MEMÓRIAS DO INSTITUTO BUTANTAN 19 6 6 VOLUME XXXIII SUPLEMENTO COMEMORATIVO SIMPÓSIO INTERNACIONAL SOBRE VENENOS ANIMAIS INTERNATIONAL SYMPÓSIUM ON ANIMAL VENOMS / / Presidente de honra — B. A. HOUSSAY Presidente efetivo — A. VALLEJO-FREIRE Secretário geral — W. BÜCHERL FASCÍCULO II í ^ SÃO PAULO-BRASIL CAIXA. POSTAL, 65 cm 2 3 4 5 6 SciELO ;lo 11 12 13 14 15 As “MEMÓRIAS DO INSTITUTO BUTANTAN” são destinadas à publicação de trabalhos realizados no Instituto ou com a sua contribui¬ ção. Os trabalhos são dados à publicidade logo após a entrega e reu¬ nidos anualmente num volume. Serão fornecidas, a pedido, separatas dos trabalhos publicados nas “MEMÓRIAS”, solicitando-se nesse caso o obséquio de enviar outras separatas, em permuta, para a Biblioteca do Instituto. Tôda a correspondência editorial deve ser dirigida ao: INSTITUTO BUTANTAN BIBLIOTECA Caixa Postal, 65 — São Paulo — Brasil Pede-se 'permuta Excha'nge desired * SciELO 10 11 12 13 14 15 16 cm SIMPÓSIO INTERNACIONAL SÔ8RE VENENOS ANIMAIS INTERNATIONAL SYMPOSIUM ON ANIMAL VENOMS INSTITUTO BUTANTAN 17 a 23 de julho de 19G6 Sob os auspícios do GOVERNO DO ESTADO DE SÃO PAULO, da FUNDAÇÃO DE AMPARO Ã PESQUISA DO ESTADO DE SÃO PAULO e do FUNDO DE PESQUISAS DO INSTITUTO BUTANTAN SUPLEMENTO COMEMORATIVO CENTENÁRIO DE NASCIMENTO DE VITAL BRAZIL COMISSÃO DE REDAÇÃO Beçak, W. — Bücherl, W. — Dessimoni v. Eickstedt, V. —• Emerson Belluomini, H. — Franco de Mello, R. — Hoge, A. R. — Lavras, A. A. C. — Leal Prado, J. — Lucas, S. M. — Mandelbaum, F. R. — Nahas, L. — Ribeiro do Valle, L. A. —■ Saliba, F. — Schenberg, S. — Vallejo-Freire, A. — Vital Brazil, O. — Zelnik, R. cm SciELO 10 11 12 13 14 15 SIMPÓSIO INTERNACIONAL SÔBRE VENENOS ANIMAIS INTERNATIONAL SYMPOSIUM ON ANIMAL VENOMS INSTITUTO BUTANTAN 17 a 23 de julho de 1966 FASCÍCULO 2 ÍNDICE — INDEX VI. Bioquímica — Biochemistry Pág. 39. Enzymes of Snake Veiioms as Tools in Biochemkal Research — E. A. Zei.ler (E.U.A.) . 349 40. Proteolytic Eiizymes of Hothrops Venom — O. B. Henri¬ ques, F. R. Mandelbaum and S. B. Henriques (Brasil) .... 359 -iL41 . Enzymes el Toxines des Venins de Serpents. Recherclies Bio- chimiques et Immunologiqnes sur le Venin de Naja nigricolUs — P. Boquet, Y. Izard, M. Jouannet et J. .Meaume (França) 371 • 42. Some Se])aralion Methods Based on Molecular Size and Charge and lheir A])|)lication to Purification of Polypeptides and Pro- teins in Snake Venoms — J. Poratii (Suécia) . 379 43. Separation Methods of Animal Venoms Constitiients — T. Suzuki (Ja])ão) . 389 .44. Some Aclions of Snake Venom on Mitochondria — V. B. Eueiott, J. M. Augustyn' and C. Gans (E.lkA.) . 411 45. The Venoms of Amphihians — J. W. Dai.y and B. Witkoi' (E.U.A.) . 425 46. Cardiotoxic Steroids froni Toads — K. Meyer (Suíça) . 433 47. Wasp Kinin — J. J. Pisano (E.U.A.) . 441 48. Purification des Xeurotoxines du Scorpion Androctonus aus- tralis — C. Rochat, H. Rochat, F. Miranda et S. Lissitzky (França) . 447 49. Chemical Properties and Biological Activity of T i t y ii s Venom — C. R. Diniz, M. V. Gomez, A. Antonio, A. P. Corrado ( Brasil j . 453 cm SciELO 10 11 12 13 14 15 ÍNDICE 1 VII. Farmacologia — Phakmacoi.ogy f>ãg. 1 ^ 50. Pharinacolojiy of Venoms — Iiilroductory llcmaiks — M. Ro¬ cha E Silva (Rrasil) . 457 1 .51. Lilieration of Pharmacologically Aclive Siilislanees from Ma.«l Cells liy Animal Venoms — E. Kaiser and W. Kaar (Áustria) 461 1 < 52. Meclianism of Hislamine Release by Animal Venoms — A. M. Rothschii.i) (Brasil) . 467 53. Use of Venoms in Stiidies on Aerve Excitation — P. Rosen- BERG (E.U.A.) . 477 54. Serotonin and Relaled Try])tamine Derivatives in Snake Venoms — J. H. Welsh (E.U.A.j . 509 55. Pharmacologically and Riochemically Active Compcnents of Ja- ])anese Ophidian Venoms — T. Suzuki (Japão) . 519 56. Sjiecific Sites of Action of Snake Venoms in tbe Central Ner- vous System — 11. 1. Bicher (Israel) . 523 . 57. Action Neuromnsciilaire des Venins de cjuelcpies CROTALIDAE, ELAPIDAE et HYDROPHIIDAE — J. CíIEYMOL. F. BoURILl.ET and M. Roch (França) . 541 58. Modes of Aclions of Purified Toxins from Elapid Venoms on Neuromuscnlar Transmission — C. Y. Lee and C. C. Chang (F ormosa) . 555 1 59. Cardiotoxic and Cardiostimnlating Faclors in Cobra Venom — A. Devi and N. K. Sarkar (índia) . 573 60. Cardiovascular Res|)onses to Snake Venoms and their Fraclions — F. E. Russell (E.U.A.) . 583 61. Pharmacology of lhe Com|)onenls of Toad Venom and Allied Snlislances — M. Okada (Ja|)ão) . 589 1 62. Some Aspects of lhe Pharmacology of lhe Venoms of African Scorpions — K. R. Adam and Cu. Weiss (Sudão e Alemanha) 603 1 63. Pharmacology of the Venoms of Mexican C e n 1 r ii r o i d c s — E. C. DEL Pozo (México) . 615 1 ^ 61. Pharmacology of lhe Polyjiejilides from the Venom of the Spider Clionriilria fera — S. Sciie.míerg and I'. A. Pereira Lima (Brasil) . 627 1 65. S[)ider Glands and Psycliolrojiics — P. N. W itt (E.U.A.) ... 639 1 O o Sjiider Wells and Psycholrojiics — C. F. Reed ( E.U.A.) . 615 1 67. The Effect of the Poison of Spider — and Diggerwasjis on their Prey (IIY.MENOPTEKA: POMPILIDAE, SPHECIDAE) _ W. Rathmaykr (Alemanha) . 651 1 cm SciELO 10 11 12 13 14 15 16 VI BIOQUÍMICA BIOCHEMISTRY Mem. Inst. Butantan Slmp. Internac. 33(2);349-35S, 1966 E. ALBERT ZELLER 349 39. ENZYMES OE SXAKE VEXÍBIS AS TOOLS EX 1?I()CHEMICAE RESEARCH E. ALBERT ZELLER Department of Biochemistry, Northwestern University Medicai Scliool, Chicago, Illinois, U.S.A. Althoiigh Riochemists have l)eeii slow in recognizing the remarkalile ])roperlie.« of snake venonis, they have recently takeii so imich advantage of this material tliat only a few typical examples of its use as a tool can he preseuted here. Some of the reasons for this later develo])ment are the followiug: (a) Snake venoms helong to the most eoncentrated enzyme soiirces in nalure; ih) tlieir com])osition is very simple in comparison with a liver homogenate; while only One oxidoreductase is found in snake venoms, it is impossible at the ])resent to say how inany occur in the liver eell; (c) they display a remarkahle stahilily when they are properly protected against light and moistnre; Id) many exeel- lent preparations of snake venoms are now commercially availahle; (e) snake venoms tend to attack key suhstances and key reactions in the animal hody; it is. therefore, j)rohahly not accidental that ive find theni of |)ractical value in the stndy of vitally important metaholic |)rocesses. t have selected three areas to demonstrate the usefulness of snake venoms as a part of analytical and preparative jtroeediires and in the stndy of certain enzyme mechanisrns. Analytical methods a. The o|)liidian E-amino acid oxidase (L-AAO) has heen applied to the determination of L-amino acids in D-aniino acid ])re])arations. Since the oxidative deamination of L-amino acids ])roceeds at |)ractically the same velocity in lhe ahsence and presence of high D-amino acid concentrations (1, 2), the occiirrence of 0.01 ])er cent of the L-form can he detected in D-amino acid prei)arations with the hei]) of L-AAO. There exists no more sensitive qiialitative or qiian- titative j)rocedure to lest the optical ])urity of D-amino acids. J. P. Greenstein and his co-workers have sharpened this tool to a high degree (3). The same enzyme has heen a])i)lied to the analysis of phenylalanine in the hlood and urine of infants suffering from phenylketonnria. This precise. ra])id and specific micromethod is hased on the measurement of ahsorptioTi of the enol-horate complex of j)henyli)yruvic acid generated from the amino acid hy the L-AAO of Crotalm adamantem [ A, o ). A specimen as small as 0.02 ml of plasma ultrafiltrate is sufficient for the test (6). W ith aj)propriate modifications, tyrosme (4, 5), tryptophan (4), and histidine (7) can he determined hy the same principie. cm 2 3 z 5 6 11 12 13 14 15 350 ENZYMES OF SNAKE VENOMS AS TOOLS IN BIOCHEMICAL RESEARCH Reccillly, L-AAO lias lieen a|)[)lie(l lo lhe idenlificalion of L-amino acids separaled hy pajier ehromalograjihy. The elironiatograni is Irealed with a veiiom solulion iC. (ídumunteus), plienazine raelhosiilfale, and a lelrazoliiim comiioiind. The laller acls as an eiectron a(a'eplor wilh |)henazine nielhosiilfale serviiig as an eleelroTi carrier. As lillle as 1 /xg of ihose amino acids, which are rajiidly deaminaled in lhe presence of lhe L-AAO, can lie reeognized liy lhe appearance of fonnazans (8). h. L-AAO can he coiipled vvilh amino acid releasing enzymes ( reaclion I). If lhe condilions are adequalely chosen, lhe former enzyme hecomes lhe rale limiling (peptldase) (L-AAO) Amino acid donor - >■ L-amino acid - >• a-kelo acid + NH3 (I) faclor of lhe syslem. On ihis hasis, a qnanlitalive melhod for peplidase aclivily has heen developed (9,10). Peplides (9, 10, 11), proleins (8), or amino acid es- lers may lie iised as amino acid donors; oxidalion of lhe lilieraled amino acid may lhen he followed manomelrically, jiholomelrically, lilrimelrically, or polaro- gra])hically. Very small aclivilies can ihiis he delermined. lí several differenl amino acids are lilieraled liy lhe peplidase, ihcse amino acids wili compele foi- lhe L-AAO. In order lo avoid lhe formalion of siich complex syslems, jieplidase syslems which release only one amino acid are preferahle. The simidtaneous produclion of glycine and L-alanine wilh olher íree amino acids does nol inlerfere wilh lhe measiirement liecanse lhe former Iwo amino acids disjilay very low af- finily for lhe enzyme and ihus can he neglecled (3). Amino acid eslers serve as excellent suhslrales hecaiise only one amino acid is involved. The lens amino peplidase, in conlrasl lo all olher amino pejilidases, acls vigorously on eslers such as L-phenylalanine elhylesler, or L-tryplojihan methylesler, and can he delecled wilh lhe help of lhe L-AAO as shown hy Palmherg (12). Ilecently, ihis principie has heen applied lo lhe delerminalion of peplidase aclivily of lhe complex formed hetween |)ancreas carhoxypejilidase and ils anlihody(8). The comjilex, localized wilh lhe hel[) of immimoeleclrophoresis, is Irealed wilh hip- puryl-L-arginine and lhe reagenls menlioncd ahove. The pejilidase aclivily of lhe antigen-anlihody comjilex is reeognized hy lhe ap])earance of lhe hlne formazan. c. There is a large hody of jnihlicalions dealing with lhe successfiil iise of [)hos[)hodiesterases and pyro|)hosphalases in lhe ehicidalion of lhe slrnctiire of niicleic acids and diniicleolide coenzymes. To single out one recent exaniple, lhe work of Pfleiderer and Woenckhans jiertaining to lhe conformalion of adenine pyridinc diniicleotide (NAD) is hrieíly summarized here(13). The aiilhors siic- ceeded in separating «-NAD from lhe ji-iorm of lhe diniicleotide from yeast NAD hy chromatography on Dowex 1 (formale form). In lhe a-isomer, which is ealalytically inactive, lhe rihosyl residue is connected lo lhe nicotinamide moiely hy an a-glycosidic linkage. Cleavage of «-NAD wilh pyrojihosphalase, jmrified from lhe venom of Naja nivea, residted in an increase in lhe e,„ax at 260 mp, from 17,900 to 19,900. This increase was of lhe same order of magnilude as jirevioiisly ohtained for lhe hydrolysis of jnire /?-NAD hy snake venom (14). This was indicalive of a similar folding of lhe moleciiles and of an inleraction hetween lhe nicotinamide and adenine rings of holh «- and ^-forms. 1 SciELO Mem. Inst. Butantan Simp. Internac. 33(2):349-358, 1966 E. ALBERT ZELLER 351 Preparative procederes a. The Work on the enzyniatic hydrolysis of «-NAD culminaled iii lhe isolation of llie «-iiicotinamide mononucleotide (aXMN). The tojdcal and chemical properlies of the new mononucleotide were thoroughly stndied. It differed very liltle spectroscopically from the ^-isoiner. hiit chemically the hydrolvsis of the a-isomer was less easily achieved than that of the y3-isomer. Without the o|)hidian enzyme, it would have l)een difficnit indeed to carry out this interesting in- vestigation on the relationship hetween the chemical slnictiire and certain properties of NAD. h. Parikh et al., with the help of dialyzed vtnom of C. adamunteus, com- pletelv destroyed the L-isomer of the racemic form of more than a dozen alijihatic and aromatic amino acids and isolated the piire D-isomers hy alcohol precipita- tion(15). This method j)ermits the rapid production of certain D-amino acid on a small scale hasis. Since the reaction is carried out in the presence of catalase, a-keto acids are formed which remain in the aqiieous solution diiring the precipitation. c. A similar idea to the resolution of racemic mixtures of amino acids has heen successfully applied to the preparation of the first pure native j)lasmalogen, phosphatidal cholinetlól. The essential step consisted in treating the crude plasmalogen with ophidian phospholi])ase A in order to destroy the accompany- ing lecithins. d. Since many L-amino acids are cpiantitatively converted into the cor- responding a-keto acids hy the comhined action of L-AAO and catalase, we have here the hasis for a simple and efficient procedure to prepare these compounds as suhstrates for enzymatic experiments (17). An example is foimd in lhe preparation of a-ketoglutaramic acid from glutaniine for studies in which the keto acids serve as a receptor in certain transaminase reactions (18). Mechamsms oe exzyme reactioxs As a fairly ])ure and easily accessihle flavoprotein, the L-AAO hecame the ohject of many studies concerning the mode of action of vellow enzymes. a. In the very first pajrer on this enzyme. it was noticed that at high leucine concentration the reaction rate was reduced(l). These rcsults were first explained hy J. B. S. Haldane’s hypothesis of a sid)strate having a two-point attachment with the enzyme, the excess suhstrate compeling with lhe initiall)' hound suhstrate for the second point (19). Douhts concerning this concept arose when it was found (20) that the pheuomenon was more marked with suhstrates undergoing rapid oxidation in the presence of L-AAO than with homologous suhstrates sluggishly degraded (Fig. 1) and that the inhihition caused hy high suhstrate concentration decreased with iucreasing oxygen density (21. 