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Full text of "Report of program activities : National Heart and Lung Institute"

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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 . 



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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. 



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° 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\ r ailable 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 

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. 



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d Sp ecialized 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 



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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. 



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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 



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clinical centers are each screening 12 5 000 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- 
ies 1 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 

LD-1 



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 

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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. A dult 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. z 01 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 hybrid s 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 u ltras 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 C 6-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 3'5 f 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. z 01 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 co mp ound 
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 a ct ion 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) Aggregat e 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 no t: (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 cell s 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: 171-17 1 + , 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 k 1 k 3 U R 
(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 U R 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 P TTA 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 (E na ) is 
described by Eq. (2), 



4* 



J na 



E n " 'M +1 ^Y" L <^r 2 + ^T~ (2) 

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

k f = k lf E l = k 2f E 2 = •'• k (n-l)f E (n-l)> where k lf> k 2 f> '" k (n-l) are 
specific rate constants for successive forward steps in the cycles and Ei, 
E 2> ' ' " E n 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, k 1 = ki Ri = k R = 

k (n-l)r R n-l' where k l r > k 2r> "" k (n-l)r are specific rate constants for 
the successive regeneration steps and Rj_ , R 2 , -•• 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 E n to 
E na , decreases in such a manner that log e 0- 5 is inversely proportional to the 
number of cycles in the cascade. Thus, when K d = 1 mM and k^/kf = 0.1, e Q 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 k cat 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 B 12 -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 . 

Me 2+ ^ 
L-Glutamate + ATP + NH 3 r; L-Glutamine + ADP + P t (1) 



£b 



Project No. Z01 HL 00202-04 LB 

Me 2+ 

L-Glutamine + NH 9 0H _ 7-glutamylhydroxamate + NHo (2) 

z s ADP, Pi or Asi 

Me 2 " 1 " ^ 
L-Glutamate + ATP ^ — pyrrolidone carboxylate + ADP + P-^ (3) 

2+ 

L-Glutamine + H 9 , ^ e > L-Glutamate + NHo (4) 

1 ADP, Asi J 

Me 2+ , Pi or Asi 
N]TP + N 2 DP ^ N 2 TP + NiDP (5) 

Glutamate, Glutamine 

The abbreviation Me 2+ , 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 v k _ 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°. 

K l k 2 k - 2 K diss 

Reaction 1 3.9 x 10 4 M" 1 90 sec" 1 9.5sec _1 3 x 10" 6 

Reaction 2 4.9 x 10 4 220 0.5 3.5 x 10" 8 

Reaction 4 230 216 1.8 3.7 x 10" 5 

K diss ls calculated from the equation K d ^ ss = k_2/Kik2- 

Reaction (3), (5), and (6) are too fast and their K diss 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 k obs 
which is independent of substrate concentration, can be estimated by using 
our home-made fast mixing cell. The values of k obs are 

k obs 

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

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

K diss 



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 k cat 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. 
P IIA ^- 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 P ILD 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 P IID 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, 
P I1D 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 + NH 2 0H 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. K m 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) > a n 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 NH 3 -Assimilatory Enzymes in E. coli 
K i2 

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 77 1 , 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 (50 1 ). 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 ~ 507 o 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 
( /W 157») 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 (E na ) is described by Eq. (1), 



5* + 1 



k^n-1 

kT 



v'\n-2 
K r> 



k' 
kT 



+ 1 



(1) 



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

k f = k lf E l = k 2f E 2 = *" k (n-l)f E ( n -l)' where k lf' k 2f> ••• k (n-l)f are 
specific rate constants for successive forward steps in the cyclas and E]^, 

E2, • • • E n 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^ r R^ = k~ Ro = 
*'■ k (n-l)r R n-l' where k^ r , k2 r , '■■ 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 E n to 
E na , decreases in such a manner that log eg 5 is inversely proportional to 
the number of cycles in the cascade. Thus, when K d = 1 mM and k r /kf =0.1, 
e Q 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 k r /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!k 3 U R 

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, Mg 2+ and Mn 2+ ) 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- . CH X CH 

CHidC 2s ch,: ^ C o 1 ch 3 ch,oh * C-Hi^c" "o£ ^CO^CH^ 



— > ok c x > ,c*^ s y c s 

CHjO^C "CH,. COjCH 3 ho^C CH,. Co v H 

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 (co x C«H& iO vo% koh-c^SO* CH 3 COCH 2 CH 2 CH CO3.C1W5- 
MHCOCH3 Co> HC*c-ioo° WHC0CH3 



