly  affect  methemoglobin  formation  by  nitrobenzene.   However,  the 
rate  of  methemoglobin  disappearance  appears  to  be  similar  in  all  cases. 

With  m-dinitrobenzene ,  the  peak  levels  of  methemoglobinemia  seems  to  be 
affected  by  the  pretreatment  with  phenobarbital  and  piperonyl  butoxide  |_Table 
2(b)j.   However,  in  this  case,  pretreatment  with  piperonyl  butoxide  and 
phenobarbital  tend  to  decrease  the  level  of  methemoglobinemia  produced  by 
m-dinitrobenzene.  More  work  is  needed  before  these  differences  in  the  be- 
havior of  these  nitrobenzenes  can  be  explained. 

Table  3(a)  shows  the  effect  of  removal  of  intestinal  flora  from  the  rat 
on  nitrobenzene -induced  methemoglobin  formation.   No  methemoglobin  was  formed 
by  administration  of  nitrobenzene  to  germ  free  rats,  whereas  the  same  rats, 
after  being  acclimatized  for  a  week  in  the  animal  room,  responded  to  nitro- 
benzene with  formation  comparable  to  normal.   Similar  results  were  also  ob- 
tained in  the  rats  whose  intestinal  flora  was  removed  by  antibiotic  treat- 
ment (neomycin,  bactrin  and  tetracycline). 

In  the  case  of  m-dinitrobenzene  i_Table  3(b)j,  it  appears  the  germ  free 
condition  also  has  an  effect  on  methemoglobin  formation.   However,  the 

b: 

Dbin 
-ompared 
corresponding  normals  and  reached  the  peak  between  2  to  3  hr  and  then  de' 
clined . 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
The  finding  that  the  intestinal  flora  markedly  influence  the  toxicity  produced 
by  nitrobenzene  adds  another  environmental  factor  in  which  the  enzyme  system 
of  the  bacteria  in  the  intestine  may  play  a  crucial  role  in  certain  drug- 
induced  diseases. 

Proposed  Course  of  Project:   In  the  light  of  the  available  data  from  the 
germ-free  and  antibiotic-treated  rats,  we  are  conducting  experiments  to  de- 
termine the  relative  contribution  of  enzyme  systems  in  liver,  intestinal 


A3  3- 


Project  No.  Z01  HL  00827-01  LCP 

musoca  and  intestinal  bacteria  to  the  formation  of  nitrobenzene -induced 
methemoglobinemia.   (1)   A  series  of  studies  are  being  conducted  in  rats 
whose  bile  flow  has  been  interrupted  in  order  to  assess  the  importance  of 
liver  in  the  formation  of  methemoglobin  by  nitrobenzene.   These  studies  will 
also  enable  us  to  assess  the  role  of  bacterial  as  well  as  intestinal  mucosal 
enzyme  system  in  the  formation  of  the  metabolite  responsible  for  the  methemo- 
globin formation  by  nitrobenzene.   (2)   We  also  shall  attempt  to  identify  the 
active  metabolite(s)  of  nitrobenzene  responsible  for  methemoglobin  formation 
by  comparing  the  spectrum  of  the  biotransformation  products  in  germ-free  and 
acclimatized  rats. 

It  is  hoped  that  this  research  will  lead  to  a  better  understanding  and 
serve  as  a  model  study  for  the  assessment  of  the  relative  importance  of  the 
intestinal  flora  in  the  metabolism  and  toxicity  of  drugs  and  environmental 
chemicals.   In  particular,  this  study  may  have  relevanc-e  to  the  hemapoetic 
toxic  properties  of  chloramphenicol,  which  contains  a  p-nitropheny 1  ring  in 
its  structure.   In  addition,  the  identification  of  the  active  metabolites  of 
nitrobenzene,  should  help  elucidate  the  general  mechanism  for  the  formation 
of  methemoglobin. 

Keyword  Description: 


germ-free  rats 
intestinal  flora 
methemoglobinemia 


nitrobenzene 
phenobarbita 1 
piperonyl  butoxide 


Honors  and  Awards:   None 


Publications:   None 


3e3 


Project  No.  Z01  HL  00827-01  LCP 
Table  1 


Effect  of  route  of  administration  on 
methemoglobin  formation 


Time    Nitrobenzene  (200  mg/kg) m-Dinitrobenzene  (20  mg/kg) 

(hr)      Oral       Intraperitoneal         Oral         Intraperitoneal 

per  cent  methemoglobin  per  cent  methemoglobin 

0.5    0.8  ±  0.8     12.3  ±  9.7  36.3  ±  3.3     47.7  ±  2.4" 

1  1.2  ±  1.2  27.0  ±  6.6*  45.0  ±  3.0  52.4  ±  1.3 

2  6.1  ±  4.3  33.4  ±  8.4"  44.4  ±  3.2  44.5  ±  1.5 
4  3.512.2  23. 0±  10.0*  21.5  +  4.3  21.0  ±  2.4 
8  0.3  ±  0.3  10.7  ±  6.9  4.7  ±  2.4        0  ±  0 

The  data  are  expressed  as  means  ±  S .E .  (N  =  3) . 
*P  <  .05 


9C# 


Project  No.    Z01  HL  00827-01  LCP 

Table    2(a) 

Effect  of  phenobarbital  and  piperonyl  butoxide  pretreatment 
on  nitrobenzene  (200  mg/kg  ip) -induced  methemoglobin  formation 


Time  Controls  Phenobarbital       Piperonyl 

treated  butoxide 

treated 

per  cent  methemoglobin 

1  hr  29.0  ±  2.7  34.0  ±  4.7*         25.3  ±  3.5 

2  hr  29.7  ±  2.6  32.7  ±  3.8  29.2  ±  3.9 
4  hr  22.9  ±  2.8  31.6  ±  3.3  28.1  ±  2.9 
6  hr  21.0  ±  1.4  25.6  ±  2.1  31.5  ±  0.8 
8  hr  15.9  ±  3.3  -  19.1  ±  2.0 

The  data  are  expressed  as  means  ±  SE  (N  =  6;  phenobarbital- 
pretreated  N  =  11) . 

*p  <  0.05 

Table  2(b) 

Effect  of  phenobarbital  or  piperonyl  butoxide  pretreatment  on 
m-dinitrobenzene  (20  mg/kg  i-p.)  induced  methemoglobin  formation, 

Time  Phenobarbital        Piperonyl 

interval  Control  pretreatment         butoxide 

pretreatment 

per  cent  methemoglobin 

1  hr  44.9  ±  4.5         40.9  +  5.2         39.7  +  3.3 

2  hr  38.4  ±  5.8  36.6  ±  8.3  37.5  ±  4.1 
4  hr  18.2  ±  3.9  9.1  ±  2.6  16.7  -  3.4 
6  hr  9.8  ±  5.6  0.8  +  0.5  5.4  ±  2.0 
8  hr  1.4  ±  1.4  0.1  ±0.1            0+0 

The    data   are   expressed  as   means   ±   SE    (N   =   6) . 


2&S 


Project  No.  Z01  HL  00827-01  LCP 

Table  3(a) 

Nitrobenzene  (200  mg/kg  i  .p  .) -induced  methemoglobin  formation 

in  germ  free,  germ  free-acclimatized  and 
antibiotic-pretreated  rats. 

Germ  free  Antibiotic 

Time             Germ  free          acclimatized  pretreated 

(hr)                rats                rats  rats 

per  cent  methemoglobin 

1  0              37.5  ±  4.2  0 

2  0              37.8+3.4  0 

4  0              26.1  ±  3.4  0 

5  0               5.0  +  0.5  0 
7                 0               4.4  ±  0.7  0 


Table  3(b) 

m-Dinitrobenzene  (20  mg/kg  i  .p  .) -induced  methemoglobin  formation  in 
germ  free,  germ  free-acclimatized  and  antibiotic-pretreated  rats. 

Germ  free  Antibiotic 

Time  Germ  free  acclimatized         pretreated 

(hr)  rats  rats  rats 

per  cent  methemoglobin 

1  6.9  ±  0.8         42.7  ±  4.2  18.7  ±  1.1 

2  22.6  +  0.7  26.9  ±  2.9  23.8  ±  3.2 

4  28.9  ±  2.5         4.96  ±  2.9  20.6  ±  0.6 

5  24.5  ±  3.7  0  17.8  +  1.3 
7              11.0  ±  6.4              0  4.5  ±2.1 

The  data  are  expressed  as  means  ±  SE  (N  =  3 ;  antibiotic-pretreated 
N  =  6). 


*o6> 


Project  No.  Z01  HL  00828-01  LCP 

1.  Chemical  Pharmacology 

2.  Drug -Tissue  Interaction 

3.  Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1971*  through  June  30,  1975 

Project  Title:   Role  of  lung  cyclic  nucleotides  in  paraquat  toxicity 

Previous  Serial  Number:   None 

Principal  Investigators:   Dr.  N.  Krishnan 

Dr .  G.  Krishna 

Other  Investigators :      None 

Cooperating  Unit :         None 

Project  Description: 

Objectives :   Paraquat  is  widely  used  as  an  herbicide.   The  plants  are 
destroyed  when  the  leaf  surfaces  are  exposed  to  paraquat  and  the  herbicide  is 
inactivated  by  absorption  onto  clay  minerals .   The  paraquat  toxicity  in 
human  has  been  well  documented,  the  pulmonary  tissue  being  the  main  target 
for  paraquat  toxicity.   Toxicity  in  man  occurs  mainly  after  drinking  solu- 
tions of  these  compounds  or  after  inhalation  of  dust.   Death  mainly  occurs 
after  2  to  5  days  with  pulmonary  edema  and  congestion  with  hyaline  membrane 
formation.   Gome  animal  species  become ihyperexcitable  after  doses  and  have 
convulsions  after  lethal  doses.   In  view  of  the  acute  toxicity  of  this  drug, 
attempts  are  being  made  in  several  laboratories  to  gain  an  insight  into  the 
mode  of  toxicity  of  this  compound  on  the  lung.   The  only  information  that  is 
presently  available  is  that  paraquat  appears  to  be  transported  by  an  energy 
dependent  process  and  stored  within  the  lung.   The  stored  paraquat  in  lung 
tissue  appears  to  not  be  metabolized  and  slowly  excreted  as  such  in  urine, 
and  thus  accounts  for  all  the  toxic  effects  observed  in  the  lungs.   Various 
attempts  are  being  made  to  understand  the  types  of  lung  cells  involved  in 
the  paraquat  toxicity.   The  cells  of  the  lung  which  has  the  highest  capacity 
to  take  up  and  bind  paraquat  would  be  the  likely  target  for  paraquat  toxicity. 
Since  paraquat  produces  pulmonary  edema  and  since  cyclic  AMP  has  been  known 
to  be  involved  in  movement  of  ions  across  various  membranes,  we  have  studied 
the  effect  of  paraquat  on  the  levels  of  cyclic  AMP  in  the  lung. 

Methods  Employed:   Paraquat  (25  mg/kg)  was  administered  intraperito- 
neally  to  male  Sprague-Dawley  rats  (150  g).   Rats  were  sacrificed  at  15  min 
and  30  min  intervals  after  administration  of  paraquat  and  the  lung  tissue 
was  removed  rapidly  and  frozen  in  liquid  nitrogen.   The  cyclic  AMP  and  cyclic 
GMP  were  extracted  with  10%  perchloric  acid  in  50%  methanol.   Cyclic  AMP  and 
cyclic  GMP  were  isolated  by  Dowex  1  chromatography  and  were  measured  using 
specific  radioimmunoassays. 


a&7 


Project  No.  Z01  HL  00828-01  LCP 


Major  Findings:   Lung  cyclic  AMP  levels  were  decreased  by  70%  over  the 
control  15  min  after  administration  of  paraquat;  while  30  min  after  admin- 
istration of  paraquat,  cyclic  AMP  levels  were  decreased  only  by  30%.      There 
were  no  changes  in  lung  cyclic  GMP  levels  at  these  times. 

Significance  to  Biomedical  Research  and  to  the  Program  of  the  Institute: 
A  marked  reduction  in  lung  cyclic  AMP  levels  within  15  min  after  administra- 
tion of  paraquat  suggests  that  the  cyclic  AMP  system  is  grossly  affected 
and  may  reflect  as  one  of  the  early  changes  that  occurs  in  the  onset  of 
toxicity  in  the  lung.   Further  studies  correlating  paraquat-induced  changes 
in  cyclic  AMP  levels  in  the  body  with  its  toxicity  are  essential  to  eluci- 
date this  point. 

Proposed  Course  of  Project:   We  propose  that  studies  on  various  neuro- 
hormonal  regulation  of  paraquat-induced  changes  in  cyclic  AMP  in  the  lung 
may  help  to  explain  if  cyclic  AMP  is  involved  in  paraquat-induced  tissue 
damage.   Improved  techniques  that  are  being  available  for  the  isolation  of 
Type  II  alveolar  epithelial  cells  will  greatly  help  to  understand  if  these 
cells  are  capable  of  concentrating  paraquat  and  thus  involved  in  paraquat- 
induced  toxicity. 

Keyword  Description: 

cyclic  AMP  lung 

cyclic  GMP  paraquat 

cyclic  nucleotides  Type  II  alveolar  epithelial 

cells 

Honors  and  Awards:   None 

Publications:   None 


^)5 


Project  No.  Z01  HL  00829-01  LCF 

1.  Chemical  Pharmacology 

2.  Drug  Tissue  Interaction 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  197^  through  June  30,  1975 


Present  Title:   Effects  of  Ca++  and  Carbachol  on  cGMP  in  Lung  Cells 

Previous  Serial  No.:      None 

Principal  Investigators:   Dr.  N.  Krishnan 

Dr.  G.  Krishna 


Other  Investigators: 
Cooperating  Unit : 
Project  Description: 


None 


None 


Objectives :   Most  studies  of  lung  function  have  been  carried  out  with 
either  whole  lung  or  lung  slices.   Because  lung  contains  at  least  1+0  differ- 
ent types  of  cells,  such  studies  are  difficult  to  interpret.   Clearly  prepa- 
rations are  needed  that  are  predominantly  of  one  single  cell  type.   However, 
as  a  first  step,  studies  with  preparations  consisting  of  mixed  population  of 
lung  cells  are  needed  to  evaluate  various  techniques  for  separating  and  iso- 
lating functional  cell  types.   Our  initial  studies  on  the  effect  of  chol- 
inergic agents  on  cyclic  nucleotide  levels  in  lung  cells  were  carried  out  on 
viable  lung  cells  prepared  by  proteolysis  of  the  lung  tissue  according  to  the 
method  of  Gould  et'al.  (Science  178:1209,  1972). 

Methods  Employed:   Male  Sprague  Dawley  rats  weighing  150  g  were 
anesthetized,  the  lungs  were  artificially  ventilated  and  perfused  at  38°C 
in  situ  with  Krebs-Ringer  phosphate  buffer  (Ca++  and  Mg++  free),  containing 
glucose  and  albumin.   The  lung  tissue,  after  removal,  was  freed  of  extra 
pulmonary  bronchi  and  connnective  tissues  and  sliced  to  1  mm  cubes  in  a 
tissue  slicer  and  incubated  in  Ca++-Mg++  free  KRP  albumin  buffer  containing 
1  mg  of  crude  collageanse,  2  mg  of  pronase,  0.5  mg  chymopapain,  10  units  of 
elastase,  0.03  mg  deoxyribonuclease  and  0.005  ml  of  crude  elastase  per  ml  of 
buffer.   The  lung  tissue  obtained  from  5  rats  was  incubated  with  the  enzyme 
mixture  (30  ml)  at  38°C  for  1+5  minutes  with  gentle  agitation  on  a  Dubnoff 
Shaker.   After  incubation,  the  contents  were  passed  through  a  silk  cloth  to 
remove  cell  debris  and  connective  tissues.   The  filtrate  was  centrifuged  for 
15  seconds  at  3,000  RPM  and  the  cells  were  washed  twice  with  Krebs-Ringer 
phosphate-albumin  buffer  and  diluted  with  the  same  buffer  and  used  for  the 
experiments .   A  small  aliquot  of  the  lung  cells  was  fixed  with  2%   glutaralde- 
hyde  for  electron  microscopic  examination. 


&>9 


Project  Wo.  Z01  HL  00829-01  LCP 

Our  studies  on  the  effect  of  calcium  and  carbamylcholine  on  cyclic 
nucleotide  levels  were  carried  out  "by  incubating  the  lung  cells  for  2 
minutes  at  38°C.   The  reaction  was  terminated  by  addition  of  10$  per- 
chloric acid.   Cyclic  AMP  and  cyclic  GMP  were  separated  by  chromatography 
on  Dowex-1  formate  columns  and  assayed  by  the  radioimmunoassay  method  of 
Steiner  (J.  Biol.  Chem.  2Vf:1106,  1972). 

Major  Findings:   Electron  microscopic  examination  of  lung  cells  showed 
that  about  50%  of  the  cells  obtained  are  Type  II  alveolar  epithelial  cells 
(Type  II  pneumocytes ) .   The  rest  of  the  cells  consist  mainly  of  macrophages, 
clara  cells ,  lymphocytes  and  Type  I  epithelial  cells . 

Last  year,  we  reported  that  carbamylcholine  did  not  activate  lung  super- 
natant guanylate  cyclase;  but  that  the  enzyme  required  .divalent  cations. 
During  the  past  year  we  tested  the  effect  of  Ca++  on  cyclic  GMP  accumulation 
in  lung  cells.   At  a  concentration  of  2.5  mM  Ca++  stimulated  cyclic  GMP 
accumulation  over  the  basal  levels  by  seventy-fold  (Basal  level,  C.U  pmoles ; 
Ca++  stimulated  levels  28  pmoles/ml  lung  cells).   Under  these  conditions, 
however,  the  addition  of  carbamylcholine  partially  prevented  the  stimula- 
tory effect  of  Ca++  on  the  accumulation  of  cyclic  GMP. 

We  also  measured  the  cyclic  AMP  levels  in  the  same  lung  cell  samples. 
Ca++  (2.5  mM)  stimulated  cyclic  AMP  accumulation  somewhat  less  markedly  to 
about  30-fold  over  the  basal  levels  ( 0 . U  pmole/ml  ceils  basal;  13  pmoles  Ca++ 
stimulated).   Addition  of  carbamylcholine  together  with  calcium  resulted  in 
further  stimulation  of  cyclic  AMP  formation  to  about  70-fold  (28  pmoles/ml 
cells).  ' 

These  results  point  to  the  crucial  role  played  by  Ca++  in  regulating 
cyclic  nucleotide  levels  within  lung  cells.   Further  experiments  are  in 
progress  to  elucidate  the  mechanism  of  these  effects  of  Ca++  and  hormone. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
A  study  on  the  effect  of  carbamylcholine  in  the  lung  may  help  to  undersstand 
the  role  of  cyclic  nucleotides  in  the  cholinergic  action  in  this  organ.   The 
profound  effect  of  Ca++  observed  in  our  studies  will  help  to  understand  the 
role  of  divalent  metal  ion  in  hormone  modulation  of  lung  cell  functions. 

Proposed  Course  of  Project:   Attempts  will  be  made  to  isolate  the  Type  II 
alveolar  epithelial  cells  (Type  II  pneumocytes)  from  other  cells  at  least  up 
to  90$  of  one  cell  type;  these  are  the  cells  that  synthesize  surfacants 
which  are  essential  in  maintaining  the  structural  integrity  of  the  pulmonary 
tissue.   The  possible  role  of  cyclic  AMP  and  cyclic  GMP  in  the  surfactant 
formation  and  the  involvement  of  cholinergic  and  adrenergic  systems  in  the 
surfactant  synthesis  will  be  investigated  In  Type' II  cells  of  the  lung  in 
order  to  elucidate  the  regulatory  role  played  by  calcium.   Studies  will  also 
be  carried  out  to  understand  the  nature  of  receptor(s)  involved  in  carbamyl- 
choline and  calcium  mediated  stimulation  of  cyclic  AMP  formation.   Moreover, 
these  cells  will  be  utilized  to  study  paraquat-induced  changes  in  cyclic 


fi/O 


Project  No.  Z01  HL  00829-01  LCP 

AMP  levels  in  the  lung  and  to  elucidate  cell  type  of  the  lung  that  has 
the  capacity  to  concentrate  paraquat. 

Keyword  Description:   Lung  cells,  calcium,  carbachol  and  cyclic  GMP 

Honors  and  Awards :   None 

Publication: 

Rodbell,  M.  and  Krishna,  G. :   Preparation  of  Isolated  Fat  Cells  and 
Fat  Cell  "Ghosts" ; "Methods  for  Assaying  Adenylate  Cyclase  Activity 
and  Levels  of  Cyclic  AMP.   In  Fleischer,  S.  and  Packer,  L.  (Eds.): 
Methods  in  Enzymology.   New  York,  Academic  Press,  197*+,  Vol.  XXXI, 
Part  A,  pp.  103-llU. 


*n 


Project  No.  Z01  HL  00830-01  LCF 

1.  Chemical  Pharmacology 

2.  Drug  Tissue  Interaction 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1971+  through  June  30,  1975 

Project  Title:   Role  of  Cyclic  Nucleotides  in  Hormone  Action 

Previous  Serial  No.:  None 

Principal  Investigators:   Dr.  Erik  K.  Frandsen 

Dr.  Gopal  Krishna 

Other  Investigator:       Mrs.  C.  McDaniels 

Cooperating  Units :        None 

Project  Description: 

Objectives :   The  role  of  cyclic  AMP  as  a  mediator  of  a  variety  of 
hormones  is  well  documented  but  the  role  of  cyclic  GMP  is  not  yet  clearly 
understood.   So  far  the  only  system  that  may  be  mediated  by  cyclic  GMP, 
appears  to  be  the  cholinergic  (muscarinic)  system.   One  of  the  main  problems 
involved  In  the  study  of  the  role  of  cyclic  GMP  in  hormone  action  appears  to 
be  the  transient  effect  that  the  hormone  produces  on  the  cyclic  GMP  system 
and  the  magnitude  of  the  effect.   Most  of  the  tissues  appear  to  contain  only 
1/100  of  cyclic  GMP  levels  as  compared  to  cyclic  .AMP  levels.   Moreover,  only 
a  small  portion  of  cells  in  the  tissue  respond  to  hormone  stimuli  and  thereby 
restricting  the  magnitude  of  overall  response. 

We  have  attempted  to  solve  these  problems  by  developing  a  sensitive  method 
for  assay  of  cyclic  GMP  and  modifying  the  methods  available  for  cell  isolation 
in  order  to  get  a  population  of  cells  which  are  enriched  with  one  type  of  cell 
from  tissues  which  consist  of  numerous  cell  types. 

Methods  Employed:   Cyclic  AMP  and  cyclic  GMP  were  extracted  with  per- 
chloric acid  from  tissues  or  cells  and  separated  on  Dowex  1  formate  (BioRad 
A.G.  IxH  200-1+00  mesh)  column.   Cyclic  AMP  and  cyclic  GMP  were  eluted  with 
2N  and  1+N  formic  acid,  respectively.   Aliquots  of  cyclic  AMP  and  cyclic  GMP 
fractions  were  lyophilized,  dissolved  in  50  yl  of  water  and  were  succinylated 
with  5  yl  of  a  mixture  containing  1  ml  of  succinic  anhydride  in  acetone  (200 
mg/ml)  and  0.36  ml  of  triethylamine.   After  10  minutes  at  room  temperature, 
the  reaction  was  terminated  by  addition  of  1  ml  of  sodium  acetate  buffer 
(0.05  M,  pH  6.2)  and  a  200  yl  aliquot  was  taken  for  radioimmunoassay. 
Standards  containing  cyclic  GMP  were  run  with  the  samples.   The  radioimmuno- 
assay was  performed  at  1+°C  overnight  (12-16  hr)  after  addition  of  50  yl  of 
125l-antigen  and  50  yl  of  cyclic  GMP  antibody  (1:25000).   The  free  125l- 
antigen  was  separated  from  the  bound  by  precipitation  with  cold  ethanol  (Q0% 

1  2/3L 


Project  No.  Z01  HL  00830-01  LC 

final  concentration).  Bovine  serum  albumin  (0.5  -  1  mg)  was  added  to  aid  a 
complete  precipitation  of  the  bound  antigen.  The  unbound  radioactivity  was 
determined  in  a  scintillation  counter. 

Pancreatic  acinar  cells  were  isolated  with  collagenase  treatment. 

Major  Findings:   The  succinylation  of  cyclic  GMP  by  succinic  anhydride 
requires  the  presence  of  triethyl  amine;  very  little  succinylation  occurs  in 
the  absence  to  triethylamine.   Table  1  shows  the  dgree  of  succinylation  with 
various  combinations  of  succinic  anhydride  and  triethylamine.   The  maximum 
succinylation  occurs  with  10  mg  of  succinic  anhydride  in  the  presence  of  20 
yl  of  triethylamine. 

Table  2  shows  that  succinylation  of  cyclic  GMP,  by  a  mixture  containing 
succinic  anhydride  and  triethylamine  is  independent  of  cyclic  GMP  concen- 
tration.  Almost  quantitive  succinylation  is  ob+ained  using  30  yl  of  mixture 
containing  1  ml  of  succinic  anhydride  (200  mg/ml  in  acetone)  and  0.36  ml 
of  triethylamine. 

The  succinylation  occurs  virtually  instantaneously  when  carried  out  in 
aqueous  medium,  but  much  more  slowly  when  carried  out  in  a  nonaqueous  medium 
like  acetone. 

The  succinylated  product  may  be  isolated  from  %-cyclic  GMP  by  thin 
layer  chromatography  on  silica  gel  (butanol: acetic  acid:water  U:l:2). 

It  has  been  identified  as  2'-0-cyclic  GMP  by  a  variety  of  techniques 
including  high  pressure  liquid  chromatography.   Moreover,  the  succinylated 
material  can  be  hydrolyzed  to  cyclic  GMP. 

The  affinity  of  the  cyclic  GMP  antibody  for  the  succinylated  cyclic  GMP 
was  found  to  be  100  times  higher  than  for  cyclic  GMP.   Thus,  succinylation 
increases  the  detectability  of  cyclic  GMP  100-fold  by  radioimmunoassay. 
Now  it  is  possible  to  quantitate  2  fentomoles  of  cyclic  GMP. 

Ethanol  at  concentrations  higher  than  80%  efficiently  precipitates  the 
bound  antigen.   Very  little  dissociation  of  antigen-antibody  complex  occurs 
in  ethanol  provided  the  mixture  is  kept  cold  ( U°C ) . 

A  similar  method  has  also  been  developed  for  assay  of  cyclic  AMP.   The 
degree  of  succinylation  as  well  as  increase  in  affinity  of  the  succinylated 
cyclic  AMP  to  cyclic  AMP  antibody  appears  to  b^  very  similar  to  that  obtained 
for  cyclic  GMP.   The  method  has  been  applied  for  the  study  of  the  role  of 
cyclic  nucleotide  in  catecholamine  and  vasoactive  intestinal  polypeptide 
(VIP)  induced  relaxation  in  the  tracheal  smooth  muscle.   Preliminary  studies 
indicate  that  cyclic  AMP  is  increased  when  tracheal  smooth  muscles  are  ex- 
posed to  either  epinephrine  or  VIP.   Theophylline  which  has  been  known  to 
potentiate  the  relaxation  of  the  tracheal  smooth  muscle  produced  by  these 
hormones  also  potentiates  the  increase  in  cyclic  AMP.   Cyclic  GMP  levels 


2/S 


Project  No.  Z01  HL  00830-01  LCP 
were  not  altered  by  these  hormones . 

The  method  for  assay  of  cyclic  nucleotides  also  has  been  utilized 
in  the  study  of  the  role  of  cyclic  GMP  in  mediating  cholinergic  response 
in  isolated  pancreatic  acinar  cells.   Preliminary  results  indicate  that 
the  cholinergic  stimulant  carbamyl  choline,  rapidly  increase  cyclic  GMP 
levels  thereby  indicating  that  cyclic  GMP  may  be  involved  in  the  cholinergic- 
ally  mediated  pancreatic  acinar  cell  functions. 

Significance  to  Biomedical  Research  and  to  the  Program  of  the  Institute: 
The  development  of  a  simple  and  sensitive  assay  of  cyclic  nucleotides  in  the 
fentomole  range  should  greatly  help  in  the  understanding  of  the  role  of  cyclic 
nucleotides  in  various  hormone  actions.   With  the  advent  of  development  of 
simple  methods  for  isolation  of  cells  of  single  cell  type  for  various  tissues 
like  liver,  lung,  pancreas,  will  greatly  help  in  the  understanding  of  the  role 
of  these  cells  and  how  they  are  modulated  by  various  hormones. 

Proposed  Course  of  Project:   The  role  of  cyclic  nucleotides  in  mediating 
sympathetic,  histaminic  and  cholinergic  functions  in  tracheal  smooth  muscles 
will  be  studied  in  greater  detail.   The  role  of  cyclic  GMP  in  mediating 
cholinergic  functions  in  pancreatic  acinar  cells  will  be  studied  in  order  to 
understand  the  mechanism  by  which  cholinergic  agents  activate  guanylate  cy- 
clase in  the  cells. 

Parasympathetic  nerve  endings  have  been  identified  in  the  islets  of 
Langerhans  in  +he  pancreas.   Stimulation  of  these  nerves  causes  increased 
secretion  of  insulin  which  is  blocked  by  atropine.   Since  in  other  systems 
cyclic  GMP  has  been  shown  to  mediate  cholinergic  function,  the  role  of  cyclic 
GMP  in  insulin  secretion  will  be  studied. 

Keyword  Description:   Cyclic  AMP,  Cyclic  GMP,  Assay  System  and  Isolated  Cells 

Honors  and  Awards :   None 

Publications:   None 


&i4 


Project  No.   Z01  HL  00830-01  LCP 


Table  1 
Effect  of  increasing  succinic  anhydride  and  triethylamine  on  the 
succinylation  of  cGMP 


yl  triethylamine 

1 

3 

mg 

succinic 
1+ 

anhydride 
10 

0 

1.8 

per  cent 
1.9 

succinylation 

1.9 

2.U 

0. 

5 

13.5 

11.0 

7-5 

8.8 

1 

52.9 

33.3 

20.2 

1U.9 

2 

71.0 

67.9 
90.  k 

52.0 

26.7 

5 

66.2 
^5.3 
U^.7 

9^.0 

78.0 

10 

73.0 

60.0 
i 

95-U 

97.0 

20 

86.5 

96.5 

-%-cGMP  (l  pmole-  5000  cpm)  was  dissolved  in  100  ul  water.   Succinic 
anhydride  was  added  in  20  pi  acetone  followed  triethylamine.   The  mixture 
was  incubated  at  room  temperature  for  30  min.   Aliquots  were  chromatographed 
on  silica  gel  and  developed  with  butanol: acetic  acid:water  (U:l:2).   The 
cGMP  and  succinylated  cGMP  spots  were  removed  and  radioactivity  was  de- 
termined. 


zrsr 


Project  No.   Z01  HL  00830-01  LCP 


Table  2 
Effect  of  increasing  cGMP  on  the  succinylation  of  cGMP 

cGMP  pinoles  pi  of  succinylation  reagents* 

5  10  30 

per  cent  succinylation 

0  51.0  66.7  9^.6 

1  hG.2  63.7  95.1 
10             50.0             6h.O  9^.7 

100  Mi. 6  63.8         96.1 

1,000  50.0  66.0         95.^ 

10,000  U7 .3  68.8  95-0 

100,000  U7.1  67.1        9^.7 

*200  mg  succinic  anhydride  in  1  ml  acetone  and  0.36  ml  triethylamine 
3h-cGMP  (l  pmole  -  5000  cpm)  was  dissolved  in  100  pi  of  aqueous  solutions 
of  various  concentrations  of  cGMP.   cGMP  was  succinylated  "by  adding  a  mixture 
containing  succinic  anhydride  and  triethylamine  (200  mg  succinic  anhydride 
in  1  ml  of  acetone  plus  0.36  ml  triethylamine).   cGMP  and  succinylated  cGMP 
were  separated  and  determined  as  described  under  Table  1. 


3fi 


Project  No.   Z01  HL  00851-02  LCP 


1.  Chemical  Pharmacology 

2.  Physiology 

3.  Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1971*  through  June  30,  1975 

Project  Title:   Paraquat  toxicity  in  rat  lung. 

Previous  Serial  Number:  NHLI-U6 

Principal  Investigators:   Dr.  Harriet  M.  Maling 

Dr.  Elise  Ann  Brandenburger  Brown 
Dr.  James  R.  Gillette 

Other  Investigators:      Mrs.  Martha  A.  Williams 

Mr.  Wilford  Saul 

Cooperating  Unit:   Dr.  Brown  is  associated  with  the  Pulmonary  Branch,  NHLI. 

Project  Description: 

Objectives :   In  this  project,  we  hope  to  elucidate  the  mechanisms 
responsible  for  the  pulmonary  toxicity  of  paraquat,  l,l'-dimethyl-U,  V- 
dipyridylium  di chloride,  an  herbicide  which  is  extensively  used  in  agricul- 
ture.  Demonstration  of  a  treatment  for  paraquat  poisoning  in  rats  should 
have  clinical  applications  in  the  treatment  of  accidental  paraquat  poi- 
sonings in  man.   An  understanding  of  the  mechanisms  involved  in  the  pulmonary 
toxicity  of  paraquat  should  give  insight  into  various  mechanisms  responsible 
for  the  development  of  pulmonary  edema  after  exposure  to  other  agents . 

Methods  Employed:   Data  has  been  collected  on  the  influence  of  various 
treatments  on  the  U8-hr  mortality  of  paraquat,  injected  i.p.   Lung  weights 
and  gross  pathologic  grading,  as  defined  in  the  Annual  Report  for  1973-7^ , 
were  used  as  indicators  of  the  extent  of  pulmonary  edema. 

Diene  conjugation  of  lung  microsomal  lipids  was  measured  by  a  modifica- 
tion of  the  method  described  by  Rao  and  Recknagel  (Exp.  Mol.  Path.  9:   271, 
1968)  for  measuring  diene  conjugation  of  liver  microsomal  lipids.   Thio- 
barbituric  acid  reactants  were  measured  as  an  index  of  lipid  peroxidation 
and  malondi aldehyde  formation.   Reversible  binding  of  paraquat  to  lung 
homogenates  was  measured  by  equilibrium  dialysis.  Uptake  of  -^C-paraquat  by 
lung  slices  was  measured  by  a  procedure  similar  to  that  described  by  Rose, 
Smith  and  Wyatt  (Nature,  252:   31^,  197*0  • 

Major  Findings:   Treatment  with  a  single  dose  of  the  beta  adrenergic 
receptor  stimulant,  1-isoproterenol,  markedly  increased  the  toxicity  of  i.p. 
paraquat  (Table  l),  as  evident  from  a  55%  reduction  in  the  LD50.   Treatment 
with  theophylline  also  increased  paraquat  toxicity.  Multiple  doses  of  the 
beta  receptor  blocking  agent,  dl-propranolol ,  protected  rats  moderately,  as 


A/7 


Project  No.  Z01  HL  00851-02  LCP 


evident  from  a  367=  increase  in  the  LD50.   These  findings  suggest  an  involve- 
ment of  the  beta  adrenergic  receptor.   A  radomized  experiment  was  therefore 
designed  to  compare  the  effectiveness  of  propranolol  isomers  in  protecting 
rats  from  paraquat  poisoning.   There  were  34  deaths  from  paraquat  in  48  rats 
treated  with  saline.   Only  17  rats  died  from  paraquat  among  48  rats  treated 
with  multiple  doses  of  dl-propranolol .   Treatment  with  1-propranolol  was 
almost  as  effective  (21  deaths  in  48  rats)  as  treatment  with  d , 1-propranolol ; 
d-propranolol  was  less  effective  (28  deaths  in  48  rats).   The  fact  that 
d-propranolol  was  less  effective  than  1-propranolol  or  D-propranolol  supports 
the  hypothesis  that  beta  adrenergic  blockade  is  at  least  part  of  the  mecha- 
nism responsible  for  protection. 

This  laboratory  has  previously  reported  the  finding  that  paraquat  does 
not  bind  covalently  to  tissue  proteins  or  other  tissue  macromolecules .  .We 
have  found,  however,  that  paraquat  does  bind  reversibly  to  lung  homogenates. 
The  7,  binding  to  a  207.  lung  homogenate  was  about  627,.   This  in  vitro  binding 
to  a  lung  homogenate  was  not  affected  by  propranolol  in  vitro . 

The  uptake  of  paraquat  by  lung  slices  in  vitro  has  been  shown  to  be 
energy-dependent  (Rose,  Smith  and  Wyatt,  Nature,  252:   314,  1974).   Our 
experiments  indicate  that  the  uptake  of  paraquat  by  lung  slices  is  inhibited 
appreciably  by  dl-propranolol  in  vitro .   The  inhibitor  constant  K^  for  dl- 
propranolol  is  approximately  1.4  x  lO'TVI.   1-Propranolol  may  be  slightly  more 
effective  than  d-propranolol  as  an  inhibitor  of  paraquat  uptake.   Dichloro- 
isoproterenol  is  about  as  effective  as  propranolol.   Other  beta  adrenergic 
blocking  agents  were  less  effective  than  propranolol  or  dichloroisoproterenol 
in  inhibiting  the  uptake  of  paraquat  by  rat  lung  slices. 

Among  other  compounds  tested  for  inhibition  of  paraquat  uptake,  imipra- 
mine  and  desipramine  (DMI)  were  as  effective  as  propranolol.   Unlike  pro- 
pranolol, however,  treatment  with  imipramine  or  desipramine  did  not  reduce 
the  48-hr  mortality  from  paraquat  i.p. 

Although  propranolol  in  vitro  inhibited  paraquat  uptake,  there  were  no 
statistically  significant  differences  in  tissue  paraquat  concentrations  6, 
24  and  48  hr  after  the  i.p.  injection  of   C-labeled  paraquat  between  rats 
treated  with  multiple  doses  of  propranolol  or  with  saline.   Decreasing  con- 
centrations of  paraquat  were  found  in  the  following  tissues:   lung  >  kidney 
»  liver  >  heart  >  muscle  >  brain  >  plasma. 

Other  investigators  have  suggested  that  paraquat-induced  formation  of 
the  superoxide  free  radical,  singlet  oxygen,  or  peroxides  is  responsible  for 
the  pulmonary  toxicity  of  paraquat.   We  have  been  unable  to  detect  a  para- 
quat-induced increase  in  diene  conjugation  of  lung  microsomal  lipid,  either 
from  lungs  of  rats  injected  with  paraquat  i.p.  or  from  lung  slices  incubated 
in  vitro  with  high  concentrations  of  paraquat,  either  in  air  or  957>  O2,  57, 
C02-   Measurements  of  thiobarbituric  acid  (TBA)  reactants,  presumably  malon- 
dialdehyde,  in  the  incubation  fluid  did  not  reveal  any  significant  increases 
resulting  from  incubation  of  lung  slices  with  high  concentrations  of  paraquat 
Thus  we  have  obtained  no  evidence  in  support  of  the  view  that  toxicity  is 


Sub 


Project  T\Tn  7,m  HL  nnfiSl-OP  LOP 

caused  by  superoxide  free  radical  formation  and  peroxide  formation. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
The  demonstration  that  1-isoproterenol  and  theophylline  treatment  increase 
the  mortality  from  paraquat  poisoning  in  rats  may  have  clinical  implications. 
It  would  seem  wise  for  physicians  to  refrain  from  use  of  these  bronchodilators 
in  the  treatment  of  paraquat  poisoning.   This  caution  is  surprising  since  the 
use  of  a  bronchodilator  would  seem  to  be  a  desirable  symptomatic  treatment 
for  a  person  experiencing  respiratory  distress. 

It  is  hoped  that  a  better  understanding  of  the  mechanisms  involved  in 
paraquat  poisoning  will  increase  our  insight  into  the  mechanisms  involved 
in  the  production  of  pulmonary  edema  from  other  causes. 

Proposed  Course  of  Project:   TBA  reactants  will  be  measured  in  incubation 
fluid  and  in  lung  homogenates  under  a  greater  variety  of  conditions,  in  a 
continuing  search  for  evidence  of  peroxide  formation  during  the  development 
of  lung  toxicity  from  paraquat.   The  studies  of  paraquat  uptake  bv  lung  slices 
will  be  extended. 

Proposed  Course  of  Project:   TBA  reactants  will  be  measured  in  incuba- 
tion fluid  and  in  lung  homogenates  under  a  greater  variety  of  conditions, 
in  a  continuing  search  for  evidence  of  peroxide  formation  during  the  develop- 
ment of  lung  toxicity  from  paraquat.   The  studies  of  paraquat  uptake  by  lung 
slices  will  be  extended. 

Keyword  Description: 

i 
diene  conjugation  of  lung  microsomal  lipids 
1-isoproterenol 
paraquat 
dl-propranolol 

pulmonary  edema,  pulmonary  toxicity 
theophylline 

Honors  and  Awards:   None 

Publications : 

Maling,  H.M.,  Webster,  M.E.,  Williams,  M.A.,  Saul,  W.  and  Anderson,W  .Jr . : 
Inflammation  induced  by  histamine,  serotonin,  bradykinin  and  compound 
48/80  in  the  rat:   Antagonists  and  mechanisms  of  action.   J.  Pharmacol. 
Exper.  Ther.  191:   300-310,  1974. 


3.19 


Project  No.   Z01  HL  00851-02  LCP 

Table  I 

The  effect  of  various  treatments  on  the  48-hr  LD50  values 
for  paraquat,  injected  i .p .  in  male  Sprague-Dawley 
rats,  as  calculated  by  probit  analysis  of  pooled  data. 

Treatment3  48-hr  LD50  for  paraquat,  injected  i.p. 


s.c.  mg/kg  (95%  confidence  interval)  umole/kg 

SALINE                        22.26  (21.61,  22.92)  119.4 

dl -PROPRANOLOL                30.27b  (27.24,  33.64)  162. 5b 

THEOPHYLLINE                  18.43b  (15.06,  22.44)  98. 9b 

1 -ISOPROTERENOL                10.04b  (7.96,  12.67)  53. 9b 


aThe  saline-treated  rats  were  723  control  rats  in  54  experiments; 
these  rats  received  1-10  doses  of  saline  s.c.  in  two  days.   Two  to  10 
doses  of  propranolol  were  injected  in  230  rats  treated  with  dl-propranolol . 
The  first  dose  of  propranolol  each  day  was  25  mg/kg  of  the  hydrochloride 
s.c.;  the  remaining  doses  were  18.75  or  20  mg/kg  s.c.,  1  1/2  to  2  1/2  hr 
apart.   Only  one  dose  of  1-isoproterenol  was  given,  0.3  mg/kg  (base)  s.c. 
in  70  rats  at  the  same  time  as  the  administration  of  paraquat  i.p.  (  3  experi- 
ments).  Only  one  dose  of  theophylline  was  given  (50  mg/kg  s.c.  in  43  rats 
at  the  same  time  as  the  i.p.  injection  of  paraquat  -  two  experiments). 

bThese  LD50  values  are  statistically  significantly  different  from  the 
corresponding  value  in  saline-treated  rats,  P  <  .05. 


aao 


Project  No.  Z01  HL  00852-03  LCP 

1.  Chemical  Pharmacology 

2.  Physiology 

3.  Bethesda,  Md . 

PHS  -NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Ethanol,  isopropanol,  and  CClA-induced  liver  toxicity. 

Previous  Serial  Number:  NHLI-1+7,  NHLI  86 

Principal  Investigator:    Dr.  Harriet  M.  Maling 

Other  Investigators:      Mr.  Wilford  Saul 

Mrs.  Martha  A.  Williams 

Cooperating  Unit:         None 

Project  Descritpion: 

Objectives :   In  our  Annual  Reports  for  1971-72  and  1973-74,  we  reported 
that  pretreatment  of  rats  with  four  oral  doses  of  ethanol  over  a  period  of 
two  days  potentiated  the  hepatotoxicity  of  a  small  dose  of  CCIa,  as  evalu- 
ated by  histologic  grading  and  levels  of  plasma  glutamic-pyruvic  trans- 
aminase and  liver  triglycerides.   Possible  mechanisms  for  this  potentiation 
have  been  examined.   We  have  also  considered  the  question  whether  the  hepato- 
toxicity of  other  compounds  is  also  potentiated  by  pretreatment  with  multiple 
doses  of  ethanol.   These  questions  seemed  important  in  view  of  the  wide- 
spread social  use  of  ethanol. 

Methods  Employed:   Standard  methods  were  employed. 

Major  Findings:   The  elevated  plasma  glutamic-pyruvic  transaminase  (GPT) 
activities  induced  by  thioacetamide  or  dimethylnitrosamine ,  as  well  as  those 
induced  by  CCIa,  were  increased  by  pretreatment  with  four  doses  of  ethanol. 
Plasma  GPT  activities  after  bromobenzene  were  not  consistently  enhanced  by 
pretreatment  with  ethanol.   The  hepatotoxicity  of  allyl  alcohol  was  not 
affected  by  pretreatment  with  ethanol.   Plasma  GPT  activities  were  not 
elevated  24  hr  after  chlorpromazine  (20-80  mg/kg  ip)  or  isoniazid  (100  and 
150  mg/kg)  in  either  untreated  rats  or  rats  pretreated  with  ethanol. 

Diene  conjugation  of  liver  microsomal  lipids  from  rats  killed  30  min 
after  the  injection  of  a  small  dose  of  CCI4  (0.1  ml/kg  i.p.)  was  potentiated 
significantly  by  pretreatment.  with  pyrazole  and  a  single  dose  of  ethanol,  or 
by  a  single  dose  of  isopropanol.   Pretreatment  with  4  doses  of  ethanol  also 
tended  to  increase  CCIa -induced  diene  conjugation. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
In  this  study,  we  have  analyzed  several  possible  mechanisms  responsible  for 
a  potentiated  toxicity  of  CCIa  and  several  other  compounds  in  rats  pretreated 


W 


Project  No.  zm  ht,  nnflsp-n?  lpp 

with  4  doses  of  ethanol.   Special  interest  arises  from  the  finding  that  this 
potentiated  hepatotoxicity  is  great  when  the  blood  alcohol  levels  are  negli- 
gible.  This  suggests  that  physicians  should  consider  the  possibility  of 
potentiated  hepatotoxicity  of  CCIa  and  possibly  other  compounds  in  persons 
with  a  history  of  recent  drinking,  even  though  no  alcohol  can  be  detected 
in  their  blood. 

Proposed  Course  of  Project:   This  project  has  been  terminated. 

Keyword  Description: 

blood  alcohol  levels  hepatotoxicity 

CCl,  isopropanol 

diene  connugation  plasma  glutamic-pyruvic  transaminase  (GPT) 

ethanol 

Honors  and  Awards:   None 

Publications : 

Maling,  H.M.,  Stripp,  B.,  Sipes,  I.G.,  Highman,  B.,  Saul,  W.  and 
Williams,  M.A.:   Enhanced  hepatotoxicity  of  carbon  tetrachloride, 
thioacetamide ,  and  dimethylnitrosamine  by  pretreatment  of  rats  with 
ethanol  and  some  comparisons  with  potentiation  by  isopropanol. 
Toxicol.  Appl.  Pharmacol.,  in  press. 

Maling,  H.M.,  Eichelbaum,  P.M.,  Saul,  W.,  Sipes,  I.G.,  Brown,  E .A  .B . 
and  Gillette,  J.R.:   The  nature  of  the  protection  against  CCl^induced 
hepatotoxicity  produced  by  pretreatment  with  dibenamine  i_N-(2- 
chloroethyl)  dibenzylaminej .   Biochem.  Pharmacol.  23:   1479-1491,  1974. 

Stripp,  B.,  Sipes,  I.G.,  Maling,  H.M.  and  Gillette,  J.R.:   Dibenamine 
impairment  of  rat  hepatic  microsomal  enzymes  and  its  relation  to  hepa- 
totoxicity induced  by  CCI4  and  dimethylnitrosamine.   Drug  Metabolism 
and  Disposition  2:   464-468,  1974. 


<w 


Project  No.  Z01  HL  00877-02  LCP 

1.  Chemical  Pharmacology 

2.  Clinical  Pharmacology 
3  .   Bethesda ,  Md . 

PHS  -NIH 

Individual  Project  Report 

July  1,  1971  through  June  30,  1972 

Project  Title:   Hydrazines  1.  Human  isoniazid  acetylation  and  metabolism. 

Previous  Serial  Number:  NHLI-U3 

Principal  Investigators:   Dr.  J.  R.  Mitchell 

Dr.  U.  P.  Thorgeirsson 
Dr .  J .  A .  Timbrell 

Other  Investigators:      Dr.  W.  R.  Snodgrass 

Dr.  W.  Z.  Potter 
Dr  .  M.  Black 
Dr.  H.  R.  Reiser  (NHLI:HE) 

Cooperating  Units:        Dr.  Thorgeirsson  is  supported  by  the  American 

Lung  Association. 
Drs  .  Potter  and  Snodgrass  are  Research  Associates 

in  the  Pharmacology-Toxicology  Program,  NIGMS 
Dr.  Black  is  a  Fogarty  International  Fellow 

Project  Description: 

i 

Objectives :   Isoniazid  and  iproniazid  can  produce  severe  and  occasion- 
ally lethal  hepatitis  in  man.   Iproniazid  was  removed  from  clinical  use 
because  of  this  hepatotoxicity ,  and  isoniazid  hepatitis  has  become  such  a 
serious  clinical  problem  that  its  use  has  been  recently  restricted. 

In  a  prospective  study  (NHLI  43,  Chest,  in  press)   we  examined  iso- 
niazid plasma  concentrations  and  monthly  liver  function  tests  in  201  patients 
during  one  year  of  isoniazid  preventive  therapy  for  tuberculosis.   About  207=, 
of  patients  showed  evidence  of  liver  injury  with  abnormal  SGOT  or  bilirubin 
values  which  subsided  while  the  patients  continued  to  take  isoniazid.   No 
correlation  was  found  between  patients  that  acetylated  isoniazid  slowly  and 
those  that  had  abnormal  liver  function  tests.   No  anti-isoniazid  antibodies 
were  found  and  no  correlation  was  seen  between  hepatic  injury  and  antinuclear 
antibodies  measured  at  the  end  of  the  study. 

Subsequently,  the  U.  S.  Public  Health  Service  conducted  a  surveillance 
study  on  13,838  patients  from  21  American  cities  to  determine  the  incidence 
of  isoniazid  hepatitis.   This  study  was  abruptly  terminated  by  then  Assistant 
Secretary  for  Health,  Dr.  Merlin  Duval,  because  of  a  high  incidence  of  hepa- 
titis (>2.37o  in  patients  over  age  50)  and  several  fatalities.   We  were  re- 
quested by  the  Tuberculosis  Research  Section,  Center  for  Disease  Control, 
U.S.P.H.S.,  to  evaluate  data  as  to  etiology  for  224  of  the  13,838  recipients 


*Si3 


Project  No.  ZQ1  HL  00877-02  LCP 

of  isoniazid  who  were  suspected  of  having  developed  isoniazid  liver  injury 
(NHLI43,  Gastroenterology,  in  press).   Some  of  the  important  findings 
from  this  retrospective  analysis  were:   1)  isoniazid-related  liver  injury  was 
indistinguishable  biochemically  (SGOT,  bilirubin,  alkaline  phosphatase)  and 
morphologically  from  iproniazid-induced  liver  damage  or  from  other  causes  of 
acute  hepatocellular  injury  such  as  viral  hepatitis;  2)  no  clinical  evidence 
for  a  hypersensitivity  mechanism  was  apparent;  3)  about  307.  of  the  patients 
with  hepatic  reactions  were  residents  of  Honolulu  and  of  Oriental  ancestry-- 
accordingly,  as  many  as  907o  of  this  group  would  be  expected  genetically  to  be 
fast  acetylators  of  isoniazid  whereas  only  457»  of  black  and  white  Americans 
are  rapid  acetylators. 

To  evaluate  a  possible  correlation  of  susceptibility  to  hepatic  injury 
and  rapid  acetylation  of  isoniazid,  we  have  now  genetically  phenotyped  26 
non-Oriental  patients  from  the  Public  Health  Service  trial  who  had  recovered 
from  isoniazid  hepatitis.   To  determine  whether  other  differences  occurred  in 
the  metabolism  of  isoniazid  by  rapid  and  slow  acetylators  of  the  drug,  radio- 
labeled isoniazid  and  acety lisoniazid  were  given  to  normal  volunteers  and 
urinary  metabolites  were  isolated  and  identified. 

Methods  Employed:   The  acetylation  phenotypes  of  patients  who  had  been 
diagnosed  as  having  isoniazid  hepatitis  were  determined  using  the  standard 
sulfamethazine  method.   In  metabolic  studies  patients  were  given  -^H-isoniazid 
or  acety 1-^H-isoniazid  and  urine  was  collected  for  48  hours  and  analyzed  by 
quantitative  radiochromatography.   Urinary  metabolites  were  identified  by 
their  Rf  values,  by  co-chromatography  with  synthesized  authentic  standards, 
by  color  reactions  with  spray  reagents,  by  reverse  isotope  dilution  analysis 
and  by  mass  spectrometry. 

Major  Findings:   Table  I  gives  the  clinical  features  and  the  acetylator 
phenotype  for  the  26  patients  who  had  previously  suffered  "probable"  or 
"possible"  isoniazid  liver  injury.   Of  the  21  individuals  in  the  "probable" 
category,  18  (867o)  of  them  displayed  the  rapid  phenotype  for  isoniazid  acety- 
lation whereas  the  expected  frequency  was  457,.   Of  the  5  subjects  in  the 
"possible"  group,  3  (607>)  were  rapid  acetylators. 

Examination  of  the  urinary  metabolites  of  isoniazid  and  acetylisoniazid 
provided  a  possible  explanation  for  the  increased  incidence  of  isoniazid 
hepatitis  in  patients  who  acetylate  the  drug  rapidly.   Fast  acetylators 
hydrolyzed  44.47.  of  a  dose  of  isoniazid  to  isonicotinic  acid,  whereas  slow 
acetylators  converted  only  30.57.  of  a  dose  to  isonicotinic  acid  (Table  2). 
This  hydrolysis,  of course,  simultaneously  liberated  stoichiometric  amounts 
of  the  h>drazino  moiety  of  isoniazid,  and  simple  hydrazines  are  well-known 
hepatotoxins ,  mutagens  and  carcinogens.   Thus,  for  any  dose  of  isoniazid, 
rapid  acetylators  are  exposed  to  467.  more  of  the  free  hydrazino  moiety  than 
are  slow  acetylators  (A4 A^3%j5%   x  100). 

It  is  also  apparent  that  the  liberated  hydrazino  moiety  is  almost  ex- 
clusively acety lhydrazine ;  i.e.,  almost  all  the  excreted  isonicotinic  acid 


5^4 


Project  No.  Z01  HL  0087-02  LCP 

came  from  acetylisoniazid  rather  than  from  isoniazid.   Rapid  and  slow  acety- 
lators  excreted  equal  amounts  of  acetylisoniazid  and  isonicotinic  acid  after 
administration  of  acetylisoniazid  (Table  2).   Accordingly,  the  differences  in 
the  excretion  of  acetylisoniazid  and  isonicotinic  acid  between  fast  and  slow 
acetylators  after  administration  of  isoniazid  must  have  resulted  from  dif- 
ferences in  the  rate  of  isoniazid  acetylation;  they  could  not  have  resulted 
from  differences  between  the  two  groups  in  the  disposition  of  these  compounds. 
Similarly,  rapid  acetylators  excreted  as  much  acetylisoniazid  and  isonico- 
tinic acid  when  receiving  isoniazid  as  when  receiving  acetylisoniazid,  demon- 
strating that  they  converted  almost  all  the  administered  isoniazid  initially 
to  acetylisoniazid.   In  contrast,  slow  acetylators  excreted  much  less  acetyl- 
isoniazid and  isonicotonic  acid  after  receiving  isoniazid ,  because  more  iso- 
niazid ascaped  acetylation  and  was  eliminated  free  or  as  isoniazid  hydrazones. 
Thus,  the  amount  of  isonicotinic  acid  formed  in  people  receiving  isoniazid, 
and  therefore  the  amount  of  total  hydrazino  material  liberated  in  the  body, 
depends  primarily  on  the  formation  of  acetylisoniazid. 

Recently  we  have  demonstrated  that  acetylisoniazid  and  acetylhydrazine , 
but  not  isoniazid,  are  converted  in  rats  iri  vivo  to  potent  acylating  agents 
that  cause  acute  hepatocellular  necrosis  (see  following  reports).   These  data 
provide  strong  support  for  the  hypothesis  that  isoniazid  hepatitis  in  pa- 
tients results  from  the  liberation  of  acetylhydrazine  in  the  body. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
The  combination  of  clinical  and  animal  studies  provides  a  satisfactory  expla- 
nation for  the  pathogenesis  of  isoniazid  hepatitis.   Given  the  seriousness  of 
this  adverse  drug  reaction  and  the  crucial  importance  of  isoniazid  in 
treating  tuberculosis,  the  significance  of  this  work  to  the  program  of  the 
institute  is  apparent. 

Proposed  Course  of  Project:   We  are  attempting  to  prove  this  hypothesis 
in  further  human  investigations  (see  following  reports).   If  successful,  we 
hope  to  develop  either  1)  alternative  methods  of  isoniazid  administration  or 
2)  a  new  isoniazid  analogue  that  could  markedly  reduce  the  hepatotoxicity 
of  isoniazid  therapy  without  altering  pharmacologic  efficicy  in  the  treat- 
ment of  tuberculosis  . 

Keyword  Description: 

acetylation  phenotypes  hepatitis 

acetylhydrazine  iproniazid 

acetylisoniazid  isoniazid 

acylating  agents  tuberculosis 

Honors  and  Awards:   None 

Publications: 

Mitchell,  J.R.  and  Jollow,  D  .J  . :   Metabolic  activation  of  drugs  to  toxic 
substances.   Progress  in  hepatology.   Gastroenterology  68:  392-410,1975. 

3  ZZS" 


Project  No.  Z01  HL  00877-02  LCP 


Mitchell,  J.R.,  Long,  M.W.,  Thorgeirsson,  U  .P .  and  Jollow,  D  .  J  .  : 
Acetylation  rates  and  monthly  liver  function  tests  during  one  year 
of  isoniazid  preventive  therapy.   Chest  >  in  press. 

Black,  M.,  Mitchell,  J.R.,  Zimmerman,  H.J.,  Ishak,  K.  and  Epler,  G.R.: 
Isoniazid-associated  hepatitis  in  114  patients.   Gastroenterology, 
in  press  . 

Mitchell,  J.R.,  Thorgeirsson,  U.P.,  Black,  M.  Timbre  11,  J  .A  .  , 
Snodgrass,  W.R.,  Potter,  W.Z.,  Jollow,  D.J.  and  Keiser,  H.R.:   Increased 
incidence  of  isoniazid  hepatitis  in  rapid  acetylators  and  possible 
explanation  by  analysis  of  urinary  metabolites  of  isoniazid.   Clin . 
Pharmacol .  Ther . ,  in  press. 

Mitchell,  J.R.  and  Potter,  W.Z.:   Drug  metabolism  in  the  production  of 
liver  injury.   Med.  Clinics  of  N.  Amer . ,  in  press. 

Mitchell,  J.R.,  Nelson,  S .D . ,  Thorgeirsson,  S .S . ,  McMurtry.  R.J.  and 
Dybing,  E.:   Metabolic  activation:  biochemical  basis  for  many  drug- 
induced  liver  injuries.   In  Popper,  H.  and  Schaffner,  F.  (Eds.): 
Progress  in  Liver  Disease,  Vol  V,  in  press. 

Gillette,  J.R.  and  Mitchell,  J.R.:   Drug  actions  and  interactions: 
theoretical  considerations.   In  Gillette,  J.R.  and  Mitchell,  J.R.  (Eds.): 
Handbook  of  Experimental  Pharmacology,  Vol.  XXVIII,  Part  3.   New  York, 
Springer-Verlag,  1975,  pp.  359-382. 

i 
Mitchell,  J.R.,  Potter,  W.Z.,  Hinson,  J  .A  . ,  Snodgrass,  W  .R .  ,  Timbrell, 
J  .A .  and  Gillette,  J.R.:   Toxic  drug  reactions.   In  Gillette,  J.R.  and 
Mitchell,  J.R.  (Eds.):   Handbook  of  Experimental  Pharmacology,  Vol. 
XXVIII,  Part  3.   New  York,  Springer-Verlag,  1975,  pp.  383-419. 

Shand,  D.G.,  Mitchell,  J.R.  and  Oates,  J .A . :   Pharmacokinetic  drug 
interactions.   In  Gillette,  J.R.  and  Mitchell,  J.R.  (Eds.):   Handbook  of 
Experimental  Pharmacology,  Vol.  XXVIII,  Part  3.   New  York,  Springer- 
Verlag,  1975,  pp.  272-314. 


25^ 


Project  No.  Z01  HL  00877-02  LCP 

Table  I 
Acetylator  phenotype  and  clinical  features  of  26  patients 
with  probable  or  possible  isoniazid  liver  injury. 


7oAcety 

lated 

Acetylator 

Age 

Peak 

Peak 

Sulfame 

thazine3 

Phenotype^ 

Diagnosis0 

Race 

SGOT 

Bilirubine 

Urine 

Blood 

Sexd 

or 

SGPTe 

93.6 

66.1 

Rapid 

Probable 

59 

BF 

700 

10.2 

95.1 

83.3 

Rapid 

Probable 

53 

WM 

1015 

35.5 

83.3 

43.7 

Rapid 

Probable 

48 

BF 

1065 

28.5 

84.9 

59.1 

Rapid 

Probable 

69 

BF 

1250 

7.1 

88.1 

72.8 

Rapid 

Probable 

56 

WM 

1420 

13.2 

74.5 

70.1 

Rapid 

Probable 

60 

WF 

1710 

27.0 

88.9 

78.3 

Rapid 

Probable 

53 

BF 

2780 

6.3 

89.9 

57.8 

Rapid 

Probable 

49 

BF 

2834 

12.0 

91.3 

69.7 

Rapid 

Probable 

59 

WF 

975 

21.2 

87.1 

61.3 

Rapid 

Probable 

65 

WM 

500 

7.4 

82.3 

51.8 

Rapid 

Probable 

26 

BF 

700 

7.5 

91.4 

58.0 

Rapid 

Probable 

40 

WM 

500 

1.6 

80.8 

49.2 

Rapid 

Probable 

35 

WF 

160f 

0.6 

77.2 

57.6 

Rapid 

Probable 

48 

BF 

1118 

7.0 

90.4 

48.1 

Rapid 

Probable 

42 

BF 

870 

0.9 

81.7 

31.4 

Rapid 

Probable 

37 

BF 

650 

1.3 

88.7 

49.3 

Rapid 

Probable 

57 

WF 

760 

1.7 

91.7 

57.9 

Rapid 

Probable 

54 

WM 

490 

1.1 

40.0 

5.8 

S  low 

Probable 

49 

WF 

660 

25.3 

65.2 

6.2 

Slow 

Probable 

61 

WF 

638 

9.5 

32.7 

4.3 

S  low 

Probable 

64 

WM 

260 

0.7 

82.1 

29.4 

Rapid 

Possible 

51 

WM 

116 

0.8 

72.6 

26.1 

Rapid 

Possible 

47 

BM 

87 

0.4 

85.9 

60.4 

Rapid 

Possible 

56 

BM 

51 

0.3 

47.7 

2.4 

S  low 

Possible 

23 

WF 

97 

0.7 

52.6 

3.9 

S  low 

Possible 

54 

WF 

68 

0.6 

aProportion  of  acety lated  sulfamethazine  6  hr  after  drug  administration  to 

patients  recovered  from  liver  injury. 
bPatients  classified  as  rapid  acetylators  if  the  proportion  of  acetylated 

sulfamethazine  was  more  than  70%  in  urine  or  more  than  257o  in  blood. 
cProbable  or  possible  isoniazid  liver  injury. 

dAge  (yr),  B  (black)  or  W  (white)  race,  M  (male) or  F  (female)  sex. 
ePeak  abnormalities  during  liver  injury  --  upper  limits  of  normal,  SGOT  (45), 

SGPT  (40),  bilirubin  (1.0). 
^Diagnosis  confirmed  by  liver  biopsy. 


A»7 


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2SS 


Project  No.  Z01  HL  00878-01  LCP 


1.  Chemical  Pharmacology 

2.  Clinical  Pharmacology 

3.  Bethesda,  Md . 

PHS  -NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Hydrazines  2.   Chemical  synthesis  of  labeled  compounds. 

Previous  Serial  Number:   None 

Principal  Investigators:   Dr.  S.  D.  Nelson 

Dr.  J .  R.  Mitchell 

Other  Investigators:      Mr.  George  Corcoran 

Mr.  Christopher  Conner 

Cooperating  Units:        Dr.  Nelson  is  a  staff  fellow  in  the  Pharmacology 

Research  Associate  Program,  NIGMS . 
Mr.  Conner  is  funded  by  the  American  Lung 
Association . 

Project  Description: 

Objectives :   Isoniazid  and  iproniazid  can  produce  hepatitis  in  man. 
Elucidation  of  the  mechanism  leading  to  this  toxic  reaction  required  the  syn- 
thesis of  several  specifically  labeled  hydrazine  derivatives. 

Methods  Employed:   Published  synthetic  procedures  were  employed  whenever 
possible  using  commercially  available  reagents,  stable  isotopes,  and  radio- 
labeled compounds.   All  radioactive  syntheses  were  preceded  by  "cold"  syn- 
thesis of  enough  material  for  melting  point  and  NMR  analysis.   Radiochemical 
purity  of  the  prepared  derivatives  was  determined  by  recrystallization  to 
constant  specific  activity  (sp.  act.)  followed  by  thin-layer  chromatography 
(tic).   The  plates  were  scraped  in  0.5  cm  bands  and  each  fraction  counted  by 
liquid  scintillation  spectrometry  in  a  Beckman  LS -355  instrument.   The  more 
unstable  hydrazines  were  purified  immediately  prior  to  use.   The  position  of 
the  tritium  radiolabel  in  certain  specifically  labeled  hydrazines  was  de- 
termined as  described  in  the  Results.   Melting  points  were  determined  on  a 
Thomas  Hoover  Uni-Melt  instrument  and  nuclear  magnetic  resonance  (NMR)  spec- 
tra were  determined  on  a  Varian  A-60. 

Results : 

1.   Acetylisoniazid  (AcINH) 

a.   N2-Acetyl-isonicotinoyl-(3H)-hydrazide  (AcINH-  H)  .   This  compound 
was  prepared  in  927o  yield  by  acetylation  of  generally  tritiated  isonicotinic 
acid  hydrazide  (INH-^H) ,  supplied  by  Amer sham-Searle ,  with  acetyl  chloride 
in  a  mixture  of  ethyl  acetate-glacial  acetic  acid  and  sodium  bicarbonate. 

1  A*? 


Project  No.  Z01  HL  00878-01  LCP 


Evaporation  of  the  mixture  yielded  a  residue  which  was  extracted  into  chloro- 
form, washed  with  a  small  amount  of  water  and  evaporated.   The  yellow  crys- 
tal mass  was  recrystallized  from  methanol rether  (3x)  to  constant  specific 
activity  and  finally  from  isopropanol: hexane  (lx).   Radiochemical  (99.8) 
purity  was  further  confirmed  by  tic  on  silica  gel  using  2-propanol rmethanol 
70:30  as  developing  solvent,  r.f.  =  0.6,  and  scraping  0.5  cm  bands  as  des- 
cribed under  Methods. 

b.  N2-Acetyl-isonicotinoyl-(l^C)-hydrazide  (AcINH-^C)  was  prepared 
in  the  same  manner  using  l^C -carbony 1-labeled  INH  (Amersham-Searle) . 

c.  N2-Acetyl-(3H) -isonicotinoyl  hydrazide  (3H-AcINH)  and  N2-acetyl- 
( l^C) -isonicotinoylhydrazide  (l^C-AcINH)  were  synthesized  by  the  same  pro- 
cedure using  H-methyl  and  -^C-carbonyl-labeled  acetyl  chloride,  respectively. 

2.  Acetylhydrazine  (AcHz) 

a.  Aeetyl-(l^C) -hydrazine  (l^C-AcHz)  was  prepared  as  the  fumarate 
salt  by  transacetylation  under  reflux  of  carbonyl-labeled  ethyl  acetate-l^C 
with  hydrazine  hydrate  dissolved  in  ethanol.   After  refluxing  several  hours 
fumaric  acid  was  added  and  upon  cooling  l^C-AcHz  fumarate  was  crystallized. 
Recrysta llization  to  constant  sp .  act.  from  ethanol  (x3)  and  then  from 
methanol :ether  (xl)  gave  the  desired  product,  m.p.  122-123°.   Radiochemical 
purity  was  confirmed  by  tic  on  Avicel  developed  in  4:1:1  n-butanol: ethanol: 
0.4N  NH4OH,  r.f.  =  0,49.   The  product  was  recrystallized  each  time  immediate- 
ly prior  to  use  since  the  compound  slowly  decomposed  even  under  a  nitrogen 
atmosphere  in  a  dessicator  maintained  at  -15°. 

b.  Acetyl-(^H) -hydrazine  (3H-AcHz)  was  prepared  as  the  hydrochloride 
salt  by  the  following  sequence  of  reactions.   t-Butyl  carbazate  (Aldrich)  was 
reacted  with  acetic-(  H) -anhydride  (New  England  Nuclear)  in  methylene  chlor- 
ide to  yield  N  -acetyl-(3H) -t-buty 1  carbazate  which  was  subsequently  hydro- 
lyzed  in  dilute  methanolic  HCl  at  room  temperature.   Evaporation  yielded  a 
hygroscopic  crystalline  mass  which  was  recrystallized  to  constant  specific 
activity  (4x)  from  methanol: ether  mixtures  to  give  white  rhombic  crystals, 
m.p.  131-133°C.   Radiochemical  purity  >  997o  was  further  confirmed  by  tic  on 
Avicel-F  using  n-butanol : ethanol: 0 .4N  NH4OH  (4:1:1)  as  developing  solvent, 
r.f.  0.48,  and  scraping  0.5  cm  bands.   Like  AcHz  fumarate,  this  salt  slowly 
decomposed  and  had  to  be  recrystallized  immediately  prior  to  use. 

3.  Nl,N2-acetylhydrazine    (DiAcHz) 

N1,N2-Acetyl-(3H) -hydrazine  (DiAcHz-3H) .   This  compound  was  synthesized 
the  acetylation  of  hydrazine  hydrate  with  a  slight  molar  excess  (2.2; 1) 
of  acetic-  H-anhydride.   The  product  was  recrystallized  to  constant  sp .  act. 
(x3)  from  methanol: ether  and  radiochemical  purity  confirmed  by  tic  as  de- 


by 


3H- 
met 
scribed    for  AcHz 

4.      N^-Isopropyl-isonicotinoylhydrazide    (IpINH) 


2  3o 


Project  No.  Z01  HL  00878-01  LCP 

a.  N2-Isopropyl-isonicotinoyl-(3H)-hydrazide  (IpINH-3H) .   This  hydra- 
zide  was  synthesized  by  the  reductive  alkylation  of  INH-^H  with  acetone  and 
hydrogen  gas  at  50  psi  in  a  Paar  hydrogenator  using  107o  Pd  on  charcoal  as  a 
catalyst.   Recrystallization  to  constant  sp .  act.  (x4)  from  methylene 
chloride-hexane  gave  the  desired  product,  m.p.  111-113°C.   Radiochemical 
purity  was  determined  to  be  >  99.8%  based  on  tic  on  silica  gel  using  chloro- 
form:ethanol:acetic  acid  15:2:0.1,  r.f.  =  0.59,  and  methanol-ethy 1  acetate 
1:1,  r.f.  =  0.68. 

b.  N2-Isopropyl-(2-14C)-isonicotinoyl  hydrazide  (2-14C-IpINH)  .   This 
radiolabeled  analog  was  prepared  and  analyzed  using  the  same  procedure  de- 
scribed for  IpINH-^H  using  unlabeled  INH  and  acetone  (2-1^C)  . 

c.  N2-Isopropyl-(l,3-14C)-isonicotinoyl  hydrazide  (l,3-14C-IpINH) . 
This  analog  was  synthesized  by  alkylation  of  the  sodium  salt  of  INH,  gener- 
ated in  situ  with  an  equimolar  amount  of  sodium  methoxide,  with  isopropyl 
iodide  (1,3-^C)  (Amersham) .   Purification  and  radiochemical  purity  determin- 
ations were  carried  out  as  previously  described. 

d.  N2-Isopropyl-(2-di)-isonicotinoyl  hydrazide  (2-di-IpINH)  .   This 
stable  isotope  analog  was  prepared  by  reacting  N  -isopropylidene-isonico- 
tinoyl  hydrazide  (synthesized  by  condensation  of  INH  and  acetone)  with 
deuterium  gas  generated  at  room  temperature  iri  situ  from  sodium  borodeuter- 
ide  and  platinum  oxide  in  deuterated  methanol.   The  reaction  took  approxi- 
mately 4  minutes.   NMR  of  the  resultant  product  showed  complete  loss  of  the 
multipletcentered  at  3.256.   Integration  showed  no  incorporation  of  deuterium 
into  any  other  portion  of  the  molecule. 

e.  N2-Isopropyl-(2-3H)-isonicotinoyl  hydrazide  (2-3H-IpINH) .   This 
material  was  prepared  using  the  same  procedure  described  for  the  deutero- 
analog  using  sodium  borotritide .   The  compound  was  purified  to  constant  sp . 
act.  as  previously  outlined.   Acid  hydrolysis  of  the  compound  and  chroma- 
tography of  the  resultant  isonicotinic  acid  and  isopropyl  hydrazine  showed 
that  all  of  the  label  was  present  in  the  isopropyl  side  chain.   Results  from 
the  deuterium  experiment  show  that  the  tritium  is  incorporated  solely  into 
the  C-2  carbon  of  the  isopropyl  group. 

5.   Isopropyl  hydrazine  (IpHz) . 

a.   Isopropyl-(2--'-^C) -hydrazine  (2-l^C-IpHz)  .   This  compound  was  pre- 
pared as  the  hydrochloride  salt  in  the  following  manner.   t-Butyl  carbazate 
(Aldrich)  was  reductively  alkylated  with  acetone  (2-l^C)  in  a  hydrogenator 
(50  psi-H  )  at  room  temperature  using  107o  Pd  on  charcoal  as  a  catalyst.   The 
intermediate  N-isopropyl-(2-14C) -t-butyl  carbazate  was  purified  by  re- 
crystallation  from  n-hexane,  m.p.  49-51°.   This  was  then  hydrolyzed  at  room 
temerature  in  methanolic  HCl  to  give  white  needles  of  ^C-IpHz  hydrochloride 
which  were  recrystallized  to  constant  sp.act.  from  methanol: ether  (x5) ,  m.p. 
110-112°C.   The  material  was  recrystallized  immediately  prior  to  use  since 
the  product  was  found  to  slowly  decompose. 


Z3( 


Project  No.  Z01  HL  00878-01  LCP 

b.  Isopropyl-(2-3H)-hydrazine  (2-3H-IpHz) .  This  tritiated  analog  was 
prepared  by  hydrogenation  with  tritium  gas  of  the  non-radioactive  isopropy- 
lidene  intermediate  at  room  temperature  and  pressure  using  tris-(tripheny 1- 
phosphine)  chlororhodium  as  catalyst.  This  soluble  catalyst  has  been  shown 
to  reduce  double  bonds  without  the  side  reactions  of  tritium  exchange.  The 
intermediate  product  was  then  hydrolyzed  and  purified  as  described  above  (5a). 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
These  syntheses  have  made  possible  the  elucidation  of  the  mechanisms  of  iso- 
niazid  and  iproniazid  hepatitis  (Z01  HL  00879-01  LCP,  Z01  HL  00881-01  LCP). 

Proposed  Course  of  Project:   Additional  syntheses  will  be  performed 
according  to  research  needs. 

Keyword  Description: 

hydrazines 
synthesis 

Honors  and  Awards:   None 

Publications:   None 


3l3* 


Project  No.  Z01  HL  00879-01  LCP 

1.  Chemical  Pharmacology 

2.  Clinical  Pharmacology 
3  .   Bethesda  ,  Md . 

PHS  -NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Hydrazines  3.  Studies  of  reactive  metabolites  in  vitro 

Previous  Serial  Number:    None 

Principal  Investigators:   Dr.  S.  D.  Nelson 

Dr.  W.  R.  Snodgrass 
Dr  .  J  .  A  .  Timbrell 
Dr  .  J  .  R.  Mitchell 

Other  Investigators:       Mr.  Kenneth  Greene 

Mr  .  George  Corcoran 

Cooperating  Units:         Drs.  Nelson  and  Snodgrass  are  staff  fellows  in 

the  Pharmacology  Research  Associate  Program,  NIGMS 

Project  Description: 

Objectives :   Both  acety lhydrazine  and  isopropy lhydrazine  are  potent 
hepatotoxins  in  male  rats.   In  vivo  studies  indicated  that  metabolic  activa- 
tion is  necessary  to  produce  the  toxic  effects  (NHLI  #  ^3  ) .   To  further 
elucidate  the  mechanism  for  activating  these  hydrazines  to  toxic  intermedi- 
ates, we  carried  out  a  series  of  i_n  vitro  experiments  with  rat  liver  micro- 
somes . 

Methods  Employed:   Rats  were  pretreated  with  phenobarbita 1  for  four  days 
(75  mg/kg,  i.p.)  before  removal  of  livers  and  purification  and  isolation  of 
microsomes.   A  second  group  was  treated  in  the  same  manner  except  that  co- 
baltous  chloride  was  administered  twice  daily  (30  mg/kg,  s.c.)  during  the 
last  two  days  of  phenobarbita 1  pretreatment .   Incubations  were  carried  out 
and  the  covalent  binding  of  reactive  intermediates  was  determined  at  various 
substrate  concentrations  and  under  varying  conditions  according  to  procedures 
previously  described  by  this  laboratory. 

Gassing  experiments  were  conducted  in  septum-sealed  vessels  using  1007o 
N2,  9:1  N2:02,  9:1  C0:02  and  air.   Propane  was  trapped  in  septum-sealed 
vessels  and  determined  by  gas -chroma tography  on  a  Perkin-Elmer  900  gas 
chroma tograph  (Poropak  Q,  column  temp.  150°;  injector  80°;  flame  ionization 
detector  200°;  N2  carrier  gas  flow  50  ml/min;  retention  time  of  propane,  1.5 
min)  .   Acetic  acid  was  determined  on  the  same  column  at  170°C  and  had  a  re- 
tention Lime  of  3.5  minutes.   Ratios  of  tritium  to  carbon-14  in  the  bound  and 
chromatographed  metabolites  was  determined  by  the  channels-ratio  method.   Gas 
chromatography-mass  spectrometry  (gc-ms)  was  performed  using  the  same  column 
conditions  already  described,  and  was  coupled  to  an  LKB-9000S  mass  spec- 

1  ail 


Project  No.  Z01  HL  00879-01  LCP 

trometer  with  an  electron  energy  of  70  eV,  accelerating  voltage  of  3.5  KV, 
and  a  trap  current  of  50  uA  . 

Specifically  radiolabeled  compounds  were  prepared  as  previously  de- 
scribed, and  the  substrates  used  included  N  -acetyl-isonicotinoyl-pH)  - 
hydrazide  (AcINH-3H) ;  N2 -acetyl-(14C) -isonicotinoy 1  hydrazide  (^4C-AcINH); 
acetyl-(14C)-hydrazine  (14C-AcHz);  acetyl-(3H) -hydrazine  (3H-AcHz)-  N2-iso- 
propyl-(2-3H)-isonicotinoyl  hydrazide  (2-3H-IpINH) :  isopropyl-(2-14C) -hydra • 
zine  (2-l^C-IpHz) ;  isopropyl-(2-3H) -hydrazine  (2-3H-IpHz) . 


Major  Findings:   In  the  presence  or  absence  of  NADPH  only  small  amounts 
of  AcINH-3H  and  ^C-AcINH  were  bound  to  liver  microsomes  in  vitro  (~  0.11 
nmoles/mg/15  min) .   In  contrast,  a  substantial  amount  of  covalent  binding 
(0.55  nmoles/mg/15  min)   occurred  with  acetyl  hydrazine  at  37°  in  the  presence 
of  microsomal  enzymes,  oxygen,  and  NADPH.   The  binding  required  NADPH  and 
oxygen.   It  was  almost  abolished  by  heat  inactivation  of  the  enzymes  and  was 
inhibited  by  a  carbon  monoxide : oxygen  atmosphere  and  SKF-525A.   Glutathione, 
a  naturally  occurring  sulfhydryl-containing  tripeptide,  was  also  found  to 
substantially  decrease  the  binding  with  concomitant  increase  in  the  formation 
of  a  glutathione  conjugate,  subsequently  indentified  as  S -acetyl  glutathione. 
Gas  chromatographic  analysis  of  the  methanol  extract  after  precipitation  of 
the  protein  showed  the  presence  of  acetic  acid  in  those  incubations  contain- 
ing NADPH  with  much  smaller  (20%)  amounts  in  incubations  without  NADPH. 

Finally,  an  antibody  against  NADPH-cytochrome  £  reductase  decreased  the 
binding  to  nearly  the  same  extent  as  it  decreased  the  rate  of  cytochrome  c_ 
reduction.   These  studies  show,  therefore,  that  the  enzyme  system  activating 
acetylhydrazine  is  a  cytochrome  P-450  mixed  function  oxidase. 

Kinetic  analysis  of  the  covalent  binding  of  acetylhydrazine  using  vari- 
ous pretreatments  showed  that  the  binding  of  radiolabel  was  markedly 
increased  by  phenobarbital  pretreatment ,  which  potentiated  necrosis  and  in 
vivo  binding,  whereas  it  was  decreased  by  pretreatment  with  cobaltous  chlo- 
ride, which  blocked  both  the  necrosis  and  in  vivo  binding. 

The  Km  for  binding  with  all  treatments  was  found  to  be  approximately 
10"3  molar  while  the  Vmax  for  acetylhydrazine  binding  was  0.03  nmoles/mg/min 
for  cobaltous  chloride  pretreated  animals,  0.06  nmoles/mg/min  for  normals, 
and  0.11  nmoles/mg/min  for  phenobarbital  pretreatment.   When  mixtures  of 
methyl  labeled  -^h-AcHz  and  carbonyl  labeled  -^C-AcHz  were  used,  the  ratio  of 
tritium  to  carbon-14  bound  was  0.92  compared  to  the  injected  material, 
illustrating  that  the  entire  acetyl  group  was  bound. 

The  same  conditions  outlined  for  the  i_n  vitro  binding  of  acetylhydrazine 
were  applied  to  isopropylhydrazine  after  first  determining  that  2--%-IpINH 
was  not  bound.   The  results  parallel  those  of  AcHz  showing  that  IpHz  is  also 
activated  by  a  P-450  system.   The  kinetic  data  for  the  binding  reaction  of 
IpHz  are  nearly  identical  to  those  found  for  AcHz  except  that  the  apparent 
Km  for  the  binding  is  one-tenth  that  of  AcHz  (10-Zf  molar  compared  to  10"3 
molar).   This  may  explain  in  part  why  IpHz  is  a  more  potent  alkylating  and 

2  3L2^ 


Project  No.  Z01  HL  00879-01  LCP 
necrotizing  agent  i_n  vivo  than  AcHz. 

As  observed  for  AcHz,  the  same  extent  of  covalent  binding  of  radiolabel 
to  tissue  macromolecules  was  found  for  both  2-3h-  and  2 -^C- la  be  led  IpHz, 
-SH/1'*C  =  0.94.   The  ratio  found  for  the  propane  evolved  in  the  In  vitro 
incubations  showed   H/   C  =  0.96.   Even  more  importantly,  pherobarbita 1  pre- 
treatment,  which  increased  in  vitro  binding,  increased  the  amount  of  propane 
evolved.   Similarly,  cobaltous  chloride  pretrea tment ,  which  decreased  the 
binding  reaction,  decreased  the  amount  of  propane  evolved. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
These  results  demonstrate  that  acetylhydrazine  and  isopropylhydrazine  are 
converted  by  hepatic  P-450  oxidases  to  potent  acylating  and  alkylating  agents 
These  chemically  reactive  metabolites  probably  cause  the  serious  hepatitis 
that  occurs  in  patients  treated  with  isoniazid  and  iproniazid.   The  possi- 
bility that  these  metabolites  may  be  carcinogenic  should  also  be  considered. 
The  propane  studies  and  the  ^h/ 14c  ratio  studies  are  consistent  with  the 
hypothesis  that  the  reactive  metabolites  are  radical  species. 

Proposed  Course  of  Project:   Completed.   Manuscripts  are  in  preparation. 

Keyword  Description: 

acetylhydrazine  reactive  metabolite 

in  vitro  covalent  binding  specific  radiolabel 

isopropylhydrazine 

Honors  and  Awards:   None 

Publications:   None 


2.3S 


Project  No.  Z01  HL  00880-01  LCP 

1.  Chemical  Pharmacology 

2.  Clinical  Pharmacology 
3  .   Bethesda,  Md . 

PHS  -NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Hydrazines  4.  Studies  of  reactive  metabolites  in  rats. 

Previous  Serial  Number:   None 

Principal  Investigators:   Dr.  J.  A.  Timbrell 

Dr  .  S  .  D.  Nelson 

Dr.  W.  R.  Snodgrass 

Dr.  J  .  R.  Mitchell 

Other  Investigators:      Mr.  Kenneth  Greene 

Mr  .  George  Corcoran 

Cooperating  Units:        Drs.  Nelson  and  Snodgrass  are  Research  Associates 

in  the  Pharmacology -Toxicology  Program,  NIGMS . 

Project  Description: 

Objectives :   We  have  previously  postulated  that  the  hepatic  necrosis 
caused  by  isoniazid  and  iproniazid  results  from  the  liberation  and  subsequent 
metabolic  activation  of  their  hydrazino  moieties  to  acylating  and  alkylating 
intermediates  in  the  body  (Z01  HL  00877-02  LCP,  Z01  HL  00878-01  LCP,  Z01  HL 
00879-01  LCP)  .  We  now  compare  the  rates  of  metabolism  of  hydrazino  compounds 
by  the  proposed  toxic  pathways  in  rats,  and  the  extent  of  hepatic  necrosis 
and  covalent  binding. 

Methods  Employed:   The  following  specifically  radiolabeled  derivatives 
have  been  prepared:   1)  Acetylisoniazid-N  -acetyl-isonicotinoy 1  (3H-ring- 
labeled)  hydrazide,  AcINH-3H;  N^-acety  1-isonicotinoyl  (   C-carbonyl-labeled) 
hydrazide,  AcINH-l^C;  N^-acetyl  (3H-methyl-labeled)  isonicotinoyl  hydrazide, 
3H-AcINH;  N2-acetyl-(1Z|'C-carbonyl-labeled)  isonictinoyl-hydrazide ,  ^C-AcINH. 
2)  Acetyl  hydrazine-acetyl-(3H_methyl-labeled) -hydrazine -hydrochloride , 
3H-AcHz;  acetyl  (^C-carbonyl-labeled)  hydrazine  fumarate,  1^+C-AcHz.   3) 
Iproniazid-N  -isopropyl-isonicotinoyl  (  H-ring  labeled)  hydrazide,  IpINH-  H; 
N^-isopropyl-isonicotinoyl  (■'■^C-carbonyl  labeled)  hydrazide,  IpINH-  C;  N  - 
isopropyl-(2-l^C)  -isonicotinoyl  hydrazide,  2-l^C-IpINH;  N^-isopro'pyl  (1,3- 
l^C)  isonicotinoyl  hydrazide,  1,3-   C-IpINH;  N  -isopropyl  (2-  H)  isonico- 
tinoyl hydrazide,  2-3H-IpINH.   4)  Isopropyl  hydrazine-isopropyl  (2--'-^C) 
hydrazine  hydrochloride,  2-14C-IpHz;  isopropyl  (2-3H)  hydrazine  hydrochloride, 
2-3fl-ipHz  (Z01  HL  00878-01  LCP).   The  derivatives  were  administered  to  rats 
intraperitoneally .   Urine  was  collected  and  metabolites  isolated  by  chroma- 
tography.  Pulmonary  expiration  of  acetone  (2-14C),   4C02  and  l^C-  or  3H- 
propane  was  trapped  in  a  coupled  series  of  three  solutions:   2,4  dinitro- 
phenylhydrazine  (14C -acetone  trapped  as  hydrazone  conjugate),  ethanolamine: 

1  3.31 


Project  No.  Z01  HL  00880-01  LCP 


2-methoxyethanol  (1'+C02),  and  diethyl  ether  at  -78°C  (propane).   Urinary 
metabolites  were  identified  by  their  Rf  values,  by  co-chromatography  with 
synthesized  authentic  standards,  by  color  reactions  with  spray  reagents,  by 
isotope  dilution  analysis  and  by  mass  spectrometry.   Propane  was  detected  in 
the  ethyl  ether  trap  by  gas  chromatography-mass  spectrometry  on  a  LKB  9000S 
using  a  Poropak  Q  column.   Tritium  to  carbon-14  ratios  (^H/l^C)  were  deter- 
mined by  the  channels  ratio  method  in  a  Beckmann  LS -355  scintillation  spec- 
trometer . 

Major  Findings;   Table  1  compares  the  pulmonary  expiration  of  -^C02  after 
administration  of  ^C-AcINH  and  ^C-AcHz  to  rats  versus  the  extent  of  acyla- 
tion  of  hepatic  macromolecules .   As  expected,  pretreatment  of  rats  with  pheno- 
barbital,  which  potentiated  hepatic  necrosis,  increased  the  amount  of  acyla- 
tion  and  the  expiration  of  ^^C02  •   Conversely,  cobalt  chloride  pretreatment, 
which  reduced  hepatic  necrosis,  decreased  the  extent  of  acylation  and  the 
expiration  of  ^  CO2 .   No  expiration  of  -^CC^  or  covalent  binding  occurred 
after  administration  of  AcINH-^C.   Thus,  measurement  of  expired  -^COo  after 
administration  of  l^C-acetyl-labeled  acetylisoniazid  (l^C-AcINH)  or  14c- 
acetyl-labeled  acetylhydrazine  (   C-AcHz)  can  be  used  as  an  index  of  the 
amount  of  acetylhydrazine  that  is  converted  to  a  chemically  reactive, 
acylating  species  in  vivo. 

This  conclusion  is  confirmed  by  analysis  of  the  urinary  metabolites  from 
14-C-AcHz  in  the  above  experiments.   Pretreatment  of  rats  with  an  inhibitor  of 
cytochrome  P-450,  cobalt  chloride,  markedly  increased  the  total  urinary  ex- 
cretion of  radioactivity  (Table  2)  and  decreased  expiration  of   CO2  (Table  1) 
because  it  apparently  inhibited  the  P-450  oxidation  of  acetylhydrazine,  as 
shown  by  the  increases  in  the  urinary  excretion  of  free  acetylhydrazine, 
diacetylhydrazine  and  the  hydrazones  of  acetylhydrazine.   The  excretion  of 
these  metabolites  are  proportional  to  the  availability  (amount)  of  total 
acetylhydrazine  in  the  body.   Conversely,  pretreatment  with  an  inducer  of 
cytochrome  P-450,  phenobarbital,  decreased  total  urinary  excretion  of  radio- 
activity and  increased  expiration  of  1^C02  because  it  induced  the  P-450  oxi- 
dation of  acetylhydrazine,  as  reflected  by  the  decreases  in  the  urinary 
excretion  of  free  acetylhydrazine,  diacetylhydrazine  and  the  hydrazones  of 
acetylhydrazine.   As  expected,  bis-p-nitrophenyl  phosphate  (BNPP)  pretreatment, 
which  prevents  the  hepatic  injury  caused  by  AcINH  as  well  as  its  hydrolysis 
to  acetylhydrazine,  had  no  effect  on  the  covalent  binding  after  the  adminis- 
tration of  AcHz  itself.   However,  the  urinary  excretion  of  AcHz  metabolites 
after  BNPP  pretreatment  is  yet  to  be  determined. 

Table  3  compares  the  expiration  of  propane  (2-3h)  after  administration 
of  isopropyl  (2-%)  hydrazine  (-%-IpHz)  to  rats  versus  the  extent  of  alkyla- 
tion  of  hepatic  macromolecules ;   Pretreatment  of  rats  with  phenobarbital, 
which  potentiated  hepatic  injury,  increased  the  extent  of  alkylation,  but 
unexpectedly  decreased  the  expiration  of  propane-2-3H.   In  contrast,  cobalt 
chloride  pretreatment  decreased  hepatic  injury,  alkylation  and  formation  of 
propane  (2-^H) . 


£37 


Project  No.  Z01  HL  00880-01  LCP 


In  an  attempt  to  explain  this  anomaly,  the  metabolism  of  IpINH-(^^C- 
carbonyl)  and  1,3-^C-IpINH  was  examined  (Tables  4,  5).   In  contrast  to 
acetylisoniazid  and  acetylhydrazine ,  metabolism  of  isopropylisoniazid 
(iproniazid)  and  isopropylhydrazine  by  cytochrome  P-450  oxidases  presumably 
occurs  at  C-2  of  the  isopropyl  group  in  addition  to  nitrogen  oxidation, 
because  acetylisoniazid  (15.7%,  Table  4),  and  acetone  (1.9%,  Table  5)  were 
formed  from  isopropylisoniazid.   Thus  the  effects  of  inducers  and  inhibitors 
of  P-450  oxidases  on  propane  expiration  will  be  quite  complex,  and  therefore 
correlations  in  vivo  between  covalent  binding  and  propane  expiration  will  be 
meaningless  without  a  complete  metabolite  profile.   This  will  necessitate  a 
more  comprehensive  metabolic  study  of  the  fate  of  both  IpINH  and  IpHz. 

To  help  elucidate  the  mechanism  leading  to  covalent  binding,  we  have 
used  the  technique  of  administering  mixtures  of  -%  and  14c  isotopes  of  iso- 
propylisoniazid and  isopropylhydrazine.  When  a  mixture  of  2-3H-IpINH  and 
l,3-14c-lplNH  was  administered  to  rats,  the  covalently  bound  material  was 
found  to  have  a  ratio  of  -%/ 14^  _  0.92  compared  to  the  injected  solution,  and 
the  expired  propane  gas  had  a  similar  ratio  of  3h/14c  (0.94).   When  a  mixture 
of  2-JH-IpINH  and  2-l^C-IpINH  was  administered,  the  covalently  bound  material 
was  determined  to  have  a  ratio  of  -%/   C  =  0.96  that  of  the  injected  material 
and  the  collected  propane  again  had  virtually  the  same  ratio  (1:1).   These 
ratios  show  that  neither  the  covalent  binding  nor  the  propane  arise  by  way 
of  C-2  oxidation.   Furthermore,  these  results,  coupled  with  the  gas  chromato- 
graphic and  mass  spectometric  data  on  the  expired  propane,  provide  strong 
evidence  that  the  covalently  bound  material  retains  the  entire  isopropyl 
group  and  probably  arises  by  the  same  enzyme  pathway  that  produces  propane. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
These  results  provide  direct  evidence  in  vivo  for  the  generation  of  chemical- 
ly reactive,  hepatotoxic  metabolites  from  isoniazid  (via  acetylisoniazid) 
and  iproniazid.   They  support  the  results  obtained  in_  vitro  on  the  mechanism 
of  hydrazine  activation  and  covalent  binding  of  reactive  metabolites. 

Proposed  Course  of  Project:   Further  studies  are  to  be  carried  out  on 
the  metabolism  in  vivo  of  IpINH  and  IpHz. 

Keyword  Description: 

gas  chromatography  metabolism  in.  vivo 

hydrazines  reactive  metabolites 

mass  spectrometry 

Honors  and  Awards:   None 

Publications:   None 


«23g 


Project  No.   7m   ttt.  nnasn-m   T.rrp 


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23? 


Project  No.   Z01  HL  00880-01  LCP 


Table  2 
Disposition  of  I'+C-acety lhydrazine  in  vivo  in  rats 


7o  dose  excreted  in  6  hr  urine 


Total  urinary 
metabolites 


None 

Phenobarbital  (PB) 

Cobalt  chloride 
+  PB 

21.7 

17.2 

39.6 

7o  dose  excreted  in  6  hr  urine 


a-Ke tog luta rate 

2.9 

2.1 

6.1 

Acetyl  hydrazone 

pyruvate  acetyl 

hydrazone 

4.9 

4.0 

8.7 

Acetyl  hydrazine 

1.5 

0.7 

1.9 

Diacetyl  hydrazine 

5.4 

3.8 

11.6 

Unknown  metabolite 

1.2 

0.8 

0.8 

Total 

15.9 

11.4 

29.1 

ato 


Project  No.  Z01  HL  00880-01  LCP 

Table  3 

Effect  of  phenobarbital  and  cobalt  chloride  on  propane  formation 

and  alkylation  of  hepatic  macromolecules  5  hours  after 

administration  of  isopropyl-(2-3H) -hydrazine  hydrochloride 

(20  mg/kg  as  free  base)  to  rats. 

Covalent  Binding  Propane-2-^H 

Treatment  nmol/mg  protein  %   of  dose 

None  0.35  ±  .025  6.7  +  0.4 

Phenobarbital  (Pb)  0.44  ±  .032  3.6  +  0.2 

Pb  +  Cobalt  chloride  0.24  ±  .034  2.2  ±  0.1 


att 


Project  No.  Z01  HL  00880-01  LCP 

Table  4 

Metabolism  and  disposition  of 
N  -is opropyl-isonicotinoyl-(l^C-carbonyl) -hydrazine  (IpINH--1-  C) 

%   dose  excreted 
(average  of  3  rats) 

24  hr  urine  67 .8 

48  hr  urine  15.7 

carcass  +  feces  4.7 

Total  88.2 


7o  of  24  hr  urinary  %   dose 

radioactivity  (average  excreted 

of  3  rats) 


Isonicotinoyl  glycine  12.5  8.2 

Isonicotinic  acid  41.0  27.6 

Acetylisoniazid  23.0  15.7 

Unidentified  metabolite  6.0  4.0 

Iproniazid  11.9  8.5 

Total  94.4  64.0 


3.4X 


Project  No.    Z01  HL   00880-01  LCP 


Table    5 


Metabolism  and  disposition  of 

N  -isopropyl-(l,3--L^'C)  -isonicotinoyl  hydra zide 

(1,3-^C-IpINH)  in  the  rat. 


7o  of  Dose  Excreted 
(average  of  3  rats) 


Urine  24  hr 
Urine  48  hr 
Expired  acetone  24  hr 
Expired  acetone  48  hr 
Expired  C02  24  hr 
Expired  C02  48  hr 
Expired  propane  24  hr 
Expired  propane  48  hr 
Carcass  and  feces 
Total 


23.9  +  1.1 

11.7  ±  0.9 

1.9  ±  0.3 

0.0 

17.1  +  1.2 
7.0  ±  0.9 

11.5  +  1.1 

1.4  ±  0.2 

13.7  ±  0.8 

88.2  ±  7.1 


2V3 


Project  No.  Z01  HL  00881-01  LCP 


1.  Chemical  Pharmacology 

2.  Clinical  Pharmacology 
3  .  Bethesda ,  Md . 

PHS  -NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Phenacetin-induced  toxicity:   N-oxidation  of  phenacetin 

Previous  Serial  Number:  None 

Principal  Investigators:   Dr.  Jack  A.  Hinson 

Dr.  Jerry  R.  Mitchell 

Other  Investigators:      Mr.  George  Corcoran 

Mr  .  Kenneth  Greene 

Cooperating  Unit:         Dr.  Hinson  holds  a  NHLI  Postdoctoral  Fellowship 

Project  Description: 

Objectives :   Previous  studies  have  shown  that  a  toxic  metabolite  of 
acetaminophen  is  formed  during  the  metabolism  of  the  drug  in  the  liver  by  a 
cytochrome  P-450-dependent  mixed  function  oxidase.   Evidence  has  been  pre- 
sented that  this  toxic  reactive  metabolite  is  N-acetyl-4-benzoimidoquinone 
which  arises  through  N-hydroxy lation  of  the  parent  drug  followed  by  non- 
enzymatic  rearrangement.   Recently  we  found  that  phenacetin  also  causes 
centrilobular  hepatic  necrosis  in  hamsters.   Since  N-hydroxyphenacetin  will 
spontaneously  rearrange  to  N-acetyl-4-benzoimidoquinone ,  a  known  arylating 
agent,  we  examined  the  capacity  of  hepatic  microsomes  to  N-hydroxy late 
phenacetin . 

Methods  Employed:   N-hydroxyphenacetin  was  synthesized  from  p-nitro 
phenetole  in  two  steps:   a)  reduction  to  the  hydroxy lamine  derivative  by  zinc 
dust  in  the  presence  of  ammonium  chloride  and  b)  acetylation  of  the  hydroxyl- 
amine  derivative  by  acetyl  chloride  in  the  presence  of  sodium  bicarbonate. 
l^C-Acetyl  chloride  was  used  to  synthesize  the  acetyl-1  C-derivative  by  a 
modification  of  the  procedure.  Microsomes  were  isolated  from  hamsters  by 
standard  procedures.   All  other  methods  were  standard  procedures. 

Major  Findings:   N-Hydroxyphenacetin  was  synthesized  and  proof  of 
structure  obtained  by  electron  impact  mass  spectroscopy. 

Incubation  of  -^H-phenacetin  with  hamster  liver  microsomes  led  to  the 
formation  of  N-hydroxyphenacetin.   Proof  of  the  structure  was  obtained  by 
electron  impact  mass  spectroscopy:   the  spectrum  of  the  isolated  material  was 
identical  to  that  of  the  synthetic  standard.   It  showed  a  molecular  ion 
having  a  molecular  weight  of  195  and  major  mass  fragments  corresponding  to 
the  losses  of  oxygen,  C0CH_2 ,  and  oxygen  plus  COCH2 . 


<2f^ 


Project  No.  Z01  HL  00881-01  LCP 


A  quantitative  assay  for  N-hydroxyphenacetin  has  been  developed  and  its 
specificity  established  by   recrystallization  of  the  product  to  constant 
specific  activity.   The  reaction  rate  of  0.5  mM  phenacetin  was  about  0.2 
nmoles  per  min  per  mg  protein  whereas  the  rate  of  de-ethylation  of  phenacetin 
was  about  2.5  nmoles  per  min  per  mg  protein.   The  reaction  was  dependent  on 
NADPH  and  molecular  oxygen  and  was  inhibited  by  a  carbon  monoxide -oxygen 
atmosphere  indicating  that  it  was  catalyzed  by  cytochrome  P-450.   Sodium 
fluoride  significantly  increased  the  rate  of  N-hydroxylation . 

Pretreatment  of  the  hamsters  with  3-methylcholanthrene  increased  the 
rate  of  N-hydroxylation  while  pretreatment  with  either  piperonyl  butoxide  or 
cobaltous  chloride  inhibited  the  reaction.   Phenobarbital  pretreatment  did 
not  affect  the  activity  of  the  enzyme. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
The  present  studies  demonstrate  that  phenacetin  is  N-hydroxylated  in_  vitro 
and  is  a  significant  metabolite. 

Proposed  Course  of  Project:   Since  N-hydroxy -phenacetin  is  an  important 
in  vitro  metabolite,  the  potential  hepotoxicity  and  nephrotoxicity  of  this 
metabolite  will  be  examined. 

Keyword  Descriptions: 

cytochrome  P-450  nephrotoxicity 

hepatotoxicity  phenacetin 

N-hydroxyphenacetin 

Honors  and  Awards:   None 

Publications : 


j»^r 


ANNUAL  REPORT  OF  THE 

LABORATORY  OF  CHEMISTRY 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

July  1,  1974  through  June  30,  1975 

The  work  of  the  Laboratory  of  Chemistry  might  be  somewhat  artificially 
divided  between  applications  (problem-solving)  and  "core"  research.   Most 
of  the  members  of  the  Laboratory  engage  in  both  in  varying  proportions  and 
again  this  year  the  emphasis  has  been  on  the  former.   Our  major  physical 
tools,  gas  and  liquid  chromatography,  mass  spectrometry,  Fourier- transform 
nuclear  magnetic  resonance,  and  X-ray  crystallography  are  usually  applied 
in  that  order  to  structural  analysis  of  compounds  of  biological  origin 
brought  to  us  from  other  groups  in  NHLI,  NIH  and  occasionally  from  the 
outside.   Success  in  such  collaborations  depends  heavily  upon  our  general 
knowledge,  as  organic  chemists,  of  the  properties  and  reactivity  of  organic 
compounds  since  several  cycles  of  purification  and  analysis  are  invariably 
required  before  meaningful  results  begin  to  appear.   We  have  found  that 
the  best  results  in  such  collaborations  are  achieved  when  both  groups  take 
the  time  to  acquaint  each  other  with  the  origin  of  the  problem,  details  of 
isolation  and,  in  our  case,  idiosyncrasies  of  the  physical  techniques  we 
use.   Special  requirements  of  the  problem  then  often  point  up  the  need 
for  new  approaches  and  dictate  the  directions  of  our  "core"  research.   Our 
interest  in  chemical  ionization-mass  spectrometry  was  one  case  in  point 
since  many  biological  compounds  refuse  to  give  molecular  ions  under 
electron  impact.   Our  laboratory,  working  on  plans  from  the  literature, 
arranged  construction  of  the  second  such  source  ever  built  in  1971.   Today 
such  sources  are  in  widespread  use  since  they  provide  superior  results, 
especially  in  such  areas  as  drug  and  metabolite  analysis.   Field  desorption, 
providing  mass  spectra  of  involatile  materials,  is  another  case — this 
year  our  shop  has  finally  completed  construction  of  a  source  and  emitter 
conditioning  apparatus. 

These  modern  spectral  techniques  provide  a  vast  amount  of  data  on  a 
compound,  and  it  must  be  processed  and  compared  to  even  more  vast 
literature  to  identify  these  compounds  with  minimum  time  and  effort.   For 
this  reason,  we  have  been  even  more  heavily  involved  with  the  Division 
for  Computer  Research  and  Technology  this  year  via  the  Chemical  Information 
System  (CIS).   This  sytem  provides,  or  will  shortly  provide:  1)  access  to 
the  CBAC  files  of  the  ACS  via  structure,  etc,,  2)  the  X-ray  crystallographic 
literature  via  the  Cambridge  Data  file,  3)    C  and  H  nmr  data,  both  to  solve 
coupling  constant/chemical  shift  problems  and  to  access  the  literature, 
and  4)  a  30,000  compound  file  of  mass  spectral  data.   Display  and  manip- 
ulation of  structures  is  also  provided.   It  is  our  hope  to  find  ways  to 
make  this  extensive  service,  which  we  have  found  so  useful  in  our  own  work, 
available  to  the  general  public  through  DCRT  facilities.   A  preliminary 
attempt  to  provide  such  a  service  with  the  mass  spectra  search  portion  of 
CIS,  operating  through  the  Aldermaston  (England)  Data  Center,  using  a  GE 
timeshare  network  has  not  lived  up  to  its  capabilities  due  to  organiza- 
tional difficulties  but  it  has  provided  us  with  valuable  experience  in 
dealing  with  such  large  timeshare  networks. 


2<S7 


In  the  area  of  nuclear  magnetic  resonance  ye  have  programs  underway 
involving  relaxation  time  measurements  on   C  nuclei  of  normal 
abundance  and  in  general  we  are  acquiring  experience  running  compounds 
in  this  beautifully  straightforward  mode.   Similar  studies  on  the   P 
nucleus  as  a  nuclear  probe  are  providing  insights  concerning  the 
nature  of  the  protein- lipid  interaction  in  phospholipids. 

The  use  of  proton  nmr  has  allowed  us  to  demonstrate  that  the  lymphatic 
node  dye  known  as  "alphazurine  2G"  varies  in  composition  according  to 
its  source.   This  may  explain  reactions  experienced  by  some  individuals 
during  lymphography. 

We  have  gained  enough  experience  in  liquid  chromatography  this  year 
to  begin  to  be  able  to  define  its  utility  in  our  group  which,  in  the 
past,  has  been  heavily  involved  in  gas  chromatography.   It  is  clear 
that  the  two  techniques  are  comparable  and  complementary  in  many  ways.  ■ 
We  have  repeatedly  collected  fractions  at  the  sub-microgram  level, 
evaporated  solvents  and  obtained  satisfactory  mass  spectra  on  the 
products.   This  technique  should  become  even  more  valuable  when  coupled 
to  field  desorption  mass  spectrometry  and  we  will  spend  more  effort 
on  the  latter  technique  next  year.   Clearly,  an  improved  detector 
would  be  very  desirable  in  liquid  chromatography  to  remove  the  need  for 
ultraviolet-absorbing  groups.   We  intend  to  attempt  to  use  an  electro- 
chemical detector  developed  last  year  for  catecholamine  assays  for  this 
purpose,  but  other  ideas  are  also  being  considered.   The  catecholamine 
assays  have  been  temporarily  shelved  until  the  return  of  the  staff 
member  who  developed  them  (E.  Whitnack) . 

Gas  chromatography-mass  spectrometry  continues  to  be  as  important  as 
ever.  With  it  we  have  been  able  to  identify  a  microscopic  amount  of 
triamcinolone  in  the  human  eye,  identify  metabolites  of  alkoxymethyl 
derivatives  of  barbiturates  and  dilantin,  metabolites  of  retinoic 
acid,  etc.,  and  in  addition,  our  instrument  continues  to  do  the  major 
portion  of  the  overdose  analysis  in  the  Washington  metropolitan  area 
(N.  Law,  Suburban  Hospital) . 

In  connection  with  last  year's  comments,  the  GT-40  interactive  oscil- 
loscope has  this  year  been  functioning  well,  as  has  the  automated 
microfiche  reader.   In  retrospect,  however,  there  seems  little  need 
for  computer  operation  of  this  latter  device. 

Our  computer  costs  continue  to  be  high,  both  due  to  storage  of  30,000 
mass  spectra  on  disk  and  because  of  the  extensive  use  of  the  IBM-360 
by  the  X-ray  facility.   The  former  should  be  helped  considerably  by 
FDA's  offer  to  support  us  at  $5, 000/month.   Clearly,  they  find  the 
system  useful  in  their  work.   Very  extensive  support  in  these 
endeavors  also  comes  from  EPA  via  Dr.  S.  Heller.   Regarding  the  X-ray 
usage  of  the  IBM-360,  minicomputers  appear  to  be  on  the  verge  of  taking 
over  the  bulk  of  this  work  and  when  the  cost  curves  intersect,  we  will 
consider  acquisition  of  the  necessary  hardware. 


The  Finnigan  GC-MS-Computer  system  bought  for  us  by  DCRT  has  been 
transferred  to  Dr.  B.  Brewer  (NHLI)  who  uses  it  exclusively  for  analysis 
of  PTH  derivatives  by  chemical  ionization. 

Work  on  insect  pheromones  has  been  more  limited  this  year  but  synthesis 
of  a  new  class  of  fire  ant  venoms  has  been  completed  and  several  such 
venoms  have  been  tested  for  antigenic  activity  (H.  Baer,  Bur.  Biol. 
Stand,  FDA) .   They  are  inactive,  however,  suggesting  that  yet  another 
component  of  the  venom  is  the  culprit  in  this  sting  which  is  becoming 
increasingly  identified  as  an  important  public  health  problem  in  Southern 
regions  of  the  United  States.   We  shall  attempt  to  separate  and  identify 
the  true  antigen  this  year. 

In  the  past  year,  members  of  our  Laboratory,  often  in  collaboration  with 
others,  have: 

1.  Determined  the  sterol  compositions  of  polyene  resistant  mutants  of 
Aspergillus  fennelliae  and  Cryptococcus  neoformans  (Kim  and  Kwon-Chung, 
NIAID) . 

2.  Determined  the  structure  of  a  pheromone  of  a  caligo  as  z-3-farnesene 
using  GC-MS  (M.  Blum,  U.  Georgia) . 

3.  Used  chemical  ionization  mass  spectrometry  to  identify  dipeptides 
released  by  the  action  of  cathepsin  C  on  large  peptides  (B.  Halpern, 
Wollongong  U. ,  Australia) . 

4.  Compared  electron  ionization,  chemical  ionization,  field  ionization, 
and  field  desorption  in  a  series  of  biologically  important  molecules 
(J.  Damico,  FDA;  H.  Beckey,  U.  Bonn) . 

5.  Further  developed  the  abilities  and  data  base  of  the  Chemical 
Information  Service  in  terms  of  mass  spectra,  etc.   Initiated  a 
worldwide  effort  to  collect   C  spectra  from  workers  in  this  field 
and  developed  a  search  routine  to  access  them.   Over  2,000  have 
been  collected  to  date  (S.  R.  Heller,  EPA;  R.  Feldman,  DCRT) . 

6.  Using   P  nmr,  showed  that  alteration  of  the  charge  on  the  protein 
portion  of  a  lipoprotein  does  not  disturb  its  basic  vesicular 
structure  (B.  Brewer,  NHLI) . 

7.  Carried  out  T  -temperature  studies  on  the   P  in  HDL  and  recombined 
HDL.   Differences  were  observed,  casting  doubt  on  the  wisdom  of 
using  recombined  particles  in  studies  on  HDL.   Paramagnetic  shifts 
of  Pr (NO  )   tend  to  prove  that  both  are  still  mycellular. 


M? 


8.  Using  H  nmr,  proved  that  commercial  preparations  of  the  lymphatic 
node  dye,  alphazurine  2G,  are  variable  in  nature,  perhaps  explaining 
reactions  to  the  dye  (L.  M.  Kleinman  and  P.  K.  Hiranaka,  Phar.  Dev. 
Service,  NIH) . 

13 

9.  Using   C  nmr,  elucidated  the  structure  of  a  mycotoxin  from 

Stachysbotrysa  alba  (E.  Mazzola  and  R.  Eppey,  FDA) . 

10.  Determined  the  structure  of  the  glyoxal-acetqnedicarboxylic  acid 
condensation  product  as  exo  vs.  endo  using  C  nmr  (U.  Weiss  and 
K.  Rice,  NIAMDD) . 

13 

11.  Established  the  structure  of  the  alkaloid  casselsine  by   C 

studies  on  its  dehydrogenation  product. 

12.  Developed  direct  methods-programs  for  X-ray  analysis. 

13.  Determined  the  structure  of  a  pyrolysis  rearrangement  product  of  a 
tetracyclic  diketone  (T.  Lee,  Walter  Johnson  High  School,  Heart 
Association  Fellow) . 

14.  Using  very  small  crystals,  solved  the  structure  of  a  benzopyrene 
derivative. 

15.  Elucidated  the  structure  of  gardmultine,  a  dimeric  indole  alkaloid 
of  m.wt.  1100,  the  largest  molecule  yet  studied  with  direct  methods 
(T.  Akiyama,  U.  of  Tokyo) . 

16.  Written  programs  to  approach  the  intractable  problem  of  triclinic 
crystals  with  2  molecules  per  assymmetric  unit. 

17.  Begun  to  convert  the  complex  IBM- 360  X-ray  programs  to  interactive 
form  for  use  by  non-specialists. 

18.  Developed  a  method  for  the  identification  and  characterization  of 
urushiol  (poison  ivy)  standards  using  specific  ion  analysis  by 
GC-MS  (H.  Baer  and  M.  Gross,  Bur.  Biol.  Stand.  FDA) . 

19.  Elucidated  the  structures  of  a  series  of  metabolites  of  methoxy- 
methyl  and  butyloxymethyl  phenobarbital  and  dilantin  using  GC-MS 

(E.  Baumel,  EPA) . 

20.  Helped  to  elucidate  the  structure  of  a  glutathione  conjugate  of 
prostaglandin  E.  (L.  Cagen  and  J.  Pisano,  NHLI) . 

21.  Synthesized  a  series  of  new  pyrollidine-ring  containing  fire  ant 
venoms  (M.  Blum,  U.  Georgia) . 

22.  Worked  out  the  GC-MS  analysis  of  all  of  the  amino  acids  in  the 
saccharopine  cycle,  in  connection  with  a  metabolic  defect  known  as 
saccharopinuria  (J.  Dancis  and  J.  Hutzler,  NYU) 


asc 


23.  Worked  on  methods  for  identifying  histidine  containing  peptides 
isolated  from  amino  acid  analyzers  in  connection  with  the 
structure  of  "elongation  factor"   (E.  Maxwell  and  E.  Tudor,  NIAMDD) . 

24.  Completed  the  structure  and  absolute  configuration  of  both  the  alkaloid 
astrocasine  and  the  drug  viminol. 

25.  Identified  a  new  pentacyclic  triterpene,  isomultiflavenol,  in 
Benincasa  hispida. 

26.  Identified  new  sulfur-containing  iridoid  glycosides  in  a  series  of 
carcinogenic  plants. 

27.  Identified  with  GC-MS ,  8  components  of  camponotus  ants  as  a  series 
of  aliphatic  secondary  alcohols  and  phenylethanol  and  its  esters 
with  aliphatic  acids  (M.  S.  Blum,  U.  Georgia)  and  synthesized 
same. 

28.  Determined  the  conformation  of  products  from  the  condensation  of 
acetylacetone  and  benzylacetophenone  (F.  H.  Greenberg,  NYU,  Buffalo) . 

29.  Isolated  with  liquid  chromatography  and  identified  with  MS  iodo- 
derivatives  of  hydroxy lpindolol  and  hydroxybenzylpropanolol.   Enough 
radioactive  material  was  collected  by  LC  for  further  studies, 

(E.  M.  Brown  and  G.  Aurbach,  NIAMDD) . 


asv 


Project  No.  Z01  HL  01001-02  LC 
1.   Laboratory  of  Chemistry 
2. 
3.   Bethesda,  Maryland  20014 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Electrochemical  Methods  of  Analysis  and  Synthesis 

Previous  Serial  Number:   NHLI-56 

Principal  Investigator:   H.  M.  Fales 

Other  Investigators:   R.  Nicholson,  National  Science  Foundation 

E.  Whitnack,  Univ.  of  Cincinnati,  Dept.  of  Medicine 

Project  Description: 

Work  has  temporarily  been  discontinued  on  the  electrochemical  catecholamine 
assay  due  to  Dr.  Whitnack1 s  departure.   Upon  her  return,  we  will  continue 
to  develop  and  apply  this  technique.   We  had  reached  the  point  of  being 
able  to  assay  epinephrine  and  norepinephrine  in  plasma  and  to  note  the 
differences  in  their  concentrations  during  stress. 

A  specially  designed  cyclic  voltammetry  sweep  generator  has  been  completed 
by  our  shop  and  with  it  we  shall  investigate  the  properties  of  a  series 
of  urushiol  (poison  ivy) -related  compounds,  as  well  as  urushiol  itself, 
to  determine  whether  differences  in  antigenicity  can  be  related  to 
structure  (H.  Baer,  Bur.  Biol.  Std. ;  FDA).   The  possibility  that  o-quinones 
react  with  sulfhydryl  groups  on  proteins  to  form  haptens  has  already  led 
to  the  demonstration  that  the  antigenic  nature  of  urushiol  on  guinea 
pigs  can  be  entirely  eliminated  by  coapplication  of  mercaptoethanol. 

In  the  near  future,  we  intend  to  study  such  oxidations  in  non-aqueous 
systems  as  models  of  drug  metabolism. 

Keyword  Descriptors:   cyclic  voltammetry,  urushiol,  poison  ivy,  drug 
metabolism 

Honors  and  Awards:   none 
Publications:   none 


ass 


Project  No.  Z01  HL  01002-03  LC 
1.   Laboratory  of  Chemistry 
2. 
3.   Bethesda,  Maryland  20014 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Nuclear  Magnetic  Resonance  of  Natural  Products 

Previous  Serial  Number:   NHLI-57 

Principal  Investigator:   E.  A.  Sokoloski 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Nuclear  Magnetic  Resonance  spectroscopy  offers  the  researcher  a  powerful 
tool  for  structural  investigations.   Application  of  this  technique  to 
various  classes  of  natural  products  has  been  the  major  endeavor  of  this 
investigator. 

Applications  and  Resulcs 

A  majority  of  time  has  been  devoted  to  investigation  of  phospholipids 
by  phosphorus-31  Fourier  Transform  NMR  spectroscopy.   By  using  the 
naturally-occurring  phosphorus  as  a  nuclear  probe  within  the  lipid  system, 
we  (collaborating  with  Dr.  Brian  Brewer-NHLI)  have  studied  the  spin 
lattice  relaxation  time  as  a  function  of  temperature  and  pH  in  several 
model  lipid  systems  and  several  lipoprotein  systems. 

pH  versus  T   studies  showed  little  or  no  alteration  in  relaxation  time 
between  pH  6.8-11.0,  suggesting  that  alteration  of  the  charge  on  the 
protein  portion  of  the  lipoprotein  molecule  does  not  disturb  the  basic 
vescicular  structure  of  the  lipoprotein.   This  could  be  interpreted  as 
confirmation  that  the  protein- lipid  interaction  is  principally  hydro- 
phobic in  nature. 

T   versus  temperature  studies  of  natural  high  density  lipoprotein  (HDL) 
and  recombined  HDL  showed  differences  in  the  nature  of  the  relaxation 
time  response  to  temperature  changes.   The  native  system  gave  increasing 
relaxation  time  with  increasing  temperature  while  the  recombined 
system  showed  the  opposite  response  in  one  case  and  a  mixed  response 
in  a  second  case.   No  final  conclusion  has  been  drawn  as  yet,  but,  since 
T   is  a  measure  of  molecular  motion  which  mirrors  molecular  organization, 
one  is  tempted  to  conclude  that  the  particle  is  altered  by  the 
recombination  process.   This  leaves  one  wondering  if  data  obtained  by 
many  individuals  on  recombined  particles  can  safely  be  extrapolated  to 
native  particles. 


Project  No.  z01  HL  01002-03  LC 

Titration  of  lipid  models  and  native  and  recombined  HDL  also  have  been  in 
progress.   The  praesodymium  ion  of  Pr (NO  )   is  paramagnetic  and  causes  a 
shift  when  complexed  to  a  molecule.   When  added  to  sphingomyelin,  a 
separation  of  the  signal  from  inner  and  outer  phosphorus  of  the  bilayer 
is  observed.   Lyso  lecithin,  native  HDL  and  recombined  HDL  do  not  show 
the  separation,  this  being  the  expected  behavior  for  a  micelle. 
Qualitatively  then,  these  seem  to  have  the  same  molecular  organization. 
However,  we  have  noted  a  quantitative  difference  in  the  interaction  of  the 
last  three  species.   Continuing  experiments  are  needed  to  confirm  this 
observation  and  delineate  its  cause. 

A  collaborative  investigation  with  Dr.  H.  Fales,  NHLI,  and  L.  M.  Kleinman 
and  P.  K.  Hiranaka  of  the  Pharmaceutical  Development  Service,  NIH  on  the 
lymphatic  node  dye  known  as  alphazurine  2G  was  concluded  during  the 
past  year.   Proton  NMR  of  several  different  samples  of  the  dye  showed 
structural  differences  in  the  materials — all  labeled  as  alphazurine  2G. 
Reports  had  been  made  that  several  patients  injected  with  the  dye  for 
the  purpose  of  obtaining  lymphograms  had  experienced  reactions  to  the 
material.   The  differences  in  structures  observed  could  account  for  the 
reactions.   A  future  publication  will  contain  particulars  of  the  investi- 
gation. 

A  structural  study  of  material  extracted  from  Melochia  tomentosa  plant 
used  by  natives  of  Curacao  to  relieve  throat  irratation  and  shown  to  be 
tumorogenic  was  completed.   The  study  undertaken  in  collaboration  with 
Drs.  Fales  and  Silverton  of  NHLI  and  Dr.  G.  Kapadia  of  Howard  University 
gave  structure  I  for  the  extracted  material. 


Structure  I 


3.S~£- 


Project  No.  ZQ1  HL  01002-03  LC 

Keyword  Descriptors:  Melochia  tomentosa,  lipoproteins,  phosphorus  nmr, 
relaxation  time,  lymphatic  node  dyes 

Publications: 

1.  Chaiken,  I.  M. ,  Cohen,  J.  S.  and  Sokoloski,  E.  A.   The  Micro- 
environment  of  histidine-12  in  ribonuclease-S  as  detected  by  C-13. 
J.  Amer.  Chem.  Soc.  96:  4703-4705,  1974. 

2.  Furie,  B.  ,  Griffin,  J.  H. ,  Feldmann,  R. ,  Sokoloski,  E.  A.  and 
Schechter,  A.  N.   The  active  site  of  staphylocaccal  nuclease: 
Paramagnetic  relaxation  of  bound  inhibitor  nuclei  by  lanthanide 
ions.   Proc.  Nat.  Acad.  Sci.  (USA)  71:  2833-2837,  1974. 

3.  Ziffer,  H.,  Seeman,  J.  I.,  Highet,  R.  J.  and  Sokoloski,  E.  A. 
Carbon-13  nuclear  magnetic  resonance  characteristics  of  3-methyl- 
cyclohexane-l,2,diols.   J.  Org.  Chem.  39:  3698-3701,  1974. 

4.  Assmann,  G. ,  Highet,  R.  J.,  Sokoloski,  E.  A.  and  Brewer,  H.  B. 

C-13  Nuclear  magnetic  resonance  spectroscopy  of  native  and  recombined 
lipoproteins.   Proc.  Nat.  Acad.  Sci.  (USA)  71:  3701-3705,  1974. 


££t 


Project  No.  Z01  HL  01003-04  LC 
1.   Laboratory  of  Chemistry 
2. 
3.   Bethesda,  Maryland  20014 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Structure  of  Natural  Products  Using  Instrumental  Methods 

Previous  Serial  Number:   NHLI-58 

Principal  Investigator:   H.  M.  Fales 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Two  interesting  pyrollidine  variants  of  the  major  component  of  fire 
ant  venom  have  been  synthesized  and  tested,  along  with  the  ordinary 
piperidine  analogs  for  antigenic  activity  (H.  Baer,  Bur.  Biol.  Std) . 
Neither  form  has  any  activity  and  it  is  clear  that  another  component 
of  the  venom  must  be  responsible  for  its  properties.   More  natural  venom 
is  being  accumulated  for  isolation  of  the  active  component  using  guinea 
pig  assays. 

In  collaboration  with  Drs.  L.  Cagen  and  J.  Pisano,  the  structure  of  a 
glutathione  adduct  of  prostaglandin  E  has  been  elucidated.   Key  points 
in  the  structure  proof  were  the  appearance  of  a  metastable  ion  in  its 
mass  spectrum  showing  loss  of  H  S  (mass  34)  and  isotope  ratios  pointing 
to  the  presence  of  sulfur.   Metastable  defocussing  should  be  especially 
helpful  in  such  experiments  and  next  year  an  effort  will  be  made  to 
automate  their  detection. 

A  series  of  dyes  used  in  lymphangiography  has  been  examined  by  H  nmr 
and  several  structures  corrected  or  authenticated.   The  variability  noted 
in  the  preparations  of  alphazurine  2G  may  be  responsible  for  the  side 
effects  noted. 

Poison  ivy  (urushiol)  is  responsible  for  a  severe  antigenic  reaction  in 
mammals,  and  in  humans  small  differences  in  the  side  chain  of  the  penta- 
decylcatechol  unit  cause  very  different  responses.   We  have  found  that 
specific  ion  analysis  of  its  trimethylsilyl  ethers  provides  a  sensitive 
and  accurate  method  for  assay  of  the  various  "standard  urushiol"  mixtures 
(H.  Baer,  FDA) 


arr 


Project  No.   z01  HL  01003-04  LC 

GC-MS  has  provided  the  structures  of  six  new  metabolites  of  a  series 
of  butoxymethylene  and  methoxymethylene  barbiturates  and  dilantins. 
Interestingly,  mass  spectra  of  the  two  possible  N-substituted  dilantins 
were  wholly  dissimilar  as  were  their  G.C.  retention  times. 

The  Suburban  Hospital  quadrupole  mass  spectrometer  has  been  modified 
by  us  in  an  attempt  to  improve  its  resolution  and  reliability.   To  date, 
its  shortcomings  in  these  regards  have  forced  the  Suburban  group  to 
continue  to  use  our  LKB  spectrometer  for  emergency  drug  identification. 

Keyword  Descriptors:   prostaglandins,  overdoses,  barbituates,  fire  ant, 
lymphagiography,  alphazurine  2G,  urushiol,  tri- 
methylsilyl  ethers 

Honors  and  Awards:   Chromatographer  of  the  Year  -  Washington  Chromatography 

Group 

Publications: 

1.  Tsai,  S.,  Fales,  H.  M. ,  and  Vaughan,  M.   Inactivation  of 
hormone- sensitive  lipase  from  adipose  tissue  with  adenosine 
triphosphate,  magnesium  and  ascorbic  acid.   J.  Biol.  Chem.  248: 
5278-5281,  1973. 

2.  Longevialle,  P.,  Milne,  G.  W.  A.  and  Fales,  H.  M.   Chemical 
ionization  mass  spectrometry  of  complex  molecules.  XI.   Stereo- 
chemical and  conformational  effects  in  the  isobutane  chemical 
ionization  mass  spectra  of  some  steroidal  amino  alcohols. 

J.  Amer.  Chem.  Soc.  95,  6666,  1973. 

3.  Heller,  S.  R. ,  Pratt,  A.  W. ,  Feldmann,  R.  J.,  Fales,  H.  M.  and 
Milne,  G.  W.  A.   A  conversational  mass  spectral  search  system 

IV.   The  evolution  of  a  system  ofr  the  retrieval  of  mass  spectral 
information.   J.  Chem.  Doc.  13:  130-133,  1973. 

4.  Brand,  J.  M. ,  Fales,  H.  M. ,  Sokoloski,  E.  A.,  MacConnell,  J.  G. , 
Blum,  M.  S.  and  Duffield,  R.  M.   Identification  of  mellein  in 
the  mandibular  gland  secretions  of  carpenter  ants.   Life  Sci. 
13:  201-211,  1973. 


ase 


Project  No.ZOl  HL  01004-04  LC 
1.   Laboratory  of  Chemistry 
2. 
3.   Bethesda,  Maryland  20014 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Isolation  and  Characterization  of  Natural  Products 

Previous  Serial  Number:   NHLI-59 

Principal  Investigator:   H.  A.  Lloyd 

Other  Investigators:   none 

Cooperating  Units: 

Project  Description: 

1.  Carcinogenic  plant  components  -  (with  G.J.  Kapadia,  Howard  University) 
Materials  isolated  from  a  number  of  plants  (ex  Acacia  villosa,  Krameri 
ixina,  Paederia  foetida,  Diospyros  virginiana)  were  examined  -  The  structures 
of  the  new  compounds  were  studied  by  mass  spectrometry  -  Novel  sulfur 
containing  iridoid  glycosides  were  found. 

2 .  Pentacyclic  triterpenes  of  Benincasa  hispida.   The  main  component 
was  the  alcohol,  isomultiflorenol,  not  previously  found  in  nature. 

3.  X-ray  structure  determination  (with  J.  V.  Silverton) 

The  structures  and  absolute  configurations  of  the  alkaloid  astrocasine  and 
of  the  drug  Viminol  were  completed. 

4.  The  NMR  and  mass  spectra  of  Michael  reaction  products  of  acetylacetone 
and  benzylacetophenone  were  studied  to  determine  the  conformation  of  the 
products  (with  F.  H.  Greenberg,  NYU,  Buffalo) . 

5.  Insect  pheromones  (in  collaboration  with  M.  S.  Blum,  University  of 
Georgia) 

The  compositions  of  glandular  extracts  of  a  number  of  insects  were 
determined  by  combined  gas  chroma tography-mass  spectrometry.   The 
suspected  unknown  components  were  synthesized  for  comparison. 

a)   Camponotus  ants  -  Among  12  species  studied,  Camponotus  clarithorax 
appeared  especially  atypical  as  to  the  variety  of  compounds  (2,6-dimethyl- 
5-hepten-l-ol,  2-phenylethanol,  citronellic,  geranic,  n-octanoic  and 
n-nonanoic  acids  and  their  esters)  not  encountered  previously  in  this 
genus. 


as? 


Project  No. Z01  HL  01004-04  LC 

b)  Termites  -  new  species  were  examined,  mainly  for  their  monoterpenes 
composition. 

c)  Millipedes  -  a  new  alkaloid  was  isolated  from  one  specie. 

d)  Myrmecocystus  ants  -  new  terpene  alcohols  were  characterized. 

e)  Myrmecia  ants  -  Several  species  of  these  primitive  Australian  ants 
were  studied  (for  C.  P.  Haskins,  Carnegie  Institution). 

6.   High  Pressure  Liquid  Chromatography  of  Natural  Products. 

A  considerable  amount  of  time  was  devoted  to  the  development  of 
preparative  HP  liquid  chromatography  techniques  for  the  separation  of 
biological  materials,  drugs,  natural  products  (terpenes,  alkaloids, 
steroids,  flavones) .   For  example,  metabolites  of  monobutoxymethyl 
dilantine  and  of  monomethoxymethylphenyl  barbiturate  were  collected 
and  identified. 

Radioactive  iododerivatives  of  hydroxypindolol  and  hydroxybenzylproponolol 
were  also  separated  and  collected  (for  E.  M.  Brown  NIAMDD) .   The 
technique  is  especially  suitable  for  air  or  heat  sensitive  materials  such 
as  the  Iridoid  glycosides  of  Paederia  foetida  or  the  carcinogenic  poly- 
phenols of  Acacia,  Krameria  and  Diospyros. 

Keyword  Descriptors:   high  pressure  liquid  chromatography,  insect 
pheromones,  mono  and  triterpenes 

Publications: 

Brand,  J.  M. ,  Blum,  M.  S. ,  Lloyd,  H.  A.  and  Fletcher,  D.  J.  C. 
Monoterpene  hydrocarbons  in  the  poison  gland  secretion  of  the 
ant  Myrmicaria  nataleusis.   Ann  Ent.  Soc.  Am.  67:  525-526  (1974). 

Lloyd,  H.  A.,  Blum,  M.  S.,  and  Duffield,  R.  M.   Chemistry  of  the 
male  mandibular  gland  secretion  of  the  ant  camponotus  clarithorax. 
Ins.  Biochem.  in  press. 

Silverton,  J.  V. ,  and  Lloyd,  H.  A.   The  crystal  and  molecular  structure 
of  the  non-morphinoid  narcotic  analgesic,  Viminol.   Acta  Cryst. 
in  press. 


.3*0 


Project  No.  201  HL  01005-04  LC 
1.   Laboratory  of  Chemistry 
2. 
3.   Bethesda,  Maryland  20014 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   X-ray  structural  R&D  for  physiologically  important 
molecules. 

Previous  Serial  Number:   NHLI-60 

Principal  Investigator:   J.  V.  Silverton 

Other  Investigators:   T.  Lee,  C.  Kabuto,  T.  Akiyama 

Cooperating  Units:   None 

Project  Description: 

PURPOSE : 

Investigations  by  X-ray  crystallography  of  molecules  of  interest  chemically 
or  physiologically  with  emphasis  on  problems  where  other  methods  are 
inconclusive.   Development  of  methods  for  extending  and  simplifying 
the  techniques  of  direct  methods. 

SUMMARY: 


Both  centrosymmetric  and  acentric  crystals  have  been  studied  by  direct 
methods  and  further  development  and  study  of  new  methods  have  been 
carried  out. 

18   2  10   3  7 

a)  A  pyrolysis  rearrangement  of  tetracyclo  (4,3,0  *  ,0  '   , 0    ) tri- 

decane-5-12-dione  (with  T.  Lee) . 

Mr.  Lee,  who  had  just  graduated  from  high  school  and  was  a  recipient 
of  a  Heart  Association  fellowship,  worked  with  the  principal 
investigator  on  this  project.   The  structure  was  solved  by  our  own 
direct  methods  programs. 

b)  The  structure  of  benzo-6, 7, 7a,8-tetrahydrobenzo [a]  pyrene. 

This  structure  was  solved  to  resolve  a  disagreement  as  to  stereochemistry 
which  could  not  be  settled  by  other  means.   Although  the  crystals  were 
very  much  smaller  than  the  optimum  and  the  X-ray  data  are  consequently 
not  as  accurate  as  usual,  the  structure  was  readily  solved  and  is  being 
refined. 


&>( 


r,   •   ^  „    Z01  HL  01005-04 
Project  No. LC 

c)  The  structure  of  gardmultine  (with  T.  Akiyama) 

This  compound,  an  unsymmetrical  dimeric  alkaloid  with  a  molecular 
weight  of  ca.  1100,  represents  the  largest  acentric  structure  we  have 
studied  and  it  is  also  one  of  the  largest  molecules  ever  attacked  by  direct 
methods.   Currently  we  have  not  solved  the  structure  but  are  carrying  out 
necessary  calculations. 

d)  The  structure  of  chaetoglobosin,  a  cytotoxic  metabolite  of  Chaetomium 
globosum  (with  T.  Akiyama) 

This  is  a  substituted  alternant  11-membered  ring  compound  of  only  partially 
known  structure  and  a  molecular  weight  of  536.   X-ray  data  has  been 
collected  and  structure  solution  is  about  to  start. 

e)  The  structure  of  imerubrine  (with  C.  Kabuto) . 

This  structure  represents  a  particularly  intractable  problem  being  a 
triclinic  crystal  with  two  parallel  but  independent  molecules  in  the 
asymmetric  unit.   Since  the  basic  assumption  of  direct  methods,  that  the 
atomic  positions  may  be  regarded  as  numerically  random,  is  far  from  true, 
the  observed  failure  of  all  previously  published  direct  methods  approaches 
is  not  unexpected.   We  are  now  using  the  structure  to  develop  and  test 
a  distinctly  new  approach  in  direct  methods--quartet  invariants 
(H.  Hauptman,  A.C.A.  Meeting,  Charlottesville,  Va.  1975).   We  have  written 
programs  to  implement  the  new  method  and  currently  we  are  working  on  the 
solution.   Since  quartet  invariant  methods  may  represent  the  greatest 
recent  advance  in  direct  methods,  we  are  hopeful  that  development  will 
radically  simplify  solution  of  structural  problems. 

f)  Job  control  language  writing  programs  (with  T.  Lee  and  G.  W.  A.  Milne) . 

A  start  has  been  made  on  eliminating  one  of  the  worst  barriers  to  the  use 
of  computer  methods  by  non-specialists;  the  control  language  for  IBM 
computers.   Results  are  promising  although  currently  incomplete. 

Keyword  Descriptors:   X-ray,  crystal,  structure,  organic 

Publications: 

Silverton,  J.  V.,  Milne,  G.  W.  A.,  Eaton,  E.  E.,  Nyi,  K. ,  Temme ,  G. 
M.   Structures  of  the  [n.2.2]  Propellanes  I.   2-hydroxy [4 . 2 . 2] 
propellane  p-nitrobenzoate.   J.  Am.  Chem.  Soc .  96:  7429-7432,  1974. 

Silverton,  J.  V.  and  Lloyd,  H.  A.   The  structure  of  viminol. 
Acta  Cryst.  B31,  in  press  1975. 


MZ 


Project  No.201  HL  01005-04 
J        LC 

Akiyama,  T.  ai?d  Silverton,  J.  V.   The  structure  of  endo-tetracyclo 
[5.5.1.0  '  .0   '   ]  tridecane  trione.   Acta  Cryst.  B31  in  press  1975. 


*43 


Project  No.ZQl  HL  01006-05  LC 
1.   Laboratory  of  Chemistry 
2. 
3.   Bethesda,  Maryland  20014 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   The  characterization  of  natural  matters 

Previous  Serial  Number:   NHLI-61 

Principal  Investigator:   R.  J.  Highet ,  Ph.D. 

Other  Investigators:   none 

Cooperating  Units: 

Project  Description: 

In  collaboration  with  Dr.  Eugene  Mazzola  and  Mr.  Robert  Eppley  of  the 
Food  and  Drug  Administration,  the  structure  of  a  mycotoxin  of 
Stachysbotrys   alba  has  been  shown  to  be  1_   by  C-13  and  proton  nmr 
studies.   The  sesquiterpene  and  unsaturated  acid  moieties  were  readily 
established  by  comparison  of  the  spectra  with  those  of  known  materials, 
but  the  novel  pyran  systems  could  only  be  demonstrated  by  double 
resonance  experiments. 


A  detailed  study  of  the  C-13  characteristics  of  potamogetonin  and 
related  materials  has  established  the  structure  as  2  and  permitted 
the  assignment  of  each  observed  resonance. 


M{ 


Project  No.ZOl  HL  01006-05  LC 

In  a  collaborative  study  with  Dr.  J.  V.  Silverton  of  this  laboratory 
and  Drs.  U.  Weiss  and  K.  Rice  of  NIAMDD,  the  products  of  the 
condensation  of  acetone  dicarboxylic  acid  and  glyoxal  have  been 
studied.   Among  the  structures  examined,  that  of  the  exo  ixomer  of  3 
was  established  by  comparison  of  the  C-13  spectrum  with  that  of  the 
known  endo  isomer. 


>=0 


CH  -'      \N^  -.  (ch  )  coch 
H  2  n     3 


The  structure  of  casselsine,  4,  n  =  12,  has  been  established  by  comparison 
of  the  C-13  spectrum  of  its  dehydrogenation  product  with  that  of  the 
corresponding  derivative  of  cassine  (4,  n=  10) .   Previously  no  facile 
method  has  been  available  to  distinguish  4  from  the  structural  isomer 
with  the  methyl  and  alkyl  groups  interchanged. 

Keyword  Descriptors:   C-13  NMR;  mycotoxin;  diterpenes,  natural  products 

Publications: 

Assmann,  G. ,  Fredrickson,  D.  S.,  Sloan,  H.  R. ,  Fales,  H.  M.  and 
Highet,  R.  J.   Accumulation  of  Oxygenated  Steryl  Esters  in  Wolman's 
Disease.   J.  Lipid  Res.  16:  28-38,  1975. 

Ziffer,  H. ,  Seeman,  J.  I.,  Highet,  R.  J.  and  Sokoloski,  E.  A. 
Carbon-13  Nuclear  Magnetic  Resonance  Characteristics  of  3-Methyl- 
cyclohexane-l,2-diols.  J.  Org.  Chem.  39:  3698,  1974. 

Highet,  R.  J.  and  Sokoloski,  E.  A.   Structural  Investigations  of 
Natural  Products  by  Newer  Methods  of  NMR  Spectroscopy.   Fortschr . 
Chem.  Organ.  Naturstoffe,  32,  120-166,  1975. 

Highet,  R.  J.  and  Edwards.  J.  M.   Analysis  of  the  Carbon-13 
NMR  Spectrum  of  Phenalenone.   J.  Mag.  Res. ,  in  press 


<^r 


Project  No.201  HL  01007-04  LC 
1.   Laboratory  of  Chemistry 
2. 
3.   Bethesda,  Maryland  20014 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Application  of  Mass  Spectrometry  to  Problems  in 
Biochemistry 

Previous  Serial  Number:   NHLI-62 

Principal  Investigator:   G.  W.  A.  Milne 

Other  Investigators:   none 

Cooperating  Units:   none 

Project  Description: 

Chemical  ionization  mass  spectrometry  is  now  firmly  established  as  an 
analytical  technique  with  considerable  utility  in  the  area  of  analytical 
biochemistry  and  work  in  this  laboratory  on  the  development  of  the  method 
has  been  largely  supplanted  by  efforts  to  apply  this  technique  to  current 
problems.   At  the  same  time,  we  have  undertaken  the  development  of  field 
desorption  mass  spectrometry,  a  newer  technique  which  has  some  advantages 
over  both  electron  ionization  and  chemical  ionization  mass  spectrometry. 
Electron  ionization  mass  spectrometry  is  still  being  used  heavily  in  a 
multitude  of  problems. 

In  collaboration  with  Drs.  Kim  and  Kwon-Chung  of  NIAID,  the  sterol  com- 
positions of  polyene  resistant  mutants  of  Aspergillus  fennelliae  and 
Cryptococcus  neoformans  have  been  established.   In  each  case,  the  various 
sterols  were  identified  by  combined  gas  chromatography-electron  ionization 
mass  spectrometry. 

The  same  analytical  technique  has  been  used  with  a  number  of  biologically 
active  insect  secretions,  supplied  by  Dr.  M.  Blum  of  the  University  of 
Georgia.   In  this  way,  for  example,  a  pheromone  from  the  scent  glands  of 
butterflies  of  the  species  caligo  has  been  identified  as  z-S-farnesene. 

In  collaboration  with  Dr.  B.  Halpern  of  Wollongong  University,  Australia, 
chemical  ionization  mass  spectrometry  has  been  used  as  a  means  of  identify- 
ing the  dipeptides  released  successively  from  large  peptide  chains  by  the 
action  of  cathepsin  C. 


344 


Project  No.ZOl  HL  01007-0-!  LC 

Keyword  Descriptors:   computers,  data  bases,  mass  spectral  data,  CMR  data, 
X-ray  diffraction  data,  computer  networks 

Publications : 

1.  Heller,  S.  R.  ,  Koniver,  D.  A.,  Fales,  H.  M.  and  Milne,  G-.  W.  A. 
Conversational  Mass  Spectra  Search  System.   Anal.  Chem. ,  46:   947-950, 
1974. 

2.  Heller,  S.  R. ,  Feldmann,  R.  J.,  Fales,  H.  M.  and  Milne,  G.  W.  A.   A 
conversational  mass  spectral  search  system.  IV.   The  evolution  of  a 
system  for  the  retrieval  of  mass  spectra  information.   J.  Chem.  Doc. 
13:   130-133,  1973. 

3.  Heller,  S.  R. ,  Fales,  H.  M. ,  Milne,  G.  W.  A.,  Feldmann,  R.  J., 
Daly,  N.  R. ,  Maxwell,  D.  C.  and  McCormick,  A.   An  experimental 
international  conversational  mass  spectral  search  system.   Adv.  in 
Mass  Spec.  6:  1037-1042,  1974. 


3£7 


Project  No.  z01  KL  01008-04  LC 
1.   Laboratory  of  Chemistry 
2. 
3.   Bethesda,  Maryland  20014 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Use  of  Digital  Computing  in  Problems  in  Biochemistry 

Previous  Serial  Number:   NHLI-63 

Principal  Investigator:   G.  W.  A.  Milne 

Other  Investigators:   none 

Cooperating  Units:   none 

Project  Description: 

Work  has  continued  in  collaboration  with  EPA  upon  the  Mass  Spectra  Search 
System  (MSSS) .   In  addition  to  this,  a  considerable  effort  has  been  invested 
in  other  aspects  of  the  NIH  Chemical  Information  System  (CIS) . 

The  MSSS  is  currently  operating  on  the  NIH  PDP-10,  which  is  used  by 
workers  in  the  Federal  Establishment  and  also  upon  the  G.E.  International 
Computer  Network,  which  is  used  by  non-Federal  and  foreign  research  groups. 
Currently,  there  are  some  140  users  of  the  system.   The  data  base  now 
contains  28,000  mass  spectra  and  should  increase  to  include  50,000  in 
the  next  calendar  year.   All  aspects  of  the  system,  even  data  collection, 
are  operating  relatively  smoothly  at  present  and  it  is  hoped  that  this  state 
of  affairs  will  continue  to  improve  so  as  to  include  the  problems  of 
expanding  the  data  base  at  regular  intervals. 

A  data  base  consisting  of  over  2,000  carbon-13  magnetic  resonance  spectra 
has  been  assembled  and  the  programs  necessary  to  search  this  data  base 
are  working.   The  number  of  spectra  in  this  data  base  should  soon  reach 
about  3,000  which  is  well  over  50%  of  all  the  spectra  published  and 
the  value  of  this  component  of  the  Chemical  Information  System  is 
already  becoming  clear.   The  X-ray  crystallography  sections  of  the  CIS 
have  been  largely  developed  in  DCRT  and  have  functioned  well  for  over 
a  year. 

An  important  link  in  the  whole  CIS  is  the  sub-structure  searching  program. 
This  will  be  used  to  facilitate  the  establishing  of  structure- literature 
and  structure-experimental  data  relationships.   This  software  was 
commenced  at  NIH  but  during  the  past  year,  the  task  of  completing  it  was 
transferred  to  an  outside  contractor  and  should  be  finished  during  the 
coming  year. 


266 


Project  No.ZOl  HL  01008-04  LC 

The  mechanism  of  hydrogen  rearrangement  in  esters  upon  electron,  chemical 
and  field  ionization  is  under  study  with  K.  Levsen  of  the  University  of 
Bonn.   Preliminary  results  suggest  that  rearrangement  takes  place  in  each 
case  with  a  unique  mechanism.   The  utility  of  various  methods  of 
ionization  in  the  mass  spectrometry  of  molecules  of  biological  importance 
has  been  studied  in  collaboration  with  colleagues  at  the  FDA  and  the 
University  of  Bonn.   This  study  confirmed  previous  suspicions  that  the 
various  methods  tend  to  complement  one  another  and  that  no  single  technique 
is  universally  superior  to  the  others. 

The  use  of  carbon-13  as  a  label  in  biosynthetic  studies  is  being  explored. 
The  label  can  be  detected  with  precision  by  mass  spectrometry  and  nuclear 
magnetic  resonance  spectroscopy  and  the  possibilities  of  this  approach  are 
very  promising. 

Keyword  Descriptors:   computers,  data  bases,  mass  spectral  data,  CMR  data 
X-ray  diffraction  data,  computer  networks 

Publications : 

1.  Kim,  S.  J.,  Kwon-Chung,  K.  J.,  Milne,  G.  W.  A.  and  Prescott:   Resistant 
Mutants  of  Aspergillus  fennelliae:   Identification  of  Sterols.   Anti- 
microb.  Ag  and  Chemother  6,  405-410,  1974. 

2.  Kim,  S.  J.,  Kwon-Chung,  K.  J.,  Milne,  G.  W.  A.,  Hill,  W.  B.  and 
Patterson:  G?   Relationship  between  polyene  resistance  and  sterol 
compositions  in  Cryptococcus  neoformans.   Antimicrob-  Ag  and  Chemother. 
7,  99-106,  1975. 

3.  Schier,  G.  M. ,  Halperr ,  B.  and  Milne.  G.  W.  A.   Characterization  of 
Dipeptides  by  electron  impact  and  chemical  ionization  mass  spectrometry. 
Biomed  Mass  Spec  1,  212-218,  1974. 

4.  Fales,  H.  M. ,  Milne,  G.  W.  A.,  Winkler,  H.  U.,  Beckey,  H.  D. ,  Damico, 
J.  N.  and  Barron,  R.   Comparison  of  mass  spectra  of  some  biologically 
important  compounds  as  obtained  by  various  ionization  techniques. 
Anal.  Chem.  47,  207-219,  1975. 


<36  f 


ANNUAL  REPORT  OF  THE 
LABORATORY  OF  KIDNEY  AND  ELECTROLYTE  METABOLISM 
NATIONAL  HEART  AND  LUNG  INSTITUTE 
July  1,  1974  through  June  30,  1975 

The  Laboratory  of  Kidney  and  Electrolyte  Metabolism  has  made 
a  number  of  significant  advances  in  the  elucidation  of  the  mech- 
anism and  control  of  electrolyte  transport  in  kidney,  toad 
bladder,  avian  erythrocyte,  and  heart  muscle.   Only  the  first 
three  will  be  summarized  in  the  annual  report.   That  concerning 
excitation-contraction  coupling  in  heart  muscle  is  discussed  in 
detail  in  an  appended  individual  summary. 

Isolated  segments  of  renal  tubules 

The  technique  of  perfusing  isolated  segments  of  renal  tubules 
in  vitro,  developed  in  this  laboratory,  has  proved  valuable  as  a 
means  of  studying  kidney  function.   As  detailed  in  previous  re- 
ports, direct  study  of  various  previously  inaccessible  nephron 
segments  has  revealed  a  surprising  diversity  of  function  among 
the  different  parts  of  the  tubule  (of  which  there  are  eight  in 
mammalia)  and  has  uncovered  a  number  of  unexpected  processes. 
Important  examples  include:  1)  an  active  chloride  transport  sys- 
tem discovered  in  the  thick  ascending  limb  of  Henle ' s  loop,  2) 
the  action  of  diuretics,  which  were  found  to  inhibit  chloride 
transport  in  this  segment,  3)  characterization  of  the  interaction 
of  vasopressin  and  the  cyclic  AMP  system  in  the  cortical  collect- 
ing tubule,  4)  analysis  of  the  active  sodium  and  potassium  trans- 
port system  in  the  cortical  collecting  tubule  and  of  the  organic 
acid  and  glucose  transporting  systems  in  proximal  tubules,  and 
5)  studies  of  the  complex  mechanism  governing  fluid  absorption  in 
the  proximal  tubule.   The  latter  continues  to  be  emphasized  in 
our  present  work. 

Transport  in  the  proximal  tubule  accounts  for  over  half  the 
reabsorption  of  glomerular  filtrate.   Despite  extensive  study  in 
vivo  using  micropuncture  techniques  there  is  little  agreement  on 
the  mechanism  of  fluid  transport.   The  isolated  preparation  pro- 
vides for  more  stringent  control  of  the  experimental  conditions 
than  does  micropuncture  and  for  this  reason  is  an  especially  use- 
ful approach  to  this  difficult  and  important  problem.   In  our 
initial  studies  of  the  isolated  perfused  rabbit  proximal  convolu- 
ted tubule,  serum  ultraf iltrate  was  used  as  perfusate  with  rabbit 
serum  in  the  bath.   Subsequently,  artificial  solutions  were  de- 
veloped which  supported  fluid  absorption  as  well  as  did  serum  and 
ultraf iltrate,  and  have  the  advantage  that  their  composition  can 
be  more  easily  manipulated.   Using  these  solutions,  we  found  that 
certain  organic  solutes  are  important  for  fluid  absorption.   Glu- 
cose and  alanine  enhance  fluid  absorption  when  added  to  the  per- 
fusate and  cause  the  voltage  to  increase,  but  they  have  no  effect 
when  added  to  the  bath.   In  our  present  studies  the  mechanism  of 
this  effect  was  investigated.   We  find  that  a-methyl-D-glucoside 


57/ 


(a  sugar)  and  cycloleucine  (an  amino  acid)  when  added  to  the  per- 
fusate, also  cause  the  rate  of  fluid  absorption  to  increase.   The 
latter  compounds  are  known  to  be  transported  by  kidney  cells,  but 
not  metabolized.   Therefore,  it  is  transport,  not  metabolism  of 
sugars  and  amino  acids  that  is  responsible  for  their  enhancement 
of  fluid  absorption.   When  glucose  and/or  alanine  are  added  to 
the  perfusate,  the  tubule  cells  swell.   Most  likely,  the  non- 
electrolytes  enter  the  cells  during  their  transport  which  increas- 
es the  intracellular  osmotic  pressure,  and  causes  water  to  enter 
the  cells.   By  analogy  to  the  small  intestine,  which  has  been 
studied  more  extensively,  the  effect  of  glucose  and  alanine  on 
fluid  absorption  probably  is  mediated  by  enhanced  sodium  trans- 
port.  It  is  believed  that  the  non-electrolytes  are  co-transport- 
ed with  sodium  into  the  epithelial  cells  across  the  lumen  surface. 
Entry  of  sodium  into  the  cells  is  believed  to  be  the  first  step 
in  its  transport  and,  though  passive,  to  be  rate  limiting.   There- 
fore, additional  sodium  entry  into  the  cells,  co-transported  with 
the  non-electrolytes,  causes  an  increase  in  the  rate  of  sodium 
transport  which  is  in  turn  coupled  to  fluid  transport.   In  pre- 
vious studies,  we  found  that  there  is  a  large  passive  back  leak 
of  glucose  into  the  tubule  lumen.   A  numerical  analysis  indicates 
that  there  is  a  significant  glucose  cycle  composed  of  glucose 
which  diffuses  into  the  tubule  lumen  and  is  pumped  out  again. 
Since  transport  of  non-electrolytes  contributes  to  fluid  absorp- 
tion from  proximal  convoluted  tubules,  this  process  is  important 
in  understanding  the  function  of  the  tubule. 

Active  transport  of  sodium  is  generally  believed  to  be  the 
driving  force  for  reabsorption  of  fluid,  as  just  discussed,  but 
the  evidence  has  been  inconclusive  and  numerous  alternative  theor- 
ies have  been  proposed.   The  importance  of  sodium  transport  was 
tested  by  removing  sodium  completely  from  the  perfusate  and  bath 
(replacement  with  choline,  tetramethyl  ammonium,  or  lithium). 
Fluid  absorption  and  voltage  fell  to  zero,  confirming  the  essen- 
tial role  of  sodium.   Removal  of  potassium  from  the  bath  has  the 
same  effect.   Potassium  is  necessary  for  active  sodium  transport, 
as  previously  demonstrated  in  other  tissues.   Taken  together, 
these  results  strengthen  the  conclusion  that  active  sodium  trans- 
port drives  fluid  absorption  in  proximal  tubules.   Complete  re- 
moval of  chloride  from  the  perfusate  and  bath  (replacement  with 
nitrate  or  perchlorate)  has  virtually  no  effect,  consistent  with 
a  passive  role  for  chloride. 

When  bicarbonate  is  removed  from  the  perfusate  and  bath,  the 
rate  of  fluid  absorpticn  decreases  by  approximately  one-third. 
Similar  results  were  noted  previously  in  micropuncture  studies  in 
rat  kidneys.   We  tested  the  theory  that  as  bicarbonate  is  reabsorb- 
ed from  the  tubule  fluid  and  its  concentration  in  the  lumen  falls, 
the  bicarbonate  concentration  gradient  itself  drives  fluid  absorp- 
tion.  The  effect  is  ascribed  to  an  osmotic  action  of  the  rela- 
tively impermeant  bicarbonate  anions.   We  are  unable  to  confirm 
this  theory  in  isolated  proximal  tubules,  however,  since  imposed 


57a 


gradients  of  bicarbonate  and  methyl  sulfate  (another  relatively 
impermeant  anion)  do  not  affect  fluid  absorption.   Carbonic 
anhydrase  is  known  to  be  important  for  bicarbonate  reabsorption 
from  proximal  tubules.   Therefore,  we  tested  the  effect  of  aceta- 
zolamide  which  is  an  inhibitor  of  carbonic  anhydrase  and  is  a 
mild  diuretic.   Acetazolamide  (10~  M)  causes  the  rate  of  fluid 
absorption  to  decrease  approximately  as  much  as  does  removal  of 
bicarbonate,  suggesting  that  the  effects  are  related.   None  of 
these  experiments  provides  an  explanation  for  the  effect  of  bi- 
carbonate, however,  which  remains  to  be  determined. 

Additional  studies  are  underway  to  elucidate  the  mechanism 
of  bicarbonate  reabsorption  and  acidification  in  proximal  tubules, 
Bicarbonate  transport  has  been  characterized  in  proximal  straight 
tubules,  using  a  new  micromethod  we  developed  for  measuring  total 
CO2 •   We  find  that  there  is  an  active  transport  process  which  re- 
absorbs bicarbonate  from  the  tubule   lumen,  despite  an  opposing 
back-leak  of  bicarbonate  into  the  lumen.   Straight  proximal 
tubules  from  superficial  and  juxtamedullary  nephrons  were  compar- 
ed, and  found  to  have  essentially  the  same  active  transport  rates, 
However,  the  tubules  from  the  juxtamedullary  nephrons  are  less 
permeable  to  bicarbonate  than  those  from  superficial  tubules  so 
that  in  the  steady  state  the  concentration  of  bicarbonate  in  the 
lumen  is  higher  in  the  latter.   The  processes  involved  will  be 
studied  further  in  these  and  other  nephron  segments  using  this 
method  as  well  as  a  microelectrode  which  measures  pH. 

Avian  Erythrocytes 

Studies  performed  in  this  laboratory  with  avian  erythrocytes 
have  added  to  our  understanding  of  the  mechanisms  underlying  the 
maintenance  of  normal  cell  volume  in  animal  cells.   It  is  gener- 
ally accepted  that  animal  cells  behave  like  osmometers  in  that 
their  volume  is  determined  by  the  amount  of  osmotically  active 
solute  that  they  contain,  especially  the  salts  of  sodium  and 
potassium.   Previously,  it  was  believed  that  the  intracellular 
sodium  and  potassium  contents  (and  thus  volume)  were  regulated  by 
active  transport  via  the  classical  ouabain-sensitive  Na  and  K 
pump.   Studies  in  this  laboratory  indicate  that  additional  pro- 
cesses are  important. 

When  the  volume  of  duck  erythrocytes  is  altered  by  changing 
the  osmolality  of  the  suspending  medium,  they  spontaneously  re- 
turn to  their  original  volume.   In  the  case  of  swollen  cells  (i.e, 
those  suspended  in  hypotonic  solutions) ,  shrinking  back  to  the 
original  size  is  accomplished  by  a  large  increase  in  potassium 
permeability  allowing  potassium  salts  to  leak  out  of  the  cell, 
followed  by  water.   Shrunken  cells  (i.e.  those  suspended  in  hyper- 
tonic media)  swell  back  to  their  original  volume,  but  the  mechan- 
ism involved  is  not  simply  explained.   Swelling  back  to  original 
volume  is  due  to  net  uptake  of  potassium  salts,  but  not  via  the 
classical  sodium  and  potassium  pump.   Ouabain  does  not  prevent 
salt  uptake  and  swelling.   The  swelling  is  also  accompanied  by  a 

■>  £73 


large  increase  in  potassium  permeability,  but  this  alone  cannot 
explain  the  result,  since  it  would  cause  potassium  salts  to  leak 
out  of,  not  into  the  cells. 

The  transport  processes  associated  with  cell  swelling,  with 
cell  shrinking  and  the  classical  ouabain-sensitive  cation  pump 
have  been  characterized  further.   Cells  incubated  with  1  mM  furo- 
semide  or  cells  in  which  intracellular  chloride  has  been  replaced 
with  sulfate  display  normal  sodium  and  potassium  transport 
through  the  ouabain  sensitive  cation  pump.   These  cells  also 
shrink  as  do  normal  cells  when  swollen  in  hypotonic  media.   In 
contrast,  furosemide  treated  cells  and  sulfate  cells  show  no  re- 
sponse to  shrinkage  in  hypertonic  media.   The  step  at  which  the 
response  is  blocked  remains  to  be  determined.   In  other  studies 
a  technique  was  developed  for  preparing  "ghosts"  of  the  nucleated 
avian  erythrocytes.   These  cells  are  normal  in  size,  can  maintain 
a  20-fold  concentration  gradient  for  potassium  compared  to  the 
extracellular  solution,  and  have  a  permeability  to  potassium  simi- 
lar to  that  of  normal  cells.   Despite  these  properties,  they  fail 
to  change  permeability  in  response  to  hypotonicity  or  hypertoni- 
city . 

Investigation  of  the  mechanism  of  recovery  of  shrunken  cells 
has  been  facilitated  by  our  previous  observation  that  the  appar- 
ently identical  process  can  be  induced  by  norepinephrine.   It  was 
found  that  compared  to  their  normal  volume  in  plasma,  duck  ery- 
throcytes shrank  when  placed  in  isoosmotic  salt  solutions.   Low 
concentrations  of  norepinephrine  in  the  medium  caused  the  shrunk- 
en cells  to  swell  back  to  their  normal  volume.   Dibutyryl  cyclic 
AMP  had  the  same  effect,  suggesting  that  the  hormone  operates 
through  cyclic  AMP.   The  role  of  cyclic  AMP  was  confirmed  by  stud- 
ies showing  that  norepinephrine  elevates  the  concentration  of 
cyclic  AMP  in  duck  red  cells,  as  it  elevates  the  permeability  of 
the  cells  to  potassium  and  the  cells  swell.   In  contrast,  there 
is  no  change  in  cell  cyclic  AMP  concentration  associated  with 
swelling  in  hypertonic  solution.   It  seems  likely  chat  hypertoni- 
city  initiates  its  effect  at  a  step  after  that  at  which  cyclic 
AMP  is  generated. 

Previously  studies  of  ion  transport  in  red  cells  have  been 
limited  for  lack  of  techniques  to  measure  directly  the  intracellu- 
lar voltage,  membrane  resistance,  and  permeability  to  ions  (espe- 
cially anions,  which  exchange  rapidly) .   We  have  now  developed  a 
technique  which  permits  direct  measurement  of  these  parameters 
in  a  single  amphibian  red  blood  cell.    The  method  involves  immo- 
bilizing the  cell  within  a  narrow  constriction  of  a  glass  pipet. 
The  immobilized  cell  is  readily  penetrated  with  microelectrodes 
to  measure  voltage  and  electrical  resistance.   The  cell  is  sealed 
well  enough  in  the  constriction  so  that  ions  pass  through  the 
cell  rather  than  around  it.   Thus  it  is  possible  to  measure  per- 
meability to  isotopes  and  electrical  resistance  across  the  whole 
cell.   In  this  experiment  the  isotopes  or  electric  current  pass 


A7f 


through  portions  of  two  membranes  of  the  cell  in  series  (one 
entering  and  one  leaving  the  cell) .   The  results  with  the  two 
methods  (puncture  and  transcellular  measurements)  are  in  good 
agreement.   The  major  findings  in  Amphiuma  red  cells  are:  1)  the 
mean  transmembrane  voltage  is  -17  mV  and  varies  directly  with  pH 
of  the  media,  2)  the  mean  specific  electrical  resistance  of  the 

2  3  6 

cell  membrane  is  lOOftcm  ,  3)  there  is  a  high  permeability  to   CI, 
but  this  is  in  large  part  electrically  silent  and  can  be  ascribed 
to  exchange  diffusion.   This  technique  represents  a  major  advance 
in  methodology  which  will  be  widely  used  to  examine  the  proper- 
ties of  red  cells  and  probably  many  other  types  of  cells,  as  well. 

Toad  Urinary  Bladder 

This  section  has  continued  to  study  the  mechanisms  by  which 
hormones  affect  salt  and  water  excretion.   Previous  work  from 
this  laboratory  has  established  the  thesis  that  vasopressin  acts 
on  responsive  epithelial  membranes  by  increasing  the  production 
and  accumulation  within  the  cell  of  cyclic  AMP.   Cyclic  AMP,  the 
"second  messenger"  of  Sutherland  and  Rail,  in  turn  elicits  the 
effects  of  the  hormone.   The  toad  urinary  bladder,  which  is  ana- 
lagous  in  many  respects  to  the  distal  portion  of  the  mammalian 
nephron,  survives  well  in  vitro.   It  has  been  used  extensively 
in  early  work  regarding  the  role  of  cyclic  AMP,  and  in  studies  of 
factors  that  modify  the  response  to  vasopressin.   For  example,  it 
has  been  shown  that  chlorpropamide,  a  sulfonylurea  derivative 
that  has  considerable  efficacy  in  the  treatment  of  diabetes 
insipidus  of  pituitary  origin,  has  an  effect  on  the  toad  urinary 
bladder  that  is  analagous  to  its  clinical  effect.   Other  studies 
have  shown  the  interaction  of  adrenal  steroid  hormones,  prosta- 
glandins, adrenergic  agents,  and  metabolic  factors  upon  the  re- 
sponse to  vasopressin.   Recent  efforts  have  been  directed  toward 
elucidating  the  mechanism  of  action  of  cyclic  AMP  in  the  epithel- 
ial cells  of  toad  bladder  and  kidney,  and  toward  gaining  an  under- 
standing of  the  function  of  the  different  types  of  epithelial 
cells  in  the  epithelial  membrane. 

The  effect  of  cyclic  AMP  in  many  tissues  is  thought  to  in- 
volve phosphoprotein  metabolism.   A  protein  kinase  that  is  stimu- 
lated by  cyclic  AMP  is  widely  distributed  and  has  been  shown  to  be 
present  in  toad  bladder  and  mammalian  kidney.   Workers  in  another 
laboratory  have  reported  that  vasopressin  (and  cyclic  AMP)  stimu- 
late a  phosphoprotein  phosphatase  in  toad  bladder.   We  have  been 
unable  to  confirm  the  report  of  stimulation  of  phosphoprotein 
phosphatase  activity  in  the  intact  bladder,  but  have  confirmed 
the  presence  of  cyclic  AMP  stimulated  protein  kinase  and  phospho- 
protein phosphatase  activity  in  homogenates  of  toad  bladder  epith- 
elial cells.   The  principle  among  many  phosphoproteins  affected 
by  cyclic  AMP  has  a  molecular  weight  of  50,000  daltons  in  sodium 
dodecyl  sulfate.   Work  has  begun  on  the  partial  purification  of 
the  50,000  dalton  phosphoprotein.   It  appears  to  be  a  cytosolic 
protein.   Our  objective  is  to  characterize  the  protein  substrate 

5  J7ST 


so  that  its  function  regarding  the  action  of  vasopressin  will  be- 
come evident.   In  addition,  the  purified  substrate  will  enable  us 
to  study  further  the  cyclic  AMP  stimulated  protein  kinase  and 
phosphoprotein  phosphatase  and  elucidate  the  role  of  these 
enzymes  in  response  to  vasopressin. 

In  other  studies  of  the  effect  of  cyclic  AMP  on  phosphopro- 
tein metabolism,  we  have  used  a  suspension  of  separated  renal 
cortical  tubules,  a  technique  developed  in  this  laboratory  sever- 
al years  ago.   The  bulk  of  cortical  tubules  are  proximal  tubules, 
one  of  the  major  sites  of  action  of  parathyroid  hormone.   It  is 
established  that  parathyorid  hormone  acts  by  stimulating  adeny- 
late cyclase,  and  many  of  the  renal  effects  of  the  hormone  can  be 
elicited  by  dibutyryl  cyclic  AMP.   We  have  found  that  parathyroid 
hormone  increases  the  incorporation  of  tracer  phosphate  into  cer- 
tain proteins  of  renal  cortical  tubules.   The  major  effect  on 
phosphorylation  involves  a  protein  with  a  molecular  weight  of  a- 
bout  50,000  daltons.   Similar  results  occur  in  homogenates  of 
renal3cortex  that  are  stimulated  with  cyclic  AMP  in  the  presence 
of  y-   P-ATP.   This  is  the  first  demonstration  of  an  effect  of 
parathyroid  hormone  on  phosphoprotein  metabolism.   No  evidence 
has  been  found  that  would  indicate  an  effect  of  cyclic  AMP  on 
phosphoprotein  phosphatase  activity  in  renal  cortex.   Ultimately 
we  hope  to  identify  the  major  phosphoproteins  affected  by  para- 
thyroid hormone  and  define  their  role  in  the  renal  response  to 
the  hormone . 

The  epithelial  cells  of  the  toad  bladder  are  morphologically 
heterogeneous.   About  70  percent  are  "granular  cells,"  20  percent 
"mitochondria  rich  cells."   If  the  cells  are  also  functionally 
heterogeneous,  as  is  likely,  it  is  obviously  important  to  know 
which  cells  are  involved  in  the  response  to  vasopressin.   Two 
laboratories  have  interpreted  electron  micrographs  of  toad  bladd- 
ers as  indicating  that  only  the  granular  cells  manifest  increased 
water  permeability  in  response  to  vasopressin.   Another  labora- 
tory has  removed  epithelial  cells  from  the  toad  bladder  and  has 
succeeded  in  separating  the  two  major  types  of  cells  by  density 
gradient  centrifugation.   They  found  that  only  the  mitochondria 
rich  cells  respond  to  neurohypophysial  hormones  with  an  increase 
in  cyclic  AMP  content.   In  view  of  the  reports  that  only  granular 
cells  manifest  increased  water  permeability  in  response  to  hor- 
mone, it  has  been  suggested  that  cyclic  AMP  or  another  signal 
from  the  mitochondria  rich  cells  stimulates  water  permeability  in 
the  granular  cells.   We  have  confirmed  the  separation  of  cell 
types  by  density  gradient  centrifugation.   In  our  experience,  the 
resulting  cells  are  not  suitable  for  study  as  intact  cells.  There- 
fore, we  studied  the  activity  in  each  type  of  cell  of  enzymes 
known  to  be  involved  in  cyclic  AMP  metabolism  in  response  to  vaso- 
pressin.  Granular  cells  are  as  rich  in  vasopressin  sensitive 
adenylate  cyclase  activity  and  cyclic  nucleotide  phosphodiester- 
ase activity  as  mitochondria  rich  cells.   We  have  concluded  that 
both  cell  types  respond  to  vasopressin  with  increased  cyclic  AMP 
production. 

6  Z7(, 


Project  No.  Z01  HL  01201-01  KE 

1.  Kidney  &  Electrolyte 

2.  Membrane  Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Study  of  the  effect  of  cholera  toxin  on  toad 
urinary  bladder 

Previous  Serial  Number:   None 

Principal  Investigators:   Joseph  S.  Handler,  M.  D. 

Agens  S.  Preston 

Other  Investigators :   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   It  is  generally  accepted  that  the  toxin  of 
Vibrio  cholera  (choleragen)  acts  on  intestinal  epithelial  cells 
and  on  other  tissues  by  stimulating  adenylate  cyclase  activity. 
The  resulting  elevation  of  intracellular  cyclic  AMP  levels  is 
responsible  for  the  typical  response  to  the  toxin,  secretion  of 
electrolyte  and  water  in  the  small  intestines,  and  in  other 
tissues,  responses  resembling  those  elicited  by  specific  hormones 
which  stimulate  adenyl  cyclase.   Recent  reports  indicate  that 
choleragen  binds  to  a  specific  glycolipid,  the  ganglioside  GMi 
which  is  the  binding  site  or  receptor  for  choleragen  in  cell 
membranes.   Once  choleragen  is  bound  to  cell  membranes,  a  tempera- 
ture and  time  dependent  reaction  appears  to  be  required  for 
adenylate  cyclase  activation.   It  is  the  purpose  of  this  study  to 
examine  the  effect  of  choleragen  on  the  toad  urinary  bladder. 
Normally,  in  vivo,  vasopressin  activates  adenylate  cyclase  in  the 
basal-lateral  (blood  surface)  plasma  membrane  of  the  epithelial 
cells  of  the  toad  bladder.   The  resultant  elevation  of  the  intra- 
cellular concentration  of  cyclic  AMP  causes  an  increased  rate  of 
active  sodium  transport  by  the  bladder  and  an  increase  in  the 
permeability  of  the  bladder  to  water.   The  objectives  of  the  study 
are  to  gain  information  about  the  role  of  the  epithelial  cell 
plasma  membrane  in  the  activation  of  adenylate  cyclase,  and  about 
the  mechanism  of  action  of  the  toxin. 

Methods:   After  18  hours  of  incubation  with  choleragen  added 
to  the  solution  bathing  the  mucosal  (urinary)  or  to   the  solution 
bathing  the  serosal  (blood)  surface  of  the  experimental  bladder, 
the  permeability  to  water  and  the  rate  of  sodium  transport  by  the 


art 


Project  No.  201  HL  01201-01  KE 

experimental  and  by  the  paired  control  tissue  are  measured  using 
standard  techniques.   Vasopressin,  cyclic  AMP,  or  theophylline, 
the  latter  an  inhibitor  of  the  enzyme  that  destroys  cyclic  AMP, 
is  added,  and  the  sodium  transport  rate  and  water  permeability 
response  measured.   GMi  or  other  agents  and  conditions  are  im- 
posed upon  the  choleragen  treated  tissue  to  assess  their  effect. 
Finally,  adenylate  cyclase  is  assayed  in  tissue  that  has  respond- 
ed to  choleragen  and  in  tissue  in  which  the  response  to  cholera- 
gen has  been  modified  in  a  significant  fashion. 

Major  Findings:   Incubation  with  choleragen  increases  the  re- 
sponse of  the  toad  bladder  to  vasopressin  and  to  theophylline, 
but  does  not  increase  the  response  to  cyclic  AMP.   Therefore,  it 
is  likely  that  choleragen  acts  by  increasing  adenylate  cyclase 
activity  in  the  toad  bladder,  as  in  other  tissues.   Choleragen- 
is  more  active  when  added  to  the  solution  bathing  the  mucosal  sur- 
face than  when  added  to  the  solution  bathing  the  serosal  surface. 

_i  1 
5X10    M  toxin  is  effective  when  added  to  the  mucosal  solution, 

_9 

but  5X10   M  in  the  serosal  solution  is  required  for  an  effect. 
No  effect  of  choleragen  is  detectable  during  the  first  30  min. 
that  it  is  present.   By  that  time,  however,  it  is  bound  to  the 
tissue  so  that  removing  it  from  the  bathing  solution  does  not 
effect  its  subsequent  action.   GMi  added  to  the  same  solution 
with  toxin  blocks  its  effect  since  it  avidly  binds  the  toxin  in 
the  solutions.   In  contrast,  the  addition  of  GMi  to  the  serosal 
solution  enhances  the  response  of  the  bladder  to  choleragen  add- 
ed to  the  mucosal  solution.   A  possible  interpretation  of  this 
observation  is  that  GMi  added  to  the  serosal  solution  is  taken  up 
by  and  incorporated  into  the  basolateral  plasma  membrane.  The  GMi 
then  migrates  within  the  membrane  to  the  mucosal  surface  where 
it  serves  as  receptor  to  bind  additional  choleragen  from  the 
mucosal  solution.   If  this  interpretation  is  correct,  the  mem- 
branes must  be  fluid  and  there  must  be  movement  of  molecules 
within  the  plasma  membrane  from  the  basolateral  (serosal)  and  to 
the  apical  (mucosal) surf ace ,  despite  the  generally  different  pro- 
perties of  these  membranes.   The  fact  that  choleragen  added  to 
the  solution  bathing  the  mucosal  surface  activates  adenylate 
cyclase,  an  enzyme  in  the  basolateral  membrane,  may  also  indicate 
movement  of  material  in  the  membranes  from  one  surface  to  the 
other . 

Proposed  course  of  project:   The  ability  of  GMi  added  to  the  solu- 
tion bathing  one  surface,  to  enhance  the  response  to  choleragen 
added  to  the  solution  bathing  the  other  surface  will  be  examined 
further,  using  other  combinations  of  agents,  and  applying  addi- 
tional controls.   If  it  is  confirmed  that  GMi  and/or  choleragen 
migrate  within  the  plasma  membrane,  the  factors  required  for  and 
affecting  the  movement  will  be  examined. 


£70 


Project  No.  Z01  HL  01201-01  KE 
Keyword  Descriptors:   cholera  toxin,  ganglioside  ,  GMi 

Honors  and  Awards :   None 
Publications : 


P7? 


Project  No.  Z01  HL  01202-02  KE 

1.  Kidney  &  Electrolyte 

2.  Membrane  Metabolism 

3.  Bethesda,  Maryland 

PHS-NHI 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Control  of  protein  phosphorylation  in  toad 
urinary  bladder 

Previous  Serial  Number:   NHLI-65 

Principal  Investigators:   Gordon  J.  Strewler,  M.D. 

Dennis  A.  Ausiello,  M.D. 
Joseph  S.  Handler,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Previous  studies  in  this  laboratory  have  estab- 
lished that  the  actions  of  the  hormone  vasopressin  on  sodium 
transport  and  on  water  permeability  in  toad  urinary  bladder  and 
in  the  renal  collecting  tubule  of  the  rabbit  are  mediated  by 
increases  in  the  cellular  levels  of  cyclic  AMP.   The  effects  of 
cyclic  AMP  in  many  tissues  are  thought  to  be  the  result  of  pro- 
tein phosphorylation  catalysed  by  cyclic  AMP-dependent  protein 
kinase.   The  state  of  phosphorylation  of  proteins  is  determined 
by  the  activity  of  protein  kinases  and  phosphoprotein  phos- 
phatases . 

In  the  initial  phases  of  this  study,  we  showed  cyclic  AMP 
dependent  phosphorylation  of  several  proteins  in  homogenates  of 
toad  bladder  epithelial  cells.   The  protein  affected  most  by  the 
cyclic  nucleotide  had  a  molecular  weight  of  ^50,000  daltons  on 
SDS  gels.   A  small  effect  of  cyclic  AMP  on  the  rate  of  dephos- 
phorylation  of  this  protein  was  also  evident,  partially  confirm- 
ing results  from  another  laboratory.   We  were  unable  to  show  any 
consistent  effect  of  vasopressin  on  protein  phosphorylation  in 
intact  cells. 

The  objectives  of  the  study  are: 

1)  To  determine  the  distribution  in  subcellular  fractions 
of  toad  bladder  epithelial  cells  of  cyclic  AMP-dependent  protein 
kinase  activity,  phosphoprotein  phosphatase  activity,  and  speci- 
fic protein  substrates  for  these  enzymes. 

2)  To  define  the  effect  of  cyclic  AMP  on  protein  dephos- 
phorylation  in  this  system. 

l  2$o 


Project  No.  Z01  HL  01202-02  KE 

3)   To  determine  the  role  of  changes  in  protein  phosphory- 
lation in  the  cyclic  AMP-mediated  effects  of  vasopressin  in  this 
tissue. 

Methods:   Phosphorvlation  of  proteins  in  intact  cells  has  been 

3  3 

accomplished  by  incubating  the  cells  with  inorganic    P.   The 
bladders  are  then  exposed  to  hormone  or  mediators  (dibutyryl 
cyclic  AMP,  theophylline).   Phosphoproteins  are  senarated  using 
polyacrylamide  gel  electrophoresis  in  sodium  dodecyl  sulfate 
(SDS) .   Protein  phosphorylation  in  homogenates  and  subcellular 

3  2 

fractions  occurs  when  the  preparation  is  incubated  with   P-ATP  in 
the  presence  of  divalant  cation. 

Subcellular  fractionation  for  these  studies  has  employed 
differential  dentrifugation  and  isopycnic  sucrose  density  grad- 
ient centrifugation.   Purity  of  fractions  has  been  assessed  by 
the  assay  of  marker  enzymes:  5'  nucleotidase,  adenylate  cyclase, 
cytochrome  oxidase,  esterase,  and  glucose-6-phosphate  dehydro- 
genase . 

Phosphoproteins  in  the  supernatant  (cytosol)  fraction  have 
been  separated  using  gel  filtration,  ammonium  sulfate  precipita- 
tion, and  ion  exchange  chromotography . 

Major  Findings:   A  fractionation  method  has  been  developed 
which  results  in  significant  enrichment  of  plasma  membrane,  mito- 
chondrial and  cytosol  marker  enzymes  in  different  fractions.   No 
satisfactory  marker  for  endoplasmic  reticulum  has  been  found. 
Autophosphorylation  stimulated  by  cyclic  AMP  (5X10-   M)  occurs 
mostly  in  the  cytosol  fraction.   The  principal  among  many  soluble 
phosphoproteins  affected  by  cyclic  AMP  is  one  of  molecular  weight 
50,000  daltons  (in  SDS).   There  is  some  autophosphorylation  of  a 
20,000  -  200,000Xg  pellet  probably  composed  of  internal  membranes, 
but  there  is  little  enhancement  by  cyclic  AMP.   No  significant 
autophosphorylation  of  plasma  membranes  or  mitochondrial  fractions 
has  been  demonstrated.   The  rate  of  dephosphorylation  in  the  cyto- 
sol fraction  is  slower  than  in  the  whole  homogenate,  but  it  appears 
to  be  enhanced  significantly  by  cyclic  AMP.   The  effects  of  cyclic 
AMP  on  dephosphorylation  in  other  fractions  has  not  been  examined. 
The  phosphoproteins  in  cytosol  have  been  separated  using  the 
techniques  mentioned  above.   The  presence  of  protein  phosphatase 
ant-ivity  in  the  system  has  made  it  impossible  to  be  certain  that 
the  label  is  not  lost  from  a  major  protein  species  during  separa- 
tion . 

It  has  not  been  possible  to  demonstrate  a  significant  effect 
of  vasopressin  on  protein  phosphorylation  in  the  whole  tissue, 
although  this  has  been  reported  by  others. 


33/ 


Project  No.  Z01  HL  02102-02  KE 
Proposed  Course: 

1)  Attempts  are  now  underway  to  partially  purify  the  soluble 
cyclic  AMP  dependent  protein  kinase  from  toad  bladder  epithelial 
cells.   Using  this  enzyme,  it  will  be  possible  to  find  potential 
protein  substrates  for  phosphorylation  in  subcellular  fractions 
that  do  not  contain  protein  kinase  activity. 

2)  Partial  purification  of  the  soluble  phosphoprotein  phos- 
phatase is  planned.   This  will  help  to  explain  the  mode  by  which 
cyclic  AMP  stimulates  dephosphoylation . 

3)  Studies  of  the  50,000  dalton  substrate  may  elucidate  its 
function.   Current  hypotheses  are  (a)  that  it  may  be  the  regula- 
tory subunit  of  a  protein  kinase;  if  so  it  should  display  cyclic 
AMP  binding  activity,  or  (b)  that  it  may  be  a  component  of  the 
microtubular  system,  tubulin.   It  should  then  bind  colchicine. 
Support  for  either  of  these  hypotheses  would  be  of  importance  in 
elucidating  the  path  through  which  cyclic  AMP  acts  as  a  "second 
messenger"  for  vasopressin. 

Keyword  Descriptors:   Toad  Bladder,  Vasopressin,  Cyclic  AMP, 

Protein  Kinase,  Phosphoprotein 
Phosphatase 

Honors  and  Awards :   None 

Publication?:   None 


£$-2. 


Project  No.  Z01  HL  01203-01  KE 

1 .  Kidney  &  Electrolyte 

2.  Membrane  Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Separation  by  morphologic  type  and  study  of 

responsiveness  to  vasopressin  of  toad  bladder 
epithelial  cells 

Previous  Serial  Number:   None 

Principal  Investigator:   Joseph  S.  Handler,  M.D. 

Agnes  S.  Preston 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Previous  work  in  this  laboratory  has  establish- 
ed the  thesis  that  vasopressin  acts  on  responsive  epithelial 
membranes  by  stimulating  the  enzyme  adenylate  cyclase,  resulting 
in  increased  production  and  intracellular  accumulation  of  cyclic- 
AMP .   The  toad  urinary  bladder  used  in  the  earlier  studies,  as 
well  as  other  anuran  and  mammalian  epithelial  membranes  that  re- 
spond to  vasopressin  are  morphologically  heterogeneous  (i.e.  - 
contain  more  than  one  cell  type) .   The  possibility  of  performing 
more  detailed  and  meaningful  study  of  cyclic  AMP  metabolism  in 
toad  bladder  cells  was  enhanced  by  a  recent  report  from  another 
laboratory  that  the  two  major  types  of  epithelial  cells  (granular 
rich-70  percent  of  total  cell  population,  and  mitochondria  rich  - 
20  percent  of  total  population)  could  be  separated  from  each  other 
by  density  gradient  centrifugation.   The  separation  is  establish- 
ed by  examination  of  cells  by  electron  microscopy  and  by  three- 
fold enrichment  of  carbonic  anhydrase  activity  in  one  band  of 
cells  on  the  gradient.   Mitochondria  rich  cells  have  been  found 
by  other  workers  to  be  rich  in  carbonic  anhydrase  activity.   It 
is  the  purpose  of  this  study  to  confirm  the  density  gradient 
separation  of  the  cells  and  to  examine  the  two  cell  types  for 
vasopressin  responsive  adenylate  cyclase  and  cyclic  nucleotide 
phosphodiesterase,  the  enzyme  that  inactivates  cyclic  AMP  by  con- 
verting it  to  5' -AMP. 

Methods:  The  cells  are  separated  by  the  method  of  Scott, 
Sapirstein  and  Yoder  (Science,  184:797,  1974).  The  epithelial 
cells  are  removed  from  the  bladder  by  incubating  the  tissue  in 
calcium  free  Ringer  solution  containing  2  mM  EDTA.   The  mixed 


Att 


Project  No.  ZQ1  HL  01203-01  KE 

cell  population  is  layered  on  top  of  a  discontinuous  gradient  of 
Ficoll  in  EDTA  Ringer.   Cells  are  spun  at  27,000  RPM  in  a  Spinco 
SW-27  for  45  min.  at  4°C  and  the  material  in  the  second  and  the 
third  bands  collected  for  further  study  after  dilution  in  EDTA 
Ringer  solution  and  centrif ugation  to  remove  the  Ficoll. 

The  cells  in  every  experiment  are  examined  by  phase  contrast 
microscopy  and  in  some  experiments  by  electron  microscopy.   Car- 
bonic anhydrase  activity  is  assayed  in  the  supernatant  solution 
of  sonicated  cells  using  an  aminco-Morrow  stop-flow  apparatus. 
The  remainder  of  the  cell  material  is  used  to  study  the  respon- 
siveness to  vasopressin  of  the  intact  cells  of  each  band  or  to 
study,  in  broken  cell  preprations,  the  activity  of  enzymes  in- 
volved in  cyclic  AMP  metabolism.   Aliquots  of  intact  cells  are 
incubated  in  regular  amphibian  Ringer  solution  with  or  without 
arginine  vasopressin.   After  5  and  10  minutes  an  aliquot  of  con- 
trol and  hormone  treated  cells  is  added  to  TCA  containing  tracer 

3 

H   -  cyclic  AMP  (for  extimation  of  recovery  -  70-80%)  and  the 
cyclic  AMP  in  the  extracts  separated  by  chromotography  and  assay- 
ed as  described  previously.   For  enzyme  assays,  aliquots  of  cells 
from  each  band  are  homogenized  in  a  tris-magnesium  buffer.   Basal 
and  vasopressin  sensitive  adenylate  cyclase  (whole  homogenate  or 
lOOOXg  pellet)  and  cyclic  nucleotide  phosphodiesterase  activity 
(lOOOXg  supernatant  solution)  are  studied. 

Major  Findings:   Electron  micrographs  reveal  that  band  2  is 
enriched  in  mitochondria  rich  cells  and  band  3  in  granular  cells, 
as  reported.   A  large  portion  (25-50  percent)  of  the  cells,  how- 
ever, are  vacuolated  or  otherwise  obviously  damaged.   The  intact 
cells  collected  from  the  gradient  have  a  variable  and  small 
increment  in  cyclic  AMP  content  in  response  to  vasopressin. 
These  observations  led  to  the  conclusion  that  many  of  the  cells 
collected  from  the  gradient  are  not  viable  and  are  a  poor  prepara- 
tion for  study  of  function  as  intact  cells.   This  is  not  surpris- 
ing since  the  cells  have  been  in  calcium-free  Ringer  solution  for 
three  hours  by  the  end  of  the  Ficoll  gradient  separation.   The 
material  in  band  two  uniformly  has  two  to  three  times  as  much 
carbonic  anhydrase  activity  (per  mg .  protein)  as  that  from  band 
three,  confirming  the  enrichment  of  each  band  seen  in  electron 
micrographs . 

Basal  adenylate  cyclase  activity  is  the  same  in  both  bands. 
Vasopressin  sensitive  adenylate  cyclase  is  slightly  enriched  in 
cells  of  band  3  (granular  cells)  which  contain  20  percent  more 
activity  per  mg.  protein  than  do  the  cells  in  band  2.   There  is 
a  similar  enrichment  in  band  3  of  cyclic  nucleotide  phcsphodies- 

_8 

terase  activity  assayed  at  a  low  (10   M)  concentration  of  cyclic 

AMP,  .but  no  difference  in  activity  between  the  two  bands  assayed 

_  •* 

at  a  high  concentration  of  cyclic  AMP  (10   M) . 

2  3$<t 


Project  No.  Z01  HL  01203-01  KE 

The  results  are  interpreted  as  confirming  that  the  two  major 
cell  types  can  be  separated  on  a  Ficoll  gradient,  but  that  the 
cells  are  too  damaged  for  meaningful  study.   In  contrast  to  the 
previous  study  (Science,  185:797,  1974)  in  which  it  was  conclud- 
ed that  only  the  mitochondria  rich  cells  respond  to  vasopressin 
with  increased  cyclic  AMP  levels,  the  enzyme  assays  of  this 
study  are  interpreted  as  indicating  that  the  granular  cells  re- 
spond to  vasopressin  with  elevation  of  cyclic  AMP  production  as 
well  as  the  mitochondria  rich  cells. 

Proposed  Course  of  Project:   Project  is  completed.   Manu- 
script is  in  preparation. 

Keyword  Descriptors:   Cyclic  AMP,  Granular  Cells,  Mitochondria 

Rich  Cells,  Adenylate  Cyclase,  Cyclic 
Nucleotide  Phosphodiesterase 

Honors  and  Awards :   None 

Publications:   None 


<wr 


Project  No.  Z01  HL  01204-01  KE 

1.  Kidney  &  Electrolyte 

2.  Membrane  Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Effect  of  parathyroid  hormone  on  protein 

phosphorylation  in  rabbit  renal  cortical  tubules 

Previous  Serial  Number:   None 

Principal  Investigator:   Dennis  A.  Ausiello,  M.D. 

Joseph  S.  Handler,  M.D. 
Jack  Orloff,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Work  in  several  laboratories  has  shown  that 
cyclic  AMP  exerts  part  or  all  of  its  effects  within  cells  by 
stimulating  the  transfer  of  phosphate  from  ATP  to  certain  pro- 
teins.  This  reaction  is  catalysed  by  a  cyclic  AMP  dependent 
protein  kinase.   For  several  years  this  laboratory  has  been 
interested  in  the  mechanism  by  which  cyclic  AMP  alters  transport 
and  permeability  and  we  are  studying  the  effect  of  cyclic  AMP  on 
protein  phosphorylation  in  toad  urinary  bladder.   This  project 
is  a  parallel  study  of  the  effect  of  parathyroid  hormone  on  (PTH) 
and  cyclic  AMP  rabbit  kidney  cortex.   In  this  study,  gel  electro- 
phoresis is  used  to  separate  phosphorylated  proteins  in  an 
attempt  to  characterize  the  endogenous  substrates  for  cyclic  AMP 
dependent  protein  kinase (s)  and  to  elucidate  their  role  in  the 
action  of  parathyroid  hormone. 

Methods:   Rabbit  kidney  cortex  homogenates  were  incubated 

3  2 

at  23°C  with    P-y-ATP  under  various  conditions.   The  reaction 
was  stopped  by  adding  samples  to  boiling  SDS  (final  concentration 
1.0%)  in  10  mM  phosphate  buffer  pH  7.2.   Samples  were  electro- 
phoresed  on  polyacrylamide  cylindrical  gels,  which  were  prepared 
as  5%-15%  gradients  for  improved  resolution.   The  gels  were  cut 
into  1  mm  slices  and  the  radioactivity  in  each  slice  determined 
by  liquid  scintillation  counting. 

In  a  second  series  cf  experiments,  separated  renal  cortical 
tubules  were  prepared  by  the  collagenase  method  previously  de- 
scribed by  this  laboratory.   The  tubules  were  incubated  at  23°C 
for  various  time  periods  with  tracer  inorganic    P04  in  standard 

1  <?g£ 


Project  No.  Z01  HL  01204-01  KE 

Krebs-Ringer  solution  without  phosphate  and  gassed  with  9  5% 
02-5%  CO2 •   Experimental  manipulations  were  performed  on  aliquots 
and  the  reactions  ended  by  the  addition  of  boiling  SDS .   Samples 
were  electrophoresed  and  processed  as  described  above. 

Major  Findings:   Studies  with  homogenates  of  cortex  revealed 
several  proteins  in  the  40,000-150,000  MW  range  whose  phosphory- 
lation was  stimulated  by  cyclic  AMP.   The  major  effect  was  a 
peptide  of  ^  50,000  daltons.   In  order  to  interpret  this  result, 
it  was  important  to  determine  whether  similar  changes  occurred 
in  intact  cells  stimulated  by  P.T.H. 

The  major  phosphorylated  peaks  observed  in  the  intact  renal 
tubule  cells  were  at  ^65,000  and  ^50,000  daltons  with  several 
smaller  peaks  between  100,000  and  150,000  daltons.   A  steady 
state  level  of  phosphorylation  was  generally  achieved  after  45  min, 
incubation  with    POi*  .   Phosphorylation  stable  through  105  min. 
Purified  bovine  PTH  at  a  concentration  of  100  U/ml  stimulated  the 
phosphorylation  of  the  50,000  dalton  peptide  (23%  increase,  p  < 
,01)  after  15  minutes  of  incubation.   The  phosphorylation  of 
several  proteins  in  the  higher  molecular  weight  range  was  also 
significantly  stimulated.   Preliminary  data  indicate  that 
dibutyry  cyclic  AMP  mimics  this  effect  of  PTH. 

Proposed  Course  of  Project:   Emphasis  will  be  placed  on 
further  characterizing  the  phosphorylated  proteins  affected  by 
PTH  in  the  intact  cell.   Attempts  will  be  made  to  see  whether 
variables  believed  to  affect  PTH  action  (eg.  changes  in  Ca++  and 
Mg++  concentration  alter  the  PTH-stimulated  phosphorylation  of 
proteins.   In  addition  homogenates  and  subcellular  fractions  will 
be  studied  in  order  to  localize  and  identify  the  phosphorylated 
substrates  and  to  correlate  them  with  the  known  actions  of  PTH. 

Keyword  Descriptors:   Cyclic  AMP,  Protein  Kinase,  Parathyroid 

Hormone 

Honors  and  Awards :   None 

Publications:   None 


H7 


Project  No.  Z01  HL  01205-01  KE 

1.  Kidney  &  Electrolyte 

2.  Electrolyte  Transport 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Regulation  of  Cation  Permeability  in  Duck 
Erythrocytes 

Previous  Serial  Number:   NHLI-135 

Principal  Investigator:   Dianne  E.  Robbie,  Ph.D. 

Other  Investigators :   None 

Cooperating  Units:   None 

Project  Description: 

In  the  erythrocytes  of  several  avian  species  cation  per- 
meability is  regulated  by  catecholamines  and  the  electrolyte 
composition  of  the  ambient  medium.   Previous  work  from  this 
laboratory  has  demonstrated  regulation  of  cell  volume  in  duck 
erythrocytes.   This  is  accomplished  through  the  control  of  cation 
permeability  by  a  volume-sensitive  mechanism.   Normal  erythrocyte 
volume  depends  in  vivo  upon  plasma  levels  of  catecholamines,  as 
indicated  by  the  shrinkage  to  a  new  steady-state  volume  of 
erythrocytes  incubated  in  catecholamine-f ree  media  or  in  plasma 
containing  the  B-adrenergic  blocking  agent,  propranolol.   Addi- 
tion of  norepinephrine  to  shrunken  "lower  steady-state"  cells 
results  in  an  immediate  increase  in  permeability  to  Na  and  K. 
In  appropriate  media  the  change  in  permeability  results  in  net 
accumulation  of  cations,  CI,  and  osmotically  obligated  water. 
Except  under  special  conditions,  these  volume  changes  are  almost 
entirely  associated  with  net  uptake  of  KC1.   Upon  restoration  of 
"normal"  volume,  cation  permeabilities  return  to  resting  levels, 
presumably  reflecting  the  intervention  of  a  "volume  sensor" 
which  reverses  the  molecular  changes  initiated  by  norepinephrine. 

A  strikingly  similar  system  is  activated  upon  osmotic  shrink- 
age of  duck  erythrocytes  in  hypertonic  media.   The  responses  to 
the  two  stimuli,  catecholamines  and  hypertonicity ,  exhibit 
similar  sensitivity  to  K  and  Na  ion  concentration  in  the  medium 
and  to  drugs.   Also,  the  maximal  effects  of  the  two  stimuli  on 
cation  permeability  are  identical,  within  experimental  error. 

Previous  evidence  (1972-73)  indicated  that  the  permeability 
changes  initiated  by  norepinephrine  were  a  consequence  of  eleva- 
tion of  cellular  cyclic  AMP  levels  caused  by  activation  of  the 
membrane-associated  adenylate  cyclase  of  these  cells.   In  con- 
trast, permeability  changes  initiated  by  hypertonicity  were  not 

1  £$2 


Project  No.  Z01  HL  01205-01  KE 

accompanied  by  detectable  changes  in  cyclic  AMP  content.   There- 
fore, the  two  stimuli  elicit  the  same  ultimate  effect,  but  a 
different  chain  of  events . 

Objectives:   I.   To  determine  whether  the  effects  of  nore- 
pinephrine and  hypertonicity  involve  separate  or  common  pathways 
for  cation  permeation; 

II.   To  examine  the  possible  interdependence  of 
the  mechanisms  activated  by  the  two  stimuli; 

III.   To  further  identify  the  biochemical  events 
initiated  by  hypertonicity  which  result  in  increased  cation 
permeability. 

Methods:   Cation  permeability  was  measured  in  suspensions  of 

duck  erythrocytes  as    K  influx  or  efflux,  according  to  methods 
which  have  been  previously  described  (Kregenow,  F.M.,  J.  Gen. 
Physiol.  58:372,  1971).   A  microcentrifugation  assay  has  also 
been  developed  which  provides  greater  speed  and  capability  in 
tracer  flux  determination. 

Major  Findings:   In  the  present  studies,  we  have  obtained 
further  evidence  that  effects  of  norepinephrine  and  hypertonicity, 
though  involving  different  initial  steps,  are  mediated  via  a 
common  final  pathway.   We  find  that  a  maximally  effective  degree 
of  hypertonicity  superimposed  upon  a  maximally  effective  concen- 
tration of  norepinephrine  does  not  cause  an  appreciably  greater 
effect  on  cation  permeability  than  either  stimulus  alone.   Fur- 
thermore, we  have  observed  that  the  permeability  response  to 
hypertonicity  is  biphasic,  decreasing  when  medium  tonicity  is 
increased  beyond  the  level  that  results  in  maximal  stimulation. 
Under  these  conditions  the  response  to  norepinephrine  is  inhibited 
in  parallel  with  the  response  to  hypertonicity.   Norepinephrine- 
dependent  cyclic  AMP  accumulation  was  also  found  to  be  progress- 
ively inhibited  under  these  conditions,  complicating  interpreta- 
tion of  the  result. 

V7e  have  investigated  the  possibility  that  cyclic  AMP  levels 
may  influence  the  response  to  hypertonicity  in  the  erythrocyte. 
As  a  first  step,  we  tested  the  effect  of  theophylline,  which 
increases  cyclic  AMP  concentration  in  many  tissues  by  inhibiting 
its  hydrolysis.   Theophylline  significantly  inhibited  the  enhance- 
ment of  K  influx  and  K  efflux  that  was  caused  by  submaximal  levels 
of  hypertonicity,  but  had  no  effect  on  the  enhancement  of  net  K 
and  water  uptake.   In  contrast,  theophylline  potentiated  the 
effect  of  submaximal  concentrations  of  norepinephrine  on  K  influx, 
and  .on  the  net  uptake  of  K,   Na  and  water.   The  theophylline 
effect  was  apparent  only  during  the  first  few  minutes  of  the 


<a$? 


Project  No.  Z01  HL  01205-01  KE 

response  to  either  stimulus.   Therefore,  if  the  cyclic  AMP  level 
is  a  determinant  of  the  response  it  influences  only  the  initial 
events  leading  to  permeability  changes. 

Proposed  Course  of  Project:   1)   In  order  to  test  further 
whether  the  level  of  cyclic  AMP  modulates  the  response  to  hyper- 
tonicity,  K  fluxes  will  be  measured  during  hypertonic  stimulation 
in  the  presence  of  exogenous  cyclic  AMP  and  of  other  agents  be- 
lieved to  alter  cellular  cyclic  AMP  levels.   Cyclic  AMP  content 
of  the  cells  will  be  measured.   2)   Other  areas  of  investigation 
under  consideration  are  the  possible  involvement  of  cyclic  GMP , 
of  phosphodiesterases  of  differing  specificity  and  of  cyclic- 
nucleo tide-activated  phosphorylation/dephosphorylation  mechanisms 
in  volume  regulation. 

Keyword  Descriptors:   Erythrocytes,  Cation  Permeability, 

Catecholamines,  Cyclic  AMP,  Hypertonicity , 
Volume  Regulation 

Honors  and  Awards :   None 

Publications:   Kregenow,  F.M.  Robbie,  D.E.,  and  Orloff,  J.: 

Effect  of  norepinephrine  and  hypertonicity  on  K 
influx  and  cyclic  AMP  levels  in  duck  erythrocytes. 
(Submitted  3/75,  Am.  J.  Physiol.) 


99o 


Project  No.  Z01  HL  01206-01  KE 

1.  Kidney  &  Electrolyte 

2.  Renal  Mechanisms 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Urinary  Acidification  by  Proximal  Straight 
Tubules 

Previous  Serial  Number:  NHLI-68 

Principal  Investigators:   David  Warnock ,  M.D. 

Maurice  Burg,  M.  D. 

Other  Investigators:  Gerald  Vurek ,  Ph.D. 

Cooperating  Unit:  Laboratory  of  Technical  Development/NHLI 

Project  Description: 

Objectives:   The  C02/bicarbonate  buffer  system  plays  a  cen- 
tral role  in  the  maintenance  of  physiologic  acid-base  balance. 
Bicarbonate  ion  is  the  principal  urinary  buffer.   Most  bicarbon- 
ate is  reabsorbed  by  the  proximal  tubule.   In  order  to  elucidate 
the  mechanisms  of  urinary  acidification,  we  have  attempted  to: 

1.  develop  the  micro-methods  to  measure  acidification  of 
the  renal  tubule  fluid; 

2.  define  the  kinetics  of  generation  and  maintainance  of 
transepithelial  bicarbonate  gradients  in  isolated  proximal  straight 
tubules . 

Methods : 

1.  The  isolation  and  perfusion  of  tubule  segments  from  the 
rabbit  kidney  has  been  described  previously.   Superficial  and 
juxtamedullary  proximal  straight  tubules  were  distinguished  on 
morphological  and  anatomical  grounds. 

2.  A  microcalorimetric  method  was  developed  to  measure  total 
C02  content  of  small  fluid  samples.   This  was  done  in  collabora- 
tion with  Dr.  Vurek  of  the  Laboratory  of  Technical  Development, 
NHL  I. 

3.  Cation/anion  permeability  ratios  were  determined  using 
the  Goldman-Hodgkin-Katz  equation  for  the  analysis  of  dilution 
and  bionic  potentials  across  the  tubule  wall. 


s?r 


Project  No.  Z01  HL  01206-01  KE 

4.   A  first-order  kinetic  model  has  been  developed  to  de- 
scribe the  transepithelial  movement  of  total  CO2  across  the 
proximal  straight  tubule.   Solution  of  the  initial  condition 
problem  provides  pump  and  leak  rate  constants,  as  well  as  a 
steady-state  level  of  C02  in  the  individual  tubule.   The  total 
CO2  content  of  the  luminal  fluid  is  satisfactorily  described  as 
a  function  of  the  transit  time  of  the  perfusate.   The  transit 
time  of  the  luminal  fluid  is  varied  by  changing  the  perfusion 
rate. 

Major  Findings: 

1.  When  the  total  CO2  concentration  in  the  perfusate  and 
bath  is  27.5  mM,  proximal  straight  tubules  reabsorb  CO2  causing 
the  concentration  in  the  lumen  to  decrease.   When  the  concentra- 
tion of  total  C02  in  the  perfusate  is  zero  and  that  in  the  bath 
is  27.5,  C02  enters  the  lumen,  causing  an  increase  in  concentra- 
tion.  At  slow  flow  rates  a  steady  state  concentration  of  CO2  is 
reached  in  the  lumen.   The  steady  state  level  differs  between 
tubule  populations.   In  proximal  straight  tubules  from  superfi- 
cial nephrons  the  mean  steady-state  luminal  total  C02  content  is 
16  mM,  while  in  proximal  straight  tubules  from  juxtamedullary 
nephrons  it  is  9  mM. 