22). Meis- ter and Vi ellner, on the hasis of detailed analysis of lhe steady State hehavior of the enzyme, proposed another inter|)retation of this ])henomenon (23). These authors had succeeded in crystallizing L-AAO from the venom of C. adarnantcus. The enzyme, with a molectdar weight of approximately 130,000, contains two moles of flavin adeniue dinucleotide (FAD). The new hy])othesis takes into consideratiou the following two points: Both FAD molecides participate in the cm SciELO 10 11 12 13 14 15 352 ENZYMES OF SNAKE VENOMS as TOOLS IN BIOCHEMICAL RESEARCH enzymalic ])rocess, and each FAD molecide is first converted inlo a lialf-reducod State; at high suhstrate tonceiitratioii, lhe FAD is further converted into the fully rediiced enzynie. FoIIowing lliis sclieine, it would he siifficient to postulate that the lialf-reduced form is reoxidized more rajjidly l)y molecular oxygen than the fully reduced enzynie. This couce])l was su])|K)rted hy recenlly jnihlished ohservatious made hy Desa and (^ilsou (24). Concentration Fig. 1 Ju order to ohtain more information ahoul the sinmitaueous parlicipation of holh FAD molecules iu the amiuo acid degradaliou and lluis ahoiit the validity of the hypolhesis lo ex])laiu the inhihitiou hy high snhstrale concentration, Well- ner compared native wilh reversihly inaclivated enzyme(25). The latler was ohlained hy heating at 38‘’C for 60 minutes. After lhe inactivation, no changes in electrophorelic mohilily, in sedimenl coefficienls, or in inleraclion wilh anti- hodies were ohserved. ITom lhese ohservalions, it can he concluded that the inactivation is nol caused hy lhe loss of FAD, nor hy dissocialion, aggregalion, or denatiiration of lhe enzyme molecule. A shifl of the ahsorption maxima of the FAD, however, indicaled an alteration in lhe mode of hinding of lhe FAD hy the |)rotein. These and other ohservatious sn|)porl the hypolhesis that con- formalional alterations in lhe environrnenl of lhe FAD molecules had laken ])lace dnring the inactivation. The jiositions of the Iwo flavin moieties relative lo each other ajiparently are changed, lluis prevenling their concerted action. AIlhough lhe conchisions are well snpjiorted hy the data, a direct ]rroof of the simultaneous action of the two FAD molecules remains lo he presented. It shoiild he men- tioned here that Massey proposed a mechanism which requires lhe action of only one FAD molecule (26). 1 SciELO Mem. Inst. Butantan Simp. Internac. 33(2);349-358, 1966 E. ALBERT ZELLER 353 I). As the last example, it can he seeii how stuclies carried out vvith L-AAO have led to the introduction of a new concept iiito the field of enzynie kinetics. After niany years of investigatioii of the sid)strate and inhihitor patteni of monoamine oxidase I MAC)), \ve were led to the assiimption that some substrales can forni two types of complexes with the active site of certain enzymes. Since the siihstrate pattern of the L-AAO, as known at that time, seemed to indicate the existence of phenomena similar to that encoimtered in the MAO reaction, and since the L-AAO could he readily crystallized from the venom of C. ada- manieus, it seemed to he a convenient tool for fiirther analysis. Our first approach consisted of determining the constants of the Michaelis- Menten relationship for more than 60 different aromatic amino acids and their ring-substituted derivativas 12). Some of data are presented grajihically in Fig. 2. 10 N.M. OMP F Cl NOz OH CH3 The maximal velocities for substituted phenylalanines and tryptojihans are given for the L-AAO of crude venom of Vipera russellii, C. adamanteus, and Naja me- Umoleuca as comjuited from manometric measiirements. It can he easily recognized that meta-suhstitution in phenylalanine and 5-snhstitiition in tryptojihan, in general, yield lietter snhstrates than other suhstitiitions. Jt seems as if the locas, size, and shape of the suhstituents rather than their effects on electron distrihiition affect the maximal rate. In trying to interpret these and related data. \ve started from an earlier observation which led us to believe that the aromatic ring of certain snhstrates of MAO and L-AAO contrihutes the major part of the binding energy between the active site and the siihstrate (21. If an aromatic system exists in the active site (27), so the henzene or heterocyclic ring of the suhstrate can he attached to it. presnmahly through 7r-orhitals and hydrophohic honding. The symmetries of the aromatic rings woiild allow the formation of more than one complex. One type of complex, called eutopic (28) or prodiictive (29), is an intermediate in the conversion of the suhstrate to the product. while the other type. named dys- topic or non-productive, is not an intermediate and, therefore, does not lead to cm SciELO 10 11 12 13 14 15 354 ENZYMES OF SNAKE VENOMS AS TOOLS IN BIOCHEMICAL RESEARCH {)ro{iucl fornialion. Tliree cases may he etivisaged: (a) The occiirreiice of only eulopic complexes, llierehy ])erniiltiiig the higliesl reaction rale of a given enzynie System; (h) the oeciirrenee of dyslopie complexes otdy, lherehy prevenling sidi- strate degradalioii; (e) lhe occiirrence of hotli productive and noii-productive complexes. Thiis, in case (c) lhe reaction velocily is a fiinclion of lhe ratio of lhe occiirrence of lhe Iwo types of complexes, ihis ratio heing delermined jiossihly hy lhe shajie of the snhstrale molecnles. If we derive in the nsiial way the relationship hetween snhslrate concenlralion and reaction rale and if we lake into consideration lhe aiipearance of dyslopie complexes, lhen we arrive at equa- tion 11. If no olher additional faclors V — V . s/[Ke + s (1 + K,,/K,i) I (II) are involved, lhen wilh increasing dyslopie character of the enzyme-siihslrate complex (i.e., as lhe ratio of occiirrence of dyslopie lo eulopic complexes in- creases), the Lineweaver-Burk plol is shifted toward increasingly higher ordinate values without a change in slope. Parallel shifts of the Lineweaver-Burk jilot have actually heen ohserved on several oceasions (2,28). More general expres- sions than thal given in equation II are fonnd in lhe papers hy Niemann (29) and hy Zeller et al. (2). Niemann independently conceived the idea of two dif- ferent types of comjilexes from his analysis of lhe suhstrate paltern of chymo- trypsin. In order to gain further informalion ahonl lhe range of ajiplicahility of the new concepl, we carried oiil additional experimental investigations. From lhe outset, we wanted lo replace lhe manometric ])rocedure of measiiring L-AA() activity with a more suitahle method hecanse the former is franght wilh several serious shorleomings. Since the measuring of oxygen consumption with the aid of lhe oxygen electrode avoids the severesl limitations of lhe manometric melhod, we adopled the polarogra|)hic melhod. Allhoiigh lhe resuits remained essentially lhe same, we feel miich more secure with oiir new data. While the interaction hetween suhstrate and flavoprotein is a rather complex process, and lhe inlerpretation of lhe kinetic data leaves many queslions un- answered, the hinding of an inhihilor hy a given enzyme is in general a more straighforward event. We attempted, therefore, to determine whether the same snhstitution rides are valid for inhihitors as well as suhstrales. Since ring- suhstiUited henzoic acids hlock L-AAO compclilively, lhey appeared lo he siiilahle ohjects for tesling this idea. For 8 sets of ortho-, rneta- and para- monosuhslituted henzoic acids, Clauss delermined lhe concenlralion which inhihiled L-AAO hv 50 per cent. In all hut one series, lhe meta-snhstiluted henzoic acids appeared lo he lhe slrongest inhihitors. In the series of hydroxylated compounds, the mela- and the para-derivatives were of eqnal inhihilory ])ower(30). We summarize onr residis in lhe following way. Il would seem as if lhe geometry of the suhstrate molecule rather than olher faclors is responaihle for the reaction rate. If this idea were correcl, the electron density at lhe íeaction cenler of the suhslrale would nol he imjiortanl as lhe rale delermining , factor. Since a qiianlilalive relalionshi|) hetween eleclron densily, as determined hy ring suhslilulion, and reaclion rale/k (or reaclion equilihria) exists in lhe fqrm of lhe Hamrnetl equalion (111), this poinl can he snhjecled to experimenlal ve- rification. The constant p is characleristic for a given reaction, while the log k — log k„ = (T . p (111) 1 SciELO Mem. Inst. Butantan E. ALBERT ZELLER 355 Slmp. Internac. 33(2) :349-35S, 1966 constanl u is defined as a measure of lhe electroii densily at lhe reaction cenler. If, afler plolling lhe left side of eqiialion III againsl o-, \ve ohlain a straight line, we consider lhe Hammelt relalionship as fulfilled. If lhe sIo])e is positive, a low electron densily favors lhe reaction at lhe rate deterinining slep, whereas a negative slope indicates that an electron shift toward lhe reaction cenler jiermits lhe enzymatic ])rocess to proceed rapidly. In a numher of enzyniatic systeins. lhe Haminett eqnation has heen found to descrihe lhe relationshij) hetween ring suhstitution and velocity adequately. To tesl L-AAO in a similar way. Clauss (30) determined polargraphically lhe reaction rates prodnced hy a niimher of ring suhstituted phenylalanines and hy the L-AAO froni C. adamunteus. The data are presented in Fig. 3. The logarithm of lhe maxinial velocity is plotted SO2NH2 2.0 1.6 O o I SOCH, • ^ SCH, • ^ 1.2 -.6 0 6 á Fig. 3 — Hammet plot for ophidian L-amino acid oxidase (see text). against o-. While a straight line niay he drawn throiigh the ])oints represent- ing several meta-snhstitiited aniino acids (open circies), it is ohvionsly iinjiossihle lo estahiish a linear relationshij) for the |)oints rejnesenting i)ara-siihstiliited |)henyl- alanines (hlack circies; the donhle circle indicates the imsuhstiluted phenylalanine). This is tantamoiint lo saying that, for the ])ara-suhstituled ])henylaianines, factors other than electron density at lhe reaction center of the snhstrate are rate de- termining. Geometric properties, therefore, may he taken into consideration instead. If lhese considerations ajtpear too hypolhetical lo he ])rescnled at this sym- posium, they may slill i)rovide iis wilh a new insight into the hiological function of L-AAO. This enzyme originally was thought lo play a role in lhe digestive action of snake venoms(31)- The low LD 50 , as determined for lhe pure enzyme hy Riissell et al. (32) suggest that it is intimately involved in the toxic action of lhe venom. This conclusion appears to he untenahle in the light of lhe ex- cm 2 3 z 5 6 11 12 13 14 15 356 ENZYMES OF SNAKE VENOMS as TOOLS IN BIOCIIEMICAL RESEARCH ])eriments rcporled liy Zvvisler vvliü foiiiul tlial monos])ecilic antiscra against L-AAO ara not ahie to reduce llu> toxicity of lhe wliole veiiom (83). However, the complex formed I)y the L-AA() atid its anlihody lias heen foimd lo he as active as the free enzyinet25). It seems eonceivahle lhat the L-AAO may destroy lhe loeal pools of |)heiiylalanine and lyrosiiie, lhe jireeiirsors of the eate- cholamines. Sinee lhe latler play an iin[)orlanl role in lhe ergolropie reaetion of the prey, this removal of lhe aromatie amino aeids should weaken lhe defense meehanism. From Fig. T and froni incomplete data iiresented in several jia- pers (34,3.8), one can see high ralios for lhe niaxinial velocities of phenylalanine lo leiicine (Vph,./Vieu) for elapid venoms as eompared with venoms of the olher two classes of snakes. If lhe aclivily loward leiicine ajipears to he representalive of the anionnl of L-AAO jiresenl (2), then low L-AAO eoncentrations often fonnd in elapid venoms are partially or entirely com|)ensaled hy a high (Vphp/Vie„) value. Aeeording to onr hypothesis, lhe relalive higher povver of phenylalanine destruclion hy elajiid venoms is due to a higher degree of enlopic eom[)lex formation hetween L-AAO and aromatie amino aeids. CONCLUSION These remarks give only a disaiipointingly small and hy necessity one-sided seetion of lhe range of studies earried oiit with venoms as an enzyme soiirce. But I do ho[)e that they demonstrate lhe tremendoiis polential of this lool. Vital Brazil, in whose honor \ve meet here, used lhe venoms to overeome their fatal effeets. Today, we are ahle lo go heyond Brazil and to iise snake venoms for the invesligalion of hiochemieal reaetions of theorelieal and jiraclical imjiortance as testificei hy many papers jiresented at this Symiiosiiim. In the future, we may add a deeper meaning to the eurious fact that the snake in the form of the caduceus is the symhol of lhe medicai profession. Befere.xces 1. ZELLER, E. A., and MARITZ, A., Helv. chim. Acta, 27, 1888, 1944. 2. ZELLER, E. A., RAMACHANDER, G., FLEISHER, G. A., ISHIMARU, T., and ZELLER, V., Biochem. J 95, 262, 1965. 3. MEISTER, A., LEVINTOW, L., KINGSLEY, R. B., and GREENSTEIN, J. B., J. biol. Chem., 192, 535, 1951. 4. LADU, B. N., and MICHAEL, P. J., J. Lab. clin. Med., 55, 491, 1960. 5. LADU, B. N., HOWELL, R. R., MICHAEL, P. J., and SOBER, E. K., Pedia- tries, 31, 39, 1963. 6. WOOLF, L. I., and GOODWIN, B. L., Clin. Chem., 10, 146, 1964. 7. BALDRIDGE, R. C., and GREENBERG. N., J. Lnb. clin. Med., 61, 700, 1963. 8. AVRAMEAS, S., and URIEL, J., Compt. Rend., 261, 584, 1965. 9. ZELLER, E. A., and MARITZ, A., Helv. physiol. Acta., 3, C 6, 1945. 10. ZELLER, E. A., Abstracts of the XVII Int. Physiol. Congr., Oxford, 1947, p. 258. cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan E. ALBERT ZELLER 357 Simp. Internac. 33(2):3-49-35S, 1966 11. ZELLER, E. A., and DEVI, A., Anier. J. Ophthal., 44, Part II, 281, 1957. 