No^HA . CH3CHCHj.CH3.CKCOiC.Hs in HCI CH 1 CHCV^CH r CHC0 3 _H 
OH NHCQCU 3 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 K D &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 MgCl , 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 107 o glycerol, 
10 mM Tris-HCl (pH 8), 10 mM MgCl 2 , 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 . 3 rd 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^ k 2 j, K cat 
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 (k 2 ) 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 -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 507 o 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 ma y 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 k l ^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/k 2 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/k 2 ) 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 H 2 S 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 



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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 SH 2 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 Ca 2+ . 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 H L 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. Biochemistr y 

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 
ATP 32 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 k ey 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, 
k cat , 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 k ca t 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 k ca t- 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 k cat ; (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: 

k l k 2 k cat 
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 V max 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 . 



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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 80-907 o -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 . 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, Acanthamoeb a 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. 



/31 



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 H 3 , y -P 32 -labeled GTP or ATP. Free nucleotides are rapid- 
ly separated from tubulin dimer ; the H 3 in the latter indicates the extent of 
binding at the E site, and the excess of P over H 3 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 i n 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 v itro 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 K m 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 Ca 4 " 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 Mg 44 " 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 Ca 44 " an d 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 K a '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 (K a =l-2 ymolar) followed by 
cyclic AMP (K a =2-4 ymolar) and cyclic XMP (K a =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 Sr 4 ^" (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 Ca 4-1 "- 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, PGE ls 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- *" a nd 
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° \v T ith 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 (V max 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 



oS 



_r = S-C-CHo = Spironolactone 
f, 
= __ S -C-C(-CH 3 ) 3 = Sc 27825 



'/ 



= __ S-C-Cl^-CHg-/ - 1 = Sc 27937 
= __S-C fS> ~ Sc 27996 




C0 2 -CH 3 



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 N o. 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 H L .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 f n 



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 14 C 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, 197 1 *. 

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. Biochemica l Pharmacology, 
in press . 

Asghar, K. , Reddy, B.G. and Krishna, G. : Hi sto chemical localization 
of glutathione in tissues. The Journal of Hist ochemistry 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 CHC1 2 
CH 2 0H 

*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 ± 

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 37.5 ± 4.2 

2 37.8+3.4 

4 26.1 ± 3.4 

5 5.0 + 0.5 
7 4.4 ± 0.7 



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 17.8 + 1.3 
7 11.0 ± 6.4 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, 197 1 * 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 ( . 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 
12 5l-antigen and 50 yl of cyclic GMP antibody (1:25000). The free 12 5l- 
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 







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 

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, 197 1 * 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. 

Treatment 3 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 30.27 b (27.24, 33.64) 162. 5 b 

THEOPHYLLINE 18.43 b (15.06, 22.44) 98. 9 b 

1 -ISOPROTERENOL 10.04 b (7.96, 12.67) 53. 9 b 



a The 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). 

b These 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 %j 5% 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 > i n 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 


thazine 3 


Phenotype^ 


Diagnosis 


Race 


SGOT 


Bilirubin e 


Urine 


Blood 






Sex d 


or 
















SGPT e 




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 


160 f 


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 



a Proportion of acety lated sulfamethazine 6 hr after drug administration to 

patients recovered from liver injury. 
b Patients classified as rapid acetylators if the proportion of acetylated 

sulfamethazine was more than 70% in urine or more than 257o in blood. 
c Probable or possible isoniazid liver injury. 

d Age (yr), B (black) or W (white) race, M (male) or F (female) sex. 
e Peak abnormalities during liver injury -- upper limits of normal, SGOT (45), 

SGPT (40), bilirubin (1.0). 
^Diagnosis confirmed by liver biopsy. 



A»7 



Project No. ?ni HL nntm-n? T.r.p 



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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. N 2 -Acetyl-( 3 H) -isonicotinoyl hydrazide ( 3 H-AcINH) and N 2 -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 ( 3 H-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-( 3 H) -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: .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,N 2 -acetylhydrazine (DiAcHz) 

N 1 ,N 2 -Acetyl-( 3 H) -hydrazine (DiAcHz- 3 H) . 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 