2.  It  is  the  "leakiness"  of  the  tubule  epithelium  to  C02 
that  accounts  for  this  difference.   Both  populations  of  tubules 
have  similar  pump  rates,  but  the  juxtamedullary  tubules  have  a 
smaller  leak  of  CO2  back  into  the  tubule  lumen  than  do  the  super- 
ficial tubules.   The  kinetic  model  predicts  the  lower  steady- 
state  C02  level  that  results  from  this  difference  in  leak  rates. 

3.  The  lower  leak  rate  in  the  juxtamedullary  tubules  is 
consistent  with  other  observations  of  relative  cation/anion  per- 
meability ratios.   On  the  basis  of  dilution  and  biionic  poten- 
tials the  tubules  from  juxtamedullary  nephrons  are  calculated  to 
be  less  leaky  to  anions  than  are  those  from  superficial  nephrons. 

4.  The  effects  on  CO2  of  acetazolamide  (which  inhibits 
carbonic  anhydrase)  was  determined.   Acetazolamide  caused  the 
steady  state  level  of  luminal  C02    content  to  increase  in  straight 
tubules  from  both  superficial  and  juxtamedullary  nephrons.   It  was 
not  possible  to  distinguish,  however,  whether  the  pump  or  leak 
process  was  affected. 

Proposed  Course  of  Project: 

1.   The  agreement  between  bicarbonate  permeability  calcula- 
ted from  the  electrical  measurements  and  the  total  C02permeability 
measured  directly  suggest  that  the  CO2  leaks  across  the  epithel- 


£f*~ 


Project  No.  Z01  HL  01206-01  KE 

ium  as  a  bicarbonate  ion.   Further  studies  are  necessary  to  con- 
firm this  point. 

2.  We  will  develop  a  pH  glass  microelectrode .   The  measure- 
ment of  pH  is  necessary  to  distinguish  dissolved  C02  from  the 
bicarbonate  anion  both  of  which  contribute  to  the  total  C02 .   It 
is  necessary  to  make  this  distinction  in  order  to  find  out  whether 
bicarbonate  ions  are  transported  per  se  or  whether  the  primary 
mechanism  is  hydrogen  ion  transport. 

3.  These  studies  will  be  extended  to  other  segments  of  the 
rabbit  nephron. 

Keyword  Descriptors:   Proximal  Tubule,  C02 /Bicarbonate ,  Acidifica- 
tion, Picapnotherm 

Honors  &  Awards :   None 

Publications : 

Warnock,  D.G.,  Burg,  M.B.,  Vurek,  G.G.:  Urinary  acidifica- 
tion by  proximal  straight  tubules.  Kidney  International  6:110A, 
1975  (Abstract,  paper  presented  to  the  7th  Annual  Meeting  of  the 
Society  of  Nephrology.  Washington,  D.  C,  1974). 

Vurek,  G.G=,  Warnock,  D.G.,  Corsey,  R. :   Measurement  of 
picomole  amounts  of  carbon  dioxide  by  calorimetry.   Analytical 
Chemistry.  47:765-767,  1975. 


a-13 


Project  No.  ZOI  HL  01207-01  KE 

1.  Kidney  &  Electrolyte 

2.  Renal  Mechanisms 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Glucose  transport  in  the  Proximal  Convoluted 
Tubule 

Previous  Serial  Number:   None 

Principal  Investigators:   David  Warnock,  M.D. 

Maurice  Burg,  M.D. 

Other  Investigators:   Clifford  Patlak,  Ph.D. 

Cooperating  Units:   Theoretical  Statistics  and  Mathematics 

Branch,  NIMH 

Project  Description: 

Objectives:   It  has  been  shown  that  in  rabbits  glucose 
transport  in  the  proximal  convoluted  tubule  is  related  to  the 
generation  of  a  transepithelial  potential  difference  and  the 
reabsorption  of  salt  and  water.   The  lumen  glucose  concentra- 
tion is  rapidly  lowerec.  in  the  proximal  convoluted  tubule, 
although  in  rabbits  there  is  a  relatively  high  permeability  of 
the  tubule  epithelium  to  glucose.   The  result  of  the  high  per- 
meability is  a  significant  influx  of  glucose  into  the  lumen  down 
the  length  of  the  proximal  convoluted  tubule.   We  have  quantified 
the  contribution  made  b   this  passive  influx  to  the  total  glu- 
cose transport  capability  of  the  proximal  convoluted  tubule  of 
rabbits. 

Methods: 

1.   The  functional,  aspects  of  glucose  transport  have  been 
previously  defined  by  this  laboratory  (Tune  and  Burg,  Amer.  J. 
Physiol  221:580-585,  1971).   This  previous  work  provided  the 
following  parameters  of  glucose  transport  in  the  rabbit  proximal 
convoluted  tubule;  passive  b  th  to  lumen  glucose  permeability, 
maximal  glucose  transport  rate  and  affinity  of  the  transport 
process  for  glucose. 

1  Sty 


Project  No.  Z01  HL  01207-01  KE 

2.   A  system  of  linear,  differential  equations  were  devel- 
oped in  collaboration  with  Dr.  Patlak  of  the  Theoretical  Statis- 
tics and  Mathematics  Branch  of  the  NIMH.   This  system  described 
the  removal  of  glucose  from  the  tubule  lumen  by  active  transport 
and  bulk  flow,  and  the  passive  entry  of  glucose  into  the  lumen 
by  transepithelial  diffusion.   The  equations  were  numerically 
integrated  with  the  MLAB  language  of  the  DEC-10  computer  facil- 
ities available  at  the  NIH. 

Major  Findings: 

1.  A  significant  load  of  glucose  is  presented  to  the  prox- 
imal convoluted  tubule  by  transepithelial  passive  glucose  influx. 
At  physiological  flow  rates  and  glucose  concentrations,  nearly 
half  of  the  glucose  load  originates  in  the  passive  transepithel- 
ial influx.   Therefore,  the  glucose  from  the  bath  is  as  impor- 
tant as  that  of  the  original  perfusate  in  any  processes  related 
to  the  active  transport  of  glucose.   We  conclude  that  the  pas- 
sive entry  of  glucose  ("leaked  load")  could  be  a  significant 
factor  in  the  reabsorption  of  salt  and  water  from  rabbit  proxi- 
mal tubules. 

2.  At  physiologic  flow  rates  and  glucose  concentrations, 
it  is  unlikely  that  solvent  drag  accounts  for  more  than  2%   of 
the  glucose  removed  from  the  luminal  compartment. 

3.  The  lumen  glucose  concentration  rapidly  falls  to  a 
steady-state  level  within  the  first  2  millimeters  of  tubule 
length.   The  steady-state  level  is  achieved  when  the  active  ef- 
flux rate  equals  the  rate  of  passive  glucose  influx.   The  steady- 
state  level  is  typically  0.5  mM  when  the  initial  perfusate  and 
bath  glucose  concentrations  are  5.5  mM.   The  lumen  glucose  pres- 
ent in  the  steady-state  originates  exclusively  from  the  passive 
glucose  influx  from  the  bath. 

Proposed  Course  of  the  Project:   The  analysis  is  essentially 
complete,  and  provides  a  means  of  analyzing  the  significance  of 
the  passive  influx  component  of  glucose  and  other  solutes. 

Keyword  Descriptors:   Proximal  Tubule,  Solute  Back  Flux 
Honors  &  Awards :   None 
Publications:    None 


2.9S- 


Project  No.  Z01  HL  01208-02  Kg 

1.  Kidney  &  Electrolyte 

2.  Renal  Mechanisms 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Mechanism  of  salt  and  water  transport  by  proximal 
renal  tubules. 

Previous  Serial  Number:   NKLI-71 

Principal  Investigators:   Maurice  B.  Burg,  M.  D. 

Nordica  Green 

Other  Investigators :   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   The  proximal  nephron  reabsorbs  approximately 
50%  of  the  glomerular  filtrate.   The  mechanisms  by  which  this 
occurs  have  been  only  partially  characterized.   There  is  a  con- 
siderable body  of  evidence  from  micropuncture  and  microperf usion 
studies  suggesting  that  sodium  is  actively  reabsorbed  providing 
the  primary  driving  force  for  fluid  and  salt  transport.   The 
process  is  complicated,  however,  and  apparently  involves  coupling 
between  the  transport  of  sodium  and  various  other  solutes  as  well 
as  coupling  to  cellular  metabolism  as  a  source  of  energy.   The 
purpose  of  these  continuing  studies  is  to  investigate  the  inter- 
relationships . 

Methods:   The  rabbit  isolated  perfused  proximal  convoluted 
tubule  preparation  developed  in  this  laboratory  and  described  in 
previous  reports  was  used  to  analyze  the  transport  processes. 

Major  Findings: 

1.   Previously  we  found  that  sugar  (glucose)  or  amino  acid 
(alanine)  in  the  perfusate  caused  a  voltage  oriented  negative  in 
the  lumen  of  the  proximal  convoluted  tubule  and  also  caused  a 
small,  but  significant,  increase  in  the  rate  of  fluid  absorption. 
It  was  conceivable  that  the  sugar  and  amino  acid  are  metabolized 
by  the  epithelial  cells  energyzing  the  sodium  pump  which  drives 
fluid  transport.   Alternatively,  the  sugar  and  amino  acid  might 
be  co-transported  with  sodium,  as  in  the  intestine,  accounting 
for  the  effect.   In  order  to  distinguish  between  these  possibili- 
ties' we  tested  the  effects  of  a  sugar  (a-methyl-D-glucoside)  and 
an  amino  acid  (cycloleucine)  known  to  be  transported  but  not 


Project  No.  Z01  HL  02108-02  KE 

metabolized,  by  proximal  tubule  cells.  When  placed  in  the  per- 
fusate, either  a-methyl-D-glucoside  or  cycloleucine  caused  the 
rate  of  fluid  absorption  and  the  voltage  to  increase,  suggesting 
that  the  transport  of  sugars  and  amino  acids  rather  than  their 
metabolism  is  coupled  to  salt  and  fluid  transport.  Additional 
evidence  for  this  view  was  provided  by  the  effect  of  phlorizin 
which  specifically  inhibits  glucose  transport.   A  low  concentra- 

_  5 

tion  (10   M)  of  phlorizin  in  the  perfusate  caused  the  rate  of 
fluid  absorption  and  the  voltage  to  decrease. 

2.  When  glucose  and/or  alanine  was  added  to  the  perfusate 
there  was  a  striking  change  in  the  appearance  of  the  epithelial 
cells.   They  swelled,  protruding  into  the  lumen.   The  change  most 
likely  is  due  to  entry  of  glucose  and  alanine  into  the  tubule 
cells  during  transport  of  non-electrolytes.   Because  of  the  addi- 
tional solute  in  the  cells,  water  enters  by  osmosis,  causing  the 
cells  to  swell. 

3.  Although  active  sodium  transport  is  generally  believed 
to  be  the  basis  of  fluid  absorption  in  this  segment,  the  evidence 
has  been  inconclusive.   There  are  numerous  other  theories,  includ- 
ing suggestions  that  there  is  no  active  sodium  transport  or  that 
sodium  and  chloride  are  co- transported  by  a  linked  mechanism.   In 
order  to  test  further  the  importance  of  sodium  transport  for  fluid 
absorption,  sodium  was  entirely  omitted  from  the  perfusate  and 
bath.   When  the  sodium  was  replaced  by  choline,  tetramethyl 
ammonium,  or  lithium,  the  rate  of  fluid  absorption  and  the  voltage 
fell  to  zero.   In  contrast,  when  chloride  was  omitted  (replaced 

by  nitrate),  there  was  no  change.   Evidently,  transport  of  sodium 
but  not  of  chloride  is  essential  for  fluid  absorption. 

4.  In  most  tissues  the  active  transport  of  sodium  is  linked 
to  that  of  potassium,  and  omission  of  potassium  results  in 
inhibition  of  the  sodium  transport.   When  potassium  was  omitted 
from  the  bath,  the  rate  of  fluid  absorption  and  the  voltage  across 
the  proximal  tubules  fell  to  zero,  additional  evidence  that 
active  sodium  transport  is  primary  and  that  it  has  a  requirement 
for  potassium  similar  to  other  tissues. 

5.  In  earlier  micropuncture  studies  it  was  found  that 
omission  of  bicarbonate  caused  the  rate  of  fluid  absorption  to 
decrease.   Therefore,  we  tested  the  affect  of  bicarbonate  on  the 
isolated  proximal  convoluted  tubules.   Omission  of  bicarbonate 
from  the  perfusate  and  bath  caused  the  rate  of  fluid  absorption 
to  decrease  by  approximately  one-third,  similar  to  the  micropunc- 
ture results.   Several  theories  have  been  advanced  to  explain 
this  bicarbonate  dependence.   One  theory  emphasizes  that  there  is 
a  change  in  tubule  fluid  chloride  and  bicarbonate  concentration 
as  bicarbonate  is  reabsorbed.   The  chloride  concentration  in  the 


#rr 


Project  No.  Z01  HL  02108-02  KE 

lumen  increases  and  that  bicarbonate  decreases.   Considering  that 
bicarbonate  permeates  the  tubule  more  slowly  than  chloride,  it 
presumably  has  a  higher  reflection  coefficient.   Therefore,  the 
concentration  gradient  for  bicarbonate  (whose  concentration  is 
higher  in  the  bath  than  in  the  lumen)  might  cause  fluid  absorp- 
tion by  osmosis.   We  tested  this  theory  by  interchanging  methyl 
sulfate  (which  permeates  the  epithelium  slowly,  as  does  bicarbon- 
ate) and  chloride  in  the  perfusate.   The  rate  of  fluid  absorption 
did  not  change,  suggesting  that  anion  concentration  differences 
are  not  important  for  fluid  absorption.   Another  theory  that 
purports  to  explain  the  dependence  of  fluid  absorption  on  bicar- 
bonate emphasizes  the  well  known  role  of  hydrogen  ion  secretion 
in  bicarbonate  reabsorption .   Bicarbonate  presumably  is  necessary 
for  hydrogen  secretion  across  the  lumen  border  of  the  tubule 
cells.   It  has  been  proposed  that  the  hydrogen  ion  secretion  is 
coupled  to  sodium  entry  into  the  cells  by  an  exchange  process. 
In  the  absence  of  bicarbonate  the  hydrogen  ion  secretion  would  be 
reduced,  limiting  sodium  transport.   The  theory  implies  recipro- 
cal dependence  of  bicarbonate  and  sodium  reabsorption.   We  intend 
to  test  for  this  by  measuring  the  effect  on  bicarbonate  reabsorp- 
tion of  removing  sodium  from  the  perfusate  and  bath. 

6.  The  enzyme  carbonic  anhydrase  is  important  for  bicarbon- 
ate transport  in  proximal  tubules.   Therefore,  we  tested  the 
effect  of  acetazolamide  which  is  an  inhibitor  of  carbonic  anhy- 
drase and  is  a  mild  diuretic.   Acetazolamide  (10~  M)  caused  the 
rate  of  fluid  absorption  by  proximal  convoluted  tubule  to  de- 
crease approximately  as  much  as  did  removal  of  bicarbonate.   It 
has  been  proposed  that  acetazolamide  has  an  action  in  proximal 
tubules  in  addition  to  inhibiting  carbonic  anhydrase,  i.e.  that 
it  inhibits  fluid  absorption  directly  and  independently  of  bi- 
carbonate transport.   We  intend  to  test  this  theory  by  seeing 
whether  acetazolamide  causes  a  further  decrease  in  the  rate  of 
fluid  absorption  in  the  tubules  in  bicarbonate-free  solutions. 

7.  In  the  studies  outlined  thus  far  the  conditions  used  were 
similar  to  those  in  the  early  proximal  tubule  in  which  the  per- 
fusate is  an  ultraf iltrate  of  serum.   Under  these  conditions  the 
rate  of  fluid  absorption  is  apparently  normal  comparable  to  the 
rate  found  in  vivo.   As  fluid  traverses  the  proximal  tubule,  how- 
ever, its  composition  changes.   Organic  solutes,  such  as  sugars, 
amino  acids,  and  lactate  are  reabsorbed,  causing  their  concentra- 
tions to  decrease  markedly  in  the  lumen.   As  noted  above,  when 
isolated  proximal  tubules  are  perfused  with  low  concentrations  of 
the  organic  solutes,  the  rate  of  fluid  absorption  is  greatly 
reduced.   The  low  rate  of  fluid  absorption  apparently  is  less 
than  the  normal  rate  in  vivo  under  what  seems  to  be  similar  con- 
ditions in  the  late  proximal  convoluted  tubule.   The  reason  for 
this  difference  is  of  interest  since  it  might  involve  a  previous- 
ly unrecognized  factor  important  for  fluid  absorption.   One 


298 


Project  No.  Z01  HL  02108-02  KE 

possibility  is  that  there  is  a  hormone  or  substrate  lacking  in 
the  in  vitro  experiments.   In  attempting  to  identify  such  a 
factor  we  have  tested  the  effect  of  added  mineralocorticoids , 
glucocorticoids,  glutamine,  and  free  fatty  acids.   The  results 
were  negative.   There  was  no  change  in  fluid  absorption.   We 
intend  to  continue  these  studies  by  testing  the  effect  of  addi- 
tional hormones  and  substrates.   Another  possibility  is  that  the 
anatomically  more  distal  parts  of  the  proximal  convoluted  tubule 
function  differently  from  the  earlier  segment.   We  have  not  been 
able  to  identify  the  anatomical  location  ("early"  vs.  "late")  of 
the  proximal  tubule  fragments  that  we  study.   We  assume  that 
since  the  fragments  are  dissected  at  random,  they  include  "late" 
as  well  as  "early"  convoluted  tubules,  but  there  is  no  proof  of 
this.   Therefore,  we  will  attempt  to  identify  and  study  "late" 
proximal  convoluted  tubules  in  order  to  see  whether  they  function 
differently  from  the  early  part  and  do  not  require  organic  sol- 
utes in  the  perfusate  for  normal  rates  of  fluid  absorption. 

Proposed  Course  of  Study:   In  addition  to  the  proposals 
listed  above,  we  intend  to  test  the  effects  of  a  number  of 
diuretic  drugs  on  the  proximal  tubules  in  order  to  discover 
whether  they  have  any  important  effect  on  this  segment  and,  if 
so,  what  the  mechanism  of  the  effect  is. 

Keyword  Descriptors:   Convoluted  Tubules,  Fluid  Absorption, 

Voltage,  Organic  Solutes,  Bicarbonate, 
Acetazolamide 

Honors  and  Awards:   None 

Publications:   Burg,  M.B.:   The  mechanism  of  fluid  absorption  by 
proximal  convoluted  tubules.  VI  International 
Congress  of  Nephrology,  Florence,  Italy,  June  8, 
1975. 

Burg,  M.  B. :  Two  Chapters  submitted  for  publica- 
tion, The  renal  handling  of  sodium  chloride  and 
Mechanisms  of  action  of  diuretic  drugs,  The  Kidney, 
Edited  by  Barry  M.  Brenner,  M.D.  and  Floyd  C. 
Rector,  M.D.,  Published  by  W.  B.  Saunders  Co. 


39? 


Project  No.  Z01  HL  01209-08  KE 

1.  Kidney  &  Electrolyte 

2 .  Renal  Mechan Lsms 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title;   Ion  Transport  in  the  Cortical  Collecting  Tubule 

Previous  Serial  Number:   NHLI  70 

Principal  Investigators:   Larry  C.  Stoner,  Ph . D 

Maurice  B.  Burg,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   We  previously  found  that  cortical  collecting 
tubules,  in  addition  to  regulating  water  reabsorption  in  response 
to  vasopressin,  also  actively  reabsorb  Na  from  and  secrete  K  into 
the  tubule  fluid.   In  the  present  studies  we  are  investigating  the 
effect  of  diuretic  agents  on  ion  transport  in  this  segment. 

Methods:   Are  the  same  as  previously  reported. 

Major  Findings:   We  had  previously  reported  that  low  concen- 
trations of  acetazolamide  in  the  bath  caused  the  transepithelial 
voltage  to  become  more  negative  in  the  cortical  collecting  tubule. 
This  was  taken  as  evidence  for  the  existence  of  a  urinary  acidi- 
fication process.   We  proposed  that  acetazolamide  inhibited  hy- 
drogen ion  secretion,  reducing  the  positive  voltage  caused  by  that 
process,  and  thus  increasing  the  observed  negative  voltage. 

In  initial  studies  we  have  found  that  the  same  concentration 
of  acetazolamide  (2X10   M)  has  little  or  no  effect  on  Na  and  K 
transport  in  the  cortical  collecting  tubule.   Higher  concentrations 

(2X10   M  and  10   M)  also  result  in  a  transient  increase  in  voltage 
which  is  followed  by  a  reduction.   The  voltage  decreases  were  30% 

(n=5)  and  50%  (n=4)  oi  Lhe  initial  voltage  at  the  two  concentra- 
tions.  The  decrease  in  voltage  occurs  between  10  and  30  min. 
after  administration  of  the  drug  and  is  not  reversible  when  the 

1  3oo 


Project  No.  Z01  HL  01209-08  KE 

_  3 

drug  is  removed.   In  addition,  10    of  acetazolamide  caused  a 

small  decrease  (-22%;  n=2)  in  the  lumen  to  bath  flux  of  Na22  and 
an  increase  in  the  bath  to  lumen  flux  of  K    (+25%;  n=7) .   Since 
these  changes  in  voltage  and  transport  were  observed  only  at 
high  concentrations  of  the  drug  their  relation  to  the  in  vivo 
diuretic  effects  of  the  drug  are  questionable. 

Proposed  Course  of  Project: 

1.  Additional  experiments  are  needed  testing  the  effect 

of  the  lower  (2X10   M)  concentration  of  acetazolamide  on  trans- 
port of  Na  and  K.   In  addition,  the  present  studies  were  carried 
out  without  bicarbonate  in  the  tubule  lumen.   It  will  be  of  inter- 
est to  see  how  acetazolamide  affects  Na  and  K  transport  when 
bicarbonate  is  present  in  the  perfusate. 

2.  Since  chlorothiazide  is  believed  to  exert  its  diuretic 
action  in  the  distal  nephron,  its  effect  on  the  cortical  collect- 
ing tubule  will  also  be  studied. 

Keyword  Descriptors:   Collecting  Tubule,  Ion  Transport,  Diuretics 

Honors  and  Awards :   None 

Publications:   None 


3©/ 


Project  No.  Z01  HL  01210-01  KE 

1.  Kidney  &  Electrolyte 

2 .  Renal  Mechanisms 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,1975 

Project  Title:   Mechanism  of  salt  transport  by  isolated  segments 
of  amphibian  distal  nephron 

Previous  Serial  Numbers:   NHLI  69 

Principal  Investigators:   Larry  C.  Stoner,  Ph.D. 

Other  Investigators:   David  Hinton,  Ph.D. 

Cooperating  Units:   None 

Project  Description; 

In  the  amphibian  distal  nephron  as  much  as  60%  of  the  NaCl 
that  is  filtered  at  the  glomerulus  is  reabsorbed,  diluting  the 
urine.   Previous  investigators  reported  lumen  negative  transepith- 
elial  voltages  in  amphibian  distal  tubules  but  did  not  report  lu- 
men positive  voltages,  such  as  we  previously  found  in  the  early 
mammalian  distal  tubule  (thick  ascending  limb  of  Henle ' s  loop) . 

Methods:   Are  identical  to  those  previously  described  for 
perfusing  isolated  renal  tubules  in  vitro. 

Major  Findings:   We  observed  that  the  "distal  convolution" 
of  the  amphibian  nephron  contains  at  least  two  functionally  dis- 
tinct segments:  The  early  segment  exhibits  a  lumen  positive  vol- 
tage (observed  in  3  species  -  frog,  toad  and  salamander)  and 
absorbs  NaCl  from  the  lumen  at  a  high  rate.   Since  chloride  moves 
out  of  the  lumen  against  an  electrochemical  gradient,  it  is  ac- 
tively transported.   In  these  properties  as  well  as  in  the  effects 
of  diuretic  drugs  this  segment  is  similar  to  the  mammalian  thick 
ascending  limb  of  Henle' s  loop.   Since  the  amphibia  lack  loops  of 
Henle  we  have  named  this  segment  the  "diluting  segment."   The 
second  segment  is  the  late  distal  tubule.   It  exhibits  a  lumen 
negative  voltage  and  absorbs  sodium  at  about  one-fourth  the  rate 
of  the  diluting  segment. 


3*3- 


Project  No.  Z01  HL  01210-01  KE 

Continuing  the  study  of  the  amphibian  distal  nephron,  we 
have  now  measured  the  permeability  to  water  of  the  diluting  seg- 
ment, the  late  distal  tubule  and  the  collecting  ducts  (salaman- 
der) .   In  all  three  segments  the  permeability  to  water  was  not 
measurably  different  from  zero.   Further,  the  water  permeability 
was  not  increased  by  the  antidiuretic  hormone  (ADH) ,  arginine 
vasotocin.   Thus,  in  this  species  of  amphibia,  the  late  distal 
nephron  differs  from  that  in  the  mammal  where  ADH  dramatically 
increases  the  water  permeability. 

We  have  also  used  the  electronmicroscope  to  ascertain  that 
the  diluting  segment  and  the  late  amphibian  distal  tubule  differ 
morphologically.   We  found  that  the  cells  of  the  diluting  segment 
are  morphologically  similar  to  those  of  the  mammalian  thick  as- 
cending limb,  and  those  in  the  late  distal  tubule  are  similar  to 
their  mammalian  counterpart  -  the  late  distal  convoluted  tubule 
or  early  cortical  collecting  tubule.   This  morphological  distinc- 
tion had  not  previously  been  made. 

Proposed  Course  of  Project:   Completed. 

Keyword  Descriptors:   Chloride  transport,  Amphibian  nephron, 

Urinary  dilution 

Honors  and  Awards:   None 

Publications:   Stoner,  L.  Isolated  segments  of  the  amphibian 

distal  nephron:  The  diluting  segment.   Manuscript 
in  preparation. 


303 


Project  No.  Z01  HL  01211-01  KE 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Anion  transport  across  individual  amphibian 
erythrocytes 

Previous  Serial  Number:   None 

Principal  Investigators:   Larry  C.  Stoner,  Ph.D. 

Floyd  M.  Kregenow,  M.  D. 

Other  Investigators:   None 

Cooperating  Units :   None 

Project  Description: 

Objectives:   To  a  large  degree,  our  knowledge  of  ion  trans- 
port in  the  red  blood  cell  has  served  as  a  basis  for  understand- 
ing ion  transport  in  other  tissues.   The  electrical  potential, 
ion  gradients,  and  permeability  to  ions  are  parameters  that  must 
be  determined  to  characterize  ion  transport.   In  red  cells  the 
methods  used  for  measurements  of  electrical  parameters  and  anion 
transport  have  been  indirect  and  lacked  precision.   The  purpose 
of  the  present  studies  is  to  develop  precise  and  direct  methods 
for  measuring  voltage,  electrical  conductance,  and  permeability 
to  ions  across  single  red  cells.   Such  methodology  should  pro- 
vide information  valuable  for  understanding  transport  in  erythro- 
cytes and  eventually  in  other  cells  as  well. 

Methods:   Individual  amphibian  red  blood  cells  are  held 
snugly  in  a  constriction  in  a  specially  prepared  glass  pipet. 
In  this  position  the  cell  is  a  cylinder  with  hemispherical  ends. 
A  second  glass  pipet  is  centered  within  the  pipet  that  holds  the 
cell.   Either  of  two  types  of  inner  pipets  is  used:  1)  a  glass 
microelectrode  ("Ling-Gerard"  type)  which  punctures  the  cell  mem- 
brane for  measurement  of  the  cellular  voltage  and  resistance,  or 
2)  a  larger  pipet  which  does  not  enter  the  red  cell,  but  is  used 
to  wash  one  end  of  the  cell  within  the  holding  pipet  with  a  radio- 
isotope containing  solution.   Permeability  to  the  isotope  is 
determined  from  the  amount  of  radioactivity  that  penetrates 
through  the  cell  and  appears  in  the  external  bath. 


3o*f 


Project  No.  Z01  HL  01211-01  KE 

Major  Findings: 

1.  Electrical  Measurements 

The  intracellular  voltage  averaged  -17  mv  (negative  in  the 
cell)  with  pH  7 .  in  the  bath.   Although  this  voltage  is  the  same 
as  that  previously  found  by  others,  we  feel  the  present  technique 
is  superior.   We  measured  stable  potentials  that  lasted  30  seconds 
or  more,  whereas  using  the  previous  techniques  the  voltages  de- 
cayed within  a  few  milliseconds  after  the  puncture.   Other  lab- 
oratories have  reported  that  variation  of  the  extracellular  pH 
leads  to  alteration  of  the  red  cell  chloride  concentration  and 
subsequently  the  intracellular  voltage.   The  present  study  con- 
firmed this  relationship.   At  pH  6.5  the  observed  cell  voltage 
was  -10  mv  and  at  pH  -8.1  the  voltage  was  -26  mv. 

Once  a  stable  voltage  was  obtained,  we  passed  small  electric 
currents  into  the  red  cell  to  measure  the  specific  resistance  of 
its  membrane.   The  results  of  29  such  attempts  provided  a  mean 

2  _1      _2 

value  of  100  ft  .  cm   (conductance  of  .01  ft    cm  )   .   In  80  other 
cells  the  same  current  was  passed  through  the  entire  cell, 
lodged  in  the  constriction  of  the  holding  pipet.   In  this  experi- 
ment the  current  passed  through  the  cell  membrane  twice  in  series, 
once  on  each  side  of  the  cell,  since  the  electrode  was  not  in- 
serted into  the  cell.   The  resistance  measured  was  exactly  twice 
that  found  when  the  electrode  was  inside  the  cell.   Thus,  the 
specific  membrane  resistance  calculated  from  the  two  experiments 
was  identical,  increasing  the  confidence  in  both  results.   Fur- 
ther, the  agreement  of  the  results  indicates  that  the  entire 
current  passed  through  the  cell  in  the  second  experiment  and 
that  the  leak  of  current  (and  ions)  around  the  cell  in  the  con- 
striction was  negligibly  small. 

2.  Radioisotope  flux: 

3  S 

When  CI    is  placed  on  one  side  of  the  cell  within  the  hold- 
ing pipet,  it  passes  through  the  cell  and  appears  in  the  bath. 

3  6  _6    _2 

The  measured  chloride    flux  is  0.84  X  10   cm   min,  equivalent 

_i     _2 
to  a  partial  chloride  conductance  of  .05  ft    cm   .   This  exceeds 

_i     _2 
the  total  electrical  conductance  (G)  of  .01  ft    cm   by  a  factor 

3  6 

of  five.   Therefore,  the  cell  of  CI    movement  is  not  electri- 
cally active  i.e.  it  cannot  be  explained  by  simple  passive  diffu- 
sion.  Two  other  observations  support  this  concentration. 

a.   When  a  voltage  (200  mv)  is  imposed  across  the  cell 

3  6 

during  a  flux  measurement,  CI    flux  increases,  but  the 
increase  is  much  smaller  than  that  theoretically  pre- 


3aST 


Project  No.  Z01  HL  01211-01  KE 

dieted  if  all  of  the  isotope  flux  were  electrically 
active.   The  observed  change  in  flux  was  used  to 
calculate  chloride  conductance.   The  partial  chloride 
conductance  was  half  of  the  total  electrical  conductance 
and  only  one-tenth  as  great  as  the  CI  conductance  that 
would  be  calculated  from  the  isotope  flux  in  the  absence 
of  imposed  voltage. 

b.   Replacement  of  90%  of  the  chloride  on  the  bath  side 

3  6 

of  the  cell  with  PAH  results  in  depression  of  the  Cl 
flux  from  the  opposite  surface  (mean  decrease  40%,  n=7 
cells) .   This  indicates  that  the  chloride  fluxes  in  the 
two  directions  are  linked.   The  results  are  consistent 
with  the  generally  accepted  hypothesis  that  chloride 
penetrates  the  red  cell  membrane  by  a  "carrier  mediated" 
mechanism. 

Proposed  Course  of  Project: 

1.  To  evaluate  the  contribution  of  other  ions  to  the  elec- 
trical conductance. 

2.  To  investigate  further  the  mechanism  of  anion  transport 
across  the  red  blood  cell. 

Keyword  Descriptors:   Anion  Transport,  Erythrocytes,  Membrane 

Resistance 

Honors  and  Awards:   None 

Publications:   None 


3<£ 


Project  No.  Z01  HL  01212-01  KE 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Volume  regulation  in  nucleated  erythrocytes 

Previous  Serial  Number:   None 

Principal  Investigators:   Floyd  M.  Kregenow,  M.D. 

Larry  C.  Stoner,  Ph.D. 

Other  Investigators:  None 

Cooperating  Units:   None 

Project  Description: 

Previous  studies  from  this  laboratory  have  shown  that  avian 
erythrocytes  contain  a  "volume  controlling  mechanism"  which  can 
regulate  cell  size  in  isotonic  or  anisotonic  media.   This  mech- 
anism returns  cells  to  their  original  volume  in  either  hypo,  hyper, 
or  isotonic  media,  even  when  the  cation  pump  is  blocked.   In  iso- 
tonic media,  the  response  is  hormone  dependent,  requiring  one  of 
the  catecholamines.   The  mechanism  allows  cells  to  dynamically 
control  their  volume,  a  property  which  is  critical  to  most  animal 
cells.   The  process  involves  the  controlled  movement  of  consider- 
able quantities  of  potassium  into  or  out  of  the  cell,  accompanied 
by  diffusable  anion,  primarily  chloride,  and  osmotically  obliged 
water.   Conceptually,  the  mechanism  consists  of  a  receptor, 
transmitter,  and  effector.   The  latter  is  presumably  located  in 
the  membrane  and  consists  of  transport  mechanisms  which  would 
have  been  previously  categorized  as  part  of  the  leak.   Previous 
evidence  indicated  that  two  transport  mechanisms  were  involved: 
one  operates  when  cells  correct  volume  by  losing  cations  (cell 
shrinkage) ,  while  the  other  functions  when  cells  correct  volume 
by  gaining  cations  (cell  enlargement) . 

Objectives : 

I.   To  provide  additional  evidence  that  the  transport  pro- 
cess associated  with  cell  enlargement  is  functionally  separate 
from  both  cation  pump  and  the  transport  process  associated  with 
cell  shrinkage. 

II.   To  determine  the  role  anions  play  in  both  transport 
processes. 

III.   To  determine  whether  the  formation  of  functional  "recon- 
stituted ghosts"  involves  the  volume  controlling  mechanism. 


2o7 


Project  No,  Z01  HL  01212-01  KE 

IV.  To  develop  methods  and  procedures  for  measuring  trans- 
port in  a  single  cell. 

Major  Findings: 

I.  To  provide  further  evidence  that  the  transport  process 
associated  with  cell  enlargement  is  functionally  separate  from 
the  classical  pump  and  also  different  from  the  transport  process 
responsible  for  cell  shrinkage,  we  studied  the  response  of  cells 
modified  in  two  ways.  The  first  method  involved  treating  normal 
cells  with  1  mM  furosemide.  In  the  second,  we  replaced  cellular 
CI  with  SOiw  producing  cells  we  have  labelled  "S04  cells."   Both 

groups  of  cells  display  normal  Na  and  K  transport  through  the 
classical  cation  pump.   In  contrast,  the  process  of  ceil  enlarge- 
ment with  its  net  uptake  of  Na,  K  and  H20  as  well  as  the  charac- 
teristic rapid  bidirectional  exchange  of  Na  and  K  is  as  the 
characteristic  rapid  bidirectional  exchange  of  Na  and  K  is  com- 
pletely inhibited.   Although  both  groups  of  treated  cells  can  no 
longer  control  their  volume  by  enlarging,  the  process  of  cell 
shrinkage  remains  intact.   The  phenomenon  of  shrinkage  in  cells 
treated  with  furosemide  is  unaltered,  i.e.  the  loss  of  K,  CI  and 
H2O  and  the  characteristic  increase  in  K  efflux  remain  the  same. 
The  shrinkage  phenomenon  also  remains  intact  in  SOt,  cells,  al- 
though the  rate  of  electrolyte  and  water  loss  is  4  times  slower. 

II.   Continuing  our  study  of  the  role  anions  play  in  the  pro- 
cess of  cell  enlargement  and  shrinkage  (see  Annual  REport  1974) , 
we  tested  whether  any  major  change  in  cellular  phosphate  occurred 
during  either  phenomenom.   Removing  phosphate  from  the  bathing 
medium  had  no  effect  on  either  process;  nor  is  the  total  organic 
phosphate  content  of  a  TCA  extract  of  cells  (37±2  mM  POit/L     - 

90%  of  which  is  ATP  and  phytic  acid)  altered  during  either  pro- 
cess . 

Sits,  a  disulfonic  acid  derivative,  is  an  amino  reactive 
agent  which  in  human  erythrocytes  is  known  to  inhibit  anion 
transport  without  affecting  cation  transport.   In  duck  erythro- 
cytes this  agent  also  inhibits  anion  transport,  as  indicated  by 
a  99.7%  reduction  of  SCU  influx  into  "SO^  cells,"  while  it  is 
without  effect  on  the  cation  fluxes  characteristic  of  cell  en- 
largement and  shrinkage. 

Two  media  have  been  developed  which  will  allow  us  to  study 
the  effects  of  pH  on  the  one  hand  and  pH  and  bicarbonate  concen- 
tration on  the  other. 

III.  Human  red  cells  can  reacquire  their  normal  cation  im- 
permeability after  undergoing  hemolysis  in  hypotonic  media.  The 
process  of  restoration  requires  changes  in  temperature  and 

2  -fee 


Project  No.  Z01  HL  01212-01  KE 

electrolyte  concentration  and  produces  an  altered  cell  form 
(called  a  ghost)  which  may  have  both  a  normal  biconcave  shape 
and  cation  content.   One  can  also  make  ghosts  from  nucleated 
cells.   To  examine  whether  the  process  of  restoration  in  these 
cells  results  in  a  predictable  adjustment  in  cell  volume,  we 
examined  ghosts  which  had  lost  3/4  of  their  intracellular 
hemoglobin . 

If  the  restoring  solution  differs  in  electrolyte  content  by 
as  much  as  100  mM  KC1,  so  that  the  resultant  ghosts  have  a  2-fold 
difference  in  KC1  content,  ghost  volume  (measured  as  the  non- 
inulin  space)  remains  the  same  ^3%.   This  volume  is  similar  to 
what  one  would  predict  if  during  restoration  the  ghosts,  despite 
their  different  electrolyte  content,  reacquired  the  same  H20  con- 
tent of  normal  cells  but  were  minus  the  volume  normally  occupied 
by  3/4  of  the  hemoglobin,  (25%  of  the  original  cell  volume) .   Ca 
must  be  present  at  the  time  of  hemolysis  for  adequate  restoration, 
and  the  process  is  also  facilitated  by  the  presence  of  POi*  . 

Whether  this  phenomenon  represents  a  form  of  volume  regula- 
tion which  is  independent  of  intracellular  protein  content  must 
await  more  precise  measurements  of  ghost  volume  and  neterogeneity 
using  a  Coulter  cell  volume  analyzer.   It  should  be  mentioned  that 
the  volume  controlling  mechanism,  mentioned  previously,  may  not 
be  involved. 

Ghosts  prepared  in  this  manner  can  maintain  a  20-fold  con- 
centration gradient  for  K,  have  a  K  permeability  comparable  to 
normal  intact  cells,  but  fail  to  respond  to  hypotonicity ,  hyper- 
tonicity,  or  norepinephrine.   Thus,  the  volume  controlling  mech- 
anism as  defined  in  intact  cells,  is  either  inoperable  or  not 
present  in  these  reconstructed  ghosts. 

IV.   Procedures  have  been  developed  using  nucleated  red 
cells  from  the  giant  salamander,  Amphiumia  for  measuring  trans- 
port in  a  single  cell  preparation.   Values  obtained  from  this 
technique  are  being  correlated  with  measurements  of  ion  transport 
on  a  large  cell  population.   (See  individual  project  report  of 
Stoner  and  Kregenow 

Proposed  Course  of  Project: 

1.  To  continue  the  objective  stated  in  II  -  IV. 

2.  To  design  experiments  to  test  for  the  presence  of  the 
mechanism  in  human  erythrocytes. 

Keyword  Descriptors:   Cation  Transport,  Volume  Controlling 

Mechanism,  Cell  Enlargement,  Single  Cell 
Measurements 


3c? 


Project  No.  Z01  HL  01212-01  KE 

Honors  and  Awards:   Symposium  talk  in  honor  of  Dr.  M.  Jacobs 
presented  at  1974  Federation  Proceedings  and  entitled,  "Charac- 
terization of  a  Mechanism  Capable  of  Regulating  Cell  Size  in 
Nucleated  Erythrocytes." 

Publications:  Effect  of  Norepinephrine  and  Hypertonicity  on  K 
Influx  and  Cyclic  AMP  in  Duck  Red  Cells..  Floyd 
M.  Kregenow,  Dianne  E.  Robbie,  and  Jack  Orloff. 
Submitted  to  Am.  J.  Physiology  for  publication. 

Two  manuscripts  in  preparation. 


3/o 


Project  No.  Z01  HL  01213-05  KE 

1.  Kidney  &  Electrolyte 

2.  Experimental  Cardiovas- 

cular Diseases 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Cardioglobulin  B-s  of  human  serum 

Previous  Serial  Number:   NHLI-67 

Principal  Investigators:   Stephen  Hajdu,  M.D. 

Edward  J.  Leonard,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   Biology  Branch,  NCI 

Project  Description: 

Objectives:   Cardioglobulin  is  the  name  we  have  applied  to 
a  mammalian  protein  system  which  regulates  calcium  entry  into 
cells.   We  reported  previously  that  3  components  of  the  system, 
cardioglobulin-A,  -B  and  -C  circulate  in  the  blood  plasma. 
Cardioglobulin-C  contains  bound  calcium,  cardioglobulin-A  has  a 
high  energy  phosphate.   The  cardioglobulin  proteins  in  the  blood 
become  activated  when  cardioglobulin-B  interacts  with  a  cell  sur- 
face component.   The  integrated  action  of  the  whole  system  is  to 
release  protein-bound  cardioglobulin-C  calcium.   We  believe  that 
the  protein  system  is  located  in  a  region  immediately  external 
to  the  plasma  membrane  and  is  capable  of  maintaining  a  local  cal- 
cium ion  concentration  which  is  higher  than  and  independent  of 
the  serum  calcium.   In  muscle,  the  calcium  of  the  cardioglobulin 
space  (immediately  external  to  the  plasma  membrane  and  in  the  T 
system)  enters  the  cell  during  depolarization  and  causes  shorten- 
ing of  the  contractile  protein.   Cardioglobulin  may  be  localized 
on  the  surface  of  other  types  of  cells  as  well  as  muscle.   Ab- 
normalities in  serum  cardioglobulin  activity  have  been  found  in 
human  diseases,  notably  systemic  lupus  erythematosus. 

Cardioglobulin  is  assayed  on  the  isolated  frog  heart.   Al- 
though the  frog  does  not  have  the  complete  cardioglobulin  system 
its  cardiac  muscle  has  a  surface  component  on  which  mammalian 
cardioglobulin  proteins  can  be  assembled  and  activated.   Activa- 
tion of  the  system  results  in  entry  of  calcium  ions  into  the 
heart  muscle,  the  reflection  of  which  is  increased  contractile 
force.   At  high  cardioglobulin  concentrations,  contracture  of 
the  heart  muscle  occurs. 


3« 


Project  No.  Z01  HL  01213-05  KE 

In  our  previous  work  we  used  rat  serum  as  a  source  of  some 
cardioglobulin  proteins  and  human  serum  for  others.   We  now 
report  the  results  of  a  study  on  human  cardioglobulin.   In  this 
report  we  show  that  human  serum  cardioglobulin  comprises  4  or 
possibly  5  components.   The  action  of  the  whole  system  can  be 
broken  down  into  3  separate  steps.   Step  1  requires  the  inter- 
action of  cardioglobulin  -Bl    and  -A.   Step  2  requires  cardio- 
globulin -B2  and  -A.   The  final  step  is  mediated  by  cardio- 
globulin -C  and  -A. 

Methods:   An  outline  of  protein  chemistry  used  for  the 
separation  of  the  individual  members  of  the  system  was  given  in 
a  previous  report  (NHLI-67). 

Major  Findings:   We  found  previously  that  the  action  of 
cardioglobulin  on  the  frog  heart  could  be  divided  into  2  steps. 
First,  diluted  human  serum  was  equilibrated  with  the  heart  for 
20  minutes  at  25°C.   During  this  time  cardioglobulin  -B  became 
bound  to  the  heart  and  was  not  removed  by  subsequent  washing 
with  Boyle-Conway  solution.   The  heart  after  washing  was  called 
a  B-  heart.   In  the  second  step,  addition  of  cardioglobulin-A 
and  -C  caused  the  cardiotonic  action  of  the  cardioglobulin  sys- 
tem. 

Our  initial  attempt  to  purify  cardioglobulin-B  was  to  separa- 
ate  serum  into  2  fractions  with  15%  sodium  sulfate.   No  -B  acti- 
vity was  found  in  either  the  0-15%  supernatant  solution  or  in  the 
precipitate,  but  activity  was  recovered  when  the  2  fractions  were 
combined.   This  showed  that  -B  activity  required  2  serum  compon- 
ents which  we  called  -Bi  and  -B2 .   The  next  advance  in  cardio- 
globulin -B  fractionation  was  based  on  our  finding  that  dextran 
sulfate  (DS) precipitates  one  of  the  -B's.   These  findings  suggest- 
ed it  might  be  possible  to  purify  one  of  the  -B  components  with 
DS.   Therefore  500,000  MW  DS  was  added  to  serum,  in  a  final  con- 
centration of  400  ug/ml.   The  precipitate  was  separated  by  cen- 
trifugation  and  dissolved  with  500  yg/ml  protamine  sulfate.   We 
then  determined  whether  this  material  could  reconstitute  B  activi- 
ty when  combined  with  the  serum  sodium  sulfate  fractions  Bi  or  B2  . 
We  found  that  DS  precipitate  produced  B  activity  when  mixed  with 
-Bj  (15%  sodium  sulfate  supernatant  solution)  but  not  with  -B2  . 
Therefore  DS  precipitated  -B2 .   To  test  the  supernatant  of  DS- 
treated  serum  for  -Bx  activity,  we  first  removed  residual  free  DS 
by  precipitating  the  serum  proteins  with  20%  sodium  sulfate. 
When  we  tested  the  DS  (0-20)  fraction  for  -Bi  activity  by  combin- 
with  DS  B2 ,  no  -Bj  activity  was  found.   This  could  occur  if  Bi  it- 
self was  inactivated  by  DS  or  if  besides  Bi  and  B2  a  third  compon- 
ent was  required  for  the  compilation  of  the  B-reaction  and  it  is 
this  which  is  DS  sensitive.   As  third  component  of  the  B-  reaction, 
we  thought  of  cardioglobulin  -A,  since  it  was  known  to  be 
DS  sensitive.   We  tested  this  latter  possibility  with  a  23%  sod- 
ium sulfate  supernatant  which  lacks  all  the  cardioglobulin 
components  except  ca  50%  cardioglobulin  A.   Test- 

o  3& 


Project  No.  Z01  HL  01213-05  KE 

ing  of  this  fraction  showed  that  it  could  reconstitute  B  activity 
when  combined  with  DS-B2  and  DS  (0-20) .   We  concluded  from  these 
experiments  that  3  components  were  required  to  make  a  B-heart: 
Bl,  found  in  a  20%  sodium  sulfate  precipitate,  and  unaffected   by 
DS;  B2,  a  euglobulin,  precipitable  with  DS;  and  a  component  in- 
activated by  DS  and  found  in  the  supernatant  of  a  23%  sodium  sul- 
fate fraction  of  whole  serum,  which  is  probably  -A.   Experiments 
were  made  to  determine  whether  all  3  of  the  B  components  had  to 
be  present  simultaneously.   It  was  found  that  a  B  heart  was  made 
when  Bl  +  A,  and  in  a  separate  step  B2   +  A  was  added  to  the 
heart.   Reverse  order  or  omission  of  A  in  either  step  led  to  nega- 
tive results. 

A-Inhibitor .   We  have  shown  that  the  cardioglobulin  reaction 
occurs  in  a  sequence  of  3  steps:  +B1  +  A;  B2  +  A;  C  +  A.   Since 
all  these  components  are  present  in  serum,  one  would  expect  the 
complete  sequence  to  occur  when  serum  is  added  to  the  frog  heart. 
However,  addition  of  as  much  as  1.0  ml  of  human  serum  at  25 °C, 
does  not  result  in  the  characteristic  action  of  cardioglobulin  on 
the  frog  heart.   Much  less  than  1.0  ml  of  serum  (0.1-0.2  ml) 
causes  contracture  if  the  serum  is  applied  in  2  steps,  with  a 
10  minute  Boyle-Conway  solution  wash  in  between  the  2  steps.   We 
asked  whether  serum  after  a  20  minute  equilibration  with  a  fresh 
frog  heart  at  25°C  had  A  +  C  activity  as  tested  on  a  B-heart. 
This  used  serum  not  only  did  not  cause  contracture  of  a  B-heart, 
but  it  could  even  inhibit  the  action  of  fresh  serum  on  a  B-heart. 
It  was  shown  that  the  addition  of  cardioglobulin-A,  but  not  -C 
overcame  the  inhibition  of  the  used  serum.   We  concluded  that 
equilibration  of  fresh  human  serum  with  a  frog  heart  caused 
elaboration  of  an  inhibitor  of  cardioglobulin-A  which  prevented 
the  cardioglobulin  reaction  from  going  to  completion. 

We  finally  succeeded  in  purifying  and  storing  human  cardio- 
globulin components  under  the  following  conditions: 

-A  free  of  -C  and  -B2 

-C  free  of  -A  and  -B2 

-B-  free  of  -B2 

-B2  free  of  -A,  -C  and  -Bl 

Possession  of  these  components  enabled  us  to  measure  any  cardio- 
globulin component  of  a  human  serum  sample  quantitatively. 

Proposed  Course  of  Project:   This  project  comes  to  an  end  by 
September  30,  1975,  due  to  the  retirement  of  one  of  us  (S.H.). 
In  the  time  remaining  an  attempt  will  be  made  to  assess  the  indi- 
vidual components  of  cardioglobulin  in  normal  subjects  and  in 
paritents  with  systemic  lupus  erythematosus. 


3(% 


Project  No.  Z01  HL  01213-05  KE 
Keyword  Descriptors:   Lupus  Erythamotosus ,  Calcium  Transport 
Honors  and  Awards :   None 
Publications:   None 


w 


ANNUAL  REPORT  OF  THE 

LABORATORY  OF  TECHNICAL  DEVELOPMENT 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

JULY  1,  1974  THROUGH  JUNE  30,  1975 


This  laboratory  continues  to  develop  new  instruments  and  methods  to 
facilitate  medical  research,  diagnosis  or  therapy.   In  many  respects,  the 
boundaries  of  our  knowledge  and  our  ability  to  apply  what  we  know  are 
limited  by  our  technology.   Methods  development  and  new  instrumentation 
serves  to  widen  our  horizons  and  to  convert  knowledge  to  practice.   We  have 
a  group  of  scientists  selected  for  their  interests  and  capabilities  to 
identify  requirements  of  biomedical  science  that  can  benefit  from  the 
application  of  instrumentation  science  and  technology.   We  develop  working 
systems  that  can  be  applied  at  N.I.H.  in  conjunction  with  ongoing  research' 
activities  make  the  results  available  to  the  scientific  community  and 
industry  by  publishing  the  method  or  instrument.   The  areas  of  our  activity 
include  analytical  methods  and  separation  techniques,  not  only  for  chemicals 
but  also  for  living  cells;  clinical  applications  include  regional  blood  flow 
measurement,  materials  and  methods  development  for  better  cardiopulmonary 
support  for  both  isolated  organs  and  whole  animals. 

An  instrument  for  the  rapid  determination  of  the  oxygen  dissociation  curve 
(ODC)  of  hemoglobin  has  been  constructed  and  is  presently  operating  in  the 
Laboratory  of  Molecular  Hematology.   The  instrument  was  developed  in 
cooperation  with  scientists  at  the  University  of  Milan  primarily  for  the 
investigation  of  the  fundamental  properties  of  the  interaction  of  hemoglobin, 
oxygen,  and  small  effector  molecules.   It  is  interesting  to  note  that 
physical  theories  based  on  the  action  of  purified  hemoglobin  have  been  shown 
to  be  invalid  from  experiments  done  on  sickle  cell  patients  with  varying 
degrees  of  irreversibly  sickled  cells  (ISC's). 

Fast,  reliable  thermistors  developed  for  use  in  the  study  of  biochemical 
reactions  have  now  been  thoroughly  tested  and  two  types  are  now  available 
commerically .   A  new  differential  amplifier  developed  to  be  used  with  them 
has  been  incorporated  into  prototypes  of  two  new  instruments.   One,  a 
differential  thermal  titration  calorimeter  is  to  be  used  in  conjunction  with 
a  new  differential  pH  meter,  to  measure  simultaneously  the  thermometric  and 
potentiometric  titration  curve  of  a  protein.   The  complete  curve,  from  pH  4 
to  10  can  be  run  in  2  minutes  on  a  2  ml  sample  containing  0.1  y  mole  of 
protein.   Such  titrations  permit  the  protein  chemist  to  identify  the  groups 
active  in  biochemical  reactions  as  well  as  those  reacting  with  inhibitors. 

The  second  instrument  is  the  stopped-flow  calorimeter  which  operates  in  the 
millisecond  range.   With  a  resolving  time  of  3  millisecond  it  supplements 
the  normal  stopped-flow  spectrophtometer  permitting  observation  of  pre- 
steady-state  reactions  that  lack  an  observable  optical  absorption.   The 
reaction  of  glutamic  dehydrogenase  and  ATPase  are  typical  samples.   The 
exploitation  of  this  system  will  be  carried  out  on  several  well  documented 
reactions  to  demonstrate  its  advantages.   In  another  development  a  high- 


3/r 


speed  quenched  flow  apparatus  has  been  constructed  and  thoroughly  tested. 
A  stepping  motor  4  syringe  drive  accurately  controlled  as  to  rate  and 
distance  of  advance  is  used  with  three  ball  mixers  and  an  automatic  sampling 
valve.   For  example,  in  the  study  of  sacroplasmic  reticulum  at  the  Institute 
of  Aging,  ATP  is  added  to  the  SR's  in  the  first  mixer,  at  different  times 
CA  is  added  in  the  second  mixer,  and  again  at  different  times  TCA  is  added 
to  quench  the  reaction  and  then  the  sample  is  quickly  collected  by  the 
automatic  sampler.   A  resolving  time  of  3  milliseconds  has  been  achieved. 

Calorimetry  using  heat  production  as  an  indicator  of  biochemical  and 
metabolic  activity  may  be  useful  in  indicating  changes  in  individual  cells 
without  harm  to  the  cells  or  modifying  their  behavior. 

In  the  host  of  cytological  tests  for  specific  biochemical  defects, 
immunoresponses  of  various  leucocytes,  and  transformation  and  oncogenesis 
induced  by  mitogens,  antigens,  specific  factors  and  organisms.   Specific 
cell  response  requires  a  destructive  or  at  best  intrusive  procedure  such  as 
staining  or  assay  of  radiothymidine  uptake  by  radioautography .   A  method 
whereby  the  heat  given  off  by  individual  cells  can  be  observed  as  inter- 
ference fringes  around  each  cell  in  the  microscope  field  requires  only  minor 
modification  of  the  microscope  optics  and  a  substage  thermal  cycling  device 
to  alternately  condense  and  remove  a  liquid  film  from  the  underside  of  a 
thin   film  supporting  the  specimen.   Granulocytes  ingesting  bacteria  show 
their  heat  production  is  related  to  the  number  of  organisms  ingested  and  on 
autolysis.   The  system  should  permit  the  same  cells  to  be  used  as  controls 
and  the  proportion  of  cells  responding  to  various  agents  determined  with  the 
test  cells  available  for  further  culture  or  other  assays. 

A  cell  growth  assay  system  explored  the  use  of  specific  spatial  filtering 
opportunities  afforded  by  the  availability  of  coherent  laser  light  sources 
and  led  to  the  development  of  a  prototype  stem  cell  colony  assay  device 
using  a  moving  grid  to  selectively  modulate  scattered  light  from  stem  cell 
colonies  and  discriminate  against  scattered  light  background  from  the 
semisolid  medium.   A  laboratory  model  for  evaluation  is  being  produced  on 
a  Department  of  Defense  contract  to  N.C.I. 

An  optical  device  made  possible  by  the  availability  of  laser  sources  is  able 
to  indicate  the  velocity  spectrum  of  moving  blood  corpuscles  in  a  capillary 
bed  and  small  vessels  by  analysis  of  the  optical  frequency  shift  produced 
by  scattering  from  the  moving  cells.   Observation  of  the  capillary  bed  of 
fingertips,  and  exposed  internal  organs  while  vasoactive  maneuvers  and 
pharmocologic  agents  are  administered  indicates  that  the  system  can  analyze 
vascular  responses  from  the  frequency  spectrum  of  backscattered  laser  light. 
Comparison  with  xenon  washout  techniques  in  a  clinical  test  indicated 
weaknesses  in  conventional  methods  and  the  desirability  of  the  new  method 
for  non- intrusive  skin  measurements  and  relatively  small  needle  probes  or 
light  pipes  for  internal  organs. 


3(C> 


Non-invasive  non-destructive  nuclear  magnetic  resonance  methods  for 
circulation  studies  is  now  ready  for  some  trials  on  clinical  materials 
and  large  animals  with  the  acquisition  of  a  large  magnet  at  the  Medical 
College  of  Wisconsin  on  our  contract  with  them  for  studies  using  electronic 
systems  developed  here  and  reduced  to  practice  there. 

Systems  of  measuring  blood  gasses  on  a  continuing  basis  for  acute  care  and 
vital  support  have  been  developed  in  conjunction  with  methods  for  providing 
extracorporeal  gas  exchange.   As  these  values  change  rapidly,  a  fast 
responding  continuous  indicator  is  desirable  without  continuous  consumption 
of  blood  for  analysis.   Following  techniques  developed  previously  for 
ultramicro  determination  of  picomolar  amounts  of  CO   and  Bicarbonate  in 
kidney  tubule  micropuncture  samples,  current  systems  sample  by  introducing 
a  micro  diffusion  cell  into  the  vascular  system  at  the  end  of  a  catheter 
and  sample  the  gasses  without  withdrawing  blood.   The  galvanic  reduction 
current  is  used  for  oxygen  and  the  heat  of  reaction  with  lithium  hydroxide 
indicates  CO  with  adequate  precision  and  accuracy.   Present  work  is 
directed  to  dependable  diffusion  sampling  beads. 

Some  separations  of  biochemical  interest  require  the  use  of  phases  with  low 
interfacial  tension.   When  low  interfacial  tension  phases  are  used  in  our 
centrifugal  countercurrent  chromatograph  emulsif ication  and  loss  of 
stationary  phase  degrades  the  system.   A  30   rotor  was  constructed  to  reduce 
emulsif ication  forces  and  the  system  tested  with  several  extraction  phases 
with  a  range  of  interfacial  tensions  to  see  the  effect  of  the  angle  rotor. 
The  single  rotor  performed  well  with  even  extremely  low  interfacial  tension 
polymer  phases  and  was  demonstrated  to  be  able  to  separate  bone  marow 
colony  stimulating  factor  at  high  efficiency  thus  contributing  to  CSF  work 
and  validating  the  concept  of  the  angle  rotor  countercurrent  chromatograph 
which  in  addition  to  the  above  has  advantages  over  other  conventional  liquid 
chromatographic  systems. 

Centrifugal  separation  systems  using  counterrotating  columns  to  avoid 
rotating  seals  while  permitting  continuous  flow  have  been  unsuitable  for 
blood  separation  due  to  the  secondary  centrifugal  force  set  up  by  the 
counterrotation.   A  new  concept  in  continuous  flow  to  rotating  systems 
published  recently  provided  the  basis  for  a  new  blood  separating  centrifuge 
that  eliminates  the  rotating  seal  that  is  generally  conceded   to  be  a  source 
of  cell  damage,  microemboli  and  generally  incompatible  with  blood  due  to 
the  limits  imposed  by  materials  suitable  for  rotating  seals.   Current  work 
uses  the  doubled  back  loop  in  which  the  rotor  speed  is  twice  the  speed  of 
the  loop  which  is  guided  by  a  tooth  belt  driven  guide.   Blood  continuously 
introduced  is  separated  into  plasma  and  cells  as  in  the  plasmapheresis 
(celltrifuge)  machine.   Platelet  survival  in  the  new  device  indicated 
minimum  damage.   Tests  with  our  sheep  perfusion  heart  preservation  system 
also  indicated  great  advantages  could  be  expected  to  result  from  the 
modification.   Applicability  to  plasmapheresis,  cell  and  platelet  trans- 
fusions and  organ  preservation  is  anticipated.   It  would  also  appear  to 
be  a  desirable  approach  to  washing  frozen  preserved  blood  cells. 


1(7 


In  order  to  advance  a  method  or  technique,  it  is  often  necessary  to  show 
how  it  can  solve  certain  problems  of  interest.   In  the  case  of  luminescence 
spectroscopy,  involving  either  fluorescence  or  phosphorescence,  there  are 
many  biochemical  problems  amenable  to  the  method.   We  choose  to  work  on 
some  intrinsically  interesting  problems  to  show  the  utility  of  luminescence. 

A  phosphorescence  study  was  performed  which  showed  that  Ag  markedly  enhances 
the  phosphorescence  of  tryptophan  (3-fold)  and  totally  quenches  the 
fluorescence;  the  study  also  showed  that  proteins  phosphorescence  was  altered 
by  Ag   in  various  ways.   The  effect  of  Ag  in  proteins  containing  sulfhydryl 
groups  could  be  attributed  to  total  luminescence  quenching  by  energy 
transfer  to  Ag  -mercaptide  absorption  bands.   However,  only  fluorescence  is 
quenched  in  non-sulfhydryl  proteins.   It  was  found  that  10%  methanol  snows 
at  77  K  were  suitable  matrices  for  the  study,  and  it  was  suggested  that 
previous  studies  on  protein  phosphorescence  done  in  glasses  of  organic  sol- 
vents may  have  studied  only  denatured  proteins.   Enzyme  activity  measurements 
showed  no  denaturation  on  10%  methanol. 

The  study  of  membranes  and  lipid  micelles  by  fluorescence  has  become 
popular  in  the  biochemical  literature.   We  have  found  that  certain  dyes 
(TNS,  ethidium  bromide,  quinacrine)  show  markedly  altered  fluorescence  in 
detergent  solutions  of  different  concentration.   In  fact,  the  critical 
micelle  concentration  (cmc)  can  be  detected  by  following  such  probe 
fluorescence.   We  have  measured  the  emission  of  some  detergent  solutions 
in  the  presence  of  various  amounts  of  salt  (which  alters  the  cmc)  and 
confirmed  the  effect. 

The  binding  of  fluorescent  compounds  by  certain  enzymes  has  yielded 
information  about  the  active  sites.   L.  Brand  reported  that  Auramine  0  was 
bound  by  liver  alcohol  dehydrogenase  but  not  by  yeast  alcohol  dehydrogenase 
as  shown  by  the  marked  enhancement  of  Auramine  0  fluorescence.   On  the  other 
hand,  we  have  examined  the  quenching  of  protein  fluorescence  of  these  enzymes 
by  Auramine  0,  and  find  that  both  enzymes  do  bind  the  dye.   In  contrast  to 
previous  reports,  therefore,  dyes  whose  fluorescence  is  not  enhanced  cannot 
be  assumed  not  to  bind  to  a  given  protein.   Similarly,  the  antimalarial  drug 
primaquine  was  reported  by  T.  Li  to  inhibit  liver  alcohol  dehydrogenase 
noncompetitively  but  not  to  inhibit  yeast  alcohol  dehydrogenase.   We  find 
that  both  enzymes  bind  the  drug,  confirm  that  yeast  ADH  is  not  inhibited, 
but  find  that  the  inhibition  is  competitive  with  the  coenzyme,  NAD. 

These  studies  have  produced  results  of  interest  to  biochemists  and  again 
illustrate  the  utility  of  fluorescence  and  phosphorescence  methods. 

The  section  on  Pulmonary  and  Cardiac  Assist  Devices  has  continued  studies 
to  imporve  the  inherent  safey  of  extracorporeal  pulmonary  support  perfusion, 
particularly  as  related  to  long-term  use. 

We  have  developed  a  method  of  coating  the  blood  contacting  surfaces  of  our 
membrane  oxygenators  with  a  pure  silicone  rubber  gum  rather  than  the  usual 


ve 


silica  reinforced  material  previously  used.   Perfusions  performed  in  sheep 
have  shown  better  platelet  sparing,  reduced  heparin  requirement  and  reduced 
general  morbidity.   Recent  clinical  experience  elsewhere  using  pure  gum 
surface  oxygenators  appears  to  confirm  our  experience  of  greater  blood 
compatability. 

The  ability  to  fabricate  multilayer  membranes  now  permits  the  production 
of  membranes  with  desirable  surface  properties  and  specific  permselectivi- 
ties.   A  membrane  incorporating  carbon  black  did  not  produce  the  usual 
transient  agranulocytosis  universally  found  at  the  beginning  of  bypass  with 
all  other  membrane  lungs  and  renal  dialysis  systems. 

We  have  recently  acquired  capability  to  cast  f luorosilicone  gum  membranes 
as  well  as  to  coat  exisiting  membranes  with  polymers  other  than  silicones.' 

For  use  in  long  term  bypass  we  have  explored  several  methods  to  measure 
cardiac  output.   Almost  all  presently  used  techniques  provide  intermittent 
data  on  cardiac  output,  the  accuracy  of  which  is  often  dubious.   The 
membrane  lung  in  the  extracorporeal  blood  circuit  serves  as  a  meter  by 
providing  a  known  quantity  of  oxygen  to  allow  continuous  determination  of 
cardiac  output  in  all  patients  undergoing  right  sided  bypass  with  the 
membrane  lung,  using  the  technique  of  Fick  cardiac  output  determination. 
Provided  all  blood  from  the  membrane  lung  is  returned  into  the  root  of  the 
aorta,  left  sided  cardiac  output  determinations  are  similarly  feasible. 

We  have  further  explored  factors  to  successful  preservation  of  the  mammalian 
heart,  using  sheep  hearts.   The  perfusion  circuit  was  coated  with  pure  gum 
silicone  rubber  by  a  technique  developed  here.   We  have  previously  shown  that 
sheep  hearts  when  perfused  ex  vivo  at  10  and  13  C  continued  to  show 
electrical  activity  and  exhibited  forceful  ventricular  contractions  for 
many  days.   In  this  study  we  sought  to  determine  which  of  the  cellular 
fractions  of  whole  blood  were  critical  to  success  of  organ  preservation. 
Separation  of  fresh  whole  blood  into  several  fractions  was  accomplihsed  in 
the  "Celltrifuge".   We  studied  platelet-poor  plasma,  platelet-rich  plasma, 
platelet-rich  plasma  with  whole  blood  added  to  give  a  hematocrit  of  1-2%, 
and  incompletely  separated  plasma,  i.e.  plasma  containing  some  red  blood 
cells,  white  blood  cells,  and  platelet-rich  plasma  (hematocrit  1-2%).   All 
studies  were  performed  at  13  C.   As  with  hearts  perfused  with  whole  blood, 
hearts  in  all  four  groups  showed  ventricular  contractions  for  various 
lengths  of  time,  with  a  peak  left  ventricular  systolic  pressure  as  high  as 
70  mm  Hg.  and  a  heart  rate  of  15-20  contractions  per  minute.   However,  the 
longest  and  most  forceful  contractions  were  in  groups  having  some  red  blood 
cells  in  the  perfusate,  and  particularly  the  group  where  red  blood  cells 
were  only  incompletely  separated.   These  hearts  on  rewarming  had  no  weight 
gain,  and  had  excellent  ventricular  function  on  rewarming.   Of  particular 
interest  in  our  study  with  a  sheep  heart  is  the  finding  that  continuous 
ventricular  contraction  at  13  C  is  an  excellent  criteria  of  cardiac 
viability.   Furthermore,  that  in  spite  of  PO   levels  above  100  mm  Hg, 
ventricular  contractions  ceased  if  flow  of  fresh  perfusate  fell  below 


3(9 


15  cc/min.   This  observation  may  suggest  a  different  mechanisms  to  cardiac 
failure  secondary  to  impaired  coronary  circulation:  "ischemia"  need  not 
only  be  the  result  of  hypoxia,  but  may  also  be  a  manifestation  of  inadequacy 
of  as  yet  undefined  substrates.   These  substrates  appear  to  be  of  molecular 
weight  5,000  to  50,000  as  determined  from  our  previous  studies  using  blood 
ultraf iltrate  as  perfusates. 


33L0 


Project  No.   Z01  HL   01401-02   LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  thorugh  June  30  1975 

Project  Title:   Angle  Rotor  Countercurrent  Chromatography 

Previous  Serial  Number:   NHLI-80 

Principal  Investigator:   Yoichiro  Ito 

Other  Investigator:   Robert  L.  Bowman 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Demonstration  of  the  potential  capability  of  the  30  angle 
rotor  countercurrent  chromatograph  in  terms  of  the  following: 

1.  Applicability  of  two-phase  solvent  systems  having  varieties  of 
interfacial  tension  and  viscosity. 

2.  Determination  of  optimum  operational  ranges  in  revolutional  speeds  and 
flow  rates. 

3.  Comparison  to  high  pressure  chromatographic  method. 

Methods  Employed  and  Major   Findings: 

The  30   angle  rotor  previously  described  was  employed  throughout.   The 
coiled  helix  column  was  first  filled  with  the  stationary  phase  and  sample 
solution  introduced  through  the  feed  line.  Then  the  apparatus  was  spun 
at  a  constant  speed  while  the  mobile  phase  was  pumped  through  the  feed 
line  using  a  syringe  driver.   The  eluates  were  either  monitored  directly 
through  a  u.v.  monitor  or  fractionated  into  test  tubes  for  further 
analysis. 

1.   Applicability  of  two-phase  solvent  systems: 

Several  two-phase  solvent  systems  were  selected  in  consideration  of  their 
varieties  in  physical  property  and  versatility  in  solute  partitioning. 
Using  these  solvent  systems,  performance  of  the  apparatus  was  evaluated 
on  separation  of  biological  compounds  as  follows: 

a.   High  interfacial  tension  phase  system:   Ethylacetate/10%  acetic  acid, 
5%NaCl  (1:1)  was  used  for  separation  of  5  catecholamine  metabolites. 

1  3A( 


Project  No.   Z01   HL   01401-02   LTD 

Using  the  same  solvent  system  abnormal  levels  of  VMA  and  HVA  were  also 
detected  from  100  microliter  of  urine  sample. 

b.  Medium  interfacial  tension  phase  system:   N-BuOH/lM  potassium 
phosphate  (pH  6.5)  (1:1)  was  used  for  separation  of  5  purines  and 
pyrimidines  using  the  organic  phase  as  a  mobile  phase.   Also  a  similar 
phase  system  of  n-BuOH/O.lM  ammonium  formate  (1:1)  was  used  for 
separation  of  7  dipeptides  all  containing  tyrosine  moiety  by  applying 
a  linear  gradient  of  dichloroacetic  acid  between  1%  and  0%. 

c.  Low  interfacial  tension  phase  systems:   Chloroform/acetic  acid/  0.1N 
HC1  (2:2:1)  was  used  for  separation  of  9  DNP  aminoacids  and  sec-BuOH/0. 3% 
dichloroacetic  acid  (1:1)  for  partition  of  bovine  insulin. 

d.  Polymer  phase  system  (extremely  low  interfacial  tension  and  high 
viscosity) :   A  polymer  phase  system  composed  of  6%  charged  polyethylene 
glycol  6000,  6%  dextran  T  500,  and  0.05M  sodium  phosphate  buffer  was 
used  for  separation  of  bone  marrow  colony  stimulating  factor  with  pH 
gradient  elution. 

In  spite  of  the  diversity  of  their  physical  properties,  all  above  solvent 
systems  showed  satisfactory  phase  retention  and  yielded  a  high  efficiency 
separation  with  elution  times  ranging  from  5  to  18  hours.   The  results 
also  indicated  that  either  organic  or  aqueous  phase  can  be  used  as  a 
mobile  phase,  and  that  the  gradient  elution  technique  was  adaptable  as  in 
the  conventional  liquid  chromatography. 

2.  Determination  of  optimum  operational  ranges  of  revolutional  speeds 
and  flow  rates: 

Using  the  chloroform/acetic  acid/0. IN  HC1  (2:2:1)  system,  separations 
of  9  DNP  aminoacids  were  performed  by  applying  combinations  of 
revolutional  speeds  (500,  700,  1000,  and  1200  rpm)  and  flow  rates 
(0.25,  0.5,  1.0  and  1.5  ml/hr)  with  a  0.3  mm  i.d.  coiled  helix  column. 
All  combinations  applied  showed  satisfactory  resolution  for  all  9  peaks. 
Although  the  highest  resolution  was  achieved  at  500  rpm  at  0.25  ml/hr 
of  flow  rate  by  an  overnight  run,  a  good  separation  was  also  obtained 
at  1200  rpm  at  1.5  ml/hr  within  6  hours.   Thus  the  results  indicate 
that  the  present  system  permits  a  wide  range  of  operational  conditions. 

3.  Comparison  to  high  pressure  liquid  chromatography: 

Twelve  isomers  of  monohydroxybenzo(a)  pyrene  (1  through  12-HOBP)  have 
previously  been  resolved  into  2  groups  by  a  high  pressure  liquid 
chromatographic  technique.   It  used  a  gradient  elution  with  methanol 
solution  35%  to  75%  through  a  column  packed  with  hydrocarbon-coated 
beads.   The  first  peak  consisted  of  2,  6,  8,  9-HOBP,  and  the  second 
peak,  1,3,4,5,7,10,11,  and  12-HOBP.   Changing  the  solvent  system  or 


:s&a. 


Project  No.  Z01   HL   01401-°^   LTD 

gradient  pattern  has  failed  to  improve  the  resolution. 

Attempts  were  made  to  separate  these  samples  with  the  30   angle  rotor 
using  a  0.3  mm  i.d.,  70  m  long  coiled  helix  column.   Using  hexane/55% 
methanol  (1:1),  or  hexane/methanol/O.OlM  sodium  phosphate  (pH8)  (100:55:45), 
8  isomers  (1,2,3,4,5,6,8,  and  9  -  HOBP)  were  resolved  distinctly  into 
3  peaks,  the  first  peak  consisting  of  2,8,  and  9-HOBP,  the  second  1  and 
7-HOBP  and  the  third,  3,4,  and  5-HOBP.   With  the  former  phase  system, 
the  first  peak  appeared  to  be  further  resolved  into  two  peaks,  probably 
2  and  8-HOBP  and  9-HOBP.   Using  a  single  solute,  column  efficiency  was 
estimated  to  be  over  15000  theoretical  plates.   Although  the  present 
method  failed  to  resolve  all  components,  it  yielded  much  higher  resolving 
power  than  a  refined  high  pressure  liquid  chromatographic  method. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

The  present  method,  in  addition  to  eliminating  complication  from  the 
solid  supports,  proposes  the  following  advantages  over  other  conventional 
liquid  chromatographic  methods.   Applicability  of  two-phase  solvent  systems 
is  almost  universal  and  covers  high  interfacial  tension  hexane/H  0  systems 
to  an  extremely  low  interfacial  tension  polymer  phase  systems  that  are 
hardly  applicable  to  the  conventional  chromatography.   The  efficiency 
yielded  with  a  fine  analytical  column  exceeds  15000  theoretical  plates 
while  that  in  the  conventional  liquid  chromatography  is  limited  to 
several  thousand  theoretical  plates.   The  method  can  be  easily  scaled  up 
for  preparative  separation  using  a  large  bore  column.   The  technique  is 
highly  reproducible  and  simple  enough  for  unskilled  technicians  to  follow. 
On  the  basis  of  these  advantages,  we  feel  that  the  method  will  be  very 
useful  in  biomedical  and  biochemical  research. 

Proposed  Course: 

Although  the  performance  of  the  present  prototype  is  satisfactory  for  our 
laboratory  use,  it  shows  loss  of  lubricant  from  the  bearings  which 
necessitates  weekly  lubrication.   Also  the  revolutional  speed  is  limited 
to  1200  rpm  because  of  the  present  design  in  that  the  lower  end  of  the 
column  holder  projecting  a  few  inches  below  the  lower  bearing  level 
tends  to  overload  the  bearing  and  damage  the  aluminum  column  holder. 
These  deficiencies  should  be  eliminated  in  the  future  model. 

Keyword  Descriptors: 

Angle  Rotor,  two-phase  solvent  systems,  separation  of  biological  compounds, 
Catecholamine  metabolites,  VMA,  HVA,  purines  and  pyrimidines, 
separation  of  7  dipeptides,  separation  of  9  DNP  aminoacids,  partition  of 
bovine  insulin,  polymer  phase  system,  and  colony  stimulating  factor. 


333 


Project  No.  Z01  HL   01401-02   LTD 


Honors  and  Awards:   None 
Publications : 

1.  Ito,  Y.,  Hurst,  R.  E. ,  Bowman,  R.  L. ,  and  Achter,  E.K.: 
Countercurrent  Chromatography,  Separation  and  Purification  Methods, 
(1),  133-165  (1974). 

2.  Ito,  Y.,  and  Bowman,  R.  L.:  Angle  rotor  countercurrent  chromatography, 
Analytical  Biochemistry,  in  press. 


33-*- 


Project  No.  Z01  HL   01402-Ql  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30  1975 

Project  Title:   New  Flow-Through  Centrifuge  Without  Rotating  Seals. 

Previous  Serial  Number:   None 

Principal  Investigators:   Yoichiro  Ito  and  Jacques  Suaudeau 

Other  Investigators:   Theodor  Kolobow,  Gerald  G.  Vurek,  and  Robert  L.  Bowman. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   1.   Designing  and  constructing  a  flow-through  centrifuge 
based  on  the  principle  reported  by  Adams. 

2.   Investigation  of  potential  capability  of  the 
centrifuge. 

Methods  Employed  and  Major  Findings: 

a.  Principle: 

Consider  a  horizontally  placed  bowl  with  a  flexible  tube  connected  at 
the  center  of  the  lower  surface.   The  other  end  of  the  tube  is  then 
supported  above  the  bowl  on  its  central  axis,  making  a  loop  of  the  tube. 
When  the  bowl  rotates  at  an  angular  velocity  of  2  w  around  the  vertical 
axis  and  the  loop  simultaneously  revolves  around  the  same  axis  at  w, 
the  tube  continuously  untwists  and  remains  always  untwisted.   In  this 
situation  the  tube  spontaneously  counterrotates  around  its  own  axis  at  - 
w  during  operation. 

b.  Design  of  the  centrifuge: 

The  prototype  has  been  constructed  by  modifying  a  conventional 
refrigerated  centrifuge.   The  frame  of  the  centrifuge  head  consists  of 
three  parallel  horizontal  rectangular  plates  ridgedly  linked  and  driven 
by  the  motor  shaft  as  a  unit.   The  frame  holds  a  centrifuge  bowl  at  the 
center,  a  contershaft  on  one  side  and  a  tube  supporting  a  tubular  guide 
on  the  opposite  side,  all  vertically  mounted  in  ball  bearings.   A 
stationary  pulley  mounted  on  the  motor  housing  on  the  axis  of  the 
centrifuge  is  coupled  through  a  toothed  belt  to  an  identical  pulley 


3Z? 


Project  No.  Z01   HL   01402-Ql   LTD 


mounted  on  the  countershaft  to  counter-rotate  the  shaft  with  respect 
to  the  rotating  frame.   This  motion  is  further  conveyed  to  the 
centrifuge  bowl  by  1:1  gearing  between  the  countershaft  and  the  bowl. 
This  arrangement  doubles  the  angular  velocity  of  the  centrifuge  bowl 
to  accommodate  the  principle  outlined  above.   To  support  the  counter- 
rotating  flow  tubes  the  tubular  guide  is  actively  counter-rotated 
at  -w  by  means  of  a  pair  of  1:1  ratio  toothed  pulleys  coupled  to  the 
hollow  shaft  and  the  countershaft. 

A  doughnut  shaped  silicone  rubber  bag  (800  ml  capacity)  equipped  with 
three  flow  lines  is  fitted  inside  the  centrifuge  bowl.   The  transparent 
lucite  cover  enables  observation  of  its  contents  under  stroboscopic 
illumination.   Three  silicone  rubber  flow  tubes  from  the  rubber  bag 
are  led  down  through  the  center  of  the  centrifuge  bowl,  then  upward 
through  the  guide  tube,  and  finally  through  the  center  hole  of  the 
stationary  centrifuge  cover.   When  properly  balanced,  the  centrifuge 
bowl  can  be  rotated  at  up  to  2000  rpm  without  detrimental  vibration. 

c.   Application  to  plasmapheresis: 

In  order  to  demonstrate  the  capability  of  the  centrifuge,  sheep  blood 
was  introduced  into  the  centrifuge  directly  from  the  animal,  while 
effluents  of  plasma  and  red  blood  cells  were  returned,  after  sampling, 
to  the  animal.   Flow  rates  through  the  individual  lines  were  controlled 
by  two  rotary  pumps,  one  set  on  the  whole  blood  line  and  the  other  on 
the  plasma  line,  the  thrid  line  having  flow  equal  to  the  difference 
between  the  two  pumps.   With  a  constant  feed  rate  of  60  ml/min  , 
plasma  free  of  red  blood  cells  was  harvested  at  12  ml/min  at  1000  rpm 
or  18  ml/min  at  1300  rpm.   During  12  hours  of  continuous  flow  of 
plasma  at  18  ml/min,  blood  and  plasma  samples  were  collected  by  intervals 
to  study  changes  in  platelet  counts.   The  results  showed  a  50%  reduction 
in  blood  platelet  count  within  the  first  one  hour,  and  a  reduction  to 
30%  of  baseline  values  by  the  12th  hour  of  operation.   These  results 
are  similar  to  reported  studies  using  a  membrane  lung  in  a  similar 
perfusion  system  without  centrifuge  and  are  much  superior  to  flow-through 
centrifuge  that  uses  rotating  seals. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

The  conventional  flow-through  centrifuge  utilizes  rotation  seals  which 
can  become  a  source  of  leaks  between  inflow  and  outflow  lines  and 
represents  a  weak  point  in  the  machinary,  in  terms  of  duration, 
complexity  and  fragility  of  the  pieces,  and  necessity  for  a  continuous 
and  equal  lubrication.   When  those  continuous  flow  centrifuges  are 
adapted  for  an  inline  blood  separation,  as  realized  for  collection  of 


354 


Project  No.  Z01   HL   01402-01   LTD 


blood  cells,  rotating  seals  become  critical  in  terms  of  platelet  injury, 
red  cell  hemolysis,  obstruction  of  the  channels  and  of  the  lubrication 
grooves  by  aggregates,  and  following  inefficiency  of  blood  separation. 
The  present  device  eliminates  all  these  complications  and  therefore 
will  contribute  to  biomedical  research  where  separation  of  vulnerable 
cells  and  intracellular  particulates  is  involved.   Thus  the  method  may 
be  useful  in  cell  washing  and  elutriation,  zonal  centrifugation  and 
countercurrent  chromatography. 

Proposed  Course: 

1.  Refinement  of  the  present  prototype  to  improve  protection  of  the 
flow  tubes  at  the  bottom  of  the  centrifuge  bowl  and  at  the  center  of 
the  centrifuge  cover. 

2.  Further  investigation  on  plasmapheresis. 

3.  Construction  of  the  second  prototype  which  is  equipped  with  an 
interchangeable  centrifuge  bowl  so  that  various  configurations  of 
the  bowl  can  be  conveniently  tested  for  cell  washing,  elutriation 
and  countercurrent  chromatography. 

Keyword  Descriptors: 

Flow-through  centrifuge  without  rotating  seals, Separation  of  vulnerable 
cells  and  intracellular  particulates,  cell  washing,  elutriation,  zonal 
centrifugation,  and  countercurrent  chromatography. 

Honors  and  Awards:   None 

Publications:   None 


35a 


Project  No.  Z01   HL   01403-02   LTD 


1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30  1975 

Project  Title:   Eye  Motion  Measurement  by  Ultrasound 

Previous  Serial  Number:   NHLI-84 

Principal  Investigator:   Frank  W.  Noble 

Other  Investigators:   Robert  L.  Bowman 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Deviant  motion  of  the  eye  at  the  first  several  harmonics  of 
the  heart  rate  may  be  indicative  of  circulatory  problems  within  the  head 
and  neck  areas.   There  is  some  evidence  that  variation  from  normal  motion 
may  warn  of  impending  stroke. 

Major  Findings:   Since  an  acoustic  wave  travelling  in  air  is  totally 
reflected  by  any  liquid  or  solid  surface,  it  is  practical  to  interrogate 
a  small  region  of  the  front  surface  of  the  eye  by  means  of  ultrasound. 
The  phase  delay  between  separate  transmitting  and  receiving  transducers 
is  modulated  by  the  eye  motion.   This  delay  is  converted  to  amplitude 
modulation  and  recorded,  resulting  in  a  record  of  eye  motion  vs.  time. 

Various  methods  of  mounting  the  transducers  with  respect  to  the  eye  of 
human  subjects  have  been  investigated  in  an  effort  to  reduce  artifacts 
due  to  extraneous  motions  of  the  subject's   head.   The  associated 
electronics  have  been  improved,  incorporating  crystal  frequency  control, 
narrow  band  receiving  equipment,  and  heavy  amplitude  limiting.   Cross- 
correlation  equipment  has  been  employed  to  compare  the  eye  motion  with 
the  patient's  electrocardiogram  to  discriminate  against  signals  which 
are  asynchronous  with  the  heart. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

If   deviant  motions  of  the  front  surface  of  the  eye  are  found  to  correlate 

with  circulatory  pathology,  the  significance  could  be  considerable. 


3*S 


Project  No.  Z01  HL   01403-02   LTD 


Proposed  Course:   Interference  problems  produced  by  random  motions  of 
the  head  and  eye  with  respect  to  the  transducers  are  of  such  magnitude 
that  even  cross-correlation  with  the  ECG  produces  only  a  barely 
recognizable  signal  at  the  heart  rate. 

A  much  better  method  of  transducer  mounting  and  signal  processing  must  be 
devised  in  order  to  make  the  system  practical. 

Keyword  Descriptors:   Eye  Motion,  Stroke  Prediction,  Ultrasonics, 
and  Circulation. 

Honors  and  Awards :   None 

Publications:   None 


3tf 


Project  No.  Z01   HL   01404-07   LTD 

1.  Laboratory  of  Technical  Development 

2.  Section  on  Pulmonary  &  Cardiac 

Assist  Devices 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Membrane  Lung  Systems  for  Long  Term  Support 

Previous  Serial  No.:  NHLI-82 

Principal  Investigator:   Theodor  Kolobow 

Other  Investigators:   Edward  Stool,  Paul  Weathersby,  Joseph  Pierce, 
Robert  L.  Bowman,  and  Jacques  Suaudeau 

Cooperating  Units:   Armed  Forces  Radiobiology  Research  Institute, 
and  Section  on  Lab  Animal  Medicine  and  Surgery 

Objectives : 

1.  Current  heart  lung  machines  cannot  be  safely  used  clinically  in  excess 
of  4-6  hours.  We  are  demonstrating  the  safety  of  the  membrane  lung  in 
long  term  use  of  many  days  and  weeks  duration. 

2.  Defects  in  membranes,  resulting  in  leaks  from  pinholes,  has  been  the 
major  stumbling  block  to  routine  clinical  use  of  the  membrane  lung.   We 
have  determined  the  cause  of  these  pinhole  defects,  and  applied  a  new 
technique  to  overcome  this  problem. 

3.  Blood  compatibility  of  synthetic  materials  is  the  most  important,  if 
not  exclusive,  limiting  factor  to  routine,  safe  use  of  artificial 
internal  organs.   Because  of  large  surface  areas  involved,  this 
limitation  applies  particularly  to  the  membrane  lung,  and  to  a  lesser 
degree,  they  also  apply  to  the  artificial  kidney,  heart  valves,  heart 
assist  devices,  and  other  implantable  organs  and  devices.   The  various 
factors  that  contribute  to  polymer  blood  compatibility  are  not  clear, 
and  very  likely  include  a  variety  of  aspects.   It  has  been  shown  that 
"impurities"  normally  present  in  synthetic  polymers  do  at  times 
contribute  to  enhanced  blood  compatibility,  and  at  other  times  to  a 
lowered  blood  compatibility.   Because  of  high  gas  transmission  we  have 
concentrated  our  investigation  on  silicone  rubber  membranes  of  different 
purities,  and  to  charged  membranes. 

4.  We  are  investigating  the  physiologic  range  of  hypothermic  temperature, 
and  the  type  of  perfusate,  for  isolated  sheep  heart  preservation. 


33o 


Project  No.  Z01   HL   01404-07  LTD 


These  studies  arise  from  the  knowledge  of  present  day  limitations  in 
preserving  hearts  beyond  24  hours  without  functional  impairment  on 
reimplantation. 

Methods  Employed  and  Major  Findings: 

1.   The  Membrane  Lung 

Membrane  casting  technology  has  been  significantly  enhanced  to  allow 
multilayer  casting  of  gas  permeable  membranes  optimally  suited  for  specific 
applications.   As  an  example,  up  to  five  separate  discrete  layers  of 
polymer  have  been  cast  with  presently  available  casting  machine,  with  a 
membrane  overall  thickness  of  50  micrometers.   It  is  now  possible  to 
custom  design,  within  limits,  a  membrane  lung  most  suitable  for  specific 
applications:  conventional  heart-lung  machine,  long  term  respiratory 
assist,  selective  enrichment  of  blood  with  oxygen,  or  preferential  removal 
of  carbon  dioxide.   With  application  of  special  high  efficiency  membranes, 
the  rating  of  the  spiral  membrane  lung  has  been  raised,  under  standard 
conditions,  to  80-100  cc/ (m  ) (min)  for  oxygen  and  carbon  dioxide.   This 
transfer  is  2-3  times  greater  than  for  other  stationary  membrane  lung 
models.   The  spiral  membrane  lung  as  of  now  is  suitable  both  for  gravity 
bypass,  as  well  as  for  pump-through  bypass. 

a.  Standard  Membrane  (3  layer  technique) 

As  presently  used,  this  membrane  has  exceptionally  strong  burst  strength, 
and  a  blood  compatibility  consistent  with  "Medical  Grade"  silicone  rubber. 
Artificial  lungs  using  this  type  of  membrane  represent  the  state  of  the  art 
for  commercial  medical  membrane  lung  devices. 

b.  Pure  Silicone  Gum  Membrane  (3  layer  technique) 

Significantly  improved  blood  compatibility  is  seen  when  no  silica  filler  is 
added  to  silicone  rubber.   Because  of  the  casting  technique  employed,  the 
burst  strength  of  this  new  membrane  is  nearly  equal  to  the  standard  membrane. 

c.  "Charged"  Membrane  (three  layer  technique) 

The  blood  contacting  surface  contains  added  acetylene  black  and  pure 
silicone  gum  rubber. 

d.  "Charged"  Membrane  with  Pure  Gum  Interface  (four  layer  Technique) 

The  charged  layer  is  physically  below  a  smooth  layer  of  pure  silicone 
gum  rubber. 


33/ 


Project  No.  Z01  HL  01404-07  LTD 


e.  Charged  Membranes  Coated  with  Fluorosilicone  Gum  Rubber 
(four  layer  technique) 

The  thin  coating  adjacent  to  blood  consists  of  fluorosilicone  gum  rubber. 

f.  Silicone  Gum  Rubber  Membrane,  Coated  with  Hypothrombogenic  and 
Nonthrombogenic  Polymer  Systems  (5  layer  technique) 

Perf luorinated  ethyl  cellulose,  and  diethylaminoethyl  cellulose 
(radiation  grafted  with  heparin)  can  be  readily  applied  to  charged 
membranes,  with  good  adherence.   Similarly,  because  of  the  physical  nature 
of  the  "charged"  membranes,  many  other  organic  coatings  can  be  applied  to 
these  surfaces  (such  as  some  hydrogels) ,  and  tested  for  use  in  the 
membrane  lung. 

A.  Clinical  Applications: 

We  believe  a  great  number  of  patients  who  ultimately  require  treatment 
with  the  membrane  lung  for  blood  respiratory  gas  exchange  represent 
complications  of  intensive  respiratory  care.   The  patient  population  at 
the  Clinical  Center  is  in  addition  burdened  by  research  patients  in  whom 
therapeutic  interventions  have  rendered  them  immunologically  susceptible 
to  pathogen  organism  invasion. 

We  have  provided  extracorporeal  respiratory  gas  exchange  with  a  membrane 
lung  in  a  young  boy  with  leukemia,  immunosuppression  with  total  body 
irradiation  followed  by  bone  marrow  transplantation,  for  12  days. 
Technically,  bypass  was  accomplished  in  a  routine  manner,  without 
complications.   The  patient  did  succumb  to  the  underlying  disease  process, 
however . 

B.  Animal  Research: 

Arteriovenous  shunt  of  blood  across  the  membrane  lung  without  the  use  of  a 
blood  pump. 

The  performance  of  new  membranes  developed  and  fabricated  in  this  laboratory 
is  presently  undergoing  animal  testing.   In  this  study,  the  common  carotic 
artery  and  the  external  jugular  vein  were  cannulated  and  connected  through 
silicone  gum  rubber  coated  silicone  rubber  tubing  across  the  test  membrane 
lung.   Heparin  was  administered  at  the  time  of  cannulation,  but  none  was 
given  further  the  moment  the  arteriovenous  shunt  was  opened.   We  measured 
changes  in  blood  flow  resistance  across  the  membrane  lung,  changes  in 
platelet  and  white  blood  cell  count,  and  white  blood  cell  differential 
counts. 


33A 


Project  No.  Z01  HL  01404-07  LTD 

1.  A  control  series  of  membrane  lungs  made  of  standard  "Medical  Grade" 
silicone  rubber  membrane  uniformly  shows  a  prompt  fall  in  platelet 
count,  white  blood  cell  count,  and  an  agranulocytosis  during  the  first  10 
minutes,  and  a  gradually  rising  perfusion  pressure. 

2.  Membrane  lungs  made  of  silicone  gum  rubber  show  no  changes  in  platelet 
count,  and  no  rise  in  perfusion  pressure  for  duration  of  the  heparin 
effect  (6  hours) . 

3.  Membrane  lungs  made  with  charged  silicone  gum  rubber  surfaces  are 
unique  as  there  is  no  change  in  platelet  count,  or  white  blood  cell  count 
at  any  time.   Unique  to  this  class  of  membranes,  the  platelet  sparing 
effect  persists  for  a  much  longer  time  compared  to  any  previous  membrane 
studied,  or  reported.   Thus,  65%  of  platelet  count  remains  in  peripheral 
blood,  compared  to  35%  with  gum  silicone  rubber  membrane,  and  20%  with 
standard  "Medical  Grade"  silicone  rubber  membrane  after  3rd  day  without  heparin 

Studies  with  other  membranes  and  membrane  coatings  produced  by  us  are 
under  way. 

4.  Studies  reported  above  were  all  done  on  lambs,  which  are  notoriously 
more  difficult  to  work  with  in  terms  of  the  coagulation  system  compared 
to  man. 

The  pure  silicone  gum  membrane  developed  by  us  was  applied  to  man  in  just 
one  single  case.   In  this  instance,  no  change  in  platelet  count,  white 
blood  cell  count  and  differential,  or  a  rise  in  perfusion  pressure 
were  observed;  virtually  no  heparin  was  given  during  the  two  day  perfusion. 

The  improved  results  in  sheep  with  new  membranes  and  membrane  coatings 
developed  by  us  leads  us  to  believe  that  even  greater  improvement  can  be 
expected  when  applied  to  man.   At  the  same  time,  the  sheep  remains  an 
excellent  model  to  find  often  minute  differences  among  candidate  membrane 
materials . 

We  feel  that  removal  of  silica  filler  and  processing  aides  was  beneficial 
to  improve  blood  compatibility.   The  addition  of  acetylene  black  into  a 
membrane  system  appears  to  exert  an  additional  blood  cellular  sparing 
effect.   Our  results  show  that  our  technical  capability  of  producing 
pinhole  free  silicone  membranes  has  allowed  us  to  use  a  material  generally 
considered  inferior  for  commercial  use,  and  to  produce  a  blood  contacting 
surface  with  improved  blood  compatibility. 

Our  findings  represent  the  first  practical  improvement  in  silicone  membrane 
technology  for  use  in  the  membrane  lung. 


33* 


Project  No.  Z01  HL  01404-07  LTD 

C.   Membrane  Lung  Priming: 

We  have  found  it  critical  to  remove  all  gas  from  the  membrane  lung  to 
reveal  the  ultimate  potential  of  a  candidate  membrane.   Blood  platelets 
typically  adhere  and  aggregate  around  gas  bubbles,  thus  initiating  a 
sequence  of  events  leading  to  thrombosis. 

In  our  newer  system,  all  priming  is  done  under  vacuum,  where  all  air  is 
removed  except  for  water  vapor.   Priming  with  degassed  prime  is  then 
accomplished  under  hydrostatic  pressure. 

Unique  to  this  system  is  speed  of  priming:  once  degassed,  the  membrane  lung 
is  primed  and  ready  for  use  in  less  than  five  minutes.   In  addition,  this 
technique  is  superior  to  the  carbon   ioxice  prime  technique  because  of 
speed  and  a  gurantee  that  no  gas  bubbles  remain  any  place  within  the 
membrane  lung. 

2.   Ex  Vivo  organ  preservation  by  hypothermic  perfusion. 

Dog  and  sheep  hearts  perfused  with  a  synthetic  medium  at  5-10  C  after  24 
hours  of  perfusion  uniformly  become  edematous,  with  a  continuous  rise  of 
their  vascular  resistances.   These  hearts  during  preservation  remain 
flaccid,  without  electrical  or  mechanical  activity. 

We  have  since  shown  that  sheep  hearts  continuously  perfused  at  10  C  and 
above  with  fresh  donor  blood  can  maintain  electrical  and  mechanical  activity, 
there  was  no  edema  and  upon  rewarming,  the  hearts  had  good  ventricular 
function.   We  have  also  shown  that  a  flow  of  at  least  15  cc/min  of  whole 
fresh  blood  was  necessary  to  assure  organ  viability.   Similarly,  an 
ultrafiltrate  enriched  solution  of  fresh  blood  (MW  cutoff  50.000) at  a 
rate  above  20  cc/min  supplied  sufficient  factors  to  assure  a  continuously 
contracting  heart,  and  excellent  cardiac  preservation. 

Blood  being  an  excellent  perfusate,  this  study  was  designed  to  determine 
which  of  the  cellular  fractions  (if  any)  of  whole  blood  were  critical  to 
success  of  organ  preservation. 

Sheep  hearts  were  excised  and  treated  in  the  same  manner  as  before.   The 
perfusate  consisted  of  various  plasma  fractions  from  an  adult  sheep, 
separated  from  blood  in  a  flow  through  centrifuge  (celltrifuge) . 

The  various  perfusates  were  as  follows: 

1.  Platelet  poor  plasma. 

2.  Platelet  rich  plasma. 


314 


Project  No.  Z01  HL  01404-07  LTD 


3.  Platelet  rich  plasma  with  whole  blood  added  to  give  a  hematocrit 
of  1-2%. 

4.  Incompletely  separated  plasma,  i.e.  plasma  containing  some  red 
blood  cells,  white  blood  cells,  and  platelet  rich  plasma 

(Hct  1-2%) 

All  studies  were  performed  at  13  C. 

As  with  hearts  perfused  with  whole  blood,  the  hearts  in  all  four  groups 
showed  ventricular  contractions  for  various  lengths  of  time,  with  a  peak 
left  ventricular  systolic  pressure  as  high  as  70  mm  Hg,  and  a  heart  rate 
of  15-20  contractions/minute.   However,  the  longest  and  most  forceful 
contractions  were  in  groups  having  some  red  blood  cells  in  the  perfusate, 
and  particularly  the  group  where  red  blood  cells  were  only  incompletely 
separated.   These  hearts  on  rewarming  had  no  weight  gain,  and  had  excellent 
ventricular  function  on  rewarming. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

Since  1970,  about  20  patients  have  been  saved  worldwide  with  the  membrane 
lung  from  acute  respiratory  failure  unresponsive  to  conventional 
treatment  using  various  types  of  membrane  lungs. 

The  complexity  of  long  term  membrane  lung  support  places  unusual  requirements 
on  blood  damage  and  membrane  lung  performance.   We  have  shown  that  silica 
free  silicone  rubber  membrane  has  superior  blood  compatibility  compared  to 
commercially  used  medical  grade  silicone  rubber  membrane  containing  silica 
fillers,  and  processing  aides;  a  much  lower  red  blood  cell  lethal  and 
sublethal  damage,  and  much  improved  compatibility  to  blood  platelets. 

We  have  shown  that  addition  of  carbon  to  the  membrane  matrix  in  a 
membrane  lung  significantly  reduces  damage  to  blood  cellular  elements. 
In  particular,  platelet  change  is  nonexistent  when  heparin  is  used,  and 
platelet  count  remained  a  high  65%  of  baseline  values  after  three  days  in 
the  absence  of  any  heparin.   It  is  likely  that  heparin  use  during  long 
term  or  short  term  bypass  in  man  could  be  severely  curtailed  if  not 
eliminated  when  these  novel  membranes  are  used.   Similarly,  these  new 
surfaces  if  applied  to  other  artificial  internal  organs  (artificial 
booster  heart,  total  artificial  heart,  heart  valves,  the  artificial 
kidney,  vascular  grafts,  etc.)  could  similarly  impart  a  new  dimension  of 
safety  and  reduce  patient  morbidity. 

Our  work  on  sheep  heart  preservation  suggests  the  importance  of  certain 
blood  cellular  fractions  to  successful  organ  preservation.   It  is  evident 
that  lessons   learned  have  great  relevance  to  other  internal  organ 
preservation. 


S3ST 


Project  No.  Z01  HL  01404-07  LTD 
Proposed  Course: 

1.  Studies  will  be  continued  to  assess  use  of  various  silicone  gum 
rubber  (including  f luorosilicone  gum)  and  additives. 

The  effects  of  carbon  concentration,  location,  and  surface  coating 
will  be  similarly  investigated. 

2.  A  program  will  be  initiated  to  develop  a  novel  type  of  membrane  lung 
suitable  for  long  term  implantation.   This  will  include  the  preparation 
of  new  types  of  membranes,  and  new  surfaces,  and  new  designs,  to  make 

a  reliable,  compact  unit. 

3.  The  merit  of  using  a  membrane  lung  in  acute  respiratory  care  will  be 
assessed  by  blood  carbon  dioxide  exchange  alone  to  the  exclusion  of 
oxygen,  in  an  arteriovenous  shunt  without  blood  pumping.   The 
simplicity  of  this  procedure  is  appealing  as  it  requires  low  blood 
flows,  and  no  blood  pump.   It  is  hoped  that  this  technique  will  lower 
the  high  incidence  of  brochopleural  fistula  in  the  treatment  of  acute 
respiratory  failure,  by  reducing  the  minute  ventilatory  volumes,  and 
peak  inspiratory  pressures.   Similarly,  it  may  be  found  useful  in  the 
treatment  of  some  states  of  hypercapnea. 

4.  We  have  the  capability  of  producing  ultrathin  (5  microns),  non- 
reinforced  or  reinforced  silicone  rubber  membrane,  which  could  be 
used  as  a  human  burn  dressing.   A  pilot  laboratory  study  will  be 
initiated  to  explore  this  possibility. 

Keyword  Descriptors: 

Artificial  lung,  membrane  lung,  perm  selective  membranes,  silicone 
rubber  membranes,  silicone  gum  rubber  membranes,  gas  exchange, 
respiratory  assist,  blood  compatibility,  arteriovenous  shunt, 
organ  perfusion,  heart  preservation,  and  ex  vivo  perfusion. 

Honors  and  Awards:   None 

Publications : 

1.  Kolobow,  T.,  Hayano,  F. ,  and  Weathersby,  P.  K. :   Dispersion-casting  thin 
and  ultrathin  fabric-reinforced  silicone  rubber  membrane  for  use  in  the 
membrane  lung,  J.  Assn.  Adv.  Med.  Instrum. ,  in  press. 

2.  Weathersby,  P.  K. ,  Kolobow,  T. ,  and  Stool,  E.  W. :  Relative  thrombo- 
genicity  of  polydimethylsiloxane  and  silicone  rubber  constituents, 
J.  Biomed.  Materials  Research,  in  press. 


334 


Project  No.  Z01  HL  01404-07  LTD 


3.   Kolobow,  T.,  Stool,  E.  W.  ,  Sacks,  K.  L. ,  and  Vurek,  G.  G.  : 

Acute  Respiratory  Failure:  Survival  Following  10  Days  Support  with  a 
Membrane  Lung,  J.  Thoracic  &  Cardiovascular  Surgery,  in  press. 


337 


Project   No.    Z01   HL   01405-02    LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Analysis  of  Microcirculation  by  Coherent  Light  Scattering 

Previous  Serial  Number:  NHLI-85 

Principal  Investigator:   Michael  D.  Stern,  Donald  L.  Lappe 

Other  Investigators:   Robert  L.  Bowman 

Cooperating  Units:   Laboratory  of  Experimental  Therapeutics,  NHLI 

Project  Description: 

Objectives:   To  continue  exploratory  development  of  a  method  of  non- 
invasively  measuring  flow  and  other  parameters  of  the  microcirculation  in 
tissues  by  means  of  analysis  of  the  spectrum  of  coherent  light  doppler- 
scattered  from  the  tissue.   The  measurement  of  these  parameters  in 
extremities  is  of  prime  importance  for  the  evaluation  of  peripheral 
vascular  diseases,  while  the  measurement  in  internal  organs  offers  an 
important  tool  for  the  study  of  pharmacodynamics  and  vascular  physiology. 
Specific  objectives  are  to  develop  a  prototype  instrument  and  method,  and 
demonstrate  the  feasibility  of  using  this  technique  in  the  physiology 
laboratory  and  clinical  setting. 

Methods : 

(a)  Theoretical  Analysis 

We  are  engaged  in  an  ongoing  theoretical  study  of  the  kinetics  of  multiple 
scattering  of  coherent  light  in  tissue  in  the  presence  of  blood  flow,  with 
the  goal  of  providing  a  framework  for  the  interpretation  of  emperical 
spectra  obtained  in  real  physiologic  settings,  and  relating  these  to  the 
true  distribution  of  blood  flow  velocities  in  the  microvascular  bed,  and 
to  the  total  tissue  flow  in  regions  of  varying  vascular  geometry. 

(b)  Instrument  Development 

A  prototype  laser  scattering  apparatus  capable  of  studying  human  skin  and 
various  tissues  in  experimental  animals  has  been  designed  and  built, 
together  with  the  associaced  signal  processing  equipment.   The  instrumental 
requirements  for  more  advanced  modifications  of  the  system,  such  as  the 
use  of  fiber-optic  light  pipes  to  measure  flow  in  inaccessible  regions, 
are  under  development. 


338 


Project  No.      Z01   HL   01405-Q2    LTD 

(c)  Experimental 

Experiments  have  been  undertaken  with  the  prototype  apparatus  to  demonstrate 
the  ability  of  this  method  to  monitor  the  real-time  dynamics  of  circulation 
in  human  skin  and  other  tissues,  to  validate  the  correlation  of  the 
doppler  spectrum  with  other  measures  of  tissue  blood  flow,  and  to  show  the 
use  of  the  laser  doppler  technique  in  studying  regional  flow  in  internal 
organs  during  pharmacologic  interventions.   A  clinical  protocol  has  been 
developed  for  the  use  of  the  instrument  to  monitor  blood  flow  in  the  skin 
of  patients  with  peripheral  vascular  diseases. 

Major  Findings: 

1.  Theoretical  analysis  indicates  that  under  a  broad  class  of  circumstances 
the  spectral  shift  of  scattered  light  can  be  analyzed  in  terms  of  a  model 

of  random  walk  of  photons  through  tissues,  suffering  repeated  doppler 
shifts  when  scattered  by  red  cells.   With  idealized  assumptions  this  model 
can  be  analyzed  analytically  and  predicts  the  general  shape  observed  for 
spectra  from  actual  tissues.   The  analysis  predicts  that  the  complete 
velocity  distribution  of  blood  flow  in  microvascular  bed  is,  in  principle, 
recoverable  from  the  spectra,  and  that  certain  weighted  average  bandwidths 
of  the  spectra  should  be  proportional  to  tissue  blood  flow,  in  fixed 
vascular  geometry. 

2.  The  prototype  instrument  has  been  designed  to  measure  the  spectra 
and  the  weighted  bandwidths  described  above.   An  appropriate  stable 
helium  neon  laser  and  photomultiplier  have  been  procured,  together  with  the 
necessary  electronics  for  real  time  spectrum  analysis  and  autocorrelation 
of  the  photodetector  signal.   With  this  system  it  has  been  possible  to 
record  spectra  with  good  signal  to  noise  ratios  from  human  skin,  and  from 
the  surface  of  internal  organs  (kidney,  liver)  of  experimental  animals. 
These  spectra  vary  in  the  expected  manner  with  interventions  which 
produce  vasomotor  changes  in  the  tissues,  and  with  occlusion  of  vascular 
supply.   A  number  of  dynamic  vascular  reflexes  associated  with  posture, 
emotion  and  respiratory  pattern,  and  with  thermoregulation  are  easily 
studied. 

3.  An  experiment  was  undertaken  in  collaboration  with  the  Department  of 
Biomedical  Engineering  of  the  University  of  Washington  to  measure  the 
correlation  of  our  method  of  measuring  the  blood  flow  in  the  forearm  with 
the     Xe  washout  technique.   Good  correlation  was  shown;  the  ability  of 
the  laser  instrument  to  monitor  continuously  made  possible  the  study  of 
the  dynamics  of  the  vascular  bed  at  the  site  of  injection  of  radioactive 
xenon,  raising  the  possiblity  of  injection  artifacts  in  the  xenon  method 
which  could  not  be  previously  documented. 

4.  An  experiment  was  undertaken  to  show  the  feasibility  of  using  the  laser 
instrument  to  monitor  continuously  microvascular  flow  in  small  regions  of 


33? 


Project  No.  Z01  HL  01405-02  LTD 

exposed  renal  cortex  in  the  rat.   Good,  reproducible  spectra  were  obtained, 
which  vanished  when  the  renal  artery  was  occluded.   Response  of  the  renal 
cortical  perfusion  to  a  number  of \asoactive  drugs  (dopamine,  angiotensin, 
norepinephrine,  isoproterenol)  was  easily  followed  in  time  and  quantitated, 
and  steady  state  dose-response  curves  measured.   At  present  this  method 
appears  very  promising  qualitatively  and  quantitatively;  it  remains  to  be 
calibrated  and  validated  against  other  methods  for  regional  organ  blood-flow. 

5.   A  clinical  protocol  has  been  established  to  use  the  prototype 
instrument  to  study  skin  microcirculation  in  patients  with  a  variety  of 
conditions  during  interventions  covered  by  other  NIH  protocols.   An 
experiment  to  study  finger  circulation  in  Raynaud's  disease,  with  the  use 
of  nitroglycerin  therapy  is  underway. 

Significance  of  the  Program  to  the  Institute: 

The  diagnosis  and  followup  of  peripheral  vascular  diseases,  the  study  of 
circulation  in  burns  and  grafts,  and  the  study  of  tissue  blood  flow  under 
dynamic  conditions  in  response  to  pharmacologic  and  hemodynamic  alterations 
are  among  the  important  applications  forseen  for  the  technique. 

Proposed  Course: 

1.  Continuation  of  theoretical  analysis. 

2.  Demonstration  of  further  applications  of  the  method  in  physiology  and 
clinical  situations. 

3.  Experiments  to  further  validate  this  method  against  other  meausres  of 
microcirculatory  parameters. 

4.  Attempts  to  extract  from  the  spectra  more  detailed  information  about 
the  distribution  of  flow  in  the  compartments  of  the  microvasculature. 

5.  Planning  for  further  advanced  development  of  prototype  instruments. 

Keyword  Descriptors:   Microcirculation,  capillaries,  blood  flow,  laser, 
doppler  effect,  renal  blood  flow,  peripheral  vascular  disease,  tissue 
perfusion,  light  scattering,  skin  blood  flow. 

Honors  and  Awards:   None 

Publications:   1.   Stern,  M. :  In  vivo  evaluation  of  the  microcirculation 
by  coherent  light  scattering.   Nature  254:  No.  5495, 
March  1975. 


3<&> 


Project  No.  Z01  HL  01406-H  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Fluorescent  Complexes  of  Proteins 

Previous  Serial  Number:   NHLI-79 

Principal  Investigator:   Raymond  F.  Chen 

Other  Investigators:   None 

Project  Description: 

Objectives:   Fluorescent  labeling  of  proteins  is  an  important  area  of 
fluorescence  spectroscopic  methods  in  biomedical  applications,  and  is 
practiced  in  histochemistry  as  well  as  biophysical  studies.   We  wish  to 
find  and  characterize  new  dyes  which  may  have  novel  properties,  and  to  show 
how  they  may  be  applied. 

Methods  Employed:   Dyes  which  are  investigated  may  bind  covalently  or 
simply  adsorb  to  a  given  protein.   In  the  case  of  covalent  binding,  the 
dye  is  reacted  with  the  protein,  and  unreacted  dye  is  separated  by  passing 
the  protein  solution  through  a  Sephadex  column.   The  properties  of  the 
fluorescent  conjugate  are  investigated  by  fluorescence  spectroscopy  and 
lifetime  measurements.   Dye-protein  adsorbates  are  similarly  studied  under 
equilibrium  conditions. 

Major  Findings:   With  Dr.  Walter  Stewart  of  NINDS  we  have  measured  the 
spectra  and  quantum  yield  of  a  number  of  compounds  he  has  synthesized  to 
label  the  proteins  of  nerve  axons.   The  most  promising  of  these  compounds, 
tentatively  named  N-110,  is  a  highly  anionic  dye  with  a  quantum  yield  of 
about  0.2  in  water  which  will  replace  Procion  Yellow,  currently  used  for 
such  neurochemical  studies. 

Two  related  dyes,  N-86  and  N-105,  were  studied  to  see  if  they  could  be  used 
to  label  proteins  for  biophysical  studies  such  as  depolarization  of 
fluorescence.   N-105  seems  to  be  promising:  it  reacts  readily  with 
proteins,  has  a  quantum  yield  near  0.2,  and  the  lifetimes  of  the  fluorescent 
conjugates  are  in  the  range  of  11  nsec.   Because  of  its  highly  negatively 
charged  character,  N-105  may  be  a  good  dye  for  labeling  basic  proteins  such 
as  histones. 

Another  class  of  dyes,  quinacrine  and  quinacrine  mustard,  was  studied. 
The  latter,  abbreviated  QM,  labels  proteins  and  gives  conjugates  with 

34t 


Project  No.  Z01  HL  01406-11  LTD 


lifetimes  ranging  from  5  to  13  nsec.   The  dye  has  a  pK  near  7.7  and  may  be 
useful  in  labeling  ampholines,  thus  giving  a  built-in  Fluorescent  pH 
indicator  in  isoelectric  focusing  experiments.   The  polarization,  lifetimes, 
corrected  spectra  of  quinacrine  and  QM  conjugates  were  written  up  in  a 
paper  to  be  submitted  shortly. 

Bilirubin-albumin  complexes  are  known  to  show  light  sensitivity.   Recent 
reports  in  the  literature  suggest  that  the  photosensitivity  is  due  to 
photooxidation  of  bilirubin  by  singlet  oxygen.   The  latter  in  turn  is 
produced  by  bilirubin  photosensitization  •  Thus , bilirubin  catalyzes  its 
own  decomposition.   A  project  has  started  showing  that  ascorbic  acid  and 
a  -tocopherol  may  slow  the  process.   This  is  in  concord  with  literature- 
reports  that  these  antioxidants  scavenge  singlet  oxygen.   The  rate  of 
decomposition  of  bilirubin  in  these  complexes  is  followed  either  by 
fluorescence  or  absorption  spectra. 

It  is  known  that  some  enzymes  are  inhibited,  or  their  response  to  activators 
altered,  after  reaction  with  various  reagents.   By  reacting  enzymes  with 
fluorescent  dyes,  we  can  probe  the  active  site  or  the  control  sites.   The 
technique  requires  specific  attachment  of  a  dye  to  a  given  area  on  a 
protein.   In  preliminary  experiments,  we  have  shown  that  f luorescamine  and 
quinacrine  mustard  produce  inhibition  of  alcohol  and  glutamate  dehydrogenases, 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

The  use  and  characterization  of  new  dyes  for  protein  studies  advances  the 
state  of  the  art  of  fluorescence  spectroscopy,  which  in  turn  is  a  major 
tool  in  histochemistry  and  biochemistry.   The  work  continues  the  institute's 
traditional  leadership  in  the  area  of  fluorescence. 

Proposed  Course: 

There  are  several  dyes  which  can  combine  covalently  with  amino  groups,  and 
which  can  serve  as  pH  markers.   Dyes  such  as  quinacrine  mustard, 
fluorescein  isothiocyanate,  and  neutral  red  could  be  incorporated  into  an 
isoelectric  focusing  matrix  to  mark  the  pH.   We  hope  to  try  this  method  out 
with  various  dyes.   We  also  wish  to  study  the  dye  distribution  in  protein 
conjugates;  i.e.,  in  a  preparation  where  the  protein  has  an  average  of  3  dyes 
bound  per  mole.  How  many  molecules  have  1,  2,  3,  4,  5,  or  6  dyes  attached? 
The  answers  will  enhance  our  understanding  of  the  accessibility  of  sites  to 
fluorescent  labeling  and  will  show  whether  it  is  a  purely  random  process 
or  not. 

Keyword  Descriptors:   Fluorescence,  dyes,  quinacrince,  quinacrine  mustard. 

Honors  and  Awards:   None 

Publications:   1.   Chen,  R.  F. :  Fluorescent  Enzyme  Inhibitors,  a  table  for 
Handbook  of  Biochemistry,  Gerald  Fasman,  Ed.,  Chemical 
Rubber  Co.,  in  press. 

3& 


Project  No.  Z01  HL  01407-12  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Applications  of  Fluorescence  in  Biochemistry 

Previous  Serial  Number:   NHLI-78 

Principal  Investigator:   Raymond  F.  Chen 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description: 

Objectives:   In  order  to  advance  a  method  or  technique,  it  is  often  necessary 
to  show  how  it  can  solve  certain  problems  of  interest.   In  the  case  of 
luminescence  spectroscopy,  involving  either  fluorescence  or  phosphorescence, 
there  are  many  biochemical  problems  amenable  to  the  method.   We  choose  to 
work  on  some  intrinsically  interesting  problems  to  show  the  utility  of 
luminescence. 

Methods  Employed:   We  have  previously  acquired  and  modfied  instrumentation 
which  permits  a  wide  range  of  measurements  to  be  made.   These  include: 
phosphorescence  and  fluorescence  excitation  and  emission  spectra,  lifetimes, 
quantum  yields,  and  stopped  flow  kinetic  measurements. 

Major  Findings: 

1.  A  phosphorescence  study  was  performed  which  showed  that  Ag  markedly 
enchances  the  phosphorescence  of  tryptophan  (3-fold)  and  totally  quenches 
the  fluorescence;  the  study  also  showed  that  proteins  phosphorescence  was 
altered  by  Ag   in  various  ways.   The  effect  of  Ag   in  proteins  containing 
sulfhydryl  groups  could  be  attributed  to  total  luminescence  quenching  by 
energy  transfer  to  Ag  -mercaptide  absorption  bands.   However,  only 
fluorescence  is  quenched  in  non-sulfhydryl  proteins.   It  was  found  that  10% 
methanol  snows  at  77  K  were  suitable  matrices  for  the  study,  and  it  was 
suggested  that  previous  studies  on  protein  phosphorescence  done  in  glasses 
of  organic  solvents  may  have  studied  only  denatured  proteins.   Enzyme 
activity  measurements  showed  no  denaturation  in  10%  methanol. 

2.  The  study  of  membranes  and  lipid  micelles  by  fluorescence  has  become 
popular  in  the  biochemical  literature.   We  have  found  that  certain  dyes  (TNS, 
ethidium  bromide,  quinacrine)  show  markedly  altered  fluorescence  in 


3*3 


Project  No.  Z01  HL  01407-12  LTD 

detergent  solutions  of  different  concentration.   In  fact,  the  critical  micelle 
concentration  (cmc)  can  be  detected  by  following  such  probe  fluorescence. 
We  have  measured  the  emission  of  some  detergent  solutions  in  the  presence 
of  various  amounts  of  salt  (which  alters  the  cmc)  and  confirmed  the  effect. 

3.   The  binding  of  fluorescent  compounds  by  certain  enzymes  has  yielded 
information  about  the  active  sites.   L.  Brand  reported  that  Auramine  0  was 
bound  by  liver  alcohol  dehydrogenase  but  not  by  yeast  alcohol  dehydrogenase 
as  shown  by  the  marked  enhancement  of  Auramine  0  fluorescence.   On  the  other 
hand,  we  have  examined  the  quenching  of  protein  fluorescence  of  these  enzymes 
by  Auramine  0,  and  find  that  both  enzymes  do  bind  the  dye.   In  contrast  to 
previous  reports,  therefore,  dyes  whose  fluorescence  is  not  enhanced  cannot 
be  assumed  not  to  bind  to  a  given  protein.   Similarly,  the  antimalarial  drug 
primaquine  was  reported  by  T.  Li  to  inhibit  liver  alcohol  dehydrogenase 
noncompetitively   but  not  to  inhibit  yeast  alcohol  dehydrogenase.   We  find 
that  both  enzymes  bind  the  durg,  confirm  that  yeast  ADH  is  not  inhibited, 
but  find  that  the  inhibition  is  competitive  with  the  coenzyme,  NAD. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

These  studies  have  produced  results  of  interest  to  biochemists  and  again 
illustrate  the  utility  of  fluorescence  and  phosphorescence  methods. 

Proposed  Course:   The  work  on  the  alcohol  dehydrogenases  will  be  completed, 
and  combined  with  measurements  made  on  the  ORTEC  nanosecond  spectrometer. 

Several  problems  reamain  in  the  use  of  fluorescent  probes  to  follow 
micellization,  as  the  curves  of  fluorescence  vs.  detergent  concentration 
show  several  inflections  indicative  of  detergent  structure  not  shown  by 
other  methods.   Further  investigations  into  this  phenomenon  are  planned. 

The  stopped-flow  fluorescence  device  will  be  used  to  follow  some  reactions 
where  DO  exchanges  with  protons  in  the  intermediate  time  range.   If 
successful,  this  would  be  the  first  use  of  stopped-flow  fluorescence  to 
follow  hydrogen  exchange. 

Key  Descriptors:    Phosphorescence,  micelles,  dyes,  alcohol  dehydrogenase. 

Honors  and  Awards:   None 

Publications:   1.   Chen,  R.  F.:  Phosphorescence  of  Tryptophan  and  Proteins 
in  the  Presence  of  Silver  Ion,  Arch.  Biochem.  Biophys. 
166,  584,  (1975). 


3#tf 


Project  No.  Z01  HL  01408-10  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Methodology  in  Fluorescence  Measurements 

Previous  Serial  No.:  NHLI-77 

Principal  Investigator:   Raymond  F.  Chen 

Other  Investigators:   None 

Cooperative  Units:   None 

Objectives:   To  advance  the  state  of  the  art  in  methods  and  instrumentation 
used  in  fluorescence  spectroscopy,  a  technique  which  is  popular  in  biomedical 
sciences,  but  which  has  many  facets. 

Methods  Employed:   As  in  the  past,  we  have  tested  commercially  available 
apparatus  to  see  what  modifications  of  hardware  or  methods  are  required  to 
obtain  data  desired  in  biophysical  chemistry.   Many  instruments  are  designed 
to  work  in  a  particular  way,  but  when  actually  in  use  by  a  biochemist,  it  is 
often  found  that  the  desired  experiment  requires  some  modification  of  the 
apparatus. 

Major  Findings: 

1.  The  ORTEC  9200  nanosecond  spectrometer  was  designed  to  investigate 
fluorescence  decay  kinetics.   However,  it  cannot  in  itself  determine 
lifetimes,  which  are  one  of  the  main  objectives  desired.   To  analyze  the 
decay  curves,  we  have  had  the  ORTEC  connected  so  that  the  digital  data 
representing  the  decay  curves  are  read  into  the  teletype  and  saved  on 
paper  tape,  or  read  onto  magnetic  tape.   The  data  then  are  analyzed  by 
convolution;  i.e.,  one  convolutes  the  lamp  flash  with  theoretical  decays 
representing  certain  lifetimes  and  then  compares  the  experimental  and 
theoretical  decay  curves  to  get  the  lifetimes.   This  is  conveniently  done 
through  our  terminal  connections  to  the  PDP-10  computer  of  the  computer 
center.   Alternatively,  one  can  use  "lifetime  standards",  consisting  of 
partially  quenched  quinine  solutions  and  compare  decay  curves  tc  see  what  the 
actual  lifetime  is.   We  have  also  obtained  a  Schoeffel  GM  100  monochromator 
and  had  a  flange  made  to  allow  emission  to  be  analyzed.   By  obtaining  decay 
curves  at  different  emission  wavelengths,  it  will  be  possible  to  obtain 
spectra  of  the  emission  at  different  times  after  the  exciting  flash. 

2.  We  tested  a  prototype  Aminco  corrected  spectra  spectrof luorometer ,  which 
was  designed  primarily  to  give  corrected  excitation  and  emission  spectra. 
The  instrument  was  loaned  to  us  for  2  months.   We  compared  excitation 

i  wr 


Project  No.  Z01  HL  01408-10  LTD 

and  absorption  spectra,  which  should  coincide;  also  we  compared  the 
instrument's  emission  spectra  with  those  we  obtained  on  another  Aminco- 
Bowman  spectrof luorometer  and  manually  corrected.   Generally,  the  instrument 
was  satisfactory,  but  areas  needing  simplification  were  pointed  out  to  the 
designers.   In  particular,  the  choice  of  time  constants  and  scanning  rates 
was  too  limited,  and  the  need  for  different  phototubes  for  excitation  and 
emission  spectra  was  criticized.   At  present,  a  phototube  has  been  found 
combining  low  noise  and  the  desired  spectral  response  characteristics  has 
been  found.   We  also  made  phosphorescence  measurements  on  the  instrument 
and  showed  that  this  could  be  done  conveniently.   No  other  commercial 
instrument  having  correction  abilities  operates  on  DC  detector  system,  so 
that  we  demonstrated  that  this  instrument  is  the  only  one  able  to  obtain 
corrected  phosphorescence  excitation  and  emission  spectra.   Some  of  these 
spectra  will  be  incorporated  into  a  paper  on  the  effect  of  denaturation  on 
the  phosphorescence  of  proteins. 

3.   We  have  been  interested  in  the  use  of  metal  ions  as  probes  of  protein 
structure  because,  when  metal  ions  interact  with  tryptophan  and  tyrosine, 
they  may  alter  the  fluorescence  and  phosphorescence.   Work  done  with  the 
phosphorescence  of  tryptophan  and  proteins  in  the  presence  of  the  heavy 
metal  ions,  Ag  ,  and  Hg   were  found  to  be  facilitated  by  the  use  of 
aqueous  snows  rather  than  to  attempt  to  make  clear  glasses.   A  paper  summari- 
zing past  work  showing  fluorescence  quenching  by  these  ions  was  prepared  and 
included  recent  work  showing  phosphorescence  enhancement  due  to  increased 
intersystem  crossing  rates  induced  by  the  heavy  metal  ions.   The  work  shows 
that  probing  wich  metal  ions  is  a  useful  method  for  studying  accessibility 
of  phosphorescent  groups  on  proteins. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

The  spectrof luorometer  was  delivered  in  this  institute  some  20  years  ago, 
and  fluorescence  spectroscopy  largely  grew  as  a  result  of  that  development. 
By  continuing  the  activity  of  these  laboratories  in  the  area  of  fluorescence 
methods,  we  continue  to  advance  a  technique  which  has  been  of  great  utility 
in  biomedical  science. 

Proposed  Course: 

The  analysis  of  decay  curves  with  the  ORTEC  is  still  not  simple  enough  to 
be  done  by  the  average  biochemist.   We  plan  to  see  whether  we  can  simulate 
2  and  3-component  decays  with  simple  model  systems  or  computer  programs 
and  to  see  what  pitfalls  one  meets  in  trying  to  compare  actual  and 
theoretical  multi-component  decay.   The  time  emission  spectral  system  will 
be  tested,  and  the  effect  of  deuterium  on  the  lamp  output  will  be  studied. 
In  the  phosphorescence  studies,  we  would  like  to  see  if  dried  material 
at  room  temperature  can  be  studied,  as  there  are  several  reports  that  this 
can  be  done.   Use  of  phosphorimetry  has  been  severely  limited  by  the  need 
to  operate  at  liquid  N   temperature. 


3*4 


Project  No.  Z01  HL  01408-10  LTD 

Key  Descriptors:   Fluorescence,  tryptophane,  silver  ions, 
spectrof luorometer . 

Honors  and  Awards:   None 

Publications: 

1.   Chen,  R.  F. :  The  Effect  of  Metal  Cations  on  Intrinsic  Protein 
Fluorescence,  in  Concepts  in  Biochemical  Fluorescence,  R.  F.  Chen  and 
H.  Edelhoch,  Eds.  M.  Dekker,  N.Y. ,  in  press. 


3+7 


Project  No.  Z01  HL  01409-04  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   An  Automated  Method  for  Rapid  Bacterial  and 
Mammalian  Cell  Growth  and  Assay 

Previous  Serial  No.:   NHLI-76 

Principal  Investigator:   Peter  Carmeci 

Other  Investigators:   None 

Cooperating  Units:   Medicine  Branch,  NCI 
Dr.  Joan  Bull 

Division  of  Oncology 
Albany  Medical  College 
Dr.  Robert  Sponzo 

American  Instrument  Co. 
Silver,  Spring,  Maryland 

Project  Description: 

The  objectives  of  this  project  are  twofold;  the  first  is  to  adapt  the 
capillary  scanning  instrument  to  the  various  requirements  of  biomedical 
research  by  cooperating  with  potential  users  of  the  method  to  develop 
the  techniques  necessary  to  facilitate  the  utilization  for  clinical  and 
research  application.   The  second  is  to  develop  other  methods  where  this 
technique  is  not  applicable.   At  the  present  time  efforts  have  been 
expended  in  the  following  areas  of  endeavor. 

A.  Developing  methods  of  pulse  height  discrimination  for 

(1)  segregating  types  of  bacterial  colonies  during  incubation,  and 

(2)  studying  the  effects  of  growth  factors  and  environment  on 
mammalian  cells. 

B.  Developing  methods  for  early  detection  of  mycoplasma  (pneumonia). 

C.  Developing  methods  for  assaying  bone  marrow  stem  cells. 

a.   A  bread-board  model  pulse  height  discriminator  has  been  built  and  the 
growth  of  stem,  myeloma,  hepatoma  cells  has  been  demonstrated.   In  addition, 
it  has  been  found  that  a  measurement  of  total  growth  of  all  viable  colonies 
in  a  capillary,  i.e.,  the  integration  of  light  scattered  pulses,  provides 

3*4 


Project  No.  Z01  HL  01409-04  LTD 

another  distinctive  and  sensitive  parameter  for  growth  and  pulse  height 
discrimination,  have  been  designed  and  fabricated  in  a  new  unit  that  is 
presently  undergoing  test.   Subsequent  work  will  be  to  characterize  the 
growth  of  myeloma,  hepatoma,  and  stem  cells  under  specific  environmental 
conditions . 

b.  Previous  indications  have  shown  that  mycoplasma  can  be  detected  by 
scattered  light  within  48  hours  (compared  to  10  day  incubation  period 
normally  required).   Growth  in  agar  has  not  been  conducive  to  growth. 
Growth  in  broth  and  in  thin  films  of  agar  has  been  successful  but  not 
optimal.   A  new  technique,  based  on  the  previous  experience  of  growing, 
mycoplasma  on  thin  films  of  agar,  has  shown  more  optimal  growth.   This 
consists  of  introducing  the  mycoplasma  in  solution  to  the  surface  of  a 
thick  (2mm)  surface  of  agar  in  a  number  of  thin  lines  that  are  optically 
aligned  to  the  capillary  scanner.   Further  testing  of  this  technique  is 
under  way. 

c.  A  new  method  of  culturing  human  bone  marrow  stem  cells  has  been 
developed.   The  cultures  are  grown  in  large  plastic  test  tubes  and  the  stem 
cells  grow  more  profusely  and  reliably  than  in  Petri  dishes  or  capillary 
tubes.   This  was  shown  in  a  series  of  tests  with  human  stem  cells  wherein 
samples  were  extracted  into  capillaries  at  various  intervals  during  their 
growth  in  large  tubes.   This  new  method  has  caused  us  to  investigate  the 
possibility  of  detection  assay  within  the  larger  test  tube  directly.   The 
colonies  are  very  few  in  relation  to  the  total  volume  of  the  culture  flask, 
therefore,  to  detect  these  by  light  transmission  would  be  very  difficult. 
Although  the  media  scatters  a  lot  of  light,  liquid  scattering  seems  to  be 
the  easiest  method  for  detection  and  assay. 

When  colonies  are  small,  grown  in  a  three  dimensional  solid  media,  and 
appear  against  a  bright  background  of  light  scattered  from  colloidal  media 
conventional  colony  counters  fail.   Computerized  image  analysis  is  elaborate 
and  expensive.   A  simple  approach  has  been  developed  for  quantitating 
colony  growth  by  using  spatial  frequency  analysis  to  detect  small  colonies 
against  a  uniform  bright  background.   Spatial  frequencies  are  converted  into 
temporal  frequencies  by  chopping  the  image  with  a  rotating  optical  pattern 
wheel,  a  method  developed  for  use  in  missile  guidance  systems  to  detect 
small  emitting  targets  against  bright  clouds.   A  prototype  system,  has 
been  developed  and  applied  to  monitoring  the  growth  of  granulocyte  colonies 
in  methylcellulose  medium  with  bone  marrow  suspensions,  with  good  results. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

The  utilization  of  capillary  tube  scanning  techniques  and  spatial  frequency 
analysis  with  a  rotating  reticle  in  bacteriology  and  mammalian  cell  cultures, 
This  will  provide  a)  means  of  detection  and  assay  early  in  the  growth  of 
colonies  and  b)  methods  that  are  readily  adaptable  to  automation.   The 
application  to  the  study  of  cell  metabolism,  metabolic  defects,  oncology, 
and  clinical  cell  sample  assay  is  anticipated. 

2  2& 


Project  No.  ZOL  HL  01409-04  LTD 
Proposed  Course: 

A.  Develop  hardware  for  pulse  height  analysis  and  investigate  its 
ultilization  for  segregation  of  two  or  more  organisms  by  their  growth 
rates,  and  characterize  cell  growth  under  varying  conditions. 

B.  Develop  techniques  to  apply  cappillary  scanning  for  detecting  and 
measuring  mycoplasma  (Pneumonia) . 

C.  Optimize  the  technique  for  bone  marrow  stem  cell  assay  and  develop 
methods  for  sizing  of  cell  colonies. 

Keyword  Descriptors: 

Automated  method,  Mammalian  cell  growth,  Cell  colony  monitoring,  Capillary 
tubes,  and  Spatial  frequency  analysis. 

Honors  and  Awards:   None 

Publications:   None 


2&> 


Project  No.  Z01  HL  01410-01  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Blood  Gas  Monitoring  for  Extended  Periods 

Previous  Serial  No.:   None 

Principal  Investigator:   Gerald  G.  Vurek 

Other  Investigators:   Theodor  Kolobow  and  D.  Warnock 

Cooperating  Units:   Laboratory  of  Kidney  and  Electrolyte  Metabolism,  NHLI 

Project  Description: 

Objectives:   There  are  clinical  situations  and  research  problems  in  which 
frequent  measurement  of  blood  PO  and  PCO   is  desirable  to  follow  the 
progress  of  therapy  or  result  of  experimental  intervention.   At  present, 
the  most  reliable  method  is  to  withdraw  blood  samples  and  make  the 
measurements  with  some  specialized  apparatus  more  or  less  remote  from  the 
source.   The  objective  of  this  program  is  to  explore  a  way  to  make  an  instru- 
ment perform  these  measurements  at  the  point  of  use  and  yet  introduce  no 
more  complicated  apparatus  than  is  presently  used  to  monitor  intravascular 
pressures. 

Methods  Employed:   Reliable  long  term  measurements  require  stable 
transduction  systems,  simple  extraction  techniques,  and  signal  processing. 
Conventional  polarographic  oxygen  sensors  and  Severinghaus  CO   sensors 
require  frequent  calibration  checks  which  make  them  unsuited   for  in  vivo 
use.   For  oxygen,  we  use  a  galvanic  cell  which  produces  a  current 
stoichiometrically  related  to  the  amount  of  oxygen  supplied  in  the  cell.   A 
flow  rate  of  10    moles  sec.    yields  a  current  of  0.3  UA  which  is  easily 
measured.   For  carbon  dioxide,  we  use  our  previously  developed  calorimetric 
measurement  of  the  heat  of  reaction  of  C0„  with  LiOH.   This  also  has  picomole 
sensitivity.   Of  course,  sample  acquisition  must  be  reliable  because  errors 
here  cannot  be  corrected  without  reference  to  an  alternative  measurement. 
Our  approach  is  to  use  a  membrane  covered  probe  which  allows  the  gases  in 
the  blood  to  diffuse  through  the  membrane  in  proportion  to  their  partial 
pressures.   By  making  the  membrane  permeability  small  compared  to  water 
(plasma)  errors  due  to  the  ever  present  stagnant  layer  can  be  reduced. 
In  addition,  the  probe  can  be  made  of  materials  which  should  resist  changes 
due  to  imbibition  of  substances  from  the  blood  and  by  a  process  which 
should  allow  the  properties  of  each  probe  to  be  measured  prior  to  use.   A 
simple  but  reliable  technique  for  converting  the  signals  to  useful  values 
is  to  convert  the  signals  to  digital  form  as  close  to  the  transducers  as 

i  35? 


Project  No.  Z01  HL  01410-0-  LTD 

possible  to  minimize  the  errors  due  to  drift  and  nonlinear  effects  of 
analog  circuitry.   Thus,  the  blood  gas  system  will  use  stable,  sensitive, 
and  specific  transducers  for  gas  analysis,  a  stable  probe,  and  digital  signal 
processing  to  obtain  reliable  long  term  blood  gas  monitoring. 

Major  Findings:   Oxygen  measurement  with  the  galvanic  cell  is  a  well 
established  procedure   and  is  used  industrially  as  well  as  in  at  least 
one  electronic  "Van  Slyke"  apparatus.   We  have  found  the  commercial 
galvanic  cell  to  be  satisfactory  although  bulky.   The  sensitivity  of  the 
apparatus  requires  that  all  the  components  be  gas  tight  to  prevent 
atmospheric  oxygen  from  leaking  into  the  system  and  swamping  the  desired 
signal.   At  present  we  use  Swagelok  fittings  which  are  satisfactory  but 
leave  room  for  improvement  in  terms  of  convenience. 

Results  from  improvement  on  the  apparatus  for  assay  of  picomole  amounts  of 
CO   (see  FY  1974  report  NHLI-87)  now  allow  usto  measure  CO  with  a 
sensitivity  +  1  s.d.  Qf  about  10    mole  sec.   .   This  has  Been  accomplished 
by  using  less  noisy  amplifiers  and  power  supplies.   Previous  work  (FY  1974 
NHLI-127) ,  had  demonstrated  that  continuous  measurement  of  CO   was  difficult 
due  to  the  problem  of  maintaining  the  proper  water  vapor  content  of  the  LiOH. 
This  has  been  eliminated  by  using  a  sampling  technique  in  which  samples  of 
the  gas  steam  from  the  probe  are  periodically  introduced  into  the  calorimeter 
chamber  which  is  a  modification  of  the  picomole  assay  apparatus.   By  using 
this  sampling  approach,  we  can  eliminate  the  overshoot  effect  observed  with 
the  earlier  steady  state  approach.   In  addition  baseline  drift  of  the 
calorimeter  is  corrected  between  samples.   Samples  can  be  taken  at  two 
minute  intervals  or  less  so  that  the  overall  system  response  time  to  changes 
in  the  patient's  PCO   is  adequate  for  monitoring  purposes. 

Sample  probe  design  is  dependent  on  several  factors  including  relative 
permeability,  response  time,  and  convenience.   In  order  to  reduce  the  error 
due  to  the  pressure  of  an  unstirred  layer  of  fluid  adjacent  to  the  probe, 
the  overall  permeability  of  the  probe  must  be  about  10%  that  of  water. 
Oxygen  is  the  limiting  gas  here  for  it  is  less  soluble  than  CO   in  most 
materials.   Silicone  rubber  has  excellent  biocompatibility  but  it  is  10 
times  as  permeable  as  water  so  that  a  very  thick,  and  thus  very  slow, 
membrane  would  be  needed.   Our  present  approach  is  to  use  a  composite 
structure  consisting  of  an  open  spring  coated  with  a  thin  layer  of  silicone 
rubber,  an  intermediate  coating  of  low  permeability  gas  phase  deposited 
para-xylylene,  and  an  outer  coating  of  silicone  rubber.   Para-x^lylene 
has  about  10   the  permeability  of  silicone  rubber  so  that  a  thin  (1  um) 
layer  has  the  needed  permeability  and  rapid  response.   The  silicone  rubber 
layers  are  support  and  protection , and  variations  in  their  properties 
due  to  manufacture  on  imbibition  produce  second  order  effects  on  the  overall 
probe  response.   At  present  we  can  use  a  1  mm  dia.  by  15  mm  long  probe  to 
obtain  adequate  gas  flux  at  expected  partial  pressures  of  80  mm  Hg  (PO  ) 
and  40  mm  Hg  (PCO  ) . 


353- 


Project  No.  Z01  HL  01410-Ql  LTD 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

Long  term  extracorporeal  oxygenation  and  other  forms  of  intensive 
respiratory  care  can  be  facilitated  by  knowledge  of  the  status  of  blood 
gases.   It  is  the  purpose  of  this  program  to  demonstrate  an  apparatus 
which  can  provide  this  information  without  the  need  for  blood  samples  and 
with  no  more  than  a  single  catheter,  comparable  to  a  pressure  manometer 
catheter,  attached  to  the  patient.   Thus  a  step  in  the  direction  of 
biochemical  monitoring  will  be  made.   This  represents  a  quantum  jump  in 
intensive  care  monitoring  because  here-to-for  only  pressures  and  bioelectric 
signals  have  been  monitored  satisfactorily. 

Proposed  Course:   The  apparatus  will  be  evaluated  in  vitro  and  its  performance 
will  be  demonstrated  in  vivo.   Additional  development  effort  may  be  under- 
taken on  the  probe  to  make  its  piping  more  flexible. 

Keyword  Descriptors:   Blood  gas  measurement,  intensive  care  monitory, 
PO  ,  PCO  ,  galvanic  cell,  and  microcalorimetry. 

Honors  and  Awards:   None 

Publications:   None 


2SI 


Project  No.  Z01  HL  01411-09  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Blood  Flow  Measurement  Using  Nuclear  Magnetic 
Resonance  Techniques 

Previous  Serial  Number:   NHLI-83 

Principal  Investigators:   Vsevold  Kudravcev,  Robert  L.  Bowman 

Other  Investigators:   Anthony  Sances,  Jr.,  Joseph  H.  Battocletti 

Cooperating  Units:   Medical  College  of  Wisconsin,  Milwaukee,  Wisconsin,  on 
contract  to  Laboratory  of  Technical  Development 

Project  Description: 

To  continue  to  develope  the  practical  aspect  of  nuclear  magnetic  labelling 
technique  for  biological  blood  flow  measurement,  especially  cerebral  and 
digital  (investigation  of  Raynaud's  phenomena). 

Progress:   The  work  is  divided  between  this  laboratory  and  a  group  under 
contract  to  this  laboratory  at  the  Medical  College  of  Wisconsin,  directed 
by  Dr.  A.  Sances,  with  Dr.  J.  H.  Battocletti  as  a  co-principal  investigator. 
This  group  conducts  the  biological  observations  using  the  NMR  labelling 
technique,  performs  theoretical  analysis  of  the  data  obtained,  and  developes 
cerebral  NMR  flowmetric  devices. 

In  this  laboratory,  the  universal  weak  detector  field  blood  flow  meter 
previously  constructed  was  investigated  using  simulated  blood  vessels  (rubber 
tubing  with  an  inside  diameter  of  1-2.5  mm).   Satisfactory  NMR  results  were 
obtained  in  the  detector  magnetic  field  with  a  strength  of  20  gauss;  a 
magnetic  field  of  2.5  kgs  was  used  for  liquid  prepolarization.   Signal-to- 
noise  ratio  was  obtained  in  values  of  20/1  and  10/1,  depending  upon  flow 
velocity  and  simulated  blood  vessel  volume,  and  without  interference  from 
surrounding  tissue  protons  (simulated  by  the  thick  rubber  tubing  walls) . 

Helmholtz  coils  previously  used  for  production  of  the  NMR  detector  field 
were  substituted  by  more  effective  and  homogenous  field  coils.   These  newer 
coils  were  constructed  to  be  large  enough  to  accommodate  human  limbs  and 
bodies  of  small  animals,  and  will  have  practical  application  in  the  near 
future.   This  application  is  dependent  on  the  development  of  proper  polariz- 
ing field  units  of  sufficient  size  and  strength,  and  the  completion  of 
suitable  NMR  probes  under  construction  at  the  present  time. 


3S* 


Project  No  Z01  HL  01411-09  LTD 

A  combined  (single  and  cross  coil)  NMR  probe  was  also  constructed  for 
digital  blood  flow  measurement  using  a  medium  strength  detector  field  (600 
gauss).   At  this  field  strength,  composite  NMR  signals  appear,  consisting 
of  strong  dominant  NMR  signals  from  polarized  protons  imbedded  in  surrounding 
tissues,  and  relatively  weak  signals  from  flowing  blood.   A  data  retrieval 
computer  (10  averages)  was  necessary  to  separate  this  interf erring,  dominant 
signal  from  the  true  flow  signal.   Blood  flow  measurement  in  the  human  finger 
was  successfully  performed  using  a  suitable  computer  during  the  author's 
recent  visit  to  the  Wisconsin  facility.   During  this  visit,  NMR  apparatus 
constructed  individually  by  the  two  laboratories  was  tested  and  compared, 
and  mutual  exchange  of  equipment  was  undertaken  with  beneficial  results  to 
both  research  units. 

During  the  past  year,  several  of  our  basic  NMR  detectors  were  improved  using 
recent  developments  in  solid  state  technqiues  and  further  modifications  were 
implemented  with  resulting  increase  in  stability  and  sensitivity.   Improved 
envelope  detector,  low  noise  RF  gates,  dual  gate  FET  preamplifiers, 
adjustable  bandpass,  L.C.  filters,  and  many  other  innovations  were  incorpo- 
rated in  the  apparatus  circuits. 

Major  Findings:   A  new  NMR  flowing  liquid  magnetization  envelope  modulator 
was  developed  and  constructed.   The  operation  of  this  modulator  is  based  on 
the  superimposition  of  three  separate  magnetic  fields:   The  DC  magnetic 
field,  the  low  frequency  AC  field,  and  the  Larmor  frequency  RF  field. 
This  modulator  allows  periodically  tagged  magnetization  envelope  of  the 
flowing  liquid  without  switching  the  transient  which  is  present  in  conven- 
tional NMR  tagging  gates.   In  addition,  due  to  the  fact  that  the  DC  field 
is  supplied  by  the  modulator  himself  the  former  may  be  moved  over  the  blood 
vessels  under  investigation  without  continuous  readjustment  of  the  Larmor 
frequency  required  to  produce  NMR  "burnout"  tags,  or  needed  degree  of 
liquid  magnetization  envelope  modulation. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
Non- intrusive  NMR  methods  can  be  used  to  mesure  peripheral  blood  flow  or 
blood  flow  from  various  organs.   This  NMR  system  is  therefore  applicable 
for  screening,  continuous  monitoring  during  surgery,  or  for  the  determination 
of  blood  flow  in  post-surgical  or  trauma  patients. 

Proposed  Course:   To  apply  the  digital  blood  flow  measurement  technique 
developed  previously  for  investigation  of  Raynaud's  phenomena;  cooperative 
research  with  the  Medical  College  of  Wisconsin  for  biomedical  application; 
development  of  a  practical  version  of  the  time-sharing  and  suppressed- 
carrier  NMR  SR  detector  described  in  previous  reports. 

Keyword  Descriptors:   Non-Invasive  Blood  Flow  Monitoring,  Nuclear  Magnetic 
Resonance,  NMR  Magnetic  Labelling  Technique. 


3sr 


Project  No.  Z01  HL  01411-09  LTD 
Honors  and  Awards:   None 
Publications: 

1.  Battocletti,  J.  H.  Linehan,  J.H.,  Wang,  O.S.,  Sances,  A.,  Larson,  S.  J., 
Evans,  S.  M. ,  Itskovitz,  H.  D.,  Bowman,  R.  L. ,  and  Kudravcev,  V.: 
Organ  Blood  Flow  Measurement  Using  NMR.   Digests  Intermag.  Conf . , 
Toronto,  Canada,  35.8,  May  1974. 

2.  Battocletti,  J.  H. ,  Sances,  A.,  Larson,  S.  J.,  Evans,  S.  M. ,  Bowman,  R.L. 
Kudravcev,  V. ,  and  Halbach,  R.  E. :   A  review  of  Nuclear  Magnetic 
Resonance  Techniques  Applied  to  Biological  Systems.  In  Llaurado,  J.  G., 
Sances,  Jr.,  A.,  And  Battocletti,  J.  H.  (Eds):  Biologic  and  Clinical 
Effects  of  Low-Frequency  Magnetic  and  Electric  Fields.  Charles  C.  Thomas, 
Springfield,  Illinois,  1974,  pp  263^-294. 

3.  Battocletti,  J.  H. ,  Evans,  S.M.,  Larson,  S.  J.,  Sances,  A.,  Bowman,  R.  L. 
Linehan,  J.  H. ,  Kudravcev,  V.,  Genthe,  W.  K. ,  Halbach,  R.  E. ,  and 
Antonich,  F.  J.  Measurement  of  Blood  Flow  by  Nuclear  Magnetic  Resonance 
Techniques.  In  W.E.  Vannoh  and  H.  Wayland ,  (Eds):  Flow,  Its 
Measurement  and  Control  in  Science  and  Industry.  Instrument  Society 

of  America,  Pittsburgh,  Pa.,  1974,  Vol.  1,  Part  3,  pp  1401-1409. 

4.  Brooks,  R.  A.,  Battocletti,  J.  H. ,  Sances,  Jr.  A.,  Larson,  S.  J., 
Bowman,  R.  L. ,  and  Kudravcev,  V.:  Nuclear  Magnetic  Relaxation  in  Blood. 
IEEE  Transactions  on  Biomedical  Eng.  22:  12-18,  Jan.  1975. 

5.  Battocletti,  J.  H.  Sances,  Jr.,  A.,  Larson,  S.  J.,  Evans,  S.  M. , 
Bowman,  R.  L. ,  Kudravcev,  V.,  and  Ackmann,  J.  J.:  Clinical  Applications 
and  Theoretical  Analysis  of  NMR  Blood  Flowmeter.  Biomed.  Eng.  (London) 
10  (1):  12-20,  January,  1975. 


3& 


Project  No.  Z01  HL  01412-03  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Discrete  Cell  Temperature  Measurement  Study 

Previous  Serial  Number:  NHLI-113 

Principal  Investigator:   Robert  L.  Bowman 

Other  Investigators:   E.  Ronald  Atkinson 

Cooperative  Units:   J.  Peterson,  Biomedical  Engineering  and  Instrumentation 
Branch,  DRS . 

Project  Description: 

Objectives:   Radioautography  and  staining  methods  are  the  means  generally 
available  for  indication  of  cell  response  to  mitogens,  immunoreagents  and 
specific  metabolic  stimulants,  i.e.  lymphocyte  transformation  tests.   We 
are  examining,  developing  instrumentation  and  methods  to  grade  cell  responses 
to  these  metabolic  modifiers  by  measuring  heat  output  from  discrete  cells. 
We  hope  to  provide  a  visual  indication  of  heat  production  from  each  cell  in 
a  field  observed  microscopically. 

Methods: 

1.  A  system  based  on  a  curve  point  transition  in  a  thin  slice  of  ferro- 
electric material  which  changed  its  optical  properties  at  35  C  was 
abandoned  when  the  reputed  transition  point  was  observed  to  be  too  high 
for  use  and  no  other  suitable  material  was  available. 

2.  A  system  based  on  the  rate  of  condensation  of  a  volatile  oil  (dimetyl 
silicone)  on  a  thin  .film  that  supports  a  film  of  cells  under  a  cover  glass 
uses  the  heat  evolved  from  each  cell  to  modify  the  thickness  of  the 
condensation  film  which  is  observed  as  interference  fringes  appearing  around 
each  cell.   A  thermal  (Peletier)  effect  heat  pump  below  the  film  is  cycled 
to  condense  and  volatilize  the  oil  below  the  film. 

3.  Cholesteric  liquid  crystals  with  a  metastable  state  in  35-37  C  range 
are  formed  in  films  to  produce  a  color  background  that  will  be  modified 
by  local  cell  heat. 

4.  Radiometric  image  formation  by  use  of  a  superconductive  bolometer 
based  on  the  point  of  superconductive  transition  of  niobium.   Direct 
radiation  from  single  cells  could  be  measured. 

i  SS7 


Project  No.  Z01  HL  01412-03  LTD 

Major  Findings:   The  evaporating  film  system  has  been  constructed  and  tested 
with  several  systems  last  year  and  discrete  cells  were  observed  to  modify 
the  oil  film  thickness  to  produce  patterns  indicating  that  some  cells 
were  better  heat  sources  than  others  but  controls  that  would  preclude 
the  possibility  that  the  fringes  were  artifacts  were  needed.   This  year 
fresh  granulocytes  were  mixed  with  bacteria  (E.  Coli)  and  the  evidence  of 
heat  compared  to  number  of  bacteria  ingested.   These  experiments  showed  a 
high  heat  production  related  to  number  of  organisms  ingested  but  on 
occasion  controls  without  bacteria  also  showed  heat  production  which  was 
presumed  due  to  degranulation  and  lysis  of  older  cells. 

A  modification  of  the  system  of  introducing  cells  that  will  permit  addition 
and  removal  of  reagents  to  the  cells  while  they  are  observed  will  be  used  to 
establish  internal  control  and  avoid  artifacts. 

The  liquid  crystal  approach  using  available  cholesteric  substances  had  a 
texture  of  color  and  crystalinity  due  to  local  color  domains  of  the  same 
order  of  magnitude  as  the  cells.  A  program  to  purify  the  liquid  crystal 
material  at  the  Chemical  Engineering  Section  of  BEIB  has  demonstrated 
remarkable  improvement  in  the  uniformity  of  films  and  suggests  that  a  37 
material  should  be  processed  and  tested  on  cell  suspensions. 

No  suitable  crystal  with  the  requisite  transparency  and  ferroelectric  curie 
transition  at  physiological  temperatures  and  suitable  mechanical  properties 
is  available  and  this  approach  has  been  suspended  until  such  material 
becomes  available.   The  superconductive  bolometer  approach  has  been  carried 
to  the  stage  of  demonstration  that  the  niobium  bolometer  performs 
with  the  sharp  change  that  was  anticipated  but  this  approach  is  expected  to 
lead  to  a  relatively  cumbersome  and  expensive  system  that  needs  more 
investment   in  time,  personnel  and  money  than  is  wise  to  invest  until  the 
simpler  systems  are  proven  inadequate  or  specific  requirements  identified 
that  can  only  be  met  by  the  radiometric  system. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
Single  cell  measurements  may  be  particularly  important  when  specific  reactor 
cells  cannot  be  separated  from  bulk  cells.   A  few  reacting  cells  in  a  larger 
batch  may  be  particularly  important  in  oncogenesis,  aging,  and  immuno- 
reactions. 

Proposed  Course:   Refinement  of  sample  handling,  determination  of  sensitivity, 
improvement  of  microsocpic  image  and  documentation  of  results  of  specific 
assays . 

Key  Descriptors:   Microcalorimetry,  Lymphocyte  Transformation,  Phagocytosis, 
Cell  Metabolism,  and  Radioautography . 

Honors  and  Awards:   None 

Publications:   None 

2  3SS 


Project  No.  Z01  HL  01413-13  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 


Project  Title: 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Instrumentation  for  the  Study  of  Pre-Steady  State  Enzyme 
Kinetics 


Previous  Serial  No.:  NHLI-74 


Principal  Investigator:   Robert  L.  Berger 


Other  Investigators: 


J.  Frolich,  NICHD 

M.  Marini,  Dept.  Biochem. ,  Northwestern  Medical  School 

N.  0.  Kaplan  and  J.  Everse,  U.  of  California,  at 

San  Diego  Medical  School 

L.  Rossi-Bernard,  University  of  Milan,  Italy 

J.  A.  McCray  and  P.  Smith,  Dept.  of  Physics,  Drexel  U. 

W.  F.  Friauf,  H.  Cascio  and  E.  Beile,  BEIB 

R.  Shrager,  DCRT,  Lab.  Physical  Sciences 

M.  Sapoff,  Thermometries,  Inc. 

B.  Balko,  Dept.  of  Physics,  Boston  University 


Cooperating  Units:   Ingold  Electrodes,  Zurich 


Project  Description: 


Objectives:   The  objectives  of  this  project  are  to  develop  new  instrumenta- 
tion methods,  data  handling  techniques  and  theoretical  treatments  for  the 
physiochemical  study  of  the  thermodynamics,  kinetics  and  thus  the  mechanisms 
of  enzyme  action  in  solutions  and  in  the  intact  cell  or  cell  membrane.   In 
particular,  to  develop  the  method  and  instruments  to  study,  in  collaboration 
with  other  laboratories,  the  reactions  of  hemoglobin  with  the  respiratory 
gases  both  in  the  normal  state  and  as  modified  by  the  changes  of  physical 
factors,  small  molecules,  various  metabolites,  and  gentically,  such  as  in 
sickle  cell  anemias.   The  reactions  of  various  cellular  enzymes,  particularly 
ATPase  and  lactate  dehydrogenase,  and  their  interactions,  and  control,  in 
the  cell  are  studied  as  they  relate  to  the  hemoglobin  reactions  in  cardiology, 
pulmonary  and  respiratory  function,  and  circulation.   Where  appropriate 
analytical  methods  are  developed  for  research  and  clinical  application. 

Methods  Employed:   The  methods  used  in  the  investigation  of  the  mechanisms 
of  enzyme  action  are  those  of  pre-steady  state  chemical  kinetics  and 
thermodynamics.   Measurements  of  the  appropriate  parameters  are  made  by 
developing  the  necessary  equipment  to  mix  solutions  rapidly  and  follow  the 
course  of  the  resulting  chemical  reactions  by  optical,  thermal,  glass 
electrode,  etc.,  detectors.   In  general,  equipment  is  not  available,  either 


3S"? 


Project  No.  Z01  01413-13-LTD 

in  the  literature  or  commercially,  for  investigations  in  this  area.   Such 
apparatus  is  conceived  and  designed  in  this  laboratory,  together  with 
consultants,  construction  being  carried  out  wherever  most  appropriate;  i.e., 
in  our  shops  or  by  commerical  f irms »  special  university  facilities,  or 
at  the  several  special  research  laboratories  such  as  the  Jet  Propulsion 
Laboratory.   In  pursuing  these  investigations,  a  wide  variety  of  physical 
parameters  must  be  studied,  which  leads  to  the  need  for  an  understanding 
of  the  underlying  physical  theory  governing  the  reactions.   Expert 
consultants  and  collaborators  are  brought  in  to  assist  in  the  design, 
analysis,  and  evaluation  of  the  equipment,  particularly  as  it  applies  to 
certain  specific  enzyme  systems  under  investigation. 

Major  Findings: 

The  optical  stopped-flow  system  has  been  further  improved  by  the  development 
of  a  new  type  of  observation  tube  which  has  eliminated  several  artifacts 
found  when  the  system  was  put  under  stringent  tests  with  a  biochemical 
system  (EGTA  &  Ca) .   Improvements  in  the  high  speed  stop  valve  have  greatly 
increased  the  reliability  of  the  instrument  and  again  eliminated  a  very 
troublesome  artifact. 

A  rapid  chemical  quench  mixing  apparatus  has  been  developed  for  the  study 
of  fast  enzymatic  reactions  containing  chemically  stable  reaction  products. 
The  essential  parts  of  the  apparatus  are:  (1)  syringe  block  and  mixers, 
(2)  stepping  motor  and  drive  assembly,  and  (3)  assay  value.   Solutions 
containing  enzyme  and  substrate  are  driven  through  a  mixing  chamber  (Berger 
ball  mixer)  and  allowed  to  react  in  an  intermediary  tube  before  passing 
into  a  second  mixer  where  a  quenching  agent  is  added  to  terminate  the 
reaction.   Alternately,  substrate  additions  can  be  made  in  the  first  and 
second  mixers  and  the  quenching  reagent  added  to  a  third  mixer.   Reaction 
time  is  varied  by  changing  the  volume  of  the  intermediary  tube  and  the 
flow  velocity  of  reactants  between  mixers.   The  syringe  pistons  are 
actuated  by  a  common  platform  attached  to  a  lead  screw  which  converts  the 
rotational  motion  of  the  stepping  motor  to  linear  translational  motion. 
Fluid  emerging  from  the  final  mixer  passes  through  an  assay  valve  which 
shunts  material  remaining  in  the  line  from  the  previous  shot  to  waste 
before  a  new  sample  is  collected.   The  apparatus,  which  was  calibrated  by 
measuring  the  pseudo-first  order  rate  constant  for  the  alkaline  hydrolysis 
of  2,4-dinitrophenylacetate,  has  a  dead  time  (minimum  quenching  time)  of 
.003  seconds. 

The  instrument  is  currently  being  used  to  study  the  pre-steady  state  time 
course  of  ATP  hydrolysis  by  the  (Na  -K  )  activated  ATPase  of  electroplax 
microsomes.   This  activity  is  part  of  an  enzyme  system  which  couples 
active  Na   extrusion  to  inward  K  flux  across  the  plasma  membrane.   In  the 
presence  of  Na   the  microsomes  are  rapidly  phosphorylated  by  ATP  resulting 
in  an  acid-stable  intermediate  complex,  E  ~  P.   If  K   is  added  with  Na 
phosphoprotein  degraded  to  an  acid-labile  intermediate  resulting  in  an 


3&> 


Project  No.  Z01  HL  01413-13-LTD 

overshoot  in  the  E  ~  P  vs  time  curve  and  an  initial  "burst"  of  inorganic 
phosphate  production.   Breakdown  of  the  acid-labile  intermediate  designed 
E-P  in  the  following  sequence  is  rate  limiting  at  low  ATP: 

E  +  S  v— *  E  S   £s£i   E  ~  P  ^      E.p  VATP, 

h  +  b  XD       WT  ^3)        (4)   E  +  Pi 

At  high  ATP  substrate  activation  of  the  final  step  is  observed.   Although 
the  precise  relation  of  steps  in  the  enzymatic  and  transport  processes  are 
yet  unknown  the  fact  that  E   P  is  activated  by  low  concentrations  of  Na 
suggests  that  it  represents  a  high  affinity  state  of  the  enzymatic  carrier 
for  Na  .   By  analogy  E-P  which  is  formed  at  low  concentrations  of  K  may 
show  high  affinity  for  K  . 

A  new  differential  thermistor  bridge  has  been  constructed  and  tested  with 
excellent  results.   It  will  be  used  on  both  the  high  speed  stopped  flow 
calorimeter,  recently  completed,  with  a  new  thermistor  probe  which  now  has 
excellent  stability  and  very  low  leakage.   Extensive  testing  will  commence 
as  soon  as  all  components  are  operating  reliably.   See  the  attached 
appendix  for  the  complete  mathematical  simulation  of  this  system  and  the 
detailed  design  of  the  calorimeter.   This  is  the  report  of  Dr.  Balko  who 
contributed  to  the  detailed  design  and  testing  of  this  system.   Preliminary 
experiments  on  glutamic  dehydrogenassreactions  have  been  carried  out  with 
Dr.  Harvey  Fisher,  V.  A.  Kansas  City,  Kansas,  furnishing  us  material  and 
biochemical  guidance. 

A  new  differential  high  speed-high  sensitivity  pH  meter  has  been  finished 
and  tested  at  Ingold  Electrodes  on  a  fluoride  detection  system  where  one 
electrode  was  a  potassium  electrode  and  the  other  a  fluoride  electrode  which 
is  also  sensitive  to  potassium.   Results  show  that  the  electrode  can  be 
used  to  +  0.0001  pF.   It  will  be  coupled  with  the  differential  thermistor 
system  to  do  thermal-potentiometric  protein  titrations  simultaneously. 

Much  work  on  the  coating  of  pH  electrodes  has  been  carried  out  using  Lycra. 
Excellent  results  with  one  set  of  electrodes  were  obtained  but  so  far  the 
work  does  not  seem  to  be  repeatable  on  other  than  a  specific  type  of  glass 
electrode. 

Significance  to  Biomedical  Research  and  The  Program  of  the  Institute: 

An  understanding  of  the  basic  mechanisms  of  disease  is  a  prerequisite  to 
prevention  and  cure.   The  investigation  of  the  reaction  of  the  respiratory 
gases  with  hemoglobin,  the  red  cell,  and  cytochrome  oxidase  in  heart 
muscle  cells  is  fundamental  to  an  understanding  of  normal  cell  respiration 
and  particularly  to  what  has  gone  wrong  as  in  the  case  of  sickle  cell  anemia, 
mycardial  infarction,  etc.   It  is  hoped  that  this  research  wil]  result  in 
instrumentation  to  permit  the  medical  scientist  to  perform  research  leading 
to  clarification  of  the  ways  in  which,  for  example,  sickle  cell  anemia  can 


3&f 


Project  No.  Z01  HL  01413-13-LTD 

be  managed  by   the  use  of  chemicals.   The  extension  of  such  investigations 
to  other  disease  systems  and  possible  results  seem  abundantly  clear  in  terms 
of  preventive  medicine  and  improvement  in  health  care. 

Prposed  Course:   Work  will  continue  on  the  various  systems  and  detectors  to 
bring  each  instrument  to  the  point  where  its  usefulness  to  the  biomedical 
scientist  has  been  established.   Efforts  will  then  be  made  to  have  it 
available  from  manufacturer. 

Keyword  Descriptors:   Fast  Thermistors,  Thermistor  Bridge,  Stopped-Flow 
Calorimeter,  Quenched-Flow  Apparatus  and  ATPase  Sacroplasmic  Reticulum. 

Honors  and  Awards:   None 

Publications: 

1.  Marini,  M.  A.,  Martin,  C.  J.,  Berger,  R.  L. ,  and  Forlani,  L: 
Biopolmers,  Vol.  13,  pp  891-902,  1974. 

2.  Marini,  M.  A.,  Martin,  C.  J.,  Berger,  R.  L. ,  and  Forlani,  L. : 

A  proposed  solution  for  the  determination  of  the  ionization  constants 
of  set  of  ionizing  groups  in  proteins,  Anal.  Cal.  Vol.  3,1974. 


Uz 


Project  No.  Z01  HL  01414-03  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Development  of  Microcalorimeters  for  Clinical  Chemistry 

Previous  Serial  No.:  NHLI-75 

Principal  Investigator:   Robert  L.  Berger 

Other  Investigators:   Edwige  Panek,  Frank  Noble,  Technical  Development,  NHLI 
Donald  Young,  Nadja  Rehak,  Clinical  Chemistry,  NIH 
Edward  Prosen,  Physical  Chem.  Div.,  NBS 
Luigi  Rossi-Bernardi,  Prof.  Enzymology,  U.  of  Milan 
Mario  Marini,  Assoc.  Prof.  Biochem. ,  Northwestern  Univ. 
Norman  Davids,  Prof.  Eng.  Mech. ,  Penn  State  Univ. 
Bohdan  Balko,  Dept.  of  Physics,  Boston  Univ. 

Cooperating  Units:   BEIB,  LMH,  NHLI 

Project  Description: 

Objectives:   Virtually  all  chemical  reactions  produce  heat  and  calorimetry 
has  long  been  used  to  investigate  them.   For  biological  use,  however,  high 
sensitivity,  small  volumes  of  reactants,  and  short  equilibration  times 
are  needed.   It  is  the  objective  of  this  project  to  develop  such  an 
instrument  for  use  in  the  time  range  of  a  few  seconds  to  1  or  2  hours. 

Methods  Employed:   Initial  designs  are  constructed  in  this  laboratory 
with  special  assistance  from  commercial  firms  in  the  construction  of  sensors; 
contracts  are  let,  where  warranted,  for  the  development  of  a  completed 
instrument  with  refinements  that  would  tax  our  own  facilities.   The 
instrument  is  then  tested  in  conjunction  with  other  interested  biochemical 
calorimetrists  utilizing  appropriate   enzymatic  and  cellular  reactions. 

Major  Findings:   The  batch  calorimeter  has  been  used  both  in  this  laboratory 
and  in  clinical  chemistry  to  investigate  its  use  and  limitations  on  several 
specific  reactions. 

The  calorimetric  system  for  measuring  the  heat  of  reactions  was  set  up  in 
the  normal  mode  (it's  measurement  of  total  heat  of  reaction)  using  an 
integrator  built  in  BEIB  and  calibrated  with  acid-base  reaction. 

Determination  of  uric  acid  in  serum  was  initiated  and  compared  with  the 
method  used  in  the  clinical  laboratory.  Effect  of  protein  level  in  the 
serum  on  the  calorimetric  determination  of  the  uric  acid  is  being  evaluated. 


363 


Project  No.  Z01  HL  01414-03  LTD 

Preliminary  investigation  of  the  hydrolysis  of  hemoglobin  by  pepsin  and 
trypsin  in  model  reaction  and  gastric  juices  established  the  feasibility 
of  the  calorimetric  method  in  the  kinetic  mode  for  determination  of  the 
activity  of  these  enzymes  in  gastric  juices  and  feces. 

The  interfacing  of  the  microcalorimeter  with  the  computer  system  is  now 
finalized  and  the  system  is  ready  to  be  used  in  the  kinetic  mode.   Using 
synthetic  substrates  which  are  specific  for  either  pepsin  or  trypsin  only, 
the  calorimetric  determinations  of  these  enzymes  is  compared  with  the  routine 
spectrophotometric  method. 

Determination  of  cholesterol  in  human  serum  using  the  cholesterol 
oxidase-catalase  coupled  reaction  was  carried  out  on  the  batch-type,  NBS- 
NIH  microcalorimeter  at  37  C.   The  experiments  were  performed  either  in 
phosphate  or  tris-HCl  buffers,  at  pH  7.4.   Because  of  the  low  specific 
activity  of  the  cholesterol-oxidase  (ranging  from  0.4  IU  to  5  IU  of  the 
enzyme  commercially  available) ,  and  its  poor  affinity  for  the  substrate, 
the  amount  of  enzyme  has  to  be  high  enough  to  transform  the  cholesterol  to 
cholest.4  en-3  one.,  therefore,  the  two  compartments  of  the  cell  have  to 
be  balanced  with  an  equal  concentration  of  albumin  in  order  to  avoid  any 
heat  of  dilution.   Under  those  conditions,  the  cholesterol-oxidase  and 
catalase  coupled  reaction  is  probably  partially  inhibited  by  the  low 
concentration  of  oxygen  available.   To  improve  this  methodology  the 
cholesterol  oxidase  (5  IU/mg)  is  being  attached  to  glass  beads. 

The  determination  of  triglycerides  was  carried  out  by  the  same  method  as 
described  for  cholesterol.   The  first  step  of  the  analysis  involves 
the  enzymatic  hydrolysis  of  the  triglycerides  by  lipase  in  a  phosphate 
buffer  at  pH  7.0.   The  AH  for  that  reaction  was  demonstrated  by  micro- 
calorimetry  to  be  close  to  zero.   The  same  result  was  detected  from  the 
heat  of  combustion  of  pure  tiplomitin.   In  the  second  step,  the  glycerol 
liberated  from  the  enzymatic  hydrolysis  was  coupled  to  glycerokinase  and 
ATP,  the  AH  of  the  reaction  was  6.5  k  cal/mole,  corresponding  to  the 
ATP  hydrolysis.   However,  it  was  observed  that  an  endothermic  reaction 
preceeded  the  ATP  hydrolysis  exothermic  reaction.   This  corresponds  to  a 
contamination  of  the  glycerokinase  by  a  low  ATPase  activity.   This 
undesirable  secondary  reaction,  limits  the  sensitivity  of  the  method  to 
0.1  M  of  glycerol  or  triglyceride. 

The  stopped-flow  microcalorimeter  is  undergoing  extensive  testing  to 
eliminate  a  number  of  artifacts  that  make  its  operation  less  reliable 
than  the  batch  system  at  present.   The  mathematical  simulation  used  in 
design  and  data  correction  of  these  systems  in  available  as  an  in  house 
technical  Report. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

The  use  of  these  methods  as  an  analytical  tool  for  clinical  chemistry  shows 


3M 


Project  No.  Z01  HL  01414-03  LTD 

considerable  promise  as  a  means  of  improving  the  accuracy,  precision,  and 
thruput  of  clinical  tests.   In  addition,  it  makes  possible  the  use  of 
many  new  tests  for  enzyme  or  substrate  tests,  antigen-antibody  reactions, 
coagulation  tests,  etc.,  which  are  not  now  able  to  be  done  due  either  to 
the  lack  of  a  suitable  detection  method  or  to  the  fact  that  the  present 
tests  are  long,  have  high  variability,  and  are  therefore  not  used. 

Perhaps  the  long-range  significance  of  this  project  are  the  possibilities 
that  the  calorimeter  offers  for  the  study  of  many  biochemical  reactions 
which  cannot  now  be  investigated  due  to  a  lack  of  a  suitable  detector  of 
the  reaction.   An  example  of  current  interest  in  the  many  steps  preceeding 
final  coagulation  that  occurs  in  the  forming  of  a  thrombus. 

Proposed  Future  Research:   The  effectiveness  of  the  batch  microcalorimeter 
as  an  instrument  suitable  for  routine  clinical  work  will  continue  to  be 
explored  in  collaboration  with  clinical  chemistry  and  molecular  hematology. 
Additional  exploration  of  other  chemical  reactions  is  planned  particularly  in 
the  area  of  fatty  acid  binding  to  proteins  and  antigen-antibody  reactions. 
Considerable  work  is  needed  to  solve  a  number  of  technical  problems 
associated  with  high  reliability  and  sensitivity  of  the  stopped-flow 
microcalorimeter  and  these  will  continue  to  be  pursued.   The  titration 
calorimeter  needs  additional  work  particularly  in  regard  to  the  inclusion 
of  pH  electrodes  for  simultaneous  pH-thermal  titrations  of  proteins  and  this 
will  be  vigorously  pursued  in  the  next  year. 

Keyword  Descriptors:   Stopped-Flow  Microcalorimetery,  Cholesterol 
Cholesterol  Oxidase,  Triglycerides,  glycerokinase,    2-3  DPG,  Batch 
Microcalorimeter . 

Honors  and  Awards:   None 

Publications: 

1.  Watt,  D.,  Berger,  R.  L. ,  Green,  D. ,  and  Marini,  M.  A.:  Thermal  Titration 
Application  of  Calorimetry  to  the  Study  of  Plasma  Coagulation,  Vol  20, 
pp.  1013-1017,  1974. 

2.  Berger,  R.  L. ,  Friauf,  W.  S.,  and  Cascio,  H.  E. :  A  Low-Noise  Thermistor 
Bridge  for  Use  in  Calorimetry,  Vol.  20,  pp  1009-1012,  1974. 

3.  Goldberg,  R.  N. ,  Prosen,  E.  J.,  Staples,  B.  R. ,  Boyd,  R.  N. ,  Armstrong, 
G.  T.,  Berger,  R.  L. ,  and  Young,  D.  S.:  Measurements  Applied  to 
Biochemical  Analysis:  Glucose  in  Human  Serum.   Anal.  Biochem.  in  press. 


3LST 


Project  No.  Z01  HL  01415-02  LTD 

1.   Laboratory  of  Technical  Development 
3.   Bethesda,  Maryland  20014 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Italy-U.S.  Cooperative  Science  Program  -  Blood  Gas 
Instruments  -  Project  78. 

Principal  Investigators:   Robert  L.  Berger 

Luigi  Rossi-Bernardi,  University  of  Milan, 
Milan,  Italy 

Other  Investigators:   M.  Luzzana,  University  of  Milan 

R.  Winslow,  Lab.  Molecular  Hematology,  NHLI 

Cooperating  Units:   Ingold  Electrodes,  Zurich 

Advanced  Products,  Milan,  Italy 


Project  Description: 

Objectives:   The  total  oxygen  needed  by  a  normal  healthy  subject  is  provided 
by  the  circulatory  system  according  to  the  well  known  equation: 

0   consumption  =  cardiac  output  x  arterial-venous  0   difference. 

Since  several  pathological  conditions  can  shift  the  oxygen  dissociation 
curve  (ODC)  and  thus  how  much  oxygen  can  be  released  to  the  tissues,  it  is 
of  considerable  clinical  and  fundamental  physio-chemical  interest  to  be  able 
to  measure  the  ODC  under  true  physiological  conditions  of  the  patient.   The 
aim  of  this  project  is  to  develop  instrumentation  to  provide  a  comprehensive 
analysis  of  the  various  chemical  factors  regulating  the  (A-V)  0   difference 
or,  more  generally,  the  oxygen  dissociation  curve  of  human  blood  under 
various  physiological  or  pathological  conditions.   ODC  position  and  shape 
is  under  control  of  various  small  molecules  or  ions,  i.e.  CO  , protons, and 
2,3-DPG,  etc. 

Methods  Employed:   A  systematic  analysis  of  the  complex  interrelationship 
among  such  variables  and  their  effect  on  ODC  requires  the  development 
of  a  simple  method  to  obtain  ODC  of  human  blood,  in  vitro,  under  conditions 
closely  simulating  the  in  vivo  situation  of  the  patient. 

Instruments  are  developed  either  at  NIH  and/or  Milan,  tests  on  pure 
hemoglobin  are  generally  conducted  first  in  Milan  where  a  large  group  is 
currently  working  on  the  purification  of  hemoglobin.   Testing  on  patient 
blood  is  then  carried  out  in  Molecular  Hematology.   Close  cooperation  exists 
with  the  medical  school  hospital  in  Milan  where  on-line  work  will  be 


ILL 


Project  No.  Z01  HL  01415-02  LTD 

carried  out  using  the  membrane  oxygenator  system,  developed  in  this 
laboratory  by  Dr.  Kolobow,  monitored  for  %  0  Hb  by  a  modification  of  the 
Optisat  also  developed  here. 

Major  Findings:   A  semi-micro  (ODC)  apparatus  has  been  constructed  and 
tested  both  in  Milan  and  here.   It  consists  of  a  tonometer  for  degassing 
the  blood  (.5  to  2  mi's  needed),  a  cell  containing  .5  or  1  ml  of  the 
degassed  blood,  an  oxygen  electrode,  a  CO   electrode,  two  syringe  drives  for 
adding  HO  and  NaOH  continuously,  and  an  x-y  recorder.   About  20  to  25 
minutes  xs  needed  for  degassing  the  blood.   . 5  ml  is  transferred  to  the  ODC 
apparatus  which  contains  10  Ul  of  catalase.   Stirring  is  started,  zero  0 
determined  and  CO  noted.   HO   is  then  added  continuously  and  this 
addition  plotted  as  the  abscissa.   The  0   electrode  reads  0   in  solution 
and  thus  is  proportional  to  the  percent  oxyhemoglobin.   NaOH  is  added  to 
keep  CO   constant.   Then  the  ODC  curve  is  run  under  near  physiological 
conditions  in  about  10  minutes.   The  system  was  carefully  checked  against 
the  Van  Slyke  manometric  apparatus.   A  number  of  corrections  for  dissolved 
oxygen,  methemoglobin  formation,  dilution  from  addition,  etc.  have  been 
worked  out.   The  system  has  been  completely  automated  on  a  PDP-8  computer 
so  that  the  HO  dirve  and  NaOH  addition  are  controlled,  calculations  made, 
and  both  printout  and  plots  carried  out.   In  the  accompanying  graph  one  sees 
that  in  a  curve  for  sickle  cell  blood  the  P50=41  mm  of  Hg,  while  for 
normal  HbA  blood  it  is  28  mm  of  Hg.   Of  greater  importance  is  the  fact  that 
the  frozen  and  thawed  hemolysate  of  the  sickle  cells  falls  on  the  same 
curve  as  normal  RBC's.   From  a  clinical  standpoint,  only  a  system  which 
measures  whole  blood,  keeping  CO.,  constant  during  the  run,  gives  an  adequate 
picture  of  the  systems  ability  to  deliver  oxygen.   Thus,  P50  or  ODC  measured 
on  an  instrument  such  as  theco-oximeter  can  give  erroneous  results.   Note 
particularly  B  of  the  figure  which  shows  the  total  oxygen  delivered  to  the 
system.   Thus,  it  is  crucial  that  a  consideration  of  what  shifts  occur  in  the 
ODC  and  how  that  affects  total  oxygen  capacity  deliverable  at  normal  venous 
partial  pressures. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute. 

The  development  of  a  simple  instrument  for  rapid  determination  of  the  oxygen 
dissociation  curve  on  4  ml  of  blood  would  be  of  great  importance  in 
estimating  the  status  of  infants  during  respiratory  failure,  operating 
room  status  of  patients  under  anesthesia,  and  what  is  happening  to  the 
blood  of  patients  with  normal  or  abnormal  hemoglobin  during  various  forms 
of  therapy,  i.e.  treatment  of  sickle  cell  patients.   In  addition,  it  allows 
us  for  the  first  time  to  conveniently  do  the  many  physiochemical  determina- 
tions necessary  to  test  models  of  hemoglobin  action. 


367 


Project  No.  Z01  HL  01415-02  LTD 

Proposed  Course:   A  prototype   of  a  new  instrument  to  determine  Hb,  Met, 
C  Hb,  0  Hb   on  10  ul  of  blood  is  undergoing  laboratory  tests  and  will  be 
put  on  clinical  trials  starting  1  July.   A  new  microprocessor  controller  to 
run  the  ODC  apparatus,  make  calculations,  and  plot,  will  be  tested  and 
added  to  the  system  thus  producing  a  complete  semi-micro  blood-gas 
apparatus  for  determining  pH,  PCO  ,  P.0  ,  ODC,  and  Hb. 

Keyword  Descriptors:   Hemoglobin,  Red  Cell,  and  ODC  Analyze 

Honors  and  Awards:   None 

Publications: 

1.   Rossi-Bernardi,  L.  ,  Rossi,  F.  ,  Luzzana,  M.  ,  Perrella,  M.  ,  and  Berger,R.L. 
Physiological  Properties  of  Sickle  Cell  Hemoglobin,  in  Proc.  of  the 
1st  Nat.  Symp.  on  Sickle  Cell  Disease,  DHEW  Publication  No.  (N.I.H.) 
75-723,  U.  S.  Gov.  Printing  Office,  1974. 


2££> 


'roject   No.    Z01   UL   01415-02    LTD 


1.5         log  P0        2.0 


U? 


Project  No  Z01  HL  01416-01  LTD 

1.  Laboratory  of  Technical  Development 

2.  Section  on  Pulmonary  &  Cardiac 
Assist  Devices 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   On-line  cardiac  Output  Measurement  During  Extracorporeal 
Membrane  Oxygenation 

Previous  Serial  Number:   None 

Principal  Investigator:   Edward  Stool 

Other  Investigators:   Theodor  Kolobow,  Gerald  Vurek,  and  Joseph  Pierce 

Cooperating  Units:   NHLI  Surgery 

Objectives:   During  extracorporeal  membrane  oxygenation  (ECMO)  cardiac 
output  must  be  measured  by  somewhat  cumbersome  intermittent  determinations 
(Fick,  Indicator-dilution,  dye  or  thermal.   These  procedures  generally 
require  additional  personnel  and  therefore  are  relatively  infrequently 
performed.   Their  intermittence  makes  them  subject  to  sampling  error  in  a 
situation  where  wide  fluctuations  in  patient  status  are  often  seen.   The 
objective  of  this  study  is  to  develop  a  technique  for  continuous  cardiac 
output  measurement  of  patients  on  extracorporeal  bypass. 

Methods: 

(a)  Theoretical  Analysis 

Utilizing  the  fact  that  the  membrane  oxygenator  is  continuously  transferring 
oxygen  into  the  patient's  blood  an  attempt  to  use  oxygen  as  a  marker  in 
veno-veno  or  pre-pulmonary  bypass  was  undertaken.   This  can  be  done  by 
either  of  two  methods.   The  first  method  relies  on  determination  of  oxygen 
saturation  in  a  continuous  fashion  across  the  membrane  oxygenator  and  in 
the  pulmonary  artery  and  is  complicated  by  the  fact  that  it  depends  on 
the  assumption  that  venous  inflow  to  the  membrane  lung  closely  approximates 
"mixed  venous"  blood  in  its  oxygen  content.   A  second  method  eliminates  this 
consideration  but  requires  stopping  pump  flow  for  5-10  sec.  and  hence  is  not 
in  an  absolute  sense  continuous. 

(b)  Experimental 

Large  closed-chest  anesthetized  sheep  are  peripherally  cannulated  in  a 
manner  similar  to  patients  undergoing  ECMO,  however,  a  large  bore  return 
catheter  is  positioned  in  their  right  ventricle.   High  flow  veno  venous 


37a 


Project  No.  Z01  HL  01416-01  LTD 

ECMO  is  carried  out  while  the  appropriate  oxygen  saturation  determinations 
are  made.   This  is  done  under  basal  conditions,  oxygen  deprivation,  B-adren- 
ergic  blockade  and  6  adrenergic  stimulation.   Simultaneous  cardiac  output 
determinations  by  conventional  technique  are  carried  out  and  compared  to 
those  obtained  by  the  oxygen  indicator  method. 

Major  Findings: 

Preliminary  experiments  have  been  directed  towards  assessing  optimal 
cannulation  techniques  and  in  determining  instrument  stability  under 
operational  conditions.   Further,  reproducible  pharmacologic  interventions 
to  safely  alter  an  anesthetized  sheep's  cardiac  output  have  been  developed. 

Significance  of  the  Program  to  the  Institute: 

ECMO  is  a  therapeutic  modality  currently  undergoing  extensive  clinical 
evaluation  in  a  NHLI  contract.   The  ability  to  measure  cardiac  output 
continuously  during  ECMO  will  not  only  improve  the  management  of  these 
patients  but  will  serve  as  a  powerful  research  tool  to  allow  for  better 
analysis  of  the  physiologic  effects  of  such  interventions. 

Proposed  Course: 

Current  experiments  wi] 1  determine  the  precision  of  the  two  methods  of 
cardiac  output  determinations  as  well  as  their  degree  of  correlation  with 
conventional  cardiac  output  techniques.   Following  a  suitable  number  of 
in  vivo  perfusions,  instrumentation  to  render  the  proposed  determinations 
truly  "on-line"  will  be  constructed. 

Keyword  Descriptors:   Cardiac  Output,  Membrane  Oxygenator,  Extracorporeal 
Circulation. 

Honors  and  Awards:   None 

Publications:   None 


271 


ANNUAL  REPORT  OF  THE 

CARDIOLOGY  BRANCH 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

July  1,  1974  through  June  30,  1975 

The  experimental  interests  of  the  Cardiology  Branch  developed  over  the 
past  few  years  have  continued.   These  relate  to  the  pathogenesis,  patho- 
physiology and  treatment  of  coronary  artery  disease;  the  ultrastructural  and 
molecular  mechanisms  responsible  for  normal  and  impaired  contractile  function 
of  the  heart;  development  of  the  diagnostic  and  investigational  capabilities 
of  echocardiography;  and  the  application  of  multidisciplinary  techniques  to 
define  the  determinants  of  irreversible  heart  failure  in  patients  with  valvu- 
lar disease  and  how  such  information  can  be  used  to  determine  optimal  time  for 
surgical  intervention. 

CORONARY  ARTERY  DISEASE 

Pharmacologic  Treatment  of  Acute  Myocardial  Infarction 

In  the  past  few  years,  we  have  shown  that  treatment  with  TNG  following 
coronary  artery  occlusion  in  dogs  diminishes  infarct  size  and  reduces  the  in- 
cidence of  ventricular  fibrillation  (VF)  occurring  spontaneously  during  AMI. 
These  actions  are  potentiated  when  a  vasoconstrictor  is  administered  to  abolish 
the  TNG- induced  fall  in  arterial  pressure  and  reflex  tachycardia. 

To  elucidate  the  mode  of  action  of  TNG  in  AMI,  we  measured  the  effects  of 
treatment  on  myocardial  blood  flow  and  on  ischemic  injury  during  coronary 
occlusion  in  dogs.   We  found  that  the  salutary  effects  of  TNG  on  ischemic 
injury  during  AMI  are  mediated  by  an  increase  in  collateral  flow  and  reduction 
in  MV02-   However,  if  TNG  causes  hypotension  or  excessive  tachycardia,  re- 
duction of  ischemic  injury  will  occur  only  when  a  vasoconstrictor  is  ad- 
ministered to  reverse  the  pressure  and  heart  rate  changes. 

We  are  now  evaluating  this  form  of  therapy  on  the  extent  and  severity  of 
myocardial  injury  sustained  during  AMI  in  man.   Thus  far  we  have  found  that  in 
pts  without  left  ventricular  failure,  TNG  alone  resulted  in  no  consistent  de- 
crease in  ischemic  injury.   However,  when  phenylephrine  was  added  to  reverse 
the  blood  pressure  lowering  and  heart  rate  speeding  effects  of  TNG,  significant 
improvement  in  myocardial  ischemia  occurred  uniformly.   In  contrast  to  the  pts 
without  failure,  TNG  alone  significantly  improved  ischemic  injury  in  all  pts 
with  failure. 

NHLI  Type  II  Coronary  Intervention  Study 

The  primary  aim  of  this  randomized,  double  blinded,  prospective  study, 
carried  out  in  collaboration  with  the  Molecular  Disease  Branch,  Section  of 
Lipoproteins,  is  to  determine  whether  lowering  LDL  cholesterol  with  diet  and 
cholestyramine  in  patients  with  premature  coronary  artery  disease  and  Type  II 
hypercholesterolemia  will  retard  the  progression  of  coronary  artery  disease. 
The  major  criterion  we  will  employ  to  answer  this  question  is  whether  there  is 
regression  of  anatomic  disease  or  evidence  of  slower  progression,  conclusions 

i  373 


that  will  be  based  on  coronary  angiograms  obtained  at  initiation  into  study 
and  after  two  years  of  treatment.   The  program  is  now  well  underway;  some  of 
the  information  that  has  emerged  to  date  is  detailed  in  another  section  of 
the  Annual  Report. 

In  addition  to  the  primary  question,  the  screening  of  numerous  patients 
for  entry  into  the  investigation  has  led  to  several  fall-out  studies.   For 
example,  the  ECG  response  to  exercise  has  heretofore  been  used  as  a  reliable 
test  to  screen  for  the  presence  or  absence  of  coronary  artery  disease.   In  our 
just  completed  analysis  of  patients  who  had  exercise  studies  and  coronary 
arteriograms,  we  found  that  the  ECG  response  to  exercise  yielded  false- 
negative  and  false-positive  results  in  over  half  of  the  subjects  tested.   This 
low  sensitivity  and  specificity  indicates  use  of  this  test  as  a  diagnostic 
tool  in  the  individual  patient  is  questionable. 

Prospective  Study  on  the  Natural  History  of  Patients  with  Coronary  Artery 
Disease  with  Only  Mild  to  Moderate  Functional  Disability 

Considerable  information,  obtained  retrospectively,  is  available  relating 
to  the  natural  history  of  coronary  artery  disease.   These  studies  suggest  that 
mortality  rate  can  be  predicted  by  the  number  of  diseased  coronary  vessels  and 
the  presence  and  magnitude  of  ventricular  dysfunction.   Using  such  data,  "pro- 
phylactic" coronary  bypass  operation  is  being  recommended  if  a  patient,  even 
if  only  mildly  symptomatic,  falls  into  a  particularly  high  risk  group.   How- 
ever, these  studies  are  based  on  data  obtained  largely  from  severely  sympto- 
matic patients  and  may  not  accurately  reflect  long-term  prognosis  of  the 
patient  with  minimal  symptoms.   Therefore,  patients  with  only  mild  to  moderate 
functional  disability  are  being  studied  by  cardiac  catheterization,  exercise 
testing,  24-hour  ECG  tape  monitoring,  etc.   Attempts  will  be  made  to  determine 
prognostic  indices.   If  high  and  low  risk  subgroups  can  be  identified,  then 
more  rational  decisions  can  be  made  regarding  which  patient  should  be  considered 
a  candidate  for  "prophylactic"  operation. 

AUTONOMIC  INNERVATION  OF  THE  HEART 

Effects  of  Cardiac  Failure  on  Ventricular  Electrical  Stability  and  Autonomic 
Innervation  of  the  Heart 

The  autonomic  nervous  system  has  profound  influences  on  the  electrical 
stability  of  ventricular  myocardium.   Moreover,  the  parasympathetic  and 
sympathetic  systems  have  opposite  effects.   Increased  vagal  tone  decreases  the 
propensity  of  the  heart  to  develop  VF,  while  enhanced  neural  sympathetic  tone 
increases  the  likelihood  of  developing  VF.   Since  heart  failure  decreases 
cardiac  neuronal  stores  of  norepinephrine,  we  have  studied  the  effects  of 
failure  on  the  electrical  stability  and  autonomic  innervation  of  the  heart.   We 
found  that  failure-induced  depression  of  cardiac  norepinephrine  stores  in- 
creases ventricular  electrical  stability.   Moreover,  cholinergic  innervation 
of  the  ventricular  septum  was  reduced  or  absent  in  most  of  the  hearts  derived 
from  failure  animals,  a  finding  that  correlated  with  impaired  capacity  of 
vagal  stimulation  to  enhance  VF  threshold.   Thus,  cardiac  failure  reduces  or 
eliminates  autonomic  neural  influences  on  the  heart.   The  relative  magnitude  of 


31¥ 


the  adrenergic  and  cholinergic  impairment  may,  in  part,  determine  the  likeli- 
hood of  heart  failure  leading  to  arrhythmic  death. 

ECHOCARDIOGRAPHIC  STUDIES 

Asymmetric  Septal  Hypertrophy,  or  ASH 

By  employing  echocardiographic  techniques,  in  the  past  two  years  we  have 
considerably  increased  our  understanding  of  the  disease  spectrum  embracing 
IHSS.   Of  note,  it  was  recognized  that  LV  outflow  obstruction  was  only  one 
manifestation  of  a  disease  that  is  basically  a  cardiomyopathy  characterized  by 
a  septum  that  is  disproportionately  thickened  in  relation  to  the  posterior 
left  ventricular  wall.   We  also  showed  the  disease  is  a  genetic  abnormality 
transmitted  as  an  autosomal  dominant  trait  with  a  high  degree  of  penetrance. 

This  past  year,  we  studied  the  clinical  characteristics  and  course  of  35 
children  with  ASH  followed  for  one  to  16  years  (average,  7.4  years).   Although 
52%  of  the  35  patients  improved  or  remained  stable,  17%  deteriorated  clini- 
cally and  31%  died  suddenly  (4%  mortality  per  year).   Two  of  the  patients  who 
died  suddenly  had  previously  undergone  operation  (6  and  13  years  previously) 
with  resultant  abolition  of  the  outflow  gradient;  4  others  were  taking  pro- 
pranolol.  No  indices  predictive  of  sudden  death  could  be  identified.   Thus, 
the  clinical  and  hemodynamic  spectrum  of  ASH  in  children  is  broad,  and,  un- 
fortunately, sudden  death  is  relatively  common  in  that  subgroup  of  children 
who  were  referred  to  the  NHLI  in  the  past  because  of  overt  manifestations  of 
cardiac  disease. 

Echocardiographic  Assessment  of  Cardiomyopathies 

We  are  continuing  our  studies  of  cardiomyopathy  by  echocardiography  begun 
last  year.   We  have  accumulated  considerably  more  patients  and  have  confirmed 
our  initial  impression  that  an  extremely  useful  clinical  classification  of  the 
cardiomyopathies  can  be  achieved  by  echocardiography.   Patients  have  been 
divided  into  those  with  dilated  cardiomyopathy,  those  with  normal  LV  volumes 
with  concentrically  hypertrophied  walls,  and  those  with  normal  LV  volumes  with 
ASH.   The  secondary  cardiomyopathies  (alcoholic,  amyloidosis,  hypereosino- 
philia,  hemochromatosis,  mucopolysaccharidoses,  etc.)  fall  into  one  of  the 
first  two  groups;  the  third  is  a  specific  genetically  determined  disease.   This 
classification  system  markedly  simplifies  diagnosis  of  patients  presenting 
with  a  cardiomyopathy. 

Pathophysiology  and  Prediction  of  Onset  of  Atrial  Fibrillation 

Systemic  embolization,  a  serious  complication  of  mitral  valve  disease  and 
of  ASH,  usually  occurs  in  patients  in  atrial  fibrillation  (AF)  and  particularly 
in  those  who  recently  have  converted  from  NSR  to  AF.   In  an  attempt  to  more 
completely  understand  the  pathophysiology  of  AF,  echocardiography  was  employed 
to  study  85  patients  with  isolated  mitral  valve  disease,  50  patients  with 
isolated  aortic  valve  disease,  and  130  patients  with  ASH.   In  all  three  groups 
of  patients,  AF  was  common  only  in  the  subgroup  of  patients  older  than  40 
years  of  age  who  in  addition  had  a  left  atrial  dimension  measured  by  echo  that 


37ST 


exceeded  45  mm.   Our  data  indicate  that  a  chronic  hemodynamic  burden  initially 
produces  left  atrial  enlargement  which  in  turn  predisposes  to  AF.   Of  note, 
12%  of  patients  who  had  AF  had  an  embolus  at  its  onset.   Thus,  "prophylactic" 
anticoagulation  may  be  indicated  in  a  patient  in  NSR  with  mitral  valve  disease 
or  ASH  who  has  a  left  atrial  dimension  exceeding  40  mm. 

Determinants  of  Ventricular  Septal  Motion 

Normally,  the  ventricular  septum  moves  posteriorly  during  systole.   Certain 
conditions,  however,  lead  to  anterior  or  "paradoxical"  movement.   To  define 
the  determinants  of  septal  motion,  echocardiographic  studies  were  performed  in 
patients  with  a  variety  of  cardiac  disorders.   We  found  that  the  direction  and 
magnitude  of  septal  motion  is  determined  by  septal  position  at  end-diastole 
relative  to  total  cardiac  transverse  dimension.   The  more  posterior  the  septum 
lies  (as  with  right  ventricular  dilatation)  the  more  likely  it  will  move 
paradoxically.   Thus,  although  paradoxic  septal  motion  is  usually  seen  in 
conditions  causing  right  ventricular  volume  overload,  it  is  not  diagnostic  of 
any  particular  hemodynamic  burden.   Additional  studies  of  cardiac  motion  em- 
ploying two-dimensional  echocardiography  are  compatible  with  the  hypothesis 
that  all  intraventricular  structures  move  during  systole  towards  the  center  of 
ventricular  mass.   This  hypothesis  has  broad  implications  in  predicting  cardiac 
motion  in  the  normal  and  diseased  heart,  since  it  provides  the  theoretical 
basis  governing  altered  patterns  of  cardiac  motion. 

Real-Time  Two-Dimensional  Echocardiography 

Over  the  past  two  years,  we  have  developed  a  sector-scanner  that  produces 
real-time,  two-dimensional  echocardiograms  that  permits  visualization  non- 
invasively  of  internal  cardiac  structures.   We  have  found  this  technique  to  be 
a  powerful  tool  for  diagnosing  and  understanding  the  anatomic  relations  of 
many  complex  congenital  anomalies.   We  also  have  found  that  this  technique 
allows  us  to  measure  mtiral  valve  area  in  patients  with  rheumatic  heart 
disease,  even  in  the  presence  of  mitral  regurgitation.   Heretofore,  accurate 
assessment  of  mitral  valve  area  could  only  be  made  by  cardiac  catheterization, 
and  only  if  mitral  regurgitation  were  not  present. 

SUDDEN  INFANT  DEATH  SYNDROME 

Sudden  infant  death  syndrome  (SIDS)  is  a  major  cause  of  mortality  in  the 
first  six  months  of  life,  but  the  primary  mechanisms  responsible  for  this  con- 
dition are  unknown.   To  investigate  possible  cardiac  mechanisms,  42  sets  of 
parents  (who  had  at  least  one  infant  die  of  SIDS)  were  studied  by  echo- 
cardiography and  ECG.   ASH  was  present  in  two  (5%)  parental  sets.   At  least 
one  member  of  13  (31%)  other  parental  sets  had  ECG  abnormalities,  the  most 
common  of  which  was  QT  interval  prolongation.   In  addition,  47%  of  the  living 
children  of  the  parental  sets  with  QT  interval  prolongation  had  the  same  ab- 
normality, consistent  with  an  autosomal  dominant  pattern  of  inheritance.   We 
also  studied  three  infants  with  "near-miss"  SIDS.   All  three  showed  pro- 
longed QT  intervals.   Thus,  our  data  suggest  that  cardiac  mechanisms, 
especially  QT  interval  prolongation,  may  play  a  role  in  a  considerable  pro- 
portion of  infant  deaths  falling  within  the  sudden  infant  death  syndrome. 


3  7^ 


VALVULAR  HEART  DISEASE 

Elucidation  of  the  Determinants  of  Irreversible  Myocardial  Failure 

Last  year  we  completed  a  retrospective  study  of  long-term  survival  in 
patients  operated  on  for  aortic  regurgitation.   We  found  that  although  absolute 
heart  size  preoperatively  did  not  influence  long-term  postoperative  survival, 
change  in  heart  size  as  assessed  over  the  first  4-6  months  following  operation 
did.   Thus,  85%  of  patients  operated  upon  for  aortic  regurgitation  whose 
cardiothoracic  ratios  decreased  survived  six  years.   In  contrast,  only  43%  of 
patients  (p<.02)  whose  heart  size  did  not  change  or  whose  heart  size  in- 
creased survived  six  years.   This  prompted  a  prospective  multidisciplinary 
study  to  define  l)whether  a  particular  grouping  of  preoperative  functional 
derangements  leads  to  prohibitive  operative  risks,  and  2)  what  type  of  de- 
rangements can  be  reversed  or  improved  by  operative  abolition  of  the  mechanical 
defect.   Evaluation  of  myocardial  function  includes  calculation  of  ventricular 
volumes,  ejection  fraction  (EF) ,  exercise  testing,  etc.   In  addition,  biopsies 
are  being  obtained  for  electron  microscopic  analysis  as  well  as  biochemical 
assessment  of  contractile  proteins.   Preliminary  analysis  of  the  pre-  and 
postoperative  data  of  one  group  of  patients  -  those  with  isolated  aortic  re- 
gurgitation, has  been  performed.   The  20  patients  studied  pre-  and  post- 
operatively were  divided  into  three  groups  based  on  preoperative  EF:   normal 
EF  (>60%),  intermediate  EF  (40-60%),  and  low  EF  (<40%) .   We  found  that 
1)  operation  does  not  improve  basal  ventricular  function,  2)  LV  volume  and 
mass  are  more  likely  to  return  toward  normal  in  patients  with  normal  or  inter- 
mediate EF,  3)  long-term  results  are  good  in  patients  with  normal  or  inter- 
mediate EF,  and  4)  long-term  results  are  poor  in  patients  with  a  low  pre- 
operative EF.   These  findings  are  now  being  applied  to  patients  followed  in 
our  OPD  to  determine  whether  echocardiographic  assessment  of  changes  in  LV 
volume  and  EF  provides  a  more  sensitive  means  than  the  traditional  clinical 
parameters  to  judge  optimal  time  for  operative  intervention. 

Effects  of  Nitroglycerin  on  Exercise  Capacity  and  on  the  Hemodynamic  Response 
to  Exercise  in  Patients  with  Valvular  Heart  Disease 

Although  the  use  of  TNG  has  been  traditionally  reserved  for  patients  with 
coronary  artery  disease,  we  have  assessed  the  potential  clinical  utility  of 
TNG  in  patients  with  valvular  heart  disease.   Thus  far,  9  patients  have  been 
studied,  most  with  mitral  and  aortic  valve  lesions.   Consistent  increases  in 
exercise  tolerance  and  hemodynamic  response  to  upright  exercise  has  been  docu- 
mented.  Our  results  suggest  that  vasodilator  therapy  may  be  a  useful  adjunct 
in  the  pharmacologic  management  of  patients  with  valvular  heart  disease  by 
reducing  exertional  symptoms  and  increasing  exercise  tolerance. 

SCINTIGRAPHY  IN  THE  ASSESSMENT  OF  HEART  DISEASE 

Newly-developed  scintigraphic  techniques  have  the  potential  of  revealing 
cardiac  anatomic  abnormalities  and  patterns  of  myocardial  perfusion  and 
motion  that  either  are  not  available  with  more  traditional  angiographic 
techniques,  or  only  can  be  obtained  invasively.   To  determine  the  applicability 
of  scintigraphic  technqiues  to  clinical  cardiology,  and  to  explore  their  limits 
in  providing  investigational  information  not  otherwise  obtainable,  several 

5  577 


studies  are  in  progress. 

For  example,  it  is  generally  accepted  that  coronary  lesions  producing  50% 
stenosis  or  less  are  of  no  functional  significance;  hence,  patients  with  such 
lesions  are  not  considered  candidates  for  bypass  surgery.   Recent  studies 
using  a  dual  isotope  technique  to  assess  relative  myocardial  perfusion,  how- 
ever, suggest  that  inadequate  perfusion  after  a  vasodilatory  stimulus  can  re- 
sult from  "subcritical"  coronary  lesions.   We  therefore  are  evaluating  by 
intracoronary  scintigraphic  techniques  the  relative  significance  of  coronary 
stenotic  lesions  of  varying  severity  by  determing  adequacy  of  regional  myo- 
cardial perfusion  at  rest  and  at  the  time  of  pacing-induced  angina. 