12. PALMBERG, P. F., unpublished data. 13. PFLEIDERER, G., and WOENCKHAUS. C., Ann. Cheyn., 690, 170, 1965. 14. SIEGEL, J. M., MONTGOMERY, G. A., and BOCK, R. M., Arcli. Biochem. Biophys., 82. 288, 1959. 15. PARIKH, J. R., GREENSTEIN, J. P., WINITZ, M., and. BIRNBAUM, S. M., J. Amer. chem. Soc., 80, 953, 1958. 16. GOTTFRIED, E. L., and RAPPORT, M. M., J. bioj. Chem., 237, 329, 1962. 17. ROULET, F., and ZELLER, E. A., Verh. Schweiz. Naturforscliges., 127, 112, ' 1947. 18. MEISTER, A., Science, 120, 43, 1954. 19. ZELLER, E. A., Advanc. Enzymol., 8. 459, 1948. 20. FLEISHER, G. A., and ZELLER, E. A., Abstracts of the llSth. Meeting Amer. chem. Soc., San Francisco, 1949, p. 20 C. 21. SINGER, T. P., and KEARNEY, E. B., Arch. Biochem. Biophys., 29, 190, 1950. 22. MARCUS, A., and FEELEY, J., Biochim. Biophys. Acta, 46, 600, 1961. 23. MEISTER, A., and WELLNER, D., in P. D. BOYER, and K. MYRBÃCK (Edi- tors), The Eiizymes, Vol. VIII, Ed. 2, Academic Press, Inc., New York, 1962, p. 615. 24. DESA, R. J., and GILSON, Q. H., Fed. Proc., 25, 649, 1966. 25. WELLNER, D., Biochemistry , 5, 1585, 1966. 26. MASSEY, V., GANTHER, H., BRUMBY, P. E., and CURTI, B., in Proc. Symp. Amherst, Mass., 1964, p. 335. 27. ZELLER, E. A., Pharmacol. Rev., 11, 387, 1959. 28. ZELLER, E. A., Biochem. Z., 339, 13, 1963. 29. NIEMANN, C., Science, 143, 1287, 1964. 30. CLAUSS, L. M., unpublished data. 31. ZELLER, E. A., in J. B. SUMNER, and K. MYRBÃCK (Editors), The Enzymes, Vol. 1, Academic Press, Inc., New York, 1951, p. 986. 32. RUSSELL, F. E., BUES, F. W., and WOO, M. Y., Toxicon, 1, 99, 1963. 33. ZWISLER, O., Abstracts of the Int. Syynp. Animal Venoms, São Paulo, July 1966, p. 96. 34. ZELLER, E. A., and MARITZ, A., Helv. chhn. Acta, 28. 365, 1945. 35. SUZUKI, T., and IWANAGA, S., Yakugaku Zasshi, 80, 1002, 1960 and Chem. Abstr., 55, 3824, 1961. cm 2 3 z 5 6 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2): 359-370, 1966 OLGA B. HENRIQUES, FAJGA R. MANDELBAUM and S. B. HENRIQUES 359 40. PROTEOLYTIC EXZVMES OF fWTHROPS VE.\0M OLGA B. HENRIQUES *, FAJGA R. MANDELBAUM and S. B. HENRIQUES * Laboratório de Bioquímica, Instituto Butantan, São Paulo, Brasil Observations on the proteolytic activity of snake venoms had aiready lieen made at the end of the last century. Studies oii this interesting hiological material, however, develojied rather slowly and purification of enzymes from snake venoms was accomplished only recently. The value of proteolytic enzymes as tools in protein chemistry has induced many scientists to search for new proteinases, and a dose study of their specificities can only he reached with purified enzymes. We are presenting the results of purification and properties of three proteo¬ lytic enzymes from the venom of Bolhrops jararaca-, one vvilh caseinase activity. a second showing hlood-clotting activity and a third with hydrolyzing activitv on arginine synthetic suhstrates. In the crnde venom. these activities differ in sensitivity to metal ions, anions and metal-hinding agents. Thus, in ])resence of sulphide and cyanide ions, the caseinase activity is strongly iidiihited whereas the henzovlarginine amidase (BAAase I and the hlood-clotting activities are nn- altered. Calcium ions increase the caseinase activity, have no action on amidase activity and slightly increase the hlood-clotting activity when assayed on oxalated plasma. The hlood-clotting activity, however, is inhihited in presence of Ca^ + ions when assayed on fihrinogen. The caseinase activity is almost completely inhihited hy Cd-+ ions. the BAAase is slightly decreased, whereas the hlood- clotting enzyme is not affected hy the same concentration of Cd-+ ions (Tahle ll. Also the effect of metal-hinding agents is different on these activities (Tahle 11). It can he seen that reduced glutathione inhihits the caseinase aiid hlood-clotting enzymes, and slightly inhihits the BAAase activity of the venom. The caseinase and hlood-clotting activities are very much decreased in the ])resence of ethyl- enediaminetetra-acetic acid. whereas the B.AAase activity is imaffected (1,2). The first attem])t to pnrify these enzymes was made hy fractional ])recipita- tion with ammoninm suli)hate and showed (1,2) already a preliminary separation of these activities (Tahle III). Fraction no. 5 had the strongest activity on casein and gelatin. The ])rotein fraction no. 6 ])resenteL-arginine methyl ester 504 230 L-Lysine ethyi ester 19 0.18 Protamine 2260 254 TABLE VI THE pll EFFECT ON THE STABILITY OF B O T H R O P 8 PROTEASE A. Each reaction mixture kept at 25° contained 1 ml. ot enzyme soiution in saline and 1 mi. of one of the foliovving soiutions: 0.005 M-tris buffer, pH 9.0; 0.005 M-tris butfer, pH 8.0; 0.002 N-HCi, 0.2 N-HCi and 2 N-HCl, to give, respectively, the toliowing pH vaiues: 9.0, 8.0, 3.0, 1.0 and 0.2. Samples of 0.2 mi. were withdrawn at the end ot 0, 2, and 26 hours. Benzoyi-L-arginine amidase and toiuene sulfonyi-L-arginine esterase specific activities are expressed as described in Tabie III and Tabie V, respectiveiy. pH Time (hours) SPECIFIC ACTIVITIES TAMEase BAAase 0 38 930 9.0 2 40 880 26 35 850 0 35 930 8.0 2 43 840 26 38 790 0 36 880 3.0 2 39 880 26 39 820 0 29 830 1.0 2 23 740 26 27 690 0 29 700 2:; 0.2 (IN-HCi) 1.5 19 690 26 19 480 cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2) :359-370, 1966 OLGA B. HENRIQUES, FAJGA R. MANDELBAUM and S. B. HENRIQUES 367 peptides 0, 1. 2 and 3. Peptide 0 remained on the initial line and peptide 1, 2 and 3 moved towards the calhode. B o t h r o p s protease A has no hvdro- Ivtic aclion on the A chain of insidin(13), a concliision supported Ity the de- monstration that the same three peptides moving to the cathode were ohserved on high voltage paper electro])horesis of incuhating mixture of H o t h r o p s protease A vvith oxidized insulin or vvilh its H chain ( Fig. 5). Fig. 4 — Paper electrophoresis of oxidized B chain of insulin incubated with B o - throps protease A. Pyridine-acetic acid-water buffer, pH 3.9. Electrophoresis for 4 h. at 1000 V. The direction of migration is from left to right towards the cathode. ta) T oriáln (b) Fig- 5 — Paper electrophoresis of oxidized insulin incubated with li o t li r o p s pro¬ tease A. Pyridine-acetic acid-water buffer, pH 3.9. Electropboresis for 2.30 h. at 1000 V. (a). 0.xidlzed insulin as control (b). Investigations of the amino acid composition and N-terniinal amino acids of these peptide fragments are siimmarized in Tafile VII. Uniess proline, lysine and ihreonine, all the other amino acids of the oxidized R chain of insulin were foimd hy acid hydrolysis of peptide 0. Since it contained only DNP-phenyl- alanine as N-tenninal amino acid, it can he concluded that this peptide resulted from hydrolysis hetween amino acids 22 (arginine) and 23 (glycine). Glycine and phenylalanine were the two amino acids foimd in peptide 1, and the DNP- derivative of this peptide proved to he DNP-glycine. After dinitrophenilation, cm SciELO 10 11 12 13 14 15 368 PROTEOLYTIC ENZYMES OF BOTHROPS V^ENOM TABLE VII — AMINO ACID COMPOSITION OF PEPTIDES OBTAINED BY THE ACTION OF BOTHROPS PROTEASE A ON OXIDIZED B CHAIN OF INSULIN Peptide Amino acids DNP-amino acid ether-soluhle 0 AIl found in oxidized B chain of insulin excepting Pro, Lys and Thr DNP — Phe 1 Giy, Phe DNP — Gly 2 Lys, Thr, Ala, Tyr, Tyr X, Pro dl DNP — Tyr 3 Lys, Thr, Ala, Pro DNP — Thr tyrosilie was fourul as the N-terminal amiiio acid of pejitide 2. Hy acid liydro- lysis il tiirned oul to ho a ])eiilajM‘[)tide of llireoniiie, i)roIine, lysiiie, alaiiiiie and tyrosine (as well as a small amount of tyrosiiie X, tlie formation of whicli coidd not he entirely prevenled). Conseqiiently ])t‘plide 2 inust have heeii formed due lo lhe hydrolysis hetweeii amino acids 25 (phenylalanine) and 26 (tyrosine) of lhe T5 chain of oxidized insniin. The fastesl electro[)horelic moving peptide (peptide 3) contained lhe foiir last amino acids (Thr, Pro, Lys and Ala) of B chain of insniin and DXP-Thr was niade evident as its DNP-derivative. Il can he concluded frorn lhese data lhat tlie sites of action of Bothrops j)iotease A on oxidized B chain are ihose indicaled in Fig. 6. B CHAIN OF OXIDIZED INSULIN Phe—Vai—Asp—Glu—Hls—Leu- CySOjH—-'dy—Ser—His—Leu—Vai—Glu—Ala—Leu— 1 2 3 4 5 0 7 8 9 10 11 12 13 14 15 -Tyr—Leu—Vai—CySO.H—Gly—Glu—Arg—Gly—Phe—Phe—Tyr—Thr—Pro—Lys—Ala 10 17 18 19 20 21 22 23 24 25 20 27 28 29 30 Fig. 6 — Summary of the speclllclty of B o t h r o p n protcase A on oxltilzed B chain of insulin. The arrows Indicate the site.s spllt hy the enzyme. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan OLGA B. HENRIQUES, FAJGA R. MANDELBAUM and QCQ Simp. Internac. S. B. HENRIQUES .S3(2):359-37ü, 1966 The action of B o t h r o p s |)rolease A oii tyrosiiie honcls must l)e associated to a very special ])eptide configuration. as oídy lhe 26-27 tyrosine liond (Tyr- Thrl was split. The olher tyrosine honds, 16-17 (Tyr-Leul in R chain as well as the 14-15 (Tyr-Ghi l and 19-20 (Tyr-CysSOsH I in A chain, were not hydro- lyzed. This selective action is also shown towards lhe l)asic amino acid honds, 22-23 (Arg-Gly) and 29-30 iLys-Ala); only Arg-Gly hond was split hy the en- zyme and confirms the jirevioiis results on lysine and arginine synthetic suhsirates. References 1. HENRIQUES, O, B., LAVRAS, A. A, C., FICHMAN, M., MANDELBAUM, F. R„ and HENRIQUES, S. B„ Biochem. J., 68, 597, 1958. 2. HENRIQUES, O. B., FICHMAN, M., and HENRIQUES, S. B., Biochem. J., 75, 551, 1960. 3. HOLTZ, P., and RAUDONAT, H. W.. Arch. exp. Path. Phurmakoh, 229, 113, 1956. 4. HAMBERG, U.. and ROCHA E SILVA, M., Arch. int. Pharmacodyn., 110, 222, 1957. 5. HENRIQUES, O. B., MANDELBAUM, F. R.. and HENRIQUES, S. B., Nature, 183, 114, 1959. 6. HABERMANN, E., Arch. exp. Path. Pharmakol., 231, 291, 1958. 7. FICHMAN, M., and HENRIQUES, O. B., A7'ch. Biochem. Biophys., 98, 95, 1962. 8. HENRIQUES, O. B., and FICHMAN, M., Personal informations. 9. BLOMBãCK, B., and VESTERMARK, A., Arkiv. for Kemi, 12, 173, 1958. 10. MANDELBAUM, F. R., and HENRIQUES, O. B., Arch. Biochem, Biophys 101. 369, 1964. 11. ROCHA E SILVA, M., BERALDO, W. T., and ROSENFELD, G., Amer. J Physiol., 156, 261, 1949. 12. DEUTSCH, H. F., and DINIZ. C. R., J. Biol, Che^n., 216, 17, 1955. 13. MANDELBAUM, F. R., CARRILLO, M., and HENRIQUES, S. B., Biochim. Biophys. Acta, 132, 508, 1967. Discussio.x T. Sueuki: “It is interesting that Bothrops protease A differs completely from the three proteinases of A. halys blomhoffii venom in their hydrolytic actions on insulin B chain”. F. R. Mandeibatwi: “In fact, the proteinases purified from the A. bjomhoffii venom hydrolyze mostiy peptide honds involving the amino groups of leucine and phenylalanine and the enzyme Bothrops protease A has no activity on these honds”. H. Michl: “Are the proteolytic enzymes of B. jararaca venom serin-enzymes?” F. R. Mandelbamn: “Yes, as trypsin, Bothrops protease A is inhihited hy diisopropylfluorophosphate (DFP), howev'er, the complete inhihition of that en¬ zyme is only reached with a 10 times higher concentration of DFP”. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):371-378. 1966 P. BOQUer, Y. IZARD, M. JOUANNET et J. MEAUME 371 41. ENZYMES ET TOXINES DES VEMNS DE SERPENTS. RECHERCHES BIOCHIMIQUES ET IMMUXOLOGIQUES SER LE VENIX DE NAJA NIGRICOLLIS P. BOQUET, Y. IZARD, M. JOUANNET et J. MEAUME Institui Pasteur, Paris, France Cet exposé ne constitue pas une revue des recherches publiées sur les en- zymes et les toxines des venins de serpents. I"ne telle entreprise dépasserait les limites dTin bref entretien en raison du nombre considérable des ])ublications parues sur ce sujetll, 2). Xotre but est de vous présenter les résultats d'ex- périences réceutes entreprises dans le dessein d établir une relatiou de causalité tntre les antigènes décelés dans un veniu par les niéthodes dJmmuno-diffusion et dlmmuno-électrophorèse et les facteurs enzymatiques et toxiques de ce mê- me veniu. Disposant de quantités relativenient importantes de venin de Naja nigricol- Us, un ELAPIDAE africain, nos recherches on été orientées vers rétude de ce poisou. D’une manière générale, on constate que les pro])riétés nocives des ve¬ nins d’ ELAPIDAE et celles des venins de VIPERIDAE ou de CROTALIDAE diffe- rent. LVxpérience nous enseigne, par exemple que la jdupart des venins neu- rotoxiques des ELAPIDAE cFAfrique contiennent ])eu ou ne contiennent pas d'en- zymes protéolytiques, alors que ceux des CROTALIDAE américains. objet de re- therclies a])profondies de la part des expérimentateurs brésiliens. dégradeut les protéines. Xotre choix étant dicté par les seules raisons matérielles, nous avous procé- dé de la manière que voici. Méthodes Dans des expériences préliminaires, le veniu de Naja nigricollis est filtré à travers un gel de dextrane, le “Sephadex C/ioo fine” choisi en raison de ses pro])riétés dVxclusion. Six fractions sont obteuues par ce procédé. Elles sont lyoj)hilisées et conservées à basse température. La dernière fraction ou “fraction VI” contient un facteur crune grande no- civité. que nous appelons “toxine «” (3) et des protéines de masses nioléculaires voisines. Pour jjuriiier cette toxine a. la fraction VI a été de nouveau filtrée .à travers un gel de “Sephadex” plus sélectif, le “Sejthadex Gso fine”. Les techniques de ces filtrations sont décrites “iii extenso” dans une précédente J)U- blication (4). cm SciELO 10 11 12 13 14 15 372 ENZYMES ET TOXINES DES VENINS DE SERPENTS. RECIIERCHES KIOCHIMIQUES ET IMMUNOLOGIQUES SUR LE VENIN DE NAJA NIGRICOLLIS (7ia(|iic fraction esl soiiinise à iiiie analyse ininuiiiologiqui- suivant Ics mé- thodes (l’imnuiiio-(liffuíion douhle cii milieii gélifié(5) et d'imnnmo-éIeclio|)ho- rèse(r)). Par ce deniier |)ro(édé oii ohtienl la sé|)aralion des aiitigèiies coiitemis daiis des éclianlillons de 5 à 10 /ig des |)rodinls exaniiiiés. IJii eouranl de 15 v/em esl maiiitemi ])eiidaiil 2 heiires et le taitipon iitilisé esl la soliitioii nsiielle de véronal sodi(jue/H(d 0,025 M (])H 8,2). Ilans les deiix séries d’ex- {.