3 H- 
met 
scribed for AcHz 

4. N^-Isopropyl-isonicotinoylhydrazide (IpINH) 



2 3o 



Project No. Z01 HL 00878-01 LCP 

a. N 2 -Isopropyl-isonicotinoyl-(3H)-hydrazide (IpINH- 3 H) . 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. N 2 -Isopropyl-(2- 14 C)-isonicotinoyl hydrazide (2- 14 C-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. N 2 -Isopropyl-(l,3- 14 C)-isonicotinoyl hydrazide (l,3- 14 C-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. N 2 -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. N 2 -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 107 o Pd on charcoal as a catalyst. The 
intermediate N-isopropyl-(2- 14 C) -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 1007 o 
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- 3 H) ; N 2 -acetyl-( 14 C) -isonicotinoy 1 hydrazide (^ 4 C-AcINH); 
acetyl-( 14 C)-hydrazine ( 14 C-AcHz); acetyl-( 3 H) -hydrazine ( 3 H-AcHz)- N2-iso- 
propyl-(2- 3 H)-isonicotinoyl hydrazide (2- 3 H-IpINH) : isopropyl-(2- 14 C) -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- 3 H 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 V max 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 
K m 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, 
- S H/ 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 ( 3 H-ring- 
labeled) hydrazide, AcINH- 3 H; N^-acety 1-isonicotinoyl ( C-carbonyl-labeled) 
hydrazide, AcINH-l^C; N^-acetyl (3H-methyl-labeled) isonicotinoyl hydrazide, 
3 H-AcINH; N 2 -acetyl-( 1Z| 'C-carbonyl-labeled) isonictinoyl-hydrazide , ^C-AcINH. 
2) Acetyl hydrazine-acetyl-( 3 H _ methyl-labeled) -hydrazine -hydrochloride , 
3 H-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- 3 H-IpINH. 4) Isopropyl hydrazine-isopropyl (2--'-^C) 
hydrazine hydrochloride, 2- 14 C-IpHz; isopropyl (2- 3 H) 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- 14 C), 4 C02 and l^C- or 3 H- 
propane was trapped in a coupled series of three solutions: 2,4 dinitro- 
phenylhydrazine ( 14 C -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 C0 2 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 
As pergillus 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 0100 1-02 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. Whitnack 1 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 01002-0 3 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 01 003-04 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 L C 
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 01005-0 4 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 ' , ) 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 H L 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 0100 6-05 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 01006-0 5 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-! L C 

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 Sp ec 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 
renal 3 cortex 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 P0 4 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 C0 2 /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 
C0 2 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 C0 2 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 C0 2 in the perfusate is zero and that in the bath 
is 27.5, C0 2 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 C0 2 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 C0 2 
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 C0 2 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 C0 2 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 C0 2 permeability 
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 C0 2 from the 
bicarbonate anion both of which contribute to the total C0 2 . 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, C0 2 /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 Na 22 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 K E 

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 "S0 4 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 H 2 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 H 2 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 -B l and -A. Step 2 requires cardio- 
globulin -B 2 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 -B 2 . 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 B 2 . 
We found that DS precipitate produced B activity when mixed with 
-Bj (15% sodium sulfate supernatant solution) but not with -B 2 . 
Therefore DS precipitated -B 2 . To test the supernatant of DS- 
treated serum for -B x 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 B 2 , no -Bj activity was found. This could occur if Bi it- 
self was inactivated by DS or if besides Bi and B 2 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-B 2 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 was 
bound by liver alcohol dehydrogenase but not by yeast alcohol dehydrogenase 
as shown by the marked enhancement of Auramine 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 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 01405-0 2 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 L TD 

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 was 
bound by liver alcohol dehydrogenase but not by yeast alcohol dehydrogenase 
as shown by the marked enhancement of Auramine 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 tryp to phan 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 01409-0 4 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. Q f 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 Clin ical 
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. Z 01 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 V ATP, 

h + b XD WT ^3) (4) E + P i 

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: 

consumption = cardiac output x arterial-venous 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) 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 % 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 
determined and CO noted. HO is then added continuously and this 
addition plotted as the abscissa. The electrode reads 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, 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 P 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. z 01 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, z m 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 141 Ce, 16 ^Yb and 85 Sr) . 
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 AT J ^p. Incorporation of 32p i n to 
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 " 9m Tc 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, 99m Tc-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 ^ 3 KC1 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; 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 PGE 2 . 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 B 2 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 B 4 B, whereas urinary kallikrein has a higher affinity for B ? . 
B 2 was shov/n to correct the coagulation defect known as Flaujeac, Williams, 
or Fitzgerald trait; B 4 B 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 th ll ^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 ^ S1 T' Pr? du ? tion of renal artery stenosis in dogs and 

r i J 6 "- th ° kSl lkrein excretion from the treated side. Changes 

flow lu?l\ ' 1 T n 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 m etab olism 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 former 1 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