MOLECULAR  MECHANISMS  RESPONSIBLE  FOR 
CARDIAC  CONTRACTION  AND  CELLULAR  PROLIFERATION 

The  Section  on  Molecular  Cardiology  has  conducted  research  in  three  areas: 
1)  phosphorylation  of  contractile  proteins  of  the  heart,  2)  the  effect  of 
phosphorylation  on  platelet  and  other  cellular  myosins,  3)  the  function  of 
contractile  proteins  in  non-muscle  cells. 

1)  Cardiac  protein  phosphorylation.   We  have  found  that  a  protein  tenta- 
tively identified  as  M-protein,  which  is  known  to  be  located  at  the  center  of 
the  myosin  filament,  can  be  phosphorylated  with  ^-labeled  AT-^P.   For  these 
studies,  canine  cardiac  myosin  and  surgical  specimens  from  patients  with  asym- 
metric septal  hypertrophy  were  utilized.   The  studies  have  shown:  a)  purified 
cardiac  myosin  contains  protein(s)  of  molecular  weight  150,000-160,000  which 
can  be  phosphorylated.   b)  This  protein  can  be  separated  from  myosin  by 
Sepharose  chromatography  in  a  high  ionic  strength  buffer.   c)  M-protein  pre- 
pared from  heart  can  be  phosphorylated  and  appears  to  be  the  same  protein  we 
isolated  that  was  bound  to  cardiac  myosin.   d)  Present  studies  are  directed 
toward  positive  identification  of  this  protein  as  being  derived  from  the  M- 
band  (utilizing  antibody  techniques)  and  uncovering  the  role  of  this  phos- 
phorylation in  cardiac  contraction. 

2)  Phosphorylation  and  actin-myosin  interaction.   We  previously  have  found 
that  the  20,000  dalton  light  chain  of  platelet  myosin  is  phosphorylated.   Re- 
cent studies  in  our  laboratory  have  resulted  in  the  purification  of  the  enzyme 
from  platelets  that  catalyze  this  phosphorylation. 

We  recently  have  shown  that  the  effect  of  phosphorylation  is  to  increase 
the  actin-activated  ATPase  activity  of  platelet  myosin  by  5-8  fold.   Dephos- 
phorylation  of  previously  phosphorylated  platelet  myosin  by  E.  coli  alkaline 
phosphatase  results  in  a  decrease  in  the  actin-activated  platelet  myosin 
ATPase  activity.   The  possibility  that  phosphorylation  of  myosin  may  serve  as 
a  switch  for  actin-myosin  interaction  in  both  non-muscle  cells  and  smooth 
muscle  cells  is  suggested  by  the  finding  that  the  platelet  kinase  can  phos- 
phorylate  the  20,000  dalton  light  chain  of  mouse  fibroblast  (a  non-muscle 
myosin)  and  chicken  gizzard  myosin  (a  smooth  muscle  myosin). 

3)  Myosin  phosphorylation  and  cell  proliferation.   Non-muscle  contractile 
proteins  are  thought  to  play  a  role  in  cell  division,  embryonic  development 
and  cell  secretion.   Myosin  has  been  isolated  from  early  myoblast  cells  prior 

6  37& 


to  cell  fusion  and  found  to  have  light  chains  similar  to  the  myosin  found  in 
adult  non-muscle  cells.   This  suggests  that  the  non-muscle  type  of  myosin 
plays  a  role  in  muscle  cells  before  they  differentiate;  i.e.,  before  the  gene 
for  skeletal  muscle  light  chains  is  turned  on.   Rhabdomyosarcoma  cells  are 
examples  of  a  differentiated  muscle  cell  that  has  de-differentiated  by  be- 
coming a  tumor  cell.   We  have  evidence  suggesting  that  these  cells  also  pro- 
duce a  non-muscle  type  of  myosin.   Hence,  two  cell  types  (myoblasts  and 
rhabdomyosarcoma  cells),  which  are  known  to  divide  at  a  much  higher  rate  than 
normal  muscle  cells  have  been  found  to  produce  a  myosin  identical  to  non-muscle 
myosin.   In  addition,  excessive  proliferation  of  medial  cells  of  the  arterial 
wall  have  been  implicated  in  the  genesis  of  atherosclerosis.   We  therefore  have 
initiated  studies  of  the  mechanism  of  medial  cell  proliferation;  in  particular, 
we  are  exploring  the  role  of  cytoplasmic  myosin  in  cell  division  and  the  effect 
of  phosphorylation  in  medial  cells  obtained  at  operation  from  patients  with  and 
patients  without  coronary  artery  disease. 

Finally,  Dr.  Marshall  Elzinga  has  sequenced  three  peptides  from  human 
platelet  actin  prepared  in  our  laboratory  and  has  compared  the  sequences  to 
the  amino  acid  sequence  of  rabbit  skeletal  muscle  actin.   Two  of  the  peptides 
comprising  20  residues  have  the  exact  same  sequence.   One  peptide  of  9  resi- 
dues has  a  single  amino  acid  substition  (threonive  for  valine) .   Further  work, 
using  human  cardiac  actin  should  answer  the  question  as  to  whether  this  sub- 
stitution is  species  specific  (rabbit  vs  man)  or  is  due  to  a  difference  in 
sequence  between  muscle  and  non-muscle  (platelet)  actin.   Further  sequence 
work  on  actin  from  human  heart  and  human  platelets  should  aid  in  uncovering 
differences  in  the  structure  and  functions  of  these  contractile  proteins. 


379 


Project  No.    Z01  HL  01601-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Effects  of  Altered  Autonomic  Innervation  of  the  Heart  on 
Ventricular  Electrical  Stability  in  Chronic  Heart  Failure 

Previous  Serial  Number:   None 

Principal  Investigator:   Kenneth  M.  Kent,  M.D. 

Other  Investigators:   Kathleen  Muth,  B.S. 

David  M.  Jacobowitz,  Ph.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Laboratory  of  Clinical  Science,  NIMH 

Project  Description:   The  autonomic  nervous  system  has  profound  influences 
on  the  electrical  stability  of  ventricular  myocardium.   Moreover,  the  in- 
fluences of  the  parasympathetic  and  sympathetic  systems  have  opposite 
effects.   Increased  vagal  tone  decreases  the  propensity  of  the  heart  to 
develop  ventricular  fibrillation,  while  enhanced  neural  sympathetic  tone 
increases  the  likelihood  of  developing  ventricular  fibrillation  (VF) .   Since 
heart  failure  is  known  to  alter  cardiac  neuronal  stores  of  norepinephrine  as 
well  as  to  alter  certain  cardiovascular  reflexes,  we  have  studied  the 
effects  of  heart  failure  on  the  electrical  stability  of  the  heart  and  on  the 
cardiac  responses  to  vagal  stimulation. 

An  infrarenal  aorto-caval  anastomosis  was  performed  on  nine  adult  male  dogs. 
An  average  of  eight  weeks  later,  the  dogs  developed  cardiac  failure.   VF 
threshold  was  assessed  in  these  animals  and  in  a  control  group  of  dogs  at 
constant  heart  rate  and  under  pentobarbital  anesthesia.   Since  barbiturates 
are  vagolytic,  this  preparation  allows  for  a  relatively  pure  assessment  of 
the  effects  exerted  by  differences  in  adrenergic  tone. 

Under  these  conditions,  the  VF  threshold  in  the  failure  animals  was 
130+12  mamp,  a  value  significantly  higher  than  in  the  control  animals, 
20+3  mamp  (p<.001).   This  enhanced  electrical  stability  of  the  dogs  in 
failure  was  associated  with  a  63%  reduction  of  cardiac  norepinephrine  content. 
Decreased  adrenergic  innervation  of  the  heart  was  confirmed  by  fluorescent 
microscopy. 

To  establish  the  causal  role  of  norepinephrine  depletion  in  elevating  VF 
threshold,  pharmacologic  depletion  of  neural  norepinephrine  was  accomplished 
with  6  hydroxydopamine  in  another  group  of  animals,  not  in  heart  failure. 
VF  threshold  measured  5  days  after  administration  of  6  hydroxydopamine,  when 
cardiac  norepinephrine  was  undetectable,  was  elevated  to  88+15  ma  (p<.01),  a 

1  3SI 


Project  No.   z01  HL  01601-01  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

value  similar  to  that  obtained  in  the  animals  with  chronic  heart  failure. 
Thus,  depletion  of  cardiac  neuronal  norepinephrine,  whether  occurring  as  a 
consequence  of  chronic  heart  failure  or  as  a  result  of  pharmacologic 
intervention,  results  in  an  elevated  VF  threshold. 

Cholinergic  innervation  of  the  heart  and  the  physiologic  effects  of  vagal 
stimulation  were  also  studied  in  the  heart  failure  preparation.   Vagal 
stimulation  decreased  the  atrial  rate  in  a  voltage  dependent  manner  in  each 
of  the  control  animals  and  7  of  9  of  the  failure  animals.   However,  in  two 
failure  animals  vagal  stimulation  caused  no  significant  reduction  in  atrial 
rate.   In  these  two  dogs,  cholinergic  fibers,  identified  by  specific  stains 
for  acetylcholines tinase  were  markedly  reduced.   Thus,  in  animals  with 
heart  failure,  cholinergic  innervation  of  the  atrium  may  be  diminished. 
Efferent  vagal  stimulation  also  increases  VF  threshold  of  the  normal  ventricle, 
an  effect  mediated  by  cholinergic  fibers  located  in  close  proximity  to  the 
ventricular  conducting  system.   These  fibers  were  absent  in  two  animals  in 
heart  failure,  decreased  in  5  and  normal  in  2.   Moreover,  VF  threshold  was 
essentially  unaltered  by  vagal  stimulation  in  2  of  6  failure  animals,  both 
of  which  had  reduced  cholinergic  innervation  of  the  H-Purkinje  system. 

Thus,  chronic  cardiac  failure  leads  to  marked  functional  abnormalities  in 
autonomic  control  of  ventricular  electrical  stability.   First,  it  appears 
that  the  well-described  depression  in  cardiac  norepinephrine  stores 
contributes  to  an  increase  in  the  electrical  stability  of  the  heart.   Such 
an  observation  is  at  variance  with  the  commonly  accepted  belief  that  failure- 
induced  depletion  of  cardiac  norepinephrine  is  invariably  deleterious, 
since  it  deprives  the  heart  of  one  of  its  important  compensatory  mechanisms 
through  which  it  can  augment  its  depressed  contractile  state.   Whether 
these  two  divergent  effects  of  norepinephrine  depletion — depressed  con- 
tractile state  and  enhanced  electrical  stability — results  in  a  net 
deleterious  or  salutary  influence,  is  unknown.   Second,  cholinergic  innervation 
of  the  ventricular  septum  was  either  reduced  or  absent  in  the  majority  of 
the  hearts  derived  from  failure  animals,  and  in  two  of  six  animals,  vagal 
stimulation  did  not  raise  VF  threshold.   Since  vagal  stimulation  enhances 
ventricular  electrical  stability,  deprivation  of  vagal  influences  by 
chronic  heart  failure  would  have  a  deleterious  effect.   Thus,  failure- 
induced  alterations  in  the  adrenergic  and  cholinergic  systems  produce 
divergent  effects  on  ventricular  electrical  stability.   The  relative 
magnitudes  of  each  of  these  changes  and  the  resultant  interactions  of  the 
adrenergic  and  cholinergic  systems  may  determine  the  likelihood  of  chronic 
hypertrophy  and  heart  failure  leading  to  arrhythmic  death. 

Keyword  Descriptors:   Heart  Failure,  Ventricular  Fibrillation,  Autonomic 
Nervous  System,  Adrenergic  Innervation,  Cholinergic  Innervation 


32* 


Project  No.   Z01  HL  01601-01  CB 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Proposed  Course  of  Project:   Completed 
Honors  and  Awards:   None 
Publications:   None 


3*3 


Project  No.  Z01  HL01602-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Enhanced  Survival  During  Acute  Myocardial  Infarction  in 
Reserpine  Treated  Dogs 

Previous  Serial  Number:   None 

Principal  Investigator:   Richard  A.  Goldstein,  M.D. 

Other  Investigators:   Kenneth  M.  Kent,  M.D. 

Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   Over  half  of  all  deaths  from  acute  myocardial  in- 
farction occur  early,  before  the  patient  obtains  medical  assistance. 
Currently,  there  are  no  preventive  measures  that  can  effectively  reduce 
the  frequency  of  sudden,  presumably  arrhythmic  deaths. 

Previous  studies  have  demonstrated  that  the  autonomic  nervous  system  has 
important  influences  on  the  incidence  of  lethal  ventricular  arrhythmias  in 
the  early  phase  of  experimental  acute  myocardial  infarction.   For  example, 
both  vagal  stimulation  and  catecholamine  depletion  (the  latter  produced 
either  surgically  or  pharmacologically)  decrease  the  incidence  of  spontane- 
ous ventricular  fibrillation  in  experimental  myocardial  infarction.   The 
purpose  of  the  present  investigation  is  to  determine  whether  reserpine,  a 
clinically  useful  catecholamine-depleting  agent,  increases  survival  during 
experimental  myocardial  infarction  when  it  is  given  chronically  in  doses 
comparable  to  those  given  clinically. 

Male  mongrel  dogs  weighing  between  21.4  and  31.3  kg  were  randomly  assigned 
to  one  of  three  treatment  groups:   control  (no  treatment),  low  dose 
(0.1  mg  im  for  6-10  days  -  equal  to  an  approximate  adult  human  dose  of 
0.25  mg  p.o.  q.d.),  and   high  dose  (0.2  mg  for  6-10  days).   Animals  were 
anesthetized  with  sodium  pentobarbital  (30  mg/kg)  and  intubated.   The  left 
anterior  descending  (LAD)  and  first  septal  coronary  arteries  were  isolated 
through  a  left  thoracotomy.   After  determining  baseline  heart  rate  and 
arterial  and  left  atrial  pressures,  the  heart  was  paced  at  180  beats/min. 
The  LAD  and  septal  coronary  arteries  were  then  ligated  and  the  animals  were 
observed  for  30  minutes  or  until  ventricular  fibrillation  occurred.   Biopsies 
of  all  four  chambers  of  the  heart  were  taken  for  norepinephrine  deter- 
minations. 


3M 


Project  No.  Z01  HL  01602-01  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Survival  times  were  14.1  minutes  for  control  animals,  16.1  minutes  for  low 
dose  reserpine  animals  (NS)  and  22  minutes  for  high  dose  reserpine  dogs 
(p<0.02  high  dose  compared  to  control).   Twenty-five  percent  of  the  control 
dogs,  22%  of  the  low  dose  reserpine  dogs  and  53%  of  the  high  dose  reserpine 
dogs  survived  the  30-minute  observation  period.   In  three  animals  re- 
ceiving high  dose  reserpine  in  which  ventricular  fibrillation  did  not  occur 
during  the  30-minute  observation  period,  observations  were  extended  for  an 
additional  70  minutes.   There  were  no  significant  arrhythmias  and  no 
hemodynamic  alterations  during  the  longer  observation  period.   Heart  rate 
prior  to  pacing  averaged  175  beats/minute  for  controls,  167  for  low  dose, 
and  138  for  high  dose  reserpine  (p<.05).     Mean  arterial  pressure  after 
10  minutes  of  ischemia  fell  3.4%  in  controls,  14%  in  low  dose  reserpinized 
animals,  and  13.6%  in  high  dose  reserpine  animals;  there  were  no  significant 
differences  in  the  left  atrial  pressures  in  the  three  groups  of  animals  during 
the  observation  period.   Neuronal  norepinephrine  concentration  in  the  left 
ventricle  averaged  0.97  ug/g  (1.28  to  .75)  in  control  animals,  to  0.06  ug/g 
in  the  low  dose  and  0.08  ug/g  in  the  high  dose  reserpine  animals. 

These  data  suggest  that  catecholamine  depletion  achieved  with  clinically 
employed  doses  of  reserpine  is  protective  against  ventricular  fibrillation 
following  experimental  myocardial  infarction.   If  these  trends  are 
supported  by  studies  in  additional  animals,  clinical  trials  might  be 
warranted  to  evaluate  the  potential  antiarrhythmic  actions  of  reserpine  in 
man. 

Keyword  Descriptors:   Acute  Myocardial  Infarction,  Ventricular  Fibrillation, 
Reserpine,  Catecholamine  Depletion,  Sudden  Death 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards :   None 

Publications :   None 


its 


Project  No.   Z01  HL  01603-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Cholinergic  Enhancement  of  Ventricular  Electrical  Stability: 
Adrenergic  Dependency  or  Primary  Action? 

Previous  Serial  Number:   None 

Principal  Investigator:   Kenneth  M.  Kent,  M.D. 

Other  Investigators:   Kathleen  Muth>  B.S. 

Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   Increased  vagal  tone  elevates  ventricular  fibrillation 
threshold,  reduces  the  incidence  of  spontaneous  ventricular  fibrillation 
after  coronary  occlusion,  and  diminishes  the  incidence  of  digitalis  toxic 
arrhythmias.   The  anatomic  pathways  that  mediate  these  beneficial  effects 
have  been  identified  in  the  ventricular  conducting  system.   In  contrast, 
increased  sympathetic  neural  stimulation  decreases  ventricular  electrical 
stability.   Since  the  cardiac  effects  of  altering  the  activity  of  the 
adrenergic  and  cholinergic  systems  in  many  instances  appears  to  be  due  to 
the  interplay  of  one  of  these  systems  on  the  other,  it  has  been  postulated 
that  the  beneficial  effects  of  increased  vagal  tone  on  ventricular 
electrical  stability  are  due  to  the  suppression  of  the  effects  of  the 
sympathetic  nervous  system.   To  test  this  hypothesis,  neuronal  norepinephrine 
was  depleted  by  the  administration  of  6  hydroxy  dopamine  in  a  group  of  six 
animals.   Three  to  five  days  later,  when  cardiac  norepinephrine  was  un- 
detectable by  chemical  analysis,  ventricular  fibrillation  threshold  was 
determined.   In  control  animals,  VF  threshold  averaged  22+6  mamp .   VF 
threshold  in  the  norepinephrine  depleted  animals  was  so  high  in  4  of  the 
animals  that  ventricular  fibrillation  could  not  be  precipitated  despite 
currents  of  120  mamps  or  more.   In  the  two  animals  in  which  ventricular 
fibrillation  could  be  induced  electrically,  vagal  stimulation  raised  VF 
threshold  from  75  to  95  mamp  in  one  animal  and  from  65  to  85  mamp  in  the 
other.   Propranolol  (one  mg/kg) ,  administered  to  block  the  cardiac  effects 
of  circulating  catecholamines,  did  not  change  ventricular  fibrillation 
threshold  caused  by  vagal  stimulation.   Myocardial  ischemia  was  induced  by 
occlusion  of  the  coronary  artery  in  the  four  treated  animals  in  which  VF 
could  not  be  precipitated  initially;  VF  threshold  fell  to  measurable 
values  in  two  animals.   Vagal  stimulation  raised  the  VF  threshold  during 
ischemia  from  28  to  38  mamp  in  one  animal  and  from  90  to  110  mamp  in  the 
other.   Propranolol,  one  mg/kg,  decreased  VF  threshold  in  the  first  animal 
from  28  to  20  mamp,  but  did  not  impair  the  vagally  mediated  response;  vagal 


386 


Project  No.   Z01  HL  01603-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

stimulation  increased  the  VF  threshold  to  42  in  this  animal.   Propranolol 
increased  VF  threshold  in  the  second  animal  such  that  VF  could  not  be 
induced  electrically.   Thus,  these  preliminary  studies,  performed  in 
animals  in  which  cardiac  adrenergic  influences  (both  intrinsic  and  circu- 
lating) were  abolished,  suggest  that  enhancement  of  ventricular  electrical 
stability  caused  by  the  cholinergic  system  does  not  occur  merely  by 
antagonizing  the  influences  of  the  adrenergic  system.   Rather,  it  appears 
that  release  of  acetylcholine  has  direct  electrophysiologic  effects. 

Keyword  Descriptors:   Ventricular  Fibrillation,  Acetylcholine,  Autonomic 
Nervous  System,  Norepinephrine 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:   None 

Publications:   None 


387 


.   Project  No.   Z01  HL  01604-03  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Clinical  Characteristics  of  Asymmetric  Septal  Hypertrophy 
Previous  Serial  Number:   NHLI-136(c) 
Principal  Investigator:   Walter  L.  Henry,  M.D. 

Other  Investigators:   Chester  E.  Clark,  M.D. 

Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   Since  the  early  descriptions  of  IHSS,  patients  have 
been  described  with  features  suggestive  of  the  disease  but  in  whom  no 
resting  or  provocable  left  ventricular  outflow  obstruction  could  be  demon- 
strated.  These  findings  have  been  interpreted  as  indicating  that  IHSS  is 
only  one  manifestation  of  a  disease  spectrum;  i.e.,  hypertrophic  cardio- 
myopathy in  which  obstruction  may  or  may  not  occur.   Recently,  we  have  con- 
firmed this  hypothesis  by  using  echocardiography  to  identify  a  specific 
anatomic  abnormality,  the  presence  of  which  is  independent  of  outflow 
obstruction.   Asymmetric  septal  hypertrophy  (ASH) ,  characterized  by  a 
ventricular  septum  at  least  1.3  times  as  thick  as  the  posterior-basal  left 
ventricular  free  wall,  was  found  in  all  patients  whose  disorder  falls 
within  the  IHSS  disease  spectrum.   One  hundred  patients  with  ASH  were 
examined.   Analysis  of  multiple  clinical  parameters  failed  to  distinguish  the 
nonobstructive  ASH  patients  from  the  obstructive  group  with  two  exceptions: 
in  nonobstructive  ASH  the  murmur  was  softer  with  little  variation  following 
provocative  maneuvers  and  a  bisferious  carotid  pulse  was  absent.   Angina 
and  syncope,  symptoms  usually  considered  characteristic  of  obstruction,  were 
also  common  in  patients  without  obstruction.   We  conclude  1)  obstructive 
and  nonobstructive  ASH  patients  cannot  be  distinguished  symptomatically, 
2)  the  absence  of  typical  physical  findings  makes  the  clinical  diagnosis  of 
nonobstructive  ASH  difficult,  and  3)  echocardiography  is  the  simplest  and 
most  reliable  means  of  establishing  the  diagnosis  in  patients  with  ASH. 

Keyword  Descriptors:   Obstructive  ASH,  Nonobstructive  ASH,  Hypertrophic 
Cardiomyopathy,  IHSS 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards :   None 

Publications:   None 


3SS 


Project  No.   Z01  HL  01605-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Comparison  of  Two-Dimensional  Echocardiographic  Systems 

Previous  Serial  Number:   None 

Principal  Investigator:   Walter  L.  Henry,  M.D. 

Other  Investigators:   David  J.  Sahn,  M.D. 

James  M.  Griffith,  M.S.E.E. 
Hugh  D.  Allen,  M.D. 
Stanley  J.  Goldberg,  M.D. 

Cooperating  Units:   Department  of  Pediatrics,  University  of  Arizona 

Medical  Center  and  Biomedical  Engineering  and  In- 
strumentation Branch,  DRS 

Project  Description:   Real-time  cross-sectional  images  of  the  heart  were 
obtained  in  44  patients  with  complex  congenital  heart  disease  using  either  a 
multiple  crystal  or  a  mechanical  sector-scanner  echocardiographic  system. 
Congenital  malformations  studied  included  single  ventricle  (6) ,  "corrected" 
transposition  (8),  d- transposition  of  great  arteries  (6),  endocardial  cushion 
defect  (8),  Ebstein's  malformation  (4),  aortic  stenosis  (6),  and  ventricular 
septal  defect  (6).   The  multiple  crystal  system  allowed  a  larger  area  of  the 
heart  to  be  visualized  simultaneously  and  resulted  in  more  rapid  demon- 
stration of  the  contour  and  positional  relations  of  atrioventricular  valves 
and  great  arteries.   The  mechanical  sector-scanner  visualized  a  smaller  area 
of  the  heart  simultaneously,  but  provided  a  higher  resolution  image  that  was 
particularly  useful  in  analyzing  the  shape  of  great  arteries  and  the  in- 
sertion of  atrioventricular  valves.   The  current  study  indicates  that  these 
two  echocardiographic  systems  provide  complimentary  information  for  the 
evaluation  of  complex  congenital  heart  disease. 

Keyword  Descriptors:   Two-Dimensional  Echocardiography,  Congenital  Heart 
Disease,  Sector-Scanner,  Multiple  Crystal  Imaging 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   None 


3&f 


Project  No.   Z01  HL  01606-02  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Echocardiographic  Findings  in  Patients  With  Hypereosinophilia 

Previous  Serial  No:   NHLI-3(c) 

Principal  Investigator:   Jeffrey  S.  Borer,  M.D. 

Other  Investigators:   Walter  L.  Henry,  M.D. 
David  C.  Dale,  M.D. 

Cooperating  Units:   National  Institute  of  Allergies  and  Infectious  Diseases 

Project  Description:   Echocardiography  (ECHO)  is  an  increasingly  important 
tool  in  the  diagnosis  and  classification  of  primary  and  secondary  cardiomyo- 
pathies.  This  report  deals  with  echocardiographic  evaluation  of  8  men,  aged 
7  to  67,  with  chronic  idiopathic  hypereosinophilic  syndromes  (IHS) .   IHS  had 
been  present  from  5  to  140  months.   No  patient  was  referred  originally 
because  of  cardiac  disease.   Only  one  had  clinical  evidence  of  cardiac  dys- 
function.  All  8  patients,  with  eosinophil  counts  ranging  from  6900  to  94,000, 
had  definite  ECHO  abnormalities.   Most  prominent  was  significant  symmetrical 
thickening  of  the  septum  and  left  ventricular  free  wall,  mean  thickness  being 
14.3  mm  ±  1.2  (SEM)  (normal  9.4  mm  ±  .2,  p<.01).   The  other  2  patients,  with 
eosinophil  counts  of  3900  and  9500,  had  no  abnormality  but  their  septal  and 
free  wall  thicknesses  were  at  the  upper  limit  of  normal.   Instantaneous  left 
ventricular  transverse  dimension  and  velocity  of  circumferential  fiber 
shortening  were  measured  in  every  patient.   No  uniform  abnormality  in  maximum 
velocity  of  circumferential  fiber  shortening  was  found.   However,  in  2 
patients,  1  symptomatic,  abnormalities  in  diastolic  relaxation  consistent  with 
a  restrictive  defect  were  seen.   The  symptomatic  patient  also  had  transverse 
dimension  slightly  below  the  lower  limit  of  normal.   In  about  1/3  of  fatal 
idiopathic  hypereosinophilic  syndrome  cases  pathologic  studies  reveal  endo- 
and  myocardial  fibrosis  mural  thrombi  and  ventricular  hypertrophy  with  either 
constricted  or  dilated  left  ventricular  cavities.   Heretofore,  it  was  believed 
that  cardiac  involvement  in  idiopathic  hypereosinophilic  syndrome  leads 
rapidly  to  death.   The  present  study  suggests  that  ECHO  may  be  of  value  in 
reassessing  the  prevalence  and  natural  history  of  cardiac  involvement  in 
idiopathic  hypereosinophilic  syndromes.   Moreover,  ECHO  may  provide  an  objective 
parameter  for  evaluation  of  therapy  in  idiopathic  hypereosinophilic  syndromes. 

Keyword  Descriptors:   Hypereosinophilia,  Echocardiography,  Cardiomyopathy 


3?<9 


Project  No.   z01  HL  01606-02  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications:   Manuscript  submitted  to  NEW  ENGLAND  JOURNAL  OF  MEDICINE 


3?/ 


Project  No.   ZQ1  HL  01608-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Congenital  Heart  Disease  Associated  with  ASH 

Previous  Serial  Number:   None 

Principal  Investigator:   Barry  J.  Maron,  M.D. 

Other  Investigators:   Jesse  E.  Edwards,  M.D. 

Victor  J.  Ferrans,  M.D.,  Ph.D. 
Chester  E.  Clark,  M.D. 
Walter  L.  Henry,  M.D. 
Edward  Lebowitz,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Section  of  Pathology,  NHLI 

Project  Description:   ASH  is  characterized  by  a  disproportionately  thickened 
ventricular  septum  that  contains  numerous  hypertrophied  bizarrely-shaped 
and  disorganized  cardiac  muscle  cells.   To  determine  whether  such  congenital 
cardiac  malformations  are  part  of  the  disease  spectrum  of  genetically 
determined  ASH,  cardiac  pathologic  observations  were  made  in  eight  patients 
with  disproportionate  septal  thickening  (ventricular  septal  to  posterobasal 
left  ventricular  free  wall  thickness  ratios  of  1.5  to  2.5)  and  the  following 
three  categories  of  associated  lesions:   1)  parachute  deformity  of  the  mitral 
valve  (occurring  either  as  an  isolated  lesion  or  with  ventricular  septal  defect, 
coarctation  of  the  aorta,  supravalvular  ring  of  the  left  atrium,  or  double 
outlet  right  ventricle;  2)  complete  interruption  of  the  aortic  arch,  and 
3)  ventricular  septal  defect.   The  arrangement  of  cardiac  muscle  cells  in  the 
disproportionately  thickened  ventricular  septum  was  normal  in  six  of  the  eight 
patients;  in  the  other  two  patients  (one  with  parachute  deformity  of  the  mitral 
valve  and  one  with  ventricular  septal  defect)  numerous  bundles  of  hypertrophied 
cardiac  muscle  cells  were  interlaced  in  a  disorganized  fashion  among  more 
normally  arranged  bundles  of  cells.   First  degree  relatives  of  six  of  the 
eight  patients  were  studied  by  echocardiography  and  found  to  have  normal 
ventricular  wall  thicknesses  and  septal-free  wall  ratios. 

It  is  concluded  that  disproportionate  ventricular  septal  thickening  may  occur 
in  patients  with  a  variety  of  congenital  heart  malformations,  but  that  such  a 
finding  is  not  necessarily  a  manifestation  of  the  disease  spectrum  of  gene- 
tically determined  ASH. 

Keyword  Descriptors:   Echocardiography,  Hypertrophic  Cardiomyopathy,  Parachute 
Mitral  Valve,  Ventricular  Septal  Defect,  Interruption  Aortic  Arch, 


3?> 


Project  No.  Z01  HL  01608-01  CB 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Coarctation  of  Aorta,  Double  Outlet  Right  Ventricle. 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications:   None 


Jf3 


Project  No.   Z01  HL  01613-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Measurement  of  Mitral  Orifice  Area  by  Two-Dimensional 
Echocardiography 

Previous  Serial  Number:   None 

Principal  Investigator:   Walter  L.  Henry,  M.D. 

Other  Investigators:   James  M.  Griffith,  M.S.E.E. 
Lawrence  L.  Michaelis ,  M.D. 
Charles  L.  Mcintosh,  M.D. 
Andrew  G.  Morrow,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Clinic  of  Surgery  and  Biomedical  Engineering  and 

Instrumentation  Branch,  Division  of  Research  Services 

Project  Description:   A  quantitative  assessment  of  mitral  valve  orifice  area 
can  be  achieved  in  pacients  with  pure  mitral  stenosis  by  cardiac  cathe- 
terization.  In  the  presence  of  mitral  regurgitation,  however,  accurate  measure- 
ment often  is  impossible  because  total  diastolic  flow  through  the  mitral 
valve  frequently  is  unknown.   Using  a  recently  developed  real-time,  two- 
dimensional  echocardiography  system,  we  were  able  to  obtain  a  cross- 
sectional  image  of  the  mitral  valve  by  scanning  the  heart  perpendicular  to  its 
long  axis  at  the  level  of  the  tip  of  the  mitral  leaflets.   Twenty  con- 
secutive patients  undergoing  operation  for  mitral  valve  disease  were  studied 
during  the  week  prior  to  operation.   In  18  of  20  (90%)  the  mitral  orifice 
was  imaged  successfully  in  early  diastole  by  two-dimensional  echo- 
cardiography so  that  mitral  valve  orifice  area  could  be  measured  directly 
in  square  centimeters.   In  14  patients  (10  with  associated  mitral  re- 
gurgitation), mitral  orifice  area  was  measured  both  by  echocardiography  and 
directly  at  time  of  operation.   In  12  of  14  (86%)  patients,  mitral  orifice 
area  by  two-dimensional  echocardiography  was  within  0.3  square  centimeters  of 
that  measured  at  operation  (correlation  coefficient  for  all  14  patients  = 
0.92;  p<0.01).  In  conclusion  the  present  study  demonstrates  that  two- 
dimensional  echocardiography  is  extremely  useful  in  the  evaluation  of 
patients  with  mitral  valve  disease  because  it  provides  a  noninvasive  method 
for  directly  measuring  the  mitral  valve  orifice  area  that  is  accurate  even 
in  the  presence  of  mitral  regurgitation. 

Keyword  Descriptors:   Mitral  Valve  Orifice,  Mitral  Stenosis,  Two- 
Dimensional  Echocardiography 


lH 


Project  No.   z01  HL  01613-01  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications:   Manuscript  to  be  published  in  CIRCULATION  (May,  1975) 


nC 


Project   No,     zm    m.  msiA-m    rn 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Mechanism  of  Beneficial  Action  of  TNG-Methoxamine  in  AMI 

Previous  Serial  No:  None 

Principal  Investigator:   Howard  J.  Smith,  MB.  CbB.MRACP 

Other  Investigators:   Richard  A.  Goldstein,  M.D. 

Kenneth  M.  Kent,  M.D.,  Ph.D. 
Roger  Aamodt,  Ph.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:  Department  of  Nuclear  Medicine,  NIH 

Project  Description:   Previous  reports  from  this  laboratory  have  shown  that 
treatment  with  nitroglycerin  (TNG)  following  coronary  artery  occlusion  in  dogs 
has  beneficial  effects  on  infarct  size  and  on  the  threshhold  at  which  electrical 
stimulation  induces  ventricular  fibrillation.   These  actions  are  potentiated 
when  methoxamine  is  administered  to  abolish  the  TNG-induced  fall  in  arterial 
pressure  and  reflex  tachycardia.   To  elucidate  the  mode  of  action  of  TNG  and 
methoxamine,  we  measured  their  effects  on  myocardial  blood  flow  (MBF)  and  on 
ischemic  injury  during  coronary  occlusion. 

Mongrel  dogs  with  chronically  implanted  myocardial  electrodes,  left  atrial 
catheters,  and  coronary  occlusive  cuffs  were  sedated  with  morphine  and 
diazepam.   MBF  was  measured  in  ischemic  and  non-ischemic  myocardium  by  use  of 
radioactive  microspheres  (15  ±  5y ,  labelled  with  141Ce,  16^Yb  and  85Sr) . 
MBF  to  the  center  of  the  ischemic  area  was  taken  to  represent  collateral  blood 
flow;  MBF  to  the  non-ischemic  myocardium  was  used  as  an  index  of  MV02-   Ischemic 
injury  was  estimated  by  summating  ST  elevations  (EST)  recorded  from  the 
myocardial  electrodes. 

MBF  at  the  center  of  the  infarct  in  14  dogs  averaged  20%  of  that  present  in 
non-ischemic  regions.   In  9  of  the  14,  collateral  flow  increased  to  33% 
following  30  minutes  of  TNG  administration.   The  enhanced  collateral  flow  was 
associated  in  7  dogs  with  a  decrease  in  ST  segment  elevation;  ischemia  was 
either  unchanged  or  increased  in  the  other  2  dogs.   In  the  2  animals  in  which 
ST  segment  elevation  did  not  diminish,  TNG  produced  excessive  tachycardia  and 
did  not  reduce  estimated  MVO2 s   Of  the  remaining  5  dogs  collateral  flow  was 
unchanged  by  TNG  in  one  and  ST  segments  diminished  only  minimally;  collateral 
flow  diminished  following  TNG  in  4  and  ST  segments  were  essentially  unchanged 
(3  dogs)  or  increased  (1  dog)  . 


3K 


Project  No.   Z01  HL  01614-01  CB 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

In  7  of  the  14  animals  that  received  TNG,  addition  of  methoxamine  20  minutes 
after  the  start  of  TNG  infusion  returned  heart  rate  towards  normal,  but 
caused  no  consistent  change  in  collateral  flow,  or  MV02-   However, 
methoxamine  uniformly  diminished  ischemic  injury  in  the  dogs  that  were  not 
responsive  to  TNG,  mainly  by  reducing  heart  rate  (and  MVO2) ,  but  also  by 
restoring  blood  pressure  in  those  dogs  that  had  developed  hypotension. 
Similar  results  were  found  when  individual  zones  of  myocardium  were  analyzed: 
methoxamine  reduced  ischemic  injury  when  prior  treatment  with  TNG  was 
associated  with  excessive  tachycardia  or  hypotension,  but  produced  little 
change  in  those  regions  that  had  responded  favorably  to  TNG. 

In  summary,  the  results  of  this  investigation  demonstrate  that  the  salutary 
effects  of  TNG  on  ischemic  injury  during  acute  myocardial  infarction  are 
mediated  by  an  increase  in  collateral  flow  and  a  reduction  in  MV02-   However, 
if  TNG  causes  hypotension  (with  resulting  diminution  in  collateral  flow)  or 
excessive  tachycardia  (with  resulting  increase  in  MVO2  and  perhaps  decrease  in 
flow)  reduction  of  ischemic  injury  will  not  occur  without  addition  of  me- 
thoxamine to  reverse  the  TNG- induced  pressure  and  heart  rate  changes. 

Keywords  Descriptors:   Nitroglycerin,  Myocardial  blood  flow,  Collateral  flow, 
Acute  myocardial  infarction,  Methoxamine. 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards :   None 

Publications:   None 


wt 


Project  No.   Z01  HL  01615-04  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Factors  Affecting  the  Operative  Mortality  in  Aortic  Valvular 
Disease 

Previous  Serial  Number:   NHLI-18(c) 

Principal  Investigator:   Walter  L.  Henry,  M.D. 

Other  Investigators:   Chester  E.  Clark,  M.D. 

Robert  E.  Golstein,  M.D. 
Samuel  B.  Itscoitz,  M.D. 
David  R.  Redwood,  M.D. 
D.  Luke  Glancy,  M.D. 
Alan  S.  Pearlman,  M.D. 
Joel  Morganroth,  M.D. 
Stephen  E.  Epstein,  M.D. 
Andrew  G.  Morrow,  M.D. 
Larry  Michaelis,  M.D. 
Charles  L.  Mcintosh,  M.D. 

Cooperating  Units:   Clinic  of  Surgery,  NHLI 

Project  Description:   The  purpose  of  this  study  is  to  define  prospectively 
those  preoperative  factors  that  indicate  an  increased  operative  risk  or  that 
irreversible  myocardial  dysfunction  has  occurred.   All  patients  18  years  old 
or  over  admitted  to  the  Cardiology  Branch  for  aortic  valve  replacement  are 
being  evaluated.   Patients  with  significant  involvement  of  other  valves  are 
excluded.   Preoperative  and  6-month  postoperative  assessment  are  based 
mainly  on  data  obtained  by  cardiac  catheterization  (including  coronary 
arteriography)  and  echocardiography.   Evaluation  of  myocardial  function 
includes  ventricular  volumes,  LV  mass,  mean  dv/dt,  mean  VCF,  and  ejection 
fraction.   These  data,  as  well  as  operative  risk,  will  also  be  correlated 
with  EKG,  x-ray,  phonocardiogram,  and  exercise  testing. 

Seventy-eight  patients  have  been  assessed  preoperatively .   Fifty-five  cf  the 
78  have  been  studied  also  at  the  6-month  postoperative  assessment  point. 
Preliminary  results  suggest  that  many  patients  who  in  the  past  would  not 
have  been  offered  surgery  because  of  a  suspected  high  risk,  in  fact  do  well 
following  operation. 

Keyword  Descriptors:   Aortic  Regurgitation,  Operative  Mortality,  Echo- 
cardiography »  Aortic  Stenosis 


318 


Project  No.  Z01  HL  01615-04  CB 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Proposed  Course  of  Project:   Continuing 
Honors  and  Awards :   None 
Publications:   None 


3?? 


Project  No.   Z01  HL  01617-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Differential  Diagnosis  of  Great  Artery  Anomalies  by  Two- 
Dimensional  Echocardiography 

Previous  Serial  Number:   None 

Principal  Investigator:   Walter  L.  Henry,  M.D. 

Other  Investigators:   Barry  J.  Maron,  M.D. 

James  M.  Griffith,  M.S.E.E. 
David  R.  Redwood,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Biomedical  Engineering  and  Instrumentation  Branch, 
Division  of  Research  Services 

Project  Description:   A  recently  developed  sector-scanner  that  produces 
real-time,  two-dimensional  echocardiograms  was  used  to  determine  whether 
visualization  of  the  great  arteries  at  their  origin  would  provide 
diagnostically  useful  information  in  patients  with  cyanotic  heart  disease. 
Thirty-one  patients  (age  2  to  31  years;  weight  5  to  50  kg)  previously 
diagnosed  by  cardiac  catheterization  were  studied.   Images  were  generated 
perpendicular  to  the  long  axis  of  the  heart  at  the  level  of  the  origin  of  the 
great  arteries.   Satisfactory  great  artery  visualization  was  obtained  in  27 
of  31  patients.   Arteries  in  cross-section  appeared  as  circles;  those 
sectioned  longitudinally  appeared  sausage-shaped.   Three  great  artery 
patterns  were  seen:   a)  a  large  single  circle,  seen  in  four  patients  with 
truncus  arteriosus  and  two  with  pulmonary  atresia;  b)  two  adjacent  circles, 
seen  in  11  patients  with  transposition  of  great  arteries,  two  patients  with 
corrected  transposition  and  one  with  double  outlet  right  ventricle;  and 
c)  a  circle  with  a  sausage-shaped  structure  curving  anteriorly  from  right 
to  left,  seen  in  five  patients  with  tetralogy  of  Fallot,  one  patient  with  a 
large  ventricular  septal  defect  complicated  by  an  Eisenmenger  reaction  and 
one  patient  with  a  ventricular  septal  defect  and  valvular  pulmonic  stenosis. 
This  latter  pattern,  also  seen  in  ten  normal  subjects,  is  characteristic  of 
normally  related  great  arteries.   We  conclude  that  two-dimensional  echo- 
cardiography is  an  accurate  noninvasive  technique  for  categorizing  in- 
dividuals with  congenital  anomalies  of  the  great  arteries. 

Keyword  Descriptors:   Great  Artery  Anomalies,  Two-Dimensional  Echocardiography, 
Transposition  of  Great  Arteries,  Truncus  Arteriosus,  Tetralogy  of  Fallot 

Proposed  Course  of  Project:   Completed 


400 


Project  No.  Z01  HL  01617-01  CB 

PKS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Honors  and  Awards:   None 

Publications:   Manuscript  published  in  CIRCULATION  51:283-291,  1975 


Ato/ 


Project  No.   Z01  HL  01618-03  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30 ,    1975 

Project  Title:   Long-Term  Effects  of  Operation  on  Obstruction  and  LV 
Hypertrophy  in  IHSS 

Previous  Serial  Number:   NHLI-21(c) 

Principal  Investigator:   Walter  L.  Henry,  M.D. 

Other  Investigators:   Stephen  E.  Epstein,  M.D. 
Chester  E.  Clark,  M.D. 
Andrew  G.  Morrow,  M.D. 

Cooperating  Units:   Clinic  of  Surgery,  NHLI 

Project  Description:   Surgical  myectomy  for  IHSS  results  in  symptomatic 
improvement  and  loss  of  LV  outflow  obstruction.   To  determine  the  mechanism 
by  which  the  obstruction  disappears,  and  the  long-term  effects  of  operation, 
we  used  echocardiography  to  measure  mitral  valve  position,  mitral  valve 
systolic  motion,  and  left  ventricular  free  wall  thickness  in  two  groups  of 
patients  with  IHSS:   13  patients  who  had  myectomy  performed  2  to  11  years 
(mean  6.5  years)  previously,  and  27  nonoperated  patients.   Preoperative 
hemodynamic  data  were  comparable  in  both  groups.   The  prominent  forward 
mitral  valve  motion  in  midsystole,  indicative  of  obstruction,  was  present  in 
each  nonoperated  and  absent  in  each  operated  patient.   Mitral  valve  position 
at  onset  of  systole  was  determined  by  calculating  the  ratio  of  mitral  valve- 
posterior  left  ventricular  wall  distance  to  septal-mitral  valve  distance. 
In  normals,  the  mitral  valve  is  positioned  near  the  posterior  left  ventricular 
wall  (ratio  0.28+.01).   While  the  mitral  valve  is  anteriorly  positioned  in 
both  IHSS  groups,  it  is  more  anterior  in  nonoperated  patients  (ratio  1.04+ 
106)  than  in  operated  patients  (ratio  .66+. 04,  p<.01).   Intraoperative  studies 
in  six  patients  revealed  the  mitral  valve  to  assume  a  more  posterior  position 
immediately  after  myectomy;  this  coincided  with  disappearance  of  the  midsys- 
tolic forward  mitral  valve  movement.   Left  ventricular  free  wall  thickness 
was  13.2+. 05  mm  in  the  nonoperated  patients  (normal  9.4+. 02),  and  11.5+.04  mm 
in  the  operated  (p<.05).   We  conclude  the  mitral  valve  is  tethered  forward 
in  IHSS.   Septal  myectomy  relieves  this  tethering  and  thereby  abolishes  the 
mid-systolic  forward  mitral  valve  motion  and  hence  left  ventricular  outflow 
obstruction.   Abnormal  mitral  valve  position  and  motion  did  not  recur  post- 
operatively during  long-term  follow-up.   Finally,  left  ventricular  free  wall 
thickening  appears  to  regress  postoperatively. 


443  3- 


Project  No.   Z01  HL  01618-03  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Keyword  Descriptors:   Myotomy,  Myectomy,  IHSS,  Obstructive  ASH,  Echo- 
cardiography 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards :   None 

Publications :   None 


m 


Project  No.   Z01  HL  01619-04  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Distribution  of  the  Cardiomyopathy  in  IHSS 

Previous  Serial  Number:   NHLI-22(c) 

Principal  Investigator:   Walter  L.  Henry,  M.D. 

Other  Investigators:   Chester  E.  Clark,  M.D. 

Stephen  E.  Epstein,   M.D. 

Cooperating  Units:   None 

Project  Description:   Patients  with  typical  idiopathic  hypertrophic  subaortic 
stenosis  (IHSS)  represent  only  one  subgroup  of  a  cardiac  disease  in  which 
the  characteristic  anatomic  abnormality  is  asymmetric  septal  hypertrophy 
(ASH).   In  most  patients  with  ASH,  left  ventricular  outflow  obstruction  is 
absent  and  cardiac  dysfunction  presumably  is  due  to  widespread  involvement 
of  the  left  ventricle  by  an  underlying  myocardial  abnormality.   In  other 
patients  with  ASH,  left  ventricular  outflow  obstruction  is  present  (typical 
IHSS)  and  constitutes  a  major  feature  of  the  hemodynamic  and  physical 
findings.   To  determine  whether  patients  with  outflow  obstruction  also 
have  the  underlying  myocardial  abnormality  diffusely  involving  the  left 
ventricle,  the  gross  morphology  of  hearts  from  patients  with  and  without 
outflow  obsturction  were  studied  both  by  necropsy  and  by  echocardiography. 
Echocardiographic  studies  revealed  that  the  ventricular  septum  was 
thicker  in  obstructive  ASH,  a  finding  confirmed  by  the  postmortem  studies. 
The  necropsy  studies  also  indicated  that  although  the  left  ventricular  free 
wall  was  thickened  in  both  obstructive  and  nonobstructive  ASH,  the  con- 
figuration of  the  left  ventricular  free  wall  was  distinctly  different  in  the 
two  groups.   In  obstructive  ASH,  the  free  wall  was  hypertrophied  and 
identical  in  appearance  to  that  seen  in  valvular  aortic  stenosis.   Moreover, 
echocardiographic  studies  indicated  that  the  thickening  of  the  free  wall 
behind  posterior  mitral  leaflet  appeared  to  regress  after  operative  relief 
of  the  outflow  obstruction.   In  contrast,  the  left  ventricular  free  wall 
of  severely  symptomatic  patients  without  outflow  obstruction  had  a 
markedly  different  and  unique  appearance;  the  free  wall  of  left  ventricle 
directly  behind  the  posterior  mitral  leaflet  was  of  normal  or  less  than 
normal  thickness,  whereas  the  remaining  free  wall  was  nonuniformly  thickened. 
On  the  basis  of  these  findings  and  the  microscopic  data  presented  in  the 
companion  paper,  we  conclude  that  the  myocardial  abnormality  in  obstructive 
ASH  (typical  IHSS)  is  localized  largely  to  the  ventricular  septum,  with 
left  ventricular  free  wall  thickening  occurring  as  a  consequence  of  outflow 


<#>* 


Project  No.  Z01  HL  01619-04  CB 

PHS-NIH 

Individual  Project  Report 
July  1,  1974  through  June  30,  197  5 

obstruction.   In  symptomatic  patients  with  nonobstructive  ASH,  however,  the 
data  suggest  that  the  left  ventricle,  including  free  wall,  is  extensively 
involved  with  a  primary  myocardial  abnormality. 

Keyword  Descriptors:   Asymmetric  Septal  Hypertrophy,  Hypertrophic 
Cardiomyopathy,  Necropsy,  Echocardiography,  IHSS 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   CIRCULATION  50:447-455,  1974 


tfcS" 


Project  No.   Z01  HL  01620-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 


Project  Title:   Pathophysiology  and  Prediction  of  Onset  of  Atrial  Fibrillation 

Previous  Serial  No:   None 

Principal  Investigator:   Walter  L.  Henry,  M.D. 

Other  Investigators:   Joel  Morganroth,  M.D. 
Alan  S.  Pearlman,  M.D. 
Chester  E.  Clark,  M.D. 
David  R.  Redwood,  M.D. 
Samuel  B.  Itscoitz,  M.D. 
Stephen  E.  Epstein,  M.D. 

Project  Description:   In  an  attempt  to  more  completely  understand  the  patho- 
physiology of  atrial  fibrillation,  echocardiography  was  used  to  study  85 
patients  with  isolated  mitral  valve  disease,  50  patients  with  isolated  aortic 
valve  disease,  and  130  patients  with  asymmetric  septal  hypertrophy.   In  all 
three  groups  of  patients,  atrial  fibrillation  was  common  only  in  the  subgroup 
of  patients  older  than  40  years  of  age  who  in  addition  had  an  echocardio- 
graphically  measured  left  atrial  transverse  dimension  exceeding  45mm.   Mean 
left  atrial  pressure  measured  at  cardiac  catheterization  did  not  provide 
nearly  as  strong  a  predictive  index  of  atrial  fibrillation.   The  results  of 
the  present  study  suggest  that  a  chronic  hemodynamic  burden  initially 
produces  left  atrial  enlargement  which  in  turn  predisposes  to  atrial  fibril- 
lation.  Of  clinical  importance,  atrial  fibrillation  was  rare  in  patients 
with  a  left  atrial  transverse  dimension  below  40mm  (3  of  117  or  3%)  but 
common  when  this  dimension  exceeded  40mm  (80  of  148  or  54%)  .   Because  10  of 
81  (12%)  patients  in  the  present  series  had  an  embolus  at  the  onset  of  atrial 
fibrillation,  it  appears  reasonable  to  consider  anticoagulation  and  anti- 
arrhythmic therapy  in  the  management  of  a  patient  in  normal  sinus  rhythm  who 
has  a  left  atrial  transverse  dimension  exceeding  40mm.   Observations  of  left 
atrial  size  in  patients  in  whom  cardioversion  was  attempted  suggest  that 
successful  cardioversion  is  uncommon  when  left  atrial  transverse  dimension 
exceeds  45mm.   Moreover,  in  patients  with  asymmetric  septal  hypertrophy  who 
have  a  left  atrial  transverse  dimension  exceeding  50mm,  the  risk  of  cardio- 
version-induced  embolization  may  well  be  greater  than  the  likelihood  of 
achieving  stable  sinus  rhythm. 

Keyword  Descriptors:   Atrial  Fibrillation,  Systemic  Embolus,  Left  Atrial 
Size,  Cardioversion,  Echocardiography 


¥(£ 


Project  No.  Z01  HL  01620-01  CB 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   Manuscript  submitted  to  CIRCULATION 


Wt 


Project  No.   Z01  HL  01621-03  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   A  Real  Time  System  for  Two-Dimensional  Echocardiography 

Previous  Serial  Number:   NHLI-26(c) 

Principal  Investigator:   Walter  L.  Henry,  M.D. 

Other  Investigators:   James  M.  Griffith*  M.S.E.E. 

Cooperating  Units:   Biomedical  Engineering  and  Instrumentation  Branch,  DRS 

Project  Description:   During  the  past  several  years  one-dimensional  pulse- 
echo  ultrasound  techniques  have  proven  extremely  useful  in  cardiac 
diagnosis.   A  one-dimensional  system,  however,  only  visualizes  structures 
lying  along  a  single  straight  line.   The  spatial  relationships  of  the  various 
cardiac  structures  are  therefore  not  so  easily  defined  as  with  two-dimensional 
systems  which  display  the  heart  by  constructing  a  plane  image  composed  of 
many  straight  lines.   We  have  developed  a  sector  scanning  system  for  ob- 
taining two-dimensional  echocardiograms  in  real  time  using  ultrasonic  pulse- 
echo  techniques.   Images  are  produced  by  angling  rapidly  a  single  transducer 
through  a  30-degree  sector  from  a  fixed  spot  (between  ribs)  on  the  patient's 
chest.   Thirty  complete  sectors  (or  frames)  are  produced  per  second.   The  use 
of  a  large  diameter  transducer  ensures  that  signal  strength  is  good  and 
cardiac  structures,  including  endocardium,  can  be  visualized.   Other  ad- 
vantages include  high  transducer  sensitivity,  real  time  imaging  and  easy 
visualization  of  various  regions  of  the  heart.   Experience  with  more  than 
100  patients  indicates  that  diagnostic  quality  two-dimensional  echo- 
cardiograms can  be  readily  obtained  in  essentially  the  same  patients  from 
whom  one-dimensional  echocardiograms  are  recorded  and  can  usually  be  per- 
formed in  less  time. 

Keyword  Descriptors:   Two-Dimensional  Echocardiography,  Mechanical  Sector- 
Scanner,  Real-Time  Imaging 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   CIRCULATION  49:1147-1152,  1974 


m 


Project  No.  Z01  HL  01622-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Effects  of  Space  Flight  on  Cardiac  Function 

Previous  Serial  Number:   None 

Principal  Investigator:   Walter  L.  Henry,  M-.Di 

Other  Investigators:   Stephen  E.  Epstein,  M.D. 

James  M.  Griffith,  M.S.E.E. 
Robert  E.  Goldstein,  M.D. 
David  R.  Redwood,  M.D. 

Cooperating  Units:   Biomedical  Engineering  and  Instrumentation  Branch,  DRS 

Project  Description:   Echocardiographic  studies  were  performed  preflight  five 
days  before  lauch  and  on  recovery  day  and  1,  2,  4,  11,  31  and  68  days  post- 
flight.   From  these  echocardiograms,  the  following  measurements  were  made: 
1)  left  ventricular  transverse  dimension  at  end-diastole,  2)  left  ventricular 
transverse  dimension  at  end-systole,  and  3)  ventricular  free  wall  thickness  at 
end-diastole.   From  these  primary  measurements,  left  ventricular  end- 
diastolic  volume,  end-systolic  volume,  stroke  volume,  and  mass  were  derived 
using  the  accepted  assumptions.   Preflight  measurements  in  the  Commander  re- 
vealed the  left  ventricular  end-diastolic  volume,  stroke  volume,  and  mass  to 
be  at  the  upper  limit  of  normal,  while  those  of  the  Scientist  Pilot   and 
Pilot  were  increased  significantly  above  the  normal  range.   These  findings 
findings  in  the  Scientist  Pilot  and  Pilot  resemble  those  seen  in  trained 
distance  runners.   Wall  thickness  measurements  were  normal  in  all  three  crew- 
members  preflight.   Postf light  basal  studies  were  unchanged  in  the  Commander 
on  recovery  day  through  68  days  postf light.   In  both  the  Scientist  Pilot  and 
Pilot,  however,  the  left  ventricular  end-diastolic  volume,  stroke  volume,  and 
mass  were  decreased  slightly.   These  decreases  were  noted  on  recovery  day 
through  11  days  postflight  but  had  returned  to  near  normal  by  31  days  post- 
flight.   Wall  thickness  measurements  were  unchanged.   Left  ventricular 
function  curves  were  constructed  for  the  Commander  and  Pilot  by  plotting 
stroke  volume  versus  end-diastolic  volume.   In  both  astronauts,  preflight  and 
postflight  data  fell  on  the  same  straight  line  demonstrating  that  no  deter- 
ioration in  cardiac  function  had  occurred.   These  data  indicate  that  the 
cardiovascular  system  adapts  well  to  prolonged  weightlessness  and  suggest 
that  alterations  in  cardiac  dimensions  and  function  are  unlikely  to  limit 
man's  future  in  space. 


¥of 


Project  No.   Z01  HL  01622-01  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Keyword  Descriptors:   Space  Flight,  Cardiac  Function,  Sky lab  4  Astronauts, 
Echocardiography 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   Manuscript  published  in  PROCEEDINGS  OF  SKYLAB  LIFE  SCIENCES 
SYMPOSIUM  (August  27-29,  1974),  pp.  711-721 


¥fo 


Project  No.   Z01  HL  01623-03  CB 

1.  Cardiology 

2.  Clinical  Physiology 
1                    3.   Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Mitral  Valve  Position  in  Patients  with  ASH 

Previous  Serial  Number:   NHLI-19(c) 

Principal  Investigator:   Walter  L.  Henry,  M.D. 

Other  Investigators:   Chester  E.  Clark,  M.D. 

Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   Left  ventricular  outflow  obstruction  in  patients  with 
IHSS  or  obstructive  asymmetric  septal  hypertrophy  (ASH)  is  due  to  abnormal 
forward  motion  during  systole  of  the  anterior  mitral  leaflet.   In  order  to 
determine  why  some  patients  with  ASH  have  left  ventricular  outflow 
obstruction  while  others  do  not,  We  studied  a  large  number  of  patients  with 
ASH  using  both  one  and  two  dimensional  echocardiography.   In  100  patients 
with  ASH  and  22  normal  subjects,  mitral  valve  position  at  onset  of  systole 
was  quantitated  by  measuring  the  distance  from  the  ventricular  septum  to 
the  mitral  valve  and  the  distance  from  the  mitral  valve  to  the  posterior 
left  ventricular  wall.   None  of  the  normal  subjects  and  only  3  of  51  non- 
obstructive ASH  patients  (6%)  had  a  septal-mitral  valve  distance  less  than 
20  mm  compared  to  23  of  35  obstructive  ASH  patients  (66%) .   Moreover,  in 
ASH  the  mitral  valve  at  onset  of  systole  was  actually  positioned  forward  in 
the  left  ventricular  cavity.   Two-dimensional  studies  in  11  patients  with 
obstructive  ASH  revealed  that  contraction  of  the  malaligned  papillary  muscles 
did  not  cause  the  abnormal  forward  mitral  valve  motion.   We  propose  that  the 
left  ventricular  outflow  obstruction  in  patients  with  obstructive  ASH  occurs 
as  a  result  of  two  factors:   1)  narrowing  of  the  left  ventricular  outflow 
tract  at  onset  of  systole,  and  2)  hydrodynamic  forces  generated  by  con- 
traction of  the  left  ventricle. 

Keyword  Descriptors:   Obstructive  ASH,  IHSS,  Outflow  Obstruction,  Echo- 
cardiography, Two-Dimensional  Imaging 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications:   AMERICAN  JOURNAL  OF  CARDIOLOGY  35:337-345,  1975 


¥tf 


Project  No.   Z01  HL  01624-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Aortic  Regurgitation:  Cardiac  Function  and  Operative  Result 


Previous  Serial  Number:   None 


Principal  Investigator:   Walter  L.  Henry,  M.D. 


Other  Investigators 


Joel  Morganroth,  M.D. 
Chester  E.  Clark,  M.D. 
Alan  S.  Pearlman,  M.D. 
Leonard  Grauer,  M.D. 
David  R.  Redwood,  M.D. 
Samuel  B.  Itscoitz,  M.D. 
Charles  L.  Mcintosh,  M.D. 
Lawrence  L.  Michaelis,  M.D. 
Andrew  G.  Morrow,  M.D. 
Stephen  E.  Epstein,  M.D. 


Cooperating  Units:   Clinic  of  Surgery,  NHLI 

Project  Description:   To  assess  the  influence  of  ventricular  function  on 
operative  results  in  patients  with  isolated  aortic  regurgitation,  20 
patients  undergoing  aortic  valve  replacement  were  studied  echocardiographically 
preoperatively  and  again  six  months  postoperatively.   Patients  were  sub- 
divided into  three  groups  based  on  preoperative  ejection  fraction  (EF) : 
normal  EF  (>60%)-ll  patients;  intermediate  EF  (40-60%)-5  patients;  low  EF 
(<40%)-4  patients.   Two  operative  deaths  occurred,  both  in  patients  with 
intermediate  EF.   EF  did  not  increase  after  operation  in  any  patient.   In 
two  patients  with  normal  EF  preoperatively,  EF  decreased;  both  experienced 
operative  complications.   Of  nine  patients  with  normal  EF  both  preoperatively 
and  postoperatively,  eight  had  reduction  in  end-diastolic  volume  (LVEDV) 
postoperatively  to  <250  ml,  7  a  decrease  in  LV  mass  to  <500  g;  all  are 
functional  class  I  or  II.   Of  patients  with  either  intermediate  EF  both 
preoperatively  and  postoperative  (3  patients),  or  normal  EF  preoperatively 
but  intermediate  EF  postoperatively  (1  patient),  3  of  4  have  LVEDV  post- 
operatively <250  ml,  4  of  4  have  LV  mass  <500  g;  3  of  4  are  clc.ss  1  or  II. 
In  contrast,  no  patient  with  either  low  EF  both  preoperatively  and  post- 
operatively (4  patients)  or  normal  EF  preoperatively  but  low  EF  post- 
operatively (one  patient)  had  LVEDV  <250  ml  or  LV  mass  <500  g.   Three  of  the 
five  died  during  long-term  follow-up;  one  is  class  III  and  one  class  II. 
Thus,  in  patients  with  aortic  regurgitation  1)  operative  does  not  improve 
vasal  ventricular  function,  2)  LVEDV  and  mass  are  more  likely  to  return  toward 
normal  in  patients  with  normal  or  intermediate  EF,  3)  long-term  results  are 


¥fX 


Project  No.   Z01  HL  01624-01  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

good  in  patients  with  normal  or  intermediate  EF  provided  an  operative  com- 
plication does  not  impair  ventricular  function,  4)  long-term  results  are 
poor  in  patients  either  with  a  low  preopeartive  EF  or  in  whom  an  operative 
complication  results  in  a  low  EF  postoperatively. 

Keyword  Descriptors:   Echocardiography,  Aortic  Regurgitation,  Ejection 
Fraction,  Operative  Results 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:   None 

Publications:   Abstract  published  in  CLINICAL  RESEARCH,  April,  1975 


4(3 


Project  No.  Z01  HL  01625-02  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

NIH-PHS 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Identification  of  Cyanotic  Heart  Disease  in  Infants  by  Two- 
Dimensional  Echocardiography 

Previous  Serial  Number:   NHLI-128(c) 

Principal  Investigator:   Barry  J.  Maron,  M.D. 

Other  Investigators:   Walter  L.  Henry,  M.D. 
Robert  Freedom,  M.D. 
David  T.  Kelly,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Department  of  Pediatrics,  Johns  Hopkins  Hospital, 
Baltimore,  Maryland 

Project  Description:   Real-time,  two-dimensional  echocardiography  was  used 
to  identify  great  artery  relations  in  23  infants  and  small  children,  in- 
cluding 16  patients  with  angiographically  documented  transposition  of  the 
great  arteries,  tetralogy  of  Fallot,  or  pulmonary  atresia.   Using  this 
tec-nique,  the  heart  was  scanned  perpendicular  to  its  long  axis  at  the  origin 
of  the  great  arteries.   Great  arteries  cross-sectioned  perpendicular  to 
their  long-axes  appears  as  circles;  when  sectioned  longitudinally,  these 
arteries  appeared  as  elongated,  sausage-shaped  structures.   In  patients  with 
normally  related  great  arteries,  a  circular  structure  (aorta)  always  was 
positioned  posterior  to  an  elongated,  sausage-shaped  structure  (distal 
right  ventricular  outflow  tract  and  proximal  main  pulmonary  artery) .   In 
transposition  of  the  great  arteries,  two  adjacent  circular  structures  were 
observed;  the  anterior  circle  (aorta)  was  located  either  to  the  right,  left 
or  directly  anterior  to  the  posterior  circle  (pulmonary  artery) .   In 
pulmonary  atresia  or  hypoplasia,  a  large  posterior  circle  (aorta)  was 
associated  with  an  anteriorly  positioned  structure  that  was  either  short  and 
small  (atretic  right  ventricular  outflow  tract)  or  elongated  with  an  area 
of  severe  narrowing  (hypoplastic  right  ventricular  outflow  tract). 

Keyword  Descriptors:   Transposition  of  the  Great  Vessels,  Tetralogy  of  Fallot, 
Pulmonary  Atresia 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications:   None 


fr* 


Project  No.   Z01  HL  01626-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Intramitochondrial  Glycogen  Deposits  in  Cardiac  Muscle 


Previous  Serial  Number:   None 

Principal  Investigator:   3arry  J.  Maron,  M.D. 

Other  Investigators:   Victor  J.  Ferrans,  M.D. ,  Ph.D. 

Cooperating  Units:   Section  of  Pathology,  NHLI 

Project  Description:   Intramitochondrial  glycogen  deposits  were  present  in 
ventricular  muscle  cells  in  4  of  16  patients  with  aortic  valvular  disease 
and  5  of  16  patients  with  asymmetric  septal  hypertrophy.   The  intra- 
mitochondrial glycogen  deposits  were  located  in  the  outer  mitochondrial 
compartment  (intracristal  space)  in  each  instance  and  were  of  the  mono- 
particulate  (B)  type  in  7  patients;  both  B-glycogen  and  a-glycogen 
rosettes  were  present  in  mitochondria  from  the  other  2  patients.   Mito- 
chondria containing  glycogen  were  present  in  about  equal  frequency  in 
hypertrophied  cells  with  otherwise  normal  ultrastructure  and  in  cells  with 
features  of  degeneration.   Intramitochondrial  glycogen  appears  to  be  a 
relatively  common  finding  in  hypertrophied  myocardium  in  a  variety  of 
cardiac  conditions. 

Keyword  Descriptors:   Myocardial  Ultrastructure,  Aortic  Valvular  Disease, 
Asymmetric  Septal  Hypertrophy,  Hypertrophic  Cardiomyopathy,  Cardiac  Muscle 
Cells,  Electron  Microscopy 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   None 


^sr 


Project  No.  Z01  HL  01627-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

NIH-PHS 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Phosphorylation  of  Cardiac  Muscle  Proteins 

Previous  Serial  Number:   None 

Principal  Investigator:   Barry  J.  Maron,  M.D. 

Other  Investigators:   Robert  S.  Adelstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   Phosphorylation  of  cardiac  muscle  proteins  is  a 
potentially  important  mechanism  in  regulating  the  interaction  of  actin  and 
myosin  during  contraction.   In  order  to  determine  the  characteristics  of 
phosphorylation  in  the  heart,  crude  preparations  of  canine  and  human  cardiac 
actomyosin  were  incubated  with  y- labeled  ATJ^p.   Incorporation  of  32p  into 
two  proteins  was  demonstrated:   1)  a  protein  with  a  molecular  weight  of 
165,000  daltons  that  was  identified  as  M-protein,  and  2)  the  light  chain  of 
mysoin  having  a  molecular  weight  of  27,000  daltons  in  the  dog  and  25,000 

daltons  in  man.   In  addition,  incubation  of  AT-^p  with  preparations  of  M- 

3  ? 
protein  produced  equal  incorporation  of  J  P  into  two  separate  protein  com- 
ponents (each  having  a  molecular  weight  of  about  165,000  daltons).   The 
phosphorylation  of  M-protein  and  of  a  light  chain  of  myosin  appears  to  be 
due  to  an  endogeneous  kinase  or  kinases. 

Keyword  Descriptors:   M-Protein,  Myosin,  Light  Chain  of  Myosin,  Protein 
Kinase 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards :   None 

Publications:   None 


4-iL 


Project  No.   Z01  HL  01628-02  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Sudden  Infant  Death  Syndrome:  Potential  Cardiac  Mechanisms 

Previous  Serial  Number:   NHLI-131(c) 

Principal  Investigator:   Barry  J.  Maron,  M.D. 

Other  Investigators:   Chester  E.  Clark,  M.D. 

Robert  E.  Goldstein,  M.D. 
Walter  L.  Henry,  M.D. 
Russell  B.  Fisher,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Coroner's  Office,  State  of  Maryland,  Baltimore,  Maryland 

Project  Description:   Sudden  infant  death  syndrome  (SIDS)  is  a  major  cause  of 
mortality  in  the  first  six  months  of  life,  but  the  primary  mechanisms 
responsible  for  this  condition  are  unknown.   To  investigate  possible  cardiac 
mechanisms,  42  sets  of  parents  (who  had  at  least  one  infant  die  of  SIDS)  were 
studied  by  echocardiography  and  electrocardiography.   Asymmetric  septal 
hypertrophy  (ASH)  was  present  in  two  (5%)  parental  sets  (one  parent  affected 
in  each  set).   At  least  one  member  of  13  (31%)  other  parental  sets  had  ECG 
abnormalities;  9  with  QT  interval  prolongation,  two  with  left  anterior  hemi- 
block,  one  with  first  degree  A-V  block  and  one  with  ST-T  abnormalities.   To 
further  study  the  role  of  QT  interval  prolongation  in  SIDS,  two  other  avenues 
of  investigation  were  pursued.   First,  QT  intervals  were  assessed  in  siblings 
of  infants  dying  suddenly  of  SIDS  (in  the  nine  families  in  which  at  least 
one  member  of  the  parental  set  showed  QT  interval  prolongation) .   Thirteen  of 
27  (47%)  of  these  siblings  showed  QT  interval  prolongation  consistent  with  an 
autosomal  dominant  pattern  of  inheritance;  the  remainder  had  normal  QT 
intervals.   Second,  three  infants  with  "near-miss  SIDS"  were  studied.   One  of 
these  infants  showed  a  markedly  prolonged  QT  interval  and  the  other  two  showed 
slightly  prolonged  QT  intervals.   In  addition,  histologic  sections  of  myo- 
cardium were  analyzed  in  another  group  of  45  infants  with  SIDS,  17  control 
infants  who  died  suddenly  from  other  causes,  and  5  normal  human  fetuses. 
Small  foci  of  normal-sized,  disorganized  myocardial  cells  were  present  in  the 
ventricular  septum  of  10  (22%)  of  the  infants  with  SIDS,  one  (6%)  of  con- 
trols and  none  of  the  fetuses.   The  foci  of  disorganized  cells  in  SIDS 
resembled  those  in  ASH,  but  were  less  marked  in  extent  and  severity.   Al- 
though the  significance  of  these  abnormally  arranged  cells  is  unknown,  they 
may  serve  as  a  nidus  for  ventricular  arrhythmias.   Furthermore,  we  studied 
five  parental  sets  of  these  infants  with  disorganized  cells;  three  had  ASH 
and  two  of  these  had  QT  interval  prolongation.   Thus,  the  results  of  the 


Yf7 


Project  No.   Z01  HL  01628-02  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

present  study  demonstrate  that  30%  of  47  parent  sets  of  infants  with  SIDS  had 
QT  prolongation  or  ASH.   Both  these  abnormalities  have  an  autosomal  dominant 
pattern  of  inheritance  and  have  been  associated  with  sudden  death  in 
children.   Therefore,  our  data  suggest  that  cardiac  mechanisms  may  play  a 
role  in  a  considerable  proportion  of  infant  deaths  falling  within  the 
sudden  infant  death  syndrome. 

Keyword  Descriptors:   QT  Interval  Prolongation,  Asymmetric  Septal  Hypertrophy. 
Elec trocardiography ,  Echocardiography 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications:   None 


H& 


Project   No.    Z01    HT,    01629-02    CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Asymmetric  Septal  Hypertrophy  in  Childhood 

Previous  Serial  Number:   NHLI-127(c) 

Principal  Investigator:   Barry  J.  Maron,  M.D. 

Other  Investigators:   Walter  L.  Henry,  M.D. 
David  R.  Redwood,  M.D. 
Chester  E.  Clark,  M.D. 
William  C.  Roberts,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Section  of  Pathology,  NHLI 

Project  Description:   Although  considerable  information  is  available  con- 
cerning the  clinical  features  and  natural  history  of  asymmetric  septal  hyper- 
trophy (ASH)  in  adults,  little  is  known  of  this  disease  in  children.   The 
clinical  characteristics  and  course  of  46  children  with  ASH,  who  were 
evaluated  at  the  National  Heart  and  Lung  Institute,  have  been  analyzed. 
Twenty-four  children  had  obstruction  to  ventricular  outflow;  22  children  had 
no  obstruction  to  ventricular  outflow,  including  11  patients  without  overt 
manifestations  of  cardiac  disease  other  than  echocardiographic  evidence  of 
ASH.   Thirty-five  of  the  46  children  have  been  followed  for  one  to  16  years 
(average,  7.4  years).   These  latter  children  represent  that  subgroup  of 
patients  with  ASH  referred  to  the  National  Heart  and  Lung  Institute  and 
diagnosed  prior  to  the  general  availability  of  echocardiography.   The 
clinical  course  of  these  patients  was  variable.   Eighteen  (52%)  of  the  35 
patients  improved  or  remained  stable,  including  two  patients  who  underwent 
left  ventricular  myotomy-myectomy  or  myotomy  and  six  patients  who  received 
propranolol.   Six  (17%)  of  the  35  patients  deteriorated  clinically  and  11 
(31%)  of  the  35  patients  died  suddenly  (4%  mortality  per  year).   Two  of  the 
patients  who  died  suddenly  had  previously  undergone  operation  (six  and  13 
years  previously)  with  resultant  abolition  of  the  outflow  gradient;  four 
others  were  taking  propranolol.   Neither  symptmatology,  electrocardiographic 
abnormalities,  heart  size,  left  ventricular  ejection  or  upstroke  time, 
magnitude  of  outflow  gradient,  or  left  ventricular  end-diastolic  pressure 
proved  predictive  of  sudden  death.   Excluding  patients  who  had  previous 
operation,  eight  (40%)  of  20  patients  with  obstruction  who  were  followed  long- 
term  and  one  (9%)  of  11  patients  without  outflow  obstruction  died  suddenly. 
Thus,  this  study  demonstrates  that  the  clinical  and  hemodynamic  spectrum  of 
ASH  in  childhood  is  broad  and  sudden  death  has  been  relatively  common  in 
that  subgroup  of  children  who  were  referred  to  the  National  Heart  and  Lung 


Vff 


Project  No.   Z01  HL  01629-02  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Institute  because  of  overt  manifestations  of  cardiac  disease. 

Keyword  Descriptors:  Sudden  Death,  Hypertrophic  Cardiomyopathy,  Idiopathic 
Hypertrophic  Subaortic  Stenosis,  Congenital  Heart  Disease,  Echocardiography. 
Cardiac  Catheterization,  Myocardium,  ASH,  Childhood 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   None 


¥%> 


Project  No.  Z01  HL  01630-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Proiect  Title:   Nitroglycerin,  Nitroprusside  and  Myocardial  Ischemia 


Previous  Serial  Number:   None 

Principal  Investigator:   Alan  S.  Pearlman,  M.D. 

Robert  Engler,  M.D. 

Other  Investigators:   Kenneth  M.  Kent,  M.D.,  Ph.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   Recent  studies  have  generated  considerable  enthusiasm 
for  the  use  of  the  nitroglycerin  (TNG)  and  nitroprusside  in  the  treatment  of 
patients  with  acute  myocardial  infarction.   These  agents  appear  to  improve 
hemodynamic  function  by  their  peripheral  dilator  effects  on  systemic 
arteries  and  veins,  which  reduce  arterial  and  left  ventricular  filling 
pressures  and  thereby  diminish  left  ventricular  work.   In  addition,  TNG 
reduces  resistance  to  coronary  collateral  flow  and  increases  coronary  flow 
to  ischemic  areas.   Thus,  TNG  appears  to  have  two  effects  that  would  con- 
tribute to  an  amelioration  of  ischemia.   However,  no  information  is 
available  relating  to  the  direct  effects  of  nitroprusside  on  coronary  arterial 
vessels.   We  therefore  examined  the  ability  of  nitroprusside  to  modify  the 
extent  of  experimentally  induced  acute  myocardial  ischemia,  and  compared  the 
effects  of  this  drug  with  those  of  nitroglycerin. 

Five  dogs  with  pre-existing  multivessel  coronary  occlusive  disease  underwent 
acute  balloon  occlusion  of  the  left  anterior  descending  coronary  artery.   The 
extent  of  ischemic  injury  was  determined  by  summating  ST-segment  elevation 
(ST)  from  7  subepicardial  electrodes  previously  placed  in  the  ischemic  zone; 
measurements  were  made  of  heart  rate,  cardiac  output,  arterial  pressure  and 
left  atrial  pressure.   Each  animal  was  subjected  to  three  occlusions  in  random 
order:   1)  control  (untreated),  2)  following  treatment  with  nitroprusside, 
3)  following  treatment  with  nitroglycerin.   Under  control  conditions,  heart 
rate,  arterial  pressure,  and  left  atrial  pressure  remained  stable  over  the 
course  of  the  15  minute  occlusion.   EST  rose  from  preocclusion  levels  to  a 
peak  value  at  10  minutes,  and  remained  stable  over  the  next  five  minutes  of 
occlusion.   Nitroprusside  caused  an  average  reduction  in  mean  arterial 
pressure  of  29%  and  an  increase  in  heart  rate  of  29%.   These  changes  were 
accompanied  by  an  increase  in  ^ST  over  control  averaging  26%  at  10  minutes 
and  34%  at  15  minutes  of  occlusion  (p<0.05).   Nitroglycerin  reduced  arterial 
pressure  21%  from  control  values  and  increased  heart  rate  29%.   Despite 

l  ¥*t 


Project  No.   Z01  HL  01630-01  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

causing  nearly  identical  hemodynamic  effects  as  nitroprusside,  however,  nitro- 
glycerin administration  was  accompanied  by  an  average  reduction  in  £ST  below 
control  values  of  13%  at  10  minutes  (p<0.05)  and  of  10%  at  15  minutes.   Thus 
in  dogs  with  pre-existing  multivessel  coronary  occlusive  disease,  none  of 
whom  had  left  ventricular  failure,  the  extent  of  ischemic  injury  following 
experimental  acute  coronary  occlusion  is  further  aggravated  by  administration 
of  nitroprusside,  in  contrast  to  the  beneficial  effect  exerted  by  nitro- 
glycerin.  While  perhaps  not  directly  applicable  to  the  clinical  situation, 
these  preliminary  results  suggest  that  the  reduction  in  left  ventricular  work 
consequent  to  nitroprusside  administration  may  not  be  accompanied  by  a  re- 
duction in  ischemic  injury;  this  finding  emphasizes  the  point  that  therapy 
for  acute  myocardial  infarction  should  be  assessed  in  relation  to  its  effects 
not  only  on  hemodynamic  function,  but  also  on  the  magnitude  of  ischemic  injury. 

Keyword  Descriptors:   Nitroglycerin,  Nitroprusside,  Acute  Myocardial  Ischemia, 
Acute  Coronary  Occlusion,  Vasodilators,  Impedance  Reduction,  Dogs 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:   None 

Publications:   None 


Viv 


Project  No.  Z01  HL  01631-02  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Nitroglycerin  Therapy  for  Acute  Myocardial  Infarction  in  Man 

Previous  Serial  Number:   NHLI-120(c) 

Principal  Investigator:   Jeffrey  S.  Borer,  M.D. 

Other  Investigators:   David  R.  Redwood,  M.D. 
Barrie  Levitt,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Cardiology  Department,  Flower  and  Fifth  Avenue 
Hospitals,  New  York,  N.Y. 

Project  Description:   Previous  investigations  in  dogs  have  demonstrated  that 
nitroglycerin  reduces  ischemic  injury  and  enhances  ventricular  electrical 
stability  during  acute  coronary  occlusion.   These  beneficial  effects  of 
nitroglycerin  are  potentiated  by  preventing  drug-induced  hypotension  with 
an  alpha-adrenergic  agonist.   Such  combination  therapy  also  has  been  found 
to  reduce  the  incidence  of  spontaneous  postocclusion  ventricular 
fibrillation  in  dogs.   The  present  study  is  designed  to  determine  the 
efficacy  of  nitroglycerin,  alone  and  with  its  hypotensive  effects  prevented 
with  phenylephrine  (an  alpha  agonist),  in  reducing  the  extent  and  severity 
of  myocardial  injury  sustained  during  acute  myocardial  infarction  in  man. 
The  efficacy  of  these  interventions  is  being  assessed  by  ST  segment  analysis 
of  35- lead  precordial  surface  maps  (experimental  studies  have  demonstrated 
that  changes  in  ST  segment  elevation  reflect  changes  in  myocardial 
ischemic  injury). 

Thus  far,  12  patients  with  acute  myocardial  infarction  have  been  studied. 
ST  segment  abnormalities  have  been  reduced  in  each  (17  to  60%)  during 
therapy  with  sublingual  nitroglycerin  and  phenylephrine.   However,  the 
optimal  therapeutic  regimen  depended  upon  whether  or  not  the  patient  was  in 
left  ventricular  failure. 

In  the  7  patients  without  left  ventricular  failure,  nitroglycerin  adminis- 
tered alone  caused  a  fall  in  blood  pressure  and  a  reflexly  mediated  in- 
crease in  heart  rate.   These  changes  were  not  associated  with  a  consistent 
decrease  in  the  degree  of  ST  segment  elevation.   In  one  patient  it  caused 
an  excessive  tachycardia,  which  led  to  an  increase  in  the  degree  of 
ischemic  injury.   In  contrast,  when  phenylephrine  was  added  and  infused  at  a 
rate  sufficient  to  reverse  the  blood  pressure  lowering  effects  of  nitro- 
glycerin, significant  improvement  in  the  degree  of  myocardial  ischemia 


423 


Project  No.   Z01  HL  01631-02  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

occurred  in  each  patient. 

In  the  5  patients  with  left  ventricular  failure,  nitroglycerin  alone  led  to 
an  average  15  mm  fall  in  PA  wedge  pressure  and  a  26  mm  Hg  fall  in  mean 
arterial  pressure.   Despite  the  fall  in  arterial  pressure,  there  was  no  re- 
flex tachycardia.   Moreover,  in  contrast  to  the  patients  without  failure, 
TNG  alone  uniformly  improved  ST  segment  abnormalities.   After  abolition  of 
the  TNG-induced  arterial  pressure  fall  with  phenylephrine,  significant  ST 
segment  improvement  was  still  present,  but  the  benefit  tended  to  be  less 
marked  than  with  nitroglycerin  alone. 

We  conclude  that  ischemic  injury  occurring  during  acute  myocardial  in- 
farction often,  but  not  always,  is  reduced  by  nitroglycerin  alone.   When 
nitroglycerin  and  phenylephrine  are  given,  ischemia  is  usually  if  not  always 
diminished,  with  minimal  risk  of  adverse  effects.   However,  response  of 
ischemia  to  therapy  is  not  homogeneous,  and  cannot  be  predicted  with 
certainty  by  monitoring  hemodynamic  variables  alone.   Therefore,  at  present 
it  appears  that  while  general  guidelines,  based  on  the  presence  or  absence 
of  failure,  can  be  employed  when  initiating  vasodilator  therapy  in  the  patient 
with  an  acute  infarct,  optimal  therapy  in  the  individual  patient  can  be 
achieved  only  if  a  method  such  as  precordial  mapping  is  utilized  for 
objective  evaluation  of  ischemia. 

Keyword  Descriptors:   Nitroglycerin,  Ischemic  Injury,  Precordial  Surface 
Maps,  Phenylephrine,  Acute  Myocardial  Infarction 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:   None 

Publications:   None 


V2V 


Project  No.  Z01  HL  01632-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Effects  of  Vasodilators  on  Coronary  Collateral  Flow 

Previous  Serial  Number:   None 

Principal  Investigator:   Norine  L.  Capurro,  Ph.D. 

Other  Investigators:   Kenneth  M.  Kent,  M.D.,  Ph.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   Augmentation  of  coronary  collateral  flow  is  potentially 
beneficial  to  ischemic  myocardium.   Studies  in  the  dog  and  man  indicate  that 
nitroglycerin  enhances  collateral  flow.   This  study  was  designed  to  test 
pharmacological  responsiveness  of  the  coronary  collateral  circulation,  and 
specifically  to  determine  the  effects  of  nitroglycerin  and  nitroprusside  on 
coronary  collateral  flow.   Dogs  of  either  sex  received  general  anesthesia, 
the  heart  was  exposed  through  a  left  thoracotomy,  and  an  ameroid  constrictor 
was  placed  around  the  anterior  descending  (LAD)  branch  of  the  left  coronary 
artery.   The  dogs  were  studied  two  to  four  weeks  post-operatively .   The  dogs 
were  anesthetized  with  sodium  pentobarbital  and  the  chest  opened.   A  poly- 
ethylene cannula  was  inserted  in  the  LAD  distal  to  the  ameroid.   A  Gregg 
cannula  was  inserted  through  the  subclavian  artery  and  secured  in  the  left 
main  coronary  artery.   Both  the  left  main  and  the  distal  LAD  were  perfused 
with  blood  shunted  from  the  dog's  carotid  artery.   When  inflow  to  the  LAD 
cannula  was  clamped,  peripheral  coronary  pressure  (PCP)  was  measured  and  retro- 
grade flow  (RF)  was  collected  from  side  arms  of  the  LAD  cannula.   Systemic 
arterial  pressure,  left  atrial  pressure,  ECG,  and  coronary  flow  were 
continuously  monitored.   Drugs  were  administered  by  constant  intracoronary 
infusion  (through  the  Gregg  cannula)  or  by  constant  intravenous  infusion. 
Since  RF  and  PCP  vary  directly  with  perfusion  pressure,  arterial  pressure  was 
held  constant  at  approximately  95  mm  Hg,  by  manipulation  of  an  inflatable  cuff 
placed  around  the  descending  aorta.   In  9  dogs  with  ameroid  constrictors, 
control  RF  ranged  from  11  to  90  ml/min  (mean  35)  and  control  PCP  ranged  from 
54  to  98  mm  Hg   (mean  70).  (In  4  normal  dogs  control  RF  ranged  from  1.1  to 
3.0  ml/min  (mean  2.0)  and  control  PCP  ranged  from  20  to  29  mm  Hg  (mean  24).) 
In  six  dogs,  intracoronary  (i.e.)  administration  of  nitroglycerin,  0.3  to 
100  Ug/min,  did  not  alter  RF  or  PCP  when  systemic  pressure  was  held  constant 
at  95  mm  Hg  and  with  an  average  heart  rate  of  160  beats/min.   In  2  dogs, 
heart  rate  was  lowered  by  vagal  stimulation  to  60/min.   In  th^se  dogs, 
nitroglycerin  (i.e.)  produced  dose-dependent  increases  in  RF  but  no  change  in 
PCP.   Intravenous  infusion  of  nitroglycerin,  10  to  300  Ug/min,  resulted  in 
progressive  increases  in  both  RF  and  PCP  at  either  rapid  (2  dogs)  or  slow 

i  VaT 


Project  No.   Z01  HL  01632-01  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

(3  dogs)  heart  rates.   In  3  dogs,  i.e.  administration  of  nitroprusside,  0.3 
to  100  ug/min  did  not  alter  RF  or  PCP .   Further  testing  of  the  effects  of 
nitroglycerin  and  nitroprusside,  as  well  as  those  adrenergic  drugs  and 
changes  of  heart  rate  and  vagal  stimulation  on  RF  and  PCP  are  planned.   These 
studies  should  help  define  the  responsiveness  of  the  coronary  collateral 
circulation. 

Keyword  Descriptors:   Coronary  Collateral  Flow,  Retrograde  Flow,  Peripheral 
Coronary  Pressure,  Nitroglycerin,  Nitroprusside 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:   None 

Publications:   None 


V^4 


Project  No.   Z01  HL  01634-02  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Determinants  of  Ventricular  Septal  Motion 

Previous  Serial  Number:   NHLI  154(c) 

Principal  Investigator:   Alan  S.  Pearlman,  M.D. 

Other  Investigators:   Chester  E.  Clark,  M.D. 
Joel  Morganroth,  M.D. 
Walter  L.  Henry,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   Echocardiographic  studies  of  ventricular  septal  (VS) 
motion  were  performed  in  patients  with  a  variety  of  cardiac  disorders  to 
define  the  determinants  of  septal  motion.   In  43  patients  with  right 
ventricular  volume  overload  secondary  to  atrial  septal  defect,  septal 
motion  was  frankly  paradoxic  in  35  (82%),  flat  in  4  (9%),  and  normal  in  4 
(9%).   Pattern  of  septal  motion  was  determined  by  the  position  of  the  VS 
relative  to  total  cardiac  diameter  (distance  from  right  ventricular  epi- 
cardium  to  left  ventricular  (LV)  epicardium  at  end-diastole) .   All  patients 
with  normal  septal  motion,  and  no  patients  with  flat  or  paradoxic  motion, 
had  a  septal  position  (defined  as  the  distance  from  right  ventricular  epi- 
cardium to  mid-septum/total  cardiac  diameter)  at  end-diastole  <.41.   When 
septal  motion  was  measured  (diastolic  minus  systolic  septal  position),  the 
direction  and  magnitude  of  VS  motion  was  linearly  related  to  end-diastolic 
septal  position  (r  =  .80,  p<.01).   Septal  motion  correlated  poorly  with  size 
of  shunt  (r  =  .13)  and  right  ventricular  pressure  (r  =  .18).   A  similar 
relation  between  septal  position  and  septal  motion  was  evident  in  1)  14 
patients  with  other  causes  of  right  ventricular  volume  overload  (r  =  .82), 
2)  19  patients  with  LV  volume  overload  (r  =  .74),  3)  10  patients  with  right 
ventricular  pressure  overload  (r  =  .93),  4)  10  patients  with  LV  pressure 
overload  (r  =  .80),  and  5)  28  normal  subjects  (r  =  .82).   We  conclude  that 
the  direction  and  magnitude  of  septal  motion  is  determined  bv  septal 
position  relative  to  total  cardiac  transverse  dimension.   Thus,  although 
paradoxic  septal  motion  is  usually  seen  in  conditions  causing  right  ventricu- 
lar volume  overload,  it  is  not  diagnostic  of  any  particular  hemodynamic 
burden.   Rather,  it  is  dependent  upon  the  geometric  position  of  the  septum 
within  the  heart. 


V£7 


Project  No.   Z01  HL  01634-02  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Keyword  Descriptors:   Echocardiography,  Atrial  Septal  Defect,  Right 
Ventricular  Volume  Overload,  Paradoxic  Septal  Motion,  Congenital  Heart 
Disease 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications:    None 


v>e 


Project  No.   ZOIHL  01636-01  CB 


1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Isolation  and  Characterization  of  Myosin  From  Patients  With 
Asymmetric  Septal  Hypertrophy 

Previous  Serial  Number:   None 

Principal  Investigator:   Barry  J.  Maron,  M.D. 

Other  Investigators:   Robert  S.  Adelstein,  M.D. 

Victor  J.  Ferrans,  M.D.,  Ph.D. 

Cooperating  Units:   Section  of  Pathology,  NHLI 

Project  Description:   Asymmetric  septal  hypertrophy  (ASH)  is  a  genetically 
transmitted  disorder  of  cardiac  muscle  characterized  by  a  disproportionately 
hyper trophied  ventricular  septum  that  contains  numerous  hyper trophied, 
bizarrely-shaped  and  disorganized  cardiac  muscle  cells.   These  disorganized 
cardiac  muscle  cells  are  presumably  the  morphologic  expression  of  the  genetic 
defect  in  ASH.   The  present  investigation  was  undertaken  to  characterize  the 
biochemical  and  molecular  characteristics  of  myosin  from  cardiac  muscle  cells 
of  patients  with  obstructive  ASH.   Biochemical  studies  were  performed  on  ven- 
tricular septal  myocardium  from  12  patients  with  obstructive  ASH,  left 
ventricular  free  wall  •myocardium  from  four  patients  with  obstructive  ASH  and 
left  ventricular  free  wall  from  four  patients  with  normal  hearts.   Myosin  was 
purified  from  preparations  of  actomyosin  by  gel  filtration  on  columns  of 
Sepharose  4B.   Specific  activity  of  myosin  from  the  ventricular  septum  of 
patients  with  ASH  ranged  from  1.2  to  2.1  ymoles  inorganic  phosphate  released 
per  mg  protein  per  minute  (average,  1.6)  and  did  not  differ  significantly  from 
the  specific  activity  of  myosin  from  the  left  ventricular  free  wall  of 
patients  with  obstructive  ASH  or  of  myosin  from  patients  with  normal  hearts. 
Polyacrylamide-SDS  gel  electrophoresis  of  myosin  from  patients  with  obstructive 
ASH  and  patients  with  normal  hearts  demonstrated  a  heavy  chain  (molecular 
weight  of  200,000  daltons)  and  two  light  chains  (molecular  weights  of  25,000 
and  20,000  daltons).   Furthermore,  characteristic  bipolar  aggregates  of  myosin 
molecules  formed  (at  low  ionic  strength)  in  preparations  of  myosin  from 
patients  with  ASH  and  from  patients  with  normal  hearts.   Therefore,  we  have 
found  no  evidence  to  suggest  that  myosin  from  patients  with  obstructive  ASH 
differs  biochemically  from  that  of  patients  with  normal  hearts  or  that  myosin 
from  the  ventricular  septum  of  patients  with  obstructive  ASH  differs  from 
myosin  from  the  left  ventricular  free  wall  of  the  same  patients. 


4>9 


Project  No.    Z01  HL  01636-01  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Keyword  Descriptors:   Myosin,  Idiopathic  Hypertrophic  Subaortic  Stenosis, 
Hypertrophic  Cardiomyopathy,  Polyacrylamide-SDS  Gel  Electrophoresis,  Ultra- 
structure;  Myosin  ATPase  activity 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications:   None 


41a 


Project  No.   Z01  HL  01637-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Indices  of  Reversibility  in  Heart  Failure 


Previous  Serial  Number:   None 

Principal  Investigator:   Barry  J.  Maron,  M.D. 

Other  Investigators:   Kenneth  M.  Kent,  M.D. 
Bruce  Reitz,  M.D. 
Jack  Copeland,  M.D. 
James  Scherer,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Clinic  of  Surgery,  NHLI  and  Radiology  Department,  NIH 

Project  Description:   To  determine  factors  responsible  for  irreversible  cardiac 
failure,  hemodynamic  studies  were  performed  in  11  dogs  with  circulatory  over- 
load (secondary  to  acrto-caval  shunt)  and  again  late  after  reversal  of  the 
shunt.   Clinical  and  hemodynamic  evidence  of  LV  failure  appeared  an  average 
of  55  days  after  exposure  to  volume  overload: 

LVEDP     LVEDV        EF  MCF 

Control  dogs     3+1      33+7      .78+. 14     .76+. 12 

Shunted  dogs    24+11*   102+42*    .49+. 2*     .31+. 06* 
*=p<.01.   (LVEDP=LV  end-diastolic  pressure;  LVEDV=LV  end-diastolic  volume; 
EF=ejection  fraction;  MCF=mean  rate  circumferential  fiber  shortening).   Late 
after  shunt  closure  (avg  73  days)  EF  (.48+. 17),  MCF  (.46+. 08)  had  not 
changed  significantly  from  preclosure  values.   LVEDP  (10+6  mm  Hg) ,  LVEDV 
(64+18  ml)  decreased  significantly  compared  to  preclosure  (p<.02)  but  were 
elevated  over  controls  (p<.01).   LVEDV  late  after  shunt  closure  was  directly 
related  to  the  magnitude  of  the  initial  shunt  (p<.05).   There  was  no  relation 
between  LVEDP,  LVEDV,  EF,  MCF,  dp/dt  measured  prior  to  and  late  after  shunt 
closure.   Thus,  in  dogs  with  volume  overload,  magnitude  of  shunt  is  a 
reliable  predictor  of  residual  LV  dilatation.   However,  usually  employed 
hemodynamic  indices  of  cardiac  function  are  of  little  value  in  predicting 
potential  for  reversibility  of  cardiac  failure. 

Keyword  Descriptors:   Heart  Failure,  Hemodynamics,  Arteriovenous  Shunt, 
Ventricular  Volume  Overload 


*v 


Project  No.  z01  HL  01637-01  CB 


PHS-NIH 

Individual  Project  Report 
July  1,  1974  through  June  30,  1975 


Proposed  Course  of  Project:   Continuing 
Honors  and  Awards:   None 
Publications:   None 


^3i- 


Project  No.    Z01  HL  01639-03  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Echocardiographic  Characteristics  of  Infiltrative  Cardiomyopathy 

Previous  Serial  Number:   NHLI-6(c) 

Principal  Investigator:   Jeffrey  S.  Borer,  M.D. 

Other  Investigators:   Walter  L.  Henry,  M.D. 

Stephen  E.  Epstein,  M.D. 

Cooperating  Units:  None 

Project  Description:  Echocardiography  (ECHO)  was  used  to  evaluate  the  cardiac 
status  of  adults  with  systemic  diseases  (amyloidosis,  4  pts;  hemosiderosis, 
5  pts;  mucopolysaccharidosis,  4  pts;  idiopathic  hypereosinophilia,  10  pts) 
known  to  be  associated  with  infiltrative  cardiomyopathy.   Though  8  (35%)  pts 
had  no  other  clinical  Or  non- invasive  laboratory  evidence  of  cardiac  disease, 
all  had  ECHO  abnormalities  consistent  with  infiltrative  disease.   LV  mass 
was  elevated  (>275  gm)  in  23  (88%),  (mean  385  gm,  nl  211,  p  <.01);  LV  free 
wall  and  septum  were  symmetrically  thickened  (  >11  mm)  in  23  pts  (mean  14.5  mm, 
nl  9.8,  p  <.01);  LV  internal  dimension  was  abnormal  in  7  pts.   Mitral  valve 
closure  slope  was  reduced  (<125  mm/sec)  in  20  pts  (mean  87  mm/sec,  nl  147, 
p<.01),  consistent  with  diminished  LV  compliance.   However,  systolic  function 
was  well  maintained  (ejection  fraction  mean  7%,  nl  71%,  n.s.).   These  ECHO 
findings  were  commonly  associated  with  each  disease  studied.   We  conclude 
that  in  adults  with  diseases  known  to  be  associated  with  infiltrative  cardio- 
myopathy 1)  ECHO  is  a  sensitive  technique  for  detecting  abnormalities  in 
patients  with  clinically  inapparent  cardiac  disease,  2)  ECHO  studies 
frequently  demonstrate  symmetrically  increased  LV  wall  thickness  and  LV  mass 
and  3)  systolic  function  is  preserved  while  diastolic  compliance  is  reduced. 

Keyword  Descriptors:   Echocardiography,  Cardiomyopathy,  Amyloidosis, 
Hemosiderosis,  Hypereosinophilia,  Mucopolysaccharidosis. 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:   None 

Publications:   None 


V33 


Project  No.  Z01  HL  01641-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Growth  of  Aortic  Smooth  Muscle  Cells  T_n  Vitro 

Previous  Serial  Number:   None 

Principal  Investigator:   Chester  E.  Clark,  M.D. 

Other  Investigators:   Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   There  is  evidence  that  the  intimal  plaque  in  athero- 
sclerosis is  initially  derived  from  smooth  muscle  cells  found  in  the 
media.   Several  investigators  have  cultivated  these  cells,  obtained  from 
animals,  and  have  shown  that  their  growth  rate  can  be  modified  by 
manipulating  their  environment,  most  notably  the  lipid  content  of  the 
culture  medium.   We  have  attempted  to  grow  cells  from  the  proximal  aorta  of 
patients  undergoing  cardiac  surgery,  in  an  effort  to  understand  some  of 
the  factors  that  control  growth  and  reproduction  of  these  cells.   Using  a 
variety  of  culture  media,  we  have  successfully  grown  human  aortic  cells, 
and  are  preparting  to  manipulate  their  growth. 

Keyword  Descriptors:   Atherosclerosis,  Aortic  Smooth  Muscle  Cells,  Lipids, 
Tissue  Culture 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:   None 

Publications:   None 


¥34 


Project  No.   Z01  HL  01642-01  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Physical  Factors  Determining  Cardiac  Motion 

Previous  Serial  Number:   None 

Principal  Investigator:   Chester  E.  Clark,  M.D. 

Other  Investigators:   Alan  S.  Pearlman,  M.D. 
Walter  L.  Henry,  M.D. 

Cooperating  Units:   None 

Project  Description:   There  have  been  numerous  studies  in  both  animals  and 
humans  providing  descriptive  data  relative  to  the  motion  of  cardiac  structures. 
Nevertheless,  there  have  been  few  efforts  to  provide  a  systematic  analysis 
based  on  physical  principles.   We  studied  18  patients  with  two-dimensional 
echocardiography:   eight  normals,  six  patients  with  right  ventricular  volume 
overload,  two  with  left  ventricular  volume  overload,  two  with  right  ventricu- 
lar pressure  overload,  one  with  left  ventricular  pressure  andright 
ventricular  volume  overload,  and  one  with  an  enlarged  right  ventricle  but  no 
volume  or  pressure  load.   The  data  allowed  us  to  test  and  validate  a  hypo- 
thesis that  overall  cardiac  movement  results  from  recoil  consequent  to 
ejection  of  blood  into  the  great  vessels  and  an  additional  internal  movement 
of  structures  during  systole  toward  the  center  of  mass  of  the  heart.   We 
hypothesized  and  subsequently  confirmed  that  the  motion  of  any  given  point 
within  the  heart  can  be  found  by  adding  the  recoil  force  to  the  movement  about 
the  center  of  mass.   This  hypothesis  accounts  for  the  observed  motion  of 
cardiac  structures,  and  in  particular  explains  the  motion  of  the  ventricular 
septum  that  is  seen  in  a  variety  of  cardiac  disorders. 

Keyword  Descriptors:   Cardiac  Motion,  Ventricular  Septum,  Center  of  Mass 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 

Publications:   Noae 


42C 


Project  No.  Z01  HL  01643-02  CB 

1.  Cardiology 

2.  Clinical  Physiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Growth  of  Cells  in  Tissue  Culture  From  Patients  with  ASH 

Previous  Serial  Number:   NHLI- 158(c) 

Principal  Investigator:   Chester  E.  Clark,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description:   We  have  shown  that  ASH  is  a  genetically  determined 
disease  that  is  transmitted  as  an  autosomal  dominant  trait.   In  an  effort 
to  understand  the  basic  cellular  defect,  we  have  attempted  to  cultivate  cells 
obtained  at  operation  from  the  ventricular  septum  of  patients  with  ASH. 
Initial  efforts  involving  enzymatic  dissociation  of  the  cells  failed.   Sub- 
sequently, we  used  an  explant  technique  that  yielded  a  very  slow  outgrowth 
of  cells.   Morphologically,  these  cells  appear  to  be  large,  bizarre 
fibroblasts.   We  have  not  positively  identified  any  cardiac  muscle  cells. 

At  the  present  time,  we  are  culturing  rat  skeletal-muscle  tumor  cells  in  an 
effort  to  select  mutants  that  are  resistant  to  metabolic  poisons.   Both  8- 
azaguanine  and  6-thioguanine  are  being  used  to  select  cells  that  lack  the 
enzyme  5-PRPP.   Our  intention  is  to  hybridize  these  mutant  skeletal  muscle 
cells  with  cardiac  cells  from  patients  with  ASH  and  study  the  process  of 
myotube  formation  as  it  is  affected  by  the  presence  of  abnormal  genetic 
material  from  the  ASH  cells.   In  this  manner,  we  hope  to  develop  some  in- 
sight into  the  nature  of  the  genetic  defect  in  ASH. 

Keyword  Descriptors:   ASH,  Skeletal  Muscle  Cells,  Hybridization 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards :   None 

Publications:   None 


436 


Project  No.  Z01  HL  01607-02  CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Dynamic  EKG— gated  Scintiangiography 

Previous  Serial  Number:   NHLI-145(c) 

Principal  Investigators:   William  R.  Brody,  M.D. 

Michael  V.  Green,  Ph .D . 

Other  Investigators:  Alan  S.  Pearlman,  M,D. 

Harold  G.  Ostrow,  B.S.E.E. 
Margaret  Douglas,  B.A. 
James  J.  Bailey,  M.D. 
Gerald  S.  Johnston,  M.D. 
Samuel  B.  Itscoitz,  M.D. 
David  R.  Redwood,  M.D. 

Cooperating  Units:   Laboratory  of  Nuclear  Medicine;  Division  of 
Computer  Research  and  Technology 

Project  Description:   Using  intravenous  "9mTc  human  albumin,  an  EKG-gated, 
computer-based,  scintigraphic  imaging  procedure  that  produces  a  sequence 
of  consecutive  10-millisecond  images  to  represent  a  single,  complete, 
average  cardiac  cycle  was  evaluated.   Repetitive  projection  of  this  image 
sequence  as  a  scintigraphic  cineangiogram  permits  visualization  of  the 
dynamic  behavior  of  the  entire  heart  and  associated  vasculature  during 
both  systole  and  diastole  and  can  reveal  defects  such  as  myocardial 
akinesis. 

The  picture  sequence  was  also  investigated  quantitatively  to  directly 
yield  ejection  duration  and  to  estimate  ejection  fraction.   Relative  (and 
potentially  absolute)  measures  of  instantaneous  ventricular  volume  and 
maximum  instantaneous  flow  can  also  be  made. 

Limitations  to  the  quantitative  aspects  of  the  procedure  include  those 
imposed  by  variable  R-R  interval  lengths,  uncertainties  in  the  calculation 
of  ventricular  background  and  various  physical  and  subject-detector 
geometry  considerations. 

The  procedure  offers  several  advantages  over  other  techniques  for 
assessing  ventricular  function.   It  is  non- invasive,  repea table,  may  be 
performed  by  technical  personnel  and  can  yield  information  not  readily 
obtained  by  other  methods.   A  series  of  30  patients  undergoing  routine 
cardiac  catheterization  has  been  studied  by  this  scintigraphic  technique. 


¥27 


Project  No.   ZQ1  HL  01607-02  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Preliminary  results  suggest  an  excellent  correlation  between  parameters  of 
ventricular  function  measured  by  the  two  methods. 

99m 
Keyword  Descriptors:   Intravenous    Tc  Human  Albumin,  Cardiac  Cycle, 

Ejection  Fraction,  Ventricular  Volume 
Proposed  Course  of  Project:   Continuing 
Honors  and  Awards:   None 
Publications:   None 


¥3& 


Project  No.   Z01  HL  01609-02  CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Scintigraphy  in  the  Assessment  of  Coronary  Artery  Disease 

Previous  Serial  Number:   NHLI-144(c) 

Principal  Investigators:   David  R.  Redwood,  M.D. 

Other  Investigators:   Harry  Agress,Jr.,  M.D. 
William  R.  Brody,  M.D. 
Michael  V.  Green,  Ph.D. 
James  J.  Bailey,  M.D. 
Alan  S.  Pearlman,  M.D. 
Samuel  B.  Itscoitz,  M.D. 
Gerald  S.  Johnston,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:     Laboratory  of  Nuclear  Medicine*  NIH 

Project  Description:   The  precise  pathophysiologic  significance  of  subcritical 
coronary  narrowing  is  unknown.   It  is  generally  accepted  that  coronary  lesions 
producing  50%  stenosis  or  less  are  of  no  functional  significance;  hence, 
patients  with  such  lesions  are  not  considered  candidates  for  bypass  surgery. 
Recent  studies  using  a  dual  isotope  technique  to  assess  relative  myocardial 
perfusion,  however,  suggest  that  inadequate  perfusion  after  a  vasodilatory 
stimulus  can  result  from  coronary  lesions  causing  as  little  as  50%  luminal 
narrowing.   These  findings  pose  important  therapeutic  questions.   We  therefore 
are  evaluating  the  relative  significance  of  coronary  stenotic  lesions  of 
varying  severity  by  studying  adequacy  of  regional  myocardial  perfusion  at  rest 
and  at  the  time  of  pacing  induced  angina  by  the  use  of  intracoronary  injections 
of  labeled  macro-aggregated  albumin  (MAA)  and  human  albumin  microspheres  (HAM) . 

Patients  investigated  are  those  who  undergo  diagnostic  cardiac  catheterization 
including  left  ventricular  angiography  and  selective  coronary  arteriography 
for  medical  indications  unrelated  to  this  study.   The  patient's  stress 
threshold  is  then  determined  by  successively  increasing  heart  rate  by  right 
atrial  pacing  until  85%  of  maximum  predicted  heart  rate  or  angina  occurs. 
Pacing  is  then  discontinued  allowing  rapid  resolution  of  angina  and  a  5-10 
min  stabilization  will  ensue.   For  the  resting  scintigram,  either  ""Re- 
labeled HAM  (consisting  of  1-1.5  mCi,  10,000  to  30,000  particles  with 
diameter  10-40  u  suspended  in  5cc  saline)  or  "lj  labeled  MAA  (100  UCi, 
200,000-400,000  particles  with  diameter  of  10-80  u  suspended  in  5cc  saline) is 
injected  into  each  coronary  artery.   Pacing  is  then  initiated  until  the  stress 
threshold  is  attained.   At  this  point,  99mTc-labeled  HAM  or    I  labeled  MAA 
is  injected  into  the  left  coronary  artery  after  which  pacing  is  discontinued. 

1  «3? 


Project  No.   Z01  HL  01609-02  CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Following  a  five-minute  rest  period,  pacing  is  repeated  as  before  and 
-L^I-MAA  injected  into  the  right  coronary  artery  at  the  point  of  maximum 
stress.   Myocardial  images  representing  resting  and  stress  scintigrams  are 
then  obtained  and  compared  to  assess  stress  induced  changes  in  regional 
perfusion.   Finally,  in  order  to  evaluate  the  functional  significance  of 
different  degrees  of  narrowing,  adequacy  of  regional  perfusion  during  pacing 
is  correlated  with  degree  of  coronary  stenosis  as  judged  from  coronary 
angiograms.  Preliminary  results  suggest  an  excellent  correlation  between 
patterns  of  perfusion  abnormality  and  the  degree  and  extent  of  disease  in 
the  coronary  arteries.   In  addition,  there  is  a  correlation  between  these 
perfusion  abnormalities  and  the  segmental  ventricular  wall  motion 
abnormalities  as  seen  on  ventricular  cine-angiography . 

Keyword  Descriptors:   Dual  isotope  technique,  Regional  myocardial  perfusion, 
Cardiac  catheterization,  Selective  coronary  arteriography,  Pacing. 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:  None 

Publications:   None 


**4> 


Project  No.    Z01  HL  01610-02   CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Stress  Myocardial  Imaging  in  the  Evaluation  of  Cardiac  Disease 

Previous  Serial  Number:   NHLI-142(c) 

Principal  Investigator:   Richard  W.  Myers,  M.D. 

Other  Investigators:   Robert  E.  Goldstein,  M.D. 
Gerald  S.  Johnston,  M.D. 
Stephen  E.  Epstein,  M.D. 
David  R.  Redwood,  M.D. 

Cooperating  Units:   Laboratory  of  Nuclear  Medicine,  NIH 

Project  Description:   The  feasibility  of  demonstrating  localized  ischemia  as 
"cold  areas"  in  a  43r  myocardial  scan  obtained  after  graded  exercise  has 
recently  been  reported.   The  underlying  mechanism  of  this  finding  probably 
involved  increased  potassium  turnover  in  ischemic  myocardium  in  addition  to 
decreased  potassium  delivery  by  diseased  vessels.   If  systematic  evaluation 
shows  acceptable  diagnostic  accuracy,  a  wide  application  to  the  assessment 
of  coronary  artery  disease  may  be  expected,  as  this  technique  would  allow 
screening  of  large  numbers  of  susceptible  patients  as  well  as  provide 
useful  information  in  evaluating  patients  for  more  definitive  techniques 
such  as  coronary  angiography. 

In  applying  stress  myocardial  imaging,  however,  consideration  must  be 
given  to  other  cardiac  diseases  that  may  involve  regional  myocardial 
ischemia  and  hence  give  false  positive  results.   Asymmetric  septal  hyper- 
trophy may  be  such  a  condition.   Its  pathologic  picture  is  that  of  non- 
uniform left  ventricular  hypertrophy.   The  frequent  clinical  findings  of 
exertional  chest  pain  and  EKG  evidence  of  ischemia  or  previous  myocardial 
infarction  further  complicate  the  differentiation  of  asymmetric  septal 
hypertrophy  from  coronary  artery  disease. 

The  purpose  of  this  study,  then,  was  first  to  evaluate  the  diagnostic  re- 
liability of  stress  myocardial  scintigraphy  in  patients  with  coronary  artery 
disease,  and  second,  to  describe  localized  abnormalities  in  patients  with 
asymmetric  septal  hypertrophy. 

Each  patient  underwent  myocardial  imaging  on  two  occasions.   At  least  four 
days  (four  half-lives  of  ^^k)  prior  to  exercise  study,  a  baseline  study 
was  performed  after  intravenous  injection  of  0.5  to  1.0  mCi  of  43kc1.   The 
patient  then  performed  a  standard  graded  exercise  protocol.   At  the  end 


W 


Project  No.  Z01  HL  01610-02  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

point  of  the  stress  test  (angina,  fatigue,  dyspnea,  or  reaching  85% 
of  maximum  predicted  heart  rate),  0.5  to  1.0  mCi  of  ^3KC1  was  injected 
intravenously.   The  patient  was  transported  to  the  Nuclear  Medicine 
Laboratory  and  myocardial  imaging  performed  as  in  the  baseline  study. 

A  comparison  of  results  of  the  independent  evaluation  of  resting  and 
stress  myocardial  imaging  to  other  diagnostic  studies  allowed  deter- 
mination of  1)  the  accuracy  of  detection  of  ischemia  secondary  to  coronary 
artery  disease,  and  2)  the  assessment  of  the  character  and  occurrence  of 
defects  in  asymmetric  septal  hypertrophy. 

The  presence  of  myocardial  defects  in  patients  with  coronary  artery  disease 
appeared  relatively  specific  when  compared  to  normal  subjects  (positive 
in  7  of  10  patients  with  coronary  artery  disease;  0  of  5  normal  patients). 
However,  3  of  7  patients  with  asymmetric  septal  hypertrophy  showed  similar 
defects.   The  technique  also  was  relatively  insensitive  in  that  3  of  10 
patients  with  coronary  artery  disease  had  negative  studies. 

43 
Keyword  Descriptors:    K  Myocardial  Scan,  Coronary  Artery  Disease, 

Asymmetric  Septal  Hypertrophy,  Graded  Exercise 
Proposed  Course  of  Project:   Completed 
Honors  and  Awards :   None 
Publications:    None 


f4> 


Project  No.   Z01  HL  01611-02  CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Pre-  and  Postoperative  Exercise  Performance  in  Patients 
with  ASH 

Previous  Serial  Number:   NHLI-163 

Principal  Investigators:   Robert  E.  Goldstein,  M.D. 

David  R.  Redwood,  M.D. 

Other  Investigators:   Samuel  B.  Itscoitz,  M.D. 
Andrew  G.  Morrow,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Clinic  of  Surgery,  NHLI 

Project  Description:   Operative  relief  of  left  ventricular  outflow  tract 
obstruction  due  to  asymmetric  septal  hypertrophy  (ASH)  frequently  produces 
subjective  improvement  in  symptoms.   However,  little  objective  data  have  been 
obtained  concerning  the  effects  of  this  operation  on  exercise  performance. 
Although  the  ability  of  left  ventricular  myotomy  and  myectomy  to  eliminate 
the  left  ventricular  outflow  tract  gradient  is  well  established,  the  ex- 
tent of  compromise  in  exercise  performance  due  to  persisting  myopathy  re- 
mains uncertain.   We  have  therefore  assessed  the  ability  of  patients  to 
perform  treadmill  exercise  by  measuring  exercise  endurance  and  maximal 
oxygen  consumption  before  and  after  left  ventricular  myotomy  and  myectomy. 
To  gain  insight  into  the  effects  of  operation  on  the  hemodynamic  response 
to  exercise,  patients  have  performed  upright  exercise  with  simultaneous 
measurement  of  cardiac  output  and  pulmonary  arterial  and  systemic  arterial 
pressures  and  oxygen  contents.   To  date,  complete  preoperative  and  post- 
operative exercise  evaluation  has  been  performed  in  ten  individuals.   Pre- 
liminary data  indicate  that  substantial  improvement  in  exercise  capacity 
is  observed  after  operation  in  some  but  not  all  individuals.   Insufficient 
data  are  available  at  present  to  relate  the  degree  of  benefit  to  clinical 
or  laboratory  findings. 

Keyword   Descriptors:   Asymmetric  Septal  Hypertrophy,  Myotomy  and  Myectomy, 
Treadmill  Exercise,  Maximal  Oxygen  Consumption,  Cardiac  Output 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards :   None 

Publications:   None 


f43 


Project  No.  Z01  HL  01612-02  CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Left  Ventricular  Function  Using  Roentgen  Videodensitometry 

Previous  Serial  Number:  NHLI-117(c) 

Principal  Investigator:   David  R.  Redwood,  M.D. 

Other  Investigators:   Leonard  E.  Grauer,  M.D. 

William  H.  Schuette,  B.E.E. 
Willard  C.  Whitehouse,  B.S. 
Samuel  B.  Itscoitz,  M.D. 

Cooperating  Units:   Biomedical  Engineering  and  Instrumentation  Branch 

Division  of  Research  Services,  Television  Engineering 
Section,  Clinical  Center 

Project  Description:   Left  ventricular  ejection  fraction,  the  ratio  between 
stroke  volume  and  end-diastolic  volume,  is  widely  used  as  an  index  of 
ventricular  function  in  patients  with  myocardial  and  valvular  heart  disease. 
However,  as  conventionally  determined,  the  calculation  of  end-diastolic  and 
end-systolic  ventricular  volumes  involves  planimetry  of  appropriate  frames  of 
the  left  ventricular  cineangiogram,  which  is  a  time  consuming  and  cumbersome 
undertaking.   Because  of  the  difficulties  of  this  method,  an  automated 
technique  has  been  developed  that  allows  for  the  direct  measurement  of 
ejection  fraction. 

This  technique  determines  the  rate  of  wash-out  of  roentgen  dense  contrast 
material  from  the  left  ventricle  during  cineangiography.   A  densitometer 
placed  over  the  image  of  the  left  ventricle  continuously  measures  the  changes 
in  contrast  density  during  the  cine  run.   The  degree  to  which  the  contrast  is 
cleared  from  the  left  ventricle  with  each  systole  is  a  function  of  the  ejection 
fraction  and  can  be  determined  by  finding  the  number  of  cardiac  cycles  (N) 
necessary  to  wash  out  50%  of  the  contrast.   The  ejection  is  then  equal  to 
l-E~0-693/N  (the  equation  for  an  exponential  curve).   Results  in  ?-9   patients 
using  this  method  show  an  excellent  correlation  with  data  obtained  by 
planimetry  techniques  (r=0.81). 

Keyword  Descriptors:   Ejection  fraction,  Ventricular  volumes. 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards:   None 


*W 


Project  No.       Z01  HL  01612-02   CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Publications:   Measurement  of  Ejection  Fraction  in  Man  by  Videodensitometry. 
Schuette,  W.H.,  Grauer,  L.E.,  Whitehouse,  W.C.,  Itscoitz,  S.B. 
Redwood,  D.R.  Catheterization  and  Cardiovascular  Diagnosis, 
in  Press. 


**ts~ 


Project  No.  Z01  HL  01616-02  CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Effect  of  TNG  during  exercise  in  Valvular  Heart  Disease 


Previous  Serial  Number:   NHLI-119(c) 

Principal  Investigator:   Jeffrey  S.  Borer,  M.D. 

Other  Investigators:   David  R.  Redwood,  M.D. 

Samuel  B.  Itscoitz,  M.D. 
Robert  E.  Goldstein,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   Nitroglycerin  reduces  pulmonary  arterial  and  left 
atrial  pressures  and  pulmonary  vascular  resistance  at  rest  in  patients  with 
mitral  stenosis.   We  have  observed  similar  changes  in  patients  with  aortic 
insufficiency  and  mitral  insufficiency.   Changes  in  cardiac  output  during 
such  studies  ha^e  been  variable.   To  assess  the  potential  clinical  utility 
of  nitroglycerin  in  patients  with  valvular  heart  disease  we  are  determining 
the  effect  of  nitroglycerin  1)  on  treadmill  exercise  capacity  and  2)  on 
the  pulmonary  artery  pressure  and  cardiac  output  response  to  exercise. 
Thus  far,  9  patients  have  been  studied,  most  with  mild  mitral  and  aortic 
valve  lesions.   Of  these,  8  have  had  technically  adequate  evaluations  of 
exercise  tolerance;  all  have  shown  increases  in  exercise  tolerance  during 
nitroglycerin  therapy  as  compared  with  placebo  therapy;  tolerance  had 
increased  an  average  of  20%  after  0.5  mg  sublingual  nitroglycerin;  four 
patients  have  undergone  technically  adequate  evaluation  of  pulmonary  artery 
pressure  and  cardiac  output  response  to  exercise.   In  all  patients,  pulmonary 
artery  pressure  has  been  lower  and  cardiac  output  higher  at  maximal 
exercise  during  nitroglycerin  therapy  than  during  placebo  therapy. 

Our  results  suggest  that  vasodilator  therapy  may  be  a  useful  adjunct  in  the 
pharmacologic  management  of  patients  with  valvular  heart  disease  by  re- 
ducing exertional  symptoms  and  increasing  exercise  tolerance. 

Key  Word  Descriptors:   Treadmill,  Cardiac  Output,  Pulmonary  Artery  Pressure 

Proposed  Course  of  Project:  Continuing 

Honors  and  Awards :   None 

Publications:   None 

1  t<& 


Project  No.  Z01  HL  01633-01  CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Persistent  Paradoxic  Septal  Motion  Following  ASD 
Closure 

Previous  Serial  Number:   None 

Principal  Investigator:   Alan  S.  Pearlman,  M.D. 

Other  Investigators:   Walter  L.  Henry,  M.D. 
Chester  E.  Clark,  M.D. 
Samuel  B.  Itscoitz,  M.D. 
David  R.  Redwood,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   None 

Project  Description:   Previous  echocardiographic  studies  have  shown  per- 
sistent paradoxic  ventricular  septal  motion  in  some  patients  following 
atrial  septal  defect  (ASD)  repair.   While  the  possibility  of  residual  shunt 
has  been  suggested  to  explain  this  observation,  the  mechanism  underlying 
persistent  abnormal  septal  motion  remains  unclear.   Recently,  we  noted  that 
paradoxic  ventricular  septal  motion  is  dependent  upon  the  relative  degree 
of  posterior  displacement  of  the  septum  within  the  total  ventricular  mass 
at  end-diastole.   We  therefore  sought  to  determine  whether  such  a  mechanism 
might  explain  persistent  postoperative  paradoxic  motion.   Nine  patients 
with  ASD  were  studied:   all  had  preoperative  and  postoperative  right  heart 
catheterization  and  echocardiographic  studies.   Preoperatively,  5  patients 
had  paradoxic,  3  flat,  and  1  normal  septal  motion.   Pulmonary : systemic  flow 
ratios  ranged  from  1.6-5.6:1,  and  right  ventricular  diastolic  diameter 
index  (RVDDI)  ranged  from  1.6-3.3  (normal  0.6-1.4).   Postoperatively,  2 
patients  had  paradoxic,  4  flat  and  3  normal  septal  motion.   No  patient  had  a 
residual  shunt.   The  3  patients  with  normal  motion  postoperatively  all  had 
return  of  RVDDI  to  normal.   One  patient  with  flat  motion  preoperatively 
developed  marked  pulmonary  hypertension,  an  increased  RVDDI,  and  paradoxic 
motion  postoperatively;  5  patients  had  only  a  small  decrease  in  RVDDI  and 
abnormal  motion  persisted  postoperatively.   There  was  a  significant  relation 
between  the  intracardiac  position  of  the  septum  at  end-diastole  and  the 
direction  and  magnitude  of  septal  motion  during  systole  in  both  pre- 
operative (r  =  0.80)  and  postoperative  (r  =  0.66)  patients.   We  conclude 
that  persistent  paradoxic  motion  after  ASD  repair  reflects  incomplete 
resolution  of  right  ventricular  dilatation  and  not  necessarily  residual 
shunt. 


¥¥T 


Project  No.   Z01  HL  01633-01  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Key  Word  Descriptors:   Echocardiography,  Atrial  Septal  Defect,  Right 
Ventricular  Dilatation,  Paradoxic  Septal  Motion,   Congenital  Heart  Disease 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:   None 

Publications:   None 


4V0 


Project  No.   Z01  HL  01635-01   CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Scintigraphic  Detection  of  Asymmetric  Septal  Hypertrophy 


Previous  Serial  Number:   None 

Principal  Investigator:   Alan  S.  Pearlman,  M.D. 

Other  Investigators:   Michael  V.  Green,  Ph.D. 
Harry  Agress,  Jr.,  M.D. 
William  R.  Brody,  M.D.,  Ph.D. 
David  R.  Redwood,  M.D. 
Gerald  H.  Johnston,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Laboratory  of  Nuclear  Medicine,  Division  of  Computer 
Research  and  Technology 

Project  Description:   Recently,  we  described  the  use  of  ECG-gated  scinti- 
graphic angiocardiography  for  the  quantitative  analysis  of  left  ventricular 
(LV)  function.   During  these  studies,  it  became  apparent  that  his  technique 
permitted  visualization  of  the  configuration  of  the  ventricular  septum. 
Since  abnormalities  of  LV  function  and  septal  configuration  characterize 
patients  with  asymmetric  septal  hypertrophy  (ASH) ,  we  investigated  the  ability 
of  scintigraphy  to  detect  abnormalities  diagnostic  of  ASH.   Based  on  echo- 
cardiographic  studies,  patients  were  assigned  to  one  of  three  groups:   ASH 
(7  pts) ,  concentric  thickening  (7  pts) ,  and  normal  septum  (7  pts)  .  Following 
peripheral  intravenous  injection  of  10  mCi  of  Tc""m-human  serum  albumin, 
scintigraphic  scans  were  obtained  in  the  left  anterior  oblique  position. 
With  the  aid  of  computer  techniques,  an  "average"  cardiac  cycle  was  generated 
for  each  patient  and  from  it  an  LV  volume  curve.   The  end-diastolic  image, 
volume  curve,  and  derived  ejection  fraction  (EF)  were  examined.   In  6  (86%) 
of  the  patients  with  ASH,  the  right  and  left  septal  surfaces  wery  not  parallel 
at  end-diastole;  in  4  (57%)  of  these  patients,  there  was  a  protrusion  of  the 
septum  high  in  the  LV  outflow  tract.   Five  patients  (71%)  with  ASH  had  an 
unusually  high  EF  (.69  or  greater);  5  (71%)  had  an  increased  atrial 
component  of  the  LV  volume  curve,  suggesting  decreased  LV  compliance.   In 
contrast  to  the  results  of  the  patients  with  ASH,  images  from  all  14  patients 
with  either  a  normal  or  concentrically  thickened  septum  demonstrated 
parallel  right  and  left  septal  surfaces,  and  EF  in  each  patient  was  less  than 
.69.   Three  (21%)  of  these  patients  had  an  increased  atrial  component  of  the 
LV  volume  curve.   We  conclude  that  scintigraphic  scanning  is  a  safe,  non- 
invasive method  for  detecting  the  lack  of  parallelism  of  septal  surfaces, 
high  EF,  and  decreased  LV  compliance  characteristic  of  ASH.   While  less 

l  4±% 


Project  No.  Z01  HL  01635-01  CB 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

sensitive  and  more  complicated  than  echocardiography,  scintigraphy  may  be 
useful  in  the  noninvasive  screening  of  patients  suspected  of  having  ASH  when 
echocardiography  is  technically  unsatisfactory.   In  addition,  since  LV 
geometry  is  usually  distorted  in  patients  with  ASH,  accurate  assessment  of 
EF  by  angiography  or  echocardiography  probably  is  unreliable.   The  technique 
we  have  described,  however,  allows  measurement  of  EF  without  recourse  to  any 
assymptions  about  LV  geometry  and  thus  may  be  helpful  in  defining  disease 
progression,  and  in  assessing  the  effects  of  therapeutic  interventions. 

Keyword  Descriptors:   Radioisotope  Imaging,  Asymmetric  Septal  Hypertrophy, 
Compliance  Cardiomyopathy,  Noninvasive  Techniques,  IHSS 

Proposed  Course  of  Project:   Continuing 

Honors  and  Awards:   None 

Publications:   None. 


*& 


Project  No.   Z01  HL  01638-01  CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   The  Interventricular  Septum  in  ASH 

Previous  Serial  Number:   None 

Principal  Investigator:   David  R.  Redwood,  M.D. 

Other  Investigators:   William  C.  Roberts,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:     Section  on  Pathology,  Division  of  Intramural  Research. 

Project  Description:   A  previous  angiographic  study  from  our  laboratory 
suggested  that  the  configuration  of  the  ventricular  septum  in  patients  with 
asymmetric  septal  hypertrophy  (ASH)  and  left  ventricular  outflow 
obstruction  (obstructive  ASH)  was  different  from  that  of  patients  without 
obstruction  (nonobstructive  ASH).   To  confirm  and  extend  these  findings,  a 
necropsy  study  of  the  septum  has  been  carried  out  in  18  patients  with  docu- 
mented ASH.   The  septum  was  cut  parallel  to  the  long  axis  of  the  heart  in 
a  plane  lying  between  the  right  coronary  and  non-coronary  aortic  leaflets. 
In  nonobstructive  ASH,  the  membranous  septum  lay  at  the  apex  of  a  triangu- 
lar muscular  septum,  the  sides  of  which  diverged  inferiorly  to  the  right 
and  left.   In  contrast,  the  membranous  septum  in  obstructive  ASH  joined 
the  muscular  septum  in  a  line  continuous  with  the  right  endocardial  border 
of  the  septum,  while  the  left  septal  border  was  convex  and  encroached  into 
the  left  ventricular  outflow  tract.   Thus,  the  septum  achieved  its  maximal 
width  more  cephalad  and  the  ratio  of  upper  to  lower  septal  width  was 
greater  (1.1+.02  vs.  .85+. 02,  p<.005)  in  obstructive  than  in  nonobstructive 
ASH.   There  was  no  difference  between  obstructive  and  nonobstructive  ASH  in 
septal  length  nor  in  septal  width  at  the  septal  equator.   This  study  is  con- 
sistent with  the  results  of  angiographic  studies  and  suggests  that 
differences  in  septal  width  and  contour  may  be  causally  related  to  the 
genesis  of  obstruction  to  left  ventricular  outflow  in  ASH. 

Keyword  Descriptors:   Asymmetric  Septal  Hypertrophy,  Ventricular  Septum, 
Left  Ventricular  Outflow  Obstruction 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   None 


fCf 


Project  No.   Z01  HL  01640-02  CB 

1.  Cardiology 

2.  Cardiovascular  Diagnosis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Limitations  of  the  Electrocardiographic  Response  to  Exercise 
in  Predicting  Coronary  Artery  Disease 

Previous  Serial  Number:   NHLI-118(c) 

Principal  Investigator:   Jeffrey  S.  Borer,  M.D. 

Other  Investigators:   John  F.  Brensike,  M.D. 
David  R.  Redwood,  M.D. 
Samuel  B.  Itscoitz,  M.D. 
Eugene  R.  Passamani,  M.D. 
Neil  J.  Stone,  M.D. 
John  M.  Richardson,  M.D. 
Robert  I.  Levy,  M.D. 
Stephen  E.  Epstein,  M.D. 

Cooperating  Units:   Molecular  Disease  Branch,  NHLI 

Division  of  Heart  and  Vascular  Disease,  NHLI 

Project  Description:   The  electrocardiographic  (ECG)  response  to  graded 
bicycle  exercise  (to  85%  of  predicted  maximal  heart  rate)  was  evaluated  prior 
to  coronary  angiography  in  89  patients,  aged  21  to  55  years,  with  type  II 
hyperlipoproteinemia.   Patients  were  studied  if  they  had  a)  a  history  of  myo- 
cardial infarction  and/or  typical  angina  (43  patients),  b)  "atypical  angina" 
(16  patients),  or  c)  a  positive  ECG  response  during  or  shortly  after  exercise, 
but  were  otherwise  without  evidence  of  cardiac  disease  (30  patients)   Of  the 
43  Group  A  patients,  39  had  _>50%  stenosis;  however,  26  (67%)  of  these  39  had 
negative  ECG  tests,  including  9  of  17  patients  with  >50%  stenosis  in  each  of  3 
vessels.   Of  the  16  Group  B  patients,  5  had  >50%  stenosis,  3  with  positive 
ECG  tests;  1  patient  had  a  positive  test  but  normal  arteriogram.   Of  the  30 
Group  C  patients,  11  (37%)  had  >50%  stenosis;  however,  9  (30%)  had  minor  steno- 
ses (<^50%)  and  10  (33%)  had  entirely  normal  coronary  arteries.   Therefore, 
exercise  electrocardiography  results  in  a  high  frequency  of  both  false 
negative  responses  (in  patients  with  clinically  suspected  coronary  disease)  and 
false  positive  responses  (in  asymptomatic  patients) .   We  conclude  that  while 
exercise  electrocardiography  may  be  of  value  in  epidemiologic  studies,  its 
applicability  as  a  diagnostic  tool  in  the  individual  patient  has  marked 
limitations. 

Keyword  Descriptors:   ECG,  Exercise,  Type  II  Hyperlipoproteinemia,  Angina, 
False  Negative  Responses,  False  Positive  Responses 


4& 


Project  No.    Z01  HL  01640-02  CB 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Proposed  Course  of  Project:   Completed 

Honors  and  Awards :   None 

Publications:   Manuscript  submitted  to  the  NEW  ENGLAND  JOURNAL  OF  MEDICINE 


4ST2 


Project   No.      Z01   HL   01644-04   CB 

1.  Cardiology 

2.  Molecular  Cardiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Phosphorylation  of  Myosin  from  Muscle  and  Non-muscle  Sources 

Previous  Serial  Number:   NHLI-18 

Principal  Investigator:   Robert  S.  Adelstein,  M.D. 

James  Daniel,  Ph.D. 
Mary  Anne  Conti,  B.S. 

Other  Investigators:   William  Anderson, Jr. ,  B.A. 

Cooperating  Units:   None 

Project  Description:   A  protein  kinase  with  a  molecular  weight  of  approxi- 
mately 80,000  daltons  has  been  isolated  from  human  platelets.   This  kinase 
catalyzes  the  phosphorylation  of  the  20,000  dalton  light  chain  of  platelet 
myosin  as  well  as  the  20,000  dalton  light  chain  of  mouse  fibroblast,  chicken 
gizzard  and  a  myosin  isolated  from  a  muscle  tumor.   Normal  human  skeletal  and 
rabbit  skeletal  myosin  are  not  phosphorylated  by  the  enzyme  but  inhibit  the 
enzymes  ability  to  phosphorylate  platelet  myosin  light  chain. 

The  effect  of  phosphorylation  on  platelet  myosin  is  to  increase  5-8  fold)  the 
actin-activated  ATPase  activity  measured  in  the  presence  of  Mg   at  low 
ionic  strength.   It  has  no  effect  on  the  platelet  myosin  ATPase  activity 
measured  in  the  presence  of  Ca   and  K+  -  EDTA  at  high  ionic  strength. 
Dephosphorylation  of  previously  phosphorylated  platelet  myosin  resulted  in  a 
decrease  in  the  actin-activated  ATPase  activity  without  alteration  of  the 
Ca^and  K+  -  EDTA  activated  myosin  ATPase  activity. 

Keyword  Descriptors:   Platelet  myosin;  non-muscle  myosin  phosphorylation; 
kinase;  actin-activation. 

Proposed  Course  of  Project:   The  mechanism  by  which  phosphorylation  of 
platelet  myosin  results  in  actin-activation  will  be  studied.   The  possibility 
that  phosphorylation  leads  to  actin-activation  in  other  non-muscle  cells  such 
as  fibroblasts  as  well  as  smooth  muscle  myosin,  will  be  investigated. 

Evidence  that  phosphorylation  of  platelet  myosin  plays  a  role  in  the  platelet 
release  reaction  will  be  studied  by  looking  for  increased  incorporation  of 
-^Pi  into  platelet  light  chain  following  treatment  of  human  platelets  with 
thrombin  or  ADP.   Recent  results  in  this  laboratory  by  Dr.  Daniel  suggest  that 
this  may  be  the  case. 

Since  the  effect  of  phosphorylation  appears  to  be  reversible,  the  phosphatase 

1  t4£*l 


Project  No.  Z01  HL  01644-04  CB 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

responsible  for  dephosphorylation  will  be  investigated  and  isolated  in  human 
blood  platelets. 

Honors  and  Awards:   None 

Publications:   Articles  published: 

1)  Adelstein,  R.S.,  Conti,  M.A. ,  Daniel,  J.L.  and  Anderson, Jr . ,W. 

The  Interaction  of  Platelet  Actin,  Myosin  and  Myosin  Light  Chain  Kinase - 
(1975) ,  Ciba  Foundation  Symposium  on  the  Biochemistry  and  Pharmacology  of 
Blood  Platelets,  New  Series  (In  press). 

2)  Adelstein,  R.S.,  Daniel,  J.L.,  Conti,  M.A.  and  Anderson, Jr . ,W. 
Platelet  Myosin  Phosphorylation:  Studies  on  the  Kinase  Substrate  and  Effect 
of  Phosphorylation.   Proceedings  of  the  9th  FEBS  Meeting  (1974,  in  press). 


vsr- 


Project   No.    Z01   HL  01645-02   CB 

1.  Cardiology 

2.  Molecular  Cardiology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Contractile  Proteins  from  Adult,  Embryonic  and  Malignant  Cells 

Previous  Serial  Number:   NHLI-164 

Principal  Investigator:   Robert  S.  Adelstein,  M.D. 

Mary  Anne  Conti,  B.S. 

Other  Investigators:   William  Anderson,  Jr.,  B.A. 
Donald  Henson,  M.D. 
John  Ziegler,  M.D. 
Marshall  Elzinga,  Ph.D. 
Guilio  Cantoni,  Ph.D. 

Cooperating  Units:   Biomedical  Research  Institute,  Boston 
NCI 

NICHHD 
NIMH 

Project  Description:   Non-muscle  myosin,  characterized  by  a  unique  set  of 
light  chains  compared  to  skeletal  muscle  and  cardiac  myosin  is  thought  to 
play  a  role  in  cell  division,  cell  secretion,  cell  motility  and  specialized 
cell  function  such  as  clot  retraction  in  platelets  and  phagocytosis  in 
white  cells.   In  order  to  elucidate  such  roles  we  have  embarked  on  a  study  of 
myosin  in  a)  myoblasts  before  and  after  fusion,  b)  muscle  tumors  such  as 
rhabdomyosarcoma,  as  well  as  non-muscle  tumors. 

The  rationale  for  these  experiments  is  the  concept  that  myoblasts  prior  to 
fusion  and  muscle  tumor  cells  having  undergone  dedif f erentiation,  would  both 
contain  a  cytoplasmic  type  myosin,  similar  to  that  found  in  fibroblasts  and 
platelets.   Evidence  for  such  a  myosin  would  be  the  presence  of  the  unusual 
light  chains  (20,000  and  15,000  daltons)  associated  with  "non-muscle"  myosin 
and  the  ability  of  the  20,000  dalton  light  chain  to  be  phosphorylated  by  the 
kinase  from  platelets  which  does  not  phosphorylate  skeletal  muscle  or  cardiac 
myosin.   Initial  experiments  in  this  laboratory  with  myoblasts  prior  to  fusion 
and  rhabdomyosarcoma  cells  removed  as  operative  specimens  and  grown  in  vitro 
have  supported  the  hypothesis.   The  molecular  weight  of  the  light  chains  of 
myoblast  myosin  prior  to  fusion  was  found  to  be  20,000  and  15,000  daltons, 
and  after  fusion  to  be  25,000,  20,000  and  18,500  (similar  experiments  with 
comparable  findings  were  carried  out  in  Dr.  Howard  Holtzer's  laboratory  at 
the  University  of  Pennsylvania  in  Philadelphia) . 

Rhabdomyosarcoma  cells  from  operative  specimens  were  found  to  contain  a 
mixture  of  light  chains  including  the  20,000  and  15,000  dalton  light  chains. 

l  ¥St 


Project  No.  Z01  HL  01645-02  CB 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Dr.  Marshall  Elzinga  in  Boston  has  been  sequencing  two  actins  prepared  in 
this  laboratory:   a)  actin  prepared  from  human  platelets  and  b)  actin 
prepared  from  human  hearts  (autopsy  specimens) .   The  purpose  of  this  work  is 
to  uncover  any  differences  between  muscle  and  non-muscle  actin.   Two  cyanogen 
bromide  peptides  of  human  platelet  actin  comprising  20  residues  have  been 
found  to  have  the  exact  sequence  as  rabbit  skeletal  muscle  myosin.   One 
peptide  of  9  residues  has  a  single  amino  acid  substitution  (threonine  for 
valine) . 

Keyword  Descriptors:   Myosin  light  chains;  cell  proliferation;  cell  division; 
cell  motility;  atherosclerosis;  rhabdomyosarcoma. 

Proposed  Course  of  Project:   Myosin  and  actin  from  a  number  of  malignant  and 
non-malignant  but  actively  proliferating  cells  (muscle  and  non-muscle)  will 
be  compared  with  regard  to  structure,  function  and  antigenic  decerminants 
with  myosin  from  cells  not  undergoing  active  cell  division.   Of  particular 
interest  (in  addition  to  the  cells  mentioned  above)  will  be  the  isolation  and 
characterization  of  myosin  from  the  media  of  aortic  cells  undergoing 
proliferation  which  is  thought  to  be  involved  in  the  genesis  of  atheroscle- 
rosis. 

Actin  and  myosin  will  be  characterized  from  cells  that  have  been  found  to  be 
abnormal  in  cell  motility  in  an  effort  to  uncover  the  cause  of  this  abnormal- 
ity. 

Honors  and  Awards :   None 

Publications:   Ostlund,  R.E.,  Pastan,  I.  and  Adelstein,  R.S.:   Myosin  in 
cultured  fibroblasts.   J.  of  Biol.  Chem  249:   3903-3907,  1974. 

Kuehl,  W.M.,  Conti,  M.A.  and  Adelstein,  R.S.:   Structural  studies  on  rabbit 
skeletal  muscle  actin:   Ordering  of  the  peptides  produced  by  cleavage  with 
cyanogen  bromide.   J.  of  Biol.  Chem.  250,  1975  (in  press). 


*S7 


ANNUAL   REPORT  OF  THE 
HYPERTENSION-ENDOCRINE   BRANCH 
NATIONAL  HEART  AND  LUNG   INSTITUTE 
July  1,  1974  throunh  June  30,  1975 

The  activities  of  the  Hypertension-Endocrine  Branch  have  been  primarily 
devoted  to  studies  of  the  chemistry> physiology  and  clinical   applications  of 
systems  known  or  thought  to  exert  controlling  influence  on  blood  pressure; 
these  include  especially  1)  the  autonomic  nervous  system,  2)  the  renin-angio- 
tensin system  3)  the  prostaglandin  system,  and  4)  the  kallikrein-kinin  system. 
In  addition,  non-related  studies  have  concerned  the  agents  responsible  for 
bronchial   asthma,   the  role  of  cyclic  AMP  in  parathyroid  function,  and  the 
chemical   mediators  of  neural   transmission  and  of  taste  sensation. 

1)  Studies  on  the  autonomic  nervous  system  have  included  studies  in 
enzymes   in  tissues,   physiologic  studies   in  animals,  and  clinical   studies  in  man. 
The  first  and  limiting  step  in  biogenesis  of  catecholamines  is  hydroxylation  of 
tyrosine  by  tyrosine  hydroxylase.     This  is  a  mixed  function  oxygenase 
requiring  tetrahydrobiopterin  as  its  natural   cofactor.     We  have  shown  that 
this  enzyme  functions  at  about  1%  of  its  total   potential   in  rat  corpus  striatum 
in  vivo  and  that  this  almost  complete  inhibition  depends  upon  end-Droduct  in- 
hibition by  dopamine  as  well   as  limitation  of  quanities  of  tetrahydrobiopterin 
in  brain  tissue.     Neuroleptic  drugs  and  lesions  in  the  nigro-striatal   area 
greatly  increase  the  rate  of  tyrosine  hydroxylation  in  vivo  without  change  in 
the  concentration  of  cofactor  or  of  dopamine.     It  was  shown  that  the  affinity 
of  tyrosine  hydroxylase  for  cofactor  was  increased  by  these  interventions.     It 
was  shown  by  chromatography  of  the  active  enzyme  on  G-25  Sephadex  that  the 
alteration  in  affinity  results  in  turn  from  a  change  in  macromolecular 
structure.     It  was  further  shown  that  the  activation  of  the  enzyme  depends 
in  turn  upon  cAMP-dependendent  protein  phosphorylation;  such  activation  is 
totally  dependent  upon  a  source  of  ATP  and  partially  dependent  upon 
cAMP  and  Mg     .      It  has  not  been  demonstrated  thus  far  that  phosphate  is 
incorporated  in  the  enzyme  under  these  conditions;  however,  since  conditions 
inducive  to  phosphorylation  increase  activity  of  tyrosine  hydroxylase  8-  to 
10-fold  in  the  presence  of  physioloaic  concentrations  of  dopamine  and  reduced 
pterin,  a  phosphorylated  intermediate  probably  exists.     Results  suggests 
that  such  phosphorylation  of  enzyme  is  important  for  the  normal   regulation  of 
neurotransmitter  synthesis.     These  studies  provide  a  molecular  explanation  of 
the  action  of  neuroleptic  drugs. 

Tyrosine  hydroxylase  production  is  regulated  by  trans-synaptic  induction, 
and  appears  to  depend  specifically  on  the  ratio  of  cAMP  to  cGMP.     A  number  of 
pharmacologic  agents  have  been  shown  to  change  this  ratio.     The  induction  of 
tyrosine  hydroxylase  in  autonomic  ganglia  is  stimulated  by  carbonydrate-active 
steroids  and  the  receptors  for  these  have  been  studied.     Induction  by 
dexamethasone,  for  example,   1s  blocked  by  11-desoxycortisol  which  has  affinity 
for  the  same  receptors.     It  was  found  also  that  beta  receptor    agonists    can 
induce     the  enzyme  when  steroid  medium  is  kept  constant. 

Dopamine-beta-hydroxylase   (DBH)   is  the  final   enzyme  in  the  biosynthetic 
pathway  of  norepinephrine.     It  was  previously  shown  in  this  laboratory  that 
this  molecule  consists  of  4  subunits,  each  with  a  molecular  weight  of  75,000 

*S1 


daltons,  and  that  the  enzyme  is  a  glycoprotein  containing  about  5%  carbohydrate. 
Since  the  protein  is  fully  active  when  bound  to  the  lectin  concanavalin  A, 
but  not  when  bound  to  antibody,  it  appears  that  the  carbohydrate  is  remote 
from  the  active  site  and  may  serve  to  orient  the  enzyme  in  the  vesicular 
membrane.  Peptide  maps  of  DBH  following  cleavage  with  cyanogen  bromide  show 

11  major  peptides.  The  4  subunits  are  probably  identical,  each  containing 

12  methionines.  These  amino  acid  analyses  of  DBH  differ  significantly  from 
those  in  the  literature. 

DBH  is  a  copper-containing  mixed  function  oxygenase  which  normally 
accepts  electrons  from  ascorbate  with  a  redox  potential  +200mV.  A  major 
advance  has  been  the  development  of  atell-free  wheat  germ  system  which  will 
measure  the  specific  translation  of  DBH  from  adrenal  polysomes,  thus  enabling 
the  study  of  DBH  synthesis  in  vitro. 

DBH  is  released  during  sympathetic  nerve  transmission  by  exocytosis,  and 
circulates  as  subunits  of  molecular  weight  75,000.  As  the  release  of  DBH  by 
monocytosis  from  synaptic  vesicles  always  parallels  the  release  of  norepine- 
phrine from  the  same  site,  circulating  DBH  has  been  studied  as  an  index  of 
norepinephrine  release.  The  cerebrospinal  fluid  was  examined  for  DBH  with 
the  use  of  a  radioimmunoassay;  the  enzyme  is  undetectable  in  CSF. 

Plasma  DBH  is  generally  high  in  pheochromocytoma;  following  successful 
surgical  intervention,  plasma  concentrations  decrease  with  a  half  life  of 
8  hours.  Further  clinical  studies  with  DBH  show  a  small,  insignificant  increase 
in  circulating  DBH  when  normal  subjects  assume  the  upright  posture;  this  paral- 
lels similar  chanqes  in  circulating  norepinephrine  and  suggest  that  neither 
measure  provides  the  index  to  the  activation  to  the  adrenergic  nervous  system 
withstanding.  The  circadian  variation  in  plasma  DBH  with  normal  activity 
showed  small,  statistically  significant  changes  of  5%  of  the  mean  which  are 
physiologically  of  no  importance.  Plasma  DBH  can  be  depressed  in  normal  man  by 
rapid  infusion  of  albumin  (the  depression  exceeding  the  dilution  effect).  In 
hypertensive  subjects,  excretion  of  epinephrine  or  norepinephrine  on  a  low- 
sodium  diet  was  significantly  greater  than  that  by  normals;  studies  are  underway 
to  measure  the  circadian  variations  of  these  pressors  in  normals  and  hyper- 
tensives. 

The  role  of  nerve  impulses  in  blood  pressure  control  was  studied  by 
measurement  of  incorporation  of  lysine  into  non-collagen  protein  of  blood 
vessel  walls  in  spontaneously  hypertensive  rats.  Such  incorporation  is 
excessive  in  these  rats  as  compared  to  normal  ones.  Whereas  this  augmented 
incorporation  could  not  be  inhibited  by  control  of  blood  pressure  with  the 
vascular  dilator  apresoline,  it  could  be  prevented  by  ganglionic  blockade  with 
hexamethonium.  These  results  suggest  that  nerve  impulses  and  not  high  pressure 
alone  are  necessary  for  the  increased  vascular  smooth  muscle  synthesis  which 
occurs  with  elevated  blood  pressure. 

2)  Control  of  renin  release  was  studied  in  animal  models  and  in  man.  Jj^ 
vitro  cultured  endothelial  cells  were  found  to  produce  an  enzyme  which  can 
produce  angiotensin  II  from  renin'  substrate,  and  also  from  anqiotensin  I. 
Studies  are  underway  to  purify  the  enzymes  involved,  which  are  different  from 
known  converting  enzyme,  and  which  may  provide  an  alternative  pathway  for 
release  of  angiotensin  II.  The  mechanisms  for  the  increase  secretion  of  renin 

4lo 


following  sinoaortic  denervation  and  vagotomy  were  further  pursued.  It  is 
apparent  that  vagal  afferent  impulses  tonically  inhibit  autonomic  efferent 
impulses  to  the  kidneys  and  the  blood  vessels,  and  that  such  efferent  impulses 
control  renin  release.  Extensive  studies  were  begun  on  the  syndrome  of  low 
renin  hypertension  to  elucidate  the  mechanism  for  renin  suppression;  these  are 
described  below.  > 

3)  Prostaglandins:  Because  prostaglandins  of  the  E  series  (PGEs)  are 
vasodilators,  we  have  studied  these  agents  to  determine  whether  deficiencies 
in  their  production  may  lead  to  hypertension  (PGFs  are  pressor  agents  which 
might  lead  to  hypertension  directly).  We  have  studied  them  by  their  adminis- 
tration to  animals  and  by  the  inhibition  of  prostaglandin  synthetase  in 
animals  and  in  nan. 

Chemically,  the  prostaglandins  are  formed  intracellular^'  from  fatty 
acid  precursors;  for  example,  arachidonic  acid  released  by  a  phospholipase  from 
the  plasma  membrane  is  converted  by  microsomes  to  the  potent  PGGo  and  PGH~ 
which  are  in  turn  rapidly  reduced  to  the  relatively  weak  prostaglandins  PGF2 
and  PGEp.  We  have  developed  radioimmunoassays  which  will  measure  PGE  in    ** 
female  urine  (results  in  male  urine  depend  largely  upon  the  contribution  of 
the  reproductive  tract).  We  have  also  measured  PGA,,,  produced  by  dehydration 
of  PGE2.  c 

We  have  discovered  two  new  prostaglandins  formed  by  addition  of 
glutathione  (GSH)  to  carbon-11  of  PGA,  and  by  enzymatic  reduction  of  the  9-keto 
group  in  PGA-11-GSN.  This  conjugate  is  found  in  human  female  urine  and 
constitutes  the  first  clear  evidence  that  PGA  may  be  produced  in  substantial 
quantities  in  the  human  kidney. 

It  has  been  established  from  previous  work  in  this  Branch  that  PGE  infused 
into  the  renal  artery  increases  sodium  excretion  and  increases  free  water 
clearance,  effects  attributed  to  a  decrease  of  sodium  reabosrption  in  the 
proximal  tubule,  possibly  related  to  the  vasodilator  effects  of  PGE.  We  have 
shown  also  that  when  indomethacin ,  an  inhibitor  of  prostaglandin  synthetase, 
is  infused  into  the  renal  artery,  this  causes  an  increase  in  sodium  excretion 
and  a  decrease  in  free  water  clearance.  These  effects  are  attributed  to  a 
decrease  by  indomethacin  of  sodium  reabsorption  in  medullary  areas,  where 
sodium  reabsorption  increases  free  formation.  This  indicates  that  prosta- 
glandins themselves  stimulate  sodium  reabsorption  in  medullary  areas. 

It  was  found  that  PGE  excretion  is  less  by  the  kidney  in  which  renal 
artery  stenosis  has  been  produced  experimentally  than  by  the  normal  kidney. 

Clinical  studies  with  prostaglandins  have  shown  that  assumption  of  the 
upright  posture  results  in  an  increase  in  urinary  PGE  of  about  ?0%.  In  patients 
with  renal  artery  stenosis,  excretion  of  PGE  on  the  side  of  the  stenosis  is 
lower  than  that  on  the  normal  side. 

It  was  found  that  infusion  of  PGE  into  the  carotid  artery  stimulates 
secretion  of  vasopressin;  in  the  water-loaded  dog,  this  results  in  decrease 
in  free  water  excretion.  Thus  PGE  is  another  substance  capable  of  releasing 
vasopressin;  studies  are  underway  to  determine  its  role  in  physiological 
control  of  ADH. 


4)   Kinins:     Plasma  pre- kail ik re in  is  a  circulating  protein  which  may  be 
converted  by  activated  Hageman  factor  or  other  activators  to  plasma  kallikrein 
whose  molecular  weight  is  about  100,000.     There  are  3  circulatina  inhibitors 
of  plasma  kallikrein. 

Kallikrein  can  liberate  bradykinin,    and  lysylhradykinin  (kallidin)   from 
circulating  kininogen.     Plasmin,  which  can  be  liberated  from  circulating 
plasminogen,  also  has  affinity  for  the  kininogens  and  can  release  kinins  from 
them.     Since  bradykinin   is  a  potent  vasodilator,  this  system  is  under  study  to 
determine  whether  elevation  of  blood  pressure  might  depend  in  part  upon  a 
deficiency  of  kinins. 

Human  urine  also  contains  kallikrein  which  is  different  from  circulating 
kallikrein,  and  whose  function  is  unknown.     We  have  shown  earlier  that  its 
excretion  increases  with  sodium-retaining  steroids  and  in  primary  aldosteronism 
and  is  decreased  in  idiopathic  hypertension.     Recent  evidence  suggests  that 
kidney  kallikrein  may  enter  the  blood  pari   passu  with  its  entry  into  urine. 

Two  highly  purified  kininogens  B2  and  B*B,  major  components  of  the  high 
and  low  molecular  weight  kininogens,  respectively,  have  been  prepared  and 
subjected  to  kinetic  studies.     Human  plasma  kallikrein  has  a  much  higher 
affinity  for  B4B,  whereas  urinary  kallikrein  has  a  higher  affinity  for  B?. 
B2  was  shov/n  to  correct  the  coagulation  defect  known  as  Flaujeac,  Williams, 
or  Fitzgerald  trait;   B4B  does  not. 

Human  urinary  kallikrein  was  further  studied.     After  treatment  with 
diisopropylfluorophosphate   (DFP)   it  can  no  longer  liberate  kinin  from  kininogen 
Type   II  which  contains  the  kinin  moiety  within  the  polypeptide  chain,  but  can 
still   release  kinin  from  type   I   in  which  the  kinin  is  at  the  C-terminal   end. 
These  results  suqgests  that  human  urinary  kallikrein  contains  two  catalytic 
sites,  and  further  that  HUK  is  unique  among  kallikreins   in  lacking  a  serine 
residue  at  the  active  site  which  cleaves  the  methionyl-lysyl   bradykinin  as  well 
as  lysylbradykinin   (Kallidin),  and  bradykinin.     As  urinary  kallikrein  will   not 
produce  methyl -lysyl bradyki nin ,  the  action  of  another  enzyme  such  as  uropepsin- 
ogen  in  the  kidney  is  probably  responsible  for  each  cleavage  from  kininogen. 

t     ^   In  clinical    studies  on  human  urinary  kallikrein  it  was  found  that  potassiui 
loads  which  stimulate  aldosterone  secretion  also  stimulate  the  release  of 
kallikrein  from  the  kidneys.     It  was  found  that  urinary  kinin  excretion   is 
significantly  correlated  with  urinary  kallikrein  excretion,  sugnestina  an 
action  of  urinary  kallikrein  on  substrate  within  the  kidney.     The  quantity  of 
methyl-lysylbradykinin   in  urine  may  exceed  that  of  lysylbradykinin  or  of 
bradykinin;  women  excrete  only  102  as  much  bradykinin  as  men.     It  has  also 
rn^oiTc   thll  ^V^ete  less  kallikrein  than  whites,  a  finding  which 
correlates  with  the  relative  incidence  of  hyoertension  in  the  two  nroups. 
Urinary  kallikrein  is  lower  in  the  urine  from  the  stenotic  kidney  in  patients 
rlfl  mllLjI  ?ry  Ste^S1T'  Pr?du?tion  of  renal   artery  stenosis  in  dogs  and 

r  i     J6"-   th°  kSl   lkrein  excretion  from  the  treated  side.     Changes 

flow    lu?l\ '  1     Tn  are.related  in  these  experiments  to  chanoes  in  renal   blocf 
now,  but  not  to  chances   in  urine  volume  or  in  urinary  sodium  and  potassium. 


**> 


Clinical   studies:     With  the  Clinical  Pharmacology  Section  of  the  Clinical 
Pharmacology  Branch,  we  have  instituted  a  screening  program  to  define  the 
characteristics  of  patients  with  hypertension  and  to  study  the  pharmacology 
and  pharmacologic  kinetics  of  blood  pressure-lowering  agents..    Hypertensives 
are  classified  according  to  salt  sensitivity  and  renin  secretion.     Patients 
in  each  group  are  studied  for  the  presence  of  sodium-retaining  steroids  other 
than  aldosterone  which  might  explain  the  syndrome.     This  involves  the  production 
of  tracer  steroids  and  determination  of  secretion  rates.     Two  patients  with   low 
renin  hypertension  were  treated  with  aminoglutetimide   ,  an  inhibitor  of  adrenal 
steroid  synthesis,  with  a  resultant  fall  of  20-30  mmHg  in  systolic  and  diastolic 
blood  pressure  after  10  days.     The  steroid  patterns  are  being  determined  again 
during  suppression.     Other  natients  were  subjected  to  suppression  of  steroid 
action  with  aldactone  (spironolactone).     This  has  proved  successful   therapy  of 
hyperaldosteronism  and  of  diagnostic  value  to  determine  dependency  of  the 
hypertension  upon  sodium-retaining  steroids. 

In  view  of  the  gynecomastia  and  loss  of  libido  which  commonly  accompany 
treatment  with  spironolactone,  we  have  studied  the  effects  of  this  agent.     In 
studies  carried  on  with  the  Laboratory  of  Chemical  Pharmacology,  we  have 
shown  that  spironolactone  destroys  microsomal   cytochrome  P450  in  the  testis, 
and  thus  lowers  17-alpha-hydroxylase  activity  required  for  the  production  of 
17-alpha-hydroxyprogesterone,  androstenedione,  and  testosterone. 

A  comprehensive  in-patient  clinical   study  was  carried  out  with  the  dual 
objective  of  defining  the  accompaniments  of  hypertension,  and  of  studying  the 
circadian  interrelations  of  blood  pressure  and  the  various  factor  with  known 
or  suspected  relationship  to  blood  pressure.     Of  fourteen  hypertensive  subjects 
studied  on  7-day  periods  of  low,  normal   and  high  sodium  intake,  9  v/ere  found 
to  have  striking  increases  of  systolic  blood  pressure  with  increase  of  sodium 
intake.     In  the  data  from  the  non-salt-sensitive  hypertensive  subjects  and 
those  from  the  normal   subjects,  compared  with  those  from  the  salt-sensitive 
hypertensive  patients,   it  was  found  that  the  salt-sensitive  hypertensive 
patients  had  higher  17-hydroxycorticosteroid  excretion  on  all   three  sodium 
intakes.     The  factors  responsible  for  this  salt  sensitivity  are  under 
investigation.     The  circadian  variations  of  17-hydroxycorticosteroids  and  of 
aldosterone  excretion  showed  the  same  peaks  and  troughs  on  all   three  intakes; 
the  amplitude  of  the  variations  of  blood  pressure  were  higher  the  higher  the 
salt  intake.     Plasma  renin  activity  in  both  groups  of  hypertensive  patients 
was  higher  than  normal   at  all  times  of  day  on  the  low-sodium  intake.     All   of 
14  patients  showed  circadian  variations  in  plasma  prekallikrein,  with  peaks  at 
4am  and  Sam  on  the  9  and  109  mEq  sodium  intake.     Two  patients  with  primary 
aldosteronism  had  higher  values  for  plasma  prekallikrein  at  all   times  of  day 
than  did  the  patients  with  "essential"   hypertension. 

Studies  of  the  function  of  the  adrenergic  nervous  system  in  hypertension 
revealed  that  when  hypertensive  patients  are  given  low-sodium  intake  they 
excrete  more  sodium  and  "norepinephrine  plus  epinephrine",  than  do  normal 
subjects  on  the  same  regimen.     The  greater  than  normal   excretion  of  norepine- 
phrine and  epinephrine  suggest  the  presence  of  hyperresponsive  adrenergic 
nervous  systems  in  these  hypertensive  patients. 

Patients  with  the  syndrome  of  juxtaglomerular  hyperplasia,  hypokalemia, 
alkalosis,  aldosteronism,  and  elevated  plasma  renin  activity     paradoxically 

*Z3 


show  normal  blood  pressure  even  with  expansion  of  vascular  volume.  We  have 
found  that  "basal"  prostaglandin  excretion  in  patients  with  this  syndrome  were 
hinher  than  normal.  Two  such  patients  were  treated  with  indomethacin,  an 
inhibitor  of  prostaolandin  synthetase,  with  resultant  increase  in  plasma 
potassium,  retention  of  sodium,  and  decrease  in  urinary  PGE.  Plasma  renin 
activity,  elevated  in  the  control  periods,  fell  to  normal  during  treatment  with 
indomethacin.  Metabolic  studies  are  beino  carried  out  to  define  the  effect  of 
indomethacin  in  this  syndrome  on  aldosterone,  prostanlandins,  nrekal 1 ikrein, 
and  kallikrein.  It  is  possible  that  excessive  production  of  vasodilators 
(PG's,  kinins)  balances  the  excessive  production  of  angiotensin  and  aldosterone 
in  this  syndrome  to  produce  the  persistently  normal  blood  pressure. 

Studies  of  calcium  and  phosphorus  metabolism  in  relation  to  metabolic 
bone  disease,  parathyroid  function,  and  the  formation  of  ren al  stones:  Studies 
which  relate  nephronenous  3,5 'cyclic  AMP  to  various  abnormalities  of  parathy- 
roid function  have  been  extended  to  normal  narathyroid  physiolony,  and  to 
patients  of  hypercalciuria  with  unknown  etiolooy.  From  the  data  available  from 
10  patients  with  hypoparathyroidism,  35  normal  subjects,  and  39  patients  with 
hyperparathyroidism,  it  was  concluded  that  measures  of  nephroaenous  cyclic  AMP 
give  a  sensitive  and  reliable  method  for  study  of  the  spectrum  of  parathyroid 
disease. 

Patients  with  nephrolithiasis  and  increased  nastrointestinal  calcium 
absorption  have  been  found  to  respond  to  treatment  with  sodium  cellulose  phos- 
phate, an  aaent  with  inhibits  calcium  absorption  from  the  out.  In  this  study, 
designed  to  evaluate  lonn-term  effects  of  this  drug,  it  has  been  shown  that  no 
new  stones  are  former1  and  no  metabolic  bone  disease  has  been  produced  by  the 
treatment  (1-5  years  in  duration),  nor  has  there  been  evidence  of  trace  metal 
depletion.  The  expected  secondary  hyperparathyroidism  has  not  been  thus  far 
apparent. 

Other  studies:  The  chemisty  and  control  of  tryptophan  hydroxylase  has  been 
studied  in  extenso.  This  enzyme  requires  tetrahydrobiopterin  and  is  a  mixed- 
function  oxygenase.  Its  distribution  in  the  central  nervous  system  is  limited 
to  serotonergic  neurons,  and  these  were  selectively  destroyed  by  administration 
of  5,6-  and  5, 7-di hydroxy tryptami no  (DHT)  which  are  specifically  taken  up  by 
these  neurons  and  subsequently  destroy  them.  Intracerebral  administration  of 
5,7-DHT  to  neonatal  animals  results  in  immediate  loss  of  tryptophan  hydroxy- 
lase in  all  brain  regions,  and  prevents  normal  developmental  increase  in  the 
enzyme.  The  nrowth  rates  of  rats  so  treated  is  rarkedly  retarded,  despite 
normal  concentrations  of  circulating  nrowth  hormone.  Studies  show  that  the 
neurotoxicity  of  these  dihydroxyindoles  depends  unon  their  tight  binding  to 
mitochondria , and  presumably  their  interuption  of  the  respiration  of  the  cell. 

It  was  shown  that  parachloroamphetamine  and  methyl  parachlcroamphetamine, 
which  deplete  brain  serotonin,  also  deplete  brain  tryptophan  hydroxylase,  and 
that  this  depletion  lasts  for  3  weeks  after  a  single  dose.  It  appears  that 
nerve  endinn  renions  of  the  brain  are  initially  destroyed,  with  retrograde 
death  of  the  cell  body.  The  initial  event  appears  to  be  the  specific  uptake 
of  the  drug  by  serotonin  nerve  endinn. 

Studies  have  continued  on  the  identification  of  mediators  released  from 
human  lung  by  antigen-antibody  reactions.  A  nreatly  simplified  method  has  been 


developed  for  the  measurement  of  histamine.     In  addition  to  histamine,  an 
arginine  esterase,  SRS-A,  and  prostaglandins  are  also  released  when  passively 
sensitized  human  lung  is  reacted  with  specific  antigen.     Human  lung  releases 
mainly  prostaglandin  E's  which  relax  human  bronchi,  and  it  appears  unlikely 
that  prostaglandins  have  a  direct  mediator  effect  in  bronchial  asthma.     However, 
they  may  act  as  modulators  of  this  disease  either  by  potentiating  the  effects 
of  other  mediators  or  by  altering  levels  of  cyclic  AMP.     The  arginine  esterase 
released  from  human  lung  is  not  a  kallikrein,  an  activator  of  plasma  kallikrein 
or  plasmin  or  a  plasminogen  activator.     The  enzyme  is  unusually  labile  to  salts, 
losing  50%  of  its  activity  on  standing  in  0.2  M  NaCl   for  one  half  hour  at  room 
temperature.     A  method  has  been  devised  for  the  purification  of  this  enzyme 
with  a  46%  yield.     Methods  have  been  devised  for  the  separation  of  human 
SRS-A's  into  four  biologically  active  fractions.     The  four  fractions  inhibited 
type  H-l   arylsulfatase  at  pH  4.5.     At  this  pH  and  at  ratios  of  arylsulfatase 
units/SRS-A  units  of  0.3  to  6.0,  the  arylsulfatase  did  not  destroy  the 
biological   activity  of  the  SRS-A's  except  for  Fraction  1.     These  and  other 
results  suggest  that  human  SRS-A's,  like  the  prostaglandins,  may  represent  a 
family  of  compounds.     Studies  have  been  initiated  on  the  biochemical  mechanism 
of  the  synthesis  of  SRS-A  in  monkey  lung.     The  conditions  required  for  optimal 
release  of  mediators  from  monkey  lung  are  similar  to  those  required  for  human 
lung,  although  higher  concentrations  of  antigen  E  and/or  longer  periods  of 
incubation  are  required.     The  amount  of  histamine  and  SRS-A  released  from 
monkey  lung  is  similar  to  that  of  human  lung  but  only  one-tenth  of  the  arginine 
esterase  activity  is  released.     Studies  of  SRS-A  formation  in  monkey  lung 
homogenates  have  been  difficult  to  interpret,  since  homogenates  contain 
substance(s)  other  than  histamine  and  SRS-A  which  contract  the  guinea  pig 
ileum.     Preliminary  experiments  indicate  the  feasibility  of  separating  SRS-A 
from  the  interfering  materials. 

Separation  of  all   but  two  of  the  20  amino  acid  phenylthiohydantoins  has 
been  achieved  by  high  performance  liquid  chromatography.     This  procedure  is 
currently  in  use  in  the  amino  acid  sequence  analysis  of  peptides  and  proteins. 
The  phenylthiohydantoins  also  have  been  separated  by  a  less  expensive  and 
potentially  more  rapid  isocratic  method  employing  two  separate  columns. 
Dinitrophenyl   derivatives  of  amino  acids  have  been  separated  by  a  similar 
procedure  which  is  more  rapid,  sensitive  and  specific  than  previous  methods. 
The  technique  is  currently  employed  in  the  N-terminal  amino  acid  analysis  of 
polypeptides.     Tryptophan,  peptides  containing  N-terminal  tryptophan, 
tryptamine  and  certain  related  indoles  react  with  Fluram  to  form  derivatives 
with  uniquely  high  fluorescence  in  strong  acid.     Fluram  also  has  been  used  to 
develop  a  membrane  filter  assay  for  proteins  in  the  submicrogram  range. 

A  zinc  protein  from  parotid  saliva  has  been  isolated  from  human  subjects 
with  normal   taste  acuity  by  oel   filtration  and  ion  exchange  chromatography. 
The  protein  has  a  molecular  weinht  of  37,000  and  does  not  appear  to  have 
subunits.     It  is  composed  of  8%  histidine  residues  and  has  2  moles  of  zinc 
per  mole  of  protein.     The  contractile  mechanism  has  been  described  in  the 
taste  buds  of  fungiform  papillae,  and  acetylcholinesterase  has  been  found 
in  the  pore  region  of  the  taste  buds  from  circumvallate  papillae  of  rats. 
Radioactive  sugars  have  been  demonstrated    to  bind  in  a  specific  manner  to 
membranes  isolated  from  taste  buds  of  circumvallate  papillae;  they  do  not  bind 
to  non-taste  bud  bearing     membranes  from  the  epithelial   tissue  surrounding 
these  papillae. 


A  double  blind  study  of  the  effects  of  zinc  sulfate  and  of  placebo  in  a 
group  of  106  unselected  patients  with  taste  and  smell  dysfunction  was  carried 
out.     The  results  indicated  placebo  and  zinc  sulfate  were  effectively 
equivalent  in  the  treatment  of  these  disorders.     The  clinical -and  pathophysio- 
logic characteristics  of  patients  with  post-influenzal   hypoguesia  and  hyposmia 
have  been  evaluated.     Biopsy  of  the  nasal  mucous  membrane  shows  inflammation 
of  the  upper  lamina  propria  in  such  patients. 

A  new  syndrome  of  acute  zinc  depletion  has  been  elucidated.     In  addition 
to  changes  in  several   sensory  modalities  these  patients  suffer  from  severe 
cerebellar  dysfunction  including  intention  tremor,  positive  Romberg  sign  and 
ataxia.     Treatment  with  zinc  ion  corrects  these  abnormalities  within  24-48 
hours. 

Human  pituitary  hormones  have  been  grown  in  vitro     in  capillary  tissue 
culture.     These  tumors  have  been  maintained  for  several  months  with  production 
of  large  amounts  of  growth  hormone  and  of  prolactin.     These  hormones  have  been 
characterized  by  physical,  chemical   and  biological   techniques  which  show  them 
to  be  indistinguishable  from  normal   human  hormones. 

The  system  whereby  the  sympathetic  nervous  system  (cervical   sympathetic 
nerves)  can  induce  serotonin  N-acetyl transferase  in  the  pineal   gland  was 
further  studied.     Nerve  impulses  appear  to  mediate  production  of  cyclic  AMP 
in  the  pineal;  large  quantities  of  cyclic  AMP-dependent  protein  kinase  in  the 
pineal   suggest  that  the  next  step  is  phosphorylation  of  chromatin-related 
protein  followed  by  transcription  of  the  messenger  RNA  for  serotinin  N-acetyl- 
transf erase.     A  number  of  protein  kinases  have  been  isolated  in  association  with 
ribosomes;  their  role  in  this  system  is  under  study. 


&6 


Project  No.      zoi  hl  018OI-01 

1.  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral  Metabolism 

3.  Bethesda,  Maryland 

PHS-MIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Outpatient  Hypertension  Diagnostic  Screening  Program 

Previous  Serial  Number: 

Principal  Investigators:  Taylor,  A.  A.,  M.D.,  Ph.D. 

Mitchell,  J.  R.,  M.D.,  Ph.D.. 
Keiser,  H.  R.,  M.D. 
Bartter,  F.  C,  M.D. 

Other  Investigators:  Snodgrass,  W.  R.,  M.D.,  Ph.D. 
Horwitz,  D.,  M.D. 
Licata,  A.  A.,  M.D.,  Ph.D. 
Vinci ,  J.,  M.D. 
Gill,  Jr.,  J.  R.,  M.D. 
Delea,  C.  S. 

Project  Description: 

An  estimated  23-24  million  Americans  or  approximately  10%  of  our 
population  have  hypertension. 

Significant  improvements  in  the  ability  to  diagnose  and  to  cure  certain 
forms  of  hypertension  have  occurred  through  the  utilization  of  increasingly 
more  sophisticated  biochemical  tests  which  permit  more  accurate  categorization 
of  hypertensive  patients  into  selected  subgroups.  The  biochemical  tests 
important  in  the  diagnostic  classification  of  hypertensive  patients  have  been 
available  in  various  laboratories  in  the  Hypertension-Endocrine  Branch  but 
an  organized  application  of  these  tests  to  the  screening  of  large  numbers 
of  outpatient  hypertensives  has  not  been  instituted  previously.  This 
project  was  designed  to  categorize  hypertensive  patients  into  established 
subgroups  in  order  to  study  various  characteristics  of  such  subgroups  more 
intensively  including  their  response  to  different  types  of  drug  therapy. 

Project  Protocol :  Each  patient  referred  to  the  Hypertension  outpatient 
clinic  is  seen  by  a  physician  who  takes  a  history  and  performs  a  physical 
examination.  A  chest  x-ray,  electrocardiogram,  urinalysis,  urine  culture 
and  routine  serum,  chemistries  are  obtained  during  the  patient's  first 
visit.  An  intravenous  pyelogram  and  radioactive  renogram  are  obtained 
between  the  first  and  second  visits.  Each  patient  is  taught  to  take  his  own 
blood  pressure  and  is  requested  to  take  it  6  times  a  day.  On  the  morning 
of  the  second  visit,  the  patient  brings  a  24  hr  urine  sample  for  sodium, 


potassium,  creatinine,  170HCS,  17KS  and  aldosterone  excretion  rate,  and 
45  min. -supine  and  3  hour-  upright  blood  samples  are  obtained  for  plasma 
renin  activity,  aldosterone  and  Cortisol.  The  same  protocol  is  followed 
on  the  third  visit  as  the  second  except  that  each  patient  is  given  Lasix 
40  mg  after  the  first  blood  sample  is  obtained. 

Based  on  these  data  the  patients  is  then  placed  in  a  diagnostic 
category  and  he  is  included  in  one  of  several  ongoing  protocol  studies 
if  appropriate. 

Consentinn  normotensive  volunteers  are  studied  in  a  similar  fashion 
except  that  they  are  not  given  Lasix. 

Major  Findings:  Since  December  1974,  25  normotensive  volunteers 
(age  range  19-61  y.o.)  and  70  hypertensive  patients  (age  range  15-74  y.o.) 
have  been  or  are  beinn  studied.  Since  initiating  this  study  there  have 
been  no  fatalities  or  morbid  cardiovascular  or  neurovascular  events. 
Among  this  patient  population  are  one  person  with  primary  aldosteronism, 
one  patient  with  suspected  renovascular  hypertension,  one  patient  with 
coarctation  of  the  aorta,  one  patient  with  supraval  vular  aortic  stenosis, 
and  8  of  34  (24%)  patients  with  suppressed  plasma  renin  activity  (upright 
plasma  renin  activity   2  ng/ml/hr.  after  40  mg  Lasix  p.o.).  No  patients 
with  pheochromocytoma  have  been  identified.  Thirty  of  45  patients  who 
have  completed  the  screeninc  studies  are  or  have  agreed  to  participate 
in  further  diagnostic  or  therapeutic  studies.  Only  one  patient  with 
spontaneous  hypokalemia  has  been  identified.  Many  of  our  patients  have 
come  from  the  NIH  employee  population. 

Keyword  Descriptors: 

Hypertension  screening,  renin-angiotensin-aldosterone,  normal  volunteers 

Honors  and  Awards: 


Publications : 


*&B 


Project  No.     zoi  hl  01802-qi 

1 .  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral   Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual   Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:     Adrenal   steroid  secretion  in  hypertension. 

Previous  Serial   Number: 

Principal    Investigator:     Mitchell,  J.   R.,  M.D.,  Ph.D. 

Other  Investicators:     Taylor,  A.  A.,  M.D.,  Ph.D. 
McMurtry,   R.  J.,  M.D.,  Ph.D. 
Bartter,   F.   C. ,  M.D. 

Cooperating  Units: 

Project  Description: 

To  investigate  the  control  mechanisms  for  secretion  of  adrenocortical 
steroids  and  their  roles  in  the  genesis  of  differnet  types  of  hypertension. 

Several    studies   have  shown  that  about  20%  of  patients  with  hypertension 
have  suppressed  plasma  renin  activity   (Woods  et  al .,  1969;  Jose,  et  al . , 
1970).     Only  a  small   number  (     1-2%)   of  these  have  primary  hyperaldosteronism. 
It  has  been  suggested  by  several   investigators  that  the  hypertensive  state 
in  the  remaining  patients  can  be  attributed  to  excessive  secretion  of  some 
other  unknown  adrenal   steroid  having  minerlocorticoid  activity  because: 
1)     they  have  low  renin  activity;  2)  administration  of  inhibitors  of 
adrenal   steroid  biogenesis,  such  as  aminoglutethimide,  ameliorates 
their  hypertension  but  not  that  of  hypertensives  with  normal   renin;   3) 
administration  of  spironolactone,  an  antagonist  of  mineralocorticoid 
actions,  lowers  their  blood  pressure;  and  4)     bilateral  adrenalectomy 
lowers  their  blood  pressure.     Three  groups  have  looked  at  individual 
steroids  in  these  patients.     Brown  et  al . ,  1972  have  reported  elevated 
plasma  desoxycorticosterone  concentrations  in  6  of  31  hypertensive 
subjects,   and  all   6  had  suppressed  plasma  renin  activity.     Mel  by  et  al • , 
(1971)  noted  3  of  12  patients  with  "low  renin"  hypertension  had 
elevated  18-hydroxy-desoxycorticosterone  secretion  rates.     Sennett 
et  al . ,    (1974)   have  recently  reported  that  15  of  15  patients  with 
"low  renin"  hypertension  and  1   of  15  patients  with  essential   hypertension 
have  elevated  16-B-hydroxy-dehydroepiandrosterone  excretion  rates. 
However,   in  none  of  these  studies  has  a  profile  of  several  adrenal 
steroid  secretion  rates  been  examined. 


*&t 


Methods  Employed 

Patient  with  hypertension  categorized  as  essential,  low  renin, 
hyperaldosterone  or  renovascular  in  type  will   be  studies  under  3  different 
regimens;     1)     Low  sodium  intake   (9  mEq  sodium,  70  mEq  potassium  for  1 
week),  2)     normal    sodium  intake   (109  mEq  sodium,  70  mEq  potassium  for 
5  days),  3)     normal   sodium  intake,  ACTH  stimulation  [250  ug  tetracosactrin 
(synthetic  G  1-24  ACTH)   intravenously  over  8  hr.   and  urine  and  plasma 
collected  for  2  days].     During  each  regimen  urine  secretory  rates  will 
be  determined  for  the  following  steroids  by  administration  of  radioactive 
tracer  doses   intravenously  followed  by  double  isotope  dilution  a^say  of 
urine  metabolites:       H-dehydroepiandrosterone   (       1   ug,  4  uCi),     H-dehydro- 
epiandrosterone  sulfate   (     1   ug,  4  uCi),  JH-16-B-hydroxydehydroepiandro- 
sterone  sulfate  (     1   ug,  4-uCi),     H-16-u-hydroxy-dehydroepiandrosterone 
sulfate   (       1   uq,   4  uCi),     H-18-hydroxy-corticosterone   (     1   ug,  4  uCi), 
H-18-hydroxy-desoxycorticosterone   (     1   ug,  4  uCi),     H-17     -hydroxy- 
pregnenolone   (     1   ug,   4  uCi).     In  addition,  5  plasma  samples   (10  ml   each) 
will   be  obtained  and  urine  will   be  collected  for  48  hr.     Plasma  DOC  and 
17-     hydroxy-progesterone  will   be  determined  by  radioimmunoassay  and 
urine  excretion  of  metabolites  of  Cortisol,  desoxycortisol ,  corticosterone 
and  aldosterone  will   be  assayed  chemically  or  by  radioimmunoassay. 

The  initial   two  patients  in  each  of  the  hypertension  groups  will   be 
given  four  of  the     H-steroids  and  the  secretory  rates  determined;  2  days 
later  the  remaining  three     H-steroids  will   be  administered  and  their 
secretory  rates  determined.     If  as  anticipated,  the  metabolites  of  the 
various  steroids  do  not  interfere  with  the  assav  of  the  other  steroids, 
all    subsequent  patients  will   receive  the  seven     H-steroids  simultaneously 
for  concomitant  evaluation  of  secretory  rates. 

Major  Findings 

Currently  6  patients,  4  with  low  renin  essential   hypertension  and 
2  with  normal    renin  essential    hypertension  have  or  are  participating  in 
this  study.     Adrenal   steroid  secretory  data  are  incomplete  at  this  time. 

Keyword  Descriptors: 

hypertension,  primary  aldosteronism,   renovascular  hypertension, 
"low  renin"  hypertension,  adrenal   steroid  secretion 

Honors  and  Awards: 
Publications: 


*&6 


Project  No.      Z01  HL  01803-01 

1.  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral  Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:     Aminoglutethirnide  in  low  renin  essential    hypertension. 

Previous  Serial   Number:     None 

Principal    Investigator:     Mitchell,  J.R.,  M.D.,  Ph.D. 

Other  Investigators:     Taylor,  A.,  M.D.,  Ph.D. 
Gill,  Jr.,  J.R.,  M.D. 
Snodarass,  W.R.,  M.D.,  Ph.D. 
McMurtry,  R.J.,  M.D.,  Ph.D. 
Dybing,  E.,  M.D. 

Cooperating  Units: 

Project  Description: 

Patients  will   be  tested  for  the  blood  pressure-lowering  effect  of 
aminoglutethirnide,  an  inhibitor  of  adrenal   steroid  synthesis.     The  pathways 
of  adrenal    steroid  synthesis  that  are  inhibited  by  aminoglutethirnide  in 
blood-pressure  responsive  patients,  and  therefore  the  pathways  that  might 
be  mediating  the  hypertension,  will    be  determined. 

Treatment  with  aminoglutethirnide  lowers  the  blood  pressure  of  patients 
with  hypertension  secondary  to  primary  aldosteronism  or  Cushing's  syndrome 
(Gaunt,  et  al_. ,  Clin.  Pharmacol.  Therap.   9:   657,  1968;  Temple  and  Liddle, 
Ann.   Rev.   Pharmacol.   10:   199,   1970;   Fishman,  et  al_. ,  J.   Clin.   Endo.  Metab. 
27:   481,   1967;  and  Gorden ,  at  al_. ,  J.   Clin.   Endo.  Metab.  28:   921,   1968). 
In  1969  Liddle's  group   (Woods,  et  al_.,  Arch.   Int.   Med.   123:   366,  1969) 
reported  that  6  of  9  hypertensive  patients  with  the  syndrome  of  low-renin 
(normal   aldosterone)  essential   hypertension  experienced  a  lowering  of  blood 
pressure  after  administration  of  aminoglutethirnide  but  patients  with  normal 
renin  hypertension  failed  to  respond.     They  noted  also  that  the  hypertension 
of  low-renin  patients  responded  to  therapy  with  spironolactone   (Carey,  et  al . , 
Arch.    Int.  Med.   130:   849,' 1972),  a  renal   antagonist  of  sodium-retaining 
steroids,  and  to  bilateral   adrenalectomy   (Gunnels,  e_t  aj_. ,  Ann.    Int.  Med. 
73:   901,   1970).     This  report  triggered  a  widespread  search  for 
the  excessive  adrenal   secretion  of  a  sodium-retaining  steroid 
other  than  aldosterone,  and  several   groups  subsequently  have 


¥71 


postulated  that  various  steroids  are  of  etiologic  sianificance  in  low- 
renin  hypertension   (Brown,  et  al . ,  Lancet  ii_:     243,   1972;  Mel  by,  et  al . , 
Circ.   Res.   23:     11-143,   1971;   Kuchel   et  al^,  Circ.   Res.   23:     11-150,  1971; 
Messerli,  et  al. ,  Proc.   56th  Meeting  of  Endocrine  Society,  p.  A-63,  1974; 
Slaton,  et~lTl.,  Clin.   Res.   23:     45A,  1975:  and  Hisahsatu,  ejt  aJL_,  J.   Clin. 
Endo.   Metab.   40:     156,   1975).     However,   in  these  studies  only  the  particular 
steroid  of  interest  was  examined.     '!o  attempt  was  made  to  determine  the 
secretion  rates  of  most  other  adrenal   steroids  nor  to  demonstrate 
cause-effect  relationships  between  increased  steroid  secretion  and 
elevation  of  blood  pressure.     Indeed,  the  direct  involvement  of  the 
proposed  steroids  in  the  pathogenesis  of  low-renin  hypertension  can 
be  questioned,   since  none  of  the  steroids  have  sufficient  intrinsic 
sodium-retaining  activity  to  be  physiolooically  effective  in  the 
amounts  apparently  secreted  by  Datients  with  low-renin  hypertension. 

The  antihypertensive  effects  of  aminoglutethimide  result  from  its 
inhibition  of  adrenal    steroid  secretion  and  the  accompanying  renal   loss 
of  sodium  and  water;   the  drug  has  no  direct  effect  on  the  kidney,  the 
adrenergic  nervous  systme  or  vascular  smooth  muscle  in  adrenalectomized 
animals  and  people   (Gaunt,  et  al . ,  Clin.   Pharmacol.   Therap.   9:     657,  1968; 
Temple  and  Liddle,  Ann.   Rev.   Pharmacol.   10:     199,  1970;   Fishman,  et  al . , 
J.   Clin.   Endocr.   Metab.   27:     431,   1967;  Gorden,  et  aL_,  J.   Clin.   Endocr. 
Metab.   28:     921,   1968;  Woods,  Liddle,  et  al^,  Arch.    Int.   Med.   123:     366,  1969) 
Thus,  the  secretion  of  a  sodium-retaininq  adrenal   steroid,  be  it 
aldosterone  or  an  unidentified  steroid,  should  play  an  important  pathogenetic 
role  in  the  hypertension  of  low-renin  patients  whose  blood  pressure 
responds  to  aminoglutethimide  therapy. 

Careful   review  of  numerous  biochemical   studies  in  dons  and  other 
animals,  with  sufficient  clinical   studies  to  confirm  that  man  responds 
similarly,   reveals  that  the  secretion  of  Cortisol    by  the  adrenal   zona 
fasciculata  is  only  minimally  affected  by  aminoolutethimide  in 
endocrinological^  normal   subjects  because  of  a  compensatory  increase 
in  ACTH  secretion   (Gaunt,  et  al.,  Clin.   Pharmacol.  Therap.   9:     657,  1968; 
Temple  and  Liddle,  Ann.   Rev.   Pharmacol.   10:     199,  1970;   Fishman,  et  al . , 
J.   Clin.   Endocr.   Metab.   27:     431,  1967;  Gorden,  etaL,  J.   Clin.   Endocr. 
Metab.   28:     921,  1968;  and  Woods,  Liddle  et  aJL  ,  Arch.   Int.   Med.   123:     366, 
1969).     In  contrast  to  the  Cortisol   pathway,   secretion  of  aldosterone  by  the 
adrenal   zona  glomerulosa  remains  inhibited  by  aminoglutethimide  even  after 
months  of  continuous  therapy  and  compensatory  increases   in  renin  do  not 
overcome  the  inhibition   (Gaunt,  et  aj_^,  Clin.   Pharmacol.  Therap  9:     657.  1968; 
Temple  and  Liddle,  Ann.   Rev.   Pharmacol.   10:     199,   1970;   Fishman,  et  al . , 
J.   Clin.   Endocr.   Metab.   27:     481,   1967;  Gorden,  et.  aJL ,  J.   Clin.   Endocr. 
Metab.  28:     921,  1968;  and  Woods,  Liddle  et  al.,  Arch.   Int.   Med.   123:     366, 
1969).  

This  phenomenon  of  preferential   inhibition  of  aldosterone  synthesis 
provides  a  powerful   tool   for  defining  the  physiologic  role  of  the  steroids 
postulated  as  having  etioloqic  significance  in  the  genesis  of  low-renin 
hypertension.      If  the  secretion  of  a  particular  steroid  is  not  inhibited 


47> 


by  aminoglutethimide  therany  when  blood  pressure  is  reduced,  then  that 
steroid  cannot  be  solely  responsible  for  the  hypertension.     By  the  same 
rationale,  one  can  determine  which  adrenal   steroid  synthetic  pathways  are 
inhibited  by  aminoglutethimide  in  blood  pressure-responsive  patients  and 
therefore  which  pathways  might  be  mediating  the  hypertension. 

Patients  with  low-renin  hypertension  on  a  normal   diet  will   be 
hospitalized  for  3  days  and  secretory  rates  will   be  determined  for 
the  following  steroids  by  administration  of  radioactive  tracer  doses 
intravenously  followed  by  double-isotope  dilution  assay  of  urinary 

3  3 

metabolites:       H-dehydroepiandrosterone   (     1   ug,  4  uCi ) ,     H-dehydro- 

3 
epiandrosterone  sulfate   (     1   ug,  4  uCi),     H-16  B-hydroxy-dehydro- 

3 
epiandrosterone   (     1   ug,  4  uC^),     H-16-B-hydroxy-dehydroepiandrosterone 
sulfate   (     1   ug,  4  uCi),  and     H-18-hydroxy-desoxycorticosterone  (     1   ug, 
4  uCi).     Five  plasma  samples   (10  ml   each)  will   be  obtained,  and  urine  will 
be  collected  for  48  hr.     Plasma  DOC,  corticosterone,  desoxycortisol , 
Cortisol,  aldosterone,  Drogesterone  and  17-     -hydroxy-progesterone  will 
be  determined  by  radioimmunoassay  and  urinary  excretion  of  metabolites 
of  Cortisol   and  aldosterone  will   be  assayed  chemically  or  by  radioimmunoassay, 

Following  these  control   determinationas,  patients  will   begin  treatment 
with  aminoglutethimide   (  1   g  per  day  in  divided  doses)  and  will   be  observed 
closely  for  clinical   evidence  of  acute  Cortisol   insufficiency.     Plasma 
Cortisol   and  aldosterone  and  urinary  free  Cortisol  will   be  monitored. 
Aminoglutethimide  has  an  immediate  onset  of  action,  and  Cortisol 
insufficiency  would  quickly  be  apparent.     Accordingly,  asymptomatic 
patients  will   be  discharged  after  4  days  of  treatment  and  be  followed 
weekly  in  the  clinic  for  another  14  days.     As  outpatients,  they  will 
record  their  blood  pressure  several   times  daily  under  the  normal 
conditions  of  their  environment.     At  each  weekly  clinic  visit,  blood 
(20  ml)  will   be  obtained  for  electrolytes,  CBC,  SGOT,  glucose,  urea 
nitrogen,  aldosterone,  Cortisol   and  renin.     Patients  will   then  be 
hospitalized  for  a  final   3  days  and  the  steroid  secretory  and 
excretory  determinations  will   be  repeated  exactly  as  above   (total 
duration  of  treatment  with  aminoglutethimide  =  3  weeks). 

Major  Findings;     Aminoglutethimide  has  been  administered  to  two 
patients  with  low  renin  essential   hypertension.     In  both  subjects,  control 
blood  pressures  of  150/100  and  140/95  had  fallen  to  mean  values  of  110/70 
and  115/75  after  10  days  of  therapy.     There  was  an  associated  fall   in 
aldosterone  excretion  rate  from  control   values  of  3.99  ug/24  hr.   to  1.49 
ug/24  hr.   in  one  patient  in  whom  the  data  has  been  analyzed.     Adrenal 
steroid  secretory  rate  data  are  incomplete  at  this  time. 

Keyword  Descriptors: 

aminoglutethimide,  low  renin  essential   hypertension; 
adrenal   steroid  secretion 


?7S 


Honors  and  Awards: 

Publications: 
None, 


«ry 


Project  No.    ZQ1  HL  01804-01 

1.  HyDertension-Endocrine  Branch 

2.  Steroid  &  Mineral   Metabolism 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual   Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:     Effects  of  Spironolactone 

Previous  Serial   Number: 

Principal    Investigator:     Taylor,  A.  A.,  M.D.,  Ph.D. 

Other  Investiaators:     Mitchell,  J.R.,  M.D.,  Ph.D. 
McMurtry,   R.J.,  M.D.,  Ph.D. 
Snodqrass,  H.R.,  M.D.,  Ph.D. 
Dybing,   E.,  M.D,  Ph.D. 
Bartter,   F.C.,  M.D. 

Cooperative  Units:     None 

Project  Description: 

To  examine  the  effects  of  spironolactone  or  renin-angiotensin- 
aldosterone  physiology  and  on  sex  steroids  in  hypertensive  patients. 

Spironolactone  is  widely  used  as  a  potassium-conserving  diuretic 
and  antihypertensive  agent.     It  has  been  suggested  by  some  as  the  drug 
of  choice  in  patients  with  low  renin  essential   hypertension,  a  subgroup 
comprising  approximately  25%  of  the  hypertensive  population.     Previous 
work  by  some  of  us  has  shown  that  spironolactone  destroys  cytochrome 
P-450  in  the  adrenal   and  testis  of  certain  experimental   animals 
(Menard  et  al . ,  Endo.   94:     1628,  1974;  Menard  et  a!.,  J.  Steroid  Biochem. 
5:     365,  1974).     Cytochrome  P-450  is  a  co-enzyme  necessary  for  normal 
function  of  the  steroid  17-hydroxylase  enzyme;  17-hydroxylase  activity 
is  decreased  in  experimental   animals  given  spironolactone.     A  recent  study 
of  the  effect  of  400  mg  spironolactone  per  day  for  five  days  in  normal   male 
volunteers  showed  only  a  transient  rise  in  plasma  LH  and  FSH  but  no 
change  in  plasma  testosterone,  estradiol,  or  prolactin.     These  findings 
failed  to  explain  the  frequently  observed  side  effects  of  gynecomastia, 
decreased  libido,  and  impotence  in  male  hypertensives  taking  spironolactone, 

Significant  interference  by  spironolactone  with  parameters  of  the 


frC 


renin-angiotensin-aldosterone  system  in  low  renin  hypertensive  patients  is 
demonstrated  by  the  recent  report  of  Lowder  et  al.,  Liddle   (NEJM  29:     243,   1974) 
that  the  increased  upright  plasma  renin  activity  induced  by  spironolactone 
in  low  renin  hypertensive  patients  persisted  for  13-36  weeks  after 
discontinuation  of  the  drua. 

Patients  previously  classified  as  havinn  essential   hypertension 
with  normal   or  suppressed  plasma  renin  activity  by  diagnostic  studies 
performed  while  off  all   antihypertensive  medications  will   be  considered 
for  inclusion  in  the  study.     Patients  with  prior  history  of  malignant 
hypertension,  evidence  of  cardiovascular  compromise  or  severe  impairment 
of  renal   function   (creatinine  clearance  =  70  cc/min  or  less)  will   be 
excluded.      Informed  consent  will   be  obtained  from  all   patients.     All 
patients  will   be  provided  with  equipment  and  taught  to  take  their  blood 
pressure.     Blood  pressure  will   be  recorded  both  by  the  patient  at  home 
and  by  physicians  in  the  outpatient  clinic  throughout  the  study. 

Following  pretreatment  measurements  of  the  plasma  and  urinary 
parameters  listed  below,  each  patient  will   receive  spironolactone, 
400  mg/day,   for  a  maximum  of  12  weeks.     After  12  weeks  of  therapy, 
spironolactone  will   be  discontinued  and  the  patient  observed  for  an 
additional   6  weeks  before  reinstitution  of  antihypertensive  therapy 
unless  development  of  si^ns  and  symptoms  of  cardiovascular  compromise 
or  accelerated  hypertension  necessitate  removal    from  the  study  and 
treatment  at  an  earlier  time.     If  there  is  no  reduction  in  blood 
pressure  after  the  initial   4  weeks  of  spironolactone  therapy,  the  patient 
will   be  removed  from  the  study  and  treated  with  other  antihypertensive 
agents.     Before,   and  at  reoular  2-4  week  intervals  during  and  after 
discontinuation  of  spironolactone,  the  followina  plasma  and  urinary 
measurements  will   be  made:     Plasma  renin  activity,  aldosterone,  Cortisol, 
deoxycorticosterone,   11-deoxycortisol ,   testosterone,  progesterone, 
17-hydroxyprogesterone,   17B-estradiol ,  prolactin,  LH,   FSH,  Ma,   K,  CI, 
CO2,  BUN ,  creatinine,   sex  hormone  binding  qlobulin;   urinary  creatinine, 
Na,   K,  aldosterone,   17-hydroxycorticosteroids,  1 7-ketosteroids, 
aldosterone  excretion  rate. 

Currently  seven  patients  have  been  included  in  this  study,   including 
two  patients  with  orimary  aldosteronism,  one  patient  with  low  renin 
essential    hypertension  and  4  patients  with  essential    hypertension  and 
normal   plasma   renin.     Only  one  patient  with  primary  aldosteronism 
has  completed  the  study.     In  this  patient  the  pretreatment  mean  BP 
was  138  +  1.1   mmMg,    95+1.4  mmHg  just  prior  to  discontinuation  of 
spiranolactone  therapy  and  110  +  1.5  mmHq  one  week  later.     Upriqht 
plasma  renin  activity  was  0.20  ng/ml/hr  prior  to  treatment;  2-21 
ng/ml/hr  just  prior  to  stoppino  spironolactone,  and  0.03  ng/ml/hr  one 
week  after  discontinuation  of  druq  therapy.     Sex  hormone  data  is 
currently  incomplete. 

Keyword  Descriptors: 

spironolactone,   renin-anoiotensin-aldosterone 

47& 


Honors  and  Awards: 


Publications: 


477 


Project  Ho.         zoi  hl  01805-01 

1.  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral  Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Adrenergic  nervous  system  function  in  hypertension. 

Previous  Serial  Number: 

Principal  Investigator:  Gill,  Jr.,  J.R.,  M.D. 

Other  Investigators:  Alexander,  R.W.,  M.D.,  Ph.D. 
Keiser,  H.R.,  M.D. 

Cooperating  Units: 

Project  Description: 

Previous  studies  in  normal  subjects  indicate  that  an  increase  in  sodium 
intake,  either  rapidly  by  infusion  of  saline  or  slowly  by  an  increase  in 
dietary  sodium,  results  in  a  decrease  in  urinary  excretion  of  norepinephrine 
plus  epinephrine  and  an  increase  in  urinary  dopamine.  These  changes  are 
consistent  with  a  decrease  in  adrenergic  activity  and  suggests  that  normally 
adrenergic  activity  may  be  inversely  related  to  the  renal  formation  and 
excretion  of  dopamine  a  potent  renal  vasodilator  and  natriuretic  catecholamine. 

The  present  studies  were  designed  to  extend  these  observations  to  include 
patients  with  hypertension. 

Patients  with  normal  renin  essential  hypertension  were  studied  on  a 
9  mEq/day  sodium  intake  for  eight  days  ,  then  on  a  249  mEq/day  sodium  intake 
for  8  days.  Daily  collections  of  urine  were  analyzed  for  norepinephrine 
plus  epinephrine  (ME+E),  dooamine  (DA)  and  sodium. 

On  a  249  mEq  sodium  intake  values  for  urinary  ME+E  and  DA  in  the 
hypertensive  patients  were  similar  to  those  in  the  normotensive  subjects. 
When  sodium  intake  was  decreased  to  9  mEq/day  the  hypertensive  patients 
excreted  significantly  more  sodium  and  more  NE+E.  Dopamine,  however,  decreased 
as,  in  the  normotensive  subjects,  but  the  values  v/ere  not  significantly 
different.  The  results  are  summarized  in  the  table  below. 


Y78 


Sodium 
Intake 

Patients 

UMaV 

mEq^d 

NE+E 
pg/day 

DA 
jjg/day 

249  mEq 

Normal 

228+1 5 

21 .1+3 

195+20 

Hypertensive 

218+8 

25.1+4.3 

204+1 8 

9  mEq 

Normal 

37+2 

37.4+5.3 

136+18 

Hypertensive 

55+7* 

60.4+7.8* 

161+22 

*P<.01 

The  results  indicate  that  when  hypertensive  patients  are  stressed  by 
a  low  sodium  intake  they  excrete  more  sodium  and  NE+E  than  normotensive 
patients.  The  greater  than  normal  excretion  of  NE+E,  possibly  a  conse- 
quence of  the  greater  sodium  loss,  suggests  the  presence  of  hyper- 
responsive  adrenergic  nervous  system  in  hypertension. 

Keyword  Descriptors: 

norepinephrine,  dopamine,  sodium  excretion,  adrenergic  nervous  system, 
hypertension 

Honors  and  Awards: 

Publications: 

1.  Alexander,  R.W.,  Gill,  Jr.,  J.R.,  Yamabe,  H.,  Lovenberg,  W.  and  Keiser, 
H.R.:  Effects  of  dietary  sodium  and  of  acute  saline  infusion  on  the 
interrelationship  between  dopamine  excretion  and  adrenergic  activity 
in  man.  J.  Clin.  Invest.  54:  194,  1974. 


¥7f 


Project  No.         z01  HL  01806-01 

1.  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral   Metabolism 

3.  Bethesda,  f'aryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Pelation  of  K  to  vascular  response  of  PP 

Previous  Serial  number: 

Principal  Investinator:  Radfar,  N. ,  M.D. 

Other  Investinators:  Rartter,  F.  C,  M.D. 

Cooperating  Units:  . 

Project  Description: 

It  is  known  that  the  patients  with  "Bartter's  Syndrome"  have 
a  vascular  hyporesponsi veness  to  angiotensin  II.  To  investigate 
the  pathogenesis  of  this  resistance,  i.e.,  to  see  whether  it  is 
primary  vascular  defect  or  whether  it  may  result  from  the  hypokalemia, 
it  was  reasonable  to  study  the  vascular  response  of  patients  with 
hypokalemia  of  diverse  orinin  including  patients  with  JG  hyperplasia.  Such 
patients  are  admitted  to  the  metabolic  ward  and  are  put  on  109  mEq  'la 
diet  and  their  response  to  angiotensin  is  determined  during  hypo- 
kalemia, and  after  the  serum  K  has  returned  to  normal  spontaneously  or 
by  K  supplementation. 

One  patient  who  had  diuretic  induced  hypokalemia  has  been  studied. 
During  hypokalemia  the  pressor  dose  required  to  increase  diastolic 
blood  pressure  by  20  mmHn  was  200  ng/kg/min.  When  serum  K  was  restored 
to  normal  tne  pressor  dose  dropped  down  to  30  ng/kg/min. 

Keyword  Descriptors: 

Bartter's  Syndrome,  hypokalemia 

Honors  and  Awards : 


Publications 


¥*o 


Project  No.   ZQ1  HL  01807-01 

1.  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral  Metabolism 

3.  Bethesda,  Maryland 


PHS-MIH 

Individual   Project  Report 
July  1,   1974  through  June  30,  1975 

Project  Title:     Studies  in  Bartter's  Syndrome 

Previous  Serial   Number: 

Principal    Investigators:     Bartter,   F.   C,  M.D. 

Gill,  Jr.,  J.   R.,   M.D. 
Taylor,  A.  A.,  M.D.,  Ph.D. 

Other  Investigators:  Bowden,  R.,  M.D. 
Vinci,  J.,  M.D. 

Project  Description: 

Since  the  initial  description  of  the  syndrome  of  hypokalemic  alkalosis, 
hyperreninism,  aldosteronism  and  juxtaglomerular  hyperplasia  with  normal 
blood  pressure,  this  branch  has  maintained  an  ongoing  interest  in  this  disorder. 
Patients  suspected  of  having  the  disorder  are  presently  being  studied 
in  extenso,  including  renal  biopsy,  thorough  evaluation  of  renal  handling  of 
Ma,  K,  and  water,  testing  of  arteriolar  sensitivity  to  pressor  agents,  red  cell 
concentration  of  fia,  and  evaluation  of  therapy.  Inhibitors  of  prostaglandin 
synthetase  are  beina  given  to  evaluate  the  role  of  prostaglandins  (pg's)  in 
this  syndrome.  Kinins  in  serum  and  urine  are  also  under  study. 