érienees on |)réci|)ile le.s aiiligèiies ])ar rimniimo-séruin 984 qiii [irovieiit (Piin cheval liyperimnumisé jiar des injeelioiis réj)élées de veniii de Naja nigricolUs. ÜÉSUI.TATS 1 — Le Iracé de la figure l. illustre le résidtat de la fillration de 500 ing de veiiin de Naja nigricollis à travers le “Sepliadex Gioo fine”. II esl défini par une mesure al)Soi|)liométrique de la teneur en protéines de eliaque éelianlil- O lun de íillrat reciieüli (A = 2.800 A). Ces éelianlillons sont réi)arlis en 6 lots corresj)ondanl aux fraelious I à VI. Le traeé de la figure 2 correspond à la fillration de la fraction VI à tra¬ vers le “Se])l)adex G 50 fine”. Les sous-fraetious Va et V la ainsi oOtenues sont définies par le second pie de ee gra|)hique. Soumis à une filtratioii de eoutrôle sur “Sepliadex G^o fine”, le produil terminal semhle homogèue. 2 — La fi gure 3 re])résenle une préj)aration dans laqiielle 25 pg de venin de Naja nigricoUis ont élé analysés |)ar immuno-électro|)liorèse et à tilre com- paralif, la re])résenlaliou seliémali(|ue des antigènes dissoeiées j)ar le même procédé. Cliaipie aniigènc préeipilé étanl désigné par un syrahole, on constate que ranligène eorrespondaut au premier arc a se déplaee vers Tanode. 11 apparait seulement dans les préparatious les ])lus riches en venin. Un second antigène que uous désignons |'ai ia lettre A est sensihlemciit iso-électriqiie à pli 8,2. I’lusieurs ares eonstituent eusuite le grou])e des antigènes B. Peu disliucts dans le itnmuuo-éleetro])liortses du venin total, ils ont élé Pobjel d’une analyse ap- rrofondie dont les lésullals sont ex])osés dans les paragraphes réservés aux frac- tions 1 et III. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):371-378, 1966 P. BOQUET, IZARD, M. JOUANNET et J. MEA UME 373 Un antigène mal cléfini. h est ohservé clans les préparations qiii contiennent L.ne forte quantité de venin. L'arc de précipilé C correspond à iin antigène hasique. Cet arc est caracté- risc par son amplitude et son a])latisspment. Enfin. trois antigènes cathodiques piécipitent selon les ares Dj, D, et D 3 . Onze parmi les treize antigènes dé- nombrés sont repartis dans les fractions I. 111 et VI. La fraction 1 est essentiellement eom[)osée de 1 antigènes du groupe B et la fraction 111 eontient Tantigène A et 3 antigènes du même groupe R. Les exiiérieuces d immuuo-diffusion selon Oucbterlony démontrent que parmi ces antigènes un seul est commun aux fractions 1 et 111. Cette oliservation est confirmé par Texperience que voici. Lorsqidon fait reagir suivant la même techni- que la fraction III sur un échantillon de sérum 984 épuisé par la fraction I ou la fraction 1 sur un échantillon du même sérum épuisé par la fraction III, trois lignes de précipité se forment dans chaque |)réparation. Au contraire le sérum 981 tel qiiel fait a])paraitre quatre lignes de précipité dans les |)réparations té- moins. Cette ohservation nous a conduit à adoj)ter la nomenclature sinvantc j)our définir les antigènes du groupe B. Fraction 1 ]R, = 3R, B, Fraction 111 cm SciELO 10 11 12 13 14 15 374 ENZYMES F:T TOXINES DES VENINS DE SEREENTS. RECIIERCIIES mOCniMIQUES et immunologiques sur le venin de NAJA NIGRICOLLÍS Li's aiiligèiies 115.. iT étaiil ideiilitjues, tious les désigiioiis ])ar lo syni- l)olc La fraclion VI coniprend les aiiligèiies forleiiietil hasiijues C. D,, el D^,. Q)iiant aiix fraelioiis II, IV et V, elles se eomporleiil eotnrne des itiélanges des fraetions voisiiies. Daiis la fraetion IV, eependant, sont assoeiés aiix aniigèiies A et C les deiix aiiligènes a el l> doiil la |)roporlion semhie faihie dans le veiiiti lel (jiiel. La fraclion «, ohlenue par deux filiralions sueeessives de la fraetion VI à travers le “Sejdiadex |)réeipile suivanl nn are eorrespondant à D-j lorsqn’on Ia soumel, à la dose de 25 à 50 /x à riinmiino-éleetro[)Iiorèse. Dans les inênies eonditions, ec))endant, des doses plus fortes de eelle fraclion soit 100 à 500 p,g, non seidement fonl apparaílre Tare de précipilation D-,, mais encore des ares eorrespondant à D^, D, el C dont la ])osilion an voisinage de Taxe de la ])ré- |)aration indicpie (pie ces aniigènes sont en faihie pro])ortion. I5ie étiide de Taction dénalurante de Ia clialeur snr les anligèiu-s qiii vien- nenl trêtre énnrnérés complete ces oOservalions. A cet effet, on chanffe à 96" dans im Oain-rnarie (2) ime soliition contenani 10 mg de venin jrar cm^ irean j)nre * et on prélève, à intervalles régniiers, des cclianlillons de eelle soinlion qni sont aiissilôt soiimis à Téprenve de rimmuno-éleclrophorèse. Dans ces eonditions en inoins (rim miniile rantigène IB, est privé de sa ])ro|)riétc de jirécipiter en [irésence de Tanticoips spccifiqne. A Texception de Fantigène 1?^, les autres antigènes dn gronpe B ne prcci- pitent pins après 5 minutes. Les délais de la perte de leur poiivoir de |)réeipiter sont de 20 minutes pour rantigène A, de 30 minutes pour rantigène Da, de 45 minutes pour Fantigène C, de 55 minutes pour Fantigène Ba el de 60 minutes pour Fantigène D^. Parallèletnent à ces recherches, nous avons entrejiris de definir les |)roprié- tés enzyrnatiques el toxiques du venin de Naja nigricolli.s et à titre comparalif, celles des fraetions ohtenues à |)arlir de ce venin. Les leclmiques utilisées dans ces essais sont rapportées dans une jmhlicaliou séparée (4). Si on admet qne la dissocialion des jiroléiues jiar la rnétliode de filtralion sur les “Se|)liadex” est fondée sur leur exclusioii dans Fordre décroissaul de leurs masses molécidaires. on est eonduit à jienser (pie la fraclion I est consti- luée ])ar les jiroléines dn venin dont la masse moléeulaire i'st plus élevée. Celle fraclion I lirise les liai.sons carhoxy-ester de Facelylcliolinc mais elle ('St sans effet sur les acides aminés estérifiés. Une correlation a été élaldie par Hamlierg et Bocha e Silva (7) entre Fajililude (pie possède le venin de Bollirops jararucd k hydrolyser le B.A.E.E et sa propriéié de lihérer de la hradykinine dans Forganisme animal. Dans cet ordre de fail, ni le venin de Nitja uigricoUis ni aucnne fraetion examinée iFonl une aclion sur le B.A.E.E, le B.A.M.E. le T.A.M.E et FA.T.E.E. La même fraclion I hydrolyse la llaison [leptidiipie de la glycyldencine, el une des deux liaisons de la glycyl-glycyl-glycine el de la glycyldeucyl-tyrosine mais elle est saus effet sur Fliémoglohiue el la caséine. • Eau cleslonlsée. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):371-378, 1966 I’. FOQUET, Y. IZARD, M. JOUANNET et J. MEAUME 375 Au contraire, elle ronipt très aisément les liaisons phospho-ester du jihospha- te de paranitro])heiiol, de l'AMF. du lii-phosphate de paranitropheiiol. de l'ADP et de rATP. Son action dépolyniérisaiite à Pégard de laeide liyaluroniqiie est faihle. Klle possède. eiifin. la propriété de [trovoquer in vitro la coagulatioii du saiig. La fraction III eontient non seideineiit la |)hos|)holipase A [troduetrice de lysoeithine aux dé[)ens de la lécithine mais eueore uu facteur lytique direct. Klle délruit le eomjtlémeiit du sérum de cohaye. diminue la vilesse de la sédi- mentalioii gloliulaire. dé])olymérise Paeide hyaluronicjue et inhilie le phénomène de Ia eoagulatiou. Knfin. celte íraetion III est légèremeut toxi(|ue, sa Ldsn pour la souris de 18-20 g. éprouvée par la voie veineuse est de 16 31 p,g alors que dans les mêmes conditions celle du venin est de 12.36 /xg. La fraction VI exerce une action anticholinestérasique sur racétyl-cholines- tcrase des glohules rouges du cheval mais elle est sans effet sur Ia eholinestérase que nous avons décelée dans la fraction 1. D’autre part, cette fraction VI est toxique et provoque des jraralysies chez Ia souris et le lapin. Le produit le [jlus ]mr que nous avons ohtenu après deux filtrations de cette fraction à travers un gel de "Sephadex Lrio”. exerce encore une action anticholinestérasique mais sa Ld.-,,, pour la souris est faihle. Elle est de 2 pg dans les conditions qui viennent (Pêtre définies. Nous 1’avons désignée sous le nom de toxine a. et nous avons conclu trime étnde préliminaire (4) que cette loxine est une j)rotéine de faihle jíoids moléculaire. Elle franchit, en effet, les memhranes d’acétate de cellulose et son coefficient de sédimenlation exprime en unités Svedherg est 0,84 (centrifugeuse Spinco; cellule standard de 12 m/m, angle 2“; concentration du produit 10 mg/cm*; vitesse de rotation 59.780 l/rn; lemirs de centrifugation 96 min; température amhiante 20" ±; 0,1" I. I ne ])remière analyse de ses acides aminés ré\èle qi/elle eontient de fortes proj)orlions d’arginine, de glycine. de lysine, (Paeide aspartique, (Paeide gluta- mique et de threonine (3). l’ne collahoration a été étahiie avec J. Porath, E. Karlsson et D. Eaker de PLniversité d l ppsala qui ont hien voulu consacrer une |)artie de leur activité à Péliide de la toxine a et entreprendre une analyse de la strueture de ce ])oly- peplide. Ces résnitats aequis, il importait (Pétahiir nne relation entre les antigènes definis ])ar les méihodes immunologiques fon(lé(‘s snr la précipitation mutuelle des antigèn(*s et des anticorps dans un gel et les diverses pro])riétés (]ui viennent (I tire énumérées. Des glohules de cheval additionnés de lécithine puis versés à la surface (Pune ])réparation de la fraction I ou du venin tel quel. soumis au préalahle à une immimo-électrojdiorèse, sont lysés entre les ares de précipité de Pantigène A qui peul ainsi ctre considéré comine étant la phospholipase. En Pahsence de léci- ihine et de la solulion tam|)onnée dont le jihosphate se comporte comine un agent inhihiteur, on ohserve une hémolyse entre les ares de Pantigène précipité 3IL, homologue vraisemhlahlement du facteur lytique direct décrit par Hahermann et Neumann l8) à propos (Pautres venins. L’em])Ioi de la technique dT riel t9) adaptée à Phvdrolyse des dérivés phos- phorés du nitrojihenol perinet de siiivre Paction des six fractions sur les liaisons cm SciELO 10 11 12 13 14 15 QTfi ENZYMKS ET TOXINES DES VENINS DE SERPENTS. RECHERCIIES BIOCIIIMIQUES ET IMMUNODOGIQUES SUR DE VENIN DE NAJA NIGEICOLLIS jjhosplio-eslpr. L’hy(lrolyse du l)i|)lios[)hatp ar raniigène D;,, (resl à cel anligène (pie correspoud le |)olype[)lide récenunent isolè dans un élal voisin de la ])urelé par Karlssou, Kaker et Poralli (10). Le talileau I résume ces oOservalious. TABLEAü I — ANTIGENES DU VENIN DE NAJA NIGRICOLLIS Cocte Funotlon pre.sumce a 9 A Phosphollpase IB, Ester hydrolase 3B, V Facteur nntlcoagulant 9 3B., Facteur hémolytique dlrcct IB, F^acteur eoagulant b 9 c Anti enzyme? D. 7 D, 9 D 3 Toxine a On peut considérer, poiir concinre, que raniigène A iso-électrique à pH 8,2 et raniigène fortemeni hasique 1);, reiirésenteni les proléines responsaldes, la pre- mière, de raclion liydrolysantc du venin sur les lecilliines et la seeonde de son cm SciELO 10 11 12 13 14 15 Mem. Inst. Butcntan Simp. Internac. S3(2):371-378, 1966 P. BOQUET, Y. IZARD, M. JOUANNET et J. MEAUME 377 action neurotoxique. On est coiiduit à admettre, d’aiitre part. qirun certain iionihre d’argumenls jtlaident eii faveiir des relations de caiisalilé que noiis avons tenlé d’étal)lir entre divers aiitigènes et certaines activités enzyniatiqiies exercées. II apjtarait cependant. que les projtriétés physiologiques et hiochimiques de plu- sieurs antigènes demeurent indétermiuées et que le nombre des autigènes révélés par le pliénonièue immunologique de la précipitatiou est inférieur au nombre des activités actuellemeut eounues du veuiu de Naja nigricollis. LVxpérienee uous enseignera daus quelle mesure une jtrotéine est capable dVxercer jdusieurs aeti- vités, dans un venin. La question stí [)Ose également de savoir quelles relations ])euvent être éta- blies entre les antigènes caractérisés dans le venin de Naja nigricollis et les an¬ tigènes des autres venins de serpeuts. Des reeherebes immunologiques en voie de dévelopjtement montrent que les venins de deiix ELAPIDAE, Naja liaje et Naja naja et eelui d'un VIPERIDAE, Echis carinatus contiennent des protéines nocives dont la eoiistitution est vraisemblablement voisine de celle de la toxine a. Dans cet ordre de faits. Tétude de la structure de cette "toxine” entreprise à Upj)sala par Karlsson, Eaker et Porath. apportera l élément essentiel d’une solu- tion à ce problème. Bibliographie 1. BOQUET, P., Toxicou, 1966, à paraitre. 2. BOQUET, P., IZARD, Y., JOUANNET, M. et MEAUME, J., Mem. Inst. Bu- tantan, SimtJ. Internac., 33, 1966. 3. BOQUET, P., IZARD, Y., JOUANNET, M, et MEAUME, J„ C. R. Acad. Sei., 2H2, 1134, 1966. 4. BOQUET, P., IZARD, Y., MEAUME, J. et .TOUANNET, M., Mem. Inst. BtiUin- tan, Simp. Internac., 33, 1966. 5. OUCHTERLONY, O., Actu Microbiol. scand., 32, 231, 1953. 6. GRABAR, P. et WILLIAMS, C. A., Biochim. Biophys. Actn, 17, 67, 1955, 7. HAMBERG, U., ROCHA E SILVA, M., Experientia, 13, 489, 1957. 8. HABERMANN, E. et NEUMANN, W., Arcii. exp. path. Phnrmak., 223, 388, 1954, 9. URIEL, J., An)i. Inst. Pusteur, 101, 104, 1961, 10. KARLSSON, E., EAKER, D. et PORATH, J., Biochim. Biophys. Acta, 1966, à paraitre. DlSCl SSIO.N C. Y. Lee: “Is there any cardiotoxic component in the venom of Naja nUjri- collisl What is the molecular size of your neurotoxin?” P. Boquet: “La présence de cardioto.xine n’a pas été recherchée. En ce qui concerne le poids moléculaire de la toxine, nous avons donné seulement le coeffi- cient de sédimentation en unités Svedberg. Le Professeur Porath vous donnera de plus amples informations sur ce point.” cm SciELO 10 11 12 13 14 15 Q7S ENZYMES P:T TOXINES DES VENINS DE SERPENTS. RECHERCMES BIOCHIMIQUES ET IMMUNOLOGIQUES SUR LE VENIN DE NAJA NIGRICOLLIS S. Minton: ‘‘In which fraction of Naja nigricoUis venom is the antigen shared with the venom of Echis carmatus?" P. Boquet: “Une communante antigcniquo a été mise en évidence entre le venin de Naja nigricoUis et réchantillon de venin (i’Echis carinatus qui nous a été adressé d’Ethiopie. Parmi les antigènes communs au venin de Naja nigricoUis et au venin d’Ecliis carinatus étudiés, il convienl de citer un antigène basique cor- respondant à l’antigène neurotoxique.” F. Kornalík: “How liave you tested the procoagulant and anticoagulant pro- perties of the Naja nigricoUis venom?” P. Boquet: “L’action du venin de Naja nigricoUis sur la coagulation du sang a été mesurée selon les techniques usuelles (détermination du temps de prothrom- bine, etc...) au moyen du plasma humain.” cm 10 11 12 13 14 15 Mem. Inst. Butantpn Simp. Internac. 