Nine  patients,  all  female,  ages  5  to  40  years,  have  been  evaluated  for 
this  syndrome.  Two  were  found  to  be  diuretic  abusers  and  vomiters,  three 
clearly  have  the  syndrome,  and  three  more  are  still  being  evaluated  for 
this  syndrome.  Two  were  found  to  be  diuretic  abusers  and  vomiters,  three 
clearly  have  the  syndrome,  and  three  more  are  still  being  evaluated.  The 
last  patient  may  represent  a  variant  of  the  disorder  in  that  she  has  hypokalemic 
alkalosis,  aldosteronism  and  hyperreninemia,  with  histologically  normal 
J-G  apparatus  and  normal  arteriolar  sensitivity  to  angiotensin  II. 

Studies  in  two  patients  with  indomethacin,  a  prostaglandin  synthetase 
inhibitor,  demonstrated  high  pg's  in  the  control  period,  low  during  the 
therapy  with  a  decrease  in  plasma  renin  activity.  Both  patients  had  an 
increase  in  plasma  potassium  concentration  and  retained  sodium.  More 
patients  are  currently  being  studied  under  highly  controlled  conditions. 

Keyword  Descriptors: 

aldosterone,  juxtaglomerular  hyperplasia,  prostaglandin 


^/ 


Honors  and  Awards: 
Publications: 


«tfa- 


Project  No.        zoi  hl  01808-01 

1.  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral   Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual   Project  Report 
July  1,   1974  through  June  30,  1975 

Project  Title:  Control   of  renin:     the  role  of  the  vagus  nerves. 

Previous  Serial   Number: 

Principal    Investigator:     Yun,  John  C.H.,  Ph.D. 

Other  Investigators: 

Cooperating  Units: 

Project  Description: 

The  mechanism  for  the  increase  in  plasma  renin  activity  (PRA)  due  to 
sinoaortic  denervation  and  cervical   vagotomy  was  examined  in  intact,  renal 
denervated,  and  renal   denervated,  adrenal ectomi zed  dogs  maintained  on  a  high- 
salt  diet. 

In  eleven  intact  animals,  PRA  increased  from  2.09  +  0.76  ng/ml/hr  in 
control   periods  to  13.09  +  2.34  ng/ml/hr   (P<0.005)  60  minutes  after 
sinoaortic  denervation.     PRA  increased  further  to  22.32  +_  3.90  ng/ml/hr 
(P< 0.005)  90  minutes  after  cervical   vagotomy.     In  eight  animals  previously 
subjected  to  renal   denervation,  PRA  increased  from  7.33  +_  1 .42  ng/ml/hr  in 
control    periods  to  14.45  +  2.39  ng/ml/hr  (P<0.01)   60  minutes  after  sinoaortic 
denervation.     Cervical   vagotomy  in  these  animals  caused  a  further  slight,  but 
not  statistically  significant,  increase  in  PRA  to  16.81   +  3.77  ng/ml/hr 
(P>0.1).     In  six  dogs  with  both  renal   denervation  and  adrenalectomy,  PRA  was 
2.07  +  1.03  ng/ml/hr  in  control   periods,  1.04  +  0.39  ng/ml/hr  60  minutes  after 
sinoaortic  denervation,  and  1 .30  +_  0.74  ng/ml/hr  90  minutes  after  cervical 
vagotomy. 

These  data  suggest  that  the  increase  in  PRA  after  sinoaortic  denervation 
is  probably  due  to  both  increased  sympathetic  discharge  to  the  kidney  and 
catecholamines  released  from  the  adrenal   medulla,  whereas  the  increase  in  PRA 
after  cervical    vagotomy  is  mediated  largely  by  increased  sympathetic  discharne 
to  the  kidney. 

Further  research  will    include  studies  on  the  mechanism(s)  by  which 
renal   nerve  stimulation  causes  renin  secretion. 


4SZ 


Keyword   Descriptors: 

vani ,   renin,    renal    nersje,   sinoaortic  nerves,   adrenal   nedulla 
Honors  and  Awards: 
Publications: 


tf# 


Project  No.     -zpi  hl  01809-01 

1,  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral  Metabolism 
-Si ..-  Bethesda,  Maryland 

PHS-MIH 
Individual   Project  Report 
July  1,   1974  through  June  30,  1975 

Project  Title:     Renal   prostaglandins,  sodium  and  blood  pressure. 

Previous  Serial   Mumber: 

Principal    Investigators:     Gill,  Jr.,  J.  R.,  M.D. 

Other  Investinators:     Alexander,  P..  W.*,  M.D.,  Ph.D. 

._      Halushka,  P.   V.,M,D.;,  Ph.D.    ":v 

Pisano,  J.  J. ,  Ph.D. 
■    .      Keiser,  H.   P.. ,  M.D. 

Cooperating  Units: 

Project  Description: 

Previous  studies  indicate  that  the  infusion  of  prostaglandins 
of  the  E  series  into  a  renal   artery  produces  an  increase  in  sodium 
excretion.     If  these  effects  are  similar  to  those  produced  by  the 
intrarenal    prostaglandins,  then  inhibition  of  intrarenal   synthesis  of 
prostaglandins  should  be  in  association  with,  a  decrease  in  renal   sodium 
excretion.     To  test  this  hypothesis,  indomethacin,  a  prostaglandin 
synthetase  inhibitor  was  infused  into  a  renal   artery  of  the  dog. 

Methods:     Mater  diuresis  was  produced  in  anesthetized  hypophysectomized 
Cortisol-  treated  dogs  by  infusion  of  2.5  per  cent  dextrose,     l-.'hen  urine 
flow  v/as  steady 'clearance  measurements  were  started.     After  three  control 
periods  were  obtained  indomethacin  0.35  mg/min  was  infused  into  the 
left  renal   artery  for  100  min  followed  by  post  control   periods  for 
2  hours.     In  a  second  series  of  studies,  the' control  period  was  followed 
by  infusion  of  Ringer's  solution  for  two  hours,  then  infsuion  of  indomethacin, 
was  superimposed  for  one  hour.     The  clearances  of  inulin   (CT„*  and 

paraaminohippurate   (CpM,)  were  determined.     Changes  in  the  clearance  of 

solute-free  water  (C„  n)  were  taken  as  an  index  of  sdium  reabsorption  by 

the  diluting  segment  of  the  nephrons.     Changes  in  the  sum  of  the  clearance 
of  solute-free  water  and  the  clearance  of  sodium  (tlL)  per  100  ml 

glomerular  filtration  rate   (GFR)  were  assumed  to  represent  changes  in 


¥$S 


proximal   tubular  sodium  reabsorption.     Urinary  PGE  and  PGF  like 
material  was  determined  by  radioimmunoassay. 

Major  Findings:     Indomethacin  increased  sodium  excretion   (UNaV) 
and  decreased  CH  Q  without  apparent  effect  on  Cr]a  +  C„  Q,  CJN  and 
CDflu.     Infusion  of  Ringer's  increased  CM    +  C„  Q,  CH  Q  and  U^V;  the 

HAH  2  2 

suprimposition  of  indomethacin  produced  a  further  increase  in  U,JaV  and 
decrease  in  Cm  q  but  no  further  change  in  C,,a  +  C(|  Q.  The  results  are 
summarized  in  the  table  below. 

«1«                     CNa+CH2°                   CH20  V 

ml/min  ml/min/100  ml   GFR      jiEq/min 

Control  33+2.8         3.2+1.0  7.8+0.9  16+2.3' 

Indomethacin  35+3.4         7.2+0.8  5.8+0.6*         63+7.6* 

Post  control  35+3.8         6.6+1.0  5.8+0.9  32+3.3 

Control  32  +_  3  6.3  +  0.4  6.0  +  0.4  11+2 

Rinaers  32+2  11.9+0.6  10.4+0.6*         66+8* 

Ringers  and  33+2  12.4+1.0  8.9+0.6*       152+16* 

Indomethacin 

♦significant  at  P  /L  0.01 

The  data  indicates  that  renal   arterial   infusion  of  indomethacin 
increases  the  renal   excretion  of  sodium.     An  increase  in  sodium  excretion 
without  an  appreciable  change  in  the  delivery  of  tubular  fluid  out  of  the 
proximal   tubular  and  with  a  decrease  in  C..  Q  suggests  that  this  effect  of 

indomethacin  on  tubular  sodium  reabsorDtion  is  located  in  the  distal   nephron 
The  decrease  in  urinary  prostaglandin-  E  like  material    from  2.43  ng/min  to 
0.3  ng/min  with  indomethacin  suggests  that  these  changes   in  sodium  handling 
by  the  tubule  could  be  attributed  to  a  decrease  in  intrarenal   prostaglandins 
These  results  suggest  that  prostaglandins  generated  within  the  kidney 
stimulate  tubular  sodium  reabsorption  rather  than  inhibit  it  as  is  the 
case  when  prostaglandins  of  the  E  series  are  infused  in  the  renal   artery. 
Further,   the  values  of  urinary  immunoreactive  prostaglandins  durinn 
infusion  of  Ringers  v/ere  similar  to  the  values  during  infusion  of  2.5 
percent  dextrose  and  suggests  that  prostaglandins  inhibition  is  not  an 
essential   element  in  the  natriuretic  response  to  Ringers.     The  overall 


&i 


results  are  therefore  consistent  with  the  hypothesis  that  renal  prostaglandins 
main  function  as  antinatriuretic  agents. 

Significance  to  Biomedical  Research  and  Institute  Program 
Prostaglandins  appear  to  exert  potent  effects  on  renal  fun ct i on 
particularly  as  regards  to  sodium  excretion  by  the  kidney.  They  could 
have  major  importance  in  normal  renal  function  and  contribute  to 
altered  renal  handling  of  sodium  which  is  a  central  feature  in  many 
cardiovascular  diseases. 

Proposed  course  of  Projects:  To  continue  to  explore  the  physiology 
of  intrarenal  prostaglandins  in  renal  physiology  and  to  determine  the 
role  of  prostaglandins  in  disordered  renal  function. 

Keyword  Descriptors: 

prostaglandins,  renal  sodium  excretion,  indomethacin 
Publications: 

None.     ■■•■:■■  , 


+rr 


Project  ::o.      zoi  hl  01810-01 

1 .  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral   Metabolism 

3.  Rethesda,  Maryland 


pns-r;iH 

July  1,  1974  through  June  30,  1975 

Project  Title:     Spironolactone  on  plasma  renin  and  don  renal   histology. 

Previous  Serial   'lumber: 

Principal    Investioators:     Taylor,  A.  A.,  M.D. 

Other  Investioators:     Bartter,   F.   C.   M.D. 

Mitchell,  J.   R.,  M.D.,   Ph.D. 

Project  Description: 

Spironolactone,  a  potassium-sparinn  diuretic  is  known  to  elevate 
peripheral  plasma  renin  activity  (PRA)  in  human  beings  and  experimental 
animals  (Vaughn,  et  aJL ,  Amer.  J.  Cardiol.  32:  523,  1973)  presumably 
by  depletion  of  extracellular  fluid  volume.  Sodium  depletion  by 
thiazide  or  mercurial  diuretics  or  dietary  sodium  restriction  increases 
both  peripheral  PRA  and  Granularity  of  the  juxtaolomerular  (JG)  cells 
in  the  afferent  arteriole  of  the  kidney  in  animals  and  man  (Hartroft, 
in  Endo.  Path. ,  Cloodworth,  ed.  Williams  and  Hi  1  kins,  1968,  p.  641.). 
The  effects  of  soironolactone  administration  on  JG  cell  histology  have 
not  been  documented  previously.  Many  patients  with  the  syndrome  of 
juxtaglomerular  cell  hyperplasia  are  treated  with  spironolactone 
because  of  profound  hypokalemia.  One  of  the  necessary  criteria  for  the 
diagnosis  of  JG  cell  hyperplasia  is  histologic  confirmation  of  such 
hyperplasia.  In  another  group  of  patients  with  low  renin  essential 
hypertension,  Lowder  and  Liddle  (NEJM  291:  1243,  1974)  have  reported 
that  PRA,  increased  by  spironolactone  therapy  remained  elevated  above 
pre-treatment  values  for  as  long  as  36  weeks  following  cessation  of 
spironolactone  therapy. 

Objectives:  1)  To  compare  spironolactone-  induced  changes  in  plasma 
renin  activity  with  changes  in  renal  histopathology  in  dogs,  both  during 
drug  treatment  and  at  regular  intervals  following  cessation  of  treatment. 

Methods  Employer!:  1)   Dons  are  housed  in  metabolic  cages,  fed  a 
diet  of  known  sodium  content  for  1  week  per  month  and  urine  collected 
daily.  2)  After  one  week  of  fixed  dietary  sodium  intake,  blood  is 
drawn  for  the  measurement  of  plasma  renin  activity  (measured  by 
radioimmunoassay  of  generated  angiotensin  I),  sodium,  potassium,  blood 
urea  nitrogen,  creatinine  aldosterone  and  Cortisol  (measured  by 
radioimmunoassay).  On  an  aliquot  of  each  daily  urine  sample  are  measured 
sodium,  potassium  and  aldosterone  (measured  as  pM,  labile  aldosterone  by 


4%% 


radioimmunoassay).     3)     The  dog  is  then  anesthetized  with  pentobarbital 
and  a  small   piece  of  one  kidney  is  removed  by  retroperitoneal   approach 
using  aseptic  techniques.     4)     For  the  next  3  weeks  each  dog  is 
allowed  ad  libitum  food  and  water  and  is  given  a  single  daily  dose  of 
spironolactone,  200  mg.     The  metabolic  balance  study  and  open  renal 
biopsy  is  then  repeated.     5)     Each  piece  of  renal   tissue  is  divided; 
one-half  is  processed  and  stained  with  hemataxylin  and  eosin,  the  other 
half  is  processed  and  stained  with  Bowie's  stain.     The  identification 
of  each  slide  is  masked;  an  estimation  of  JG  hyperplasia  is  made  on  the 
Hematoxyline-eosin  sections  and  an  index  of  JG  cell   granularity 
determined  on  the  Bowie  stained  sections  by  a  previously  described 
method   (Hartroft  in  Endo.   Path . ,  Bloodworth,  ed.,  Williams  and  Wilkins, 
1968,  p.   641). 

After  one  month  of  spironolactone   (200  mg/day)  treatment  of  6 
dogs,  plasma  renin  activity  has  increased  from  0.99  +  0.31  ng/ml/hr 
(mean  +  SEH)   (pretreatment)  to  4.92  +  1.04  ng/ml/hr.     After  2  months 
of  drug  treatment,  the  mean  value  in  3  dogs  is  5.05  +  1.37  ng/ml/hr. 
Urinary  aldosterone  excretion   (  measured  daily  for  th~e  last  3  days  of 
each  balance  period)  was  0.074  +  0.022  ug/24  hrs.     (n  =  6)  in  the 
pretreatment  period  and  was  0.585  +  0.070  ug/  24hrs.  after  one  month 
of  therapy.     No  significant  changes  in  plasma,  BUN,  creatinine,  sodium 
or  potassium  had  occurred  after  one  month  of  therapy  in  6  dogs  and  after 
2  months  of  therapy  in  3  dogs.     The  renal   histology  has  been  reviewed 
and  a  JGI  determined  for  each  slide  but  the  code  will  not  be  broken 
until   the  study  is  completed. 

Keyword  Descriptors:    :    .; 

plasma  renin  activity  (PRA),  spironolactone 

Honors  and  Awards:        ,~,    ..    .. 


Publications 


¥tf 


Project  No.  ZQ1  HL  01811-01 

1.  Hypertension- Endocrine  Branch 

2.  Steroid  &  Mineral  Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual   Project  Report 
July  1,  1974  through  June  30,   1975 

Project  Title:     Metabolism  of  albumin  on  patients  with  idiopathic  edema. 

Principal    Investigators :     Dominguez,  M.,  M.D. 

Gill,  Jr.,  J.   R.,  M.D. 

Other  Investigators: 

Cooperating  Units: 

Project  Description: 

Our  previous  work  has  shown  that  total  circulating  pool  of  albumin 
(TCA)  is  significantly  less  than  in  normal  women.  The  low  value  for 
TCA  was  associated  with  an  increase  in  fractional  catabolic  rate  per  day  of 
albumin  and  occasionally  with  a  decreased  rate  of  synthesis  of  albumin. 
The  presence  or  absence  of  hypoalbuminemia  is  being  correlated  with  the 
morning  to  evening  weight  gain  as  outpatient. 

Patients  are  evaluated  as  regards  to  thyroid,  liver  or  kidney  functions, 

When  sodium  excretion  is  stable  on  a  sodium  take  of  109  mEq  per  day,  5  uCi 

131 
of   I  Serum  albumin  is  given  intravenously  and  aliquots  of  blood  and 

urine  are  collected  for  seven  days. 

a)  Plasma  volume:  Injected  doses  in  cp  10  minutes/serum  cp  lO'-BKG. 

b)  Plasma  volume  nl/kg=  Plasma  volume/TBW 

c)  Total  circulating  albumin=  P.V.  ml/kg  X  albumin  concentration. 

d)  Fractional  CataboTic  Rate  is  calculated  as  the  total  of  counts  in 
the  urine  divided  by  the  plasma  counts  divided  by  plasma  volume. 

e)  Synthetic  rate  is  obtained  as  TCA  X  FCR. 


&o 


Results 


Numbers 


TCA 
gm/kg 


FCR 


SR 
gm/kg/ day 


1 .  Normal 
women 
mean 

2.  Idiopathic 
edema 


10 


a)  normal 
3 


1.61 


1.58 


b)  hyper- 
catabolic 
hypo- 
alb  uminemi  a 

2        1.25 

c)  hypospynthetic 
hypo- 
albuminemia 

fl        1 .37 


,11 


108 


144 


,096 


.18 


17 


.18 


.132 


Three  of  six  patients  with  a  complaint  of  fluids  retention  have  a 
decrease  in  TCA,  The  cause  of  the  decrease  was  an  increase  in  catabolism 
of  albumin  in  tw©;and  a  decrease  in  synthesis in-one.  One  of  the 
patient  with  normal  albumin  metabolism  had  hypoplasia  of  the  lymphatics 
in  both  legs.  The  basis  for  tne  complaint. ofj edeme  in  the  other  two 
is  uncertain  but  tabulation  of  morning  and  evening  weights  indicate  little 
postural  weight  gain  (2  lbs.  or  less)  which  is  the  hallmark  of  the  disorder. 

Keyword  Descriptors:  r        y   - 

idiopathic  edema,  albumin,  hypoalbuminemia 

Honors  and  Awards: 

Publications: 


¥9f 


Project  No.   z01  HL  01812-01 

1 .  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral  Metabolism 

3.  Bethesda,  Maryland 


PHS-rilH 
Individual   Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:     Coronary  artery  disease  and  urinary  steroids 

Previous  Serial   Number: 

Principal    Investigator:     Licata,  A.  A.,  M.D.,  Ph.D. 

Other  Investigators:     Vestergaard,  P.,  M.D. 
Bartter,   F.  C,  M.D. 

Project  Description: 

It  has  been  reported  that  patients  with  myocardial   infarctions 
excrete  elevated  urinary  levels  of  11-ketoetiocholanolone,  11-B-hydroxy- 
androsterone,   11-B-hydroxyetiocholanolone.     This  project  attempts 
to  verify  these  findings  and  to  determine  if  patients  with  coronary 
artery  disease,  but  without  myocardial   infarct,  also  excrete  increased 
amounts  of  steroid  in  comparison  to  a  control   group.     Thirty  patients 
have  volunteered  for  the  project  and  comprise  one  of  three  groups- 
normals,  coronary  artery  disease  without  infarct,  coronary  artery  disease 
with  infarct.     Preliminary  data  from  two  patients  in  each  group  have 
been  collected.     These  data  are  being  analyzed  to  determine  the  optimum 
number  of  urine  specimens  needed  to  insure  statistical   validity  for  the 
remainder  of  the  groups. 

Keyword  Descriptors: 

Coronary  artery  disease;  steroids 

Honors  and  Awards: 

Publications: 


*{9> 


Project  No.        ZQ1  HL  Q^l-ou 

1 .  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral   Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual   Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:     Nephrogenous  cyclic  AMP  as  a  parathyroid  function 
test. 

Previous  Serial   Number:     None. 

Principal    Investigator:     Broadus,  A.E.,  H.D.,  Ph.D. 

Other  Investigators:     Bartter,   F.C.,  M.D. 

Cooperating  Units:     Mahaffey,  J.E.,  M.D.  and  Neer,  P.M.,  M.D., 

Endocrine  Unit,  Massachusetts  General   Hospital, 
Boston,  Mass. 

Project  Description: 

The  central  objective  of  this  research  has  been  to  demonstrate 
that  the  measurement  of  nephrogenous  cyclic  AMP  is  a  valid  index  of 
parathyroid  function.     The  physiologic  rationale  of  this  analysis 
was  provided  in  detail   in  a  previous  Project  Report  (1973-74)  and 
will   not  be  reviewed  here. 

Initially,  we  examined  the  diagnostic  utility  of  nephrogenous 
cyclic  AMP  in  a  pilot  series  of  approximately  TO  hyperpara thyroid 
patients  and  several   chronically  hypoparathyroid  patients.     The 
results  were  sufficiently  encouraging  that  our  objective  has  changed 
to  the  collection  of  a  large  clinical   series  designed  to  firmly 
establish  the  usefulness  of  nephrogenous  cyclic  AMP  in  parathyroid 
disease   (see  Major  Findings,  below). 

During  the  course  of  these  investigations  it  has  become  apparent 
that  nephrogenous  cylcic  AMP  might  be  uniquely  useful   in  studying 
normal   parathyroid  physiology  and  subtle  errors  in  calcium  metabolism 
(see  below).     Therefore,  in  the  past  4  months  an  objective  of  our 
work  has  been  the  study  of  patients  with  hypercalciuria  as  well   is 
normal   subjects  under  a  variety  of  circumstances. 

Methods  Employed: 

Plasma  and  urinary  cyclic  AMP  are  measured  by  radioligand  and 
assay  after  column  chromatography.     Plasma  immunoreactive  PTH  is 
measured  by  a  well -character!' zed  antiserum  (GP-1).     Other  analyses 


are  routine. 

Standard  calcium  infusions  (4  mg/kg/hour)  are  performed  in  selected 
mild  cases  of  hyperparathyroidism  and  in  occasional  hypercalciuric 
patients,  with  determinations  of  nephrogenous  cyclic  AMP  and  PTH. 

Major  Findings: 

Data  is  presently  available  from  10  patients  with  hypoparathyroidism, 
35  control  subjects  and  39  cases  of  hyperparathyroidism.  The 
completed  series  will  contain  10,  50  and  45-50  patients  in  the  3  groups, 
respectively.  In  addition,  approximately  8  patients  with  non-parathyroid 
hypercalcemia  have  been  studied.  The  clearance  ratio  of  cyclic  AMP 
to  creatinine  is  the  simplest  expression  of  nephrogenous  cyclic  AMP, 
values  progressively  greater  than  unity  indicating  increasing  renal 
contributions  of  the  nucleotide.  The  results  +  SEM  are: 


Patients  (n) 


HypoPT   (10) 
Normal    (35) 
HyperPT   (39) 
HyperPT,  mild   (14) 
HyperPT,  azotemic   (8) 


Serum  Ca 

PTH# 

CcAMP/Ccr 

Urinary  cAMP 

(mq/dl ) 

(uleq/ml ) 
34  +  2** 

(nmole/min) 

7.9  +  0.3 

1.23  +  0.03 

1.91  +  0.15 

9.5+  0.1 

59  +  3 

1.81  +  0.12 

2.82  +  0.21 

11.4  +  0.2 

156  +  28 

3.64  +  0.13 

4.80  +  0.34 

10.5  +  0.1 

93  +  6 

3.42  +  0.11 

4.43  +  0.37 

12.1  +  0.6 

299  +  76 

3.71  +  0.24 

3.03  +  0.57 

#normal    range  PTH  30-30  uleq/ml 
♦♦undetectable  in  5  patients 

In  the  table  above,  the  hyperparathyroid  group  is  subdivided  into 
14  patients  with  mild  disease   (serum  calcium       10.8  mg/dl )  and  8  patients 
with  moderate  renal    failure   (creatinine  clearance  39  +3  ml/min).      In 
all   cases,  the  clearance  ratio  was  greater  than  2.8;  PTH  was  in  the 
normal    range  in  about  20%  of  cases.     From  this  series  we  have  concluded 
that  nephrogenous  cyclic  AMP  is  a  sensitive,  reliable  and  useful   tool 
for  study  of  the  spectrum  of  parathyroid  disease. 

Although  urinary  cyclic  AMP  is,  on  the  average,  derived  in  roughly 
equal   parts  from  the  circulation   (by  glomerular  filtration)  and  from 
the  nephrogenous  pool,  we  have  found  that  plasma  cyclic  AMP  varies  over 
only  a  2  1/2  to  3  -  fold  range  in  spite  of  filtration  rates  which  vary 
over  a  10-fold  range  (15-150  ml/min).     This  suggested  that  correlation 
of  cyclic  AMP  excretion  with  simultaneously  determined  creatinine 
clearance   (nmoles  cyclic  AMP  per  100  ml   GFR)  might  provide  a  strikingly 
simple  test   (plasma  cyclic  AMP  analysis   is  not  required),  which  would 
be  of  wide  availability  and  utility.     Our  data  indicates  that  this  simple 
determination  is  extremely  useful   and  will   likely  become  the 
preferred  analysis  and  means  of  data  expression  in  the  near  future. 


¥?¥ 


Patients  with  mild  hyperparathyroidism  may  offer  extreme  difficulties 
in  differential  diagnosis,  in  large  measure  because  of  their  tendency  to 
present  with  only  infrequent  episodes  of  frank  hypercalcemia.- 
In  5  such  patients,  we  have  found  that  a  standard  calcium  infusion 
fails  to  suppress  nephrogenous  cyclic  AMP  normally.  PTH  analyses 
during  these  infusions  were  difficult,  or  impossible,  to  interpret 
(as  noted  by  others). 

There  is  much  controversy  concerning  the  pathogenesis  and 
treatment  of  idiopathic  hypercalciuria,  owing  largely  to  conflicting  and 
inadequate  methology  in  the  study  of  such  patients.  Although  our 
findings  in  these  patients  are  preliminary,  it  appears  that  nephrogenous 
cyclic  AMP  offers  the  most  sensitive  approach  to  the  study  of  this 
disorder.  There  investigations  will  form  a  major  part  of  our  objective 
during  the  coming  year. 

Keyword  Descriptors: 

parathyroid,  cyclic  AMP 

Honors  and  Awards: 


Publications: 

Two  manuscripts  are  currently  in  preparation. 


y?r 


Project  M.  Z01  HL  01822-01 


1 .  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral  Metabolism 

3.  Rethesda,  Maryland 

PHS-MIH 
Individual    Project  P.eoort 
July  1,   1974  through  June  30,   1975 

Project  Title:   Aminoglycoside  effects  on  urinary  calcium. 

Previous  Serial   dumber: 

Principal    Investigator:     Licata,  A.,  M.D.,  Ph.D.;  ^cMurtry,  R.,  M.D.,  Ph.D. 

Other  Investinators:     Bartter,   F.C.,  M.D. 

Cooperatinn  Units: 

Project  Description: 

Preliminary  in  vivo  data  from  studies  with  rats  showed  that  acute  dose 
of  aminoolycosides  altered  urinary  calcium  clearance.     We  investigated  this 
possibility  in  normal    volunteers  by  placina  them  on  a  constant  calcium  diet 
and  then  monitoring  their  urinary  Ion  of  electrolytes  after  a  24-hour  dose 
of  gentamycin  and  kanamycin.     Preliminary  results  sungest  that  these  drugs 
caused  a  decrease  in  urinary  calcium  and  sodium. 


Day 

iia 
120+ 

Gentamycin 
K 

58 

Ca 
267 

'la 
171 

Kanamycin 

V 

t4 

Ca 
143 

2 

99 

60 

236 

93 

74 

136 

3 

58 

70 

199 

67 

70 

141 

*4 

47 

57 

194 

90 

69 

140 

5 

44 

63 

163 

52 

71 

94 

6 

43 

62 

169 

67 

68 

110 

*drug  given 
values  are  mean  results  from  two  volunteers 

Keyword  Descriptors: 

aminoglycosides,   renal   electrolytes 

Honors  and  Awards: 

Publications: 


4<& 


Project  No.        ZQ1  HL  01823-01 

1.  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral   Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Vitamin  D  metabolism  in  renal  stone  disease. 

Previous  Serial  Number: 

Principal  Investigator:  Licata,  A.,  M.D.,  Ph.D.;  Bartter,  F.C.,  M.D. 

Other  Investigators: 

Cooperating  Units: 

Project  Description: 

A  number  of  clinic  patients  with  nephrolithiasis  are  being  seen,  some  of 
whom  have  hyperabsorption,  and  others  idiopathic  hypercalciuria.  We  propose 
to  look  at  the  vitamin  D  status  of  these  two  classes  of  patients  by  developing 
a  suitable  vitamin  D  assay.  This  project  consists  of  the  establishment  of  a 
25-hydroxycholecalciferol  receptor  assay  and  its  subsequent  use  in  screening 
these  patient  populations.  Although  the  development  of  the  assay  is  underway 
there  are  no  data  to  report  at  this  time. 

Keyword  Descriptors: 

vitamin  D  status  in  nephrolithiasis 

Honors  and  Awards: 
Publications: 


497 


Project  Mo.   Z01  hl  01824-01 

1.  Hypertension-Endocrine  Branch 

2.  Steroid  &  Mineral   Metabolism 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Chemical  regulators  of  parathyroid  gland  secretion. 

Previous  Serial  Number: 

Principal  Investigator:  Licata,  A.,  M.D.,  Ph.D.;  Bartter,  F.C.,  M.D. 

Other  Investigators: 

Cooperating  Units: 

Project  Description: 

Recent  scientific  literature  has  suggested  the  possibility  that  the 
secretion  of  parathyroid  hormone  can  be  controlled  by  certain  vitamin  D 
metabolites.  This  implies  that  therapeutically  it  may  be  feasible  for 
specifically  designed  drugs  to  modify  disease  of  the  parathyroid  glands. 
A  preliminary  step  in  this  direction  is  the  establishment  of  an  in  vitro 
culture  system  to  test  the  effect  of  specific  drugs  on  parathyroid  gland 
function.  This  has  been  commenced.  No  data  available  yet. 

Keyword  Descriptors: 

parathyroid  gland  metabolism  in  vitro 
Honors  and  Awards: 
Publications: 

None 


m 


Project  No.   zoi  hl  01825-01 

1.  Hypertension -Endocrine  Branch 

2.  Steroid  &  Mineral  Metabolism 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Evaluation  of  sodium  cellulose  phosphate  (S.C.P.) 

Previous  Serial  Number: 

Principal  Investigator:  Dominguez,  M.,  M.D. 

Other  Investigators:  Licata,  A.  A.,  M.D.,  Ph.D. 

Cooperating  Units: 

Project  Description: 

To  determine  the  efficacy  of  S.C.P.  in  a  long-term  study  (one  year), 
of  patients  with  hypercalciuria  secondary  to  intestinal  hyperabsorption 
of  calcium. 

The  patients  were  characterized  in  the  basis  of  24  hour  urinary 
calcium  over  20C  mg,  on  a  calcium  diet  of  600  mg.  Normal  or  low  PTH 
serum  concentration.  Low  cAMP  urinary  excretion  and  high  calcium  absorption 
after  the  administration  of  47-Ca. 

Nine  patients  were  identified  with  this  disorder  and  started  on  S.C.P. 
5  gm  three  times  a  day.  Every  four  months  they  have  been  evaluated  as 
outpatient  and  at  the  end  of  one  year  treated  as  inpatient. 

The  evaluations  included  some  determinations  in  blood  such  as  Ca, 
P,  Mg,  alkaline  phosphatase,  MS,  Ht.,  WBC,  liver  function  test,  creatinine 
clearance,  cooper,  iron,  zinc,  PTH  and  in  urine  Ca,  P,  Mg,  Ma,  K,  creatinine, 
cAMP,  and  zinc.  A  plain  film  of  abdomen  was  taken  at  the  same  time,  too. 

Major  Findings:  Good  tolerance  to  S.C.P.,  no  evidence  of  new  stones 
were  found  during  this  period,  and  no  side  effects  in  relation  with  the 
treatment. 

The  study  will  be  completed  in  August,  1975. 


¥ff 


Keyword  Descriptors: 

sodium  cellulose  phosphate,  hypercalciuria,  nephrolithiasis 
Honors  and  Awards : 

Publcations: 

Pak,  C.Y.C.,  Delea,  C.S.,  and  Bartter  F.C.  Treatment  of  recurrent 
nephrolithiasis  with  cellulose  phosphate.  New  England  J.  Med.  290: 
175-187,  1974. 


gco 


Project  No.   Z01  HL  01841-01  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Binding  and  Metabolic  Effects  of  Neurotoxic  Drugs 

Previous  Serial  Number:   None 

Principal  Investigator:   Donald  F.  Bogdanski,  Ph.D. 

Other  Investigators:   Walter  Lovenberg,  Ph.D. 
Hans  G.  Baumgarten,  M.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives :   Neurotoxic  drugs  are  currently  in  wide  use  in  pharmacological 
laboratories  because  of  their  ability  to  destroy  specific  cells  in  the  central 
nervous  system.   The  cells  destroyed  belong  to  specific  cell  groups  having  a 
particular  neurotransmitter.   At  present,  the  mechanism  of  action  of  the 
neurotoxic  drugs  is  unknown.   Evidence  reported  from  this  laboratory  suggests 
that  specificity  is  a  matter  of  uptake  by  the  cell.   The  drugs  may  be 
generally  toxic  to  all  cells  provided  they  penetrate  to  the  interior.   The 
purpose  of  the  present  investigations  was  to  determine  the  mechanism  of 
toxicity  and  describe  it  at  a  molecular  level  if  possible.   It  was  thought 
that  the  toxicity  might  be  related  to  the  formation  of  quinones.   If  so,  the 
site  of  a  generalized  toxicity  might  be  mitochondria,  in  which  the  transport 
of  electrons  from  NADH  is  inhibited  by  certain  quinones,  such  as  rotenone. 
This  idea  served  as  the  starting  point  for  the  experiments  to  be  described. 

14 
Materials  and  Methods:   Radioactive  C  -labeled  5,6-dihydroxytryptamine  and 

5, 7-dihydroxytryptamine,  and  C-^-parachloroamphetamine  were  synthesized  and 

supplied  by  Dr.  K.  Schlossberger  of  the  Max  Planck  Institute,  Germany. 

The  subcellular  distribution  of  labeled  drugs  in  rat  brain  was  investigated 
by  density  gradient  centrifugation.   The  12,000  x  g  (P2)  pellet  was  re- 
suspended  and  layered  over  a  discontinuous  density  gradient  consisting  of  1.4, 
1.2,  1.0,  0.8  M  sucrose  and  the  P2  suspension  in  0.32  M  sucrose.   After  90 
minutes  centrifugation  at  56,000  x  g,  the  particles  fractionate  at  the  various 
interphases,  from  low  to  high  density,  as  follows:   A,  myelin;  B,  membranes 
and  small  synaptosomes;  C,  synaptosomes  and  small  mitochondria;  D,  mitochondria 
and  large  synaptosomes;  E,  nonspecific  debris. 

The  gradient  is  drained  into  counting  vials,  5  drops  per  vial,  and  the  radio- 
activity in  each  vial  determined  by  scintillation  counting. 


Shf 


Project  No.   Z01  HL  01841-01  HE 

Major  Findings:   The  different  drugs  used  thus  far  can  be  classified  according 
to  their  binding  in  specific  cellular  subfractions .   Three  distinct  classifi- 
cations were  apparent:  1)  both  5,6-  and  5,7-dihydroxytryptamine  were  localized 
in  the  mitochondrial  fraction.   Moreover,  5,7-dihydroxytryptamine  was 
apparently  covalently  bound  as  determined  by  exhaustive  extraction  with  ether 
and  methanol.   Because  of  the  tenacious  binding,  these  compounds  remained  in 
the  mitochondrial  fraction   for  at  least  24  hours,  at  which  time  the  radio- 
activity in  the  other  fractions  had  fallen  to  low  levels.   2)  Colchicine 
was  present  in  higher  concentrations  in  the  synaptosomal  fraction  than  in 
other  fractions.   The  rate  of  disappearance  at  1,  4,  and  24  hours  was  pro- 
portional in  all  fractions.   3)  After  24  hours  para-chloroamphetamine  was 
bound  to  the  membrane  and  the  myelin  fraction. 

In  contrast,  5-hydroxytryptamine  disappeared  from  all  fractions  within  24 
hours.   Thus,  all  neurotoxic  drugs  are  tightly  bound  to  specific  cell  frac- 
tions in  brain  tissue. 

The  data  from  experiments  with  the  dihydroxytryptamines,  together  with  earlier 
morphological  data,  suggest  a  mitochondrial  site  of  action  for  these  drugs. 
In  preliminary  experiments  5,7-dihydroxytryptamine  and  6-hydroxydopamine 
inhibited  reduction  of  cytochrome  b  in  the  presence  of  NADH  and  cyanide  in 
rat  liver  and  bovine  adrenal  medullary  mitochondria. 

Significance  to  Biomedical  Research  and  Institute  Program:   Interest  in  these 
compounds  is  related  to  their  ability  to  destroy  specific  groups  of  cells  in 
the  brain.   For  example,  the  tryptamines  can  eliminate  serotonergic  cells 
whereas  6-hydroxydopamine  can  eliminate  adrenergic  neurons.   The  specificity 
of  neurotoxicity  can  thus  be  used  to  study  the  functions  of  a  specific  group 
of  neurons  in  the  central  nervous  system. 

The  mechanism  of  toxicity  is  currently  the  object  of  a  lively  controversy  in 
biomedical  research.   The  investigations  reported  here  suggest  that  the 
dihydroxytryptamines  destroy  cells  directly  by  their  mitochondrial  toxicity. 
The  manufacture  of  vital  energy  resources,  or  other  mitochondrial  functions, 
might  be  blocked.   This  would  cause  the  death  of  the  cell.   Specificity  of 
cell  toxicity  is  determined  by  the  entrance  of  the  compound  into  the  cell. 
Serotonergic  cells  transport  these  compounds,  hence  they  are  destroyed. 

Proposed  Course  of  Project:   The  main  objective  of  future  research  is  to 
extend  observations  on  mitochondrial  toxicity.   The  inhibitory  effect  of 
neurotoxic  drugs  on  electron  transport,  respiration,  oxidative  phosphoryla- 
tion, lipid  metabolism  and  protein  metabolism  will  be  studied.   The  possible 
usefulness  of  the  dihydroxytryptamines  in  carcinoid  tumors  should  be  con- 
sidered. 

Keyword  Descriptors:   neurotoxicity      5,6-dihydroxytryptamine 
5,7-dihydroxytryptamine      synaptosomes      mitochondria 

Honors  and  Awards :   None 

Publications:   None 

2  Se» 


Project  No.   Z01  HL  01842-01  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  19  74  through  June  30,  1975 

Project  Title:   Electron  Transport  in  Bovine  Adrenal  Medullary  Vesicles 

Previous  Serial  Number:   None 

Principal  Investigator:   Donald  F.  Bogdanski,  Ph.D. 

Other  Investigators:   John  W.  Greenawalt,  Ph.D. 
Glen  Decker 

Cooperating  Units:   Johns  Hopkins  Medical  School 

Department  of  Physiological  Chemistry 
Baltimore,  Maryland 

Project  Description: 

Objectives:   This  work  was  based  on  previous  investigations  of  metabolically 
dependent  retention  of  norepinephrine  by  adrenergic  neurons  in  rat  heart 
slices.   Retention  may  be  more  complex  than  usually  described,  that  is,  a 
simple  binding  of  amine  with  ATP,  divalent  cation  and  protein.   Moreover, 
extravesicular  Mg  -ATP  is  probably  not  sufficient  to  account  for  vesicular 
retention  of  NE  as  proposed  by  the  Scandinavian  School  of  Neuro chemists. 
Their  conclusion  was  based  upon  studies  with  isolated  bovine  adrenal  medullary 
or  splenic  nerve  vesicles.   The  isolated  nerve  vesicles  do  not  retain  NE  at 
37°  even  in  the  presence  of  Mg-ATP.   Moreover,  our  investigations  revealed 
metabolic  dependencies  that  could  not  be  anticipated  from  the  results  of 
studies  with  isolated  vesicles.   Thus,  retention  was  studied  in  auto-dialyzed 
heart  slices  in  which  the  plasma  membrane  is  the  effective  dialysis  membrane. 
During  auto-dialysis,  endogenous  substrates  are  removed  from,  and  exogenous 
substrates  together  with  metabolic  inhibitors,  were  added  to  the  cytosol. 
The  vesicles  are  metabolically  isolated  and  controlled  in  their  in  vivo 
location  within  the  neuron. 

Retention  is  dependent  upon  intermediary  metabolism  and  respiration,  but  is 
independent  of  oxidative  phosphorylation.   In  the  absence  of  glycolysis, 
inhibitors  which  eliminate  oxidative  phosphorylation  may  or  may  not  block 
metabolically  dependent  retention  depending  upon  the  occurrence  of  electron 
transport.   Thus,  electron  transport  is  required  for  metabolically  dependent 
retention  (blocked  by  antimycin  A) .   Electron  transport  apparently  is  the  basis 
for  the  generation  of  energized  state  (dissipated  by  dinitrophenol)  but 
not  oxidative  phosphorylation  (not  blocked  by  oligomycin  or  by  atractyloside) . 
Based  on  these  studies,  a  decision  was  made  to  study  electron  transport  direct- 
ly in  isolated  adrenal  medullary  vesicle  membranes. 


St>3 


Project  No.   Z01  HL  01842-01  HE 

Major  Findings:   Our  studies  have  added  significantly  to  the  findings  of 
Flatmark  et  al.,  who  proposed  a  conventional  electron  transporting  chain  as 
follows : 

Dehydrogenase  -+■  Flavoprotein  -»■  Cytochrome  b^l 

-»■    Oxidase  (presumed)   ■*  Oxygen 

Our  studies  indicate  the  existence  of  a  new  cytochrome  in  the  membranes  having 
an  a  absorption  maximum  at  555  to  556  nm  and  which  is  distinct  from  cytochrome 
be  which  has  a  similar  a  absorption  band.   The  evidence  for  the  existence  of 
this  cytochrome  is  as  follows:   1)  at  room  temperature,  the  asymmetric  a  band 
is  split  in  the  presence  of  some  Krebs  cycle  intermediates,  by  low  concentra- 
tion of  dithionite  or  by  oxygenation  of  membranes  completely  reduced  by 
dithionite.   2)  The  two  absorption  maxima  show  an  independent  temporal  develop- 
ment in  the  presence  of  cyanide,  or  Ca"^  plus  cyanide,  and  3)  their  relative 
steady  state  maximum  absorption  differs  at  various  pH. 

A  second  new  finding  was  the  existence  of  a  cyanide  sensitive  factor  absorbing 
at  444  nm  in  the  presence  of  cyanide.   This  factor  may  be  similar  to  that 
earlier  reported  in  liver  microsomes  by  Sato  and  by  Gaylor.   It  also  may 
correspond  to  the  presumed  oxidase  of  Flatmark. 

A  third  new  finding  was  the  response  of  the  cytochromes  to  oxygen  and  to  Krebs 
cycle  intermediates.   Of  the  latter  group,  the  response  may  be  absent  (citrate), 
slight  (pyruvate,  isocitrate,  succinate,  fumarate  and  malate) ,  or  moderate 
(ct-ketoglutarate,  oxaloacetate) .   It  is  not  yet  known  whether  these  responses 
represent  specific  dehydrogenase  activity. 

The  cytochromes  are  oxidized  in  the  presence  of  dopamine  but  not  norepinephrine. 
The  cytochromes  are  reduced  by  pyridine  nucleotides  and  oxidized  by  ATP. 
Generally,  responses  were  not  inhibited  by  antimycin  A,  rotenone  or  dinitrophenol 
but  studies  are  incomplete. 

A  new  electron  transport  pathway  is  proposed: 

Endogenous  reducing  equivalents  ->  Cytochrome  b,-^,  -> 

Cytochrome  C^rr    c     "*"     Cyanide  sensitive  factor  -*■            O2 

The  enzymes  discussed  here  are  absent  from  storage  granules  in  mast  cell  tumors. 

Significance  to  Biomedical  Research  and  Institute  Program:   These  studies  of 
the  storage  organelles  of  the  synapse  have  the  same  general  significance  for 
the  biochemistry,  physiology  and  medical  applications  of  synaptic  junctions 
outlined  in  the  report  on  the  efflux  of  norepinephrine  from  ra';  heart  slices. 
The  finding  of  a  new  enzyme  is,  of  course,  extremely  significant  in  biochemis- 
try'. 

Proposed  Course  of  Project:   The  relationship  between  the  cyanide  sensitive 

2  $0/ 


Project  No.   Z01  HL  01842-01  HE 

factor,  oxygen,  endogenous  reducing  equivalents  and  cytochromes  will  be 
established.   Evidence  for  the  occurrence  of  these  factors  in  other  storage 
granules  will  be  sought.   The  function  of  these  cytochromes  and  electron 
transporting  factors  will  be  investigated.   Obvious  roles  such  as  the 
electron  donor  for  the  8  hydroxy lation  of  dopamine  will  be  explored.   A 
possible  relationship  between  the  electron  transporting  pathways  and 
metabolically  dependent  retention,  and  the  metabolism  of  membrane  constituents 
will  be  investigated. 

Keywords:   adrenal  medullary  vesicles       membranes      cytochromes 
electron  transport      catecholamines      cytochrome  br/i 
cyanide  sensitive  factor      cytochrome,-!-,-  c 

Honors  and  Awards:   None 

Publications :   None 


SdC 


Project  No.   Z01  HL  01843-02  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Release  of  Norepinephrine  from  Neuronal  Vesicles 

Previous  Serial  Number:   NHLI-37 

Principal  Investigator:   Donald  F.  Bogdanski,  Ph.D. 

Other  Investigators:   None 

Cooperating  Units :   None 

Project  Description: 

Objectives :  These  investigations  were  designed  to  study  some  characteristics 
of  the  amine  pump  located  at  the  plasma  membrane  of  adrenergic  nerve  endings, 
and  to  apply  the  findings  to  mechanisms  of  synaptic  transmission,  if  possible. 

Previous  publications  of  work  at  the  Laboratory  of  Chemical  Pharmacology,  NHLI, 
have  described  some  properties  of  a  saturable,  Na+-dependent  transport 
mechanism  for  norepinephrine  (NE)  in  peripheral  and  isolated  brain  nerve 
endings  (syraptosomes) .   A  modei  synapse  was  devised  based  upon  the  Ca 
dependent  efflux  of  stored  ^h-NE  from  heart  ventricle  slices  incubated  in 
Na  -deficient  (choline"1")  Krebs  bicarbonate  medium.   The  efflux  was  found  to 
be  Na+-dependent  and  inhibitable  by  desipramine,  an  inhibitor  of  the  amine 
pump.   Published  work  has  shown  that  synaptic  transmission  can  be  inhibited 
by  desipramine.   The  present  work  extends  these  findings. 

Major  Findings:   The  efflux  of  Ca"1""1"  mobilized  3H-NE  from  rat  heart  slices 
incubated  in  sodium  deficient  Krebs-HC03  (choline"1")  was  studied.   Slices  were 
prepared  from  rats  injected  with  %-NE  18  hours  previously.   The  slices  were 
incubated  60  minutes  in  standard  Krebs-HCO-j  medium  in  order  to  re-establish 
physiological  conditions.   The  slices  were  then  transferred  to  the  Na+-def icient 
media.   Previous  work  has  established  that  the  %-NE  is  bound  within  the 
endogenous  pool  after  18  hours.   This  amine  is  mobilized  by  Ca"1""1"  and  appears 
in  the  Na  -deficient  medium  from  which  85%  of  the  released  radioactivity  is 
recovered  as  the  free  base. 

The  rate  of  efflux  was  greatly  reduced  by  1  to  3  uM  desipramine  and  1  to  3  uM 
cocaine.   In  contrast,  phenoxyb en z amine  at  1  to  100  uM  slightly  reduced  the 
rate  of  efflux.   The  negative  result  of  the  phenoxybenzamine  experiment  is  of 
interest  because  all  three  drugs  are  reported  to  be  potent  inhibitors  of  amine 
transport. 

At  this  point  there  are  two  main  considerations  for  the  observed  inhibition 

1  SB6 


Project  No.   Z01  HL  01843-02  HE 

of  efflux.  The  inhibitors  act  either  upon  the  storage  vesicle  or  upon  the 
amine  pump,  which,  as  we  have  published,  may  transport  intracellular  NE  to 
the  extracellular  medium. 

The  possibility  that  the  above  results  are  accounted  for  by  a  vesicular  site 
of  action  can  be  minimized  by  reports  of  other  laboratories.   Thus,  cocaine, 
at  100  uM,  produces  only  25%  inhibition  of  the  efflux  of  catecholamine  from 
isolated  splenic  nerve  vesicles.   This  concentration  is  far  greater  than  the 
range  of  concentrations  that  inhibit  efflux.   Generally,  a  disparity  in 
effective  concentrations  applies  to  desipramine  as  well.   In  contrast, 
phenoxybenzamine  is  a  potent  inhibitor  of  efflux  from  isolated  vesicles,  but 
had  little  effect  in  the  above  experiments.   Thus,  the  site  of  inhibition- of 
efflux  produced  by  desipramine  and  cocaine  is  the  amine  pump  in  the  plasma 
membrane.   The  weak  effect  of  phenoxybenzamine  indicates  that  this  drug  does 
not  block  the  amine  pump  operating  in  an  outward  direction.   Since  attachment 
of  inhibitor  to  carrier  at  the  outer  surface  of  the  membrane  would  slow  trans- 
location in  either  direction,  it  appears  likely  that  phenoxybenzamine  has  only 
a  weak  effect  on  the  transport  mechanism.   This  is  in  direct  contrast  to 
current  thinking. 

Significance  to  Biomedical  Research  and  Institute  Program;   Investigations  of 
the  physiological  and  biochemical  characteristics  of  nerve  endings  generally 
are  of  great  interest  because  synaptic  transmission  is  crucial  to  either  the 
initiation,  control  or  maintenance  (or  all  three)  of  all  bodily  functions, 
both  physical  and  mental.   Synaptic  transmission  is  the  point  of  attack  for 
many  drugs  currently  used  for  a  wide  variety  of  illnesses  ranging  from  hyper- 
tension to  mental  disease.   Fundamental  knowledge  of  the  nature  of  the  bio- 
chemical processes  controlling  synaptic  transmission  is  crucial  to  the 
understanding  of  the  actions  of  existing  drugs,  and  the  development  of  drugs. 

The  findings  reported  above  indicate  that  one  aspect  of  the  currently  proposed 
mechanism  of  the  action  of  one  drug  was  erroneous.   Simultaneously,  these 
findings  open  a  door  to  new  knowledge.   Thus,  why  does  phenoxybenzamine  inhibit 
uptake  of  amine  if  it  neither  blocks  amine  transport,  nor  deplete  stores  of  amine 
from  the  nonstimulated  nerve  like  reserpine?   Is  the  answer  to  this  question 
related  to  the  mechanism  by  which  phenoxybenzamine  stimulates  overflow  of  NE 
from  electrically  stimulated  adrenergic  nerves? 

The  findings  reported  above  together  with  published  reports  of  inhibition  of 
synaptic  transmission  by  desipramine  and  apparent  inhibition  of  overflow  by 
cocaine,  suggest  that  synaptic  transmission  includes  a  component  of  outward 
transport  of  NE,  which  can  be  blocked  by  transport  inhibitors. 

Proposed  Course  of  Project:   The  differences  between  the  effects  of  desipramine 
and  cocaine,  on  the  one  hand,  and  phenoxybenzamine  on  the  other  will  be  ex- 
ploited to  establish  the  existence  of  an  outward  transport  capability  in 
adrenergic  nerve  endings.   Experiments  with  ouabain  and  reserpine,  together 
with  the  drugs  will  help  characterize  the  amine  transport  mechanism. 


SOT 


Project  No.   Z01  HL  01843-02  HE 

Keyword  Descriptors:   adrenergic  nerve,  endings      storage  of  norepinephrine 
outward  transport  of  nerve  transmitter      desipramine      cocaine 
phenoxybenzamine 

Honors  and  Awards :   None 

Publications : 

Bogdanski,  D.F.  and  Blaszkowski,  T.P.:   Role  of  extravesicular  adenosine 
triphosphate  and  apparent  vesicular  energy  conservation  reactions  in 
retention  of  norepinephrine  by  adrenergic  nerve  endings.   Neuropharmacology 
14:  11-20,  1975. 


$36 


Project  No.   Z01  HL  01844-01  HE 


1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Receptors  Participating  in  the  Induction  of 
Tyrosine  Hydroxylase 

Previous  Serial  Number:   None 

Principal  Investigator:   Ingeborg  Hanbauer,  Ph.D. 

Other  Investigators:   A.  Guidotti,  M.D. 
E.  Costa,  M.D. 

Cooperating  Units:   Laboratory  of  Preclinical  Pharmacology 
National  Institute  of  Mental  Health 
St.  Elizabeth's  Hospital 
Washington,  D.C. 

Project  Description: 

Objectives :   In  superior  cervical  ganglia  an  increased  impulse  traffic  in  the 
preganglionic  nerve  fibers  causes  an  induction  of  TH  about  48  hours  after  the 
stimulation.   Decentralization  of  the  superior  cervical  ganglion  or  pretreat- 
ment  with  nicotinic  receptor  blockers  curtails  this  increase  in  TH  activity. 
This  project  was  carried  out  to  define  the  role  of  nicotinic  and  muscarinic 
receptors  in  the  induction  of  TH  in  intact  and  decentralized  superior  cervical 
ganglia. 

Methods :   cAMP  and  cGMP  concentration  and  tyrosine  hydroxylase  activity  were 
measured  as  described  in  the  previous  report. 

Major  Findings:   Utilizing  pharmacological  agonists  and  antagonists  specific 
for  various  receptors  it  has  been  possible  to  obtain  a  better  understanding 
on  the  participation  of  cholinergic  receptors  in  the  regulation  of  TH 
induction.   Nicotine  causes  a  marked  decrease  of  cGMP  content  in  SCG.   Direct 
activation  by  nicotine  (50  ymol/kg)  of  nicotinic  receptors  elicits  an  increase 
in  TH  activity  48  hours  after  injection.   However,  a  higher  dose  of  nicotine 
(100  ymol/kg),  which  presumably  also  activates  muscarinic  receptors,  fails  to 
induce  TH.   We  therefore  studied  the  effect  of  methacholine,  a  muscarinic 
receptor  agonist,  and  found  that  it  prevented  the  increase  of  TH  activity. 
If  atropine  is  used  to  block  muscarinic  receptors  higher  concentrations  of 
nicotine  also  induce  TH.   Moreover,  atropine  facilitates  TH  induction  by 
nicotine  in  decentralized  superior  cervical  ganglia.   A  scheme  of  the  pertinent 
synaptic  connections  involved  in  the  regulation  of  TH  induction  is  shown: 


SD? 


Project  No.   Z01  HL  01844-01  HE 


Nicotinic 


Neuron 


Interneuron 


Muscarinic 


Reserpine  stimulates  indirectly  the  nicotinic  receptor.   However,  in  the  case 
of  reserpine  this  interaction  by  muscarinic  receptor  agonists  is  difficult  to 
demonstrate,  since  reserpine  depletes  the  catecholamines  from  their  storage 
sites. 

Proposed  Course  of  Project:   Studies  on  guanylate  cyclase  during  stimulation 
of  muscarinic  and  nicotinic  receptors  will  be  undertaken  to  elucidate  the 
molecular  mechanisms  of  these  receptor  functions.   More  detailed  studies  on 
a-  and  ^-adrenergic  receptor  agonists  and  antagonists  will  be  required. 


Keyword  Descriptors: 
sympathetic  ganglia 


tyrosine  hydroxylase 
nicotinic  receptors 


enzyme  induction 

muscarinic  receptors 


Honors  and  Awards :   None 


Publications:   None 


ST/0 


Project  No.   Z01  HL  01845-01  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Regulation  of  Tyrosine  Hydroxylase  by  Steroid 

Receptors 
Previous  Serial  Number:   None 
Principal  Investigator:   Ingeborg  Hanbauer,  Ph.D. 

Other  Investigators:   Walter  Lovenberg,  Ph.D. 
A.  Guidotti,  M.D. 
E.  Costa,  M.D. 

Cooperating  Units:   Laboratory  of  Preclinical  Pharmacology 
National  Institute  of  Mental  Health 
St.  Elizabeth's  Hospital 
Washington,  D.C. 

Project  Description: 

Objectives :   It  has  been  reported  that  enzyme  induction  in  various  tissues  is 
under  the  control  of  corticosteroids  and  it  is  now  widely  accepted  that 
responses  to  carbohydrate  active  steroids  are' mediated  by  specific  steroid 
receptor  molecules  in  the  cytoplasm.   The  steroid-receptor  complex  is  then 
transported  to  the  nucleus,  where  subsequent  interaction  with  repressor 
proteins  presumably  alters  transcriptional  processes. 

Methods :  cGMP  concentration  was  measured  by  -^P-incorporation  into  histones 
catalyzed  by  a  cGMP  dependent  protein  kinase.  TH  activity  was  measured  by  a 
radiochemical  technique  with  6-methyltetrahydropterin  as  cof actor. 

Major  Findings:   Dexamethasone  induces  TH  in  sympathetic  ganglia,  but  not  in 
adrenal  medulla.   This  induction  is  preceded  by  a  sharp  decrease  in  cGMP 
concentration.   Reserpine  elicits  an  increase  of  plasma  corticosterone  levels 
lasting  for  at  least  6  hours.   Similarly  to  dexamethasone,  reserpine  causes  a 
fall  in  cGMP  content  immediately  after  its  administration.   The  induction  of 
TH  elicited  by  dexamethasone  or  reserpine  was  blocked  by  pretreatment  with 
cortexolone,  a  metabolite  of  corticosterone  with  high  affinity  for  the  steroid- 
receptor  protein  but  without  intrinsic  glucocorticosteroid  activity. 

Significance  to  Biomedical  Research  and  Institute  Program:   Our  results 
demonstrate  that  corticosteroids  can  reduce  the  cGMP  levels  in  superior 
cervical  ganglion.   Considering  an  antagonistic  role  of  cGMP  on  cAMP 
dependent  processes  in  several  cellular  systems,  we  can  propose  an  inter- 
action of  glucocorticosteroids  with  the  cyclic  nucleotide  system.   This 
may  be  important  in  elucidating  mechanisms  whereby  catecholaminergic  cells 
change  their  macromolecular  composition  to  adapt  to  persistent  environmental 
changes . 

i  S'lt 


Project  No.   Z01  HL  01845-01  HE 

Proposed  Course  of  Project:   We  intend  to  elucidate:   1)  the  role  of  carbo- 
hydrate active  steroids  on  the  guanylate  cyclase  system,  2)  the  role  of  cGMP 
in  the  regulation  of  the  phosphorylated  state  of  non-histone  nuclear  pro- 
teins . 

Keyword  Descriptors:   tyrosine  hydroxylase      cortexalone 
corticosterone 

Honors  and  Awards:   None 

Publications: 

1.  Hanbauer,  I.,  Guidotti,  A.,  and  Costa,  E. :   Dexamethasone  induces 
tyrosine  hydroxylase  in  sympathetic  ganglia  but  not  in  adrenal 
medulla.   Brain  Res.  85:  527-531,  1975. 

2.  Hanbauer,  I.,  Lovenberg,  W. ,  Guidotti,  A.,  and  Costa,  E. :   Role  of 
cholinergic  and  glucocorticosteroid  receptors  in  the  tyrosine 
hydroxylase  induction  elicited  by  reserpine  in  superior  cervical 
ganglion.   Brain  Res. ,  in  press. 


&> 


Project   No.      Z01  HL   01846-01  HE 


1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Regulation  of  Tyrosine  Hydroxylase  in  Carotid  Body 

Previous  Serial  Number:   None 

Principal  Investigator:   Ingeborg  Hanbauer,  Ph.D. 

Other  Investigators:   Walter  Lovenberg,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives :   The  carotid  body  senses  physiological  oscillations  of  arterial 
p02,  PCO2  and  [H+] .   Fluorescence  histochemistry  has  shown  that  the  carotid 
body  contains  catecholamines,  mainly  dopamine,  and  indolealkylamines . 
The  transmitters  are  located  in  vesicles  in  the  type  I  cells.   It  appears 
that  the  chemoreception  for  p02,  pC02  and  [H+]  is  carried  out  by  three 
distinct  receptor  mechanisms.   Our  strategy  to  obtain  information  on  specific 
transmitters  involved  in  the  various  types  of  chemoreception  was  to  study 
compensatory  mechanisms  by  specific  rate  limiting  enzymes  for  the  bio- 
synthesis of  each  neurotransmitter. 

Methods :   Rats  are  kept  in  a  sealed  chamber  with  a  defined  volume  of  atmosphere 
selected  as  appropriate  to  elicit  severe  hypoxia  and  hypercapnia  in  about  30 
minutes.   Tyrosine  hydroxylase  activity  was  measured  using  a  modification  of 
the  method  described  by  Waymire  et  al.  (Analyt.  Biochem.  43:588-600,  1971) 
with  6-methyltetrahydropterin  as  cofactor. 

Maj or  Findings :   When  rats  are  kept  in  hypoxic  conditions  tyrosine  hydroxylase 
activity  is  increased  20  hours  later  reaching  about  twice  the  values  of  control 
rats.   If  rats  are  pretreated  with  apomorphine  (5  mg/kg  i.p.),  a  dopamine- 
receptor  agonist,  the  increase  in  tyrosine  hydroxylase  activity  elicited  by 
hypoxia  and  hypercapnia  is  completely  abolished.   However,  the  increase  in 
tyrosine  hydroxylase  activity  is  not  abated  by  pretreatment  of  rats  with  3- 
receptor  agonists,  cholinergic  receptor  agonists  or  by  specific  antagonists 
for  dopamine,  norepinephrine  or  acetylcholine  receptors. 

Significance  to  Biomedical  Research  and  Institute  Program:   From  these  studies 
the  working  hypothesis  can  be  adopted  that  dopamine  is  involved  in  the  function 
of  chemoreceptors,  which  are  stimulated  by  either  hypoxia  or  hypercapnia,  or 
both  simultaneously.   Since  tyrosine  hydroxylase  is  the  rate  limiting  enzyme 
in  the  biosynthesis  of  dopamine  the  regulation  of  its  induction  or  activation 


r/j 


Project  No.   Z01  HL  01846-01  HE 

must  be  controlled  by  hypoxia  and  hypercapnia. 

Proposed  Course  of  Project:   The  following  experimental  approaches  will  be 
undertaken:   1)  Further  examination  of  nature  of  the  increase  in  tyrosine 
hydroxylase  activity.   By  an  immunoprecipitation  technique  it  will  be 
established  whether  new  synthesis  of  tyrosine  hydroxylase  molecules  is 
elicited  by  hypoxia  or  hypercapnia.   The  involvement  of  second  messengers  in 
tyrosine  hydroxylase  induction  which  may  be  activating  specific  protein 
kinases  will  be  investigated. 

2)  Studies  on  the  role  of  type  II  cells  in  the  regulation  of  chemoreceptors 
with  the  possible  involvement  of  specific  amino  acids  such  as  GABA,  glycine 
and  taurine  will  be  done. 

3)  The  responsiveness  of  chemoreceptors  to  hypoxia  in  carotid  body  of 
spontaneous  hypertensive  rats  (SHR)  will  be  studied. 

Keyword  Descriptors:   carotid  body      hypoxia      tyrosine  hydroxylase 
dopamine 

Honors  and  Awards :   None 

Publications:   None 


&<* 


Project  No.   Z01  HL  01847-01  HE 

1.  Hypertension- Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:    Role  of  Second  Messengers  in  Tyrosine  Hydroxylase 
Induction 

Previous  Serial  Number:   None 

Principal  Investigator:   Ingeborg  Hanbauer,  Ph.D. 

Other  Investigators:   E.  Costa,  M.D. 

A.  Guidotti,  M.D. 

Cooperating  Units:   Laboratory  of  Preclinical  Pharmacology 
National  Institute  of  Mental  Health 
St.  Elizabeth's  Hospital 
Washington,  D.C. 

Project  Description: 

Objectives :   The  goal  of  these  studies  is  to  understand  the  effect  of  neuro- 
transmitters on  the  control  of  tyrosine  hydroxylase  (TH)  induction.   In 
adrenal  medulla  trans-synaptic  induction  of  TH  is  elicited  by  direct  or  in- 
direct stimulation  of  nicotinic  receptors.   This  stimulation  causes  an 
immediate  increase  in  cAMP  content  and  a  delayed  induction  of  TH.   We  applied 
this  model  to  studies  on  the  superior  cervical  ganglion  of  rats. 

Methods :   cAMP  and  cGMP  concentrations  were  estimated  by  measuring  the  incor- 
poration of  ->^P  into  histones  using  cAMP  or  cGMP  dependent  protein  kinases. 
TH  was  measured  by  a  radiochemical  technique  with  6-methyltetrahydropterin  as 
cofactor. 

Major  Findings:   When  adrenal  demedullated  rats  are  exposed  to  cold  the  cAMP 
concentration  in  SCG  is  increased  3  to  5  times  and  this  increase  lasts  for 
several  hours.   The  increase  of  cAMP  content  is  accompanied  by  a  decrease  in 
cGMP  concentration.   Since  the  increase  in  cAMP  could  be  prevented  by  pro- 
pranolol we  studied  the  effect  of  various  beta  receptor  agonists,  dopamine, 
norepinephrine,  and  carbamylcholine.   Repeated  injections  of  isoproterenol  and 
a  single  injection  of  epinephrine  caused  a  significant,  long  lasting  increase 
of  cAMP  associated  with  a  delayed  increase  of  TH  activity.   Both  changes  could 
be  blocked  by  propranolol.   In  contrast,  dopamine  and  norepinephrine  caused  a 
minute  and  short  lasting  increase  of  cAMP  which  was  not  followed  by  a  delayed 
TH  induction.   Carbamylcholine  failed  to  change  cAMP  and  cGMP  concentration 
and  TH  activity.   Decentralization  of  the  ganglion  48  hours  before  injection 
of  isoproterenol  or  epinephrine  prevented  neither  the  increase  in  cAMP  nor  the 
induction  of  TH. 

i  r/r 


Project  No.   Z01  HL  01847-01  HE 

Significance  to  Biomedical  Research  and  Institute  Program:   It  can  be  con- 
cluded from  these  studies  that  TH  induction  can  be  elicited  by  stimulation 
of  beta  adrenergic  receptors  (2  hours).   This  regulatory  mechanism  for  TH 
induction  obtains  physiological  importance  in  adrenal  demedullated  rats, 
where  adrenal  medullary  compensation  mechanisms  are  absent. 

Proposed  Course  of  Project:   We  intend  to  elucidate  in  superior  cervical 
ganglion  the  role  of  cAMP  in  the  regulation  of  the  phosphorylated  state  of 
non-histone  nuclear  proteins.   This  project  will  be  mainly  based  on  the 
observation  that  the  phosphorylated  state  of  acidic  nuclear  proteins  play  an 
important  role  in  the  regulation  of  gene  expression  in  eukariotic  cells  and 
in  the  modulation  of  messenger  RNA. 

Keyword  Descriptors:   cyclic  AMP      cyclic  GMP      sympathetic  ganglia 
tyrosine  hydroxylase 

Honors  and  Awards :   None 

Publications : 

1.  Hanbauer,  I.,  Kopin,  I.J.,  Guidotti,  A.,  and  Costa,  E. :   Induction  of 
tyrosine  hydroxylase  by  beta  adrenergic  receptor  agonists  in  normal  and 
decentralized  sympathetic  ganglia.   Role  of  c-AMP.   J.  Pharmacol.  Exp. 
Ther.   193:  95-104,  1975. 

2.  Hanbauer,  I.,  Guidotti,  A.,  and  Costa,  E. :   Involvement  of  cyclic  nucleo- 
tides in  the  long  term  induction  of  tyrosine  hydroxylase.   In 
Collegium  Internationale  Congress  du  Neuropsychopharmacologicum.   Paris, 
France,  Excerpta  Medica,  in  press. 

3.  Guidotti,  A.,  Hanbauer,  I.,  and  Costa,  E.:   Role  of  cyclic  nucleotides 
in  the  induction  of  tyrosine  hydroxylase.   In  Drummond,  Robison  and 
Greengard  (Eds.):   Advances  in  Cyclic  Nucleotide  Research.   Raven  Press, 
1975,  Vol.  5. 

4.  Costa,  E.,  Guidotti,  A.,  and  Hanbauer,  I.:   Do  cyclic  nucleotides  promote 
the  trans-synaptic  induction  of  tyrosine  hydroxylase?   Life  Sci.  14: 
1160-1188,  1974. 

5.  Hanbauer,  I.  and  Guidotti,  A.:   Cyclic  AMP  dependent  regulation  of  tyro- 
sine-3-monoxygenase  in  adrenal  medulla.   Effect  of  denervation.   Naunyn 
Schmiedeberg's  Arch.  Pharmacol.   287:  213-217,  1975. 


iftf 


Project  No.   Z01  HL  01848-01  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  19  74  through  June  30,  19  75 

Project  Title:   A  Radioimmunoassay  for  Dopamine-g-Hydroxylase 

Previous  Serial  Number:   None 

Principal  Investigator:   Pauline  Lerner,  Ph.D. 

Other  Investigators:   Walter  Lovenberg,  Ph.D. 

Cooperating  Units :   None 

Project  Description: 

Objectives:   Dopamine-beta-hydroxylase  (DBH)  is  the  enzyme  which  catalyzes  the 
final  reaction  in  the  biosynthesis  of  norepinephrine.   The  determination  of  DBH 
levels  is  of  interest  both  for  in  vitro  studies  of  neurochemical  systems  and 
for  clinical  studies  covering  a  variety  of  physiological  and  disease  states. 
Several  assays  for  DBH,  which  are  based  on  the  catalytic  activity  of  the 
enzyme,  are  now  in  use.   However,  interpretation  of  results  from  these  assays 
is  made  difficult  by  the  presence  of  endogenous  DBH  inhibitors.   In  addition, 
enzymatic  assays  do  not  detect  enzymatically  inactive  DBH,  which  is  present 
along  with  the  functional  enzyme  in  serum.   For  these  reasons,  several  workers 
have  turned  to  radioimmunoassay  for  DBH.   A  radioimmunoassay,  besides  being 
sensitive  and  specific,  would  be  unaffected  by  DBH  inhibitors  and  would  detect 
enzymatically  inactive  DBH.   Our  objective  is  to  develop  such  an  assay  for  use 
in  our  laboratory. 

Methods :  Antiserums  have  previously  been  produced  in  rabbits  by  injection  of 
pure  bovine  DBH  or  highly  purified  human  DBH.  For  iodination  of  the  antigen, 
chloramine  T  and  125i  were  reacted  with  purified  bovine  DBH. 

The  coated  tube  method  of  radioimmunoassay  has  been  explored.   This  method  is 
especially  well  suited  for  large  antigens,  such  as  DBH  (molecular  weight 
300,000),  and  has  been  the  only  radioimmunoassay  method  to  be  reported  for 
DBH.   In  this  method,  diluted  antiserum  is  placed  in  a  plastic  tube,  and  the 
antibodies  are  allowed  to  bind  directly  to  the  tube.   Next,  an  albumin  solu- 
tion is  used  to  cover  up  nonspecific  protein  binding  sites  on  the  plastic. 
After  this,  a  solution  containing  radioactive  DBH  (and  sometimes  also  unlabeled 
DBH)  is  introduced  into  the  tube,  and  the  DBH  is  allowed  to  bind  to  the  anti- 
body which  is  attached  to  the  tube  walls.   After  unreacted  material  is  washed 
out,  the  amount  of  bound  DBH  can  be  determined  by  counting  the  empty  tube. 

Major  Findings:   Experimental  techniques  have  been  developed  for  performing 
radioimmunoassay  of  DBH  by  the  coated  tube  method.   The  chloramine  T  method 

1  S7r 


Project  No.   Z01  HL  01848-01  HE 

of  radioiodination  has  been  adapted  to  use  with  DBH.   DBH  can  be  iodlnated 
to  a  relatively  high  specific  activity  without  loss  of  immunoreactivity  of 
the  protein,  and  the  iodinated  antigen  can  be  recovered  without  significant 
contamination  from  unreacted  iodide.   Several  experimental  parameters  for  the 
assay  itself,  including  type  of  plastic,  type  and  concentration  of  antiserum, 
and  incubation  time,  have  been  investigated  and  optimized.   A  method  for 
measuring  tritiated  protein  bound  to  tubes  has  been  worked  out.   The  radio- 
active part  of  the  tube  is  cut  off,  broken  up  physically,  and  counted  with  a 
counting  fluor  in  a  liquid  scintillation  counter. 

This  assay  can  detect  immuno reactive  material  in  normal  human  serum.   Pre- 
liminary experiments  with  human  cerebrospinal  fluid  have  not  enabled  us  to 
detect  immunoreactive  material  in  this  fluid.   The  assay  is  currently  being 
used  in  experiments  on  protein  biosynthesis  by  bovine  adrenal  medullae. 

Significance  to  Biomedical  Research  and  Institute  Program:   DBH  is  found, 
along  with  epinephrine  and  norepinephrine,  in  vesicles  in  the  adrenal  medulla 
and  sympathetic  and  central  noradrenergic  neurons.   During  synaptic  trans- 
mission, the  vesicles  release  their  contents  by  exocytosis.   DBH  from  the 
sympathetic  nervous  system  eventually  appears  in  serum  and  several  investiga- 
tors have  suggested  that  serum  DBH  may  be  a  useful  indicator  of  sympathetic 
activity.   Likewise,  DBH  from  the  central  nervous  system  might  be  found  in 
cerebrospinal  fluid.   If  this  is  the  case,  DBH  levels  in  cerebrospinal  fluid 
of  patients  may  give  useful  information  on  the  biochemistry  of  normal  and 
diseased  mental  states.   The  adrenal  medullary  DBH  is  also  of  great  interest 
because  its  synthesis  can  be  affected  by  various  pharmacological  agents.   The 
radioimmunoassay   for  DBH,  which  is  sensitive,  specific,  and  unaffected  by 
factors  which  alter  DBH  enzymatic  activity,  promises  to  be  a  useful  tool  in 
studying  these  phenomena. 

Proposed  Course  of  Project:   We  plan  to  produce  more  antiserum  directed  against 
human  DBH.   We  hope  to  obtain  an  antiserum  which  has  both  high  specificity  and 
high  avidity  for  DBH.   This  would  enable  us  to  refine  our  assay  for  human  DBH 
and  make  it  more  sensitive.. 

We  would  also  like  to  increase  the  sensitivity  of  the  assay  for  bovine  DBH  so 
that  we  can  analyze  smaller  amounts  of  tissue.   We  plan  to  investigate  the  use 
of  very  small  polystyrene  beads,  rather  than  tubes,  as  a  means  of  increasing 
the  sensitivity  of  the  assay. 

Keyword  Descriptors:   dopamine-B-hydroxylase      immunoassay      cerebrospinal 
fluid      schizophrenia      hypertension 

Honors  and  Awards:   None 

Publications:   None 


r# 


Project  No.   Z01  HL  01849-01  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  19  75 

Project  Title:   Synthesis  of  Dopamine-3-Hydroxylase  in  a  Cell 
Free  System 

Previous  Serial  Number:   None 

Principal  Investigators:   Pauline  Lerner,  Ph.D. 

Paul  MacDonnell,  Ph.D. 

Other  Investigators:   Walter  Lovenberg,  Ph.D. 
Gordon  Guroff,  Ph.D. 

Cooperating  Units:   Section  on  Intermediary  Metabolism 
Laboratory  of  Biomedical  Sciences 
National  Institute  of  Child  Health  and 
Human  Development 

Project  Description: 

Objectives :   Dopamine-beta-hydroxylase  (DBH)  is  the  enzyme  which  synthesizes 
the  neurotransmitter  noradrenalin  from  its  precursor,  dopamine.   The  adrenal 
medulla  is  a  rich  source  of  this  enzyme.   Several  studies  have  shown  that  the 
adrenal  medulla  regulates  the  rate  of  synthesis  of  DBH  in  response  to  neuronal 
factors.   Nerve  impulses  arriving  at  synapses  on  the  adrenal  gland  can  mediate 
an  increase  in  synthesis  of  DBH.   The  chemical  link  between  activity  at  the 
synapse  and  protein  synthesis  within  the  cell  is  not  yet  understood.   If  DBH 
synthesis  can  be  achieved  in  a  cell  free  system,  it  would  be  possible  to 
systematically  investigate  the  effects  of  various  chemical  and  pharmacological 
agents  on  DBH  synthesis.   Our  objective  is  to  develop  a  cell  free  system  which 
can  synthesize  DBH  and  to  study  factors  which  affect  this  system. 

Methods:   Fresh  bovine  adrenal  glands  are  used  to  prepare  RNA.   The  polysome 
fraction  is  first  isolated  from  the  adrenal  medullae.   RNA  is  obtained  from 
the  polysomes  by  phenol  extraction.   RNA  which  contains  polyadenylic  acid 
(poly  A)  is  then  separated  by  use  of  an  oligodeoxythymidylic  acid  column. 
The  poly  A  RNA  (messenger  RNA)  is  used  as  the  template  for  RNA  synthesis  in  a 
cell  free  system  derived  from  untoasted  wheat  germ. 

The  protein  products  of  the  cell  free  synthesis  are  assayed  for  DBH  by  means 
of  a  radioimmunoassay.   The  coated  tube  method  of  radioimmunoassay  is  employed, 
using  an  antiserum  raised  against  purified  bovine  DBH. 

Ma j o r  F ind ings :   Preliminary  work  was  aimed  at  establishing  optimum  conditions 

i  r/f 


Project  No.   Z01  HL  01849-01  HE 

for  recovery  of  biosynthetically  active  RNA  from  bovine  adrenal  medullae. 
Extreme  freshness  of  the  adrenal  glands  was  found  to  be  essential  for  high 
activity.   The  poly  A  containing  fraction  of  RNA  from  the  polysomes  proved 
to  be  the  most  active  RNA  fraction  for  protein  synthesis.   This  fraction  of 
RNA,  isolated  from  fresh  adrenal  medullae,  routinely  stimulates  protein 
synthesis  to  a  level  four  to  seven  times  as  high  as  the  background  from  the 
wheat  germ  system  alone. 

Immunological  evidence  indicates  that  some  of  the  material  synthesized  in  this 
manner  is  indeed  DBH.   It  appears  that  0.2  to  1  percent  of  the  protein 
synthesized  in  vitro  from  adrenal  medullary  RNA  is  DBH.   RNA  preparations  from 
two  tissues  which  do  not  synthesize  DBH,  the  salivary  gland  and  the  adrenal 
cortex,  have  also  been  used  in  the  cell  free  protein  synthesis  system.   The 
radioimmunoassay  indicates  that  no  DBH  is  synthesized  with  either  of  these  two 
RNAs. 

Significance  to  Biomedical  Research  and  Institute  Program:   DBH  is  necessary 
for  the  in  vivo  synthesis  of  the  catecholamines  epinephrine  and  norepinephrine. 
The  catecholamines  are  of  particular  interest  because  of  their  role  in  the 
regulation  of  blood  pressure.   Several  workers  have  suggested  a  possible 
relationship  between  serum  DBH  and  hypertension.   Brain  DBH  is  essential  for 
the  biosynthesis  of  norepinephrine,  which  functions  as  a  neurotransmitter  in 
the  central  nervous  system.   There  is  some  evidence  for  a  deficiency  in  brain 
norepinephrine  and  DBH  in  schizophrenics.   In  both  the  central  and  the 
peripheral  nervous  systems  DBH  may  be  related  to  disease  states.   Our  studies 
are  designed  to  elucidate  factors  which  affect  the  rate  of  synthesis  of  this 
crucial  enzyme . 

Proposed  Course  of  Project:   We  now  have  immunological  evidence  that  DBH  is 
being  synthesized  in  the  cell  free  system  which  we  use.   We  plan  to  collect 
other  evidence,  using  chromatographic  techniques,  supporting  the  identity 
of  this  material  as  DBH.   We  also  plan  to  compare  RNA  from  membrane  bound  and 
nonmembrane  bound  polysomes  for  their  ability  to  direct  DBH  synthesis.   Since 
DBH  is  a  protein  destined  for  export  from  the  cell,  it  may  be  synthesized 
preferentially  on  membrane  bound  polysomes. 

Another  goal  is  to  scale  down  our  assay  so  that  we  can  work  with  smaller 
tissue  samples.   If  possible,  we  will  continue  our  study  with  tissues  from 
smaller  animals  which  can  be  subjected  to  pharmacological  treatments. 

Keyword  Descriptors:   protein  synthesis      dopamine-3-hydroxylase 
hypertension       schizophrenia 

Honors  and  Awards:   None 

Publications:   None 


STxs 


Project  No.   Z01  HL  01850-06  HE 


1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Biochemistry  of  the  Spontaneously  Hypertensive  Rats 

Previous  Serial  Number:   NHLI-270 

Principal  Investigator:   Walter  Lovenberg,  Ph.D. 

Other  Investigators:   Yukio  Yamori,  M.D. 

Akinobu  Nagaoka,  M.D. 
Teruhiro  Nakada,  M.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives :   The  strain  of  rats  developed  in  Kyoto,  Japan,  that  exhibit  uni- 
form and  severe  hypertension  have  been  used  as  an  animal  model  of  human 
essential  hypertension.   These  spontaneously  hypertensive  rats  (SHR)  have  been 
studied  by  numerous  investigators  for  deviations  in  various  metabolic  systems. 
Previous  work  in  this  laboratory  has  established  that  there  appears  to  be  an 
inverse  relationship  between  catecholamine  metabolism  in  central  nervous  system 
and  blood  pressure.   It  was  also  observed  that  biochemical  differences  in 
various  normotensive  rat  strains  were  marked  and  that  the  only  strain  of  any 
value  as  a  control  strain  was  that  of  Kyoto /Wistar  colony,  the  parent  strain 
of  the  SHR.   We  also  observed  that  there  appeared  to  be  an  early  increase  in 
the  rate  of  lysine  incorporation  into  vascular  non-collagen  protein.   The 
objective  of  the  current  experiments  is  to  examine  some  of  the  factors  that 
may  be  involved  in  the  early  increase  in  the  incorporation  of  lysine  into  the 
non-collagen  proteins. 

Methods :   Male  SHR  were  obtained  from  the  NIH  animal  colony  and  control  Kyoto/ 
Wistar  rats  were  obtained  from  laconic  Farms,  Inc.   The  SHR  were  divided  into 
4  groups  at  6  weeks  of  age.   One  group  had  the  splanchnic  nerve  resected, 
one  group  was  treated  with  Hexamethonium  (50  mg/kg  s.c.  twice  daily),  and  a 
third  group  received  Apresoline  (80  mg/liter  in  the  drinking  water) .   A  group 
of  SHR  and  Wis  tar/Kyoto  were  nontreated  controls.   The  incorporation  of  ^E- 
lysine  into  proteins  of  the  heart,  thoracic  aorta,  and  the  mesenteric  artery 
was  measured  2  hours  following  I.V.  injection  of  50  yc  of  %- lysine.   In  heart, 
the  actomyosin  was  specifically  isolated  by  standard  techniques.   In  the  vessels 
the  non-collagen  proteins  were  isolated  following  extraction  with  hot  5% 
trichloroacetic  acid.   The  rate  of  incorporation  was  estimated  by  determining 
the  specific  activity  of  the  protein  (dpm/mg)  and  comparing  it  with  the 
measured  specific  activity  of  the  serum  lysine.   Wistar/Kyoto  rats  (the  parent 


ray 


Project  No.   Z01  HL  01850-06  HE 

strain  of  SHR)  were  used  as  control  animals.   Collagen  and  elastin  were  also 
isolated. 

Major  Findings:   The  experiments  were  performed  at  8  weeks  of  age  after  two 
weeks  of  treatment  and  the  following  data  obtained: 

Blood  Pressure       Mesenteric  Artery  Protein 
mm  Hg  Collagen     Non-Collagen 

dpm/gm  tissue 

Control  SHR  168+2  49.0+4.9  13.7+0.2 

Apresoline  136  +  3  52.0  +  3.0  15.1  +  1.5 

Hexamethonium  137  +  7  47.3  +  3.8      8.2  +  1.0 

Splanchnectomized  136+3  58.8+2.7      7.9+0.3  , 

Wistar/Kyoto  131+2  34.8+5.8      7.8+0.5 

Protein  synthesis  was  also  measured  in  the  heart  and  aorta  and  no  significant 
differences  were  observed.   The  synthesis  of  elastin  was  similar  in  all  groups. 

Significance  to  Biomedical  Research  and  Institute  Program:   The  current  work 
confirms  our  earlier  findings  that  incorporation  of  lysine  into  non-collagen 
proteins  of  the  small  vasculature  is  increased  in  young  SHR.   The  possibility 
that  this  increase  in  protein  synthesis  is  neuronally  mediated  is  suggested  by 
the  return  to  control  levels  in  animals  given  hexamethonium  or  denervated. 
Apresoline,  a  vasodilator  which  was  effective  in  controlling  blood  pressure, 
did  not  reduce  incorporation.   These  findings  would  provide  common   ground  for 
those  investigators  who  suggest  that  the  primary  cause  of  hypertension  is 
neurogenic  and  those  that  suggest  a  primary  abnormality  in  vessel  structure. 

Proposed  Course  of  Project:   1)  Further  investigation  on  the  relationship 
of  nerve  activity  and  vascular  protein  synthesis  are  planned. 

2)  These  studies  will  be  extended  to  the  new  stroke-prone  strain  of  rat  that 
has  just  been  established  at  the  NIH. 

3)  The  peripheral  and  central  aminergic  systems  will  be  reevaluated  in  the 
stroke-prone  rats  with  particular  attention  devoted  to  the  prehypertensive 
stage. 

Keyword  Descriptors:   hypertension       spontaneously  hypertensive  rats 
catecholamines      sympathetic  nerves      vascular  protein  synthesis 

Honors  and  Awards :   None 

Publications:   None 


ra> 


Project  No.   Z01  HL  01851-01  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 
3-  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Regulation  of  Catecholamine  Synthesis 

Previous  Serial  Number:   None 

Principal  Investigator:   Walter  Lovenberg,  Ph.D. 

Other  Investigators:   Ingeborg  Hanbauer,  Ph.D. 

Eleanor  A.  Bruckwick,  B.S. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   The  synthesis  of  catecholamines  in  the  central  nervous  system 
is  controlled  by  the  activity  of  tyrosine  hydroxylase.   Factors  which  regulate 
this  enzyme  therefore  may  affect  the  responsiveness  of  these  neuronal  systems. 
It  is  known  that  catecholamine  synthetic  rates  in  vivo  respond  to  various 
drugs  and  stimulants.   The  objective  of  this  work  is  to  determine  the  molecular 
mechanism  by  which  tyrosine  hydroxylase  can  alter  its  catalytic  activity. 

Methods:  The  striatal  area  of  brains  from  male  Sprague-Dawley  rats  were  used 
as  a  source  of  tyrosine  hydroxylase.  This  enzyme  was  assayed  by  the  standard 
tritium  release  assay. 

Major  Findings:   As  reported  by  others  the  K_  of  tyrosine  hydroxylase  for  its 
cofactor  was  reduced  to  about  1/3  the  control  value  following  treatment  with 
haloperidol  and  other  neuroleptics.   This  activation  appeared  to  be  due  to 
change  in  the  macromolecule  since  it  was  retained  following  chromatography  on 
Sephadex.   Since  these  drugs  are  known  to  be  inhibitors  of  the  dopamine 
sensitive  adenylate  cyclase  in  brain,  the  effect  of  cyclic  AMP  and  protein 
phosphorylating  conditions  on  the  enzyme  in  vitro  were  examined.   Tyrosine 
hydroxylase  activity  was  measured  in  40,000  x  g  supernatant  fractions  of  rat 
striata  solubilized  in  0.2%  Triton  X  100.   Addition  of  ATP,  cAMP,  Mg++,  EGTA, 
NaF,  and  theophylline  in  concentrations  optimal  for  protein  phosphorylation 
to  the  tyrosine  hydroxy lation  reaction  mixture  reduces  the  apparent  Kjjj  for  6- 
methyltetrahydropterin  (0.5  mM  to  0.15  mM) .   The  reduction  in  cofactor  Km 
appears  to  be  similar  to  that  seen  in  vivo  following  dopamine  receptor  blockade 
and  may  represent  an  important  regulatory  mechanism.   This  change  in  cofactor 
Km  is  sufficient  to  account  for  a  2-  to  3-fold  increase  in  tyrosine  hydroxylase 
activity  at  estimated  physiological  cofactor  levels  (0.1  mM) .   The  increase  in 
activity  is  totally  dependent  upon  ATP  and  partially  dependent  upon  cAMP  and 
Mg++.   Neither  ATP  (0.5  mM) ,  cAMP  (0.1  mM) ,  nor  Mg*4"  when  added  alone  shows  any 
effect  on  the  kinetic  properties  of  tyrosine  hydroxylase.   Cyclic  GMP  does  not 

l  ra3 


Project  No.   Z01  HL  01851-01  HE 

replace  cAMP  in  this  system.   The  above  experiments  suggest  but  do  not  pro- 
vide direct  evidence  for  tyrosine  hydroxylase  phosphorylation.   Immunoradio- 
chemical  studies  have  thus  far  been  unable  to  show  the  direct  incorporation 
of  32p  from  ATP-y-^P.   The  rapid  removal  of  phosphate  by  phosphatases, 
however,  can  not  be  exluded. 

Significance  to  Biomedical  Research  and  Institute  Program:   The  importance  of 
the  above  findings  from  a  physiological  point  of  view  is  that,  since  reduced 
biopterin  is  in  suboptimal  concentration  in  brain  tissue,  a  rapid  and  sub- 
stantial change  in  the  catecholamine  synthetic  rate  can  occur  even  when  con- 
centrations of  catecholamines  remain  unchanged.   If  it  is  assumed  that  the 
tyrosine  hydroxy lating  system  is  in  the  dephosphorylated  configuration  in  the 
resting  control  animal  and  that  the  levels  of  tetrahydrobiopterin  and  dopamine 
are  approximately  0.1  mM,  the  system  would  be  operating  at  less  than  1%  of  its 
capacity.   The  enzyme  activity  measured  in  the  above  experiments  is  over  700 
ng/gram  of  tissue/hour,  whereas  the  values  observed  for  in  vivo  synthesis  are 
about  1  to  2%  of  this  rate.   A  decrease  in  the  K^  of  the  cof actor  would  re- 
sult in  a  proportional  increase  in  in  vivo  synthesis.   Activation  of  a  protein 
kinase  or  inactivation  of  a  protein  phosphatase  would  result  in  a  rapid 
decrease  in  the  Km  for  tetrahydrobiopterin.   This  may  be  the  phenomenon 
which  occurs  when  a  lesion  is  placed  in  the  nigro-striatal  pathway  or  when  a 
neuroleptic  drug  is  given.   Based  on  such  a  molecular  mechanism,  receptor 
mediated  feedback  inhibition  could  easily  be  explained. 

Proposed  Course  of  Project:   Further  attempts  will  be  made  to  determine  whether 
the  tyrosine  hydroxylase  is  directly  phosphorylated  or  whether  an  activator 
molecule  is  involved.   Experiments  will  also  be  directed  toward  establishing 
the  molecular  commonality  between  the  in  vitro  phosphory lating  effects  and  the 
in  vivo  drug  effects. 

Keyword  Descriptors:   tyrosine  hydroxylase      catecholamines      dopamine 
neuroleptic 

Honors  and  Awards: 

Publications : 

1.  Lovenberg,  W.  and  Bruckwick,  E.A. :   Mechanisms  of  receptor  mediated 
regulation  of  catecholamine  synthesis  in  brain.   In:  Usdin,  E.  and 
Bunney,  W.E.  (Eds.):   Pre  and  Postsynaptic  Receptors,  presented 

at  the  American  College  of  Neuropsychopharmacology ,  Puerto  Rico, 
Dec.  1974,  Marcel  Dekker,  1975,  pp.  149-169. 

2.  Lovenberg,  W. ,  Bruckwick,  E.A. ,  and  Hanbauer,  I.:   The  effect  of 

ATP,  cyclic  AMP  and  magnesium  on  the  affinity  of  rat  striatal  tyrosine 
hydroxylase  for  its  cof actors.   Proc .  Nat.  Acad.  Sci.,  in  press. 


£"*/ 


Project  No.   Z01  HL  01852-04  HE 

1.  Hypertension-Endocrine  Branch 

2-.    Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Regulation  of  Hydroxyindole  Pathway  in  the 
Pineal  Gland 

Previous  Serial  Numbers:   NHLI-269,  NHLI-273 

Principal  Investigator:   Walter  Lovenberg,  Ph.D. 

Other  Investigators:   Jay  S.  Skyler,  M.D. 

Ingeborg  Hanbauer,  Ph.D. 
William  Merrick,  Ph.D. 

Cooperating  Units:   Molecular  Hematology  Branch,  NHLI 

Project  Description: 

Objectives :   The  hydroxyindole  pathway  of  the  pineal  gland  offers  a  unique 
opportunity  to  examine  mammalian  enzyme  regulation  and  regulation  of  protein 
synthesis.   The  synthesis  of  melatonin  is  controlled  by  the  levels  of 
serotonin-N-acetyltransf erase  (NAT)  which  in  turn  is  under  neuronal  control. 
Beta  receptor  stimulation  causes  a  50-fold  increase  in  NAT  activity.   Cyclic 
AMP  is  the  second  messenger  in  this  system.   The  objective  of  this  project  is 
to  define  at  a  molecular  level  the  mechanism  by  which  cAMP  is  eliciting  the 
large  increase  in  NAT  activity. 

Methods:   Protein  kinases  were  isolated  by  ammonium  sulfate  fractionation,  gel 
filtration  and  DEAE  cellulose  chromatography.   Calf  thymus  pineal  chromatin 
were  isolated  by  standard  techniques.   Protein  kinase  activity  was  determined 
by  the  incorporation  of  32p  into  protein  using  y~^P~ATP  and  millipore 
filtration.   Binding  of  cAMP,  and  actinomycin  D  were  measured  by  similar 
techniques.   Template  capacity  was  measured  using  E.  coli  polymerase  and  JH- 
UTP.   Serotonin-N-acetyltransf erase  was  measured  by  radio-techniques. 

Major  Findings:   Both  rat  and  bovine  pineal  glands  have  substantial  amounts  of 
a  cAMP-dependent  protein  kinase.   This  cAMP-dependent  protein  kinase  was  puri- 
fied to  homogeneity  and  was  found  to  consist  of  a  catalytic  and  regulatory 
(cAMP  binding)  subunit.   It  was  then  reasoned  that  if  protein  phosphorylation 
was  mediating  the  changes  in  NAT  then  at  least  one  of  three  processes  must 
occur:   1)  stimulation  of  gene  transcription,  2)  increase  in  race  of  trans- 
lation of  -RNA  by  ribosomal  protein  synthesis,  or  3)  a  direct  activation  of 
the  apoenzyme  by  phosphorylation. 

Work  on  characterization  of  ribosomal  protein  kinases  has  continued.   Using 
reticulocyte  ribosomes  the  kinase  activity  has  been  examined  by  DEAE  cellulose, 

i  s>r~ 


Project  No.   Z01  HL  01852-04  HE 

Sephadex,  and  phosphocellulose  chromatography.   Multiple  peaks  of  protein 
kinase  activity  are  seen.   Preliminary  characterization  of  these  enzymes 
showed  them  to  have  differing  dependencies  upon  cyclic  AMP  and  cyclic  GMP 
for  full  activity.   Some  differences  in  substrate  specificity  are  observed. 
In  addition  to  the  usual  substrates  histone,  protamine,  and  casein,  we  also 
examined  reticulocyte  fractions  that  were  enriched  in  regard  to  each  of  the 
protein  synthetic  initiation  factors.   One  of  the  peaks  of  kinase  activity 
eluted  from  the  DEAE  cellulose  column  preferentially  used  GTP  as  the  phosphate 
donor.   This  may  be  particularly  significant  in  view  of  the  role  of  GTP  in 
the  protein  synthetic  mechanism. 

Significance  to  Biomedical  Research  and  Institute  Program;   It  can  be  con^ 
eluded  from  these  studies  that  cAMP  may  exert  an  affect  on  NAT  by  stimulating 
protein  kinase  which  in  turn  activates  both  transcription  and  translation. 
Activation  of  a  precursor  protein  by  phosphorylation  does  not  occur.   There- 
fore, the  sequence  of  events  by  which  neuronal  impulses  can  be  translated  into 
changes  in  enzyme  can  be  hypothesized  as  follows: 

Sympathetic  nerve  ->  Norepinephrine  ■*  Pineal     -*■     Adenyl  cyclase 

3  Receptor 

cAMP  ->  Protein  -*■  Increased     ■*■  Elevated 
kinase    transcription    NAT 
+ 
Translation 

Proposed  Course  of  Project:   The  following  experimental  approaches  will  be 
undertaken: 

1.  Further  definition  of  the  role  of  phosphorylation  in  initiation  of 
polypeptide  synthesis,  especially  initiation  factors  per  se, 

2.  Characterization  of  the  various  reticulocyte  ribosomal  kinases  and 
development  of  a  fractionation  strategy  for  purification, 

3.  Further  examination  of  the  products  of  chromatin  phosphorylation 
particularly  the  acidic  proteins,  using  pineal  chromatin  rather  than 
the  heterologous  cAMP  thymus  chromatin, 

4.  Direct  measurement  of  incorporation  of  amino  acid  into  NAT  and  in  vivo 
experiments  with  protein  synthesis  inhibitors. 

Keyword  Descriptors:   pineal  gland      melatonin      serotonin-N-acetyl- 
transterase      protein  kinase      cyclic  AMP 

Honors  and  Awards :   None 

Publications:   None 


r^ 


Project   No.      Z01  HL  01853-12  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Studies  on  the  Properties  of  Iron-Sulfur  Proteins 

Previous  Serial  Number:    NHLI-268 

Principal  Investigator:   Walter  Lovenberg,  Ph.D. 

Other  Investigators:   Larry  F.  Bennett 
Peter  DeBrunner 

Cooperating  Units:   Dept.  of  Chemistry,  University  of  California, 
San  Diego 
Dept.  of  Physics,  University  of  Illinois, 
Urbana 

Project  Description: 

Objectives :   Iron-sulfur  proteins  constitute  a  broad  class  of  enzymes  and 
electron  carriers  that  are  distinguished  by  the  presence  of  one  or  more  iron 
atoms  coordinated  exclusively  by  sulfur  atoms  at  the  active  center.   Although 
these  types  of  redox  centers  may  be  considered  primative  from  an  evolutionary 
point  of  view,  they  function  innumerable  key  and  sophisticated  roles  in 
bacteria,  plants  and  animals  today.   Previous  work  from  this  laboratory  has 
described  the  discovery,  isolation,  and  extensive  chemical  and  physical 
characterization  of  these  proteins.   The  current  objectives  of  this  project 
are  to  further  refine  physical-chemical  knowledge  of  certain  Iron-sulfur 
electron  carriers  and  to  search  for  new  roles  for  iron-sulfur  enzymes  in 
mammalian  physiology. 

Methods:   Clostridium  pasteurianum  was  used  as  a  source  of  the  two  proteins 
under  consideration.   Pure  ferredoxin  and  rubredoxin  were  isolated  from  these 
organisms  by  techniques  devised  in  this  laboratory  and  given  in  previous 
reports . 

Major  Findings:   Little  new  experimentation  was  done  on  this  project  during 
the  past  year,  however,  collaborative  work  continued  with  Drs .  DeBrunner  and 
Bennett.   Techniques  have  been  developed  for  preparation  of  5^Fe  rubredoxin 
samples  that  are  indistinguishable  from  native  rubredoxin.   Using  this  better 
quality   Fe  rubredoxin,  magnetic  Mossbauer  spectra  have  been  obtained  with 
large  paramagnetic  splitting  and  excellent  resolution.   Such  studies  give  us 
valuable  information  on  the  molecular  environment  of  the  iron  atom  and  the 
distortion  of  its  ligands.   They  now  can  be  coordinated  with  other  types  of 
spectroscopic  data. 


£x7 


Project  No.   Z01  HL  01853-12  HE 

Significance  to  Biomedical  Research  and  Institute  Program:   The  work  described 
above  is  part  of  our  continuing  program  to  elucidate  the  structure  and  mechanism 
of  iron-sulfur  redox  centers  in  proteins.   Although  such  centers  have  only 
been  recognized  for  a  little  over  a  decade,  it  is  now  apparent  that  they 
represent  one  of  the  most  important  mechanisms  for  the  transfer  of  electrons 
in  biology.   While  the  biological  importance  of  rubredoxin  is  not  well  under- 
stood, this  protein  from  Clostridium  pasteurianum  is  now  probably  the  most 
completely  resolved  protein  molecule  from  both  a  structural  and  reaction 
mechanism  point  of  view.   As  such  it  has  become  a  model  for  the  study  of  other 
proteins,  particularly  iron-sulfur  proteins. 

Proposed  Course  of  Project:     1)  Complete  Mossbauer  spectroscopic  study  of 
rubredoxin.   2)  Establish  a  new  study  using  detailed  electron  exchange  methods 
to  obtain  greater  understanding  of  the  redox  mechanism.   3)  Start  a  new 
series  of  experiments  using  a  new  improved  laser  Raman  instrument  to  probe  the 
atomic  properties  of  the  active  site  iron. 

Keyword  Descriptors:   ferredoxin      rubredoxin      iron-sulfur  proteins 
Mossbauer  spectroscopy 

Honors  and  Awards :   None 

Publications :   None 


s-xB 


Project  No.   Z01  HL  01854-04  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Characterization  of  Human  Dopamine-$-Hydroxylase 

Previous  Serial  Number:   NHLI-272(c) 

Principal  Investigator:   Robert  C.  Rosenberg,  Ph.D. 

Other  Investigators:   Walter  Lovenberg,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives :   The  objective  of  this  project  is  to  isolate  and  characterize 
dopamine-8-hydroxylase  (DBH)  from  human  plasma. 

Methods :   1)  Purification:   Previous  work  in  this  laboratory  has  shown  that 
by  using  standard  isolation  techniques  highly  purified  DBH  can  be  obtained 
from  pooled  human  plasma.   These  techniques  include  ammonium  sulfate  fractiona- 
tion, binding  to  and  elution  from  DEAE  cellulose  columns  and  gel  filtration 
on  agarose  columns.   Additional  purification  steps  include:  affinity 
chromatography  on  tyramine-sepharose  columns  or  concanavalin  A  sepharose 
columns. 

2)  Characterization:   DBH  from  bovine  adrenals  has  been  isolated  and 
characterized  as  to  its  molecular  weight,  subunit  content,  amino  acid  analysis, 
carbohydrate  content,  copper  content  and  steady  state  kinetic  properties. 
Similar  characterization  can  be  done  with  human  DBH. 

Major  Findings:   Purification:   As  previously  reported  human  serum  contains 
up  to  10  yg/ml  of  DBH.   Although  the  purification  procedure  developed  pre- 
viously yielded  a  highly  purified  enzyme  (~2000-fold) ,  the  recovery  was  poor 
and  the  final  product  showed  significant  contamination  by  three  or  four  other 
proteins.   Attempts  at  refining  the  procedure  to  both  increase  the  recovery 
of  enzymatic  activity  and  to  eliminate  the  contaminating  proteins  have  been 
pursued.   Modification  of  the  conditions  used  in  the  ion  exchange  and  gel 
filtration  steps  have  resulted  in  substantial  increases  in  the  recovery  of 
enzymatic  activity  from  these  steps.   It  has  also  been  found  that  the 
conditions  previously  reported  for  Con  A-sepharose  affinity  chromatography  of 
DBH  cause  considerable  loss  of  enzymatic  activity.   The  use  of  these  unfavorable 
conditions  of  ionic  strength  can  explain  the  poor  recoveries  of  DBH  activity 
found  in  this  and  other  laboratories.   Efforts  to  obtain  reasonable  quantities 
of  electrophoretically  homogenous  human  plasma  DBH  are  continuing. 


&? 


Project  No.   Z01  HL  01854-04  HE 

Characterization :   The  use  of  inhibitors  to  study  the  kinetics  of  the  DBH 
catalyzed  conversion  of  tyramine  to  octopamine  is  considerably  complicated  by 
the  requirement  for  catalase  in  reactions  where  ascorbate  serves  as  the 
electron  donor.   Many  if  not  all  of  the  compounds  that  have  been  reported  to 
inhibit  DBH  also  inhibit  catalase.   Thus  a  reaction  system  for  DBH  which  does 
not  require  catalase  would  prove  invaluable  in  elucidating  the  detailed 
mechanism  of  DBH  activity.   Other  laboratories  have  reported  that  K4Fe(CN)6 
can  serve  as  the  electron  donor  in  DBH  catalyzed  reactions .   Our  preliminary 
results  indicate  that  when  Fe(CN)g   is  used  as  the  electron  donor  substantial 
DBH  activity  can  be  observed  even  when  catalase  is  not  included  in  the  reaction 
mixture.   Thus,  use  of  a  DBH  assay  system  with  Fe(CN)g   serving  as  the  electron 
donor  permits  reevaluation  of  the  compounds  that  have  been  reported  to  inhibit 
or  activate  DBH.   Towards  this  end  we  have  found  that  one  of  the  standard  assay 
procedures  for  measuring  DBH  in  plasma  facilitates  removal  of  the  Fe(CN)£~ 
produced  in  the  assay  reaction.   Fe(CN)?-  would  otherwise  interfere  with  the 
spectrophotometric  determination  of  the  reaction  product.   Further  preliminary 
results  have  shown  that  azide  ion  is  a  true  inhibitor  of  DBH.   Spectroscopic 
study  of  the  azide-DBH  complex  should  provide  information  about  the  number  and 
nature  of  active  sites  in  DBH. 

Significance  to  Biomedical  Research  and  Institute  Program:   DBH  is  essential 
for  the  function  of  adrenergic  neurons  and  chromaffin  cells  since  it  catalyzes 
the  final  step  in  the  synthesis  of  the  neurotransmitter,  norepinephrine.   DBH 
has  been  found  within  the  synaptic  and  chromaffin  granule  vesicles,  and  upon 
nerve  stimulation  it  is  released  along  with  the  neurotransmitter  by  an 
exocytotic  process.   Hyperactivity  of  the  sympathetic  nervous  system  has  been 
implicated  in  the  development  or  maintenance  of  essential  hypertension  in  man 
and  experimental  forms  of  hypertension  in  animals.   Detailed  molecular  knowledge 
of  the  structure,  function  and  regulation  of  DBH  is  important  for  a  complete 
understanding  of  the  functioning  of  the  adrenergic  nervous  system  in  both  nor- 
mal and  disease  states.   There  is  a  large  body  of  work  on  the  in  vivo  inhibition 
of  DBH  by  various  chelating  and  thiol  compounds,  some  of  which  have  been  used 
as  antihypertensive  agents  in  man.   A  detailed  understanding  of  the  mechanism 
by  which  these  inhibitors  modulate  DBH  activity  can  provide  valuable  information 
on  the  functioning  of  the  native  enzyme  as  well  as  suggestions  for  potentially 
useful  new  antihypertensive  agents. 

Proposed  Course  of  Project:   Work  on  purifying  human  DBH  from  plasma  is  con- 
tinuing.  Simultaneously,  steady  state  kinetic  studies  on  the  effects  of  both 
chemical  and  pharmacological  inhibitors  and  activators  of  DBH  are  being  pursued 
using  partially  purified  enzyme.   These  studies  are  and  will  be  carried  out 
under  conditions  where  the  enzyme  activity  is  not  catalase  dependent. 

Keyword  Descriptors:   serum  dopamine-B-hydroxylase      hypertension 
adrenal  glands 

Honors  and  Awards :   None 

Publications:   None 


?2o 


Project  No.   Z01  HL  OI855-OI  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 
3»  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Catalogue  of  Isohormones  of  Human  Growth  Hormone 
Previous  Serial  Number:   None 

Principal  Investigator:   Jay  S.  Skyler,  M.D» 

Other  Investigators:   Andreas  C.  Chrambach,  Ph.D. 

Cooperating  Units:   Reproduction  Research  Branch,  NICHD 

Project  Description: 

Objectives:   l)  To  develop  a  catalogue  of  the  various  isohormone  species  of 
human  growth  hormone  (hGH)  in  relation  to  molecular  structure. 

Methods:   The  catalogue  is  based  on  results  obtained  by  three  techniques: 
l)  quantitative  polyacrylamide  gel  electrophoresis  in  a  multiphasic  buffer 
system  optimized  for  the  characterization  of  hGH;  2)  isoelectric  focusing  in 
polyacrylamide  gel;  3)  polyacrylamide  gel  electrophoresis  in  sodium  dodecyl 
sulfate  (SDS-PAGE)  in  the  presence  and  absence  of  reducing  agents.  Poly- 
acrylamide gel  electrophoresis  and  SDS-PAGE  are  responsive  to  variation  in 
molecular  size,  while  polyacrylamide  gel  electrophoresis  and  isoelectric 
focusing  in  polyacrylamide  gel  detect  variation  in  molecular  net  charge.   In 
polyacrylamide  gel  electrophoresis,  the  joint  95%  confidence  envelope  of 
retardation  coefficient  (a  parameter  of  molecular  size)  and  relative  free 
mobility  (a  parameter  of  molecular  net  charge  in  a  given  buffer  milieu),  serves 
as  the  best  single  way  of  identifying  and  defining  isohormone  species. 

In  addition,  reactivity  in  immunologic  assay  or  bioassay  is  required  by 
definition  before  any  species  can  be  designated  an  "isohormone". 

Major  Findings:   Standard  hGH  preparations,  from  the  National  Pituitary  Agency 
of  the  U.S.A.  and  from  AG  KABI  of  Stockholm,  exhibit  multiple  isohormone 
species.   These  are  both  "many"  and  "few".  They  are  "many"  in  that  any 
preparation  contains  2  to  5  recognizable  isohormones.  They  are  "few"  in  that 
only  the  same  group  of  5  isohormones  has  been  found  in  all  preparations 
examined.  hGH  iodinated  by  a  lactoperoxidase  procedure  showed  preservation 
of  gross  confirmation  by  polyacrylamide  gel  electrophoresis  criteria.  hGH 
iodinated  by  a  gentle  "monoiodo"  chloramine-T  procedure  showed  slight  altera- 
tion by  polyacrylamide  gel.  electrophoresis  criteria,  despite  showing  full 
activity  in  both  radioimmunoassay  and  radioreceptor  assay. 


s?s 


Project  No.   Z01  HL  OI855-OI  HE 

The  related  hormones,  human  prolactin  and  human  chorionic  somatomammotropin, 
have  been  compared  with  the  multiple  hGH  forms.   Several  human  prolactin  and 
human  chorionic  somatomammotropin  isohormone  species,  some  of  which  are 
indistinguishable  from  some  of  the  hGH  isohormone s,  have  been  identified. 

Significance  to  Biomedical  Research  and  Institute  Program:   Growth  hormone 
is  important  in  growth  retardation  and  in  acromegaly.   In  addition,  it  may 
play  a  role  in  the  vascular  complications  of  juvenile  diabetes  mellitus,  and 
its  secretion  is  markedly  decreased  in  obesity.  Furthermore ,    it  may  play  a 
role  in  anabolic  processes  in  general;  e.g.,  post -myocardial  infarction,  post- 
operatively, etc.   The  current  attempts  to  catalogue  the  multitude  of  growth 
hormone  species  known  will  allow  better  insight  into  structure -function  rela- 
tionships in  terms  of  these  various  actions  of  growth  hormone . 

Proposed  Course  of  Project:   l)  Characterization  of  additional  known  species 
of  growth  hormone,  including  circulating  "big"  growth  hormone,  fatty  acid 
hound  growth  hormone,  synthetically  produced  growth  hormone. 

Keyword  Descriptors:      growth  hormone      gel  electrophoresis 
isohormone s 

Honors  and  Awards:   None 

Publications:   None 


s$> 


Project  No.   Z01  HL  OI856-O2  EE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Control  of  Growth  Hormone  Secretion 

Previous  Serial  Number:   NHLI-27J+ 

Principal  Investigator:   Jay  S.  Skyler,  M.D. 

Other  Investigators:   Hans  G.  Baumgarten,  M.D.,  Ph.D. 
Walter  Lovenberg,  Ph.D. 

Cooperating  Units:   None 

Project  Description:- 

Objectives:   The  secretion  of  growth  hormone  by  the  adenohypophysis  is  under 
the  control  of  hypothalamic  peptides,  growth-hormone  releasing  hormone  and 
growth-hormone  release-inhibiting  hormone  (somatostatin).   These  hypothalamic 
peptides  in  turn  are  under  control  of  dopaminergic  and  serotonergic  neurons 
in  the  hypothalamus  and/or  other  brain  areas.  Recent  evidence  has  shown  that 
serotonergic  activity  stimulates  release  of  growth  hormone  and  that  dopaminer- 
gic activity  may  either  stimulate  or  inhibit  growth  hormone  release,  with 
some  species  differences. 

Serotonin  production  is  regulated  by  tryptophan  hydroxylase  and  dopamine  pro- 
duction by  tyrosine  hydroxylase.  An  accompanying  report  discusses  specific 
inhibitors  of  these  enzymes. 

The  objective  of  this  project  is  to  define  further  the  hypothalamic  regulation 
of  growth  hormone  secretion  by  serotonergic  and  dopaminergic  mechanisms. 

Methods:   Drugs  which  are  specifically  cytotoxic  for  serotonergic  and  catechol- 
aminergic  neurons  are  injected  intraventricular ly  into  newborn  or  adult  rats. 
Growth  of  rats  is  measured  by  weight  change.   Growth  hormone  is  measured  by 
standard  radioimmunoassay  and  destruction  of  neurons  are  monitored  by  measuring 
changes  in  tryptophan  and  tyrosine  hydroxylase. 

Major  Findings:   A  single  dose  of  5>7-dihydroxytryptamine  (5,7-IHT)  to  a  new- 
born rat  causes  a  reduction  of  growth.   Likewise,  intraventricular  administra- 
tion of  75  M-g  5^7-DHT  to  rats  of  8  weeks  of  age  causes  a  reduction  in  weight 
gain  over  the  next  several  weeks.   This  dose  of  5,7-DHT  causes  a  permanent 
destruction  of  about  20fo  of  the  serotonergic  neurons  in  the  hypothalamus. 
Circulatory  basal  growth  hormone  concentrations,  however,  are  unaltered. 


?33 


Project  No.   Z01  HL  018^6-02  HE 

Significance  to  Biomedical  Research  and.  Institute  Program:   Understanding  the 
monoaminergic  control  of  growth  hormone  secretion  may  allow  better  insight 
into  regulation  of  growth,  including  such  aspects  as  the  mechanism  of  psycho- 
logically induced  growth  retardation.   Likewise,  there  is  potential  for 
development  of  pharmacologic  agents  to  either  enhance  growth  hormone  secretion 
(in  the  case  of  some  growth-hormone  deficient  patients)  or  depress  its 
secretion  (in  the  case  of  acromegalic  patients  or  patients  with  diabetes 
mellitus) . 

X 

Growth  hormone  levels  in  patients  with  juvenile  diabetes  are  elevated,  and 
may  be  related  to  diabetic  complications.   The  ability  to  suppress  this 
elevated  growth  hormone  could  potentially  help  diminish  these  vascular  com- 
plications of  diabetes. 

In  addition,  these  studies  may  promote  the  fundamental  understanding  of  neuro- 
endocrine regulation,  especially  of  the  hypothalamic-hypophyseal  axis. 
Growth  hormone  levels  may  also  potentially  serve  as  an  index  of  central f 
serotonergic  activity. 

Proposed  Course  of  Project:   The  following  experimental  approaches  will  be 
undertaken: 

1.  further  examination  of  the  effects  of  inhibitors  of  tryptophan 
hydroxylase  and  tyrosine  hydroxylase  on  growth  hormone  secretion, 
with  specificity  increased  for  specific  CMS  activity, 

2.  attempts  to  overcome  the  changes  with  other  substances  that  stimulate 
or  inhibit  growth  hormone  secretion,  including  acute  administration 
of  agonists, 

3.  attempts  to  examine  secretion  of  other  pituitary  hormones  to  determine 
specificity  of  effects  for  growth  hormone  secretion,  specifically 
prolactin,  thyroid  hormones,  gonadal  hormones. 

Keyword  Descriptors:   neurotoxin      serotonin      growth  hormone 
acromegaly 

Honors  and  Awards:   None 

Publications:   None 


-TiV 


Project  Wo.  Z01  HL  018^7-02  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  197^  through  June  30,  1975 

Project  Title:   Growth  Hormone  Secretion  and  Structure  in  Cultured  Cells 

Previous  Serial  Number:   NHLI-275(c) 

Principal  Investigator:   Jay  S.  Skyler,  M.D. 

Other  Investigators:   Richard  A.  Knazek,  M.D. 
Walter  Lovenberg,  Ph.D. 

Cooperating  Units:   Laboratory  of  Pathophysiology,  NCI 

Project  Description: 

Objectives:   l)  To  develop  a  method  of  production  of  large  quantities  of  growth 
hormone  and  prolactin  for  therapeutic  purposes.   2)  To  gain  insight  into  con- 
trol of  hormone  secretion  in  tissue  culture. 

Methods:   Tumors  taken  from  patients  undergoing  hypophysectomy  are  grown  in 
tissue  culture  both  in  monolayer  and  in  artificial  capillaries.  Growth  hormone 
and  prolactin  secretion  into  tissue  culture  media  is  monitored  by  radioimmuno- 
assay. Agents  to  stimulate  or  decrease  hormone  secretion  are  added  to  the 
tissue  culture  apparatus  and  secretion  monitored. 

Growth  hormone  and  prolactin  produced  by  the  tumor  are  compared  with  their 
"normal"  serum  and  pituitary  counterparts  by  immunological  dilution  in  radio- 
immunoassay, dilution  in  radioreceptor  assay,  gel  filtration,  and  quantitative 
polyacrylamide  gel  electrophoresis. 

Major  Findings:   We  have  been  able  to  maintain  production  of  human  prolactin 
in  artificial  capillaries  for  as  long  as  five  months,  and  obtained  a  total  of 
3  mg  human  prolactin  from  a  single  culture  unit.  A  continuous  line  of  rat 
pituitary  cells  inoculated  into  multiple  artificial  capillary  units  has  pro- 
duced amounts  of  growth  hormone  and  prolactin  as  high  as  100  meg/day/ culture 
unit.  Both  human  growth  hormone  (hGH)  and  human  prolactin  have  also  been 
obtained  in  culture,  in  excess  of  2000  ng/ml.   In  confirmation  of  the  work  of 
others,  we  have  found  hGH  secretion  is  increased  significantly  in  culture  by 
the  addition  of  hydrocortisone  to  the  media.   By  using  single-pass  perfusion 
for  human  pituitary  cells  cultured  on  artificial  capillaries,  we  have  found 
both  an  eight-fold  immediate  and  a  lesser  prolonged  response  of  increased 
human  prolactin  secretion  to  TRH. 

We  have  established  four  criteria  which  must  be  met  by  hormone  produced  in 
tissue  culture  in  order  to  be  considered  indistinguishable  from  "standard" 

1  £?r 


Project  No.   Z01  HL  01857-02 

hormone  obtained  by  pituitary  extraction:   l)  parallel  dose-response  immuno- 
dilution  curves  in  radioimmunoassay;  indicating  that  specific  antibody  fails 
to  recognize  structural  differences;  2)  parallel  dose-response  curves  in 
specific  radioreceptor  assay,  "receptodilution",  indicating  that  receptor 
binding  sites  do  not  discriminate  between  the  compared  materials;  3)  identical 
partition  coefficients  on  gel  filtration;  h)   indistinghishable  joint  95</«  con- 
fidence envelopes  of  retardation  coefficient  and  relative  free  mobility  in 
quantitative  polyacrylamide  gel  electrophoresis  in  a  multiphasic  buffer  system 
optimized  for  characterization  of  the  hormone  in  question.   Tissue  culture 
produced  hGH  has  met  these  four  criteria.   Human  prolactin  from  tissue  culture 
has  met  the  three  criteria  thus  far  available  in  our  laboratory  (all  except 
receptodilution) . 

Significance  to  Biomedical  Research  and  Institute  Program:   The  production  of 
large  quantities  of  growth  hormone  and  prolactin  would  make  these  available  for 
both  investigative  and  clinical  purposes.   Prolactin  may  be  important  in  breast 
cancer.   Growth  hormone  is  important  in  growth  retardation.   It  may  additionally 
be  important  in  anabolic  processes  in  general;  e.g.,  after  myocardial  infarc- 
tion, postoperatively  in  osteoporosis,  etc.   Its  usefulness  in  these  conditions 
could  be  tested  if  supplies  were  available. 

Proposed  Course  of  Project:   l)  Demonstration  of  biological  activity  of 
culture-derived  hormone,  both  in  vitro  and  in  vivo, 

2)  increased  scale  of  production, 

3)  development  of  a  purification  scheme  for  the 
culture -produced  hormones, 

k)   demonstration  of  freedom  of  contamination  with 
other  materials, 

5)  further  use  of  the  artificial  capillary  system  for 
physiologic  and  pharmacologic  studies. 

Keyword  Descriptors:   growth  hormone      prolactin      pituitary  tumors 
capillary  tissue  culture 

Honors  and  Awards:   None 

Publications:   None 


£U 


Project  Wo.   ZOl  HL  OI858-O2  HE 


1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 
3«  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  197^  through  June  30,  1975 

Project  Title:   Juvenile  Diabetes  Mellitus 

Previous  Serial  Number:   NHLI-276(c) 

Principal  Investigator:   Jay  S.  Skyler,  M.D. 

Other  Investigators:   George  J.  Ellis,  M.D. 

Franc ine  L.  Levobitz,  R.N. 
Stanley  Slater,  M.D. 

Cooperating  Units:   Division  of  Endocrinology,  Department  of  Medicine, 
■Duke  University,  Durham,  North  Carolina;  Carolina's 
Camp  for  Diabetic  Children;  Laboratory  of  Clinical 
Sciences,  NIMH 

Project  Description: 

Objectives:  l)  To  improve  education  of  health  personnel  about  diabetes 
mellitus,  2)  to  improve  education  of  children  with  diabetes  mellitus  about 
their  condition,  and  3)  to  better  understand  juvenile  diabetes,  especially 
complications  of  diabetes. 

Methods:   Approximately  110  youths  with  diabetes  mellitus  each  year  attend 
Carolina's  Camp  for  Diabetic  Children.   Medical  and  nursing  students  live  in 
cabins  with  groups  of  8  to  12  children  with  diabetes  to  both  learn  about  the 
condition  and  help  in  instructing  the  children  in  proper  diabetic  management. 

Evaluation  forms  are  completed  by  staff  to  monitor  diabetes  education  of 
patients.  Follow-up  questionnaires  are  completed  by  children  and  parents. 

Major  Findings:   l)  Children  can  learn  proper  techniques  of  insulin  administra- 
tion and  urine  testing,  and  continue  to  utilize  these  techniques  for  many 
months  after  being  taught.   They  tend  not  to  continue  to  adhere  to  proper 
dietary  habits. 

2)  Complications  of  diabetes  may  be  detected  even  in  young  patients  if  somewhat 
sophisticated  screening  (fundus  photography,  vibratory  perception  threshold) 
is  used.   These  seem  to  correlate  with  elevated  blood  lipids  and  a  history  of 
poor  diabetic  control. 

3). Diabetic  camp  appears  to  be  the  single  best  setting  for  education  of  health 
personnel  about  diabetes  mellitus. 


T37 


Project  No.   ZOI  HL  OI858-O2  HE 

k)   Data  were  gathered  concerning  the  format  and  teaching  programs  of  k^ 
camps  for  children  with  diabetes  in  the  United  States.   This  data  is  to  serve 
as  a  background  for  developing  guidelines  for  such  camps.   Two  international 
workshops  have  been  held  for  that  purpose. 

Significance  to  Biomedical  Research  and  Institute  Program:   That  control  of 
diabetes  is  related  to  vascular  complications  has  become  increasingly  apparent 
in  light  of  many  recent  studies.   Since  in  the  past  (and  even  today)  this  con- 
cept has  been  controversial,  normalization  of  blood  sugar  has  not  been  the 
goal  of  management  of  most  patients  with  diabetes. 

The  attainment  of  that  goal  is  difficult  to  achieve  and  requires  much  effort 
on  the  part  of  physician  and  patient  alike.   However,  neither  has  been  well- 
educated  in  the  methods  needed  to  achieve  that  goal.   Since  camps  for  children 
with  diabetes  provide  a  format  for  education  of  health  personnel  and  patients, 
they  may  provide  one  of  the  best  formats  for  achieving  the  goal  of  normaliza- 
tion of  blood  sugar. 

Vascular  complications  of  diabetes  by  no  means  account  for  all  of  the  morbidity 
associated  with  the  condition.  Indeed  the  marked  increase  in  incidence  and 
severity  of  atherosclerotic  disease  associated  with  diabetes  is  equally  im- 
portant. Our  findings  indicate  there  may  be  a  correlation  between  elevated 
lipids,  early  signs  of  vascular  complications,  and  poor  control.  Thus 
education  in  achievement  of  normalization  of  sugar  may  help  to  decrease  the 
incidence  and  severity  of  atherosclerotic  disease  and  of  vascular  complica- 
tions of  diabetes. 

Proposed  Course  of  Project:   l)  Develop  teaching  aids  for  patients  with 
diabetes. 

2)  Develop  teaching  materials  to  improve  education  of  health  personnel 
about  diabetes. 

3)  Monitor  the  effectiveness  of  these  teaching  materials. 

k)   Expand  studies  of  incidence  of  vascular  complications  in  childhood 
diabetes. 

5)  Correlate  emotional  reaction  of  campers  with  diabetes  and  the  complexity 
and  success  of  their  treatment  regimen. 

Keyword  Descriptors:   diabetes      diabetic  camps 

Honors  and  Awards:   Dr.  Skyler  was  appointed  Chairman  of  the  Committee 
on  Camps,  American  Diabetes  Association. 

Publications:   None 


530 


Project  No.   Z01  HL  Ol859-04  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  197^  through  June  30,  1975 

Project  Title:   Characterization  of  the  Active  Site(s)  of  Dopamine-p- 
Hydroxylase 

Previous  Serial  Number:   NHLI-279 

Principal  Investigator:   Gustavus  A.  Walker,  Ph.D. 

Other  Investigators:   Walter  Lovenberg,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   The  objective  of  this  project  is  to  determine  the  number  of  active 
sites  on  dopamine -^-hydroxylase  (DBH)  indirectly  by  first  determining  the  total 
number  of  electrons  transferred  when  DBH  goes  from  the  physiologically  reduced 
state  to  the  physiologically  oxidized  state,  and  secondly,  by  determining 
the  number  of  electrons  transferred  as  a  unit  through  potentiometric  titra- 
tions. 

Methods :   DBH  was  isolated  by  a  modification  of  previously  published  procedures. 
The  modification  utilized  agarose  gel  filtration  as  the  final  purification 
step.   The  enzyme  was  demonstrated  to  be  electrophoretically  homogeneous  with 
a  specific  activity  50$>  greater  than  the  best  preparation  of  DBH  yet  published. 

Dichloroindophenol  and  potassium  ferrocyanide  were  selected  as  suitable  redox 
mediators.   The  oxidized  or  reduced  form  of  each  mediator  was  determined 
spectrophotometrically. 

Major  Findings:   A  major  obstacle  in  the  investigation  of  the  redox  potential 
of  DBH  is  the  requirement  for  relatively  large  amounts  of  the  purified  protein. 
The  reported  yields  of  DBH  from  previous  investigators  are  low  with  equally 
low  specific  activities.   For  example,  Foldes  et  al.  isolated  from  600  grams 
(wet  weight)  bovine  adrenal  medulla  less  than  5  mg  of  DBH  with  a  specific 
activity  of  less  than  2  pinoles  of  octopamine  produced  per  minute  per  mg  DBH; 
Wallace  and  Loveriberg  improved  the  efficiency  of  yields  from  a  comparable  amount 
of  starting  medulla  to  about  10  mg  of  pure  DBH  with  a  specific  activity  of  about 
20  umoles  per  minute  per  mg  DBH.  By  substituting  two  consecutive  agarose  gel 
filtration  steps  for  a  Sephadex  G-200  step  and  final  DEAE  gradient,  we  are  able 
to  isolate  from  600  grams  of  adrenal  medulla  about  l8  mg  of  DBH  with  a  specific 
activity  of  30  umoles  per  minute  per  mg  DBH.  The  amino  acid  analysis  performed 
on  the  DBH  purified  by  this  technique  gave  identical  results  with  that  per- 
formed on  DBH  purified  by  the  technique  of  Wallace  and  Lovenberg. 

1  ssf 


Project  No.  Z01  HL  01859-04  HE 

The  temperature  at  which  DBH  is  stored  can  also  affect  the  specific  activity 
with  time.   For  example,  purified  DBH  loses  about  50/0  of  its  specific  activity 
in  a  week  if  stored  at  K°C   in  plastic  containers  but  only  1%   of  the  specific 
activity  is  lost  in  a  week  if  DBH  is  stored  in  similar  containers  at  -20°C 
(part  of  the  loss  under  the  latter  conditions  may  be  due  to  the  effects  of 
freezing  and  thawing).   Quite  obviously  choice  and  length  of  purification  pro- 
cedures as  well  as  storage  time  are  important  considerations  in  evaluating 
protein  yields  and  specific  activity.) 

Only  preliminary  findings  have  resulted  from  the  use  of  dichloroindophenol 
and  ferrocyanide  as  redox  mediators  with  DBH.  While  both  compounds  can  couple 
with  DBH  only  reduced  dichloroindophenol  can  significantly  donate  electrons 
to  oxidized  DBH  at  equimolar  concentration  of  dye  to  protein.   Several 
thousand-fold  excess  of  ferrocyanide  to  DBH  are  required  to  transfer  electrons 
to  DBH  from  ferrocyanide.   This,  of  course,  suggests  that  the  midpoint 
potential  of  DBH  is  closer  to  dichloroindophenol  (Eq  =  +2l8  mv)  than  to 
ferrocyanide  (Eq  =  +36O  mv). 

Significance  to  Biomedical  Research  and  Institute  Program:   Dopamine-p- 
hydroxylase  catalyzes  the  final  step  in  the  synthesis  of  the  neurotransmitter 
norepinephrine,,  Upon  nerve  stimulation  DBH  together  with  norepinephrine  is 
released  by  an  exocytotic  process  from  sympathetic  nerve  endings  and  adrenal 
vesicles  to  appear  in  the  serum  of  man.   The  release  of  DBH  and  catecholamines 
is  a  process  intimately  involved  in  the  mechanism  of  nerve  transmission  of 
impulses.   In  order  to  fully  understand  the  process  of  nerve  impulse  trans- 
mission and  the  physiological  effects  of  released  DBH  a  complete  elucidation 
of  the  molecular  properties  of  this  enzyme,  especially  the  active  site(s),  is 
necessary. 

Proposed  Course  of  Project:   Experiments  are  planned  to  determine  the  stoichio- 
metry  of  electron  transfer  using  2,6-dichloroindophenol  and  potassium 
ferrocyanide  as  the  respective  electron  donor  and  acceptor,  and  monitor  the 
changes  in  the  Cu(ll)  electron  spin  resonance  signal.  Estimates  of  the  mid- 
point potential  of  DBH  can  be  made  through  dye  equilibration  with  DBH  by 
determining  the  relative  percentage  of  reduced  and  oxidized  forms  of  each 
reactant  at  equilibrium.   Values  for  the  midpoint  potential  of  DBH  can  be  more 
precisely  determined  by  potentiometric  titrations.   This  technique  also  allows 
the  determination  of  a  number  of  electrons  transferred  to  DBH  as  a  unit. 
Samples  could  be  removed  to  electron  spin  resonance  tubes  at  various  points 
during  the  titration  to  correlate  changes  in  E^  with  changes  in  DBH  oxidation. 

Another  interesting  area  to  be  explored  is  the  equivalence  of  DBH  subunLts. 
Although  a  few  investigators  have  examined  this  question,  subsequent  evaluation 
of  their  results  suggests  that  they  were  working  with  impure  preparations  of 
DBH.  End  group  analysis  and  cyanogen  bromide  cleavage  are  the  methods  of 
choice  to  investigate  the  equivalence  of  DBH  subunits. 

Keyword  Descriptors:   glycoprotein      dopamine -p-hydroxy las e      copper 
redox  potential 


s*k> 


Project  No.   Z01  HL  0l8 59-04  HE 

Honors  and  Awards:   None 

Publications: 

Wallace,  E.F.  and  Lovenberg,  W. :   Studies  on  the  carbohydrate  moiety 
of  dopamine  3-hydroxylase:   Interaction  of  the  enzyme  with  concanavalin 
A.   Proc.  Nat.  Acad.  Sci.  USA  71:  3217-3220,  1974. 


S# 


Project  No.  Z01  HL  01860-01  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 
3«  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Mitochondrial  Monoamine  Oxidase 

Previous  Serial  Number:   None 

Principal  Investigator:   Margaret  Walker,  Ph.D. 

Other  Investigators:   Walter  Lovenberg,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Mitochondrial  monoamine  oxidase  (MAO)  catalyzes  the  deamination 
of  neurohumoral  and  vasoactive  amines.   Inhibitors  of  this  enzyme  have  been 
to  be  effective  therapeutic  agents  in  depression  and  high  blood  pressure, 
however,  the  mechanisms  by  which  MAX)  inhibitors  exert  their  therapeutic  effect 
on  these  diverse  diseases  are  not  known. 

MAO  has  been  found  in  at  least  two  major  forms  with  different  substrate  and 
inhibitor  specificities.   Whether  the  two  classes  of  MAO  are  distinct  enzymes 
or  whether  the  same  enzyme  exists  in  different  chemical  environments  (i.e., 
with  different  amounts  of  bound  lipid)  has  not  been  distinguished.  There  is 
evidence  that  MAO  may  be  an  iron-flavoprotein,  however,  it  has  not  been  well 
characterized. 

The  purpose  of  this  project  is  to  determine  the  amount  of  iron  in  pure  MAO, 
the  nature  of  its  binding,  and  its  functional  significance  in  the  major  forms 
of  the  enzyme.   Characterization  of  the  substrate  binding  sites  will  also  be 
explored. 

Methods :   MAO  is  being  purified  from  beef  liver.   Initially  the  procedure 
of  Youdim  and  Sourkes  (Canadian  J.  Biochem.  hk:    1397;  1966)  is  being  used. 
The  iron  content  will  be  monitored  throughout  the  purification  procedure, 
and  both  serotonin  and  benzylamine  deamination  will  be  assayed  to  determine 
whether  either  the  A  or  B  form  of  MAO  is  being  purified  preferentially. 
Additional  procedures  which  will  be  attempted,  include  affinity  chromatography, 
and  carrying  out  purification  in  the  presence  of  iron  and/or  sulfhydryl 
reagents  to  see  if  activities  can  be  improved. 

MAO  is  assayed  for  its  ability  to  deaminate  serotonin  (MAO  type  A),  benzyl- 
amine (type  B),  and  kynuramine  (A  and  B)  by  standard  methods.   Total  iron  will 
be  determined  using  the  ferrous  iron  chelator  O-phenanthroline  or  aa '-dipyridyl 
under  reducing  conditions.   It  may  be  necessary  to  release  iron  with  sodium 
mersalyl. 

1  $V3- 


Project  No.  Z01  EL  01860-01  HE 

Major  Findings:   Work  on  the  purification  of  MAO  from  beef  liver  has  just  begun. 
The  enzyme  at  this  point  has  a  specific  activity  of  12,000  umoles  kynuramine 
per  mg  per  hour.   This  is  comparable  to  the  preparations  reported  from  many 
laboratories,  but  is  much  less  than  the  highest  specific  activity  reported. 
The  visible  spectrum  of  the  MAO  shows  peaks  at  approximately  4l0  nm  and  ^90 
nm  typical  of  flavoproteins.   Iron  analysis  gives  a  value  of  6Fe/l50,000  mw. 
This  high  value  suggests  that  the  MAO  may  still  be  contaminated  with  some 
mitochondrial  iron-sulfur  protein. 

Significance  to  Biomedical  Research  and  Institute  Program:   The  relevance  of 
this  study  to  biomedical  research  lies  in  the  importance  of  understanding  mono- 
aminergic  neuronal  systems  and  the  role  they  play  in  certain  types  of  diseases. 
It  appears  that  these  monoaminergic  systems  may  be  involved  in  the  regulation 
of  blood  pressure.  Work  from  this  laboratory  and  others  strongly  implicate 
a  role  for  central  noradrenergic  neurons  in  the  development  of  hypertension. 
The  role  of  MAO  in  regulating  amounts  and  availability  of  neurotransmitter 
amines  is  known,  however,  the  fact  that  many  MAO  inhibitors  are  effectvie 
antihypertensive  agents  is  not  well  understood.  The  hypotensive  effect  'of  in- 
hibiting an  enzyme  which  inactivates  pressor  amines  appears  paradoxidal 
but  may  reflect  a  mechanism  of  the  central  nervous  system.   Examining  MAO  at 
the  molecular  level  may  provide  insight  as  to  the  factors  that  determine  the 
substrate  and  inhibitor  specificity  of  the  different  forms  of  this  enzyme. 
This  knowledge  will  certainly  be  of  value  in  designing  new  and  better  inhibitors 
of  MAO  to  be  used  as  antihypertensive  agents. 

Proposed  Course  of  Project:   Work  is  continuing  on  the  purificatL  on  of  MAO. 
If  highly  purified  MAO  still  contains  appreciable  amounts  of  iron  the  next 
steps  are: 

1)  to  determine  which  ligands  are  involved  in  iron  binding.   MAO  is  reported 
to  contain  8  cysteine  residues  per  100,000  mw,  and  these  appear  to  be  essential 
for  activity.  MAO  would  be  titrated  with  sodium  mersalyl  to  determine  whether 
iron  was  released  in  this  process, 

2)  to  determine  the  function  of  iron  -  MAO  preparations  have  been  reported 
which  have  good  activity  but  widely  varying  amounts  of  iron.  Possibly  in 
vitro  assays  do  not  measure  the  role  of  iron  in  this  enzyme.  For  example, 
iron  may  participate  in  electron  transfer  to  an  intermediate  carrier  in  the 
mitochondria  rather  than  to  02« 

3)  to  determine  whether  the  activity  of  purified  MAO  can  be  improved  by  using 
conditions  which  might  reconstitute  an  iron  or  iron-sulfur  center. 

Keyword  Descriptors:   monoamine  oxidase      iron-sulfur  protein 
deamination      hypertension 

Honors  and  Awards:   None 

Publications:   None 


&3 


Project  No.   Z01  HL  Ol86l-03  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Biochemical  Pharmacology 
3«  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  197^  through  June  30,  1975 

Project  Title:   P-Chloromethamphetamine  and  Tryptophan  Hydroxylase 

Previous  Serial  Numbers:   NHLI-277,  NHLI-278 

Principal  Investigator:   Margaret  Walker,  Ph.D. 

Other  Investigators:   Walter  Lovenberg,  Ph.D. 

Hans  G.  Baumgarten,  M.D.,  Ph.D. 

Cooperating  Units:   None 

Project  Description:' 

Objectives:   Tryptophan  hydroxylase  is  the  rate-limiting  enzyme  in  the 
synthesis  of  serotonin.   Inhibition  of  tryptophan  hydroxylase  activity  by 
chlorinated  amphetamines  has  been  shown  to  be  one  reason  for  the  serotonin 
depletion  caused  by  these  drugs.   Because  tryptophan  hydroxylase  inhibition 
and  serotonin  depletion  are  very  long  lasting  despite  the  short  half-life 
of  these  drugs  in  the  body,  it  -was  thought  that  the  chlorinated  amphetamines 
might  have  a  neurotoxic  effect  similar  to  that  caused  by  5,6-  or  5>7- 
dihydroxytryptamine.   Since  p-chloromethamphetamine  (PCMA)  has  been  used  as 
an  antidepressant  in  the  past,  and  since  structurally  similar  drugs  are  still 
in  use  as  appetite  suppressants,  it  seemed  necessary  to  further  examine  its 
effect  on  serotonergic  neurons. 

Methods:   Male  Sprague -Dawley  rats  ( 150  to  200  gm)  were  given  i.p.  injections 
of  either  saline  or  PCMA  (10  mg/kg).  Rat  brains  were  removed  and  dissected 
as  described  by  Victor,  Baumgarten  and  Lovenberg  (J.  Neurochem.  22:  ^hl,    197^-) • 
Brain  regions  were  stored  in  liquid  Ng  until  assayed,  and  tryptophan  and 
tyrosine  hydroxylase  were  assayed  as  described  in  previous  reports. 

Major  Findings:   Although  chlorinated  amphetamines  have  been  shown  to  cause 
an  immediate  and  long-lasting  Inhibition  of  tryptophan  hydroxylase  activity,  a 
comparison  of  the  effects  in  various  brain  regions  had  not  been  done.   In  our 
studies  we  observed  the  following: 

1)  When  the  time  course  of  tryptophan  hydroxylase  inhibition  was  examined 

in  brain  regions  rich  in  serotonergic  nerve  endings  (i.e.  the  septum,  striatum 
and  cerebral  cortex)  there  was  approximately  30%  inhibition  after  only  3  hours, 
and  maximum  inhibition  occurred  at  approximately  2k   hours. 

2)  In  brain  regions  containing  cell  bodies  of  serotonergic  neurons  (the 
mesencephalic  tegmentum  and  pons  medulla)  inhibition  of  tryptophan  hydroxylase 
was  not  seen  until  Zk   or  48  hours  after  injection  of  PCMA. 

5W 


Project  No.  Z01  HL  Ol86l-03  HE 

3)  Animals  pretreated  with  chlorimipramine,  a  serotonergic  uptake  inhibitor, 
did  not  show  tryptophan  hydroxylase  inhibition  in  any  region  at  either  3  or 
6  hours  following  PCMA  treatment  (longer  time  intervals  were  not  examined). 

4)  Tyrosine  hydroxylase  activity  was  not  significantly  affected. 

These  studies  suggest  that  PCMA  is  being  taken  up  into  serotonergic  neurons 
specifically.   It  appears  to  be  concentrated  at  the  nerve  endings,  where  it 
has  an  immediate  effect.   The  delayed  effect  on  cell  body  areas  may  be  due  to 
retrograde  degeneration  of  axons  and  cell  bodies. 

Significance  to  Biomedical  Research  and  Institute  Program:   l)  The  use  of 
chlorinated  amphetamines  as  antidepressants  in  the  past  and  the  fact  that 
structurally  related  drugs  are  presently  being  used  as  appetite  suppressants 
make  it  essential  to  determine  the  mode  of  action  of  these  drugs  and  their 
possible  neurotoxicity. 

2)  Compounds  which  can  selectively  inactivate  serotonergic  neurons  would  be 
useful  tools  in  elucidating  the  role  of  serotonin  in  the  brain.   PCMA.  does 
not  completely  inactivate  tryptophan  hydroxylase.   It  shows  a  different 
pattern  of  inhibition  than  5,7-d-ihydroxytryptamine,  for  instance,  and  there- 
fore might  be  used  in  combination  with  this  or  other  drugs  to  further  deplete 
serotonin. 

Proposed  Course  of  Project:   The  appetite  suppressant  chlorphentermine  is 
structurally  similar  to  PCMA.,  and  it  is  an  effective  inhibitor  of  serotonin 
uptake. 

PCMA  CHLORPEKN'i'KKMINE 

CH2-C-NHCHo  HI      '  3 

V       CH3 


It  has  been  reported  that  chlorphentermine  does  not  deplete  serotonin  in  whole 
rat  brain,  however,  and  the  effect  on  tryptophan  hydroxylase  has  not  been 
studied. 

We  will  examine  the  effect  of  chlorphentermine  on  tryptophan  hydroxylase 
activity  and  serotonin  levels  in  various  regions  of  the  rat  brain  to  determine 
whether  there  is  an  effect  which  is  masked  in  the  whole  brain. 

Keyword  Descriptors:   p-chloroamphetamine      serotonin      tryptophan 
hydroxylase      chlorphentermine      neurotoxin 

Publications:   None 

2  s^r 


Project  No.  Z01  HL  01900-02  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Experimental  Therapeutics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Prostaglandins  in  Renal  and  Vascular  Physiology 

Previous  Serial  Numbers:  NHLI-260(c),  265(c) 

Principal  Investigator:   Robert  E.  Bowden,  M.D. 

Other  Investigators:   Harry  R.  Keiser,  M.D. 
John  J.  Pisano,  Ph.D. 
John  R.  Gill,  Jr.,  M.D. 
Michael  J.  Andrews,  Jr.,  M.D. 
Robert  A.  Guyton,  M.D. 

Cooperating  Unit:   Surgery  Branch,  National  Heart  and  Lung  Institute 

Project  Description: 

Objectives:   1)  Further  development  of  methodology  for  measuring  prostaglandins 
(PG)  in  biologic  fluids  in  subnanogram  range,  and  2)  apply  these  methods  to 
the  study  of  the  role  of  prostaglandins  in  renal  physiology  and  pathology  in 
hypertension. 

Methods:   1.   Radioimmunoassay.   Assays  have  been  developed  as  reported  pre- 
viously from  this  laboratory  which  can  measure  reliably  PGAi,  PGE  and  PGF  with 
a  sensitivity  of  150  to  200  pg. 

2.   Conventional  methods  of  extraction  and  chromatography  of  prostaglandins 
proved  to  be  unsuitable  for  the  radioimmunoassay  of  many  prostaglandins.   Con- 
sequently, methods  utilizing  Sephadex  LH-20  chromatography  for  the  purifica- 
tion of  urinary  extracts  were  developed  and  proved  to  be  satisfactory  for  the 
radioimmunoassay  of  PGE  and  PGF. 

Major  Findings:   A.   Humans 

1.   PGE  excretion  in  normal  and  hypertensive  subjects:   The  range,  of  urinary 
PGE  excretion  for  normals  in  our  laboratory  is  from  24  to  45  ng/hr  when  the 
subject  is  eating  a  diet  with  109  mEq  sodium  daily.   PGE  excretion  increases 
20  to  30%  on  changing  from  supine  to  standing  position.   Patients  with 
essential  hypertension  (2),  renal  artery  stenosis  (2),  Bartter's  syndrome  (5) 
and  idiopathic  postural  hypotension  (1)  were  studied. 


&r 


Age 

Supine 

Standing 

(yr) 

24  hr 
20.1 

(4  hr) 
10.3 

(4  hr) 

30 

7.3 

25 

26.1 

29.3 

19.5 

32 

34.2 





40 

17.5 





36 

52.5 





15 

25.0 

11.0 

19.5 

Child 

50.2 

12.5 

34.0 

Child 

40.0 

25.2 

43.0 

32 

105.2 





22 

39.5 

.42.9 

15.6 

Project  No.   Z01  HL  01900-02  HE 

PGE  Excretion  (ng/hr) 
Age 
Patient        Diagnosis 

L.G.  Renal  artery  stenosis 

R.A.  Renal  artery  stenosis 

G.A.  Essential  hypertension 

O.H.  Essential  hypertension 

B.B.  Bartter's  syndrome 

D.D.  Bartter's  syndrome 

M.J.  Bartter's  syndrome 

K.B.  Bartter's  syndrome 

J.E.  Bartter's  syndrome 

L.H.  Idiopathic  postural  LBP 

These  data  show  that  patients  have  a  wider  range  of  PGE  excretion  than  nor- 
mals.  Patients  with  renal  artery  stenosis  tend  to  excrete  lower  amounts ■ of 
PGE,  as  did  one  patient  with  essential  hypertension.   Patients  with  Bartter's 
syndrome  tend  to  excrete  higher  levels  of  PGE  than  normal.   Of  considerable 
interest  is  the  finding  that  3  patients  had  a  decrease  in  PGE  excretion  when 
changing  from  the  supine  to  the  standing  position.   Two  of  these  patients  had 
renal  artery  stenosis,  and  one  had  idiopathic  postural  hypotension,  conditions 
in  which  one  would  expect  a  greater  than  normal  decrease  in  renal  blood  flow 
in  changing  from  the  supine  to  the  standing  position. 

2.  Studies  using  P-113,  a  competitive  antagonist  of  angiotensin,  were  performed 
in  the  two  patients  with  renal  artery  stenosis. 

PGE  Excretion  (ng/hr) 
Patient         Supine  (4  hr)       P-113  (4  hr) 

L.G.  10.3  26.0 

R.A.  29.3  21.6 

One  patient  had  a  marked  increase  in  PGE  excretion  with  P-113  while  the  other 
had  a  26%  decrease.   The  former  response  is  unexpected.   The  latter  response 
is  the  expected  one  since  angiotensin  II  has  been  reported  to  increase  PGE 
release.   P-113  by  blocking  the  action  of  angiotensin  should  decrease  PGE  ex- 
cretion.  However,  the  latter  patient's  response  is  complicated  by  the  fact 
that  she  was  also  receiving  propranolol. 

3.  One  patient,  L.G.,  was  studied  under  identical  conditions  5  months  apart. 
PGE  excretion  (ng/hr)  on  8/14/74  was  18.5  (supine),  6.3  (standing)  and  on 
1/1/75  was  10.3  (supine)  and  7.3  (standing).   Thus  the  level  of  PGE  excretion 
appears  to  remain  the  same  from  month  to  month  in  the  same  patient. 

4.  Two  patients,  L.H.  (postural  hypotension)  and  B.B.  (Bartter's  syndrome), 
were  treated  with  Indomethacin,  a  prostaglandin  synthetase  inhibitor,  at  a  dose 

2  SV& 


Project  No.   Z01  HL  01900-02  HE 

of  150  mg  daily.   This  decreased  the  PGE  excretion  by  50%.   It  had  no  effect 
on  the  postural  hypotension  of  the  first  patient  but  was  associated  with  a 
significant  increase  in  serum  potassium  in  the  second  patient. 

5.   Levels  of  PGA  and  PGE  were  measured  in  renal  venous  blood  from  patients 
undergoing  catheterization  of  the  renal  veins  for  the  diagnosis  of  renal 
artery  stenosis.   Two  patients  have  been  studied,  one  with  classic  renal 
artery  stenosis  and  a  second  with  an  ectopic  kidney  but  with  low  renin  hyper- 
tension.  Plasma  PGE  and  PGA  were  obtained  from  the  right  and  left  kidneys 
and  the  inferior  vena  cava  in  the  initial  control  period  and  after  stimulation 
with  Hydralazine  (a  potent  vasodilator  which  stimulates  the  production  of 
renin) .   Plasma  levels  of  50  to  70  pg/ml  of  both  PGE  and  PGA  were  found  and 
were  not  different  in  the  3  sites,  before  or  after  Hydralazine.   These  values 
are  close  to  the  lower  limits  of  detectability  of  our  assay. 

B.   Dogs 

1.  Effects  of  saline  infusions  and  Indomethacin  on  PGE  excretion  in  hypo- 
physectomized  mongrel  dogs  anesthetized  with  pentobarbital.   As  detailed  last 
year  the  infusion  of  lactated  Ringer's  solution  produced  a  20  to  50%  decrease 
in  PGE  excretion  compared  to  control  values  when  the  animal  was  receiving  2.5% 
D/W.  When  Indomethacin  (4  mg/kg  I.V.)  was  administered  PGE  excretion  fell  50 
to  75%  and  there  were  increases  in  renal  blood  flow,  urine  volume,  sodium 
excretion  and  free  water  clearance.   These  experiments  have  been  extended  to 
include  a  control  infusion  of  2.5%  dextrose  solution  throughout  the  collection 
periods.   There  is  a  decrease  in  PGE  excretion  of  25  to  30%  from  control  levels 
throughout  the  course  of  the  experiment.   These  data  indicate  that  the 
prostaglandin  activity  decreases  not  in  response  to  Ringer's  solution,  but 
possibly  secondary  to  volume  expansion  or  time  after  the  operative  stresses 

at  the  start  of  the  experiment. 

2.  Effects  of  Indomethacin  in  conscious  dogs  with  stenosis  of  the  left  renal 
artery.   This  is  a  continuation  of  the  studies  started  and  reported  last  year 
(NHLI  265c) .   Dogs  were  prepared  with  a  75%  stenosis  of  the  left  renal  artery 
and  bilateral  ureterostomies.   After  the  dogs  had  recovered  for  several  weeks 
differential  renal  function  studies  were  performed  while  the  animals  were 
unanesthetized.   We  now  have  complete  studies  in  8  dogs.   Urine  volume,  PAH 
clearance,  inulin  clearance,  kallikrein  excretion  and  free  water  clearance  were 
all  reduced  significantly  (mean  reduction  of  50%)  on  the  left  or  stenotic  side. 
There  was  a  highly  significant  correlation  between  the  differences  in  kalli- 
krein excretion  between  the  normal  and  the  stenotic  kidney  and  the  differences 
in  renal  blood  flow  (PAH  clearance)  in  these  animals.   There  was  a  similarly 
significant  correlation  between  differences  in  kallikrein  excretion  and 
differences  in  inulin  clearance.   There  were  no  such  correlations  of  kallikrein 
excretion  with  urine  volume  or  with  excretion  of  sodium  or  potassium.   There 
was  also  a  significant  reduction  in  excretion  of  PGE  and  PGF  on  the  stenotic 
side.   There  was  a  correlation  between  PGE  and  PGF  levels  for  both  kidneys. 
When  Indomethacin  (2  to  10  mg/kg  I.V.)  was  administered  there  was  an  average 
decrease  of  80%  in  PGE  and  PGF  excretion.   There  was  a  significant  increase  in 
urine  volume  and  free  water  clearance  from  both  sides,  a  significant  fall  in 

3  S*f 


Project  No.   Z01  HL  01900-02  HE 

renal  blood  flow  on  only  the  stenotic  side,  and  no  significant  changes  in 
inulin  clearance  or  excretion  of  kallikrein,  sodium  or  potassium. 

3.   Bioassay.   Attempts  at  bioassay  for  PGE  in  samples  of  human  urine  have 
produced  levels  that  are  30  to  40%  of  our  radioimmunoassay  results.   However, 
samples  are  toxic  to  assay  organs.   This  toxicity  appears  to  be  secondary 
to  residues  of  organic  solvents. 

Significance  to  Biomedical  Research  and  Institute  Program;   The  control  of  the 
excretion  of  prostaglandins  is  poorly  understood.   The  data  presented  here  are 
very  tentative  but  suggest  that  PGE  is  involved  either  primarily  or  secondarily 
in  renal  physiology.   PGE  levels  fall  with  standing  in  certain  patients  in 
whom  one  would  expect  a  strong  vasoconstrictive  reaction  in  the  kidney.   In 
one  patient  given  an  angiotensin  blocker,  the  levels  of  PGE  rose,  perhaps 
correlating  with  a  competitive  reduction  in  this  vasoconstrictive  response. 

Our  data  with  Indomethacin  in  conscious  dogs  are  most  important.   They  con- 
tradict current  dogma  which  says  that  PG's  are  mediators  of  natriuresis  and 
diuresis  in  the  normal  kidney.   Those  studies  were  done  in  either  isolated 
perfused  kidneys  or  in  kidneys  after  extensive  surgery.   Our  studies  show  that 
inhibition  of  PG  synthesis  in  unanesthetized  animals  does  not  produce  profound 
changes  in  blood  pressure  and  renal  function.   In  fact  the  changes  we  found, 
while  significant,  were  relatively  small  inspite  of  85%  mean  reductions  in  PG 
levels.   Thus  PG's  are  not  the  mediators  of  the  excretion  of  salt  and  water 
excretion. 

Proposed  Course  of  Project:   1)  Studies  of  PG  excretion  in  patients  with 
various  types  of  hypertension  before  and  during  treatment.   2)  Studies  of 
PG  excretion  in  patients  and  normals  in  whom  the  renin-angiotensin  system  is 
either  stimulated  or  inhibited  by  either  physiologic  or  pharmacologic  means. 

3)  Study  of  the  effects  of  Indomethacin  in  patients  with  Bartter's  syndrome. 

4)  Study  of  the  interrelationships  between  the  PG's,  the  kallikrein-kinin 
system  and  the  renin-angiotensin  system. 

Keyword  Descriptors:  prostaglandins  urine  blood  radioimmunoassay 
column  chromatography  man  dogs  hypertension  Bartter's  syndrome 
indomethacin 

Honors  and  Awards :   None 

Publications: 

Gimbrone,  M.A. ,  Jr.  and  Alexander,  R.W. :   Angiotensin  II  stimulation  of 
prostaglandin  production  in  cultured  human  vascular  endothelium.   Science, 
in  press. 


rW 


Project  No.   Z01  HL  01901-01  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Experimental  Therapeutics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Kinins  in  Urine 

Previous  Serial  Number:   None 

Principal  Investigator:   Valdemar  Hial,  M.D. ,  Ph.D. 

Other  Investigators:   John  J.  Pisano,  Ph.D. 
Harry  R.  Keiser,  M.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Kinins  are  potent  endogenous  peptides  which  exert  their  effects 
in  at  least  three  broad  areas:   the  vascular  system,  inflammatory  processes 
and  in  smooth  muscle.   There  are  three  important  kinins:  bradykinin,  Lys- 
bradykinin  and  Met-Lys-bradykinin.   The  enzymes  responsible  for  the  production 
of  kinins  from  kininogens  are  known  as  kininogenases  (kallikreins) .   Kalli- 
krein  from  plasma  releases  bradykinin  and  that  from  glandular  tissues  releases 
Lys-bradykinin.   The  Met-Lys-bradykinin  is  of  obscure  origin.   Project  objec- 
tives were  to  investigate  the  presence  of  three  kinins  in  urine  giving  special 
emphasis  to  estimation  and  biosynthesis  of  Met-Lys-bradykinin. 

Methods :   We  developed  a  method  to  estimate  the  three  kinins  in  urine  using 
IRC-50  in  a  batch  technique  and  a  SP-Sephadex  C-25  column.   The  kinins  were 
assayed  on  an  isolated  guinea  pig  ileum.   Dipeptidyl  aminopeptidase  I  (DAP  I) 
as  well  as  amino  acid  analysis  was  used  to  characterize  Met-Lys-bradykinin. 
Collections  of  urine  for  24  hours  were  made  from  normal  subjects  into  plastic 
containers  using  6N  HC1  to  maintain  a  pH  about  2. 

Major  Findings:   The  results  are  summarized  in  the  following  table: 

Men  (n=10) 

yg/24  hr  (mean  +  SEM) 
Bradykinin  3.4  +  0.7 

Lys -Bradykinin  7.6  +  1.1 

Met-Lys-Bradykinin  9.5  +  1.6 

Total  Kinins  20.6  +  2.47 

Women  (n=6) 

Bradykinin  2.0  +  0.5 


sr/ 


Project  No.   Z01  HL  01901-01  HE 


Lys-Bradykinin  4.8  +  1.1 

Met-Lys-Bradykinin  1.0  +  0.4 

Total  Kinins  7.8  +  1.6 

In  men  total  kinins  average  20.6  yg/24  hr  and  the  amounts  of  Lys-bradykinin 
and  Met-Lys-bradykinin  are  essentially  the  same,  each  constitutes  approxi- 
mately 40%  of  the  total  kinins.   In  women  total  kinins  average  only  7.8  yg/24 
hr  and  while  the  amount  of  all  kinins  is  reduced,  the  major  reduction  is  in 
Met-Lys-bradykinin.   These  differences  between  men  and  women  in  total  kinins 
and  in  Met-Lys-bradykinin  are  both  highly  significant  with  p  <  0.001. 

We  have  some  preliminary  evidence  from  in  vitro  studies  which  indicate  that 
Met-Lys-bradykinin  is  released  by  action  of  uropepsin  on  kininogen  fragments 
in  human  urine  when  stored  at  pH  2.0. 

Significance  to  Biomedical  Research  and  Institute  Program:   This  is  the  first 
time  the  individual  kinins  in  human  urine  have  been  quantitated.   It  is  also 
the  first  time  that  Met-Lys-bradykinin  has  been  demonstrated  in  human  urine 
using  several  different  techniques.   These  data  have  shown  a  striking 
difference  between  normal  men  and  women.   This  focuses  our  attention  on  the 
possible  role  of  other  enzyme  systems  in  the  production  of  these  potent  vaso- 
dilators.  It  also  suggests  possible  controlling  influences  for  the  production 
of  Met-Lys-bradykinin. 

Proposed  Course  of  Project:   Studies  to  prove  the  actual  source  of  Met-Lys- 
bradykinin.   These  will  involve  investigation  of  other  urinary  enzymes  as 
possible  sources  of  kinins  and  of  the  relationships  between  changes  in  these 
enzymes  (including  kallikrein  and  urokinase)  and  the  levels  of  the  different 
urinary  kinins. 

Keyword  Descriptors:   human  urinary  kinins    bradykinin    Lys-bradykinin 
Met-Lys-bradykinin     dipeptidyl-aminopeptidase  I     column  chromatography 
bioassay 

Honors  and  Awards:   None 

Publications:   None 


5T> 


Project  No.      Z01  HL  01902-06  HE 


1.  Hypertension-Endocrine  Branch 

2.  Section  on  Experimental  Therapeutics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Clinical  Investigations  of  Vasoactive  Systems 

Previous  Serial  Number:   NHLI-264(c) 

Principal  Investigator:   David  Horwitz,  M.D. 

Other  Investigators:   Walter  Lovenberg,  Ph.D. 
Michael  F.  Roizen,  M.D. 
Horst  Brobecker,  M.D. 
Joseph  M.  Vinci,  M.D. 
Harry  R.  Keiser,  M.D. 

Cooperating  Units:   Laboratory  of  Clinical  Science,  NIMH 

Project  Description: 

A.   Studies  of  Dopamine-g-Hydroxylase  (DBH)  in  Hypertension 

Objectives:   DBH  is  the  enzyme  that  catalyzes  the  final  step  in  the  synthesis 
of  norepinephrine  the  sympathetic  neuromediator.   DBH  is  released  from  sym- 
pathetic nerves,  can  be  measured  in  plasma  and  has  been  proposed  as  an  index 
of  sympathetic  activity  in  man.   It  has  been  reported  that  plasma  DBH  levels 
are  higher  in  patients  with  essential  hypertension  than  in  subjects  with  pri- 
mary etiologies  for  their  hypertension  and  that  such  differences  are  of 
diagnostic  value.   Our  past  observations  suggested  that  plasma  levels  of  DBH 
activity  were  an  insensitive  reflection  of  sympathetic  activity;  further, 
patients  with  essential  hypertension  showed  low  as  well  as  higher  levels  of 
DBH  activity.   Our  recent  studies  were  intended  to  determine  whether  sympathet- 
ic activity  differs  in  essential  hypertensives  in  comparison  with  subjects 
with  normal  blood  pressure  or  with  secondary  hypertension.   At  the  same  time 
the  usefulness  of  DBH  activity  was  evaluated  by  comparison  with  an  established 
and  sensitive  test,  that  of  plasma  norepinephrine  levels. 

Methods:   Blood  pressure  was  determined  in  supine  and  erect  postures  on  eight 
separate  visits  to  establish  mean  values  and  variability  for  each  subject. 
On  one  visit  blood  samples  were  drawn  after  30  and  40  minutes  oJ:  rest  and  two 
and  ten  minutes  of  standing.   Plasma  DBH  activity  was  assayed  by  the  Nagatsu 
method  utilizing  the  enzymatic  conversion  of  tyramine  to  octopamine  and  photo- 
metric assay  after  oxidation  of  octopamine  to  p-hydroxybenzalclehyde.   Plasma 
norepinephrine  was  measured  by  a  newly  developed  method,  that  of  Roizen  et  al. ; 
the  norepinephrine  was  enzymatically  N-methylated  and  tritium-labeled  through 
use  of  [3H-methyl]-S-adenosylmethionine  and  phenylethanolamine-N-methyltrans- 


iT3 


Project  No.   Z01  HL  01902-06  HE 

f erase;  the  radioactive  end-product  was  thereafter  extracted  and  assayed  by 
liquid  scintillation  spectrometry. 

Major  Findings:   Twenty-eight  subjects  have  been  studied  (20  with  intermittent 
or  sustained  essential  hypertension,  5  secondary  hypertensives,  2  normals,  1 
with  idiopathic  orthostatic  hypotension) .  Mean  plasma  norepinephrine  levels 
for  the  20  essential  hypertensives  were  .25  ng/ml  while  supine  and  .52  ng/ml 
after  10  minutes  of  standing  (+108%);  the  corresponding  values  for  DBH 
activity  were  43  and  46  (+7%)  units.   The  correlation  between  resting  levels 
of  plasma  norepinephrine  and  DBH  activity  was  weak  and  not  statistically 
significant  (Spearman  rank  correlation  coefficient  0.4,  probability  of  chance 
occurrence  >  .05).   Six  subjects  underwent  the  stimulus  of  a  four  hour  period 
of  standing;  at  the  end  of  the  period  plasma  DBH  levels  had  risen  only  14% 
(51  units  pre,  58  post)  whereas  plasma  norepinephrine  levels  were  111%  higher 
(0.21  ng/ml  pre,  .45  post).   The  findings  in  a  patient  with  advanced  idiopathic 
orthostatic  hypotension  were  of  special  interest  in  that  they  revealed 
negligible  and  unresponsive  plasma  norepinephrine  levels  (.05,  .07,  .06  ng/ml) 
in  the  presence  of  normal  DBH  activity  (48  Nagatsu  units) . 

Dopamine- 3-hydroxylase  levels  have  been  determined  in  nine  patients  with  pheo- 
chromocytoma;  three  of  the  subjects  showed  values  which  were  above  the  normal 
range  and  four  were  at  the  upper  limits  of  normal.   Postoperative  values  were 
determined  for  six  subjects  and  revealed  a  56%  fall  from  an  initial  mean  level 
of  82  Nagatsu  units.   Serial  blood  samples  drawn  after  removal  of  the  tumor 
revealed  an  exponential  decline  of  plasma  DBH  activity  with  a  half-life  of  8 
hours. 

Significance  to  Biomedical  Research  and  Institute  Program:   The  recent  results 
support  our  previous  conclusion  that  plasma  DBH  activity  is  a  relatively 
insensitive  index  of  sympathetic  nervous  system  function  in  man.   The  half- 
life  of  plasma  DBH  has  been  measured  for  the  first  time  in  patients  with  pheo- 
chromocy tomas .   The  prolonged  half-life  of  DBH  correlates  with  the  muted 
responsiveness  of  this  index. 

Proposed  Course  of  Project:   Additional  subjects  will  be  studied  to  permit 
valid  comparisons  of  essential  hypertensives  with  normal  subjects  and  patients 
with  secondary  forms  of  hypertension. 

B.   Effect  of  Dietary  Potassium  on  Urinary  Excretion  of  Kallikrein 

Objectives :   Urinary  kallikrein  is  an  enzyme  that  forms  the  vasodilator  kinin, 
kallidin,  from  a  plasma  substrate.   We  have  previously  shown  that  urinary 
kallikrein  is  increased  by  salt  deprivation  and  by  administration  of  sodium- 
retaining  steroids.   The  object  of  the  present  study  was  to  determine  whether 
urinary  excretion  of  kallikrein  is  influenced  by  dietary  intake  of  potassium 
and  whether  this  vasodilator  system  responded  similarly  in  normotensive  and 
hypertensive  subjects. 

Major  Findings:   The  effects  of  three  levels  of  potassium  intake  (85  mEq  for 
5  days,  185  mEq  for  7  days  and  25  mEq  for  10  days)  on  kallikrein  excretion 

2  -TSV 


Project  No.   Z01  HL  01902-06  HE 

were  observed  in  16  normal  subjects  and  eight  patients  with  uncomplicated 
essential  hypertension;  intake  of  sodium  was  kept  at  110  mEq  throughout. 
Urinary  kallikrein  was  measured  by  the  radiochemical  assay  of  Beaven  et  al. 
Urinary  excretion  of  kallikrein  was  higher  with  the  185  mEq  than  with  25  mEq 
potassium  intake  for  each  subject,  was  usually  proportional  at  intermediate 
levels  of  intake,  and  paralleled  aldosterone  excretion.   Changes  in  kallikrein 
excretion  were  gradual  reaching  maximum  levels  after  5  to  7  days  of  high 
potassium  intake  and  minimum  levels  after  6  to  10  days  of  low  potassium  intake. 
Responses  were  qualitatively  similar  for  normotensive  and  hypertensive  sub- 
jects.  Mean  urinary  excretion  on  the  last  day  of  each  period  is  shown  in  the 
following  table: 


85  mEq 


185  mEq    25  mEq 


16  Normals 

10.8 

19.1 

5.8 

8  Hypertensives 

8.8 

13.9 

6.1 

n  =  19 

16.1 

28.8 

4.2 

n  =  24 

82 

155 

26  , 

Kallikrein  (units /day) 
Kallikrein  (units /day) 
Aldosterone  (pg/day) 
Potassium  (mEq/day) 

Mean  values  for  kallikrein  or  aldosterone  differed  significantly  (p  <  .01)  for 
each  period.   In  one  additional  subject  the  high  potassium  intake  was  pro- 
longed to  two  weeks  and  the  low  potassium  period  to  four  weeks;  the  correspond- 
ing levels  of  kallikrein  excretion  were  sustained  for  the  duration  of  each 
period. 

Significance  to  Biomedical  Research  and  Institute  Program:   Potassium  is  shown 
to  be  a  major  determinant  of  the  urinary  excretion  of  both  kallikrein  and 
aldosterone.   These  findings  support  the  hypothesis  that  alterations  in 
aldosterone  levels  mediate  changes  in  kallikrein  excretion. 

Proposed  Course  of  Project:  Direct  measurements  of  urinary  and  plasma  kinin 
levels  will  be  attempted  and  relationships  to  renal  function  and  alterations 
in  salt  metabolism  and  sympathetic  nervous  system  activity  will  be  investigated. 


Keyword  Descriptors:  dopamine-3-hydroxylase  norepinephrine  sympathetic 
nerve  activity  human  hypertension  urinary  kallikrein  aldosterone 
dietary  potassium 

Honors  and  Awards :   None 

Publications: 

1.  Margolius,  H.S.,  Horwitz,  D.,  Pisano,  J.J.,  and  Keiser,  H.R. :   Urinary 
kallikrein  excretion  in  hypertensive  man.   Relationships  to  sodium  intake 
and  sodium-retaining  steroids.   Circulation  Res.  35:  820-825,  1974. 

2.  Lovenberg,  W. ,  Bruckwick,  E.A. ,  Alexander,  R.W. ,  Horwitz,  D.,  and  Keiser, 
H.R. :   Evaluation  of  serum  dopamine-3-hydroxylase  activity  as  an  index  of 
sympathetic  nervous  activity  in  man.   In  Usdin,  E.  (Ed.):   Neuropsycho- 
pharmacology  of  Monoamines  and  Their  Regulatory  Enzymes .   New  York,  Raven 
Press,  1974,  pp.  121-128. 


£TS~ 


Project  No.   Z01  HL  01903-01  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Experimental  Therapeutics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Plasma  Prekallikrein  in  Hypertension 

Previous  Serial  Number:   None 

Principal  Investigator:   Teruhiro  Nakada,  M.D. 

Other  Investigators:   Harry  R.  Reiser,  M.D. 

Cooperating  Units :   None 

Project  Description: 

Objectives :   A  low  level  of  urinary  kallikrein  excretion  in  patients  with 
essential  hypertension  was  first  reported  by  Elliot  and  Nuzum  as  long  ago  as 
1934.   Since  then,  literature  dealing  with  the  relationship  between  high  blood 
pressure  and  the  kallikrein-kinin  system  has  been  scanty.   The  main  objective 
of  this  research  was  to  clarify  the  relationship  between  plasma  prekallikrein 
and  blood  pressure  with  special  reference  to  the  influence  of  dietary  sodium 
intake. 

Methods :   Seventeen  normotensive  volunteers  and  22  hypertensive  patients  were 
studied.   Blood  samples  were  drawn  from  each  subject  when  he  was  taking  either 
an  ad  lib  diet  or  one  with  a  controlled  level  of  sodium.   In  patients  with 
renovascular  hypertension,  Hydralazine  (a  potent  vasodilator)  was  administered 
to  examine  its  effect  on  the  level  of  plasma  prekallikrein. 

In  animal  experiments,  plasma  prekallikrein  was  assayed  in  dogs  eating  either 
a  regular  diet  or  a  diet  high  in  sodium  (180  mEq/day)  and  in  dogs  with  uni- 
lateral renal  artery  stenosis.   In  Wistar/NIH  rats  experimental  hypertension 
was  produced  by  administration  of  desoxycorticosterone  (DOC)  and  tap  water 
with  1%  NaCl.   Groups  of  rats  on  either  DOC  or  1%  NaCl  or  normal  diet  were 
used  as  controls.   Plasma  prekallikrein  was  determined  by  a  modification  of 
the  radiochemical  assay  using  p-tosyl-L-arginine  methylester  (TAMe)  as 
originally  developed  by  Beaven  and  Pisano.   One  unit  of  kallikrein  activity 
is  that  amount  of  TAMe  esterase  activity  found  in  1.0  ml  of  a  standard  acetone- 
activated  human  plasma  incubated  for  30  minutes  at  room  temperature. 

Major  Findings:   There  was  a  small  but  significantly  (p  <  .05)  lower  level  of 
plasma  prekallikrein  in  patients  with  essential  hypertension  (2.22  +  0.08 
units/ml,  mean  +  S.E.,  n=9)  when  compared  with  normotensive  subjects  (2.56  + 
0.13  units/ml,  n=17) .   There  was  no  significant  difference  in  the  level  of 
plasma  prekallikrein  between  normotensive  subjects  and  patients  with  reno- 
vascular hypertension  (2.52  +  0.07  units/ml,  n=ll) .   Plasma  prekallikrein  values 

l  £SZ 


Project  No.   Z01  HL  01903-01  HE 

were  the  same  in  normotensive  men  (2.61  +  0.12  units/ml,  n=10)  and  women 
(2.48  +  0.26  units/ml,  n=7) .   The  effect  of  dietary  sodium  intake  on  plasma 
prekallikrein  was  as  follows  (n=2) :   low  sodium  intake  (9  mEq/day) ,  3.48  + 
0.33  units/ml;  normal  sodium  intake  (109  mEq/day),  2.42  +  0.15  units/ml; 
high  sodium  intake  (259  mEq/day),  2.63  units/ml.   Intravenous  injection  of 
20  mg  of  Hydralazine  reduced  the  plasma  prekallikrein  level  significantly 
(p  <  0.005)  (pre-infusion  value  2.42  +  0.07  units/ml,  n=6;  post-infusion 
value  1.85  +0.09  units/ml,  n=6) . 

In  dog  studies  there  was  no  significant  difference  of  plasma  prekallikrein 
among  the  three  groups:   normal  controls  0.45  +  0.10  units/ml,  mean  +  S.E., 
n=8;  high  sodium  diet  0.56  +  0.04  units /ml,  n=4;  and  renovascular  hyperten- 
sion 0.47  +  0.04  units /ml,  n=6.   In  rat  experiments  repeated  administration 
of  DOC  (12.5  mg/100  g  body  weight)  elevated  plasma  prekallikrein  from  2.99  + 
0.28  units/ml  (n=8)  to  3.84  +  0.13  units/ml, (n=13)  on  the  tenth  experimental 
day  and  this  value  increased  further  to  4.29  +  0.16  units/ml  (mean  +  S.E., 
n=12)  on  the  30th  experimental  day  (p  <  0.005).   However,  administration  of 
1%  sodium  chloride  with  DOC  did  not  increase  plasma  prekallikrein  levels  on 
the  tenth  experimental  day  (3.09  +  0.13  units /ml,  n=10)  and  the  slight  in- 
crease on  the  30th  experimental  day  (3.42  +  0.15  units /ml,  n=7)  was  not 
statistically  significant.   A  high  sodium  diet  did  not  significantly  change 
levels  of  plasma  prekallikrein  in  rats.   On  the  other  hand,  a  low  sodium  diet 
increased  plasma  prekallikrein  significantly,  from  control  values  of  2.99  + 
0.28  units/ml,  n=8,  to  3.82  +  0.14  units/ml,  mean  +  S.E.,  n=9,  on  the  tenth 
experimental  day  (p  <  0.025),  however,  the  values  returned  to  control  level 
on  the  30th  experimental  day  (2.89  +  0.11  units/ml,  mean  +  S.E.,  n=8) 

Significance  to  Biomedical  Research  and  Institute  Program:   The  finding  of  a 
lower  level  of  plasma  prekallikrein  in  hypertensive  patients  is  important  since 
it  suggests  that  the  plasma  kallikrein-kinin  system  is  more  active  in  hyperten- 
sion.  Review  of  this  data  indicates  that  many  of  these  hypertensives  were  on 
therapy  and  had  normal  blood  pressures  when  the  blood  sample  was  drawn.   Thus, 
this  finding  will  need  further  verification.   It  is  clear  that  a  low  sodium 
diet  produces  a  significant  increase  in  plasma  prekallikrein.   This  raises 
an  important  point.  We  have  shown  previously  that  urinary  kallikrein  is  in- 
creased by  a  low  sodium  diet.   The  higher  plasma  prekallikrein  could  reflect: 
1)  decreased  activity  of  the  plasma  kallikrein-kinin  system,  2)  presence  of 
some  renal  kallikrein  in  plasma,  or  3)  an  increase  in  formation  of  plasma 
prekallikrein  due  to  the  action  of  aldosterone  (as  ve  have  shown  for  urinary 
kallikrein) .   The  fall  in  plasma  prekallikrein  after  Hydralazine  suggests  that 
the  plasma  kallikrein-kinin  system  may  be  a  mediator  in  drug  induced  vaso- 
dilation.  These  factors  all  have  important  bearing  on  the  role  of  the  kalli- 
krein-kinin system  in  normal  physiology  and  in  hypertension. 

Proposed  Course  of  Project:   Plasma  prekallikrein  will  be  assayed  in  more 
untreated  patients  with  essential  hypertension.   Plasma  kininogen,  another 
indicator  of  the  activity  of  the  plasma  kallikrein-kinin  system,  will  be  assayed 
in  the  same  patients  to  help  explain  our  findings  in  human  hypertension.   The 
effects  of  various  vasodilator  drugs  on  plasma  prekallikrein  will  be  studied 
in  man  and  rat. 

2  ST7 


Project  No.   Z01  HL  01903-01  HE 

Keyword  Descriptors:   plasma  prekallikreln      man     dog       rat 
hypertension      dietary  effects      dexoxycorticosterone      renal 
artery  stenosis 

Honors  and  Awards:   None 

Publications:   None 


S5B 


Project  No.      Z01  HL   01904-01  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Experimental  Therapeutics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  19  75 

Project  Title:   Renal  Kallikrein  and  Plasma  Kininogen  in  Hypertension 

Previous  Serial  Number:   None 

Principal  Investigator:   Frank  Perez  Acuna,  M.D. 

Other  Investigators:   Harry  R.  Keiser,  M.D. 

Cooperating  Units :   None 

Project  Description: 

Objectives:   1.   Develop  a  method  for  the  assay  of  kallikrein  in  renal  tissue. 
2.  Determine  if  there  is  a  correlation  between  amounts  of  kallikrein  in  renal 
tissue  and  in  urine  in  several  different  animal  models  of  hypertension.   3. 
Determine  if  values  of  plasma  kininogen  are  abnormal  in  hypertensive  humans 
and  animals. 

Methods :   After  extensive  experimentation  the  following  technique  was 
developed  for  measuring  kallikrein  in  renal  tissues.   Rats  were  anesthetized, 
the  aorta  was  ligated  above  the  renal  arteries  and  the  kidneys  were  perfused 
in  situ  for  10  minutes  with  cold  isotonic  saline  and  3.1%  sodium  citrate  to 
flush  out  all  plasma  and  its  kallikrein.   The  kidneys  were  removed  and  the 
cortex  was  isolated,  weighed,  and  homogenized  in  0.25  M  sucrose,  pH  7.4. 
After  centrifugation  at  40,000  rpm  for  30  minutes  the  supernatant  was  filtered 
through  Sephadex  G-25.   The  filtrate  was  assayed  for  kallikrein  activity  by 
incubation  with  semipurified  dog  plasma  kininogen  and  detection  of  kinin  re- 
leased by  bioassay  on  guinea  pig  ileum.   The  incubations  were  performed 
either  directly  in  the  bioassay  bath  or  in  separate  tubes,  the  contents  of 
which  were  boiled  and  later  added  to  the  bioassay  bath. 

Urinary  kallikrein  was  measured  via  the  radiochemical  esterolytic  assay  using 
3H-tosylarginylmethyl  ester  (^H-TAME)  as  described  in  previous  reports. 

Plasma  kininogen  was  assayed  by  incubation  of  aliquots  of  human  or  rat  plasma 
with  appropriate  excesses  of  either  partially  purified  human  or  rat  urinary 
kallikrein.   The  kinins  generated  were  assayed  on  the  guinea  pig  ileum. 

Major  Findings:   A  surprisingly  large  amount  of  kininase  activity  was  associated 
with  the  kallikrein  from  rat  kidneys.   This  made  assays  of  kallikrein  invalid 
because  of  rapid  destruction  of  kinins.   This  kininase  activity  was  only 
partially  removed  by  gel  filtration.   Various  inhibitors  of  kininase  were  tried 
and  8-hydroxyquinoline  (3  x  10-3M)  was  able  to  inhibit  about  70%  of  the 

1  &r? 


Project  No.   Z01  HL  01904-01  HE 

kininase  activity.   Using  the  inhibitor  we  were  unable  to  find  any  differences 
in  kallikrein  activity  in  kidneys  of  spontaneously  hypertensive  rats  and  the 
normotensive  Kyoto/Wistar  control  rats.   When  the  esterolytic  assay  for 
kallikrein  was  applied  to  the  renal  cortical  homogenates  it  was  apparent  that 
esterolytic  activity  was  about  5  times  greater  than  biologic  activity.   Thus 
the  nonspecific  esterolytic  method  could  not  be  used.   Similar  studies  using 
kidneys  from  rats  made  hypertensive  by  a  clip  on  one  renal  artery  or  their 
sham-operated  controls  showed  no  differences  in  levels  of  kidney  kallikrein. 
Again  the  problems  with  kininases  were  only  partially  overcome  and  the  overall 
recovery  of  kallikrein  activity  was  poor. 

When  the  activity  of  the  kallikrein-kinin  system  changes  there  should  be 
appropriate  changes  in  the  substrate,  i.e.  plasma  kininogen.  Thus  the  level 
of  plasma  kininogen  should  provide  an  inverse  indicator  of  kallikrein  system 
activity.  Plasma  kininogen  levels  averaged  2.41  +  0.45  micrograms  kallidin/ 
ml  plasma  (mean  +  S.E.M.)  in  normal  humans  (n=4) .  In  two  subjects  kininogen 
did  not  change  significantly  after  the  subject  stood  for  4  hours.  There  were 
no  apparent  differences  in  plasma  kininogen  levels  when  subjects  were  tested 
for  5  to  7  days  each  on  diets  of  109,  9,  or  259  mEq  of  sodium  daily. 

Hypertensive  patients  (n=5)  on  therapy  had  levels  of  plasma  kininogen  (1.69 
+  0.12)  that  averaged  29%  less  than  normal  controls.   When  2  patients  with 
essential  hypertension  were  given  acute  infusions  of  Hydralazine  (20  mg  I.V.) 
they  had  15  and  50%  increases  in  plasma  kininogen  within  30  minutes,  during 
the  acute  antihypertensive  effect  of  the  drug. 

Significance  to  Biomedical  Research  and  Institute  Program:   The  measurement 
of  renal  kallikrein  is  vital  to  our  understanding  of  this  vasodilator  system 
in  salt  and  water  homeostasis  and  in  hypertension.   These  studies  of  renal 
kallikrein,  while  negative,  have  indicated  that  other  approaches  must  be 
used  to  solve  this  problem. 

The  studies  of  plasma  kininogen  are  very  preliminary.   However,  they  indicate 
that  plasma  kininogen  may  provide  a  useful  index  of  the  activity  of  the 
kallikrein-kinin  system  in  man  and  animals.   The  finding  of  lower  levels  of 
kininogen  in  treated  hypertensives  must  be  extended  to  untreated  hypertensives. 
But  it  suggests  that  this  vasodilator  system  may  be  attempting  to  compensate 
for  the  hypertension  in  these  patients.   The  increase  in  kininogen  to  infusion 
of  a  vasodilator  suggests  that  this  system  may  be  responding  or  buffering 
acute  changes  in  the  sympathetic  nervous  system  or  in  the  renin-angiotensin 
system.   This  provides  further  clues  to  interactions  between  these  potent 
vasoregulatory  systems. 

Proposed  Course  of  Project:  The  principal  investigator  is  returning  to 
Venezuela.  The  work  on  renal  kallikrein  will  stop.  The  work  on  plasma 
kininogen  will  be  continued  by  others  in  our  group  using  newer  technology. 

Keyword  Descriptors:   rat  renal  kallikrein      bioassay      kininase  activity 
human  plasma  kininogen      dietary  effects      hydralazine 


Publications:   None 


sto 


Project  No.   Z01  HL  01905-05  HE 

1.  Hypertension-Endocrine  Branch 

2 .  Section  on  Experimental  Therapeutics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Urinary  Kallikrein  and  Kinin 

Previous  Serial  Number:   NHLI-266(c) 

Principal  Investigator:   Joseph  M.  Vinci,  M.D. 

Other  Investigators:   David  Horwitz,  M.D. 

Harry  R.  Keiser,  M.D. 
John  J.  Pisano,  Ph.D. 
Robert  Brown,  M.D.* 
Robert  Rhamey,  M.D.* 
John  Foster,  M.D.* 

Cooperating  Units:   *Department  of  Medicine 
Vanderbilt  University 

Project  Description: 

Objectives:   To  study  the  role  of  the  kallikrein-kinin  system  in  normal  and 
hypertensive  humans  and  animals. 

Methods:   1.   Urinary  kallikrein  excretion  is  measured  with  a  previously 
described  radiochemical  assay  [NHLI-79(c) ] . 

2 .  Urinary  kinin  is  measured  by  a  radioimmunoassay  developed  by  this 
laboratory  using  an  antibody  which  binds  bradykinin,  kallidin  and  methionyl- 
lysyl-brady kinin,  thus  giving  a  measure  of  total  immunoreactive  kinins. 

3.  Urinary  kallikrein  was  measured  in  normal  and  hypertensive  man  on  109 
mEq,  9  mEq  and  259  mEq  daily  sodium  intake. 

4.  On  a  109  mEq  Na  diet,  fludrocortisone,  0.5  mg/day,  was  given  and  kallikrein 
excretion  measured.   Spironolactone,  400  mg/day,  was  given  to  noi-nals  on  a 

9  mEq  sodium  diet  and  to  patients  with  primary  aldosteronism. 

5.  Urinary  kallikrein  and  kinin  were  measured  in  normal  subjects  and  in 
patients  with  essential  hypertension  during  a  potassium  intake  of  85  mEq,  185 
mEq  and  25  mEq  daily  with  a  constant  sodium  intake. 

6.  Urinary  kallikrein  was  determined  in  samples  from  31  patients  undergoing 
differential  renal  function  studies  as  part  of  an  evaluation  for  renovascular 
hypertension  at  the  Hypertension  SCORE  Program  at  Vanderbilt  University.   The 
patients  also  had  complete  evaluation  including  rapid  sequence  intravenous 

1  stt 


Project  No.   Z01  HL  01905-05  HE 

pyelography,  radioisotopic  renography,  renal  arteriography  and  determination 
of  plasma  renin  activity  in  renal  veins. 

Dogs :   1.   Measurements  of  urinary  kallikrein  and  kinin  were  performed  from 
each  ureter  in  unanesthetized  dogs  with  bilateral  ureterostomies  and  stenosis 
of  the  left  renal  artery  and  related  to  simultaneous  measurements  of  PAH 
clearance,  inulin  clearance,  urine  flow,  osmolality,  urinary  sodium  and 
potassium  excretion. 

2.   The  effects  of  intravenous  administration  of  minoxidil  and  the  angio- 
tensin converting  enzyme  (SQ  20,881)  on  the  urinary  kallikrein-kinin  system 
were  determined. 

Major  Findings:   Humans 

1.  Relationships  to  Sodium  Intake  and  Sodium-Retaining  Steroids 

a)  Normal  man:   When  sodium  intake  was  changed  from  109  mEq/day  to  9  mEq/ 
day,  daily  kallikrein  increased  progressively  in  each  of  13  subjects  to  a 
mean  maximal  value  that  was  271%  of  control  by  day  7.   An  increase  in  sodium 
intake  to  259  mEq/day  resulted  in  the  return  of  kallikrein  excretion  to  con- 
trol values.   Administration  of  fludrocortisone  to  4  subjects  on  a  109  mEq 

Na  diet  for  10  days  resulted  in  increased  kallikrein  excretion  to  a  mean 
maximal  value  that  was  203%  of  control.   In  two  subjects  on  a  diet  containing 
9  mEq  Na/day,  elevated  kallikrein  excretion  decreased  on  400  mg  spironolactone 
daily.   These  findings  demonstrate  that  the  kallikrein-kinin  system  responds 
to  sodium-retaining  steroids  whether  of  endogenous  or  exogenous  origin. 

b)  Hypertensives :   Eleven  patients  with  essential  hypertension  excreted 
significantly  less  (p  <  0.C01)  kallikrein  than  did  13  normal  subjects  on  a 
109  mEq  diet.   On  a  9  mEq  Na  diet  kallikrein  excretion  increased  in  the 
majority  of  patients  with  essential  hypertension  but  remained  significantly 
less  (p  <  0.001)  than  in  normal  subjects.   Three  patients  with  primary  aldo- 
steronism exhibited  a  kallikrein  excretion  that  was  seven-fold  higher  (p  < 
0.001)  than  patients  with  essential  hypertension  and  their  kallikrein  excretion 
was  unchanged  when  dietary  sodium  was  changed.   Their  kallikrein  excretion, 
however,  was  decreased  by  treatment  with  spironolactone. 

2.  Relationship  to  Potassium  Intake 

Thirteen  normals  receiving  a  185  mEq  K  and  109  mEq  Na  diet  showed  a  216% 
mean  maximal  increase  in  urinary  kallikrein  excretion  compared  to  control 
values  obtained  on  an  85  mEq  K  and  109  mEq  Na  diet.   With  reduction  of  K  intake 
to  25  mEq,  there  was  a  66%  mean  maximal  decrease  in  kallikrein  excretion  from 
control  values.   Seven  essential  hypertensives  showed  a  maximum  increase  in 
kallikrein  excretion  of  198%  on  high  potassium  intake  and  an  80%  mean  maximum 
reduction  in  urinary  kallikrein  excretion  on  low  potassium  excretion.   Again 
urinary  kallikrein  appears  responsive  to  the  effective  level  of  circulating 
mineralocorticoid  activity,  this  time  when  the  latter  is  altered  by  changes  in 
potassium  intake  a  known  potent  stimulus  to  aldosterone  production. 

2  $t* 


Project  No.   Z01  HL  01905-05  HE 

In  preliminary  observations  when  urines  from  the  control  period  of  this  study 
were  assayed  for  kinins  there  was  a  highly  significant  (p  <  0.01)  correlation 
(r  =  0.785)  between  kinin  excretion  and  kallikrein  excretion  (n=18) . 

3.   Renovascular  Hypertension 

Very  little  or  no  kallikrein  was  detected  in  the  urine  from  10  of  the  31 
patients.   Five  of  these  patients  had  unilateral  renovascular  disease  and  5 
had  disease  which  was  considered  to  be  bilateral  (in  3  it  was  of  equal  severity 
from  side  to  side  and  in  2  it  was  unequal).   In  addition,  there  were  4 
patients  with  low  or  undetectable  kallikrein  in  the  urine  from  only  one 
kidney.   In  all  4  of  these  patients  the  renovascular  disease  was  unilateral 
and  the  involved  kidney  was  the  one  with  low  or  undetectable  kallikrein.   This 
finding  of  low  or  undetectable  levels  of  kallikrein  is  very  rare  in  our 
experience.   Of  over  1000  adults  tested  only  3  had  very  low  levels  of  urinary 
kallikrein  and  all  3  were  hypertensives.   Only  2  of  603  normal  children  had 
very  low  urinary  kallikrein.   These  studies  are  preliminary  but  they  show 
that  in  a  significant  number  of  patients  there  is  little  or  no  kallikrein 
detectable  in  the  urine  from  one  or  both  kidneys.   In  addition,  they  show 
that  the  kidney  affected  with  renovascular  disease  generally  excretes  reduced 
or  undetectable  amounts  of  kallikrein. 

Dogs 

Unanesthetized  dogs  with  bilateral  ureterostomies  and  stenosis  of  the  left 
renal  artery  of  greater  than  75%  showed: 

1)  2.31  times  the  urine  flow  on  the  right  compared  to  the  left,  2)  2.11 
times  the  PAH  clearance  on  the  right  compared  to  the  left,  3)  correlation  of 
changes  in  renal  blood  flow  and  changes  in  kallikrein  with  a  correlation 
coefficient  of  0.94  (p  <  0.01),  4)  no  change  in  kallikrein  excretion  in 
response  to  Indomethacin. 

Significance  to  Biomedical  Research  and  Institute  Program:   Collectively  the 
above  data  support  the  hypothesis  that  the  renal  kallikrein-kinin  system  is 
involved  in  hypertensive  disease  and  in  salt  and  water  homeostasis. 

The  above  data  show  that  urinary  kallikrein  is  correlated  with  renal  blood 
flow  and  that  both  are  reduced  in  renovascular  hypertension  in  man  and  dogs. 
Drugs  which  increase  renal  blood  flow  appear  to  increase  urinary  kinin 
excretion  acutely  without  affecting  kallikrein  excretion. 

Proposed  Course  of  Project:   1.  Continue  to  test  the  hypothesis  that  the 
kallikrein-kinin  system  is  dependent  on  mineralocorticoid  activity  and  con- 
sequently on  the  renin-angiotensin  system.   This  will  involve  extension  of 
our  measurements  to  the  plasma  kallikrein-kinin  system  and  the  study  of  this 
and  the  urinary  kallikrein-kinin  system  in  induced  hyper-reninemic  states. 

2.   Further  characterization  of  the  kallikrein-kinin  system  in  low,  normal 
and  high  renin  patients. 

3  5*3 


Project  No.   Z01  HL  01905-05  HE 

3.  Study  the  effects  of  hydralazine,  a  peripheral  vasodilator,  on  the 
kallikrein-kinin  system  in  dog  and  man. 

4.  Study  the  effects  of  the  angiotensin  converting  enzyme  inhibitor,  SQ  20,881, 
on  the  kallikrein-kinin  system  in  normal  renin  hypertensives. 

5.  Study  the  diagnostic  potential  of  kallikrein  excretion  in  diseases  affecting 
renal  cortical  blood  flow. 

6.  Determine  more  precisely  the  relationship  between  the  kallikrein-kinin 
system  and  the  prostaglandins. 

Keyword  Descriptors:  human  hypertension  urinary  kallikrein  renal 
artery  stenosis  potassium  intake  aldosterone  urinary  kinins 
sodium  intake 

Honors  and  Awards :   None 

Publications : 

1.  Margolius,  H.S.,  Horwitz,  D. ,  Geller,  R.G.,  Alexander,  R.W. ,  Gill,  J.R. , 
Jr.,  Pisano,  J.J.,  and  Keiser,  H.R. :   Urinary  kallikrein  excretion  in 
normal  man.   Relationships  to  sodium  intake  and  sodium-retaining  steroids. 
Circulation  Res.   35:  812-819,  1974. 

2.  Keiser,  H.R.,  Margolius,  H.S.,  Brown,  R. ,  Rhamey,  R. ,  and  Foster,  J.: 
Urinary  kallikrein  in  patients  with  renovascular  hypertension.   In 
Pisano,  J.J.  and  Austen,  K.F.  (Eds.):   Chemistry  and  Biology  of  Kallikrein- 
Kinin  System  in  Health  and  Disease.   Washington,  D.C.,  United  States 
Government  Printing  Office. 


&¥■ 


Project  No.   Z01  HL  01906-02  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Experimental  Therapeutics 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Urokinase  Excretion  and  Function 

Previous  Serial  Number:   NHLI-267(c) 

Principal  Investigator:   Gilbert  M.  Wilcox,  M.D. 

Other  Investigators:   Harry  R.  Keiser,  M.D. 
John  J.  Pisano,  Ph.D. 
Valdemar  Hial,  M.D.,  Ph.D. 
David  Horwitz,  M.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Urokinase  (UK)  is  a  urinary  proteolytic  enzyme  which  activates 
plasminogen  to  plasmin  and  thereby  dissolves  fibrin  clots.  Methods  to  measure 
UK  have  utilized  either:   measurement  of  the  area  of  fibrin  clot  dissolved 
when  a  drop  of  UK  containing  solution  is  applied  to  a  fibrin-coated  slide 
which  contains  an  excess  of  plasminogen  or  measurement  of  UK  esterolytic 
activity  using  an  appropriate  ester  substrate.   A  new  radiochemical  esterolytic 
method  has  been  developed  using  H^-acetyl-glycyl-lysine  methyl  ester.   The 
first  objective  was  to  determine  the  validity  of  this  simple  and  highly 
sensitive  assay  by  comparison  with  the  fibrin  plate  assay.   Subsequent  project 
objectives  were  to:   1)  investigate  UK  stability  in  solutions  of  varying  pro- 
tein and  salt  content,  2)  determine  urokinase  excretion  in  normal  subjects, 
3)  determine  UK  excretion  under  the  following  experimental  conditions: 
nephrectomy,  experimental  hypertension,  renal  vein  thrombosis  and  in  human 
kidney  transplant  rejection,  and  4)  study  UK  excretion  in  normal  subjects 
during  various  maneuvers  to  determine  its  relationships  with  sympathetic 
nervous  system  activity  and  the  kallikrein-kinin  system. 

Methods :   Urokinase  was  assayed  using  H-^-acetyl-glycyl-lysine  methyl  ester  as 
enzyme  substrate;  UK  action  on  this  synthetic  ester  splits  off  H^-methanol  which 
was  measured  in  a  scintillation  counter.   Timed  urine  collections  were  made 
under  various  experimental  conditions  and  bovine  serum  albumin  was  added  to 
each  sample  to  enhance  stability.   Whole  urine  was  assayed,  the  radioactivity 
generated  being  proportional  to  the  enzyme  activity. 

Major  Findings:   1)  Assay  of  duplicate  aliquots  of  human  and  rat  urine 
demonstrated  that  data  obtained  with  the  radiochemical  assay  was  analogous 
with  urokinase  measured  with  the  fibrin  plate  assay. 


srr 


Project  No.   Z01  HL  01906-02  HE 

2)  The  radiochemical  assay  was  highly  reproducible  with  a  interassay 
variability  of  less  than  10%  and  measured  small  amounts  of  UK. (lower  limit 
of  sensitivity  of  0.2  CTAU/ml) . 

3)  Urokinase  was  unstable  in  solutions  of  low  protein  or  salt  concentration; 
stability  was  greatly  enhanced  by  raising  the  protein  concentration  (addition 
of  albumin  or  lysozyme)  and  was  essentially  independent  of  salt  concentration 
if  albumin  concentration  was  greater  than  1  mg/ml. 

4)  Urokinase  excretion  in  normal  volunteers  on  109  mEq  Na  /100  mEq  K  diet 
and  ad  lib  activity  was:   men  8131  +  4454  CTAU/24  hr,  n=6,  (mean  +  SEM) ; 
women  7686  +  3464  CTAU/24  hr,  n=5-   Recovery  of  UK  from  human  urine  was  78%  + 

3.5%. 

5)  Urokinase  excretior  was  measured  in  control  (sham  operation)  and  in  rats 
made  hypertensive  by  unilateral  renal  artery  stenosis:   control  530  +  96  CTAU/ 
24  hr,  n=5,  RAS  hypertensive  1034  +  404  CTAU/24  hr,  n=6. 

6)  The  effect  of  posture  on  urokinase  excretion  was  studied.   Eleven  subjects 
were  fed  a  109  mEq  Na   100  mEq  K+  diet.   Four  hour  urine  specimens  were 
collected  on  consecutive  days  always  between  5  am  and  9  am;  only  posture  was 
varied.   Urokinase  excretion  was  728  +  197  (supine)  and  1270  +  209  CTA  units/ 
4  hr  (standing)  (mean  +  SEM).   This  increase  was  observed  in  10  of  11  subjects 
and  was  highly  significant  (p  <  .005).   These  data  show  a  possible  correlation 
between  urinary  plasminogen  activator  excretion  and  sympathetic  nervous  system 
activity. 

Significance  to  Biomedical  Research  and  Institute  Program:   The  radiochemical 
assay  has  been  validated  as  a  simple  and  accurate  assay  for  urokinase.   The 
conditions  for  the  stability  of  urokinase  in  urine  have  been  established.   Our 
values  for  urokinase  excretion  in  normals  are  similar  to  those  reported  by 
others  using  the  fibrin  plate  assay.   Urokinase  excretion  was  increased  in 
rats  with  renal  artery  stenosis.   This  may  represent  a  compensatory  response 
since  urokinase  may  be  an  additional  source  of  urinary  kinins  (potent 
vasodilators).   The  study  of  the  effects  of  posture  on  urokinase  excretion 
suggest  a  direct  relationship  with  sympathetic  nervous  system  activity.   Science 
continues  to  search  for  a  good  integrated  indicator  of  sympathetic  nervous 
system  activity.   Urokinase  excretion  may  provide  such  an  indicator. 

Proposed  Course  of  Project:   Studies  will  be  performed  to  pursue  the  relation- 
ship between  urokinase  excretion  and  sympathetic  nervous  system  activity.   These 
will  include  infusion  of  specific  vasoactive  amines  into  normal  volunteers  and 
study  of  the  effects  of  sympatholytic  drugs  in  hypertensives.   The  role  of 
urokinase  in  producing  kinins  will  be  pursued  by  measuring  both  substances 
under  these  various  experimental  procedures. 

Keyword  Descriptors:   urokinase      plasminogen  activator      human  and  rat 
urine      postural  effects      renal  artery  stenosis      sympathetic  nervous 
system      fibrin  plate  assay 

Publications:   None 

2  SZ6 


Project  No.      Z01  HL   01941-04  HE 

1.  Hypertension-Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Studies  on  the  Structure  of  Villikinin 

Previous  Serial  Number:   NHLI-217 

Principal  Investigator:   John  J.  Pisano,  Ph.D. 

Other  Investigators:   Eszter  Kokas,  Ph.D. 

Cooperating  Units:   Physiology  Department 

University  of  North  Carolina 
Medical  School 

Project  Description: 

Objectives:   To  isolate  and  determine  the  structure  of  villikinin,  a  substance 
obtained  from  intestinal  mucosa  which  has  a  specific  stimulant  action  on 
intestinal  villous  motility. 

Methods  Employed:   Canine  and  porcine  intestinal  mucosa  samples  were  extracted 
with  trichloroacetic  acid  (TCA)  and  the  extracts  applied  to  Dowex  50,  Bio- 
Gel  P-4  and  Bio-Gel  P-10  columns.   Active  fractions  were  bioassayed  in  dogs 
in  vivo,  by  the  usual  method  of  noting  the  increase  in  intestinal  villous 
motility. 

Major  Findings:   At  least  four  peaks  with  kallikrein-like  activity  were  ob- 
served when  crude  canine  villikinin  was  gel  filtered  on  Bio-Gel  P-4  columns. 
The  high  molecular  weight  substance (s)  and  the  two  unstable  low  molecular 
weight  substances  were  not  examined  further  since  their  chemical  and  biological 
properties  proved  to  be  different  from  villikinin.   The  main  fraction  having 
an  approximate  MW  2000-3000  was  inactivated  by  leucinaminopeptidase  and  Narg- 
ase.   Experiments  with  carboxypeptidase  A  were  inconclusive  because  the  enzyme 
unexpectedly  affected  the  bioassay.   Upon  rechromatography  of  the  active  ma- 
terial on  Bio-Gel  P-10,  a  single  peak  of  activity  was  obtained  with  an  apparent 
MW  2000.   This  material  was  inactivated  by  Pronase.   A  dose-response  curve  has 
been  determined  for  villikinin  by  both  topical  and  intravenous  administration. 
Porcine  mucosa  contains  material  biologically  and  chemically  similar  to  canine 
villikinin. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:   Villi- 
kinin appears  to  be  a  specific  hormone  which  controls  intestinal  villous 


SZ7 


Project  No.   Z01  HL  01941-04  HE 

motility.   Contraction  of  villi  promotes  lymph  flow  and  absorption  of  nutrients 
from  the  GI  tract. 

Proposed  Course:   To  determine  if  porcine  mucosa  is  a  richer  source  of  villi- 
kinin.   To  isolate  and  determine  the  structure  of  villikinin. 

Keyword  Descriptors:   villikinin    intestinal  villi   villous  motility 
polypeptide   intestinal  mucosa 

Honors  and  Awards:   None 

Publications:   None 


Slg 


Project  No.   Z01  HL  01942-05  HE 

1.  Hyper tens ion- Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 


Project  Title:   Clinical  Biochemistry  of  the  Kallikrein-Kinin  System 

Previous  Serial  Number:   NHLI-280 

Principal  Investigator:   John  J.  Pisano,  Ph.D. 

Other  Investigators:   Jorge  A.  Guimaraes,  Ph.D. 
Kerin  Yates,  B.S. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   To  develop  procedures  for  the  assay  of  components  of  the  kalli- 
krein-kinin  system.   To  establish  the  role  of  the  kallikrein-kinin  system  in 
health  and  disease.   To  determine  if  plasmin  generates  peptides  in  plasma 
which  potentiate  kinin  actions. 

Methods  Employed:   Bioassay  and  radioimmunoassay  of  kininogen  and  kinins. 
Enzymatic  digestions  of  plasma  with  trypsin,  kallikrein  and  plasmin. 

Ma j or  F ind ing s :   1)  Kininogen  Assay:   The  classical  method  for  the  assay  of 
kininogen  involves  the  bioassay  of  bradykinin  generated  by  the  action  of  an 
excess  of  trypsin  added  to  heated  plasma.   It  has  been  suspected  by  some  but 
not  generally  recognized  that  trypsin  also  generates  peptides  which  potentiate 
the  action  of  bradykinin  when  bioassayed  with  the  rat  uterus  of  guinea  pig 
ileum.   To  determine  if  trypsin  does,  in  fact,  produce  potentiating  peptides 
we  first  liberated  all  the  kinin  from  kininogen  in  a  plasma  sample  by  prolonged 
incubation  with  an  excess  of  human  urinary  kallikrein.   Liberated  kinin  was 
inactivated  in  the  process  by  kininases  in  the  plasma.   Trypsin  or  plasmin  was 
then  added  to  the  sample  to  produce  the  suspected  potentiating  peptides  and 
the  recovery  of  added  bradykinin  determined.   A  potentiation  factor  was  cal- 
culated as  PF  =  recovered  bradykinin.   Both  trypsin  and  plasmin  uroduced  pep- 
added  bradykinin 
tides  which  potentiated  the  action  of  bradykinin  on  the  guinea  pig  ileum; 
PF  =  2.85.   Taking  this  factor  into  consideration  3.00  yg  of  bradykinin  is  re- 
leased by  the  kininogen  in  1  ml  plasma.   Other  laboratories  no;  aware  of  the 
extent  of  potentiation  have  erroneously  reported  8-10  yg  bradykinin.   The 
finding  that  physiological  levels  of  plasmin  generates  potentiating  peptides 
has  considerable  significance  since  it  is  likely  that  kinin  generation  and 


st? 


Project  No.   Z01  HL  01942-05  HE 

plasminogen  activation  simultaneously  occurs  in  a  number  of  pathophysiological 
conditions. 

Human  plasma  contains  a  kininogen  type  preferred  by  plasma  kallikrein.   It 
accounts  for  15-38%  of  the  total  kininogen.   Glandular  kallikrein  is  about 
equally  active  on  all  the  kininogen  types.   See  report  #  Z01  HL  1944-18  HE. 

Kinin  released  from  kininogens  was  also  determined  by  radioimmunoassay. 
The  good  agreement  with  the  bioassay  indicates  that  the  more  practical  radio- 
immunoassay technique  now  can  be  used  to  assay  kininogens. 

Plasma  kallikrein  has  been  determined  in  12  normal  subjects.   The  mean 
plasma  level  of  four  females  taking  oral  contraceptives  was  0.65  TU/ml,  two 
females  not  on  the  pill,  1.22  and  6  males,  1.14.   The  lower  level  of  plasma 
kallikrein  in  females  on  the  pill  is  of  considerable  interest  because  of  the 
high  incidence  of  hypertension  in  this  group. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:   Assay 
of  total  kininogens  and  the  plasma-kallikrein-preferral  kininogen  type  will  be 
essential  to  the  evaluation  of  the  pathophysiologic  role  of  the  kallikrein- 
kinin  system.   The  generation  of  bradykinin  potentiating  peptides  by  plasmin 
points  to  an  important  interrelationship  of  the  fibrinolytic  and  kallikrein- 
kinin  systems. 

Proposed  Course:   To  use  the  kininogen  assay  in  studies  on  the  pathophysiologic 
role  of  the  kallikrein-kinin  system  especially  in  hypertensive  diseases.   To 
study  the  interrelationship  of  the  kallikrein-kinin  fibrinolytic  and  sympa- 
thetic nervous  systems.   To  study  the  plasma  kallikreinin  females  taking  oral 
contraceptives . 

Keyword  Descriptors:   kininogen   kinin   plasmin   kallikrein   radioimmuno- 
assay 

Honors  and  Awards:   None 

Publications:   None 


fro 


Project  No.  Z01  HL  01943-05  HE 

1.  Hypertension-Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 


Project  Title:   Peptide  Biochemistry 

Previous  Serial  Number:   NHLI-281 

Principal  Investigator:   John  J.  Pisano,  Ph.D. 

Other  Investigators:   Carl  L.  Zimmerman,  M.S. 
Hiroshi  Nakamura,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   To  develop  necessary  methodology  for  the  micro  characterization 
and  assay  of  peptides  and  amino  acids. 

Methods  Employed:   High  pressure  liquid  chromatography,  spectrophotof luorom- 
etry. 

Major  Findings:   Previous  methods  for  the  HPLC  analysis  of  PTH-amino  acids 
formed  in  the  Edman  sequence  analysis  of  peptides  has  been  improved  by  the  use 
of  new  microparticle  columns.   Separation  of  all  PTH  amino  acids  with  the  ex- 
ception of  PTH-met  from  PTH-val  has  been  achieved  by  gradient  elution  from  a 
25  cm  DuPont  Zorbax-ODS  column.   This  system  is  currently  in  use  in  the  analysis 
of  PTH  samples  from  both  manual  and  automated  degradation. 

In  addition  to  gradient  elution,  a  method  has  been  developed  for  the  iden- 
tification of  PTH-amino  acids  by  the  less  expensive  and  potentially  more  rapid 
isocratic  elution  from  two  columns:   Group  A,  the  most  polar  PTH  derivatives 
of  asp,glu,asn,gln,thr,ser,gly,ala  and  tyr  are  resolved  on  a  25  cm  12  mm 
column  of  Zorbax-ODS.   The  non-polar  Group  B  derivatives:   met,val,phe  ,ile,leu, 
trp  and  lys  are  separated  on  a  DuPont  ETH  (100  cm  x  12  mm) .   Dinitrophenyl 
(DNP)  amino  acids  formed  by  reaction  with  1-f luoro-2,4-dinitrobenzene  have 
been  analyzed  by  HPLC.   This  procedure  is  the  most  sensitive  and  specific 
method  for  the  analysis  of  DNP-amino  acids  and  will  have  its  greatest  appli- 
cation in  the  determination  of  the  N-terminal  amino  acid  of  polypeptides. 

Tryptophan,  peptides  containing  N-terminal  tryptophan,  tryptamine  and 
certain  related  compounds  react  with  Fluram  to  form  derivatives  with  uniquely 
high  fluorescence  in  strong  acid. 


rrf 


Project  No.    201  HL  0194.3-05  HK 

A  membrane  filter  assay  has  been  developed  for  the  determination  of  pro- 
teins in  the  submicrogram  range.   Proteins  are:   (1)  treated  with  Fluram  in 
sodium  borate  buffer  (pH  9.0)  containing  0.03  M  MgCl2,  (2)  collected  on  mem- 
brane filters,  and  (3)  desorbed  from  the  filters  by  an  acetone-sodium  berate 
buffer  (pH  9.0).   By  measuring  the  fluorescence  intensity  of  the  extract,  as 
little  as  0.3  ug  of  protein  can  be  determined  in  the  presence  of  reactive  low 
molecular  weight  substances  which  do  not  interfere  because  they  are  not  re- 
tained by  the  filter. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:   The 
development  of  improved  methods  for  the  determination  of  amino  acid  sequences 
of  polypeptides,  will  increase  the  feasibility  of  determining  the  structures 
of  countless  naturally  occurring  biologically  active  peptides.   Better  methods 
for  the  detection  and  assay  of  vasoactive  peptides  will  facilitate  the  study  of 
their  biological  roles. 

Proposed  Course:   To  develop,  perfect  and  apply  essential  new  methods  of  the 
analysis  for  polypeptides. 

Keyword  Descriptors:   amino  acid  sequence   PTH  amino  acids   DNP  amino  acids 
N-Group  analysis   amino  acid  analysis   fluorometric  assay   protein  assay 

Honors  and  Awards:   None 

Publications: 

Tamura,  A.,  Nakajima,  T.,  Nakayama,  T. ,  et  al. :   Identification  of  pep- 
tides with  l-dimethylaminoaphthalene-5  sulfonyl  chloride.   Anal.  Biochem. 
52:595-606,  1973. 

Zimmerman,  C.L.,  Pisano,  J.J.,  and  Appella,  E. :   Analysis  of  amino  acid 
phenylthiohydantoins  by  high  speed  liquid  chromatography.   Biochem. 
Biophys.  Res.  Comm.  55:1220-1224,  1974. 


T73- 


Project  No.   Z01  HL  01944-18  HE 

1.  Hypertension-Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Biochemistry  of  the  Kallikrein-Kininogen-Kinin  System 

Previous  Serial  Number:   NHLI-284 

Principal  Investigator:   Jack  V.  Pierce,  Ph.D. 

Other  Investigators:   Jorge  A.  Guimaraes,  Ph.D. 
Marion  E.  Webster,  Ph.D. 
John  J.  Pisano,  Ph.D. 
Allen  P.  Kaplan,  M.D. 

Cooperating  Units:   Allergic  Diseases  Section 

Laboratory  of  Clinical  Investigation 

National  Institute  of  Allergy  and  Infectious  Diseases 

Project  Description: 

Objectives:   Purification  of  glandular  kallikrein  and  components  of  the  plasma 
kinin  system  for  characterization  purposes  and  for  production  of  specific  anti- 
serums.  Preparation  of  purified  specific  antibodies  for  biochemical  and  chemi- 
cal studies.   Preparation  of  affinity  adsorbents  from  purified  antibodies  and 
antigen  for  purification  and  other  purposes,  such  as  devising  specific  bio- 
chemical and  radioimmunochemical  assays.   Applications  of  these  purified  pro- 
teins, affinity  adsorbents,  and  assay  methods  to  studies  of  human  disease 
states,  such  as  hypertension. 

Methods  Employed:   Protein  purification;  enzyme  kinetics  ;  bioassay. 

Major  Findings:   1)  Human  Plasma  Kininogens.   a)  Kinetics:   Two  of  the  highly 
purified  kininogens,  B2aand  B4  3,  described  in  the  previous  report  (Serial 
No.  NHLI-284),  were  used  for  kinetic  analyses  of  human  urinary  and  plasma 
kallikreins,  bovine  trypsin,  and  human  plasmin.   The  results  are  summarized 
below: 


ST3 


Project  No.   Z01  HL  01944-18  HE 


Enzyme 


Human  urinary  kallikrein 
Human  plasma  kallikrein 
Bovine  trypsin 
Human  plasmin 


Kininogen 


B2a* 


Km  (uM) 

9.0 
115 
23.1 
23.1 


k ~ (sec~l) 
cat 

4.5 

2.7 

0.67 

0.015 


B4B** 


K   (uM) 
m 

5.8 

3.7 

27.0 


k "  (sec~l) 
cat 

4.2 

3.2 

0.52 


*  1.0  mole  kinin/72,400  g 

**   1.0  mole  kinin/106,000  g 

These  two  kininogen  forms  were  chosen  because  B2a  appears  to  be  the  major  LMW 
kininogen  in  human  plasma  (35%  of  total)  and  the  form  probably  isolated  by 
other  workers;  and  because  B4B  comprises  the  major  part  of  the  HMW  kininogens. 
As  shown,  human  plasma  kallikrein  sharply  discriminates  between  them.   It  has 
about  thirty  times  the  affinity  for  B43  as  for  B2a;  the  latter  is  a  very  poor 
substrate  for  this  kallikrein  compared  with  other  kallikreins  and  even  with 
the  non-kallikrein,  trypsin.   B4a  and  B4y  were  like  B43  in  being  very  good 
substrates  for  plasma  kallikrein. 

b)  HMW  Kininogen  (B4) ,  a  New  Clotting  Factor:   The  above  results  assume 
a  much  greater  interest  in  view  of  the  recent  discovery  that  certain  patients 
with  "a  prolonged  activated  partial  thromboplastin  time  and  an  inability  to 
form  plasmin.  eradykinin  or  PF/d:'l"  (K.D.  Wuepper  et  al.,  Flaujeac  Trait: 
Deficiency  of  Kininogen  in  Man.  Fed.  Proc.  34:859  Abs.,  1975)  lack  both  LMW 
and  HMW  kininogens,  but  only  the  latter  form  can  correct  the  clotting  and  other 
defects:   "The  kinin-forming,  intrinsic  coagulation  and  fibrinolytic  pathways 
of  plasma  and  the  formation  of  the  permeability  globulin  PF/dil  are  dependent 
upon  plasma  HMW-kininogen"  (op  cit.).   Two  other  groups  have  found  patients 
with  a  similar  defect  which  is  also  corrected  by  fractions  containing  HMW 
kininogen.   Part  of  the  initial  evidence  for  this  hypothesis  was  obtained  by 
means  of  monospecific  antiserums  to  LMW  human  kininogens  prepared  in  our  lab- 
oratory.  We  had  earlier  found  immunological  identity  between  LMW  and  HMW 
kininogens  with  these  antiserums. 

Sheep  antiserums  to  both  B43  and  B4y  contain  at  least  two  classes  of  anti- 
bodies, one  directed  against  the  kininogens  and  the  other  directed  against  a 
non-kininogen  group  called  X,  in  B4a,  B,  and  Y.   We  have  isolated  this  second 
class  of  antibodies,  Abx,  by  affinity  chromatography  of  the  antiserums  on 
columns  of  insolubilized  LMW  kininogen,  followed  by  affinity  chromatography 
of  the  filtrate  on  another  column  of  insolubilized  B4B  +  y.   There  appears  to 
be  a  third  class  of  antibodies  still  to  be  investigated. 

Our  various  crude  and  highly  purified  kininogen  fractions  have  been  tested 
in  three  ways:   1)  by  an  assay  developed  by  M.E.  Webster  for  an  unknown  factor 
required  for  the  activation  of  unactivated  Hageman  factor  (HF^) ;  2)  by  the 
classical  clotting  assay  (performed  by  A. P.  Kaplan,  using  Williams  plasma 


&? 


Project  No.   Z01  HL  01944-18  HE 


which  lacks  the  Flaujeac  trait);  and  3)  by  Ouchterlony  double  diffusion, 
using  Abx.   The  results  are  summarized  below: 


Activity 

HF± 

Clotting 

Kininogen 

Activator 

Time 

Ouchterlony 

Bla,  B2a,  B3.1a  and  6 

_ 

_ 

_ 

B3.2a  and  6 

B4a,  g,  and  y 

+ 

+ 

+ 

Peak  Al 

- 

- 

- 

Peak  A2 

- 

- 

- 

Peak  A3 

+ 

+ 

+ 

Peak  A4 

+ 

+ 

+ 

B4y  incubated  with: 

Human  plasma  kallikrein 

+ 

+ 

+ 

Human  urinary  kallikrein 

+ 

N.T.* 

+ 

Hog  pancreatic  kallikrein 

+ 

N.T. 

+ 

Bovine  trypsin 

- 

N.T. 

+** 

Human  plasmin 

- 

N.T. 

+** 

Porcine  pepsin 

- 

N.T. 

- 

*  N.T.  =  not  tested. 

**  Spurs  indicating  the  presence  of  kininogen  antigen  +  Kgn  -X,  instead  of 
only  Kgn  -X  as  in  B4y  or  B4y  treated  with  kallikreins. 

c)  Methionyl-lysyl-bradykinin  (MLBK) ,  the  Peptide  Kinin  from  Human 
Kininogens :   The  reaction  of  several  purified  human  kininogens  at  pH  2-6  with 
crystalline  porcine  pepsin  very  rapidly  gave  a  kinin  peptide  which  was  stable 
for  extended  periods  to  further  digestion  by  pepsin.   The  peptic  peptide  had 
about  one-eighth  the  guinea  pig  ileum  activity  of  the  same  amount  of  kininogen 
treated  with  trypsin.   Like  MLBK,  the  activity  of  the  peptide  increased  about 
8-fold  when  treated  with  human  plasma  aminopeptidase  or  dipeptidyl  peptidase 
I  to  give  BK.   Amino  acid  analysis  of  the  purified  peptide  derived  from  kinino- 
gen B3.2a  was  consistent  with  its  being  MLBK.   The  peptic  kinin  also  had  the 
same  retention  volume  as  MLBK  on  an  analytical  SP-Sephadex  C-25  column  which 
separates  MLBK,  LBK,  and  BK. 

2)  Human  Urinary  Kallikrein.  a)  Two  Catalytic  Sites:   Since  human  urinary 
kallikrein,  like  other  glandular  kallikreins,  cleaves  two  dissimilar  peptide 
bonds,  Met-Lys  and  Arg-Sers  in  kininogens  to  form  lysyl-bradykinin,  we  have 
for  several  years  thought  it  likely  that  these  kallikreins  would  have  two 
non-identical  catalytic  centers.   We  treated  human  urinary  kallikrein  with  DFP 
until  all  of  the  arginine  esterase  (TAME)  activity  had  been  destroyed.   The 
DIP-enzyme  was  unable  to  release  kinin  from  LMW  kininogen  II  (bradykinin  se- 
quence inside  the  molecule),  but  was  still  able  to  form  kinin  from  LMW  kinino- 
gen I  (bradykinin  at  the  C-terminus) .   It  is  therefore  practically  certain 


S7T 


Project  No.   Z01  HL  01944-18  HE 

that  human  urinary  kallikrein  has  two  different  active  sites.   On  the  other 
hand,  DFP  treatment  of  rat  and  horse  urinary  kallikreins,  hog  pancreatic  kalli- 
krein, and  human  plasma  kallikrein  destroyed  both  active  sites.   Thus,  human 
urinary  kallikrein  may  be  unique  in  lacking  a  serine  residue  in  site  2,    the 
active  center  responsible  for  cleaving  the  Met-Lys  bond. 

b)  Kinetics:   Human  urinary  kallikrein,  like  trypsin  and  several  other 
serine  proteinases, -  shows  substrate  activation  with  TAME,  but  not  with  BAEE. 
The  kinetics  of  human  urinary  kallikreins  A  and  B  (see  Serial  No.  NHLI-134) 
were  carefully  studied  at  25°  between  0.15  and  20  mM  TAME.   When  the  data  were 
analyzed  according  to  an  equation  developed  for  the  hydrolysis  of  TAME  by 
trypsin  (Trowbridge  et  al. ,  Biochemistry  2:843,  1963),  an  excellent  fit  was  ob- 
tained.  As  with  trypsin  and  TAME,  two  sets  of  kinetic  parameters  were  obtained, 
one  between  0.15  and  1.0  mM  TAME  and  the  other  between  2.0  and  20  mM. 

c)  Bioassay  of  Kallikrein  in  Urine:   Incubation  of  from  10  to  25  ul  of 
untreated  urine  with  about  0.15  mg  of  kininogen  Prep  B  for  15  min  at  37°  yields 
about  150  ng  of  LBK,  an  amount  readily  detected  by  the  isolated  guinea  pig 
ileum.   Quantitation  in  TAME  units  can  be  achieved  by  means  of  an  LBK  standard 
and  a  curve  relating  a  standard  human  urinary  kallikrein  to  the  amount  of  LBK 
produced  under  the  above  conditions.   Those  urine  samples  in  which  kininases 
and  aminopeptidases  are  high  relative  to  kallikrein  and  might  therefore  inter- 
fere with  the  bioassay  by  destroying  the  LBK  formed  or  by  converting  it  to 
bradykinin,  respectively,  can  be  treated  at  pH  4.4/1  hr/37°  to  destroy  the 
kininase  and  with  1,10-phenanthroline  to  inhibit  the  aminopeptidase  activity. 
This  method  is  useful  for  measuring  urinary  and  other  kallikreins  in  very  dilute 
solutions  and  for  checking  the  usual  TAME  and  BAEE  assay  methods  for  determining 
kallikrein  content. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:   The 
purification  of  the  components  of  the  plasma  kallikrein  system  is  crucial  to 
investigations  of  its  physiological  function(s).   This  system  is  activated  si- 
multaneously with  the  intrinsic  blood  coagulation  and  lysis  systems.   It  now 
appears  that  HMW  kininogen,  which  we  had  previously  isolated  in  highly  puri- 
fied form,  is  necessary  for  the  activation  of  Hageman  factor,  which  in  turn 
initiates  these  three  systems.   It  will  therefore  be  of  great  importance  to 
study  the  chemistry  of  pure  HMW  kininogens  and  the  mechanism  whereby  they  act. 

Proposed  Course:   HMW  Human  Kininogens:   We  plan  to  do  further  experiments 
designed  to  discover  the  conditions  under  which  B4  kininogens  acquire  the 
capacity  to  activate  Hageman  factor  and  to  look  for  ways  to  separate  the  X 
antigen  from  the  kininogen  part. 

Human  Urinary  and  Other  Kallikreins:   Further  work  will  be  done  on  the 
two  active  sites  of  kallikreins,  using  various  active  site  reagents. 

Keyword  Descriptors:   plasma  kallikrein    urinary  kallikrein   kininogen 
kinins   coagulation 

Honors  and  Awards:   None 


Project  No.   Z01  HL  01944-18  HE 


Publications: 


Nustad,  K. ,  Pierce,  J.V.,  and  Vaaje,  K.  :   Synthesis  of  kallikreins  by- 
rat  kidney  slices.   Brit.  J.  Pharmacol.  53:229-234,  1975. 


T77 


Project  No.   Z01  HL  01945-03  HE 

1.  Hypertension- Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland" 


PHS-NHLI 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Histamine,  Prostaglandins  and  Esterase  From  Human  Lung 

Previous  Serial  Number:   NHLI-286(c) 

Principal  Investigator:   Marion  E.  Webster,  Ph.D. 

Other  Investigators:   Hidenobu  Takahashi,  Ph.D. 
Harold  H.  Newball,  M.D. 

Cooperating  Units:   Johns  Hopkins  University  School  of  Medicine 
Baltimore,  Maryland 

Project  Description: 

Objectives:   The  passive  sensitization  of  human  lung  fragments  as  first  de- 
scribed by  Parish  (Nature  215:738,  1967)  provided  a  laboratory  model  for  the 
study  of  allergic  asthma  in  man  and  permits  investigation  of  the  role  of 
mediators  in  chis  immediate  type  of  allergy.   The  purposes  of  this  study  are 
to  identify  mediators  released  from  human  lung  during  sensitization;  to  de- 
termine the  mechanism  of  their  release;  to  assess  the  role  of  pharmacological 
compounds  used  in  the  treatment  of  allergy;  and  to  provide  a  rational  basis 
for  the  development  of  new  drugs. 

Methods  Employed:  Human  lung  obtained  during  surgery  is  cut  into  fragments 
(30-100  mg),  sensitized  with  sera  obtained  from  individuals  allergic  to  rag- 
weed, washed  free  of  serum  proteins  and  incubated  with  a  preparation  of  pure 
antigen  E  (5-500  ng) .  Release  of  arginine  esterase  was  determined  by  sensi- 
tive radiochemical  assays  developed  in  this  laboratory  (Beaven  et  al. ,  Clin. 
Chim.  Acta  32:67,  1971). 

Major  Findings:   An  improved  method  for  the  measurement  of  histamine  has  been 
developed  by  combining  the  procedure  of  Thithapanda  et  al.  (Comp.  Gen. 
Pharmacol.  3:139,  1972)  with  that  of  the  method  previously  employed  (Beaven 
et  al. ,  Clin.  Chim.  Acta  37:91,  1972).   This  greatly  simplified  method  has 
been  applied  routinely  to  the  measurement  of  the  release  of  histamine  in  lung 
supernatants  and  the  results  correlate  well  with  the  earlier  method. 

In  addition  to  histamine,  an  arginine  esterase  and  SRS-A,  prostaglandins 
are  also  released  when  passively  sensitized  human  lung  is  reacted  with  spe- 
cific antigen.   Measurement  of  the  prostaglandins  released  by  radioimmunoassay 


T7g 


Project  No.   Z01  HT,  01945-03  HE 

(NHLI-99c)  indicated  that  the  increase  in  prostaglandin  F  was  much  smaller 
than  that  previously  reported  (Piper  and  Walker,  Br.  J.  Pharmacol.  47:291, 
1973).   Prostaglandin  E-like  substances,  on  the  other  hand,  increased  in  a 
manner  similar  to  that  of  histamine  and  arginine  esterase.   As  prostaglandin 
F's  constrict  and  prostaglandin  E's  relax  human  bronchi,  it  appears  unlikely 
that  prostaglandins  have  a  direct  mediator  effect  in  bronchial  asthma. 
However,  they  may  act  as  modulators  of  this  disease  either  by  potentiating 
the  effects  of  other  mediators  or  by  altering  the  levels  of  cyclic  AMP. 

Our  earlier  results  (Webster  et  al.,  Ciencia  e  Cultura  26:372,  1974)  had 
indicated  that  the  arginine  esterase  released  from  human  lung  by  antigen- 
antibody  interaction  was  neither  a  kallikrein  nor  an  activator  of  plasma  ■ 
kallikrein.   This  esterase  is  also  neither  plasmin  nor  a  plasminogen  acti- 
vator.  After  antigen  challenge,  the  lung  incubation  medium  contained  no 
detectable  plasmin  (<0.2  ng)  and  the  small  amount  of  plasminogen  activator 
found  increased  only  two-fold  under  conditions  which  caused  a  four-fold  or 
greater  increase  in  histamine  and  arginine  esterase  activity. 

The  arginine  esterase  activity  is  found  in  the  precipitate  of  an  80% 
ethanol  extraction  of  SRS-A  (Slow  Reacting  Substance  of  Anaphylaxis)  (NHLI- 
285(c)).   When  this  precipitate  was  suspended  in  0.04  M  Tris,  pH  8.0,  the 
enzyme  was  insoluble  and  could  be  removed  by  slow-speed  centrifugation.   The 
addition  of  3  mM  EDTA-2Na  solubilized  the  enzyme  suggesting  that  its  insolu- 
bility was  due  to  formation  of  an  insoluble  salt.   The  enzyme  was  stable  in 
0.25  M  Tris  pH  5.0  to  8.0,  but  unstable  at  pH  4.0  and  below  losing  50%  of  its 
activity  in  4  hrs  at  4°  at  pH  4.0  and  greater  than  90%  at  pH  3.0.   The  enzyme 
also  showed  unusual  lability  with  various  salts.   For  example,  in  one-half 
hour  at  room  temperature  it  lost  40,  50  and  80%  of  its  activity,  respectively, 
on  standing  in  0.2  M  LiCl,  NaCl  and  KC1  dissolved  in  0.14  M  Tris  buffer, 
pH  8.0. 

Initial  attempts  to  purify  the  arginine  esterase  by  chromatography  on 
DEAE-cellulose  were  unsuccessful.   Although  the  enzyme  readily  adsorbed,  it 
could  not  be  removed  under  the  above  constrictions  of  pH  and  ionic  strength. 
However,  the  enzyme  did  adsorb  to  CM-cellulose  at  pH  5.0  and  could  be  eluted 
by  applying  a  linear  gradient  from  0.05  M  Tris-acetate,  pH  5.0,  to  0.15  M 
Tris-acetate,  pH  8.0.   Under  these  conditions  46%  of  the  activity  could  be 
recovered  as  a  single  peak,  which  paralleled  the  protein  concentration.   Two 
additional  small  peaks  of  activity  were  recovered,  represent  12  and  8%  of 
the  starting  activity.   Despite  the  number  of  purification  steps,  the  argi- 
nine esterase  recovered  in  the  main  peak  had  only  been  purified  9-15  fold, 
suggesting  that  this  esterase  may  represent  a  significant  portion  of  the 
protein  released  following  antigen-antibody  interaction.   However,  further 
evidence  to  establish  the  purity  of  these  preparations  will  be  required. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:   The 
identification  of  mediators  released  from  human  lung  by  antigen-antibody 
reaction  coupled  with  an  investigation  of  the  mechanism  of  their  release  and 
the  compounds  which  inhibit  this  release,  should  provide  a  rationale  for  the 
development  of  therapeutic  agents  for  bronchial  asthma. 

2  STTf 


Project  No.  Z01  HL  01945-03  HE 

Proposed  Course:   Continued  investigation  into  the  mediators  and  modulators 
released  from  human  lung  by  antigen-antibody  interaction. 

Keyword  Descriptors:   histamine   prostaglandins   arginine  esterase 
lung   bronchial  asthma 

Honors  and  Awards:   None 

Publications: 

Maling,  H.M. ,  Webster,  M.E.,  Williams,  M.A. ,  Saul,  W.  and  Anderson,  W.  , 
Jr.:   Inflammation  induced  by  histamine,  serotonin,  bradykinin  and 
compound  48/80  in  the  rat.   J.  Pharm.  Exp_.  Therap.  191:300-310,  1974. 

Miller,  R.L.,  Webster,  M.E.  and  Melmon,  K.L. :   Interaction  of  leukocytes 
andotoxin  with  the  plasmin  and  kinin  systems.   J_.  Europ .  Pharm.  (In 
press),  1975. 

Oh-ishi,  S.  and  Webster,  M.E.:   Vascular  permeability  factors  (PF/Nat 
and  PF/Dil) :   their  relationship  to  Hageman  factor  and  the  kallikrein- 
kinin  system.   Biochem.  Pharm.  24:591-598,  1975. 

Oh-ishi,  S.  and  Webster,  M.E.:   Formation  of  prekallikrein  activator 
and  TAME  esterase  activity  by  dilution  of  human  plasma.   In  Pisano,  J.J. 
(Ed.):   Chemistry  and  Biology  of  the  Kallikrein-Kinin  System  in  Health 
and  Disease.   Washington,  D.C.,  U.S.  Government  Printing  Office,  1975, 
(In  press) . 

Webster,  M.E.  and  Oh-ishi,  S.:   Activation  of  Hageman  factor  (Factor 
XII):   requirement  for  activators  other  than  prekallikrein.   In  Pisano, 
J.J.  (Ed.):   Chemistry  and  Biology  of  the  Kallikrein-Kinin  System  in 
Health  and  Disease.   Washington,  D.C.,  U.S.  Government  Printing  Office, 
1975,  (In  press). 


SBc 


Project  No.  Z01  HL  01946-02  HE 

1.  Hypertension-Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Purification  of  SRS-A  from  Human  Lung 

Previous  Serial  Number:   NHLI-285(c) 

Principal  Investigator:   Hidenobu  Takahashi,  Ph.D. 

Other  Investigators:   Marion  E.  Webster,  Ph.D. 
Harold  H.  Newball,  M.D. 

Cooperating  Units:   Johns  Hopkins  University  School  of  Medicine 
Baltimore,  Maryland 

Project  Description: 

Objectives:   SRS-A  (Slow  Reacting  Substance  of  Anaphylaxis)  is  released  from 
passively  sensitized  human  lung  by  addition  of  specific  antigen  and  has  been 
proposed  as  a  mediator  of  bronchial  asthma.   First  described  in  1940,  a 
number  of  attempts  have  been  made  to  purify  this  substance  from  guinea  pig, 
cat  and  rat  tissue.   Although  much  progress  has  been  made,  the  precise  chem- 
ical structure  of   this  compound  has  still  not  been  determined.   In  the 
present  study  we  are  again  attempting  to  isolate  SRS-A  and  to  determine  its 
chemical  structure. 

Methods  Employed:   Human  lung  fragments  (30-100  mg)  were  passively  sensitized 
with  ragweed  antisera  (1:80  dilution),  washed  free  of  serum  proteins,  and 
incubated  for  15-30  min  at  37°  with  specific  antigen  E  (500  ng/tissue  piece). 
The  reaction  was  terminated  by  removal  of  the  tissue.   Biological  activity 
was  measured  by  bioassay  using  the  isolated  guinea  pig  ileum  in  the  presence 
of  atropine  and  triprolidine. 

Major  Findings:   Previous  studies  from  this  laboratory  (NHLI-285 (c) )  had 
shown  that  SRS-A  from  human  lung  could  be  separated  into  four  distinct  frac- 
tions by  extraction  with  ethanol,  base  hydrolysis  (0.1  M  NaOH  for  30  min  at 
37°),  filtration  and/or  elution  through  Amberlite  XAD-2  and  chromatography 
on  silicic  acid.   Yields  were  50-75%  of  the  starting  activity,  provided 
tyramine  (500  ug/ml)  was  used  to  protect  SRS-A  from  destruction  by  ultraviolet 
light.   This  procedure  was  similar  to  that  devised  by  Orange  and  coworkers 
(J.  Immunol.  110:760,  1973)  for  the  purification  of  rat  SRS-A.   However,  in 
their  experiments,  rat  SRS-A  appeared  to  be  homogenous  since  it  adsorbed  to 
XAD-2  and  eluted  from  silicic  acid  only  in  the  final  solvent. 


S2t 


Project  No.  Z01  HL  01946-02  HE 

Additional  studies  have  shown  that  SRS-A's  can  be  further  purified  by 
chromatography  on  DEAE-cellulose.   The  column  is  similar  to  that  described  by 
Rouser  and  coworkers  (J.  Amer.  Oil  Chenist's  Soc.  38:544,  1961)  for  the  sepa- 
ration of  lipids  except  that  methanol: ammonium  carbonate  was  used  as  the  final 
solvent  rather  than  glacial  acetic  acid,  which  destroyed  the  biological 
activity  of  the  SRS-A's.   In  the  final  procedure,  the  SRS-A's  are  separated 
into  the  same  four  biologically  active  fractions  by  extraction  with  ethanol, 
base  hydrolysis  and  chromatography  on  silicic  acid  and  DEAE-cellulose.   This 
procedure  has  been  applied  to  a  number  of  preparations  of  SRS-A  from  different 
lungs  and  results  have  been  reproducible  with  an  overall  recovery  through  all 
steps  of  about  50%. 

Orange  and  coworkers  (J.  Immunol.  113:316,  1974)  reported  that  arylsul- 
fatases  (Type  H-I,  II  and  III)  destroyed  the  biological  activity  of  rat  and 
human  SRS-A.   We  have  found  that  SRS-A  fractions  inhibit  the  activity  of  type 
H-I  arylsulfatase  at  pH  4.5,  utilizing  p-nitrocatechol  sulfate  as  substrate. 
Under  these  conditions  this  arylsulfatase  is  inhibited  by  both  sodium  sulfate 
and  sodium  phosphate  to  the  same  extent.   SRS-A  fractions  I,  II,  III  and  IV 
inhibited  5,  3,  46  and  78  ug  sulfate  equivalent  per  SRS-A  unit,  respectively. 
Fractions  III  and  IV  still  contain  appreciable  amounts  of  salts  derived  from 
the  original  Tyrode's  solution  and  these  salts  may  have  contributed  to  the 
inhibition  seen  in  these  fractions.   Nevertheless,  measurement  of  inhibition 
of  arylsulfatase  during  silicic  acid  and  DEAE-cellulose  chromatography  gave 
results  which  correlated  well  with  biological  activity.   For  example,  those 
fractions  which  inhibited  arylsulfatase  also  contained  biological  activity  to 
SRS-A.   At  this  pit  and  at  ratios  of  arylsulfatase  units/SRS-A  units  of  0.3 
to  6.0,  type  II— 1  arylsulfatase  failed  to  destroy  the  biological  activity  of 
these  SRS-A's  except  for  Fraction  I. 

These  results  suggest  that  human  SRS-A's,  like  the  prostaglandins,  may 
represent  a  family  of  compounds.   Alternatively,  human  SRS-A's  may  be  bound  to 
compounds  which  differ  in  charge  and  mobility.   This  latter  possibility  would 
appear  unlikely  since,  prior  to  separation,  the  crude  SRS-A  was  extracted  with 
ethanol  to  prevent  its  binding  to  protein  and  hydrolyzed  with  base  to  prevent 
its  binding  to  phospholipids.   The  additional  possibility  that  the  alkali 
treatment,  itself,  had  produced  the  multiple  forms  deserves  further  consider- 
ation, although  similar  treatment  of  rat  SRS-A  did  not  result  in  cleavage. 
Whether  SRS-A  contains  a  sulfate  group  also  remains  to  be  established  with 
certainty.   Further  studies  on  the  chemical  structure  are  indicated. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:   SRS-A 
is  one  of  the  few  remaining  mediators  of  bronchial  asthma  whose  chemical 
structure  remains  unknown.   The  purification  of  SRS-A  and  the  determination  of 
its  structure  should  greatly  aid  the  development  of  antagonists  for  this  vaso- 
active substance  which  may  well  be  clinically  useful. 

Proposed  Course:   Efforts  will  continue  to  devise  methods  for  the  further 
purification  and  chemical  characterization  of  human  SRS-A's. 

Keyword  Descriptors:   lung   SRS-A   bronchial  asthma 

2  SB> 


Project  No.  Z01  HL  01946-02  HE 


Honors  and  Awards:   None 


Publications 


Webster,  M.E.,  Newball,  H.H. ,  Oh-ishi,  S.,  Takahashi,  H.,  Horakova,  Z., 
Atkins,  F.L.  and  Beaven,  M.A. :   Release  of  histamine  and  arginine 
esterase  activity  from  passively  sensitized  human  lung  by  ragweed 
antigen.   Ciencia  e  Cultura  26:372-376,  1974. 


SQ3 


Project  No.   Z01  HL   01947-01  HE 

1.  Hypertension- Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Biosynthesis  of  SRS-A  in  Monkey  Lung 

Previous  Serial  Number:   None 

Principal  Investigator:   Barbara  Davis,  M.S. 

Other  Investigators:   Marion  E.  Webster,  Ph.D. 

Cooperating  Units:   None 

Project  Description: 

Obj  ectives:   Although  SRS-A  (Slow  Reacting  Substance  of  Anaphylaxis)  was  first 
described  in  1940  by  Kellaway  and  Trethewie  and  shown  to  be  released  from 
monkey  lung  in  1966  by  Goodfriend  et  al. ,  (Int.  Arch.  Allergy  30:511),  the 
biochemical  mechanism  of  its  synthesis  remains  unknowns.   The  precursors  of 
SRS-A  and  the  enzymes  involved  in  its  formation  have  not  yet  been  described. 
In  this  project  efforts  will  be  undertaken  to  study  the  mechanism  of  biosyn- 
thesis of  SRS-A  in  monkey  lung. 

Methods  Employed:   Monkey  lung  fragments  (50-100  mg)  or  homogenates  are  sensi- 
tized with  human  ragweed  antisera,  washed  free  of  serum  proteins,  and  incu- 
bated for  5-30  min  at  37°  with  varying  concentrations  of  antigen  E  (5-5,000 
ng).   The  reaction  is  stopped  by  removal  of  the  tissue  or  by  chilling  to  4°. 
The  biological  activity  of  SRS-A  is  determined  by  bioassay  utilizing  the 
isolated  guinea  pig  ileum  in  the  presence  of  anticholinergic  and  antihista- 
minic  agents.   Histamine  and  arginine  esterase  are  determined  by  radiochemical 
techniques  described  elsewhere  (Webster  et  al. ,  Ciencia  e  Cultura  26:372, 
1974;  project  report  #Z01  HL  1945-03  HE). 

Major  Findings:   Previous  studies  in  this  laboratory  demonstrated  the  release 
of  an  arginine  esterase,  as  well  as  SRS-A  and  histamine,  from  passively  sensi- 
tized human  lung  on  addition  of  specific  antigen.   Monkey  lung  also  releases 
an  arginine  esterase  although  at  concentrations  approximately  1/10  that  of 
human  lung. 

The  amount  of  antiserum  required  to  passively  sensitize  monkey  lung  is 
similar  to  that  previously  found  for  human  lung.   With  monkey  lung,  however, 
higher  concentrations  of  antigen  E  and/or  longer  periods  of  incubation  at 
37°  are  required  to  effect  similar  release  of  histamine  and/or  SRS-A.   SRS-A 
is  released  from  monkey  lung  at  a  significantly  slower  rate  than  histamine 
when  less  than  maximal  amounts  of  antigen  E  are  employed,  suggesting  intra- 

1  &{ 


Project  No.   Z01  HL  01947-01  HE 

cellular  synthesis  of  SRS-A  as  opposed  to  the  immediate  release  of  preformed 
histamine. 

As  with  human  lung,  the  optimal  concentration  of  antigen  E  required  for 
release  varies  with  each  tissue.   Preliminary  experiments  suggest  that  the 
release  of  arginine  esterase  may  correlate  with  synthesis  of  SRS-A.   In  order 
to  select  the  proper  antigen  concentration,  therefore,  each  tissue  will  be 
tested  with  varying  concentrations  of  antigen  employing  the  rapid  radiochemical 
method  for  arginine  esterase. 

A  biochemical  method  for  the  detection  of  SRS-A  activity,  such  as  inhi- 
bition of  arylsulfatase  activity  (Z01  HL  1946-02  HE),  is  desirable.   Also, 
studies  of  SRS-A  formation  in  monkey  lung  homogenates  have  been  difficult  to 
interpret,  since  homogenates  contain  substance (s)  other  than  histamine  and 
SRS-A  which  contract  the  guinea  pig  ileum.   In  monkey  lung,  as  in  human  lung, 
SRS-A  occurs  in  the  80%  ethanol  extracts.   Thin-layer  chromatography  of  these 
extracts  on  silicic  acid  resulted  in  nearly  complete  loss  of  activity  even 
though  a  nitrogen  atmosphere  and  the  absence  of  light  was  employed  to  reduce 
lability.   Present  evidence  suggests  that  the  salts  in  the  ethanol  extracts 
can  be  successfully  removed  by  chromatography  on  Sephadex  LH-20  in  80%  ethanol 
(Kuritzky  and  Goodfriend,  Int.  Arch.  Allergy  46:522,  1974)  without  major  loss 
of  activity.   Further  studies  will  be  required  to  determine  whether  a  bio- 
chemical method  can  be  developed  for  measurement  of  SRS-A  activity. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:  The 

determination  of  the  mechanism  of  biosynthesis  of  SRS-A  should  assist  in  the 

development  of  antagonists  of  SRS-A  which  may  be  of  therapeutic  value  in 
bronchial  asthma. 

Proposed  Course:   To  continue  investigation  into  alternate  methods  for  the 
determination  of   SRS-A  activity  and  to  initiate  studies  on  the  mechanism  of 
biosynthesis  of  SRS-A  in  monkey  lung. 

Keyword  Descriptors:   SRS-A   lung   monkey   bronchial  asthma   biosynthesis 

Honors  and  Awards:   None 

Pub 1 i  ca  t  i  ons :   None 


ser 


Project  No.    Z01  HL  01948-03  HE 

1.  Hypertension-Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 
Individual  Project  Report 
July  1,  1974  through  June  30,1975 

Project  Title:   The  Role  of  Prostaglandins  in  the  Vascular  System 

Previous  Serial  Number:   NHLI-283 

Principal  Investigator:   Lauren  M.  Cagen,  Ph.D. 

Other  Investigators:   John  J.  Pisano,  Ph.D. 
Henry  M.  Fales,  Ph.D. 
Robert  E.  Bowden,  M.D. 
William  Jakoby,  Ph.D. 
Marjorie  P.  Peyton,  B.S. 

Cooperating  Units:   Laboratory  of  Chemistry,  NHLI 

Section  on  Enzyme  and  Cellular  Biochemistry 
National  Institute  of  Arthritis,  Metabolism  and  Digestive 
Diseases 

Project  Description: 

Objectives:   To  determine  the  structure  of  polar  forms  of  prostaglandin  formed 
by  exposure  of  PGA  to  human  red  blood  cells  and  to  assess  the  significance  of 
this  mode  of  metabolism  in  the  physiology  and  pharmacology  of  PGA. 

Methods  Employed:   Metabolites  of  PGA  were  purified  by  thin-layer  and  column 
chromatography  and  analyzed  by  gas  chromatography  and  mass  spectrometry. 

Major  Findings:   A  method  for  extracting  prostaglandins  from  plasma  has  been 
modified  to  give  consistent  >85%  yields  of  prostaglandin  A.   Formerly  yields 
were  sometimes  as  low  as  23%.   Modifications  consisted  of,  1)  thoroughly  washing 
glassware  with  methanol,  2)  careful  control  of  silicic  acid  column  length, 
3)  washing  column  with  2  ml  benzene  after  sample  application  to  remove  non- 
polar  lipid  material,  4)  dissolving  the  final  sample  in  a  small  amount  of 
methanol  (amounting  to  10%  of  intended  final  volume)  before  attempting  to  take 
it  up  to  buffer.   Using  methanol  in  the  final  sample  necessitates  using  10% 
methanol  in  the  radioimmunoassay.   This  presented  no  problem.   Straight  lines 
with  slopes  very  near  1.0  were  obtained  for  log-logit  plots  of  standard  curves 
in  which  all  tubes  contained  10%  methanol.   Sensitivity  was  equal  to  that  ob- 
tained when  no  methanol  was  used. 


$&> 


Project  No.   Z01  HL  01948-03  HE 

Using  this  modified  method,  1  ml  samples  of  normal  plasma  were  extracted 
in  triplicate  and  assayed  for  prostaglandin  A.   Values  of  250+200  pg/ml  were 
obtained.   The  large  error  with  minute  quantities  necessitates  the  testing  of 
larger  plasma  samples.   Extraction  of  1  ml  water  or  buffer  and  radioimmunoassay 
for  PGA  consistently  gave  values  of  0. 

PGAi  is  converted  to  whole  humn  blood  and  by  suspensions  of  human  red 
cells  to  a  polar  form(s)  without  vasodepressor  activity.   The  polar  products 
formed  when  ^h-PGAi  was  incubated  with  tris-saline  suspensions  of  saline  washed 
human  red  blood  cells  were  purified  by  solvent  extraction  and  XAD  column  and 
cellulose  thin- layer  chromatography.   Combined  gas  chromatography-mass  spectro- 
metry revealed  the  presence  of  2  prostaglandin  derivatives  in  the  purified 
samples.   The  first  of  these  thermolyzes  to  PGA^  during  gas  chromatography; 
the  second  compound  thermolyzes  to  a  novel  prostaglandin  derivative  which  con- 
tains 2  hydroxyl  groups,  no  keto  group,  and  the  elements  of  H2S  and  which  was 
assigned  the  structure:   9,15-dihydroxy-ll-mercaptoprost-13-enoic  acid.   This 
compound  was  also  obtained  by  treating  PGA^  with  H2S  and  NaBH4  successively. 

Amino  acid  analysis  of  samples  of  purified  red  cell  product  hydrolyzed  in 
6  N  HC1  showed  the  presence  of  glutamic  acid  and  glycine  in  quantities  stoich- 
iometric with  prostaglandin,  and  of  lesser  amounts  of  cystine.   No  other  amino 
acids  were  observed.   This  strongly  implies  that  the  polar  red  cell  products 
are  glutathione  conjugates  of  PGAi,  containing  one  molecule  of  glutathione  per 
molecule  of  prostaglandin.   NMR  of  the  red  cell  product  showed  that  the  13,14 
double  bond  was  still  present  while  the  10,11  double  bond  had  disappeared. 
Thus  the  first  of  the  red  cell  products  results  from  conjugate  addition  of  GSH 
to  the  11  position  of  PGA-^;  the  second  product  arises  from  the  first  by  enzy- 
matic reduction  of  the  9-keto  group. 

GSH  reacts  rapidly  and  nonenzymically  with  PGA^  at  physiological  pH  and 
37°.  The  product  of  this  reaction  was  identical  in  chromatographic  mobility 
to  the  red  cell  product  in  all  systems  of  TLC  and  paper  chromatography  tested. 
Samples  of  synthetic  GSH  conjugate  were  reduced  with  NaBH4.  The  reduced  and 
unreduced  GSH  conjugate,  and  the  red  cell  product  all  had  the  same  chromato- 
graphic mobility. 

The  reaction  of  PGA-^  with  GSH  may  also  be  enzymically  catalyzed.   Four 
highly  purified  GSH  S  transferases  were  tested  for  activity  towards  PGA^;  all 
were  found  to  be  active.   No  difference  in  the  enzymatic  and  non-enzymatic 
product  was  observed. 

As  expected,  the  GSH  conjugate  of  PGAi  thermolyzes  to  PGA^  during  gas 
chromatography,  while  the  NaBH^  reduced  GSH  conjugate  thermolyzes  to  the  same 
diol  mercaptan  derivative  observed  during  gas  chromatography  of  red  cell  product. 
Thus  the  synthetic  GSH  conjugates  and  the  red  cell  products  appear  identical. 

The  same  PGFj-like  material  is  liberated  by  treatment  of  the  reduced  GSH 
conjugate  and  the  polar  red  cell  product  with  BrCN,  further  confirming  the 
identity  of  these  2  substances.   However  the  BrCN  fragment  appears  from  its 


ser 


Project  No.   ZQ1  HL  01948-03  HE 

mass  spectrum  and  from  its  sensitivity  to  periodate  cleavage  to  be  a  glycol- 
containing  rearrangement  product  of  PGF^  rather  than  PGFi  itself. 

In  experiments  performed  in  dogs,  -%-PGA^  was  infused  into  the  renal 
artery.   About  75%  of  the  radioactivity  appearing  in  the  urine,  (20%  of  that 
added)  and  more  than  25%  of  the  radioactivity  appearing  in  the  renal  venous 
effluent,  48%  of  that  added,  were  converted  to  highly  polar  forms.   Almost  no 
unaltered  PGA-,  was  recovered.   In  experiments  with  guinea  pigs  35%  of  the 
^H-PGAi  injected  appeared  in  the  urine  after  24  hrs,  and  almost  100%  of  this 
had  been  converted  to  polar  metabolites.   These  metabolites  formed  in  dogs  and 
in  guinea  pigs  are  more  polar  than  any  of  the  previously  described  metabolites 
of  PGA^,  but  are  consistent  with  the  high  polarity  of  glutathione  conjugates 
of  PGA]^. 

Samples  of  human  urine  were  examined  for  the  possible  presence  of  prosta- 
glandins.  Since  seminal  plasma  contains  high  levels  of  prostaglandins,  urine 
samples  from  female  subjects  were  employed.   Significant  levels  of  immuno- 
reactive  PGI?2  (IPGB2)  were  liberated  from  the  aqueous  residue  of  samples  of 
human  urine  after  removal  of  primary  prostaglandins  and  their  less  polar  metab- 
olites by  solvent  extraction.   Levels  of  iPGB2  obtained  ranged  from  140  to  300 
pg/ml  and  corresponded  to  at  least  50%  of  the  PGE2  content  of  the  same  urine 
samples.   Since  it  is  not  yet  possible  to  estimate  the  recovery  of  prostaglandin 
by  this  procedure,  it  is  probable  that  the  actual  level  in  urine  is  considerably 
higher. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:   Prosta- 
glandins of  the  A  series  have  potent  vasodilator  and  natriuretic  effects  and 
have  been  used  experimentally  in  the  treatment  of  hypertension;  these  compounds 
are  able  to  maintain  normal  levels  of  sodium  excretion  at  the  same  time  that 
blood  pressure  is  lowered.   It  has  been  reported  that  PGA  may  be  formed  in  vivo 
and  reach  physiologically  significant  levels  in  human  plasma  in  normal  and 
pathological  states. 

Little  is  known  about  the  metabolism  of  PGA  in  humans  or  other  animals. 
We  have  shown  that  PGA^  is  converted  by  human  red  blood  cells  to  PGAi-GSH 
conjugate.   Although  PGA^  reacts  readily  with  GSH  nonenzymically,  this  reaction 
is  also  catalyzed  by  enzymes  present  in  high  concentration  in  liver  and  kidney. 

Preliminary  experiments  indicate  that  significant  amounts  of  prostaglandin 
may  exist  in  urine  in  conjugated  form.   This  is  the  first  report  that  prosta- 
glandin conjugates  form  in  vivo.   Although  further  work  is  necessary  to  es- 
tablish the  chemical  nature  of  these  conjugates,  this  result  is  .strongly 
suggestive  that  PGA2,  a  substance  known  to  have  potent  effects  on  renal  blood 
flow  and  diuresis,  is  formed  in  the  human  kidney  in  vivo. 

It  was  found  that  %-PGAi  injected  into  canine  renal  artery  was  almost 
entirely  converted  into  other  chemical  forms  in  one  passage  through  the  kidney. 
The  ability  of  the  kidney  to  rapidly  metabolize  PGA  may  account  for  past  fail- 
ures to  observe  PGA  in  renal  venous  effluent  after  infusion  of  chemical  stimu- 
lators of  prostaglandin  synthesis. 

3  SB8 


Project  No.  Z01  HL  01948-03  HE 

In  the  course  of  this  work,  it  was  found  that  PGA  is  highly  susceptible  to 
Michael- type  addition  reactions  by  sulfur  nucleophiles.   This  reactivity  is  of 
potential  physiological  significance,  since  many  enzymes,  including  such  key 
membrane  enzymes  as  Na-K  ATPase  and  adenyl  cyclase,  are  inhibited  by  alkylating 
agents.   It  is  of  interest  in  this  regard  that  PGA  shares  some  of  the  renal 
effects  of  the  diuretic  agent  ethacrynic  acid,  a  substance  which  also  contains 
an  a,8-unsaturated  ketone.   It  is  conceivable  that  some  of  the  biological  effects 
of  PGA  are  due  to  its  ability  to  alkylate  protein  sulfhydryl  groups. 

Proposed  Course:   The  chemical  nature  of  the  conjugated  forms  of  prostaglandin 
found  in  human  urine  and  of  adducts  formed  from  PGA^  by  passage  through  dog 
kidney  will  also  be  determined. 

The  levels  of  this  substance (s)  in  urine  will  be  assessed  by  radioimmuno- 
assay of  the  PGB2  liberated  by  treatment  with  KOH.   levels  in  normal  human 
female  urine  will  be  established  and  compared  with  levels  in  pathological  states 
thought  to  affect  prostaglandin  production,  e.g.,  renal  artery  stenosis, 
essential  hypertension,  and  malignant  tumors. 

The  natural  occurrence  of  polar  conjugates  of  PG  in  urine  raises  the 
question  of  the  ability  of  kidney  to  generate  PGA.   The  ability  of  kidney  of 
various  species  including  human  to  convert  PGE^  to  PGA^  will  be  assessed,  using 
assays  for  PGA^  in  the  conjugated  and  unconjugated  forms. 

Bradykinin  is  known  to  be  a  potent  stimulator  of  prostaglandin  synthesis 
in  mammalian  kidney  and  in  vascular  endothelium.   It  has  been  suggested  that 
bradykinin  acts  to  stimulate  an  endogenous  lipase  which  liberates  the  unsatur- 
ated fatty  acid  precursors  of  prostaglandins.   Experiments  to  test  this  hypo- 
thesis will  be  performed,  using  homogenates  of  rabbit  renal  medulla.   The 
effect  of  bradykinin  on  unsaturated  fatty  acid  release  and  on  the  generation 
of  possible  mediators  of  hormone  action,  such  as  cAMP  and  cGMP  will  be  measured. 
If  preliminary  experiments  are  successful,  the  characterization  of  the  renal 
hormone-sensitive  lipase  will  be  undertaken. 

Keyword  Descriptors:   prostaglandins   glutathione   red  blood  cells   urine 

Honors  and  Awards :   None 

Publications: 

Cagen,  L.M.,  Pisano,  J.J.,  and  Fales,  H.M.  :   Glutathione  adducts  of  PGA-^. 
Fed.  Proc.  34:790,  1975. 


S89 


Project  No.      Z01  HL     01949-02  HE 

1.  Hypertension-Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Amino  Acid  Sequence  Determination  of  Polypeptides 

Previous  Serial  Number:   NHLI-282 

Principal  Investigator:   Henry  C.  Krutzsch,  Ph.D. 

Other  Investigators:   John  J.  Pisano,  Ph.D. 
Henry  M.  Fales,  Ph.D. 

Cooperating  Units:   Laboratory  of  Chemistry,  NHLI 

Project  Description: 

Objectives:   To  continue  the  successful  development  of  a  new  method  for  the 
determination  of  the  amino  acid  sequence  of  polypeptides  with  emphasis  on 
improvement  in  scope  and  sensitivity.   The  technique  utilizes  digestion  of  the 
polypeptide  T,\Lth  dipeptidyl  aminopeptidase  (DAP)  ,  followed  by  gas  chromatogra- 
phy-mass  spectrometry  (GC-MS)  to  identify  the  suitably  derivitized  dipeptide 
products. 

Methods  Employed:   The  method  generally  employed  in  the  DAP/GC-MS  method  of 
polypeptide  sequencing  is  as  follows: 

1)  Separate  DAP  digests  of  the  polypeptide  and  its  single  Edman  degraded 
cogener. 

2)  GC-MS  of  the  volatile  dipeptide  derivative  mixtures  for  separation  and 
identification  of  the  dipeptides. 

3)  Combination  of  the  two  sets  of  dipeptides  to  yield  the  polypeptide 
structure. 

Major  Findings:   1)  Enzymology:   A  major  thrust  has  been  centered  on  improving 
and  expanding  the  enzymology  involved  in  the  DAP/GC-MS  method  of  polypeptide 
sequencing.   As  a  result  of  this  effort,  the  scope  of  polypeptides  which  can 
be  sequenced  by  this  technique  has  been  enlarged.   Previously,  when  DAP-I  was 
the  sole  enzyme  employed,  digestion  of  polypeptides  containing  proline  came  to 
a  halt  at  the  point  when  this  moiety  appeared  one  or  two  residues  distal  to  the 
amino  terminus.   Digestion  also  ceased  when  a  lysine  or  arginine  residue  ap- 
peared at  the  amino  terminus. 

1  !       S*> 


Project  No.  Z01  HL  01949-02  HE 

The  proline  problem  was  surmounted  by  the  application  of  two  other  more 
specific  DAP  enzymes,  DAP-IV  and  V.   DAP- IV  has  the  ability  to  cleave  the  pep- 
tide bond  involving  the  carboxyl  group  of  proline.   This  enzyme  had  been  pre- 
iously  reported  but  much  more  work  was  required  before  it  could  be  used  suc- 
cessfully in  polypeptide  sequencing.   Efforts  were  directed  at  removal  of  pre- 
viously unrecognized  contaminating  proteases,  elimination  of  GC  background- 
forming  material  and  further  enzyme  characterization,  chiefly  defining  the 
scope  of  the  enzyme's  activity  toward  various  peptide  substrates. 

DAP-V,  a  previously  unreported  DAP  enzyme,  has  the  ability  to  cleave  the 
peptide  bond  involving  the  amino  group  of  proline.   Thus,  the  B-naphthylamides 
of  the  polypeptides  val-ala-pro-ala,  gly-pro-pro-ala,  pro-ala-pro-pro,  and 
gly-pro-pro-pro  were  successfully  digested.   The  possibility  that  these  diges- 
tions were  effected  by  monopeptidases  or  endopeptidases  was  ruled  out  in  other 
experiments.   Final  confirmation  of  the  presence  of  the  expected  dipeptide 
products  was  obtained  via  GC-MS.   Work  is  presently  under  way  to  purify  large 
amounts  of  this  enzyme  to  establish  its  usefulness  in  the  sequencing  of  poly- 
peptides. 

The  problem  with  lysine  and  arginine  residues  was  solved  through  further 
investigation  of  the  enzymology  of  DAP-I.   Thus,  by  further  alterations  in 
reaction  conditions,  chiefly  in  pH  and  amounts  of  activators  used,  polypeptides 
containing  lysine  or  arginine  at  the  amino  terminal,  or  in  a  position  allowing 
them  to  appear  at  the  amino  terminal  during  digestion,  could  be  digested  by 
DAP-I.   These  changes  caused  some  GC  background,  but  it  was  not  of  any  conse- 
quence. 

Another  result  of  these  investigations  was  to  further  increase  the  yield 
of  dipeptides  produced  from  DAP  digestion.   Coupled  with  some  procedural  im- 
provements in  dipeptides  derivitization,  the  lower  limit  of  sensitivity  has 
been  extended  down  to  the  range  of  1-2  nanomoles  of  polypeptides.   Previously, 
10-20  nanomoles  of  peptide  was  required  for  total  sequence  analysis. 

2)  Volatile  dipeptide  derivatives  and  gas  chromatography:   The  trimethyl- 
silyl  derivative  is  now  the  standard  volatile  dipeptide  derivative  used. 
Several  GC  columns  have  been  tried  but  the  2  mm  x  0.6  m  1%  OV-1  column  pre- 
viously described  has  proven  to  be  the  best.   One  useful  finding  was  that  a 

2  mm  x  0.6  m  1%  Dexsil  300  column  can  be  used  as  a  supplemental  column  giving 
slightly  different  resolution  characteristics. 

3)  GC-MS:   The  number  of  dipeptides  observed  has  now  increased  to  over  150 
out  of  the  400  possible.   This  increase  has  provided  at  least  several  represen- 
tatives of  dipeptides  containing  a  given  member  of  the  common  20  aminoacids  in 
either  the  amino  terminal  or  carboxyl  terminal  position  of  the  dipeptide.   This 
additional  information  will  permit  the  confident  identification  of  any  newly 
encountered  dipeptides.   The  need  for  direct  MS  probe  introduction  for  identi- 
fication of  dipeptides  containing  His, Gin  and  Asn  has  been  partially  removed 
through  better  masking  of  active  sites  in  the  GC,  but  still  remains  an  absolute 
requirement  for  those  dipeptides  containing  arginine.   Techniques  for  direct 
probe  identification  of  such  dipeptides  have  also  been  improved  by  several 
changes  in  the  experimental  procedure  employed  in  this  operation. 

2  5?/ 


Project  No.   Z01  HL  m 949-02  HE 

4)  Polypeptide  unknowns:   Seven  more  polypeptide  unknowns  were  successfully 
sequenced.   One  contained  amino  terminal  arginine,  another  contained  proline. 
Six  of   the  seven  were  sequenced  in  two  days. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:   Greater 
sensitivity  and  confidence  is  required  for  elucidating  the  structures  of  minute 
quantities  of  biologically  important  polypeptides  including  hormones,  neuro- 
transmitters, cell  growth  factors,  transplantation  antigens,  etc.   The  inves- 
tigation described  above  has  further  demonstrated  and  expanded  the  scope  and 
utility  of  the  promising  DAP/GC-MS  technique  for  polypeptide  sequence  determi- 
nations. 

Proposed  Course:   Further  work  will  continue  to  center  on  improving  the  enzy- 
mology  involved  in  the  DAP/GC-MS  sequencing  method.   In  addition,  the  list  of 
polypeptide  unknowns  sequenced  by  the  method  will  be  expanded  in  order  to 
further  demonstrate  the  scope  and  usefulness  of  the  DAP/GC-MS  method  in  protein 
sequencing. 

Keyword  Descriptors:   polypeptide  sequencing   gas  chromatography-mass  spectro- 
scopy  dipeptidyl  amino  peptidases 

Honors  and  Awards:   None 

Publications:   None 


$?X 


Project  No.    Z01  HL   01950-01  HE 

1.  Hypertension- Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland 


PHS-NHLI 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:   Fibrinolytic  Inhibitors  and  Vascular  Endothelium 

Previous  Serial  Number:   None 

Principal  Investigator:   Gilbert  M.  Wilcox,  M.D. 

Other  Investigators:   Valdemar  Hial,  M.D.,  Ph.D. 
John  J.  Pisano,  Ph.D. 
Michael  A.  Gimbrone,  Jr.,  M.D. 

Cooperating  Units:   Department  of  Pathology 

Peter  Bent  Brigham  Hospital 
Harvard  Medical  School 
Boston,  Massachusetts 

Project  Description: 

Objectives:  To  characterize  and  determine  the  level  of  the  components  of  the 
fibrinolytic  system  in  pure  cultures  of  endothelial  cells  produced  from  human 
veins. 

Methods  Employed:   Endothelial  cells  (HEC)  and  smooth  muscle  cells  (SME)  were 
obtained  from  human  umbilical  cord  veins  by  collagenase  treatment  and  dis- 
section.  Confluent  monolayers  of  endothelial  cells  and  dense  subcultures  of 
smooth  muscle  cells  were  grown,  pooled,  washed,  resuspended,  frozen  and 
stored.   Before  testing,  samples  were  freeze-dried  and  resuspended  in  Tris 
buffer  0.2  M  pH  7.5  to  give  approximately  8  x  10"  cells/ml.   The  following 
components  of  the  fibrinolytic  system  were  studied:   plasminogen,  plasmin, 
plasminogen  activator,  inhibitors  of  plasminogen  activation  and  antiplasmin. 
The  measurements  involved  testing  the  samples  before,  and  after  activation  by 
urokinase  trypsin  or  kaolin.   In  addition,  we  investigated  the  activation  of 
purified  plasminogen  by  urokinase  and  the  recovery  of  plasmin  added  to  HEC. 
The  samples  were  assayed  by  the  fibrin  plate  method  or  by  the  radiochemical 
esterolytic  method  using  ^H-acetyl-glycyllysine  methyl  ester  (ACLME)  or  3h- 
tosyl  arginine  methyl  ester  (TAME) . 

Major  Findings:   1)  No  plasminogen  was  detected  in  the  HEC. 

2)  No  plasminogen  activator  was  detected  in  the  HEC  when  assayed  with 
fibrin  plates,  however,  low  AGLME  and  TAME  esterase  activity  (which  could  be 


S?3 


Project  No.    7.01  HT.  mQSO-01  HE 


due  to  activator  bound  to  an  inhibitor)  was  detected  in  HEC.   The  esterase 
activity  was  not  increased  by  kaolin  or  trypsin  but  was  slightly  inhibited  by 
soybean  trypsin  inhibitor. 

3)  A  potent  inhibitor  of  human  urokinase  was  found  in  the  HEC.   Equal 
volumes  of  HEC  8  x  106  cells/ml  and  urokinase  (50  CTAU/ml)  gave  100%  inhibition 
of  the  activator  (measured  by  the  fibrin  plates). 

4)  No  antiplasmin  was  detected  in  the  HEC  preparation  by  the  fibrin  plate 
assay. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:   Little 
is  known  about  the  nature  and  source  of  inhibitors  of  plasminogen  activators 
in  plasma.   The  present  findings  point  to  the  endothelial  lining  as  a  source. 
Endothelial  cells  classically  have  been  viewed  as  a  source  of  plasminogen 
activators  and  the  finding  of  an  inhibitor (s)  in  endothelial  cells  changes 
our  understanding  of  the  role  of  the  vascular  lining  in  fibrinolysis.   It  is 
possible  that  the  fibrinolytic  activity  of  blood  vessels  is  controlled  by  the 
level  of  inhibitor  rather  than  the  activator.   This  new  concept  could  be  of 
great  importance  in  our  understanding  of  the  pathogenesis  of  thrombotic 
diseases. 

Proposed  Course:   To  isolate  and  characterize  the  inhibitor  of  plasminogen 
activator  in  HEC. 

Keyword  Descriptors:   human  vascular  endothelial  cells   human  vascular  smooth 
muscle  cells   urokinase   urokinase  inhibition   plasmin   fibrinolysis  coagu- 
lation 

Honors  and  Awards:   None 

Publications:   None 


STf 


Project  No.ZOl  HL  01951-01  HE 

1.  Hypertension- Endocrine  Branch 

2.  Physiological  Chemistry 

3.  Bethesda,  Maryland* 


PHS-NHLI 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Angiotensin  Converting  Enzymes  (ACE)  in  Endothelial  Cells 

Previous  Serial  Number:   None 

Principal  Investigator:   Valdemar  Hial,  M.D.,  Ph.D. 

Other  Investigators:   Gilbert  M.  Wilcox,  M.D. 
John  J.  Pisano,  Ph.D. 
Michael  A.  Gimbrone,  Jr.,  M.D. 

Cooperating  Units:   Department  of  Pathology 

Peter  Bent  Brigham  Hospital 
Harvard  Medical  School 
Boston,  Massachusetts 

Project  Description: 

Objectives:   To  determine  the  ACE  and  kininase  activity  in  pure  cultures  of 
human  vascular  endothelial  cells  and  human  smooth  muscle  cells. 

Methods  Employed:   Human  endothelial  cells  (HEC)  and  smooth  muscle  cells  (SME) 
were  obtained  from  human  umbilical  cords  by  collagenase  treatment  and  dis- 
section.  Confluent  monolayers  of  endothelial  cells  and  dense  subcultures  of 
SME  were  grown,  harvested,  pooled,  washed,  resuspended  and  frozen.   They  were 
subsequently  freeze-dried  and  resuspended  in  Tris  buffer  0.2  M  pH  7.5  or 
borate-phosphate  buffer  .05  M  pH  8.0  containing  approximately  8  x  10°  cells/ 
ml.   Aliquots  of  10-50  yl  of  these  cells  were  incubated  with  pure  AI  or 
bradykinin  at  37°C  pH  8.0  x  5-60  min;  control  samples  were  incubated  with 
buffer  alone.   The  effect  of  chloride  and  cobalt  ions  and  various  inhibitors 
of  ACE  and  kininase  were  studied.   The  amount  of  angiotensin  I  or  II  was 
assayed  on  the  rat  uterus  and  rat  blood  pressure.   Bradykinin  was  assayed  on 
guinea  pig  ileum  and  dog  blood  pressure.   An  increase  in  All  or  decrease  in 
bradykinin  during  an  incubation  would  be  indicative  of  ACE  or  kininase 
activity  respectively. 

Major  Findings:   1)  Both  HEC  and  SMC  contain  AEC  and  kininases.   The  SMC 
contain  relatively  less  of  each  enzyme  compared  to  HEC. 

-)  The  inhibitory  characteristics  of  several  substances  on  ACE  from  HEC 
are  summarized  in  the  following  table: 


§fr 


Project  No.         Z01  HL   01951-01  HE 


Substance 

ACE 

HEC 

Kinlnase 

Absence  of 
chloride 

0 

In 

NT 

Cobalt  (C0++) 
(1  mM) 

0 

NT 

EDTA 
(1  mM) 

0 

NT 

BPF 
(1  mM) 

0 

+ 

8-HOQ 
(4  mM) 

0 

+ 

Mercaptoethanol 
(1  mM) 

0 

NT 

SBTI 

(0.05  mg/ml) 

+ 

NT 

SMC 
ACE  Kinlnase 


Inhibition* 

NT 

NT 
NT 
NT 


NT 


NT 


NT 


NT 


NT 


NT 


NT 


*  +  =  90-100%  inhibition 
+  =  20-50%  inhibition 
0  =  no  effect 
NT  =  not  tested 

3)  This  enzyme  releases  active  products,  directly  from  tetradecapeptide 
(renin  substrate)  and  this  reaction  is  not  inhibited  by  pepstatin,  a  potent 
inhibitor  of  renin. 

Abbreviations  used:   BPF  -  Bothrops  Potentiator  Factor;  8-HOQ  -  8-hydrox- 
yquinoline;  SBTI  -  soybean  trypsin  inhibitor. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:   It  is 
well  established  that  the  renin-angiotensin  system  is  involved  in  regulation 
of  blood  pressure,  and  that  angiotensin  converting  enzyme  is  a  central  com- 
ponent of  this  system.   The  present  study  shows  that  vascular  endothelium 
is  a  rich  source  of  ACE.   The  vessel  wall  (smooth  muscle)  also  has  activity. 
The  enzyme  contained  in  these  locations  appears  to  be  different  from  that 
isolated  from  lung  or  kidney  by  other  investigators  (based  on  inhibition 
characteristics).   Since  this  ACE  is  present  in  the  vessel  lining  and  wall, 
it-  may  be  involved  in  the  local  production  of  All  which  could  regulate  the 
tone  of  the  vessels  and  ultimately  blood  pressure.   There  is  the  possibility 


9* 


Project  No.   Z01  HL  01951-01  HE 

that  this  ACE  is  identical  to  tonin,  an  enzyme  able  to  split  the  tetradeca- 
peptide  (renin  substrate)  releasing  All  directly  since  tonin  is  inhibited  by 
SETI,  does  not  require  chloride  or  cobalt  and  is  not  inhibited  by  BPF,  EDTA 
and  mercaptoethanol-characteristics  shared  by  our  converting  enzyme.   These 
cell  cultures  provide  a  unique  opportunity  to  investigate  ACE's  and  kininases 
for  the  following  reasons:   (a)  the  cultures  are  pure,  allowing  a  separation 
of  the  component  parts  of  the  vascular  wall  not  before  possible;  (b)  the 
cell  types  are  widely  distributed  in  the  body  and  results  would  effect  inter- 
pretation of  enzyme  activities  found  in  any  organ  by  previous  investigators; 
(c)  all  conclusions  are  applicable  to  humans. 

Proposed  Course;   Purification  and  characterization  of  the  pure  enzyme.  .To 
study  the  possible  physiological  role  of  the  enzyme  in  blood  pressure  and 
arterial  tone  regulation. 

Keyword  Descriptors:   human  vascular  endothelial  cells   human  vascular 
smooth  muscle  cells   urokinase   urokinase  inhibitor   plasmin   fibrinolysis 
coagulation 

Honors  and  Awards:   None 

Publications:   None 


577 


Project  No.  Z01  HL  01981-13  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Neuroendocrine logy 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Taste  and  Olfaction 

Previous  Serial  No.:   NHLI-106(c) 

Principal  Investigator:   Robert  I.  Henkin,  M.D.,  Ph.D. 

Other  Investigators:   Clark  Lum,  Ph.D. 
A.L.  Larson,  M.D. 
C.F.T.  Mattern,  M.D. 
H.  Edelhoch,  Ph.D. 
M.L.  Swenberg,  Ph.D. 

Cooperating  Units:   Campbell  Institute  for  Food  Research,  NIAMDD,  NIAID,  NCI, 

Dqartment  of  Biochemistry,  Washington  University,  St.  Louis, 
Missouri 

Project  Description: 

Objectives :   To  investigate  in  a  systematic  manner  the  anatomical,  physiologi- 
cal, pharmacological,  pathological  and  chemical  correlates  of  taste  and  olfaction. 

Major  Findings:   Taste 

Anatomy. 

1.   Histochemical  investigations  of  the  mechanisms  of  taste  function. 
The  distribution  of  acetylcholinesterase  (AChE)  in  the  taste  bud  of  the  rat 
circumvallate  papilla  was  investigated  by  histochemical  electron  microscopy. 
Previous  reports  from  this  and  other  laboratories  of  specific  AChE  activity 
around  subgemmal  and  intragemmal  nerves  and  between  some  taste  bud  cells  were 
confirmed.   In  addition  dense  precipitation  of  AChE  between  microvilli  in  the 
taste  bud  pore  was  observed.   These  studies  suggest  that  acetylcholine  may  be 
involved  in  some  of  the  early  events  in  the  taste  process  which  are  believed  to 
occur  in  the  pore  area.   This  hypothesis  is  based  upon  several  previous  obser- 
vations.  (1)  There  are  neurosecretory  granules  in  Type  I  cells  of  the  taste 
bud,  granules  which  may  represent  storage  vesicles  of  acetylcholine;  this  hy- 
pothesis has  not  yet  been  documented  by  systematic  investigation,  (2)   many  more 
cholinergic  fibers  than  adrenergic  fibers  innervate  the  taste  bud  in  rat  and  in 
man,  (3)  in  Type  I  familial  dysautonomia,  in  which  taste  buds  are  not  present, 
parenteral  administration  of  methacholine  restores  taste  function  to  or  toward 
normal,  (4)  I1^5  a-bungaro toxin  appears  to  bind  to  the  microvilli  of  taste  bud 
membranes  suggesting  the  presence  of  an  acetylcholine  receptor.   The  most  likely 
anatomical  candidate  for  this  receptor  is  the  Type  III  cell  of  the  bud  which  is 
the  only  cell  in  which  neural  connections  appear  to  be  directed  from  the  bud 

i  svf 


Project   No.  ZOl  HL  01981-13  HE 


to  the  brain. 


2.  Evidence  of  a  contractile  mechanism  In  the  taste  bud  of  the  mouse 
fungiform  papilla.    By  means  of  a  epimicroscope,  a  television  screen  and  a 
video  tape  recorder,  the  pore  of  the  taste  buds  of  fungiform  papillae  of 
anesthetized  mice  were  observed  directly  and  recorded.   After  exposure  to  the 
vapor  from  over  a  high  concentration  of  HC1  the  extrusion  of  fluid  through  the 
pore  of  the  taste  buds  was  observed.   This  extrusion  occurred  within  seconds 
after  the  exposure  to  the  HC1  vapor.   The  fluid  then  receded  leaving  a  ring  of    ( 
material.   By  electron  microscopy  the  pore  was  seen  to  be  filled  with  small 
vesicles  which  communicated  with  the  oral  cavity.   The  possible  contractile 
mechanism  by  which  this  process  occurred  was  suggested  by  the  observation  of  a 
long  bundle  of  tonofilaments  in  Type  II  cells  of  the  taste  bud.   The  retraction 
and  subsequent  extension  of  this  bundle  could  account  for  these  observations. 

3.  Investigations  of  the  architecture  of  the  taste  buds  of  human  fungiform 
and  circumvallate  papillae.   Taste  buds  from  human  fungiform  papillae  have  not 
been  previously  studied  in  any  systematic  manner.   Taste  buds  in  fungiform 
papillae  open  directly  into  the  oral  cavity;  they  are  narrower  and  somewhat  ■ 
more  elongated  than  taste  buds  from  circumvallate  papillae.   Taste  buds  in  human 
fungiform  papillae  number  between  0-10  and  usually  appear  in  multiples.   Taste 
buds  in  human  circumvallate  papillae  lie  in  crypts,  do  not  directly  open  to  the 
oral  cavity  and  number  betseen  20-100.   The  three  major  cell  types  previously 
observed  in  taste  buds  from  circumvallate  papillae  have  also  been  observed  in 
each  of  these  taste  buds;  however,  the  dense  core  vesicles  seen  in  Type  I  cells 
of  taste  buds  from  circumvallate  papillae  are  absent  in  Type  I  cells  of  taste 
buds  from  fungiform  papillae.   Also  absent  is  the  dense  extra  cellular  material   i 
normally  found  in  taste  buds  from  circumvallate  papillae.   Functionally,  taste 
buds  in  human  fungiform  papillae  subserve  all  taste  qualities  but  are  most  sen- 
sitive to  salt  and  sweet  tastants.   Taste  buds  in  human  circumvallate  papillae 
also  subserve  all  taste  qualities  but  are  most  sensitive  only  to  sweet  tastants. 

Chemistry: 

1.  Purification  and  properties  of  miraculin,  a  glycoprotein  from  Synsepalum 
dulcificum   which  provokes  sweetness  and  blocks  sourness.    In  the  annual  report 
from  this  section,  1972-73,  the  process  of  purification  of  miraculin  developed  in 
this  laboratory  was  elucidated.   Of  further  note  in  this  regard  is  the  following: 

(1)  The  sugar  composition  of  this  glycoprotein,  confirmed  by  thin  layer 
chromatography  and  by  gas  liquid  chromatography,  is  xylose,  fucose,  galactose  and 
mannose  which  differs  significantly  from  that  reported  by  other  groups. 

(2)  These  sugars  number  15  per  mole  of  protein  and    appear  in  the  ratio 
of  (1:1:1:2)  for  fucose,  xylose,  galactose  and  mannose,  respectively. 

(3)  The  amino  acid  composition  of  native  miraculin  was  determined  on  3 
samples  of  the  protein  on  3  occasions  after  24,48  and  72  hours  of  hydrolysis.     I 
These  results  differ  from  those  previously  published  by  another  investigator 
whose  results  were  obtained  from  one  24  hour  hydrolysis „ 

(4)  The  action  of  miraculin  in  man  differs  from  that  suggested  by  several 
investigators  but,  in  part,  was  similar  to  that  noted  by  Dr.  Linda  Bartoshuk. 
Oral  application  of  miraculin  for  1-3  minutes  makes  all  sour  tastants  taste  sweet 
although  the  miraculin  itself  is  tasteless.   Miraculin  also  blocks  the  taste  of 

I 
2  l>ao 


Project  No.  Z01  HL  01981-13  HE 

sour  itself,  all  sour  tastants  tasting  less  sour.  A  similar  blocking  effect  for 
bitter  tastants  has  also  been  observed. 

(5)  The  locus  of  this  effect  appears  to  be  the  palatal  taste  buds  for 
the  effect  of  miraculin  on  the  tongue  alone  was  minimal  whereas  the  palatal 
taste  buds  (which  primarily  subserve  the  sour  and  bitter  taste)  participated 
in  this  reaction. 

(6)  An  intact  system  for  recognizing  the  taste  of  sweet  was  important 
for  this  effect  to  occur.  In  patients  with  aglycogeusia  and  application  of 
miraculin  blocked  sourness  and  bitterness  but  did  not  produce  any  sensation 
of  sweetness. 

(7)  Further  modification  of  the  purification  process  of  miraculin  has 
yielded  a  significant  improvement  in  purity  of  the  glycoprotein.  The  initial 
chromatography  on  Biogel  CM  30  has  been  modified  such  that  the  elution  process 
is  carried  out  with  0.1  M  sodium  phosphate  buffer  with  a  linear  pH  gradient  of 
6.5  to  7.2.   Previously  the  gradient  extended  to  7.8  and  much  non-specific 
material  was  eluted  at  the  higher  pH.   The  purified  elute  was  then  lyophillized 
and  dialyzed  against  0.1  M  carbonate  buffer  which  also  resulted  in  the  removal 
of  some  additional  non-specific  material.  The  resultant  material  was  then 
placed  on  a  QAE  Sephadex  A-50  column  at  pH  9.0  (previously  10.5)  and  eluted 
with  a  linear  NaCl  gradient  from  0.05  -  0.3  M  (previously  0.05  -  0.65  M) .   The 
eluate  from  this  column  yielded  constant  specific  activity  as  indicated  by  the 
constancy  of  several  indicators  of  protein  concentration  over  the  eluted  peak. 

(8)  By  these  modifications  miraculin  was  eluted  as  a  single  peak  which 
was  homogeneous  on  disc  gel  electrophoresis  in  SDS  buffer  and  by  equilibrium 
dialysis  sedimentation.   On  isolectric  focusing  however  7  subunits  were  observed 
between  pH  3-8.5,  the  major  active  sub unit  observed  at  pH  8.02.   It  had  a  UV 
maximum  at  278  nm  and  a  minimum  at  250  nm.   The  maximum  fluorescence  emission 
peaks  were  at  250  nm  and  350  nm  with  excitation  at  280  nm  in  0.1  M  carbonate 
buffer'  at  pH  9.0  or  0.1  M  phosphate  buffer  at  pH  7.0.   The  sedimentation  constant 
of  miraculin  itt  0.1  M  phosphate  buffer  was  2.80. 

(9)  Miraculin  apparently  binds  to  taste  bud  membranes  for  its  sweetness 
provoking  and  sourness  blocking  effects  are  not  altered  by  5  M  urea  but  are 
obviated  following  rinsing  the  mouth  with  a  0.1%  solution  of  SDS. 

2.   Further  work  on  the  modification  of  miraculin  by  the  action  of  sodium 
periodate  (NalO, )  and  sodium  borohydride  (NaBH, ) .   After  treatment  of  miraculin 
with  low  concentrations  of  NalO/  (3  x  10-bM)  only  galactose  was  selectively 
oxidized.   Following  this  procedure  the  blocking  effect  of  miraculin  was  pre- 
served, i.e.,  the  sourness  of  0.02  M  citric  acid  was  reduced,  but  no  sweetness 
provoking  effect  was  observed.   At  higher  concentrations  of  NalO,  the  oxidation 
of  the  other  sugars  occurred  with,  xylose  being  affected  next  readily  (after 
galactose  as  noted),  then  mannose  and  finally  fucose.   These  studies  suggest 
that  (a)  some  specific  sequence  of  sugars  may  be  important  in  forming  a  code  by 
which  binding  of  miraculin  to  the  receptor  occurs  and  (b)  that  galactose  binding 
to  the  taste  bud  membrane  may  play  a  special  role  in  the  sweetness  provoking 
effect  of  the  glycoprotein. 


4>ef 


Project  No.    Z01  HL  01981-13  HE 

Analyses  of  the  polysaccharide  moeities  from  miraculin  treated  with  NaBH^ 
and  3n^NaB  indicated  that  the  fucose  moeity  was  reduced  to  fucitol.   This  sug- 
gested an  unusual  oligosaccharide  linkage  in  this  glycoprotein.   The  sweetness 
provoking  activity  of  miraculin  was  enhanced  2-3  fold  by  this  procedure  and  may 
relate  to  the  reduction  of  fucose  by  NaBH^  at  a  concentration  of  10_4m.   These 
observations  further  confirm  the  suggestion  that  the  sweetness  provoking  effect 
of  miraculin  may  relate  to  the  nature  of  its  polysaccharide  moeities  and  to 
their  molecular  arrangement. 

3.   Demonstration  of  non-specific  and  specific  binding  of  sugars  and 
mouse  2.5  S  nerve  growth  factor  to  bovine  taste  bud  membranes.   Taste  bud 
membranes  were  isolated  from  taste  buds  from  bovine  circumvallate  papillae, 
and  non-taste  bud  bearing  epithelial  tissue  membranes  were  isolated  from  the 
epithelium  surrounding  these  papillae  by  techniques  previously  described.   Both 
tissues  were  evaluated  with  respect  to  their  ability  to.  bind  various  sugars 
under  several  conditions. 

14 
Non-specific  and  specific  binding  of   C  labelled  sucrose,  fructose,'  glu- 
cose, saccharin  and  cyclamate  to  taste  bud  and  non-taste  bud  bearing  membranes 
was  studied.   For  non-specific  binding,  glucose,  fructose,  and  sucrose  exhibited 
greater  sugar  binding  capacity  to  the  taste  bud  membrane  fraction  P^f-nx  than 
did  the  corresponding  ^(Bl  anc^  P3(B)  fractions.   For  non-taste  bud  membrane 
fractions,  there  was  no  difference  in  sugar  binding  capacity  to  any  of  these 
membrane  fractions  (Table  I).   Binding  to  any  purified  fraction  obtained  from 
taste  bud  membranes  was  greater  than  that  for  the  original  filtrate.   For  the 
membranes  obtained  from  the  epithelial  tissue  no  increase  in  relative  specific 
activity  over  that  of  the  original  filtrate  was  observed. 

For  specific  or  competitive  binding,  labelled  sucrose,  fructose,  glucose, 
cyclamate  and  saccharine  bound  specifically  to  taste  bud  membranes  but  only 
non-specifically  to  corresponding  non-taste  bud  bearing  membranes.   Labelled 
lactose  bound  non-specifically  to  both  taste  bud  and  non-taste  bud  membranes 
and  no  specific  binding  could  be  obtained. 

Binding  of  labelled  sugar  to  taste  bud  membranes  was  both  dose  and  temper- 
ature dependent,  and  was  inhibited  by  increasing  concentrations  of  unlabelled 
sugar,  EDTA  (79%),  NaCl  (54%),  neuraminidase  (40%),  phospholipase  A  (38%), 
and  urea  (30%).   Dissociation  constants  were  in  the  range  of  10~3m,  consistent 
with  the  preference  thresholds  in  cow  (sucrose  >  fructose  >  glucose;  lactose 
no  response).   High  dissociation  of  constants  for  saccharine  and  cyclamate  are 
consistent  with  the  lack  of  preference  responses  to  these  substances  in  cow. 

125 
Using  bovine  taste  bud  membranes  obtained  from  the  P^rg)  fraction  I 

2.5  S  mouse  nerve  growth  factor  (NGF)  exhibited  dose  dependent  binding.   Specific 

displacement  of  I125  NGF  by  native  NGF  was  also  observed. 


(SoV 


Filtrate 
P2(B) 
P3(B) 
P4(B) 


Project   No.ZOl  HL  01981-13  HE 


TABLE  I 

BUD     14 
FRACTION     [C] -GLUCOSE 


14 


[C] -FRUCTOSE 


(  x  10   cpm/mg  Protein) 
2.23  (1)  8.81  (1) 

2.92  (1.3)  9.47  (1.1) 


1.74  (.8) 


7.81  (.9) 


14 


[C] -SUCROSE 


(  x  10" 

-4 

cpm/mg  Protein) 

Filtrate 

3.68  (1) 

15.30  (1) 

3.64  (1) 

P2(B) 

11.98  (3.2) 

56.90  (3.7) 

11.27  (3.1) 

P3(B) 

35.91  (9.7) 

130.00  (8.5) 

25.54  (7.0) 

P4(B) 

34.84  (9.4) 

272.50  (17.8) 

86.76  (23.8) 

GP1 

FRACTION 

14 [C] -GLUCOSE 

14 [C] -FRUCTOSE 

14 [C] -SUCROSE 

3.33  (1) 
3.66  (1.1) 

1.78  (.5) 


(  ) Relative  specific  activity 


663 


Project  No.   Z01  HL  01981-13  HE 

4.   Isolation,  purification  and  determination  of  some  of  the  chemical 
characteristics  of  gustin,  a  zinc  containing  protein  obtained  from  human 
parotid  saliva.   In  my  annual  report  1972-73  the  existence  of  a  zinc  containing 
protein  in  saliva  was  hypothesized.   Work  during  this  year  has  resulted  in  the 
isolation,  purification  and  determination  of  some  of  the  chemical  characteris- 
tics of  this  protein. 

The  basis  for  this  hypothesis  was  based  upon  several  physiological  observa- 
tions.  Saliva  plays  an  important  role  in  taste.   Patients  with  xerostomia 
exhibit  hypogeusia  and  associated  pathological  changes  in  taste  bud  structure. 
Similar  <  bservations  of  adverse  changes  in  taste  acuity  have  been  made  in 
desalivate  rats.   Oral  administration  of  various  non-protein  electrolyte  solu- 
tions has  been  unsuccessful  in  restoring  taste  function  to  normal  in  patients 
with  xerostomia  whereas  treatment  of  these  patients  with  systemic  agents  which 
restore  salivary  function  to  normal  has  been  associated  with  the  recovery  of 
normal  taste  acuity  and  the  appearance  of  normal  taste  buds.   Zinc  is  another 
important  factor  in  taste  perception.   Patients  with  hypogeusia  of  various 
etiologies  shoitf  pathological  changes  in  taste  buds  which  are  similar  to  those 
observed  in  patients  with  xerostomia.   Patients  with  hypogeusia  in  whom  salivary 
flow  rates  are  normal  exhibit  lower  than  normal  concentrations  of  zinc  in  serum 
and  in  parotid  saliva.   Oral  administration  of  zinc  to  some  of  the  patients 
with  hypogeusia  has  resulted  in  the  normalization  of  serum  and  parotid  salivary 
zinc  concentration,  taste  bud  anatomy  and  taste  perception.   From  these  studies 
and  observations  we  hypothesized  that  a  zinc  containing  protein  was  a  normal 
constituent  of  parotid  saliva  and  its  function  was,  in  some  manner,  related  to 
the  growth,  nutrition  and  function  to  taste  buds.   In  order  to  verify  these 
hypotheses  a  zinc  containing  protein  in  parotid  saliva  was  isolated,  purified 
and  characterized. 

Whole  parotid  saliva  was  collected  from  6  men  and  3  women  with  normal  taste 
acuity.   Saliva  was  lyophylized,  dissolved  in  zinc  free  water  and  centrifuged 
at  20,000  x  g  for  30  minutes,  the  clear  supernatant  fluid  used  for  all  subse- 
quent assays. 

Gel  filtration  chromatography  was  carried  out  in  Sephadex  G  150  and 
DEAE-A50  and  in  carboxymethylcellulose  columns.   The  relative  concentration  of 
protein  in  parotid  saliva  was  measured  by  three  spectroscopic  methods  which  re- 
flect distinctive  protein  properties.   The  absorption  at  280  nm  is  accounted 
for  by  the  amount  of  tyrosine  and  tryptophan  in  the  protein.   The  intensity  of 
fluorescence  at  340  nm  is  dependent  upon  the  tryptophan  quantum  yield  of  the 
protein.   The  absorption  at  215  nm  is  determined  principally  by  the  peptide 
chromophore  although  minor  differences  will  arise  if  aromatic  chromophores  are 
present  in  unusual  amounts.   We  have  used  the  difference  in  absorption  between 
215  and  225  nm  since  this  method  largely  eliminates  any  absorbance  due  to 
turbidity.   Zinc  was  measured  by  flameless  atomic  absorption  spectrophotometry. 
Polyacrylamide  gel  electrophoreses  of  each  column  elute  were  also  performed  as 
was  amino  acid  analysis.   Molecular  weight  determinations  by  equilibrium  sedi- 
mentation was  performed  after  the  protein  was  thoroughly  dialyzed  against 
0.1  M  NaCl  or  6  M  guanidine  hydrochloride. 


*©f 


Project       No.       Z01  HL     01981-13  HE 

(I.)  Chromatography 

A:   Sephadex  G  150.   Elution  with  0.01  M  PO  buffer  pH  6.8  produced  pro- 
files using  each  of  the  three  methods  of  protein  determination  which  are  quite 
distinct  since  different  protein  properties  are  measured  by  each  technique 
(Fig.  1).   The  215  nm  profile  was  divided  into  five  major  fractions  which  were 
labelled  II  to  VI.   An  additional  fraction,  referred  to  as  I,  was  evident  in  the 
solvent  front  which  contained  very  little  215  nm  absorption  but  was  highly 
fluorescent. 

B:   DEAE-A50.   Fraction  II  was  divided  into  two  parts  since  the  protein 
in  the  first  part  (IIA)  contained  a  much  higher  Zn/215  nm  ratio  than  did  the 
remaining  tubes  (IIB).   Fraction  IIA  were  lyophilized,  dissolved  in  zinc  free 
water,  dialyzed,  placed  on  a  DEAE-A50  column,  and  initially  eluted  with  0.01  M 
PO,  buffer  pH  6.8  followed  by  elution  with  a  linear  gradient  of  NaCl  (0-0.5  M) 
in  the  same  buffer.   A  major  separation  of  the  215  nm  absorption  peak  from  the 
zinc,  fluoresence  and  280  nm  absorption  peaks  occurred  during  elution  with 
phosphate  buffer.   Most  of  the  zinc  appeared  in  two  neighboring  peaks  which  were 
eluted  before  the  NaCl  gradient.   Moreover  the  zinc/280  nm  ratio  was  very  similar 
in  these  two  peaks  suggesting  that  the  proteins  resembled  each  other  rather 
closely. 

C:   CMC.   The  two  major  and  one  minor  zinc  peaks  obtained  from  the  DEAE- 
A50  column  were  pooled  and  lyophilized.   The  product  was  dissolved  in  water, 
dialyzed,  placed  on  a  CMC  column,  and  eluted  with  the  same  0.005  M  PO,  buffer, 
pH  5.9  in  a  linear  gradient  of  NaCl  (0-0.30  M) .   The  zinc  was  largely  concen- 
trated in  a  single  peak  and  the  ratio  of  zinc  to  protein  in  this  peak  was  con- 
stant and  independent  of  the  parameter  used  to  evaluate  protein  concentration. 
In  the  absence  of  two  proteins  having  all  four  identical  properties  the  protein 
in  the  eluted  peak  must  represent  a  single  molecular  species. 

The  increase  in  specific  activity  of  the  zinc  protein  (the  Zinc/OD   ;  and 
Zinc/0D9R  ),  as  obtained  from  the  maximum  value  in  the  zinc  peaks  in  the  three 
chromatographic  columns,  is  shown  in  Table  II.   It  is  evident  that  the  elimina- 
tion of  the  contaminating  280  nm  absorbing  material  is  accomplished  mainly  on 
the  Sephadex  G  150  column  whereas  the  elimination  of  the  contaminating  215  nm 
material  occurs  mainly  on  DEAE-A50.   There  is  obviously  a  great  disparity  in  the 
215/280  ratio  of  many  of  the  proteins  present  in  saliva.   The  value  used  to 
calculate  the  specific  activity  of  the  starting  solution  was  the  zinc  and  215  nm 
absorption  of  whole  saliva.   Yields  were  necessarily  low  since  the  yield  was 
sacrificed  for  purity  in  selecting  the  most  active  fraction  for  further  purifi- 
cation on  the  DEAE-A50  and  CMC  columns.   These  studies  suggest  that  a  2  00-fold 
purification  from  the  whole  parotid  saliva  was  accomplished  by  these  procedures. 

(II)  Molecular  Characterization 

A:   Polyacrylamide  gel  electrophoreses.   Electrophoresis  of  the  purified 
human  parotid  zinc  protein  revealed  one  broad  band.   Electrophoresis  in  0.1  M 
sodium  phosphate  with  0.1%  SDS  showed  one  major  band  and  one  faint,  more  slowly 
migrating  band.   After  reduction  in  2-mercaptoethanol  no  significant  change  was 
noted  in  the  migration  of  the  major  band  although  some  diffuse  staining  was  evident 


&>r 


Project     No.    ym    m   n-\9KL_u 


HE 


200  (CD) 


cm  cp  ^3; 

^(G0)SI2V 


£o£ 


Project  No.  Z01  HL  01981-13  HE 

adjacent  to  this  major  band.   The  molecular  weights  of  the  major  and  minor  com- 
ponents in  the  SDS  gels  determined  by  comparison  with  appropriate  standards, 
were  44,000  and  84,000,  respectively. 


,E  II 


SPECIFIC  ACTIVITY  AND  YIELD  OF  PAROTID  ZINC  PROTEIN 


Specific  Activity  Specific  Activity  Yield  of    Recovery  of 
Zn/OD  215  ppb/OD  Zn/OD  280  ppb/OD  Parotid  Zn  Total  Protein 

Protein  %       % 


Substance 


Whole  parotid  saliva  0.05 

Sephadex  G  150  0.58 

DEAE-A50  65 . 5 

CMC  268 


0.19 

— 

-- 

678 

5.9 

95 

850 

6.5 

84 

1040 

16.5 

100 

B:   Sedimentation  equilibrium.   The  molecular  weight  of  the  purified  sali- 
vary zinc  protein  determined  by  sedimentation  equilibrium  was  37,000  as  calcu- 
lated from  the  Svedberg  equation.   Equilibrium  centrifugation  in  6.0  M  guanidine 
hydrochloride  gave  a  molecular  weight  of  35,000  indicating  that  this  molecule 
does  not  dissociate  into  equal  subunits  in  the  presence  of  a  reducing  agent. 

C:  Amino  acid  composition.  The  amino  acid  analysis  of  the  purified  parotid 
zinc  protein  was  determined.   The  histidine  content  of  the  parotid  zinc  protein 
is  high  (8  %  per  mole)  while  the  half-cysteine  content  is  quite  low. 

D:   Zinc-protein  ratios.   Based  upon  a  molecular  weight  of  37,000  present 
estimates  suggest  that  there  is  two  moles  of  zinc  per  mole  of  protein. 

PHYSIOLOGY 

1.   Salt  preference  and  blood  pressure  response  in  male  rats  given  estrogen, 
progesterone  and  estrogen  and  progesterone.   Preference  for  various  concentrations 
of  Na  and  K  salts  and  other  tastants  was  determined  in  36  normal  Sprague-Dawley 
rats  in  which  (A)  estrogen,  (B)  progesterone,  (C)  estrogen  and  progesterone  or 
(D)  vehicle  alone  was  administered  parenterally,  daily  for  periods  of  12  weeks. 
Weight,  food  intake  and  blood  pressure  were  measured  at  least  weekly.   Results 
indicated  that  on  estrogen  alone  male  rats  exhibited  significantly  greater  pref- 
erence for  NaCl,  HaHC03,  NaAcetate  (NaAc)  and  KC1  than  did  control  rats  given 
vehicle  alone.  This  change  occurred  within  two  weeks  of  estrogen  administration. 
However,  both  estrogen  treated  and  control  rats  rejected  HC1  and  quinine  to  a 
similar  degree.  Rats  given  progesterone  alone  exhibited  a  significantly  greater 
preference  for  NaCl  and  KC1  than  did  controls  only  after  prolonged  administration 
of  this  hormone  and  both  groups  rejected  HC1  and  quinine  to  a  similar  degree. 


&e>t 


Project   No.  Z01  HL  01981-13  HE 

Rats  given  estrogen  and  progesterone  together  exhibited  an  enhancement  over 
those  given  either  estrogen  or  progesterone  alone  in  their  preference  for  NaCl, 
NaHC03,  NaAc,  and  KC1.   These  effects  occurred  prior  to  those  noted  for  rats 
given  estrogen  alone;  however,  these  rats  also  rejected  HC1  and  quinine  to  a 
similar  degree  as  the  control  rats.   No  change  in  blood  pressure  was  observed 
in  any  of  these  rats  over  the  course  of  the  study. 

2.  Salt  taste  in  patients  with  essential  hypertension  and  with  hypertension 
due  to  primary  hyperaldosteronism.   It  is  widely  believed  that  excessive  salt 
intake  plays  a  role  in  the  development  of  hypertension  in  man.   Conversely,  it 
has  been  demonstrated  that  deprivation  of  salt  and  water  is  efficacious  in  the 
treatment  of  hypertension.   Since  salt  intake  in  man  is  under  voluntary  control 
several  investigators  have  measured  salt  taste  acuity  in  patients  with  hyper- 
tension in  an  attempt  to  evaluate  the  possible  role  this  variable  might  play 

in  the  development  of  this  disease.   Their  results  suggested  that,  as  a  group, 
patients  with  hypertension  exhibited  lower  than  normal  salt  taste  acuity.   From 
this  observation  one  group  reasoned  that  these  patients  used  excessive  amounts 
of  salt  on  their  food  to  achieve  the  preferred  salty  taste  and  thereby  unmasked 
whatever  genetic  propensity  they  might  have  had  to  develop  hypertension. 

Although  this  reasoning  is  quite  attractive  critical  studies  of  salt  taste 
acuity  in  patients  with  hypertension  have  not  yet  been  carried  out.   In  an 
effort  to  do  this  specific  measurements  of  salt  taste  acuity,  i.e.,  detection 
and  recognition  thresholds  for  NaCl,  were  evaluated  in  50  patients  with  well 
documented  essential  hypertension  and  in  10  patients  with  hypertension  due  to 
primary  hyperaldosteronism.   In  some  patients  thresholds  were  measured  before 
and  after  therapy  which  was  efficacious  in  lowering  blood  pressure  to  or  toward 
normal.   Results  of  these  studies  indicate  that,  as  a  group,  taste  thresholds 
for  NaCl  in  patients  with  either  essential  hypertension  or  hypertension  due  to 
primary  hyperaldosteronism  do  not  differ  from  normal. 

3.  Salt  preference  in  patients  with  untreated  and  treated  essential 
hypertension.     Sodium  chloride  preference  was  studied  in  16  patients  with 
essential  hypertension  and  26  normotensive  volunteers  over  a  2  day  period.   Each 
exhibited  normal  detection  and  recognition  thresholds  for  the  taste  of  NaCl. 
Each  was  placed  on  a  constant  dry  diet  containing  9  mEq  Na+  and,  as  the  only 
source  of  fluid,  given  a  choice  of  drinking  either  distilled  water,  0.15  M  NaCl, 
or  some  combination  of  the  two  fluids.   Patients  with  essential  hypertension 
drank  a  significantly  greater  proportion  of  their  total  fluid  as  saline 

(day  1:  34.9%  versus  12.6%;  day  2:  34.1%  versus  13.5%)  and  drank  a  greater  total 
volume  of  fluid  (day  1:  1332  versus  669  cc;  day  2:  1419  versus  824  cc)  than 
did  the  normotensive  volunteers.   The  tdal  amount  of  Na+  consumed  by  the  patients 
was  4.8  to  7.3  times  greater  than  that  of  the  normotensive  volunteers.   Effective 
treatment  of  hypertension  lowered,  mean  NaCl  preference,  Na+  intake  and  total 
fluid  intake  in  the  four  subjects  studied  under  these  conditions.   These  latter 
findings  suggest  that  treatment  with  antihypertensive  agents  may  play  some  role 
in  altering  salt  appetite. 


10 


&>fi 


Project  No.  Z01  HL  01981-13  HE 

4.   Effects  of  various  orally  placed  ganglionic  blocking  agents  on  taste 
acuity  in  man.    In  order  to  investigate  the  neuropharmacology  of  taste  in  man 
various  drugs  were  placed  into  the  oral  cavity  and  taste  acuity  was  measured 
by  determination  of  taste  detection  and  recognition  thresholds  and  of  forced 
scaling.   Drugs  included  succinylcholine  chloride  (10,20  and  40  mg)  methylcholine 
chloride  (20  mg)  curare  (9  mg) ,  gallamine  (9  mg) ,  reserpine  (5  mg)  and  pro- 
pranolol (50  mg) .   Each  drug  was  placed  into  the  oral  cavity  for  2-3  minutes 
and  then  expectorated.   Measurements  of  taste  acuity  were  determined  immediately 
prior  to  oral  placement  of  each  drug  and  for  varying  time  periods  after  ex- 
pectoration.  Results  indicated  that  succinylcholine,  methacholine  and  curare 
significantly  inhibited  taste  acuity  for  all  tastants.   Effects  of  curare  per- 
sisted for  up  to  18  hours  whereas  the  effects  of  succinylcholine  and  metha- 
choline lasted  over  a  period  of  one  to  two  hours.   Gallamine  inhibited  taste 
acuity  for  bitter  and  sour  tastants  and  altered  forced  scaling  somewhat  for 
each  tastant . 

PATHOPHYSIOLOGY 

1.  Localization  of  99   technetium  in  the  region  of  the  nose  in  Sjogren's 
syndrome.   Patients  with  Sjogren's  syndrome  accumulated  abnormal  amounts  of 
99m  technetium  pertechnitate  in  the  region  of  the  nose  during  isotopic  salivary 
flow  studies.   It  was  concurrently  and  independently  observed  that  many  patients 
with  Sjogren's  syndrome  had  hyposmia  and  pathological  changes  in  their  nasal 
mucous  membranes.   Fourteen  patients  with  Sjogren's  syndrome  were  studied  for 
the  relationship  of  the  above  observations  and  the  nasal  accumulation  of 
radionuclide  was  compared  with  a  control  group  of  16  subjects.   Eleven  of  the 

14  patients  with  Sjogren's  syndrome  (78%)  has  nasal  accumulation  of  the 
radionuclide;  14  had  hyposmia  and  13  of  14  had  chronic  inflammation  of  the 
nasal  mucous  membrane.   One  of  the  16  controls  (6%)  localized  radionuclide  in 
the  nasal  region.   Results  suggest  that  hyposmia,  inflammatory  changes  in  the 
nasal  mucous  membrane  and  nasal  accumulation  of  99m  technetium  pertechnitate 
are  interrelated  aspects  of  Sjogren's  syndrome.  This  nasal  accumulation  of 
radionuclide  has  been  termed  Rudolph  Sign. 

2.  Nasal  mucous  membrane  biopsy  in  Sjogren's  syndrome:  A  new  diagnostic 
technique.   A  procedure  was  developed  for  biopsy  and  light  microscopic  eval- 
uation of  the  nasal  mucous  membrane.   This  procedure  was  applied  in  15  female 
patients  with  Sjogren's  syndrome  and  the  results  compared  with  those  of  a 
similar  procedure  applied  to  the  lip.   Results  indicated  that  the  pathological 
changes  found  in  the  nose  were  similar  to  those  found  in  the  lip.   The  most 
characteristic  pathological  feature  in  the  nose  and  in  the  lip  was  periglandu- 
lar infiltration  by  chronic  inflammatory  cells  with  glandular  atrophy.   These 
changes  in  the  nose  have  been  compared  with  similar  biopsies  obtained  in  160 
other  female  patients  with  hyposmia  of  varying  etiologies  and  these  changes  were 
observed  only  in  patients  with  Sjogren's  syndrome.   Biopsy  of  the  nasal  mucous 
membrane  is  a  simple  technique  which  can  be  employed  repeatedly  and  has  little 
morbidity.   As  such,  it  is  a  useful  technique  to  aid  in  the  diagnosis  and  eval- 
uation of  therapy  of  Sjogren's  syndrome  and  in  the  differential  diagnosis  of 
hyposmia. 


11  **1 


Project  No.  Z01  HL  01981-13  HE 

3.  Abnormalities  of  taste  and  smell  after  head  trauma.   Abnormalities  of 
taste  and  smell  were  studied  in  29  patients  after  head  trauma.   These  abnor- 
malities included  decreased  taste  acuity  (hypogeusia) ,  a  distortion  of  taste 
acuity  (dysgeusia) ,  decreased  smell  acuity  (hyposmia) ,  and  a  distortion  of  smell 
acuity  (dysosmia) .   This  syndrome  can  occur  even  after  minimal  head  trauma  and 
can  begin  months  after  the  moment  of  injury.   The  patients  exhibited  a  signifi- 
cant decrease  in  total  serum  zinc  concentration  (patients,  77  *  3  yg/100  ml, 
mean  ±  1  SEM,  vs  controls,  99  ±  2   yg/100  ml,  P  >  0.001)  and  a  significant  in- 
crease in  total  serum  copper  concentrations  (113  *  4  yg/100  ml  vs  100  *  2  yg/100 
P  <  0.001)  compared  with  control  subjects.   Mean  salivary  zinc  levels  in  these 
patients  are  also  significantly  lower  than  in  normal  subjects.   Symptoms  of  hy- 
pogeusia, dysgeusia,  and  dysosmia  are  frequent  sequelae  of  head  injury  and  are 
important  to  the  patients  and  to  their  care  after  trauma. 

4.  Clinical  aspects  of  post  influenzal  hypogeusia,  dysgeusia,  hyposmia 
and  dysosmia.    We  have  recently  described  a  syndrome  characterized  by  hypo- 
geusia, with  or  without  dysgeusia,  and  hyposmia,  with  or  without  dysosmia. 
Eighty-seven  of  143  consecutive  patients  studied  at  the  Taste  and  Smell  Clinic 
at  the  NIH  developed  the  above  symptoms  following  an  influenza-like  illness 

a  clinical  entity  which  we  have  named  post  influenzal  hypogeusia  and  hyposmia 
(P1HH) .   The  clinical  findings  of  these  87  patients  were  defined  and  related 
to  specific  pathophysiological  changes. 

Patients  ranged  in  age  from  19-83  years  (mean,  54  years),  were  of  both 
sexes,  of  varied  ethnic  and  racial  groups  and  residents  of  various  geographical 
regions  in  the  world.   In  each  subject  the  onset  of  PIHH  followed  an  upper 
respiratory  illness  which  was  clinically  of  viral  origin  and  usually  occurred 
in  the  winter  or  spring  months .   The  illness  began  as  a  severe  coryza  with 
fever,  chills,  malaise,  anorexia,  arthralgia  and  myalgia.   Lower  respiratory 
and  gastrointestinal  symptoms  were  occasionally  present  but  not  prominent. 
At  the  onset  of  the  coryza  the  patients  usually  noted  an  abrupt  decrease  in 
taste  acuity  (hypogeusia)  and  smell  acuity  (hyposmia).   At  first,  most  patients 
were  not  overly  concerned  about  these  changes  since  many  had  experienced  similar 
hypogeusia  and  hyposmia  with  previous  influenza-like  illnesses.   However,  during 
the  recovery  period,  they  noted  that  their  taste  and  smell  acuity  did  not  re- 
turn to  normal.   Approximately  one-half  of  the  patients  also  developed  symptoms 
of  dysgeusia  and  dysosmia.   These  symptoms  usually  began  during  the  recovery 
period.   In  about  one-half  of  these  latter  patients  the  dysgeusia  and  dysosmia 
became  increasingly  severe  with  time  whereas  in  the  other  half  they  fluctuated 
in  intensity,  usually  decreasing  and  disappearing  over  the  next  six  months. 

Following  the  influenza  and  the  development  of  PIHH  patients  noted  dryness 
of  their  nasal  passages  and  decreased  frequency  and  quantity  of  nasal  discharge. 
Neither  nasal  crusting  nor  epistasis  was  prominent  and  nasal  stuffiness  was  un-   , 
common.   There  was  no  associated  decrease  in  tearing,  salivary  flow  or  secretion 
from  other  glands.   Patients  never  complained  of  any  associated  oral  ulcerations, 
gingivitis,  "cold"  or  "canker"  sores.   Swelling  of  the  parotid  or  other  salivary 
glands  was  never  evident  and  lymphadenopathy  in  the  head  and  neck  region  was  not 
a  common  feature  either  during  the  acute  or  recovery  phase  of  the  influenza. 


12  £(0 


Z01  HL  01981-13  HE 
Project 


No, 


Patients  were  referred  to  our  clinic  by  physicians  who  had  exhausted  all 
their  knowledge  of  diagnosis  or  treatment  of  these  distressing. symptoms.   Patient 
were  referred  from  local  family  physicians,  neurologists,  otorhinolaryngolosists 
and  psychiatrists. 

Physical  examination  of  the  head  and  neck: 

Patients  were  examined  3  months  to  10  years  following  their  initial  ill- 
ness.  The  most  prominent  consistent  clinical  feature  of  PIHH  was  in  the  nose 
where  the  tenacious,  clear,  occasionally  white  mucous  blanket  normally  found 
was  not  observed.  The  nasal  mucous  membranes  were  moist  but  neither  shiny  nor 
glistening.  The  membranes  were  often  pale  in  color,  but  not  similar  to  the 
pale,  boggy  bluish  tinge  commonly  observed  in  allergic  rhinitis  or  the  reddish 
tinge  seen  in  infectious  rhinitis.  Nasal  membranes  looked  similar  to  those 
following  the  topical  application  of  a  vasoconstrictor  agent  such  as  neo- 
synephrine.  A  second  consistent  feature  was  the  patency  of  the  nasal  airway. 
The  deeper  structures  of  the  nasal  cavity  were  easily  seen  by  anterior 
rhinoscopy  without  the  use  of  vasoconstrictor  agents.  The  openings  of  the  glands 
of  the  nasal  mucous  membranes  were  not  prominent.  Membranes  were  not  edematous 
and  there  was  little  vascular  congestion  or  turgor.   Purulent  secretions, 
crusting  or  bleeding  were  not  commonly  observed.  Two  patients  (2/87)  had 
unilateral  nasal  polyps. 

There  were  no  distinctive  changes  observed  in  the  oral  cavity,  external 
auditory  canal  or  tympanic  membranes  or  in  the  neck.  No  significant  adenopathy 
was  present  and  indirect  mirror  laryngeal  examination  was  within  normal  limits 
in  all  patients. 

Roentgenographic  examinations 

Paranasal  Sinuses:  Roentgenographs  of  the  paranasal  sinuses  including 
Waters,  Caldwell,  submento-vertex  and  lateral  views  were  obtained  in  each 
patient.   Roentgenographs  were  considered  to  be  abnormal  in  16  patients  (18%). 
Of  these,  a  rudimentary  frontal  sinus  was  the  only  abnormality  present  in  2. 
Of  the  remaining  14,  2  had  evidence  of  acute  sinusitis  manifested  by  air  fluid 
levels,  one  in  the  left  antrum,  the  other  in  the  right  frontal  sinus.   Four 
exhibited  thickening  of  the  mucosa  of  the  antrum  of  mild  to  moderate  degree 
without  evidence  of  an  air-fluid  level.  One  had  an  antral  osteoma  and  3  had 
mucocoeles,  one  in  the  left  antrum,  one  in  the  right  antrum  and  one,  bilaterally. 
Four  exhibited  bilaterally  small,  sclerotic  well-aerated  antra.  Although  there 
was  no  antecedent  history  of  chronic  sinusitus  or  treatment  of  sinus  disease 
the  radiographic  interpretation  of  these  changes  in  these  latter  four  patients 
was  that  of  chronic  maxillary  osteitis.   One  of  the  patients  with  nasal  polyps 
had  an  air-fluid  level  in  the  frontal  sinus.  The  other  patient  with  nasal  polyps 
had  no  abnormalities  of  the  paranasal  sinuses  on  roentgenographic  examination. 
Of  these  16  patients,  only  three  had  local  unilateral  disease  of  the  sinuses 
which  in  time  of  onset  and  degree  of  severity  could  be  related  to  the  PIHH. 
These  patients  were  included  in  this  study  because  their  local  disease  was  un- 
ilateral . 


13  «" 


Project  No.  Z01  HL  01981-13  HE 

Skull:   Roentgenographs  of  the  skull  including  frontal  and  lateral  views 
were  obtained  in  each  patient.   Two  patients  exhibited  calcification  of  the 
falx  cerebri  while  one  exhibited  hyperostosis  frontalis  interna.   Roentgeno- 
graphs of  all  other  patients  were  considered  to  be  within  normal  limits.   No 
evidence  of  an  olfactory  cleft  tumor  was  found  in  any  patient. 

Radionuclide  studies:   Brain  scanning  following  an  intervenous  injection  of 
99m  technetium  was  carried  out  in  each  patient;  while  no  intrinsic  pathology 
of  the  brain  was  observed  in  any  patient  accumulation  of  99m  technetium  in  the 
nasal  region  was  observed  on  both  frontal  and  lateral  views  in  40%  of  the 
patients.   This  accumulation  was  previously  observed  in  78%  of  a  group  of  patien 
with  Sjogren's  syndrome,  has  been  termed  Rudolph  Sign  and  has  been  previously 
been  related  to  the  intensity  of  inflammation  in  the  nasal  mucous  membrane. 

Taste:   Median  detection  and  recognition  thresholds  for  the  patients  with  PIHH 
were  elevated  above  normal  levels  for  salt,  sour  and  bitter  whereas  for  sweet, 
median  detection  and  recognition  thresholds  were  at  the  upper  limit  of  normal. 
Forced  scaling  patterns  for  the  patients  were  significantly  different  from 
those  of  the  controls,  for  each  taste  quality  tested. 

Smell:   Median  detection  and  recognition  thresholds  for  the  patients  with  PIHH 
for  representatives  of  each  of  three  vapors  (pyridine,  nitrobenzene  and  thio- 
phene)  were  elevated  above  normal  levels  for  each  vapor  tested. 

Hearing:   Air  and  bone  conduction  thresholds  were  obtained  in  79  of  the  87 
patients  on  at  least  one  occasion.   Twenty-six  of  79  patients  (31%)  had  no     ^ 
history  of  hearing  loss  and  had  normal  hearing  thresholds  upon  testing.   Thirty- 
five  of  the  79  (44%)  gave  a  history  of  hearing  loss  prior  to  the  onset  of  their 
influenza  and  had  abnormalities  demonstrated  by  testing.   Eighteen  of  the  79 
(25%)  noted  the  onset  of  hearing  abnormalities  at  or  during  the  time  of  the 
influenza  and  hearing  losses  of  various  types  were  observed. 

None  of  the  18  patients  with  hearing  losses  related  in  time  to  the  in- 
fluenza gave  a  history  of  otitis  media  at  the  time  of  the  acute  illness.   Each 
of  these  18  patients  developed  a  sensorineural  hearing  loss,  8  with  unilateral 
losses,  10  with  bilateral  losses.   Ten  (56%)  exhibited  a  mild  high  frequency 
loss  (loss  of  30  dB  or  less  in  the  speech  range  with  further  losses  in  the 
frequencies  above  2000  Hz),  2  (11%)  exhibited  a  moderate-severe  high  frequency 
loss  (loss  of  30-60  dB  in  the  speech  range) ,  4  (22%)  exhibited  a  "notch"  at 
4000  Hz  and  2  (11%)  exhibited  a  "peaked  curve". 


* 


Microscopic  examinations 

Taste  buds:    Taste  buds  from  patients  with  PIHH  were  examined  by  light 
and  electron  microscopy  and  compared  with  taste  buds  removed  from  normal  vol- 
unteers and  from  patients  with  various  acute  and  chronic  diseases  in  whom  taste 
acuity  was  normal.   Taste  buds  from  patients  with  PIHH  showed  severe  disruption 
of  the  pore  region  of  the  bud  with  marked  diminution  in  the  number  and  organi- 
zation of  the  neurosecretory  granules,  absence  of  the  dense,  extracellular 
material,  amputation  of  the  finger-like  projections  from  cells  which  extend  intc 
the  pore  region  and  the  presence  of  many  large  and  small  vacuoles. 


14 


6.CX- 


' 


Project  No.   Z01  HL  01981-13  HE 

Nasal  mucous  membranes :   Biopsies  from  the  nasal  mucous  membranes  of 
patients  with  PIHH  were  examined  by  light  microscopy  and  compared  with  similar 
tissue  taken  from  normal  volunteers  undergoing  local  nasal  procedures  for 
cosmetic  reasons  and  from  patients  with  various  acute  and  chronic  diseases  in 
whom  olfactory  acuity  was  normal. 

Biopsies  from  patients  demonstrated  thickening  of  the  surface  epithelium 
with  mild  cellular  atypia,  moderate  to  marked  thickening  of  the  basement  mem- 
brane and,  most  characteristically,  infiltration  of  the  upper  lamina  propria 
with  chronic  inflammatory  cells.   These  cells  are  mainly  lymphocytes  but 
plasma  cells  were  also  observed.   Relatively  little,  if  any,  inflammation 
was  present  in  the  submucosal  glands.   Significant  sclerosis  and  fibrosis  of 
the  upper  lamina  propria  was  also  commonly  observed.   Both  serous  and  mucous 
glands  were  decreased  in  number  but  serous  glands  were  decreased  to  a  much 
greater  degree. 

The  diagnosis  of  PIHH  may  be  made  by  an  appropriate  history,  observing 
clinical  findings  in  the  nose,  finding  abnormalities  in  taste  and  smell  per- 
ception, demonstrating  specific  changes  in  nasal  mucous  membrane  biopsies  and 
by  demonstrating  the  presence  of  Rudolph  Sign  following  intravenous  injection 
of  99m  technetium.   Together  these  findings  are  relatively  specific  for  this 
disorder  which  is  surprisingly  common. 

5.   Treatment  of  patients  with  taste  and  smell  dysfunction.  A  randomized, 
double  blind  crossover  study  of  the  effects  of  zinc  sulfate  and  placebo  was 
carried  out  in  106  patients  with  taste  and  smell  dysfunction  secondary  to  a 
variety  of  etiological  factors.   In  the  patient  group  prior  to  treatment  mean 
serum  zinc  concentration  and  leukocyte  alkaline  phosphatase  activity  were  sig- 
nificantly lower  than  normal.   Results  indicate  that  placebo  was  effectively 
equivalent  to  zinc  sulfate  in  the  treatment  of  these  disorders  although  there 
was  no  change  in  mean  serum  zinc  concentration  or  urinary  zinc  excretion  among 
the  placebo  responders.   Although  these  results  demonstrate  abnormalities  of 
zinc  metabolism  in  patients  with  taste  and  smell  dysfunction  they  fail  to  pro- 
vide evidence  for  a  systematic,  therapeutic  approach  to  the  many  disorders 
underlying  taste  and  smell  dysfunction.   However,  the  methods  and  procedures 
developed  in  this  study  demonstrate  that  taste  and  smell  dysfunction  can  be 
studied  in  a  quantitative,  systematic  manner. 

Significance: 

1.   Anatomy.   Details  of  the  anatomical  characteristics  and  functional  sig- 
nificance of  Type  I,  II  and  III  cells  in  taste  buds  from  fungiform  and  circum- 
vallate  papillae  have  been  confirmed.   Type  III  cells  appear  to  be  the  receptor 
cell  of  the  taste  buds  as  determined  by  the  direction  of  its  synaptic  connections, 
the  Type  II  cell  appears  to  function  in  a  manner  which  protects  the  taste  bud 
through  its  contractile  mechanism  and  the  Type  I  cell  may  supply  the  neurotrans- 
mitter material  necessary  for  the  initial  neural  events  of  the  taste  process  to 
occur.   The  presence  of  acetylcholinesterase  in  the  pore  region  of  the  taste  bud 
confirms  earlier  hypotheses  relating  to  the  significance  of  the  cholinergic 
nervous  system  in  the  taste  bud. 


15 


<U3 


Project  No.  Z01  HL  01981-13  HE 

2.   Chemistry.   A  simpler  process  to  purify  miraculin  has  been  developed 
which  results  in  the  yield  of  a  much  more  purified  glycoprotein.   The  properties 
of  this  glycoprotein  havebeen  studied.   The  sugar  moeities  of  this  glycoprotein 
have  been  confirmed  as  fucose,  xylose,  mannose  and  galactose  and  their  ratios 
confirmed  by  thin  layer  and  gas-liquid  chromatography.   The  utilization  of  this 
glycoprotein  in  the  control  of  sugar  intake  in  man  has  been  investigated  and 
found  to  be  particularly  useful  in  the  intake  of  sweetened  beverages  where  it 
can  be  adapted  to  supply  all  the  sweetness  required  for  normal  hedonic  purposes. 

The  functional  significance  of  taste  bud  membranes  has  been  confirmed 
through  the  demonstration  of  specific  binding  and  displacement  of  several  sugars 
to  these  membranes.   Each  ^C  sugar  studied,  behaved  as  did  unlabelled  sugar  with 
respect  to  binding  activity;  specific  binding  of  l^C  sugars  to  the  taste  bud 
membranes  occurred  but  did  not  occur  with  the  non-taste  bud  bearing  epithelial 
membranes  where  only  non-specific  binding  occurs.   Inhibitors  which  bind  metals 
or  attack  sialic  acid  residues  significantly  interfere  with  this  binding  in- 
dicating the  importance  of  these  two  moeities  in  the  binding  process. 

The  role  cf  nerve  growth  factor  (NGF)  in  the  taste  process  has  been  in- 
vestigated.  Specific  displacement  of  this  substance  in  isolated  taste  bud 
membranes  has  been  achieved  suggesting  that  a  role  for  NGF  can  be  identified 
at  the  taste  receptor.   NGF  activity  has  been  isolated  in  human  parotid  saliva 
as  measured  by  bioassay,  in  chick  sympathetic  ganglia,  and  by  radioreceptor 
assay,  using  dorsal  root  ganglia.   This  activity  has  been  related  to  a  purified 
fraction  of  human  parotid  saliva  and  is  the  first  demonstration  of  a  NGF 
type  of  activity  in  man  not  identifiable  with  tumor  activity. 

A  zinc  containing  protein  present  in  human  parotid  saliva  was  predicted  in 
the  annual  report  1972-73.   This  protein  has  been  identified,  isolated,  purified 
and  some  of  its  chemical  characteristics  identified.   It  has  a  molecular  weight 
of  37,000  and  contains  8%  histidine  per  mole.   The  protein  has  been  labelled 
in  vivo  with  Zn^5  ana  constant  specific  activity  with  native  Zn  has  been 
achieved.   This  protein  has  some  properties  in  common  with  mouse  NGF  and  has 
been  shown  to  displace  1^-25  mouse  NGF  in  binding  studies  with  bovine  taste  bud 
receptor  membranes.   This  protein  is  often  significantly  reduced  in  the  saliva 
of  patients  with  hypogeusia.   Therapy  with  zinc  ion  may  induce  the  formation 
of  this  protein  and  result  in  the  alleviation  of  the  observed  symptoms  of  taste 
loss  and  taste  dysfunction.   This  protein  has  been  named  gustin. 

A  total  protein  fractionation  of  human  parotid  saliva  has  been  accomplished 
for  the  first  time.   This  fluid  has  been  fractionated  into  6  major  fractions 
with  gustin  a  major  component  of  Fraction  II.   A  glycoprotein  with  several  un- 
usual molecular  characteristics  has  also  been  identified  in  Fraction  II.   This 
protein  is  made  up  primarily  of  proline  (1/3)  and  lysine  (1/3)  and  may  represent  \ 
a  form  of  procollagen.   Studies  in  which  gustin  and  the  glycoprotein  were  com- 
bined have  shown  an  enhancement  of  NGF  activity  suggesting  that  these  proteins 
may  exhibit  some  functional  significance  in  combination  which  neither  has  in- 
dividually.  Protein  markers  have  also  been  used  to  characterize  each  of  the 
other  fractions  obtained  from  human  parotid  saliva.   NGF  activity  is  not  present 
in  these  other  fractions. 


16  *'< 


Project  No.  Z01  HL  01981-13  HE 

3.  Physiology.   The  relationship  between  salt  intake,  estrogen  and  proges- 
terone administration  and  hypertension  was  systematically  evaluated.   In  the 
male  rat  although  estrogen  or  estrogen  plus  progesterone  administration  produces 
an  increase  in  salt  intake  it  does  not  appear  to  be  associated  with  the  pro- 
duction of  hypertension.   On  the  other  hand  in  preliminary  studies  administra- 
tion of  estrogen  or  estrogen  plus  progesterone  to  female  rats  is  associated 
with  both  the  increase  in  salt  intake  and  the  production  of  hypertension.   These 
studies  suggest  that  some  differences  in  endocrine  function  may  be  important  in 
understanding  the  mechanism  by  which  these  hormones  produce  these  different 
effects  in  male  and  female  rats.   These  results  could  lead  to  information  of 
importance  in  understanding  the  mechanism  by  which  various  drugs  used  to  con- 
trol fertility  influence  blood  pressure. 

Studies  in  man  clearly  demonstrate  that  patients  with  essential  hyperten- 
sion exhibit  a  significantly  greater  preference  for  NaCl  than  do  normotensive 
subjects.   This  preference  is  not  related  to  taste,  per  se.   Following  effec- 
tive treatment  with  antihypertensive  agents  preliminary  studies  suggest  that 
salt  preference  in  these  patients  decreases.   Whether  these  changes  are 
causally  related  are  not  presently  known  but  these  studies  offer  a  useful, 
novel  manner  in  which  to  obtain  important  information  of  usefulness  to  our 
knowledge  of  the  physiology  of  salt  intake  and  to  its  possible  relationship  to 
blood  pressure. 

4.  Pathophysiology.   An  approach  by  which  patients  with  disorders  of  taste 
and  smell  can  be  systematically  investigated  in  order  to  evaluate  the  etiologi- 
cal factors  responsible  for  their  disorders  has  been  established.   It  is  now 
possible  to  provide  a  rational  biochemical  basis  upon  which  to  establish  a 
differential  diagnosis  for  these  disorders.   These  novel  techniques  have  been 
applied  to  several  systemic  disorders  and  offer  aid  in  the  differential  diagno- 
sis of  these  disorders  (e.g.,  Sjogren's  syndrome,  allergic  rhinitis,  Wegener's 
granulomatosis  and  midline  granuloma  of  the  face) .   The  diminution  of  the 
protein  gustin  in  some  patients  with  disorders  of  taste  and  smell  has  proven  to 
be  a  useful  clinical  tool  in  these  investigations  and  has  allowed  the  diagnosis 

of  central  lesions  affecting  taste  acuity  to  be  distinguished  from  that  of  more 
local  lesions  which  affect  taste.   The  importance  of  metabolic  factors  in  the 
production  of  abnormalities  of  taste  following  head  trauma  has  been  suggested 
for  the  first  time  and  this  hypothesis  is  substantiated  by  the  finding  of  de- 
pressed levels  of  gustin  in  patients  of  this  type. 

Proposed  Course  of  Project: 

1.  The  identification  of  an  acetylcholine  receptor  at  the  taste  bud  re- 
ceptor membrane  will  be  sought,  in  part  through  the  utilization  of  the  binding 
of  a-bungaro toxin  to  the  taste  bud  receptor  membrane.   Other  biochemical 
techniques  will  be  utilized  to  investigate  the  role  of  neurotransmitters  at 
the  taste  bud  membrane  in  vivo,  in  man  and  animals,  and  in  vitro. 

2.  The  systematic  investigation  of  the  relationships  between  salt  intake, 
hormones  and  hypertension  will  be  continued  in  studies  to  be  performed  in  man 
and  in  animals. 


17 


4>t*- 


Project  No.   Z01  HL  01981-13  HE 

3.  The  molecular  characteristics  of  gustin  will  be  investigated  further. 
Its  relationship  with  the  salivary  glycoprotein  will  be  systematically  studied. 

4.  A  radioimmunoassay  and  a  radioreceptor  assay  for  gustin  and  for  the 
salivary  glycoprotein  will  be  obtained.   In  this  manner  a  definitive  clinical 
test  for  the  quantitation  of  these  proteins  and  their  relationship  to  taste 
acuity  will  be  established. 

5.  A  double-blind  study  will  be  undertaken  (once  the  identification 
of  an  appropriate  patient  population  is  established  through  the  utilization 
of  the  radioimmunoassay  and  radioreceptor  assay)  to  establish  the  efficacy  of 
zinc  ion  in  the  treatment  of  an  appropriate  population  of  patients  with 
taste  disorders. 

6.  The  characteristics  of  human  nerve  growth  factor  will  be  established 
in  relationship  to  the  characteristics  of  gustin  and  the  salivary  glycoprotein. 
These  characteristics  will  be  compared  to  that  of  other  growth  factors  inclu- 
ding insulin,  NSILA  and  epidermal  growth  factor. 

Keyword  Descriptors;   Taste,  Smell,  Saliva,  Parotid  Proteins,  Gustin 

Publications : 

Henkin,  R.I.,  Schechter,  P.J.,  Raff,  M.S.,  Bronzert,  D.A.  and 
Friedewald ,  W.T.:   Zinc  and  taste  acuity:   A  clinical  study  including  a 
laser  microprobe  analysis  of  the  gustatory  receptor  area.   In  Clinical 
Applications  of  Zinc  Metabolism,  Pories,  W. ,  Strain,  W. ,  Hsu,  J.M.  and 
Woosley,  R.L.  (Eds.),  C.C.  Thomas,  Springfield,  111.,  1975,  Chapter  19, 
pps.  204-228. 

Giroux,  E.L.  and  Henkin,  R.I.:   Purification  and  some  properties  of 
Miraculin,  a  sweetness-provoking  glycoprotein  from  miracle  fruit.  J. 
Agri.  Food  Chem.  22:  595-601,  1974. 

Schechter,  P.J.  and  Henkin,  R.I.:   Abnormalities  of  taste  and  smell 
following  head  trauma.   J.  Neurol.  Neurosurg.  Psychiat.  37:  802-810,  1974. 

Henkin,  R.I.:  Salt  taste  in  patients  with  essential  hypertension  and  with 
hypertension  due  to  primary  hyperaldosteronism.   J.  Chron.  Dis .  27: 
235-244,  1974. 

Henkin,  R.I.:   Taste  in  man.   In  Scientific  Foundations  of  Otolaryngology, 
Harrison,  D.  and  Hinchcliffe,  R.  (Eds.),  Wm  Heinemann  Medical  Books,  Ltd. 
London,  England,  1975  (In  press). 

Catalanotto,  F. ,  Slavik,  M. ,  Danilson,  D.,  Reiser,  H.  and  Henkin,  R.I.: 
Changes  in  preference  for  NaCl  following  administration  of  6-azauridine 
and  6-azauridine  triacetate.   Pharmacology  and  Therapeutics  in  Dentistry, 
1975  (In  press) . 


18  &£ 


Project  No.  Z01  HL  01981-13  HE 

Henkin,  R.I.,  Lippoldt,  R.E.,  Bilstad,  J.  and  Edelhoch,  H. :   A  zinc 
containing  protein  isolated  from  human  parotid  saliva.   Proc .  Nat. 
Acad.  Sci.,  72:  488-492,  1975. 

Henkin,  R.I.,  Larson,  A.L.  and  Powell,  R.D.:   Clinical  aspects  of  post 
influenza-like  hypogeusia,  dysgeusia,  hyposmia  and  dysosmia.   Annals 
Oto.  Rhin.  Laryn. ,  1975  (In  press). 

Paran,  N.,  Mattern,  C.F.T.  and  Henkin,  R.I.:   Ultrastructure  of  the  taste 
bud  of  the  human  fungiform  papilla       Cell  and  Tissue  Research, 
1975  (In  press) . 


19  6'7 


Project  No.   Z01  HL  01982^08  HE 

1.  Hypertension-Endocrine  Branch  |! 

2.  Section  on  Neuroendocrinology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Trace  Metal  Metabolism 

Previous  Serial  Number:   NHLI  105(c) 

Principal  Investigator:   Robert  I.  Henkin,  M.D.,  Ph.D. 

Other  Investigators:   R.  Aamodt,  Ph.D. 

W.  Mallette,  M.D.,  Ph.D. 

G.  Aurbach,  M.D. 

D.  Tormey,  M.D. ,  Ph.D. 

C.  Gillin,  M.D. 

B.  Patten,  M.D. 

W.  D.  Grover,  M.D. 

Cooperating  Units:   National  Cancer  Institute,  Nuclear  Medicine  Branch,  Clinical 
Center,  NINDS,  NIMH,  Metabolic  Disease  Branch,  NIAMDD, 
Baylor  University,  School  of  Medicine,  Houston,  Texas, 
St,  Christopher's  Hospital  for  Children,  Philadelphia,  Pa. 

Project  Description: 

Objectives :   To  study  the  physiology,  metabolism,  biochemistry  and  pathology 
of  copper,  zinc  and  other  trace  metals  in  physiological  fluids  and  tissues  of 
normal  subjects,  in  patients  with  various  diseases  and  in  animals.   These 
studies  include  the  interaction  between  metals  and  their  binding  proteins. 

Major  Findings: 

Biochemistry:   Estimation  of  zinc  concentration  in  parotid  saliva  by  f lameless 
atomic  absorption  spectrophotometry  in  normal  subjects  and  in  patients  with 
idiopathic  hypogeusia.   A  relationship  between  taste  acuity  and  zinc  metabolism 
has  been  previously  established  in  that  zinc  depleted  patients  exhibit  hypo- 
geusia (loss  of  taste  acuity) ;  treatment  with  zinc  ion  returns  this  hypogeusia 
toward  or  to  normal  in  some  of  these  patients.   These  changes  in  zinc  metabolism 
were  reflected  initially,  in  some  patients  in  the  untreated  state,  in  lower     j 
than  normal  levels  of  zinc  measured  in  serum  and  hair  and  with  a  return  to 
normal  following  treatment  with  zinc  ion.   Although  these  general  relationships 
were  clearly  established  in  some  patients  there  were  many  untreated  patients 
with  hypogeusia  in  whom  serum  zinc  concentrations  were  within  normal  limits 
yet  whose  hypogeusia  was  amenable  to  treatment  with  zinc  ion.   These  results 
suggested  that  serum  and  hair  zinc  reflected  changes  in  taste  acuity  and  zinc 
metabolism  in  only  a  very  general  manner,  primarily  in  cases  of  severe  zinc 
depletion.  d 

1  4ft 


Project  No.  Z01  HL  01982-08  HE 

A  close  relationship  between  taste  acuity  and  the  presence  of  saliva  has 
been  previously  established  in  that  patients  with  xerostomia  of  several 
etiologies  exhibit  hypogeusia;  treatment  of  these  underlying  conditions  with 
several  agents  which  returned  salivary  flow  toward  or  to  normal  was  associated 
with  the  return  of  taste  acuity  toward  or  to  normal. 

Since  saliva  and  zinc  both  appeared  to  be  important  factors  in  the  control 
of  taste  acuity,  it  seemed  useful  to  measure  the  zinc  concentration  of  saliva 
in  subjects  with  normal  taste  acuity  and  in  patients  with  hypogeusia  in  an 
attempt  to  obtain  a  closer  correlation,  if  it  did  exist,  between  zinc  levels 
and  taste  acuity. 

Estimation  of  salivary  zinc  concentration  by  conventional  flame  aspiration 
atomic  absorption  spectrophotometry  (AAS)  was  attempted  first  but  it  proved 
impractical  since  salivary  zinc  concentrations  were  often  below  or  near  the 
sensitivity  of  the  instrument  and  a  rather  large  amount  of  sample  was  required 
since  it  was  necessary  to  measure  the  sample  without  dilution.   To  overcome 
these  problems  we  measured  salivary  zinc  concentration  by  flameless  AAS.  '  A 
method  by  which  salivary  zinc  concentrations  were  estimated  by  flameless  AAS 
was  developed  and  applied  to  subjects  with  normal  taste  acuity  and  in  patients 
with  hypogeusia.   By  this  technique  zinc  was  determined  rapidly  and  reproducibly 
in  concentrations  below  1  ppb  (1  ng/ml)  in  a  sample  as  small  as  5  ul.   By  this 
technique  significantly  less  zinc  was  measured  in  the  saliva  of  patients  with 
hypogeusia  compared  with  that  in  subjects  with  normal  taste  acuity  (p  <  0.001, 
Table  I). 

TABLE  I 

No  of  Age 

Group  Subjects         Range 

Normal  Volunteers 34 16-65 

Men  11  23-64 

Women  23  16-65 

All  Caucasians  29  16-65 

All  Blacks  4  34-55 


Patients  with  47  19-73  10  ±  6* 

hypogeusia 

Mean  ±  1  SD 

* 

Significantly  below  mean  of  normal  volunteers  (p  <  0.001) 


Zn 
ppb 

51 

+ 

14+ 

53 

+ 

11 

50 

+ 

16 

49 

+ 

14 

58 

+ 

14 

«f 


Project  No.  Z01  HL  01982-08  HE 
Physiology 

1.  Role  of  zinc  in  taste,  food  intake  and  growth.   Preference  for  HC1, 
food  intake,  growth  and  changes  in  zinc  metabolism  were  studied  in  4  groups 
of  rats  fed  either  zinc  deficient  or  zinc  replete  diets  ad_  libitum  or  by 
force.   Group  A  was  fed,  ad  libitum,  a  zinc  deficient  diet;  group  B  was  pair 
fed  with  group  A  a  zinc  supplemented  diet;  group  C  was  fed,  ad  libitum,  a 
zinc  supplemented  diet;  group  D  was  force-fed  a  zinc  deficient  diet  in  an       i 
amount  similar  to  that  taken  by  group  C.   Mean  plasma  zinc  concentrations  and 

24  hour  urinary  zinc  excretion  was  measured  in  each  group.   At  the  termination 
of  the  experiment  organ  weights  of  each  group  were  also  measured. 

Plasma  zinc  and  urinary  zinc  excretion  were  significantly  lower  in  groups 
A  and  D  (receiving  zinc  deficient  diets)  than  in  the  controls  (group  B  and  C) . 
HC1  preference  for  each  group  (A  and  D)  given  zinc  deficient  diet  was  signifi- 
cantly greater;  i.e.,  they  demonstrated  decreased  aversion,  than  did  the 
controls  suggesting  decreased  taste  acuity  in  the  zinc  deficient  groups. 
Growth  rates  of  the  rats  of  group  A  was  significantly  below  that  of  the  rats 
fed  zinc  supplemented  diets  (groups  B  and  D)  but  also  below  that  of  rats 
force-fed  zinc  deficient  diet.   Testicular  weight  was  significantly  lower 
than  in  controls  in  rats  fed  zinc  deficient  diet  ad_  libitum  or  by  force. 
These  observations  indicate  that  some  sequellae  of  zinc  deficiency  relate  to 
the  metal  deficiency,  per  se,  (i.e.,  taste  changes,  anorexia)  whereas  others 
relate  more  closely  to  changes  in  food  intake  (body  weight,  testicular  changes). 

2 .  Cerebellar  dysfunction,  mental  changes ,  anorexia,  and  taste  and  smell   i 
dysfunction  associated  with  acute  zinc  loss  ;   a^  new  syndrome.    L-histidine  was 
administered  orally  in  graded  doses  to  4  women  and  2  men  with  progressive 
systemic  sclerosis.   During  the  administration  of  this  amino  acid  a  constellation 
of  clinical  changes  occurred  following  a  decrease  in  serum  zinc  concentration 
and  an  increase  in  urinary  zinc  excretion.   These  changes  were  the  production 

of  anorexia,  taste  and  smell  dysfunction,  organic  mental  and  psychiatric 
symptoms  including  depression  and  frank  psychosis  and  cerebellar  dysfunction 
including  intention  tremor,  positive  Romberg  sign  and  ataxia.   Following  dis- 
continuation of  the  amino  acid  no  rapid  changes  of  these  symptoms  occurred. 
Following  administration  of  zinc  ion,  with  or  without  the  continued  adminis- 
tration of  the  amino  acid  L-histidine,  all  symptoms  and  signs  and  metabolic 
changes  were  consistently  reversed  within  24-48  hours.   These  studies  suggest 
that  zinc  plays  an  important  role,  not  only  in  appetite  and  taste  and  smell 
function  but  also  in  cerebellar  function  as  well.   These  studies  have  also  been 
now  carried  out  in  patients  with  chronic  zinc  depletion  with  similar  results. 

3.  Zinc   metabolism  in  man.    Zinc  absorption,  distribution  and  excretion 
was  studied  in  11  patients  with  hypogeusia  of  various  etiologies,  parents  of  a  I 
patient  with  Wilson's  disease  and  a  patient  with  cystinuria.   Zinc   was  ad- 
ministered orally  (l-2yCi)  or  intravenously  (IV) (15-24  yCi)  while  patients  were 
on  a  constant  diet.   Patients  with  hypogeusia  were  given  Zn"*-*"  (100  mg/day)  or 
placebo  during  the  study.   Zinc^5  activity  in  blood,  urine,  liver  and  thigh 

was  determined  and  total  body  retention  was  measured  for  207  to  800  days  after 
administration.   Following  IV  injection,  the  concentration  of  zinc  5  in  whole- 


6X> 


i 


Project  No.  z01  Hl  01982-08  HE 

blood  and  plasma  decreased  rapidly  (<2%  in  plasma,  <5%  in  whole-blood  after 
24  hours) .   Red  blood  cell  concentration  reached  maximum  7-10  days  after  in- 
jection, then  gradually  decreased.   External  gamma-ray  measurements  over  the 
liver  area  showed  maximum  value  (50-75%  of  injected  zinc")  between  5  and 
24  hours,  then  gradually  decreased.   Activity  measured  over  the  thigh  area 
decreased  initially,  then  increased  to  maximum  values  during  the  next  20-40 
days.   Following  oral  administration  of  zinc65}  three  absorption  patterns  were 
observed:   6  patients  absorbed  65  ±4%  (M  ±  SEM)  ;  3  patient?  r.bsorbed  signifi- 
cantly less  (28  ±  9%,  p  <  0.01)  and  2  patients  absorbed  significantly  more 
(99  ±  1%,  p  <  0.001).   Total  body  retention  fit  a  two  component  exponential 
pattern  with  biological  half-time  of  8  and  300  days.   The  longer  half-time 
component  was  shortened  in  1  patient  with  congenital  hypogeusia  and  2  patients 
while  receiving  exogenous  zinc.   This  effect  was  not  seen  in  other  patients 
given  exogenous  zinc. 

These  results  may  be  applied  directly  to  an  understanding  of  zinc  metab- 
olism in  several  disease  states.   Lombeck  et  al. ,  reported  the  reduction  of 
oral  absorption  of  Zn   in  patients  with  acrodermatitis  enteropathica  and 
suggested  this  abnormality  as  the  cause  of  the  signs  and  symptoms  of  observed 
zinc  deficiency  in  this  condition.   In  the  two  untreated  patients  they  studied 
absorption  of  Zn  5  was  indeed  low  (patients;  15%,  30%:  normals;  mean,  66%,  fit. 
range,  58%-77%) .   They  also  stated  that  the  elimination  rate  of  absorbed  Zn 
was  similar  in  both  patients  and  controls  clearly  delineating  an  absorptive 
not  a  retentive  defect  of  zinc.   However,  in  the  one  treated  patient  they 
studied  Zn   absorption  was  much  higher,  42%.   Thus,  following  treatment, 
patients  with  acrodermatitis  enteropathic  may  exhibit  an  increased  oral  ab- 
sorption of  Zn  ^  in  contrast  to  the  usually  observed  decreased  Zn"  absorption 
observed  in  normal  subjects  given  exogenous  zinc. 

Results  of  our  total  body  absorption  indicated  three  distinct  absorptive 
patterns  as  noted  above.   One  group  (A),  absorbed  99  ±  1%  (Mean  ±  1  SEM)  of  the 
administered  dose;  one  group  (B) ,  absorbed  65  ±  4%  of  the  dose,  range  51-77%; 
a  third  group  (C) ,  absorbed  28  ±  9%  of  the  dose,  range  8-37%). 

Group  B,  which  comprised  the  largest  number  of  patients,  exhibited  ab- 
sorption in  a  range  quite  similar  to  that  noted  by  Lombeck  et  al.  in  their 
small  group  of  normal  subjects.   However,  absorption  in  Group  C  was  quite  simi- 
lar to  that  noted  in  their  patients  with  untreated  acrodermatitis  enteropathica 
yet  our  patients  exhibited,  as  their  only  functional  complaint,  disorders  of 
taste  and  smell;  although  the  mean  level  of  serum  zinc  in  our  patients  was 
significantly  lower  than  normal  as  was  the  alkaline  phosphatase  activity  of 
their  blood  leukocytes  none  had  any  of  the  clinical  manifestations  of  acro- 
dermatitis enteropathica. 

Evaluation  of  these  results  suggest  that  impaired  zinc  absorption  is  not 
the  primary  defect  in  acrodermatitis  enteropathica.   Data  from  Lombeck  et  al. 
and  from  our  group  would  suggest  that  the  defect  in  acrodermatitis  enteropathica 
lies  in  the  genetic  lack  of  an  appropriate  gastrointestinal  transport  moeity 
(polypeptide  or  protein) ,  a  lack  which  is  somehow  corrected  through  the  ad- 
ministration of  oral  zinc,  even  in  very  small  quantities  which  may  be  only 

4  £>/ 


Project  No.   Z01  HL  01982-08  HE 

slightly  higher  than  the  presently  accepted  Recommended  Daily  Allowances. 
This  would  suggest  that  zinc  acts  in  this  disease  to  either  induce  the  formation 
of  this  transport  moeity,  to  inhibit  its  repression,  etc.   If  this  hypothesis 
were  correct  then  the  pathological  sequellae  of  acrodermatitis  enteropathic 
may  be  explained  in  full.   Absorption  in  the  presence  of  small  amounts  of 
dietary  zinc  would  be  low  producing  the  sequellae  of  zinc  deficiency  in  blood 
and  tissues;  absorption  in  the  presence  of  larger  amounts  of  zinc  would  in- 
crease gradually,  correcting  these  abnormalities  since  the  remainder  of  the 
binding  and  transport  system  on  the  blood  side  of  the  gut  wall  appears  to  be 
intact.   This  hypothesis  would  also  explain  the  sometimes  observed  lag  phase 
in  the  production  of  symptoms  with  the  stopping  of  oral  zinc  supplementation. 

The  role  of  impaired  zinc  absorption  in  patients  with  hypogeusia  and  hy- 
posmia  is  more  complex.   In  these  patients  there  appears  to  be  at  least  two 
defects  in  zinc  metabolism,  the  one  involving  an  impairment  absorption  across 
the  gut  wall  in  some  patients  and  another  involving  an  impairment  in  the  for- 
mation of  the  zinc  containing  parotid  protein  gustin,  a  protein  which  has  been 
suggested  to  act  by  stimulating  growth  and  development  of  taste  buds. 

Continuation  of  research  into  these  important  areas  have  been  hindered 
by  several  difficulties  such  as  the  practical  measurement  of  zinc  in  several 
body  tissues,  the  difficulty  of  serum  or  urine  zinc  levels  to  reflect  accurately 
body  zinc  levels,  sparsity  of  total  body  counting  equipment  with  which  to  carry 
out  dynamic  studies  of  zinc  metabolism  and  vagaries  in  the  measurement  tech- 
niques themselves.   Lombeck  et  al.,  reported  no  measurable  Zn   excreted  in 
the  urine  in  their  patients  with  acrodermatitis  enteropathica  over  the  first 
4  days  of  their  studies.   These  results  may  be  simply  due  to  the  technical 
failure  to  measure  radioactivity  in  the  entire  24  hour  urine  sample  collected 
since  in  our  patients  with  hypogeusia  and  hyposmia,  even  among  those  who  ab- 
sorbed less  than  10%  of  the  administered  dose,  measurable  amounts  of  Zn^5  were 
excreted  within  the  first  24  hours.   Indeed,  measurement  of  urinary  zinc  ex- 
cretion over  the  first  24  hours  of  our  studies  after  a  small  oral  load  of  Zn°^ 
appears  to  supply  an  useful  approximation  of  zinc  absorption.   Comparison  of 
the  total  body  absorption  of  Zn^5  calculated  on  the  basis  of  balance  data  with 
the  simple  urinary  excretion  of  Zn^5  during  the  first  24  hours  after  adminis- 
tration indicates  the  correlation  between  these  two  techniques  is  highly  sig- 
nificant (r  =  +0.810,  p  <  0.001).   Calculations  of  zinc  absorption  on  the  basis 
of  balance  data  compared  with  that  using  only  the  data  obtained  from  the  uri- 
nary excretion  of  Zn°5  over  the  first  24  hours  agrees  very  closely  over  a  range 
of  absorption  from  8%-100%;  signed  differences  between  absorption  calculated 
by  these  two  methods  are  less  than  1%  whereas  on  an  absolute  basis  differences 
are  approximately  15%. 

4.   Role  of  parathyroid  hormone  in  copper  metabolism.   Copper  metabolism 
was  studied  in  8  patients  with  well  documented  hyperparathyroidism  before  and 
after  removal  of  their  parathyroid  adenomas.   In  each  patient  prior  to  surgery 
serum  copper  levels  were  significantly  elevated  above  normal  (patients,  155  ± 

6  yg/dl,  M  ±  1  SEM;  controls,  106  ±  2)  whereas  urinary  copper  excretion  was 
approximately  3-10  times  normal  (patients,  range  50-400  yg/24  hours;  controls, 
20-70  ug/24  hours).   Within  three-five  days  following  surgical  removal  of  the 

5  £** 


Project  No.  ZQ1  HL  01982-08  HE 


parathyroid  adenoma  urinary  copper  excretion  returned  to  normal  levels.   This 
return  was  independent  of  urinary  changes  in  adenyl  cyclase,  calcium,  phos- 
phorus, sodium  or  potassium.   These  studies  suggest  that  parathyroid  hormone 
may  play  some  specific  role  in  the  control  of  urinary  copper  excretion  and 
thereby  in  the  control  of  copper  metabolism. 

5 .  Copper  and  zinc  metabolism  in  patients  with  breast  carcinoma.   Serum 
copper  and  zinc  concentrations  were  measured  in  seventy  patients  with  breast 
carcinoma  at  varying  stages  of  the  disease  and  under  varying  conditions  of 
treatment.  Mean  serum  copper  concentrations  were  significantly  elevated  above 
normal  (patients,  162  ±  10  yg/dl,  mean  1  1  SEM;  controls,  106  ±  2)  whereas 
serum  zinc  concentration  was  significantly  lower  than  normal  (patients,  72-  ±  5 

yg/dl;  controls,  96  ±  2).   Many  of  these  patients  exhibited  anorexia  and  taste 
and  smell  dysfunction.   Whether  elevated  serum  copper  and  lowered  serum  zinc 
concentrations  may  serve  as  a  diagnostic  or  therapeutic  aid  in  the  understanding 
of  this  disease  is  at  present  under  investigation. 

6.  Copper  and  zinc  metabolism  in  hypothyroidism.   Serum,  urine  and  sali- 
vary concentrations  of  zinc  and  copper  have  been  measured  in  5  patients  with 
hypothyroidism  before  and  after  therapy  with  thyroid  hormone.   Serum  and  urine 
concentrations  of  zinc  and  copper  have  also  been  meastired  in  15  rats  made  hypo- 
thyroid with  ll31  an(j  in  15  control  rats.   In  man  serum  zinc  and  saliva  concen- 
trations were  below  normal  off  treatment  and  returned  toward  normal  after 
therapy  with  thyroid  hormone.   In  rats  serum  zinc  concentration  was  signifi- 
cantly lower  than  normal  whereas  urinary  zinc  excretion,  expressed  per  mg  of 
creatinine,  was  significantly  higher  than  normal. 

7.  Copper  and  zinc  metabolism  in  unmedicated  acute  and  chronic 
schizophrenia.   Patients  with  schizophrenia  have  been  reported  to  exhibit 
several  abnormalities  of  zinc  and  copper  metabolism  and  these  changes  have 
been  related  to  various  therapies  to  aid  in  the  treatment  of  these  disorders. 
Each  of  these  studies  suffers  from  severe  procedural  limitations  and  the  con- 
clusions were  not  justified  on  the  basis  of  the  data  obtained.   In  order  to  ob- 
tain a  systematic  evaluation  of  this  important  problem  copper  and  zinc  levels 
were  studied  in  30  patients  with  unmedicated  acute  and  chronic  schizophrenia 

in  various  tissues  and  related  to  their  disease  states  where  possible. 

Copper  and  zinc  levels  were  measured  in  5  tissues  as  noted  in  Table  II. 


TABLE  II 

SERUM 

URINE 

GASTRIC 

CSF      FLUID    HAIR 

Condition 

Number 

Zn 

yg/dl 

Cu 

yg/dl 

Cerulo- 

plasmin 

ng/dl 

Zn     Cu 
yg/24° 

Zn   Cu 

yg/dl 

Zn  Cu 
yg/dl 

Zn 

yg/dg 

Patients 

20 

92  ±3 

93  ±5 

34±  1 

286135   30±3 

3+1  411 

4214  712 

13616 

Controls 

85 

96  ±2 

106  ±2 

30±  2 

353123  3615 

712  812 

4015  1013 

18014 

6A3 


Project  No.   Z01  HL  01982-08  HE 

The  results  of  these  studies  indicated  that  patients  with  schizophrenia 
do  not  exhibit  any  change  from  normal  in  copper  or  zinc  levels  in  serum,  urine, 
CSF  or  gastric  fluid.   However,  levels  of  zinc  in  hair  were  uniformly  below  the 
normal  mean  and  the  mean  level  was  significantly  lower  than  normal  (p  <  0.001). 

In  order  to  evaluate  these  findings  further  a  second  study  was  begun  in 
which  10  additional  patients  were  studied  and  also  in  which  a  group  of  patients 
with  schizophrenia  treated  with  various  tranquilizing  agents  were  studied.   This 
work  was  undertaken  since  it  is  possible  that  long  term  therapy  with  some  tran- 
quilizing agents  may  itself  produce  zinc  loss.   This  problem  arises  since  any 
drug  with  an  imidazole  group  may  act  as  a  zinc  chelator  and  produce  a  long 
term  loss  of  total  body  zinc  as  measured  by  hair  zinc.   These  studies  are  in 
their  final  stages. 

8.  The  Environmental  Protection  Agency  (EPA)  Panel  on  Zinc.    The  report 
on  zinc  for  the  EPA  has  been  completed  and  the  draft  returned  from  its  first 
reviews.   The  first  draft  of  this  work  was  distributed  to  the  parent  panel  with- 
in the  National  Academy  of  Science  in  March.   Initial  results  indicate  that 
fish  are  significantly  sensitive  to  low  levels  of  zinc  which  may  appear  in  the 
water  secondary  to  the  improper  handling  of  untreated  or  even  treated  human 

and  animal  waste  products;  this  effect  produces  a  respiratory  death  and  may  be 
one  cause  of  the  rapid  depletion  of  fish  in  areas  where  they  were  previously 
plentiful.   In  man,  in  children  a  new  syndrome  of  zinc  toxicity  has  been  de- 
scribed consisting  of  lethargy,  fatigue  and  anemia.   Treatment  with  chelating 
agents  has  been  useful  in  the  reversal  of  these  symptoms.   The  role  of  zinc 
in  growth  and  development  in  man  has  been  outlined. 

9 .  Investigation  of  the  copper  defect  in  Menkes  Kinky  Hair  Disease  (MKHD) . 
MKHD  is  a  genetic,  X-linked  disorder,  characterized  by  failure  to  thrive, 
seizures,  progessive  cerebral  deterioration,  pili  torti,  degeneration  of  blood 
vessel  walls,  deficiency  of  tissue  copper  and  decreased  activity  of  several 
copper  dependent  enzymes.   Serum  albumin  and  renal  function  are  normal.   Three 
patients  with  MKHD  were  investigated  under  four  conditions:  (1)  untreated, 

(2)  treated  with  1.5  mg  CUSO4  intravenously,  (3)  treated  with  10  mg  CuS04 
orally  for  10  days  and  (4)  treated  with  10  mg  copper  nitrilotriacetic  acid 
(CuNTA)  orally  for  10  days.   Without  treatment  serum  copper  and  ceruloplasmin, 
respectively,  were  less  than  1/4  normal  (patients,  M  ±  1  SEM,  24  ±  1  ug/dl, 
4  ±  1  mg/dl;  normal,  110  ±  2  ug/dl,  34  ±  2  mg/dl) ,  while  urinary  copper  ex- 
cretion was  more  than  four  times  normal  (patients,  34  ±  5  ug/24°;  normal, 
7+1  ug/24°).   Intravenous  CUSO4  rapidly  increased  serum  copper  and  cerulo- 
plasmin slightly,  as  previously  reported,  transiently  increased  further  the 
already  increased  urinary  copper  excretion  but  produced  little  if  any 
amelioration  of  the  existing  tissue  copper  deficiency.   Oral  CUSO4  did  not 
alter  significantly  either  serum  or  urinary  copper  or  ceruloplasmin  because 
it  is  mainly  absorbed  into  the  gastrointestinal  mucosa,  rather  than  across 
the  gastrointestinal  tract.   Oral  CuNTA  increased  serum  copper  and  cerulo- 
plasmin.  Since  copper  is  transported  into  cells  by  first  order  kinetics  only 
after  forming  a  relatively  small  molecular  weight  organometallic  complex, 
copper  in  ceruloplasmin,  due  to  its  high  association  constant,  is  unavailable 


6a# 


Project  No.  z01  ^  01982-08  HE 

for  this  transport  whereas  CuNTA  is.   Conceivably  CuNTA  could  fulfill  the 
necessary  requirements  for  tissue. 

Significance: 

1 .  Transition  metal  ion  absorption  in  the  gas tro intestinal  tract. 
Based  upon  studies  in  normal  man  with  Zn65  and  in  patients  with  Menkes-Kinky 
Hair  Disease  an  unified  theory  of  the  manner  by  which  transition  metal  ions 
are  absorbed  across  the  gut  has  been  proposed.   This  involves  the  absorption 
of  a  small  molecular  weight  organometallic  complex  across  the  gut  mucosa  in 
a  manner  which  follows  first  order  kinetics,  is  then  passively  absorbed  into 
the  blood  and  transported  by  mechanisms  previously  shown. 

2.  Role  of  zinc  in  growth  and  development.   The  immediate  major  role  of  zinc 
in  growth  is  through  its  control  of  food  intake.   This  hypothesis  was  suggested 
previously  from  our  earlier  work  and  has  been  confirmed  by  studies  in  which 

rats  were  force  fed  zinc  deficient  diet  and  results  compared  with  rats  fed 
zinc  deficient  diet  ad  libitum.   Results  demonstrate  that  serum  zinc  concen- 
trations in  rats  fed  zinc  deficient  diet  ad  libitum  or  by  force  were  similar 
and  significantly  below  those  of  rats  fed  zinc  supplemented  diets.   Similarly 
both  groups  of  rats  fed  zinc  deficient  diet  exhibited  hypogeusia.   However, 
the  force  fed  rats  grew  at  a  significantly  greater  rate  than  did  the  rats 
fed  zinc-deficient  diet  ad  libitum  indicating  the  role  of  anorexia  in  this 
process.   In  addition  testicular  weights  of  the  force-fed  rats  were  signifi- 
cantly greater  than  those  of  the  ad  libitum  fed  rats  indicating  that  food  in- 
take rather  than  zinc  deficiency  per  se  is  an  important  factor  in  this  finding. 
These  studies  are  the  first  to  separate  the  specific  roles  of  zinc  affecting 
food  intake  and  tissue  changes. 

3.  Role  of  copper  in  Menkes-Kinky  Hair  Disease.   This  disease  is  at 
present  difficult  to  treat  since  the  mechanisms  underlying  this  disease  are 
poorly  understood.   We  have  proposed  an  hypothesis  for  the  etiology  of  this 
disease,  including  the  observed  blood  vessel  changes,  based  upon  biochemical 
considerations;  i.e.,  these  patients  lack  the  small  molecular  weight  organo- 
metallic complex  which  is  necessary  to  produce  normal  copper  utilization  by 
tissues.   To  verify  this  hypothesis  copper  was  conjugated  with  nitrilotriacetic 
acid  and  this  complex  was  given  orally  to  2  patients  with  this  disease.   In 
each  case  there  was  an  increase  in  serum  copper  concentration  suggesting  that 
copper  crossed  the  gut  wall.   These  studies  identify  a  possible  biochemical 
defect  which  could  be  responsible  for  some  of  the  symptoms  of  the  disease  and 
are  the  first  to  identify  a  possible  oral  mode  of  treatment  for  this 
disorder. 

4.  Measurement  of  zinc  in  saliva  and  its  meaning  in  health  and  disease. 
The  development  of  this  technique  represents  the  first  successful  attempt  to 
measure  zinc  accurately  in  human  saliva.   The  technique  is  simple,  rapid,  in- 
expensive, accurate  and  reproducible.   This  tool  allows  the  screening  of  large 
numbers  of  patients  with  various  oral  and  metabolic  abnormalities.   It  also 
allows  for  a  rational  differential  diagnosis  of  hypogeusia  to  be  made  in  that 
patients  with  normal  salivary  zinc  levels  may  not  exhibit  hypogeusia  on  the 

8  £iT 


Project  No.  ZQ1  HL  01982-08  HE 

basis  of  salivary  factors  which  influence  taste  buds.   With  this  technique  it 
is  possible  to  specify  local  disease  of  the  oral  cavity  or  specific  protein 
abnormalities  of  the  saliva  factors  in  the  production  of  altered  taste  acuity. 

5.  Possible  control  of  copper  metabolism  by  parathyroid  hormone.   Studies 
in  patients  with  hyperparathyroidism  secondary  to  parathyroid  adenomas  suggest 
that  parathyroid  hormone  per  se  influences  urinary  copper  excretion.   This 
effect  may  be  either  (1)  through  the  mobilization  of  copper  from  its  sites  in 
bone  and  its  passive  excretion  in  the  urine,  (2)  a  specific  action  of  para- 
thyroid hormone  on  the  renal  tubule  related  to  the  tubular  reabsorption  of 
copper  or  (3)  a  specific  action  of  parathyroid  hormone  on  metallothionine  or 
another,  as  yet  unidentified,  enzyme  important  in  copper  conservation  in  the 
kidney.   Although  the  site  of  action  is  not  yet  understood  these  studies 
demonstrate  for  the  first  time  a  role  for  parathyroid  hormone  in  copper  metab- 
olism and  allow  for  a  study  of  the  specific  locus  of  action  of  this  hormone 

on  copper  metabolism. 

6.  Role  of  zinc  in  cerebellar  and  sensory  function.   The  acute  loss  of 
zinc  from  the  body  is  associated  with  the  production  of  cerebellar  changes  in- 
cluding intention  tremor  and  ataxia  as  well  as  changes  in  sensory  function. 
These  changes  are  all  rapidly  and  completely  reversed  following  the  adminis- 
tration of  zinc  ion.   Recent  studies  in  Houston  have  indicated  that  hypogeusia, 
hyposmia  and  cerebellar  dysfunction  associated  with  zinc  loss  may  appear  on  a 
chronic  as  well  as  on  an  acute  basis  and  that  some  of  these  signs  and  symptoms 
may  be  reversed  by  zinc  administration.   Other  studies  indicate  that  zinc, 
which  is  the  fourth  most  prevalent  ion  in  the  brain,  may  be  readily  removed 
and  replaced  from  its  sites  in  brain.   These  sites  include  hippocampus  and 
cerebellum.   The  role  of  zinc  in  neural  function  has  received  much  attention 
recently  by  Barbeau  and  others  who  have  suggested  that  zinc  metabolism  may  be 
important  as  a  factor  in  epilepsy.   Since  the  cerebellum  appears  to  play  an 
important  role  in  the  control  of  seizure  activity  the  role  of  zinc  in  these 
tissues  may  lead  to  a  new  understanding  of  the  interrelationships  between 
cerebellar  function,  seizure  activity  and  zinc  metabolism. 

7.  Possible  role  of  histidine  and  zinc  metabolism  in  schizophrenia . 
Experimental  production  of  schizophrenic  like  symptoms  in  man  have  been  in- 
vestigated by  several  workers  in  an  attempt  to  understand  the  mechanisms  of 
these  puzzling  diseases.   Administration  of  the  amino  acid  methionine  to 
patients  with  schizophrenia  exaccerbate  the  symptoms  of  these  already  effected 
patients.   However,  administration  of  the  amino  acid  histidine  produces  symp- 
toms which  may  be  interpreted  as  those  of  an  acute  psychosis  in  previously 
mentally  normal  subjects;  these  symptoms  are  quickly  and  completely  reversed 
by  administration  of  zinc  ion.   These  findings  may  lead  to  a  new  biochemical 
approach  to  the  understanding  of  these  diseases. 

/-r  8.   A  simple  test  for  measurement  of  zinc  absorption  in  man.   Urinary 
Zn   excretion  after  a  small  oral  dose  of  Zn65  is  directly  proportional  to  the 
absorption  of  the  metal  over  a  range  of  absorption  from  8%-100%.   This  tech- 
nique may  make  the  evaluation  of  zinc  absorption  in  man  a  practical  and  simple 
test  by  which  the  present  vagaries  of  zinc  metabolism  may  be  systematically 
evaluated. 

9  **£ 


Project  No.  ZQ1  H1  01982-08  HE 
Proposed  Course  of  Project; 

1.  The  kinetics  studies  of  Zn   and  Zn   in  man  will  be  continued  in 
order  to  quantitate  in  more  detail  the  manner  by  which  zinc  is  absorbed,  dis- 
tributed and  excreted  in  man  in  health  and  in  disease. 

2.  The  specific  interrelationships  between  copper  and  parathyroid  hormone 
will  be  investigated  and  the  locus  of  its  action  identified  and  characterized. 

3.  The  treatment  of  patients  with  Menkes-Kinky  Hair  Disease  with  copper 
-NTA  on  a  long  term  basis  will  be  carried  out  through  collaboration  with 

Dr.  W.D.  Grover,  Temple  University,  Philadelphia,  Pa.  Problems  of  therapy- 
will  be  evaluated  and  mechanisms  of  action  will  be  verified..  Blood  vessel 
changes  will  be  studied  in  particular  detail. 

4.  The  book  Zinc  written  under  the  auspices  of  the  National  Academy  of 
Sciences  and  the  Environmental  Protection  Agency  will  be  completed.   It  will 
represent  the  state  of  the  art  of  our  present  knowledge  of  zinc  at  all  levels 
of  biological  importance.   It  will  be  published  by  the  National  Academy  of 
Sciences  in  1975. 

5.  Salivary  zinc  levels  will  be  measured  in  patients  with  various  diseases 
and  correlated  with  taste  acuity  in  order  to  evaluate  the  correlation  between 
these  two  measurements  and  to  evaluate  the  use  of  salivary  zinc  levels  as  a 
useful  index  in  the  differential  diagnosis  of  hypogeusia. 

6.  Further  studies  in  the  role  of  zinc  in  cerebellar  function  will  be 
carried  out  at  the  clinical  level.   The  ability  of  zinc  to  cross  the  blood 
brain  barrier  and  bind  to  specific  brain  sites  will  be  investigated  in  an 
effort  to  understand  the  role  this  ion  plays  in  brain  function. 

7.  A  systematic  investigation  of  the  potential  role  of  histidine  and 
zinc  in  schizophrenia  will  be  evaluated  in  a  double  blind  study  to  be  under- 
taken within  the  NIMH  this  fall. 

Keyword  Descriptors;   Copper,  Zinc,  Zinc  metabolism,  Zinc  absorption,  Menkes- 
Kinky  Hair  Disease. 

Honors  and  Awards :   None 

Publications: 

1.   Henkin,  R.I.:  Metal-albumin-amino  acid  interactions:   Chemical  and 
physiological  interrelationships.   In  Protein-Metal  Interactions, 
Friedman,  Mendel  (Ed.),  Plenum  Publishing  Co.,  N.Y.,  1974,  pps.  299-328, 


10  6XT 


Project  No.  Z01  HL   01982-08  HE 

2.  Henkin,  R.I.:   On  the  role  of  adrenocorticosteroids  in  the  control  of 
zinc  and  copper  metabolism.   In  Trace  Elements  in  Man  and  Animals  II, 
Hoekstra,  W.G.,  Suttie,  J.W.,  Ganther,  H.  and  Mertz,  W.  (Eds.) 
University  Park  Press,  Baltimore,  Md. ,  Chapter  91,  1974,  pps.  647-651. 

3.  Henkin,  R.I.:   Growth  hormone  dependent  changes  in  zinc  and  copper 
metabolism  in  man.   In  Trace  Elements  in  Man  and  Animals  II, 
Hoekstra,  W.G.,  Suttie,  J.S.,  Ganther,  H.  and  Mertz,  W.  (Eds.) 
University  Park  Press,  Baltimore,  Md.,  Chapter  94,  1974,  pps.  652-655. 

4.  Henkin,  R.I.,  Patten,  B.M.,  Re,  Peter,  and  Bronzert,  D. :  Cerebellar 
dysfunction,  mental  changes,  anorexia  and  taste  and  smell  dysfunction 
associated  with  acute  zinc  loss:   A  new  syndrome.   Arch.  Neurology, 
1975  (In  press) . 

5.  Henkin,  R.I.,  Mueller,  C.  and  Wolf,  R. :   Estimation  of  zinc  concen- 
tration of  parotid  saliva  by  flameless  atomic  absorption  spectro- 
photometry in  normal  subjects  and  in  patients  with  idiopathic  hypo- 
geusia.   J.  Lab.  Clin.  Med.,  1975  (In  press). 


11 


6*9 


Project  No.       Z01  HL  01983-12  HE 

1.  Hypertension-Endocrine  Branch 

2.  Section  on  Neuroendocrinology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Hormonal  Effects  on  Sensory  and  Neural  Function 

Previous  Serial  Number:   NHLI-104(c) 

Principal  Investigator:   Robert  I.  Henkin,  M.D.,  Ph.D. 

Other  Investigators:   M.  Buchsbaum,  M.D. 
J.  Gillin,  M.D. 
F.  Catalanotto,  D.D.S. 

Cooperating  Units:   National  Institute  of  Mental  Health,  University  of 
Connecticut,  School  of  Dental  Medicine,  Farmington, 
Connecticut,  College  of  Physicians  &  Surgeons,  N.Y.,  N.Y. 

Project  Description: 

Objectives:   To  investigate  systematically  the  interrelationships  between 
steroid  hormones,  thyroid  hormones,  electrolytes  and  psychodelic  agents  on 
neural  and  sensory  function  with  respect  to  the  manner  by  which  sensory  signals 
are  detected  and  integrated  by  sensory  receptors,  neuraxons  and  brain.   In 
addition,  to  investigate  the  relationship  between  steroid  and  protein  hormones 
and  amino  acids  and  sleep  patterns. 

Major  Findings:  1.   Central  and  peripheral  nervous  system  changes  related  to 
changes  in  endogenous  secretion  of  adrenal  corticosteroids  in  man. 
Averaged  evoked  cortical  responses  (AER)  using  a  graded  intensity  series  of 
visual  and  auditory  stimuli  were  obtained  in  9  patients  with  adrenal  cortical 
insufficiency  and  in  3  with  panhypopituitarism  while  each  was  maintained  on 
adequate  replacement  therapy,  while  each  was  off  all  adrenocorticosteroid  re- 
placement therapy  for  4-9  days  and  while  each  was  replaced  with  deoxycortico- 
sterone acetate,  20  mg  daily  for  2-4  days.   Patients  with  adrenal  cortical  in- 
sufficiency were  also  studied  off  all  replacement  therapy  while  receiving 
intravenous  infusions  of  ACTH  (40  units)  in  5%  dextrose  and  water  or  5%  dextrose 
and  water  without  ACTH  over  an  8  hour  period.   Under  the  same  conditions  measure- 
ments of  eye  movements,  light  touch,  manual  and  oral  stereognosis .,  and  rod  and 
frame  manipulation  were  obtained.   Results  indicated  that,  as  a  group,  while 
untreated,  AER  were  greater  in-  amplitude  than  while  treated  with  adequate  re- 
placement adrenocorticosteroids .   Treatment  with  deoxycorticosterone  (DOCA) 
acetate  did  not  alter  these  changes  in  any  manner.   Administration  of  ACTH 
during  the  period  off  all  steroid  replacement  was  accompanied  by  a  decrease  in 
amplitude  of  the  AER.   Treatment  with  replacement  carbohydrate-active  steroid 
alone  returned  these  changes  to  normal  within  24-48  hours. 


*2? 


Project  No.  Z01  HL  01983-12  HE 

While  off  all  adrenocorticosteroid  therapy  these  patients  also  showed 
inability  to  perform  oral  stereognosis  tasks,  manipulate  rod  within  a  frame 
to  obtain  a  perpendicular  placement  yet  were  extremely  sensitive  to  the  place- 
ment of  nylon  filaments  upon  their  skin,  recognizing  skin  deformation  at  a 
significantly  lower  pressure  than  that  of  normal  volunteers.   Treatment  with 
DOCA  did  not  alter  these  findings  in  any  manner  but  replacement  therapy  with 
carbohydrate-active  steroid  returned  these  changes  to  or  toward  normal  in  each 
patient . 

2.  The  effects  of  ACTH  on  human  sleep  patterns.   The  effects  of  ACTH  on 
human  sleep  was  studied  in  9  healthy  normal  volunteers  by  administration  of 
ACTH  (40  units  intravenously)  beginning  either  at  8  a.m.,  3  p.m.  or  11:30  p.m. 
and  continuing  over  a  period  of  8  hours.   ACTH  was  also  administered  to  3 
patients  with  treated  and  untreated  adrenal  cortical  insufficiency  in  the  same 
manner  over  8  hours  beginning  at  8  a.m.   ACTH  produced  a  significantly  greater 
reduction  in  REM  sleep  in  the  normal  volunteers  than  in  the  patients  with 
Addison's  disease,  as  expected.   During  the  overnight  infusion  beginning  at 
11:30  p.m.,  approximately  4  hours  of  continuous  infusion  of  ACTH  was  required 
before  REM  sleep  was  reduced.   The  REM  suppressive  effect  of  ACTH  appeared  to 
attenuate  about  12  hours  following  the  termination  of  the  ACTH  infusion.   In 
addition,  ACTH  significantly  reduced  total  sleep  time  in  the  volunteers  fol- 
lowing infusions  beginning  at  8  a.m.  and  3  p.m.  but  not  when  ACTH  was  infused 
during  sleep.   Delta  sleep  was  also  reduced  following  the  8  a.m.  infusion 

in  the  volunteers. 

These  results  suggest  that  ACTH  affected  sleep  in  the  normal  volunteers 
through  its  effect  on  adrenal  corticosteroid  secretion.   Time  appears  to  be  an 
important  aspect  of  the  effect  of  ACTH  and  adrenocorticosteroids  upon  the 
central  nervous  system.   The  effects  of  ACTH  on  the  sleep  patterns  of  patients 
with  Addison's  disease,  off  treatment,  suggests  that  the  protein  hormone  may 
play  some  direct  role  in  altering  central  nervous  system  function. 

3.  The  effects  of  L-histidine  on  the  sleep-waking  cycle  in  man. 
L-histidine  was  administered  to  3  patients  with  intractable  narcolepsy  at  a 
dose  of  20  gm/day  for  2  weeks,  to  4  normal  volunteers  at  a  dose  of  32.4  gm/day 
for  5  days  and  to  one  patient  with  scleroderma  at  a  dose  of  48.6  gm/day  for 

16  days.   No  effect  on  nocturnal  sleep  patterns  in  any  of  the  patients  or  sub- 
jects or  on  the  symptoms  of  narcolepsy  was  observed.   Because  of  the  metabolism 
of  histidine  to  histamine  these  results  fail  to  support  the  hypothesis  that 
histamine  is  a  "waking  factor". 

4.  The  role  of  thyroid  hormone  in  taste  and  smell  sensation.   In  order  to 
study  the  specific  role  of  thyroid  hormone  in  taste  function  fifteen  adult  male 
rats  were  made  hypothyroid  by  treatment  with  l!31  (300  pCi/lOO  gm  body  weight) 
matched  with  15  control  rats,  each  caged  individually  and  offered  a  choice  be- 
tween water  and  graded  concentrations  of  sucrose,  quinine,  NaCl  and  HC1.   Hypo- 
thyroid rats  exhibited  increased  preference  for  NaCl  (p  <  0.001)  and  decreased 
aversion  for  quinine  (p  <  0.001)  than  did  the  untreated  controls  but  exhibited 
an  increased  preference  for  sucrose  (p  <  0.001).   There  were  no  significant 


aid 


Project  No.  Z01  HL  01983-12  HE 

differences  between  the  controls  and  the  hypothyroid  rats  for  HC1.   After 
parenteral  administration  of  thyroxine  to  the  hypothyroid  rats.,  10  yg/100  gm 
body  weight  for  2-3  weeks  preference  for  NaCl,  sucrose  and  quinine  returned 
to  normal. 

Following  treatment  with  exogenous  thyroid  hormone  each  of  these  taste 
changes  reverted  to  normal.   The  changes  occurred  within  1  week  of  hormone 
administration  and  persisted  as  long  as  the  hormone  was  administered.   These 
studies  indicate  that  hypothyroidism  produced  by  I^-^l  in  rats  is  associated 
with  significant  alterations  in  taste  and  may  serve  as  a  useful  model  for 
studies  in  man. 

In  patients  with  hypothyroidism  taste  and  smell  function  had  been  previ- 
ously evaluated.   Hypogeusia,  hyposmia,  dysgeusia  and  dysosmia  had  been  previ- 
ously observed  while  off  treatment  reverting  to  or  toward  normal  during  re- 
placement.  In  an  effort  to  evaluate  the  pathological  changes  which  might 
underlie  these  findings  surgical  removal  of  lingual  circumvallate  papillae 
was  carried  out  in  3  patients  with  untreated  hypothyroidism  and  examined  by 
light  and  electron  microscopy.   Whereas  as  many  as  100  taste  buds  may  be  ob- 
served in  circumvallate  papillae  from  subjects  with  normal  taste  acuity  no 
taste  buds  have  been  observed  in  any  of  the  papillae  examined  in  the  patients 
with  untreated  hypothyroidism. 

Significance:   Role  of  steroid,  ACTH  and  thyroid  hormones  in  neural  function. 

Steroid  hormones:   Carbohydrate  active  steroids  affect  neural  function 
at  each  locus  of  the  nervous  system,  at  the  receptor,  the  nerve  and  the  cen- 
tral nervous  system.   These  effects  have  also  been  observed  in  sleep. 

ACTH:   ACTH  has  an  effect  on  central  nervous  system  function  apparently 
independent  of  its  action  on  the  secretion  of  adrenocorticostercids.   This  has 
been  observed  in  patients  with  adrenal  cortical  insufficiency,  off  treatment, 
who  exhibited  significant  changes  in  their  sleep  patterns  after  receiving 
ACTH  without  any  change  in  their  secretion  of  adrenocorticosteroids.   ACTH 
effects  on  sleep  were  not  observed  in  normal  volunteers  in  whom  ACTH  was  ad- 
ministered during  the  sleep  period  itself  but  significant  effects  were  recorded 
when  ACTH  was  administered  8  hours  prior  to  the  sleep  recordings.   These  results 
in  normal  subjects  suggest  that  ACTH  can  effect  sleep  through  its  effects  on 
adrenocorticosteroid  secretion  but  that  time  is  an  important  aspect  of  these 
effects . 

Thyroid  Hormones :   Decreased  thyroid  hormone  produces  dysfunction  in 
taste  and  smell  acuity  (hypogeusia,  dysgeusia,  hyposmia  and  dysosmia)  as  well 
as  decreased  auditory  acuity,  decreased  axonal  conduction  velocity  and  increased 
synaptic  delay.   These  results  are  similar  in  all  respects  to  those  observed  in 
patients  with  Cushing's  syndrome  in  whom  excessive  secretion  of  carbohydrate- 
active  steroid  has  been  measured.   Replacement  with  thyroid  hormone  is  associated 
with  the  return  of  each  of  these  sensory  and  neural  abnormalities  toward  or  to 
normal.   Studies  on  taste  were  also  carried  out  in  hypothyroid  animals  untreated 
and  after  treatment  with  replacement  thyroid  hormone.   Results  were  similar  to 

3  63t 


Project  No.  Z01  HL  01983-12  HE 


those  observed  in  man. 

Sleep  studies;   ACTH  and  carbohydrate-active  steroid  play  significant 
roles  in  the  control  of  delta  sleep  as  well  as  REM  sleep. 

Proposed  Course  of  Project: 

1.  To  define  further  the  specific  role  of  ACTH  and  other  protein 
hormones  on  sensory  and  neural  function. 

2.  To  demonstrate  the  specific  role  of  thyroid  hormone  on  taste  receptor 
and  neural  function. 

Keyword  Descriptors:   Adrenocorticosteroids ,  Thyroid  Hormone,  ACTH,  Sleep, 
Averaged  Brain  Evoked  Responses,  Taste,  Smell. 


Honors  and  Awards 


None 


Publications:   1.   Biichsbaum,  M.S.,  Henkin,  R.I.  and  Christiansen,  R.  :   Age 
sex  differences  in  averaged  evoked  responses  in  a  normal 
population  with  observations  on  patients  with  gonadal  dys- 
genesis.  J.  Electroenceph.  Clin.  Neurophys.  37:  137-144, 
1974. 

2.  Gillin,  J.C.,  Jacobs,  L.S.,  Snyder,  F.  and  Henkin,  R.I.: 
Effects  of  decreased  adrenal  corticosteroids:   Changes  in 
sleep  in  normal  subjects  and  patients  with  adrenal  cortical 
insufficiency.   J.  Electroenceph.  Clin.  Neurophys.  36: 
283-289,  1974. 

3.  Gillin,  J.C.,  Jacobs,  L.S.,  Snyder,  F.  and  Henkin,  R.I.: 
Effect  of  ACTH  in  sleep  of  normal  volunteers  and  of  patients 
with  Addison's  disease.   Neuroendocrin.  15:  21-31,  1974. 

4.  Henkin,  R.I.:   Sensory  changes  during  the  menstrual  cycle. 

In  Biorhythms  and  Human  Reproduction,  Ferin,  M. ,  Halberg,  F., 
Richart,  R.M.  and  VanderWiele,  R.  (Eds.),  John  Wiley  &  Sons, 
N.Y.,  1974,  pp.  277-285. 

5.  Henkin,  R.I.:   A  study  of  circadian  variation  in  taste  and 
smell  in  normal  man  and  in  patients  with  adrenal  cortical  in- 
sufficiency:  the  role  of  adrenal  cortical  steroids.   In 
Biorhythms  and  Human  Reproduction,  Ferin,  M. ,  Halberg,  F. , 
Richart,  R.M.  and  VanderWiele,  R. (Eds . ) ,  John  Wiley  &  Sons , 
N.Y.,  1974,  pp.  397-408. 

6.  Henkin,  R.I.:   Effects  of  ACTH,  adrenocorticosteroids  and 
thyroid  hormone  on  sensory  function.   In  Anatomical  Neuro- 
endocrine logy,  Stumpf,  W.E.  and  Grant,  L.D.  (Eds.),  Karger, 
AG,  Basel,  1975  (In  press). 


£3> 


Project  No.  Z01  HL  01983-12  HE 

7.  McConnell,  R.J.,  Menendez,  C.E.,  Rivlin,  R.S.  and  Henkin,  R.I.: 
Taste  and  smell  in  patients  with  hypothyroidism.   Amer.  J.  Med., 
1975  (In  press). 

8.  Gillin,  J.C.,  Fram,  D.H.,  Wyatt,  R.J.,  Henkin,  R.I.  and  Snyder, F.: 
L-Histine:  Failure  to  affect  the  sleep-waking  cycle  in  man. 
Psychopharmacologia  40:  305-311,  1975. 

9.  Henkin,  R.I.,  Stillman,  I.S.,  Gilbert,  D.L.  and  Lipicky,  R.J.: 
Ineffectiveness  of  Lysergic  acid  diethyl  amide-25  (LSD)  on  altering 
Na-K  currents  in  squid  giant  axon.   Experientia  30:  916-917,  1974. 


6  IS 


ANNUAL  REPORT  OF  THE 
MOLECULAR  DISEASE  BRANCH 
NATIONAL  HEART  AND  LUNG  INSTITUTE 
July  1,  19 74  through  June  30,  1975 

The  research  program  of  the  Molecular  Disease  Branch  is  as  in  the  past 
directed  primarily  toward  the  elucidation  of  the  chemistry  and  metabolism  of 
the  plasma  lipoproteins  with  the  objective  of  defining  and  ultimately  treating 
the  derangements  of  lipid  transport  and  metabolism  that  lead  to  atherosclerosis 
and  numerous  other  diseases  in  man.     Some  of  the  specific  areas  of  progress  in 
the  past  year  are  outlined  below.     In  addition,  we  are  continuing  a  collabora- 
tive investigation  of  the  structural,  biological  and  immunochemical  properties 
of  parathyroid  hormones  and  related  peptides. 

Basic  to  any  understanding  of  the  structure  and  function  of  the  plasma 
lipoproteins,  normal  and  abnormal,  is  a  knowledge  of  the  chemistry  of  the 
smaller  molecules  that  they  contain.     The  protein  components   (apoproteins) 
which,  as  a  group,  serve  to  promote  the  transfer  of  lipid  from  cells  and  to 
stabilize  it  in  the  circulation  obviously  present  the  greatest  challenge  in 
this  regard.     The  primary  structure  of  the  A  apoproteins  from  human  plasma 
have  been  established,  A-II  here  and  A-I  elsewhere.     The  three  C  apoproteins 
were  discovered  here  and  the  primary  structure  of  C-I  and  C-III  determined  in 
past  years.     Work  on  the  structure  of  C-II  has  now  progressed  to  the  point 
where  sequencing  and  alignment  of  peptide  fragments   (generated  enzymatically 
and  chemically)  is  about  75%  completed.     It  has  been  found  that,  unlike  the 
intact  C-II  apoprotein,  none  of  these  peptides  is  capable  of  activating  lipo- 
protein lipase. 

The  quaternary  structure  of  the  intact  lipoproteins  is  determined  by  the 
nature  of  the  interactions  between  their  constituent  apoproteins  and  lipids. 
We  reported  last  year  several  important  observations  on  the  configuration  and 
orientation  of  the  A  apoproteins  in  human  high  density  lipoproteins   (HDL) . 
These     studies,  employing  physical  methods  for  measurement  of  molecular  prop- 
erties    of  proteins  and  lipids   (-^C  and  ^  P  nuclear  magnetic  resonance,  circu- 
lar dichroism,  fluorescence,  difference  absorption  spectroscopy),  have  continued 
in  collaboration  with  members  of  the  Laboratory  of  Chemistry,  NHLE ,  and  the 
Clinical  Endocrinology  Branch,  NIAMDD.     From  investigations  of  the  character- 
istics of  the  individual  A  apoproteins  in  aqueous  solution,  after  recombination 
in  vitro  with  specific  phospholipids  and  in  their  normal  state  in  native  HDL 
we  have  obtained  considerable  new  evidence  in  support  of  the  molecular  struc- 
ture for  HDL  that  was  suggested  last  year.     In  this  model  HDL  is  a  spherical 
micelle  (ca.   120  A  in  diameter)   consisting  of  lipids  with  the  polar  groups  of 
the  phospholipids  at  the  surface  (aqueous  interface)   and  with  the  globular  A 
apoproteins  which  exhibit  a  high  degree  of  ordered  (helical)  structure ,  par- 
tially imbedded  or  "floating"  in  a  "sea"  of  lipid.     It  appears  that  the  major 
forces  in  the  protein- lipid  interactions  are  hydrophobic  and  that  interactions 
between  the  A-I  and  A-II  proteins  could  be  of  special  importance  in  the  organ- 
ization of  the  native  HDL  molecule.     Observations  on  the  self-association  of 
apoprotein  A-II  suggest  that  it  may  be  an  A-II  dimer  that  is  specifically  in- 
volved in  lipid  binding. 


*3r 


We  had  found  in  the  past  that  A-I  in  the  absence  of  A-II  was  unable  to 
bind  the  phospholipids  lecithin  and  sphingomyelin  to  any  significant  extent. 
In  recent  studies,  however,  it  has  been  shown  that  A-I  avidly  binds  lysoleci- 
thin  in  a  fashion  seemingly  related  to  the  primary  structure  of  this  apoprotein. 
The  functional  significance  of  this  association  is  unknown  at  present,  but  it 
is  noteworthy  that  apoprotein  A-I  is  reported  to  be  an  activator  of  the  enzyme 
lecithin: cholesterol  acyl  transferase  which  catalyzes  the  transfer  of  a  fatty- 
acid  from  lecithin  to  cholesterol  in  a  reaction  that  generates  lysolecithin. 

In  continuing  investigation  of  factors  that  influence  synthesis  and 
degradation  of  the  human  plasma  lipoproteins  through  kinetic  analyses  of  data 
obtained  after  administration  of  radioactively  labeled  apolipoproteins ,  we 
have  benefitted  greatly  from  a  close  collaboration  with  the  Laboratory  of 
Theoretical  Biology,  NCI.     Last  year  it  was  shown  that  the  B  and  C  apoproteins 
of  the  very  low  density  lipoproteins   (VLDL)   turn  over  independently  and  trans- 
fer separately  through  two  distinct  compartments  of  the  circulating  lipopro- 
teins.    In  addition,  it  was  found  that  in  patients  with  type  III  hyperlipopro- 
teinemia production  of  apoprotein  B  is  accelerated,  exceeding  the  capacity  of 
the  body  to  metabolize  VLDL  through  the  normal  pathway  and  probably  causing 
the  accumulation  of  abnormal  VLDL  that  is  characteristic  of  this  disorder. 
Current  studies  are  designed  to  obtain  data  that  will  enable  us  to  describe 
HDL  metabolism,  or  at  least  the  metabolism  of  its  A-I  and  A-II  apoproteins , 
in  terms  of  the  multicompartmental  model  of  human  lipoprotein  metabolism  that 
has  been  developed  from  the  earlier  work.      Computation  of  rates  of  synthesis 
and  fractional  catabolic  rates  for  the  HDL  apoproteins  has  indicated  that 
apoproteins  A-I  and  A-II  turn  over  at  the  same  rate.     These  studies  are  con- 
sistent with  the  view  that  the  HDL  macromolecule  is  metabolized  as  a  whole 
without  preferential  removal  of  individual  apoproteins.     In  addition,  it  has 
been  shown  that  the  fall  in  plasma  HDL  content  that  occurs  in  normal  humans 
following  ingestion  of  a  diet  high  in  carbohydrate  is  the  result  of  increased 
catabolism  while  the  rate  of  synthesis  remains  unchanged.     The  metabolism  of 
HDL  is  of  particular  interest  because  A-I ,  a  putative  activator  of  lecithin : 
cholesterol  acyl  transferase,  is  the  predominant  protein  component  and  it  has 
been  suggested  that  high  density  lipoproteins  may  play  a  critical  role  in  the 
removal  of  cholesterol  from  tissues.     The  HDL  further  serves  as  a  reservoir 
for  the  C  apoproteins   (one  of  which  (C  II)  is  the  activator  of  lipoprotein 
lipase)  between  tides  of  triglyceridemia  during  which  they  move  to  the  VLDL. 

The  patients  with  Tangier  disease,  a  rare  familial  deficiency  of  HDL 
that  was  discovered  at  the  NHLI  in  1960,  provide  us  with  a  unique  opportunity 
to  learn  from  an  experiment  of  nature  more  about  the  functions  of  HDL.     Last 
year,  study  of  the  small  amounts  of  HDL  obtainable  from  Tangier  homozygotes 
had  led  to  the     conclusion  that  it  lacked  the  apoprotein  A-I.     More  recently, 
however,  it  was  found  using  immunochemical  methods  that  there  is  no  selective 
reduction  in  the  total  amount  of  A-I   (relative  to  A-II)  in  the  plasma  of  these 
patients.     Although  we  cannot  rule  out  the  possibility  that  the  Tangier  A-I 
is  abnormal,  the  data  available  at  present  yield  no  evidence  of  differences 
between  the  composition  of  Tangier  A-I   (or  A-II)  and  that  of  normals.     Thus, 
all  of  the  findings  to  date  are  consistent  with  the  hypothesis  that  Tangier 
disease  is  the  result  of  a  regulatory  rather  than  a  structural  gene  defect. 


£U 


During  the  past  year  the  abnormalities  of  the  low  density  lipoprotein 
(LDL)   and  VLDL  previously  noted  in  the  Tangier  patients  have  been  investigated 
in  detail.     All  of  the  four  homozygotes  studied  had  LDL  of  grossly  abnormal 
lipid  composition,  undoubtedly  secondary  to  the  HDL  deficiency.     It  appears 
that,  in  the  absence  of  HDL,  LDL  does  not  function  normally  as  a  carrier  of 
cholesterol.     Its  cholesterol  content  is  low  and  triglyceride  content  markedly 
elevated.     Preliminary  experiments  suggest  that  such  triglyceride-rich  LDL 
can  serve  as  a  substrate  for  lipoprotein  lipase  and  this  may  account  for  the 
low  levels  of  LDL  in  Tangier  plasma.     The  VLDL  of  Tangier  homozygotes , 
although  morphologically  and  chemically  normal,  may  exhibit  an  altered  electro- 
phoretic  mobility.     This  was  found  to  be  attributable  to  a  relative  deficiency 
of  the  C  apoproteins.     When  VLDL  synthesis  was  increased  by  carbohydrate  feed- 
ing, the  C  protein  content  of  the  VLDL  was  remarkably  low  in  all  four  patients. 
On  a  normal  dietary  regimen,  however,  the  C  protein,  although  low  in  the  two 
male  patients ,  was  normal  in  the  two  women.     Perhaps  this  is  related  to  the 
fact  that  HDL  concentrations  are  normally  higher  in  women  than  in  men  (this 
is  one  of  the  few  known  sex  differences     in  plasma  lipoproteins ) .     In  any 
case,  all  of  the  observations  in  the  Tangier  patients  are  consistent  with  the 
hypothesis  that  the  HDL  functions  as  a  reservoir  or  sink  for  the  C  apoproteins 
that  move  between  it  and  VLDL.     Whether  the  accumulation  of  lipids  in  nerve 
and  other  tissues  in  Tangier  disease  is  due  to  impaired  removal  of  cholesterol 
secondary  to  the  deficiency  of  HDL  or  to  endocytosis  of  the  abnormal  circulat- 
ing lipoproteins  that  are  found  in  this  disorder  remains  to  be  determined. 

Further  information  on  the  movement  of  the  C  apoproteins  between  HDL  and 
VLDL  has  been  obtained  by  quantifying  the  changes  in  plasma  lipoprotein 
distribution  and  composition  following  heparin-induced  lipolysis  in  patients 
with  hypertriglyceridemia.     It  appears  that  the  C  proteins  preferentially 
associate  with  triglyceride-rich  VLDL  and  accumulate  in  HDL  only  when  the 
amount  of  VLDL  and  its  associated  triglyceride  is  markedly  reduced.     The  data 
from  these  studies  further  suggest  that  the  capacity  of  the  HDL  "reservoir" 
for  C  proteins  may  be  relatively  limited. 

The  partition  of  C  proteins  among  the  lipoproteins  of  different  density 
classes  is  apparently  distorted  in  several  types  of  diseases.     Because  of  the 
unique  distribution  of  C  apoproteins  in  patients  with  biliary  cirrhosis  the 
characteristic  abnormal  lipoprotein  (LP-X)   found  in  these  patients  is  of 
particular  interest.     Until  recently  no  combination  of  conventional  separation 
methods  had  yielded  preparations  free  of  contamination  with  B  apoprotein. 
Using  zonal  block  electrophoresis,  however,  we  have  recently  obtained  prepara- 
tions of  LP-X  that  should  be  suitable  for  definitive  analysis  of  its  apoprotein 
content. 

Certain  important  kinds  of  studies  of  lipoprotein  metabolism  can  be 
carried  out  only  with  experimental  animals  or  with  isolated  organ  systems. 
The  rat  is  a  convenient  subject  for  such  work  and  characterization  of  the  rat 
plasma  lipoproteins  was  therefore  undertaken  several  years  ago.     Last  year 
it  was  reported  that  homologues  for  all  of  the  major  human  apolipoproteins 
had  been  identified,  purified  and  partial ly  characterized.     Significant  quan- 
titative differences  between  the  rat  and  the  human  in  terms  of  the  distribution 
of  individual  apoproteins  in  lipoproteins  of  different  densities  have  now  been 


*37 


defined.     Studies  of  the  apoproteins  in  the  plasma  and  in  the  liver  liposomes 
of  rats  fed  orotic  acid  have  enabled  us  to  learn  in  which  lipoprotein  form 
the  several  C  apoproteins  are  released  from  the  liver  and  have  led  to  the 
conclusion  that  the  absence  of  one  sialylated  form  of  apoC-II-I  in  human 
abetalipoproteinemia  reflects  the  failure  of  the  liver  to  elaborate  VLDL 
rather  than  a  defect  in  sialylation. 

It  has  been  shown  in  this  Institute  and  elsewhere  that  patients  with 
type  II  hyperlipoproteinemia  (familial  hypercholesterolemia)  have  a  defect  in 
the  feed-back  regulation  of  cholesterol  synthesis.     This  defect  which  can  be 
demonstrated  in  skin  fibroblasts  cultured  from  affected  individuals  consists 
in  a  failure  of  the  cells  to  respond  to  the  presence  of  LDL  with  a  decrease 
in  the  activity  of  hydroxyl-methylglutaryl  coenzyme  A  (HMG  CoA)  reductase , 
the  enzyme  that  catalyzes  the  rate- limiting  step  in  cholesterol  synthesis-. 
Thus  far,  cultured  fibroblasts  from  five  patients  homozygous   for  type  II 
hyperlipoproteinemia  have  been  studied;  seven  others  have  been  initiated. 
In  order  to  define  further  the  mechanisms  through  which  HMG  GoA  reductase 
activity  is  regulated  -  in  normal  and  in  type  II  cells  -  it  will  be  necessary 
to  measure  the  amounts  of  enzyme  protein  as  well  as  enzyme  activity  in  future 
studies.       A  highly  purified  HMG  CoA  reductase  has  been  prepared  by  affinity 
chromatography  and  it  is  hoped  that  an  antibody  developed  against  this  material 
will  be  usable  for  the  immunoassay  of  the  reductase  protein.     The  purified 
reductase  will  also  be  used  for  investigation  of  the  mechanisms  through  which 
its  activity  is  apparently  regulated  rapidly  and  reversibly,  perhaps  by 
phosphorylation  and  (^phosphorylation. 

The  type  II  Coronary  Intervention  Study,  designed  to  test  the  hypothesis 
that  reducing  the  concentration  of  plasma  LDL  can  favorably  affect  the  pro- 
gression of  established  coronary  artery  disease,  has  been  in  progress   for 
three  years.     In  this  study  250  suitable  patients  with  type  II  hyperlipo- 
proteinemia and  coronary  artery  disease  demonstrated  by  angiography,  will  be 
treated  with  dietary  control  alone  or  with  diet  plus  cholestyramine  and 
followed  in  a  double-blind  fashion  for  2  to  5  years.     Of  the  total  of  about 
15,000  patients  that  have  been  referred  for  consideration  for  this  study, 
47%  have  been  referred  in  the  past  year.     There  are  at  present  98  patients 
in  the  study,   54  who  have  completed  their  one  year  evaluations  and  35  who 
have  completed  their  two  year  evaluations.     It  is  expected  that  a  number  of 
changes  that  have  been  made  in  the  programs  for  referral  and  screening  will 
expedite  enrollment  of  patients  in  the  study  and  make  it  possible  to  begin 
during  the  coming  year  to  analyze  some  of  the  extensive  data  that  have  al- 
ready been  collected. 


&S 


Project  No.  Z01  HL  02001-15  MDB 

1.  Molecular  Disease  Branch 

2.  Section  on  Lipoprotein 
Structure 

3.  Bethesda,  Maryland  20014 
PHS-NIH 

Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  The  Lipoproteins  of  Tangier  Disease 

Previous  Serial  Number:       NHLI-288(c) 

Principal  Investigators:      Peter  N.  Herbert,  M.D. 

Robert  J.  Heinen,  B.S. 
Eve  C.  Church,  M.S. 

Other  Investigators:  None 

Cooperating  Units:  Trudy  Forte,  Ph.D.,  Donner  Labs.,  Berkeley, 

California 

Victor  J.  Ferraris,  M.D. ,  NHLI 
Donald  S.  Fredrickson,  M.D. ,  Institute  of 
Medicine,  National  Academy  of  Sciences, 
Washington,  D.C. 

Project  Description: 

Objectives: 

Tangier  disease,  a  familial  deficiency  of  high  density  lipoprotein  (HDL), 
should  provide  unique  insight  into  the  obscure  physiological  role  of  plasma 
high  density  lipoproteins.  This  disorder  represents  a  natural  biochemical 
ablation  experiment.  Affected  subjects  demonstrate  impressive  storage  of 
cholesteryl  esters  in  reticuloendothelial  cells  and  transient  or  severe 
peripheral  neuropathy.  This  project  was  designed  to  elucidate  the  relation- 
ships between  the  plasma  HDL  deficiency,  the  abnormalities  of  plasma  lipo- 
proteins and  the  observed  pathology. 

Methods : 

Large  quantities  of  plasma  were  obtained  by  plasmapheresis  from  patients 
homozygous  for  Tangier  disease.  The  methods  of  lipoprotein  and  apolipoprotein 
isolation  and  chemical  analyses  have  been  described  in  previous  annual 
reports . 

Major  Findings: 

1)  The  beta  (rather  than  the  normal  alpha- 2)  electrophoretic  mobility 
of  Tangier  very  low  density  lipoproteins  (VLDL)  was  shown  to  be  attributable 
to  a  relative,  though  not  absolute,  C-apoprotein  deficiency,  a  defect 


63? 


Project  No.  Z01  HL  02001-15  MDB 

paralleling  that  found  in  type  III  hyperlipoproteinemia.  A  peculiar, 
apparently  sex  and  diet  related,  variation  in  C-protein  composition  was 
observed.  When  VLDL  synthesis  was  induced  by  carbohydrate  feeding  al]  four 
Tangier  homozygotes  studied  had  remarkably  low  VLDL  C-protein  content  - 
10-30%  (normal  40-50%).  The  two  female  patients,  in  contrast  to  the  males, 
had  a  normal  C-protein  content  on  normal  or  high  fat  diet.  These  findings 
confirm  the  supposition  that  HDL  serves  as  an  important  reservoir  for  the  C- 
apoproteins  but  suggests  that  significant  sex  differences  in  C-protein 
metabolism  may  exist. 

2)  All  Tangier  homozygotes  studied  had  low  density  lipoproteins  (LDL) 
of  grossly  abnormal  chemical  composition.  Triglyceride  was  increased  from 

the  normal  6-10%  to  30-35%  and  cholesterol  decreased  to  15-20%  (normal  38-45%). 
This  abnormality  of  LDL  composition  undoubtedly  is  related  to  the  HDL 
deficiency  and  suggests  that  cholesteryl  esters  of  HDL  origin  normally  ex- 
change for  triglyceride  in  the  metabolic  conversion  of  VLDL  to  LDL.  In  the 
absence  of  HDL,  LDL  does  not  serve  its  normal  role  as  the  repository  of  plasma 
cholesterol.  The  very  high  triglyceride  content  of  Tangier  plasma  LDL,  in 
addition,  may  account  for  the  low  levels  of  LDL  characteristic  of  this  dis- 
order since  preliminary  experiments  suggest  that  such  triglyceride  rich  LDL 
can  serve  as  a  substrate  for  lipoprotein  lipase. 