33(2):379-388, 1966 JERKER PORATII 379 12. SOME SEPAHATIOX METHODS HASED OX MOLECELAH SIZE AXl) CHARGE AXD THEIR APPLICATIOX TO PERIEICATIOX OE POLYPEPTIDES AXD PROTEIXS IX SXAKE VEXOMS JERKER PORATH Institute of Biochemistry, University of UppsaUi. Uppsala, Sweden Intkoduction A inalhematical jirohlem can often he solved in many ways. One of iheni niighl he tlie mo?[ effective. another the most informative, while perhaps all olhers niight appear disgraceful to the mathematicians. An analogous sitiiation often arises when lhe hioehemist is faced with the proldem of isolating a particular snh.stance froni a eomplieated mixlure siich as, for exam])le, an animal venoni. Many sej)aration melhods are availahle hut a ])ro])er choice among theni is dif- ficult. Another question coneerns the selection of order when several metliods are required. I eannot give general answers hut only express some persoiial opinions in the ho|)e to guide those with limited ex])erienee in these matlers. íl is a commonplaee misunderstanding that a method which most effeclively resolves a eomplieated mixture in a single ste]) of neeessity also is superior to those of less separation power. Although ihis is usually true when the purpose is entirely analytieal it is not neeessarily the case in preparative fractionation. 1 can mention gel electrophoresis as an example. Analytieal electro))horesls in thin layers of stareh gel or ])olyacrylamide has heen extremely usefui for studies of genetically delermined variations in protein patterns of hiological fluids and ex- tracts. Xevertheless. in spite of the impressivo resolving ])ower of this lechniqne it is nsnally far more practical to use other methods for large seale se])arations hecause of the difficnlties involved in the transfer of gel electrophoresis lo a preparative seale. Gel electrophoresis is hased on at least three |)hysieal faclors or ])arameters. viz., eharge, molecular size and shape. Eractionation in two or more sejiarate ])rocesses often gives hetter resnits and affords more information ahonl the physieal or Chemical properties of the snhstance lo he i)nrified. Actnal ap])lication of these methods may he fonnd in references 11,2). Provided lhal the gel filtration step is carried ont in a colnmn calihrated with respect to the molecular size — retention relationships a rongh estimation is possihie of lhe molecular dimensions of the snhstance under sludy. Electro|)horesis ])erformed in an adsor|)tion-free médium permits conclusions ahout elecirical eharge of the migrant species. Since electrophoresis and gel filtration are highly reliahie and reproducihle methods, holh can he em|)loyed easily on any seale retpiired in lahoratory work. Moi.ECUl.AR SIEVING Several kinds of gel materiais are availahle that provide se])aration hased on molecular size. Sephadex and Riogel. cross-linked dextran and polyacrylamide, respeclively, are lhe most importani commercially availahle gel snhstance ai)plieahle Sephadex and Riogel. cross-linked dextran and polyacrylamide. cm 2 3 z 5 6 11 12 13 14 15 Qon SOMK SEPARATION METIIODS BASED ON MOLECULAR SIZE AND CHARGE AND THEIR APPLICATION TO PURIFICATION OF POLY- PEPTIDES AND PROTEINS IN SNAKE VENOMS lo fraclionatioii in aqucous soliilions. IJolli of llifse gcis are availahlo iii dif- fercnl degree of crossdiiikage, tluis providitig for inolcciilar wcighls froin a fcw huiidred iip lo several lumdred tfioiisaiids. l'or siil)slaiices of cvcn liiglicr iiiole- fular sizc and for paiiicles agarose gel can lie used. I'or snake venom fractioii- alion Sepliadox (ílOO, CTS, (',50 and (;25 luive lieen fouiul lo Oe exireniely useful. An almost complete list of referenees lo lhe gel filtraiion nuTiiod ean he ohtained from Pharmacia Fine Chemicals, U|)pfala, Sweden. Ellctuoimiouesis The nnex[)cricnced rnay feei discomfori when faced vvilh lhe jirohlem of ehoosing among lhe very many versions of eleetro|)horeli(' eipiipinenl and tech- niqnes now availahie. Yet eleelrophoresis is simple in jiractice ailhough not in theoretical delails. This is certaiidy trne even for column eleelrophoresis lhe melhod I [jrefer myself. Among lhe numerons ap|)aratuses for eleetro|)horetic fractionation al my dis])osai al lhe Instilnie of Pioehemistry in U|)|)sala I vvonld seleel lhe ai)])araliis of Steilan Hjerlén |8) for orienting and analylieal purposes and lhe cohimns de- scrihed hy myself for preparations on any scale in lhe range of 25 milligrarns (4) to 25 grams |5). The lechnique developed hy Hjerlén is a kind of free zone eleelrophoresis hased on a principie first descrihed hy Alexander Kolin (6) and inde])endently somevvhat laler hy Hjerlén (7,8). The fractionalion chamhcr consisis of a qnartz luhe a fevv millimeter in diameter placed helvveen two eleclrode chamhers and sejjaraled from lhem hy memhranes. Dnring lhe rnn, lhe luhe is rotated con- linually ahonl its longitudinal axis. Samples can he transferred to or wilhdrawn from lhe luhe hy a capiliary tuhing. FfficienI cooling is achieved hy immersion of lhe luhe iu a water hath. The [irogressing separation eau he followed hy a scanning arrangeinent which measnres ahsorption al 280 ni/j,. The salient fealure of lhe melhod is lhe revolving luhe. P>y sleady rolalion of lhe luhe al a conslanl speed conveclion is virtualiy eliminaled. A sinall sample projierly inlroduced (vvhile luhe is revolving) gives a slahie slarling zone. II no voltage is a|)|)lied, lhe zone vvill hroaden oídy very slovvly, ehiefly hy molecular diffiision. A prolein zone remains sharp for honrs. When an eleelrie field is ajiplied lhe varioiis componenls slari lo move aeeording lo lheir eleclrophorelic mohilities. Al iniervals lhe se])aralion pallern is cheeked hy scanning. Al any time can he renioved from lhe rotating luhe and analyzed for hiological aclivily, Chemical |)ro|)erties, etc. Sam|)les rangi ng in size from a fevv /xg lo a fevv mg ean thus he analyzed. Unforltmalely ihis apiiaralus is noi yel commereially prodiieed. Iu eerlain ap|)liealions some of lhe ihindayer eleclro])horesis teehniípies are effeelive suhstilules. The Hjerlén eleelrophoresis inelhod unforiunalely cannol yel he sealed iqi lo allovv fractionalion of lhe hundreds of milligrarns and more required in mosi |)i'eparalion |rroccdnres. Hovvever, eleelrophoresis in free soluliou (i.e. eonlaining no solid su|)porl I can he performed on a larger scale lhan is afforded hy lhe cm SciELO 10 11 12 13 14 15 i«£er.í. Itist. Butantan JERKER PORATU 381 Simp. Internae. 33(2):379-388, 1966 *• Hjeríéii ajiproach. This is acconiplished in flowing-filni eleclroplioresis. a coii- tir.uous separatiüii process (9). Hecause sample can he inlroduccd (■oiitimiously. the lailer melhod can piovide separations on any desired scale if lhe componeiits difíer widely in inohility. Since this niethod is mainly useful for group separa- tion and does not have lhe resolving |)Ower reqnired for se|)araling very elosely related suhstances it wili nol l)e fiirlher (hscnssed here. Instead reference is tnadc' lo lhe lhesis of HannigllO). In slationary tolumn eleclroplioresis lhe advantage of operating in a uniform. free solution must he sacrificed. A ])oroiis povvder is introduced inlo tlie hnffer or a density gradient is jirodiiced ( 11 I lo snppress niacroconvection. Only lhe first technique wilI he descrihed here in some delail. It wilI suhseqnenlly lie referred to siinply as “colunin electro|)horesis'’. Column eleclroplioresis is a discontiniious melhod. As in lhe case of re- volving tiilie eleclroplioresis the sample is introduced in the cohinin hefore an electric ficld is applied. The rini may he continued for several days at a safe temperature provided lhat the tuhe is surrounded with a cooling jacket and the evoliition of joule heat is kejit low enongh to avoid the creation of a large radial temperature gradient. Evidently, column eleclroplioresis permits the fractionation of suhstances with similar iiiohilities. In my opinion the potentialities of column eleclroplioresis have heeii iinder- estimated. I should üke to discuss hriefly some of the most important drawhacks and supjiosed inconveniences inherent or adhereiil lo the method. The first and niosl important prohlem is lhe interference hy the supiiort. Adsorptioii has soinetimes heen considered to play a suhstantial role in lhe frac¬ tionation as indicated hy the term ■TIectrochromatography”. If charged suhstances adhere to the supporl hy adsorption or precipitation the dattiage can he disastroiis. for this not only increases electroosmosis hut aiso promotes fiirther adsorption. Fortnnately. siqiports with very high degrees of "inertness'’ are now availahie, siicli as specially puriíied cellulose I from Grycksho Pap|)ershruk. (irycksho, Svvedenj and Sephadex. Another ohjeclion to column eleclroplioresis concerns lhe difficully of irionilor- ing lhe advaucemenl of lhe migraling zones. Unless lhey are colorcd, suhstances Iravelling in lhe noii-lransparenl column caiinol he ohserved during a run. How- ever, suhstances that have reached lhe end of the column can he removed con- linuously hy a washing lechnique and analysed hy a rccorder hefore collection (12). riiis jirocedure allows monilored withdrawal of the fast-nioving suhstances after which lhe eleclroplioresis can he proloiiged to separate lhe componenls with low uiohilities. Hy |)ro|)er seleclion of column length lhe eleclroplioresis may he pcr- foriiied very efficientiy. I ncharged and charged colorcd markers may of iour.se he introduced lo serve as indices of electroosmosis and lo facililalc esliniations ol lhe stage of fractionation. ■■(ichiniu eleclroplioresis is complicalcd ' is an oficn heard slalcment. 1 hose who once have tried lhe melhod in oiir institute never use lhat argunienl. It is an extremelv simple melhod whether employed on a moderate (4) or a large scale (5). The LKB column for preparative eleclroplioresis on a large scale is indeed so simple that I can operate it myself when my tcchnician is ill, although I admit that reading the descriptioii in the manual demands intellectual effoii I Manual and a|)|)aralus are availahie from LKB Instrumenl AB. Bromma 1. Svveden I. cm 2 3 z 5 6 11 12 13 14 15 382 SOMp; SKPARATION METHODS RASED ON MOLECULAR SIZE AND CIIARGE AND THEIR APPLICATION TO PURIFICATION OF I>OLY- PEPTIDES AND PROTEINS IN SNAKE VENOMS l().\ EXCHANGE ClIliOMATOCliAPIIY 'riie grcat l)n*ak-lliroiin;h for cliromalograjiliy of proleiiis caiiio vvilli lhe iii- trodtictioii of lhe eelhilose ion excliangers hy Soher and I’elerson |13). Dielhyl- aniinoelhyl ethers of eelhdose I DKAE-eellulose) liave proved lo he ])arlicidarly iiseful. More reeenlly, aiialogoiis ioii-exehangers have heen |)roduced wilh Se- phadex as nialrix for exainple, lhe DKAh]-. CM-(earhoxytnelhyl), and SE-(snl])ho- elhyl) derivalives. The Sephadex excliangers have lhe advanlage over llie cor- responding celluloses in iieing more insoinhie. This is parlicniarly evideni wilh lhe ealionic types. The large volume changes lhal accompany allerations in lhe ionic slrength of lhe mediurn oflen com|)licale ehromalography on long ion ex- change Sephadex eolnmns. The pojmlarily lhal prolein ehromalogra])hy has enjoyed lhe lasl deeade is dne lo lhe easy lechnical o|)eralions and lhe simple ir.px])ensive eqnipmenl reqnired — at leasl for crnde gronp separalions. The more effeclive elulion ehromalo- graphic leclmiques, enqiloying an eluanl of conslanl or conlinnonsly ehanging eomposilion, are more dernanding however, hecanse small varialions in lhe experi- menlal condilions oflen ehange lhe elulion |)allern heyond reeognilion. Lack of re[)roducihilily is oflen experienced nnless exlreme eare is laken in lhe preparalion of Imffers, in mainlaining a conslanl s])eed of elulion, lemiieralure, elc. Occasionally, lhe lime and efforl expended in searching for ojilimnm con¬ dilions for elulion ehromalography of prolein mixlures nsing an ion exehanger is amply rewarded. 1’olycarhoxylic ion excliangers such as Amherlile IHC-5() have heen used wilh greal success lo fraclionale hasic [lolyjieplides and proleins of small molecular size. Application of gel filtration and ion exchance ciihomatogiiapiiy TO THE PLIlilEICATION OE SNAKE NELUÍOTOXINS Isolulion procrdurex 1 like lo examplify lhe melhods descrihed ahove wilh some cxperimenls reeenlly riiade hy l)r. Kverl Karisson and Dr. David Eaker in my lahoralory. More delailed deseriplions of lhe procediires wili ajipear laler (1-1 ). (iohmin eleclrojilioresis has heen a|)plied only for orienlalion [iiirposes, sinee in ihis par- licnlar case, lhe isolalion of iieuroloxins, ion exehange ehromalography has a higher resolving power. Hnl eleclrophoresis of crnde venoni gives a good groiiii sejiaralion of iieiiro- loxins and phospholipases (Fig. Ij- ophcol dfnsity 280 m/j Fig. 1 -- Column: Celluto.se, MunktelTs, 103X3 cm. Huffer: O.OIS M Acelute buí- for, 1.50 M Glycine'-- pll 5.0. Sample: 150 mg cruíle venom. Run: 21 hours, 1200 volt, 35 mA. Fiotd strength 8 volt/cm. Fractions: 5 ml per 30 min. N and P Indicate the fractions vvith the highest neuroloxic and phospholipase activiiies. (Run made by Dr. Kvert Karlsson). cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan JERKER PORATH 383 Simp. Internac. 