3)  The  lipid  accumulation  in  Tangier  tissues  has  been  evaluated  by 
light  and  electron  microscopic  studies  of  bone  marrow,  skin  and  gastro- 
intestinal tissues  from  affected  subjects.  Most  striking  was  the  accumulation 
of  neutral  lipid  in  Schwann  cells  and  even  nerve  axons,  a  finding  undoubtedly 
related  to  the  peripheral  neuropathy  in  these  patients.  It  is  not  known 
whether  this  lipid  arises  from  endocytosis  of  abnormal  circulating  lipo- 
proteins or  reflects  a  •  failure  of  HDL  to  remove  lipid  from  nerves  by  net 
transfer.  The  former  mechanism  gains  support  from  the  demonstration  of 
bizarre  lipoprotein  forms  in  Tangier  HDL  which  appear  to  be  generated  during 
chylomicron  catabolism  and  which  disappear  when  the  diet  contains  no  fat. 

4)  Immunochemical  studies  of  Tangier  plasma  failed  to  support  earlier 
claims  that  the  A-I  apoprotein  content  was  selectively  reduced.  Preliminary 
studies  have  failed  to  demonstrate  compositional  differences  between  Tangier 
and  normal  A-I  and  A-II.  If  this  finding  is  confirmed  when  Tangier  A-I  and 
A-I  are  isolated  in  quantity,  the  data  will  point  toward  a  regulatory  rather 
than  structural  genetic  defect  in  this  disorder. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

HDL  concentrations  are  higher  in  women  than  in  men,  one  of  the  few 
lipoprotein  differences  between  the  two  sexes.  HDL  concentrations  are  also 
very  high  in  species  resistant  to  atherosclerosis.  The  functions  of  HDL  are 
only  partially  known.  Study  of  Tangier  disease  offers  unique  opportunities 
for  determining  the  structure  and  function  of  all  lipoproteins. 

Proposed  Course: 

1)  The  lipid  and  apoprotein  composition  of  the  abnormal  lipoproteins 


Project  No.  Z01  HL  02001-15  MDB 
generated  during  chylomicron  catabolism  in  Tangier  disease  will  be  defined. 

2)  The  plasma  lipoproteins  of  Tangier  heterozygotes  will  be  examined  on 
normal  diets  and  after  carbohydrate  feeding  using  the  techniques  applied  in  the 
studies  of  the  Tangier  homozygotes. 

3)  Tangier  apoA-I  and  apoA-I  will  be  isolated  and  characterized 
chemically  and  immunologically  to  resolve  the  issue  of  a  structural  defect  in 
this  disorder. 

Publications : 

1.  Ferrans,  V.  J.,  and  Fredrickson,  D.  S. :  Pathology  of  Tangier  Disease. 
Amer.  J.  Path.  78:  101-136,  1975. 


Ht 


Project  No.  Z01  HL  02001-15  MDB 
Key  Words: 

1)  lipoproteins 

2 )  apolipoproteins 

3)  HDL  -  high  density  lipoproteins 

4)  VLDL  -  very  low  density  lipoproteins 

5)  LDL  -  low  density  lipoproteins 

6 )  atherosclerosis 

7)  lipoprotein  deficiencies 

8 )  dyslipoproteinemias 

9 )  hyperlipoproteinemia 
10)  Tangier  disease 


£&L 


Project  No.  Z01  HL  02007-08  MDB 

1.  Molecular  Disease  Branch 

2.  Section  on  Lipoprotein 
Structure  ■ 

3.  Bethesda,  Maryland  20014 
PHS-NIH 

Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Characterization  of  the  Human  Plasma 

Apolipoproteins 

Previous  Serial  Number:       NHLI-296  and  NHLI-298(c) 

Principal  Investigators:      Peter  N.  Herbert,  M.D. 

Linda  L.  Bausserman,  B.S. 
J.  Roger  Lee,  B.A. 
Marguerite  J.  LaPiana,  B.A. 
Robert  J.  Heinen,  B.S. 
Richard  S.  Shulman,  M.D. 

Other  Investigators:  None 

Cooperating  Units:  Trudy  Forte,  Ph.D.,  Prank  Lindgren,  Ph.D.,  and 

Alexander  Nichols,  Ph.D.,  Dormer  Laboratories, 
Berkeley,  California 

Project  Description: 

Ob j  ectives : 

Studies  in  recent  years  have  revealed  that  abnormalities  of  plasma  lipid 
concentrations  are  frequently  associated  with  altered  distributions  of  the 
individual  apolipoproteins  among  different  density  classes  of  lipoproteins, 
It  is  not  known  whether  these  are  cause  or  effect  relationships.  This  pro- 
ject is  part  of  a  continuing  effort  to  characterize  the  major  and  minor  apo- 
lipoproteins and  to  relate  their  distribution  to  various  types  of  well- 
defined  clinical  disorders. 

Methods : 

Plasma  was  obtained  from  patients  with  types  I,  II,  III  and  V  hyper- 
lipoproteinemia, biliary  cirrhosis  and  familial  homozygous  hypobetalipoprotein- 
emia.  The  techniques  of  lipoprotein  isolation  by  ultracentrifugation  and 
agarose  column  chromatography,  and  apolipoprotein  purification  by  rapid-flow- 
gel  and  ion-chromatography  have  been  described  in  previous  annual  reports. 
Preparative  polyacrylamide  gel  electrophoresis  has  been  added  to  the  immuno- 
chemical and  electrophoretic  techniques  employed  in  earlier  studies. 

Major  Findings: 

1)  Investigation  of  the  apoprotein  content  of  the  6-VLDL  of  type  III 

1  6{l 


Project  No.  Z01  HL  02007-08  MDB 

subjects  has  continued.  It  was  reported  last  year  that  the  C-protein 
contribution  to  type  III  VLDL  was  reduced  by  approximately  20%.  The  apo- 
proteins from  the  VLDL  of  several  additional  patients  have  been  fractionated 
and  this  conclusion  seems  partially  inaccurate.  Three  patients  with  type  III 
had  VLDL  C-protein  contents  within  the  normal  range  and,  of  greater  interest, 
the  VLDL  had  normal  electrophoretic  mobility.  This  finding  underscores  the 
unreliability  of  electrophoresis  in  lipoprotein  phenotyping  and  supports  our 
contention  that  the  cholesterol  content  of  type  III  VLDL  is  a  more  specific 
marker  in  this  disorder.  In  addition,  the  published  report  that  type  III 
VLDL  is  disproportionately  rich  in  the  "high-arginine"  apolipoprotein  was  not 
confirmed . 

2)  The  lipoproteins  and  apolipoproteins  from  two  subjects  homozygous  for 
the  gene  of  hypobetalipoproteinemia  were  isolated  and  compared  with  normals 
and  with  a  subject  having  the  more  common  autosomal  recessive  abetalipopro- 
teinemia.  The  VLDL  and  LDL  fraction  from  the  one  homozygous  hypobetalipopro- 
teinemic  (H-H)  subject  extensively  evaluated  contained  no  immunochemical 
traces  of  beta  lipoprotein,  but  did  contain  apoA-I,  apoA-II  and  the  C-apopro- 
teins.  Barrel-shaped  structures  identical  to  those  seen  in  the  LDL  fraction 
of  the  common  variety  of  abetalipoproteinemia  were  observed  on  electron 
microscopy.  Polyacrylamide  gel  electrophoresis  of  apoHDL  from  both  H-H 
subjects  demonstrated  reduction  but  not  complete  absence  of  the  C-III-1  apo- 
protein, a  finding  previously  reported  in  abetalipoproteinemia. 

3)  Studies  of  the  effects  of  in  vivo  heparin- induced  triglyceride 
hydrolysis  on  the  distribution  of  the  C-apoproteins  were  completed.  One 
subject  was  studied  twice,  at  plasma  triglyceride  concentrations  of  2700  mg% 
and  2400  mg%,  and  triglycerides  fell  45%  and  25%  respectively  in  response  to 
heparin  injection.  The  C-protein  content  of  the  VLDL,  however,  was  unchanged 
after  lipolysis  in  both  experiments  and  in  neither  experiment  did  the  VLDL 
hydrolysis  lead  to  significant  increases  in  the  S^  0-20  lipoprotein  class. 
Two  additional  subjects  were  studied  at  triglyceride  levels  of  364  mg%  and 
426  mg%.  Their  plasma  triglycerides  fell  by  64%  and  53%  with  heparin- induced 
lipolysis.  In  contrast  to  the  patient  with  higher  triglyceride  levels, 
accumulation  of  lipoprotein  in  the  S^  0-20  class  was  observed  and  decreases 
of  47  and  64%  in  the  total  VLDL  protein  occurred.  One  patient  showed  no 
change  in  the  relative  C-protein  content  in  VLDL  while  the  second  had  a  50% 
drop.  There  was  an  8%  increase  in  the  HDL  C-protein  in  the  latter  subject. 
These  results  suggest  that  over  a  wide  range  of  plasma  triglyceride  concen- 
trations the  C-proteins  selectively  associate  with  the  triglyceride-rich  VLDL 
and  redistribute  to  HDL  only  when  VLDL  (and  triglyceride)  concentrations  are 
drastically  reduced.  They  also  suggest  that  HDL  may  have  a  limited  capacity 
to  accommodate  C-proteins  after  VLDL  lipolysis. 

4)  Work  has  progressed  on  the  primary  structure  of  apoC-II.  In  contrast 
to  the  two  C-proteins  which  have  been  sequenced,  apoC-II  presents  special 
problems  in  obtaining  adequate  quantities  for  this  analysis.  Nevertheless, 
enzymatic  and  chemical  peptides  have  been  generated  from  this  apoprotein  and 
the  sequential  degradation  and  alignment  of  these  peptides  is  about  75% 
complete.  In  contrast  to  the  intact  apoprotein,  none  of  these  peptides  has 


&*- 


Project  No.  Z01  HL  02007-08  MDB 

been  found  capable  of  lipoprotein  lipase  activation. 

5)  The  LP-X  lipoprotein  of  primary  biliary  cirrhosis  is  being  in- 
vestigated because  of  the  unique  redistribution  of  C-apoproteins  which 
characterizes  this  disorder.  No  combination  of  preparative  ultracentrifugation 
and  agarose  gel  chromatography  satisfactorily  resolved  the  LP-X  lipoprotein 
from  contamination  with  LDL  containing  B-apoprotein.  A  system  of  zonal  block 
electrophoresis  has  been  developed  which  appears  capable  of  this  separation. 
Preliminary  results  suggest  that  all  previous  estimates  of  the  quantitative 
and  qualitative  protein  content  of  the  LP-X  lipoprotein  were  in  error. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

The  apolipoprotein  serves  a  critical  role  in  the  stabilization  of  plasma 
lipids,  provides  co-factor  activities  for  lipolysis  and  may  well  modulate  the 
fine  controls  that  govern  plasma  lipoprotein  concentrations.  The  characteri- 
zation should  contribute  greatly  to  our  understanding  of  physiological  and 
pathological  lipoprotein  metabolism. 

Proposed  Course: 

1)  The  characterization  of  the  lipid  and  apolipoprotein  distributions 
in  type  III  hyperlipoproteinemia  and  primary  biliary  cirrhosis  will  be 
completed. 

2)  Current  critical  analyses  of  the  available  preparative  techniques 
for  lipoprotein  and  apolipoprotein  purification  will  be  continued. 

3)  The  primary  structure  determination  of  apoC-II  will  be  finished. 

4)  Isolation  and  characterization  of  the  various  "arginine-rich" 
apolipoproteins  will  continue  with  an  effort  to  relate  their  occurrence  and 
distribution  to  abnormalities  of  plasma  lipid  transport. 

Publications : 

1.  Brewer,  H.  B. ,  Jr.,  Shulman,  R.  S. ,  Herbert,  P.  N. ,  Ronan,  R. ,  and 
Wehrly,  K. :  The  complete  amino  acid  sequence  of  alanine  apolipoprotein 
(apoC-III) ,  an  apolipoprotein  from  human  plasma  very  low  density 
lipoproteins.  J.  Biol.  Chem.  249:  4975-4984,  1974.   (Cited  as  "in  press" 
in  1974  project  report  NHLT-296). 

2.  Shulman,  R.  S.,  Herbert,  P.  N. ,  Wehrly,  K. ,  and  Fredrickson,  D.  S. :  The 
complete  amino  acid  sequence  of  C-I  (apoLp-Ser),  an  apolipoprotein  from 
human  very  low  density  lipoproteins.  J.  Biol.  Chem.  250:  182-190,  1975. 
(Cited  as  "in  press"  in  1974  project  report  NHLI-296). 

3.  Herbert,  P.  N. ,  Forte,  T.  M. ,  Shulman,  R.  S. ,  LaPiana,  M.  J.,  Gong,  E.  L. , 
Levy,  R.  I.,  Fredrickson,  D.  S. ,  and  Nichols,  A.  V.:  Structural  and 


6<*r 


Project  No.  Z01  HL  02007-08  MDB 

compositional  changes  attending  the  ultracentrifugation  of  very  low 
density  lipoproteins .  Prep.  Biochem. ,  in  press . 


£<& 


Project  No.  Z01  HL  02007-08  MDB 
Key  Words: 

1)  lipoproteins 

2)  apolipoproteins 

3)  HDL  -  high  density  lipoproteins 

4)  VLDL  -  very  low  density  lipoproteins 

5)  LDL  -  low  density  lipoproteins 

6 )  atherosclerosis 

7)  lipoprotein  deficiencies 

8 )  dyslipoproteinemias 

9 )  hyperlipoproteinemia 
10 )  abetalipoproteinemia 


6*7 


Project  No.  Z01  HL  02008-06  MDB 

1.  Molecular  Disease  Branch 

2.  Section  on  Lipoprotein 
Structure 

3.  Bethesda,  Maryland  20014 
PHS-NIH 

Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Rat  Plasma  Lipoproteins  and  Apo lipoproteins 

Previous  Serial  Number:       NHLI-297 

Principal  Investigators:      Peter  N.  Herbert,  M.D. 

Lloyd  0.  Henderson,  Ph.D. 

Other  Investigators:         Marguerite  J.  LaPiana,  B.A. 

J.  Roger  Lee,  B.A. 
John  Stonik,  B.S. 

Cooperating  Units:  •  Herbert  G.  Windmueller,  Ph.D.,  Laboratory  of 

Nutrition  £  Endocrinology,  NIAMDD 

Project  Description: 

Ob j  ectives : 

The  study  of  lipoprotein  metabolism  and  interrelations  is  most  con- 
veniently undertaken  in  non-human  species  were  isolated  organ  systems  are 
available  and  extensive  perturbation  is  possible.  The  rat  has  been  widely 
employed  for  such  studies,  but  rat  lipoproteins  and  apolipoproteins  remain 
incompletely  characterized.  Such  characterization  of  the  rat  apolipoproteins 
has  been  the  subject  of  study  in  this  laboratory  for  the  last  six  years. 
Employing  orotic  acid  fed  rats,  studies  have  commenced  regarding  the  lipo- 
protein origins  of  the  apolipoproteins. 

Methods : 

The  techniques  of  lipoprotein  isolation  and  apolipoprotein  fractionation 
and  characterization  have  been  outlined  in  previous  annual  reports.  In 
addition,  hepatic  liposomes  from  orotic  acid  fed  rats  have  been  isolated  as 
described  by  others  (J.  Lipid  Res.  12:  450-459,  1970). 

Major  Findings: 

1)  As  detailed  in  the  1974  project  report,  homologues  of  all  of  the 
human  apolipoproteins  have  been  identified  in  the  rat  and  purified  to  homo- 
geneity. They  have  been  characterized  by  amino  acid  analysis,  COOH  and  NH2 
terminal  residues,  carbohydration  and  molecular  weight  by  gel  chromatography 

2)  Significant  quantitative  differences  between  the  distribution  of 
apolipoproteins  in  the  human  and  rat  were  identified. 

1  6t6 


Project  No.  Z01  HL  02008-06  MDB 

a)  The  C-protein  group  was  shown  to  constitute  20%  of  the  mass  of 
rat  high  density  apolipoprotein  contrasted  to  10%  in  the  human. 

b)  The  C-II  apolipoprotein,  the  activator  of  lipoprotein  lipase  in 
the  rat  and  human,  was  found  in  higher  concentration  in  rat  HDL  than  in  VLDL. 
This  relative  distribution  is  reversed  in  the  human. 

c)  The  arginine-rich  apolipoprotein  was  found  to  comprise  10-15%  of 
the  total  rat  apoHDL,  whereas  its  human  counterpart  is  found  in  only  trace 
amounts  in  this  density  class. 

d)  The  A-II  apoprotein  which  constitutes  20-30%  of  human  apoHDL  was 
a  relatively  minor  component  (<  5%)  of  rat  apoHDL. 

It  remains  to  be  demonstrated  whether  these  differences  in  apolipoprotein 
distribution  can  be  related  to  the  well-known  differences  in  plasma  lipo- 
protein metabolism  in  the  rat  and  human. 

3)  Rats  fed  a  diet  containing  1%  orotic  acid  develop  hepatic  steatosis 
and  a  plasma  lipoprotein  profile  resembling  that  in  patients  with  abetalipo- 
proteinemia.  The  plasma  and  hepatic  lipoproteins  and  apolipoproteins  of 
these  animals  were  extensively  investigated.  The  plasma  from  75%  of  the 
treated  rats  contained  no  immunochemical  traces  of  the  betalipoprotein  after 
7-14  days. 

Studies  of  the  plasma  apolipoproteins  and  those  associated  with  liver 
liposomes  suggested  that: 

a)  ApoC-I  and.  apoC-II  are  secreted  by  the  liver  in  association  with 
both  HDL  and  VLDL. 

b)  C-III-0  is  secreted  with  VLDL  and  C-III-3  only  with  HDL. 

c)  The  absence  of  one  sialylated  form  of  apoC-III  (C-III-1)  in  human 
abetalipoproteinemia  reflects  failure  of  the  liver  to  elaborate  VLDL,  rather 
than  a  defect  in  sialylation. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

The  completion  of  the  studies  described  should  greatly  facilitate 
extrapolation  of  results  of  physiological  experiments  in  the  rat  to  problems 
of  lipoprotein  physiology  and  pathology  in  the  human.  The  orotic  acid  fed 
rat,  specifically,  has  already  contributed  to  our  knowledge  of  the  lipo- 
protein origins  of  the  apolipoproteins. 

Proposed  Course: 

1)  Characterization  of  rat  apoLDL,  arginine-rich  protein  and  the  apo- 
proteins of  all  density  classes  in  intestinal  lymph  will  be  completed. 


6& 


Project  No.  Z01  HL  02008-06  MDB 

2)  The  mechanism  of  the  rat  C-II  apoprotein  activation  of  lipoprotein 
lipase  will  be  further  defined. 

3)  The  work  on  the  orotic  acid  fed  rat  will  be  extended  in  studies  of 
the  apolipoprotein  elaboration  by  the  isolated  perfused  liver. 

Publications : 

1.  Herbert,  P.  N. ,  Windmueller,  H.  G. ,  Bersot,  T.  P.,  and  Shulnan,  R.  S. : 

Characterization  of  the  rat  apolipoproteins .  I.  The  low  molecular  weight 
proteins  of  rat  plasma  high  density  lipoproteins.  J.  Biol.  Chem.  249: 
5718-5724,  1974.   (Cited  as   "in  press"  in  1974  project  report  NHLT-297). 


£&> 


Project  No.    Z01  HL  02008-06  MDB 
Key  Words: 

1)  rat 

2 )  lipoproteins 

3 )  apolipoproteins 

4)  HDL  -  high  density  lipoproteins 

5)  VLDL  -  very  low  density  lipoproteins 

6)  LDL  -  low  density  lipoproteins 

7)  orotic  acid 

8 )  abetalipoproteinemia 

9)  organ  perfusion 
10)  hepatic  steatosis 


6<r/ 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  No.  Z01  HL  02002-03  MDB 

1.  Molecular  Disease  Branch 

2.  Section  on  Lipoproteins 

3.  Bethesda,  Maryland  20014 


Project  Title: 
Previous  Serial  Number: 
Principal  Investigators: 


Other  Investigators: 


NHLI  Type  II  Coronary  Intervention  Study 

NHLI-289(c) 

Robert  I.  Levy,  M.D. 
Stephen  E.  Epstein,  M.D. 
John  F.  Brensike,  M.D. 
Eugene  R.  Passamani,  M.D. 
John  M.  Richardson,  M.D. 
Irving  K.  Loh,  M.D. 

Patricia  Strobell 
Marjorie  Myrianthopoulos 
Beverly  Rogers 
Margaret  Kremer 
James  Curtsinger 
John  Sloane 
Ann  Horn 
Shirley  Shanks 
Kunjlata  Shah,  M.D. 

Cardiology  Branch,  NHLI 

Lipid  Metabolism  Branch,  NHLI 

Data  Management  Branch,  Division  of  Computer, 

NTH 
Research  and  Technology,  NTH 
Biometrics  Research  Branch,  NTH 
Clinical  Center  Administration,  NIH 

Project  Description: 

Objectives: 

The  primary  aim  of  this  study  is  to  determine  whether  loweri:ig  the  LDL 
cholesterol  in  patients  with  premature  coronary  artery  disease  and  type  II 
will  slow,  stop  or  reverse  the  progression  of  coronary  artery  disease  (i.e., 
to  test  the  lipid  hypothesis  in  a  specific  group  of  high  risk  patients). 

Methods : 

Two  hundred-fifty  patients  with  coronary  artery  disease  demonstrated  by 
coronary  angiography  who  meet  all  the  criteria  outlined  in  the  protocol  will 
be  randomly  split  into  two  equal  groups  (treatment  -  24  grams  cholestyramine 
and  diet  control  -  control  -  24  grams  placebo  and  diet  control).  They  will 


Cooperating  Units: 


6CA 


Project  No.  Z01  HL  02002-03  MDB 

be  followed  in  a  double  blind  fashion  monthly  for  2-5  years.  Repeat  coronary 
angiography  will  be  performed  at  2  years  and  at  5  years  (if  the  study 
continues ) . 

The  end  point  will  be:  Da  significant  difference  in  the  progression 
of  coronary  disease  as  shown  on  angiography,  or  2)  a  significant  difference 
in  new  myocardial  infarctions  or  death,  or  3)  lack  of  any  of  the  above  at  5 
years. 

Major  Findings: 

1)  To  date  (April  1,  1975)  the  NHLi  Type  II  Coronary  Intervention  Study 
has  had  approximately  15,000  patients  referred  for  consideration  for  the 
study.  Of  these,  about  7,000  or  47%  have  been  referred  within  the  last  year. 
At  current  rates  of  intake,  it  is  estimated  that  10,000  to  15,000  new  patients 
will  be  screened  within  the  next  year. 

Of  the  15,000  patients  referred,  approximately  8,250  were  not  eligible 
for  the  program  after  the  lab  screening  (they  were  not  cholesterol  eligible 
or  had  elevated  triglycerides).  Of  the  remaining  6,750,  approximately  600 
are  pending  further  processing;  of  the  6,150  processed  beyond  this  point, 
about  3,150  were  found  to  be  ineligible  over  the  telephone.  Of  the  remaining 
3,000,  750  have  not  been  interested  in  being  considered  for  the  program,  and 
another  30%  or  900  have  been  found  to  not  meet  the  IDL  requirements  (not  type 
II)  and  were  therefore  ineligible  for  the  program. 

The  remainder  (approximately  1,350)  have  either  been  seen  (1,200)  or  are 
scheduled  to  be  seen  and  evaluated  in  detail,  including  measurement  of 
approximately  20  risk  factors,  determination  of  presence  or  absence  of  evidence 
for  underlying  coronary  artery  disease  by  history,  physical  and  non- invasive 
testing,  determination  of  dietary  effect  on  their  lipid  abnormality  and 
classification  as  type  II. 

Currently  there  are  98  patients  in  the  program  and  on  medication,  54 
patients  who  have  completed  their  year  evaluation  and  35  patients  who  have 
completed  their  two  year  evaluation. 

Results  to  date  include: 

1)  Less  progression  of  coronary  artery  disease  in  study  patients  than 
expected  and  probable  regression  (an  unexpected  finding)  in  at  least  two 
patients . 

2)  Changes  in  angiography  (better  or  worse)  were  seen  in  approximately 
40%  of  the  patients  who  have  returned  for  their  two  year  catheterization. 

3)  Improved  or  unchanged  symptomatic  status  has  been  observed  in  all 
patients  will  in  the  program  with  objective  improvement  (exercise  test,  etc.) 
in  many. 


&C2 


Project  No.   Z01  HL  02002-03  MDB 

4)  Four  deaths  and  two  documented  ME's  have  occurred  in  patients  in  the 
program. 

An  additional  year's  experience  has  confirmed  the  preliminary 
observations  made  last  year.  Namely  - 

5)  An  approximate  10%  incidence  of  positive  exercise  tests  in 
asymptomatic  type  II 's  has  been  observed  (about  3-5  times  greater  than  that 
reported  in  the  literature). 

6)  An  approximate  13%  incidence  of  coronary  calcifications  has  been 
observed  in  asymptomatic  type  II 's  giving  an  overall  incidence  of  approxi- 
mately 20%  of  asymptomatic  patients  21-55  with  type  II  who  have  evidence  of 
occult  coronary  artery  disease  (positive  exercise  test  and/or  coronary 
calcifications) . 

7)  Approximately  4-0%  of  our  patients  have  normalized  on  diet  and  in 
these  the  average  cholesterol  drop  is  25%.  The  overall  cholesterol  drop  is 
13-15%  including  all  patients  (non-adherers  as  well  as  adherers). 

8)  Exercise  testing  does  not  appear  to  be  as  sensitive  or  specific  in 
an  asymptomatic  population  (even  a  high  risk  population  like  this  one)  as 
many  previously  thought  and  our  data  raise  real  questions  about  its  useful- 
ness in  individual  patient  care  decisions. 

9)  Coronary  calcifications  on  fluoroscopy  is  a  readily  available, 
inexpensive  procedure  with  little  patient  morbidity  and  appears  to  be  a 
better  screening  procedure  for  occult  coronary  artery  disease  in  our 
asymptomatic  type  II  population  than  the  more  expensive  and  elaborate  exercise 
testing. 

10)  Evaluation  of  a  sub-group  of  our  patients  who  were  psychologically 
typed  (type  A  vs.  type  B)  does  not  substantiate  the  hypothesis  that  type  A 
personalities  are  more  likely  to  have  coronary  artery  disease  than  type  B 
personalities . 

11)  The  Safety  Monitoring  Board  has  not  informed  us  of  any  untoward  or 
new  reactions  to  the  medication  nor  have  we  noted  any  reactions  not  pre- 
viously described  except  for  a  few  episodes  of  tinnitus. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

The  answer  to  the  question  posed  by  this  study  is  one  of  the  most 
important  in  the  field  of  heart  research.  It  has  implications  not  only  for 
those  patients  who  are  type  II  with  coronary  artery  disease,  but  also  for 
type  II' s  without  disease,  and  for  the  general  population.  Also  the  know- 
ledge gained  about  the  natural  history  of  coronary  artery  disease  and  the 
factors  affecting  it  will  significantly  advance  our  current  knowledge  and 
have  implications  for  the  treatment  of  the  major  cause  of  mortality  in  the 
adult  population  of  the  United  States. 


W 


Project  No.  Z01  HL  02002-03  MDB 
Proposed  Course: 

1)  Our  major  effort  will  continue  to  be  to  expedite  patient  enrollment 
into  the  program.  This  is  possible  because  of  changes  which  have  been  made 
in  the  program  office.  Seven  months  ago  it  was  decided  that,  in  the  interest 
of  completing  the  program  in  a  reasonable  period  of  time  and  of  ensuring  the 
quality  and  accuracy  of  the  essential  information  for  the  program,  a)  some  of 
the  individualized  attention  which  patients  had  formerly  received  would  have 
to  be  sacrificed  (realizing  that  this  might  increase  patient  dropouts)  and 

b)  some  of  the  patient's  service  and  information  of  lesser  scientific 
importance  (diet  information,  general  patient  laboratory  and  history  infor- 
mation) would  have  to  be  dropped  in  order  to  allow  us  to  process  two  to  three 
times  the  number  of  patients  which  we  were  currently  seeing  with  the  same 
personnel . 

Over  the  last  seven  months,  this  new  system  has  evolved  and  is  currently 
being  used.  Approximately  twice  the  number  of  people  are  being  processed 
and  it  appears  to  be  working.  Our  proposed  course  is  to  continue  to  expand 
the  system  as  much  as  outside  facilities  allow  to  the  limit  of  approximately 
three  times  previous  enrollment. 

2)  In  addition,  where  possible,  we  will  attempt  to  make  our  referrals 
more  selective  and  will  begin  to  analyze  and  process  the  extensive  results 
obtained  to  date.  Currently,  we  have  98  people  in  the  program,  35  of  whom 
have  received  re-catheterization  at  two  years.  These  35  individuals  have 
had  over  30  risk  factors  measured  on  them  periodically,  as  well  as  the 
invasive  and  non- invasive  cardiovascular  tests  performed.  In  addition,  we 
have  54  people  who  have  been  through  their  one  year  evaluation  and  have 
documented  coronary  artery  disease,  as  well  as  repetitive  measurement  of 
over  30  risk  factors  and  baseline  invasive  and  non- invasive  test  information. 
Finally,  we  have  approximately  1,050  patients  with  type  II  who  have  been 
studied  in  detail  with  over  approximately  20  risk  factors  measured  and  pre- 
and  post-diet  information  available. 

Publications : 

None 


*rr 


Project  No. :  Z01  HL  02002-03  MDB 


Key  Words: 

1)  type  II 

2)  coronary  artery  disease 

3)  LDL  cholesterol 

4)  angiography 

5)  intervention  study 

6)  catheterization 

7)  cardiovascular  tests 

8)  high  risk  patients 

9)  epidemiology 

10)  double  blinded  trial 


6SL 


Project  No.  Z01  HL  020Q3-Q5  MDB 

1.  Molecular  Disease  Branch 

2.  Section  on  Lipoproteins 

3.  Bethesda,  Maryland  20014 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  The  Biochemistry  and  Metabolism  of  Plasma 

Lipoproteins 

Previous  Serial  Number:      NHLI-291(c) 

Principal  Investigators:      Robert  I.  Levy,  M.D. 

Conrad  B.  Blum,  M.D. 

Other  Investigators:         Leslie  Jenkins,  M.A. 

Cooperating  Units:  Frank  T.  Lindgren,  Ph.D.,  University  of 

California,  Berkeley,  California 
Mones  Berman,  Ph.D.,  NCI 
Marshall  Hall,  M.D. ,  NCI 
Shlomo  Eisenberg,  M.D. ,  Hadassah  Medical 
Center,  Jerusalem,  Israel 

Project  Description: 

Objectives: 

125 

1)  To  determine  the  metabolic  behavior  of    I-VLDL  in  various  types  of 

hyperlipoproteinemia  under  basal  conditions  and  under  various  metabolic  and 
nutritional  perturbations. 

2)  To  establish  the  metabolic  behavior  of  plasma  HDL,  extending  the 
already  developed  multicompartmental  model  of  apolipoprotein  metabolism  to 
include  the  two  major  apoproteins  of  HDL. 

3)  To  elucidate  the  factors  involved  in  exchange  and  equilibrium  of 
C-peptides  between  VLDL  and  HDL. 

Methods : 

The  isolation,  purification,  and  iodination  of  VLDL,  LDL,  and  HDL  have 
been  described  previously.  The  VLDL,  LDL,  and  HDL  apoproteins  are  separated 
electrophoretically  on  15%  polyacrylamide  gels  using  a  continuous  buffer 
system  with  0.1%  sodium  lauryl  sulfate. 

Major  Findings: 

125 
Methodology  for  performing    I-HDL  turnover  studies  has  been  developed 


6S7 


Project  No.  Z01  HL  02003-05  MDB 

and  eight  studies  have  been  completed  in  five  normal  volunteers.  Another 
seven  studies  are  underway  and  should  be  completed  by  July  1,* 1975.  A 
number  of  insights  into  high  density  lipoprotein  metabolism  have  resulted: 

(a)  the  two  major  peptides  of  high  density  lipoprotein  seem  to  be 
metabolized  homogeneously  in  contrast  to  the  B  and  C  peptides  of  VLDL; 

(b)  the  kinetics  of  high  density  lipoprotein  and  its  two  major  peptides 
approximate  a  simple  two-compartmental  model;  (c)  synthesis  rates  and 
fractional  metabolic  rates  for  HDL  protein  have  been  computed  for  normal 

and  perturbed  conditions,  showing  that  (d)  the  fall  in  HDL  levels  accompanying 
carbohydrate  feeding  is  solely  a  result  of  altered  catabolism  with  synthesis 
remaining  constant. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

Since  the  predominant  protein  of  HDL  is  the  activator  for  lecithin- 
cholesterol  acyl  transferase  and  HDL  is  the  natural  substrate  for  this 
enzyme,  a  knowledge  of  the  biological  behavior  is  crucial  to  a  full  under- 
standing of  cholesterol  metabolism.  The  hypothesis  of  some  that  HDL  is 
responsible  for  removal  of  cholesterol  from  tissues  heightens  interest  in 
its  biological  behavior. 

Furthermore,  since  HDL  acts  as  a  reservoir  for  the  C-peptides  (including 
apoC-II,  the  activator  for  lipoprotein  lipase)  it  plays  an  important  role  in 
regulating  triglyceride  metabolism. 

Proposed  Course: 

1)  Studies  of  VLDL  metabolism  will  continue,  especially  focusing  on 
effects  of  nutritional  and  pharmacologic  perturbations. 

2)  Studies  of  the  turnover  of  HDL  peptides  will  be  extended  to  include 
additional  perturbations  in  testing  and  further  developing  the  multi- 
compartmental  modeling. 

Publications : 

1.  Blum,  C. ,  and  Levy,  R.  I.:  Interconversions  of  apolipoprotein 
fragments .  Ann.  Rev.  Med. ,  in  press . 

2.  Eiseriberg,  S. ,  and  Levy,  R.  I.:  Lipoprotein  metabolism.  Advan.  Lipid 
Res. ,  in  press. 


6  SB 


Project  No.  Z01  HL  02003-05  MDB 
Key  Words: 

1)  VLDL  -  very  low  density  lipoproteins 

2)  LDL  -  low  density  lipoproteins 

3)  HDL  -  high  density  lipoproteins 

4 )  apoproteins 

5)  turnover  studies 

6)  125I 


<£$r 


Project  No.  Z01  HL  02004-08  MDB 

1.  Molecular  Disease  Branch 

2.  Section  on  Genetics  and 
Lipid  Biochemistry 

3.  Bethesda,  Maryland  20014 
PHS-NTH 

Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Lipid  Constituents  of  Human  Tissues 

Previous  Serial  Number:      NHLI-293(c) 

Principal  Investigators:      Howard  R.  Sloan,  M.D. ,  Ph.D. 

Gerd  Assmann,  M.D. 

Other  Investigators:         Barbara  Davis,  M.S. 

Stephen  Demosky,  B.S. 
Anne  Flory,  B.S. 

Cooperating  Units:  •         None 

Project  Description: 

The  principal  objective  of  this  study  is  the  development  of  inclusive 
methods  for  the  determination  of  simple  and  complex  lipids,  including  the 
neutral  glycolipids,  the  gangliosides ,  the  plasmalogens ,  complex  sterols  and 
steryl  esters,  and  partial  glycerides  in  plasma  and  tissues,  for  purposes  of 
improved  diagnosis  of  the  already  established  lipidoses  and  evaluation  of 
heretofore  unknown  lipid  storage  diseases.  The  human  liver  has  been  used  as 
the  prototype  tissue  source;  the  spleen,  lymph  nodes  and  adrenal,  however, 
have  also  been  studied.  The  methodology  is  particularly  needed  in  order  to 
gain  an  insight  into  the  biochemical  interrelationships  of  each  of  these 
lipid  classes  and  sub-classes  with  each  other  and  with  the  other  major  lipid 
classes. 

The  methods  of  extracting,  separating  and  analyzing  the  various  lipids  in 
human  tissues  have  been  thoroughly  described  in  PHS-NTH  Individual  Project 
Report,  July  1,  1973  through  June  30,  1974,  Serial  No.  NHLI-293(c).  New 
methods,  including  conversion  to  acetylated  derivatives,  have  been  developed 
for  the  identification  and  quantification  of  the  carbohydrate  components  of 
lipids.  In  addition,  methods  have  been  developed  for  the  identification  of 
several  new  lipids  employing  the  techniques  of  gas-liquid-mass  spectrometry 
and  high  resolution  mass  spectrometry  as  well  as  liquid-liquid  chromatography. 

Techniques  have  been  developed  for  analyzing  all  of  the  major  lipid 
classes  and  sub-classes  in  human  tissue.  The  normal  values  and  ranges  for 
these  lipids  have  been  presented  in  a  prior  annual  report  (NHLI-264 [cj ) .  The 
use  of  the  techniques  developed  in  this  study  have  permitted  the  post-abortion 
confirmation  of  the  in  utero  diagnosis  of  one  case  of  Niemann-Pick  disease, 
type  A  (in  addition  to  the  three  cases  correctly  diagnosed  last  year).  Two 
cases  of  Gw-,  gangliosidosis  have  been  confirmed  by  chemical  examination  of 

1  <^o 


Project  No.  Z01  HL  02004-08  MDB 

the  aborted  tissues  as  well  as  an  additional  case  of  metachromatic  leuko- 
dystrophy (total  now  three). 

Analyses  of  post-mortem  samples  of  liver,  spleen  and  adrenals  of  two  more 
patients  with  Wolman's  disease  (in  addition  to  the  one  studied  last  year) 
revealed  the  accumulation  of  the  following  sterols:  7-alpha-hydroxycholesteryl 
ester,  7-beta-hydroxy- ,  7-keto-,  5-6-alpha-epoxy-  and  5-6-beta-epoxy-choles- 
teryl  esters.  These  compounds  were  isolated  and  identified  by  gas  chromatog- 
raphy, mass  spectroscopy,  and  NMP  spectrometry. 

Tissue  storage  of  lipids  is  a  principal  process  in  atheromata  formation. 
Studies  which  elucidate  abnormalities  in  lipid  metabolism  and  provide  new 
means  for  the  detection  of  biochemical  defects  offer  useful  new  approaches 
which  may  yield  specific  clues  to  the  process  of  atherogenesis .  Investigation 
of  tissues,  particularly  liver,  spleen  and  adrenal,  from  patients  who  have 
died  of  previously  unclassified  lipidoses  is  in  progress. 

Publications : 

1.  Kaback,  M.  M. ,  Sloan,  H.  R. ,  Sonneborn,  M. ,  Herndon,  R.  M. ,  and  Percy,  A. 
K. :  CLp -gangliosidosis  type  I:  in  utero  detection  and  fetal  mani- 
festations. J.  Pediat.  82:  1037-1041,  1973. 

2.  Assmann,  G. ,  Fredrickson,  D.  S. ,  Sloan,  H.  R. ,  Fales,  H.  M. ,  and  Highet, 
R.  J.:  Accmmulation  of  oxygenated  steryl  esters  in  Wolman's  disease. 

J.  Lipid  Res.  16:  28-38,  1975. 


Ul 


Project  No.  Z01  HL  02004-08  MDB 


Key  Words: 

1)  lipids 

2)  human 

3)  tissues 

4)  neutral  lipids 

5)  phospholipids 

6 )  glycolipids 

7 )  sphingolipids 


££V 


Project  No.    Z01  HL  02005-07  MDB 

1.  Molecular  Disease  Branch 

2.  Section  on  Genetics  and 
Lipid  Biochemistry 

3.  Bethesda,  Maryland  20014 
PHS-NIH 

Individual  Project  Report 
July  1,  1974-  through  June  30,  1975 

Project  Title:  Tissue  Lipidoses  and  Hyperlipoproteinemias 

Previous  Serial  Number:      NHLI-294(c) 

Principal  Investigators:      Howard  R.  Sloan,  M.D. ,  Ph.D. 

Gerd  Assmann,  M.D. 

Other  Investigators:         Barbara  Davis,  M.S. 

Stephen  Demosky,  B.S. 
Briston  Williamson 
Arthur  Pace 

Cooperating  Units:  None 

Project  Description: 

The  principal  objective  of  this  study  is  to  improve  the  knowledge  of  the 
biochemical  and  the  enzymatic  basis  of  the  genetically  determined  tissue  lipid 
storage  disorders  as  well  as  to  improve  diagnostic  techniques  for  both  the 
patients  and  possible  heterozygous  carriers.  Findings  in  this  and  other 
laboratories  have  demonstrated  that  tissue  culture  cells  derived  from  the  bone 
marrow  and  skin  of  patients  with  all  of  the  lipid  storage  diseases  have  the 
same  genetic  profile  as  the  patient  from  whom  they  were  obtained.  It  is, 
therefore,  possible  to  diagnose  all  of  these  diseases  by  the  appropriate 
enzymatic  analysis  of  cells  derived  from  tissue  culture  of  the  skin  rather 
than  by  resorting  to  biopsy  of  the  liver  or  some  other  organ.  The  study  of 
these  diseases  offers  a  model  of  great  interest  for  a  study  of  the  storage  of 
lipid  within  tissues. 

The  discovery  of  the  new  technique  of  cell  hybridization  has  opened  an 
additional  path  for  the  study  of  these  inherited  metabolic  disorders.  Hybrids 
of  particular  interest  include:  hybrid  cell  lines  produced  by  crosses  between 
fibroblast-like  cells  from  patients  with  different  forms  of  Niemann-Pick 
disease,  different  forms  of  Gaucher 's  disease,  and  different  forms  of  GL^. 
gangliosidosis.  Hybrids  are  also  being  prepared  with  cells  from  individuals 
with  the  various  lipid  storage  diseases  and  with  mouse  L-cells.  Additional 
hybrids  between  mouse  L-cells  and  fibroblasts  derived  from  patients  with 
homozygous  type  II  and  type  III  hyperlipoproteinemia  have  been  produced. 

In  annual  report  NHLE-294(c)  the  methods  of  obtaining,  processing,  storing 
and  placing  into  culture  tissues  from  patients  with  the  various  lipid  storage 
disorders  and  the  hyperlipoproteinemias  have  been  described.  In  addition, 
methods  have  been  developed  for  assaying  and  purifying  several  of  the  enzymes 


Project  No.  Z01  HL  02005-07  MDB 

that  catabolize  sphingolipids .  These  enzymes  are  present  in  decreased 
amounts  in  the  sphingolipidoses . 

Normal  ranges  for  many  of  the  enzymes  involved  in  the  catabolism  of  the 
sphingolipids  have  been  established  for  normal  human  fibroblasts  and  fibro- 
blasts obtained  from  normal  amniotic  fluid.  It  is  now  possible  to  make  a 
diagnosis  (including  a  pre-natal  diagnosis)  of  all  of  the  lipid  storage 
diseases.  Many  successful  pre-natal  diagnoses  have  been  made  and  no  false 
positives  or  false  negatives  have  been  obtained. 

Cholesteryl  ester  hydrolase  (acid)  has  been  purified  over  1,000-fold. 
Minimal  amounts  of  triglyceride  lipase  activity  remain  in  these  preparations 
and  two  bands  are  seen  on  polyacrylamide  gel  electrophoresis.  More  than  99.9% 
of  the  triglyceride  lipase  activity  has,  however,  been  removed. 

Cell  lines  from  12  patients  with  homozygous  type  II  hyperlipoproteinemia 
and  10  patients  with  type  III  hyperlipoproteinemia  have  been  initiated.  In 
five  of  the  type  II  lines  serum  causes  an  insignificant  decrease  in  HMG  CoA 
reductase  activity  compared  with  normal  cell  lines.  It  has  been  observed  that 
when  some  cells  are  treated  with  the  detergent  Kyro  EOB,  significant  amounts 
of  HMG  CoA  hydrolase  are  released,  leading  to  the  formation  of  acetoacetic 
acid  and  then  acetone  during  the  course  of  the  reductase  assay.  The  formation 
of  the  latter  product  has  been  conclusively  demonstrated  by  classical  organic 
chemical  techniques.  The  release  of  the  undesired  HMG  CoA  hydrolase  can  be 
avoided  by  extracting  the  enzyme  from  the  tissue  culture  cells  in  the 
following  manner.  The  temperature  of  a  suspension  of  the  cells  is  gradually 
lowered  from  +5°C  to  -50°C  at  a  rate  of  5°C  per  minute.  The  tube  containing 
the  cell  suspension  is  then  placed  in  air  at  25°C  and  allowed  to  thaw  slowly. 
This  procedure  is  repeated  twice. 

Tissue  storage  of  lipids  is  a  principal  process  in  atheroma  formation. 
Studies  which  elucidate  abnormalities  in  lipid  metabolism  and  which  provide 
new  means  for  the  differentiation  of  biochemical  defects  offer  useful  new 
approaches  which  may  yield  specific  clues  to  the  process  of  atherogenesis . 
Moreover,  a  perpetuation  of  metabolic  disorders  in  its sue  culture  cells  makes 
possible  a  detailed  approach  to  the  pathogenesis  and  control  of  metabolic 
disorders.  In  addition,  this  perpetuation  may  facilitate  the  study  of  the 
genetic  defect  in  the  various  lipid  storage  disorders.  The  purification  of 
the  various  sphingolipid  hydrolases  may  permit  detailed  understanding  of 
several  lipid  storage  disorders  at  the  molecular  level,  and  thereby  facilitate 
our  understanding  of  the  process  of  lipid  accumulation  within  tissues. 

Future  studies  will  be  directed  at  attempting  to  delineate  differences  in 
cholesterol  metabolism  between  normal  cultured  ceils  and  those  derived  from 
patients  with  the  various  hyperlipoproteinemias.  The  differential  effects  of 
serum,  plasma,  and  various  fractions  of  lipoproteins  on  the  activity  of  HMG 
CoA  reductase  will  be  evaluated  in  these  cell  lines.  In  addition,  detailed 
studies  of  HMG  CoA  reductase  activity  in  the  various  hybrids  will  be  conducted 
in  an  attempt  to  determine  the  nature,  and  possibly  the  chromosomal  location, 
of  those  factors  that  are  responsible  for  the  normal  feedback  inhibition 

2  tW 


Project  No.  Z01  HL  02005-07  MDB 

exerted  by  cholesterol  on  HMG  CoA  reductase.  Similar  studies  will  be  employed 
to  differentiate  more  clearly  the  differences  between  homozygous  type  II 
hyperlipoproteinemia  and  type  III  hyperlipoproteinemia. 

Publications : 

1.  Breslow,  J.  L. ,  Lux,  S.  E. ,  Spaulding,  D.  R. ,  and  Sloan,  H.  R. :   3-hydroxy- 
3-methyl  glutaryl  coenzyme  A  reductase  activity  in  fibroblasts  from  pheno- 
typic  homozygous  type  II  hyperlipoproteinemia.  Pediat.  Res.  8:  386-388, 
1974. 


US' 


Project  No.  Z01  HL  02005-07  MDB 


Key  Words: 

1)  lipid  storage  disorders 

2 )  lipoproteins 

3 )  hyperlipoproteinemias 

4)  tissue  culture 

5)  hybridization 

6)  enzymes  of  lipid  metabolism 

7)  HMG  CoA  reductase 

8)  cholesterol  biosynthesis 

9)  fibroblasts 


£U 


Project  No.    Z01  HL  02006-03  MDB 

1.  Molecular  Disease  Branch 

2.  Section  on  Genetics  and 
Lipid  Biochemistry 

3.  Bethesda,  Maryland  20014 


PHS-NIH 
Individual  Project  Report 
July  1,  19 74  through  June  30,  1975 


Project  Title: 

Previous  Serial  Number: 
Principal  Investigators : 
Other  Investigators : 

Cooperating  Units : 

Project  Description: 


Microscopic  Studies  in  Tissue  Lipid  Storage 
Diseases 

NHLI-295(c) 

Howard  R.   Sloan,  M.D. ,  Ph.D. 

Stephen  Demosky,  B.S. 
Briston  Williamson 

Victor  J.   Ferrans,  M.D. ,  Ph.D. 
Section  of  Pathology,  NHLI 


Microscopic  study  of  tissues  obtained  from  patients  with  tissue  lipid 
storage  disorders  can  contribute  to  the  understanding  of  the  biochemical 
basis  of  these  genetically  determined  diseases  and  to  the  improvement  of 
diagnostic  techniques  which  may  benefit  potential  carriers  as  well  as  those 
afflicted  carriers.     These  techniques  can  also  help  to  elucidate  the  nature 
of  the  cellular  abnormalities  in  the  hyperlipoproteinemias.       Tne  light  and 
electron  microscopic  appearance  of  these  macrophages  may  provide  clues  to  "the 
processes  by  which  lipid  is  stored  within  tissues  in  pathological  conditions. 

To  obtain  tissues  for  these  studies ,  patients  with  lipid  storage  diseases 
and  hyperlipoproteinemias  are  admitted  for  evaluation  including  biopsy  of  "the 
bone  marrow  and/or  liver.     Tissues  are  fixed  in  Baker's  formalin,  phosphate- 
buffered  glutaraldehyde ,  and  digitonin-glutaraldehyde  and  prepared  for  exam- 
ination with  light  and  electron  microscopes. 

We  have  now  completed  electron  microscopic  examination  of  fibroblasts 
derived  from  patients  with  various  lipid  storage  diseases  and  the  hyperlipo- 
proteinemias ,  including  patients  who  are  homozygous  for  type  II  hyperlipopro- 
teinemia.    There  are  significant  differences  between  the  different  diseases 
and  an  initial  subclassification  now  seems  possible. 

Significant  differences  have  been  demonstrated  at  the  electron  micro- 
scopic level  between  the  lipid-laden  macrophages  observed  in  various  forms 
of  Niemann-Pick  disease.     These  differences  do  not  at  this  time,  however, 
permit  a  definitive  diagnosis.     The  macrophages  from  patients  with  Gaucher' s 
disease,  G^-gangliosidosis  and  Tangier  disease  are  clearly  distinguishable 


ur 


Project  No.     Z01  HL  02006-03  MDB 

on  the  basis  of  appearance  in  electron  micrographs.     Study  of  the  macrophages 
from  patients  with  various  hyperlipoproteinemias  suggest  that  at  least  a 
tentative  diagnosis  of  Tangier  disease  and  possibly  of  type  III  hyperlipopro- 
teinemia could  be  made  in  this  way. 

Auto- fluorescence  analysis  of  macrophages  in  various  tissue  lipid  storage 
diseases  demonstrates  the  presence  of  fluorescent  material  in  almost  all  of 
the  lipid  storage  diseases.     It  is  not  possible  at  this  time  to  make  a  defini- 
tive diagnosis  of  any  of  the  lipid  storage  diseases  based  on  the  auto- 
fluorescence  spectrum  obtained  from  examination  of  the  lipid  laden  macrophages. 
Significant  difference  between  tissues  from  patients  with  the  various  lipid 
storage  diseases  have,  however,  been  demonstrated. 

In  other  studies ,  cultured  cells  were  grown  on  cover  slips  and  then  incu- 
bated with  5-bromo-4-chloro-3-indolyl-g-galactopyranoside  (BCI-gal).      Cultures 
of  peripheral  blood  leukocytes  and  thick  sections   (10  y)   of  human  liver, 
spleen  and  kidney  were  also  incubated  with  this  substrate.     The  indigo  pro- 
duced following  cleavage  of  the  terminal  galactose  of  this  molecule  makes  it 
possible  to  visualize  the  subcellular  localization  of  several  g-galactosidases. 
Use  of  the  BCT-gal  assay  has  enabled  us  to  make  the  intrauterine  diagnosis  of 
five  cases  of  G»«, -gangliosidosis ,  including  the  first  and  second  cases. 
Chemical,  enzymatic,  microscopic,  and  histochemical  studies  of  tissues  from 
the  abortuses  have  verified  the  diagnoses.     The  technique  has  also  been  uti- 
lized to  predict  that  twelve  fetuses,  known  to  be  at  risk  for  &., -gangliosidosis  , 
would  be  normal.      The  clinical  course  of  the  twelve  children  adjudged  by  histo- 
chemical and  enzymatic  tests  to  be  normal,  have  verified  the  in  utero  diag- 
nosis . 

Tissue  storage  of  lipids  is  a  principal  process  in  atheroma  formation. 
Light  microscopic  and  electron  microscopic  studies  may  help  to  elucidate  the 
mechanism  by  which  lipid  is  stored  within  tissues  in  various  pathological 
conditions  and  may  provide  specific  clues  to  the  study  of  the  process  of 
atherogenesis .     The  differences  already  noted  may  help  to  explain  ~&ie  differ- 
ences between  several  different  types  of  atheromata  formation. 

These  further  electron  microscopic  studies  of  cells  and  tissues  from 
patients  with  the  lipid  storage  disorders  are  essentially  completed  and  will 
be  terminated  when  all  samples  presently  on  hand  have  been  examined. 

Publications : 

1.      Sloan,  H.    R.  ,  and  Breslow,  J.  :      Foam  cells.      In  Hematology  of  Infancy  and 
Childhood  (Nathan,  D.    and  Oski,  F.  ,  eds.),  Saunders,  Phila.  ,   1974,  pp. 
761-780.      (Cited  as  "in  press"  in  project  report  NHLI-295(c)  ,  1974. 


6ib 


Project  No.  Z01  HL  02006-03  MDB 


Key  Words; 

1)  light  microscope 

2)  electron  microscope 

3)  lipid  storage  disorders 

4 )  hyperlipopro  teinemL  as 

5)  in  utero  diagnosis 

6)  lipid- laden  macrophages 

7)  auto- fluorescence 


Uf 


Project  No.  Z01  HL  02009-08  MDB 

1.  Molecular  Disease  Branch 

2.  Section  on  Peptide  Chemistry 

3.  Bethesda,  Mary land  20014 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Structure  and  Function  of  Parathyroid  Hormone 

Previous  Serial  Number:      NHLI-299 

Principal  Investigators:      H.  Bryan  Brewer,  Jr.,  M.D. 

Thomas  Fairwell,  Ph.D. 

Other  Investigators:         Rosemary  Ronan,  B.A. 

Ann  LaRue,  B.A. 

Cooperating  Units:  Claude  Arnaud,  Mayo  Clinic,  Rochester,  Minn. 

Werner  Rittel,  Ciba  Geigy  Pharmaceutical  Co. , 
Basle,  Switzerland 

Project  Description: 

1)  Objective: 

Isolation,  characterization,  and  sequence  analysis  of  human  parathyroid 
hormone  (HPTf  I). 

Methods  Employed: 

HPTH  has  been  isolated  from  parathyroid  adenomas  obtained  from  patients 
undergoing  surgery  for  hyperparathyroidism  in  the  United  States  and  Western 
Europe.  The  adenomas  are  extracted  with  8M  urea  in  0.2M  hydrochloric  acid, 
and  fractionated  with  ether,  acetic  acid,  sodium  chloride,  and  trichloro- 
acetic acid  (TCA  powder).  The  TCA  powder  was  further  purified  by  gel  and  ion 
exchange  chromatography. 

Major  Findings: 

HPTH  has  been  isolated  in  homogeneous  form  by  the  methods  outlined 
above,  and  detailed  in  last  year's  report.  During  the  last  year  sufficient 
hormone  was  isolated  to  permit  the  isolation  and  characterization  of  the 
peptide  fragments  obtained  from  tryptic  digestion  of  the  intact  molecule.  In 
addition,  procedures  were  developed  for  the  selective  modification  of  the 
lysine  residues,  and  subsequent  restricted  cleavage  of  the  hormone  at  the 
arginine  residues.  These  procedures  permitted  the  reinvestigation  of  the 
amino  terminal  sequence  of  the  biologically  active  region  of  the  hormone 
which  we  had  previously  determined  on  a  relatively  small  amount  of  material. 
An  amino  acid  sequence  identical  to  our  previous  report  was  obtained.  Thus, 


670 


Project  No.  Z01  HL  02009-08  MDB 

there  appears  to  be  no  evidence  for  isohormones  for  HPTH  despite  the  use  of 
adenomas  from  over  a  thousand  different  patients.  During  the  next  year  we 
plan  to  complete  the  remainder  of  the  sequence  of  human  parathyroid  hormone 
by  automated  and  manual  Edman  degradations  of  the  intact  hormone,  and  peptide 
fragments  obtained  from  tryptic  and  citroconylated  digestions  of  the  native 
hormones . 

2)  Objective: 

Immunoassay  of  parathyroid  hormone  (in  collaboration  with  Dr.  Arnaud 
and  colleagues). 

Methods  Employed: 

The  methods  utilized  in  these  studies  have  been  detailed  over  the  years 
in  the  annual  reports,  and  involve  labeling  of  the  hormone  by  the  chloramine 
T  method  of  Hunter  and  Greenwood,  and  separation  of  the  antibody-bound  and 
free  hormone  by  dextran-coated  charcoal. 

Major  Findings: 

Two  groups  of  antisera  were  prepared  in  order  to  investigate  the 
immunological  properties  of  the  synthetic  1-34  peptide  (HPTH  1-34)  and  the 
intact  hormone  (1-84).  Four  antisera  were  prepared  ft>om  goats  immunized  with 
HPTH  (1-34),  while  the  other  antisera  were  obtained  from  three  guinea  pigs 
immunized  with  crude  human  PTH  extracted  from  parathyroid  adenoma  tissue. 
These  antisera  were  studied  by  competitive  radioimmunoassays  under  strictly 
equilibrium  conditions.  All  four  goat  anti-HPTH  (1-34)  sera  had  2-15  times 
higher  affinity  for  HPTH  (1-34)  than  for  HPTH  (1-84),  regardless  of  the 
labeled  antigen  (1-34  or  1-84)  used.  However,  the  three  guinea  pig  anti-HPTH 
sera  showed  2-12  times  higher  affinity  toward  HPTH  (1-34)  than  toward  HPTH 
(1-84)  only  when  HPTH  (1-34)  was  the  labeled  antigen.  With  HPTH  (1-84)  as 
labeled  antigen,  HPTH  (1-84)  exhibited  2-100  times  higher  affinity  than  HPTH 
(1-34).  These  studies,  therefore,  indicate  that  the  antigen  used  for 
development  of  the  antisera,  and  the  labeled  tracer  used  in  the  immunoassay 
significantly  influence  the  type  of  results  obtained  in  the  assessment  of  the 
immunological  activity  of  the  intact  hormone  and  the  amino  terminal  synthetic 
peptide.  These  observations  may  be  explained  at  least  in  part  by  the  con- 
formational properties  of  the  two  polypeptides  (see  section  4). 

3)  Objective: 

Determination  of  the  biological  activities  of  HPTH  and  BPTH,  and  their 
respective  synthetic  amino  terminal  tetratriacontapeptides  (HPTH  1-34, 
BPTH  1-34)  (in  collaboration  with  Dr.  Arnaud  and  colleagues). 

Methods  Employed: 

Adenylate  cyclase  assays  on  purified  plasma  membranes  and  in  vivo  rat 


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Project  No.  Z01  HL  02009-08  MDB 

bioassays  were  performed  as  outlined  in  previous  annual  reports. 

Major  Findings: 

Adenylate  cyclase  assays  were  performed  with  purified  plasma  membrane 
fractions  obtained  from  rat,  chicken  and  human  kidney  cortex.  The  ratio  of 
biological  activity  utilizing  the  cyclase  assay  for  the  intact  bovine  and 
human  hormone  is  as  follows:  HPTH  (1-84):BPTH  (1-34);  rat  1:25;  chicken  1:3; 
and  human  1:1.  The  synthetic  1-34  human  peptide,  however,  was  15  times  more 
active  than  the  bovine  (1-34)  peptide  in  all  three  systems.  The  synthetic 
HPTH  (1-34)  fragment  had  10%  of  the  activity  of  HPTH  (1-84)  with  the  chicken 
and  human  cortex  system,  but  had  65%  the  activity  in  the  membranes  isolated 
from  the  rat  kidney. 

In  the  in  vivo  rat  hypocalcemic  bioassay  HPTH  (1-84)  and  BPTH  (1-34)  had 
almost  equal  biological  activity.  However,  the  BPTH  (1-34)  fragment  was  four 
times  more  active  than  the  HPTH  (1-34)  polypeptide. 

These  results,  therefore,  indicate  that  the  relative  biological  potency 
of  HPTH  and  BPTH  and  their  synthetic  fragments  are  significantly  dependent  on 
the  assay  systems  employed.  The  relative  response  may  reflect  differences  in 
molecular  structure  of  the  receptors  in  various  species,  variation  in  minimal 
chain  length  required  to  activate  the  cyclase  systems,  or  differences  in 
degradation- inactivation  of  the  intact  hormones  and  fragments  in  the  various 
systems  studied. 

4)  Objective: 

Comparison  of  the ' conformation  of  HPTH,  BPTH,  and  the  synthetic  HPTH 
(1-34)  and  BPTH  (1-34)  fragments. 

Methods  Employed: 

Analysis  of  the  secondary  or  a-helical  structure  by  circular  dichroism 
(CD) ,  and  tertiary  structure  by  fluorescence  spectroscopy  has  been  detailed 
in  previous  annual  reports. 

Major  Findings: 

The  CD  spectra  of  HPTH,  BPTH,  HPTH  (1-34),  and  BPTH  (1-34)  at   acidic  pH 
revealed  a  major  trough  near  200  nm,  which  is  characteristic  of  polypeptides 
in  unordered  or  random  conformation.  At  neutral  pH,  a  significant  red  shift 
was  observed  in  the  spectra  of  all  four  polypeptides.  The  spectra  contained 
two  major  troughs  at  204  to  208,  and  at  222  nm,  which  are  the  characteristic 
electronic  transitions  associated  with  polypeptides  in  a-helical  conformation. 
These  studies  indicate  that  there  is  a  significant  increase  in  ordered 
structure  in  all  the  polypeptides  following  titration  from  acid  to  physiologic 
pH. 


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Project  No.  Z01  HL  02009-08  MDB 

The  fluorescence  spectra  of  the  individual  hormones  and  synthetic 
fragments  revealed  significant  differences.  The  spectra  of  HPTH  revealed  a 
peak  with  a  maximum  at  344  nm,  which  did  not  change  in  6M  guanidine  hydro- 
chloride. In  HPTH  (1-34)  the  wavelength  of  tryptophanyl  fluorescence  was 
343  nm,  which  was  normalized  to  348  nm  in  6M  guanidine  hydrochloride.  In 
addition,  HPTH  showed  no  loss  of  tryptophanyl  fluorescence  during  titration 
between  pH  8.0  and  11.0  in  either  aqueous  solution  or  with  denaturing 
reagents.  In  contrast,  HPTH  (1-34)  demonstrated  a  25%  loss  of  tryptophanyl 
fluorescence  in  aqueous  solution  which  was  normalized  in  6M  guanidine 
hydrochloride . 

Alkaline  titration  of  BPTH  revealed  a  20-25%  loss  of  tryptophanyl 
fluorescence  in  aqueous  solution,  which'  was  eliminated  in  solutions  containing 
denaturing  reagents.  However,  BPTH  (1-34)  showed  no  loss  of  tryptophanyl 
fluorescence  in  aqueous  solution,  and  was  therefore  comparable  to  the  native 
1-84  human  hormone. 

The  combined  results  of  these  studies  indicate  these  polypeptide  hormones 
and  synthetic  fragments  are  not  rigid,  but  can  undergo  a  variety  of  con- 
formational changes  dependent  on  the  aqueous  environment.  In  addition,  the 
conformation  of  the  intact  hormones  and  the  synthetic  fragments  in  both  HPTH 
and  BPTH  appear  to  be  different  in  the  region  near  the  tryptophan  residue  at 
position  23.  These  findings  may  be  of  major  importance  with  regard  to  the 
immunological  cross-reactivity  of  antibodies  prepared  against  the  native 
hormone  or  synthetic  fragments  since  conformational  differences  between  the 
antigens  may  influence  the  antibodies  produced,  and  the  cross-reactivity  of 
the  individual  polypeptides.  They  also  provide  an  explanation  for  differences 
in  the  biological  properties  of  the  individual  hormones  and  fragments  since 
degradation  or  inactivation  and  receptor  site  interactions  may  be  profoundly 
altered  by  the  conformational  properties  of  the  constituent  polypeptides. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

This  work  is  directed  toward  a  better  understanding  of  the  structure, 
function,  and  physiological  role  of  polypeptides  in  cellular  metabolism. 
The  determination  of  the  covalent  structure  of  human  PTH,  and  the  structure- 
function  determinants  of  the  primary  and  three-dimensional  structure  of  the 
hormone  will  permit  the  synthesis  of  selected  biologically  active  and 
inactive  regions  of  the  hormone  which  may  be  used  for  physiological  studies, 
as  well  as  the  development  of  immunological  assays  for  use  in  the  clinical 
assessment  of  patients  with  a  variety  of  disorders  of  calcium  metabolism. 

Proposed  Course: 

During  the  next  year  it  is  planned  to  complete  the  covalent  structure  of 
human  parathyroid  hormone.  The  synthesis  of  the  carbaxyl  terminal  portion  of 
the  hormone  will  then  be  undertaken  by  a  commercial  company  in  order  to 
develop  antibodies  against  this  segment  of  the  structure.  It  is  ultimately 
planned  to  develop  an  immunoassay  for  the  amino  as  well  as  the  carboxyl 


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Project  No.  Z01  HL  02009-08  MDB 

terminal  region  of  the  hormone  which  will  then  be  distributed  to  the  bio- 
medical community  for  immunoassays  in  clinical  medicine. 

Publications : 

1.  Brewer,  H.  B. ,  Jr.,  Fairwell,  T. ,  Rittel,  W. ,  and  Arnaud,  C.  D. :  The 
chemistry  of  the  parathyroid  hormone.  Amer.  J.  Med.  56:  759,  1974. 

2.  Elson,  N.  A.,  Brewer,  H.  B. ,  and  Anderson,  W. :  Hemoglobin  switching  in 
sheep  and  goats.  III.  Cell-free  initiation  of  sheep  globin  synthesis. 
J.  Biol.  Chem.  249:   5227,  1974. 

3.  Arnaud,  C.  D. ,  Dibella,  F.  P.,  Brewer,  H.  B. ,  Zawistowski,  K. ,  and 
Verheyden,  J. :  Human  parathyroid  hormone:  biologic  and  immunologic 
activities  of  its  synthetic  (1-34)  tetratriacontrapeptide  and  the 
utility  of  a  carboxy-terminal  specific  radioimmunoassay  in  assessment  of 
hyperparathyroid  syndromes.  Proceedings  of  the  Fifth  Parathyroid 
Conference,  Excerpta  Medica,  in  press. 

4.  Brewer,  H.  B. ,  Jr.,  Fairwell,  T. ,  Ronan,  R. ,  Rittel,  W. ,  and  Arnaud,  C. : 
Human  parathyroid  hormone.  Proceedings  of  the  Fifth  Parathyroid 
Conference,  Excerpta  Medica,  in  press. 

5.  Arnaud,  C.  D. ,  and  Brewer,  H.  B. ,  Jr.:  Parathyroid  hormone:  structure 
and  immunoheterogeneity.  In  Simmons,  I.  L. ,  and  Ewing,  G.  W.  (Eds.): 
Methods  in  Radioimmunoassay,  Toxicology,  and  Related  Areas,  New  York, 
Plenum  Publishing  Corp.,  1974,  p.  45. 


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Project  No.  Z01  HL  02009-08  MDB 


Key  Words: 

1)  peptide 

2)  parathyroid  hormone 

3 )  irarnunoassay 

4)  circular  dichroism 

5)  biological  activity 

6)  adenyl  cyclase 

7)  amino  acid  sequence 

8 )  conformation 

9)  fluorescence 

10)  tertiary  structure 


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Project  No.     Z01  HL  02010-05  MDB 

1.  Molecular  Disease  Branch 

2.  Section  en  Peptide  Chemistry 

3.  Bethesda,  Maryland  20014 

PHS-NIH 
Individual  Project  Report 
July  1,   1974  through  June  30,  1975 

Project  Title:  Structure  and  Function  of  the  Plasma  Lipo- 

proteins 

Previous  Serial  Number:  None 

Principal  Investigators:  H.   Bryan  Brewer,  Jr.,  M.D. 

Gerd  Assmann,  M.D. 

Other  Investigators:  Ann  LaRue  ,  B.A. 

Anne  Flory,  B.S. 

Cooperating  Units:  ■  Harold  Edelhoch,  Ph.D. ,  John  Gwynne,  M.D. , 

and  James  Osborne,  Ph.D.  ,  Clinical  Endo- 
crinology Branch,  NIAMDD 

Edward  Sokoloski,  B.S.  and  Robert  Highet,  Ph.D. 
Chemistry  Branch,  NHLI 

Project  Description: 

1)  Objective: 

Determination  of  the  molecular  organization  of  the  phospholipids  in 
native  high  density  lipoprotein  (HDL) . 

Methods  Employed: 

The  spatial  orientation  of  the  phospholipids  of  HDL  was  assessed  by 
phosphorus  nuclear  magnetic  resonance  (^p  NMR)  utilizing  Fourier  transform 
technique. 


Major  Findings : 


31p 


VLDL,  LDL,  and  HDL  all  exhibited  characteristic  T3  NMR  specxra  con- 
taining two  major  resonances.  These  two  resonances  were  separated  by  0.6  ppm 
and  could  be  assigned  to  phosphatidylcholine  and  sphingomyelin.  The  relative 
intensities  of  the  NMR  spectra  of  ihe   two  phospholipids  agreed  very  well  with 
chemical  determinations  of  'the   individual  lipids.  In  order  to  evaluate  the 
solvent  accessibility  of  the  polar  headgroups  of  the  phospholipids ,  native 
HDL  and  synthetic  phospholipids  were  titrated  with  the  paramagnetic  ion, 
europrium.  When  phospholipids  in  bilayered  structure  are  titrated  with 
paramagnetic  ions ,  the  electronic  transitions  of  the  polar  headgroups  that 
are  exposed  to  the  aqueous  solvent  are  shifted  up field.  The  inner  phosphorus 


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Project  No.    Z01  HL  02010-05  MDB 

groups  are  shielded  and  remain  uncomplexed  with  the  titrating  reagent. 
Phospholipids  in  micellar  structure  have  all  the  polar  headgroup     exposed. 
Titration  of  native  HDL  revealed  complete  broadening  and  shifting  of  the 
phosphorus  peaks  suggesting  that  all  of  the  phospholipid  polar  headgroups 
were  available  for  binding.     These  studies  suggest  that  the  lipid  in  HDL  is 
organized  into  a  spherical  micelle,  rather  than  in  a  classical  membrane  bi- 
layer.     These  results  are  consistent  with  recent  low  angle  x-ray  diffraction 
studies  which  indicate  that  HDL  has  an  organized  symmetrical  structure  with  a 
relatively  electron-poor  (nonpolar)   central  region,  and  a  relatively  electron- 
rich  (polar)  outer  region   (Shipley,  et  al.  ,  J.   Supramolecular  Struc.   1,  18, 
1972). 

2)  Objective: 

Lipid-protein  interactions  of  A-I  and  A-II  with  the  phospholipids , 
phosphatidylcholine   (PC)   and  sphingomyelin  (SPM). 

Methods  Employed: 

The  apo lipoproteins  were  recombined  with  phospholipid  in  aqueous  solu- 
tion without  soni cation.     The  recombined  particles  were  isolated  by  gel 
filtration  or  preparative  ultracentrifugation.     The  isolated  complex  was 
evaluated  for  the  molar  ratio  of  lipid  to  protein,  and  by  circular  dichroism 
for  changes  in  conformation  concomitant  with  lipid-protein  binding. 

Major  Findings: 

ApoHDL,  apoA-II,  and  reduced  and  carboxymethylated  apoA-II  readily  re- 
combined with  PC  or  SPM  to  form  particles  that  were  similar  in  size  to  native 
HDL.     The  carboxyl  terminal,  but  not  the  amino  terminal  cyanogen  bromide 
fragment  of  A-II ,  recombined  with  lipid.     ApoA-I ,  and  the  carboxyl  terminal 
cyanogen  bromide  fragment  of  A-I,  however,  recombined  with  PC  or  SPM  to  only 
a  very  limited  extent.     A-I  in  the  presence  of  A-II,  however,  readily  re- 
combined.    These  studies  suggest  that  A-II  may  influence  the  structure  of 
A-I,  permitting  lipid- lipid  interaction,  or  that  A-I  may  be  held  in  the  re- 
combined particle  by  protein-protein  interactions. 

Analysis  of  the  recombined  particles  by  circular  dichroism  indicated 
that  there  was  an  increase  in  helical  structure  concomitant  with  lipid- 
protein  interaction. 

3)  Objective: 

Determination  of  the  quaternary  structure  of  native  HDL. 

Methods  Employed: 

The  quaternary  structure  of  HDL  has  been  evaluated  by  a  variety  of 
techniques  including  ^C  NMR,  CD,  molecular  modeling,  and  recombination  of 
the  constituent  apoproteins  with  isolated  phospholipids. 

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Project  No.     Z01  HL  02010-05  MDB 
Major  Findings : 

A  variety  of  approaches  have  been  employed  in  order  to  gain  some  in- 
sight into  the  molecular  organization  of  lipids  and  proteins  in  HDL.     The,  „ 
major  force  involved  in  lipid-protein  interactions  has  been  evaluated  by       C 
NMR  of  PC  and  SPM     specifically  enriched  in  the  polar  headgroups.     Spin  lat- 
tice relaxation  times  of  recombined  apoproteins,  and  model  compounds  were 
identical,  indicating  that  the  polar  headgroups  of  PC  and  SPM  were  in  the 
same  hydrophilic  environment  in  either  sonicated  lipid  particles  or  reassembled 
lipoproteins.     These  studies  suggest  that  ionic  binding  is  of  minor  importance 
in  lipid-protein  interactions.     The  major  force,  therefore,  is  hydrophobic  in 
nature.     A  significant  increase  in  helical  structure  has  been  observed  follow- 
ing the  recombination  of  apolipoproteins  with  lipids.     Models  of  apolipopro- 
teins  in  a-helical  conformation  have  demonstrated  the  presence  of  amphipathic 
surface  areas.     One  surface  is  hydrophilic,  while  the  other  surface  is  hydro- 
phobic in  nature. 

The  combined  results  from  these  and  other  studies  have  permitted  the 
development  of  a  molecular  model  for  HDL.     In  this  model  HDL  is  a  spherical 
micelle  with  the  globular  proteins  visualized  as  "icebergs"  in  a  "sea"  of 
lipid.     The  major  force  involved  in  lipid-protein  interaction  is  hydrophobic 
in  nature.     The  specific  orientation  of  the  A-I  and  A-II  in  the  particle     is 
as  yet  unknown.      Recombination  studies  outlined  above  would  suggest,  however, 
that  protein-protein  interactions  between  A-I  and  A-II  may  be  of  importance 
in  the  molecular  organization  of  HDL.     This  model  is  quite  different  than 
the  "classic"  model  of  lipoproteins  in  which  the  lipid  portion  of  the  par- 
ticle was  conceptualized  as  an  "oil  droplet"  with  the  protein  covering  the 
surface  of  the  droplet.     The  spatial  organization  of  the  neutral  lipids , 
cholesteryl  ester  and  triglyceride  are  as  yet  unknown.     However,  x-ray  dif- 
fraction data  would  suggest  that  they  are  "core"  constituents  of  the  lipo- 
protein particle. 

4)     Objective: 

Determination  of  the  molecular  properties  of  A-I  in  aqueous  solution 
and  in  native  HDL. 

Methods  Employed: 

The  molecular  properties  of  A-I  have  been  evaluated  by  fluorescence, 
difference  absorption  spectroscopy,  and  circular  dichroism. 

Maj  or  Findings : 

Native  A-I  has  a  high  degree  of  helical  content  in  aqueous  solution  at 
neutral  pH.     This  helical  content  is  partially  eliminated  in  acid,  more  ex- 
tensively lost  in  alkali,  and  almost  destroyed  in  1.7M  guanidine  hydrochlo- 
ride.    The  fluorescence  behavior  of  A-I  revealed  a  high  degree  of  ordered 
structure  with  the  major  fluorescence  peak  near  333,  indicating  that  the 


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Project  No.     Z01  HL  02010-05  MDB 

tryptophanyl  residues  are  shielded  from  the  aqueous  environment.  Denatura- 
tion  of  A-I  with  1.  7M  guanidine  hydrochloride  caused  a  normalization  of  the 
fluorescence  spectra.  Thus ,  A-I  behaves  as  a  typical  globular  protein  with 
a  high  degree  of  ordered  structure. 

The  unique  absence  of  tryptophanyl  residues  in  A-II  has  permitted  "the 
direct  analysis  of  the  molecular  behavior  of  A-I   (contains  4  tryptophan 
residues)  in  native  HDL.     Analysis  of  A-I  by  the  procedures  outlined  above 
has  demonstrated  a  marked  increase  in  stability  of  A-I  in  HDL  to  changes  in 
temperature,  pH  and  guanidine  hydrochloride.     Therefore,  the  tertiary  struc- 
ture of  A-I  is  markedly  stabilized  when  incorporated  into  native  HDL.     The 
ndcroenviornment  of  A-I  in  HDL  which  imparts  the  unique  stability  to  the   ■ 
intact  lipoprotein  particle  is  as  yet  unknown.  -  These  forces  may  involve 
polar  phospholipids,  non-polar  lipids  or  other  apolipoproteins .     Further 
studies  will  be  required  to  clarify  the  specificity  of  the  interaction  of 
A-I  with  lipids ,  and  with  other  apolipoproteins . 

5 )  Objective : 

Determination  of  the  molecular  properties  of  A-II  from  HDL. 

Methods  Employed: 

The  evaluation  of  the  molecular  properties  of  A-II  was  performed  by  the 
procedures  outlined  above. 

Major  Findings: 

A  variety  of  physical  studies  have  clearly  indicated  that  A-II ,  similar 
to  A-I,  has  a  high  degree  of  ordered  structure  in  aqueous  solution.     The 
molecule,  however,  is  not  a  rigid  structure,   and  undergoes  extensive  loss  of 
structure  with  extremes  of  pH  and  low  concentrations  of  guanidine  hydro- 
chloride.    A  unique  feature  of  A-II,  however,  was  discovered  which  indicated 
that  A-II  self-associates  under  various  protein  concentrations.     In  the  con- 
centration range  investigated,  A-II  self-associates  to  an  oligomeric  species 
which  is  consistent  with  a  monomer-dimer  equilibrium.     The  self-association 
of  A-II  is  associated  with  significant  increases  in  secondary  and  tertiary 
structure.     Tnis  reversible  association  was  found  to  be  dependent  on  temper- 
atures between  5  and  50  C,  showing  a  maximum  in  association  near  25°C.     The 
observation  that  A-II  self-associates  is  undoubtedly  of  major  importance  in 
the  molecular  organization  of  A-II  in  HDL,  and  may  indicate  that  a  specific 
oligomeric  species  may  be  the  active  molecular  form  which  will  recombine  with 
lipids. 

6)  Objective: 

Evaluation  of  the  binding  of  A-I  to  lysolecithin. 


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Project  No.      Z01  HL  02010-05  MDB 

Methods  Employed: 

Similar  to  those  outlined  above. 

Major  Findings: 

The  molecular  binding  of  A-I  to  lysolecithin  was  investigated  for  two 
reasons:     first  -  A-I  is  known  to  be  a  cofactor  for  the  enzyme  lecithin  cho- 
lesterol acyl  transferase  (LCAT)  which  generates  lysolecithin  following  the 
transfer  of  the  fatty  acid  side  chain  of  lecithin  to  the  hydros/I  group  of 
cholesterol;  second  -  A-I  is  unable  to  bind  PC  or  SPM  to  any  significant  de- 
gree without  the  presence  of  A-II  in  the  recombination  mixture.     A-I  in  these 
studies 7was  able  to  bind  lysolecithin  with  an  association  constant  greater 
than  10   .       The  binding  appears  to  be  to  the  lysolecithin  monomer,  with  ulti- 
mate formation  of  a  lipid-protein  complex  that  could  be  isolated  by  gel  per- 
meation chromatography.     Of  major  interest  was  the  ability  of  lysolecithin  to 
bind  to  A-I  in  the  presence  of  1.  8M  guanidine  hydrochloride.     A  25  amino  acid 
cyanogen  bromide  fragment  of  A-I  also  binds  lysolecithin  as  strongly  as  the 
native  molecule.     These  studies  would,  therefore,  suggest  that  the  binding  of 
A-I  to  lysolecithin  may  be  related  to  the  primary  structure  of  the  protein. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 

This  work  is  directed     toward  a  general  understanding  of  the  molecular 
forces  involved  in  lipid-protein  interactions ,  with  the  ultimate  goal  of 
understanding  the  molecular  architecture  of  the  plasma  lipoproteins.     It  is 
assumed  that  a  fundamental  understanding  of  the  quarternary  structure  of  the 
plasma  lipoproteins  will  permit  more  sophisticated  studies  to  be  performed  on 
the  functions  of  the  circulating  lipoproteins.     The  mode  of  lipid  transport 
of  these  lipoproteins ,  the  metabolism,  and  intercon  vers  ions  of  the  various 
lipoprotein  families  will  be  of  fundamental  importance  in  the  understanding 
of  lipid  transport  and  metabolism  in  normal  individuals ,  and  in  patients  with 
disorders  of  lipid  metabolism  and  atherosclerosis. 

Proposed  Course : 

During  the  next  year  it  is  planned  to  continue  a  detailed  examination  of 
the  molecular  properties  of  the  individual  apoproteins,   and  to  initiate  stu- 
dies on  the  interactions  between  the  apoprotein  both  in  aqueous  solution  and 
in  recombined  particles.     Systematic  studies  will  also  be  continued  on  the 
nature  of  the  lipid  organization  in  recombined  lipoprotein  particles.     The 
molecular  model  for  HDL  presented  in  this  report  will  be  tested  by  a  variety 
of  different  approaches  including  isosteric  modifications  of  the  apolipopro- 
tein  as  well  as  structural  perturbations  of  the  lipid  structure.     It  is 
hoped  in  the  near  future  to  be  able  to  begin  synthetic  studies  on  i±ie  apolipo- 
proteins  which  would  permit  more  detailed  studies  on  the  nature  of  lipid- 
protein  interactions. 

As  in  the  past ,  we  plan  to  continue  our  studies  on  the  isolation  and 

5  6S° 


Project  No.    Z01  HL  02010-05  MDB 

characterization  of  the  human  apolipoproteins ,  as  well  as  the  apolipoproteins 
from  a  variety  of  animal  species.     In  addition,  it  is  planned  to  complete  the 
amino  acid  sequence  of  human  A-I.     retailed  analysis  of  the  different  poly- 
morphic forms  of  A-I  is  nearly  complete.     Theses  studies  will  permit  mors  de- 
tailed analysis  of  the  role  of  polymorphism  in  the  structure  and  immunological 
properties  of  the  plasma  lipoproteins. 

Publications : 

1.  Assmann,  G.  ,     and  Brewer,  H.   B.  ,  Jr.  :     Lipid-protein  interactions  in  high 
density  lipoproteins.     Proc.  Natl.   Acad.   Sci.    71:     989-993,  19 74. 

1  3 

2.  Assmann,  G.  Highet,  R.  J.,  Sokoloski,  E.  A.,  and  Brewer,  H.   B. ,  Jr.:         C 

nuclear  magnetic  resonance  spectroscopy  of  native  and  recombined  lipopro- 
proteins.     Proc.  Natl.   Acad.   Sci.    71:    3701-3705,  1974. 

3.  Assmann,  G.  ,  Sokoloski,  E.   A.  ,  and  Brewer,  H.   B.  ,  Jr.  :       "T  nuclear  mag- 
netic resonance  spectroscopy  of  native  and  recombined  lipoproteins. 
Proc.   Natl.  Acad.   Sci.    71:   549-553,  1974. 

4.  Gwynne,  J. ,  Brewer,  H.  B. ,  Jr. ,  and  Edelhoch,  H. :  The  molecular  proper- 
ties of  apoA-I  from  human  high  density  lipoprotein.  J.  Biol.  Chem.  249: 
2411-2416. 

5.  Assmann,  G.,  and  Brewer,  H.  B.  ,  Jr.  :     A  molecular  model  of  high  density 
lipoproteins.     Proc.  Natl.   Acad.   Sci.    71:     1534-1538,  1974. 

6.  Shulman,  R.   S.  ,  Herbert,  P.   N.  ,  Eredrickson,  D.   S.,  Wehrly,  K.  ,  and 
Brewer,  H.   B.  ,  Jr.  :•     Isolation  and  alignment  of  the  tryptic  peptides  of 
alanine  apolipoprotein,   an  apolipoprotein  from  human  plasma  very  low  den- 
sity lipoproteins.      J.   Biol.    Chem.    249:      4969-4974,   1974. 

7.  Brewer,  H.   B.  ,  Jr. ,  Shulman,  R.  ,  Herbert,  P.  ,  Ronan,  R.  ,  and  Wehrly,  K.  : 
The  complete  amino  acid  sequence  of  alanine  apolipoprotein  (apoC-III),   an 
apolipoprotein  from  human  plasma  very  low  density  lipoproteins.     J.   Biol. 
Chem.    249:      4975-4984,   1974. 

8.  Gwynne,  J.  ,  Palumbo,  G.  ,  Brewer,  H.   B.  ,  Jr. ,  and  Edelhoch,  H. :     The  inter- 
action of  apoA-I  from  human  high  density  lipoproteins  with  lysolecithin. 
J.   Biol.   Chem.  ,  in  press. 

9.  Gwynnne,  J.  ,  Brewer,  H.   B.  ,  Jr.  ,  and  Edelhoch,  H.  :     The  molecular  behavior 
of  apoA-I  in  human  high  density  lipoproteins.     J.   Biol.   Chen.   250:     2269- 
2274,   1975. 

10.      Gwynne,  J.,  Palumbo,  G. ,  Osborne,  J.    C. ,  Brewer,  H.   B.,  Jr.,  and  Edelhoch, 
H. :     The  self- association  of  apoA-II,  an  apoprotein  of  human  high  density 
•    lipoprotein  complexes.     Arch.   Biochem.   Biophys.  ,  in  press. 


w 


Project  No.     Z01  HL  02010-05  MDB 

11.  Osborne,  J.    C.  ,  Palumbo ,  G.  ,  Brewer,  H.   B.  ,  Jr.  ,  and  Edelhoch,  H.  :     The 
self-association  of  the  reduced  apoA-II  apoprotein  from  the  human  high 
density  lipoprotein  complex,  in  press. 

12.  Mahley,  R.   W.  ,  Weisgraber,  K.  H.  ,  Innerarity,  T.  ,  Brewer,  H.   B.  ,  Jr.  , 
and  Assmann,  G.  :     Swine  lipoproteins  and  atherosclerosis.      Changes  in 
the  plasma  lipoproteins  and  apoproteins  induced  by  cholesterol  feeding. 
Biochem. ,     in  press. 


*a> 


Project  No.     Z01  HL  02010-05  MDB 

Key  Words : 

1)  lipid 

2 )  sequence 

3 )  ccn formation 

4)  NMR  (nuclear  magnetic  resonance) 

5 )  lipoprotein 

6)  self-association 

7)  lysolecithin 

8)  lecithin 


£&3 


Annual  Report  of  the 

Molecular  Hematology  Branch 

National  Heart  and  Lung  Institute 

July  1,  1974  through  June  30,  1975 

The  factors  influencing  the  regulation  of  hemoglobin  gene  expression  are 
being  studied  in  human  and  animal  erythroid  cells.   The  objective  is  to  under- 
stand the  molecular  basis  of  hereditary  anemias  (specifically  the  thalassemias 
and  hemoglobinopathies)  in  order  to  be  able  to  devise  effective  methods  for 
treating  these  diseases.   The  general  approach  has  been  to  fractionate  normal 
and  diseased  erythroid  cells  into  the  various  components  required  for  hemoglobin 
synthesis  and  then  to  reconstitute  the  cellular  components  in  such  a  way  as  to 
reproduce  the  activity  of  the  original  intact  cell.   The  function  of  each  cellu- 
lar component  from  the  diseased  cell  can  then  be  compared  with  its  counterpart 
from  a  normal  cell.   The  first  phase  of  this  program  has  been  successfully 
completed,  namely,  the  fractionation  of  the  cytoplasm  of  normal  and  diseased 
reticulocytes  and  the  reconstitution  of  a  cell-free  system  capable  of  dupli- 
cating the  normal  and  disturbed  functions  of  reticulocyte  cytoplasm.   A  de- 
tailed characterization  of  purified  cytoplasmic  components  (initiation  factors, 
elongation  factors,  ribosomes,  messenger  RNA,  etc)  is  being  carried  out  in  order 
to  understand  fully  the  mechanism  of  protein  synthesis  in  mammalian  cells. 

Fractionation  of  the  nucleus  of  erythroid  precursors  is  the  second  phase 
of  the  program.   During  the  past  year  the  cell-free  system  which  is  capable 
of  transcribing  globin  messenger  RNA  sequences  from  bone  marrow  chromatin  has 
been  refined.   Although  this  assay  is  still  relatively  crude,  it  should  allow 
a  search  for  the  factors  involved  in  the  regulation  of  the  synthesis  of 
specific  globin  messenger  RNA. 

The  Molecular  Hematology  Branch  has  as  its  goal  the  treatment  of  human 
genetic  diseases,  specifically,  thalassemia  and  sickle  cell  anemia.   Several 
approaches  have  been  initiated:   First,  as  described  above,  is  the  complete 
fractionation  of  the  erythroblast  so  as  to  allow  a  study  of  the  factors  in- 
volved in  messenger  RNA  and  protein  synthesis.   With  this  knowledge  ways  will 
be  sought  for  influencing  globin  messenger  RNA  production  in  thalassemic  cells. 
Second,  the  mechanism  of  globin  gene  switching  is  under  active  investigaton 
using  the  animal  model  system  of  the  hemoglobin  A  to  hemoglobin  C  gene  switch 
in  sheep  and  goats.   The  goal  is  to  learn  the  mechanism  of  this  switch  in 
order  to  understand  how  to  obtain  adult  to  fetal  gene  switching  in  patients 
who  have  normal  fetal  but  abnormal  adult  hemoglobin.   Third,  somatic  cell 
hybrids  generated  by  fusion  of  erythroid  cells  and  established  cell  lines 
have  been  used  in  an  attempt  to  establish  replicating  cells  capable  of  globin 
synthesis.   These  hybrids  are  being  utilized  to  study  regulation  of  expression 
of  the  globin  genes.   In  addition,  human-mouse  hybrids  containing  one  or  more 
human  chromosomes  are  being  studied  in  order  to  determine  the  localization  of 
the  human  alpha  and  beta  globin  structural  genes. 

The  Clinical  Service  of  the  Molecular  Hematology  Branch,  opened  two  years 
ago,  has  greatly  expanded.   Patients  with  various  blood  cell  disorders  in- 
cluding thalassemia,  sickle  cell  anemia  and  other  hemolytic  anemias  are 

i  ^es* 


diagnosed,  treated  and  studied.   Several  clinical  protocols  are  under  investi- 
gation. 

A  summary  of  our  results  obtained  over  the  past  twelve  months  for  pro- 
jects continued  from  past  years  follows: 

1)  Initiation  factors:   An  intensive  effort  continues  to  be  made  to 
identify,  purify,  and  characterize  the  eukaryotic  initiation  and  elongation 
factors.   Of  the  seven  known  initiation  factors,  five  have  been  purified  to 
homogeneity  and  have  been  fully  characterized.   An  intensive  effort  is  now 
under  way  to  locate  possible  messenger  RNA  specific  initiation  factors  which 
might  play  a  role  in  translational  control  of  protein  synthesis. 

2)  Mechanism  of  hemoglobin  initiation:   As  the  various  initiation  and 
elongation  factors  have  been  purified  they  have  been  utilized  to  determine  the 
exact  step-by-step  mechanism  of  hemoglobin  synthesis.   The  overall  picture  of 
initiation  of  eukaryotic  protein  synthesis  is  now  reasonably  well  established. 
The  essential  role  of  initiation  factor  MP  has  been  elucidated. 

3)  Molecular  basis  of  thalassemia:   Patients  with  various  types  of 
thalassemia  have  been  studied  and  shown  to  have  a  quantitative  decrease  in 
the  amount  of  messenger  RNA  for  the  affected  globin  chain.   Messenger  RNA 
from  these  patients  have  been  utilized  to  prepare  human  globin  chain  comple- 
mentary DNA  for  several  studies  (see  section  6  and  7,  below). 

4)  Hemoglobin  A  ->  C  switch  in  sheep  and  goats:   Sheep  and  goat  bone 
marrow  cells  capable  of  generating  erythroid  colonies  in  tissue  culture  have 
been  separated  by  unit  gravity  sedimentation.   The  population  of  colony  forming 
cells  responsible  for  the  A  ->  C  switch  appears  to  have  been  identified. 
Utilizing  the  experience  gained  in  tissue  culture  of  sheep  and  goat  erythroid 
cells,  human  erythroid  colonies  have  been  grown  from  bone  marrow  of  patients 
with  various  hemolytic  anemias.   The  concepts  and  techniques  developed  for 
study  of  A  ->  C  switch  in  cells  of  sheep  and  goats  are  being  applied  to  the 
study  of  regulation  of  human  hemoglobin  synthesis. 

5)  Synthesis  of  globin  DNA  gene:   The  mechanism  of  action  of  the  viral 
enzyme,  RNA-directed  DNA  polymerase,  is  under  investigation.  Using  short 
single-base  oligonucleotides  of  defined  length  it  has  been  shown  that  the 
enzyme  transcribes  various  ribonucleotide  bases  in  different  ways:   slippage 
with  oligo(rA),  faithful  transcription  with  oligo(rC),  and  poor  transcription 
with  oligo(rU)  and  oligo(rG).   This  variability  in  enzyme  behavior  may 
account  for  the  fact  that  the  enzyme  does  not  transcribe  natural  messenger 
RNA  templates  completely. 

6)  Cell-free  synthesis  of  globin  messenger  RNA:   A  hybridization  assay 
has  been  devised  which  reproducibly  quantitates  globin  messenger  RNA  sequences 
transcribed  in   vitro  from  bone  marrow  chromatin  using  the  enzyme  RNA  poly- 
merase.  This  assay  system  should  permit  the  systematic  investigation  of  the 
variables  affecting  specificity  of  in  vitro  transcription.   Chromatin  from 
human  bone  marrow  can  now  be  utilized  for  studies  to  quantitate  the  synthesis 
of  alpha  and  beta  globin  messenger  RNA  sequences. 

7)  Globin  gene  expression  in  somatic  cell  hybrids:   Hybrid  cells  have 
been  obtained  from  the  fusion  of  human  marrow  cells  with  Friend  mouse  erythro- 
leukemia  cells  which  exhibit  either:   co-expression  of  human  and  mouse  globin, 


6S6 


expression  of  mouse  globin  only,  or  total  absence  of  glob in  gene  expression. 
In  one  somatic  cell  hybrid,  human  beta  globin  messenger  RNA  but  no  human 
alpha  globin  messenger  RNA  was  detected.  These  data  indicate  that  the  alpha 
and  beta  globin  structural  loci  are  on  separate  chromosomes.  In  addition, 
somatic  hybrid  cells  have  been  generated  which  contain  a  complete  complement 
of  rodent  chromosomes  but  only  one  or  more  human  chromosomes.  These  hybrids 
are  being  utilized  with  the  technique  of  complementary  DNA-DNA  hybridization 
to  localize  the  human  alpha  and  beta  structural  globin  genes. 

8)   Clinical  investigations.   (a)   Iron  chelation  therapy  in  trans- 
fusional  hemosiderosis  is  being  evaluated  using  the  agent  Desferal  administered 
either  with  or  without  ascorbic  acid.   It  has  been  shown  that  iron-loaded 
patients  can  be  placed  in  negative  iron  balance  with  Desferal  and  that  this 
can,  under  some  circumstances,  be  markedly  enhanced  by  the  simultaneous 
administration  of  ascorbic  acid.   (b)   Cardiac  hemolytic  anemia.   The  degree 
of  anemia  has  been  shown  to  have  no  consistent  effect  on  the  hemolytic 
rate  in  patients  with  cardiac  hemolysis.   In  addition,  several  patients  have 
been  shown  to  have  mild  or  moderate  hemolysis  but  severe  anemia;  this  re- 
flects inadequate  bone  marrow  compensation. 

A  report  on  the  new  programs  established  during  the  past  12  months 
follows: 

1)  Regulation  of  the  respiratory  function  of  blood.   A  program  has  been  esta- 
blished which  is  aimed  at  understanding  the  mechanisms  which  regulate  the 
respiratory  function  of  blood  at  molecular,  cellular  and  physiologic 
levels.   Structure-function  relationships  are  studied  in  normal  and  mutant 
human  hemoglobins.   A  new  hemoglobin  variant  has  been  discovered  which 
appears  to  be  the  first  human  protein  whose  structural  alteration  has 
arisen  by  mutation  which  corresponds  to  a  "nonsense"  mutation  in  bacteria. 
Two  amino  acids  are  lacking  at  the  mutant's  carboxyl  terminal  end.   This 
hemoglobin  has  the  highest  oxygen  affinity  of  any  known  human  variant, 
nonetheless  carriers  of  it  are  asymptomatic. 

2)  Evaluation  of  alteration  of  blood  oxygen  affinity  as  the  basis  for  the 
treatment  of  sickle  cell  anemia.   Thiszrecently  established  project  is 
designed  to  study  the  relationship  between  sickling  and  the  oxygen 
transport  of  blood.   Patients  with  sickle  cell  anemia  are  admitted  to  the 
Clinical  Center  and  studied  intensively  using  a  number  of  biochemical  and 
physiologic  techniques. 


rt7 


Project  No.   Z01  HL  02201-03  MHB 
1.  Molecular  Hematology  Branch 
2. 
3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Mechanism  of  hemoglobin  biosynthesis  in  cell-free  systems 

Previous  Serial  Number:   NHLI-88 

Principal  Investigator:   William  C.  Merrick,  Ph.D. 

Other  Investigators:   Brian  Safer,  M.D. ,  Ph.D. 
Sherrill  Adams,  Ph.D. 
Wayne  Kemper,  M.A. 
Irving  London,  Ph.D. 
W.  French  Anderson,  M.D. 

Cooperating  Units:   Molecular  Disease  Branch,  NHLI 

Laboratory  of  Nutrition  and  Endocrinology,  NIAMDD 
Development  Immunology  Branch,  NICHHD 
Roche  Institute  of  Molecular  Biology 
Massachusetts  Institute  of  Technology 

Project  Description: 

Objectives:   The  mechanism  and  regulation  of  the  initiation  of  mammalian  pro- 
tein synthesis  is  being  .investigated  by  utilizing  artificial  template  or  hemo- 
globin mRNA-directed  protein  biosynthesis.   Components  from  the  rabbit  reticulo- 
cyte have  been  fractionated  and  highly  purified.   Reactions  of  either  the 
separate  components  or  combinations  of  components  are  being  studied  to  eluci- 
date the  sequential  steps  involved  in  protein  synthesis. 

Methods:   Purification  of  separate  components  has  been  achieved  using  standard 
techniques  such  as  ion-exchange  column  chromatography,  gel  filtration,  salt 
fractionation,  and  sucrose  density  gradient  centrifugation.   Physical  char- 
acterization of  initiation  and  elongation  factors  (IF  and  EF,  respectively) 
have  been  performed  using  gel  electrophoresis  in  various  buffers,  gel  fil- 
tration, analytical  centrifugation,  and  amino  acid  analysis.   Biological 
characterization  of  components  of  protein  biosynthesis  has  been  developed  or 
modified  from  techniques  described  in  the  literature. 

Ma j or  Findings :   1)  IF-M2A  has  been  purified  to  homogeneity  and  characterized. 
IF-M2A  is  active  as  a  single  polypeptide  chain  of  molecular  weight  125,000. 
The  amino  acid  composition  is  unusual  in  that  glutamic  acid  (plus  glutamine) 
constitutes  18.8  mole  percent  while  cysteine  and  tryptophan  consititute  only 
0.7.  and  0.4  mole  percent,  respectively.   IF-M2A  has  a  frictional  ratio  of 
1.66  and  a  pi  of  6.45.   The  expression  of  GTPase  activity  by  IF-M2A  has  been 
shown  to  require  both  40S  and  60S  subunits. 

1  £21 


Project  No.  Z01  HL  02201-03  MHB 

2)  IF-M2Ba  has  been  purified  to  homogeneity.   IF-M2Ba  self  aggregates  at  pro- 
tein concentrations  greater  than  0.1  mg/ml  and  consequently  limited  physical 
studies  have  been  possible.   IF-M2Ba  has  molecular  weight  of  16,500  and  is 
active  as  a  single  polypeptide  chain.   Although  the  precise  role  for  either 
IF-M2Ba  or  IF-M2B8  in  protein  synthesis  is  still  unclear,  these  proteins 
appear  to  be  involved  mostly  with  subunit  joining  and  not  with  binding  of  the 
initiator  tRNA. 

3)  IF-MP  has  been  purified  to  homogeneity.   This  initiation  factor  is  dif- 
ferent from  the  others  which  have  been  purified  in  this  laboratory  in  that  it 
is  the  only  one  composed  of  subunits  (35,000  and  55,000  daltons).   Physical 
studies  indicate  that  IF-MP  may  exist  as  a  dimer  (90,000  daltons)  or  a 
tetramer  (180,000  daltons);  however,  the  biologically  active  form  has  not  been 
determined.   IF-MP  forms  a  ternary  complex  with  GTP  and  initiator  tRNA  which 
can  be  subsequently  bound  to  40S  subunits. 

4)  A  comparison  of  the  initiation  factors  which  have  been  physically  char- 
acterized indicate  several  common  features:   1)  IF-M1,  IF-M2A,  and  IF-MP  all 
have  frictional  ratios  greater  than  1.5;   2)  the  ratio  of  lys:his:arg  is  , 
approximately  10:2:5  with  the  total  basic  amino  acid  content  being  15  to  20 
mole  percent;  3)  all  of  these  factors  are  inactivated  by  N-ethylmaleimide. 

5)  A  complex  of  initiator  tRNA  and  40S  subunits  can  be  formed  either  by  IF-MP 
or  IF-M1.   This  40S  complex  subsequently  requires  IF-M2A,  IF-M2B,  60S  subunits 
and  GTP  to  generate  an  80S  initiation  complex  which  can  react  with  puromycin 
(however,  the  template  AUG  must  also  be  present  during  the  formation  of  the 
40S  or  80S  complex).   Similar  studies  utilizing  salt-washed  ribosomes  with 
endogenous  mRNA  indicate  that  IF-MP,  but  not  IF-M1,  is  capable  of  generating  a 
puromycin  sensitive  80S  complex. 

6)  IF-MP  has  been  shown  to  be  able  to  relieve  the  inhibition  of  globin  syn- 
thesis in  crude  lysate  systems  which  contain  either  double-stranded  RNA  or 
oxidized  glutathione  or  which  are  hemin-def icient.   The  inhibition  of  globin 
synthesis  does  not  appear  to  be  a  direct  effect  on  IF-MP,  but  rather  an  in- 
direct effect  mediated  by  a  protein  which  is  present  only  at  times  when  globin 
synthesis  is  inhibited.   IF-MP  is  also  capable  of  selectively  binding  globin 
mRNA  and  poly(A)  (in  addition  to  Met-tRNAf ) .   This  protein  has  also  been  iso- 
lated from  mRNP  particles  suggesting  that  IF-MP  may  play  a  role  in  both  mRNA 
and  Met-tRNA  recognition  in  the  formation  of  an  initiation  complex. 

7)  Treatment  of  rabbit  reticulocyte  EF-1  with  either  phospholipase  C  or  phos- 
pholipase  AB  causes  a  change  in  the  molecular  weight  of  EF-1  from  800,000  to 
200,000  without  loss  of  biological  activity.   This  observation  supports  the 
suggestion  that  rabbit  reticulocyte  EF-1  (like  EF-1  from  other  tissues)  is  com- 
posed of  subunits  (in  this  case  47,000  and  50,000  daltons)  which  are  held  to- 
gether to  some  extent  by  phospholipids. 

Significance  to  Biomedical  Research  and  Institute  Program 

An  understanding  of  the  regulation  of  gene  expression  is  essential  to  evaluate 
normal  and  abnormal  cell  function.   The  observation  that  IF-MP  may  play  some 
regulatory  role  in  globin  biosynthesis  lends  support  to  the  idea  that  regu- 
lation of  the  initiation  of  protein  synthesis  may  be  an  important  part  of 
globin  gene  expression.   Similar  mechanisms  may  also  be  operative  in  other 
tissues.  o  W° 


Project  No.  Z01  HL  02201-03  MHB 

Proposed  Course  of  Project:   We  plan  to  continue  purification  and  characteri- 
zation of  those  factors  which  have  not  been  fully  evaluated.   With  the  homo- 
geneous factors  available  and  those  to  be  obtained,  studies  with  exogenous  mRNA 
are  planned  to  explore  fully  the  differences  in  factor  requirements  for  protein 
synthesis  initiation  with  natural  mRNA  as  opposed  to  artificial  templates.   In 
addition,  studies  are  planned  to  examine  the  differences  between  mRNA  and  mRNP 
particles  as  well  as  factors  which  might  effect  the  ratio  of  a  and  g  globin 
chain  synthesis. 

Keyword  Descriptors:   reticulocyte,  initiation  factors,  elongation  factors, 
protein  biosynthesis,  protein  purification,  molecular  weight,  methionyl- 
puromycin,  hemin  regulation,  mRNA,  poly(A). 

Honors  and  Awards :   None 

Publications: 

1.  Anderson,  W.F.:   Cell-free  synthesis  of  globin  chains:   An  overview.  '  Ann. 

N.Y.  Acad.  Sci.  _241:  142-155,  1974. 

2.  Elson,  N.A. ,  Brewer,  B. ,  and  Anderson,  W.F. :   Hemoglobin  switching  in  sheep 

and  goats:  III.  Cell-free  initiation  of  sheep  globin  synthesis.   J.  Biol. 
Chem.  249:  5227-5235,  1974. 

3.  Anderson,  W.F.,  Bosch,  L. ,  Gros,  F. ,  Grunberg-Manago,  M. ,  Ochoa,  S.,  Rich, 

A. ,  and  Staehelin,  T. :   Initiation  of  protein  synthesis  in  prokaryotic  and 
eukaryotic  systems.   FEBS  Lett.  4^:  1-6,  1974. 

4.  Merrick,  W.C.,  and  Anderson,  W.F. :   Purification  and  characterization  of 

homogeneous  protein  synthesis  initiation  factor  Ml  from  rabbit  reticulo- 
cytes.  J.  Biol.  Chem.  250:  1197-1206,  1975. 

5.  Merrick,  W.C.,  Kemper,  W.M. ,  Kantor,  J. A. ,  and  Anderson,  W.F.:   Purifi- 

cation and  properties  of  rabbit  reticulocyte  protein  synthesis  elongation 
factor  2.   J.  Biol.  Chem.  250:  2620-2625,  1975. 

6.  Clemens,  M.J.,  Safer,  B.,  Merrick,  W.C.,  Anderson,  W.F.,  and  London,  I.M.  : 

Initiation  of  protein  synthesis  in  rabbit  reticulocytes  by  double-stranded 
RNA  and  oxidized  glutathione:   Evidence  for  an  indirect  mode  of  action  on 
polypeptide  chain  initiation.   Proc.  Nat.  Acad.  Sci.  USA  (in  press). 

7.  Merrick,  W.C.,  Kemper,  W.M. ,  and  Anderson,  W.F. :   Purification  and  charac- 

terization of  homogeneous  initiation  factor  M2A  from  rabbit  reticulocytes. 
J.  Biol.  Chem.  (in  press). 


6V 


Project  No.  Z01  HL  02202-03  MHB 
1.  Molecular  Hematology  Branch 
2. 
3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Evolution  of  the  Protein  Synthesizing  Machinery 

Previous  Serial  Number:   NHLI-89 

Principal  Investigator:   William  C.  Merrick,  Ph.D. 

Other  Investigators:   Norton  Elson,  M.D. 

Brian  Safer,  M.D.,  Ph.D. 
Sherrill  Adams,  Ph.D. 
Severo  Ochoa,  Ph.D. 
W.  French  Anderson,  M.D. 

Cooperating  Units:   Roche  Institute  Molecular  Biology 

Project  Description: 

Objectives:   To  evaluate  the  interchangeability  of  components  from  various 
organisms  which  are  required  for  protein  biosynthesis  and  thus  determine  the 
level  of  evolutionary  homology  in  both  the  components  and  mechanisms  of 
protein  biosynthesis. 

Methods:   Protein  and  nucleic  acid  components  from  different  sources  are  iso- 
lated and  compared  in  cell-free  assay  systems. 

Ma j or  Findings :   1)  A  comparison  has  been  made  of  the  ability  of  the  formylated 
and  unformylated  initiator  tRNAs  of  E^  coli  and  rabbit  liver  to  participate  in 
a  number  of  model  reactions  of  protein  synthesis.   The  results  indicate  that 
the  prokaryotic  initiator  tRNA  species  functions  efficiently  in  partial  re- 
actions which  involve  only  binding,  but  that  the  methionine  donated  by  the  pro- 
karyotic tRNA  is  not  incorporated  efficiently  into  peptide  linkage.   The  effect 
of  formylation  on  the  effectiveness  of  the  initiator  tRNA  is  not  clear;  it  re- 
duces activity  in  ternary  complex  formation,  does  not  affect  AUG-directed 
binding  to  40S  subunits,  and  increases  the  rate  or  extent  of  incorporation  of 
methionine,  or  both,  into  methionyl-puromycin  and  globin  chains. 

2)   In  the  reticulocyte  lysate,  AUG-directed  methionyl-puromycin  synthesis  re- 
quires methionyl-tRNAf,  40S  and  60S  subunits,  IF-M1,  IF-M2A,  IF-M2B  and  GTP. 
In  the  Artemia  salina   (brine  shrimp)  system,  AUG-directed  methionyl-puromycin 
synthesis  requires  only  methionyl-tRNAf,  40S  and  60  S  subunits,  and  EIF-1  (an 
initiation  factor  corresponding  to  IF-M1) .   In  crosses  between  these  two  sys- 
tems, it  has  been  shown  that  the  requirement  for  IF-M2A,  IF-M2B  and  GTP  is  due 
to  the  differences  between  rabbit  and  Artemia  salina  60S  subunits.   When  rabbit 
reticulocyte  60S  subunits  are  used  with  either  rabbit  or  Artemia  salina  40S 


Project  No.ZOl  HL  02202-03  MHB 

subunits  (with  either  IF-M1  or  EIF-1) ,  IF-M2A,  IF-M2B  and  GTP  are  required. 
IF-M2A,  IF-M2B,  and  GTP  are  not  required  if  Artemia  salina  60S  subunits  are 
used.   These  results  suggest  that  Artemia  salina  60S  subunits  contain  proteins 
corresponding  to  IF-M2A  and  IF-M2B. 

Significance  to  Biomedical  Research  and  Institute  Program:   The  ability  of 
prokaryotic  components  to  interact  in  eukaryotic  protein  synthesis  details 
both  the  ways  in  which  the  process  is  similar  as  well  as  the  manner  in  which 
they  are  distinct.   The  ability  to  distinguish  between  bacterial  and  mammalian 
cell  functions  is  a  large  part  of  the  basis  for  the  formulation  of  therapeutic 
treatment  of  bacterial  infection.   The  continued  study  of  such  differences  as 
well  as  differences  between  eukaryotic  organisms  may  lead  to  new  and  unique 
treatments  for  human  diseases  (bacterial,  viral  or  parasitic). 

Proposed  Course  of  Project:   The  species  specificity  of  the  cell  component  re- 
quired for  protein  biosynthesis  will  continue  to  be  investigated  using  both 
artificial  and  natural  mRNA  templates. 

Keyword  Descriptors:  .  evolution,  E^  coli,  rabbit  reticulocyte,  Met-tRNA  , 
fMet-tRNAf,  initiation  complex,  globin  biosynthesis,  methionyl-puromycin, 
initiation  factors. 

Honors  and  Awards :   None 

Publications: 

1.  Elson,  N.A.,  Adams,  S.L.,  Merrick,  W.C.,  Safer,  B.S.  ,  and  Anderson,  W.F.: 
Comparison  of  fMet-tRNAf  and  Met-tRNAf  from  E_;_  coli  and  rabbit  liver  in 
initiation  of  hemoglobin  synthesis.   J.  Biol.  Chem.  250:  3074-3079,  1975. 

2.  Nombela,  C. ,  Nombela,  A.,  Ochoa,  S.,  Merrick,  W.C.,  and  Anderson,  W.F. : 
Nature  of  eukaryotic  proteins  required  for  joining  of  40S  and  60S  ribo- 
somal  subunits.   Biochem.  Biophys.  Res.  Commun.  6^3:  409-416,  1975. 


693 


Project  No.  Z01  HL  02205-02  MHB 
1.   Molecular  Hematology  Branch 
2. 
3.   Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Mechanism  of  Action  of  the  Enzyme  RNA-Directed  DNA  Polymerase 

Previous  Serial  Number:   NHLI-92 

Principal  Investigator:   Gary  Weiss,  M.D.,  Ph.D. 

Other  Investigators:   Judith  Kantor,  Ph.D. 
Amy  Falvey,  Ph.D. 
W.  French  Anderson,  M.D. 

Cooperating  Units:   Viral  Oncology  Branch,  NCI 

Project  Description: 

Objectives:   The  enzyme  RNA-directed  DNA  polymerase,  also  known  as  Reverse 
Transcriptase,  from  avian  myeloblastosis  virus  (and  other  sources)  provides  a 
possible  means  for  synthesizing  a  DNA  gene  directly  from  isolated  messenger 
RNA.   Unfortunately,  the  DNA  product  so  far  obtained  is  only  a  partial  copy  of 
the  messenger  RNA  template.   It  is  desired,  therefore,  to  learn  the  mechanism 
of  action  of  the  enzyme  in  order  to  establish  conditions  whereby  a  complete 
(and  active)  DNA  gene  can  be  transcribed  from  a  globin  messenger  RNA. 

Methods:   The  enzyme  is  purified  by  standard  procedures  from  avian  myeloblas- 
tosis virus  obtained  through  the  National  Cancer  Institute.   Globin  messenger 
RNA  is  isolated  from  rabbit  reticulocytes;  DNA  product  analysis  is  by  CeSO, 
density  gradient  centrifugation.   Primers  and  single  base  oligonucleotides  are 
prepared  by  enzymatic  and/or  chemical  means  followed  by  purification  by 
standard  nucleic  acid  fractionation  techniques.   Artificial  block  polynucleo- 
tide templates  are  being  synthesized  by  means  of  various  enzymatic  and  chemical 
techniques  including  use  of  the  enzyme  polynucleotide  phosphorylase. 

Major  Findings:   1)  The  synthesis  of  DNA  products  complementary  to  a  range  of 
artificial  templates  by  the  enzyme  RNA-directed  DNA  polymerase  has  been  studied. 
Of  the  single  base  ribopolynucleotides,  poly(C),  poly(A),  and  poly(I)  were 
active  while  poly(G)  and  poly(U)  were  almost  inactive.   Of  the  deoxypolynucleo- 
tides  tested,  only  poly(dC)  showed  significant  activity. 

2)   In  order  to  examine  the  fidelity  of  transcription,  single  base  oligonucleo- 
tides of  defined  length  were  studied.   The  minimum  length  showing  activity  for 
an  oligo(rC)  template  was  9;  the  minimum  primer  length  of  oligo(dG)  was  3  or  4. 
Using  (rC)^3  as  template  and  (dG)g  as  primer,  the  oligo(dG)  product  coelectro- 
phoresed  with  the  template.   However,  using  (rA) 20  as  template  and  (dT)^o  as 
primer,  a  very  large  (10-16S)  product  was  formed.   No  significant  activity  was 
obtained  with  oligo(rU)  templates.   It  therefore  appears  that  RNA-directed  DNA 
polymerase  transcribes  the  various  ribonucleotide  bases  in  different  ways: 
slippage  with  oligo(rA),  faithful  transcription  with  oligo(rC),  and  poor 


Project  No.  Z01  HL  02205-02  MHB 

transcription  with  oligo(rU). 

3).  The  Km  has  been  determined  for  each  substrate.  By  increasing  the  concen- 
tration of  nucleotide  triphosphate  in  the  reaction  mixture  a  higher  percentage 
of  large  (10S)  cDNA  can  be  obtained.  Studies  to  determine  if  this  10S  cDNA  is 
a  faithful  copy  of  the  mRNA  template  are  underway. 

Significance  to  Biomedical  Research  and  Institute  Program:   The  ability  to 
synthesize  a  DNA  gene  jin  vitro  would  be  a  major  step  towards  the  goal  of 
successful  therapy  of  human  genetic  diseases. 

Proposed  Course  of  the  Project:   Detailed  studies  on  the  mechanism  of  action 
of  the  enzyme  RNA-directed  DNA  polymerase  are  being  carried  out. 

Keyword  Descriptors:  Reverse  transcriptase,  complementary  DNA,  messenger  RNA, 
enzyme  kinetics,  oligonucleotides 

Honors  and  Awards :   None 

Publications: 

1.   Falvey,  Amy  K. ,  Kantor,  Judy  A.,  Robert,  M.G.,  Picciano,  Dante  J.,  Weiss, 
Gary  B. ,  Vavich,  Joel  M. ,  and  Anderson,  W.  French:   Mechanism  of  action 
of  RNA-directed  DNA  polymerase.   I.  Transcription  of  globin  messenger  RNA. 
J.  Biol.  Chem.  249:  7049-7056,  1974. 


6fr 


Project  No.ZOl  HL  022Q6-Q2  MHB 
1.  Molecular  Hematology  Branch 
2. 
3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Mechanism  of  Globin  Messenger  RNA  Transcription  in  Bone 
Marrow  Cells 

Previous  Serial  Number:   NHLI-107 

Principal  Investigators:   Alan  W.  Steggles,  Ph.D. 

Golder  Wilson,  M.D.,  Ph.D. 

Other  Investigators:   Judith  Kantor,  Ph.D. 
Arthur  Nienhuis,  M.D. 
W.  French  Anderson,  M.D. 
Joseph  E.  Pierce,  D.V.M. 
Leonard  Stuart 

Cooperating  Units:   Section  on  Laboratory  Animal  Medicine  and  Surgery,  NIH 
Ungulate  Unit,  NIH  Animal  Center 

Project  Description: 

Objectives:   The  objective  of  this  project  is  to  determine  the  normal  mechanism 
of  regulation  of  globin  messenger  RNA  synthesis  in  bone  marrow  cells.   Specific 
regulatory  factors,  either  protein  or  RNA  molecules,  will  be  sought  which  in- 
fluence the  transcription  of  the  individual  human  or  sheep  globin  genes.   These 
studies  will  be  applied  to  chromatin  from  cells  of  patients  with  beta  thalas- 
semia to  provide  further  insight  into  molecular  defect  in  this  disease. 

Methods:  Young  sheep  are  the  source  of  bone  marrow  and  other  tissues.  Chromatin 
and  chromatin  fractions  (histones,  acidic  proteins,  DNA)  are  obtained  by 
standard  biochemical  techniques.   DNA-dependent  RNA  polymerase  is  purified  from 
sheep  liver.   Messenger  RNA  is  detected  by  hybridization  with  globin  comple- 
mentary DNA.   Detection  of  the  hybrid  is  by  analysis  with  single  strand 
specific  nuclease.   Alternatively,  after  purification  of  the  cDNA*RNA  hybrid 
by  preliminary  ribonuclease  digestion  and  hydroxy lapatite  chromatography,  it  is 
detected  by  cesium  sulfate  buoyant  density  centrif ugation.   The  chromosomal 
proteins  are  dissociated  from  DNA  by  high  salt  in  concentrated  urea  and  the 
non-histone  protein  fraction  purified  by  ion  exchange  chromatography.   These 
non-histone  proteins  are  reconstituted  to  liver  chromatin. 

Maj or  Findings :    1)  Globin  gene  transcription  by  bacterial  polymerase  has  been 
definitively  confirmed  by  demonstration  of  labeled  RNA  sequences  which  are 
synthesized  ^n  vitro  and  hybridize  to  globin  cDNA. 

2)   A  hybridization  assay  has  been  devised  which  reproducibly  quantitates 
globin  sequences  permitting  systematic  investigation  of  the  variables  affecting 
the  specificity  of  in  vitro  transcription. 

l  616 


< 


Project  No.  Z01  HL  02206-02  MHB 

3)   Initial  evidence  suggests  that  RNA  transcription  in  vivo  is  asymmetrical 
(only  one  DNA  strand  is  transcribed)  but  that  the  ±a   vitro  reaction  with 
bacterial  polymerase  appears  to  be  symmetrical  in  that  there  is  transcription 
of  both  the  globin  gene  and  the  DNA  strand  complementary  to  it. 

Significance  to  Biomedical  Research  and  Institute  Program:   Understanding 
the  mechanism  of  messenger  RNA  transcription  in  eucaryotic  cells  is  essential 
to  understanding  gene  action. 

Proposed  Course  of  the  Project:   Reconstitution  of  bone  marrow  non-histone 
proteins  to  non-erythroid  chromatin  may  provide  a  means  to  assay  specific  non- 
histone protein  fractions  for  regulatory  factors.   Purification  of  the  globin 
genes  by  fractionation  of  chromatin  will  be  pursued  in  an  effort  to  obtain  DNA 
fragments  also  containing  sequences  of  nucleotides  which  may  interact  with 
regulatory  proteins. 

Keyword  Descriptors:   Gene  regulation,  histones,  non-histone  chromosomal 
proteins,  chromatin,  nucleic  acid  hybridization,  RNA  polymerase,  messenger  RNA 
synthesis,  globin  genes  erythroid  cells. 


Honors  and  Awards:   None 
Publications: 

1.  Anderson,  W.F. ,  Steggles,  A.,  Wilson,  G.,  Kantor,  J.,  Velez,  R., 
Picciano,  D.,  Falvey,  A.,  and  Nienhuis,  A.:   Cell-free  transcription 

of  human  bone  marrow  chromatin.   Ann.  N.Y.  Acad.  Sci.  241:566-581,  1974. 

2.  Anderson,  W.F.,  Barker,  J.E.,  Elson,  N.A. ,  Merrick,  W.C.,  Steggles,  A.W. 
Wilson,  G.N.,  Kantor,  J. A.,  and  Nienhuis,  A.W. :   Activation  and  inacti- 
vation  of  genes  determining  hemoglobin  pheno types.   J.  Cell  Physiol. 
85:477-494,  1975. 


197 


Project  No.  Z01  HL  0220Z-Q2  MHB 

1.  Molecular  Hematology  Branch 

2. 

3.  Bethesda,  Maryland  20014 

PHS-NHLI 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Globin  Gene  Expression  in  Somatic  Cell  Hybrids 

Previous  Serial  Number:   NHLI-94 

Principal  Investigators:   Albert  Deisseroth,  M.D. ,  Ph.D. 

Arthur  W.  Nienhuis,  M.D. 

Other  Investigators:    Ramon  Velez,  M.D. 
Jane  Barker,  Ph.D. 
W.  French  Anderson,  M.D. 

Cooperating  Units:   Laboratory  of  Biochemical  Genetics,  NHLI 
Department  of  Biology,  Yale  University 

Project  Description: 

Objectives:   Somatic  cell  hybrids,  generated  by  fusion  of  erythroid  cells  and 
established  cell  lines,  will  be  used  to  obtain  continuously  replicating  cells 
capable  of  globin  synthesis.   These  would  then  be  utilized  to  study  regulation 
of  expression  of  the  globin  genes.   In  addition,  human-mouse  hybrids  containing 
one  or  more  human  chromosomes  would  be  studied  for  localization  of  human  globin 
structural  genes. 

Methods:   Erythroid  cells  are  obtained  from  human  and  animal  bone  marrow,  mouse 
spleen,  or  fetal  liver  and  purified  by  differential  immune  lysis,  buoyant 
density  centrif ugation,  or  unit  gravity  sedimentation.   These  cells  are  fused 
to  established  cell  lines  using  inactivated  Sendai  virus.   Selective  media  are 
constructed  which  favor  the  growth  of  the  hybrid  cells  over  the  parent  lines 
and  the  hybrid  nature  of  the  resulting  clones  is  confirmed  by  analysis  of  the 
chromosomal  composition  and  isozyme  content.   Globin  messenger  RNA  is  detected 
by  hybridization  of  the  hybrid  cell  RNA  to  appropriate  complementary  DNA 
probes  and  the  presence  of  globin  is  detected  by  radioactive  precursor  incor- 
poration.  Gene  localization  studies  require  DNA-cDNA  hybridization  using 
fractionated  and  species  specific  globin  cDNA  probes. 

Major  Findings:   1)  Hybrids  were  obtained  from  fusion  of  human  marrow  cells  with 
Friend  mouse  erythroleukemia  cells  which  exhibit  either:  co-expression  of  human 
and  mouse  globin,  expression  of  mouse  globin  only,  or  total  absence  of  globin 
gene  expression.   Two  of  the  human  X  mouse  hybrid  cell  lines  exhibited  DMSO 
induction  of  both  human  and  mouse  globin  synthesis  as  determined  by  annealing 
of  the  cellular  RNA  to  the  specific  mouse  and  human  globin  complementary  DNAs 
(cDNA) .   Alpha  enriched  and  beta  enriched  human  cDNAs  were  prepared  using 
beta  thalassemia  and  HbH  disease  reticulocyte  mRNAs,  respectively.   One  of  the 
two  positive  human  X  mouse  hybrids  contained  human  beta  mRNA  but  not  alpha. 
Purified  hemoglobin  labeled  by  incubation  of  these  hybrid  cells  with  [^H] leu- 
cine contained  [^H]  beta  globin  and  mouse  globins,  but  no  human  alpha  globin. 

1  698 


Project  No.  Z01  HL  02207-02  MHB 

These  data  indicate  that  the  alpha  and  beta  structural  loci  are  on  separate 
chromosomes . 

2)  The  pattern  of  globin  gene  expression  was  correlated  with  the  chromosomal 
composition  of  the  hybrid  cells  by  karotyping,  Giemsa  banding  and  isozyme 
analysis.   Loss  of  the  human  biarmed  chromosomes  present  in  cell  hybrids 
exhibiting  co-expression  of  human  and  mouse  globin  genes  resulted  in  loss  of 
human  but  not  mouse  globin  mRNA  synthesis. 

3)  In  order  to  identify  the  specific  chromosomes  bearing  human  globin  genes, 
homogeneous  stable  populations  of  Chinese  hamster  X  human  cell  hybrids  con- 
taining one  human  chromosome  and  a  full  complement  of  Chinese  hamster  chromo- 
somes were  generated.   In  the  several  hybrid  cells  studied  thus  far,  human  ■ 
globin  genes  were  not  detected  by  DNA-cDNA  hybridization. 

Significance  to  Biomedical  Research  and  Institute  Program;   An  understanding 
of  the  mechanism  of  mammalian  gene  regulation  is  of  fundamental  Importance  in 
understanding  human  genetic  disease.   This  program  offers  opportunity  for  in- 
sight into  genetic  regulation  by  exploiting  our  knowledge  of  and  ability  to 
study  the  expression  of  the  globin  genes  in  stable  somatic  cell  hybrids. 

Proposed  Course  of  the  Project:   The  studies  leading  to  the  chromosome  assign- 
ment of  the  alpha  and  beta  globin  structural  genes  will  be  continued.   Somatic 
cell  hybrids  which  coexpress  both  mouse  and  human  globin  chains  will  be  studied 
to  attempt  to  learn  the  factors  controlling  expression  of  the  globin  genes. 

Keyword  Descriptors:   Erythroleukemia,  Friend  cell,  virus,  molecular  hybridi- 
zation, complementary  DNA,  messenger  RNA,  gene  mapping,  erythroid  cells, 
human  bone  marrow,  Chinese  hamster. 

Honors  and  Awards :   None 

Publications : 

1.  Deisseroth,  A.,  Burk,  R. ,  Picciano,  D.,  Minna,  J.,  Anderson,  W.F.,  and 
Nienhuis,  A.W. :   Expression  of  globin  genes  in  somatic  cell  hybrids:   I. 
Synthesis  in  human  marrow-mouse  erythroleukemia  hybrid  cells.   Proc.  Nat. 
Acad.  Sci.  USA,  22:1102-1106,  1975. 

2.  Deisseroth,  A.,  Barker,  J.,  Anderson,  W.F. ,  and  Nienhuis,  A.:   Hemoglobin 
synthesis  in  somatic  cell  hybrids:   II.  Coexpression  of  mouse  with  human 
or  Chinese  hamster  globin  genes  in  interspecific  somatic  cell  hybrids  of 
mouse  erythroleukemia  cells.   Proc.  Nat.  Acad.  Sci.  (in  press). 


499 


Project  No.  Z01  HL  02210-01  MHB 
1.  Molecular  Hematology  Branch 
2. 
3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   The  Mechanisms  of  Regulation  of  the  Respiratory  Function  of 
Blood 

Previous  Serial  Number :   None 

Principal  Investigator:   Robert  M.  Winslow,  M.D. 

Other  Investigators:   Mei-Lie  Swenberg,  Ph.D. 
R.  L.  Berger,  Ph.D. 
Ehrhard  Gross,  Ph.D. 

Cooperating  Units:   Laboratory  of  Technical  Development,  NHLI 

Section  of  Molecular  Structure,  Reproduction 
Research  Branch,  NICHD 

Project  Description: 

Objectives:   The  objective  of  this  project  is  the  understanding  of  the 
mechanisms  which  regulate  the  respiratory  function  of  blood  at  molecular, 
cellular,  and  physiologic  levels.   The  oxygenation  of  normal  human  hemoglobin, 
variant  human  hemoglobins,  and  hemoglobins  of  animals  are  studied,  along  with 
the  cellular  factors  which  modify  these  properties.   The  ultimate  objective  of 
the  project  is  to  understand  the  relationships  among  these  properties,  pul- 
monary gas  exchange  and  tissue  oxygen  demands,  under  normal  and  pathologic 
conditions. 

Methods:   1)   A  continuous  automatic  oxygenation  apparatus  based  on  a  Cary 
118C  Spectrophotometer  has  been  assembled,  and  allows  precise  measurement  of 
oxygen  equilibria  of  purified  hemoglobin  solutions.   The  effects  of  each  of  the 
cellular  factors  which  are  known  to  modify  hemoglobin  function  can  be  investi- 
gated systematically  (i.e.,  temperature  pH,  salt,  2,3-DPG,  and  hemoglobin  con- 
centration) with  hemoglobin  which  has  been  purified  by  column  chromatographic 
methods. 

2)  A  new  apparatus  for  the  precise  measurement  of  whole  blood  oxygen  equi- 
libria which  has  been  developed  by  Drs.  Berger  (NHLI,  Laboratory  of  Technical 
Development)  and  L.  Rossi-Bernardi  (University  of  Milan)  is  currently 
operating  in  our  laboratory. 

3)  A  rapid,  convenient  method  for  the  identification  of  mutant  human  hemo- 
globins has  been  developed  and  employs  cellulose  thin  layers  for  "fingerprint" 
analysis,  using  extremely  small  samples  and  minimal  space  and  time. 

Major  Findings:   1)  A  hemoglobin  variant  (hemoglobin  McKees  Rocks  0145-KTerm) 
has  been  characterized  in  regard  to  structure  and  function.   It  is  the  first 
mammalian  protein  whose  structural  alteration  has  arisen  by  mutation  which 
corresponds  to  a  "nonsense"  mutation  in  bacteria:   two  amino  acids  are  lacking 

1  Yoo 


Project  No.   Z01  HL  0221Q-Q1  MHB 

at  its  carboxyl  terminal  end.   It  has  the  highest  oxygen  affinity  of  any  known 
human  variant,  yet  carriers  of  it  are  asymptomatic. 

2)   The  properties  of  the  blood  of  sheep  and  goats  after  an  anemia- induced 
"switch"  to  hemoglobin  C  is  currently  under  study.   We  have  demonstrated  that 
after  the  switch,  goat  blood  has  an  extraordinary  sensitivity  to  C02,  such 
that  it  promotes  the  unloading  of  oxygen.   A  correlation  between  this  unique 
property  and  the  known  structure  of  goat  hemoglobin  is  underway. 

Significance  to  Biomedical  Research  and  Institute  Programs:   The  control  of  the 
respiratory  function  of  blood  is  important  in  any  condition  in  which  tissue 
oxygen  delivery  may  be  compromised.   In  addition,  the  capacity  of  the  blood  to 
deliver  oxygen  plays  an  important  role  in  the  compensatory  physiology  of  anemia 
and  the  regulation  of  the  size  of  the  red  cell  mass. 

Proposed  Course  of  Project:   1)  A  systematic  characterization  of  the  properties 
of  normal  blood  will  be  continued;  the  availability  of  the  new  equipment 
renders  existing  data  obsolete. 

2)  Additional  human,  hemoglobin  variants  may  be  studied.   Of  special  interest 
is  the  compensatory  mechanisms  which  are  employed  by  subjects  with  high 
affinity  hemoglobins. 

3)  The  characterization  of  the  role  of  hemoglobin  C  in  the  adaptation  of 
sheep  and  goats  to  their  environment  will  be  pursued.  Other  animals  with 
unique  environmental  demands  might  be  studied  also. 

4)  New  equipment  is  being  designed  by  the  Laboratory  of  Technical  Development 
(Dr.  Berger)  which  will  enable  the  study  of  the  kinetic  properties  of  blood 
oxygenation  and  deoxygenation.   It  is  hoped  that  this  study  will  lead  to  better 
understanding  of  the  relationships  between  capillary  blood  flow  and  oxygenation 
which  occurs  iji  vivo. 

Honors  and  Awards :   None 

Publications :   None 


To  I 


Project  No.  Z01  HL  02211-01  MHB 
1.  Molecular  Hematology  Branch 
2. 
3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Evaluation  of  Alteration  in  Blood  Oxygen  Affinity  as  a  Basis 
for  the  Treatment  of  Sickle  Cell  Anemia 

Previous  Serial  Number:   None 

Principal  Investigator:   Robert  M.  Winslow,  M.D. 

Other  Investigators:   Jack  Fulmer,  M.D. 

Arthur  W.  Nienhuis,  M.D. 
Ronald  G.  Crystal,  M.D. 
Mei-Lei  Swenberg,  Ph.D. 

Cooperating  Units:   Pulmonary  Branch,  NHLI 

Laboratory  of  Technical  Development,  NHLI 

Project  Description: 

Objectives:   This  project  is  designed  to  study  the  relationship  between 
sickling  and  the  oxygen  transport  of  blood.   Sickling  is  a  function  of  blood 
oxygen  saturation  and  is  therefore  affected  by  factors  which  modify  oxygenation. 
The  overall  objective  is  to  define  the  extent  of  organ  involvement  and  some 
parameters  of  blood  oxygen  transport  (pulmonary  functions,  response  to  exercise, 
blood  oxygen  affinity,  and  sickling)  in  a  small  number  of  patients  with  sickle 
cell  anemia.   The  effects  of  agents  which  modify  one  or  more  of  the  known 
properties  of  blood  oxygenation  or  sickling  can  then  be  investigated.   The 
current  protocol  is  designed  to  test  the  effect  of  low  dose  carbon  monoxide  on 
sickling  and  exercise  tolerance.   In  addition,  in  vitro  studies  are  directed 
toward  an  understanding  of  the  molecular  and  cellular  mechanisms  which  regulate 
oxygenation  and  gellation  of  hemoglobin  within  sickled  cells. 

Methods:   The  life  span  of  red  cells  is  measured  by  labelling  a  cohort  of 
reticulocytes  with  radioactive  amino  acids.   The  differential  response  of  young 
and  old  cells  to  a  therapeutic  maneuver, can  then  be  observed  by  labelling  a 
second  cohort  with  a  different  label  (  C)  or  (  H)  after  an  appropriate  inter- 
val.  A  battery  of  clinical  tests  is  administered  which  defines  the  degree  of 
organ  involvement  in  individual  patients.   Blood  oxygen  affinity  is  measured 
using  new  equipment  designed  in  the  Laboratory  of  Technical  Development,  NHLI. 
Pulmonary  functions  and  exercise  testing  are  carried  out  in  collaboration 
with  the  Pulmonary  Branch,  NHLI. 

Major  Findings:   1)   A  relationship  has  been  found  between  the  number  of 
irreversibly  sickled  cells  (ISC's)  degree  of  anemia,  and  blood  P50:   patients 
with  the  most  ISC's  frequently  have  low  hematocrits  and  high  P5o's.   These 
findings  suggest  that  ISC's  have  low  oxygen  affinity  and  in  spite  of  their 
obvious  rheologic  disadvantage  may  facilitate  oxygen  delivery. 

2)   There  is  very  poor  correlation  between  the  subjective  assessment  of  the 

l  ~7ex 


Project  No.   Z01  HL  02211-01  MHB 

severity  of  symptoms  of  sickle  cell  anemia  and  the  objective  measurements  of 
organ  damage. 

Significance  to  Biomedical  Research  and  Institute  Program:   Sickle  cell  anemia 
is  a  serious  genetic  disease  which  has  a  long  history  of  therapeutic  failure. 
Before  rational  therapies  can  be  undertaken,  greater  understanding  of  patho- 
physiology is  required.   Little  attention  has  been  paid  to  the  relationships 
between  sickling  and  oxygen  supply  and  demand  and  the  effect  of  a  given  therapy 
thereon. 

Proposed  Course  of  Project:   1)  We  plan  to  continue  the  Clinical  Protocol  and 
study  the  effect  of  the  alteration  of  blood  oxygen  affinity  on  sickling,  by  the 
administration  of  carbon  monoxide  in  low  doses  to  selected  patients . 

2)  We  plan  to  investigate  the  mechanism  of  the  low  oxygen  affinity  of  ISC's 
and  to  attempt  to  understand  whether  these  unique  cells  are  harmful  or  bene- 
ficial in  sickle  cell  anemia  patients. 

3)  A  model  of  the  respiratory  function  of  normal  blood  will  be  built  up  and 
applied  to  the  problem  of  sickle  cell  anemia.   To  this  end,  the  kinetic  and 
equilibrium  oxygenation  properties  of  sickle  blood  will  be  carefully  studied, 
using  the  new  equipment  described  above. 

Honors  and  Awards:   None 

Publications:   None 


7*3 


Project  No.  Z01  HL  02203-03  MHB 

1.  Molecular  Hematology  Branch 

2.  Clinical  Hematology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Regulation  of  Hemoglobin  Chain  Synthesis  in  Beta- Thalassemia 

Previous  Serial  Number:   NHLI-109(c) 

Principal  Investigator:   Arthur  W.  Nienhuis,  M.D. 

Other  Investigators:   Ramon  Velez,  M.D. 

Judith  Kantor,  Ph.D. 
Alan  Steggles,  Ph.D. 
Golder  Wilson,  M.D.,  Ph.D. 
W.  French  Anderson,  M.D. 

Cooperating  Units:   None 

Project  Description: 

Objectives:   Beta-thalassemia,  also  known  as  Cooley's  Anemia,  is  an  hereditary 
disease  characterized  by  severe  anemia.   The  anemia  is  a  consequence  of  a  low 
level  of  beta  chains  of  hemoglobin  being  produced  by  the  patient's  red  blood 
cells.   This  results  in  an  excess  of  alpha  glob in  chains.   These  excess  alpha 
chains  precipitate  in  the  cell  and  lead  to  the  destruction  of  the  cell.   No 
amino  acid  change  has  been  found  in  the  hemoglobin  beta  chains  of  thalassemic 
patients.   It  is  generally  assumed,  therefore,  that  the  molecular  abnormality 
resides  in  the  regulation  of  beta  chain  synthesis.   Analysis  of  globin  mRNA 
from  thalassemia  cells  has  indicated  that  there  is  a  reduced  amount  of  beta 
globin  mRNA  relative  to  that  for  the  alpha  globin,  so  that  the  defect  must  be 
specifically  in  the  production  of  messenger  RNA.   The  normal  mechanisms  for 
regulation  of  globin  mRNA  production  and  the  nature  of  the  defect  in  beta 
thalassemia  cells  are  being  investigated. 

Methods:   Reticulocytes  and/or  bone  marrow  are  obtained  both  from  patients  with 
beta-thalassemia  and  from  patients  displaying  a  high  reticulocyte  count  second- 
ary to  another  cause  (for  example,  recovery  from  folic  acid  deficiency,  sickle 
cell  anemia,  etc).   Globin  mRNA  is  isolated  from  reticulocytes  and  used  as  a 
template  for  synthesis  of  globin  complementary  DNA  (cDNA)  by  the  viral  enzyme, 
RNA  directed  DNA  polymerase.   Alpha  and  beta  globin  cDNAs  are  made  using  mRNA 
from  reticulocytes  of  patients,  with  beta  thalassemia  and  hemoglobin  H  disease, 
respectively,  and  these  are  further  purified  by  hydroxylapatite  chromatography. 
DNA  and  nuclear  RNA  are  isolated  from  cells  of  normal  patients  and  those  with 
beta  thalassemia  and  the  relative  amounts  of  alpha  and  beta  nucleotide  se- 
quences in  these  components  is  determined.   Chromatin  is  utilized  for  cell- 
free  synthesis  of  globin  mRNA. 


7a¥ 


Project  No.  Z01-HL  02203-03 
MHB 

Major  Findings:   1)  DNA  from  beta  thalassemic  cells  hybridized  equally  to  both 
alpha  and  beta  human  cDNA  in  a  manner  quantitatively  similar  to  the  hybridi- 
zation of  these  probes  to  normal  human  DNA.   Hence,  gene  deletion  does  not 
appear  to  be  responsible  for  the  deficiency  of  beta  globin  mRNA  production  in 
the  patients  we  have  studied. 

2)  Methodology  has  been  established  for  the  isolation  of  high  molecular  weight 
nuclear  RNA  from  a  small  number  of  bone  marrow  cells.   Application  of  these 
techniques  to  thalassemia  cells  will  permit  determination  of  the  relative 
number  of  alpha  and  beta  sequences  in  the  immediate  product  of  transcription. 

Significance  to  Biomedical  Research  and  Institute  Program:   The  thalassemias 
are  severe  hereditary  diseases  which  as  a  group  affect  more  individuals  world 
wide  than  any  other  single  gene  defect.   A  method  is  needed  for  treating  these 
diseases  which  will  reduce  or  eliminate  the  frequent  blood  transfusions  that 
are  often  required.   In  addition,  the  techniques  utilized  to  learn  how  the 
rate  of  hemoglobin  synthesis  is  regulated  can  then  be  applied  to  studying  other 
diseases  where  the  defect  is  also  in  faulty  regulation  in  the  synthesis  of  a 
gene  product. 

Proposed  Course  of  Project:   The  relative  amount  of  alpha  and  beta  globin 
messenger  RNA  sequences  synthesized  from  thalassemic  and  non-thalassemic 
chromatin  in  the  cell-free  RNA  synthesizing  system  will  be  quantitated. 
Fractionation  of  the  nuclear  components  of  thalassemic  erythroid  cells  will  be 
carried  out  to  determine  the  molecular  defect  in  thalassemia. 

Keyword  Descriptors:   Thalassemia,  globin  messenger  RNA,  globin  genes,  nuclear 
RNA,  nucleic  acid  hybridizations,  anemia,  gene  regulation,  bone  marrow  cells, 
reticulocytes. 

Honors  and.  Awards:   None 

Publications: 

1.  Anderson,  W.F.:   Isolation  and  translation  of  messenger  RNA  from  beta- 
thalassemia  red  cells.   Ann.  N.Y.  Acad.  Sci.  232:15-32,  1974. 

2.  Nienhuis,  A.W. ,  and  Anderson,  W.F.:   The  molecular  defect  in  thalassemia. 
Clinics  in  Haematology  _3: 437-466,  1974. 

3.  Velez,  R. ,  Kantor,  J.A.,  Picciano,  D.J.,  Anderson,  W.F.,  and  Nienhuis,  A.W. : 
Alpha  and  beta  complementary  DNAs  of  human  and  rabbit:   Specificity  of 
hybridization.   J.  Biol.  Chem.  in  press. 


y&r 


Project  No.  Z01  HL  02204-03  MHB 

1.  Molecular  Hematology  Branch 

2.  Clinical  Hematology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   The  Mechanism  of  Hemoglobin  Switching  in  Sheep  and  Goats 

Previous  Serial  Number:   NHLI-287 

Principal  Investigators:   Jane  Barker,  Ph.D. 

Arthur  W.  Nienhuis,  M.D. 

Other  Investigators:   Thomas  Musliner,  M.D. 
Joseph  E.  Pierce,  M.D. 
Leonard  Stuart 
W.  French  Anderson,  M.D. 

Cooperating  Units:   Section  on  Laboratory  ANimal  Medicine  and  Surgery,  NHLI 
Ungulate  Unit,  NIH  Animal  Center 

Project  Description: 

Objectives:   This  project  utilizes  the  erythropoietin- induced  switch  in  hemo- 
globin phenotype  which  occurs  in  sheep  and  goats  as  a  model  system  for  under- 
standing factors  which  regulate  globin  gene  expression  in  erythroid  cells. 
Both  the  cellular  events  accompanying  differentiation  and  hemoglobin  synthesis 
and  the  subcellular  events  related  to  the  regulation  of  transcription  will  be 
explored. 

Methods :   Bone  marrow  is  obtained  from  normal,  anemic,  or  transfused  phlethoric 
animals.   The  cells  are  grown  in  vitro,  directly  or  after  fractionation  by  unit 
gravity  sedimentation,  under  conditions  in  which  erythroid  colonies  form. 
Globin  synthesis  is  quantitated  and  the  type  and  amount  of  each  globin  chain 
determined  by  chromatography  or  gel  electrophoresis.   Chromatin  extracted  from 
bone  marrow  is  assayed  ±n   vitro  for  its  ability  to  support  synthesis  of  globin 
gene  sequences  as  measured  by  nucleic  acid  hybridization  to  complementary  DNA 

Major  Findings:   1)   Cells  capable  of  generating  erythroid  colonies  are  large, 
presumably  immature  cells  and  may  be  separated  from  the  differentiating  ery- 
throid cells  by  unit  gravity  sedimentation.   These  colony  forming  cells  respond 
to  high  concentrations  of  erythropoietin  by  producing  colonies  active  in  the 
synthesis  of  hemoglobin  C.   Initial  results  indicate  that  a  fraction  of  colony 
forming  cells  of  intermediate  sedimentation  velocity  have  the  greatest  potential 
for  hemoglobin  C  production.   This  differential  sensitivity  to  erythropoietin 
may  reflect  the  position  of  the  cell  in  the  erythroid  maturation  sequence. 

2)   Sheep  colony  forming  cells  appear  to  require  an  approximately  ten-fold 
higher  concentration  of  erythropoietin  for  stimulation  of  hemoglobin  C  pro- 
duction than  do  goat  cells.   However,  colony  formation  occurs  at  equivalently 
low  erythropoietin  concentrations  for  the  cells  of  both  species. 


7a£ 


Project  no.  Z01  HL  02204-C3  MHB 

3)   Human  erythroid  colonies  have  been  grown  from  bone  marrow  of  patients 
with  various  hemolytic  anemias.   Thus,  the  concepts  and  techniques  developed 
for  study  of  the  A  to  C  switch  in  the  cells  of  sheep  and  goats,  may  prove  use- 
ful in  studying  regulation  of  human  hemoglobin  synthesis. 

Proposed  Course  of  the  Project: 

The  marrow  fractionation  studies  will  be  extended  to  determine  if  a  popu- 
lation of  precursor  cells  can  be  obtained  which  is  capable  of  generating 
colonies  producing  only  hemoglobin  C.   The  relationship  between  the  duration 
of  exposure  to  high  concentration  erythropoietin  and  the  production  of  hemo- 
globin C  will  be  defined  to  determine  if  commitment  to  hemoglobin  phenotype  is 
an  irreversible  event.   Synthesis  of  specific  hemoglobins  in  human  colonies 
will  be  examined. 

Significance  to  Biomedical  Research  and  Institute  Program:   Activation  of  hemo- 
globin F  synthesis  in  the  cells  of  patients  suffering  from  sickle  cell  anemia 
or  thalassemia  should  totally  alleviate  the  symptoms  of  the  disease.   The  sheep 
and  goat  model  system  will  be  useful  in  defining  conditions  under  which  hemo- 
globin phenotype  can  be  influenced  thereby  facilitating  the  search  for  agents 
which  regulate  the  proportion  of  fetal  hemoglobin  synthesis  in  human  erythroid 
marrow  cells. 

Keyword  Descriptors:   Erythropoieses,  regulation  of  hemoglobin  synthesis, 
erythropoietin,  erythroid  colony,  bone  marrow  fractionation,  human  bone  marrow 
in  vitro,  glob in  gene  regulation. 

Honors  and  Awards :   None 

Publications: 

1.  Nienhuis,  A.W. ,  Elson,  N.A.,  Barker,  J.E.,  and  Anderson,  W.F. :   Hemoglobin 
switching  in  sheep  and  goats:   Aspects  of  the  molecular  mechanism.   Ann. 
N.Y.  Acad.  Sci.  241:  566-581,  1974. 

2.  Nienhuis,  A.W. ,  and  Bunn,  H.F.:   Hemoglobin  switching  in  sheep  and  goats: 
Occurrence  of  the  hybrid  hemoglobin,  o^B  6   in  the  same  red  cell.   Science 
185:946-948,  1974. 

3.  Barker,  J.E.,  Anderson,  W.F.,  and  Nienhuis,  A.W. :   Hemoglobin  switching  in 
sheep  and  goats:   V.  Effect  of  erythropoietin  concentration  on  in  vitro 
erythroid  colony  growth  and  globin  synthesis.   J.  Cell  Biol.  64:515-527, 
1975. 

4.  Nienhuis,  A.W. ,  Barker,  J.E.,  Deisseroth,  A.,  and  Anderson,  W.F.:  Regu- 
lation of  globin  gene  expression.  In  Weatherall,  D.J.  (Ed):  Disordered 
Erythropoieses  in  Childhood.   CIBA  Symposium,  in  press. 


7*7 


Project  No. Z01  HL  02208-02  MHB 

1.  Molecular  Hematology  Branch 

2.  Clinical  Hematology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Iron  Chelation  Therapy  in  Transfusional  Hemosiderosis 

Previous  Serial  Number:   NHLI-95(c) 

Principal  Investigator:   Arthur  W.  Nienhuis,  M.D. 

Other  Investigators:   Walter  Henry 

Frederic  Bartter,  M.D. 
Roger  Aamodt,  M.D. 

Cooperating  Units:   Cardiology  Branch,  NHLI 

Endocrinology  Branch,  NHLI 

Section  on  Total  Body  Counting,  Nuclear  Medicine  Department 

Project  Description: 

Objectives:   The  object  of  this  study  is  to  evaluate  available  iron  chelators, 
to  maximize  their  effectiveness,  and  to  test  new  chelators  as  they  become 
available.   The  current  protocol  is  designed  to  evaluate  the  effect  of  Desferal 
on  total  iron  balance.   The  added  effect  of  ascorbic  acid  on  urinary  iron 
excretion  and  GI  absorption  is  also  being  evaluated. 

Methods :   Patients  are  studied  under  metabolic  balance  conditions.   A  standard 
diet  of  measured  calcium,  phosphorus,  and  iron  content  is  eaten  daily  and  the 
total  excretion  of  these  elements  is  measured  in  the  stool  and  urine.   Analysis 
of  iron  absorption  is  obtained  by  administration  of  tracer  doses  of  Fe-59  and 
measurement  of  absorption  by  the  total  body  counter. 

Major  Findings:    1)  Ascorbic  acid  causes  a  marked  increase  in  urinary  iron  ex- 
cretion in  younger,  less  heavily  iron-loaded  patients.   Iron  absorption  and  the 
excretion  of  iron  in  the  stool  are  unchanged.   Thus  negative  iron  balance  in 
response  to  desferal  is  markedly  enhanced  by  ascorbic  acid  in  these  patients. 

2)   Older,  more  heavily  iron- loaded  patients,  treated  with  desferal,  show  a 
more  varied  response  to  ascorbic  acid.   One  patient  showed  a  doubling  of  urinary 
iron  excretion,  but  an  equivalent  reduction  in  stool  iron,  so  that  net  iron 
balance  was  unaffected  by  ascorbic  acid.   Withdrawal  of  ascorbic  acid  was 
followed  by  a  fall  in  the  urinary  iron  over  several  weeks  which  paralleled  a 
reduction  in  white  cell  ascorbic  acid  to  subnormal  levels.   Thus,  the  effect  of 
ascorbic  acid  administration  in  this  patient  is  apparently  related  to  its  intra- 
cellular concentration  and  raises  the  possibility  that  the  tissue  source  of 
chelated  iron  may  also  be  influenced  by  ascorbic  acid.   Another  patient  showed 
an  increase  in  both  urinary  and  fecal  iron  excretion  in  response  to  ascorbic 
acid.   Hence  net  negative  iron  balance  was  markedly  increased. 


Project  No.  Z01  HL  02208-02  MHB 

3)  A  clinical  pathological  correlative  study  was  performed  on  a  23  year  old 
patient  with  congenital  anemia,  transfused  from  birth,  who  died  from  cardiac 
hemachromatosis.   The  biochemical  form  of  iron  was  found  to  be  different  for 
various  tissues;  ferritin  iron  was  concentrated  in  the  liver  and  the  pancreas, 
while  the  metabolically  less  active  form,  hemosiderin  was  concentrated  in  the 
heart  and  endocrine  tissues.   Heavy  deposits  of  iron  were  present  in  all  the 
endocrine  glands  although  function  was  variably  affected.   Parathyroid  function 
was  abolished  but  the  plasma  concentrations  of  several  of  the  pituitary 
hormones  was  elevated. 

4)  Echocardiographic  studies  were  performed  on  several  iron-loaded  patients 
at  the  NIH,  and  also  at  two  large  thalassemia  clinics  in  New  York  City.   Left 
ventricular  wall  thickening  and  an  increase  in  cardiac  weight  presumably  re- 
flected myocardial  iron  deposition  and  was  present  in  even  the  youngest  patients, 
despite  lack  of  clinical  evidence  of  cardiac  hemachromatosis.   These  data  will 
be  useful  in  the  subsequent  evaluation  of  chelation  therapy. 

Significance  to  Biomedical  Research  and  Institute  Program:   Transfusional  hemo- 
siderosis is  a  major  cause  of  morbidity  and  mortality  in  patients  requiring 
prolonged  transfusion  therapy.   The  role  of  iron  chelators  in  improving  the 
clinical  course  of  these  patients  must  be  ascertained. 

Proposed  Course  of  Project:   This  project  will  be  continued  until  a  suitable 
iron  chelator  is  found  and  evaluated  or  until  the  need  for  transfusion  in 
thalassemia  and  other  congenital  hemolytic  anemias  is  removed. 

Keyword  Descriptors:   Hemachromatosis,  cardiac  hemosiderosis,  iron  chelation, 
Desferal,  ascorbic  acid,  ferritin,  hemosiderin,  metabolic  balance  study, 
endocrine  gland  dysfunction,  thalassemia,  echocardiogram. 


Honors  and  Awards:   None 

Publications: 

1.   Arnett,  E.N. ,  Nienhuis,  A.W. ,  Henry,  W.L.,  Ferrans,  V.J.,  Redwood,  D.R., 

and  Roberts,  W.C.:   Massive  myocardial  hemosiderosis:   A  structure-function 
Conference  at  the  National  Heart  and  Lung  Institute.   Am.  Heart  J.   in  press, 


7«? 


Project  No.  ZQ1  HL  02209-02  MHB 

1.  Molecular  Hematology  Branch 

2.  Clinical  Hematology 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Cardiac  Hemolytic  Anemia. 

Previous  Serial  Number:   NHLI-96(c) 

Principal  Investigator:   Arthur  W.  Nienhuis,  M.D. 

Other  Investigators:   Gary  Weiss,  M.D. 

Laurence  Corash,  M.D. 
Harvey  Gralnick,  M.D. 
Roger  Aamodt,  M.D. 

Cooperating  Units:   Cardiac  Surgical  Branch,  NHLI 

Clinical  Pathology  Branch,  NIH  Clinical  Center 
Section  on  Total  Body  Counting,  Nuclear  Medicine 
Department. 

Project  Description: 

Objectives:   The  objectives  of  this  study  are: 

1)  to  evaluate  various  parameters  of  hemolysis  as  to  their  usefulness  in 
measuring  hemolytic  rate  in  patients  with  cardiac  hemolytic  anemia; 

2)  to  determine  the  state  of  iron  balance  in  these  patients  when  on  thera- 
peutic doses  of  oral  iron;  and 

3)  to  seek  ways  to  minimize  the  rate  of  hemolysis  in  patients  with  cardiac 
lesions,  or  prosthetic  valves. 

Methods:   Patients  are  admitted  to  the  Clinical  Center  for  the  following 
studies:   red  cell  chromium  survival,  carbon  monoxide  red  cell  survival,  iron 
absorption  and  urinary  iron  excretion.   Cardiac  output  is  estimated  with  the 
echocardiogram. 

Major  Findings:   1)   The  degree  of  anemia  has  no  consistent  effect  on  the 
hemolytic  rate  in  patients  with  cardiac  hemolysis.   Thus,  certain  patients 
show  reduced  rate  of  red  cell  destruction  following  transfusion  while  others 
show  no  change  in  hemolytic  rate. 

2)   Several  patients  have  been  shown  to  have  mild  or  moderate  hemolysis  but 
severe  anemia,  reflecting  inadequate  bone  marrow  compensation.   Elevated  sedi- 
mentation rate,  elevated  gamma  globulins  and  poor  response  to  oral  iron  sug- 
gests that  a  chronic  inflammatory  process  may  be  responsible  for  relative  bone 
marrow  failure,  although  intensive  diagnostic  evaluation  has  defined  such  a 
process  in  only  two  or  seven  patients. 


7  to 


Project  No.   Z01  HL  02209-02  MHB 

Significance  to  Biomedical  Research  and  Institute  Program:   Cardiac  hemolytic 
anemia  is  a  significant  complication  of  prosthetic  valve  replacement.   To 
maximize  the  clinical  benefit  of  this  surgical  procedure  to  these  patients 
efforts  must  be  made  to  minimize  the  severity  of  the  anemia. 

Proposed  Course  of  the  Project:   Patients  currently  followed  by  the  Hematology 
Branch  will  be  re-evaluated  periodically,  in  an  effort  to  identify  factors 
responsible  for  the  inadequate  bone  marrow  response  to  hemolysis.   Additional 
patients  will  be  studied  to  define  further  the  relationship  between  hemolytic 
rate,  degree  of  anemia,  and  bone  marrow  function. 

Keyword  Descriptors:   Cardiac  hemolytic  anemia,  valve  prosthesis,  red  cell 
survival,  bone  marrow  function,  iron  absorption,  valvular  heart  disease. 

Honors  and  Awards:    None 

Publications:  None. 


7N 


Annual  Report  of  the 
Section  on  Pulmonary  Biochemistry 

Pulmonary  Branch 
National  Heart  and  Lung  Institute 
July  1,  1974  through  June  30,  1975 

The  connective  tissue  of  the  lung  is  fundamental  to  lung  structure  and 
function.  In  human  pulmonary  disease  there  is  a  broad  spectrum  of  connective 
tissue  involvement  from  an  apparent  excess  of  connective  tissue  in  the  inter- 
stitial lung  disorders  to  an  apparent  destruction  of  the  connective  tissue 
frame  work  in  the  emphysematous  disorders.  The  overall  objective  of  this 
work  is  to  understand  and  eventually  control  the  mechanisms  of  lung  connective 
tissue  synthesis  and  destruction  so  that  ultimately  we  can  selectively  in- 
hibit or  reverse  the  f  ibrotic  process  in  the  interstitial  disorders  and  pro- 
mote the  preservation  and  generation  of  functional  alveolar  units  in  the 
emphysematous  disorders.  The  initial  objectives  of  this  laboratory  since  its 
inception  in  July  1972  have  been  to:  (1)  develop  the  in  vitro  technology  by 
which  these  problems  could  be  attacked;  (2)  set  up  a  clinical  chest  service 
to  evaluate  patients  with  connective  tissue  related  lung  disorders;  (3)  apply 
the  in  vitro  technology  to  the  study  of  these  disorders  in  animal  models  and 
in  human  disease.  All  three  objectives  have  been  achieved.  Specifically,  it 
is  now  possible  to  examine  the  synthesis  and  proteolysis  of  connective  tissue 
in  explant  cultures  of  animal  and  human  lung,  in  diploid  cell  lines  derived 
from  animal  and  human  lung  and  in  homologous  and  heterologous  cell- free  pro- 
tein synthesizing  systems.  A  clinical  pulmonary  service  has  been  established 
using  sophisticated  methods  to  evaluate  patients  with  connective  tissue  re- 
lated lung  disorders.  In  many  instances,  the  methods  of  explant  culture, 
cell  culture  and  cell-free  protein  synthesis  can  be  used  to  evaluate  these 
clinical  problems.  The  following  is  a  summary  of  the  ongoing  projects  in  the 
laboratory  this  past  year. 

I.  Models  of  Lung  Growth 

Two  models  are  being  used  to  investigate  the  control  of  synthesis  and 
proteolysis  of  lung  connective  tissue  components:  (1)  changes  during  normal 
lung  growth  and  (2)  "compensatory"  lung  growth  following  unilateral  pneumo- 
nectomy. 

Just  prior  to  birth,  approximately  3-5%  of  the  total  amino  acids  incor- 
porated into  lung  protein  per  hour  go  into  collagen.  In  the  neonatal  period, 
there  is  a  shift  in  the  emphasis  of  the  proteins  synthesized  by  lung  so  that 
this  figure  goes  to  12-15%.  During  this  period  there  is  a  rapid  accumulation 
of  the  total  amount  of  collagen  in  the  lung  as  well  as  the  relative  amount  of 
collagen  per  unit  lung  mass.  By  adulthood,  the  level  of  collagen  synthesis 
returns  to  its  low  level  of  3-5%  and  the  accumulation  of  collagen  in  the  lung 
stops.  Within  one  month  after  left  pneumonectomy  in  the  adult  rabbit,  the 
mass  of  the  right  lung  doubles.  This  growth  can  be  characterized  by  a  period 
of  rapid  collagen  accumulation  preceded  by  a  shift  in  emphasis  of  total  pro- 
tein synthesis  toward  collagen  synthesis.  As  a  result,  the  total  amount  of 
collagen  doubles,  but  the  density  of  collagen  remains  constant.  This  is  in 


7/$ 


contrast  to  normal  neonatal  lung  growth  in  which  the  shift  in  protein  syn- 
thesis toward  collagen  accumulation  results  in  an  increased  concentration  of 
collagen.  Studies  with  mechanical  alteration  of  the  chest  cavity  following 
unilateral  pneumonectomy  have  suggested  that  mechanical  factors  may  be 
partially  responsible  for  the  initiation  of  the  complex  biochemical  events 
associated  with  lung  growth. 

The  adult  rabbit  lung  synthesizes  approximately  1  mg  of  collagen  per  day, 
yet  the  amount  of  collagen  per  unit  lung  mass  does  not  change.  Thus  the  syn- 
thesis of  collagen  must  be  matched  by  an  equivalent  proteolysis  of  collagen 
so  as  to  prevent  abnormal  collagen  accumulation.  Methods  have  been  developed 
to  define  the  degradation  of  newly  synthesized  lung  collagen  as   early  proteo- 
lysis (0-48  hours)  and  late  proteolysis  (greater  than  48  hours) .  in  the 
newborn,  25%  of  newly  synthesized  collagen  is  degraded  within  four  hours,  but 
there  is  no  further  proteolysis  of  this  collagen  over  the  next  two  days.  In 
the  adult,  although  synthesis  of  collagen  is  2-3  fold  less,  the  same  per- 
centage of  collagen  is  degraded  in  the  same  time.  Pharmacologic  agents,  such 
as  colchicine  can  significantly  increase  the  amount  of  early  proteolysis  of 
newly  synthesized  lung  collagen.  Thus,  it  appears  that  (1)  25%  of  newly  syn- 
thesized lung  collagen  is  degraded  very  rapidly;  (2)  early  proteolysis  of 
newly  synthesized  lung  collagen  does  not  appear  to  be  important  in  controlling 
collagen  accumulation  in  lung  growth;  (3)  most  likely,  late  proteolysis  is 
responsible  for  the  degradation  of  the  remainder  of  the  newly  synthesized 
lung  collagen;  and  (4)  the  proteolysis  of  collagen  can  be  significantly  altered 
by  pharmacologic  agents.  The  latter  may  become  very  important  in  therapeutic 
intervention  of  the  fibrotic  lung  disorders. 

Although  the  glycx>saminoglycans  contribute  less  than  1%  to  total  lung 
dry  weight,  they  are  potentially  of  major  importance  in  lung  growth  and  dis- 
ease because  of  their  influence  on  connective  tissue  accumulation  and  phy- 
sical state.  There  are  significant  changes  in  glycosaminoglycans  as  the 
lung  grows.  For  example,  chondroitin  4-sulfate  is  relatively  more  important 
in  the  fetal  lung  while  dermatan  sulfate,  heparin  and  heparan  sulfate  be- 
come significant  in  adult  life.  These  alterations  in  lung  glycosaminoglycan 
composition  are,  in  part,  controlled  by  age  related  changes  in  glycosamino- 
glycan synthesis.  These  changes  may  have  importance  in  controlling  alter- 
ations in  lung  function  during  development. 

II.  Control  of  Collagen  Synthesis  in  Lung  Cell-Free  Systems 

An  active,  partially  fractionated  cell-free  protein  synthesizing 
system  has  been  established  from  rabbit  lung.  Lung  polysomes  can  also  be 
translated  in  heterologous  cell-free  systems  including  the  rabbit  reticulo- 
cyte and  wheat  embryo  systems.  Collagen  in  lung  is  synthesized  on  the  class 
of  large  polysomes  associated  with  the  endoplasmic  reticulum.  During  lung 
development  there  are  significant  changes  in  the  relative  proportion  of  lung 
polysomes  that  synthesize  collagen  suggesting  that  at  least  some  of  the  con- 
trol of  lung  collagen  synthesis  may  be  due  to  changes  in  lung  collagen  gene 
expression  or  to  changes  in  the  number  of  cells  that  synthesize  collagen. 
These  methods  are  being  applied  to  tissue  culture  systems  of  animal  and 
human  lung  cells  utilized  in  in  vitro  models  of  fibrotic  lung  disease. 


7(4 


The  cell-free  system  is  currently  being  utilized  to  identify  vulnerable  steps 
in  the  process  of  connective  tissue  synthesis  which  may  be  influenced  to 
treat  specific  lung  disorders. 

III.  Heterogeneity  of  Lung  Collagen 

Collagen  is  the  most  abundant  protein  in  lung,  comprising  10-15% 
of  the  adult  lung.  This  collagen  is  heterogeneous;  there  are  probably  four 
different  types  of  collagen  synthesized  by  the  lung.  Using  animal  and 
human  lung  and  cultured  lung  cells,  the  following  has  been  determined: 

The  lung  parenchyma  and  blood  vessels  synthesize  al(I)  and  a2  chains 
(Type  I  collagen)  and  al (III)  chains  (Type  III  collagen) ,  while  the  tracheo- 
bronchial tree  synthesizes  al (II)  chains  (Type  II  collagen) .  These  collagen 
chains  have  distinct  primary  amino  acid  sequences  as  demonstrated  by  cyano- 
gen bromide  peptide  mapping  techniques.  All  evidence  to  date  suggests  that 
while  lung  collagen  is  markedly  heterogeneous,  each  collagen  type  is  iden- 
tical to  collagen  elsewhere  in  the  body  (e.g.,  lype  I  is  the  same  from  lung, 
skin,  bone,  tendon,  blood  vessels;  Type  II  is  the  same  from  tracheobronchial 
tree  and  sternal  cartilage;  and  Type  III  is  the  same  from  lung,  aorta  and 
skin) . 

The  f  ibrotic  lung  diseases  have  many  primary  stimuli  including  inhala- 
tion of  toxic  materials,  hypersensitivity  states,  radiation  injury  and  assoc- 
iated with  systemic  disorders  such  as  scleroderma.  It  is  likely  that  these 
disorders  are  associated  with  injury  to  different  lung  cells,  suggesting  the 
possibility  that  there  may  be  a  heterogeneity  in  the  types  of  collagen  syn- 
thesized in  these  different  diseases.  It  may  be  possible  to  classify  the 
interstitial  lung  diseases  on  the  basis  of  the  types  of  collagen  synthesized 
in  a  similar  fashion  to  the  lipoprotein  disorders  and  certain  hemoglobin 
disorders. 

IV.  Collagen  Synthesis  in  Cultured  Lung  Cells 

The  application  of  biochemical  technology  to  human  lung  disease  is 
limited  by  the  complexity  of  the  organ  and  the  unavailability  of  large  quan- 
tities of  lung  cells  from  patients  with  lung  disease.  There  are  more  than  40 
cell  types  in  lung,  and  some  of  these  cells  are  responsible  for  the  synthesis 
of  5  different  types  of  collagen  chains.  When  the  lung  synthesizes  abnormal 
amounts  of  collagen  in  response  to  injury,  the  same  cells  normally  producing 
collagen  may  proliferate;  they  may  synthesize  relatively  more  collagen  or 
cells  that  do  not  normally  synthesize  collagen  may  be  "turned  on"  to  do  so. 
One  of  the  primary  objectives  of  this  laboratory  is  to  develop  the  technology 
to  culture  the  cells  from  lung  that  are  important  in  collagen  synthesis  in 
health  and  disease. 

Fibroblasts  from  animal  and  human  lung  synthesize  both  Type  I  and  Type 
III  collagen.  An  epithelial  cell  cultured  from  fetal  cat  lung  also  syn- 
thesized Type  I  and  Type  III  collagen  but  not  as  actively  as  the  fibroblast. 
Pulmonary  alveolar  macrophages  do  not  synthesize  collagen.  Methods  have  been 
developed  to  use  molecular  weight  maps  of  the  proteins  secreted  by  these  cells 


7tS~ 


as  specific  "fingerprints"  of  each  cell  type.  The  "fingerprint"  of  the 
fibroblast, epithelial  cell  and  macrophage  are  markedly  different. 

These  methods  are  being  adapted  to  establish  model  systems  of  human 
lung  diesase  in  which  the  interaction  of  human  or  animal  lung  cells,  alveolar 
macrophages,  lymphocytes  and  serum  components  can  be  studied  in  vitro. 

V.  The  Accumulation  of  Collagen  in  the  Human  Lung 

The  in  vitro  technology  has  been  established  to  quantitate  human  lung 
collagen  composition  and  synthesis  from  biopsy  specimens  in  normal  and  dis- 
eased lungs.  Normal  human  lung  parenchyma  synthesizes  at  least  two  collagen 
chains  (al (I) ,  a2)  and  probably  a  third  (al (III) ) .  Fibroblasts  cloned  from 
human  fetal  lung  synthesize  al(I) ,  a2  and  al(III)  chains.  In  the  16-20 
week  human  fetal  lung  and  in  the  adult  human  lung,  approximately  4%  of  the 
total  amino  acids  incorporated  into  protein  are  incorporated  into  collagen. 
Biopsies  from  three  patients  with  idiopathic  pulmonary  fibrosis  have  been 
examined  and,  interestingly,  none  had  increases  in  the  percentage  of  protein 
synthesis  that  was  collagen,  suggesting  that  the  molecular  pathology  in 
fibrotic  lung  disease  is  more  than  simply  an  increase  in  fibrous  tissue 
synthesis . 

Methods  developed  in  animal  systems  to  quantitate  collagen  proteolysis 
are  being  utilized  with  human  lung  biopsies  to  establish  the  significance  of 
control  of  collagen  degradation  on  human  lung  collagen  accumulation.  These 
techniques  are  adaptable  to  quantitating  the  influence  of  pharmacologic  agents 
on  the  fibrotic  process  and  thus  predicting,  for  each  patient,  which  agents 
are  most  efficacious. 

VT.  Experimental  Models  of  the  Interstitial  Lung  Disorders 

A  major  goal  of  this  laboratory  is  to  understand  and  eventually  control 
the  mechanisms  involved  in  connective  tissue  synthesis  and  degradation  in 
human  lung  disease  involving  these  components.  Because  the  availability  of 
human  lung  biopsy  material  is  limited,  it  necessitates  that  any  biochemical 
techniques  applied  to  this  tissue  is  worked  out  first  in  animal  models,  thus 
insuring  the  maximum  amount  of  information  and  the  maximum  benefit  to  the 
patient.  For  that  reason  two  groups  of  models  of  fibrotic  diseases  are  being 
developed:  (1)  animal  models;  and  (2)  tissue  culture  models. 

Animal  models  -  Although  collagen  accumulation  takes  place  in  silica 
exposed  animals,  it  is  a  difficult  model  to  work  with  because  the  silicotic 
"nodules"  are  discrete  and  inhcmogeneous .  Radiation  induced  fibrosis  has 
been  a  much  more  satisfactory  model  and  preliminary  studies  have  demonstrated 
increased  collagen  density  6  weeks  after  6000  R  to  the  lung. 

Tissue  culture  models  -  The  pulmonary  alveolar  macrophage  can  be  induced 
to  secrete  collagenase,  a  unique  and  specific  enzyme  for  mammallian  collagen. 
Rabbit  macrophages  activated  with  BGC  (in  vivo)  will  secrete  collagenase  after 
1-2  days  in  culture.  Exposure  of  these  macrophages  to  protein  antigens  de- 
rived from  mycobacterium  will  further  augment  this  finding.   Sufficient 


7/4 


quantities  of  mammallian  coliagenase  are  being  purified  to  produce  antibodies 
specific  for  it.  Soon,  it  should  be  possible  to  have  tissue  culture  systems 
involving  lung  cells,  macrophages,  lyrnphocytes  and  serum  from  animals, 
normal  humans  and  humans  with  f  ibrotic  lung  disease  in  which  the  rates  of 
synthesis  and  degradation  of  specific  collagen  types  can  be  quantitated  and 
manipulated. 

VTI.   Studies  of  Patients  with  Fibrotic  Lung  Disease 

The  fibrotic  lung  disorders  represent  15-20%  of  the  non-infectious 
disorders  of  lung.  The  mean  survival  of  these  patients  is  45  months  from  the 
onset  of  symptoms.  Although  approximately  5-10%  of  these  patients  respond  to 
corticosteroids,  there  is  essentially  no  treatment  for  the  remainder.  The. 
natural  history,  etiology  and  pathogenesis  of  "idiopathic"  fibrotic  lung 
disease  is  poorly  studied.  The  Pulmonary  Branch,  NHLI,  has  undertaken  a  de- 
tailed study  of  these  patients  with  several  major  objectives: 

(1)  To  determine  the  etiology  of  idiopathic  fibrotic  lung  disease. 

(2)  To  follow  patients  longitudinally  to  determine  (a)  the  natural 
history  of  this  disorder  and  (b)  which  pulmonary  function  parameters 
are  most  sensitive  to  the  disease  process. 

(3)  To  correlate  pulmonary  alveolar  constituents  (fluid  and  pulmonary 
alveolar  macrophages)  with  fibrotic  disease. 

(4)  To  correlate  radioisotopic  monitors  of  lung  function  (ventilation, 
perfusion  and  gallium  scans)  with  the  disease  process. 

(5)  To  correlate  lung  pathogenic  alterations  with  biochemical  and 
functional  changes. 

(6)  To  study  the  pharmacologic  therapy  of  this  disease  process. 

Preliminary  studies  suggest: 

(1)  Obstruction  to  airflow  is  a  significant  part  of  the  pathology  of 
this  disorder. 

(2)  Classic  immune  mechanisms  (at  least  as  defined  by  serologic  para- 
meters) are  not  frequent  occurrences. 

(3)  Rare  patients  can  be  classified  as  having  environmental  etiologies 
(defined  by  electron  probe  analyses  of  biopsies) . 

(4)  The  biochemical  pathology  is  much  more  than  simply  an  increase  in 
connective  tissue  synthesis. 

(5)  Ventilatory  and  arterial  blood  gas  parameters  with  exercise  are 
probably  the  most  sensitive  monitors  of  the  disease  process. 

(6)  Patients  with  this  disorder  have  marked  abnormalities  in  ventilation- 
per fusion  iiusmatching. 

These  disorders  are  almost  uniformly  fatal  and  affect  a  significant  pro- 
portion of  the  population.  Up  to  this  time,  there  has  been  no  information 
on  pathogenesis  and  there  is  essentially  no  cure.  By  ccmbining  studies  on 
patients  with  these  disorders  with  our  large  basic  research  program  in  the 
control  of  connective  tissue  accumulation  in  lung,  we  expect  to  make  major 
inroads  into  understanding  and  eventually  curing  these  disorders. 


V/7 


Project  No.  Z01  HL  02401-03  PB 

1.  Pulmonary  Branch 

2.  Section  on  Pulmonary 

Biochemistry 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Models  of  Lung  Growth 

Previous  Serial  Number:  NHLI  110 

Principal  Investigators:  Ronald  G.  Crystal,  M.D. 

Morton  J.  Cowan,  M.D. 
Allen  L.  Hbrwitz,  M.D. 
Robert  Bienkowski,  Ph.D. 

Other  Investigators:.  None 

Cooperating  Units:  William  M.  Thurlbeck,  M.D. 

Midhurst  Medical  Research  Institute 
Midhurst  Sussex,  England 

Project  Description: 

Objectives:  The  major  function  of  the  lung  is  to  transfer  oxygen  from  the 
atmosphere  to  the  blood  and  carbon  dioxide  from  the  blood  to  the  atmosphere. 
This  function  is  provided  by  the  unique  structure  of  lung  and  its  mechanical 
properties  in  relation  to  changes  in  intrapleural  pressure.   The  structure 
and  mechanical  properties  of  lung  are  intimately  dependent  on  the  connective 
tissue  composition  of  the  lung.  The  presence  of  these  connective  tissue 
components  is  controlled  by  a  balance  of  synthesis  and  degradation.  Two 
models  are  being  used  to  investigate  the  control  of  connective  tissue  syn- 
thesis and  degradation:  (1)  changes  during  normal  lung  growth  and  (2) 
"compensatory"  lung  growth  following  unilateral  pneumonectomy. 

Methods:   (1)  Connective  tissue  synthesis  and  degradation  during  lung  develop- 
ment. Methods  have  been  developed  to  keep  explants  of  lung  tissue  viable  for 
more  than  24  hours  under  defined  culture  conditions.  Minces  from  rabbit 
lungs  obtained  at  different  ages  are  incubated  at  varying  times  and  the  rates 
of  collagen  synthesis,  non-collagen  protein  synthesis  and  glycosaminoglycan 
synthesis  are  determined.  In  parallel  studies,  the  density  of  lung  collagen, 
total  glycosaminoglycans  and  specific  types  of  glycosaminoglycans  are  quanti- 
tated.  Other  methods  are  used  to  quantitate  the  fraction  of  newly  synthesized 
collagen  that  is  subsequently  degraded. 

(2)  Lung  growth  following  pneumonectomy.  Adult  rabbits  undergo  unilateral 
pneumonectomy  while  paired  litter  mate  controls  undergo  (a)  thoracotomy 


1/f 


without  pneumonectomy  or  (b)  pneumonectomy  and  immediate  filling  of  the 
empty  chest  cavity  with  wax.  At  intervals  following  surgery,  the  lungs  are 
examined  in  explant  culture  as  described  above. 

Major  Findings:  (l)  Connective  tissue  synthesis  and  degradation  during  lung 
development.  Last  year  the  normal  pattern  of  lung  collagen  and  non-collagen 
protein  synthesis  were  described  in  normal  rabbit  lung  growth  (see  individual 
project  report  #  NHLI  110) .  The  collagen  synthesis  methods  have  been  extended 
so  the  lung  explants  can  be  kept  viable  for  more  than  24  hours.  This  has 
permitted  examination  of  the  early  proteolysis  (0-24  hours)  of  newly  syn- 
thesized lung  collagen.  In  the  newborn  lung,  25%  of  the  collagen  synthesized 
in  four  hours  is  degraded  to  less  than  10,000  molecular  weight  (  from  original 
100,000  molecular  weight).  Under  the  conditions  used,  there  is  no  further 
degradation  of  newly  synthesized  collagen  from  4  to  24  hours  of  incubation. 
In  the  adult  lung,  although  synthesis  of  collagen  is  2-3  fold  less,  25%  of 
the  newly  synthesized  collagen  is  degraded.  Thus  it  appears  that:  (a)  one- 
fourth  of  newly  synthesized  lung  collagen  is  degraded  very  rapidly  (probably 
intracellular ly  or  immediately  following  secretion  from  the  cell) ;  (b)  this 
mechanism  does  not  appear  to  be  important  in  the  control  of  lung  collagen 
accumulation  during  lung  growth;  and  (c)  since  collagen  density  does  not 
change  in  the  adult,  75%  of  the  collagen  synthesized  by  the  adult  lung  must 
be  present  for  greater  than  24  hours  before  it  is  degraded. 

The  density  of  lung  glyoosaminoglycans  (GAG)  in  lung  varies  significantly 
with  age.  Parenchymal  GAG  content  ranged  between  0.2  -  0.4%  (w/w)  of  dry 
weight  with  highest  density  in  adult  lung.  Fetal  lung  contains  a  relatively 
high  proportion  of  chondroitin  4-sulfate  while  the  GAG  in  lung  parenchyma 
of  older  animals  was  predominantly  dermatan  sulfate,  heparan  sulfate  and 
heparin.  The  rate  of  synthesis  of  total  GAG  per  cell  increased  with  develop- 
ment to  a  maximum  in  lung  from  weanling  rabbits  and  fell  to  low  rates  of 
synthesis  in  mature  rabbits.  Fetal  rabbit  lung  parenchyma  synthesized  mostly 
hyaluronic  acid  and  heparan  sulfate  while  in  weanling  rabbit  parenchyma  hyal- 
uronic acid  and  chondroitin  4/6  sulfate  synthesis  was  greatest.  In  mature 
animals,  the  rates  of  synthesis  of  all  types  of  GAG  were  relatively  low, 
but  there  is  a  relatively  greater  emphasis  on  synthesis  of  dermatan  sulfate 
and  heparin. 

(2)  Lung  growth  following  pneumonectomy.  Within  one  month  after  left  pneu- 
monectomy in  the  adult  rabbit,  the  mass  of  the  right  lung  doubles.  This 
growth  can  be  characterized  by  a  period  of  rapid  collagen  accumulation  pre- 
ceded by  a  shift  in  emphasis  of  total  protein  synthesis  toward  collagen 
synthesis.  As  a  result,  the  total  amount  of  collagen  doubles  but  the  concen- 
tration of  collagen  remained  constant.  This  is  in  contrast  to  neonatal  lung 
growth  in  which  the  shift  in  protein  synthesis  toward  collagen  accumulation 
resulted  in  an  increased  concentration  of  collagen. 

When  wax  is  used  to  obliterate  the  space  left  by  pneumonectomy,  the  "compensa- 
tory" lung  growth  (  changes  in  lung  cells,  collagen  synthesis  and  accumu- 
lation) is  completely  obviated.  Hence,  it  appears  that  mechanical  factors 
(i.e.,  size  of  the  chest  cavity)  are  crucially  important  in  determining  lung 


73* 


growth  in  this  model. 

Significance  to  Biomedical  Research  and  Institute  Program;  These  two  models 
provide  a  means  to  study  the  lung  during  periods  of  "turning  off"  or 
"turning  on"  of  connective  tissue  synthesis  and  degradation.  Through  a 
comprehensive  approach  at  the  cellular  and  sub-cellular  levels,  it  should  be 
possible  to  understand  the  control  of  structural  and  non-structural  protein 
synthesis  in  the  lung.  This  has  obvious  applications  to  the  control  of 
fibrosis  in  the  interstitial  lung  diseases.  An  understanding  of  lung  growth 
will  hopefully  provide  means  to  approach  the  regeneration  of  functional 
alveolar  units  in  the  emphysematous  disorders. 

Proposed  Course  to  Project:  The  methods  to  quantitate  the  density,  synthe- 
sis  and  early  degradation  of  lung  collagen  and  the  density  and  synthesis  of 
lung  glycosaminoglycans  in  models  of  lung  growth  are  now  complete.  The 
next  steps  include: 

(1)  Quantitation  of  "late"  degradation  of  collagen  present  for  more  than 
24  hours.  Investigation  into  manipulation  of  lung  collagen  degradation 
(both  "early"  and  "late")  with  serum  and  pharmacologic  agents. 

(2)  Continued  investigation  into  the  factors  responsible  for  the  growth  of 
the  lung  following  unilateral  pneumonectomy.  There  is  preliminary  evidence 
that  serum  "growth"  factors,  as  yet  undefined,  may  play  an  important  role. 

(3)  Continued  investigation  of  the  subcellular  mechanisms  involved  in  the 
control  of  the  synthesis  of  connective  tissue  components  during  lung  growth 
(see  report  on  mechanisms  of  collagen  and  non-collagen  protein  synthesis  in 
lung  cell-free  systems,  project  report  no.  Z01  HL  02402-03  PB) . 

(4)  Investigations  into  the  interactions  of  connective  tissue  components 
(collagen,  elastin,  glycosaminoglycans)  between  themselves  and  lung  cells 
in  controlling  lung  connective  tissue  accumulation. 

(5)  Role  of  immunologic  and  proteolytic  mechanisms  (lymphocytes,  macrophages, 
immunoglobulins,  complement)  in  the  maintenance  of  normal  lung  connective 
tissue. 

(6)  Quantitate  the  morphcmetric  changes  in  post-pneumonectomy  lung  growth 
(with  Dr.  William  Thurlbeck,  Midhurst  Medical  Research  Institute,  Midhurst 
Sussex,  England) . 

Keyword  Descriptors:  Lung,  Collagen,  Glycosaminoglycans,  Pneumonectomy, 
Lung  Growth,  Proteolysis,  Explant  Culture. 

Honors  and  Awards:  None 

Publications: 

Crystal,  R.G.  Lung  Collagen:  Definition,  Diversity  and  Development.  Fed.  Proc. 
33:  2248,  1974. 


72/ 


Cowan,  M.J.  and  Crystal,  R.G.  Lung  Growth  After  Unilateral  Pneumonectomy: 
Quantitation  of  Collagen  Synthesis  and  Content.  Am.  Rev,  of  Rasp.  Pis.  Ill: 
267,  1975. 

Cowan,  M.J. ,  Collins,  J.F.  and  Crystal,  R.G.  Collagen  and  Lung  Growth:  A 
Prototype  of  Connective  Tissue  Differentiation.  In:  J.  Last  (Ed.): 
Eukaryotes  at  the  Subcellular  Level:  Development  and  Differentiation. 
M.  Dekker,  New  York,  1975  (in  press) . 


7<5L^ 


Project  No.  Z01  HL  02402-03  PB 

1.  Pulmonary  Branch 

2.  Section  on  Pulmonary 
Biochemistry 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  3,  1974  through  June  30,  1975 

Project  Title:   Control  of  Collagen  Synthesis  in  Lung  Cell-Free  Systems 

Previous  Serial  Number:  NHLI  113 

Principal  Investigators:  Ronald  G.  Crystal,  M.D. 

James  F.  Collins,  Ph.D. 
Morton  J.  Cowan,  M.D. 

Other  Investigators:  None 

Cooperating  Units:  None 

Project  Description: 

Objectives:  While  explant  culture  systems  are  adequate  for  the  description 
of  protein  synthesis  and  degradation  by  lung,  it  does  not  have  the  potential 
to  understand  the  molecular  control  of  these  events.  Since  a  primary  goal  of 
this  laboratory  is  to  understand  and  eventually  control  the  mechanisms  of 
lung  connective  tissue  accumulation,  it  is  necessary  to  develop  systems  in 
which  specific  identification  of  control  mechanisms  can  be  identified.  For 
this  reason,  a  cell-free  protein  synthesizing  system  is  desirable  and  affords 
an  opportunity  to  understand  control  of  lung  protein  synthesis  at  several 
levels. 

Methods:  The  components  necessary  for  a  partially  fractionated  cell-free 
protein  synthesizing  system  have  been  isolated  from  rabbit  lung  and  human 
lung  fibroblast  cultures  using  conventional  techniques.  These  components 
include  polyribosomes,  0.5M  KCl  polysome  wash  fraction  (including  initiation 
factors,  elongation  factors,  synthetases,  and  tRNA.)  and  post-trans lational 
enzymes  (e.g.,  prolyl  hydroxylase).  Polysomes  are  separated  into  two  classes: 
those  bound  to  the  endoplasmic  reticulum  and  those  free  in  the  cytoplasm. 
The  identification  of  collagen  in  a  cell-free  system  is  complex,  but  a  rapid 
assay  using  purified  bacterial  collagenase  has  been  used  with  success. 

Major  Findings:  Collagen  in  lung  is  synthesized  in  polysomes  isolated  from 
the  endoplasmic  reticulum  of  lung.  These  polysomes  can  be  translated  ef- 
ficiently in  a  heterologous  system  (rabbit  reticulocyte  and  liver)  where  the 
only  components  from  lung  are  the  polysomes.  Synthesis  of  collagen  in  the 
cell-free  system  has  been  identified  by  molecular  sieve  and  ion-exchange 
chromatography,  acrylamide  gel  electrophoresis,  sensitivity  to  collagenase 
and  synthesis  of  hydroxyproline  containing  peptides.  During  lung  development 


7*23 


there  are  significant  changes  in  the  relative  proportion  of  lung  polysomes 
that  synthesize  collagen.  The  percentage  of  collagen  (relative  to  other  lung 
proteins)  in  the  cell-free  system  derived  from,  the  newborn  rabbit  is  twice 
that  in  the  cell-free  system  derived  from  the  adult  rabbit.  These  changes 
parallel  the  findings  in  control  of  collagen  synthesis  in  lung  explants  sug- 
gesting that  at  least  some  of  the  control  of  lung  collagen  synthesis  may  be 
due  to  changes  in  lung  collagen  gene  expression  or  due  to  changes  in  the  num- 
ber of  lung  cells  that  synthesize  collagen. 

Significance  to  Biomedical  Research  and  Institute  Program:   At  each  stage  of 
development  of  the  other  projects  in  this  laboratory,  the  cell-free  system  can 
be  applied  to  understand  the  control  of  synthesis  of  structural  and  non-struc- 
tural proteins  in  the  lung.  Once  the  normal  mechanisms  are  identified,  com- 
parison with  human  pathologic  states  will  be  made.  For  example,  identification 
of  an  increase  in  a  specific  collagen  mRNA  in  a  certain  fibrotic  lung  disease 
may  help  identify  the  primary  pathology.   In  addition,  the  cell-free  system 
is  readily  adaptable  to  studying  molecular  control  mechanisms  and  identifying 
vulnerable  steps  in  the  process  of  connective  tissue  synthesis  which  may  be 
influenced  to  treat  specific  human  lung  disorders. 

Proposed  Course  to  Project;  As  the  cell-free  system  is  refined,  it  will  be 
used  to  quantitate  the  messenger  RNA  for  specific  collagen  chains.  A  purified 
collagen  mRNA,  will  be  used  to  make  a  reverse  DNA  copy  to  be  used  as  a  probe 
for  quantitating  the  number  of  collagen  genes  during  normal  lung  growth  and  in 
the  fibrotic  lung  disorders.   Techniques  will  be  developed  to  establish  cell- 
free  systems  from  human  lung  including  human  lung  cells  maintained  in  culture. 
Investigations  will  concentrate  on  identifying  the  molecular  mechanisms  con- 
trolling normal  lung  growth  and  the  fibrosis  of  lung  following  injury. 

Keyword  Descriptors:   Lung,  Protein  Synthesis,  Collagen,  Cell-free,  Messenger 
RNA,  Translational  Control,  Polysomes,  Initiation  Factors. 

Honors  and  Awards:   None 
Publications: 

Collins,  J.F.  and  Crystal,  R.G.   Protein  Synthesis.   In:  R.G.  Crystal  (ed.). 
The  Biochemical  Basis  of  Pulmonary  Function,  M.  Dekker,  New  York,  1975. 

Collins,  J.F.  and  Crystal,  R.G.   Characterization  of  Cell-Free  Synthesis  of 
Collagen  By  Lung  Polysomes  in  a  Heterologous  System.   J.  Biol.  Chem.  (Sub- 
mitted) 


7A¥ 


Project  No.  Z01  HL  02403-02  PB 

1.  Pulmonary  Branch 

2.  Section  on  Pulmonary 
Biochemistry 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Collagen  Synthesis  in  Cultured  Lung  Cells 

Previous  Serial  Number:  NHLI  109 

Principal  Investigators:  Ronald  G.  Crystal,  M.D. 

Allan  Hance,  M.D. 
Allen  Horwitz,  M.D.,  Ph.D. 
Norton  Elson,  M.D. 
Kathryn  Bradley,  M.S. 
Sally  McConnell-Breul,  M.S. 

Other  Investigators:  None 

Cooperating  Units:  None 

Project  Description: 

Objectives:   The  application  of  biochemical  technology  to  human  lung  disease  is 
limited  by  the  complexity  of  the  organ  and  the  unavailability  of  large  quan- 
tities of  lung  cells  from  patients  with  lung  disease.   There  are  more  than  40 
cell  types  in  lung,  and  some  of  these  cells  are  responsible  for  the  synthesis 
of  5  different  types  of  collagen  chains.  When  the  lung  synthesizes  abnormal 
amounts  of  collagen  in  response  to  injury,  the  same  cells  normally  producing 
collagen  may  proliferate;  they  may  synthesize  relatively  more  collagen  or  cells 
that  do  not  normally  synthesize  collagen  may  be  "turned  on"  to  do  so.  One  of 
the  primary  objectives  of  this  laboratory  is  to  develop  the  technology  to  cul- 
ture the  cells  from  lung  that  are  important  in  collagen  synthesis  in  health  and 
disease. 

Methods:  Fibroblasts  and  epithelial  cells  are  cloned  from  primary  cultures  of 
rabbit,  cat,  rat  or  human  lung  cells  dispersed  from  lung  with  trypsin.  Macro- 
phages are  removed  from  lung  by  lavage.  Methods  have  been  developed  to  quan- 
titate  absolute  rates  of  collagen  and  non-collagen  protein  synthesis  in  these 
cells  utilizing  intracellular  measurements  of  specific  activity  or  labeled 
amino  acids  and  by  quantitating  labeled  collagen  with  collagenase  or  hydroxy- 
proline  measurements.  The  types  of  collagen  synthesized  are  determined  by 
ion-exchange  chromatography  and  cyanogen  bromide  peptide  mapping  techniques. 

Major  Findings:  Fibroblasts  from  fetal  or  newborn  rabbit,  cat,  rat  and  human 
lung  actively  synthesize  Type  I  and  Type  III  collagen.  The  collagen  secreted 
into  the  media  of  these  cultures  is  in  a  precursor  form  called  pro-a  chains. 
An  epithelial  cell  cultured  from  fetal  cat  lung  also  synthesizes  Type  I  and 

1  W 


Type  III  collagen  but  not  as  actively  as  the  fibroblast.  Pulmonary  alveolar 
macrophages  do  not  synthesize  collagen.  A  method  has  been  developed  to  prepare 
a  "fingerprint"  of  the  proteins  secreted  by  cultured  lung  cells  to  be  used  as 
biochemical  markers  to  identify  the  cell  types.  The  "fingerprint"  of  the 
fibroblast,  epithelial  cell  and  macrophage  are  markedly  different.  Purified 
collagen  chains  isolated  from  the  media  of  these  cultures  are  being  utilized 
to  prepare  antibodies  against  specific  types  of  collagen.  These  antibodies 
are  being  used  to  quantitate  collagen  synthesis  in  culture,  cell-free  systems 
and  biopsy  specimens  (by  immunof luorescent  methods). 

Significance  to  Biomedical  Research  and  Institute  Program:   The  mechanisms  by 
which  collagen  is  synthesized  and  degraded  in  the  lung  are  fundamental  to  the 
control  of  normal  lung  structure  and  function.   In  the  fibrotic  lung  disorders 
these  mechanisms  are  presumably  deranged  so  that  collagen  is  synthesized  in 
either  abnormal  amounts  and/or  in  abnormal  regions  of  the  lung.   The  establish- 
ment of  diploid  cell  lines  of  the  cells  responsible  for  collagen  synthesis  in 
health  and  disease  will  allow  investigations  at  the  molecular  level.   This  in- 
cludes identification  of  the  normal  mechanisms  and  how  these  are  varied  fol- 
lowing lung  injury. 

Proposed  Course  of  Project:   There  will  be  continued  establishment  of  dif- 
ferent cell  lines,  the  investigation  of  the  type  of  collagen  synthesized  by 
each  and  inquiry  into  the  mechanisms  of  collagen  synthesis  in  each.  One  area 
of  particular  interest  is  the  synthesis  of  three  different  collagen  chains  by 
an  apparent  homogeneous  cell  fibroblast  line.   Through  cloning  techniques,  it 
should  be  possible  to  determine  if  this  is  in  fact  a  single  cell  line.   If  so, 
investigations  into  the  quantitative  control  of  synthesis  and  degradation  of 
each  collagen  type  will  be  done.   Once  these  techniques  are  established  in 
normal  animal  lung  cells,  they  will  be  applied  to  the  study  of  human  cells  from 
normal  and  abnormal  lungs.   Immunof luorescent  methods  should  enable  the  iden- 
tification of  collagen  types  in  biopsy  specimens  from  patients  with  different 
types  of  lung  disease. 

Keyword  Descriptors:  Lung,  Collagen,  Collagen  Heterogeneity,  Lung  Cells, 
Macrophage,  Fibroblasts,  Epithelial  Cell,  Cell  Culture. 

Honors  &  Awards:  None 

Publications:   Hance,  A.J.,  Bradley  K.  and  Crystal,  R.G.   Lung  Collagen 
Heterogeneity  II.   Synthesis  of  Type  I  and  Type  III  Collagen  by  Rabbit  and 
Human  Lung  Cells  in  Culture.   J.  Clin.  Invest.  (Submitted) 


r& 


Project  No.  Z01  HL  02404-03  PB 

1.  Pulmonary  Branch 

2.  Section  on  Pulmonary 
Biochemistry 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  The  Accumulation  of  Collagen  in  Human  Lung 

Previous  Serial  Number:  NHLI  111  (c) 

Principal  Investigators:  Ronald  G.  Crystal,  M.D. 

Morton  J.  Cowan,  M.D. 
Jack  D.  Fulmer,  M.D. 
Kathryn  Bradley,  M.S. 
Sally  McConnell-Breul,  M.S. 
Allan  J.  Hance,  M.D. 

Other  Investigators:  None 

Cooperating  Units:  None 

Project  Description: 

Objectives:  The  connective  tissue  of  the  lung  is  fundamental  for  lung  struc- 
ture and  mechanical  properties.  In  addition,  the  interstitial  lung  diseases, 
many  of  which  result  in  pulmonary  fibrosis,  represent  approximately  20%  of 
lung  diseases  (other  than  the  infectious  diseases) .  Almost  nothing  is  known 
about  the  composition  of  human  lung  collagen  nor  its  synthesis  and  regulation. 
With  the  ultimate  aim  being  an  ability  to  control  pulmonary  fibrosis  through 
molecular  mechanisms,  our  laboratory  is  developing  the  technology  to  quanti- 
tate  human  lung  collagen  composition  and  synthesis. 

Methods:  Lung  tissue  is  obtained  from  patients  undergoing  thoracotomy  or 
from  fetuses  following  therapeutic  abortion.  Intact  collagen  chains  are  ex- 
tracted with  conventional  techniques.  Lung  collagen  is  synthesized  in  short- 
term  explant  cultures  of  human  lung  and  compared  and  identified  with  the  ex- 
tracted collagen  chains  by  ion-exchange  chromatography,  SDS  and  acidic  acry- 
lamide  gels,  sensitivity  to  collagenase  and  hydroxyproline  content.  Conditions 
have  been  developed  to  quantitate  and  compare  the  rate  of  collagen  synthesis 
in  biopsies  from  human  lung. 

Major  Findings:  Normal  human  lung  synthesizes  at  least  two  collagen  chains 
(al (I) ,  a2)  and  probably  a  third  (al (III) ) .  Fibroblasts  cloned  from  human 
lung  synthesize  al(I),  a2  and  al(III)  chains.  In  the  16-20  week  human  fetal 
lung  and  in  the  adult  human  lung  approximately  4%  of  the  total  amino  acids 
incorporated  into  protein  are  incorporated  into  collagen.  Biopsies  from  three 
patients  with  idiopathic  pulmonary  fibrosis  have  been  examined.  Interestingly, 


7*2/ 


none  had  increases  in  the  percentage  of  protein  synthesis  that  was  collagen, 
suggesting  that  the  molecular  pathology  in  f ibrotic  lung  disease  is  more  than 
simply  an  increase  in  fibrous  tissue  synthesis.  Methods  have  been  developed 
to  quantitate  human  lung  collagen  proteolysis  as  well  as  synthesis  to  deter- 
mine the  influence  of  rates  of  degradation  of  collagen  on  accumulation  of  col- 
lagen in  these  disorders. 

Significance  to  Biomedical  Research  and  Institute  Program:  In  the  interstitial 
lung  disorders,  the  lung  responds  to  injury  by  producing  fibrosis  resulting 
in  functional  pathology  in  gas  exchange.  Through  the  techniques  developed  in 
our  laboratory,  it  should  be  possible  to:  (1)  guantitate  the  degree  of  the 
f ibrotic  process  (i.e.,  rate  of  collagen  synthesis  relative  to  collagen  de- 
struction) (2)  develop  an  in  vitro  system  for  testing  drug  efficacy  in  these 
disorders. 

In  the  emphysematous  disorders  there  is  destruction  of  the  connective  tissue 
comprising  the  lung  with  either  absent  or  ineffective  remodeling  of  the  con- 
nective tissue.  Of  particular  interest  is  the  al  antitrypsin  group  of 
patients,  who  have  an  inherited  disorder  of  the  serum  al  globulins  associated 
with  emphysema.  The  techniques  described  above  can  be  used  directly  to  test 
the  influence  of  the  serum  proteins  on  protecting  the  lung  from  proteolysis. 

Proposed  Course  of  Project:  The  normal  patterns  of  collagen  synthesis  and 
HegrgrJa-i-jnn  will  hp  qnan-t-i  ta-t-ftfl  for  different  age  groups.  Once  a  baseline  is 
firmly  established,  it  will  be  possible  to  quantitate  the  influence  of  other 
factors  (drugs,  al  globulins,  a2  macroglobulins)  on  the  f ibrotic  process  in 
biopsy  specimens  from  patients  with  interstitial  disease.  It  should  then  be 
possible  to  predict,  for  each  patient,  which  drug(s)  will  be  most  efficacious. 

Keyword  Descriptors:  Collagen,  Human  Lung,  Pulmonary  Fibrosis,  Collagen 
Synthesis,  Collagen  Degradation. 

Honors  and  Awards:  None 

Publications:  Bradley,  K. ,  McConnell-Breul,  S.  and  Crystal,  R.G.  Collagen 
in  the  Human  Lung:  Composition  and  Quantitation  of  Rates  of  Synthesis. 
J.  Clin.  Invest.,  55:  543,  1975. 

Crystal,  R. ,  Bradley,  K. ,  McConnell-Breul,  S.,  Collins,  J.,  Hance,  A.,  and 
Cowan,  M.  Collagen  in  the  Lung:  Development  of  a  Technology  Applicable  to 
Human  Lung  Disease.  Chest,  67:  305,  1975. 


7J& 


Project  No.  Z01  HL  02405-02  PB 

1.  Pulmonary  Branch 

2.  Section  on  Pulmonary 
Biochemistry 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Experimental  Models  of  the  Interstitial  Lung  Disorders 

Previous  Serial  Number:   NHLI  112 

Principal  Investigators:  Ronald  G.  Crystal,  M.D. 

Sally  McConnell-Breul,  M.S. 

Allan  J.  Hance,  M.D. 

Allen  L.  Horwitz,  M.D.,  Ph.D. 

Norton  Elson,  M.D. 

William  Wagner,  M.S. 

Jack  D.  Fulmer,  M.D. 

Other  Investigators:   None 

Cooperating  Units:  None 

Project  Description: 

Objectives:  A  major  goal  of  this  laboratory  is  to  understand  and  eventually 
control  the  mechanisms  involved  in  connective  tissue  synthesis  and  de- 
gradation in  human  lung,  in  diseases  involving  these  components.   Because  the 
availability  of  human  lung  biopsy  material  is  limited,  it  necessitates  that 
any  biochemical  techniques  applied  to  this  tissue  is  worked  out  first  in 
animal  models,  thus  insuring  the  maximum  amount  of  information  and  the  maxi- 
mum benefit  to  the  patient.  For  that  reason  two  groups  of  models  of  fibrotic 
disease  are  being  developed:  (1)  animal  models;  and  (2)  tissue  culture  models. 

Methods:   (1)  Animal  models:  Silica  particles,  1-5  microns  in  diameter  are 
injected  intratracheal ly  in  6  week  old  rabbits.  At  intervals  following  ex- 
posure, the  animals  are  killed  and  measurements  are  make  of  lung  of  collagen 
content,  rates  of  collagen  synthesis  and  the  relative  proportion  of  the  pro- 
teins are  being  synthesized  that  are  collagen.  These  are  compared  to  litter 
mate  paired  controls.  Other  animal  models  being  developed  include:  radiation 
(external)  injury,  hypersensitivity  to  inhaled  antigens  and  chemical  mediated 
lung  injury.  In  all  animal  models,  the  biochemical  measurements  are  being 
compared  to  morphologic  (light  and  electron  microscopy)  and  functional  altera- 
tions. The  latter  are  being  studied  in  a  newly  developed  in  vitro  plethysmo- 
graph  which  can  be  used  to  measure  pulmonary  function  parameters  of  the  paren- 
chyma and  airways. 

(2)  Tissue  culture  models:  The  development  of  methods  to  culture  and  contin- 
ually passage  lung  cells  in  culture  has  enabled  us  to  begin  the  development  of 

i  rsff 


in  vitro  models  of  lung  cell  injury.  Cultured  lung  cells  can  be  interacted 
with  other  cells  (macrophages,  lymphocytes)  or  their  products.  They  can  also 
be  studied  in  relation  to  direct  irradiation  or  with  chemical  and  pharmaco- 
logical mediators  of  lung  injury.  Rates  of  connective  tissue  .synthesis  and 
degradation  can  be  quantitated  and  cell-free  methods  can  be  applied.  Mor- 
phologic alterations  at  the  electron-microscopic  level  are  also  studied. 

Major  Findings:   (1)  Animal  models  -  Although  collagen  accumulation  takes 
place  in  silica  exposed  animals,  it  is  a  difficult  model  to  work  with  because 
the  silicotic  "nodules"  are  discrete  and  inhomogeneous .  Radiation  induced 
fibrosis  has  been  a  much  more  satisfactory  model  and  preliminary  studies  have 
demonstrated  increased  collagen  density  6  weeks  after  6000  R  to  the  lung. 

(2)  Tissue  culture  models  -  The  pulmonary  alveolar  macrophage  can  be.  induced 
to  secrete  collagenase,  a  unique  and  specific  enzyme  for  mammallian  collagen. 
Rabbit  macrophages  activated  with  BCG  (in  vivo)  will  secrete  collagenase  after 
1-2  days  in  culture.  Exposure  of  these  macrophages  to  protein  antigens  de- 
rived from  mycobacterium  will  further  augment  this  finding.  Sufficient  quan- 
tities of  mammallian  collagenase  are  being  purified  to  produce  antibodies 
specific  for  it.  Soon,  it  should  be  possible  to  have  tissue  culture  systems 
involving  lung  cells,  macrophages,  lymphocytes  and  serum  from  animals,  normal 
humans  and  humans  with  fibrotic  lung  disease  in  which  the  rates  of  synthesis 
and  degradation  of  specific  collagen  types  can  be  quantitated  and  manipulated. 

Significance  to  Biomedical  Research  and  Institute  Program:  As  we  learn  to 
define  the  animal  and  tissue  culture  models  of  fibrotic  lung  disease,  we 
should  be  able  to  understand  the  sensitive  control  points  where  we  can  inter- 
rupt the  fibrotic  process  with  pharmacologic  agents. 

Proposed  Course  to  Project;   Continued  development  of  these  models  with  par- 
ticular emphasis  on  the  quantitative  description  of  the  fibrotic  process 
including  the  type  of  collagen  synthesized  and  how  this  may  be  influenced  with 
drugs.  Other  projects  in  our  laboratory  such  as  cell-free  collagen  synthesis 
will  be  applied  to  these  models  as  the  technology  develops. 

Keyword  Descriptors:   Lung,  Fibrosis,  Animal  Models,  Cultured  Cells,  Lung 
Injury,  Macrophages,  Lymphocytes,  Fibrotic  Lung  Disease,  Collagen  Synthesis, 
Collagen  Degradation. 

Honors  and  Awards:  None 

Publications:   None 


73a 


Project  No.  Z01  HL  02406-02  PB 

1.  Pulmonary  Branch 

2.  Section  on  Pulmonary 
Biochemistry 

3.  Bethesda,  Maryland 

PHS  -  NIH 
Individual  Project  Eeport 
July  1,  1974  through  June  30,  1975 

Project  Title:  Heterogeneity  of  Lung  Collagen 

Previous  Serial  Number:  NHLI  108 

Principal  Investigators:  Ronald  G.  Crystal,  M.D. 

Allan  Hance,  M.D. 
Kathryn  Bradley,  M.S. 

Other  Investigators:  None 

Cooperating  Units:  None 

Project  Description: 

Objectives:  Collagen  is  the  most  abundant  protein  in  lung,  comprising  10-15% 
of  the  adult  lung  by  dry  weight.  There  are  more  than  40  cell  types  in  lung 
including  mesenchymal  cells,  epithelial  cells  and  smooth  muscle  cells,  all  of 
which  have  been  implicated  in  other  organs  to  synthesize  collagen.  The 
primary  objective  of  this  project  is  to  identify  the  heterogeneous  collagen 
chains  in  lung. 

Methods:  Because  the  majority  of  collagen  synthesized  in  lung  is  synthesized 
early  in  life,  the  collagen  in  the  adult  is  mostly  cross-linked  and,  there- 
fore, almost  impossible  to  extract  intact.  However,  conditions  have  been 
developed  so  that  100%  of  the  collagen  synthesized  in  short-term  explant  lung 
cultures  can  be  extracted  and  subsequently  analyzed.  Different  lung  structures 
(bronchial  tree,  blood  vessels  and  peripheral  lung)  are  isolated  under  a  dis- 
secting microscope  and  incubated  in  the  presence  of  labeled  amino  acids. 
The  collagen  chains  synthesized  are  extracted,  purified  and  subsequently 
compared  by  cyanogen  bromide  peptide  mapping  techniques. 

Antibodies  to  collagen  chains  purified  by  tissue  culture  methods  are  being 
used  to  relate  each  of  the  heterogeneous  collagen  chains  to  specific  lung 
structures. 

Major  Findings:  Prior  studies  demonstrated  that  the  lung  parenchyma  and 
blood  vessels  synthesized  al(I)  and  a2  chains  (Type  I  collagen)  while  the 
tracheobronchial  tree  synthesized  al (II)  chains  (Type  II  collagen) .  More 
recent  studies  have  demonstrated  od(III)  chain  (Type  III  collagen)  synthesis 
in  the  parenchyma.  These  collagen  chains  have  distinct  primary  amino  acid 
sequences  as  demonstrated  by  cyanogen  bromide  peptide  maps  on  ion  exchange 

1  73/ 


columns  and  acrylamide  gels.  All  evidence  to  date  suggests  that  while  lung 
collagen  is  markedly  heterogeneous,  each  type  collagen  is  identical  to 
collagen  elsewhere  in  the  body  (e.g.,  Type  I  is  the  same  from  lung,  skin, 
bone,  tendon,  blood  vessels;  Type  III  is  the  same  from  lung,  aorta,  skin; 
Type  II  is  the  same  from  tracheobronchial  tree  and  sternal  cartilage. 

Significance  to  Biomedical  Research  and  Institute  Program;  Normal  lung 
structure  and  function  depends  on  the  collagen  comprising  it.  During  the 
developmental  process  there  undoubtedly  are  changes  in  the  control  of  collagen 
synthesis  as  these  structures  change.  An  understanding  of  these  mechanisms 
will  help  toward  an  understanding  of  the  pathologic  process  in  diseased  lung. 

The  fibrotic  lung  diseases  have  many  primary  stimuli  including  inhalation  of 
toxic  materials,  hypersensitivity  states,  radiation  injury  and  associated 
with  systemic  disorders  such  as  scleroderma.  It  is  likely  that  these  dis- 
orders are  associated  with  injury  to  different  lung  cells,  suggesting  the 
possibility  that  there  may  be  a  heterogeneity  in  the  types  of  collagen  syn- 
thesized in  these  different  diseases.  It  may  be  possible  to  classify  the 
interstitial  lung  diseases  on  the  basis  of  the  types  of  collagen  synthesized 
in  a  similar  fashion  to  the  lipoprotein  disorders  and  certain  hemoglobin 
disorders. 

The  heterogeneity  of  lung  collagen  may  also  be  a  crucially  important  deter- 
minant of  lung  mechanical  properties  in  the  normal  development  of  lung. 

Proposed  Course  of  Project:   The  al(I),  a2,  al(II)  and  al(III)  collagen 
chains  from  lung  have  now  been  isolated,  purified  and  mapped.  It  is  known 
that  there  is  greater  than  70  sq.  meters  of  basement  membrane  (al(IV)  col- 
lagen chains)  in  lung;  the  next  step  will  be  to  isolate  and  purify  these 
chains.  Once  that  is  accomplished,  it  should  be  possible  to  quantitate  the 
rate  of  synthesis  and  degradation  of  each  lung  collagen  type  in  normal  lung 
development  and  in  human  lung  disease. 

Keyword  Descriptors:  Lung,  Collagen,  Collagen  Heterogeneity,  Peptide 
Mapping,  Collagen  Synthesis,  Collagen  Degradation,  Tracheobronchial  Tree, 
Blood  Vessels,  Lung  Parenchyma. 

Honors  and  Awards:  None 

Publications : 

Hance,  A.J.  and  Crystal,  R.G.  Collagen.  In:  R.G.  Crystal  (Ed.).  The  Bio- 
chemical Basis  of  Pulmonary  Function,  M.  Dekker,  New  York,  1975  ^in  Press) . 


7J& 


Project  No.  Z01  02407-01  PB 

1.  Pulmonary  Branch 

2.  Section  on  Pulmonary 
Biochemistry 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Studies  of  Patients  with  Fibrotic  Lung  Disease 

Previous  Serial  Number:  None 

Principal  Investigators:  Ronald  G.  Crystal,  M.D. 

Jack  D.  Fulmer,  M.D. 
Norton  Elson,  M.D. 

Other  Investigators:  None 

Cooperating  Units:  Bruce  Line,  M.D. ,  Clinical  Center,  Nuclear  Medicine 

Herbert  Reynolds,  M.D. ,  Allergy  and  Infectious  Diseases, 

Laboratory  of  Clinical  Investigation 
Irving  J.  Selikoff,  M.D.,  Mount  Sinai  School  of  Medicine, 

University  of  New  York 
Frederick  J.  Vfenzel,  M.D. ,  Marshfield  Clinic, 

Marshfield,  Wisconsin 
William  Roberts,  M.D.,  Heart  Institute,  Intramural 

Research  -  CD 
Victor  Ferrans,  M.D.,  Heart  Institute,  Intramural 

Research  -  OD 
Ralph  Dolin,  M.D.,  Allergy  and  Infectious  Diseases 

Laboratory  of  Clinical  Investigation 

Project  Description: 

Objectives:  The  fibrotic  lung  disorders  represent  15-20%  of  the  non-infectious 
disorders  of  the  lung.  The  mean  survival  of  these  patients  is  45  months  from 
the  onset  of  symptoms.   Although  approximately  5-10%  of  these  patients  re- 
spond to  corticosteroids,  there  is  essentially  no  treatment  for  the  remainder. 
The  natural  history,  etiology  and  pathogenesis  of  "idiopathic"  fibrotic  lung 
disease  is  poorly  studied.  The  Pulmonary  Branch,  NHLI,  has  undertaken  a  de- 
tailed study  of  these  patients  with  several  major  objectives: 

(1)  To  determine  the  etiology  of  idiopathic  fibrotic  lung  disease. 

(2)  To  follow  patients  longitudinally  to  determine  (a)  the  natural  history 
of  this  disorder  and  (b)  which  pulmonary  function  parameters  are  most 
sensitive  to  the  disease  process. 

(3)  To  correlate  pulmonary  alveolar  constituents  (fluid  and  pulmonary 
alveolar  macrophages)  with  fibrotic  disease. 

(4)  To  correlate  radioisotopic  monitors  of  lung  function  (ventilation,  per- 
fusion and  gallium  scans)  with  the  disease  process. 

1  733 


(5)  Tb  correlate  lung  pathogenic  alterations  with  biochemical  and  functional 
changes. 

(6)  To  study  the  pharmacologic  therapy  of  this  disease  process. 

Methods:  Patients  admitted  to  the  Pulmonary  Branch  Clinical  Service  enter 
an  extensive  protocol  which  includes:  detailed  medical  and  pulmonary  history 
and  physical  exam,  routine  serologic,  roenteographic  and  EKG  studies,  sero- 
logic studies  aimed  at  inrnune  processes;  pulmonary  function  studies  including 
lung  volumes,  flow  rates,  diffusing  capacity,  flow-volume  curves,  closing 
volume,  closing  capacity,  body  plethysmograph  functional  residual  volume  and 
lung  resistance  at  all  lung  volumes,  static  and  dynamic  pressure-volume  curves, 
maximum  flow  static  recoil  curves,  iso-volume  pressure-flow  curves,  ventila- 
tory and  arterial  blood  gas  studies  at  rest  and  exercise.  Dong  lavage  for 
macrophage  function  and  morphology,  secreted  proteins  of  macrophages  and  con- 
stituents of  alveolar  wash  fluid.  Technetium  vascular  scans,  133^  ventila- 
tion scans  and  85gallium  scans.  When  indicated,  lung  biopsy  is  done  either 
through  the  fiberoptic  bronchscope  or  via  open  thoracotomy.  Tissue  is  studied 
by  light  microscopy,  electron  microscopy,  culture  (routine,  fungal,  mycobac- 
teria and  virus) ,  collagen  synthesis  and  degradation  (see  Project  Report'  No. 
Z01  HL  02401-03  PB) ,  electron  probe  analysis  for  mineral  content  and  tissue 
culture  (see  Project  Report  No.  Z01  HL  02403-02  PB) .  Selected  patients  are 
then  entered  into  a  drug  treatment  protocol  where  all  patients  are  treated 
with  corticosteroids  (standard  therapy)  and  50%  of  the  patients  are  treated 
(in  addition)  with  azothioprine  (in  a  double  blind  fashion) . 

Major  Findings:  Preliminary  studies  suggest: 

(1)  Obstruction  to  airflow  is  a  significant  part  of  the  pathology  of  this 
disorder. 

(2)  Classic  immune  mechanisms  (at  least  as  defined  by  serologic  parameters) 
are  not  frequent  occurrences. 

(3)  Rare  patients  can  be  classified  as  having  environmental  etiologies 
(defined  by  electron  probe  analyses  of  biopsies) . 

(4)  The  biochemical  pathology  is  much  more  than  simply  an  increase  in  con- 
nective tissue  synthesis. 

(5)  Ventilatory  and  arterial  blood  gas  parameters  with  exercise  are  probably 
the  most  sensitive  monitors  of  the  disease  process. 

(6)  Patients  with  this  disorder  have  marked  abnormalities  in  ventilation- 
per fusion  mismatching. 

Significance  to  Biomedical  Research  and  Institute  Program:  These  disorders 
are  almost  uniformly  fatal  and  affect  a  significant  proportion  of  the  pop- 
ulation. Up  to  this  time,  there  has  been  no  information  on  pathogenesis  and 
there  is  essentially  no  cure.  By  combining  studies  on  patients  with  these 
disorders  with  our  large  basic  research  program  in  the  control  of  connective 
tissue  accumulation  in  lung,  we  can  expect  to  make  major  inroads  into  under- 
standing and  eventually  curing  these  disorders. 

Proposed  Course  to  Project:  Studies  as  outlined  will  be  continued.  As 
methods  are  developed  in  the  basic  laboratory,  they  will  be  applied  to  study 
the  biopsy  specimens  from  human  lung.  Particularly  important  are  the  studies 
in  lung  explants  and  tissue  culture  where  the  manipulation  and  control  of  lung 

73* 


ooHagen  synthesis  and  degradation  can  be  studied  with  pharmacologic  agents. 
As  results  with  pharmacologic  agents  become  promising,  they  will  be  studied 
in  patients  when  applicable. 

Keyword  Descriptors:  Fibrotic  Lung  Disease,  Lung  Collagen,  Lung  Fibrosis, 
Pulmonary  Function  Testing,  Corticosteroids,  Azothioprine,  Macrophage. 

Honors  and  Awards:  None 

}?ublications : 

Fulmer,  J.D.  and  Crystal,  R.G.  The  Biochemical  Basis  of  Pulmonary  Function. 
In:  R.G.  Crystal  (Ed.):  The  Biochemical  Basis  of  Pulmonary  Function.  M. 
Dekker,  New  York,  1975  (in  Press) .  ~ 


7-3T 


Annual  Report  of  the 
Section  on  Molecular  Pharmacology 

Pulmonary  Branch 
National  Heart  and  Lung  Institute 
July  1,  1974  through  June  30,  1975 

Receptors:   Dysfunctions  of  airway  and  vascular  smooth  muscle  are  most  read- 
ily brought  under  pharmacological  control  by  drugs  acting  at  neuromuscular 
junctions  where  signals  are  transmitted  to  receptors  in  muscle  membrane  by 
chemical  agents.   As  an  aid  to  the  design  of  new  drugs  and  to  understanding 
the  selectivity  of  certain  drugs  for  receptors  in  specific  organs,  the  molecu- 
lar events  associated  with  receptor  activation  are  being  studied  in  this 
Section.   Over  the  long  term  emphasis  will  be  placed  on  a-adrenergic,  sero- 
tonin and  other  receptors  that  initiate  contraction,  since  relatively  less  is 
known  about  these  receptors  than  about  the  3-adrenergic  relaxing  mechanisms 
mediated  by  cyclic  AMP.   Ultimately  attention  must  be  directed  to  the  possible 
role  of  calcium  release  from  membrane  stores,  activation  of  phospholipid  turn- 
over and  stimulation  of  guanyl  cyclase  activity,  all  of  which  have  been  postu- 
lated to  play  a  role  in  contractile  events.   At  the  moment,  however,  interest 
centers  on  the  initiating  event,  the  adsorption  of  agonist  on  a  recognition 
site,  and  research  has  been  directed  primarily  to  the  design  of  techniques 
for  receptor  isolation. 

The  successful  synthesis  of  [3H]-sulfanilic  acid  of  high  specific  activity  and 
the  availability  of  35S-labeled  material  should  make  it  possible  to  use  double 
isotope  procedures  to  identify  those  components  of  receptors  which  undergo 
agonist-dependent  changes  in  conformation.   Sulfanilic  acid  was  selected  as  a 
reagent  because  of  the  ease  with  which  it  can  be  diazotized  for  direct  incor- 
poration into  membrane  proteins  or  into  other  reagents  that  in  turn  can  be 
reacted  with  membranes.   Preparation  of  a  suitable  SH  reagent  is  nearing  com- 
pletion and  feasibility  studies  on  the  application  of  the  technique  to  aortic 
receptors  are  encouraging. 

Magnetic  resonance  and  the  reaction  of  drugs  with  biomolecules :   Previous  re- 
ports have  described  the  search  for  those  physical  techniques  most  likely  to 
be  useful  for  exploring  receptor  topography  and  the  evolution  of  electron 
spin  resonance  as  the  method  of  choice.   The  systematic  study  of  the  inter- 
action of  spin-labeled  compounds  with  model  surfaces  that  vary  considerably 
in  topography  has  continued.   There  has  been  some  shift  in  emphasis  toward 
studies  of  the  fluid  properties  of  phospholipid  membranes.   Changes  in  mem- 
brane fluidity  with  concomitant  increases  in  the  ease  of  association  of  pro- 
teins embedded  in  the  membrane  have  been  postulated  to  play  a  role  in  receptor 
response,  neoplastic  transformation  and  a  variety  of  other  membrane  functions. 

The  most  complicated  surface  thus  far  delineated  by  these  studies  is  that  of 
the  biotin  binding  protein,  avidin.   ESR  studies  of  its  complexes  with  spin- 
labeled  biotin  analogues  of  variable  length  suggest  a  quadrivalent  site  with 
two  pairs  of  clefts,  the  members  of  each  pair  being  separated  by  about  16  A. 
Between  each  pair  of  clefts  there  exist  two  lipophilic  sites  and  there  is  a 
more  peripheral  lipophilic  site  in  the  vicinity  of  each  cleft.   Other  studies 
of  'this  character  have  shown  that  immobilization  of  aminoacridine  spin  labels 
in  DNA  complexes  is  consistent  with  intercalation  of  the  amines  in  the  helix 


737 


and  that  ESR  can  be  used  to  study  the  effects  of  substituents  on  the  mobility 
of  acridines  in  such  complexes.   By  inserting  into  cell  membranes  stearic 
acid  analogues  bearing  spin  labels  at  various  positions  along  the  side  chain, 
it  has  been  possible  to  measure  changes  in  the  fluid  character  of  the  phospho- 
lipid bilayer.   Such  studies  have  shown  that  neoplastic  transformation  of  sev- 
eral cell  types  by  a   temperature  sensitive  mutant  of  Rous  sarcoma  virus  and 
by  SV-40  do  not  cause  any  change  in  membrane  fluidity.   Studies  of  the  H+ 
transporting  purple  membrane  of  Halobacterium  halobium  have  disclosed  a  bio- 
logical rarity,  an  extremely  rigid  membrane.   Spectral  evidence  suggested  two 
populations  of  membrane  lipids,  one  of  which  is  presumably  tightly  associated 
with  the  membrane's  single  protein.   An  interesting  new  feature  of  the  ESR 
studies  has  been  the  use  of  spin  trapping,  i.e.  the  conversion  of  a  biologic- 
ally generated  unstable  free  radical  into  a  stable  free  radical  sufficiently 
long  lived  to  be  easily  studied.   This  procedure  has  been  used  to  measure  the 
temperature-dependent  formation  of  free  radical  intermediates  in  the  metabo- 
lism of  organic  halides. 

Ion  transport:   The  sodium-and  potassium-dependent  ATPase  of  cell  membranes 
is  of  interest  as  the  putative  receptor  for  cardiac  glycosides,  which  act  ex- 
tracellularly  to  inhibit  the  intracellular  hydrolysis  of  ATP.   The  system  is 
also  of  interest  because  of  its  general  role  in  maintaining  excitability  of 
nerve  and  muscle  membranes.   It  remains  probably  the  most  useful  paradigm  for 
theoretical  studies  of  the  relationship  between  lipoprotein  organization  in 
the  membrane  and  transmembrane  control  of  intracellular  enzymic  functions. 
Various  aspects  of  the  system  are  under  investigation  in  many  laboratories; 
the  emphasis  in  this  section  is  on  structural  studies  of  the  system's  major 
protein. 

Current  studios  with  a  conveniently  prepared  purified  ATPase  system  from  kid- 
ney have  shown  that  cyanylation  of  protein  SH  groups  by  2-nitro-5-thiocyano- 
benzoic  acid  gives  a  stepwise  inactivation,  consistent  with  at  least  two  popu- 
lations of  SH  groups.   Protection  by  the  substrates,  ATP  and  sodium,  occurs 
with  only  the  more  slowly  reactive  sulfurs.   Efforts  to  find  evidence  for 
hidden  SH  groups  that  become  exposed  upon  phosphorylation  of  the  system  have 
not  yet  succeeded  with  the  cyanylating  reagent,  although  such  groups  can  be 
revealed  when  4C-cyanylating  reagent  has  been  synthesized  and  13C- cyanylating 
reagent  is  undergoing  synthesis.   These  compounds  should  make  possible  the 
determination  of  the  relative  labeling  of  the  two  protein  components  of  ATPase. 
Since  ligand-dependent  conformational  changes  associated  with  the  ion  trans- 
port cycle  are  limited  to  the  larger  protein,  future  emphasis  will  be  placed 
on  the  use  of  I3C  NMR  to  explore  conformational  changes  in  this  protein  and 
on  the  base  catalyzed  cleavage  of  the  cyanylated  protein  to  detect  conforma- 
tionally  mobile  loci. 

Studies  in  lung:   Studies  on  the  mechanisms  for  storage  and  release  of  hista- 
mine and  serotonin  in  the  lungs  of  Wistar  rats  continue.   The  rapid  appearance 
of  serotonin  storage  mechanisms  at  4-5  weeks  of  age  was  reported  earlier  but 
the  physiological  role  of  serotonin  storage  sites  remains  unclear.   Release 
of  this  amine  from  subcellular  organelles  has  been  offered  as   one  of  several 
possible  explanations  for  the  rapid  vasoconstriction  induced  by  hypoxia.   Up- 
take of  circulating  serotonin  has  been  variously  reported  to  lead  to  immediate 
metabolic  degradation  in  epithelial  cells  or  to  partial  storage  in  unchanged 

2  736 


form,  depending  on  the  laboratory  reporting  and  on  the  amounts  used.   Further 
study  of  these  questions  seems  desirable. 

Current  studies  with  histof luorescent  techniques  have  shown  serotonin  but  not 
histamine  to  be  present  in  lung  in  large  cells,  somewhat  resembling  mast  cells, 
that  are  clustered  in  the  vicinity  of  blood  vessels  and  intimately  associated 
with  sympathetic  nerve  fibers,  although  no  synapses  have  been  seen.   Isolated 
slices  of  lung  tissue  will  concentrate  serotonin  but  not  histamine  to  levels 
4-5  times  those  in  the  medium  when  nanomolar  concentrations  are  used.   Studies 
of  the  effects  of  drugs  on  the  kinetics  of  release  from  these  model  prepara- 
tions have  not  yet  been  accomplished.   In  germ-free  animals  maintained  up  to 
11  months,  the  levels  of  histamine  but  not  of  serotonin  remain  at  less  than 
1/10  those  observed  in  normal  animals. 

Surfactant  isolated  from  rabbit  lung  has  been  examined  by  nuclear  magnetic 
resonance  at  the  31P  frequency.   Since  no  signals  were  detectable  except  after 
sonication  of  the  preparation,  the  phospholipid  must  be  highly  immobilized  in 
the  intact  complex.   Paraquat,  a  drug  originally  investigated  because  of  the 
peculiar  delayed  pulmonary  edema  that  it  causes  in  man,  has  some  structural 
similarity  to  decamethonium.   A  search  for  possible  anticholinergic  effects 
disclosed  that  it  has  some  neuromuscular  blocking  properties  in  chicks.   The 
turnover  of  membrane  phospholipids,  particularly  phosphatidic  acid  and  phos- 
phatidyl inositol,  is  often  increased  in  tissues  exposed  to  acetylcholine  or 
a-adrenergic  agonists  and  might  be  expected  to  be  diminished  by  paraquat.  The 
effects  of  this  drug  on  incorporation  of  labeled  phosphate  into  phospholipids 
of  rat  lung  slices  were  equivocal,  amounting  to  small  reductions  in  the  turn- 
over of  phosphatidylglycerol  and  phosphatidylcholine  and  a  modest  increase  in 
phosphatidic  acid.   Propranolol,  which  has  been  observed  to  give  a  small  but 
significant  reduction  in  paraquat  toxicity  in  vivo,  had  much  larger  effects  on 
phosphate  incorporation  as  previously  reported  in  the  literature.   Effects  of 
the  two  drugs  appeared  to  be  additive. 

Miscellaneous  studies  with  biogenic  amines:   The  sensitive  (10-50  pg  range) 
and  specific  assay  methods  for  histamine,  serotonin  and  catecholamines  that 
were  developed  in  this  laboratory  and  used  in  the  studies  of  amine  turnover 
in  lung  have  also  made  possible  for  the  first  time  the  accurate  measurement  of 
biogenic  amines  in  plasma  and  urine.   The  methods  have  proved  useful  in  the 
diagnosis  and  study  of  disorders  in  which  amines  are  thought  to  play  a  role. 
Although  histamine  has  been  long  suspected  as  the  mediator  of  conditions  such 
as  cold-  and  exercise-induced  urticaria,  the  present  studies  provide  the  first 
direct  evidence  that  histamine  is  released  during  such  conditions.   Gastric 
ulcers  associated  with  basophilic  leukemia  and  gastric  tumors  have  been  shown 
also  to  be  due  to  circulating  histamine.   Treatment  with  the  H2  blocker  meti- 
amide  has  produced  remission  of  the  ulcers  in  these  patients. 

In  previously  reported  studies  of  histamine  metabolism,  salicylates  in  thera- 
peutic doses  were  found  to  inhibit  the  riboside  conjugation  of  imidazole  acet- 
ic acid  in  man  and  various  animal  species.  Studies  in  vitro  have  since  shown 
that  the  salicylates  inhibit  directly  (Kj  t»  10~5)  the  conjugating  enzyme.  The 
enzyme  has  been  isolated  from  liver  and  kidney  and  is  unusual  in  that  it  re- 
quires both  ATP  and  PP-ribose-P  as  well  as  magnesium.   The  need  for  this 


73? 


enzyme  in  the  body's  economy  is  not  apparent,  since  imidazole  acetic  acid  it- 
self is  inactive  pharmacologically  and  is  readily  excreted  from  the  kidney 
without  need  for  additional  conjugation.   The  enzyme  may  be  involved  in 
nucleotide  salvage  pathways.   There  are  indications  in  the  literature  that 
the  salicylates  inhibit  nucleic  acid  synthesis  in  bacteria  and  lymphocytes 
during  blast  transformation.   These  observations  would  suggest  an  inhibitory 
action  of  aspirin  on  rapidly  growing  tissues,  and  indeed  aspirin  administration 
was  found  to  inhibit  the  growth  of  a  transplantable  ascites  tumor  in  mice  and 
^C-thymidine  incorporation  into  the  nucleic  acid  precipitable  fraction  of 
this  tumor.   Similar  studies  are  underway  with  Lewis  lung  cell  carcinoma,  a 
rapidly  growing  solid  tumor  which  produces  death  by  metastasis  to  lung.   The 
possible  effect  of  aspirin  on  other  tumors  will  be  explored  with  research 
workers  in  the  Cancer  Institute.   Our  major  interest  in  this  phenomenon  is 
that  aspirin  may  exert  its  effect  in  inflammation  by  inhibition  of  leukocyte 
proliferation  in  inf lammed  tissues.   It  is  hoped  to  use  such  models  as  turpen- 
tine-induced lung  edema  in  rats  for  this  purpose. 


7*(0 


Project  No.  Z01  HL  02501-02  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Influence  of  Age,  Infection  and  Drugs  on  Lung 

Amines 

Previous  Serial  Number:   NHLI-106 

Principal  Investigators:   Dr.  Michael  A.  Beaven 

Dr.  Zdenka  Horakova 
Mr.  Richard  E.  Shaff 

Other  Investigators:,      Dr.  David  Jacobowitz 

Dr.  David  Small 

Cooperating  Units:        Dr.  Jacobowitz  is  a  member  of  the  Laboratory  of 

Clinical  Science,  Section  on  Histopharmacology, 
NIMH 

Dr.  Small  is  with  the  Division  of  Research 
Resources,  Veterinary  Resources  Branch 

Project  Description: 

Introduction:   Our  previous  studies  (see  project  report  NHLI-106,  1974) 
have  shown  that  in  rats  histamine  and  serotonin  levels  in  lung  are  less  than 
0.1  ug/g  at  birth.   The  levels  of  histamine  in  lung  increase  progressively 
with  age  and  attain  levels  of  7-17  ug/g  by  18  months  of  age.   Serotonin  lev- 
els increased  to  a  maximum  of  4-6  ug/g  by  10  weeks  of  age  and  then  remained 
constant  for  the  remainder  of  the  rat's  life.   The  studies  suggested  that 
accumulation  of  the  amines  was  due  to  the  appearance  of  storage  mechanisms  in 
the  older  animal.   The  two  amines  were  resistant  to  agents  which  selectively 
destroyed  sympathetic  and  serotonergic  nerves,  and  they  reside  presumably  in 
extraneuronal  stores.   The  histamine  and  serotonin  stores  were  shown  to  dif- 
fer in  several  respects.   Studies  with  monoamine  oxidase  inhibitors  and  amino 
acid  precursors  indicated  that  the  serotonin  turns  over  rapidly  and  hista- 
mine slowly  in  the  lung.   Both  amines  also  differed  in  their  susceptibility 
to  reserpine  and  compound  48/80;  serotonin  but  not  histamine  was  depleted  by 
reserpine  treatment;  and  histamine  but  not  serotonin  was  partially  depleted 
by  compound  48/80  administration.   It  seemed  unlikely  therefore  that  the  two 
amines  were  stored  in  the  same  type  of  cell. 

This  year's  study  has  been  concerned  with  the  histological  investigation  of 
storage  sites  of  the  amines  and  of  the  influence  of  infection  on  the  levels 
of  the  amines  in  lung. 

1  7W 


Project  No.  Z01  HL  02501-02  PB 

Histological  Studies:   Histological  studies  using  the  formaldehyde  fluo- 
rescent techniques  developed  in  Sweden  showed  that  serotonin  Was  localized 
exclusively  in  large  cells  surrounding  the  blood  vessels .   These  cells  were 
present  along  all  blood  vessels,  and  they  had  the  characteristic  morphology 
of  mast  cells.  An  unusual  feature  was  the  cells  appeared  to  be  intimately 
associated  with  sympathetic  nerve  fibers.   Synapses  of  nerve  fibers  to  the 
mast  cell  were  not  discernible,  however;  these  cells  were  absent  in  young 
rats  (9-day-old)  as  might  be  expected  from  the  low  levels  of  serotonin  in 
lung  at  this  age.   Histological  examination  of  histamine  stores  has  not  been 
made  at  this  time. 

Pharmacological  Studies;   Continued  studies  in  vivo  and  iri  vitro  point 
to  further  differences  in  the  storage  of  histamine  and  serotonin  in  lung. 
Experiments  with  lung  slices  ±n   vitro  have  confirmed  our  earlier  impression 
that  lung  possesses  the  ability  to  take  up  serotonin  but  not  histamine.   Tri- 
tiated  serotonin,  for  example,  is  rapidly  taken  up  into  lung  slices  by  a 
saturable  process  that  is  temperature  dependent  and  which  is  inhibited  by 
metabolic  inhibitors,  such  as  iodoacetate,  N-ethylmaleimide  and  cyanide. 
Concentration  gradients  of  1:3.5  to  4.5  (medium  to  slice  ratio)  were  con- 
sistently obtained.   The  uptake  was  blocked  by  agents  that  are  known  to  block 
the  uptake  of  serotonin  into  brain  slices.   In  contrast  to  serotonin,  passage 
of  tritiated  histamine  into  lung  slices  was  slow,  and  the  ratio  of  histamine 
in  medium  to  tissue  did  not  exceed  1.   These  studies  suggested  serotonin  but 
not  histamine  is  taken  up  by  a  transport  system  into  lung  tissue. 

Studies  with  Germ-Free  Rats:   An  important  aspect  of  this  year's  work 
has  been  the  finding  that  histamine  levels  are  very  low  in  lung  of  germ-free 
rats.   In  these  rats,  lung  histamine  ranged  from  0.08  +  .02  (+  SEM,  n  =  10) 
yg/g  at  birth  to  0.87  +  0.13  yg/g  (n  =  8)  at  11  months  of  age.   After  removal 
of  rats  from  the  germ-free  barrier,  there  is  a  gradual  and  progressive  in- 
crease in  histamine  levels  in  lung.   For  example,  after  1,  3,  7,  11,  20,  30 
weeks  and  11  months,  the  lung  histamine  was  1.1+0.1,  2.0+0.2,  2.3+0.8, 
4.8  +0.9,  7.5  +  2.6  and  11.7  +  5.4  yg/g,  respectively. 

Presently,  studies  in  collaboration  with  Dr.  David  Small  (Veterinary  Resources 
Branch)  are  underway  to  identify  the  specific  organisms  that  are  responsible 
for  the  increase  in  lung  histamine.   Germ-free  rats  are  being  exposed  to 
organisms  known  to  infect  rat  lung.   Histamine  levels  and  histidine  decarboxyl- 
ase activity,  the  enzyme  responsible  for  the  synthesis  of  histamine  in  tissues, 
are  being  followed.   Subsequent  studies  will  be  concerned  with  the  mechanism 
by  which  bacteria  increase  lung  histamine  and  whether  antigen-induced  release 
of  histamine  is  increased  in  infected  rats  compared  to  germ-free  rats,  using 
the  in  vitro  lung  model  of  Webseter  and  collaborators . 

The  current  findings  raise  two  important  questions.   Firstly,  that  there  ap- 
pear to  be  differences  in  the  storage  of  histamine  and  serotonin.   It  has 
been  assumed  that  the  two  amines  coexist  in  mast  cells  in  rat.   Secondly, 
histamine  but  not  serotonin  is  responsive  to  the  bacterial  environment.   Our 
results  suggest  that  infection  raises  the  level  of  histamine  in  lung  tissue. 
This  would  presumably  lead  to  more  pronounced  symptoms  during  allergic  release 

2  7** 


Project  No.  Z01  HL  02501-02  PB 

of  histamine.   It  is  possible  that  the  role  of  serotonin  in  lung  is  physio- 
logical and  histamine  pathological.   The  proximity  of  serotonin  to  blood 
vessels  suggests  that  this  amine  may  be  important  in  the  regulation  of  blood 
vessels. 


Keyword  Descriptors: 


Rat  lung,  histamine,  serotonin,  germ-free  rats, 
infection,  amine-storage 


Honors  and  Awards : 


None 


Publications : 


Webster,  M.E. ,  Newball,  H.H.,  Oh-Ishi,  S., 
Takahashi,  H.,  Horakova,  Z.,  Atkins,  F.L..and 
Beaven,  M.A. :   Release  of  histamine  and  arginine 
esterase  activity  from  passively  sensitized  human 
lung  by  ragweed  antigen.   Cienc.  Cult.  26:   372- 
376,  1974. 


Baxter,  J.H. ,  Beaven,  M.A.  and  Horakova,  Z.:  Ef- 
fects of  adrenergic  agents,  theophylline,  and 
other  drugs  on  dextran  edema  and  histamine  release 
in  rats.   Biochem.  Pharmacol.  23:   1211-1217,  1974. 

Atkins,  F.L.  and  Beaven,  M.A. :   Studies  of  ornithine 
decarboxylase  and  histaminase  (diamine  oxidase) 
activities  in  rat  thymus  and  their  relationship  to 
the  thymus  lymphocyte.   Biochem.  Pharmacol.  24: 
763-768,  1975. 

Beaven,  M.A.  and  Shaff,  R. :   Identification  of  his- 
taminase and  diamine  oxidase  activities  in  various 
tissues  of  rat  by  sensitive  isotopic  assay  proce- 
dures.  Biochem.  Pharmacol.,  in  press. 


7& 


Project  No.  Z01  HL  02502-02  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Influence  of  Aspirin  on  Amine  and  Purine  Metabolism 

in  Tumor  Cells 

Previous  Serial  Number:    NHLI-107 

Principal  Investigators:   Dr.  Michael  A.  Beaven 

Dr.  Zdenka  Horakova 
Dr.  Maria  Christina  de  Mello 

Other  Investigators:      Dr.  Valdemar  Hial 

Cooperating  Units:        Dr.  de  Mello  is  the  recipient  of  a  fellowship  from 

the  Brazilian  National  Research  Council 
Dr.  Hial  is  a  member  of  the  Hypertension-Endocrine 
Branch,  NHLI 

Project  Description: 

Introduction:   The  present  studies  stemmed  from  our  earlier  findings 
that  aspirin  and  salicylates  in  man  and  other  animal  species  inhibit  the 
formation  of  ribosyl  imidazole  acetic  acid  in  vivo  and  ±n   vitro  (see  current 
project  report  Z01  HL  00604-01  LCM) .   Salicylates  also  inhibit  nucleotide 
synthesis  and  growth  of  aerobic  and  anaerobic  bacteria  in  concentrations  that 
can  be  considered  pharmacological  (Schwartz  and  Mandel,  Biochem.  Pharmacol. 
21:   771,  1972) .   Similar  effects  on  nucleotide  synthesis  have  been  noted  in 
mammalian  cell  systems  in  studies  reported  by  Janakidevi  and  Smith  (J.  Pharm. 
Pharmacol.  22:   51,  58  and  249,  1970).   These  findings  led  us  to  investigate 
the  possibility  that  aspirin  might  preferentially  inhibit  growth  of  rapidly 
growing  tissues.   Aspirin  in  moderate  doses  was  found  to  inhibit  growth  of  a 
transplantable  mouse  ascites  tumor  and  of  a  solid  tumor,  Lewis  lung  cell 
carcinoma.   These  two  tumors  were  chosen  for  study  because  of  their  rapid 
growth  and  the  ability  to  measure  their  volume.   The  production  of  histamine 
by  the  ascites  tumor  was  also  followed.   Studies  with  labeled  thymidine  and 
uridine  were  undertaken  to  assess  the  effect  of  aspirin  on  tumor  RNA  and 
DNA  synthesis. 

Methods:   Six-week-old  CDfj  mice  were  housed  in  isolators  under  con- 
trolled lighting  conditions  and  temperature.   The  transplantable  ascites 
tumor  of  Dunn  and  Potter,  obtained  from  Mr.  Sidney  Yancey  of  the  Laboratory 
of  Chemical  Pharmacology,  NCI,  and  subsequently  maintained  in  our  laboratory, 
was  transferred  to  new  animals  weekly.   Aliquots  of  0.1  ml  of  ascites  fluid 

l  7** 


Project  No.  Z01  HL  02502-02  PB 

were  diluted  to  10  ml  with  sterile  Locke's  solution  and  0.1  ml  of  this  sus- 
pension was  injected  i.p.  into  each  mouse.   Lewis  lung  cell  carcinoma  was 
obtained  from  Dr.  D.  S.  Zaharko,  Laboratory  of  Chemical  Pharmacology,  NCI. 
This  tumor  was  transplanted  by  means  of  a  trocha  #13  under  the  surface  of 
the  skin. 

Aspirin  or  vehicle  was  administered  by  stomach  tube  twice  daily  on  the  day 
before  and  the  days  following  inoculation  with  tumor.   Body  weight  was  mea- 
sured daily.   Tumor  volume  was  measured  by  opening  the  abdomen  and  draining 
the  fluid  from  the  abdominal  cavity  into  a  glass  tube.   Thymidine  and  uri- 
dine incorporation  into  nucleic  acids  was  determined  by  injection  of  C1  - 
labeled  nucleotides  directly  into  the  intraperitoneal  cavity  and  removal  of 
samples  of  tumor  at  different  intervals  after  the  injection  of  label.   The 
nucleic  acids  were  isolated  by  precipitation  with  trichloroacetic  acid. 
Nucleotide  incorporation  was  also  studied  in  vitro  in  cultures  of  the  ascites 
tumor  cells.   Histamine  and  serotonin  were  assayed  by  enzymatic  derivative 
isotope  techniques  described  in  previous  project  reports. 

Results:   Studies  in  vivo:   Aspirin  treatment  inhibited  the  increase  in 
body  weight  which  accompanied  development  of  the  ascites  tumor.   The  differ- 
ences in  body  weight  between  aspirin  and  vehicle-treated  groups  of  mice  were 
highly  significant  (p  <  0.01)  in  four  separate  experiments.   In  mice  that 
were  not  inoculated  with  ascites  tumor,  aspirin  treatment  did  not  alter  body 
weight.   Tumor  volume  and  a  number  of  tumor  cells  were  reduced  significantly 
(approximately  40%,  p  <  0.001)  in  all  experiments.   The  size  and  number  of 
cells  per  unit  volume  of  ascites  tumor  remained  unchanged.   Although  the  con- 
centration of  histamine  in  the  tumor  was  unchanged,  the  total  amount  of  hist- 
amine excreted  in  urine  was  reduced  by  45%  in  mice  treated  with  aspirin. 
This  reduction  would  be  expected  from  the  reduced  tumor  size. 

ll*C-Thymidine  incorporation  into  the  TCA  insoluble  fraction  was  reduced  in 
mice  treated  with  aspirin,  although  the  difference  was  not  significant  due 
to  a  large  variation  in  individual  values  for  both  vehicle-treated  and 
aspirin-treated  mice.    C-Uridine  incorporation  was  unaffected  by  aspirin 
treatment . 

Studies  with  Ascites  Tumor  Cells  in  vitro:  The  uptake  and  incorporation 
of  ll+C- thymidine  and  uridine  into  TCA  precipitable  RNA  and  DNA  is  being  stud- 
ied in  vitro  in  suspensions  of  the  ascites  tumor  cells.  In  Locke's  solution, 
the  incorporation  of  the  labeled  nucleotides  into  nucleic  acids  is  rapid  when 
the  cells  are  initially  removed  from  the  animal  but  diminishes  as  the  incuba- 
tion continues  and  increases  after  2  hours.  The  rate  of  incorporation  of 
labeled  thymidine  is  reduced  significantly  in  the  presence  of  aspirin  in  con- 
centrations of  1-3  mM.  These  findings  are  preliminary,  and  currently  studies 
are  being  carried  out  with  more  complete  media  (Eagle's)  in  which  the  rate 
of  nucleotide  incorporation  into  nucleic  acid  does  not  diminish  with  time. 

Conclusions:   These  studies  indicate  a  possible  effect  of  aspirin  on 
tumor  growth.   The  possibility  that  aspirin  acts  on  growth  of  other  tumors 
will  be  explored  with  research  workers  in  the  National  Cancer  Institute.   Our 

2  7*S~ 


Project  No.  Z01  HL  02502-02  PB 

major  interest  in  this  phenomenon  is  whether  aspirin  exerts  similar  effects 
in  inflammation  and  whether  the  major  effect  of  aspirin  is  in  reduction  of 
monocyte  proliferation  in  the  inf lammed  tissue.   It  is  hoped  to  use  such 
models  as  the  rat  paw  and  terpentine- induced  lung  edema  in  rat.  Aspirin  is 
known  to  be  particularly  effective  in  chronic  inflammatory  conditions,  such 
as  arthritis,  where  macrophage  activity  may  be  an  important  component. 


Keyword  Descriptors: 


Aspirin,  salicylates,  inhibition  of  tumor  growth, 
ascites  tumor,  Lewis  lung  cell  carcinoma,  aspirin 
and  nucleic  acid  synthesis. 


Honors  and  Awards: 
Publications : 


None 


Beaven,  M.A. ,  Horakova,  Z.  and  Keiser,  H. :   Inhibi- 
tion by  aspirin  of  ribose  conjugation  in  the  metab- 
olism of  histamine.   Eur.  J.  Pharmacol.  29:   138- 
146,  1974. 


Horakova,  Z.  and  Beaven,  M.A. :   Time-course  of  hist- 
amine release  from  rat  paw  after  thermal  injury. 
Eur.  J.  Pharmacol.  27:   305-312,  1974. 

Markley,  K. ,  Horakova,  Z.,  Smallman,  E.T.  and 
Beaven,  M.A.:   The  role  of  histamine  in  the  produc- 
tion of  traumatic  shock.   Eur.  J.  Pharmacol.,  in 
press . 


-7*4 


Project  No.  Z01  HL  02503-03  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Disorders  of  Amine  Metabolism  in  Human  Disease 

Previous  Serial  Numbers:  NHLI-263(c),  NHLI-94(c)  and  NHLI-95(c) 

Principal  Investigators:   Dr.  Zdenka  Horakova 

Dr.  Michael  A.  Beaven 


Other  Investigators: 


Cooperating  Units: 


Allen  Kaplan,  M.D. 
Morton  Grossman,  M.D. 
Harry  Keiser,  M.D. 
George  P.  Canellos,  M.D. 
Richard  E.  Shaff 

Dr.  Kaplan  is  with  the  Laboratory  of  Clinical 
Invesigation,  NIAID 

Dr.  Grossman  is  with  the  Veterans  Administration 
Hospital,  Center  of  Digestive  Diseases,  Los 
Angeles,  Calif. 

Dr.  Keiser  is  Deputy  Chief  of  the  Hypertension- 
Endocrine  Branch  and  Head  of  the  Section  on 
Experimental  Therapeutics,  HE,  NHLI 
Dr.  Canellos  is  with  the  Medicine  Branch,  NCI 


Project  Description: 

The  development  of  sensitive  enzymatic  isotope  assay  procedures  for  the  mea- 
surement of  biogenic  amines  now  makes  it  possible  to  assay  the  amines  in 
plasma  and  urine.   These  assays  are  specific  and  100-200  times  more  sensitive 
than  the  older  fluorometric  procedures.   Over  the  past  few  years  (see  project 
reports  1972-1974),  isotopic  assays  for  histamine,  serotonin  and  most  re- 
cently the  catecholamines  have  been  established  in  this  laboratory.   These 
have  been  used  to  study  a  variety  of  diseases  associated  with  defects  of 
amine  storage  and  metabolism  and  include  immediate  hypersensitivity  reactions 
(studies  conducted  with  Dr.  Allen  Kaplan) ,  diseases  of  the  gastrointestinal 
tract  in  which  histamine  or  serotonin  may  be  involved  (Dr.  Morton  Grossman) 
and  amine-producing  tumors ,  such  as  basophilic  leukemia  carcinoid  and  medul- 
lary thyroid  carcinoma  (Drs.  Canellos  and  Keiser).   In  addition  to  these 
studies,  urine,  plasma  and  tissue  samples  are  received  (about  200  per  year) 
from  outside  hospitals  with  requests  for  assays  of  the  amines  or  of  the  vari- 
ous enzymes  associated  with  amine  metabolism.   The  principal  assays  were 
shown  in  a  table  in  last  year's  report. 


7^7 


Project  No.  Z01  HL  02503-03  PB 

Assay  Procedures:   The  assays  for  the  biogenic  amines  take  advantage  of 
the  fact  that  the  major  route  of  metabolism  of  the  amines  is  methylation, 
either  of  hydroxyl  or  the  alkylamine  groups,  and  that  radioactive  metabolites 
of  the  amines  can  be  formed  by  incubation  of  the  sample  with  radioactive 
methyl  donor,  S-adenosyl-L -methionine  (3H  or  ll*C-methyl  label),  and  the 
appropriate  methyltransf erase  enzyme.   These  enzymes  are  readily  prepared  by 
ammonium  sulfate  fractionation  of  extracts  of  pineal,  brain,  or  liver.   The 
radioactive  metabolite  is  then  separated  from  the  excess  S-adenosylmethionine 
and  assayed  for  radioactivity.   When  the  enzymes  are  specific,  as  in  the  case 
of  histamine-N-methyltransf erase  and  hydroxyindole-O-methyltransf erase,  rela- 
tively simple  extraction  procedures  can  be  used.   When  the  enzymes  are  less 
specific,  as  in  the  case  of  catechol-O-methyltrans f erase ,  additional  isola- 
tion steps  are  required.   The  techniques  for  separation  of  the  enzymes  and 
the  design  of  this  type  of  assay  stem   largely  from  the  work  of  Dr.  Axelrod. 

Tritium  release  assays  have  been  developed  in  this  laboratory  for  histaminase, 
tryptophan  hydroxylase  and,  recently,  dopamine-3-hydroxylase.   These  assays 
utilize  substrates  with  tritium  label  attached  to  points  where  proton  exchange 
is  expected  to  occur  during  the  enzymatic  reaction.   The  released  tritium  is 
measured  in  water  by  sublimation  of  water  directly  from  the  incubation  mix- 
ture.  The  assays  are  simple,  precise  and  are  sufficiently  sensitive  to  de- 
tect the  conversion  of  10-15  moles  of  substrate.   The  first  assay  to  be  de- 
veloped in  this  laboratory,  that  of  histaminase,  has  been  used  extensively 
in  investigations  of  medullary  carcinoma  of  the  thyroid  and  other  disorders 
(see  earlier  project  reports) . 

Studies  in  Patients  with  Cold-  and  Exercise-Induced  Urticaria:   A  total 
of  8  patients  with  cold-  or  cholinergic-induced  urticaria  have  been  studied. 
In  all  patients,  exposure  of  one  arm  to  cold  or  vibration  resulted  in  release 
of  histamine  into  plasma  of  blood  reaching  the  brachial  vein.   In  most  pa- 
tients, plasma  histamine  levels  increased  from  <  1  to  8-13  ng/ml.   In  one 
patient,  plasma  histamine  levels  rose  to  over  30  ng/ml  in  the  brachial  vein 
blood  and  also  rose  (to  5  ng/ml)  in  the  systemic  circulation  (blood  from  the 
opposite  arm).   This  patient  experienced  mild  shock  and  dizziness.   In  all 
patients,  the  symptoms  ameliorated  as  the  histamine  levels  declined.   No 
histamine  could  be  detected  in  plasma  in  control  patients  subjected  to  the 
same  treatment.   The  release  of  histamine  in  a  cold-induced  urticaria  ap- 
peared to  be  mediated  by  IgE  antibody,  since  transfer  of  an  IgE  fraction  from 
one  of  these  patients  to  a  normal  subject  resulted  in  the  appearance  of  symp- 
toms and  release  of  histamine  in  this  subject  when  exposed  to  cold. 

A  ninth  patient  (an  ex-professional  wrestler)  with  exercise-induced  urticaria 
was  unusual  in  that  his  symptoms  were  not  relieved  by  the  customary  adminis- 
tration of  antihistaminics .  This  patient  showed  no  release  of  histamine  or 
bradykinin  but  was  found  to  have  elevated  serotonin  plasma  levels.  Adminis- 
tration of  methysergide  aborted  his  attacks  much  to  his  relief,  since  he  was 
troubled  by  his  attacks  during  his  sexual  activities.  This  problem  had  been 
of  particular  concern  to  him  and  his  wife  during  the  past  year. 


7*0 


Project  No.  Z01  HL  02503-03  PB 

Studies  in  Patients  with  Histamine-Producing  Tumors  Associated  with  Se- 
vere Gastric  and  Peptic  Ulcers;   One  patient  who  has  a  gastric  carcinoma  and 
elevated  plasma  histamine  levels  had  been  located   (patient  of  Dr.  Grossman). 
The  patient  has  severe  gastric  and  peptic  lesions.   Histamine  levels  in 
plasma  of  this  patient  have  been  consistently  5-7  ng/ml.   This  was  the  first 
patient  in  which  we  have  detected  histamine  in  plasma  under  normal  resting 
conditions.   Histamine  excretion  in  his  urine  was  also  elevated,  300-400  ug/ 
24  hr,  compared  to  a  urine  excretion  of  17  +14  (n  =  36,  range  <  5-51  yg/24 
hr)  in  normal  subjects  and  patients  with  miscellaneous  diseases.   Since  stud- 
ies with  labeled  histamine  in  humans  have  shown  that  only  1-3%  of  the  hist- 
amine entering  the  blood  stream  is  excreted  unchanged  in  urine,  this  elevated 
urine  excretion  could  represent  a  total  excretion  of  over  10  mg  of  histamine 
per  day.   Currently,  the  patient  is  being  treated  with  metiamide,  one  of  the 
new  H2  histamine  blocking  drugs. 

In  addition  to  the  above  patient,  two  other  patients  (of  Dr.  Canellos,  NCI) 
with  Philadelphia  chromosome  chronic  granulocytic  leukemia  developed  an 
accelerated  phase  of  the  disease  with  high  white  blood  cell  count,  high  hist- 
amine levels  in  blood  (10.5  and  11.5  yg/ml,  respectively,  compared  to 
0.08+0.03  [n  =  17]  yg/ml  in  whole  blood  of  normal  subjects)  and  high  urine 
excretion  of  histamine,  326  and  1011  yg/24  hr.   One  patient  had  symptoms  of 
hyperhistaminemia  associated  with  wheezing,  urticaria  and  pruritis.   The 
second  had  peptic  ulcers.   The  second  patient  also  had  elevated  histamine 
levels  in  plasma  (up  to  40  ng/ml) .   Treatment  with  metiamide  resulted  in 
improvement  in  both  patients  and  a  reduction  in  gastric  secretion  in  the 
second  patient. 

Studies  in  other  Diseases  with  High  Histamine  or  Serotonin  Excretion; 
To  facilitate  these  studies,  the  isotopic  serotonin  assay  has  been  adapted 
for  measurement  of  serotonin  in  urine  (see  Project  Report  Z01  HL  02504-01  PB). 
To  date  elevated  histamine  excretion  has  been  detected  in  3  basophilic  leu- 
kemics average  861,  range  323-1400  yg/24  hr;  5  mastocytosis,  average 
121  +  74,  range  30-231  yg/24  hr;  and  one  patient  with  urticaria  pigmentosa. 
Apart  from  the  patients  with  basophilic  leukemia,  histamine  levels  were  not 
elevated  in  blood.   Raised  histamine  or  serotonin  has  not  been  detected  in  a 
variety  of  other  diseases  tested  to  date. 

These  studies  will  be  continued  using  the  isotopic  assays  and  will  be  used 
to  evaluate  compounds  that  are  known  to  antagonize  or  alter  the  metabolism 
of  histamine  and  serotonin. 

Keyword  Descriptors:      Enzymatic  isotope  derivative  assays,  histamine, 

serotonin,  catecholamines,  gastric  carcinoma, 
immediate  hypersensitivity  reactions ,  basophilic 
leukemia,  hyperhistaminemia 

Honors  and  Awards:        None 


7*7 


Project  No.  Z01  HL  02503-03  PB 

Publications:  Beaven,  M.A. :   Assay  of  Neurotransmitters  and  Drugs 

by  Isotope  Dilution  Derivatization  Techniques.   In 
Iverson,  I.L.  and  Snyder,  S.  (Eds.):   Handbook  of 
Neuropharmacology,  Vol.  I.   New  York,  Plenum  Press, 
in  press. 

Beaven,  M.A.  and  Horakova,  Z.:   Assay  of  Histamine 
and  Histamine  Metabolizing  Enzymes  by  Enzymatic 
Isotope  Dilution  Analysis.   In  Rocha  e  Silva,  M. 
(Ed.):   Handbook  of  Experimental  Pharmacology. 
Berlin,  Springer-Verlag,  in  press. 

Kaplan,  A. P.,  Gray,  L.,  Horakova,  Z.,  Shaff,  R.E. 
and  Beaven,  M.A. :   Mediator  release  in  cold  urti- 
caria and  cholinergic  urticaria.   J.  Lab.  Clin. 
Invest. ,  in  press. 

Beaven,  M.A. ,  Baylin,  S.B.,  Marshall,  J.R.  and 
Sjoerdsma,  A. :   Rise  of  plasma  histaminase  activ- 
ity during  early  human  pregnancy.   J.  Clin.  Endo- 
crinol. ,  in  press. 

Baylin,  S.B.,  Beaven,  M.A.  and  Horakova,  Z.: 
Increased  appetite  and  weight  gain  after  aminoguan- 
idine  treatment.   Experientia,  in  press. 


rsv 


Project  No.  Z01  HL  02504-01  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:  Sensitive  Assay  for  Serotonin  in  Tissues 

Previous  Serial  Number:  None 

Principal  Investigator:  Mr.  Richard  E.  Shaff 

Other  Investigator:  Dr.  Michael  A.  Beaven 


Cooperating  Units: 
Project  Description: 


None 


The  purpose  of  the  present  work  was  to  develop  an  enzymatic  double  isotope 
assay  for  serotonin  in  which  H-serotonin  is  added  as  an  internal  standard 
and  ^C-methyl  labeled  S-adenosylmethionine  (SAMe-  C)  is  used  as  a  methyl 
donor.   A  single  isotope  assay  in  which  H-methyl  labeled  S-adenosylmethio- 
nine (SAMe-  H)  i.<=  used  as  a  methyl  donor  has  been  previously  described 
(Saavedra  et  al. ,  J.  Pharmacol,  Exp.  Ther.  186:   508,  1973).   In  our  experi- 
ence, there  is  considerable  variation  in  recovery  of  serotonin  from  tissue 
to  tissue,  and  there  is  a  need  to  monitor  recovery  with  an  internal  standard. 
It  was  decided  to  use  a  side  chain  label  p-  H-serotonin  for  this  purpose, 
since  the  generally  labeled  3H-serotonin  that  is  commercially  available  is 
too  unstable. 

Rat  liver 


1)    Serotonin   (f3-3H-serotonin  )   +  Acetyl-CoA 


>  N-acetylserotonin   (^H) 


acetylating 
enzyme 
Pineal 

2)   N-Acetylserotonin   (%)   +  SAMe-lltC  >  llfC-melatonin   (%) 

HIOMT 

In  the  first  step,  the  sample  is  incubated  with  a  tracer  amount  of  B-3H-sero- 
tonin  (side  chain  label) ,  acetyl-CoA  and  an  ace tyltransf erase  system  prepared 
from  rat  liver  (Reaction  1) .   After  a  30-min  incubation,  the  mixture  is  fur- 
ther incubated  with  SAMe-^C  and  hydroxyindole-O-methyltransferase  (HIOMT)  to 
form  14,C-labeled  melatonin  (Reaction  2)  .   Unlabeled  melatonin  is  added  as 
carrier,  and  the  melatonin  is  then  separated  from  excess  SAMe-llfC  by  extrac- 
tion into  toluene.   The  first  reaction  is  necessary  because  serotonin,  unlike 
N-acetylserotonin,  is  a  poor  substrate  for  HIOMT.   A  standard  curve  is  pre- 
pared by  taking  known  amounts  of  serotonin  through  the  procedure.   The  assay 
is  specific  and  sufficiently  sensitive  to  measure  1-2  pmoles  (20-40  pg)  of 
serotonin. 


rsrt 


Project  No.  Z01  HL  02504-01  PB 

The  assay  has  been  used  extensively  in  our  studies  of  lung  amines  (see  Project 
Report  No.  Z01  HL  02501-02  PB)  and  in  studies  with  human  subjects  (see  Project 
Report  No.  Z01  HL  02503-03  PB) .   The  assay  has  also  been  modified  to  measure 
serotonin  and  N-acetylserotonin  in  urine  specimens.   For  the  latter,  the 
urine  is  split  into  two  samples.   The  first  sample  is  assayed  by  the  normal 
procedure  to  obtain  values  for  serotonin  and  N-acetylserotonin;  the  other 
specimen  is  assayed  by  omitting  the  first  reaction.   This  measures  the  amount 
of  N-acetylserotonin  present.   These  assays  have  shown  that  significant  amounts 
of  both  serotonin  and  N-acetylserotonin  are  excreted  in  normal  human  urine. 

Surveys  of  various  rat  tissues  have  shown  that  lungs  contain  the  highest  level 
of  serotonin  in  the  body  (4-6  yg/g) .   Intestine,  whole  blood  and  spleen  also 
contain  relatively  high  levels  (1-2  yg/g)  of  amine  as  has  been  observed  in 
studies  with  the  fluorometric  assays  of  serotonin.   Unlike  the  fluorometric 
assays,  the  isotope  procedure  detected  no  serotonin  in  the  heart. 

The  use  of  side  chain  labeled  3-  H-serotonin  has  allowed  us  to  study  the  ki- 
netics of  the  two  reactions  outlined  above.   The  second  reaction  is  quantita- 
tive and  goes  to  completion,  whereas  the  first  reaction  proceeds  to  about  30% 
completion.   Addition  of  N-acetylserotonin,  the  product  of  the  reaction,  fur- 
ther reduces  the  rate  of  conversion  of  serotonin  to  N-acetylserotonin  in  a 
competitive  manner,  but  the  reaction  proceeds  to  the  same  equilibrium  point 
irrespective  of  the  amount  of  serotonin  originally  present.   We  have  attempted 
to  overcome  this  inhibition  by  coupling  both  reactions,  i.e.  adding  both 
enzymes  and  cofactors  in  the  same  incubation,  but  this  results  in  higher 
values  for  the  blanks . 

Keyword  Descriptors:      Serotonin,  N-acetylserotonin,  urine  excretion, 

enzymatic  isotope  derivative  assay. 

Honors  and  Awards :        None 

Publications:  None 


-rsra. 


Project  No.  Z01  HL  02505-01  PB 

1 .  Pulmonary 

2 .  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-N1H 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title: 


Interaction  of  Adrenochrome  with  Red  Cell  Ghost 
Membranes 


Previous  Serial  Number : 


None 


Principal  Investigator:   Dr.  David  B.  Millar 


Other  Investigator: 
Cooperating  Unit: 


Dr.  Colin  F.  Chignell 

Dr.  Millar  is  Chief  of  the  Laboratory  of  Physical 
Biochemistry,  Environmental  Sciences  Department, 
Naval  Medical  Research  Institute,  Bethesda,  Md. 


Project  Description: 

Certain  musculoskeletal  traumatic  injuries  have  been  observed  to  lead  to  a 
hemolytic  process  known  as  chronic  anemia  of  trauma.   Although  the  precise 
mechanism  of  hemolysis  is  at  present  unknown,  it  has  been  suggested  that 
adrenochrome  and  perhaps  additional  other  serum  factors  may  be  involved 
(Valeri  et  al.,  J.  Med.  3:   20,  1972).   If  adrenochrome  is  indeed  the  causa- 
tive agent,  then  it  seems  reasonable  to  postulate  the  interaction  of  this 
metabolite  with  the  erythrocyte  membrane  may  ultimately  lead  to  destruction 
of  the  membrane  and  lysis  of  the  cell.   We  have  therefore  examined  the  inter- 
action of  adrenochrome  semicarbazide  (ADCS)  with  human  red  cell  ghost  mem- 
branes by  means  of  several  spectroscopic  techniques  including  circular  dichro- 
ism,  fluorescence  spectroscopy,  and  electron  spin  resonance. 

Human  red  cell  ghosts  were  prepared  from  outdated  blood  bank  blood  by  the 
method  of  Dodge  et  al.  (Arch.  Biochem.  Biophys.  100:   119,  1963).   Ghost 
preparations  were  repeatedly  washed  and  dialyzed  until  hemoglobin  was  spectro- 
photometrically  undetectable.   The  ghosts  were  sonicated  in  an  ice  bath,  then 
stored  at  4  and  used  within  2  days  of  sonication.   Fluorescence  titrations 
were  performed  at  25  in  an  Aminco-Bowman  fluorometer.   Circular  dichroism 
measurements  were  made  at  27   in  a  Cary  60  spectropolarimeter  equipped  with 
a  6001  attachment.   Electron  spin  resonance  (ESR)  measurements  were  made  with 
a  Varian  E-4  spectrometer.   The  erythrocyte  ghost  membranes  were  reacted  with 
spin  labels  I  or  II  (10-1+  M)  at  4  overnight.   Unreacted  spin  label  was  re- 
moved by  repeated  centrifugation  and  resuspension  in  buffer.   Erythrocyte 
ghost  membranes  were  labeled  with  stearic  acid  probe  III  by  the  addition  of 
a  methanolic  solution  (2  x  10~3  M)  to  the  membranes. 


7T3 


Project  No.  Z01  HL  02505-01  PB 


NHC0CH2I 


CH3(CH2)m  —  C  —  (CH2)nC00H 
0^  ^N  —0 


III 


In  the  presence  of  sonicated  ghost  membranes,  l-anilino-8-naphthalene  sul- 
fonic acid  (ANS)  showed  a  strong  symmetrical  fluorescence  emission  band  with 
an  apparent  maximum  at  485  nm  when  activated  at  either  265  nm  or  380  nm. 
The  addition  of  ADCS  to  solutions  containing  the  membranes  and  ANS  resulted 
in  quenching  of  the  fluorescence  of  the  probe.   Analysis  of  the  quenching 
data  by  means  of  the  Stern-Vollmer  relationship  indicated  that  the  quenching 
of  the  ANS  fluorescence  by  ADCS  was  probably  due  to  a  collision  of  the 
adrenochrome  derivative  with  the  membrane  bound  probe.   In  contrast  to  the 
intact  ghost  membranes  in  which  approximately  40%  of  the  proteins  are  present 
in  the  a-helical  structure  (Lenard  and  Singer) ,  the  proteins  of  the  sonicated 
ghost  membranes  existed  to  the  extent  of  about  90%  in  the  a-helical  form. 
ADCS  (2  x  10~5  M)  had  no  detectable  effect  on  the  tertiary  structure  of  the 
sonicated  membranes  as  measured  by  circular  dichroism. 

ADCS  also  had  no  effect  on  the  ESR  spectrum  of  sonicated  ghosts  labeled  with 
spin  labels  I  or  II.   These  observations  again  suggest  that  ADCS  has  no  ef- 
fect on  the  conformation  of  ghost  membrane  proteins.   ADCS  also  did  not 
change  the  electron  spin  resonance  spectrum  of  stearic  acid  label  III  (12,3) 
incorporated  into  the  sonicated  ghost  membrane.   This  would  indicate  that 
ADCS  does  not  alter  the  organization  of  the  lipids  of  ghost  membranes. 

These  results  suggest  that  while  adrenochrome  does  bind  to  human  red  cell 
ghost  membranes  it  is  unlikely  to  be  the  sole  causative  agent  responsible  for 
post  trauma  hemolysis.   No  further  studies  on  the  interaction  of  ADCS  with 
ghost  membranes  are  contemplated  at  this  time. 


Keyword  Descriptors: 


Honors  and  Awards : 


Musculoskeletal  traumatic  injury,  hemolysis,  adreno- 
chrome, fluorescence,  electron  spin  resonance, 
spin  labels,  circular  dichroism. 


None 


Publications : 


Millar,  D.B.  and  Chignell,  C.F.:   Partial  character- 
ization of  the  l-anilino-8-naphthalene  sulfonate- 
adrenochrome  semicarbazide  interaction  site  in 
erythrocyte  ghost  membrane  fragments.   J.  Biophys . , 
in  press . 


7S& 


Project  No.  Z01  HL  02505-01  PB 

Chignell,  C.F.:   A  topographical  study  of  the  active 
site  of  erythrocyte  carbonic  anhydrase  by  means  of 
spin  labeled  drugs.   J.  Pharm.  Sci.  64:   512-515, 
1975. 

Chignell,  C.F.:   The  Fluorescence  of  Drug-Protein 
Interactions.   In  Chen,  R.F.  and  Edelhoch,  H. 
(Eds.):   Concepts  in  Biochemical  Fluorescence  Spec- 
troscopy.  New  York,  Marcel  Dekker,  Inc.,  in  press. 

Chignell,  C.F.:   Fluorescence  Spectroscopy  as  a 
Tool  for  Monitoring  Drug-Albumin  Interactions .  -In 
Morselli,  P.L.,  Garattini,  S.  and  Cohen,  S.N. 
(Eds.):   Drug  Interactions .   New  York,  Raven  Press, 
1974,  pp.  111-122. 

Chignell,  C.F.:   Protein  binding  and  drug  action. 
Ann.  Rep.  Med.  Chem.  9:   280-289,  1974. 

Chignell,  C.F.:   Ligand  Binding  to  Plasma  Albumin. 
In  Sober,  H.A.  (Ed.):   Handbook  of  Biochemistry. 
Cleveland,  Ohio,  The  Chemical  Rubber  Co.,  in  press. 

Chignell,  C.F.:   Protein  Binding.   In  Garrett,  E.R. 
and  Hirtz,  J.  (Eds.):  Methods  in  Drug  Metabolism 
Research.   New  York,  Marcel  Dekker,  Inc.,  in  press. 


7sr 


Project  No.  Z01  HL  02506-02  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:  Spin  Trapping  Studies  of  the  Microsomal  Metabolism 

of  CCl3Br 

Previous  Serial  Number:  NHLI-102 

Principal  Investigator:  Dr.  Victorio  Wee 

Other  Investigator:  Dr.  Colin  F.  Chignell 

Cooperating  Unit:  Dr.  Wee  is  a  Visiting  Fellow  of  the  NHLI 

Project  Description: 

It  has  been  postulated  that  CC11+  is  metabolized  by  the  liver  to  give  free 
radical  species  which  are  thought,  in  turn,  to  be  responsible  for  the  hepato- 
toxicity  of  this  compound.   The  high  chemical  reactivity  of  free  radical 
intermediates  makes  their  detection  at  room  temperature  very  difficult.   In 
previous  studies,  we  have  attempted  to  detect  the  formation  of  free  radicals 
during  the  metabolism  of  CCl3Br  by  rat  liver  microsomes  with  the  aid  of  spin 
labeled  analogues  of  stearic  acid  and  choline.   One  of  the  problems  with 
this  approach  is  that,  although  we  were  able  to  observe  chemical  destruction 
of  these  nitroxides  during  the  metabolism  of  CCl3Br  by  rat  liver  microsomes, 
it  was  difficult  to  establish  that  their  disappearance  was  due  to  a  reaction 
with  free  radical  intermediates.   Spin  trapping  is  a  technique  in  which  short- 
lived free  radicals  react  with  a  nonparamagnetic  compound  to  produce  a  stable 
free  radical  (Janzen,  Accounts  Chem.  Res.  4:   31,  1971).   We  therefore  de- 
cided to  attempt  to  trap  free  radical  intermediates  produced  during  the  metab- 
olism of  CCl3Br  with  the  aid  of  phenyl-_t-butyl  nitrone  (I)  and  2-methyl-2- 
nitrosopropane  (II) .   It  has  been  shown  that  when  these  compounds  react  with 
chemically  generated  free  radicals  they  produce  the  relatively  stable  nitrox- 
ide  analogues  III  and  IV,  respectively. 

Rat  liver  microsomes  were  prepared  from  male  Sprague-Dawley  rats  that  had 
been  pretreated  with  phenobarbital  (80  mg/kg)  for  5-7  days.   Electron  spin 
resonance  (ESR)  measurements  were  made  with  a  Varian  E-4  spectrometer  equipped 
with  a  variable  temperature  accessory. 

When  trapping  agent  I  (.01  M)  was  incubated  with  rat  liver  microsomes  in  the 
presence  of  CCl3Br,  TPNH,  and  a  TPNH  regenerating  system,  a  free  radical 
signal  consisting  of  a  triplet  (aj  =  15  G)  in  which  each  of  the  major  hyper- 
fine  lines  was  split  into  a  doublet  (a2  =  4.5  G) .   This  triplet  could  only  be 

l  7Si 


Project  No.  Z01  HL  02506-02  PB 


0 


(\)/         CH=N-C(CH3)3   +  R*  — *■  ff       jV  ch-n 


-N-C(CH3)3 


I 


CH: 


CHq  C  CHc 


III 
CH3 

I 

CH3  C  CH. 


N  =  0  N_i_0 

I 

R 


II 


IV 


observed  when  the  sample  was  incubated  at  37  .   If  the  temperature  of  the 
sample  was  decreased  to  25  ,  the  ESR  signal  disappeared  and  could  be  regen- 
erated by  warming  the  sample  again  for  37  .   No  signal  was  observed  in  the 
absence  of  either  CCl3Br  or  TPNH  with  its  accompanying  regenerating  system. 

Experiments  with  spin  trap  II  were  less  successful.   One  of  the  problems  with 
this  label  is  that  under  the  influence  of  light  it  spontaneously  reacts  with 
solvent  molecules  to  produce  stable  free  radical  species.   It  was  impossible 
to  adjust  experimental  conditions  so  that  these  free  radical  intermediates 
were  absent.   The  presence  of  a  solvent  generated  free  radical  species  from 
spin  label  II  made  experiments  in  the  presence  of  microsomes  and  CCl3Br 
difficult  to  interpret.   No  further  studies  were  therefore  carried  out  on 
this  compound . 

Halogenated  hydrocarbons  are  extensively  used  as  solvents  in  many  industrial 
processes.  Therefore  it  becomes  extremely  important  to  understand  the  mech- 
anism whereby  these  compounds  cause  toxicity. 

Studies  with  spin  trap  I  will  be  continued  and  extended.  Attempts  will  be 
made  to  isolate  and  characterize  the  stable  free  radical  species  formed  in  the 
presence  of  microsomes,  CCl3Br  and  TPNH. 

Keyword  Descriptors:      Spin  traps,  electron  spin  resonance,  carbon  tetra- 
chloride, bromotrichlorome thane,  phenyl-t-butyl 
nitrone,  and  2-methyl-2-nitrosopropane. 

Honors  and  Awards:        None 


7S7 


Project  No.  Z01  HL  02506-02  PB 

Publications:  Chignell,  C.F.  and  Starkweather,  D.K.:   A  spin  label 

study  of  human  erythrocyte  ghost  membranes  damaged 
by  methyl  phenyldiazene  carboxylate.   Life  Sci.  14: 
641-652,  1974. 


7se 


Z01  HL  02507-02  PB 


Project  No. 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesd,a,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:  Acetylcholinesterase  from  Torpedo  californica 

Previous  Serial  Number:   NHLI-101 

Principal  Investigator:   Dr.  Colin  F.  Chignell 

Other  Investigators:      Dr.  Victorio  Wee 

Dr.  Birandra  K.  Sinha 
Dr.  Palmer  W.  Taylor,  Jr. 

Cooperating  Units:        Dr.  Wee  is  a  Visiting  Fellow  of  NHLI 

Dr.  Sinha  held  a  Visiting  Fellowship,  NHLI,  and  is 
now  a  Guest  Worker  supported  by  the  Microbiological 
Associates,  Bethesda,  Md. 

Dr.  Taylor  is  Associate  Professor  of  Pharmacology 
at  the  University  of  California,  San  Diego 

Project  Description: 

It  is  the  aim  of  this  study  to  examine  the  topography  of  acetylcholinesterase 
and  the  acetylcholine  receptor  protein  isolated  from  Torpedo  with  the  aid  of 
spin-labeled  inhibitor  ligands  (I,  II). 


CH. 


CH3  CH3  CHo 

01  "  ©  I  ©I  ©1 

CH3  —  N  (CH2)10  N  —  CH3  CH3  —  N  — -  (CH2)10 N  CH3 

I 
CH3 


II 


Acetylcholinesterase  was  isolated  from  the  electroplax  of  Torpedo  californica 
by  previously  published  procedures  (Mol.  Pharmacol.  10:   78,  ibid  10:   93, 
1974) .   The  cholinergic  receptor  was  isolated  from  the  same  tissue  using  the 
procedure  of  Schmidt  and  Raftery  (Biochemistry  12:   852,  1973).   The  spin- 
labeled  ligands  (I,  II)  were  prepared  by  previously  reported  procedures 


TS"? 


Project  No.  Z01  HL  02507-02  PB 

(NHLI-104) .   Electron  spin  resonance  (ESR)  measurements  were  made  with  a 
Varian  E-A  spectrometer  equipped  with  a  quartz  aqueous  sample  cell. 

Previous  studies  have  shown  that  spin  labels  I  and  II  were  more  effective 
inhibitors  of  Torpedo  acetylcholinesterase  than  decamethonium  itself.   The 
ESR  spectrum  of  a  dilute  aqueous  solution  of  spin  label  I  consisted  of  three 
sharp  lines.   However,  the  measurable  spin  concentration  of  I  was  only  one- 
third  of  that  which  would  be  expected  from  an  equimolar  solution  of  noninter- 
acting  spin  label.   This  suggests  that  the  nitroxide  groups  at  each  end  of 
the  molecule  come  close  enough  to  permit  an  exchange  of  spin  states.   By  in- 
creasing the  temperature,  a  more  rapid  exchange  rate  occurred  and  a  five-line 
ESR  spectrum  was  observed.   The  spin  concentration  calculated  from  this  spec- 
trum closely  approximated  that  of  a  noninteracting  label.   When  spin  label  I 
bound  to  Torpedo  acetylcholinesterase,  its  ESR  spectrum  was  broad  and  highly 
asymmetric.   A  splitting  of  69  G  between  high  and  low  field  extrema  suggested 
that  the  drug  was  strongly  immobilized.   The  spin  concentration  of  I  bound  to 
acetylcholinesterase  was  the  same  as  that  of  a  noninteracting  nitroxide  which 
suggested  that  the  distance  between  the  nitroxide  groups  was  large  enough  to 
prevent  spin  exchange  from  taking  place.   The  ESR  spectrum  of  spin  label  I 
and  Torpedo  acetylcholinesterase  appeared  to  have  a  single  bound  component 
which  suggests  that  both  ends  of  the  molecule  are  immobilized  to  the  same  ex- 
tent.  The  ESR  spectrum  of  spin  label  II  bound  to  acetylcholinesterase  was 
similar  to  that  described  for  spin  label  I. 

Attempts  to  demonstrate  any  interaction  between  spin  labels  I  and  II  and  the 
acetylcholine  receptor  have  been  so  far  unsuccessful.   The  main  problem  with 
these  kinds  of  measurements  is  the  low  concentration  of  receptor  which  we  are 
currently  able  to  obtain.   However,  Dr.  Taylor  has  developed  a  new  technique 
involving  partitioning  of  receptor  rich  membranes  between  two  phases  which  it 
is  hoped  will  enable  him  to  isolate  the  purified  acetylcholine  receptor  in 
greater  quantities.   This  should  be  possible  to  study  the  interaction  of 
labels  I  and  II  with  the  receptor. 

Acetylcholinesterase  provides  a  very  useful  model  system  for  examining  the 
molecular  basis  for  drug-receptor  interactions.   In  addition,  this  enzyme  is 
functionally  important,  since  it  is  responsible  for  the  destruction  of  acetyl- 
cholinesterase that  diffuses  away  from  the  receptor  surface.   The  possibility 
of  isolating  the  acetylcholine  receptor  in  sufficiently  high  quantities  will 
make  it  feasible  to  study  directly  on  a  molecular  level  the  interaction  be- 
tween a  receptor  and  its  agonist. 

The  electron  spin  resonance  studies  with  labels  I  and  II  will  be  extended  to 
chemically  modified  acetylcholinesterase  in  which  the  catalytic  serine  has 
been  sulfonylated  or  phosphorylated.   Studies  with  these  modified  enzymes  and 
spin  label  II  should  be  of  interest,  since  it  may  be  possible  to  detect 
whether  the  nitroxide  is  binding  to  the  catalytic  site  or  to  the  peripheral 
anionic  site.   Spin  label  studies  of  complexes  between  spin  labels  I  and  II 
and  acetylcholinesterase  in  the  presence  of  ligands  such  as  propidium  diiodide 
which  are  known  to  bind  to  the  peripheral  anionic  site  should  be  also  of 
interest.   With  the  possibility  of  obtaining  pure  membrane  fragments  containing 

9  "740 


Project  No.  Z01  HL  02507-02  PB 

high  amounts  of  the  acetylcholine  receptor,  it  should  be  possible  to  carry 
out  spin-labeled  studies  of  the  receptor  molecule  as  it  exists  in  the  membrane 
Studies  of  the  solubilized  receptor  should  also  make  it  possible  to  compare 
the  conformation  of  the  acetylcholine  receptor  in  the  membrane  and  as  it  is 
isolated  in  solution. 

Keyword  Descriptors:      Spin  labels,  electron  spin  resonance,  acetylcholin- 
esterase,  decamethonium,  acetylcholine  receptor. 

Honors  and  Awards:        None 

Publications:  Sinha,  B.K.  and  Chignell,  C.F.:   The  synthesis  and 

pharmacology  of  some  spin-labeled  analogs  of  biotin, 
hexamethonium,  decamethonium,  dichlorisoproterenol 
and  propranolol.   J .  Med .  Chem . ,  in  press. 


W 


Z01  HL  02508-01  PB 


Project  No. 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title: 


Membrane  Fluidity  in  Contact  Inhibited  and  Trans- 
formed Cells 


Previous  Serial  Number: 
Principal  Investigator: 
Other  Investigator: 
Cooperating  Unit: 

Project  Description: 


None 

Dr.  Colin  F.  Chignell 

Dr.  John  Bader 

Dr.  Bader  is  head  of  the  Section  on  Cell  Growth 
Regulation  in  the  Chemistry  Branch,  NCI 


Barnett  and  co-workers  (Proc.  Natl.  Acad.  Sci.  U.S.A.  71:   1992,  1974)  have 
carried  out  spin  label  studies  of  the  membranes  from  contact  inhibited  mouse 
embryo  fibroblast  3T3  cells  and  3T3  cells  transformed  by  oncogenic  RNA  and 
DNA  viruses  and  by  a  chemical  carcinogen.   Their  experiments  suggested  that 
the  membranes  of  transformed  cells  have  a  higher  fluidity  than  do  the  mem- 
branes from  contact  inhibited  cells.   Inbar  and  Shinitsky  (Proc.  Natl.  Acad. 
Sci.  U.S.A.  71:   2128,  1974)  have  used  a  fluorescent  probe  technique  to  demon- 
strate that  membranes  isolated  from  an  ascites  form  of  mouse  malignant  trans- 
formed lymphoma  cells  have  a  higher  degree  of  fluidity  than  membranes  isolated 
from  normal  lymphocytes.   Our  studies  were  undertaken  to  determine  whether 
such  differences  in  membrane  fluidity  could  be  demonstrated  in  chick  embryo 
fibroblasts  infected  with  a  temperature  sensitive  mutant  of  Rous  sarcoma 
virus.   These  cellsj which  appear  normal  when  cultured  at  41j  undergo  trans- 
formation when  the  incubation  temperature  is  shifted  from  41   to  37  . 

Cells  were  incubated  with  stearic  acid  label  I  (12,3)  at  a  concentration  of 
1  x  10   M  for  30  min  at  37  .   The  cells  were  then  washed  twice  in  buffer  and 
then  placed  in  a  glass  capillary  tube.   The  electron  spin  resonance  spectrum 
of  the  spin-labeled  cells  was  recorded  in  a  Varian  E-4  spectrometer  equipped 
with  a  variable  temperature  accessory.   The  fluidity  of  the  cell  membranes 
was  expressed  in  terms  of  an  order  parameter  S  (Seelig,  J.  Am.  Chem.  Soc.  92: 
3881,  1970),  which  is  defined  by  the  relationship 


0.568  (T,,'  -  T  ') 


S  = 


7*A 


Project  No.  Z01  HL  02508-01  PB 


where 


a»  =|  (T./+2V) 

and  T„',  Tj_'  are  the  separations  of  the  inner  and  outer  hyperfine  extrema  of 
the  membrane  bound  stearic  acid  label  I  (12,3). 


CH3(CH2)m -/C^ #  (CH2)nC00H 

0      N  -1-  0 


I  (m,n) 

The  membrane  fluidity  of  chick  embryo  fibroblasts  transformed  by  a  number  of 
viruses  is  shown  in  Table  1.   It  can  readily  be  seen  that  there  is  no  differ- 
ence in  the  membrane  fluidity, as  measured  by  the  order  parameter^of  the  nor- 
mal and  transformed  cells.   We  then  attempted  to  reproduce  the  experiments 
of  Barnett  and  co-workers  using  the  same  tumor  cell  lines.   It  can  be  seen 
from  Table  1  that  the  membrane  fluidity  of  mast  cells  from  Balb/3T3  mice  is 
unaffected  by  transformation  with  either  murine  sarcoma  virus  or  SVAO  virus. 
Since  the  completion  of  this  work,  other  investigators  have  also  reported 
their  inability  to  demonstrate  differences  in  membrane  fluidity  between  nor- 
mal and  transformed  cells  using  the  spin  label  technique  (Gaffney,  Proc.  Natl, 
Acad.  Sci.  U.S.A.  72:   664,  1975). 


Transformed  cells  are  known  to  exhibit  certain  differences  in  their  physical 
and  biochemical  characteristics.   Nevertheless,  the  suggestion  by  Barnett  and 
co-workers  and  Inbar  and  Shinitsky  that  the  membranes  of  transformed  cells 
are  more  fluid  than  those  from  contact  inhibited  cells  is  not  supported  by 
these  investigations.   No  further  studies  on  the  membrane  properties  of  nor- 
mal and  transformed  cells  are  contemplated  at  this  time. 


Keyword  Descriptors: 


Spin  labeling,  electron  spin  resonance,  membranes, 
membrane  fluidity,  chick  embryo  fibroblasts,  Rous 
sarcoma  virus,  murine  sarcoma  virus,  SV40  virus, 
mouse  Balb/3T3  mast  cells. 


Honors  and  Awards ; 


None 


Publications: 


None 


y&s 


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Project  No.  Z01  HL  02509-01  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title: 
Previous  Serial  Number: 
Principal  Investigator: 
Other  Investigators: 

Cooperating  Units:  , 


Acridine  Spin  Labels  as  Probes  for  Nucleic  Acids 

None 

Dr.  Birandra  K.  Sinha 

Dr.  Colin  F.  Chignell 
Dr.  David  B.  Millar 

Dr.  Sinha  held  a  Visiting  Fellowship  in  the  NHLI 
and  is  now  a  Guest  Worker  supported  by  the  Micro- 
biological Associates,  Bethesda,  Md. 
Dr.  Millar  is  Chief  of  the  Laboratory  of  Physical 
Biochemistry,  Environmental  Biosciences  Department, 
Naval  Medical  Research  Institute,  National  Naval 
Medical  Center,  Bethesda,  Md. 


Project  Description: 

The  nature  of  the  interaction  between  amino  acridines  and  nucleic  acids  is 
of  considerable  interest  because  of  the  mutagenicity  of  these  compounds  and 
their  reported  antitumor  activity.   The  biological  properties  of  the  acri- 
dines are  believed  to  arise  either  from  their  intercalation  into 
DNA  (or  RNA)  or  from  their  association  with  phosphate  groups  on  the  outside 
of  the  double  helix  of  nucleic  acids.   We  have  therefore  synthesized  amino 
acridine  spin  labels  I  and  II  and  studied  their  interaction  with  RNA  and  DNA 
by  means  of  electron  spin  resonance  and  other  spectroscopic  techniques. 


>V 


0CH3 


7*r 


Project  No.  Z01  HL  02509-01  PB 

Acridine  spin  labels  I  and  II  were  synthesized  from  their  corresponding 
9-chloro-acridines  and  4-amino-2,2,6,6-tetramethyl-l-piperidinyloxyl .   Calf 
thymus  DNA  and  calf  liver  RNA  were  purchased  from  a  commercial  source  and 
used  as  received.   Electron  spin  resonance  (ESR)  spectra  were  recorded  with 
a  Varian  E-4  spectrometer  operating  at  9.5  GHz.   Samples  were  introduced  into 
the  cavity  in  quartz  micro  flat  cells.   The  temperature  of  the  samples  was 
maintained  with  a  Varian  variable  temperature  accessory  and  was  measured  with 
a  Yellow  Springs  Instrument  Co.  telethermometer .  A  Cary  15  spectrophotometer 
equipped  with  a  water  jacket  was  used  for  the  Tm'  determinations.   Sedimenta- 
tion velocity  measurements  were  made  with  a  Beckman  Spinco  Model  E  analytical 
ultracentrif uge . 

The  ESR  spectrum  of  acridine  spin  label  I  bound  to  DNA  was  broad  and  asym- 
metric with  a  maximal  hyperfine  splitting  (2TM)  of  58.7  G.   Neither  heat 
denaturation  of  the  DNA  nor  the  addition  of  salt  (0.1  M  NaCl)  altered  the 
2T„  value  of  bound  label  I  (Table  1) .   The  2T„  value  of  acridine  spin  label 
II  bound  to  DNA  was  55.5  G.   The  addition  of  0.1  NaCl  to  the  DNA-II  complex 
did  not  significantly  alter  the  ESR  spectrum  of  the  probe  (Table  1) . 

TABLE  1 
The  maximal  hyperfine  splittings  (2Tm)  of  acridine  spin  labels  I  and  II 


bound  to  calf  thymus  DNA 

Sample                            NaCl 

2T„ 

(0.1  M) 

(G) 

DNA  +1 

58.7 

DNA  (heat  denatured)  +1 

59.0 

DNA  +1                                     + 

59.0 

DNA  +11                                    - 

55.5 

DNA  +11                                    + 

55.0 

In  the  absence  of  spin  label,  DNA  was  found  to  have  a  sedimentation  coeffi- 
cient of  11.2  S  while  on  the  addition  of  spin  label  I,  the  sedimentation  co- 
efficient decreased  to  8.92.   This  observation  suggests  that  label  I  is  in- 
deed intercalated  into  the  DNA.   The  effect  of  the  acridine  spin  labels  on 
the  melting  temperature  (Tm')  of  DNA  was  studied  by  means  of  absorption 
spectroscopy  and  electron  spin  resonance.   The  Tm'  value  for  DNA  alone  was 
found  to  be  66.5   as  determined  by  the  increase  in  the  optical  density  of 
the  solution  at  260  nm.   In  the  presence  of  spin  label  I,  the  melting  temper- 
ature of  DNA  was  raised  to  70  .   It  was  also  found  possible  to  measure  the 
Tm'  value  for  complexes  between  the  spin  labels  I  and  II  and  DNA  by  monitor- 
ing the  concentration  of  free  spin  label  in  solution  as  a  function  of  tem- 
perature.  The  Tm'  value  of  a  complex  between  DNA  and  I  was  found  to  be  69° 

2  7U 


Project  No.  Z01  HL  02509-01  PB 

by  this  technique.   The  complex  between  DNA  and  II  exhibited  Tm'  values  of 
76  by  the  spectrophotometric  assay  and  78  by  the  ESR  method. 

The  binding  parameters  for  the  interaction  between  I  and  II  and  DNA  were 
determined  by  titrating  a  fixed  amount  of  nucleic  acid  with  increments  of 
spin  label  and  measuring  the  concentration  of  free  probe  by  ESR.   Both  spin 
labels  exhibited  at  least  two  kinds  of  binding  sites  on  DNA  (Table  2) .  At 
low  ionic  strength,  it  is  known  that  binding  occurs  both  by  intercalation  and 
an  electrostatic  interaction  between  the  dye  and  the  nucleic  acid.   The  addi- 
tion of  a  high  concentration  of  salt  is  reported  to  abolish  binding  due  to 
the  electrostatic  process.   It  will  be  seen  from  Table  2  that  in  the  presence 
of  0.1  M  NaCl  the  affinity  of  both  spin  labels  for  DNA  is  drastically  reduced. 

TABLE  2 


Sample 

Binding  Parameters 

Nj 

Kl 

N2 

K2 

(M  x  10"5) 

(M  x  10~5) 

I 

0.150 

120.0 

2.11 

0.74 

I  +  0.1  M  NaCl 

0.127 

16.1 

1.88 

0.079 

II 

0.186 

93.0 

0.18 

0.27 

II  +  0.1  M  NaCl 

0.163 

19.7 

0.22 

0.22 

From  the  ESR  spectra  of  complexes  between  labels  I  and  II  and  DNA,  it  is  ap- 
parent that  the  piperidine  ring  that  bears  the  nitroxide  has  greater  mobility 
in  the  complex  between  DNA  and  II.   This  suggests  that  there  is  a  fundamental 
difference  in  the  way  that  these  two  spin  labels  interact  with  DNA.   This 
difference  is  also  reflected  in  the  melting  temperatures  of  complexes  between 
I  and  II  and  DNA  with  spin  label  II  producing  a  greater  increase  in  the  Tm' 
value.   Molecular  models  of  labels  I  and  II  demonstrate  a  pronounced  steric 
effect  of  the  methoxyl  group  in  the  2  position  which  forces  the  acridine 
ring  into  a  conformation  in  which  it  is  perpendicular  to  the  flat  piperidine 
ring.   This  observation  may  explain  the  differences  in  the  mobility  of  the 
nitroxide  group  of  labels  I  and  II  bound  to  DNA.   It  is  also  possible  that 
the  conformational  differences  between  the  acridine  labels  may  explain  why  I 
is  more  effective  in  raising  the  melting  temperature  of  DNA. 

The  ESR  spectrum  of  label  I  bound  to  calf  liver  RNA  indicated  that  the  spin 
label  had  a  higher  degree  of  mobility  than  when  bound  to  DNA.   It  is  likely 
that  the  calf  liver  RNA  exists  in  a  single  stranded  form.   It  therefore 
appears  possible  that  these  spin  labels  may  be  useful  in  detecting  double 
helical  and  single  stranded  regions  in  nucleic  acids. 


7*7 


Project  No.  Z01  HL  02509-01  PB 

Acridines  are  of  interest  for  two  reasons:   firstly,  because  of  their  bio- 
logical and  pharmacological  effects  and  secondly,  because  they  may  be  useful 
tools  for  probing  the  conformation  of  nucleic  acids  in  biologically  important 
complexes  such  as  chromatin.   In  both  these  areas,  it  is  to  be  expected  that 
spin  labels  I  and  II  will  provide  useful  information  not  only  on  the  pharma- 
cological and  toxicological  effect  of  acridines  in  biological  systems  but 
also  in  the  area  of  gene  transcription.   For  future  studies,  9-aminoacri- 
dines  will  be  prepared  in  which  different  chain  lengths  are  inserted  between 
the  9-amino  function  and  the  acridine  moiety.   The  binding  of  spin  labels  I 
and  II  to  nucleic  acids  isolated  from  normal  and  transformed  cells  and  to 
chromatin  will  also  be  studied. 

Keyword  Descriptors:      Electron  spin  resonance,  spin  labels,  9-aminoacri- 

dines,  DNA,  RNA. 

Honors  and  Awards :        None 

Publications:  None 


768 


Project  No.  Z01  HL  02510-01  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:  Spin  Label  Studies  of  Halobacterium  halobium  Purple 

Membranes 

Previous  Serial  Number:   None 

Principal  Investigator:   Dr.  Colin  F.  Chignell 

Other  Investigator:       Dr.  Derek  A.  Chignell 

Cooperating  Unit:   .      Dr.  Derek  A.  Chignell  is  a  Lecturer  in  Biochemistry 

in  the  Department  of  Biochemistry,  The  University, 
Dundee,  Scotland 

Project  Description: 

The  isolation  of  a  purple  colored  fragment  from  the  cell  membrane  of  the  ex- 
treme halophile  H^_  halobium  represents  one  of  the  first  successful  attempts 
to  obtain  simple  membrane  system  capable  of  translocating  hydrogen  ions 
(Oesterhelt  and  Stoeckenius,  Proc.  Natl.  Acad.  Sci.  U.S.A.  70:   2853,  1973). 
These  purple  membranes  (PM)  contain  a  single  protein,  bacteriorhodopsin, 
which  constitutes  about  75%  of  their  dry  weight.   The  purple  color  (emax  570 
nm)  of  PM  is  due  to  retinaldehyde  bound  covalently  to  bacteriorhodopsin,  and 
evidence  has  accumulated  that  this  light-induced,  proton-locating  membrane 
bears  a  close  relationship  to  the  visual  receptor  membrane,  although  the  ions 
translocated  are  different.   Cone  (Nature  New  Biol.  236:   39,  1972)  and 
Brown  (Nature  New  Biol.  236:   35,  1972)  have  demonstrated  that  the  rhodopsin 
molecules  in  rod  outer  segments  are  highly  mobile,  the  viscosity  of  the  phos- 
pholipids being  low  enough  to  allow  rotation  and  translation  of  the  protein 
molecules  within  the  membrane.   This  is  consistent  with  a  low  degree  of  satu- 
ration of  the  fatty  acid  side  chains  found  in  the  rod  outer  segment  phospho- 
lipids.  By  contrast,  the  side  chains  of  PM  phospholipids  are  highly  saturated, 
consisting  of  dihydrophytol  groups  linked  to  phosphatidylglycerol  by  ether, 
instead  of  ester,  linkages.   The  degree  of  fluidity  of  PM  therefore  is  of 
some  interest. 

Spin  labels  have  been  found  to  be  extremely  useful  as  probes  of  membrane 
structure  (Jost  and  Griffith,  Methods  in  Pharmacology,  Vol.  2,  223,  1972). 
In  particular,  spin-labeled  fatty  acids  with  the  general  formula  I  (m,n)  have 
provided  information  concerning  the  molecular  organization,  phase  transitions 
and  fluidity  of  various  natural  and  synthetic  membranes.   The  structure  of 
mammalian  visual  receptor  membranes  has  also  been  studied  with  the  aid  of 

.  1  749 


Project  No.  Z01  HL  02510-01  PB 


CH3  (CH2)m C  ^—   (CH2)nCOOH 

<f  N 0 


I  (m,n) 

spin  labels  (Hong  and  Hubbell,  Proc.  Natl.  Acad.  Sci.  U.S.A.  69:   2617,  1972). 
We  have  therefore  studied  the  molecular  organization  of  PM  with  the  aid  of 
stearic  acid  spin  labels  (I  12,3;  5,10;  1,14)  and  a  palmitamide  spin  label  (II) 


CH3(CH2)iitCONH  — <     N — 0 


II 


Stearic  label  I  (12,3)  exhibited  maximum  hyperfine  splittings  (2Tm)  of  62  G 
and  59  G  when  bound  to  PM  at  25  and  37  ,  respectively.   A  comparison  of  the 
2Tm  of  I  (12,3)  bound  to  PM  with  the  2Tm  of  the  same  label  bound  to  other  mem- 
brane systems,  such  as  human  erythrocytes  and  lymphocytes,  influenza  virus, 
sarcoplasmic  reticulum  vesicles,  E.  coli  membrane  vesicles,  and  submito- 
chondrial  particles,  indicates  that  the  PM  is  the  most  rigid  of  all  the  mem- 
branes hitherto  probed  with  this  label.   An  Arrhenius  plot  of  the  2Tm  values 
of  I  (12,3)  bound  to  PM  showed  a  single  discontinuity  at  about  29  .   When 
the  PM  were  crosslinked  with  glutaraldehyde,  the  Arrhenius  plot  became  mono- 
phasic.   Thus,  it  would  appear  that  discontinuity  in  the  Arrhenius  plot  of 
untreated  PM  is  due  to  a  phase  change  in  bacteriorhodopsin  rather  than  to  an 
alteration  in  the  molecular  organization  of  the  phospholipids. 

Stearic  acid  spin  label  I  (5,10)  was  also  highly  immobilized  when  bound  to 
PM  with  2Tm  values  of  62.7  G,  60.7  G  and  58.7  G  at  5°,  25°  and  37°,  respec- 
tively.  In  other  membrane  systems,  the  motion  of  stearic  spin  label  I  (5,10) 
has  been  found  to  be  almost  isotropic.   In  contrast,  at  37   the  2Tm  values 
of  spin  label  I  (12,3)  and  I  (5,10)  bound  to  PM  are  almost  identical.   Thus, 
it  would  appear  that  the  rigidity  of  the  PM  system  exists  deep  into  the  in- 
terior of  the  membrane.   The  electron  spin  resonance  (ESR)  spectrum  of  stearic 
acid  label  I  (1,14)  bound  to  PM  at  25   revealed  the  presence  of  two  popula- 
tions of  spin  labels,  one  of  which  was  more  highly  immobilized  than  the  other. 
It  seems  likely  that  the  highly  immobilized  labels  are  bound  to  boundary  lip- 
id which  is  closely  associated  with  the  bacteriorhodopsin  (Biochim.  Biophys . 
Acta  311:   141,  1973). 

Verma  and  co-workers  have  been  able  to  demonstrate  a  light-dependent  change 
in  the  viscosity  of  beef  retinal  rods  with  the  aid  of  a  stearamide  spin  label 
analogous  to  II  (Biochem.  Biophys.  Res.  Commun.  55:   704,  1973).   We  were 
unable  to  detect  any  such  changes  in  PM  with  labels  I  (1,14)  or  II.   This  is 

2  770 


Project  No.  Z01  HL  02510-01  PB 

perhaps  not  surprising,  since  it  is  known  for  PM  the  "dark  reaction"  in  which 
the  "bleached"  form  (emax  415  nm)  returns  to  the  "unbleached",  form  (emax  570 
nm)  is  very  fast  (Nature  New  Biol.  233:   149,  1971).   The  dark  reaction  can 
be  slowed  by  suspending  the  PM  in  a  high  salt  solution  saturated  with  ether. 
We  found,  however,  that  such  treatment  causes  a  "fluidization"  of  the  membrane 
lipids  as  reflected  by  a  dramatic  increase  in  the  mobility  of  PM  bound  label 
I  (1,14).   While  illumination  of  such  PM  did  result  in  bleaching,  no  change 
in  the  ESR  spectrum  of  I  (1,14)  was  detected.   The  dark  reaction  can  also  be 
slowed  by  cooling  the  PM  in  liquid  nitrogen.   At  this  temperature,  the  ESR 
spectrum  of  I  (1,14)  approached  the  "rigid  glass"  limit  and  was  not  affected 
by  illumination. 

The  evidence  presented  in  these  studies  points  to  an  extremely  rigid  structure 
for  PM  with  two  populations  of  phospholipids,  one  of  which  is  tightly  bound 
to  the  protein  in  the  membrane.   Immobilization  of  the  protein  by  glutaralde- 
hyde  has  very  little  effect  on  this  rigidity.   Consistent  with  the  findings 
of  Racker  and  Hinkle  (J.  Membrane  Biol.  17:   181,  1974),  these  experiments 
suggest  that  proton  translocation  occurs  via  a  pore  mechanism  rather  than 
being  dependent  upon  the  mobility  of  the  bacteriorhodopsin  within  the  PM.   No 
change  in  the  molecular  organization  of  PM  could  be  detected  upon  illumination. 

This  work  has  now  been  completed  and  no  further  studies  on  the  purple  membrane 
system  from  Halobacterium  halobium  are  contemplated. 

Keyword  Descriptors:      Purple  membranes,  Halobacterium  halobium,  spin 

labels,  electron  spin  resonance. 

Honors  and  Awards :        None 

Publications:  Chignell,  C.F.  and  Chignell,  D.A. :  A  spin  label 

study  of  purple  membranes  from  Halobacterium 
halobium.   Biochem.  Biophys.  Res.  Commun.  62:   136- 
143,  1975. 


77/ 


Project  No.  Z01  HL  02511-02  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title: 

Previous  Serial  Number: 
Principal  Investigator; 
Other  Investigators: 

Cooperating  Unit: 


Physicochemical  Studies  of  Lung  Surfactant  Lipo- 
protein 

NHLI-217 

Dr.  Colin  F.  Chignell 

Mr.  Robert  Sik 

Mr.  Edward  Sokoloski 

Mr.  Sokoloski  is  in  the  Laboratory  of  Chemistry. 
NHL  I 


Project  Description: 

Lung  surfactant  is  a  lipoprotein  which  is  known  to  reduce  the  surface  tension 
of  the  alveolar  lining  of  the  lung,  thereby  preventing  the  collapse  of  this 
organ.   Considerable  controversy  still  persists  concerning  the  precise  func- 
tion of  the  apoprotein  portion  of  lung  surfactant.   This  study  has  been 
undertaken  to  determine  whether  the  apoprotein  modifies  the  physical  proper- 
ties of  lung  surfactant. 

Lung  surfactant  has  been  isolated  from  a  lavage  of  the  lungs  of  both  dogs  and 
rabbits.   The  lung  surfactant  lipoprotein  was  purified  by  means  of  density 
gradient  centrifugation  on  NaBr  gradients  (Am.  J.  Physiol.  223:   707,  1972). 

The  surfactant  lipoprotein9isolated  from  both  rabbit  and  dog  lungsjwas  delip- 
idated  by  repeated  treatment  at  -20  with  ether  ethanol  (3:1).   Disc  gel 
electrophoresis  of  the  apoprotein  indicated  the  presence  of  two  main  compo- 
nents with  masses  of  about  approximately  30,000  daltons  and  10,000  daltons. 
Both  preparations  were  found  to  be  contaminated  with  plasma  proteins,  of 
which  serum  albumin  seemed  to  be  the  main  component. 

Electron  spin  resonance  studies  with  the  aid  of  stearic  acid  spin  labels 
I  (m,n)  have  indicated  that  the  molecular  organization  of  the  intact  lipopro- 
tein is  highly  ordered  at  or  near  the  interface  with  the  aqueous  environment 
but  that  the  interior  of  the  lipoprotein  is  extremely  fluid.   In  these  re- 
spects, lung  surfactant  lipoprotein  resembles  the  erythrocyte  ghost  membrane 
in  terms  of  its  molecular  organization. 


77* 


Project  No.  Z01  HL  02511-02  PB 


CH3  (CH2)m C  ^ ;  (CH2)nC00H 

0      N  —  0 


I  (m,n) 

Nuclear  magnetic  resonance  (NMR)  measurements  of  the  P   resonance  of  the 
phospholipid  phosphate  groups  present  in  the  lipoprotein  have  been  attempted. 
However,  it  was  found  that  the  P3   resonance  of  lung  surfactant  was  broadened 
to  the  point  where  it  was  undetectable  in  the  NMR  spectrometer.   In  contrast, 
when  the  phospholipids  were  extracted  from  the  lipoprotein  and  resonicated 
into  deuterium  oxide,  a  sharp  P31  resonance  was  observed.   These  results  sug- 
gest that  in  the  lung  surfactant  lipoprotein  the  phosphate  groups  of  the 
phospholipids  are  highly  immobilized. 

Lung  surfactant  plays  an  important  role  in  lung  physiology.   Furthermore, 
alterations  in  lung  surfactant  in  certain  disease  states  and  after  exposure 
to  environmental  pollutants  are  known  to  cause  respiratory  problems .   It  is 
therefore  important  to  understand  more  precisely  the  physical  characteristics 
of  this  important  lipoprotein. 

The  physical  characteristics  of  the  lung  surfactant  lipoprotein  will  be  fur- 
ther characterized  by  means  of  other  spin  labels.   In  addition,  the  physical 
properties  of  the  lung  surfactant  lipoprotein  will  be  compared  with  those  of 
isolated  sonicated  phospholipids  in  an  attempt  to  determine  what  effect,  if 
any,  the  apoproteins  have  on  the  physical  characteristics  of  this  complex. 

Keyword  Descriptors:      Lung  surfactant,  apoprotein,  phospholipids,  elec- 
tron spin  resonance,  spin  labels,  nuclear  magnetic 
resonance,  gel  electrophoresis. 

Honors  and  Awards :        None 

Publications:  None 


773 


Project  No.  Z01  HL  02512-02  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Further  Spin  Label  Studies  of  Egg  White  Avidin 

Previous  Serial  Number:  NHLI-100 

Principal  Investigator:  Dr.  Colin  F.  Chignell 

Other  Investigator:  Dr.  Birandra  K.  Sinha 

Cooperating  Unit:         Dr.  Sinha  held  a  Visiting  Fellowship,  NHLI,  and  is 

now  a  Guest  Worker  supported  by  the  Microbiological 
Associates,  Bethesda,  Md. 

Project  Description: 

Avidin  is  a  tetrameric  protein  (mass  68,000  daltons)  that  binds  four  mole- 
cules of  vitamin  biotin  (I).   The  biotin  binding  sites,  one  per  subunit,  are 
grouped  in  two  pairs  at  opposite  ends  of  the  avidin  molecule  (Green  e_t  al.  , 
Biochem.  J.  ]25:   781,  1971).   We  have  studied  the  topography  of  the  avidin 
binding  sites  with  the  aid  of  four  spin-labeled  analogs  of  biotin  (II-V) . 
Fluorescence  and  optical  absorption  spectroscopy  indicated  that  labels  II-V 
occupied  the  same  binding  sites  on  avidin  as  did  biotin.   The  electron  spin 
resonance  spectrum  (ESR)  of  the  4:1  complex  between  II  and  avidin  contained 
broad  line  components  characteristic  of  a  highly  immobilized  spin  label 
(Table  1) .   Dipole-dipole  interactions  between  spin  labels  bound  to  adjacent 
sites  split  each  of  the  major  hyperfine  lines  into  doublets  with  a  separation 
of  13.8  G.   The  distance  between  adjacent  bound  nitroxide  groups  was  calcu- 
lated from  the  splitting  to  be  16  A  (Table  1).   The  dissociation  of  the  4:1 
complex  between  II  and  avidin  was  biphasic  with  approximately  half  of  the 
spin  labels  dissociating  at  a  rate  (kdiss  =  2.51  x  10-1+  sec--1)  that  was  much 
faster  than  the  remainder  (kai.ss  =  1.22  x  10~5  sec-1).   The  electron  spin 
resonance  spectrum  of  the  2:1  complex  between  II  and  avidin  clearly  showed 
that  immediately  after  mixing  spin  labels  were  distributed  in  a  random  fashion 
among  the  available  binding  sites  but  that  they  slowly  redistributed  them- 
selves so  that  each  label  bound  to  a  site  which  was  adjacent  to  an  occupied 
site.   The  final  time- independent  electron  spin  resonance  spectrum  exhibited 
a  splitting  of  69  G  between  the  low  and  high  field  hyperfine  lines  which  is 
characteristic  of  highly  immobilized,  noninteracting  spin  label.   Spin  labels 
III  and  IV  interacted  with  avidin  in  a  similar  fashion  to  that  described  for 
II  with  the  exception  that  their  dipolar  splittings  were  11.9  G  and  14.2  G, 
respectively.   From  these  splittings  it  was  estimated  that  the  distance  be- 
tween adjacent  avidin  bound  nitr oxides  was  16.7  A  for  label  III  and  15 . 7  A 

1  77f 


Project  No.    Z01  HL  02512-02  PB 


TABLE  1 

The 

ESR 

parame 

ters  of  complexes  between  the  spin- 
and  avidin 

lab< 

sled  biotin  analogs 

Compound 

Maximum  hyperf. 
splitting  of  2 
complex* 

me 
1 

Dipolar  splitting 
of  4:1  complex 

Calculated  distance 

between  adjacent 

nitroxides 

(G) 

(G) 

(A) 

II 

69.0 

13.8 

16.0 

III 

64.9 

11.9 

16.7 

IV 

63.5 

14.2 

15.7 

V 

62.0 

— 

— 

*  Measured  at   least   3  hr  after  mixing. 


0*> 

I 

HN- 


(CH2)itC0R 


R  =  H 


II 


III 


IV 


R  = 


R  = 


R  = 


R  = 


NH- 


>^ 


nA 


0 

— NH-CH2 


33 


— NHCH2  CONH  -<  N  ~  0 


775- 


Project  No.  Z01  HL  02512-02  PB 

for  label  IV  (Table  1) .   The  electron  spin  resonance  spectrum  of  label  V 
bound  to  avidin  was  characteristic  of  a  noninteracting,  highly  immobilized 
nitroxide  with  a  maximum  splitting  of  62  G.   The  electron  spin  resonance  spec- 
trum of  V  bound  to  avidin  was  independent  of  both  time  and  the  amount  of 
bound  label.   The  rate  of  dissociation  of  V  from  a  4:1  complex  with  avidin 
was  monophasic  (k^igg  =  3.85  x  10   sec-1).   These  studies  support  a  model 
for  avidin  in  which  the  recognition  site  of  the  heterocyclic  ring  system  of 
biotin  is  represented  as  a  cleft  located  within  a  hydrophobic  depression  in 
the  surface  of  the  protein. 

These  studies  clearly  show  that  spin-labeled  ligands  can  provide  useful  in- 
formation on  the  topography  of  receptors  and  other  binding  sites  found  in 
proteins  and  other  biologically  important  macromolecules .   The  biotin  avidin 
system  is  the  first  in  which  it  has  been  possible  to  estimate  molecular  dis- 
tances using  the  electron  spin  resonance  technique  and  spin  labels.   It  is 
hoped  that  a  more  precise  knowledge  of  binding  site  topography  may  make  it 
possible  to  design  more  specific  drug  molecules. 

This  project  has  now  been  completed  and  no  further  work  on  this  system  is 
anticipated. 

Keyword  Descriptors:  Avidin,  biotin,  electron  spin  resonance,  fluores- 
cence spectroscopy,  absorption  spectroscopy,  spin 
labels. 

Honors  and  Awards:        None 

Publications:  Chignell,  C.F. ,  Starkweather,  D.K.  and  Sinha,  B.K.: 

A  spin  label  study  of  egg  white  avidin.   J.  Biol. 
Chem. ,  in  press. 


774 


Project  No.  Z01  HL  02513-03  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md . 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Purification  and  Characterization  of  Na  +  K- 

ATPase 

Previous  Serial  Number:   NHLI-97 

Principal  Investigators:   Dr.  Clyde  A.  Takeguchi 

Dr.  Ueli  Honegger 
Dr.  Elwood  0.  Titus 

Other  Investigator: .      Mr.  Wallace  W.  Holland 

Cooperating  Units:        Dr.  Takeguchi  is  a  Research  Associate  in  the 

Pharmacology-Toxicology  Program,  NIGMS 
Dr.  Honegger  holds  a  Visiting  Fellowship,  NHLI 

Project  Description^ 

Objectives  :   The  Mg^-dependent ,  Na+  +  K+-stimulated,  ouabain- inhibit- 
able  ATP  hydrolyzing  system  is  an  integral  part  of  the  active  transport 
mechanism  that  moves  cations  across  biological  membranes  against  electrical 
and  chemical  gradients.   The  enzyme  is  a  lipoprotein  complex  which  is  itself 
a  part  of  the  cell  membrane.   The  goals  of  this  project  are  to  describe  the 
molecular  events  (presumably  reversible  changes  in  conformation  of  ATPase) 
that  are  associated  with  ion  transport  and  to  ascertain  those  structural 
features  which  enable  the  ionophoric  portions  of  the  system  to  distinguish 
between  sodium  and  other  monovalent  cations.   Previously  this  laboratory  has 
demonstrated  that  both  agents  that  act  extracellularly  (cardiac  glycosides 
and  potassium)  and  those  which  act  intracellularly  (sodium  and  ATP)  alter 
the  conformation  of  a  single  protein  which  appears  to  traverse  the  whole 
membrane.   Certain  of  these  conformational  changes  apparently  expose  normally 
inaccessible  protein  sulfhydryl  groups.   The  present  study  was  therefore  de- 
signed to  seek  reagents  that  could  specifically  label  the  conformationally 
mobile  sulfur-containing  moieties  or  that  might  initiate  the  cleavage  of 
peptide  chains  at  these  sites.   Since,  in  theory,  both  of  these  objectives 
could  be  obtained  by  cyanylation  of  the  SH  groups  of  conformationally  mobile 
cysteine  residues  (Degani  and  Patchornick,  Biochemistry  13:   1,  1974),  the 
reaction  of  the  ATPase  system  with  cyanylating  reagents  is  being  examined. 
Methods  for  obtaining  highly  purified  enzyme  in  bulk  are  also  being  examined. 

Methods  Employed:   The  outer  medulla  of  the  rabbit  kidney,  which  is 
particularly  rich  in  transport  sites  associated  with  sodium  reabsorption, 


777 


Project  No.  Z01  HL  02513-03  PB 

has  been  used  as  the  enzyme  source.  A  modification  of  the  Jorgensen  proce- 
dure (Methods  in  Enzymology  32:   277,  1974)  was  employed  to  obtain  purified 
ATPase.   TCNB  (5-thiocyano-2-nitrobenzoic  acid)  was  synthesized  according  to 
the  method  of  Degani  and  associates  (J.  Am.  Chem.  Soc.  92:   6969,  1970). 

Major  Findings:   A  modification  of  the  method  of  Jorgensen  consistently 
resulted  in  high  yields  of  ATPase  with  high  specific  activity  from  rabbit 
kidneys  obtained  from  commercial  sources. 

5  x  10   M  Ellman's  reagent  (5,5-bisdithio-2-nitrobenzoic  acid)  and 
5  x  10-3  M  TCNB  inhibited  ATPase  specific  activity  by  54%  and  68%,  respec- 
tively.  Kinetic  experiments  with  these  sulfhydryl  reagents  also  indicated 
that  there  may  be  two  populations  of  sulfhydryl  groups  in  the  active  enzyme, 
one  with  a  reaction  t-1/2  of  a  few  seconds,  and  the  second  with  a  t-1/2  of 
about  20  to  30  minutes.   Na  and  ATP  seem  to  protect  the  enzyme  from  inactiva- 
tion  by  these  sulfhydryl  reagents.   This  protection  is  lost  with  the  addition 
of  Mg++. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
The  ionic  gradients  across  cell  membranes  are  maintained  by  this  cation  trans- 
port system.   These  gradients  are  a  prerequisite  for  all  cellular  functions 
involving  neuronal  conduction,  the  production  of  electrical  currents,  the 
excitability  of  muscle  and  nerve,  the  storage  of  neurotransmitters  that  convey 
impulses  from  nerve  to  smooth  muscle  and  the  bulk  transport  of  electrolytes. 
Attempts  to  characterize  fully  the  enzymatic  basis  for  this  transport  system 
are  fundamental  to  an  understanding  of  the  regulatory  mechanisms  of  the  cardio- 
vascular system.   Purification  and  characterization  of  this  enzyme  system 
would  permit  further  investigations  into  the  role  of  various  drugs,  such  as 
ouabain  and  other  cardiac  glycosides,  upon  this  enzyme  at  the  molecular  level. 

Proposed  Course  of  Project:   Further  studies  will  focus  on  labeling  the 
purified  ATPase  with  11+C-  and  13C-labeled  TCNB.   Since  cyanylation  initiates 
chain  breakage,  patterns  of  peptide  fragments  should  reflect  labeling  differ- 
ences due  to  ligand-dependent  conformational  changes.   13C  NMR  spectroscopy 
may  enable  us  to  see  the  micro-environment  of  the  labeled  sulfhydryl  groups 
as  it  undergoes  conformational  changes.   Protein  degradative  studies  will  be 
undertaken  in  an  effort  to  determine  the  location  of  the  two  populations  of 
SH  groups  revealed  by  the  kinetic  studies. 

Keyword  Descriptors:      ATPase,  ion  transport,  membranes,  cardiac  glyco- 
sides, sulfhydryl  groups 

Honors  and  Awards :        None 

Publications:  Titus,  E.O.  and  Hart,  W.M.,Jr.:   The  use  of  sulf- 

hydryl reagents  to  identify  proteins  undergoing 
ligand-dependent  conformational  changes  associated 
with  the  function  of  (Na+  +  K+) -ATPase.   Ann.  N.Y. 
Acad.  Sci.  242:   246-254,  1974. 


770 


Project  No.  Z01  HL  02514-01  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:  Chemical  Characterization  of  Pharmacological 

Receptors 

Previous  Serial  Number:  None 

Principal  Investigator:  Dr.  Elwood  0.  Titus 

Other  Investigator:  Mr.  Wallace  W.  Holland 

Cooperating  Units:  None 

Project  Description: 

Objectives :   The  molecular  mechanisms  by  which  neurohumoral  transmitters 
activate  receptors  in  mammalian  smooth  muscle,  secretory  glands,  etc.,  are 
unknown.   The  first  step  is  almost  certainly  adsorption  to  a  recognition 
site  which  exhibits  high  affinity  for  a  limited  range  of  organic  structures 
related  to  the  natural  agonist.   Conformational  changes  associated  with  this 
adsorption  initiate  the  response.   The  goal  of  this  project  is  to  isolate 
those  components  of  adrenergic  and  other  receptors  which  serve  as  agonist- 
recognition  sites  in  smooth  muscle  and  other  autonomically  innervated  mamma- 
lian tissues. 

Studies  of  the  nicotinic  cholinergic  receptor  isolated  from  the  electro- 
plax  of  electric  fish  together  with  studies  of  insulin,  angiotensin  and  other 
receptors  from  mammalian  tissues  indicate  that  individual  receptors  are 
characterized  by  specific  proteins.   The  introduction  of  site-directed,  co- 
valently  bound  radioactive  labels  into  such  proteins  should  be  useful  in  iso- 
lating receptors.   Although  the  specif ic  agonist-binding  properties  of  the 
electroplax  receptor  survive  solubilization  in  detergents  and  separation  from 
the  membrane  matrix,  studies  with  the  mammalian  muscarinic  receptor  indicate 
that  this  may  not  always  be  the  case.   Even  if  it  is  impossible  to  purify  a 
receptor  without  denaturation,  the  isolation  of  a  labeled  polypeptide,  un- 
equivocally identifiable  as  derived  from  the  receptor,  is  of  interest,  since 
the  information  which  defines  the  receptor  is  inherent  in  the  amino  acid 
sequence  of  the  protein. 

Methods  Employed:   The  fractionation  of  membrane  proteins  will  be  fol- 
lowed by  radioactive  labels  covalently  attached  to  SH,  COOH,  or  tyrosyl  moi- 
eties since,  with  appropriate  choice  of  reagents,  these  functional  groups  can 
be  labeled  in  intact  tissue  under  physiological  conditions.   Since  the 

l  77? 


Project  No.  Z01  HL  02514-01  PB 

adsorption  of  an  agonist  causes  conformational  perturbation  of  the  receptor 
substance  and  since  conformational  change  is  frequently  manifested  in  altered 
reactivity  of  the  above-mentioned  moieties,  ligand-dependent  changes  in 
labeling  patterns  will  be  used  for  the  identification  of  receptor-related 
proteins.   The  procedure  is  best  controlled  by  a  double  isotope  procedure  in 
which  identical  preparations  are  treated  with  the  same  reagent,  that  reagent 
being  labeled  with  different  isotopes  as  it  is  used  in  the  presence  or  the 
absence  of  the  agonist  which  alters  the  conformation.   The  two  preparations 
are  mixed  and  the  individual  proteins  are  isolated  from  membranous  subtrac- 
tions.  In  theory,  a  constant  ratio  of  the  two  isotopes  should  be  observed 
for  all  proteins  except  those  affected  by  the  agonist.   The  procedure  has 
been  successfully  used  for  the  identification  of  the  M  protein  of  bacterial 
membranes  (Fox  and  Kennedy,  Proc.  Natl.  Acad.  Sci.  U.S.A.  54:   891,  1965), 
the  ouabain-sensitive  component  of  ion  transport  systems  (Hart  and  Titus, 
J.  Biol.  Chem.  248:   4674,  1973),  the  glucagon-sensitive  components  of  the 
adenylate  cyclase  system  in  rat  liver  (Storm  and  Chase,  J.  Biol.  Chem.  250: 
2539,  1975)  and  one  of  the  proteins  in  the  cholinergic  receptor  of  electro- 
plax  (Reiter  et  al . ,  Proc.  Natl.  Acad.  Sci.  U.S.A.  69:   1168,  1972). 

Major  Findings:  A  double  isotope  procedure  applicable  to  the  minute 
quantities  of  receptors  in  mammalian  tissues  will  require  radioactive  labels 
with  specific  activities  of  the  order  of  Curies  per  mmole.   Both  experience 
in  this  laboratory  and  a  survey  of  the  literature  indicated  that  changes  in 
the  rate  of  reaction  of  protein  sulfhydryl  groups  with  electrophilic  reagents 
were  likely  to  be  the  most  sensitive  indicators  of  conformational  alteration. 
First  priority  was  therefore  given  to  synthesis  of  reagents  that  reacted  with 
SH  groups,  that  could  be  easily  labeled  with  two  isotopes  and  that  would  re- 
act preferentially  with  extracellular  sites. 

In  collaboration  with  the  New  England  Nuclear  Corp.,  3H-sulfanilic  acid 
was  prepared  by  reaction  (1)  and  purified  to  a  specific  activity  of  3  Ci/nmole 


-\0/~  S°3H  +  3H  >NH2  -(O/ 


(1)  NH2  -\  (  )  V  SO3H  +  3H >  NH2  -\  [)   >-  SO3H 

CI  H3 

Since  S35-labeled  sulfanilic  acid  of  equivalent  specific  activity  is  avail- 
able, and  since  coupling  of  diazotized  anilic  acid  to  protein  SH,  tyrosyl  and 
imidazole  groups  occurs  readily  at  physiological  pH,  sulfanilic  acid  will  be 
used  in  double  labeling  experiments.   Preliminary  experiments  on  the  relative 
effects  of  d-  and  ^-norepinephrine  on  the  labeling  of  intact  rabbit  aortas  by 
diazotized  S^-sulfanilic  acid  indicate  that  individual  proteins  isolated 
from  the  membrane  fraction  accept  from  0.03  to  0.9  pmols  of  label  and  that 
two  proteins  from  this  fraction  may  be  sensitive  to  norepinephrine. 

The  label  from  sulfanilic  acid  may  be  introduced  into  a  reagent  more 
specific  for  SH  groups  by  the  following  reaction: 


7f0 


Project  No.  Z01  HL  02514-01  PB 
0 

N-CH2-CH2-/Q\-0H  +  NEN-/(^j\-S03H 

0 

A            /^N=N-<S>-S°3H 
>     ||    N-CH2-CH2-/rj\-OH  N ' 

0 

A  synthesis  of  the  starting  material,  N-hydroxyphenylethylmaleimide,  has  been 
devised  and  is  nearly  completed. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
Clinically  important  differences  in  response  to  drugs  of  essentially  similar 
structure  probably  reflect  the  existence  of  different  receptor  subtypes  in 
different  tissues.   Specific  examples  are  the  32_adrenergic  agonists  such  as 
salbutamol  which  selectively  act  on  the  bronchodilatory  mechanisms  in  lung 
and  the  H2-histamine  antagonists  which  act  selectively  on  histamine-dependent 
gastric  acidification.   The  molecular  basis  for  these  distinctions  and  a  more 
rational  basis  for  the  design  of  organ  selective  drugs  would  be  provided  by 
information  on  the  chemical  nature  of  receptors. 

Proposed  Course  of  Project:   Double  isotope  studies  of  the  labeling  of 
adrenergic  receptors  in  rabbit  aorta  and  other  tissues  will  be  carried  out 
using  S35-  and  H3-labeled  reagents.   Reaction  schemes  specific  for  conforma- 
tionally  sensitive  C00H-  groups  will  be  designed. 

Keyword  Descriptors:      Receptors,  norepinephrine,  radioactive  labeling. 

Honors  and  Awards:        None 

Publications:  Titus,  E.O.:   Characterization  of  pharmacological 

receptors.   Naunyn-Schmiedeberg's  Archiv.  Pharma- 
kol. ,  in  press. 


7g/ 


Project  No.  Z01  HL  02515-02  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:  Biochemical  Effects  of  Paraquat  on  the  Lung 

Previous  Serial  Number:    NHLI-105 

Principal  Investigators:   Dr.  Elise  Ann  Brandenburger  Brown 

Dr.  Harriet  M.  Maling 

Other  Investigator:       Mr.  Wilfred  Saul 

Cooperating  Unit:         Dr.  Maling  and  Mr.  Saul  are  with  the  Laboratory  of 

Chemical  Pharmacology,  NHLI 

Project  Description: 

This  project  is  a  study  of  some  biochemical  effects  which  might  be  involved 
in  the  pulmonary  toxicity  of  the  herbicide,  paraquat  (Pq) .   This  year  we 
continued  our  studies  on  the  effects  of  Pq  and  related  bipyridinium  herbi- 
cides on  acetylcholinesterase  (ACE)  of  the  lung.   With  acetylthiocholine 
bromide  as  a  substrate,  as  described  by  Ellman  e_t  al.  (Biochem.  Pharmacol.  7: 
89,  1961),  the  apparent  Ki  for  Pq  in  rat  lung  homogenates  was  2.9  x  10-3  M. 
The  Ki  values  for  diquat  and  morfamquat  were  2.4  x  10-lt  M  and  1.4  x  10-1*  M. 
Since  the  concentration  of  Pq  found  in  rat  lung  under  toxic  conditions  is 
more  than  an  order  of  magnitude  less  than  the  apparent  Ki  value,  it  is  im- 
probable that  inhibition  of  ACE  is  responsible  for  the  pulmonary  edema  induced 
by  this  compound. 

Since  some  ACE  inhibitors  also  block  the  neuromuscular  junction  and  since  Pq 
produces  neuromuscular  weakness  in  rats,  we  observed  the  effects  of  Pq  in 
chicks.   The  i.p.  injection  of  Pq  (15  mg/kg)  produced  extensor  paralysis  and 
death  within  6  hr;  no  obvious  changes  occurred  after  10  mg/kg.   A  subthresh- 
old dose  of  d-tubocurarine  chloride  (1.5  mg/kg)  produced  a  transient  flaccid 
paralysis  when  administered  90  min  after  10  mg/kg  of  Pq.   Agents  that  de- 
polarize the  neuromuscular  junction  cause  an  extensor  paralysis,  and  competi- 
tive blocking  agents  produce  a  flaccid  paralysis  in  birds  (Paton  and  Zaimis, 
Pharmacol.  Rev.  4:   219,  1952).   Our  observations  suggest  that  Pq  can  produce 
a  dual  block  of  neuromuscular  function. 

Many  compounds  have  been  tested  for  protection  against  the  pulmonary  toxicity 
of  Pq  (H.M.  Maling,  unpublished) .   Among  the  few  effective  compounds  were 
beta  adrenergic  receptor  blocking  agents,  especially  dl-propranolol  (Pr) .   In 
order  to  explain  why  Pr  increases  the  LD50  for  Pq  in  rats,  we  examined  the 


7*2 


Project  No.  Z01  HL  02515-02  PB 

effects  of  Pr,  Pq,  and  Pr  +  Pq  on  the  incorporation  of  32P-phosphate  into  the 
phospholipids  of  rat  lung  slices.  A  study  of  phospholipid  synthesis  seemed 
desirable,  since  Pq  has  been  reported  to  lower  the  quantity  of  dipalmitoyl 
phosphatidylcholine,  the  predominant  pulmonary  surfactant  (Fisher  et  al. , 
J.  Appl.  Physiol.  35:   268,  1973).   Furthermore,  the  pulmonary  edema  produced 
by  Pq  could  result  from  alterations  in  membrane  permeability,  which  is  main- 
tained in  part  by  phospholipids.   Rat  lung  slices  were  preincubated  with  Pr 
(5  x  10-1*  M)  ,  Pq  (5  x  lO-1*  M)  ,  or  Pr  +  Pq  for  30  min  before  the  addition  of 
32P-phosphate  and  continued  incubation  for  an  additional  30  min.   The  pattern 
of  incorporation  into  phospholipids  was  altered  by  both  Pr  and  Pq  but  more 
markedly  by  Pr.   The  incorporation  into  phosphatidylglycerol  was  doubled  by 
Pr,  reduced  slightly  by  Pq,  and  increased  about  50%  by  Pr  +  Pq.   Incorporation 
into  phosphatidylcholine  was  reduced  slightly  by  Pq,  about  70%   by  Pr,  and 
about  80%  by  Pr  +  Pq.   In  contrast,  incorporation  into  phosphatidic  acid  was 
increased  about  50%  by  Pq,  fivefold  by  Pr,  and  sixfold  by  Pr  +  Pq.   Incorpora- 
tion into  another  phospholipid,  presumably  cytidine  diphosphate-diglyceride 
(Hauser  and  Eichberg,  J.  Biol.  Chem.  250:   105,  1975),  was  increased  more,  than 
twelvefold  by  Pr  or  Pr  +  Pq  but  was  unaffected  by  Pq  alone.   These  changes  in 
metabolism  may  be  related  to  the  protective  effects  of  treatment  with  Pr. 

A  variety  of  radiolabeled  precursors,  such  as  cytidine,  myoinositol,  and 
sodium  palmitate,  will  be  utilized  in  further  studies  of  the  alterations  in 
pulmonary  phospholipids  produced  by  Pr  and  Pq .   Anenoic  phospholipids  will  be 
determined  because  of  their  probable  relationship  to  pulmonary  surfactant. 


Keyword  Descriptors: 


Paraquat,  diquat,  morfamquat,  propranolol,  lung 
phospholipids,  acetylcholinesterase  of  lung,  phos- 
phatidic acid,  phosphatidylglycerol,  cytidine 
diphosphate-diglyceride,  pulmonary  surfactant, 
phosphatidylcholine 


Honors  and  Awards: 


None 


Publications : 


Maling,  H.M. ,  Eichelbaum,  F.M. ,  Saul,  W.  ,  Sipes, 
I.G.,  Brown,  E.A.B.  and  Gillette,  J.R.:   The  nature 
of  the  protection  against  CCli^  toxicity  produced  by 
pretreatment  with  Dibenamine  (N-(2-chloro-ethyl) 
dibenzylamine) .   Biochem.  Pharmacol.  23:   1479- 
1491,  1974. 


Trams,  E.G.  and  Brown,  E.A.B. :   The  activity  of  2', 
3' -cyclic  adenosine  monophosphate  3'-phosphoester- 
hydrolase  in  elasmob ranch  and  teleost  brain.   Comp. 
Biochem.  Physiol.  48B:   185-189,  1974. 


733 


Project  No.  Z01  HL  02516-01  PB 

1 .  Pulmonary 

2.  Molecular  Pharmacology 

3.  Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


Project  Title:  Information  Retrieval  in  Pharmacology 

Previous  Serial  Number:  None 

Principal  Investigator:  Dr.  Elise  Ann  Brown 

Other  Investigator:  Dr.  Harry  Keiser 

Cooperating  Unit:         Dr.  Keiser  is  Deputy  Chief  of  the  Hypertension- 
Endocrine  Branch  and  Head,  Section  on  Experimental 
Therapeutics,  HE,  NHLI 

Project  Description: 

In  initiating  new  research  projects,  information  must  be  obtained  on  prior 
efforts  in  the  field,  on  the  feasibility  of  available  procedures  and  on  the 
extent  to  which  the  phenomena  of  interest  have  been  satisfactorily  explored. 
Such  assessments  of  the  state  of  the  art  require  a  professional  background 
not  supplied  by  computer  personnel  or  technical  librarians .   This  is  espe- 
cially true  in  clinical  and  pharmacological  studies,  where  judgments  must 
frequently  be  made  concerning  dose,  species,  routes  of  administration  of 
drugs,  etc.   Information  retrieval  and  appraisal  activities  have  therefore 
been  established  in  NHLI. 

Extensive  use  was  made  of  the  MEDLINE  and  TOXLINE  computer  files  on  line  from 
the  National  Library  of  Medicine,  the  personal  files  of  various  investigators 
and  interviews  with  informed  investigators.   I  attended  a  course  on  the  use 
of  TOXLINE  in  order  to  improve  my  facility  with  the  computer  systems. 

Major  inquiries  were  requested  by  ten  investigators  and  minor  inquiries  by 
four  investigators.   All  but  one  were  personnel  of  the  National  Heart  and 
Lung  Institute. 

The  areas  of  biomedical  investigation  which  were  explored  included 

1)  Propranolol,  i.v.  administration  and  half-life  in  man 

2)  Paraquat — mechanism  of  toxicity 

3)  Diquat  toxicity 

4)  Slow  reacting  substance  of  the  lung  (SRS-A) — history,  identity, 
source 

5)  Electron  spin  resonance  studies 


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Project  No.  Z01  HL  02516-01  PB 

6)  Intercalation  of  DNA  and  RNA  with  acridine  compounds 

7)  Adenosine  pharmacodynamics  in  tissue  cell  systems 

8)  Phospholipid  formation  in  the  lung 

a)  formation  of  dipalmitoyl  lecithin 

b)  formation  of  cytidine  diphosphatediglyceride 

c)  formation  of  phosphatidylethanolamine 

d)  effects  of  5-hydroxytryptamine,  dopamine  and  acetylcholine 

e)  changes  with  changes  in  cell  membrane  permeability 

9)  Sulfur  compounds — metabolism  in  the  lung 

10)  Drug-metabolizing  enzymes  in  the  lung 

11)  Effect  of  isoproterenol  on  DNA  synthesis. 

12)  Glutathione  levels  in  the  lung  as  altered  by  drugs 

13)  Mucopolysaccharides  and  protein  binding  sites  in  lung 

14)  Pharmacology  and  adverse  effects  of 

a)  Phenformin 

b)  Ibuprofen 

c)  Primaquine 

d)  Ozone 

e)  Minoxidil 

f)  Pindalol 

15)  Spectral  analysis  of  retinal  pigments 

16)  Use  of  superoxide  dismutase  in  therapy 

17)  Protein  synthesis  in  the  lung 

18)  Antihypertensive  effects  of  SQ  20881 

19)  Biological  effects  of  Pepstatin 

20)  Interaction  of  hexamethonium  and  acetylcholinesterase 

21)  Verbenol  as  an  hypertensive  agent 

22)  Effects  of  conconavalin  A  and  plant  agglutinins  on  cell  membrane 
permeability  and  on  ATPase 

23)  Enzymes  of  histamine  metabolism  in  monkey  lung 

24)  Lipoperoxidation  in  lung 

The  volume  of  medical  literature  has  increased  so  that  individuals  cannot 
handsearch  for  topics  easily.   Rapid  access  to  information  on  the  usage  and/ 
or  toxicology  of  compounds  is  essential  for  the  most  cost  effective  utiliza- 
tion of  biomedical  personnel. 

Collaboration  has  been  actively  sought  with  several  NHLI  laboratories.   I 
have  instructed  several  investigators  in  search  procedures  so  that  they  can 
expedite  their  own  work. 

In  order  to  double  the  output  rate  of  computer  searches,  a  proposal  for  a 
new  cathode  ray  terminal  system  was  submitted  in  January  to  Dr.  H.  Keiser. 

The  proposed  system  will  double  the  rate  for  entering  commands  to  the  MEDLINE 
and  TOXLINE  systems  and  increase  by  8-fold  the  rate  of  interaction  with  DCRT 
computers .   This  should  encourage  more  use  of  the  facility  and  reduce  the 
time  needed  to  obtain  information. 

Keyword  Descriptors:      Information  retrieval,  drug  metabolism,  pharmacology 

2  7sr 


Project  No.  Z01  HL  02516-01  PB 
Honors  and  Awards:        None 

Publications:  Brown,  E.A.B.:   The  localization,  metabolism,  and 

effects  of  drugs  and  toxicants  in  lung.   Drug  Metab. 
Rev.  3:   33-87,  1974. 


ANNUAL  REPORT  OF  THE 

CLINIC  OF  SURGERY 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

July  1,  1974  through  June  30,  1975 

The  clinical  and  laboratory  programs  of  the  Surgery  Branch  have,  as 
in  past  years,  largely  centered  upon  the  study  of  operative  methods  for 
the  correction  of  congenital  and  acquired  heart  and  lung  diseases,  assess- 
ment of  the  results  of  such  operations,  and  laboratory  studies  related  to 
cardiovascular  physiology  and  pharmacology. 

Operative  Treatment  of  Acquired  Valvular  Heart  Disease.  The  first 
successful  prosthetic  replacement  of  the  mitral  valve  was  carried  out  in 
this  Clinic  in  1960.   Since  that  time  the  study  and  treatment  of  patients 
with  acquired  valvular  heart  disease  has  continued  as  one  of  the  principal 
clinical  research  efforts  of  the  Surgery  Branch.  The  majority  of  patients 
who  have. had  mitral  valve  replacement  have  had  insertion  of  a  prosthesis 
with  a  moving  poppet  of  ball  or  disc  configuration.  The  hemodynamic 
function  of  such  artificial  valves  is  generally  satisfactory,  but  their 
use  has  been  associated  with  an  extremely  high  incidence  of  systemic  emboli. 
The  incidence  of  emboli  can  be  reduced  but  not  abolished  by  the  administration 
of  anticoagulants  or  agents  which  decrease  platelet  adhesiveness. 
Laboratory  investigations,  here  and  at  other  centers,  indicated  that  a  valve 
constructed  of  autologous  or  homologous  tissue  would  be  superior  to  a 
mechanical  device.   Experimental  and  clinical  use  of  tissue  valves,  however, 
soon  proved  that  the  attachment  of  the  tissue  to  the  supporting  framework 
was  insecure  and  valve  failure  from  detachment  universally  occurred. 
Studies  in  this  laboratory  proved  that  if  the  supporting  frame  for  the 
tissue  was  made  flexible  rather  than  rigid  that  the  stresses  on  the  attach- 
ments were  greatly  reduced. 

Thus,  in  the  summer  of  1965,  the  first  porcine  xenograft  aortic 
valve  was  inserted  in  the  mitral  position.  That  patient  is  living  and  well. 
Since  that  time  110  additional  patients  have  undergone  mitral  and/or 
tricuspid  valve  replacement  with  the  porcine  xenograft.  Eighty- six  patients 
are  alive,  cumulative  followup  approximates  2000  patient -months,  and  15 
patients  have  been  followed  more  than  4  years.  Anticoagulants  have  not  been 
administered  postoperatively  unless  their  use  was  required  by  a  rigid 
prosthesis  in  the  aortic  position.  One  patient  only  has  had  a  systemic 
embolus  and  this  occurred  in  the  immediate  postoperative  period  presumably 
from  the  Teflon  valve  base.  The  function  of  the  xenograft  has  been  studied 
by  postoperative  cardiac  catheterization  in  44  patients.  Following  mitral 
replacement  the  average  left  atrial  mean  pressure  was  17  mm.  Hg,  the 
average  atrio-ventricular  diastolic  gradient  4  mm.  Hg,  and  the  average 
mitral  valve  area  was  2.3  cm. 2.  After  tricuspid  replacement  the  average 
right  atrial  mean  pressure  was  11  mm.  Hg,  and  the  right  atrio-ventricular 
pressure  gradient  averaged  4  mm.  Hg.  Valve  failure,  with  severe  regurgita- 
tion through  the  xenograft  has  occurred  in  one  patient,  57  months  following 
operation.  The  defective  valve  was  removed  and  dysfunction  proved  to  be 

i  797 


the  result  of  infection  with  a  fungus,  probably  histoplasmosis.  Two  patients 
have  had  bacteremia  and  have  been  successfully  treated  with  antibiotics; 
valve  dysfunction  did  not  result. 

This  clinical  experience  indicates  that  at  this  time  the  porcine 
xenograft,  preserved  in  stabilized  glutaraldehyde  and  mounted  on  a  flexible 
stent,  is  the  prosthesis  of  choice  when  replacement  of  the  mitral  or 
tricuspid  valve  is  necessary.   Such  xenografts  will  not  be  utilized  in  the 
aortic  position  in  this  Clinic  until  evidence  is  available  that  the  valves 
can  withstand  the  additional  mechanical  stress  which  accompanies  implantation 
in  the  aortic  root. 

Operative  Management  Qf  Acquired  Tricuspid  Valve  Disease.   Tricuspid 
regurgitation  commonly  accompanies  acquired  mitral  stenosis  or  regurgitation, 
and  optimal  management  of  the  tricuspid  disease  has  never  been  defined 
precisely.   In  1967  it  was  the  opinion  of  the  surgeons  in  this  Clinic  that 
in  virtually  all  patients  tricuspid  regurgitation  would  regress  spontaneously 
if  the  mitral  disease  was  dealt  with  effectively.   Since  that  time  clinical 
observations  have  indicated  that  that  premise  was  often  incorrect  and  a 
number  of  patients  in  whom  tricuspid  regurgitation  was  not  treated  have 
continued  to  experience  severe  symptoms  of  tricuspid  regurgitation  after 
mitral  valve  replacement.  For  this  reason,  the  operative  management  of 
tricuspid  disease  has  become  more  aggressive,  and  an  increasing  number  of 
patients  have  undergone  either  tricuspid  valve  replacement  or  tricuspid 
annuloplasty . 

During  the  years  1972-1974,  76  patients  with  mitral  and/or  aortic 
valve  disease  were  proved  to  have  tricuspid  regurgitation  or  stenosis  when 
the  tricuspid  valve  was  palpated  at  the  time  of  operation.   In  21  of  the  76 
patients  no  operative,  procedure  on  the  tricuspid  valve  was  carried  out;  in 
30  patients  a  tricuspid  annuloplasty  was  performed;  in  the  remaining  25 
patients  the  tricuspid  valve  was  replaced  with  a  procine  xenograft.  The 
operative  mortality  among  patients  in  whom  the  tricuspid  valve  was  not 
treated  was  107<,;  in  those  having  an  annuloplasty  mortality  was  23%;    and 
the  operative  mortality  was  12%  in  the  patients  among  the  patients  having 
tricuspid  valve  replacement.   Clinical  followup  and  hemodynamic  evaluations 
indicate  that  tricuspid  valve  replacement  gives  a  long-term  result  superior 
to  that  provided  by  tricuspid  annuloplasty.   Evaluation  of  those  patients 
in  whom  tricuspid  regurgitation  was  present  but  was  not  treated  is  difficult, 
since  it  is  likely  that  the  magnitude  of  regurgitation  was  less  in  this 
clinical  subgroup  than  in  those  who  had  annuloplasty  or  valve  replacement. 
It  is  clear,  however,  that  an  aggressive  surgical  approach  is  indicated 
when  the  patient  with  mitral  valve  disease  is  found  to  have  significant 
tricuspid  regurgitation  as  well,  and  is  likely  that  the  operation  of  choice 
is  tricuspid  valve  replacement. 

Intimal  Proliferation  in  Venous  Autografts.  This  year  it  seems 
likely  that  more  than  200,000  patients  will  undergo  operative  treatment  for 
obstructive  coronary  atherosclerosis.  The  operative  procedure  usually 
performed  is  the  insertion  of  multiple  segments  of  autologous  saphaneous 
vein  as  grafts  between  the  ascending  aorta  and  the  coronary  arteries  distal 
to  the  sites  of  principal  obstruction.  The  benefit  provided  by  such  an 

2  m 


operation  is  largely  dependent  upon  patency  of  the  vein  grafts.  The 
application  of  precise  microsurgical  techniques  will  provide  assurance 
of  early  patency,  but  in  a  significant  number  of  patients  the  grafts 
become  occluded  after  6-18  months  by  progressive  proliferation  of  the 
intima  of  the  graft. 

An  experimental  study  was  made  of  the  effects  of  two  pharmacologic 
agents  on  intimal  proliferation  in  venous  autografts.   In  dogs,  36  segments 
of  the  femoral  artery  were  replaced  with  segments  of  autologous  femoral 
vein.  The  animals  were  then  divided  into  three  groups:   no  specific  post- 
operative treatment;  postoperative  administration  of  dipyridamole  (Persantine) ; 
postoperative  treatment  with  methylprednisolone.   Six  weeks  after  operation 
femoral  arteriography  was  carried  out  to  determine  graft  patency,  and  the 
animals  were  then  killed  and  the  grafts  removed  and  studied  histologically. 
Overall,  94%  of  the  grafts  were  patent  and  the  patency  rate  did  not  differ 
among  the  three  groups.  The  thickness  of  the  intima  was  measured  in  the 
proximal,  middle,  and  distal  thirds  of  the  grafts  to  quantify  the  extent 
of  intimal  proliferation.   Intimal  proliferation  was  similar  in  the  control 
and  dipyridamole  treated  groups.  Dogs  which  had  been  treated  with 
methylprednisolone  had  significantly  less  intimal  thickening  in  the  middle 
third  of  the  grafts' than  did  control  animals  (125  microns  vs.  214). 

The  experiment  shows  that  at  six  weeks  the  extent  of  intimal 
proliferation  is  not  influenced  by  dipyridamole  treatment.  While  the 
steroid  effectively  decreased  thickening  in  the  middle  of  the  grafts, 
substantial  proliferation  was  still  evident  near  each  anastomosis.  This 
suggests  that  unknown  factors  relating  to  blood  flow  and  the  technique  of 
anastomosis  may  also  be  of  importance.  Furthermore,  observations  of  this 
nature  carried  out  over  a  period  of  one  year  would  more  closely  approximate 
the  clinical  problem.  The  feasibility  of  such  long-term  studies  is 
presently  under  investigation. 

Experimental  and  Clinical  Studies  of  the  Contribution  of  Atrial 
Contraction  to  Right  Heart  Function  Before  and  After  Right  Ventriculotomy. 
Many  studies  have  been  made  of  the  effects  of  left  atrial  contraction  on 
left  ventricular  function.   Little  attention,  however,  has  been  given  to 
the  contribution  of  right  atrial  systole  to  right  ventricular  function. 
The  studies  outlined  were  stimulated  by  the  observation  that  many  patients 
evidenced  right  heart  failure  when  they  developed  ineffective  atrial 
contraction  after  operations  requiring  right  ventriculotomy  (repair  of 
tetralogy  of  Fallot,  closure  of  ventricular  septal  defect) . 

The  effects  of  atrial  contraction  were  studied  in  open  chest  dogs 
before  and  after  a  vertical  right  ventriculotomy  was  made.   Effective  atrial 
contraction  was  abolished  by  simultaneous  atrial  and  ventricular  (A-V)  pacing. 
Before  ventriculotomy,  and  when  cardiac  output,  aortic  pressure,  and  heart 
rate  were  kept  constant,  the  mean  right  atrial  pressure  rose  only  slightly 
(1.4  mm.  Hg)  when  A-V  pacing  was  instituted.   In  the  same  animals,  however, 
right  atrial  pressure  rose  9.5  mm.  Hg  after  a  right  ventriculotomy  had  been 
made  by  a  technique  which  did  not  require  bypass  or  inflow  occlusion. 
Right  ventricular  failure  with  gross  evidences  of  tricuspid  regurgitation 
was  easily  induced  after  ventriculotomy  by  volume  overload  and  A-V  pacing. 

3  Iff 


With  the  blood  volume  unchanged  restoration  of  effective  atrial  contraction 
by  sequential  A-V  pacing  eliminated  palpable  tricuspid  regurgitation  and 
lowered  the  average  mean  right  atrial  pressure  from  22  to  4  mm.  Hg.  After 
right  ventriculotomy  the  right  atrial  pressure  was  maintained,  constant  by 
adjustments  of  circulating  blood  volume.   In  these  animals  loss  of  atrial 
contraction  was  followed  by  a  42%  reduction  in  cardiac  output. 

Additional  observations  were  made  in  13  patients  who  were  studied 
in  the  immediate  postoperative  period.   Both  atrial  and  ventricular  pacing 
electrodes  had  been  placed  at  operation,  permitting  simultaneous  or 
sequential  pacing  of  the  right  atrium  and  right  ventricle.   In  8  patients 
who  had  had  right  ventriculotomies  abolition  of  atrial  contraction  caused 
an  average  reduction  in  cardiac  output  of  227o  (thermal  dilution  method)  . 
In  five  other  patients  in  whom  the  right  ventricle  had  not  been  incised 
simultaneous  A-V  pacing  caused  a  trivial  fall  in  cardiac  output  of  only 
5%. 

The  therapeutic  implications  of  the  study  are  clear.  The  right 
ventricular  failure,  which  is  commonly  observed  after  repair  of  the  tetralogy 
can  be  ameliorated/ Insuring  that  effective  atrial  contraction  is  maintained. 
This  requires  that  both  atrial  and  ventricular  electrodes  be  placed  at 
operation  so  that  during  periods  of  nodal  rhythm  or  complete  heart  block 
appropriately  timed  atrial  contractions  can  be  supplied  artifically. 


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Project  No.  Z01  HL  02601-01  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Cardiac  valve  replacement  with  the  Hancock  porcine: 
A  five  year  clinical  experience 

Previous  Serial  Number:   None 

Principal  Investigators:   Charles  L.  Mcintosh,  M.D. 

Lawrence  L.  Michael is,  M.D. 
Andrew  G.  Morrow,  M.  D. 

Project  Description:   The  first  clinical  implantation  of  a  glutaraldehyde- 
fixed  porcine  xenograft  mounted  on  a  flexible  stent  was  performed  in  July 
1970  at  the  NHLI.   Since  then,  110  patients  have  undergone  single  or 
multiple  valve  replacement  with  this  prosthesis;  86  patients  are  alive. 
Cumulative  followup  totals  1732  patient  months,  with  10  patients  followed 
more  than  four  years.  Anticoagulants  have  not  been  administered  routinely 
po  stoperatively . 

Results:   Pre-  and  postoperative  hemodynamic  assessments  have  been 
performed  in  44  patients.  Following  mitral  valve  replacement  the  average 
left  atrial  mean  pressure  was  17  mm.  Hg;  the  average  left  atrio- 
ventricular mean  diastolic  gradient  was  4  mm.  Hg;  the  average  calculated 
mitral  valve  area  was  2.3  cm  .  After  tricuspid  valve  replacement  the 
average  right  atrial  mean  pressure  was  11  mm.  Hg,  and  the  right  atrio- 
ventricular mean  pressure  gradient  was  4  mm.  Hg.  Prosthetic  regurgitation 
secondary  to  prosthetic  dysfunction  has  occurred  in  one  patient  57  months 
following  mitral  valve  replacement  with  a  xenograft.  Preliminary  pathological 
examination  revealed  the  valve  to  be  infected  with  a  fungus  organism,  most 
likely  histoplasmosis.   One  patient  has  had  an  arterial  embolus  (10  days 
postoperative,  good  recovery) .  Two  patients  with  documented  bacteremia 
were  successfully  treated  with  antibiotics,  and  did  not  require  replacement 
of  their  xenograft  prosthesis. 

Keywords:  Porcine  Xenograft,  Long-term  followup,  heterograft 

Proposed  Course:   This  study  will  be  presented  at  the  Vascular  Society 
Meeting  in  June  1975. 


79/ 


Project  No.  Z01  HL  02602-01  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Cardiac  patient  data  profile 

Previous  Serial  Number:   None 

Principal  Investigators:   Charles  L.  Mcintosh,  M.D. 

Andrew  G.  Morrow,  M.  D. 

Cooperating  Unit:         Data  Management  Branch,  DCRT 

Project  Description:   Approximately  4000  patients  have  undergone  operation 
for  acquired  and  congenital  heart  disease  in  this  Clinic.   Pre-  and 
postoperative  hemodynamic,  clinical  assessment  and  long-term  followup  has 
generated  valuable  data  concerning  survival,  complications,  and  functional 
improvement  following  operative  intervention.   Individuals  who  have  under- 
gone single  or  multiple  valve  replacement  (n=1000)  are  being  used  as  a  pilot 
cohort  of  patients  in  this  study.   Survival,  functional  class  improvement, 
hemodynamic  results,  specific  incidences  of  emboli,  prosthetic  malfunction, 
infection,  and  reoperation  are  being  analyzed.   Autopsy  findings  or  clinical 
cause  of  death  is  also  being  recorded.   In  addition  to  functioning  as  a 
bookkeeping  device,  the  system  also  provides  a  more  thorough  followup 
system  than  currently  exists.   Whenever  new  data  is  acquired  from  a  hospital 
admission,  outpatient  visit,  or  communication  with  the  patient  or  their 
physician,  the  individuals  file  is  updated.   The  data  is  also  being  trans- 
ferred to  microfiche  for  easy  acquisition  and  storage. 

Results:  The  1000  series  Starr»- Edwards  aortic  valve  patients  (n=122)  have 
been  reviewed  and  entered  into  the  program  and  results  tabulated.   Of  the 
original  122  patients,  29  are  knovnto  be  alive,  74  are  dead  and  19  have  been 
lost  to  followup.   The  living  patients  now  have  a  cumulative  followup  of 
5042  months.   The  incidence  of  leading  complications  is:   1)  thromboembolism 
25%;  2)  ball  variance  23%;  and  3)  anemia  15%.  Myocardial  infarction  and 
arrhythmia  were  the  leading  cause  of  death  in  this  series.   The  1200  (aortic) 
and  6000  (early  mitral)  series  patients  have  been  reviewed  and  entered  into 
the  computer  program,  but  final  analyses  is  not  yet  available.   In  addition 
all  other  valvular  patients  have  been  reviewed  and  currently  are  being 
entered  into  the  program  for  print  out  and  analyses. 

Proposed  Course:   The  utility  of  the  system  is  being  assessed  with  these 
valvular  patients,  and  a  decision  will  be  made  as  to  whether  the  system  will 
be  expanded  to  include  other  operated  groups.   The  Cardiology  Branch  is 
considering  a  similar  program  for  their  unoperated  patients.   If  such  a 
system  is  adopted  a  complete  natural  history  profile,  with  the  operation 
being  the  variable,  will  be  generated  on  each  patient  seen  at  the  NHLI. 

Keywords:  Long-term  valve  followup,  Computer  valve  followup 

7fi 


Project  No.   Z01  HL  02603-01   SU 
1.      Clinic  of  Surgery 
3.      Bethesda,   Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Surgical  management  of  acquired  tricuspid  valve  disease 

Previous  Serial  Number:   None 

Principal  Investigators:   Robert  H.  Breyer,  M.  D. 

James  McClenathan,  M.  D. 

Other  Investigators:      Charles  L.  Mcintosh,  M.  D,  Ph.D. 

Project  Description:   The  management  of  tricuspid  valve  disease  associated 
with  mitral  or  mitral-aortic  disease  continues  to  be  a  controversial 
subject.   In  1967  the  argument  for  conservative  management  was  presented 
by  this  Clinic.   Since  that  time,  our  approach  to  tricuspid  valve  disease 
has  become  more  aggressive  and  an  increasing  number  of  patients  undergo 
tricuspid  valve  replacement  or  annuloplasty . 

A  retrospective  review  was  undertaken  to  compare  the  results  of 
conservative  management  vs.  tricuspid  annuloplasty  vs.  tricuspid  valve 
replacement.   During  the  years  1972-1974  seventy- six  patients  were 
diagnosed  to  have  tricuspid  regurgitation  and/or  stenosis  by  palpation  of 
the  valve  at  the  time  of  operation.  Twenty-one  patients  did  not  undergo 
any  tricuspid  procedure,  30  patients  underwent  tricuspid  annuloplasties, 
25  underwent  tricuspid  valve  replacement  with  porcine  xenografts. 

Results:  Operative  mortality  for  the  three  respective  groups  was  10%, 
23%,  and  127».  By  July  1,  1975  six  month  postoperative  catheterization 
studies  will  be  complete  for  all  patients.  At  that  time  postoperative 
functional  class  and  pre-  and  postoperative  hemodynamics  will  be  analyzed. 

Preliminary  data  indicate  that  an  aggressive  surgical  approach  is 
indicated  in  the  management  of  tricuspid  valve  disease.  Tricuspid  valve 
replacement  is  shown  to  be  superior  to  tricuspid  annuloplasty. 

Proposed  course:   Upon  completion  of  analyses  of  hemodynamic  and  clinical 
evaluation  a  paper  will  be  submitted  for  publication. 

Keywords:   Tricuspid  Valve  Replacement,  Tricuspid  Annuloplasty. 


7?3 


Project  No.  Z01  HL  0260*-02  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  197*+  through  June  30,  1975 

Project  Title:   Phonocardiographic  detection  of  ball  variance 

Previous  Serial  No:   NHLI-I69 

Principal  Investigator:   Lee  C.  Amsler,  M.  S. 

Other  Investigators:   Harry  W.  Seipp 
Robert  Romanoff 
Charles  L.  Mcintosh,  M.  D. 

Cooperating  Unit:   Division  of  Computer  Research  and  Technology,  Computer 
Systems  Laboratory,  NIH 

Project  Description:   Phonocardiograms  from  patients  with  Starr-Edwards 
aortic  prostheses,  series  1000  and  1200,  have  been  analyzed  with  the  aid 
of  a  hybrid  computer  system.   The  following  parameters  are  obtained  from 
the  phonocardiogram  for  evaluation:   l)   a  correlation  coefficient  (Rxy)  for 
the  opening  prosthetic  aortic  valve  sound,  2)  a  prosthetic  aortic  opening 
sound  intensity  to  closing  sound  intensity  ratio  (A0/AC),  and  3)  a  prosthetic 
aortic  opening  sound  energy  to  closing  sound  energy  ratio  within  a  high 
frequencey  window  (Hi-Freq  A0/AC).   A  spectrographic  analysis  was  performed 
on  a  representative  sample  of  our  patient  population  to  evaluate  the  use 
of  this  technique  in  the  detection  of  silastic  ball  variance. 

Results:   Sixty-five  silastic  aortic  valve  patients,  Starr-Edwards  1000  and 
1200  series,  have  been  studied  via  phonocardiography  and  fourteen  patients 
with  recorded  phonocardiograms  have  come  to  reoperation  or  autopsy  with 
findings  of  ball  variance  at  the  NIH  since  1969-   Twelve  of  these  "variant" 
patients  had  indications  of  ball  variance  by  one  or  more  of  the  criteria 
being  utilized.   The  remaining  two  "variant"  patients  were  diagnosed  pre- 
operative^ via  a  percent  stroke-length  determination.   Positive  diagnoses 
of  ball  variance  were  obtained  in  36%   of  the  "variant"  patients  by  the  Rxy 
and  in  72%   of  the  "variant"  patients  by  both  the  A0/AC  ratio  and  Hi-Freq 
A0/AC  ratio.   The  results  from  the  spectral  analysis  evaluation  supported 
the  diagnostic  use  of  prosthetic  valve  frequency  patterns  in  the  detection 
of  ball  variance. 

Proposed  Course:   Phonocardiograms  are  routinely  recorded  on  all  Starr- 
Edwards  aortic  series  valve  patients  and  on  all  Starr-Edwards  6000,  6l00, 
and  6120  series  mitral  valve  patients  in  the  NIH  outpatient  clinic.   The 
functional  status  of  the  prosthetic  valves  in  vivo  are  determined  whenever 
possible  in  conjunction  with  the  continual  evaluation  of  the  diagnostic 
techniques.   Reports  indicate  that  the  spectrographic  analysis  technique  is 
one  of  the  most  reliable  and  comprehensive  methods  for  the  evaluation  of  all 

1  7fY 


Project  No.   Z01  HL  02604-01  SU 
1 .   Clinic  of  Surgery 
3-   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  197*+  through  June  30,  1975 

prosthetic  heart  valves.   Our  preliminary  findings  with  this  technique  are 
very  encouraging.   We  believe  that  a  routine  spectral  analysis  of  phono- 
cardiograms  can  be  a  valuable  adjunct  to  attaining  a  more  complete  under- 
standing of  prosthetic  valve  dysfunction  and  the  techniques  currently 
employed  for  their  detection. 

Keywords:  Phonocardiogram,  prosthetic  dysfunction,  opening-closing  ratio 


w 


Project  No.   Z01  HL  02605-0?  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  19lh   through  June  30,  1975 

Project  Title:   NU-5  atomic  powered  pacemaker  -  experimental  and  clinical 
evaluation 

Previous  Serial  No.   NHLI  *+9  (c) 

Principal  Investigator:   Charles  L.  Mcintosh,  M.  D. 

Other  Investigators:   Peter  Frommer,  M.  D. 

Joseph  E.  Pierce,  D.V.M. 

Cooperating  Units:   Office  of  the  Director  and  Section  on  Lab  Animal 
•  Medicine  &  Surgery,  NHLI 

Project  Description:   Efforts  began  several  years  ago  to  develop  a  cardiac 
pacemaker  energy  source  (Plutonium  238)  suitable  for  human  implantation. 
Since  May  19&9i  sixty-five  atomic  powered  pacemakers  have  been  implanted  in 
dogs  for  in  vivo  testing;  of  these,  30  are  the  currently  used  NU-5  series. 
The  first  human  implant  was  done  April  9,   1973  and  the  second  implant  in 
June  of  1973-   Thirty  dogs  were  implanted  with  NU-5  series  pacemakers 
between  July  and  September  1972. 

Results:   One  pacemaker  in  the  30  animals  implanted  with  the  NU-5  series 
pacemakers  has  intermittently  failed  and  was  removed  for  examination  by 
Arco.   A  second  long-term  implant  animal  died  suddenly,  but  no  malfunction 
of  the  pacemaker  was  found.   The  remaining  twenty-eight  units  are  function- 
ing well  at  this  time.   The  first  unit  implanted  in  a  human  continues  to 
function  well.   The  second  patient   required  two  additional  units  because  of 
persistent  infection  of  the  pacemaker  sites.   A  third  human  implant  was 
performed  November  5>  197^-   The  three  clinically  implanted  NU-5  series 
pacemaker  continues  to  function  well  at  this  time. 

Proposed  Course:   Animal  evaluation  will  continue  for  an  indefinite  time 
period  with  those  now  implanted.   Human  implants  will  be  evaluated  over  the 
next  decade. 

Keywords:   Plutonium  238  pacemaker,  long-life  pacemaker 


1W 


Project  No.   Z01  HL  02606-06  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 
July  1,  197^  through  June  30,  1975 

Project  Title:   Silastic  hall  variance  detection 

Previous  Serial  Number:   NHLI-52  (c) 

Principal  Investigator:   Charles  L.  Mcintosh,  M.  D. 

Other  Investigators:   William  Schuette,.  B.  S.,  E.  E. 
Andrew  G.  Morrow,  M.  D. 

Cooperating  Unit:   Biomedical  Engineering  &  Instrumentation  Branch,  NIH 

Project  Description:   The  technique  for  determining  percent  stroke-length 
of  the  poppet  of  a 'prosthetic  valve  ( Starr-Edwards)  was  developed  in  1970 
at  the  NIH.   Other  techniques  for  diagnosing  ball  varinace  have  a  low 
yield  (approximately  25%)    for  detecting  this  possible  fatal  complication. 
The  limiting  factor  in  utilizing  our  technique  is  the  ability  to  visualize 
the  barium  impregnated  ball  on  cine. 

Results:   Fourteen  patients  have  been  studied  and  operated  upon  at  the  NIH 
for  aortic  ball  variance  since  1970.   Twelve  of  these  patients  had  other 
criteria  of  ball  variance,  but  two  patients  were  thought  "normal"  by 
existing  criteria.   The  first  patient  required  operation  for  a  diseased 
mitral  valve,  and  inspection  revealed  obvious  ball  variance  of  the  aortic 
ball.   The  second  patient  was  operated  on  for  aortic  ball  variance  determined 
by  this  technique  and  the  poppet  was  found  to  be  variant  at  the  time  of 
operation. 

Proposed  Course:   This  technique  and  the  phonocardiographic  analysis  of  all 
patients  with  silicone  rubber  poppets  are  being  routinely  screened  for 
possible  valve  dysfunction  or  ball  variance  in  our  clinic.   A  cine  camera  is 
being  installed  in  the  Department  of  Radiology  so  that  this  study  may  be 
performed  on  routine  outpatient  visits.   Plans  are  being  made  with 
Dr.  Benedict  Kinglsey  of  Hahnemann  Medical  College  to  organize  a  conference 
dealing  with  diagnosis  of  valve  dysfunction  to  be  held  in  the  Spring  of  1976. 

Keywords:  Ball  variance,  noninvasive  technique,  prosthetic  dysfunction 


ft  7 


Project  No.   Z01  HL  026CT-01  SU 
1.   Clinic  of  Surgery 
3-   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  197^  through  June  30,  1975 

Project  Title:   Topical  hypothermia  system 

Previous  Serial  Number:   None 

Principal  Investigator:   Charles  L.  Mcintosh,  M.  D. 

Cooperating  Unit:   Biomedical  Engineering  &  Instrumentation  Branch 

Project  Description:   Topical  hypothermia  utilizing  "1+°"  c  iced  saline 
has  become  an  important  adjuvant  in  myocardial  preservation  during  open 
heart  operations.   The  major  disadvantages  of  iced  saline  are:   l)  dilution 
of  pump  prime  with  saline;  2)  difficulty  maintaining  desired  h°   C  temperature; 
3)  need  for  continuous  suctioning  of  saline  and;  h)   cost  of  large  volumes 
of  sterile  saline. 

In  an  attempt  to  optimize  the  topical  hypothermia  concept  and  eliminate 
some  of  the  above  disadvantages,  cooling  coils  of  two  sizes  have  been 
constructed.   Each  side  of  the  coil  is  covered  with  a  teflon  shield  to 
prevent  direct  myocardial  contact.   An  ARTE-8  Nes  labs  refrigeration  unit 
is  used  to  cool  and  circulate  30%  alcohol  solution  through  the  coil.   The 
coil  is  placed  in  the  posterior  pericardial  sack  and  enough  sterile  saline 
is  added  to  cover  the  ventricles.   The  temperature  of  the  saline  is  then 
controlled  by  a  thermocouple  sevomechanism  to  maintain  the  desired  ^°  C 
temperature. 

A  series  of  dogs  being  used  for  a  hypothermia  protection  study  have 
provided  us  with  the  experimental  model  to  evaluate  this  system.   Myocardial 
thermocouple  probes  have  been  used  to  compare  myocardial  temperature 
achieved  with  the  conventional  vs.  this  cooling  system. 

Results:   Preliminary  results  show  that  we  are  able  to  obtain  comparable 
myocardial  cooling  with  both  systems;  but  have  eliminated  the  disadvantages 
of  running  iced  saline. 

Proposed  Course:   Further  animal  studies  are  being  completed  and  plans  are 
being  made  to  utilize  this  system  for  topical  hypothermia  protection  during 
cardiac  operations.   A  paper  will  be  submitted  for  publication  when  the 
system  has  been  evaluated  clinically. 

Keywords:   Topical  hypothermia,  myocardial  preservation,  cooling  device 


79£ 


Project  No.   Z01  HL  02608-01   SU 
1.      Clinic   of  Surgery 
3.      Bethesda,   Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Symposium  on  Intraoperative  Protection  of  the  Myocardium 

Previous  Serial  Number:   None 

Principal  Investigators:   Lawrence  L.  Michaelis,  M.D. 

Victor  J.  Ferrans,  M.D. 
Robert  A.  Guy ton,  M.D. 
Paul  -R.  Hickey,  M.D. 
Bruce  A.  Reitz,  M.D. 
William  R.  Brody,  M.D. 

Other  Investigators:      Charles  L.  Mcintosh,  M.D. 

Andrew  G.  Morrow,  M.D. 

Cooperating  Unit:   Section  on  Pathology,  NHLI 

Project  Description:   On  June  11  and  12,  1974,  the  Clinic  of  Surgery  of  the 
NHLI  hosted  a  symposium  on  Intraoperative  Protection  of  the  Myocardium. 
The  symposium  was  sponsored  and  funded  by  the  John  E.  Fogarty  International 
Center  for  Advanced  Study  in  the  Health  Sciences  and  participants  included 
the  intramural  investigators  listed  above  as  well  as  numerous  scientists 
from  outside  the  NIH. 

The  symposium  was  organized  as  a  forum  for  surgical  investigators  to  become 
familiar  with  each  others  work  and  to  discuss  their  goals  and  techniques 
with  investigators  of  different  disciplines  who  are  interested  in  this 
general  area.   Participants  representing  the  fields  of  physiology,  pharma- 
cology, biochemistry,  and  pathology  were  included  in  the  symposium. 

Results  and  Proposed  Course:  The  proceedings  of  the  symposium  have  been 
transcribed  and  edited  and  will  be  published  in  a  special  edition  of  The 
Annals  of  Thoracic  Surgery  in  July,  1975. 

Keywords:  Myocardial  Protection,  Cardiopulmonary  Bypass,  Cardiovascular 
Surgery 


w 


Project  No.  Z 01  HL  02609-02  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  effect  of  dipyridamole  and  methylprednisolone  on 
intimal  proliferation  in  venous  autografts  used  for 
arterial  bypass 

Previous  Serial  Number:   NHLI-191 

Principal  Investigators:   William  R.  Brody,  M.D. 

John  W.  Brown,  M.D. 
Bruce  A.  Reitz,  M.D. 
Paul  R.  Hickey,  M.D. 

Other  Investigators:      Donald  L.  Fry,  M.D. 

Lawrence  L.  Michaelis,  M.D. 

Cooperating  Unit:   Section  on  Experimental  Atherosclerosis,  NHLI 

Project  Description:   Eighteen  dogs  underwent  36  femoral  artery  bypass 
procedures  using  autologous  femoral  vein  and  were  placed  into  three 
groups;  no  treatment;  postoperative  treatment  with  dipyridamole;  and 
postoperative  treatment  with  methylprednisolone.   After  six  weeks,  femoral 
angiography  was  performed  to  assess  graft  patency,  the  animals  were  killed, 
and  the  grafts  removed  and  prepared  for  histologic  examination. 

Results:   Overall  graft  patency  was  94  percent,  with  no  significant  dif- 
ferences among  groups.   Mean  intimal  thickness  measurements  were  taken  in 
the  proximal,  middle,  and  distal  tissues  of  the  grafts  to  quantitate 
intimal  proliferation.   The  differences  between  control  and  dipyridamole 
treated  groups  were  not  significant.   Dogs  treated  with  methylprednisolone 
had  significantly  less  intimal  thickening  in  the  middle  third  than  controls 
(125  microns  vs.  214). 

These  data  show  no  detectable  response  of  intimal  proliferation  to 
dipyridamole  therapy.   While  corticosteroids  effectively  decreased 
thickening  in  the  middle  of  the  vein  grafts,  substantial  thickening 
remained  near  the  anastomotic  sites,  suggesting  that  factors  relating  to 
blood  flow  and  operative  technique  are  of  importance. 

Proposed  Course:   These  results  have  been  submitted  in  abstract  form  for 
presentation  to  the  Surgical  Forum  of  the  American  College  of  Surgeons. 

Keywords:   Intimal  Proliferation,  Coronary  Bypass  Grafts,  Dipyridamole, 
Methylprednisolone 


Boo 


Project  No.  Z01  HL  02610-02  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Protection  of  the  myocardium  during  anoxic  arrest 

Previous  Serial  Number:   NHLI-189 

Principal  Investigators:   William  R.  Brody,  M.D. 

Bruce  A.  Reitz,  M.D. 
Paul  R.  Hickey,  M.D. 
Michael  J.  Andrews,  M.D. 
Lawrence  L.  Michaelis,  M.D. 

Other  Investigator:       William  C.  Roberts,  M.D. 

Cooperating  Unit:   Section  on  Pathology,  NHLI 

Project  Description:   Numerous  studies  have  shown  acute  alterations  in  left 
ventricular  function  and  morphology  after  cardiopulmonary  bypass  (CPB), 
but  long-term  changes  occurring  in  chronic  survivors  have  not  been  documented, 
This  study  assesses  the  long-term  effects  of  CPB  in  combination  with  popular 
methods  of  myocardial  protection. 

Thirty-seven  dogs  were  placed  on  CPB  for  100  minutes  using  a  bubble 
oxygenator  without  hemodilution,  maintaining  mean  aortic  pressure  at 
80  mm.  Hg.   The  left  ventricle  was  vented.   The  animals  were  divided  into 
three  groups : 

I.  Normothermic  anoxic  arrest  for  60  minutes  (aortic  occlusion). 

II.   Continuous  coronary  perfusion  with  an  empty,  beating  heart  for 
60  min.  at  35°  C. 

III.  Hypothermic  anoxic  arrest  (aortic  occlusion)  for  60  min.  with 
topical  cold  saline  lavage  (4°  C). 

Survival  in  Groups  II  and  III  was  significantly  better  than  Group  I 
(82%  and  92%  versus  45%).  The  22  survivors  of  all  groups  underwent  left 
heart  catheterization  and  LV  cineangiography  five  months  later,  and  were 
compared  to  a  control  group  of  eight  normal  dogs . 

Results:   All  three  groups  had  significant  elevation  of  LVEDP  (p<_.01), 
depression  of  maximum  dp/dt  (p  4.   .05),  and  signs  of  LV  dysfunction  on  angi- 
ography when  compared  to  the  normal  dogs.   Significant  differences  in 
function  between  Groups  II  and  III  were  not  observed.   Gross  subendocardial 
fibrosis  was  present  in  the  hearts  of  all  survivors.   The  infusion  of 
solutions  similar  to  extracellular  fluid  (Ringers  solution),  or  intracellular 


0Ot 


Project  No.  Z01  HL  02610-02  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 


fluid  (Sacks  solution)  during  anoxic  arrest  in  Groups  I  and  III  did  not 
affect  survival  or  chronic  function. 

These  data  indicate  that  although  survival  is  greatly  enhanced  when 
coronary  artery  perfusion  or  topical  hypothermia  is  used,  neither  method 
prevents  chronic  deterioration  in  ventricular  function  nor  the  development 
of  subendocardial  fibrosis. 

Proposed  Course:   This  study  will  be  presented  at  the  first  meeting  of  the 
Samson  Thoracic  Surgical  Society  in  May,  1975  and  will  be  published  in  the 
Journal  of  Thoracic  and  Cardiovascular  Surgery.   This  paper  has  been 
awarded  the  Prize  Manuscript  in  the  Intern/Resident  Division  of  the  Samson 
Thoracic  Surgical  Society. 

Keywords:   Cardiopulmonary  Bypass,  Myocardial  Protection,  Subendocardial 
Fibrosis 


fai. 


Project  No.  SOI  HL  02611-01   SU 
1.      Clinic   of  Surgery 
3.      Bethesda,   Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   The  contribution  of  atrial  contraction  to  right  heart 
function  before  and  after  right  ventriculotomy: 
Experimental  and  clinical  observations 

Previous  Serial  Number:   None 

Principal  Investigators:   Robert  A.  Guyton,  M.D. 

Michael  J.  Andrews,  M.D. 
Paul  R.  Hickey,  M.D. 

Other  Investigators:      Lawrence  L.  Michaelis,  M.D. 

Andrew  G.  Morrow,  M.D. 

Project  Description:   The  effects  of  loss  of  effective  atrial  contraction 
and  of  right  ventriculotomy  upon  right  heart  function  were  studied  in  a 
hemodynamically  controlled  canine  preparation.  A  clinical  study  was  then 
undertaken  of  the  effects  of  loss  of  effective  atrial  contraction  in  two 
groups  of  postoperative  patients,  one  group  with  right  ventriculotomy  and  a 
control  group  without  ventriculotomy. 

Results:   In  open-chest  dogs,  loss  of  effective  atrial  contraction  had 
little  effect  upon  right  heart  function.   Similarly,  ventriculotomy  alone 
had  little  deleterious  effect,  but  loss  of  atrial  contraction  after  ven- 
triculotomy caused  a  marked  elevation  in  right  atrial  pressure  (9.5  mg.  Hg, 
p<.01)  at  a  constant  cardiac  output,  aortic  pressure  and  heart  rate. 
Restoration  of  atrial  contraction  led  to  a  dramatic  reversal  of  volume- 
induced  right  heart  failure  (right  atrial  pressure  of  22  mm.  Hg  decreased 
to  4  mm.  Hg,  p<_.001)  after  ventriculotomy. 

In  eight  patients  who  had  had  right  ventriculotomies,  abolition  of 
effective  atrial  contraction  caused  an  average  reduction  in  cardiac  output 
of  22%,  whereas  cardiac  output  fell  only  5%   in  five  control  patients  (p<  .01) 

Either  ventriculotomy  or  loss  of  effective  atrial  contraction  is  well 
tolerated  by  the  right  heart,  but  the  combination  of  ventriculotomy  and 
loss  of  atrial  contraction  may  cause  clinically  significant  impairment  of 
right  heart  function. 

Proposed  Course:   This  study  will  be  presented  at  the  Annual  Meeting  of  the 
American  Association  for  Thoracic  Surgery  in  April  1975,  and  will  be  sub- 
mitted for  publication  to  the  Journal  of  Thoracic  and  Cardiovascular  Surgery. 

Keywords:   Atrial  Contraction,  Right  Heart  Function,  Ventriculotomy 


go3 


Project  No.  Z01  HL  02612-01  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   A  mechanical  device  for  sutureless  aortosaphenous  vein 
anastomosis 

Previous  Serial  Number:   None 

Principal  Investigators:   Robert  A.  Guyton,  M.D. 

Michael  J.  Andrews,  M.D. 
James  H.  McCIenathan,  M.D. 

Other  Investigator:       Lawrence  L.  Michaelis,  M.D. 

Project  Description:   Previously  developed  techniques  for  sutureless  vascular 
anastomosis  have  required  eversion  of  both  the  distal  and  proximal  vessel. 
Since  the  human  aorta,  particularly  when  it  is  pathologically  thickened,  is 
not  easily  everted,  a  mechanical  device  has  been  developed  for  sutureless 
anastomosis  of  a  vein  segment  to  a  non-everted  aorta.   This  device  was  used 
in  twenty  dogs  for  chronic  interposition  of  a  reversed  autologous  vein 
segment  between  the  aorta  and  the  distal  portion  of  the  divided  left  sub- 
clavian artery. 

Results:   Angiographic  examination  two  to  three  weeks  after  operation 
revealed  that  17  of  20  grafts  were  patent.   Four  of  these  17  animals  were 
killed  at  one  month  after  operation  and  four  were  killed  three  months  after 
operation.   All  eight  autopsy  specimens  revealed  widely  patent  anastomosis 
with  an  intact  endothelial  covering  over  the  mechanical  device.   Corrosion 
of  portions  of  the  stainless  steel  device  was  evident. 

Proposed  Course:   Data  from  this  study  will  be  compiled  and  presented  for 
publication  to  report  the  feasibility  of  this  type  of  anastomosis.   The 
surviving  animals  will  be  killed  one  year  after  operation  for  pathologic 
study. 

Keywords:   Sutureless  Anastomosis 


3o^ 


Project  No.  Z 01  HL  02613-01  SU 
1.  Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Subendocardial  ischemia  during  partial  coronary  occlusion 
in  dogs:   The  significance  of  S-T  segment  elevation  in 
subendocardial  electrograms 

Previous  Serial  Numbers:  None 

Principal  Investigators:   Robert  A.  Guyton,  M.D. 

Michael  J.  Andrews,  M.D. 
James  H.  McClenathan,  M.D. 
Glenn  E.  Newman,  M.D. 

Other  Investigators:      Victor  J.  Ferrans,  M.D. 

Lawrence  L.  Michaelis,  M.D. 

Cooperating  Unit:   Section  of  Pathology,  NHLI 

Project  Description:   Partial  coronary  occlusion  was  evaluated  in  open 
chest  animals  by  recording  epicardial  and  endocardial  electrograms  from 
the  area  of  distribution  of  the  occluded  artery.   Radioactive  micro- 
sphere measurements  of  regional  coronary  flow  were  made  before,  during, 
and  two  to  four  minutes  after  partial  coronary  occlusion.   Finally,  animals 
subjected  to  three  hours  of  partial  coronary  occlusion  were  killed  two 
weeks  later  for  pathological  examination. 

Results:   As  partial  coronary  occlusion  is  gradually  increased,  endocardial 
S-T  segment  elevation  occurs  at  a  higher  distal  coronary  pressure  than  does 
epicardial  S-T  segment  elevation.   Coronary  occlusion  was  adjusted  to  effect 
endocardial  without  epicardial  S-T  elevation  at  a  distal  coronary  pressure 
of  40-50  mm.  Hg.  Microsphere  measurements  of  regional  coronary  flow  in 
this  situation  revealed  no  change  in  epicardial  flow  (.90  to  .99  cc/min/gm) 
and  a  76%  reduction  in  endocardial  flow  (1.00  to  .24  cc/rain/gm,  p<.001). 
Measurement  of  regional  myocardial  flow  at  2  to  4  minutes  after  release  of 
the  occlusion  demonstrated  no  change  from  pre-occlusion  levels  in  epi- 
cardial flow  (.97  to  1.02  cc/min/gm)  and  reactive  hyperemia  in  the  sub- 
endocardial layer  (1.02  to  2.80  cc/min/gm,  p<.01).   Endocardial  without 
epicardial  S-T  segment  elevation  was  maintained  for  three  hours  in  a 
second  group  of  dogs.  A  full -thickness  myocardial  biopsy  was  taken  for 
electron  microscopic  examination  at  the  end  of  the  three  hour  period. 
Results  of  electron  microscopy  are  pending.   Pathologic  examination  of 
these  animals  two  weeks  after  operation  revealed  focal  subendocardial 
infarction  in  all  animals  without  full-thickness  infarction.   It  is  con- 
cluded that  endocardial  S-T  segment  elevation  is  associated  with  subendo- 
cardial ischemia  and  infarction  and  that  epicardial  S-T  segment  elevation 
may  be  absent  during  subendocardial  ischemia  which  is  sufficiently  severe 
to  cause  infarction. 

.    !  00? 


Project  No.  z 01  HL  02613-01  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Proposed  Course:   After  histologic  evaluation  is  completed,  this  study 
will  be  submitted  for  publication. 

Keywords:   Subendocardial  Ischemia,  Endocardial  Electrograms,  Regional 
Coronary  Flow,  Partial  Coronary  Occlusion 


8c4 


Project  No.   ?01  HL  02614-01    SU 
1.      Clinic   of  Surgery 
3.      Bethesda,   Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Subepicardial  and  subendocardial  ischemia  following 
coronary  occlusion 

Previous  Serial  Number:  NHLI-193 

Principal  Investigators:   Robert  A.  Guyton,  M.D. 

Michael  J.  Andrews,  M.D. 

Other  Investigator:       Lawrence  L.  Michaelis,  M.D. 

Project  Description:   Epicardial  and  endocardial  electrodes  have  been 
used  to  record  S-T  segment  elevation  in  the  area  of  an  acute  myocardial- 
infarction  in  open  chest  dogs,  with  careful  control  of  hemodynamic 
variables.   Five  minute  occlusions  of  the  distal  left  anterior  descending 
coronary  artery  under  constant  hemodynamic  conditions  caused  reproducible 
epicardial  and  endocardial  S-T  segment  elevation. 

Results:   The  endocardial  and  epicardial  S-T  segment  elevation  caused  by 
a  five  minute  coronary  occlusion  is  increased  by  increasing  cardiac  output, 
increasing  heart  rate  or  decreasing  aortic  pressure  when  other  hemodynamic 
variables  remain  constant  (p  <  .05).   Elevation  of  aortic  pressure  caused  a 
greater  decrease  in  epicardial  S-T  elevation  than  in  endocardial  S-T 
elevation  (p  <.  .05)  while  changes  in  cardiac  output  or  heart  rate  caused 
alterations  in  epicardial  S-T  elevation  which  were  quantitatively  similar 
to  changes  in  endocardial  elevation.   Nitroglycerin  infusion  in  these 
animals,  at  a  constant  heart  rate,  aortic  pressure,  and  cardiac  output 
caused  no  change  in  endocardial  or  epicardial  S-T  segment  elevation  with  a 
five  minute  coronary  occlusion. 

Proposed  Course:   Preliminary  results  of  this  study  were  presented  in  a 
Symposium  on  Intraoperative  Protection  of  the  Myocardium  to  be  published 
in  the  July  issue  of  Annals  of  Thoracic  Surgery.   Final  results  will  be 
compiled  and  submitted  for  publication. 

Keywords:   Coronary  Artery  Occlusion,  Subendocardial  Ischemia,  Endocardial 
and  Epicardial  Electrograms 


ec7 


Project  No.    Z01  HL   02615-01    SU 
1.      Clinic  of  Surgery 
3.      Bethesda,   Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Alterations  in  regional  ischemia  following  partial  coronary 
occlusion:  The  effects  of  changes  in  blood  pressure,  heart 
rate,  and  cardiac  output 

Previous  Serial  Number:   None 

Principal  Investigator:   Robert  A.  Guyton,  M.D. 

Other  Investigators:      Lawrence  L.  Michaelis,  M.D. 

Project  Description:   In  an  open  chest  canine  preparation,  endocardial  and 
epicardial  S-T  segment  elevations  were  recorded  as  circumflex  coronary 
pressure  was  decreased  (in  steps  of  10  mm.  Hg)  at  a  constant  aortic  pressure, 
heart  rate  and  cardiac  output.   Recordings  were  then  repeated  at  a  different 
aortic  pressure,  heart  rate,  or  cardiac  output. 

Results:   As  circumflex  coronary  pressure  is  decreased  endocardial  S-T 
segment  elevation  gradually  increases  as  coronary  pressure  is  decreased 
from  70  to  50  mm.  Hg  and  generally  fails  to  increase  further.   Epicardial 
S-T  segments,  however,  are  depressed  as  coronary  pressure  is  decreased 
from  70  to  50  and  then  rise  rapidly  as  coronary  pressure  is  decreased 
further.   Preliminary  results  indicate  that  increases  in  aortic  pressure, 
heart  rate,  or  cardiac  output  tend  to  increase  the  level  of  endocardial 
S-T  segment  elevation  present  at  any  circumflex  coronary  artery  pressure 
and  tend  to  increase  the  pressure  at  which  epicardial  S-T  segment  elevation 
occurs . 

Proposed  Course:   Additional  experiments  are  necessary  to  provide  sufficient 
data  for  statistical  analysis.   Further  studies  may  be  conducted  upon  the 
effects  of  nitroglycerin  during  partial  coronary  occlusion  with  constant 
aortic  pressure,  heart  rate,  and  cardiac  output. 

Keywords:   Partial  Coronary  Occlusion,  Regional  Coronary  Ischemia,  Blood 
Pressure,  Heart  Rate,  Cardiac  Output 


sm 


Project  No.  Z01  HL  02616-01  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Temporal  changes  in  regional  coronary  flow  after  partial 
coronary  occlusion 

Previous  Serial  Number:  None 

Principal  Investigators:   Robert  A.  Guyton,  M.D. 

James  H.  McClenathan,  M.D. 

Other  Investigators:      Lawrence  L.  Michaelis,  M.D. 

Cooperating  Units:   Radiation  Safety  Office 

Project  Description:   Regional  myocardial  blood  flow  was  measured  using 
radioactive  microspheres  during  partial  coronary  occlusion  in  dogs.  Measure- 
ments were  made  before,  during,  and  after  a  variable  occlusion  which  main- 
tained a  constant  distal  coronary  pressure  of  40  to  45  mm.  Hg.  A  second 
group  of  dogs  was  studied  at  5,  30,  90,  and  180  minutes  after  an  occlusion 
which  maintained  a  constant  proximal  circumflex  coronary  artery  resistance 
for  three  hours . 

Results:   In  five  dogs  at  a  constant  distal  circumflex  coronary  artery 
pressure,  regional  coronary  flow  was  unchanged  over  a  three  hour  period 
in  the  unoccluded  area  (.76  to  .71  cc/min/gm)  and  in  the  epicardial  layer 
of  the  occluded  area  (.56  to  .48  cc/min/gm)  but  fell  by  more  than  50%  in 
the  endocardial  layer  of  the  occluded  area  (.22  to  .09  cc/min/gm,  p4».02). 
Resistance  to  coronary  flow  increased  in  the  ischemic  endocardial  layer 
by  more  than  100%,  suggesting  that  ischemia  may  be  self -propagating.   Release 
of  the  partial  occlusion  led  to  full  restoration  of  flow  in  all  layers  of 
the  occluded  area. 

A  second  group  of  twelve  dogs  was  studied  during  a  three  hour  occlusion 
simulating  a  constant  proximal  coronary  resistance.   Distal  coronary  pressure 
and  flow  to  the  occluded  area  increased  in  all  12  dogs  during  the  first  30 
minutes  of  occlusion,  but  changes  at  90  and  180  minutes  varied  from  dog  to 
dog.   In  seven  animals  in  which  maximum  flow  to  the  endocardial  layer  of 
the  occluded  area  was  less  than  0.7  cc/min/gm,  changes  in  distal  coronary 
pressure  and  in  flow  to  the  occluded  area  were  linearly  related  (r  =  .81, 
p<.001  and  r  =  .80,  p  <  .  001.)  to  directionally  opposite  changes  in  flow  to 
the  nonoccluded  area.   This  result  suggests  that  a  coronary  steal  phenomenon 
may  be  operative  in  these  animals.   In  the  five  animals  with  maximum  endo- 
cardial flow  greater  than  0.7  cc/gm/min,  no  relationship  between  flow  to  the 
occluded  area  and  flow  to  the  nonoccluded  area  was  observed.   In  all  12  dogs, 
flow  to  the  endocardial  layer  of  the  occluded  area  was  directly  proportional 
to  distal  coronary  pressure  (r  =  .93,  p c   .001),  but  epicardial  flow  tended 


8o9 


Project  No.  Z01  HL  02616-01  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md . 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

not  to  decrease  until  coronary  pressure  fell  below  about  40  ram.  Hg. 

Results:   This  study  has  identified  two  positive  feedback  systems,  or 
"vicious  cycles",  which  may  be  important  in  the  temporal  evolution  of 
myocardial  infarction:   1)  A  time-related  increase  in  coronary  resistance 
in  ischemic  areas  and  2)  decrease  in  flow  to  an  ischemic  area  as  flow  to 
adjacent  nonischemic  areas  is  increased. 

Proposed  Course:   This  study  is  complete  and  a  manuscript  is  in  preparation, 

Keywords:   Regional  Coronary  Flow,  Partial  Coronary  Occlusion,  Coronary, 
Resistance,  Subendocardial  Ischemia 


Slo 


Project  No.  Z01  HL  02617-01  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Extended  storage  of  the  canine  heart  for  transplantation 

Previous  Serial  Number:   None 

Principal  Investigators:   Donald  C.  Watson,  M.D. 

David  L.  Gregg,  M.D. 

Other  Investigator:       Charles  L.  Mcintosh,  M.D.,  Ph.D. 

Project  description:   Canine  hearts  are  removed  from  donor  dogs  and  perfused 
for  48  hours  in  a  specially  constructed  chamber  with  a  solution  which  is 
free  of  calcium  and  saline  and  rich  in  potassium,  glucose,  and  oxygen.  The 
perfusion  pressure  is  constant  at  18  mm.  Hg.   Coronary  flow,  fluid  pH  and 
electrolyte  concentrations  are  monitored.  At  48  hours  of  perfusion  graft 
viability  is  tested  by  orthotopic  homotransplantation  into  a  suitable 
recipient. 

Results:   Most  of  the  hearts  of  recent  experiments  have  been  able  to  maintain 
the  circulation  of  the  recipient  for  4-72  hours  without  inotropic  support. 
The  graft  viability  has  been  directly  related  to  the  amount  of  coronary 
flow  during  the  perfusion.  The  determinants  of  coronary  flow  are 
established  at  the  initial  arrest  and  are  currently  under  investigation. 

Proposed  Course:   Various  methods  of  elective  cardioplegia  are  being  tested 
and  their  effects  on  coronary  resistance  measured.   The  effects  of  coronary 
vasodilators  are  also  being  ascertained.  Once  low  coronary  resistance 
has  been  established,  cardiac  preservation  for  periods  longer  than  48  hours 
will  be  attempted. 

Keywords:   Cardiac  Transplantation,  Myocardial  preservation 


ett 


Project  No.  Z01  HL  02618-01  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Anatomic  correction  of  transposition  of  the  great  vessels 

Previous  Serial  Number:   None 

Principal  Investigator:   David  L.  Gregg,  M.D. 

Other  Investigator:      Charles  L.  Mcintosh,  M.D.,  Ph.D. 

Project  Description:   Under  total  cardiopulmonary  bypass,  a  model  of 
transposition  of  the  great  vessels  is  made  in  dogs  by  the  construction  of 
an  intra-atrial  baffle.   The  transposition  complex  is  then  corrected  at  the 
great  vessel  level  by  constructing  a  truncus  arteriosus  utilizing  the 
proximal  segments  of  the  aorta  and  pulmonary  artery  and  distal  aorta.   The 
pulmonary  blood  flow  is  then  established  between  the  left  ventricle  and 
distal  pulmonary  artery  by  means  of  an  extra  cardiac  shunt. 

Results:   Of  15  dogs,  only  one  has  been  successfully  separated  from  cardio- 
pulmonary bypass;   the  remaining  dogs  dying  from  acute  right  ventricular 
failure. 

Proposed  Course:   Currently  a  population  of  puppies  who  have  undergone 
pulmonary  artery  banding  are  growing  into  adult  size.   At  that  time  further 
attempts  will  be  made  to  establish  this  correction  in  the  hope  that  the 
developed  right  ventricular  hypertrophy  will  enable  the  right  ventricle  to 
sustain  systemic  work. 

Keywords:   Transposition  of  Great  Vessels,  Operative  Procedure 


etx 


Project  No.  Z01  HL  02619-01  SU 
1.   Clinic  of  Surgery 
3.   Bethesda,  Md. 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Study  of  the  rate  of  development  and  severity  of  arterial 
atheromas  in  high  and  normal  flow  states  of  hyper- 
cholesterolemic  animals 

Principal  Investigators:   Michael  S.  Cann,  M.D. 

Robert  J.  Breyer,  M.D. 

Other  Investigators:      Charles  L.  Mcintosh,  M.D.,  Ph.D. 

Donald  Fry,  M.D. 
Donald  Watson,  M.D. 

Cooperating  Unit:   Section  of  Experimental  Atherosclerosis,  NHLI 

Project  Description:   Three  groups  of  adult  mongrel  dogs  were  prepared  in 
the  following  manner:   the  first  underwent  creation  of  unilateral  carotid- 
jugular  and  femoral -femoral  arterio-venous  fistulae,  followed  by  total 
thyroidectomy  and  ingestion  of  a  high  cholesterol  diet;  the  second  underwent 
simultaneous  shunting  and  thyroidectomy  followed  by  the  special  diet;  the 
final  group  had  thyroidectomy  and  special  diet  followed  by  shunting  at  a 
later  date.  All  animals  were  carried  on  a  high  cholesterol  diet  for  six 
months  and  followed  with  weekly  or  biweekly  serum  cholesterol  and  thyroid 
function  studies. 

Results:   None  of  the  animals  has  been  sacrificed,  but  all  will  have  been 
within  the  next  two  months. 

Proposed  course:   Animals  will  be  sacrificed  after  six  months  of  special 
diet,  and  shunted  vessels  as  well  as  contralateral  normal  vessels  removed 
for  gross  and  microscopic  study. 

Keywords:   Arterio-venous  fistulae,  Thyroidectomy. 


8/3 


Project  No.   Z01  HL  02620-01    SU 
1.      Clinic  of   Surgery 
3.      Bethesda,   Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   The  effects  of  isoproterenol  and  dopamine  on  regional 

coronary  flow  distribution  in  dogs  with  partial  coronary 
occlusion 

Previous  Serial  Number:   None 

Principal  Investigators:   James  H.  McClenathan,  M.D. 

Robert  A.  Guyton,  M.D. 

Other  Investigator:       Lawrence  L.  Michaelis,  M.D. 

Project  Description:   In  11  dogs,  partial  occlusion  of  the  circumflex 
coronary  artery  was  created.   Left  atrial,  aortic,  and  distal  circumflex 
coronary  artery  pressure,  cardiac  output,  heart  rate  and  left  ventricular 
dp/dt  were  monitored.   Pharmacologic  doses  of  isoproterenol  and  dopamine 
were  given.   Regional  coronary  flow  was  measured  with  radioactive  micro- 
spheres . 

Results:   Dopamine  caused  an  increase  in  aortic  and  distal  circumflex 
coronary  artery  pressure,  cardiac  output  and  peak  dp/dt.   There  was  no 
redistribution  of  regional  coronary  flow. 

Isoproterenol  caused  an  increase  in  heart  rate,  cardiac  output  and 
peak  dp/dt,  while  aortic  and  distal  coronary  pressure  decreased.  Total 
flow  to  the  myocardium  distal  to  the  occlusion  was  unchanged.  However, 
there  was  redistribution  of  flow  away  from  the  subendocardium  (p <  .01). 
We  are  proposing  that  the  redistribution  of  coronary  flow  distal  to  the 
occlusion  which  follows  administration  of  isoproterenol  is  related  to  the 
decrease  in  perfusion  pressure  and  the  increase  in  myocardial  contractility, 

Proposed  Course:   A  manuscript  is  in  preparation. 

Keywords:   Regional  Coronary  Flow,  Partial  Coronary  Occlusion,  Dopamine, 
Isoproterenol 


eif 


Project  No.   Z01  HL  02621-01   SU 
1.      Clinic  of  Surgery 
3.      Bethesda,   Md. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Distribution  of  blood  flow  before  and  after  repair  of 
coarctation  of  aorta 

Previous  Serial  Number:   None 

Principal  Investigator:   Glenn  E.  Newman,  M.D. 

Michael  J.  Andrews,  M.D. 

Other  Investigators:      Lawrence  L.  Michaelis,  M.D. 

Project  Description:   Repair  of  aortic  coarctation  is  sometimes  associated 
with  two  hazardous  postoperative  complications;  a  severe  exacerbation  of 
the  pre-existing  systemic  hypertension  and  mesenteric  vasculitis.  A  sudden 
change  in  pressure  and  flow  distal  to  the  coarctation  has  been  suggested 
as  the  initiator  of  these  postoperative  events,  but  laboratory  assessment 
of  the  clinical  situation  has  been  hampered  by  the  difficulty  in  creating 
an  appropriate  laboratory  model  and  by  accurate  methods  of  measuring  flow 
to  various  organs  over  a  several  day  period. 

We  have  developed  a  successful  method  of  producing  aortic  coarctation 
in  young  dogs  by  constructing  the  descending  thoracic  aorta  with  a  tape 
tied  over  felt  pads.   After  these  animals  have  matured  we  will  repair  the 
coarctation  and  will  perform  chronic  flow  measurements  by  injecting  radio- 
active microspheres  via  a  catheter  implanted  in  the  left  atrium.   Flow 
to  the  myocardium,  gut,  kidney,  and  skeletal  muscle  will  be  assessed  in 
these  animals  (unanesthetized)  prior  to  repair,  in  the  immediate  post- 
operative period,  and  after  postoperative  equilibration  occurs.   Chronic 
aortic  pressure  measurements  will  be  made  proximal  and  distal  to  the 
coarctation. 

Results:   Retrograde  catheterization  has  demonstrated  a  significant  coarc- 
tation in  these  animals  (six  weeks  after  operation  -  average  gradient 
55  mm.  Hg). 

Proposed  Course:   After  the  animals  have  reached  a  stable  condition 
following  coarctation  repair  (10-14  days),  they  will  be  killed  and  the 
above  mentioned  organs  will  be  analyzed  for  the  various  microspheres 
injected  before  and  after  the  operation.   Thus,  flow  to  these  organs 
before  and  after  repair  can  be  quantitated.   It  is  hoped  that  this 
information,  pressure  changes  that  occur,  and  renin  and  angiotension 
assays  in  these  animals  will  help  identify  the  etiology  of  these  poten- 
tially lethal  postoperative  events. 

Keywords:   Coarctation  of  Aorta,  Repair,  Distribution  of  Blood  Flow 

etsr 


Project  Wo.   Z01  HL  02622-01  SU 

1.   Clinic  of  Surgery 

PHS"NIH  3.   Bethesda,  Md. 

Individual  Project  Report 

July  1,  197^  thorugh  June  30,  1975 

Project  Title:   Study  of  arterial  wall  permeability  as  a  function  of 
flow  rate 

Previous  Serial  No: 

Principal  Investigators:   Lynn  H.  Harrison,  M.  D. 

Michael  S.  Cann,  M.  D. 

Other  Investigators:   Charles  L.  Mcintosh,  M.  D. 
Donald  L.  Fry,  M.  D. 

Cooperating  Unit:   Section  of  Experimental  Atherosclerosis,  NHLI 

Project  Description:   Adult  mongrel  dogs  were  subjected  to  creation  of 
ipsilateral  carotid- jugular  and  femoral-femoral  arteriovenous  fistulas  as 
well  as  corresponding  arteriotomies  on  the  contralateral  side.   Animals 
were  then  sacrificed  at  2U,  U8,  and  72  hours  as  well  as  at  k   weeks  to  one 
year  following  shunting.   All  were  given  an  intravenous  dose  of  Evan's 
Blue  Dye,  U.S. P.,  1.5  cc/Kg,  2\   hours  prior  to  sacrifice.   At  sacrifice, 
vessels  were  harvested  on  both  shunted  and  nonshunted  sides  and  scanned 
with  a  photosensitive  cell  capable  of  detecting  Evan's  Blue  Dye  con- 
centration.  Microscopic  sections  of  vessels  were  also  taken. 

Results:   Arterial  wall  permeability  appeared  to  be  greatest  during  the 
first  2U-1+8  hours  following  shunting,  and  tapered  off  exponentially  in 
the  ensuing  days  and  weeks.   Preliminary  microscopic  examination  suggests 
a  thickening  of  intima  and  possibly  media  in  shunted  arteries  as  well  as 
a  realignment  of  intimal  cells  in  response  to  increased  flow. 

Proposed  Course:   Several  additional  animals  have  been  shunted  to  fill 
out  groups  of  long-term  animals  in  order  to  make  the  sample  group  large 
enough  for  statistical  comparison  with  the  other  groups.   These  animals 
await  sacrifice  at  the  appropriate  intervals. 

Keywords:   arterial  wall  permeability 


8/6 


ANNUAL  REPORT  OF  THE 
SECTION  ON  EXPERIMENTAL  ATHEROSCLEROSIS 
NATIONAL  HEART  AND  LUNG  INSTITUTE 
July  1,  1974  through  June  30,  1975 

The  program  for  the  Section  on  Experimental  Atherosclerosis  has  continued 
to  be  targeted  on  the  mechanisms  leading  to  the  genesis  of  the  atherosclerotic 
plaque,  the  lethal  lesion  of  atherosclerosis.   The  ultimate  aim  of  this  program 
is  to  identify  each  of  the  steps  in  the  evolutionary  sequence  of  this  disease 
process  in  terms  of  cellular  metabolic  mechanisms  and  physical  chemical  laws 
so  that  deliberate  strategies  can  be  developed  to  interupt  this  sequence  at 
various  selected  points  of  vulnerability.   This  broad  objective  has  required 
the  development  of  a  multidisciplinary  program  consisting  of  experimental 
pathology,  human  pathology,  lipoprotein  chemistry,  physical  chemistry,  arterial 
tissue  culture,  vascular  physiology  and  data  processing.   The  present  report 
deals  only  with  achievements  in  our  most  active  areas  this  year  and,  therefore, 
a  brief  review  of  certain  background  information  and  the  overall  program  plan 
will  help  the  reader  to  place  current  efforts  in  clearer  perspective. 

Background  Information  and  Overall  Program  Plan 

A  variety  of  experimental  observations  from  this  section,  which  have  been 
covered  in  previous  annual  reports,  are  consistent  with  the  following  working 
hypothesis  upon  which  the  program  of  this  section  is  based.   We  have  shown  that 
macromolecular  (albumin)  transport  into  the  vascular  interface  is  not  uniform 
along  the  arterial  tree  but  tends  to  vary  with  a  typical  topographic  pattern  in 
normal  animals.   This  pattern  appears  qualitatively  identical  to  the  pattern 
of  intimal  lipid  deposition  (sudanophilia)  that  develops  in  the  same  species 
when  serum  cholesterol  is  elevated  by  dietary  means.   This  set  of  observations 
can  be  explained  by  the  following  hypothetical  sequence. 