33(2):379-38S, 1966 The procedure of isolatioii of the neurotoxin « of Naja nigricoilis, the loxiii most extensively studied so far, was in short the following: A sample of 1.1 grani of dry crude Naja nigricoilis venoni dissolved in 10 nil of sodiuni phosphate huffer of pH 7.3, 0.03 M in sodinm ion. This solution was suhjected to gel filtration in a Sephadex G75 column (3X80 cm I with elulion S])eed kept at a rale of ahoiit 18 ml jier hoiir. 10 ml fractions wcre collected. Fig. 2 shows a diagram of the same general paltern ohtained from an analytical experiment. Fig. 2 — Gel filtration of 150 mg crutle venom on a 3.2X80 cm column of Sepha¬ dex G-75 in 0.03 M .sodium-phosphate buf- fer, pH 7.3. Flow rate: 17.5 ml/hr, 3.5 ml/traction. Tn and T/5 indicate the areas with highest toxic activity. Pooled fractions containing T* (see the shaded area in the diagram) were transferred to a 3.2X45 cm column of Amherlite lRC-50 equilihrated with 0.28 M sodium phosphate huffer of pH 7.3. Elution was performed at a rale of 30 ml per hour. Fig. 3 shows the elution pattern ohtained in this experiment. Ahout 25 mg of jiure neurotoxin Tj was ohtained. Fig. 3 — IRC-50 chromatography of Ta from gel filtration (Fig. 2). Sample ap- plied to 3.2X45 cm column in 250 ml 0.03 M fiodium-phosphate huffer. Elution at .30 ml/hr with 0.28 M sodium-phosphate buf- fer, 6 ml/traction. Fraction 0 = 150 ml. 30 30 r'acUon No 10 Karlsson and Eaker have adopted this technique for fractionation of other elapidian venoms. Eig. -1 shows the gel filtration diagram of the venom of Hemachatus haemachatus. The neurotoxins have ahout the same elution volume as those of Naja nigricoilis. When chromatograiihed on Amherlite IH(i-50 with cm 2 3 z 5 6 11 12 13 14 15 384 SOME SEPAKATION METIIODS BASED ON MOLECULAR SIZE AND CHARGE AND THEIR APPLICATION TO PURIFICATION OF POLY- PEPTIDES AND i>ROTEINS IN SNAKE VENOMS gradieni elulion llu- neiiroloxiii fractioii was n-solved inio a large iniiiiher oí ilislinclly differeiil eomponeiiLs. iiiaiiy of vvliicli were showii lo possess neiiroloxie activity (Fig. 5). *254 EFFLUENT, ml Fig. 4 — Gel flltration of 1.3 g of erude Hemachatus haemachatus venom on a 3.2X74.5 cm column of Sephadex G-75 In 0.06 M sodium-phosphate buffer, pll 7.3. Flovv rate: 18.8 ml/hour. Effluent monitored contlnuou.sly at 254 m/j with an LKB Uvicord. The last peak ha.s Aj,, hlgher than Aj„. Fig. 5 — Chromatography on Amberlite IRC-50 of fraction “Neurotoxin.s" (Fig. 4). Column: 3.2X21 cm equilibrated with 0.20 M sodium-phosphate buffer, pll 7.3, and eluted with 50 ml 0.06 molar buffer before applicatlon of samplo. Run: Performed with a Beckman Model 130 Spectrochrom Analyzer whlch permits a contlnuous monitoring of absorbancy, pll and conductivity. After applicatlon of sample elutlon with 0.06 M sodium-buffer was continued until no more unadsorbed material carne out. Exponential concave gradient from 0.15 M to 1.50 M sodium. Flow rate 40 ml/hour. SoiiK’ propcrlirs of lho nvuroloxiiis Neiiroloxiii Ta of Naja iii^rivollis lias a molecular weiglil of 6737. It coii- si.sts of 61 amino aeid residiies liiied iip iii a single jieplide ehain eross-linked liy foiir disulfide liridges. The neurotoxin Ta is liiglily liasie, as are all lhe snake nenroloxins sludied so far iii Up|)sala. Thiis il moves rajiidly lo lhe ealhode al |)il 8.7 in lhe HjerUhi eleelrophoresis (l'’ig. 6). The IJ),,io has lieen fonnd lo he 1.8 /j.g, as delermined in miee of 18-20 g hody weighl. In Tahie I are eom|)iled amino aeid analysis dala for IVaja nisrrwoUis Ta , for llcniarlialii.s huornacliiiliis eomponenis .'5, 5 and 12 (Fig. .SI, and for lhe erahii- loxins a and h i.solaled hy 'Faniiya and Arai (16|. Peaks 3 and ,3 represeni highly aclive nenroloxins (I^l),,,,, 1..5 and 2.0 /tg). lhe similarily in amino aeid eomposilion is remarkahie. Fnriher fnel for exeiled speenlalion comes from lhe very similar fignres for lhe Naja naja atra loxin of Yang and indeed aiso for lhe seorpion loxin de.serihed al lhe Symposinm liy Dr. Li.ssitzky. All of ihese dala seern lo indieale lhal a single sjieeifie (or a fevv elosely similar) rnoleenlar pallerns evolved in widely differeni rpeeies is ea|)ahle of hloeking some fnnda- menlal physiologie.al proeess. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):379-388, 1966 JERKER PORATH 385 • y ' 9 ~ -- - r 7 í • i . « i • ' B e i . . . ' * ■i • < . • r . A \ V,8 -£-r B B i Fig. 6 — Free zone electrophoresis showing electrophoretic homogeneity. Sample: 10 of Tit in 0.03 M buffer, pH 7.3, optlcal density at 279 mii 1.5. 0.1 M Tris-acetic acicl buller. Run: pH 8.7 (measured at 22»), 3 mA, 1,520 V and 1.2°. Exposures: 0.60, and 132 min. atter the start. Distance of migratlon: 17.5 cm. Arrow Indlcates the .starting zone. cm SciELO 10 11 12 13 14 15 386 SOME SEPARATION METIIODS BASED ON MOLECULAR SIZE AND CIIARGE AND THEIR APPLICATION TO PURIFICATION OF POLY- PEI>TIDES AND PROTEINS IN SNAKE VENOMS TABLE I N.n* Il.h** H.h Il.h E.a»** E.b*»*» Amino acid residues Ta 3 5 12 Lysine 6 4 6 11 4 4 Histidine 2 2 2 1 1 2 Amkie 7 10 8 4 10 10 Arginlne 3 5 4 1 3 3 Cy.stelc acid — — — — Aspartic acid 7 9 5 6 5 4 Methlonine Sulfonc — — — -- Threonine 8 7 9 3 5 5 Serine 2 3 4 3 7 7 Glutamic acid ü 5 S 1 8 8 Proline 5 T) 4 Tj 4 4 Glycinc 5 5 5 2 5 9 Alanine Ü 0 0 1 0 0 Vi Cystlne 8 8 8 8 8 o Valine 2 1 1 4 2 Methionine 0 0 0 3 0 0 Isoleucine 3 3 1 2 4 4 Leucine 2 o •1 7 1 1 Tyrosine 1 .1 1 1 1 1 Phenylalanine 0 0 0 1 2 2 Tryptophan 1 1 1 0 1 or 2 1 or 2 Total number of amino aci(i residues dl dl 01 60 61 61 * Naja nigricoltis ♦* Hemachatus haemachatnn *»• Erabutoxin a «»»» Erabutoxin b Min. mol. WGight (i787 6828 6823 6707 LD 100, iig (intrnvenousiy in mice, 18-20 g) 1.8 1.5 2 50 Acknowledgements — Dr. Paul Boquet directed my interest to lhe snake venoms. I am most thankful to him and his associates for fruitful collaboration over some years, for the supply of venom and their help in the assays of our fractions vvith respect to their neurotoxic activity. The Swedish Natural Science Research Foundation spon.sored the snake venom research and paid my travelling expenses. SciELO Mem. Inst. Butantan Simp. Internac. 33(2):379-38S, 1966 JERKER PORATH 387 Kefekenciís 1 . 2 . 3. 4. 5. 6 . 7. 8 . 9. 10 . 11 . 12 . 13. 14. 15. 16 . DELIN, S., SQUIRE, P. G., and PORATH, J., Bioclüm. Biophys. Acta, 89, 398, 1964. MAKONNEN, B., PETTERSSON, C., and PORATH, J., to be published in Biochim. Biophys. Acta. HJERTÉN, S., Fr-ee Zone Electrophoresis, Thesis, Uppsala, 1967. PORATH, J., Methods in immunology and hnmunocheniistry, Vol. I, in press. PORATH, J., Sei. Tools, 11, no. 2, 21, 1964. KOLIN, A., J. Appl. Physics, 25, 1442, 1954. HJERTÉN, S., Arkiv fôr Kemi, 13, 151, 1958. HJERTÉN, S., in Protides Biol. Fluids, Proc. 7th. Colloqu., Bruges, 1959, Else- vier, Amsterdam, 1960, p. 28. BARROLIER, J., WATZKA, E., and GIBIAN, H., Z. Naturforseh., 13b, 754, 1958. HANNIG, K., Eine Neuentwicklung der tràgerfreien Ablenkungselektrophorese iind ihre Anwendung auf zytologische Probleme, 1964. SVENSSON, H., HAGDAHL, L., and LERNER, K. D., Sei. Tools, 4, 1, 1957. PORATH, J., LINDNER, E. B., and JERSTEDT, S., Nature, 182, 144, 1958. PETERSON, E. A., and SOBER, H. A., J. Amer. Chem. Soc., 78, 751, 1956. KARLSSON, E., EAKER, D. L., and PORATH, J., Biochim. Biophys. Acta, in press. PORATH, J., and UI, N., Biochim. Biophys. Acta, 90. 324, 1964. TAMIYA, N., and ARAI, H., Biochem. J., 99, 624. 1966. 1 . 2 . 3. 4. 5. 6 . 7. 8 . 9. 10 . 11 . 12 . 13. 14. 15. 16 . cm 2 3 L. 5 6 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):389-410, 1966 TOMOJI SUZUKI 389 13. SEPARATIO-X METHODS OE AMiMAL VENOMS CONSTITUENTS TOMOJI SUZUKI Institute for Protein Research, Osaka University, Joancho, Kitaku, Osaka, Japan Al)oiit twentv vears ago, the qiiestion was raisecl in oiir laEoratory of whether zinc, which was l)ecoming the ohject of attentioii as a metal of hiological im- portance, was present in varioiis snake venoms. At that time, Dr. Delezene re- ported that many snake venoms contain a considerahle amount of zinc. On the other hand, Japanese workers had reported that zinc could not he detected in the venoms of Japanese and Formosan snakes. W’e re-examined this in thirteen kinds of venom of Japanese and Formosan snakes hy emission spectro])hotometry, and it was foimd that all the venoms examined contained considerahle amounts of zinc and calcium, a small amount of magnesium, and traces of manganese and other metais. Afterwards. when the effects of cations on the purified en- zymes were studied. it was shown that zinc ions markedly inhihit the activities of alkaline phosphatases of the venoms. such as 5’-micleotidase, phosphomono- esterase, and that calcium ion is an essential component for maintaining the tertial structures of some physiologically active proteinases and toxic proteins in the venoms. In this way, step hy step, our efforts were concentrated on separating the different enzymes in snake venoms, and the prohlems arising from studies on the pharmacological actions of snake venoms stimulated our interest to elucidate the homeostatic mechanisms which were disturhed hy the injection of snake venom enzymes. At present, we are making systematic studies on the purification of enzymes in the venoms of snakes in connection with their physiological actions. At the heginning of these studies, we applied the venoms to paper electro- phoresis(l). We found that ELAPIDAE snake venoms generally contain hasic jiroteins while CROTALIDAE snake venoms contain acidic proteins (Fig. 1). The purification of the phosphodiesterase in these venoms was our next suh- ject. It was essential to separate phosphodiesterase from 5’-nucleotidase to study the structure of nucleic acid and to elucidate the structure of new nucleotides which were isolated from various hiological materiais (2,3,4. 5), and this se- jiaration was also necessary for development of fundamental studies on the pro- duction of inosinic acid in Japan. Ratch tests were carried out to select suitahle adsorhents for chromatographic separation of snake venom phosphodiesterases, with reference to the results of [laper electroiihoresis. Results of two experiments using 2 mg. of each venom can he seen in Figure 2. The enzyme of the venom of the Formosan Cohra, Naja naja atra as we had expected, was readily adsorhed on alumina C-y-gel. calcium phosphate gel, CM-cellulose or Amberlite CC-50. On the other hand. the enzyme of the venom of the CROTALIDAE snake Agkistro- don halys blomhofjii (“Mamushi” in Japanese), was readily adsorhed on calcium [ihosphate gel, alumina C-y-gel and DEAE-cellulose (6). Therefore, ELAPIDAE cm 2 3 z 5 6 11 12 13 14 15 390 SEPARATION METÜODS OF ANIMAL VENOMS CONSTITUENTS 0 0 Naja naja atra Naja naja naja Naja hannah Bun(jarus multicinctus Vipera 7'usselUi forínosensis Trimeresurns mucrosquaynatns Trimeresiirus stejnegeri Trimeresurns flavoviridis Trimeresurns o7ciíiaa;e«sis Agkistroãoyi acutus Agkistrodon halys bloynhof- fii 0 ^ G Flg, 1 _ Paper electrophoresis ot snake venoms. Conditions: Phosphate buffer pH 6.0 fi = 0.1, Toyo Roshi No. 50. FlR. 2 — Adsorptlon of Mamushi and Cobra venom Phosphodlesterase on variou.s adsorbents. 1. CM-Cellulo.se, 2. Cellulose powder, 3. Amborlite CG50. 4. Cal- cium phosphate gel, 5. Alumina C-,gel, 6. DEAE-Cellulose (Mamushi). cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):389-410, 1966 TOMOJI SUZUKI 391 5 nake venoms wert* appüecl to Ci\I-colliilo?e. alumina C-y-gol and calciiini pliosjjliate gel. Therefore. any of these ion exchangers could lie used to olitain ])hos])ho- diesteiase free froni 5’-nucleotidase. On the other hand. “Mamushi” venom was chromatographed on DEAE-cellulose. Mlien the venom was chromatogra|)hed on DEAE-cellulose using gradient elution, the ])hos])hodiesterase was sejjarated into three entities. These partially pnrified enzymes were purified fnrther hy chroma- togra])hy on CM-cellulose. or Sephadex-gel. Thus the three kinds of phosi)ho- diesterases were isolated free from 5 -nncleotidases (7, 8, 9). Tahle I shows the pnrified enzymes which were olitained dnring our stiidies in the last twelve years. TABLE I SNAKE VENOM ENZYMES Phospholipase A Hyaluronidase 5’-Nucleotidase Endonuclease ATPase Enzymes fountl in all venoms L-Amlno acid oxldase Phosphodiesterase Phosphomonoesterase Glycerophosphatase Enzymes found especiaíly in CROTALIDAE venoms Proteinase Enzymes connected with physiologica! activities of CROTALIDAE venoms Arginlne ester hydrolase Enzymes found especiaíly in ELAPIDAE venoms Acetylchollnestera.se Peptidase NADase \\ hen the venom of Agkisírodon halys blomlwHii was applied to a DE.AE- cellnlose coltimn, jihospliolijiase .A was separated into two entities. Similarly other snake venoms contain two kinds of jiliospliolipase A. Eor example, phosjiholipase A in Cohra {Naja naja atra) venom was also sejiarated into two entities liy chromatography on CM-cellnlose (101. Moreover, the proteinase and arginine ester hydrolase of ‘Alamuslii*’ venom were hoth separated chromatographieally into three entities 111.12 ). In the cotirse of these sttidies. it was foimd that the nucleotide jiyrojihos- phatases. which had heen reported to hydrolyze the ])yro])hos])hate linkage of AAD. EAD and coenzvme A. were identical with phos[)hodiesterases (9). and so. nucleotide |)vrophos|)halase is not shown in the Tahle 1. The two activities in the “jVIamiishi” venom were always eluted in the same fraction in chromatographic procednres. So. it is thought that ATPase in this venom corresponds lo jdiospho- diesterase. N.ADase which decomposes NAD to form nicotinamide and adenosine diphosphate rihose was |)resent in the venom of Bungaras rmilticinctas and Tri- meresiirus graniineus. The NADase in the venom of Trinieresurus gramineiis was purified hy columu chromatographies ou CM-cellulose and DEAE-cellulose 113). cm SciELO 10 11 12 13 14 15 392 SEPARATION METUODS OF ANIMAL VENOMS CONSTITUENTS In this paper, 1 will rejiorl lhe procedures íor piirificalion of proteiiiases and arginine ester hydrolases, and also some hioehemical pro])erties of these ])urified enzymes. The most eharaeteristic j)hysiological sym])tonis prodnced hy lhe injeclion of lhe vcnoms of A. halys hlomhofjii are connecled vvilh proleinases and arginine eslerases. The dislriljiilion of ])roleinases and arginine eslerases in Formosan and Japanese snake venoms can he seen in Tahie II. It was found lhat proleinase aelivity is present oídy in venoms of lhe CROTALIDAE and lhal lhe venoms of ELAPIDAE do not exhihil ap])recial)le proleinase aclivily. And, il was also found lhal lhe hydrolytic aclivilies for henzoylarginine-elhylesler (HAEE) are ])resent wilh lhe proteitiase aclivily. When lhe venom was Irealed wilh EDTA, lhe pro¬ leinase aclivily was comj)lelely losl while lhe esterolylic aclivily remained un- changed. Therefore lhe enzyme responsil)le for eslerolylic aclivily musl he dislinguished from lhe jjroleinase aclivily. And conversely, on Irealmenl wilh diisopropyl flnorophosjihale (DFP), esterolylic aclivily was completely losl while proleinase aclivily remained unchanged. The venom of A. halys hlomhojjii, containing considerahie amounts of proleinases and arginine eslerases iTahIe III, was used in furlher sludies. TABLE II — PROTEINASE AND ARGININE ESTERASE ACTIVITIES IN SNAKE VENOMS VENOMS Ca.sein (PU) cas. FR, m- tyr. AzocolI BAEE /X mole/min CROTALIDAE A. halyH hlomhoffii 20.2 0.96 13.3 A. acutns 27.0 3.38 8.8 T. flavoviriílis 19.2 1.14 4.3 T. imicrosqnarnatus 26.9 1.4 128 T. okÍ7i(ive7isis 13.2 0.98 11.8 T. oramlneuH 9.0 0.14 30 ELAPIDAE N. najd atra 0.12 0,02 0 N, naja naja 0 0,02 0 N. hannah 0 0.05 0 li. inulticinctus 0.8 0 0 All the activities were converted to the value for 1 mg. venom. A lypical elulion jiallerti of proleinases of ihis venom from a DEAE-celluIose coliimn is shown in lhe Figure 3. Most of the caseinolytic aclivily was distrihuted into ihree fraclions and designated as jiroleinase a, h and c. The recovery of tolal protein from the coinmn was 98 jjer cent and approximately 85 per cent of lhe total caseinolytic aclivily was recovered in the ehiale. The contents of proleinases a, h and c in the crude venom were estimaled lo he ahout 0.5, 8.0 and 7.5 per cent, respectively. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):389-410, 1966 TOMOJI SUZUKI 393 UJ Fig. 3 — Separatlon of three Proteinases írom the Venom of A. haliis blornhoffii by Chromatography on DEAE-cellulose Column. Tube Number (10 ml/tube) Column size : 2.5X70 cm Buffer : 0.005 M ~ 0.1 M ~ 0.2 M ~ 0.5 M CH^COONa (pH 7.0) Sample applled : 3 g of lyophillzed venom Among the three fractions with proteinase activity. only the proteinase h fraction caiised hemorrhage wlien injected intracutaneousiy iii alhino raldiits (14). As the hemorrhagic activity of proteinase h could he estimated quantitatively l)y the niethod of Kondo and co-workers (151. \ve attempted to piirify proteinase li to verify the correlation of the hemorrhagic and the ])roteinase activities. After gel-filtration on a Sephadex G-25 column for desalting, the proteinase h fraction was rechromatographed on DEAE-cellulose iising gradient elution, and this chro¬ matography gave a single imiform peak of hemorrhagic and proteinase activity (Eig. 4). However, as shown in Figure 4. the curve of ahsorhancy at 280 mp did not coincide with the curve of hemorrhagic or proteinase activity. After lyo])hilization, the sample was ajiplied to a hydroxylapatite column and the hemor¬ rhagic activity was also eluted together with the caseinolytic activity (Fig. 5l. To purify the resulting proteinase h pre])aration. chromatograjihy on DEAE-Sephadex A-25 column was used (Fig. 6). I5y this procedure, some im])urities were removed. The ])otencies of proteinase h at each purification step are summarized in the Talde III. The hemorrhagic and caseinolytic activities of |)roteinase h were not separated hy these purification procedures. The increase in potency of the he¬ morrhagic activity in each ste]) was essentially in parallel with that of the caseinolytic activity of jiroteinase h. The purified proteinase h thus ohtained was ])hysicochemically homogeneous. Tahle III shows that the s])eeific activity of the purified preparation is 2.5-fold that of the proteinase h preparation ohtained from the first DEAE-cellulose column. The recovery of the proteinase h activity from the crude venom was only 30 to 35 per cent. Therefore. with reference to the chromatographic patterns in Tahle III, we attempted to simplify the purifica- lion method to increase the yield. The proteinase h fraction eluted from the first DEAE-cellulose column was applied to a Se])hadex G-lOO column (Fig. 7). By cm SciELO 10 11 12 13 14 15 Fig. 4 — Rechromatography of Proteinase b (HR-II) on DEAE-celluIose. Tube Number (6 ml/tube) O m Column size Buffer Sample appiied 1.5X25 cm 0.005 M~ 0.2 M CH,COONa (pH 7.) IIR-II fraction separateci on DEAE-cellulo.se at 280 m;ii = 127) (total absorbancy Flg. 5 Purlficatlon ot Proteinase b (IlR-II) on Hydroxylapatlte Column. Tube Number (9.3 ml/tube) Column size ; 2.5X50 cm Buffer : 0.001 M~ 0.1 M phosphate (pll 7.5) Sample appiied : IIR-II fraction eluted from DEAE-cellulose (total ab.sorbancy at 280 míi = 253) cm SciELO 10 11 12 13 14 15 -ABSORBANCY AT 280 m>J ABSORBANCY (at 280 fn;j) Mem. Inst. Butantan Simp. Internac. 33(2):3S9-410. 1966 TOMOJI SUZUKI 395 u Fig. 6 — Purification of Proteinase b on DEAE-Sephadex A-23 Column. Tube Number (3 ml/tube) Zolumn size : 1.5X20 cm Buffer : 0.005 M~0.4 M CH,COONa (pH 7.0) sample applied : HR-II purified on hydroxylapatite (total absorbancy at 280 m/i = 127) ÜJ Flg. 7 — Gel Filtration oí Proteinase b Fraction on Sephadex G-lOO. Tube Number (5.0 ml/tube) Ten mllliliter of 3.48% proteinase b fraction was applied to a column of Sephadex G-lOO (3.2X88 cm), equllibrated wlth 0.05 M AcONa (pH 7.0), and eluted with same buffer. Flow rate was 60 ml per hour at 40C. 2 3 4 SciELO cm 10 11 12 13 14 15 396 SEPARATION METHODS OF ANIMAL VENOMS CONSTITUENTS TABLE III — IIEMORRHAGIC AND ENZYME ACTIVITIES DURING PURIFICATION OF PROTEINASE b FRACTION FROM Ilemorrhagic actlvity Proteinase activity MHD* and Its íldu- Specific ciai llmlts (//g) activity (PU) . cas. FR )ig. tyr. /mg protein First DEAE-cellulose column Second DEAE-cellulose column Hydroxylapatlte column DEAE-Sephadex column 0.48 (0.42-0.57) 2.10 20.2 0.42 (0.39-0.49) 2.38 33.0 0.26 (0.22-0.30) 3.84 40.6 0.19 (0.17-0.23) 5.25 70.5 •MIID: Minlmum Hemorrhage Dose when injected In the skin oí the back of albino rabbit. I this procedure, proteinase b was eliited in a symmetrical peak. When lhe pro- leinase h fraction separated from Üie column was applied to DEAE-Sej)hadex A-50, lhe al)sorl)ancy curve at 280 m/i completely coincided wilh the proteinase activity (Eig. 8). Thus, a simple pnrification melhod was achieved increasing the yield of lhe purified proteinase 1» (Tahie IV) and lhe recovery of proteinase 1) activity from the criide venom was ahout 90 per cent. Tahie V shows the analytical data of the purified proteinase h. Calcium ion is the component of this metal-protein. Fig. 8 — Concave Gradient Chromatography of the purified Proteinase b (IIR-II) from Sephadex G-lOO on DEAE-Sephadex A-50. Tube Number (3 ml/tube) Column slze Mlxing chamber Reservolr Sample applied 1.5X29 cm 250 ml of 0.005 M 0.4 M CIIjCOONa 112.5 mg CII.COONA (pfl 7.0) cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan ôimp. Internac. 33(2):3S9-410, 1966 TOMOJI SUZUKI 397 TABLE IV _ SUMMARY OF PURIFICATION PROCEDURES OF PROTEINASE b FRACTION Protein (g.) Total units Specific activity Crude venom 38.4 DEAE — cellulose eiuate 6.238 212,700 34.1 Sephadex G-lOO eiuate 2.856 197,500 69.2 TABLE V — AMINO ACID COMPOSITION AND CARBOHYDRATE CONTENT OF PROTEINASE b (HR-II) Per cent of amino acid residue Per cent of carbohydrate residue Gly 2.58 Tyr 3.86 Galactose 6.0 Ala 2.82 Try 1.83 Ser 2.74 Cys/2 4.54 Mannose 2.0 Thr Pro 3.95 3.25 Met Asp 8.00 11.79 Fucose trace V'al Ileu 4.10 4.55 Glu Amido N 8.43 2.06 Glucosamine 6.5 Leu 5.44 Arg 4.12 Sialic acid 3.0 Phe 2.67 His 2.37 Lys 3.99 Calcium 0.32 Total A.A. residue 77.69 Total sugar residue and calcium 17.82 The hemorrhagic and caseinolytic activities of proteinase h was inhihited by EDTA (Tal)le VI), and the decrease in biological activity was parallel wilh the aniount of EDTA added. The proteinase activity decreased as the calcium ions were removed hy electrodialysis ÍEig. 9), and consequentiy a conformational change of proteinase h was ohserved hv measurement of the difference spectriini of the protein. The activity was not regained even wlien calcium ion was added to this dialyzed deionized protein. TABLE VT EFFECT OF EDTA ON PROTEINASE b (HR-II) EDTA (Final M) HEMORRHAGIC ACTIVITY Proteolytic activity Relative value (%) MHD//ig. protein Relative value (%) None 3.6 100 100 1X10-* 2.4 67 29 5 X 10-^ 0.8 22 6 1X10-3 0.5 14 2 5 X 10-3 0.1 3 0 1X 10-= 0 0 0 130 íig of proteinase b were treated with the indicated amounts of EDTA for 30 minutes at 37°C in a totai volume of 1.0 ml. cm SciELO 10 11 12 13 14 15 398 SEPARATIOM M?:TnODS OF ANIMAR VENOMS CONSTITUENTS 100 o Electrociialysis Time (hours) Protein concn: 0.44%, Voltage: 400 V, Cunent 1.9 ~ 0.9 mA Flovv rate of cielonized water was 25 ml/hr at 40C \Ve aiso altempled to purify proteiiiase c. The coolent of proleiiiase c in tlie enide venom of A. Iialys hlomhojjii was al>oiil 7.5 ])er cent. Similar ])ro- cediires lo tliose iised iii the |)iirifi(atioii of |)roleiiiase I) were ap|)lied. The residis can he seen in Tahie VII. The (ireparalion ohtained froiti a Sephadex TABLE VII PURIFICATION METIIOD OF PROTEINASE FRACTION Protein (g.) Total units * Specilic activity ** Crude venom 30.0 DEAE-cellulose eluate 6.93 163,548 23.6 Sephadex G-lOO eluate 2.346 174,308 74.3 * One unit w-a.s ciefined as the amount oI enzymc whith yields a color equivalent to 1.0 ítg oI tyroslne per minute using casein as substrate. •• Specilic activity is expres.sed as unlts per mg protein. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Slmp. Internac. 33(2):3S9-410, 1966 TOMOJI SUZUKI 399 (MOO columii was eleítro|)lioreticaIly and ultraceiitrifugally honiogeneous and so proteinase c was also purified by this simple nielhod. When 10 /ig of proteinase c were injected subcutaneously into lhe skin of a depilated albino ral)bit. a marked edema was observed. Injection of cqual amounts of l)romelain, papain, plasmin, a-chymotrypsin or bacterial proteinase obtained from Bacillus suhtilis, did not cause edema. It is not clear by vvhat mechanism proteinase c causes this edema. Only 0.5 per cent of the crude venom of A. Iialys hlomhojjü corres])onds to lhe proteinase a. Phospholipase A, L-amino acid oxidase, hyaluronidase and various olher pro- teins were found as contaminants of protease a in the eluate of lhe first DEAE- cellulose column (Fig. 3). To remove these contaminants, this fraction was sub- mitted to a purification procedure (Table VII1). The recovery of proteinase a TABLE VIII PURIFICATION PROCEDURES OF PROTEINASE a FROM THE VENOM OF A. HALYS BLOMHOFFII 1. DEAE-Cellulose Chromatography oí Crucie Venom, Proteinase a Fraction 1 2. Phospho-Ceilulose Chromatography i 3. Sephadex G-25 Gel Filtration i 4. Seconci DEAE (pH 8.5) Chromatography I 5. Thirci DEAE (pH 7.0) Chromatography 1 6. Sephadex G-lOO Ge) Filtration I Puriíied Proteinase a activity was about 70 per cent of lhat in the crude venom. The jihysiological activity of jiroteinase a is not yet known. The electrophoretic and ultracentrifugal ])atterns showed that each purified ])roteinase appears as a single protein (Fig. 10 and 111. In addition to these criteria of |)urily, the homogeneity of each ])roteinase was also given by lhe overlapping curves of absorbancy at 280 m/x and proteinase activity (Fig. 12). Moreover. these purified proteinases were honiogeneous on polyaerylamide gel electrojilioresis. The optimum jill values of each [noteinase can he seen in Figure 13. The sidistrate specificities of purified proteinases a, b and c were examined using synthetic glucagon and B chain of oxidized insulin from bovine origin (Fig. 14). These proteinases are considered as digestive enzymes of the snake, however, they differ from the usual mammalian digestive enzymes. trypsin and chymotrypsin in their hydrolylic actions. Allhough the venom jiroteinases have S])ecificities similar to ihose of bacterial proteinases, their hydrolytic sites are restricted within narrow limits. Proteinase b is the hradvkinin destroving en- zyme in the venom. and it readily hydrolyzes the glycylphenylalanyl linkage of bradykinin and of the B chain of insulin. Although proteinase c hydrolyzes the glycylphenylalanyl linkage of the B chain easily, it did not hvdrolvze the glycylphenylalanyl linkage of bradykinin. cm SciELO 10 11 12 13 14 15 400 SEPARATION METHODS OF ANIMAL VENOMS CONSTITUENTS JU^ d b c Fig. 10 — Electrophoretic Patterns of Purified Proteinase a, b and c. Electrophoresis was carricd out at condltions described belovv; proteinase a: 1.0%, pH 8.GO, = 0.1, G.14 mA, 3 . 30 C; proteinase b: 0.92%, pH 8.51, = 0.1, 6.10 mA, 4.C0C; proteinase c: 0.80%, pH 6.00, fi ~ 0.1, 6.00 mA, 6 .O 0 C. The pictures were taken after 150, 120 and 125 minutes on proteinase a, b and c, respectively. abc Flg. 11 — Ultracentrifugal patterns of purified proteinase a, b, and c. A soiution of sampie in sodium phosphate buffer (pH 7.0, /i = 0.1) was run at conditions described beiow; proteinase a: 0.751, 55,430 rpm, 25.7°C; proteinase b; 0.65%, 56,100 rpm, 16.6°C; proteinase c: 0.77%, 56,100 rpm, 16.6“C. The pictures were taken after 48, 64 and 64 minutes on proteinase a, b and c, respectively. Fig. 12 — Linear Gradient Elution of Purified Proteina.se a, b and c by DEAE- cellulo.se Chromntography. Column size Buffer Sampie appiied 1.5X24 cm a 0.001 M Tris (pH 8.5) to 0.06 M CH,COONa (pH 7.0) b 0.01 M CHjCOONa (pH 6.2) to 0.2 M CII,COONa (pH 6.2) c 0.05 M CII^COONa (pH 6.2) to 0.5 M CH^COONa (pH 6.2) a - 80.4 mg. b = 99.0 mg, c = 69.7 mg cm SciELO 10 11 12 13 14 15 Per cent activity Mem. Inst. Butantan Simp. Internac. 