Endothelial  permeability  is  altered  by  a  local  hemodynamic  or  structural 
factors  such  that  there  is  a  greater  flux  of  plasma  substances  across  the 
endothelial  surface  into  the  intimal  tissues  at  these  sites.   This  increased 
flux  results  in  increased  tissue  concentrations  of  certain  of  the  plasma 
substances.   The  concentration  profile  across  the  wall  will  depend  not  only 
on  the  magnitude  of  the  flux  but  also  upon  the  locations  and  nature  of  the 
barriers  to  this  flux.   Generally  speaking,  the  concentration  will  be  highest 
in  the  intimal  region,  will  decrease  across  the  wall,  and  become  minimal  in 
the  lymphatic  spaces  of  the  adventitia.   Smooth  muscle  cells  have  been  shown 
to  proliferate  in  the  presence  of  increased  concentration  of  plasma  substances. 
These  cells  metabolize  the  lipoproteins  by  hydrolizing  the  triglyceride  present. 
However,  they  cannot  metabolize  the  associated  cholesterol  in  the  lipoproteins 
and  therefore  these  cholesterol  substances  tend  to  accumulate  in  the  form  of 
intracellular  inclusions  and  extracellular  aggregates.   These  aggregates  of 
cholesterol  products  apparently  cannot  be  resolublized  and  transported  out  of 
the  vascular  tissue  rapidly  enough  to  prevent  their  progressive  accumulation. 
The  tissue  responds  to  the  accumulation  as  to  any  other  "foreign"  body, 
ultimately  walling  it  off  with  fibrous  tissue  and  chronic  inflamatory  cells 
thereby  producing  the  mature  atheromatous  plaque. 


&7 


In  accordance  with  the  above  scheme,  the  program  has  been  structured 
around  study  of  the  atherogenic  precursors,  study  of  the  transport  processes 
by  which  these  precursors  are  carried  into  the  intimal  space,  and  studies 
of  the  various  tissue  responses  to  the  presence  of  these  atherogenic  precursors. 
Progress  in  these  three  important  areas  over  the  past  year  are"  outlined  below. 

Tissue  Responses 

During  the  past  year  we  have  continued  to  examine  the  response  of  arterial 
tissue  components  to  increase  concentration  of  plasma  substances.   These  studies 
have  been  carried  out  in  tissue  culture  as  well  as  in  acute  and  chronic  studies 
in  normal  and  hyperlipidemic  animals. 

The  removal  of  the  endothelial  surface  in  normal  animals  using  an  intra- 
vascular balloon  technique  has  been  shown  to  result  in  a  greatly  increased  flux 
of  plasma  substances  into  the  eroded  intimal  surface  which  is  followed  by  an 
enormous  proliferative  response  of  the  smooth  muscle  cells  at  the  vascular 
interface.   This  proliferative  response  continues  until  the  surface  becomes 
covered  again  with  a  normal  endothelial  cell  layer  which  may  take  from  weeks 
to  months.    With  the  reappearance  of  the  endothelial  cell  layer,  the  increased 
transport  of  plasma  substances  into  the  intimal  space  is  returned  to  normal  and 
the  smooth  muscle  cell  proliferative  response  subsides,  resulting  in  a  residual 
intimal  thickening  indistinguishable  from  that  seen  in  older  animals.   The 
same  studies  carried  out  in  hyperlipidemic  animals  are  also  associated  with  a 
proliferative  response  of  the  smooth  muscle  cells,  however,  with  marked  intra- 
cellular and  extracellular  lipid  deposition  resulting  in  an  atheromatous 
lesion. 

These  observations  are  consistent  with  the  tissue  culture  work  reported 
in  last  year's  annual  report  and  which  have  shown  that  smooth  muscle  cells  in 
tissue  culture  proliferate  rapidly  in  the  presence  of  the  low  density  lipo- 
proteins and  become  laden  with  intracellular  lipid  vacuoles   particularly 
when  exposed  to  VLDL.   The  above  observations  suggest  that  the  initial  lesion 
of  atherosclerosis  is  a  proliferative  response  of  arterial  smooth  muscle  cells 
when  they  are  exposed  to  an  increase  in  the  tissue  concentration  of  certain 
plasma  substances,  e.g.,  lipoproteins. 

Further  insights  into  tissue  responses  were  gained  from  study  of  our 
animal  colonies.   Three  important  animal  colonies  were  harvested  and  revealed 
some  new  and  interesting  results.   The  first  of  these  was  the  primate  colony 
(Erythrocebus  Patas  monkeys)  which  was   studied  in  collaboration  with  the 
Veterinary  Resources  Branch  in  DRS.   The  primates  were  studied  in  three  groups: 
one  on  normal  diet,  one  on  a  high  fat  diet,  and  one  on  a  high  fat  diet  to  which 
one-half  of  one  percent  cholesterol  was  added.   The  control  animals  developed 
insignificant  disease,  whereas  the  high  fat  animals  developed  occasional 
fatty  streaking  and,  only  rarely,  developed  plaques.   In  sharp  contrast  to 
these,  the  cholesterol  fed  animals  developed  florid  proliferative  atherosclerosis 
progressing  from  fatty  streaks  to  raised  plaques  and  finally  complicated  plaques 
with  occasional  thrombosis. 

The  second  study,  which  was  carried  out  under  contract  with  the  University 
of  Missouri,  was  designed  to  examine  the  influence  of  age,  sex  and  diet  on  the 
arterial  tissue   changes  in  miniature  swine.   Histologic  examination  of  these 

( 

2  8?Q 


tissues  revealed  a  histologic  pattern  similar  to  that  seen  in  the  monkeys, 
but  differed  in  that  the  lesions  were  slightly  more  f ibrotic  and  did  not 
show  any  evidences  of  thrombotic  activity.   The  severity  of  the  disease  both 
in  the  monkey  and  swine  colonies  appeared  to  correlate  principally  with  the 
level  of  serum  cholesterol  and  not  with  other  variables  such  as  age  or  sex. 
The  disease  in  both  species  was  principally  intimal  with  only  moderate, 
secondary  medial  changes  and  was  in  every  respect  indistinguishable  from  the 
human  counterpart. 

The  third  colony  was  composed  of  NIH  foxhounds  which  were  studied  under 
contract  to  the  Colorado  State  University  with  a  protocol  similar  to  the  above 
swine  study,  i.e.,  to  determine  the  effect  of  age,  sex,  and  dietary  cholesterol 
on  the  development  of  atherosclerosis.   The  histologic  characteristics  of  the 
disease  produced  in  these  animals  was  different  from  that  seen  in  the  above 
primate  and  swine  studies  as  well  as  different  from  that  seen  in  previous 
studies  with  foxhounds  carried  oat  at  NIH.   For  a  given  serum  cholesterol 
level  the  lesions  produced  in  these  animals  were  much  more  severe  both  in 
extent  and  by  histologic  criteria  than  were  the  lesions  examined  in  the  pre- 
vious studies.   There  was  a  marked  tissue  destruction  with  invasion  of 
histiocytes  and  polymorphonuclear  leukocytes.   Spread  of  the  process  from 
the  intima  to  involve  the  entire  media  of  the  vessel  was  common  at  many 
locations.   There  was  frequent  evidence  of  prior  thrombosis  with  recanalization 
of  the  vessels.   Seven  clinically  significant  thrombotic  accidents  occurred 
in  this  colony,  including  myocardial   infarcation  and  gangrene  of  an  extremity. 
Moreover,  the  overall  topographic  distribution  of  the  lesions  was  more  peripheral 
and  less  central  than  in  the  previous  studies  in  the  foxhounds  as  well  as  in 
the  swine  and  primate  studies. 

The  principal  difference  in  the  experimental  protocol  governing  this 
experimental  colony  was  the  inclusion  of  beef  tallow  and  taurocholic  acid 
(a  naturally  occurring  bile  salt)  in  the  diet  of  these  animals.   We  conclude 
tentatively  from  these  important  observations  that  while  the  serum  cholesterol 
appears  to  be  a  common  governing  variable  in  this  disease  process,  other 
factors  such  as  bile  acid  metabolism  and  the  composition  of  the  triglyceride 
appear  to  be  important  modulating  influences  on  the  tissue  response  to  the 
increase  tissue  concentration  of  the  atherogenic  precursors. 

Study  of  the  Atherogenic  Precursors 

Evidence  has  mounted  from  our  studies  this  year  that  the  atherogenic 
precursors  in  all  species  are  closely  related  to  the  cholesterol  carried  in 
the  low  density  serum  lipoproteins.   Two  sorts  of  studies  support  this.   The 
first  are  the  colony  studies  which,  as  indicated  above,  continue  to  show  a 
strongly  positive  correlation  between  the  serum  cholesterol  level  in  a  given 
species  and  both  the  extent  and  severity  of  the  associated  atherosclerotic 
processes.   The  second  are  the  detail  studies  of  the  changes  in  the  lipoproteins 
that  occur  concomitant  with  the  phase  of  the  rapid  progression  of  the  athero- 
sclerotic process.   Detailed  analysis  of  the  plasma  lipoproteins  indicates  that 
dietary  cholesterol  induces  a  characteristic  hyperlipoproteinemia  with  a 
lipoprotein  pattern  consistent  among  the  various  species.   The  rapid  prolif- 
erative phase  of  atherosclerosis  is  associated  with  an  increased  cholesterol 
and  cholesterol  ester  content  of  several  different  lipoproteins  in  the  low 


8(9 


and  very  low  density  fractions.   Analysis  of  the  apoprotein  content  of  these 
lipoproteins  has  revealed  an  important  new  correlation,  i.e.,  the  appearance 
of  a  characteristic  arginine-rich  apoprotein.   This  apoprotein  has  been  found 
in  all  species  that  have  been  examined  (monkey,  pig,  dog,  rabbit,  rat  and 
type  III  human  hyperlipoprotenemia)   and  may  function  in  the  transport  of 
cholesterol  between  lipoproteins  and  the  arterial  wall.    Thus,  it  appears 
that  an  atherogenic  factor  is  associated  in  all  species  with  the  appearance 
of  certain  cholesterol  carrying  lipoproteins  that  have  a  common  arginine- 
rich  apoprotein.   The  structural  characteristics,  physical  properties,  and 
metabolism  of  these  lipoproteins  are  currently  under  intensive  study  in 
this  laboratory. 

Studies  of  Transvascular  Macromolecular  Transport 

It  is  highly  probable  that  the  atherogenic  precursors  are  either  macro- 
molecules   or  are  carried  on  macromolecular  vehicles  from  the  blood  into  the 
intimal  space.   As  discussed  above,  we  are  hopeful  that  we  are  converging 
on  the  chemical  identity  of  these  and, in  anticipation  of  this,  have  spent 
considerable  effort   this  year  developing  the  necessary  technical,  physical 
chemical,  and  mathematical  tools  to  quantify  these  important  transport 
processes.   The  serum  albumin  and  the  Evans  blue  dye  albumin  complex  transport 
systems  have  been  developed  as  prototype  models  for  this  purpose  to  devise 
the  necessary  theory,  experimental,   and  analytic  protocols  for  the  future 
studies. 

Experiments  were  carried  out  using  a  specially  devised  organ  support 
system  in  which  it  was  possible  to  study  the  freshly  excised  aorta  under 
controlled  conditions  of  mechanical  constraint,  temperature,  bathing  milieu, 
and  transmural  pressure.   Using  iodine  tagged  albumin  the  following  observations 
were  made.    Stirring  of  the  albumin  solution  over  the  vascular  interface  does 
not  appreciably  increase  the  uptake  of  albumin.   From  this  it  was  concluded 
that  a  significant  diffusion  barrier  does  not  normally  exist  at  the  vascular 
interface  and,  therefore,  transport  properties  are  determined  only  by  the  wall. 
The  second  set  of  observations  demonstrated  that  the  accumulation  of  tagged 
albumin  in  the  intimal  space  varied  as  the  square  root  of  time.   This  obser- 
vation plus  corroborative  measurements  of  the  concentration  distribution  across 
the  wall  (using  autoradioagraphic  techniques)  indicated  that  the  albumin 
transport  system  was  following  the  mathematical  solution  of  Fick's  second  law 
of  diffusion  as  applied  to  diffusion  in  a  two-phase  system.   Studies  using 
doubly  tagged  albumin  (covalently  tagged  with  iodine  and  "complexed"  with 
Evans  blue  dye)  indicated  that  the  accumulation  of  Evans  blue  dye  in  the 
intimal  space  was  approximately  four  times  greater  than  the  accumulation  of 
tagged  albumin.   Moreover,  studies  of  the  rate  of  elution  of  Evans  blue  dye 
back  out  of  the  intimal  space  indicated  that  the  elution  rate  into  a  saline 
milieu  was  less  than  one-tenth  of  that  into  an  albumin-saline  milieu. 

We  conclude  from  these  two  sets  of  studies  that  albumin  is  transported  into 
the  intimal  space  in  accordance  with  classic  diffusion  theory  and  that  Evans 
blue  dye  is  transported  into  and  through  the  intimal  space  on  albumin  as  a 
vehicle.   However,  in  the  intimal  space  the  Evans  blue  dye  enters  into  a  new 
competitive  equilibrium  between  tissue  albumin  and  some  fixed,  binding  sub- 
stance in  the  wall.   Various  binding  relationships  were  explored  and  it  was 


€9js> 


found  that  a  simple  linear  adsorption  isotherm  described  the  binding  process 
adequately.   Measurements  of  the  partition  coefficient  for  albumin  between 
blood  and  the  tissue  space  were  carried  out  using  iodine  tagged  albumin  and 
measuring  the  plateau  on  the  uptake  curve  for  the  tissue.   This  plateau 
occurred  at  about  fifteen  hours  and  the  value  of  the  partition  coefficient 
was  approximately  .20,  that  is  to  say,  the  equilibrium  volume-concentration 
of  albumin  in  the  tissue  space  is  about  one-fifth  of  that  in  the  blood.   The 
albumin  uptake  curves  were  then  evaluated  using  this  partition  coefficient 
to  determine  the  diffusion  coefficient  of  albumin  in  arterial  tissue.   This 
was  found  to  be  approximately  2  x  10~°  square  centimeters  per  second.   With 
this  information  it  was  possible  then    by  comparison  of  Evans  blue  dye  up- 
take and  the  tagged  albumin  uptake  to  calculate   the  binding  constant  for 
Evans  blue  dye  in  the  wall.   This  was  found  to  be  about  15,  i.e.,  the  amount 
of  Evans  blue  dye  bound  to  structural  substances  in  the  wall  is  about  15  times 
that  free  to  diffuse  on  tissue  albumin.   Thus,  it  has  been  possible  to  define 
the  diffusion  of  albumin  as  well  as  the  diffusion  and  chemical  complexing  of 
a  substance  carried  on  albumin  in  rather  complete  and  unambiguous  physical 
chemical  terms.   This  represents  a  significant  advance  in  a  previously 
obscure  area  of  transport  mechanics . 

The  transmural  pressure  acts  as  an  added  driving  force  for  this  macro- 
molecular  flux.   The  magnitude  of  this  was  evaluated  by  creating  a  transmural 
pressure  of  100  mm  of  mercury  and  measuring  the  resultant  increment  increase 
in  flux.   This  increment  increase  represented  the  "convective  flux"  related 
directly  to  the  imposed  transmural  pressure.   Under  steady  state  conditions, 
this  was  found  to  be  approximately  one-half  of  the  diffusive  flux  across  the 
wall.  (In  calculating  the  steady  state  diffusive  flux  values  from  the 
literature  were  used  for  the  concentration  of  the  diffusing  species  in  the 
adventitial  lymphatic  space,) 

Normally,  the  principal  barrier  to  transport  resides  in  the  endothelial 
surface  itself.   As  indicated  earlier,  we  have  shown  that  transmural  trans- 
port increases  enormously  in  regions  of  endothelial  injury  and  even  under 
normal  conditions  is  also  increased  at  certain  characteristic  sites  along  the 
arterial  tree. 

Detailed  electron  microscopic  studies  are  being  carried  out  to  discover 
the  ultrastructural  features  of  endothelial  cells  that  might  correlate  with 
increased  transendothelial  transport.   When  viewed  by  scanning  electron 
microscopy,  the  endothelial  surface  in  areas  of  increased  transport  is  more 
disordered,  has  poorly  defined  cell  borders  and  nuclei,  and  appears  to  have 
a  "soft"  or  "boggy"  texture.   Transmission  electron  microscopy  shows  surpris- 
ingly little  evidence  of  abnormal  endothelial  structure  but  does  show  moderate 
subendothelial  edema  in  these  regions.   Although  the  above  are  by  far  the  most 
common  morphologic  features  of  regions  of  increased  transport,  discrete  areas 
of  endothelial  cell  erosion  are  also  observed  occasionally,  suggesting  that 
these  areas  may  be  populated  by  a  more  fragile  cell  surface  or  that  these 
areas  are  episodically  exposed  to  abnormally  high  hemodynamic  forces. 

Methods  have  been  devised  to  measure  the  discrete  rheologic  properties  of 
these  areas  using  small  calibrated  jets  of  fluid  to  establish  the  cell  "yield 
stress"  and  using  a  device  with  a  small  indenting  probe  to  establish  subsurface 


ex 


"hardness."  Although  the  jet  studies  are  still  in  the  developmental  stage, 
preliminary  data  from  the  indentation  studies  suggest  that  areas  of  increased 
transport  are  "softer."  Hemodynamic  studies  (detailed  in  previous  reports) 
have  been  carried  out  in  a  continuing  effort  to  define  the  de.tailed  stress 
pattern  to  which  this  fragile  surface  is   exposed. 

In  conclusion,  it  has  been  possible  to  define  certain  fundamental  trans- 
port forces  and  the  associated  fluxes  for  albumin  and  albumin  Evans  blue  dye 
complex  across  the  intimal-medial  space.   These  relationships  have  been  shown 
to  follow  classical  non-equilibrium  thermodynamic  relationships  thereby 
establishing  the  fundamental  physical  chemical  behavior  of  the  albumin  trans- 
port system  into  the  intima  and  media.   With  these  prerequisite  determinations 
accomplished,  we  are  now  in  the  position  to  approach  the  question  of  trans- 
endothelial  transport  and,  as  the  atherogenic  precursors  become  better  defined, 
to  begin  applying  these  same  techniques  more  directly  to  study  of  atherogensis. 

Concluding  Remarks 

As  indicated  above,  the  work  this  year  has  added  further  evidence  supporting 
the  working  hypothesis  presented  at  the  beginning  of  this  report,  namely,'  that 
arterial  smooth  muscle  cell  proliferation  and  the  metabolism  of  certain  low 
density  lipoproteins  occur  in  regions  of  increased  concentration  of  certain 
atherogenic  precursors  arising  from  the  blood,  particularly  in  areas  of 
increased  transendothelial  transport.   Much  of  our  effort  over  the  ensuing  years 
will  be  directed  to  a  more  detailed  identification  of  these  atherogenic 
precursors,  to  establishing  the  physical  chemical  laws  governing  their  trans- 
port and  accumulation  at  certain  sites  of  the  wall,  and  to  designing  studies 
to  gain  deeper  insight  into  the  cellular  responses  to  these  transmural  concen- 
tration distributions. 

These  efforts  have  required,  and  will  continue  to  require  collaborative 
ties  among  the  B/l/D  (VRB  (DRS),  Clinical  Pathology  Dept.  (CC) ,  BEIB  (DRS) , 
DCRT,  Molec.  Dis.  (H) ,  SLAM  (H) ,  and  Heart  Surgery  (H) ) ,  as  well  as  extramural 
contractural  support. 

We  have  reviewed  the  economics  of  contract  support  for  this  program  in 
an  effort  to  seek  alternative,  less  expensive  approaches  to  our  objectives. 
In  view  of  the  very  real  constraints  on  both  positions  and  space  that  are 
likely  to  continue  for  the  foreseeable  future,  we  shall  have  a  continuing  need 
for  our  service  contracts,  particularly  in  the  areas  of  electron  microscopy, 
preparative  lipoprotein  chemistry  and  radio  labelling  of  proteins.   Thus,  for 
the  present  at  least,  the  service  contracts  appear  to  be  the  only,  if  not  the 
most  economical  source  for  these  essential  program  supports. 

In  the  area  of  research  contracts,  which  we  use  for  much  of  our  colony 
work,  certain  alternatives  appear  possible  in  the  near  future.   We  are  eagerly 
seeking  scientific  collaboration  with  certain  members  of  the  Veterinary  Resources 
Branch  (DRS)  in  an  effort  to  develop  a  more  efficient  and  scientifically 
manageable  solution  to  our  projected  need  for  experimental  animal  colonies. 
The  soaring  costs  for  these  essential  program  support  areas  are  of  major 
concern  to  us  and  will  be  under  continual  review  for  more  economical  alter- 
natives. 


B-2.X 


Project  No.  Z01  HL  02801  03  SEA 

1.  Office  of  Director  on  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   The  arterial  intimal  tissue  responses  following  endothelial 
injury. 

Previous  Serial  Number:   NHLI-199 

Principal  Investigator:   Donald  L.  Fry,  M.D. 

Other  Investigators:   Victor  J.  Ferrans,  M.D.,  Ph.D. 

Cooperating  Units:   Section  on  Pathology,  NHLI 

Project  Description 

Objective:   To  study  the  functional  and  structural  changes  in  the 
arterial  wall  following  the  erosion  of  the  endothelial  surface  by  increased 
fluid  shear  stress. 

Methods  Employed:   Detailed  methodology  for  this  project  appears  in 
last  year's  report.   Briefly,  the  endothelial  surface  of  the  descending 
thoracic  aorta  in  a  group  of  dogs  was  discretely  removed  using  an  inflated 
arterial  balloon.   The  animals  were  allowed  to  recover  and  were  examined  at 
selected  times  following  endothelial  cell  erosion  using  both  light  micro- 
scopic and  electronmicroscopic  techniques  to  examine  the  sequences  of  events 
leading  to  repair  of  the  damaged  arterial  surface.   Concomitant  estimates  of 
trans-endothelial  macromolecular  transport  were  carried  out. 

Major  Findings:  The  sequence  of  repair  were  as  follows:   There  was 
immediate  generalized  platelet  adhesion  to  the  eroded  surface  which  was 
followed  in  a  matter  of  days  by  a  rapid  proliferation  of  modified  smooth 
muscle  cells.   Albumin  transport  into  the  arterial  wall  was  increased  from 
10  to  15  fold  both  in  the  acutely  eroded  surface  as  well  as  the  surface 
subsequently  covered  with  smooth  muscle  cell  proliferation.   At  a  point  in 
time  corresponding  roughly  with  the  reappearance  of  a  normal  endothelial  cell 
cover,  both  the  proliferative  response  and  the  increased  trans-intimal 
transport  of  albumin  decreased. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  atherosclerotic  processes  are  classically  characterized  by  f ibromuscular 
hyperplasia  and  lipid  deposition.   The  sequence  of  events  leading  to  this 
thickened  intima  are  not  understood.   The  topography  of  intimal  prolifera- 
tive processes  in  the  diseased  state  frequently  corresponds  to  areas  that 


e*i 


Project   No,    Z01   HL  02801   03   SEA 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

may  have  been  exposed  to  increased  mechanical  stress  and/or  injury.   If  the 
mechanisms  of  these  naturally-occurring  processes  in  this  important  disease 
are  to  be  understood,  it  is  essential  that  we  understand  in  greater  detail 
the  factors  inducing  and  controlling  the  reparative  processes  occurring  at 
the  vascular  interface. 

Proposed  Course  of  the  Project:   A  large  amount  of  histologic  and 
electron  microscopic  material  has  been  generated  from  the  above  studies  and 
is  still  in  the  process  of  analysis.   The  major  problem  in  the  interpretation 
of  these  data  is  the  lack  of  precision  with  which  the  exact  extent  and  area 
of  the  initial  injury  can  be  defined,  particularly  in  the  chronic  preparations, 
It  is  not  possible  without  this  information  to  establish  unequivocally  the 
exact  relationship  between  the  reappearance  of  the  normal  endothelial  sur- 
face, the  "shutting-off "  of  the  smooth  muscle  cell  proliferative  response, 
and  the  reappearance  of  normal  macromolecular  transport  into  the  vascular 
interface.   A  variety  of  new  techniques  are  being  devised  which  will  allow 
us  to  create  well-defined,  controlled,  discrete  regions  of  endothelial  cell 
erosion  thus  allowing  us  to  define  this  important  set  of  feedback  mechanisms 
which  play  a  central  role  in  the  atherogenic  process. 

Keyword  Descriptors 

Endothelial  erosion;  albumin  transport  mechanics;  Evans  blue  dye; 
endothelial  and  intimal  repair;  atherosclerosis. 

Honors  and  Awards 

1.  Invited  speaker,  American  Heart  Association,  Council  for  High  Blood 
Pressure  Research  conference  entitled,  "Arteriosclerosis  and  Hypertension: 
Observations  and  Speculation,"  in  Cleveland,  Ohio,  October  12,  1974. 

2.  Invited  speaker  at  meeting  on  Fluid  Dynamic  Aspects  of  Arterial 
Diseases,  Ohio  State  University,  September  18,  1974. 

3.  Invited  as  discussion  leader  on  Gordon  Research  Conference  on 
Atherosclerosis,  June  1975,  entitled  "Fluid  mechanics,  transport  phenomenon 
and  the  arterial  wall," 

Publications 

None  this  year. 


&*Y 


Project  No.   Z01  HL  02802  03  SEA 


1.  Office  of  Director  of  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   The  relationship  of  arterial  intimal  Evans  blue  dye 

accumulation  to  surface  reflectance  and  light  absorption. 

Previous  Serial  Number:   NHLI-200 

Principal  Investigator:   Donald  L.  Fry,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description 

Objective:  To  establish  and  validate  the  relationship  between  the  con- 
centration of  Evans  blue  dye  in  the  vascular  interface  and  the  absorption  of 
red  light. 

Methods  Employed:   A  special  reflectance  measuring  system  was  designed 
and  constructed,  such  that  opened  vessel  surfaces  could  be  viewed  under  uni- 
form illumination  through  a  lens  system  on  a  large  glass  screen.   A  small 
aperture  in  the  center  of  this  screen  led  to  the  sensing  surface  of  a  photo- 
multiplier   tube.   Appropriate  light  filters  could  be  inserted  in  this  light 
path  as  needed.      The  opened  vessel  surfaces  were  held  in  a  specially 
designed  servo-motor-driven  tray  submerged  in  saline,  both  to  act  as  a 
metabolic  supporting  milieu  for  the  vessel  surface,  as  well  as  providing  a 
nonreflecting  optical  coupling  to  the  photo-sensing  system.   Various  regions 
of  the  vessel  surface  could  then  be  maneuvered  by  the  operator  under  visual 
control  such  that  a  very  small  region  on  the  vessel  surface  could  be  placed 
under  the  aperture  in  the  screen.   With  a  Wratten  29  filter  in  the  light 
path  it  was  possible  to  measure  the  amount  of  red  light  that  is  absorbed  and 
reflected  from  the  vessel  surface.   By  staining  various  regions  on  the  vessel 
surface  to  varying  depths  it  was  possible  to  establish  a  monotonic  relation- 
ship between  the  optical  absorbance  p  (negative  log  of  the  ratio  of  light 
intensity  from  stained  surface  to  that  from  adjacent  unstained  surface)  of 
the  stained  surface  and  time  (duration  of  exposure  to  EBD-albumin  complex)  . 
A  reflectance  model  based  on  the  Lambert-Beer  law  of  light  absorption  was 
evaluated  to  represent  this  relationship  by  fitting  model  parameters  to  data 
obtained  as  dye  was  eluted  back  out  of  the  surface  using  an  untagged  albumin 
solution.   The  final  model  that  was  chosen  implicitly  predicts  the  optical 
absorption  coefficient  for  EBD  from  these  fitted  parameters  and  also  the 
change  in  vessel  surface  area  that  occurs  with  stretch  of  the  wall.   The 
accuracies  of  these  implications  were  tested  by  measureing  the  EBD  absorption 
coefficient  directly  in  an  Aminco  microphotometer  and  by  measuring  the 
correlation  between  measured  changed  in  wall  stretch  with  those  predicted 
from  reflectance  measurements. 

1  8AST 


Project  No.  Z01  HL  02802  03  SEA 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Major  Findings:   It  was  found  that  the  accumulation  of  EBD  in  the 
vascular  intima  bears  an  approximately  logarithmic  relationship  to  the 
amount  of  light  absorbed  as  given  by  M  =  kjp  +  k3p3,  where  M  is  the 
surface  accumulation  in  n •moles/cm2  and  p  is  the  optical  absorbance. 
The  constants  k^  and  k2  were  found  to  be  3.92  and  0.80,  respectively. 

The  validity  of  the  model  was  established  by  noting  that  the  predicted 
value  of  the  absorption  coefficient  (from  the  fitted  parameters)  was  0.128- 
as  compared  to  0.126  for  the  directly  measured  value.   The  linear  regression 
of  the  predicted  wall  stretch  data  on  directly  measured  values  had  a  slope 
of  0.99,  a  negligible  intercept,  and  a  correlation  coefficient  of  0.99.   We 
conclude  that  the  model  represents  the  actual  physical  state  with  good 
precision. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
When  Evans  blue  dye  is  injected  intravenously  into  an  animal  virtually  all 
of  the  dye  becomes  bound  to  serum  albumin.   Knowing  the  molar  ratio  of  Evans 
blue  dye  to  albumin  permits  the  use  of  Evans  blue  dye  as  a  measurable  visual 
tag  for  albumin.   Measurement  of  the  amount  of  Evans  blue  dye  in  the  intima 
thus  can  be  used  as  a  measure  of  transvascular  macromolecular  transport.   We 
have  shown  that  the  atherosclerotic  process  is  greatly  enhanced  in  regions  of 
increased  transport.   The  ability  to  quantify  the  topography  of  permeability 
in  the  opened  vascular  interface  with  this  relatively  simple  ref lectometric 
scanning  technique  opens  many  new  areas  in  the  study  of  atherogenesis  here- 
tofore closed. 

Proposed  Course  of  the  Project:   This  methodology  will  be  applied  first 
to  studies  designed  to  quantify  the  correlation  between  the  topography  of 
vascular  permeability  and  the  topography  of  the  atherosclerotic  lesions  in 
animals.   The  second  major  area  of  application  will  be  to  quantify  the 
permeability  changes  that  occur  with  various  altered  hemodynamic  events 
known  also  to  be  related  to  an  increased  atherosclerotic  process.   A  manuscript 
is  in  preparation. 

Keyword  Descriptors 

Evans  blue  dye;  intimal  Evans  blue  dye  accumulation;   optical  absorption 
coefficient  (Evans  blue  dye) . 

Honors  and  Awards 

1.  Invited  speaker  at  meeting  on  Fluid  Dynamic  Aspects  of  Arterial 
Diseases,  Ohio  State  University,  September  18,  1974. 

2.  Invited  speaker  American  Heart  Association,  Council  for  High  Blood 
Pressure  conference  entitled,  "Arteriosclerosis  and  Hypertension:   Observations 
and  Speculation,"   in  Cleveland,  Ohio,  October  12,  1974. 

2  6X1, 


Project  No.  Z01  HL  02802  03  SEA 

3.   Invited  as  discussion  leader  on  Gordon  Research  Conference  on 
Atherosclerosis,  June  1975,  entitled  "Fluid  mechanics,  transport  phenomenon 
and  the  arterial  wall." 

Publications 

None  this  year. 


e&7 


Project  No.  Z01  HL  02803  02  SEA 


1.  Office  of  Director  of  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   The  study  of  arterial  transport  processes  in  an  _in  vitro  life 
support  system. 

Previous  Serial  Number:   NHLI-197 

Principal  Investigator:   Donald  L.  Fry,  M.D, 

Other  Investigator:   Robert  W.  Mahley,  M.D. ,  Ph.D.,  and  Robert  J.  Lutz,  Ph.D. 

Cooperating  Unit:   Biomedical  Engineering  and  Instrumentation  Branch,  Division 
of  Research  Services 

Project  Description 

Objective:   This  is  a  continuing  effort,  the  objective  of  which  is  to 
develop  an  in  vitro  arterial  support  system  for  study  of  transvascular  trans- 
port mechanics  and  short  range  metabolic  mechanisms. 

Methods  Employed:   A  device  has  been  developed  which  consists  of  a  leaded 
glass  chamber,  the  environment  of  which  is  thermostatically  and  humidistatically 
controlled  by  using  appropriate  electronic  feedback  systems.   A  carefully 
removed  arterial  segment  is  placed  in  a   specially  designed  holding  device 
which  permits  control  of  the  mechanical  constraints  on  the  blood  vessel  as 
well  as  anchoring  the  vessel  for  application  of  a  "well  assembly"  and  "pressure 
manifolding  system"  in  the  above  chamber  to  permit  selected  areas  of  the 
opened  vascular  interface  to  be  studied  under  controlled,  chemical,  thermal 
and  mechanical  conditions. 

Major  Findings:   Tissues  studied  in  the  above  system  have  been  found  to 
remain  viable  by  both  physiological  and  ultras true tural  criteria  at  least  two 
hours.   The  tissue  should  be  viable  for  periods  in  excess  of  8  to  12  hours 
with  provision  for  profusion  of  the  adventitial  surface  of  the  vessel  and 
refreshment  of  the  milieu  in  contact  with  the  endothelial  surface.    The 
macromolecular  conductance  of  the  vascular  interface  for  albumin  has  been 
found  to  be  greatly  increased  by  subtle  mechanical  injury,  temperatures  in 
excess  of  40  C,  stretching  the  surface  in  excess  of  200%,  exposing  the  surface 
to  saline,  exposure  to  certain  foreign  proteins,  brief  exposure  to  air,  and 
exposure  to  various  simple  polar  solvents  such  as  ethanol.   Moderate  increases 
in  conductance  were  produced  by  concentrations  of  epinephrine,  noradrenalin, 
and  histamine  in  levels  of  approximately  one  order  of  magnitude  above 
"physiologic"  serum  levels.   Serotonin,  adenosine  diphosphate  do  not  appear 
to  alter  the  wall  conductance. 


aa« 


Project  No.  Z01  HL  02803  02  SEA 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
This  is  an  in  vitro  technique  that  has  been  developed  in  which  the  trans- 
endothelial,  intimal  and  medial  transport  processes  of  arterial  tissue  can 
be  studied  under  controlled  mechanical,  thermal  and  chemical  conditions  for 
periods  of  at  least  two  hours  and  probably  considerably  longer  with  continuous 
refreshment  of  the  milieu  and  purging  of  the  adventitial  surface.   This  degree 
of  experimental  control  is  not  possible  in  the  ±n   vivo  situation  and,  there- 
fore, this  new  methodology  opens  many  new  avenues  for  studying  transport 
mechanics  otherwise  not  available.   Evidence  described  elsewhere  from  this 
laboratory  strongly  suggests  that  increased  interfacial  transport  of  macro- 
molecules  is  one  of  the  central  factors  in  the  atherogenic  process.   Conse- 
quently, this  new  methodology  should  lead  to  important  new  insights  in  this 
disease  process. 

Proposed  Course  of  the  Project:   The  above  device  will  be  further 
developed  so  that  the  studies  may  be  carried  out  for  extended  periods  of 
time  to  permit  evaluation  of  transport  processes  of  substances  having  very 
low  transvascular  flux  such  as  lipoproteins.   The  studies  described  above 
in  which  a  number  of  vasoactive  substances  have  been  screened  for  their 
activity  will  be  continued.   These,  as  well  as  other  substances,  such  as 
angiotension,  other  catecholamines,  divalent  cations,  f ibrinolysin,  various 
proteases,  lipases,  hyaluronidase,  chondroitenase,  neuraminidase  and  so 
forth,  must  be  evaluated  on  a  quantitative  basis  in  view  of  their  potential 
atherogenic  activity.   The  exact  methodology  for  adventitial  purging  is  in 
the  process  of  development  and  will  be  continued. 

Keyword  Descriptors 

Atherosclerosis;  transvascular  transport  mechanics;  endothelial  surface, 
endothelial  permeability;  organ  support  system. 

Honors  and  Awards 

1.  Invited  speaker  at  meeting  on  Fluid  Dynamic  Aspects  of  Arterial 
Disease,  Ohio  State  University,  September  18,  1974. 

2.  Invited  speaker  American  Heart  Association,  Council  for  High  Blood 
Pressure  Research  conference  entitled,  "Arteriosclerosis  and  Hypertension: 
Observations  and  Speculation,"  in  Cleveland,  Ohio,  October  12,  1974. 

3.  Invited  as  discussion  leader  on  Gordon  Research  Conference  on 
Atherosclerosis,  June  1975,  entitled  "Fluid  mechanics,  transport  phenomenon 
and  the  arterial  wall." 

Publications 

None  this  year. 


8*? 


Project  No.  Z01  HL  02804  01  SEA 


1.  Office  of  Director  of  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  arterial  ultrastructure  in  areas  of  increased  and 
decreased  transvascular  transport. 

Previous  Serial  Number:   None 

Principal  Investigator:   Donald  L.  Fry,  M.D. 

Other  Investigators:   None 

Cooperating  Unit:   Tousimis  Research  Laboratory  under  contract  #NIH  73-C-535-CC 

Project  Description 

Objective:  To  establish  the  ultrastructural  characteristics  of  the 
endothelial  surface  and  subendothelial  tissues  in  areas  of  increased  and 
decreased  transendothelial  transport  of  Evans  blue  dye  tagged  albumin. 

Methods  Employed:   The  topographic  distribution  of  increased  trans- 
endothelial albumin  flux  was  determined  using  the  Evans  blue  tagging  technique 
in  four,  two  year  old  foxhounds  and  in  two,  one  year  old  minipigs  by  admin- 
istering the  dye  twenty-four  hours  prior  to  study.   At  the  time  of  study 
each  animal  was  anesthetized  with  pentobarbital,  and  both  the  axillary  arteries 
and  inferior  vena  cava  were  cannulated  for  pressure  profusion  with  buffered 
saline  and  fixation  with  three  percent  buffered  glutaraldehyde  for  one  hour. 
The  arterial  trees  were  then  carefully  dissected  free  and  sets  of  specimens 
were  removed  for  transmission  and  scanning  electron  microscopy  from  areas  of 
increased  transendothelial  albumin  transport  and  from  areas  of  normal 
transport. 

Major  Findings:   By  scanning  electron  microscopy,  the  tissues  from  normal 
transport  areas  showed  a  highly  ordered  endothelial  surface  pattern  with 
distinct  cell  borders  while  tissues  from  high  transport  areas  showed  a  high 
degree  of  disorder  and  pleomorphism  with  poor  definition  of  cell  borders. 
Transmission  electron  microscopy  showed  normal  endothelial  architecture  in 
both  types  of  regions  but  differences  in  the  subendothelial  space.   Normal 
regions  demonstrated  a  more  ordered  and  densely  packed  pattern  of  intimal 
connective  tissue  (collagen,  elastic  fibers,  and  elastin)  whereas  tissues 
from  high  transport  areas  showed  a  much  looser  connective  tissue  pattern 
with  many  open  spaces  characteristic  of  moderate  interstitial  edema. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  localized  development  of  the  atherosclerotic  process  has  been  shown  to 
correspond  to  regions  of  antecedent  increased  transendothelial  transport  of 

1  e& 


Project  No.  Z01  HL  02804  01  SEA 

albumin  and  perhaps  of  other  macromolecules  such  as  lipoproteins.   The  above 
observations  suggest  that  this  disease  process  may  result  from  increased  flux 
of  atherogenic  precursors  across  regions  of  structurally  and  functionally 
abnormal  endothelial  cells. 

Proposed  Course  of  the  Project:   These  data  are  still  in  the  process 
of  analysis.   Studies  are  in  progress  to  define  the  nature  of  the  endothelial 
surface  changes  and  factors  causing  these  changes. 

Keywork  Descriptors 

Endothelial  surface;  atherosclerosis;  endothelial  ultras true ture; 
endothelial  permeability. 

Honors  and  Awards 

1.  Invited  speaker  at  meeting  on  Fluid  Dynamic  Aspects  of  Arterial 
Diseases,  Ohio  State  University,  September  18,  1974. 

2.  Invited  speaker  American  Heart  Association,  Council  for  High  Blood 
Pressure  Research  conference  entitled,  "Arteriosclerosis  and  Hypertension: 
Observations  and  Speculation,"  in  Cleveland,  Ohio,  October  12,  1974. 

3.  Invited  as  discussion  leader  on  Gordon  Research  Conference  on 
Atherosclerosis,  June  1975,  entitled  "Fluid  mechanics,  transport  phenomenon 
and  the  arterial  wall." 

Publications 

None  this  year. 


&/ 


Project  No.   Z01  HL  02805  01  SEA 


1.  Office  of  Director  of  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  mechanics  of  albumin  transport  into  the  arterial  intimal 
medial  space. 

Previous  Serial  Number:   None 

Principal  Investigator:   Donald  L.  Fry,  M.D. 

Other  Investigators:   Robert  W.  Mahley,  M.D.,  Ph.D.,  and  Karl  Weisgraber,  Ph.D. 

Cooperating  Units:   None 

Project  Description 

Objective:   To  establish  a  valid  physical  chemical  model  for  macro- 
molecular  transport  into  the  arterial  wall. 

Methods  Employed:   A  specially  designed  in  vitro  test  system  described 
elsewhere  was  used  to  measure  the  flux  of  125  Iodine  tagged  albumin  and  Evans 
blue  dye  tagged  albumin  into  the  arterial  wall.   This  system  permitted  the 
arterial  surface  to  be  exposed  to  the  tagged  albumin  under  controlled  condi- 
tions for  various  selected  periods  of  time.   Special  experimental  maneuvers 
were  devised  such  that  it  was  possible  to  study  the  uptake  of  albumin,  not 
only  as  a  function  of  time,  but  also  as  a  function  of  wall  stretch  and  of 
transmural  pressure. 

Major  Findings:   The  accumulation  of  tagged  albumin  in  the  arterial  wall 
under  conditions  of  zero  transmural  pressure  was  found  to  vary  with  the 
square  root  of  time.   This  relationship  was  found  not  only  for  1*25  tagged 
albumin  but  also  for  the  accumulation  of  Evans  blue  dye.   In  some  studies 
uptake  was  measured  using  doubly  tagged  albumin  so  that  the  relationship 
between  albumin  uptake  and  Evans  blue  dye  accumulation  could  be  defined 
explicitly.    It  was  found  that  Evans  blue  dye  does  not  alter  the  uptake  of 
jl25  albumin  and  that,  although  both  follow  a  square  root  law,  the 
accumulation  of  Evans  blue  dye  for  all  time  periods  was  approximacely  four 
to  five  times  greater  than  for  the  I 125  tagged  albumin.   Moreovev,  studies 
of  the  rate  with  which  Evans  blue  dye  diffuses  back  out  of  the  intimal 
surface  showed  that  the  rate  -of  elution  in  albumin  was  over  ten  times  that 
in  a  saline  milieu  indicating  that  albumin  acts  as  the  principal,  if  not 
sole,  vehicle  for  transport  of  Evans  blue  dye  in  the  intimal  space  as  well 
as  in  the  blood  phase.   These  data  indicate  that  Fick's  Second  law  of 
Diffusion  in  a  two-phase  system  can  be  used  to  explain  all  of  the  above 
observations,  provided  that,  in  the  case  of  the  albumin-Evans  blue  dye 
complex,  one  incorporates  a  binding  coefficient  to  represent  the  ratio  of 


83> 


Project  No.  Z01  HL  02805  01  SEA 

bound  Evans  blue  dye  to  diffusible  (on  albumin)  Evans  blue  dye  into  the 
intimal  space.   Thus  the  model  for  albumin  transport  in  the  intimal  space 
can  be  represented  by  3c/8T  =  D/ (3+1) 92c/8x2  where  c  is  the  concentration 
of  the  diffusing  molecular  species  of  interest,  D  is  the  coefficient  of 
diffusion  for  albumin  in  the  tissue  space,  3  is  the  aforementioned  binding 
coefficient  which  for  the  Evans  blue  dye-albumin  complex  in  the  wall  would 
be  approximately  15   (6  would  be  zero  for  the  l!25  albumin).   This  model 
represents  the  diffusive  flux  into  the  arterial  wall  that  is  driven  by  an 
electrochemical  gradient. 

In  the  arterial  system,  a  second  driving  force  in  addition  to  the  above 
exists,  i.e.,  the  pressure  acting  across  the  wall.   This  was  studied  by 
measuring  the  uptake  of  tagged  albumin  as  a  function  of  imposed  transmural 
pressure  differences  both  in  positive  and  negative  directions-   Comparison 
of  the  uptake  curve  for  the  positive  transmural  pressure  to  that  for  the 
negative  transmural  pressure  allowed  calculation  of  the  increment  increase 
in  flux  into  the  wall  related  to  the  pressure  alone.   The  pressure  driven 
flux  into  the  wall  was  found  to  be  of  the  same  order  as  that  for  the  steady 
state  diffusive  flux  into  the  wall. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute:  A 
large  body  of  evidence  both  from  this  laboratory  as  well  as  from  others 
indicate  that  the  atherogenic  precursors  probably  come  from  the  blood  phase 
into  the  wall  as  various  complexes  with  proteins.   There  is  also  some  evidence 
that  dissociation  in  the  intimal  space  may  occur  with  preferential  binding 
of  lipid  substances  in  the  tissue  matrix.   While  albumin  itself  can  carry 
both  fatty  acids  and  some  cholesterol,  its  major  interest  to  us  is  related 
to  its  use  as  a  prototype  model  for  macromolecular  transport  and  binding  in 
the  intimal  space.   Thus,  while  of  inherent  interest  in  albumin  and  fatty 
acid  transport,  the  above  studies  begin  for  the  first  time  to  place  macro- 
molecular  transport  in  vascular  tissue  on  firm  physical-chemical  grounds  and 
allow  us  to  sharpen  our  technical,  physical,  and  mathematical  tools  which 
will  be  essential  in  subsequent  studies  of  the  transport  processes  for  the 
atherogenic  precursors  (as  these  become  better  defined) . 

Proposed  Course  of  the  Project:   The  above  studies  will  be  expanded  to 
include  arteries  other  than  the  aorta,  to  explore  the  influence  of  vasoactive 
substances  on  the  albumin  transport  process,  and  to  establish  more  firmly  the 
influence  of  other  physical  parameters  such  as  temperature,  pH,  etc. 

Keyword  Descriptors 

Transvascular  macromolecular  transport  mechanics;  atherosclerosis. 

Honors  and  Awards 

1«   Invited  speaker  at  meeting  on  Fluid  Dynamic  Aspects  of  Arterial 
Disease,  Ohio  State  University,  September  18,  1974. 

2,   Invited  speaker  American  Heart  Association,  Council  for  High  Blood 
Pressure  Research  conference  entitled,  "Arteriosclerosis  and  Hypertension: 
Observations  and  Speculation/'  in  Cleveland,  Ohio,  October  12,  1974. 

2  en 


Project  No.  Z01  HL  02805  01  SEA 

3.   Invited  as  discussion  leader  on  Gordon  Research  Conference  on 
Atherosclerosis,  June  1975,  entitled  "Fluid  mechanics ,  transport  phenomenon 
and  the  arterial  wall." 

Publications 

None  this  year. 


4 

6i* 


Project  No.  Z01  HL  02806  02  SEA 


1.  Office  of  Director  of  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Blood  velocity  profiles  and  hemodynamic  stresses  in  the  aorta 
and  its  major  branches. 

Previous  Serial  Number:   NHLI-208 

Principal  Investigator:   Dali  J.  Patel,  M.D.,  Ph.D. 

Other  Investigators:   H,  Bulent  Atabek,  Ph.D.,  and  Sung  C.  Ling,  Ph.D. 

Cooperating  Units:   The  Department  of  Aerospace  and  Atmosphere  Sciences, 
The  Catholic  University  of  America 

Project  Description 

Objective:   1)  To  measure,  in  vivo,  blood  flow  fields  in  the  left  cir- 
cumflex coronary  artery  (LCCA) ,  and  2)  to  study  the  geometry,  pressure  area 
variations, and  flow  patterns  at  the  aortic  trifurcation  area  in  dogs. 

Methods  Employed:   1)  Pressure,  pressure-gradient,  pressure-radius 
relationships  and  arterial  taper  were  measured  on  the  LCCA  of  anesthetized 
and  open-chested  dogs  under  wide  range  of  blood  pressures  and  flow  rates 
produced  by  intravenous  infusion  of  Persantine.   Velocity  profile,  wall 
shear  and  flow  were  computed  from  these  data  using  a  nonlinear  theory  of 
blood  flow.   Flow  was  also  measured  with  an  electromagnetic  flow  meter  to 
provide  an  independent  check. 

2)   To  determine  geometry  and  pressure-area  relationship  in  the  aortic 
trifurcation  area,  a  16  mm  cine  camera  equipped  with  a  high-power  telephoto 
lens  was  employed.   Motion  pictures  of  the  pulsating  arteries  were  taken. 
These  pictures  also  contained  the  image  of  a  strip  chart  (reflected  by  a  mirror 
into  the  view  field  of  the  camera)   indicating  the  arterial  pressure.   At  the 
end  of  filming  the  animal  was  sacrificed  and  the  region  of  interest  was  filled 
with  a  casting  material  under  the  mean  value  of  the  in  vivo  pressure.   The 
casts  were  taken  out  from  the  animal  after  hardening  and  sliced  at  regular 
intervals.   Enlarged  projections  of  these  slides  were  used  to  determine 
variation  of  shape  and  cross-sectional  area  along  the  aorta,  at  the  junction 
and  along  the  branches.   The  developed  films  were  mounted  on  glass  slides. 
Diameters  were  measured  (with  a  calibrated  reticle  inserted  in  the  microscope 
eyepiece)  and  the  corresponding  pressures  were  determined  from  these  films. 
Resulting  information  was  used  for  determining  the  pressure-area  relation. 

Major  Findings:   Results  for  the  LCCA  indicated  that  (1)  the  flow  profiles 
although  less  blunt  than  those  in  the  descending  thoracic  aorta  were  still 


eisr 


Project  No.  Z01  HL  02806  02  SEA 

nonparabolic;  (2)  wall  shear  in  coronary  arteries  maintained  a  high  value 
through  diastole,  and  (3)  during  intravenous  infusion  of  Persantine  both 
coronary  flow  and  wall  shear  increased.   The  peak  values  of  the  shear  stress 
during  these  infusions  could  approach  the  yield  stress  values  reported  by  Fry 
for  the  endothelial  cells. 

The  study  of  the  trifurcation  area  is  still  in  progress.   The  geometry 
and  the  pressure-radius  relationships  of  this  region  have  been  experimentally 
determined.   The  pressure-flow  relationships  are  currently  under  study. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  role  of  hemodynamic  stresses  in  the   etiology  of  early  atherosclerosis  is 
well  established  by  Fry.   In  order  to  study  this  role  quantitatively  we  need 
to  measure,  ±a   vivo,  flow  fields  in  the  critical  areas  of  the  circulatory 
system.   The  present  study  of  LCCA  and  aortic  trifurcation  area  signifies 
a  modest  step  towards  this  goal. 

Proposed  Course  of  the  Project:   The  LCCA  study  is  completed  and  will  be 
submitted  to  Circulation  Research  for  publication.   The  propagation  of  pressure 
and  flow  in  the  aortic  trifurcation  area  will  be  studied. 

Keyword  Descriptors 

Mechanical  factors  in  atherosclerosis;  local  blood  flow  field;  geometry 
of  critical  areas  of  circulatory  system. 

Honors  and  Awards 

1.  Visited  Bulgaria  as  a  member  of  the  American  delegation  sent  by  the 
National  Academy  of  Sciences  to  visit  various  institutions  and  discuss  common 
research  problems  in  Biomechanics  with  members  of  Bulgarian  Academy  of  Sciences. 

Publications 
None 


636 


Project  No.  Z01  HL  02807  02  SEA 


1.  Office  of  Director  of  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Vascular  mechanics:   arterial  wall  properties 

Previous  Serial  Number:   NHLI-207 

Principal  Investigator:   Dali  J.  Patel,  M.D.,  Ph.D. 

Other  Investigators:   R.  N.  Vaishnav,  Ph.D. 

Cooperating  Units:   Department  of  Civil  and  Mechanical  Engineering,  The 
Catholic  University  of  America,  Washington,  D.  C. 

Project  Description  ' 

Objective:   To  study  local  and  overall  viscoelastic  properties  of  the 
blood  vessel  wall  under  physiologic  condition. 

Methods  Employed :   Two  different  methods  were  developed  to  study  two 
aspects  of  local  rheology  of  the  vascular  intima  and  the  endothelial  surface. 

1)  The  first  method  was  designed  to  study  the  local  compliance  of  the  vascular 
intima.   To  this  end,  a  segment  of  canine  middle  descending  thoracic  aorta 
(MDTA)  was  excised,  slit-open longitundinally  and  stretched  to  in  vivo  dimen- 
sions in  a  stretch-rack.   The  vessel  was  supported  on  the  advential  side  by 

a  plaster-of-Parris  slurry  which  was  allowed  to  harden.   Small  forces  (120  mg. 
typically)  were  then  applied  to  various  locations  on  the  intimal  surface  using 
a  modified  analytical  balance,  one  pan  of  which  was  replaced  by  a  metal  rod  with 
a  prodder  tip  with  0.19  mm  diameter.   The  displacement  due  to  indentation 
caused  by  the  tip  was  measured  as  a  function  of  time  by  means  of  a  differential 
transformer.   The  indentation  at  the  end  of  30  seconds  (630)  was  considered  to 
be  indicative  of  the  local  compliance  of  the  intima  and  its  substructure.   A 
considerable  number  of  segments  were  tested  using  this  method  at  various  sites 
on  the  intima  including  the  regions  adjacent  to  intercostal  orifices. 

2)  The  second  method  was  designed  to  study  the  strength  of  the  endothelium 
using  saline  jets.   An  instrument  was  designed  to  apply  jets  at  specific  sites 
on  the  endothelium  by  modifying  a  microscope  with  turret-mounted  objectives, 
one  of  which  was  replaced  by  a  jet  nozzle  (diam.  0.4  mm).   A  carefully 
handled  aortic  segment  was  stretched  as  above  in  a  stretch  rack  and  backed  by 
a  piece  of  lucite.   Controlled  jets  of  saline  were  then  applied  to  the  endo- 
thelium for  various  durations  and  the  resulting  lesions  made  visible  by 
staining  the  tissue  subsequently  with  Evans  blue  dye. 

The  overall  anisotropic,  nonlinear  viscoelastic  properties  of  tissue 
were  studied  using  segments  of  canine  MDTA  and  carotid  artery  and  canine 


837 


Project  No.  Z01  HL  02807  02  SEA 

jugular  and  human  saphenous  veins.   In  some  instances  the  veins  had  been 
transplanted  in  the  arterial  circulation  for  varying  periods  prior  to 
study.   The  method  in  this  case  consisted  of  applying  various  levels  of 
intravascular  pressure  and  longitudinal  force  to  the  segment  and  measuring 
photographically  the  resulting  dimensions. 

Major  Findings;   The  findings  on  the  local  compliance  of  the  vascular 
intima  have  been  published  (see  publications).   Specifically,  the  regions 
adjacent  to  the  intercostal  orifices  were  found  to  be  less  compliant  than 
the  regions  far  away  from  the  orifices.   This  confirms  the  histological 
finding  of  the  existence  of  collagen-rich  intimal  pads  in  the  orifice  regions. 
From  the  study  of  the  lesions  created  by  saline  jets  it  was  found  that  the  ' 
endothelium  could  resist  high  normal  stress  but  was  susceptible  to  damage  by 
relatively  low  shear  stress.   This  was  evident  by  the  fact  that  an  intermediate 
jet  strength  gave  rise  to  an  annular  lesion  consisting  .of  an  undamaged  core 
where  the  jet  hit  directly  and  a  ring  of  damaged  endothelium  where  the  shear- 
ing stress  due  to  jet  efflux  exceeded  the  endothelial  yield  strength. 
Experiments  are  underway  to  refine  the  method  and  to  quantify  the  endothelial 
yield  strength  using  this  method. 

The  work  on  the  overall  viscoelastic  properties  of  artery  yielded  10 
arterial  relaxation  functions  which  fully  describe  the  nonlinear  viscoelastic 
properties  of  arteries  under  physiologic  loading  conditions.   From  the  work 
on  veins,  it  was  found  that  the  elastic  behavior  of  veins  is  markedly  different 
from  that  of  artery,  veins  becoming  highly  nondistensible  at  arterial  pressure 
but,  nonetheless,  being  capable  of  resisting  these  pressures.   A  paper  on  elastic 
properties  of  venous  segments  has  been  submitted  for  publication  to  Circulation 
Research. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
Recent  findings  of  Fry  indicate  that  hemodynamic  and  local  tissue  factors 
probably  play  a  significant  role  in  the  pathogenesis  of  atherosclerosis.   Hemo- 
dynamic shear  stresses,  for  example,  can  alter  the  permeability  of  the  endo- 
thelium to  the  macromolecules  in  the  blood.   The  jet  studies  should  provide 
valuable  information  on  the  strength  of  the  endothelial  surface  and  its 
permeability  to  macromolecules.   Likewise,  it  has  been  observed  that  regions 
of  the  vascular  intima  subjected  to  chronic  high  undirectional  stresses  appear 
to  develop  densely  oriented  collagen   in  the  subendothelial  region  which 
appears  to  protect  them  from  subsequent  lipid  deposition.   The  studies  on 
local  compliance  of  the  vascular  intima  reported  above  are  significant  in 
that  they  provide  a  rheological  counterpart  to  the  histological  finding 
stated  above.   The  work  on  overall  viscoelastic  properties  is  the  most  general 
characterization  of  the  mechanical  properties  of  arteries  available  to  date 
and  should  help  in  studying  effect  of  aging,  disease  process  and  pharmacologic 
agents  on  tissue  properties.   The  work  on  veins  has  considerable  clinical 
significance  in  surgical  procedures  to  replace  diseased  arterial  segments. 

Keywork  Descriptors 

Mechanical  factors  in  atherosclerosis;  endothelial  surface  strength; 
local  blood  vessel  properties. 

2  €38 


Project  No.  Z01  HL  02807  02  SEA 

Honors  and  Awards 

1.  Participated  in  a  symposium  on  "Recent  Advances  in  Blood  Rheology 
and  Hemodynamics,"  held  during  the  26th  International  Congress  on  Physiological 
Sciences  in  New  Delhi,  India,  1974. 

2.  Participated  in  a  satellite  conference  to  the  26th  International 
Congress  of  physiological  sciences  entitled   "Circulatory  and  Metabolic 
Adaptations  to  Stress,"  in  Bombay,  India,  1974. 

3.  Invited  to  present  a  paper  entitled  "Nonlinear  Viscoelastic 
Properties  of  Large  Blood  Vessels,"   International  Conference  on  Cardio- 
vascular System  Dynamics,  Valley  Forge,  Pa.,  April  8,  1975. 

Publications 

1.  Janicki,  J.S.,  Patel,  D.J.,  Young,  J.T.,  and  Vaishnav,  R.N. : 
Rheologic  properties  of  blood  vessels.   In  Research  Animals  in  Medicine. 

DHEW  Publication  No.  (NIH)  72-333,  Wash.,  D.C.,  pgs.  573-582,  1973. (Publ. , 1974) 

2.  Gow,  B.S.,  Schonfeld,  D.,  and  Patel,  D.J.:   The  dynamic  elastic' 
properties  of  the  canine  left  circumflex  coronary  artery.   J.  Biomechanics, 
7:389-395,  1974. 

3.  Patel,  D.J.  and  Vaishnav,  R.N. :  Rheology  of  blood  vessels.  Pro- 
ceedings of  the  International  Union  of  Physiological  Sciences,  Vol.  X,  XXVI 
International  Congress,  New  Delhi,  1974,  pp.  82-83. 

4.  Gow,  B.S.  and  Vaishnav,  R.N. :  A  microindentation  technique  to 
measure  rheological  properties  of  the  vascular  intima.   J.  Appl.  Physiol., 
Vol.  38,  No.  2,  February  1975,  p.  344-350, 

5.  Plowman,  F. ,  Young,  J.T.,  and  Janicki,  J.S.  :   An  instrument  for 
dynamic  measurement  of  longitudinal  stresses  and  strains  in  a  blood  vessel 
in  vivo.    Biorheology  (in  press). 


esf 


Project  No.  Z01  HL  02808  02  SEA 


1.  Office  of  Director  of  Intramural  Res. 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Trial  of  psychophysiologic  techniques  for  the  amelioration 
of  hypertension. 

Previous  Serial  Number:   NHLI  206  (c) 

Principal  Investigators:   Bernard  L.  Frankel,  M.D.;  Dali  J.  Patel,M.D. ,Ph.D. ; 

David  Horwitz,  M.D. 

Other  Investigators:   William  T.  Friedewald,  M.D.;  Edward  Freis,  M.D. 

Cooperating  Units:   Hypertension  Endocrine  Branch,  NHLI;  Clinical  Trials 
Branch,  NHLI;  Veterans  Administration  Hospital. 

Project  Description 

Objective:   Studies  reporting  that  blood  pressure  (BP)  can  be  altered 
by  psychophysiologic  techniques  such  as  biofeedback,  autogenic  exercises,  and 
hypnosis  have  not  provided  definitive  information  on  how  long  the  effects 
were  sustained  at  useful  levels  and  usually  were  not  adequately  controlled. 
The  present  study  is  designed  to  determine  whether  a  combination  of  psycho- 
physiologic techniques  can  produce  a  sustained,  therapeutically  useful 
reduction  in  BP  in  patients  with  essential  hypertension. 

Methods:   The  study  group  will  consist  of  30  patients  with  uncomplicated 
essential  hypertension  who  show  mean  diastolic  BP  levels  of  90  to  105  while 
supine.   Patients  undergo  an  initial  eight  week  period  of  evaluation  during 
which  BP  is  taken  at  least  once  a  week  by  a  nurse-observer  blind  to  the 
patient's  experimental  status  throughout  the  study.   Thereafter,  patients  enter 
a  sixteen-week  study  period  during  which  weekly  BP  determinations  continue. 
They  are  randomly  allocated  to  one  of  three  groups: 

1)  Active  Treatment  (AT)  Group  -  In  20  laboratory  sessions,  AT 
patients  are  trained  in  the  use  of  electromyographic  and/or  skin  temperature 
biofeedback  and  diastolic  BP  feedback.   They  also  learn  autogenic  exercises, 
relaxation  training  and  auto-suggestion.   During  laboratory  training  sessions, 
pulse,  BP,  finger  temperature,  and  frontalis  muscle  activity  are  monitored 
noninvasively.    These  patients  also  pursue  a  monitored  daily  home  practice 
program  to  reinforce  and  generalize  laboratory  learning. 

2)  Pseudo- Treatment  (PT)  Group  -  Patients  receive  only  pseudo- 
diastolic  BP  feedback  arranged  to  convey  a  sense  of  success  to  the  patient. 
They  are  seen  as  frequently  as  the  AT  patients.   Physiologic  monitoring  is 
the  same  as  for  the  AT  patients-  but  home  practice  is  omitted. 


Project  No.  Z01  HL  02808  02  SEA 

3)   No-Treatment  Control  (NTC)  Group  -  NTC  patients  participate  in  a 
minimal  program  consisting  of  weekly  BP  determinations  by  the  nurse-observer 
for  16  weeks. 

Second  Phase  Studies:   PT  and  NTC  patients  are  offered  the  opportunity 
to  participate  in  the  AT  protocol  after  completing  their  initial  sixteen-week 
period,  each  patient  serving  as  his  own  control.   Patients  in  any  of  the 
three  groups  having  at  least  a  ten  percent  fall  in  mean  diastolic  BP  are 
studied  for  an  additional  two  months  to  determine  if  this  decrease  is  sus- 
tained.  Comparisons  are  based  on  the  findings  in  the  last  six  weeks  of 
each  period. 

Associated  Observations;   Prospective  observations  of  psychological  and 
hemodynamic  status  are  performed  initially.   Patients  undergo  a  clinical 
interview,  standard  psychological  tests,  and  determinations  of  cardiac  output 
utilizing  a  CO„-rebreathing  technique.   Findings  will  be  correlated  with 
clinical  success  in  an  attempt  to  delineate  the  characteristics  predictive 
of  success. 

Maj or  Findings :   Studies  of  ten  patients  have  been  completed;  two 
were  in  the  active  treatment  group,  three  in  the  pseudo-treatment  group,  and 
five  in  the  no-treatment  control  group.   Of  the  two  AT  patients,  one  has 
shown  a  modest  decrease  in  mean  post-AT  diastolic  BP  (from  93.7  to  90.5  mm  Hg) 
whereas  the  other  was  a  treatment  failure.   One  of  the  three  PT  patients 
showed  a  modest  decrease  in  mean  post-PT  diastolic  BP  (from  104.3  to  100  mm  Hg); 
he,  thereafter,  participated  in  active  treatment  for  sixteen  more  weeks  and 
had  a  further  comparable  decrease  (from  100  to  95.8  mm  Hg) .   The  two  other 
PT  patients  showed  no  post-PT  changes  in  diastolic  BP;  one  continued  with  the 
AT  protocol  and  showed  an  appreciable  post-AT  clinical  response  (from  94.7  to 
86.7  mm  Hg) .   There  were  no  BP  changes  in  the  five  NTC  patients.   The  BP  of 
one  of  these  five  then  also  remained  unchanged  after  subsequent  participation 
in  the  AT  protocol.   Ten  additional  patients  are  actively  involved  in  the 
s  tudy . 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
These  preliminary  findings  suggest  that  psychophysiologic  techniques  can 
reduce  BP  in  some  subjects.   This  is  the  first  study  designed  with  adequate 
controls  to  evaluate  the  specificity  of  such  BP  alterations  and  to  establish 
whether  they  have  the  magnitude  and  persistence  to  make  them  clinically  use- 
ful. 

Keyword  Descriptors 

Hypertension,  psychophysiology,  biofeedback. 

Honors  and  Awards :   None 

Publications:   None 


gv/ 


Project  No.  Z01  HL  02809  01  SEA 


1.  Office  of  Director  on  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Quantitation  of  the  apolipoproteins  in  plasma  by  two- 
dimensional  Immunoelectrophoresis . 

Previous  Serial  Number:   None 

Principal  Investigator:   Robert  W.  Mahley,  M.D. ,  Ph.D. 

Other  Investigator:   Thomas  P.  Bersot,  M.D.,  Ph.D, 

Cooperating  Units:   None 

Project  Description 

Objective:   To  determine  changes  in  the  distribution  of  apolipoproteins 
in  the  plasma  of  control  and  cholesterol  fed  animals  and  man.   Also  to  monitor 
changes  in  cholesterol  induced  hyperlipoproteinemia  following  hypolipidemic 
drug  therapy. 

Methods  Employed:   Monospecific  antisera  has  been  prepared  to  the 
'arginine-rich,'r  A-l,  and  B-apoproteins  of  the  rat  lipoproteins  and  are  in 
preparation  for  dog,  miniature  swine  and  human  apolipoproteins.   Laurell's 
two-dimensional  quantitative  immunoelectrophoretic  procedures  as  revised  by 
Versey  and  Davis  has  been  modified  for  lipoprotein  apoprotein  quantitation. 
Our  modification  of  the  method,  which  includes  delipidation  of  samples  by 
triton,  has  overcome  a  major  difficulty  of  apoprotein  quantitation,  i.e., 
failure  to  analyze  hypertriglyceridemic  plasma  because  of  the  presence  of 
large,  poorly  migrating  lipoproteins.   In  addition  to  allowing  quantitation 
of  all  plasma  samples,  delipidation  also  reduced  the  possibility  of  masked 
antigenicity. 

The  rat  plasma  lipoproteins  are  the  principal  lipoproteins  under  inves- 
tigation at  this  time.   The  plasma  lipoproteins  of  rats  are  fractionated  by 
ultracentrifugation  into  four  major  density  classes  -  d< 1.006  (VLDL) ; 
1.006-1.019  (intermediate);  1.019-1.063  (LDL  and  HDI^  or  HDLC)  and  1.063- 
1.21  (HDL2) .   The  quantitative  distribution  of  the  various  apoproteins  is 
determined  in  each  ultracentrif ugal  fraction  for  control  chow  fed  animals 
vs.  rats  on  various  hypercholesterolemic  diets.   The  hypercholesterolemic 
diets  contain  lard  and  cholesterol  plus  a  bile  acid  (either  cholic  acid  or 
taurocholate) .   In  addition,  some  of  the  animals  also  receive  propyl 
thiouracil  (PTU) .   The  alterations  in  lipoprotein  metabolism  following 
administration  of  hypolipidemic  drugs  is  determined  by  analysis  of  changes 
in  the  apolipoprotein  distribution. 


g<^ 


Project  No.  Z01  HI  02809  01  SEA 

Major  Finding:   Using  the  two-dimensional  quantitative  electrophoretic 
procedure  as  modified,  it  has  been  determined  that  cholesterol  feeding  in 
association  with  taurocholate  and  PTU  results  in  a  five-fold  elevation  of 
the  "arginine-rich"  apoprotein  in  plasma.   The  increase  in  the  "arginine- 
rich"  apoprotein  is  associated  with  the  occurrence  of  the  beta-VLDL  in  the 
d<1.006  fraction.   Administration  of  an  experimental  drug  supplied  by  the 
Upjohn  Company  (U-41,792)  results  in  a  reduction  of  the  "arginine-rich" 
apoprotein  in  the  d<1.006  fraction  and  an  increase  in  the  HDL  lipoprotein. 

Significance  to  Biomedical  Research  and  the  Program  of  the  Institute: 
A  relatively  simple  and  rapid  quantitative  method  for  apolipoprotein  analysis 
provides  a  useful  tool  to  monitor  changes  in  plasma  lipoproteins  induced  by 
dietary  manipulations  and  drug  therapy.   These  studies  will  provide  insight 
into  changes  in  lipoprotein  metabolism  associated  with  the  development  of 
atherosclerosis  following  cholesterol  feeding  or  the  reversal  of  athero- 
sclerosis following  drug  therapy. 

Proposed  Course:   The  project  will  continue  along  the  lines  indicated 
above.   The  methods  will  be  extended  to  the  analysis  of  human  lipoprotein 
changes  induced  by  diet  and  drug  therapy. 

Keyword  Descriptors 

Quantitative  Immunoelectrophoresis;  apolipoproteins;  dietary  manipulation; 
drug  therapy;  experimental  animals;  hypolipidemic  drugs. 

Honors  and  Awards 

None 
Publications 

None 


e>& 


Project  No.  Z01  HL  02810  04  SEA 


1.  Office  of  Director  on  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Hyperlipoproteinemia  and  atherosclerosis:   changes  in  plasma 
lipoproteins  and  apolipoproteins  induced  by  cholesterol 
feeding  in  dogs,  swine,  rats,  rabbits,  and  Patas  monkeys . 

Previous  Serial  Number:   NHLI  201 

Principal  Investigator:   Robert  W.  Mahley,  M.D, ,  Ph.D. 

Other  Investigators:   Karl  H.  Weisgraber,  Ph.D.,  and  Donald  L.  Fry,  M.D. 

Cooperating  Units:   NHLI  Contract  #N01  HI-3-2926,  Meloy  Laboratories, 
Springfield,  Virginia. 

Project  Description 

Objectives:   1)  To  characterize  the  lipoproteins  and  apoproteins  from 
control  dogs,  miniature  swine,  Patas  monkeys,  rats,  and  rabbits,  and  to 
compare  these  to  changes  induced  by  cholesterol  feeding.   2)  To  correlate  the 
type  of  hyperlipoproteinemia  with  the  type,  distribution  and  degree  of 
atherosclerosis . 

Methods  Employed:   The  various  animal  species  indicated  above  are  fed 
diets  which  contain  0.5  to  2.0%  cholesterol  as  described  previously. 
Isolation  of  the  plasma  lipoproteins  is  accomplished  by  the  combination  of 
ultracentrifugation  and  Geon-Pevikon  block  electrophoresis.   The  purified 
lipoproteins  were  characterized  with  respect  to  electrophoretic  mobility, 
immunochemical  reactivity,  size  by  electron  microscopy,  chemical  composition 
and  apoproteins.   The  apoproteins  are  isolated  and  purified  by  Sephadex 
and  DEAE  column  chromatography.   Analyses  of  the  apoproteins  include  amino 
acid  analysis,  N-  and  C-terminal  amino  acids,  and  molecular  weights. 

Major  Findings:   Dogs,  miniature  swine,  rats,  rabbits  and  Patas  monkeys 
fed  high  cholesterol  diets  have  a  similar  lipoprotein  response  which  is 
associated  with  the  development  of  atherosclerosis.   Animals  on  a  low 
cholesterol  diet  serve  as  controls.   The  characteristics  of  the  Hyperlipo- 
proteinemia associated  with  atherosclerosis  are  as  follows:   1)  The  beta-VLDL 
become  prominent  lipoproteins.   The  B-VLDL  are  beta  migrating  lipoproteins 
in  the  d< 1.006  fraction  which  resemble  the  beta-VLDL  of  human  type  III 
hyperlipoproteinemia  particularly  with  respect  to  the  prominence  of  the 
"arginine-rich"  apoprotein.   2)  LDL  and  the  intermediate  lipoproteins  (IDL) 
are  present  in  increased  concentrations  and  are  variably  enriched  in  the 
"arginine-rich"  apoprotein.   These  lipoproteins  and  the  beta-VLDL  may 


&w 


Project  No.  Z01  HL  02810  04  SEA 


represent  remnants  of  intestinal  lipoproteins  induced  to  transport  the 
dietary  lipid.   3)  A  unique  class  of  lipoproteins,  which  we  have  called 
HDL  ,  are  a  consistent  feature  following  cholesterol  feeding.   These  lipo- 
proteins   are  cholesterol-rich  and  contain  the  "arginine-rich"  apoprotein 
and  A-l.   They  lack  the  B-apoprotein.   The  HDL  are  important  regulators  of 
sterol  synthesis  in  aortic  smooth  muscle  cells  and  fibroblasts  (See  project 
report  entitled, "Tissue  culture  studies  of  aortic  smooth  muscle  cells  and  skin 
fibroblasts:  cell  growth  and  metabolism  in  response  to  incubation  with  various 
lipoprotein  classes." 

Characterization  of  the  "arginine-rich"  apoprotein  suggest  homology  of 
this  protein  among  the  species.   They  have  a  similar  amino  acid  analysis 
and  contain  12  moles%  arginine.   The  "arginine-rich"  apoprotein  appears  to 
play  an  essential  role  in  cholesterol  transport  between  lipoproteins  and 
possibly  between  lipoprotein  and  the  aortic  wall  (atherosclerotic  lesion) . 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
Characterization  of  cholesterol  induced  hyperlipoproteinemias  and  develop- 
ment of  animal  models  resembling  human  disease  will  enable  us  to  better 
understand  human  lipoprotein  metabolism.   In  addition,  these  studies  are 
designed  to  correlate  the  type  of  hyperlipoproteinemia  with  the  type, 
distribution  and  degree  of  experimentally  induced  atherosclerosis. 

Keyword  Descriptors 

Atherosclerosis;  lipoproteins;  cholesterol;  diet;  dogs;  swine;  rats; 
rabbits;  monkeys. 

Honors  and  Awards 

Invited  speaker  at  Lipid  Metabolism  Gordon  Research  Conference, 
entitled  "Atherogenic  and  Non-Atherogenic  Hyperlipoproteinemia  Induced  by 
Cholesterol  Feeding  in  Dogs." 

Invited  lecturer  at  Bowman-Gray  Medical  Center,  entitled  "Cholesterol 
Induced  Hyperlipoproteinemia  and  Atherosclerosis," 

Publications 

1.  Mahley,  R.W. ,  and  Weisgraber,  K,H, :  Canine  lipoproteins  and  athero- 
sclerosis.  I.   Isolation  and  characterization  of  plasma  lipoproteins  from 
control  dogs.   Circ.  Res.  35:  713,  1974. 

2.  Mahley,  R.W.,  Weisgraber,  K.H.,  and  Innerarity,  T. :   Canine  lipo- 
proteins and  atherosclerosis.   II.   Characterization  of  the  plasms  lipoproteins 
associated  with  atherogenic  and  non-atherogenic  hyperlipidemia.   Circ.  Res. 

35:  722,  1974. 

3.  Mahley,  R.W.,  Weisgraber,  K.H.,  Innerarity,  T.,  Brewer,  H.B.  and 
Assmann, -G. :  Swine  lipoproteins  and  atherosclerosis.   Changes  in  the  plasma 
lipoproteins  and  apoproteins  induced  by  cholesterol  feeding.   Biochemistry 
(In  press) . 


8f5~ 


Project  No.  Z01  HL  02811  04  SEA 

1.  Office  of  Director  and  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Aortic  metabolism  of  plasma  lipoproteins. 

Previous  Serial  Number:   NHLI  202 

Principal  Investigator:   Robert  W.  Mahley,  M.D.,  Ph.D. 

Other  Investigators:   Donald  L.  Fry,  M.D.,  Karl  H.  Weisgraber,  Ph.D. 

Cooperating  Units:   None 

Project  Description 

Objective:   To  determine  1)  which  classes  of  plasma  lipoproteins  are 
involved  in  aortic  transport;  2)  whether  these  lipoproteins  are  transported 
as  intact  macromolecules  or  hydrolyzed  at  the  surface  and  components  trans- 
ported separately;  3)  the  fate  of  the  lipoprotein  components  metabolized  by 
the  aorta. 

Methods  Employed:   The  in  vitro  transport  method  (described  by  Dr.  D.  L. 
Fry)  is  used  to  study  aortic  endothelial  transport  of  the  plasma  lipoproteins 
under  controlled  conditions.   Presently  the  dog  is  being  used  as  the  experi- 
mental model  but  we  will  soon  extend  this  to  the  miniature  swine  as  well. 
The  components  of  canine  plasma  lipoproteins  (VLDL,  LDL,  HDL}  and  HDL2)  are 
labelled  with  various  radioisotopes.   Initially  we  attempted  to  use 
*2->I  as  our  protein  tag.   After  exhaustive  studies  we  conclude  that  it  is 
impossible  to  limit  the  125i  to  the  protein  moieties, and  variable  amounts 
of  lipids  are  labelled.   The  125j  lipid  label  is  unstable.   We  are  now  able 
to  label  the  protein  moieties  of  the  canine  lipoproteins  with  35s-methionine 
in  vivo.   At  the  same  time  we  are  able  to  label  the  phospholipids  with  ->^P 
orthophosphate.   l^C-cholesteryl-esters  and  -%-free  cholesterol  moieties  of 
these  lipoproteins  are  labelled  in  vitro  by  the  exchange  method  of  Avigan. 
Uptake  of  label  and  the  metabolism  of  the  lipoproteins  are  followed  by 
analysis  of  changes  in  the  incubation  media,  examination  of  the  aorta  by 
direct  isotope  counting  following  oxygen  combustion  (as  described  in  a 
separate  project  report)  and  by  light  and  electron  microscopic  autoradio- 
graphy . 

Major  Findings:   Preliminary  findings  indicate  the  feasibility  of  this 
approach  to  the  study  of  aortic  endothelial  transport  and  metabolism  of 
plasma  lipoprotein.   Methodologic  problems  and  validation  of  techniques 
continues  to  be  a  major  component  of  this  project.   In  addition  to  validation 
of  the  in  vitro  technique  (described  in  a  separate  project  report  by  Dr.  D. 
L.  Fry),  methodology  for  the  quantitation  of  four  separate  radioisotopes 

1  e<& 


Project  No.  Z01  HL  02811  04  SEA 

(  C,   H,    S  and   P)  has  been  established.   The  labelled  plasma  lipopro- 
teins or  a  dried  portion  of  the  aorta  following  an  in  vitro  transport  study 
are  placed  in  an  oxygen  combustion  flask  and  ignited,   35s-methionine  which 
is  the  protein  tag  and  ^2p  which  is  the  phospholipid  tag  are  converted  to 
inorganic  sulfate  and  phosphate,  respectively.   These  isotopes  remain  in  the 
flask  and  are  quantitated  together  by  standard  double  label  liquid 
scintillation  counting.   The  flask  is  heated  to  drive  off  the  tritium  in 
the  form  of  3H2°  and   c  in  the  form  of  1^C02.   The  14C02  is  collected  by 
bubbling  the  gas  through  a  base  converting  it  to  an  insoluble  carbonate  and 
the  3H20  is  collected  on  a  condenser  in  an  ice  bath.   Isotope  recovery  is 
greater  than  90%  and  modifications  are  being  made  to  increase  the  efficiency 
of  the  method. 

Significance  to  Bio-medical ^Research  and  the  Program  of  the  Institute; 
It  is  agreed  by  most  that  1)  cholesterol  within  atheromata  is  derived  largely 
from  plasma  lipoproteins  and  that  2)  plasma  lipoproteins  can  be  detected 
within  the  same  lesions.   However,  the  mode  of  transport  and  the  quantitative 
significance  of  lipoproteins  in  the  lesions  are  far  from  clear.   Serious  ' 
questions  remain  as  to  whether  the  lipoproteins  cross  the  endothelial  surface 
intact  or  whether  they  are  hydrolyzed  at  the  surface  with  only  some  of  the 
components  entering  the  tissue.   Our  in  vitro  approach  to  this  problem  should 
shed  light  on  this  most  difficult  problem  of  atherosclerosis  research  as  well 
as  add  to  our  knowledge  of  lipoprotein  metabolism. 

Proposed  Course  of  the  Project:   The  project  will  be  continued  along 
the  lines  indicated  above.   It  will  also  be  extended  to  the  miniature  swine 
and  nonhuman  primates  for  comparative  studies. 

Keyword  Descriptors 

Atherosclerosis;  lipoproteins;  endothelial  transport;  aorta  transport; 
lipid  metabolism. 

Honors  and  Awards 

None 
Publications 

None 


e*7 


Project  No.  Z01  HL  02812  06  SEA 


1.  Office  of  Director  on  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 


PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Animal  models  for  study  of  atherosclerosis. 

Previous  Serial  Number:   NIH  198 

Principal  Investigators:   Robert  W.  Mahley,  M.D.,  Ph.D.;  Donald  L.  Fry,  M.D. 

Other  Investigators:   Victor  J.  Ferrans,  M.D. ,  Ph.D.;  Joseph  E.  Pierce,  DVM; 
Jere  M.  Phillips,  DVM  and  David  K.  Johnson,  DVM 

Cooperating  Units:   Section  on  Pathology,  NHL I;  Section  on  Laboratory  Animal 
Medicine  and  Surgery,  NHLI;  Veterinary  Resources  Branch, 
Division  of  Research  Services;  University  of  Missouri 
(Research  Contract  #N01-HI- 3-2947) ;  and  Colorado  State 
University  (Research  Contract  #N01-HI-4-2903) 

Project  Description 

Objective:   To  determine  the  suitability  of  a  variety  of  animals  as  models 
for  atherosclerosis  as  compared  to  the  human  disease. 

Methods  Employed:   The  animal  models  which  have  been  studied  in  varying 
detail  are  the  Patas  monkey,  miniature  pig,  dog,  rabbit,  and  rat.   The 
experimental  conditions  under  which  the  pathologic  processes  in  these  animals 
can  be  made  to  resemble  those  in  man  have  been  detailed  in  previous  project 
reports,   including  those  referenced  above,  and  therefore  will  not  be 
described  in  detail  here.   Briefly,  the  disease  can  be  induced  in  the  rabbit, 
pig,  and  monkey,  by  feeding  diets  high  in  cholesterol  and  fat  (lard);  whereas 
disease  can  be  induced  in  the  dog  and  rat  to  a  comparable  extent  only  if 
hypothyroidism  is  also  induced.   In  dogs  the  types  of  dietary  fats  are  varied 
and  included  Wesson  oil,  pork  lard,  beef  tallow,  safflower  oil  and  peanut  oil. 

Blood  chemistries,  which  includes  detailed  lipoprotein  studies,  are 
monitored  druing  the  experimental  period.   At  termination  each  animal  is 
examined  in  detail  using  the  standardized  necropsy  procedures  as  described 
previously.   Topographic  distribution  and  histologic  characteristics  of  aortic, 
coronary,  and  peripheral  arteries  are  compared  to  human  atherosclerosis.   The 
comparative  human  material  is- derived  from  young  adults  dying  tramatic  deaths, 
unselected  hospital  cases,  and  patients  with  documented  types  of  hyperlipopro- 
teinemia. 

Major  Findings:   Dietary  induced  atherosclerosis  in  our  principal  animal 
models  -  the  dog,  swine  and  monkey  -  have  features  which  are  strikingly  similar 
to  that  in  man.   The  topographic  distribution  of  lesions  in  all  species  formed 

i  we 


Project  No.  Z01  HL  02812  06  SEA 

a  relatively  characteristic  and  predictable  pattern  similar  to  that  in  man. 
Microscopic  analysis  showed  early  disease  to  be  characterized  by  fatty 
streaking  with  only  moderate  intimal  f ibromuscular  hyperplasia;  whereas  with 
longer  duration  of  elevated  serum  lipids  marked  intimal  f ibromuscular  hyper- 
plasia occurred  with  the  development  of  cell  death,  deposition  of  extra- 
cellular lipids  and  cholesterol  crystals.   Complicated  disease  appeared  with 
the  formation  of  atheromatous  gruel  under  fibrotic  caps,  calcification  and 
monocytic  infiltration.   The  degree  of  disease  in  all  the  animals  is  directly 
correlated  with  the  plasma  cholesterol  level  and  the  appearance  of  a  distinctive 
hyperlipoproteinemia.   (Subject  of  progress  report  entitled,  "Hyperlipo- 
proteinemia and  atherosclerosis.   Changes  in  plasma  lipoproteins  and  apolipo- 
proteins  induced  by  cholesterol  feeding  in  dogs,  swine,  rats,  rabbits,  and 
Patas  monkeys. ") 

The  source  of  the  dietary  fat  appears  to  be  an  important  determinant  of  the 
distribution  and  morphologic  characteristics  of  the  disease  in  the  dog.   When 
beef  tallow  is  substituted  for  pork  lard  as  the  principal  dietary  fat,  the 
disease  becomes  much  more  severe  and  complicated  in  a  shorter  period  of  time 
with  the  development  on  many  of  the  secondary  complications  as  described  in 
man.   These  complications  include  ulceration,  thrombosis  and  embolism.   Coronary 
artery  disease  with  arterial  occlusion  and  thrombosis  is  also  accentuated  when 
beef  tallow  is  the  source  of  dietary  fat.   The  role  of  dietary  fat,  as  a 
determinant  of  the  type  and  severity  of  atherosclerosis,  is  being  explored 
further  in  dogs  and  extended  to  swine,  monkeys  and  rats. 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute: 
The  "atherosclerotic  process"  is,  in  fact,  an  ensemble  of  processes  occurring 
at  the  cellular,  physicochemical,  biochemical,  and  biophysical  level  in  the 
arterial  intima.   The  purpose  of  this  program  is  to  identify  as  many  of  these 
fundamental  processes  as  possible,  establish  which  are  relevant  to  those  in 
man,  and  study  these  in  great  detail  in  the  animal  model,  wherein  the  pertinent 
variables  can  be  measured  or  controlled  with  a  rigor  not  possible  in  man. 

A  clear  definition  of  the  role  of  diet  in  the  development  of  atherosclerosis 
in  man  is  of  utmost  importance. 

Proposed  Course  of  the  Project:  The  pursuit  of  the  above  objectives  will 
continue  both  at  NIH  in  collaboration  with  the  above  mentioned  cooperating 
units  and  with  our  contractor  at  Colorado  State  University. 

Keyword  Descriptors 

Animal  models;  atherosclerosis;  swine;  dogs;  rats;  rabbits;  monkeys; 
lipoproteins;  cholesterol. 

Honors  and  Awards 

Invited  speaker  at  the  Deuel  Conference  on  Lipid  Metabolism,  Carmel,  Calif, 
entitled,  "Suitability  of  Animal  Models  for  Studies  of  Atherosclerosis," 
(Robert  W.  Mahley) . 

Publications:   None 

.   2  8*1 


Project  No.  Z01  HL  02813  03  SEA 


1.  Office  of  Director  of  Intramural  Research 

2.  Section  on  Experimental  Atherosclerosis 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Tissue  culture  studies  of  aortic  smooth  muscle  cells  and  skin 
fibroblasts:   cell  growth  and  metabolism  in  response  to 
incubation  with  various  lipoprotein  classes. 

Previous  Serial  Number:   NHLI-255 

Principal  Investigator:   Thomas  F.  Bersot,  M.D.,  Ph.D. 

Other  Investigators:   Robert  W.  Mahley,  M.D.,  Ph.D.,  Donald  L.  Fry,  M.D. 

Cooperating  Units:   None 

Project  Description 

Objectives :   1)  To  study  the  effect  of  various  lipoprotein  classes  upon 
aortic  smooth  muscle  cell  proliferation.   2)   To  study  the  effects  of  various 
lipoprotein  classes  upon  smooth  muscle  cell  and  skin  fibroblasts  cholesterol 

metabolism. 

Methods  Employed:   1)   Standard  techniques  were  used  in  determining  cell 
proliferation  in  response  to  various  classes  of  lipoproteins.   2)   Electron 
microscopy  was  used  to  establish  that  cells  were  similar  to  smooth  muscle  cells 
as  reported  in  the  literature.   3)   Cellular  cholesterol  production  was 
assessed  by  measuring  the  activity  of  3-hydroxy-3-methyl-glutaryl-coenzyme  A 
reductase,  the  rate  limiting  enzyme  in  cholesterol  synthesis. 

Major  Findings:   1)   Cultures  of  aortic  smooth  muscle  cells  from  swine 
and  dogs  have  been  maintained  up  to  ten  generations  without  alteration  of 
growth  potential  or  morphologic  characteristics.   2)   When  added  to  growth 
limiting  medium  in  equal  amounts  with  respect  to  cholesterol  concentration 
lipoproteins  from  normo-  and  hyperlipidemic  animals  stimulated  cell  prolif- 
eration to  the  same  extent.   Very  low  density  (VLDL)  and  low  density  (LDL) 
lipoproteins  stimulated  smooth  muscle  cell  proliferation  7.8-  and  6.1-  fold 
respectively.   High  density  lipoprotein  (HDL)  and  a  high  density  lipoprotein 
(HDL  )  induced  by  cholesterol  feeding  of  swine  stimulated  cell  proliferation 
3.3-  and  3-  fold  respectively.   3)   In  skin  fibroblasts  from  normal  human 
controls  and  swine  aortic  smooth  muscle  cells  HMG  CoA  reductase  activity  was 
equally  suppressed  by  human  LDL  and  swine  VLDL,  LDL  and  HDL  when  each 
lipoprotein  class  was  added  in  equal  amounts  with  respect  to  cholesterol 
concentration.   HDL  suppressed  enzyme  activity  only  when  added  in  50  fold 
greater  concentrations  with  respect  to  cholesterol. 


eso 


Project  No,  Z01  HL  02813  03  SEA 

Significance  to  Bio-medical  Research  and  the  Program  of  the  Institute; 
In  growth  limiting  medium  lipoproteins  containing  the  B-apoprotein  are  more 
effective  in  stimulating  aortic  smooth  muscle  cell  proliferation.   Animals 
and  humans  with  high  concentrations  of  these  lipoprotein  develop  atherosclerosis. 
This  predilection  to  develop  atherosclerosis  may  be  related  to  the  ability  of 
B-apoprotein  containing  lipoproteins  to  stimulate  smooth  muscle  cell  prolif- 
eration ^n  vivo. 

The  ability  of  HDL  to  suppress  HMG  CoA  reductase  is  of  interest  because 
this  lipoprotein  contains  no  B-apoprotein.   Previously  it  had  been  postulated 
that  the  B-apoprotein  was  essential  and  acted  via  a  specific  receptor  on  the 
cell  surface  of  fibroblasts.   The  mechanism  whereby  lipoproteins  suppress 
cellular  HMG  CoA  reductase  remains  to  be  elucidated. 

Proposed  Course  of  the  Project:   Further  studies  with  smooth  muscle  cells 
will  be  done  to  elucidate  the  mechanism  of  exogenous  cholesterol  metabolism. 

Keyword  Descriptors 

Atherosclerosis;  smooth  muscle  cells;  cholesterol  metabolism. 
Honors  and  Awards 

None 

Publications 

B.  Greg  Brown,  Robert  W.  Mahley,  and  G.  Assman:   Swine  Aortic  Smooth 
Muscle  in  Tissue  Culture:   Cell  Growth  and  Regulation  of  Cholesterol 
Synthesis  in  the  Presence  of  Purified  Swine  Lipoproteins.   Circulation 
Research  (in  press) 


857 


Annual  Report  of  the  Section  of  Pathology 

Office  of  the  Director 

of  the  Division  of  Intramural  Research 

National  Heart  and  Lung  Institute 

July  1,  1974  through  June  30,  1975 

This  section  is  concerned  primarily  with  structural  alterations  produced 
by  various  cardiovascular  and  pulmonary  diseases.   Structural  alterations  are 
studied  at  gross,  light  microscopic,  ultrastructural  and  histochemical  levels. 
Studies  during  this  period  focused  on  coronary,  hypertensive,  valvular  and 
myocardial  heart  diseases. 

CORONARY  HEART  DISEASE 

A  continuing  major  undertaking  of  this  laboratory  is  the  examination  of 
coronary  arteries  in  a  number  of  different  conditions.   Previous  studies  have 
focused  primarily  on  the  status  of  these  arteries  in  patients  with  fatal  acute 
myocardial  infarction.   During  the  past  year  or  so  the  status  of  these  arteries 
in  patients  with  fatal  pure  angina  pectoris  and  in  individuals  in  whom  the 
first  coronary  event  was  sudden  death  were  also  examined  systematically.  A 
major  conclusion  from  these  studies  is  that  the  myocardial  response  to  similar 
degrees  of  coronary  narrowing  is  quite  different.   For  example,  the  degrees  of 
coronary  luminal  narrowing  are  similar  in  patients  with  fatal  angina  pectoris, 
in  patients  with  fatal  acute  myocardial  infarction,  and  in  patients  in  whom 
death  is  the  first  coronary  event.   A  major  mystery  of  coronary  heart  disease 
is  to  determine  why  the  myocardium  responds   so  differently  to  similar  degrees 
of  coronary  luminal  narrowing.   Studies  to  attempt  to  answer  this  question  are 
continuing  in  this  laboratory. 

In  addition  to  studying  the  coronary  arteries  in  patients  with  fatal 
ischemic  heart  disease,  the  coronary  arteries  are  being  examined  systematically 
in  patients  of  varying  ages  and  sex  who  die  from  automobile  accidents.   Thus, 
it  will  be  possible  to  have  controls  of  coronary  arteries  for  comparison  with 
the  patients  with  ischemic  heart  disease. 

A  previous  study  from  this  laboratory  showed  unequivocally  that  athero- 
sclerosis is  accelerated  in  patients  on  long-term  corticosteroid  therapy. 
There  has  been  much  discussion  in  the  past  couple  of  years  regarding  use  of 
aspirin  as  a  preventive  of  intraarterial  thrombosis  or  specifically  platelet 
aggregation.  Presently,  the  coronary  arteries  in  a  number  of  patients  who  died 
of  rheumatoid  arthritis  but  who  were  treated  for  many  years  with  high  doses  of 
aspirin  are  being  examined  to  see  if  these  individuals  have  less  atheroscler- 
osis than  patients  of  similar  age  and  sex  not  treated  with  aspirin  or  cortico- 
steroid therapy. 

.  In  recent  years,  a  number  of  reports  have  appeared  describing  patients 
with  "myocardial  infarction  and  angiographically  normal  coronary  arteries." 
Patients  reported  with  this  combination  were  reviewed  along  with  patients 

i  grj 


studied  in  our  laboratory  at  necropsy  who  had  large  myocardial  scars  but 
morphologically  normal  coronary  arteries.  A  major  implication  of  most  of  the 
previous  reports  was  that  acute  myocardial  infarction  may  occur  in  the  presence 
of  normal  coronary  arteries.   This  thesis  was  re-examined.   Among  40  patients 
previously  described  with  non-catheter  induced  myocardial  infarction  and 
angiographically  normal  coronary  arteries,  in  none  was  coronary  angiography 
performed  at  the  time  of  myocardial  infarction.   Indeed,  the  interval  between 
the  onset  of  infarction  and  the  performance  of  coronary  angiography  was  longer 
than  1  month.   In  5  additional  patients,  however,  in  whom  coronary  angiography 
was  performed  at  the  time  of  acute  myocardial  infarction,  coronary  angiography 
in  each  showed  an  obstructed  coronary  artery  but  repeat  catheterization  at 
later  times  disclosed  angiographically  normal  coronary  arteries.   Thus,  it  was 
apparent  that  the  status  of  the  coronary  arteries  at  the  time  of  actual  myo- 
cardial necrosis  was  uncertain  in  most  reported  patients.   Indeed,  a  normal 
coronary  arterial  tree  has  never  been  demonstrated  by  angiography  at  the  time 
of  acute  myocardial  infarction.   It  was  reasoned  that  there  were  at  least  5 
explanations  to  explain  why  a  coronary  tree  might  be  entirely  normal  by 
angiography  after  healing  of  an  acute  myocardial  infarction:  1)  the  acute  myo- 
cardial infarction  never  occurred.   Evidence  is  presented  that  this  is  quite 
unlikely.   2)  Too  large  a  myocardial  mass  or  too  little  hemoglobin  or  tod  low 
a  profusion  pressure  was  present  to  supply  the  myocardium  by  a  normal  coronary 
tree.   Although  this  explanation  does  explain  the  presence  of  myocardial  scars 
in  some  patients  with  big  hearts,  the  patients  reported  as  having  normal 
coronary  arteriograms  and  acute  myocardial  infarction  all  had  normal  sized 
hearts,  normal  blood  hematocrits  and  either  normal  or  elevated  blood  pressures 
preceding  the  myocardial  infarction.   This  explanation,  therefore,  is 
unlikely.   3)  The  coronary  angiograms  were  misinterpreted.   It  is  well  known 
that  angiography  tends  to  underestimate  the  degree  of  coronary  luminal 
narrowing  but  this  explanation  did  not  appear  adequate  for  several  reasons  to 
explain  the  occurrence  of  myocardial  infarction  in  most  of  the  patients. 
4)  Coronary  spasm  caused  the  acute  myocardial  infarction.   Patients  with 
Prinzmetal's  angina  were  reviewed  and  it  was  clearly  shown  that  spasm  has 
never  been  documented  to  cause  acute  myocardial  infarction.   5)   Acute 
myocardial  infarction  was  caused  by  an  occluding  embolus  which  subsequently 
lysed  or  recanalized.   Evidence  was  presented  that  this  was  the  most  likely 
explanation  for  the  occurrence  of  "myocardial  infarction  and  angiographically 
normal  coronary  arteries." 

There  have  been  many  reports  in  the  past  describing  histologic  features  of 
tuberous  xanthomas  but  it  has  not  been  known  whether  or  not  the  patients  in 
whom  the  xanthomas  were  examined  had  normal-  or  hyper-lipoproteinemia.   Thus, 
it  was  not  known  whether  or  not  these  xanthomas  differed  structurally  in 
patients  with  type  II  versus  let's  say  type  III  hyperlipoproteinemia.   A 
tuberous  xanthoma  was  examined  histochemically  and  ultrastructurally  from  a 
patient  with  homozygous  type  II  hyperlipoproteinemia.   It  was  found  that  all 
the  lipid  was  contained  in  foam  cells.   The  cell  types  identified  in  the 
xanthoma  were  primitive  mesenchymal  cells,  elongated  perivascular  and 
fibroblast-like  cells,  and  macrophages  which^filled  with  lysosomes.   The  lipid 
was  present  in  the  xanthoma  in  4  different  forms,  primarily  in  non-membrane- 
bound  forms.   It  appeared  that  non-lysosomal  lipid  storage  in  foam  cells  is  a 
characteristic  tissue  response  to  the  underlying  metabolic  defect  in  type  II 
hyperlipoproteinemia. 


ss*/ 


Much  has  been  written  from  this  Institute  on  Tangier's  disease  where  it 
was  initially  described,  hut  little  structural  information  has  appeared  on 
this  entity.  Histologic,  histochemical  and  electronmicroscopic  studies  were 
made  of  bone  marrow,  tonsil  and  jejunum  from  patients  with  Tangier  disease. 
Four  morphologically  distinct  types  of  lipid  inclusions  were  observed  in  foam 
cells  observed  in  these  3  types  of  tissues:  1)  crystals  of  cholesteryl  esters; 

2)  droplets  composed  of  mixtures  of  cholesteryl  esters  and  triglycerides; 

3)  ceroid,  and  4)  particles  which  corresponded  in  size  to  plasma  chylomicrons 
and  very  low  density  lipoproteins  (VLDL) .   Comparisons  were  made  of  the 
ultrastructure  and  histochemistry  of  foam  cells  in  Tangier  disease  and  in 
other  lipid  storage  diseases.   Plasma  chylomicrons  and  VLDL  were  considered 
the  important  sources  of  lipid  accumulated  in  foam  cells  in  Tangier  disease. 

SYSTEMIC  HYPERTENSION 

During  recent  years  in  this  section  about  400  hearts  are  accessioned 
annually  from  patients  with  various  fatal  cardiovascular  disorders.  Most  of 
the  hearts  are  enlarged  and  by  far  the  most  common  cause  of  the  cardiomegaly 
has  been  hypertension.   A  number  of  conditions  termed  "the  hypertensive 
diseases"  were  reviewed  to  see  the  frequency  of  a  history  of  systemic  hyper- 
tension and  secondly  to  see  the  percent  with  cardiomegaly  unexplained  by  any 
mechanism  other  than  hypertension.   The  conditions  reviewed  were  sudden 
coronary  death,  angina  pectoris,  acute  myocardial  infarction,  cardiac 
complications  particularly  cardiac  rupture  of  acute  myocardial  infarction, 
aneurysm  of  aorta,  atherothrombotic  obstruction  of  the  abdominal  aorta  or  of 
its  branches,  cerebrovascular  accidents  including  atherothrombotic  cerebral 
infarction,  primary  intracerebral  hemorrhage,  lacunar  softenings,  and  Charcot- 
Bouchard  aneurysms.   It  was  clear  from  analyzing  the  numbers  from  patients 
with  each  of  these  various  conditions  that  systemic  hypertension  is  an  even 
more  common  precursor  of  symptomatic  vascular  disease  than  previously  realized 
from  obtaining  a  history  of  hypertension  or  from  recording  one's  blood 
pressure.   In  other  words,  the  heart  weight  is  a  better  indicator  of  hyper- 
tension than  the  history.   It  was  clear  that  hypertension  acts  as  a  major 
risk  factor  to  development  of  cardiovascular  disease  in  two  ways:  1)  by 
increasing  the  deposition  of  atherosclerotic  plaques  in  major  arteries  most 
commonly  by  causing  luminal  narrowing  with  resulting  organ  ischemia  or 
infarction  or  both,  and  2)  by  weakening  the  media  of  certain  arteries  causing 
aneurysms  which  may  or  may  not  rupture.   Its  effect  on  the  arterial  media  is 
direct  whereas  its  effect  on  the  arterial  intima  is  indirect.  Hypertension  is 
the  only  known  major  underlying  factor  in  2  conditions:   intracerebral  micro- 
aneurysm (with  or  without  rupture)  and  dissecting  aortic  aneurysm  (excluding 
patients  with  the  Marfan  and  Marfan-like  syndrome) ,  both  of  which  are  associated 
with  medial  weakening  or  disruption.  The  more  common  consequence  of  hyper- 
tension, however,  is  its  ability  to  increase  the  amount  of  atherosclerotic 
plaquing  in  various  arteries.  This  effect,  however,  is  indirect  because 
population  groups  with  normal  (<  200  mg  per  100  ml)  serum  cholesterol  levels 
do  not  have  more  or  larger  atherosclerotic  plaques  than  do  normotensive 
persons  in  those  populations. 


girr 


VALVULAR  HEART  DISEASE 

A  continuing  activity  in  this  section  has  been  study  of  patients  with 
valvular  heart  disease  to  gain  more  information  regarding  the  natural 
history  of  various  conditions  and  secondly  to  gain  information  regarding 
prosthetic  valves.  Many  reports  are  available  regarding  hemodynamic  evaluation 
of  various  prosthetic  valves  but  few  have  appeared  evaluating  these  valves 
from  a  morphologic  standpoint.   Previous  studies  from  this  laboratory  have 
focused  primarily  on  the  caged-ball  prosthesis  and  on  various  tissue  valves. 
During  the  past  year  a  study  of  61  patients  with  a  poppet-disc  valve  was 
carried  out.   Detailed  study  of  the  early  and  late  deaths  of  these  patients 
clearly  showed  that  the  disc  poppet  prosthesis  is  not  an  ideal  substitute 
cardiac  valve.   It  clots,  despite  anticoagulant  therapy,  it  is  intrinsically 
stenotic,  portions  of  it,  i.e.,  the  disc,  degenerate  and  it  causes  hemolysis 
to  erythrocytes.   Currently,  the  porcine  or  pig  valve  is  being  studied  and 
data  will  be  accumulated  on  it  during  the  following  year. 

An  opportunity  presented  itself  to  study  2  patients  who  had  had  Hufnagel 
prostheses,  the  original  "cardiac"  valve  inserted  into  the  descending  aorta. 
One  of  these  patients  died  11  and  the  other  one  13  years  after  implantation  of 
the  Hufnagel  prosthesis.   Neither  of  these  2  patients  nor  any  of  the  3  previ- 
ously reported  long-term  survivors  with  Hufnagel  prostheses  in  descending 
aorta  had  prosthetic  related  complications.   Because  of  the  danger  of  excising 
the  descending  aortic  prosthesis,  it  appears  most  reasonable  not  to  remove  the 
descending  aortic  prosthesis  at  any  time  in  these  patients  if  aortic  valve 
replacement  is  subsequently  performed. 

Extensive  studies  in  the  past  have  been  carried  out  in  this  laboratory  on 
patients  with  valvular  aortic  stenosis.   These  previous  studies  showed  that 
most  individuals  aged  15  to  65  with  aortic  stenosis  had  congenitally  bicuspid 
valves  and  that  75%  of  them  were  male.   During  the  past  decade,  73  hearts  have 
been  collected  with  atretic  aortic  valves.   This  anomaly  is  the  most  common 
cause  of  death  from  congenital  malformations  during  the  first  week  of  life. 
Among  the  73  hearts,  the  patients  ranged  in  age  from  1  to  17  days  (average  5) 
and  74%  were  boys.   Among  the  73  patients,  4  had  normal  or  near  normal  sized 
left  ventricles  and  69  had  hypoplastic  ones.  Among  the  4  with  normal  or  near 
normal  sized  left  ventricles,  all  had  ventricular  septal  defects  and  3  had 
normally  developed  mitral  valves.   In  contrast,  among  the  69  with  hypoplastic 
left  ventricles  none  had  ventricular  septal  defects.   A  review  of  previous 
reports  on  aortic  atresia  failed  to  disclose  any  reports  of  ventricular  septal 
defect  or  normal-sized  left  ventricle  associated  with  aortic  atresia  and 
therefore  a  new  classification  of  this  condition  was  proposed. 

In  1962  the  specific  cardiovascular  lesion  of  carcinoid  heart  disease  was 
described  from  this  laboratory  for  the  first  time.   Its  description  at  that 
time  was  limited  to  its  gross  and  histologic  features.   During  the  past  year 
the  endocardial  lesion  in  carcinoid  heart  disease  was  studied  by  electron- 
microscopy.   The  cellular  elements  of  the  carcinoid  plaque  consist  primarily 
of  smooth  muscle  cells  and  the  extracellular  elements,  of  collagen  fibrils, 
microfibrils,  layers  of  fibrillar  material  and  dense  spicules.   No  elastic 
fibers  or  fibrin  deposits  are  present.   These  observations  suggest  that 
carcinoid  plaques,  which  occur  exclusively  in  patients  with  the  carcinoid 

4  2& 


syndrome,  result  from  stimulation  of  endocardial  smooth  muscle  cells  to 
produce  collagen  and  basement  membrane-like  material. 

The  condition,  rupture  of  a  sinus  of  Valsalva  aneurysm,  is  well  known, 
but  there  is  virtually  no  information  on  the  occurrence  of  aneurysm  of  1  of 
the  3  sinuses  of  Valsalva  without  rupture.  An  opportunity  to  study  such  a 
patient  occurred  during  this  past  year.   The  patient,  an  83-year-old  man,  had 
a  large  aneurysm  involving  1  of  the  3  sinuses  of  Valsalva.   It  was  entirely 
an  incidental  necropsy  finding.   Thus,  these  aneurysms  do  not  always  rupture. 

A  common  observation  at  necropsy  in  older  hearts  is  papillary  nodules  on 
the  valvular  endocardium  and  occassionally  on  the  mural  endocardium.   These 
lesions  were  examined  in  3  patients  by  electronmicroscopy  and  a  new  term  for 
this  condition  was  presented,  namely  "endocardial  papillary  elastof ibroma, "  to 
emphasize  the  features  which  are  most  conspicuous  and  which  serve  to  differ- 
entiate this  tumor  from  myxoma.   These  lesions  had  not  been  looked  at 
ultrastructurally  previously. 

Diffuse  endocardial  fibroelastosis  (EFE)  is  a  rare  congenital  malformation 
of  the  heart,  but  focal  EFE  is  common.  Most  patients  with  healed  myocardial 
infarction  have  dense  endocardial  thickening  over  the  area  of  myocardial 
scarring.   The  left  ventricular  endocardium  from  10  patients  with  EFE  was 
studied  by  electronmicroscopy.   The  elastic  fibers  were  much  larger  in  the  4 
patients  with  congenital  EFE  than  in  the  6  patients  with  acquired  EFE.   The 
explanation  for  this  difference,  unfortunately,  was  not  determined. 

MYOCARDIAL  HEART  DISEASE" 

During  the  past  several  years  a  number  of  myocardial  biopsies  have  been 
submitted  to  this  unit  primarily  from  D.C.  General  Hospital  and  primarily 
from  patients  with  idiopathic  cardiomyopathy.   These  biopsies  have  been 
evaluated  from  several  angles  but  one  not  previously  utilized  was  comparing 
the  morphologic  ultrastructural  observations  in  the  patients  who  were  known 
to  be  habitual  alcoholics  to  those  patients  known  not  to  be  habitual 
alcoholics.   Examination  of  the  2  groups  blindly  showed  no  ultrastructural 
differences. 

At  cardiac  operations  performed  at  the  Clinical  Center  biopsies  of  myo- 
cardium are  taken  routinely  for  electronmicroscopic  and  histologic  studies. 
Some  of  these  biopsies  are  derived  from  papillary  muscle  or  left  atrial 
appendage  during  mitral  valve  replacement  and  others  are  taken  by  direct 
bippsy  from  left  ventricular  free  wall  at  the  time  of  aortic  valve  replacement. 
In  patients  with  hypertrophic  cardiomyopathy  a  portion  of  left  ventricular 
outflow  myocardium  is  excised.  As  a  consequence  there  is  a  large  data  base 
available  of  myocardial  biopsies  to  study.  Among  134  patients  with  cardiac 
hypertrophy  of  various  causes  the  nuclear  membranes  of  the  cardiac  muscle  cells 
were  studied  and  3  distinctive  abnormalities  were  observed:  1)  increased 
foldings  and  convolutions;  2)  nuclear  pseudoinclusions  formed  by  cytoplasmic 
organelles  protruding  into  saccular  invaginations  of  the  nuclear  membranes, 
and  3)  intranuclear  tubules.  The  increased  foldings  and  convolutions  of  the 
nuclear  membranes,  and  the  nuclear  pseudoinclusions  appear  to  result  from 
synthesis  of  nuclear  membranes  in  excess  of  that  needed  to  accomodate  the 

5  «T 


increase  In  nuclear  volume  which  occurs  in  hypertrophy.   Intranuclear  tubules 
in  cardiac  muscle  cells  probably  represent  an  extreme  cellular  response  to 
the  stimulus  of  hypertrophy. 

In  other  myocardial  biopsies  intranuclear  glycogen  in  myocardium  was 
studied.   Glycogen  deposits  within  nuclei  of  hypertrophied  and  normal-sized 
cardiac  muscle  cells  were  observed  in  6  (7%)  of  90  patients  with  various 
cardiac  diseases.   The  cells,  however,  containing  intranuclear  glycogen  did 
not  show  damage  or  degeneration.   It  appears  that  under  certain  conditions 
the  nuclei  of  cardiac  muscle  cells  can  acquire  the  capacity  to  synthesize 
glycogen  but  its  exact  function  within  the  nucleus  is  as  yet  undetermined. 

Bone  marrow  transplantation  has  been  a  recent  intervention  in  the  National 
Cancer  Institute  in  treating  patients  with. leukemia,  aplastic  anemia,  immune 
deficiency  disease  or  metastatic  cancer.   The  hearts  of  20  patients  with  these 
various  disorders  were  studied  both  histologically  and  ultrastructurally.   All 
had  received  bone  marrow  transplantation.   The  cardiac  alterations  were  found 
to  be  similar  to  those  which  occur  in  patients  with  similar  hematologic  and 
neoplastic  disorders  who  had  not  been  treated  with  bone  marrow  transplantation. 

PULMONARY  PATHOLOGY 

There  has  been  considerable  interest  for  a  number  of  years  in  pulmonary 
disease  by  members  of  the  pathology  section  of  the  National  Heart  and  Lung 
Institute.   Interest  has  focused  primarily,  however,  on  various  pulmonary 
vascular  changes  produced  by  cardiac  diseases.   During  the  past  year  the 
clinical  program  of  the  Pulmonary  Branch  of  the  NHLI  has  gotten  underway  and, 
consequently,  approximately  30  lung  biopsies  were  submitted  to  the  pathology 
section  of  the  NHLI  for  examination.   These  biopsies  were  studied  extensively 
histologically  and  samples  of  each  were  also  studied  by  electronmicroscopy. 
In  addition,  some  tracheobronchial  washings  were  studied  cytologically .   These 
morphologic  findings  are  correlated  with  the  pulmonary  function  studies  done 
during  life  on  these  patients.   The  opportunity  to  study  extensively 
morphologically  pulmonary  parenchymal  disease  has  added  a  new  and  very  welcomed 
dimension  to  the  NHLI  pathology  section.   In  addition,  pulmonary  parenchymal 
disease  in  patients  from  other  institutes  at  NIH  is  also  being  studied  much 
more  thoroughly  by  the  NHLI  pathology  section.   These  observations  will  form 
the  nucleus  of  hopefully  many  future  presentations. 


8£8 


Project  No.  Z01-HL-03001-01-OD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  Coronary  Arteries  in  Ischemic  Heart  Disease 

Previous  Serial  Number:   None 

Principal  Investigator:  William  C.  Roberts,  M.D. 

Other  Investigators:  None 

Cooperating  Units:   None 

Project  Description:   This  article  summarizes  observations  on  coronary  arteries 
in  fatal  ischemic  heart  disease  and  also  summarizes  observations  on  acute  or 
recent  lesions  particularly  in  these  patients:  1)  among  patients  with  fatal 
ischemic  heart  disease,  thrombi  are  infrequent  in  patients  dying  suddenly  and 
in  those  in  whom  the  necrosis  was  limited  to  subendocardium;  2)  thrombus  is 
found  in  a  coronary  artery  in  about  60%  of  patients  with  fatal  transmural  acute 
myocardial  infarction;  3)  among  patients  with  transmural  myocardial  necrosis, 
the  major  determinant  of  the  presence  of  coronary  thrombosis  appears  to  be 
cardiogenic  shock;  4)  the  larger  the  area  of  myocardial  necrosis  the  greater 
the  likelihood  of  coronary  thrombosis;  5)  when  coronary  thrombosis  is  associated 
with  acute  myocardial  infarction,  the  thrombus  is  always  located  in  the  artery 
responsible  for  profusing  the  area  of  myocardial  necrosis;  6)  thrombi  occur  in 
fatal  ischemic  heart  disease  in  coronary  arteries  which  are  already  severely 
narrowed  by  old  atherosclerotic  plaques;  7)  coronary  thrombi  in  fatal  acute 
myocardial  infarction  are  usually  occlusive,  short,  and  located  entirely  in 
the  major  trunks. 

Keyword  Descriptors:   Coronary  thrombosis,  coronary  atherosclerosis. 

Honors  and  Awards:   None 

Publication:  Accepted  for  publication  in  Cardiovascular  Clinics 


8& 


Project  No.  ZO1-HL-030Q2-Q1-QD-P 

1 .  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  Coronary  Arteries  in  Fatal  Coronary  Events 

Previous  Serial  Number:   None 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description:   This  report  emphasizes  certain  aspects  of  the  coronary 
tree  in  patients  with  various  types  of  fatal  coronary  events.   Points  empha- 
sized are  the  role  of  coronary  thrombosis  in  acute  myocardial  infarction.   Two 
factors  implicate  coronary  thrombosis  as  the  precipitating  cause  of  acute  myo- 
cardial infarction:  1)  the  occurrence  of  coronary  arterial  thrombi  in  many 
patients  with  fatal  acute  myocardial  infarction  and  2)  the  location  of  the 
thrombus  in  the  coronary  arteries  responsible  for  supplying  the  area  of 
myocardial  necrosis.   Five  factors,  however,  tend  to  indicate  that  coronary 
thrombosis  is  a  consequence  rather  than  the  precipitating  cause  of  acute 
myocardial  infarction:  1)  the  low  frequency  of  thrombi  in  patients  dying 
suddenly  with  or  without  previous  evidence  of  cardiac  disease;  2)  the  increasing 
frequency  of  thrombi  with  increasing  intervals  between  the  onset  of  symptoms  of 
acute  myocardial  infarction  and  death;  3)  the  absence  of  thrombi  in  fatal 
transmural  acute  myocardial  infarction  nearly  as  often  as  they  are  present; 
4)  the  near  absence  of  thrombi  in  fatal  subendocardial  infarction;  5)  the 
occurrence  of  thrombi  in  high  percentage  only  in  patients  with  cardiogenic 
shock,  most  of  whom  have  large  transmural  infarcts.   It  appears  from  study  of 
107  patients  with  fatal  acute  myocardial  infarction  in  this  laboratory  that  the 
key  to  coronary  thrombosis  just  as  the  key  to  thrombosis  occurring  anywhere  in 
the  body  is  slow  blood  flow  or  relative  stasis  and  sufficient  time  for  the 
thrombus  to  form.   The  absence  of  these  2  factors  may  explain  the  absence  of 
coronary  thrombosis  in  the  sudden  coronary  death  cases  and  the  increasing 
frequency  of  thrombosis  as  the  interval  from  onset  of  symptoms  of  myocardial 
ischemia  to  death  increases. 

Keyword  Descriptors:   Coronary  thrombosis,  coronary  atherosclerosis. 

Honors  and  Awards:   None 

Publications:   This  report  will  appear  as  a  chapter  in  a  book  entitled 

Cardiac  Controversies  to  be  published  in  1975  by  Springer- 
Verlag  Publishers,  New  York. 


B6t> 


Project  No.  ZO1-HL-03003-01-OD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Reports 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  Coronary  Arteries  In  Coronary  Heart  Disease 

Previous  Serial  Number:   None 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description:   This  report  summarizes  certain  characteristic  changes  in 
the  coronary  arteries  observed  at  necropsy  in  patients  with  fatal  ischemic  heart 
disease:  1)  the  coronary  arteries  are  diffusely  involved  by  atherosclerotic 
plaques;  2)  with  rare  exception,  at  least  2  of  the  3  major  coronary  arteries 
are  narrowed  >75%  by  old  atherosclerotic  plaque;  3)  the  atherosclerotic  process 
is  limited  to  the  epicardial  coronary  arteries;  4)  certain  portions  of  the 
coronary  tree  tend  to  develop  larger  atherosclerotic  plaques  and,  therefore, 
more  narrowed  lumens  than  other  portions;  5)  of  the  so-called  3  types  of 
atherosclerotic  plaques,  namely  lipid,  fibrous,  complicated,  only  the  complica- 
ted plaque  is  responsible  for  causing  significant  (>75%)  narrowing  of  the  lumens 
of  the  coronary  arteries;  6)  the  degree  of  coronary  arterial  luminal  narrowing 
by  atherosclerotic  plaques  and  the  extensiveness  of  the  plaquing  are  similar  in 
patients  with  fatal  ischemic  heart  disease  irrespective  of  the  type  of  fatal 
coronary  event;  7)  the  composition  of  coronary  atherosclerotic  plaques  and  the 
degree  of  coronary  arterial  luminal  narrowing  in  patients  with  fatal  ischemic 
heart  disease  appear  similar  irrespective  of  whether  or  not  the  blood  lipo- 
protein pattern  is  normal  or  abnormal;  8)  the  shapes  of  lumens  of  atherosclerotic 
coronary  arteries  are  quite  variable;  9)  and,  although  the  number  1  risk  factor 
to  development  of  atherosclerosis  in  the  western  world,  advanced  age  does  not 
necessarily  indicate  the  presence  of  severe  coronary  atherosclerosis. 

Keyword  Descriptors:   Ischemic  heart  disease,  atherosclerosis,  hyperlipo- 
proteinemia. 

Honors  and  Awards:   None 

Publications:   This  piece  is  being  published  as  a  chapter  in  a  book  entitled 
Pathobiology  Annals  1975  (Vol.  5)  to  be  published  in  1975  by 
Appleton-Century-Crof ts . 


Sit 


Project  No.  Z01-HL-Q3Q04-01-OP-? 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  Coronary  Arteries  in  Ischemic  Heart  Disease 

Previous  Serial  Number:   None 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators:   Bernadine  H.  Bulkley,  M.D. 

Victor  J.  Ferrans,  M.D.,  Ph.D. 

Cooperating  Units:   None 

Project  Description:   This  report  summarizes  chronic  lesions  observed  in  the 
coronary  arteries  in  patients  with  fatal  ischemic  heart  disease  and  calls 
attention  to  acute  lesions  in  coronary  arteries  other  than  thrombi.   These 
latter  lesions  are  1)  hemorrhage  into  an  old  atherosclerotic  plaque.   These 
were  observed  in  20%  of  nearly  200  patients  studied  in  this  laboratory  with 
fatal  ischemic  heart  disease.   There  was  evidence  in  none  of  the  patients, 
however,  that  hemorrhage  caused  any  additional  narrowing  of  the  coronary 
lumen.   2)  Coronary  artery  embolism.   This  was  observed  in  15  patients  and 
criteria  for  the  diagnosis  of  embolism  was  established.   It  was  emphasized 
that  clot  nearly  always  is  present  in  an  intramural  coronary  artery  when 
embolism  is  present  and  that  in  addition  it  occurs  in  the  distal  portions  of 
the  extramural  coronary  arteries.   3)  Dissecting  aneurysm  (hematoma)  of  a 
coronary  artery  with  and  without  associated  dissection  of  aorta.   Three 
patients  were  summarized  in  whom  dissection  was  isolated  to  a  coronary  artery. 

Keyword  Descriptors:   Dissecting  aneurysm,  coronary  embolism. 

Honors  and  Awards:   None 

Publications:   This  was  published  in  La  Revue  de  Medicine,  Vol.  16:15-20, 

January,  1975.   Complete  title:  Necropsy  Observations  on  the 
Coronary  Arteries  in  Ischemic  Heart  Disease. 


£*<?■ 


Project  No.  Z01-HL-Q3005-01-OD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Coronary  Thrombosis  in  Myocardial  Infarction 

Previous  Serial  Number:   None 

Principal  Investigator:   A.  Bleakley  Chandler,  M.D. 

Other  Investigators:   Irving  Chapman,  M.D. 
Leif  R.  Erhardt,  M.D. 
William  C.  Roberts,  M.D. 
Colin  J.  Schwartz,  M.D. 
D.  Sinapius,  M.D. 
David  M.  Spain,  M.D. 
Sol  Sherry,  M.D. 
Paul  M.  Ness,  M.D. 
Toby  L.  Simon,  M.D. 

Cooperating  Units:   Multiple  ones 

Project  Description:   In  recent  years  the  widely  held  concept  that  coronary 
thrombi  cause  mvocardial  infarcts  has  been  seriously  questioned.   On  the  basis 
of  pathologic  studies,  several  reports  have  suggested  that  coronary  thrombi  do 
not  cause  infarcts  but  instead  are  the  result  of  infarction.   Should  these 
findings  become  generally  substantiated,  the  antithrombotic  approach  to  the 
prevention  and  therapy  of  ischemic  heart  disease  must  be  revised.   This 
workshop  was  organized  to  examine  more  closely  this  issue  and  to  sort  out 
reasons  for  such  divergent  views  of  the  role  of  thrombosis  in  the  pathogenesis 
of  myocardial  infarction. 

Keyword  Descriptors:   Coronary  thrombosis. 

Honors  and  Awards:   None 

Publications:  American  Journal  of  Cardiology  34:823-833,  December  1974. 


etz 


Project  No.  Z01-HL-030Q6-01-QD-? 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Thrombosis,  Atherosclerosis  and  Ischemic  Heart  Disease 

Previous  Serial  Number:   None 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators:   Victor  J.  Ferrans,  M.D.,  Ph.  D. 

Cooperating  Units:   None 

Project  Description:   This  report  summarizes  observations  learned  during  the 
past  couple  of  years  in  this  laboratory  suggesting  that  atherosclerotic 
plaques  result,  at  least  in  part,  from  organization  of  thrombi:  1)  the  presence 
of  known  components  of  thrombi — namely  fibrin  and  platelets — within  athero- 
sclerotic plaques;  2)  the  occurrence  of  known  atherosclerotic  plaques — namely 
foam  cells,  cholesterol  clefts,  pultaceous  debris,  calcium — in  organized 
hematomas  or  known  thrombi  wherever  they  occur  in  the  body;  3)  the  presence  of 
multiluminal  channels  in  vessels,  a  recognized  consequence  of  organization  of 
pulmonary  thromboemboli — and  presumably  also  coronary  thrombi  or  emboli; 

4)  the  major  component  of  the  complicated  atherosclerotic  plaque,  i.e.,  that 
capable  of  causing  significant  luminal  narrowing,  in  the  coronary  arteries  of 
patients  with  fatal  ischemic  heart  disease  is  fibrous  tissue  or  collagen,  not 
lipid  and  this  is  true  whether  or  not  hyperlipidemia  is  or  was  present; 

5)  experimentally  induced  thrombi  under  proper  conditions  may  be  transformed 
into  atherosclerotic  plaques  closely  resembling  those  observed  in  human  coronary 
arteries.   The  above  factors  do  not  prove  that  thrombosis  is  the  cause  of 
atherosclerosis,  but  together  they  strongly  suggest  that  organization  of  thrombi 
plays  a  major  role  in  the  development  of  the  complicated  atherosclerotic  plaque. 

Keyword  Descriptors:   Coronary  thrombosis,  coronary  atherosclerosis. 

Honors  and  Awards:   None 

Publications:   Accepted  for  publication  as  a  chapter  in  the  book  entitled 
Thrombosis,  Platelets,  Anticoagulation  and  Acetylsalicylic 
Acid  to  be  published  by  Stratton  Intercontinental  Medical 
Book  Corporation  in  1975. 


B& 


Project  No.  Z01-HL-Q3Q07-01-3D-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Acute  Myocardial  Infarction  and  Angiographically  Normal 
Coronary  Arteries 

Previous  Serial  Number:   None 

Principal  Investigator:   Ernest  N.  Arnett,  M.D. 

Other  Investigators:   William  C.  Roberts,  M.D. 

Cooperating  Units:   None 

Project  Description:   This  report  examines  previous  reports  of  myocardial 
infarction  and  angiographically  normal  coronary  arteries  and  discusses 
possible  explanations  for  the  occurrence  of  "myocardial  infarction  and  normal 
coronary  arteriograms."  There  have  been  45  reported  patients  with  "myocardial 
infarction  and  angiographically  normal  coronary  arteries:"  in  5  the  acute 
myocardial  infarction  was  produced  at  the  time  of  cardiac  catheterization  and 
in  the  other  40  patients  acute  myocardial  infarction  was  unrelated  to  cardiac 
catheterization.   In  39  of  the  40  patients  with  non-catheter  related  infarction 
the  interval  between  the  onset  of  acute  myocardial  infarction  and  performance 
of  coronary  angiography  was  longer  than  2  months.   Thus,  the  status  of  the 
coronary  arteries  at  the  time  when  a  portion  of  left  ventricular  myocardium 
was  noted  is  uncertain.   Indeed,  a  normal  coronary  arterial  tree  has  never 
been  demonstrated  by  angiography  at  the  time  of  acute  myocardial  infarction. 
This  report  examines  then  how  a  coronary  tree  may  be  entirely  normal  by 
angiography  after  healing  of  an  acute  myocardial  infarction.  Five  explanations 
are  considered  and  evidence  is  presented  that  the  acute  myocardial  infarction 
was  caused  by  an  occluding  embolus  which  subsequently  lysed  or  recanalized. 

Keyword  Descriptors:   Coronary  embolism,  coronary  spasm,  coronary  angiography. 

Honors  and  Awards:   None 

Publications:   Accepted  for  publication  in  Circulation. 


OUT 


Project  No.  ZOl-HL-03008-Ql-OD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,.  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Myocardial  Embolus  to  Coronary  Artery 

Previous  Serial  Number:   None 

Principal  Investigator:   William  J.  Hammer,  M.D. 

Other  Investigators:   Victor  J.  Ferrans,  M.D.,  Ph.D. 
William  C.  Roberts,  M.D. 

Cooperating  Units:   Division  of  Cardiology,  Department  of  Medicine, 
Georgetown  University,  Washington,  D.C. 

Project  Description:   Although  infrequent,  emboli  to  coronary  arteries 
usually  consist  of  fibrin  and  platelets.   Other  dislodged  material  in  coronary 
arteries  have  included  calcific  debris,  clumps  of  neoplastic  cells,  suture  and 
other  foreign  materials,  and  colonies  of  microorganisms.   The  usual  consequence 
of  coronary  embolism  is  acute  myocardial  infarction.   Of  over  200  hearts 
examined  systematically  at  necropsy  in  patients  with  fatal  coronary  heart 
disease,  one  was  observed  to  have  an  embolus  of  necrotic  myocardium  in  a 
coronary  artery.   The  patient,  a  73-year-old  woman,  died  suddenly  10  hours 
after  onset  of  symptoms  (chest  pain)  of  acute  myocardial  infarction.   A 
precordial  murmur  was  never  audible.   Necropsy  disclosed  rupture  of  one  left 
ventricular  papillary  muscle  and  a  clump  of  myocardium  in  the  lumen  of  the 
right  coronary  artery.   The  embolized  myocardium  was  similar  to  that  observed 
in  the  ruptured  papillary  muscle. 

Myocardial  embolus,  to  our  knowledge,  has  not  been  reported  previously 
in  a  coronary  artery.  The  coronary  embolus  in  the  present  patient  probably 
would  have  been  missed  had  not  the  entire  major  coronary  tree  been  examined 
histologically,  a  method  used  to  study  the  coronary  arteries  at  necropsy  in 
all  of  our  over  200  fatal  cases  of  coronary  heart  disease. 

Keyword  Descriptors:   None 

Honors  and  Awards:   None 

Publications:   Accepted  for  publication  in  Chest. 


eu 


Project  No.  ZO1-HL-030Q9- Q1-0D-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland  ■ 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Tuberous  Xanthoma  in  Type  II  Hyperlipoproteinemia 

Previous  Serial  Number:  None 

Principal  Investigator:   Bernadine  H.  Bulkley,  M.D. 

Other  Investigators:   L.  Maximilian  Buja,  M.D. 

Victor  J.  Ferrans,  M.D.,  Ph.D. 
Gregory  B.  Bulkley,  M.D. 
William  C.  Roberts,  M.D. 

Cooperating  Units:   Surgery  Branch,  National  Cancer  Institute 

Project  Description:  Histologic,  histochemical  and  ultrastructural  studies 
of  a  tuberous  xanthoma  from  a  patient  with  homozygous  type  II 
hyperlipoproteinemia  showed  that  virtually  all  of  the  lipid  was  within 
histiocytic  foam  cells;  no  lipid  was  identified  in  interstitial  regions 
or  in  blood  vessels.   Primitive  mesenchymal  cells,  elongated  perivascular 
and  fibroblast-like  cells,  and  lysosome-filled  macrophages  also  were 
present  within  the  xanthoma,  indicating  possible  stages  in  the  evolution 
of  dermal  mesenchymal  cells  into  mature,  cholesterol -rich  foam  cells. 
Morphologically  the  lipid  was  in  4  different  forms:  large  droplets,  which 
were  the  dominant  form,  and  membrane-bound  crystals,  concentric  lamellar 
bodies,  and  ceroid.  The  paucity  of  membrane -bound  lipid  forms,  relative 
to  the  abundant  free  lipid  droplets,  indicated  that  lysosomal  digestion 
was  a  minor  metabolic  pathway  for  the  intracellular  metabolism  of  lipid 
in  the  xanthoma.   Thus,  non-lysosomal  lipid  storage  in  foam  cells  is  a 
characteristic  tissue  response  to  the  underlying  metabolic  defect  in  type 
II  hyperlipoproteinemia. 

Keyword  Descriptors:   xanthoma,  skin,  type  II  hyperlipoproteinemia, 
pathology,  ultrastructure 

Honors  and  Awards:   None 

Publications:   Bulkley,  B.H.,  Buja,  L.M.,  Ferrans,  V.J.,  Bulkley,  G.B., 

and  Roberts,  W.C.:  Tuberous  Xanthoma  in  Homozygous  Type  II 
Hyperlipoproteinemia.  Archives  of  Path,  (in  press) 


067 


Project  No.  ZO1-HL-03Q10-02-QD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   The  Pathology  of  Tangier  Disease 

Previous  Serial  Number:   NHLI-243(c) 

Principal  Investigator:   Victor  J.  Ferrans,  M.D. ,  Ph.D. 

Other  Investigators:   Donald  S.  Fredrickson,  M.D. 

Cooperating  Units:   Molecular  Disease  Branch,  National  Heart  and  Lung 
Institute 

Project  Description:   Histologic,  histochemical  and  electron  microscopic 
studies  were  made  of  bone  marrow,  tonsil  and  jejunum  from  patients  with 
Tangier  disease.  The  foam  cells  in  these  3  types  of  tissues  contained  4 
morphologically  distinct  types  of  lipid  inclusions:  1)  crystals  of 
cholesteryl  esters;  2)  droplets  which  were  composed  of  mixtures  of 
cholesteryl  esters  and  triglycerides;  3)  ceroid,  and  4)  particles  which 
corresponded  in  size  to  plasma  chylomicrons  and  very  low  density 
lipoproteins  (VLDL) .   Detailed  comparisons  were  made  of  the  ultrastructure 
and  histochemistry  of  foam  cells  in  Tangier  disease  and  in  other  lipid 
storage  diseases.   Examination  of  small,  unmyelinated  nerves  in  jejunal 
mucosa  and  submucosa  revealed  the  presence  of  lipid  deposits  to  the 
polyneuropathy  which  develops  in  patients  with  Tangier  disease  was  discussed 
in  detail.   Plasma  chylomicrons  and  VLDL  are  considered  to  be  important 
sources  of  the  lipid  which  accumulates  in  foam  cells  in  Tangier  disease. 

Keyword  Descriptors:   Tangier  disease,  pathology,  ultrastructure,  bone 
marrow,  intestine,  skin,  nerves,  tonsils 

Honors  and  Awards :   None 

Publications:   Ferrans,  V.J.,  and  Fredrickson,  D.S.:   The  Pathology  of 

Tangier  Disease.  American  J.  of  Path.  78:   101-158,  1975 


844 


Project  No.  Z01-HL-Q3Q11-Q1-QD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  Hypertensive  Diseases.   The  Extent  of  Hypertension  as 
a  Risk  Factor. 

Previous  Serial  Number:   None 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description:   This  report  summarizes  clinical  frequencies  of  systemic 
hypertension  and  necropsy  evidence  of  cardiomegaly  in  various  cardiovascular 
conditions  termed  "the  hypertensive  diseases"  because  of  their  frequent 
association  with  systemic  hypertension.   Although  long  recognized  as  a  major 
risk  factor,  systemic  hypertension  appears  to  be  an  even  greater  risk  factor 
to  development  of  various  cardiovascular  conditions  than  previously 
appreciated.   Hypertension  by  itself  appears  to  be  the  sole  underlying  factor 
in  most  cases  of  non-traumatic  cerebral  arterial  or  aortic  rupture.   In 
association  with  hyperlipidemia,  hypertension  clearly  accelerates  athero- 
sclerosis and  its  devastating  consequences. 

This  report  demonstrates  that  systemic  hypertension  acts  as  a  major  risk 
factor  to  development  of  cardiovascular  disease  in  2  ways:  1)  by  increasing 
the  deposition  of  atherosclerotic  plaques  (intimal  lesions)  in  major  arteries 
most  commonly  causing  luminal  narrowing  with  resulting  organ  ischemia  or 
infarction  or  both,  and  2)  by  weakening  the  media  of  certain  arteries  causing 
aneurysms  which  may  or  may  not  rupture.   Its  effect  on  the  arterial  media  is 
direct  whereas  its  effect  on  the  arterial  intima  is  indirect.  Hypertension  is 
the  only  known  major  underlying  factor  in  2  conditions:   intracerebral  micro- 
aneurysm (with  or  without  rupture)  and  dissecting  aortic  aneurysm,  both  of 
which  are  associated  with  medial  weakening  or  disruption.   Reduction  in  blood 
pressure  clearly  leads  to  a  reduction  in  the  incidence  of  non-traumatic 
intracerebral  hemorrhage  and  of  dissecting  aneurysm.  The  more  common  conse- 
quence of  hypertension,  however,  is  its  ability  to  increase  the  amount  of 
atherosclerotic  plaquing  in  various  arteries.  This  effect,  however,  is  not 
direct  because  hypertension  in  population  groups  with  normal  serum  cholesterol 
levels  do  not  have  more  or  larger  atherosclerotic  plaques  than  do  the  normo- 
tensive  persons  in  those  populations.  Thus,  hypertension  accelerates  or 
increases  atherosclerosis  only  in  hypercholesterolemic  population  groups  and 
not  in  those  with  normal  serum  cholesterol  levels. 


&? 


Keyword  Descriptors:   Sudden  coronary  death,  angina  pectoris,  acute  myocardial 
infarction,  aortic  aneurysm,  atherothrombotic  obstruction 
of  abdominal  aorta  or  its  branches,  cerebrovascular 
accident,  renal  failure. 

Honors  and  Awards:   None 

Publications:   American  Journal  of  Medicine  (in  press). 


eye 


Project  No.  ZOl-HL-03012-Ol-OD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  3Q,  1975 

Project  Title;   Cardiac  Pathology  after  Valve  Replacement  Using  Disc 
Prostheses 

Previous  Serial  Number:   None 

Principal  Investigator:  William  C.  Roberts,  M.D. 

Other  Investigators:  Michael  C.  Fishbein,  M.D. 
Abner  Golden,  M.D. 

Cooperating  Units:  Department  of  Pathology,  Georgetown  University, 
Washington,  D.C. 

Project  Description:   Clinical  and  necropsy  observations  are  described  in  61 
patients  in  whom  one  or  more  cardiac  valves  had  been  replaced  with  discoid 
prostheses  (Hufnagel  type) .  The  most  common  (31%)  cause  of  death  among  the  45 
patients  dying  early  (<65  days  from  operation)  appeared  to  be  prosthetic  dis- 
proportion, i.e.,  the  prosthesis  was  too  big  for  the  aorta  or  ventricular 
cavity  into  which  it  was  inserted  so  that  inadequate  space  was  present  between 
the  margins  of  the  disc  and  the  endocardium  of  ventricle  or  intima  of  aorta. 
Prosthetic  thrombosis  occurred  in  only  3  of  the  45  patients  dying  early,  but 
in  each  poppet  movement  appeared  considerably  altered.   In  contrast,  thrombi 
were  observed  on  a  prosthesis  in  14  of  the  16  patients  dying  late  (from  4  to 
47  months  [avg  21]  postoperatively) ,  but  in  none  did  the  thrombi  appear  of 
sufficient  size  to  alter  poppet  function.   Excessive  bleeding  occurred  in  11 
of  the  45  (24%)  early  deaths  and  was  primarily  related  to  the  insertion  of  a 
patch  in  the  root  of  aorta.  Uncorrected  valvular  disease  either  by  itself  or 
by  its  ability  to  alter  function  of  the  prosthesis  appeared  responsible  for 
death  in  6  (13%)  of  the  45  patients  dying  early  and  in  2  (6%)  of  the  16  dying 
late.   Insertion  of  a  mitral  poppet  disc  in  a  patient  with  uncorrected  aortic 
regurgitation,  even  of  mild  degree,  may  be  hazardous  because  the  aortic  regur- 
gitant jet  stream  may  interfere  with  proper  function  of  the  mitral  disc. 
Likewise,  insertion  of  a  poppet  disc  only  in  the  aortic  valve  position  in  a 
patient  with  combined  aortic  and  mitral  regurgitation  may  considerably  increase 
the  degree  of  mitral  incompetence  because  the  aortic  prosthesis  is  intrinsically 
obstructive. 

Disc  wear  or  variance  was  observed  in  all  but  one  prosthesis  in  place  for 
>1  year.  Although  hemolytic  anemia  of  significant  degree  was  not  observed  in 
any  of  the  16  patients  dying  late,  the  occurrence  of  renal  hemosiderosis  in  13 
of  the  16  patients  indicates  that  the  poppet  disc  prosthesis  is  considerably 
traumatic  to  erythrocytes. 


07/ 


Thus,  the  disc-poppet  prosthesis  is  not  an  ideal  substitute  cardiac  valve. 
It  clots,  despite  anticoagulant  therapy,  it  is  intrinsically  stenotic, 
portions  of  it,  i.e.,  the  disc,  degenerate,  and  it  causes  hemolysis  to  erythro- 
cytes. 

Keyword  Descriptors:   None 

Honors  and  Awards:   None 

Publications:   American  Journal  of  Cardiology,  May  1975. 


e>7a- 


Project  No.  ZO1-HL-03013-01-OB-F 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Observations  After  Insertion  of  Hufnagel  Prostheses  in 
Descending  Aorta 

Previous  Serial  Number:   None 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators:   Michael  C.  Fishbein,  M.D. 

Cooperating  Units:   Department  of  Pathology,  Georgetown  University, 
Washington,  D.C. 

Project  Description:   Clinical  and  necropsy  observations  are  described  in  2 
patients  who  died  11  and  13  years,  respectively,  after  implantation  of 
Hufnagel  prostheses  (the  first  ever  used  successfully  in  humans  to  treat 
cardiac  valve  disease)  in  the  descending  thoracic  aorta.  Despite  the  long 
implantation  periods,  there  was  no  evidence  of  prosthetic  degeneration  or 
thrombosis  or  intravascular  hemolysis.   Although  approximately  4000  Hufnagel 
descending  aortic  prostheses  were  distributed  by  the  manufacturer  for  human 
use,  data  in  only  55  patients  in  whom  these  prostheses  were  inserted  were 
found  in  previous  publications,  and  26  of  them  had  died.   Of  the  13  late 
deaths  previously  reported,  no  evidence  of  prosthetic  degeneration  or 
thrombosis  was  described  in  the  3  patients  surviving  >3  years  (8,  10,  12.5 
years,  respectively).   Since  neither  our  2  patients  nor  the  other  3  reported 
long-term  survivors  had  prosthetic-related  complications  and  since  the 
dangers  of  excising  the  descending  aortic  prosthesis  are  considerable,  it 
appears  most  reasonable,  as  a  rule,  not  to  remove  the  descending  aortic 
prosthesis  at  any  time  in  these  patients  if  aortic  valve  replacement  is 
subsequently  performed. 

Keyword  Descriptors:   None 

Honors  and  Awards:   None 

Publications:   Chest  (in  press). 


073 


Project  No.  ZO1-HL-03014-Q1-OD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda, .Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Aortic  Valve  Atresia:   A  Necropsy  Study  of  73  Cases. 

Previous  Serial  Number:   None 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators:   Lowell  W.  Perry,  M.D. 
Roma  S.  Chandra,  M.D. 
Stephen  R.  Shapiro,  M.D. 
Lewis  P.  Scott,  M.D. 

Cooperating  Units:   Division  of  Pediatric  Cardiology,  Department  of  Child 
Health  and  Development  and  Department  of  Pathology, 
Children's  Hospital  National  Medical  Center,  Washington,  D.C. 

Project  Description:   Although  a  relatively  uncommon  lesion,  aortic  valve 
atresia  is  an  important  congenital  cardiovascular  malformation  because  it 
represents  the  least  tolerated  cardiac  anomaly  and,  therefore,  one  extreme  of 
the  spectrum  of  congenital  heart  disease.   Among  infants  with  congenital  mal- 
formations of  the  heart  or  great  vessels  during  the  first  week  of  life,  aortic 
valve  atresia  is  the  most  common  cause  of  death,  the  most  common  cause  of 
congestive  heart  failure,  and  the  cause  of  the  largest  hearts.   This  report 
describes  the  morphologic  features  of  73  hearts  examined  at  necropsy  with 
aortic  valve  atresia.   The  patients  ranged  in  age  from  1  to  17  days  (average  5) 
and  74%  were  boys.   Previous  reports  on  aortic  valve  atresia  described  only  the 
occurrence  of  a  hypoplasitc  left  ventricle  with  or  without  atretic  mitral 
valves.   In  the  present  study  of  73  cases  4  were  found  to  have  normal  or  near- 
normal  sized  left  ventricles  rather  than  hypoplastic  ones  and  3  of  them  had 
normally  developed  mitral  valves.   All  4  patients  with  normal  or  near -normal 
sized  left  ventricles  had  ventricular  septal  defects  whereas  all  the  remaining 
69  with  hypoplastic  left  ventricles  had  intact  ventricular  septae.   Because  no 
previous  descriptions  have  appeared  of  ventricular  septal  defect  or  normal-sized 
left  ventricles  associated  with  aortic  atresia  a  new  classification  of  this 
condition  was  proposed.   In  essence,  there  are  2  types  of  aortic  valve  atresia: 
in  type  I  the  left  ventricle  is  hypoplastic  and  the  ventricular  septum  is 
intact.   Among  our  69  patients  with  this  type,  25  had  atretic  mitral  valves  and 
44  had  hypoplastic  mitral  valves.   In  type  II,  the  left  ventricular  cavity  is 
of  normal  size  or  near-normal  size  and  one  or  more  ventricular  septal  defects 
are  present.  Among  our  4  patients  with  this  type,  1  had  an  atretic  mitral 
valve  and  3  had  a  normally  developed  mitral  valve.   Although  operative  inter- 
vention in  patients  with  hypoplastic  left  ventricles  associated  with  aortic 
atresia  is  fruitless,  it  appears  that  operative  intervention  may  be  fruitful 
in  the  individuals  with  normal  or  near -normal  sized  left  ventricular  cavities. 


8r+ 


Keyword  Descriptors:  Ventricular  septal  defect,  mitral  valve  atresia. 

Honors  and  Awards:  None 

Publications:   Accepted  for  publication  in  the  American  Journal  of  Cardiology. 


87S- 


Project  No.  Z01-HL-03015-Q2-QD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Endocardial  Structure  in  Carcinoid  Heart  Disease 

Previous  Serial  Number:   NHLI -234(c) 

Principal  Investigator:  Victor  J.  Ferrans,  M.D.,  Ph.D. 

Other  Investigators:   William  C.  Roberts,  M.D. 

Cooperating  Units:   None 

Project  Description:  The  carcinoid  syndrome  is  associated  with 
pathognomonic  cardiac  lesions  which  consist  of  plaque-like  fibrous 
thickenings  in  mural  and  valvular  endocardium.  Ultrastructural  study  of 
these  plaques  in  right  atrium  (2  patients)  and  tricuspid  and  pulmonic 
valve  (1  patient)  showed  similar  features.  Most  cellular  elements  were 
mature  smooth  muscle  cells  which  varied  from  fusiform  to  stellate  in  shape 
and  had  greatly  thickened,  reduplicated  basement  membranes.   Extracellular 
components  consisted  of:   layers  of  normal -appearing  collagen  fibrils 
oriented  parallel  to  the  surfaces  of  the  plaques  and  arranged  in  a  cross- 
weaving  pattern;  100  to  200  A  diameter  microfibrils;  layers  of  fibrillar 
material  similar  to  basement  membranes  of  smooth  muscle  cells,  and  dense 
spicules,  150  A  diameter  and  up  to  800  A  in  length.   No  elastic  fibers 
or  fibrin  deposits  were  found.   These  findings  suggest  that  carcinoid 
plaques  result  from  stimulation  of  endocardial  smooth  muscle  cells  to 
produce  collagen  and  basement  membrane-like  material;  such  stimulation  may 
be  intermittent,  as  evidenced  by  the  layered  arrangement  of  the  plaques. 

Keyword  Descriptors:   endocardium,  pathology,  ultrastructure,  carcinoid 
syndrome 

Honors  and  Awards :  None 

Publications:   Ferrans,  V.J.,  and  Roberts,  W.  C:   Ultrastructure  of 
Endocardial  Plaques  in  Carcinoid  Heart  Disease. 
Human  Pathology  (in  press) 


81i 


Project  No.  Z01-HL-03016-01-OD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  Unruptured  Sinus  of  Valsalva  Aneurysm 

Previous  Serial  Number:   None 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators:   Michael  C.  Fishbein,  M.D. 
Robert  T.  Obma,  M.D. 

Cooperating  Units:   Skemp-Grandview  Clinic,  La  Crosse,  Wisconsin 

Project  Description:   An  unruptured  congenital  sinus  of  Valsalva  aneurysm 
(behind  the  right  aortic  valve  cusp)  is  described  as  an  incidental  necropsy 
finding  in  an  82-year-old  man.   Review  of  previous  reports  on  aneurysms 
involving  only  1  of  3  aortic  sinuses  disclosed  that  few  cases  have  been 
described  and  that  these  lesions  are  rarely  diagnosed  during  life.   It  is 
probable,  however,  that  unruptured  aortic  sinus  aneurysm  (involving  only  1 
sinus)  is  more  common  than  previous  reports  indicate,  but  that  among 
patients  with  congenital  sinus  aneurysm,  more  likely  than  not,  rupture 
will  occur. 

Keyword  Descriptors:   None 

Honors  and  Awards:   None 

Publications:   Accepted  for  publication  in  the  American  Journal  of  Cardiology 
to  appear  probably  June  1975. 


«77 


Project  No.   ZO1-HL-Q3Q17-01-OD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland  . 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Endocardial  Papillary  Elastofibromas 

Previous  Serial  Number:   None 

Principal  Investigator:  Michael  C.  Fishbein,  M.D. 

Other  Investigators:  Victor  J.  Ferrans,  M.D. ,  Ph.D. 
William  C.  Roberts,  M.D. 

Cooperating  Units:  None 

Project  Description:  The  organization  of  cellular  and  extracellular 
components  appeared  similar  and  was  distinctive  in  3  endocardial  papillary 
elastofibromas  studied.   Each  tumor  papilla  contained:   1)  a  dense,  central 
core  of  collagen  and  elastic  tissue;  2)  a  peripheral,  myxomatous  layer  with 
deposits  of  acid  mucopolysaccharides,  and  3)  an  overlying,  hyperplastic 
layer  of  endothelial  cells.   Ultrastructural  study  of  1  tumor  showed  that 
the  cells  in  all  3  zones  had  numerous  cytoplasmic  filaments,  100  A  in 
diameter,  and  dilated  cisterns  of  endoplasmic  reticulum;  endothelial 
cells  also  had  intercellular  junctions  and  numerous  pinocytotic  vesicles. 
The  myxomatous  stroma  varied  from  amorphous  to  fibrillar,  and  the 
collagenous  cores  showed  focal  degeneration.  The  name  endocardial 
papillary  elastofibroma  is  suggested  to  emphasize  those  features  which  are 
most  conspicuous  and  which  serve  to  differentiate  this  tumor  from 
myxoma . 

Keyword  Descriptors:   myocardium,  valves,  neoplasm,  elastofibroma, 
pathology,  ultrastructure 

Honors  and  Awards:  None 

Publications:   Fishbein,  M.C.,  Ferrans,  V.J.,  and  Roberts,  W.C.: 
Endocardial  Papillary  Elastofibromas.  Archives  of 
Path,  (in  press) 


ei<b 


Project  No.   ZO1-HL-03018-Q1-QD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Ultrastructural  Features  of  Endocardial  Fibroelastosis 

Previous  Serial  Number:  None 

Principal  Investigator:  Michael  C.  Fishbein,  M.D. 

Other  Investigators:  Victor  J.  Ferrans,  M.D.,  Ph.D. 
William  C.  Roberts,  M.D. 

Cooperating  Units:  None 

Project  Description:  Histological  and  ultrastructural  studies  of  left 
ventricular  endocardium  from  10  patients  with  endocardial  fibroelastosis 
(EFE)  revealed  that  the  average  size  of  elastic  fibers  in  thickened 
endocardium  was  much  larger  in  the  4  patients  with  congenital  EFE  than 
in  the  6  patients  with  acquired  EFE  (secondary  to  ischemic  heart  disease 
in  2  patients,  to  prosthetic  cardiac  valves  in  3,  and  to  irradiation  of  the 
chest  in  1) .   Both  components  of  normal  elastic  tissue  (central,  amorphous 
cores  and  peripheral  microfibrils)  were  present  in  endocardial  elastic 
fibers  of  each  patient.  Ultrastructural  identification  of  elastic  fibers 
was  greatly  facilitated  by  staining  with  silver  tetraphenylporphin  sulfonate. 

Keyword  Descriptors:   endocardium,  fibroelastosis,  pathology,  ultrastructure 

Honors  and  Awards:  None 

Publications:   Fishbein,  M.C.,  Ferrans,  V.J.,  and  Roberts,  W.C.: 

Histologic  and  Ultrastructural  Features  of  Primary  and 
Secondary  Endocardial  Fibroelastosis.   Submitted  to 
Archives  of  Path.   Laboratory  Investigation  (abstract) 
(in  press) 


erf 


Project  No.  ZQ1-HL-Q3Q19-01-OB-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Morphologic  Evaluation  of  Myocardial  Protection 

Previous  Serial  Number:  None 

Principal  Investigator:  Victor  J.  Ferrans,  M.D.,  Ph.D. 

Other  Investigators:  None 

Cooperating  Units:  None 

Project  Description:   The  purpose  of  this  project  is  to  review  histologic, 
histochemical  and  electron  microscopic  methods  considered  useful  in  the 
morphologic  evaluation  of  procedures  designed  to  prevent  myocardial 
ischemic  or  metabolic  injury  during  cardiac  operations.  Conclusions  of 
this  review  are: 

1.  Transmural  samples  of  myocardium  should  be  studied,  as  the  response  of 
the  ventricular  walls  to  ischemic  injury  is  not  homogeneous. 

2.  Collection  of  samples  should  be  continued  until  the  injury  reaches  a 
stable  end  point. 

3.  Studies  of  myocardial  protection  should  take  into  account  the  fact  that 
ischemic  injury  is  modified  considerably  by  reflow  phenomena. 

4.  Ultrastructural  studies  are  indispensable,  and  histologic  methods  are 
of  limited  value  in  the  morphologic  evaluation  of  early  myocardial  injury. 

Keyword  Descriptors:   myocardium,  metabolism,  ischemia,  pathology, 
ultrastructure,  histochemistry 

Honors  and  Awards :   None 

Publications:   Ferrans,  V.J.:  Morphologic  Methods  for  Evaluation  of 
Myocardial  Protection.  Annals  of  Thoracic  Surgery, 
July,  1975 


<8&o 


Project  No.   ZO1-HL-03020-Q2-OB-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Cardiac  Morphologic  Changes  Produced  by  Ethanol 

Previous  Serial  Number:  NHLI -235(c) 

Principal  Investigator:  Victor  J.  Ferrans,  M.D.,  Ph.D. 

Other  Investigators:   L.  Maximilian  Buja,  M.D. 
William  C.  Roberts,  M.D. 

Cooperating  Units:  None 

Project  Description:  This  study  presents:   1)  new  ultrastructural 
observations  (showing  varying  degrees  of  swelling  of  sarcoplasmic 
reticulum,  mitochondrial  damage,  dilatation  of  T  tubules,  lipid 
accumulation,  myofibrillar  lysis  and  interstitial  fibrosis)  made  on 
myocardial  biopsies  from  patients  with  congestive  cardiomyopathy  and 
chronic  alcoholism,  and  .2)  a  comprehensive  review  of  all  cardiac 
morphologic  changes  produced  by  the  acute  and  chronic  ingestion  of  large 
amounts  of  alcohol  alone  and  alcohol  plus  cobalt-containing  compounds 
(cobalt-beer  cardiomyopathy)  in  humans  and  experimental  animals. 

Keyword  Descriptors:  myocardium,  pathology,  ultrastructure, 
cardiomyopathies,  ethanol,  drug  effect 

Honors  and  Awards:   None 

Publications:   Ferrans,  V.J.,  Buja,  L.M.,  and  Roberts,  W.C.:  Cardiac 

Morphologic  Changes  Produced  by  Ethanol.   In  Rothschild, 
M.,  Schreiber,  S.  and  Oratz,  M.  (Eds.):  Alcohol,  Nutrition 
and  Protein  Synthesis.  New  York,  Pergamon  Press,  1975, 
pp.  139-185. 


est 


Project  No.  Z01-HL-03021-01-3D-P 

1.  ODIR 

2.  Pathology  Section 

3 .  Bethesda , . Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Massive  Myocardial  Hemosiderosis 

Previous  Serial  Number:   None 

Principal  Investigator:   Ernest  N.  Arnett,  M.D. 

Other  Investigators:   Arthur  W.  Nienhuis,  M.D. 
Walter  L.  Henry,  M.D. 
Victor  J.  Ferrans,  M.D..  Ph.D. 
David  R.  Redwood,  M.D. 
William  C.  Roberts,  M.D. 

Cooperating  Units:   Molecular  Hematology  and  Cardiology  Branches, 
National  Heart  &  Lung  Institute 

Project  Description:   This  report  discusses  a  23-year-old  man  with  Blackfan- 
Diamond  anemia.   He  received  over  500  transfusions  during  his  life  and  the 
effects  of  the  massive  deposition  of  iron  in  the  myocardium  is  reviewed. 
This  report  emphasizes  that  heavy  deposition  of  iron  in  myocardial  fibers 
causes  the  myocardium  to  function  abnormally  and  the  present  patient 
developed  evidence  of  congestive  failure  and  various  arrhythmias.   The 
distribution  of  iron  in  myocardium  was  described  and  the  amount  was  quantitated 
biochemically  and  also  studied  ultrastructurally. 

Keyword  Descriptors:   None 

Honors  and  Awards:   None 

Publications:   Accepted  for  publication  in  the  American  Heart  Journal. 


802. 


Project  No.  Z01-HL-03022-01-OD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Cardiac  Ultrastructure  in  the  Cardiomyopathies 

Previous  Serial  Number:  None 

Principal  Investigator:  Victor  J.  Ferrans,  M.D.,  Ph.D. 

Other  Investigators:  None 

Cooperating  Units:   None 

Project  Description:  This  communication  summarizes  investigations  on 
cardiac  morphology  in  patients  with  hypertrophic  cardiomyopathy  (asymmetric 
septal  hypertrophy,  ASH)  and  in  patients  with  congestive  cardiomyopathies 
of  various  causes.   In  the  ventricular  septum  of  patients  with  ASH  the 
muscle  cells  are  severely  disorganized  and  often  run  in  different  directions 
instead  of  in  parallel.  These  cells  are  wider  and  shorter  than  in 
hypertrophy  due  to  other  causes  and  show  increased  cellular  branching, 
extensive  side-to-side  intercellular  junctions,  widened  Z  bands,  and 
evidence  of  formation  of  new  sarcomeres.  Some  myofibrils  are  oriented 
obliquely  or  perpendicular  to  the  longitudinal  axis  of  the  cell,  and  some 
myofilaments  that  originate  from  a  single  Z  band  of  a  given  myofibril  insert 
into  Z  bands  of  other  myofibrils.  These  observations  have  led  to  the 
conclusions  that:  1)  ASH  is  a  disease  that  involves  the  architectural 
arrangement  of  the  muscle  cell,  particularly  that  of  their  contractile 
elements,  and  2)  this  abnormal  arrangement  may  lead  to  the  generation  of 
abnormal  mechanical  forces,  which  in  turn  may  be  responsible  for  the  bizarre 
type  of  hypertrophy.   In  patients  with  obstructive  ASH  these  abnormalities 
are  either  absent  or  present  only  to  a  very  limited  extent  in  muscle  from 
the  free  walls  of  the  left  and  right  ventricles.   In  contrast  to  this, 
these  changes  were  extensively  distributed  throughout  both  free  walls  in 
severely  symptomatic  patients  with  non-obstructive  ASH.  These  observations 
suggest  that  the  hypertrophy  that  develops  in  the  ventricular  free  walls  of 
patients  with  obstructive  ASH  is  secondary  to  obstruction  to  outflow,  and 
that  cardiac  functional  limitation  in  these  patients  is  due  largely  to  this 
obstruction.  The  more  diffuse  abnormalities  in  patients  with  non-obstructive 
ASH  suggest  that  these  changes  have  a  direct,  important  contribution  to  the 
cardiac  functional  impairment  (failure  of  diastolic  compliance)  in  these 
patients. 

No  distinctive  or  diagnostic  lesion  was  demonstrated  by  histologic  or 
ultrastructural  study  in  myocardium  of  patients  with  congestive  cardio- 
myopathies. These  studies  revealed  degenerative  changes,  the  severity  of 
which  generally  correlated  with  the  duration  and  degree  of  symptoms  of 

l  083 


cardiac  dysfunction.  The  ultrastructural  changes  in  patients  with 
congestive  cardiomyopathies  were  found  to  occur  to  a  variable  extent 
in  the  late  stages  of  ventricular  hypertrophy  due  to  other  causes. 

Keyword  Descriptors:   myocardium,  cardiomyopathies,  pathology, 
ultrastructure 

Honors  and  Awards :  None 

Publications:   Ferrans,  V.J.:   Cardiac  Ultrastructure  in  the 

Cardiomyopathies.   Part  of  the  Symposium  on  the 
Cardiomyopathies  in  the  Proceedings  of  the  VII  World 
Congress  of  Cardiology,  Excerpta  Medica,  Amsterdam  (in  press) 


ag«f 


Project  No.   Z01-HL-03023-01-OD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Cardiac  Structure  in  Hypertrophy 

Previous  Serial  Number:  None 

Principal  Investigator:  Victor  J.  Ferrans,  M.D. 

Other  Investigators:   None 

Cooperating  Units:  None 

Project  Description:  This  project  consisted  of  a  detailed  review  of 
cardiac  structure  in  hypertrophy,  with  emphasis  on  ultrastructural 
alterations  of  myocardium  in  the  three  stages  of  hypertrophy  (developing 
hypertrophy,  stable  hyperf unction  and  cellular  exhaustion)  and  on 
quantitative  data  derived  from  stereological  analysis  of  electron 
micrographs  from  animal  models  of  hypertrophy. 

Keyword  Descriptors:  myocardium,  hypertrophy,  ultrastructure 

Honors  and  Awards :  None 

Publications:   Ferrans,  V.J. :  Cardiac  Structure  in  Hypertrophy. 

To  be  published  as  a  book  chapter  in  Cardiac  Hypertrophy, 
Morkin,  E.  (Ed.),  John  Wiley  &  Sons 


e§sr 


Project  No.  ZO1-HL-03024-O1-QD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:  Nuclear  Membranes  in  Hypertrophied  Human  Myocardium 

Previous  Serial  Number:   None 

Principal  Investigator:  Victor  J.  Ferrans,  M.D.,  Ph.D. 

Other  Investigators:  Michael  Jones,  M.D. 
Barry  J.  Maron,  M.D. 
William  C.  Roberts,  M.D. 

Cooperating  Units:   Clinic  of  Surgery  and  the  Cardiology  Branch,  National 
Heart  and  Lung  Institute 

Project  Description:  Nuclear  membranes  of  cardiac  muscle  cells  were 
studied  in  134  patients  with  cardiac  hypertrophy  of  various  causes. 
Abnormalities  observed  consisted  of:  1)  increased  foldings  and  convolutions; 
2)  nuclear  pseudoinclusions  formed  by  cytoplasmic  organelles  protruding 
into  saccular  invaginations  of  the  nuclear  membranes,  and  3)  intranuclear 
tubules.  The  increased  foldings  and  convolutions  of  the  nuclear  membranes, 
and  the  nuclear  pseudoinclusions,  appear  to  result  from  synthesis  of 
nuclear  membranes  in  excess  of  that  needed  to  accomodate  the  increase  in 
nuclear  volume  which  occurs  in  hypertrophy.   Intranuclear  tubules  were 
found  in  6  patients  and  consisted  of  tubular  invaginations,  400  to  650  A 
in  diameter,  of  the  inner  nuclear  membranes  into  the  nucleoplasm.   Some  of 
these  tubules  were  straight  and  cylindrical,  and  were  associated  with  a 
peripheral  layer  of  marginated  chromatin;  others  were  not  associated  with 
chromatin,  appeared  coiled  and  followed  irregular  courses.   Intranuclear 
tubules  in  cardiac  muscle  cells  probably  represent  an  extreme  cellular 
response  to  the  stimulus  of  hypertrophy. 

Keyword  Descriptors:   myocardium,  hypertrophy,  cardiomyopathies,  nuclei, 

nuclear  membranes,  pathology,  ultrastructure,  nuclear 
tubules 

Honors  and  Awards:   None 

Publications:   Ferrans,  V.J.. ,  Jones,  M.,  Maron,  B.J.  and  Roberts,  W.C.  : 

The  Nuclear  Membranes  in  Hypertrophied  Human  Cardiac  Muscle 
Cells.  Am.  J.  Pathol.  78:   427-460,  1975 


B& 


Project  No.  ZO1-HL-03025-Q2-OD-? 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland. 

PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Intranuclear  Glycogen  in  Myocardium 

Previous  Serial  Number:  NHLI -237(c) 

Principal  Investigator:  Victor  J.  Ferrans,  M.D.,  Ph.D. 

Other  Investigators:   Barry  J.  Maron,  M.D. 

L.  Maximilian  Buja,  M.D. 

Nayab  Ali,  M.D. 

William  C.  Roberts,  M.D. 

Cooperating  Units:   Cardiology  Branch,  National  Heart  and  Lung  Institute 
Cardiac  Laboratory,  District  of  Columbia  General 
Hospital 

Project  Description:   Ultrastructural  and  cytochemical  studies  of 
myocardial  biopsies  disclosed  the  presence  of  glycogen  deposits  within 
nuclei  of  hypertrophied  and  normal -sized  cardiac  muscle  cells  in  6  (7%) 
of  90  patients  with  various  cardiac  diseases.   Intranuclear  glycogen 
appeared  as  g- particles,  160  to  360  A  in  diameter,  which  either  formed 
small  aggregates  or  were  dispersed  in  the  nucleoplasm.   Cells  containing 
intranuclear  glycogen  did  not  show  damage  or  degeneration.  Glycogen 
particles  in  the  cytoplasm  of  these  cells  were  of  the  same  size  and 
appearance  as  those  in  the  nuclei.  Criteria  for  the  ultrastructural 
identification  of  intranuclear  glycogen  are  proposed,  and  it  concluded 
that  under  certain  conditions  the  nuclei  of  cardiac  muscle  cells  can 
acquire  the  capacity  to  synthesize  glycogen. 

Keyword  Descriptors:  myocardium,  pathology,  ultrastructure,  hypertrophy, 
glycogen 

Honors  and  Awards:   None 

Publications:   Ferrans,  V.J.,  Maron,  B.  J.,  Buja,  L.  M.,  Ali,  N.,  and 

Roberts,  W.  C:  Intranuclear  Glycogen  Deposits  in  Human 
Cardiac  Muscle  Cells:  Ultrastructure  and  Cytochemistry. 
Journal  of  Molecular  and  Cellular  Cardiology  (in  press) 


067 


Project  No.  Z01-HL-03026-Q2-QD-P 

1.  ODIR 

2.  Pathology  Section 

3.  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   Cardiac  Lesions  in  Bone  Marrow  Transplantation 

Previous  Serial  Number:   NHLI-231(c) 

Principal  Investigator:   L.  Maximilian  Buja,  M.D. 

Other  Investigators:   Victor  J.  Ferrans,  M.D.,  Ph.D. 
Robert  G.  Graw,  M.D. 

Cooperating  Units:   Experimental  Hematology  Section,  Pediatric  Oncology 
Branch,  National  Cancer  Institute 

Project  Description:   Cardiac  pathologic  findings  were  analyzed  in  20 
necropsied  patients  from  a  series  of  26  patients  with  leukemia,  aplastic 
anemia,  immune  deficiency  disease  or  metastatic  cancer  who  had  been  treated 
with  bone  marrow  transplantation.   Most  cardiac  alterations  were  similar  to 
those  which  occur  in  patients  with  hematologic  and  neoplastic  diseases  who 
have  not  been  treated  with  bone  marrow  transplantation,  and  consisted  of: 
hemorrhage  (12  patients),  foci  of  necrosis  associated  with  sepsis  (9  patients), 
myocardial  abscesses  (4  patients),  infective  endocarditis  (1  patient)  and 
hemosiderosis  (1  patient) .   Other  cardiac  alterations  were  more  specifically 
related  to  bone  marrow  transplantation.   Six  patients  exhibited  a 
distinctive  interstitial  reactive  change  characterized  by  the  presence  of  a 
pleomorphic  population  of  lymphoid,  histiocytic  and  Anitschkow  cells.   This 
alteration  may  have  been  induced  by  abnormal  immune  mechanisms,  as  suggested 
by  the  observation  that  5  of  the  6  patients  with  interstitial  change  had 
clinical  evidence  of  graft-versus-host  disease.   Two  patients  developed 
fatal  cardiac  failure  in  the  post-transplant  period,  and  exhibited  myocardial 
damage  with  histologic  and  ultrastructural  features  indicative  of  severe 
acute  injury.   Findings  in  these  2  patients  consisted  of:  1)  necrotic 
muscle  cells  which  exhibited  multiple  contraction  bands,  diastase-resistant 
PAS  staining  and  intracellular  fibrin  deposits;  2)  microthrombi  which  were 
composed  of  fibrin  and,  occasionally,  of  fibrin  and  platelets,  and  3) 
extravasated  erythrocytes  and  fibrin  strands  in  interstitium.  Clinico- 
pathologic  analysis  strongly  suggested  that  the  fatal  cardiotoxicity  in 
both  patients  resulted  primarily  from  effects  of  high  doses  of 
cyclophosphamide  (180  mg/kg  and  270  mg/kg)  which  were  administered  as  part 
of  a  newly  developed  regimen  of  combination  chemotherapy-immunosuppression 
(B.A.C.T.).  Our  findings  emphasize  the  need  for  more  effective  and  less 
toxic  antineoplastic  and  immunosuppressive  therapy  for  patients  who  require 
bone  marrow  transplantation. 


aes 


Keyword  Descriptors:   transplantation,  bone  marrow,  myocardium,  pathology, 
ultrastructure,  drug  effects 

Honors  and  Awards:  None 

Publications:   Buja,  L.M.,  Ferrans,  V.J.,  and  Graw,  R.  G. :  Cardiac 

Pathologic  Findings  in  Patients  Treated  with  Bone  Marrow 
Transplantation.  Human  Pathology  (in  press) 


08? 


Project  No.  ZO1-HL-03027-01-QD-P 

1.  ODIR 

2.  Pathology  Section 

3 .  Bethesda,  Maryland 

PHS-NIH 
Individual  Project  Report 
July  1,  1974  through  June  30,  1975 

Project  Title:   The  Structural  Basis  of  Abnormal  Cardiac  Function 

Previous  Serial  Number:   None 

Principal  Investigator:   William  C.  Roberts,  M.D. 

Other  Investigators:   None 

Cooperating  Units:   None 

Project  Description:  This  report,  in  essence,  is  a  review  of  morphologic 
observations  made  in  this  laboratory  during  the  past  10  years  regarding 
coronary,  hypertensive,  valvular,  idiopathic  myocardial  and  pericardial 
heart  diseases.  The  status  of  the  major  extramural  coronary  arteries  in 
patients  with  fatal  ischemic  heart  disease  is  summarized.   Specifically,  for 
fatal  or  even  symptomatic  ischemic  heart  disease  to  occur,  at  least  2  of  the  3 
major  coronary  arteries  must  be  >75%  narrowed  by  old  atherosclerotic  plaques. 
The  myocardial  response  to  severe  coronary  narrowing  is  highly  variable  and 
indeed  no  differences  in  degrees  of  luminal  narrowing  were  observed  among 
fatal  cases  of  angina  pectoris,  sudden  coronary  death,  and  acute  myocardial 
infarction.   The  frequency  of  histories  of  systemic  hypertension  in  many 
cardiovascular  conditions  was  reviewed  and  secondly  the  percent  of  patients 
with  various  cardiovascular  conditions  who  have  cardiomegaly  were  reviewed. 
It  was  emphasized  that  systemic  hypertension  is  a  far  greater  risk  factor  than 
previously  emphasized  by  history  of  hypertension  or  measurement  of  elevated 
blood  pressure  if  left  ventricular  hypertrophy  is  used  as  an  indicator  of 
hypertension  rather  than  history  or  actual  measurement  of  blood  pressure.   The 
morphologic  features  of  various  valvular  lesions  were  reviewed  and  emphasis 
was  placed  on  the  fact  that  rheumatic  heart  disease  is  a  less  common  cause  of 
valvular  heart  disease  than  are  non-rheumatic  etiologies.   Stress  was  placed 
on  classifying  valvular  heart  disease  into  the  purely  regurgitant   lesions 
and  into  those  with  elements  of  stenosis  because  the  etiology  of  the  former 
are  only  3  in  number  whereas  the  etiology  of  the  latter  were  numerous. 
Morphologic  features  of  the  idiopathic  cardiomyopathies,  i.e.,  both  the 
ventricular  dilated  and  the  non-ventricular  dilated  type  (ASH),  were  reviewed. 
Likewise,  various  morphologic  features  of  pericardial  heart  disease  were 
reviewed . 

Keyword  Descriptors:   Coronary  heart  disease,  hypertension,  valvular  heart 
disease,  myocardial  heart  disease,  pericardial  heart  disease. 

Honors  and  Awards:   None 

Publication:   Chapter  in  a  book  entitled  Clinical  Cardiovascular  Physiology 
edited  by  Herbert  Levine  to  be  published  in  iy/^  by  Grune  and 
Stratton.  ^Q 


ANNUAL  REPORT  OF  THE 

SECTION  ON  THEORETICAL  BIOPHYSICS 

OFFICE  OF  THE  DIRECTOR  OF  INTRAMURAL  RESEARCH 

NATIONAL  HEART  AND  LUNG  INSTITUTE 

July  1,  1974  through  June  30,1975 

The  primary  interest  of  the  Section  on  Theoretical  Biophysics  is  the 
theory  of  transport  processes  in  biological  systems,  with  particular  reference 
to  problems  in  cardiovascular,  renal,  and  membrane  physiology.  The  section 
is  concerned  both  with  the  formulation  of  theoretical  models  and  with  the 
development  of  mathematical  and  computational  methods  for  their  analysis. 
Currently  much  of  the  research  in  the  section  centers  on  the  mechanism  of 
urine  formation  in  the  mammalian  kidney. 

Mathematical  theory  of  renal  function: 

Since  this  report  marks  the  fifteenth  anniversary  of  our  work  on  the 
theoretical  analysis  of  renal  function,  a  brief  retrospective  seems  in  order. 
From  1960  to  1970  we  were  primarily  concerned  with  the  theoretical  analysis 
of  solute  cycling  models  of  the  renal  counterflow  system.   In  these  models, 
originally  proposed  by  Wirz  and  theoretically  analyzed  by  Kuhn  and  Ramel> 
concentration  of  urine  occurs  by  salt  being  actively  transported  out  of 
ascending  limb  of  Henle  into  the  interstitium,  whence  it  enters  descending 
Henle's  limb  to  be  recycled.   In  this  early  work  we  analysed  both  the  steady 
state  and  transient  behavior  of  solute  cycling  systems  and  established  that  in 
single  solute  systems  active  salt  transport  out  of  ascending  Henle's  limb 
was  necessary  for  concentration. 

A  large  body  of  data,  both  from  micropuncture  and  isolated  tubule 
experiments,  suggests  that  concentration  of  fluid  in  descending  Henle's 
limb  occurs  primarily  by  water  extraction  rather  than  solute  cycling.   In 
this  process  solute  from  ascending  Henle's  limb  and  collecting  duct  enters 
the  interstitium  raising  its  osmotic  pressure  and  so  withdrawing  water  from 
descending  Henle's  limb  and  collecting  duct.   The  water  and  salt  supplied 
by  the  tubules  are  taken  up  by  the  blood  vessels  of  the  medulla  and  returned 
to  the  systemic  circulation.  Until  1970,  attempts  to  model  the  water  extracting 
process  were  singularly  unsuccessful.   The  basic  difficulty  was  the  failure 
of  solutions  of  equations  describing  such  models  to  give  simultaneous  solute 
and  water  balance  —  one  or  the  other  accumulating  in  the  medullary 
interstitium. 

i   In  1970  we  discovered  both  the  cure  and  the  cause  —  in  that  order.   In 
earlier  models  medullary  capillaries  had  been  assumed  to  play  a  subsidiary 
role  in  the  concentrating  mechanism,  conserving  solute  supplied  by  the  renal 
tubules  by  a  parallel  vascular  counterflow  system  that  allows  equilibration 
of  solute  in  ascending  and  descending  flows.   In  solute  cycling  models  the 
classic  view  is  essentially  correct,  but  in  models  that  concentrate  by  water 
extraction  the  capillaries  are  an  integral  part  of  the  counterflow  system. 
The  difference  between  ascending  and  descending  vascular  volume  flows  must 
equal  water  taken  up  from  the  renal  tubules  and  the  difference  between 
ascending  and  descending  axial  solute  flows  must  equal  solute  taken  up  from 
the  tubules.   If  one  assumes  that  concentrations  in  ascending  and  descending 

i  89/ 


flows  are  identical  one  is  led  to  a  central  core  model  of  the  medullary 
counterflow  system  in  which  blood  vessels  and  interstitium  are  merged  into 
a  single  tube,  closed  at  the  papillary  end  and  open  at  the  cortical  where  it 
is  assumed  to  empty  into  the  systemic  circulation.   Three  parallel  flow  tubes, 
which  can  exchange  with  the  core  and  with  each  other  correspond  to  ascending 
limbs  of  Henle's  loop,  descending  limbs  of  Henle's  loops  and  collecting 
ducts.   With  this  model  we  made  the  first  coherent  analysis  of  salt,  water 
and  urea  movement  and  of  free  energy  balance  in  the  medullary  counterflow 
system. 

More  or  less  simultaneously  with  the  development  of  the  central  core 
model  we  discovered  that  the  equations  for  the  earlier  models  were  inconsis- 
tant  under  the  usual  assumptions  that  were  introduced  to  solve  them.   There 
are  various  ways  to  relieve  this  inconsistency.   One  is  to  permit  volume  flow 
in  the  interstitium  parallel  to  the  other  flow  tubes.   With  large  hydraulic 
and  solute  permeabilities  of  the  vasa  recta  this  leads  to  the  central  core 
model.   Alternatively,  one  can  include  hydrostatic  and  colloid  osmotic 
pressure  (neglected  in  earlier  models)  as  a  driving  force  for  the  transmural 
movement  of  solutes  and  water  in  medulla.   This  alternative  was  not  feasible 
in  1970,  because  numerical  methods  for  solving  the  equations  describing  such 
a  complicated  flow  system  were  not  available. 

During  the  years  1970-71  and  1971-72  the  analytical  theory  of  the 
central  core  model  was  intensively  developed  and  essentially  completed.   Among 
the  principal  results  of  this  study  were:   1)  an  analysis  of  the  behavior 
of  the  medullary  counterflow  system  for  solute  cycling,  water  extracting, 
and  mixed  modes  of  operation,  2)  a  study  of  concentration  profiles  and 
energetic  requirements  for  different  pump  kinetics  in  ascending  Henle's 
limb,  3)  a  new  understanding  of  the  role  of  urea  in  the  concentrating 
mechanism. 

Early  in  1972  a  detailed  five  year  research  plan  was  developed  with  the 
two  closely  related  goals  of  expanding  and  deepening  the  general  theoretical 
analysis  of  renal  function  and  of  developing  a  realistic  computer  simulation 
of  renal  function,  which  would  relate  membrane  transport  properties  of  the 
nephron  to  macroscopic  function  of  the  kidney.  This  program  required  a  sub- 
stantially increased  committment  of  NHLI  resources,  which  was  justified  on 
the  basis  of  the  following  goals. 

1)  Increased  understanding  of  renal  function. 

2)  Improved  diagnostic  methods. 

3)  Better  treatment  of  renal  and  cardiac  disease. 

4)  Improvement  in  the  design  of  artificial  kidneys. 

In  carrying  out  the  program  the  primary  need  was  to  develop  numerical 
methods  for  solving  the  equations  describing  kidney  models.   To  do  this 
required  expertise  in  numerical  analysis  and  computer  programming  not 
possessed  by  NHLI  at  the  outset.   A  cooperative  program  was  initiated  with 


8^ 


a  DCRT  mathematician  who  subsequently  transferred  to  NHLI.   We  also  have 
utilized  cooperative  and  collaborative  programs  with  mathematicians 
possessing  required  expertise  at  the  University  of  Maryland,  Louisiana 
Polytechnic  Institute,  and  SUNY,  Stony  Brook,  N.  Y.   This  program  has 
progressed  extremely  well  and  we  have  now  developed  several  very  efficient 
algorithms  for  solving  difference  equations  describing  water  and  solute 
transport  in  flow  systems.   These  methods  have  been  applied  to  a  variety 
of  models  of  the  medullary  counterflow  system  and  have  now  been  applied  to 
a  model  of  the  whole  kidney.   With  this  model  it  has  been  possible  for  the 
first  time  to  simulate  behavior  of  the  whole  kidney  as  a  function  of 
hydrostatic  pressure  in  renal  artery,  vein,  and  pelvis,  protein  concentration 
in  arterial  blood,  and  phenomeno logical  equations  describing  transport  of 
salt  and  water  across  nephron  and  capillary  walls.   The  extension  of  our 
numerical  method  to  models  that  consider  several  solutes  and  the  distribution 
of  nephrons  into  cortical  and  juxta  medullary  seems  straight  forward 
and  with  continued  support  we  expect  to  meet  our  goal  of  developing  a  realistic 
computer  simulation  of  the  kidney  by  1977. 

Although  our  present  models  of  the  kidney  and  its  various  subsystems  are 
at  an  intermediate  level  of  sophistication ,  their  study  has  already  trans- 
formed our  concepts  of  renal  function.   In  the  classic  view,  the  functional 
unit  of  the  kidney  is  the  individual  nephron;  the  one  million  or  so  nephrons 
making  up  the  kidney  act  strictly  in  parallel  to  function  as  one  giant 
nephron.   Until  the  idea  of  countercurrent  multiplication  was  introduced, 
it  was  thought  urine  formation  could  be  understood  by  the  sequential  series 
processing  of  glomular  filtrate  by  the  various  tubular  segments.   Folding 
the  nephron  in  the  countercurrent  models  introduced  the  concept  that  the  more 
distal  parts  of  the  nephron  can  interact  with  the  more  proximal .   In  the 
solute  cycling  models  this  interaction  is  restricted  to  direct  pairwise 
interaction  of  contiguous  segments.   This  is  reflected  in  the  linearity  of 
the  equations  describing  these  models  and  the  sparseness  of  their  connectivity 
matrix.   In  the  central  core  models  a  new  level  of  interaction  is  introduced. 
Functionally  the  counterflowing  solution  in  the  core  links  solute  and  water 
transport  in  noncontiguous  segments  of  the  nephron.   Thus,  solute  transport 
out  of  ascending  limb  and  collecting  duct  in  the  inner  medulla  induces  water 
movement  out  of  descending  limb  in  the  outer  medulla;  urea  transport  out  of 
collecting  duct  is  coupled  to  salt  transport  out  of  ascending  Henle's  limb; 
and  active  transport  in  cortex  and  outer  medulla  is  coupled  to  passive  trans- 
port in  the  inner  medulla. 

The  numerical  analysis  of  the  detailed  models,  with  tubules  and  capil- 
laries exchanging  either  directly  or  via  the  interstitial  space,  have  con- 
firmed the  insight  gained  from  the  central  core  model.   It  has  also  suggested 
the  probable  importance  of  the  coupling  of  different  types  of  nephrons  via 
the  vascular  interstitial  space.   In  addition,  study  of  these  detailed  models 
has  shown  the  importance  of  the . transport  properties  of  the  capillaries. 

The  general  thesis  we  have  drawn  from  these  studies  is  that  the  func- 
tional unit  of  the  kidney  is  not  the  individual  nephron  but  a  nephro- 
vascular  unit  consisting  of  a  group  of  nephrons  and  their  tightly  coupled 
vasculature.   The  operation  of  this  unit  depends  both  on  the  segmental 
transport  characteristics  of  the  individual  nephron  and  capillaries  and  the 
way  in  which  these  are  coupled  by  the  architecture  of  the  unit.   Our  mathe- 

3  &?3 


matical  techniques  have  reached  the  point  where  for  the  first  time  a  synthesis 
of  transport  characteristics  and  coupling  is  possible. 

A  summary  of  specific  results  over  the  past  twelve  months- follows: 

Improvement  of  numerical  methods  for  solving  the  difference  equations 
describing  various  models  continued  as  a  major  project.   During  the  year 
a  variety  of  partitioning  schemes  have  been  developed  for  solving  models 
piecewise.   A  generalized  sparse  matrix  routine  was  adopted  for  Newton - 
Raphson  solutions.   In  addition  several  special  sparse  matrix  inversion 
methods  were  developed.   The  efficiency  of  these  schemes  was  compared  for 
parallel  flow  tubes  exchanging  via  an  interstial  space.   Piecewise  solutions 
were  found  to  be  accurate  and  most  efficient  in  utilization  of  computer 
storage  and  time. 

The  improved  numerical  methods  were  used  to  obtain  solutions  for  a 
model  of  the  whole  kidney  and  various  models  of  the  medullary  counterflow 
system  as  described  above. 

Earlier  work  on  solution  of  the  time  dependent  equations  by  quasi- 
linearization  was  extended  and  compared  with  Newton  type  methods. 

Previous  work  on  analytical  solutions  of  the  time  dependent  equations 
of  counter  current  systems  with  only  solute  exchange  has  been  extended  to 
give  solutions  for  systems  with  differing  ascending  and  descending  flow 
velocities.   These  solutions  have  been  compared  with  those  obtained  by 
numerical  inversion  of  Laplace  transforms. 

A  major  effort  has  been  directed  toward  establishing  conditions  for 
the  existence  and  uniqueness  of  differential  equations  describing  water  and 
solute  transport  in  flow  systems.   For  a  single  tube  exchanging  water  and  a 
single  solute  with  an  external  bath  it  has  been  possible  to  establish  such 
conditions.   It  is  anticipated  that  the  analytical  tools  developed  for  the 
problem  will  be  useful  in  studying  more  complicated  flow  equations. 

The  work  on  free-energy  balance  in  renal  counterflow  systems  has  ap- 
peared (Math.  Bioscience  21:299-310,  1974).   This  thermodynamic  analysis 
has  been  extended  to  include  the  effects  of  viscous  dissipation. 


W  + 


Project  No.   Z01  HL  03201-15   STB 

1 .  ODIR 

2 .  Section  on  Theoretical  Biophysics 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:  Mathematical  Theory  of  Renal  Function 

Previous  Serial  Number:   NHLI-239 

Principal  Investigator:   John  L.  Stephenson,  M.D. 

Other  Investigators:   Raymond  Mejia 

Alan  M.  Weinstein  (Student) 
Harvard  Medical  School 
Boston,  Massachusetts 

Bruce  Kellogg,  Ph.D. 
Professor  of  Mathematics 
IFDAM,  University  of  Maryland 
College  Park,  Maryland 

Kenny  Crump,  Ph.D. 
Professor  of  Mathematics 
Louisiana  Polytechnic  Institute 
Ruston ,  Louisiana 

Jack  Garner,  Ph.D. 
Professor  of  Mathematics 
Louisiana  Polytechnic  Institute 
Ruston,  Louisiana 


Cooperating  Units : 


Project  Description: 


NIAMDD,  Office  of  Mathematical  Research 

Institute  of  Fluid  Dynamics  and  Applied  Mathematics 
University  of  Maryland 

Department  of  Mathematics 
Louisiana  Polytechnic  Institute 
Ruston,  Louisiana 


Objectives:   The  primary  purpose  of  this  project  is  to  develop  the  general 
theory  of  the  transport  and  flow  processes  taking  place  in  the  kidney.   This 
theory  provides  the  general  basis  for  guantitative  models  of  renal  function. 

Major  findings:   1)   The  thermodynamic  analysis  of  flow  through  an 
isothermal  system  of  tubes  was  extended  (by  Alan  Weinstein)  to  include  a 

i  e?s- 


Project  No.    ZQ1  HL  03201-15  STB 

term  representing  dissipation  of  energy  due  to  viscous  flow.   The  dissipation 
per  unit  length  was  found  to  be  approximately  R. (F.  )  ,  where  R.  is  the 
hydrodynamic  flow  resistance  of  the  ith  tube  in  the  system  and  P.   is  the 
axial  volume  flow.   This  energy  dissipation  was  computed  for  a  particular 
collection  of  models  and  its  variation  with  changes  in  the  parameters  of 
the  model  studied. 

2) Earlier  work  on  analytical  solutions  of  the  time  dependent  equations  of 
countercurrent  systems  with  only  solute  exchange  has  been  extended  to  give 
solutions  for  systems  with  differing  ascending  and  descending  flow  velocities. 
These  solutions  have  been  compared  with  those  obtained  by  numerical  inversion 
of  Laplace  transforms  (with  J.  Garner  and  K.  Crump) . 

3)   In  a  collaborative  program  with  IFDAM,  University  of  Maryland,  a 
study  was  undertaken  by  Dr.  Bruce  Kellogg  on  the  existence,  uniqueness,  and 
other  mathematical  properties  of  solutions  of  the  differential  equations 
describing  kidney  models.   The  main  effort  has  been  on  the  transport  of  fluid 
and  solute  in  a  single  tube,  with  prescribed  functions  giving  the  transport 
of  fluid  and  solute  in  or  out  of  the  tube. 

One  goal  of  the  research  was  to  determine  conditions  on  these  transport 
functions  which  guarantee  the  existence  and  uniqueness  of  a  solution  to 
the  problem.   In  this  study,  a  distinction  was  made  between  the  equations 
with  and  without  diffusion.   The  general  conclusions  of  the  study  may  be 
summarized  as  follows:   For  the  problem  with  diffusion  D  >  0,  a  set  of 
conditions  were  found  which  guarantee  the  existence  and  uniqueness  of 
solutions.   For  the  problem  with  D  =  0,  the  solution  may  not  exist.   This 
is  due  to  the  possible  presence  of  a  "stagnant"  point  in  the  tube;  that 
is,  a  point  where  flow  becomes  0.   A  modified  formulation  of  the  boundary 
conditions  was  found  with  which  the  existence  and  uniqueness  of  solutions 
of  the  equations  could  be  insured.   This  modified  formulation  gives  rise 
to  a  boundary  condition  of  an  unusual  kind,  in  that  concentrations  must  be 
specified  at  both  ends  of  the  tube,  provided  the  flow  is  entering  both  ends 
of  the  tube.   The  conditions  on  the  fluid  and  solute  fluxes  that  are  required 
for  the  analysis  of  the  equations  without  diffusion  are  somewhat  more 
restrictive  than  was  required  for  the  equations  with  diffusion.   Finally, 
the  limiting  behavior  of  the  solution  as  D  tends  to  0  was  studied.   (This 
limiting  process  is  known  as  a  singular  perturbation  problem,  and  is  frequ- 
ently studied  in  other  parts  of  applied  mathematics.)   It  was  found  that, 
under  appropriate  hypotheses,  the  solutions  of  the  problem  with  D  >  0 
converges  to  the  solution  with  D  =  0,  thereby  justifying  the  unusual  boundary 
condition  that  was  needed  to  analyze  the  presence  of  a  stagnant  point . 

The  main  analytical  tools' used  in  this  study  were  the  method  of 
continuation;  that  is,  following  a  curve  in  a  Banach  space,  and  a  modified 
form  of  the  maximum  principle  that  was  used  to  establish  the  nonsingularity 
of  the  linearized  problem.   It  is  anticipated  that  these  tools  will  be 
useful  in  studying  more  complicated  flow  equations. 


e?6 


Project  No.   Z01   HL  03201-15   STB 


4)   Earlier  work  on  kidney  models  was  generalized  to  give  equations  for 
solute  and  water  transport  in  an  arbitrary  network  of  exchanging  flow  tubes. 
The  motivation  for  this  was  primarily  economy  in  constructing  algorithms 
describing  a  growing  array  of  kidney  models;  but  in  developing  a  meta- 
theory  for  a  hierarchical  set  of  kidney  models,  it  became  obvious  that  except 
for  specific  vocabulary  the  theory  described  solute  and  water  exchange  in  an 
arbitrary  network  of  flow  tubes  in  which  fluxes,  concentration,  pressures  and 
spatial  configuration  can  be  described  by  a  suitably  indexed  set  of  functions, 
boundary  conditions,  and  parameters.   Such  a  description  leads  to  a  set  of 
differential-integral  equations  for  conservation  of  matter  and  the  equations 
of  motion.   When  combined  with  phenomenological  equations  for  transmural  flux, 
these  equations  can  occasionally  be  solved  analytically,  but  usuallly  must 
be  approximated  by  difference  equations  and  solved  numerically. 

In  this  work  we  formulated  this  set  of  differential-integral  equations 
and  the  approximating  set  of  difference  equations.   A  general  scheme  for 
solving  these  equations  by  the  Newton-Raphson  methods  we  have  used  for  kidney 
models  and  a  general  theory  of  partitioning  the  equations  were  developed. 
This  theory  of  partitioning  is  basic  in  the  development  of  specific  com- 
putational algorithms. 

A  concept  which  has  emerged  from  this  work  is  that  from  our  point  of 
view  a  model  of  a  network  of  exchanging  flow  tubes  is  a  set  of  difference 
equations  that  determine. a  state  vector  of  the  system  r  implicitly  as  a 
function  of  a  vector  of  boundary  values  T    ,   and  a  vector  of  parameters  T    . 

Symbolically  we  have 

W  V  V  =  °'  (1) 

where  if)  denotes  the  set  of  equations, 
s 


In  our  modeling  work  to  date  we  have  regarded  T     as  unknown  and  solved 
the  set  of  equations  (1)  for  T  as  a  function  of  T     and  T    .      The  goal  of  this 
work  has  been  to  find  a  set  of  equations  such  that  selected  entries  in  T 
correspond  to  experimental  output  data  for  given  boundary  conditions  r  , 
corresponding  to  experimental  input  data,  and  some  reasonable  set  of 
parameters  V    .   This  type  of  simulation  has  reached  the  point  where  our 
models  are  behaving  in  a  qualitatively  reasonable  way.   We  are  now  in  a 
position  to  look  at  the  inverse  problem.   Namely,  given  a  set  of  experimental 
input  output  pairs,  corresponding  to  selected  entries  in  T     and  T    ,   to  find 
T   ,  so  that  input  output  pairs  computed  from  the  model  will  correspond  as 
closely  as  possible.   From  this  viewpoint  each  experimental  input  output  pair 
(Te,  T  )  will  correspond  to  a  model  input  output  triple  (T  ,  r  ,  T   ) .  Within 
thl  limits  of  experimental  error  we  can  make  experimental  and  model  inputs 
identical,  i.e. 

rm  =  r*  (2) 

B      B 


697 


Project  No.   Z01   HL   03201-15   STB 
Each  experiment  will  then  yield  a  set  of  equations 

6     =  re  -  rm  (3)  . 

Ye      s      s 

Ideally  we  seek  to  determine  T      such  that  <J>  =0.   Usually  this  is  impossible 
and  we  must  be  content  with  minimizing  (J>  with  respect  to  some  norm.   Com- 
putationally such  parameter  evaluation  is  more  difficult  than  simulation, 
and  in  the  past  would  have  been  pointless  because  various  models  of  the 
kidney  and  its  subsystems  failed  on  a  qualitative  level,  but  the  present 
generation  of  models  is  beginning  to  permit  quantitative  comparison  of  model 
and  experimental  outputs. 

Significance  to  biomedical  research:   This  work  gives  conceptual  insight  into 
renal  function  and  provides  the  basis  for  quantitative  models  of  renal 
function. 

Proposed  Course:   We  plan  to  continue  work  in  the  following  general  areas: 

1)  The  general  theoretical  analysis  of  the  non-linear  partial  and 
ordinary  differential  equations  occurring  in  mathematical  models  of  the 
kidney. 

2)  The  development  of  methods  of  parameter  analysis  for  mathematical 
models  of  the  kidney. 

3)  Thermodynamic  analysis  of  models  of  the  kidney. 
Keyword 

Descriptors:   Mathematical  theory  of  transport,  thermodynamics,  kidney, 

numerical  inversion  of  Laplace  transforms,  viscous  flow,  differential 
equations,  differential-integral  equation,  central  core  model  of  the 
kidney,  mathematical  model,  solute  and  water  transport  in  a  network 
of  exchanging  flow  tubes,  membrane  transport. 


Honors  and  Awards :   None . 

Publications : 

Stephenson,  J.  L.   Free  Energy  Balance  in  Renal  Counterflow  Systems. 
Math  Biosciences  21:299-310,  1974. 


S98 


Project  No.    Z01  HL  03202-04   STB 

1 .  ODIR 

2 .  Section  on  Theoretical  Biophysics 

3.  Bethesda,  Maryland 


PHS-NIH 

Individual  Project  Report 

July  1,  1974  through  June  30,  1975 

Project  Title:   Computer  Simulation  of  Renal  Function 

Previous  Serial  Number:   NHLI-240 

Principal  Investigator:   John  L.  Stephenson,  M.D. 

Other  Investigators:     Raymond  Mejia 

Bert  Hubbard,  Ph.D. 
Professor  of  Mathematics 
University  of  Maryland 


Kenny  Crump,  Ph.D. 
Professor  of  Mathematics 
Louisiana  Polytechnic  Institute 
Ruston,  Louisiana 

Reginald  Tewarson,  Ph.D. 

Professor  of  Applied  Mathematics  and  Statistics 

SUNY 

Stony  Brook ,  L .  I . ,  New  York 

Walter  Gross 
Graduate  Student 
Department  of  Mathematics 
University  of  Maryland 

Project  Description: 

Objectives:   The  purpose  of  this  project  is  to  develop  a  computer, 
simulation  of  the  mammalian  kidney  that  gives  a  realistic  description  of 
steady  state  and  transient  transport  of  electrolytes,  non-electrolytes,  and 
water.   This  will  allow  the  correlation  of  micropuncture  and  macroscopic 
clearance  data  with  membrane  transport  characteristics. 

Methods:   The  numerical  methods  for  solving  the  steady  state  and 
transient  equations  of  the  medullary  counterflow  system  described  in  last 
year ' s  report  have  been  refined  and  extended .   We  now  have  computational 
methods  that  are  sufficiently  efficient  to  permit  solution  of  models  of 
the  whole  kidney.   This  work  has  included  improvement  of  the  Newton-Raphson 
methods  by:   1)  The  development  of  specific  sparse  matrix  routines  (in  col- 
laboration with  R.  Tewarson,  SUNY) .   2)  The  adaptation  of  a  general  purpose 
sparse  matrix  algorithm.   3)  The  development  of  partitioning  schemes  that 


8?? 


Project  No.   Z01  HL  03202-04   STB 

permit  individual  flow  tubes  to  be  solved  against  assumed  interstitial 
concentrations  and  pressures.   The  interstitial  concentration  and  pressure 
are  then  adjusted  by  Newton's  method  to  give  solute  and  water  balance  for 
each  interstitial  compartment.   Solution  of  the  equations  for  the  individual 
tubes  can  either  be  a)  by  Newton's  method,  or  b)  a  stepwise  solution  pro- 
ceeding in  the  direction  of  flow.   A  comparative  study  has  shown  this  last 
method  to  be  accurate  and  the  most  efficient  in  the  utilization  of  computer 
storage  and  time.   Its  extension  to  multinephron  models  of  the  whole  kidney 
seems  feasible. 

Several  quasi-linearization  schemes  for  solving  the  time  dependent 
equations  of  the  kidney  model  have  now  been  studied.   (B.  Hubbard  and  W. 
Gross.)  The  schemes  are  characterized  by  the  fact  that  in  advancing  one  time 
step,  the  solution  of  a  linear  system  of  equations  is  required;  thus  they 
give  a  potential  improvement  over  fully  implicit  schemes,  which  require  the 
solution  of  a  set  of  nonlinear  equations  at  each  time  step.   The  schemes  can 
be  used  both  to  follow  the  changes  in  concentrations  as  a  function  of  time, 
and  to  calculate  the  steady  state  values  of  the  concentrations  after  the 
solution  settles  down. 

These  quasi-linear  methods  have  been  programmed  for  the  three  tube  and  six 
tube  models  of  the  medullary  counterflow  system,  and  various  numerical  experi- 
ments have  been  conducted.   For  sufficiently  small  time  steps,  all  the  schemes 
gave  accurate  results  for  the  transient  problem.   As  the  time  step  increased, 
sooner  or  later  all  the  schemes  developed  spurious  oscillations.   They 
differed  considerably  (by  as  much  as  a  factor  of  100)  in  the  size  of  the 
time  step  where  this  occurred.   The  best  scheme  was  found  to  be  competitive 
with  Newton's  method  in  calculating  the  steady  state  solution. 

Solution  of  the  time  dependent  equations  by  numerical  inversion  of  the 
Laplace  transforms  of  the  system  has  been  extended  (with  K.  Crump) .   The 
method  should  be  very  useful  in  the  analysis  of  problems  in  which  volume 
flow  is  not  changing,  e.g.  isotope  uptake  and  washout. 

Major  findings :  The  equations  describing  a  model  of  the  whole  kidney 
were  solved  using  a  Newton-Raphson  method.   The  method  permits  both  steady 
state  and  transient  solutions.   With  this  model  it  has  been  possible  to 
simulate  behavior  of  the  whole  kidney  as  a  function  of  hydrostatic  pressures 
in  renal  artery,  vein,  and  pelvis,  protein  concentration  in  arterial  blood, 
and  phenomenological  equations  describing  transport  of  salt  and  water  across 
nephron  and  capillary  walls.   Concentrations  and  hydrostatic  pressures  were 
computed  for  the  various  nephron  segments  and  in  cortical  and  medullary 
capillaries  and  inter stitium. 

In  most  ways  calculations  with  the  whole  kidney  model  met  intuitive 
expectations.   Thus,  increased  proximal  tubule  delivery  to  the  medulla 
increased  urine  flow  and  decreased  urine  concentration;  increased  proximal 
tubule  and/or  distal  reabsorption  increased  glomerular  filtration;  auto- 
regulation  of  the  model  could  be  obtained  by  adjusting  afferent  arteriolar 
resistance;  and  the  behavior  of  the  whole  kidney  model  was  in  general  agree- 
ment with  models  of  the  cortex  or' medulla  alone. 

2  <jdO 


Project  No.    Z01  HL  03202-04   STB 


New  insights  were  obtained  from  the  model  with  respect  to  the  role  of 
interstitial  hydrostatic  pressure.   In  the  model  this  adjusts  so  as  to 
give  fluid  balance  in  cortical  and  medullary  interstitium.   For  this  pressure 
to  remain  within  reasonable  physiological  limits,  hydraulic  permeabilities 
of  cortical  and  medullary  capillaries  must  be  greater  than  some  critical 
minimum  value. 

We  also  have  used  our  improved  numerical  methods  to  analyze  more  detailed 
models  of  the  medullary  counterflow  system.   These  studies  have  confirmed  our 
hypothesis  that  the  central  core  model  is  the  prototype  for  medullary  function. 
In  addition,  they  have  suggested  the  probable  importance  of  the  cascaded 
structure  of  the  medullary  counterflow  system.   Calculations  on  a  single 
nephron  model  of  the  medulla  in  which  all  nephrons  are  assumed  to  descend 
to  the  papilla  have  shown  that  enough  urea  is  not  available  to  drive  the 
hypothesized  passive  salt  transport  system  in  the  inner  medulla.   Calculations 
in  a  two  stage  model  in  which  the  urea  entering  the  inner  medulla  via  col- 
lecting duct  reflects  a  5/1  ratio  of  cortical  to  juxta  medullary  nephrons 
relieves  this  problem;  but  so  far  we  have  not  been  able  to  assign  values  to 
the  various  parameters  in  the  model  that  will  permit  the  inner  medulla  to 
concentrate  passively  and  cycle  the  urea  load  observed  at  the  loop.   This  has 
led  us  to  hypothesize  that  urea  concentration  in  ascending  limbs  of  Henle 
that  are  returning  from  the  depths  of  the  inner  medulla  rises  above  urea 
concentration  in  the  core,  and  urea  diffuses  out  of  these  ascending  loops 
and  combine  with  urea  supplied  by  the  collecting  duct  to  drive  the  passive 
mechanism  for  loops  turning  at  an  intermediate  point.   Equations  have  been 
formulated  for  this  cascaded  model,  but  we  have  not  as  yet  made  any  detailed 
computations  on  it. 

The  studies  on  the  whole  kidney  model  and  the  medullary  models  have  given 
further  support  to  our  thesis  that  the  functional  unit  of  the  kidney  is  not 
the  individual  nephron,  but  a  nephrovascular  unit  consisting  of  a  group  of 
nephrons  and  their  tightly  coupled  vasculature. 

Proposed  Course:   We  have  now  developed  our  numerical  methods  to  the  point 
where  it  appears  feasible  to  formulate  and  solve  a  multinephron  multisolute 
model  of  the  kidney  that  accounts  for  salt,  water,  and  urea  transport  and 
hydrostatic  and  oncotic  pressure.   Simulation  with  this  model  should  give  us 
new  insight  into  the  role  of  urea  in  the  concentrating  mechanism  and  of  the 
very  intricate  hydrostatic  and  oncotic  pressure  relationships  in  the  kidney. 
In  addition  we  believe  that  such  a  model  will  be  sufficiently  realistic  to 
permit  the  first  quantitative  interpretation  of  overall  renal  function  in 
terms  of  membrane  transport  characteristics. 

Keyword 

Descriptors:   Computer  simulation  of  renal  function,  kidney,  transport 
of  electrolytes,  non-electrolytes  and  water,  numerical  methods, 
Newton-Raphson  method,  sparse  matrix  algorithms,  guasi-linearization 
countercurrent  system,  hydrostatic  and  oncotic  pressure,  partition- 
ing, flow  processes,  nephrovascular  unit,  steady  state  and  transient 
'  transport. 

Honors  and  Awards:   None 

3  W 


Project  no.   Z01   HL  03202-04   STB 
Publications: 

Stephenson,  John  L.,  R.  P.  Tewarson,  and  Raymond  Mejia.   Quantitative 
analysis  of  mass  and  energy  balance  in  non-ideal  models  of  the  renal 
counterflow  system.   Proc.  Nat.  Acad.  Sci.  USA.  71:1618-1622,  1974. 


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