33(2):389-410, 1966 TOMOJI SUZUKI 401 PH Fig. 13 — Optimum pH of Venom protelnases. NHj NHj SOjH Phe-Val -Àsp-Glu -His -Leu-Cys-Gly-Ser-His - Leu-Val-Glu - Ala - Leu Mamushi a Ha bu SO,H Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-LyS'Ala Ha bu Fig. 14 — Amlno Acid Sequence of Oxidized Bovine Insulin B-chaln and the Sites of Hydrolysis by Snake Venom Proteinases. cm 2 3 4 5 6 SciELO ;lo 11 12 13 14 15 402 SEPARATION METIIODS OF ANIMAL VENOMS CONSTITUENTS Fig. 13 — Chromatography of the Venom of A. lialys blovüioffii on DEAE-cellulose. Tube Number (20 ml/tube) Column size: 5.0X33 cm. Burter, CH^COONa pH 7.0. Venom applled, 6 g. Wo also aUt'm|)te(l lo piirify llie “cloUiog eiizyiiie” and the "‘('ai)illary-|)er- meahility-inereasing eiizyme” wliicli vvere present in eonsiderahle aniounts in lhe eluate of the DEAK-cellulose eohmiii (Fig. 15). The hlood clottiiig eiizyme frac- tion free from lhe hexnorrhagie proteiiis and ollier sniall iminirities was ohlained after reehromalograjxhy on a DFAF-celIuIose eolnnin (Talile IX) followed l)y fractionation on a hydroxyla|)alile eoluinn (Fig. 16). Afler eoneenlralion and desailitig hy Se|)hadex (1-25 fillialion, lhe hlood elotling enzyine fraetion was ap|)Iied to a eohiinn of DFAF-Se|)iiadex A-25, and elnled hy eoneave gradienl elntion (Fig. 17). Ily ihis |)rocednre llie elotling enzyine was purified lo a TABLE IX — SUMMARY OF PUlUFICATION PROCEDURES USED FOR THE “CLOTTING ENZYME” FRACTION FROM Total protein A. nt 280 mu Enzyme unils * Speciflc activlty First DEAE-cellulose column 1,242 1,849 1.5 Seconti DEAE-cellulose column 106 1,541 14.5 Hydroxylapatite column 42 1,392 33.2 DEAE-Sephadex column 22.3 788 33.3 • Tosylarginlne mcthylestcr was used as substrato cm SciELO 10 11 12 13 14 15 ABSORBANCY (at 280 Tube Number (5.5 ml/tube) Column size : 1.5X20 cm Buffer : 0.005 M 0.C5 M ~ 0.1 M phosphate (pH 6.8) Sample appiled : Clotting enzyme fraction (total absorbancy at 280 mu = loS) Column slze Sample appiled Bufíer Tube Number (5 ml/tube) 1.8X30 cm 0.005 M ^ 0.4 M CHjCOONa (pH 7.0) Purified clotting enzyme (total absorbancy at 280 m/i = 42) cm SciELO 10 11 12 13 14 15 404 SEPARATION METIIODS OF ANIMAL VENOMS CONSTITUENTS physicofiiemirally lioniogeneous slate and Kig. 18 shows thf ullracenlrifiigal pattern. As menlioned hcfora, among the arginine ester liydrolases in lhe eluale froni lhe firsl DEAE-cellidose coluinn (Eig. 8), an enzynie vvas foiitid which lias a hypotensive aetion and increases tapillary permeahility. The arginine esler liydro- lytic activity of ihis enzyme amounted 80 lo 40 per eenl of llie tolal arginine esler hydrolytic aclivity of lhe venom. This physiological aclive arginine hydro- lase was jiurified liy a similar inelhod lo thal used for lhe clotling enzyme. The results are summarizeil in Tahle X. The sjiecific activity of this enzyme increased TABLE X SUMMARY OF PURIFICATION PROCEDERES USED FOR THE “CAPILLARY PERMEABILITY INCREASING ENZYME” FRACTION FROM Total protein A. at 280 m/i Enzyme unlt.s • Specific activity First DEAE-cellulose column 1,030 2,440 2.37 Second DEAE-cellulose column 314 1,916 6.1 Hydroxylapatlte column 65.8 1,363 20.7 DEAE-Sephadex column 29.0 837 28.9 • Tosylarginlne methylester was used as substrate. markedly and lhe jmrified jireparalion was homogencoiis on iillracenlrifugation (Eig. 19), and aiso on cyanogum eleclro|)horesis at varions jiH valnes. When 3 /ag of ihis purified preparalion wcre injecled inlo lhe skin of an alliino ralihit, capillary permeahilily was distinctly increased, as jiulged hy lhe Evans Illue lesl. When this preparation was incuhated with [lurified liradykininogen, no release of hradykinin was detected liy assay on gninea-jiig ilenm, and so it is not clear hy whal mechanism the permeahility of the ca|)illaries is increased. In olher experiments, we foimd lhat a consideralde amoiml of this ca[)illary permeahilily incrcasing enzyme was also jiresenl in Crolalua udumunteus venom and Trimere- siirus jlavoviridis venom. The fractions in lhe eluate from the first DEAli-celhilose cohimn (Eig 8) which contained “hradykinin releasing”, and “clotling” activities were collected, and rcchromatographed. Then the fraclion of the ehiate wilh “hradykinin re¬ leasing activity” was jmrified further on a CM-celhilose cohimn (Tdg. 20). In this way the “hradykinin releasing enzyme” was ohtained free from clotting en¬ zyme, hiit oniy 5 jier cent of the lolal nnits of arginine esler hydrolytic activity of lhe venom were recovered in this jiartially jmrified enzyme jirejiaralion, and no furlher jmrification was altemjited. Althongh, the arginine esler hydrolytic activity of ihis jmrified enzyme was imexjiectedly low, lhe aclivity in this jire- paration seems to he linked lo lhe hradykinin releasing activity (16). On heat- treatment, the hradykinin releasing aclivity decrcased jiarallcl with the arginine esler hydrolytic activity. Also, in DEP-inhihition exjierimenls, lhe hradykinin releasing aclivity decrcased jiarallel wilh lhe arginine esler hydrolytic activity, with increase in lhe concenlration of DEI’. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):389-410, 1966 TOMOJI SUZUKI 405 Fig. 18 — Ultracentrlfugal Pattern of the “Clotting Enzyme”. A solution of the purified “clotting enzyme” at a concentration of 1.0 per eent In sodlum phosphate buffer, pH 7.5, ionic strength, 0.1 ii was run at 58,100 rpm at 19.4°C and the plcture 'vas taken after 52 minutes centriíu- gatlon. Fig. 19 — Uitracentrifugai Pattern of the “Capiliary Permeabiiity In- creasing Enzyme”. A soiution of the purified “capiliary permeabiiity increasing enzyme” at a concentration of 1.0 per cent in sodium phosphate buffer, pH 7.5, ionic strength, 0.1 ii was run at 57,800 rpm at 21.2<>C, and the picture was taken alter 55 minutes centri- fugation. Column Buffer Protein applied Tube Number (5 ml/tube) 1.5X32.5 cm CHjCOONa, pH 6.0 total absorbancy at 280 m/i = 134 cm SciELO 10 11 12 13 14 15 ARGININE ESTER HYDROLYTIC ACTIVITY (unit8/ml.) 406 SEPARATION METHODS OF ANIMAL VENOMS CONSTITUENTS The effecls of synllietic siihstrale.s caii he seeii in Tal)le XI. The inliil)ition eauFcd hy henzoylarginiiie elhylesler depeiuled oii its conceiitialion, and lhe lirady- kinin releasing aeliviiy was com|)leleIy inhihited when a suffieienl amounl ol henzoylargininc elhylesler was added. Lysine elhylesler and henzoylargininaniide, which were nol hydrolyzed hy lhe hradykinin releasing enzyine of lhe venoni, did not itdiihil ils activily. TABLE XI — EFFECT OF SYNTIIETIC SUBSTRATE ON THE PURIFIED BRADYKININ RELEASING ENZYME Synthetic substrate Final concentration of Per cent of bradykinin added substrate (M) releasing activity None 100 * BAEE 2.5 X 10 -’ 100 2.5 X 10 -» 68 2.5 X 10 -' 40 2.5 X 10 -* 10 2.5 X 10 -* 0 LysEE 2.5 X 10 -» 100 BAA 2.5 X 10 -’ 100 When inhlbitor was not acUied, the amount of hradykinin was estlmated to be 0.8 lig upon guinea-pig ileum. '25 /ig of the enzyme was used. Trasylol, which is a polent itdiihilor of nrinary and ])ancreatic kallikreins, caused dislinel inhihition of lhe aeliviiy of lhe hradykinin releasing enzyme of lhe venoin. However, il inhihiled lhe arginine esler hydrolylic aeliviiy of lhe crude venorn only slighlly. We lhoughl lhal Irasylol woiild prohahly nol inhihil lhe aelivilies of lhe “elolling” and “eajiillary jiermeahilily inereasing” enzynies of lhe venorn, and ihis was nexl exaniined and foinid lo he lhe ease. Thns, lhe inhihilory aelion of Irasylol is speeifie for lhe hradykinin releasing enzyme. From lhese residis and lhe sjieeifieilies of lhe enzymes for varioiis synlhelic suhslrales, il seems lhal lhe hradykinin releasing enzyme in lhe venorn is a salivary kallikrein of lhe snake. I wonld also like lo menlion two Iri-pyroghilamyl peplides which were re- cenlly isolalcd from lhe vcnoms of CROTALIDAE (17). \V'hen lhe venorn of A. halys hlomhojjii was applied lo a DEAK-eelhilose eohimn and eluled wilh 0.00.5 M lo 0.1 M aeelale hnffer al pH 7.0 a ihird main ])eak wilh ahsorplion al 280 m/a was ehiled. This fraelion eonlained jteplides of low molecular weighl and ils ahsorhancy al 280 m/i was ahoul 10 [ler cenl of lhal of lhe linfraetionaled venorn. The llV-sjieelriím of lhe pejrlide fraelion was similar lo lhal of lry|)lophan. Tahie XII, shows lhe |)roeedure used lo purify lhe pe|)lides. Afler removing |)rolein eonlaminanls hy gel-fillralion on a eohimn of Sejihadex G-25. lhe eluled [leplide fraelion was applied lo a eohimn of DFAE- cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 3S(2):389-410, 1966 TOMOJI SUZUKI 407 TABLE XII — PURIFICATION PROCEDURES OF TRYPTOPIIAN- CONTAINING PEPTIDES FROM THE VENOM OF A. HALYS HLOMHOFFII Step Total Dry Weight 1 Grude Venom 40875 32.72 g 2 DEAE-cellulose Column 5040 3 Sephadex G-25 3090 4 DEAE-Sephadex A-25 2200 5 DEAE-cellulose Column 2010 6 DEAE-Sephade.x A-25 1696 160.00 mg Sephadex A-25, and al)sorl)e(l materiais were eluted hy 0.5 M pyridine acetate huffer at pH 5.0. Fiiial piirificalion was achieved l)y rechromatograpliies of tlie jiartially purified jieptide fraction on DEAE-cellulose and DEAE-Se|)hadex A-25. Al lhe heginniiig of this work, as only a single spot was seen on pajier or ihin layer chromatography or ])aper electrojihoresis of this pe[)tide fraction, whicli stained vvith Ehrlich’s reagent, we thouglit there was oídy one jieptide. Acid hydrolysis of the sample in conslant hoiling HCl in an evacnated sealed tnhe. yielded as])artic and glntamic acids, ammonia and traces of tryptophan. Sanger’s teclmiqiie failed to reveal a free N-terininal residne, and C-terminal analysis hv hydrazinolysis gave only tryptophan. Pyroghitamic acid was identified in jrarlial hydrolyzates prepared with N-NaOH or 0.1 N-HCl. Afler digestion with carhoxy- jreptidase A, tryptojrhan and two fragments, ])yroghitamylasparagine and pyro- glntaniylglntarnine, weie separated froni the reaclion mixture, and these dipejrtides were separated froni each other hy high voltage eleclrojrhoresis (Fig. 21). They were identified hy compari.son with synthetic saniples. 1 % o O 1 o# 9 Z 0 3 + \ __j 'Try Fig. 21 — Compari.son with CPa.se Digest of Tiyptophan-contain- ing Peptides and Synthetic Samples. High voltage electrophoresia was carried out at pH 3.5 at 3000 V per 60 cm íor 120 min 1. Synthetic Pyroglu- Asp(NHj) -l-Try 2. CPase Digest of the Sample 3. Synthetic Pyroglu- GlutNHJ -l-Try The ahove restdts snggesl thal there aie two [leptides of similar electrophorelie mohilily in lhe |)e|)lide fraction. These two com|)onents were se])arated from the peplide fraction hy high voltage electro|)horesis (Fig. 22). Using ninhvdrin and cm SciELO 10 11 12 13 14 15 408 SEPARATiON METHODS OF ANIMAL VENOMS CONSTITUENTS x r z 3 1. Synthetic PyrgIu-AspíNHJ — Try 2. Natural Tryptophan- containing peptides 3. Synthetic Pyrglu-GluíNH^) -Try Fig. 22 — Comparlson vvith Synthetic and Natural Peptides. High voltage electrophoresis vvas carrled out at pH 3.5 at 4000 V per 60 cm for 240 min. microhiological methocls, lhe molar ratio of amino ackls iii lhe acid hydrolyzate of One of these two jieplides vvas estalilished as L-Tryploplian :L-Giutaniic acid >= 1:2 and thal of lhe olher as L-Tryj)tophan :L-Glntamic acid :L-As])artic = 1:1:1. Tluis, lhe seqiiences of lhe two peptides were deduced lo he Pyro-Ghi (NH2)-Try and Fyro-As[) (NH2)-Try, respectively. Next, the distrilnilion of these pe])tides in various snake venoms was examined (Tahle XIII). The venoms of Crolaliis adanianteus, Bothrops jararaca and Tri- meresiirus jlavoviridis containeti hoth peptides, vvhile the venom of Vipera russellii contained only one of them, and in the venom of Naja naja atra neither of the two peptides was foimd. The significance of these pejitides in the salivary gland of poisonotis snakes is ohscure. It is templing to speeulate that these peptides are originated from [ireciirsors of enzymes or liiologically active peptides, which are present in especially high concentrations in CROTALIDAE and VIPERIDAE venoms, dnring activating processes. TABLE XIII — DISTRIBUTION OF TRYPTOPHAN-CONTAINING PEPTIDES IN VARIOUS SNAKE VENOMS SNAKE PyrgIu-Glu(NHj)-Try Pyrglu-AspINHjl-Try A. halys hlomhoffii (CROTALIDAE) + + A. piscivorus piscivorus (CROTALIDAE) + + T. flavoviridis (CROTALIDAE) + + C. adarncijiteus (CROTALIDAE) + “T B. jararaca (CROTALIDAE) + + V. russeUii (VIPERIDAE) — + N. naja naja (ELAPIDAE) — — lhe Work I have reporled in ihis pajier is maiidy coimected wilh lhe jiliar- macological and enzymatic aclivities of Jajianese snake venoms. I feel thal phar- macological investigations on snake venoms are eomplicaled hy the facl thal these venoms are what yoii mighl eall Solutions of toxins in saliva, and snake venom cm SciELO 10 11 12 13 14 15 Mcm. Tnst. Butantan TOMOJI SUZUKI Simp. Internac. 33(2):389-410, 1966 foiitains niany com])oneiUs with physiological activities. Therefore, I l)elit‘ve that for lhe elucidatioii of tlie jihysiological aclions of snake veiioms on a molecular l)asis, it is importaiil lo piirify each coinjionent from each siiake venom. We now liave al hand several lechniqiies for this purpose. Cliromatography on ion-exchaiigers and gel-filtration are excellent melhods for lhe piirification of jiliarinacologically active proteins. All these tnethods have their limitations, hiil fortunatelly lhe limitations are not the same for the various materiais. By com- liining appropriate cliromatographical and gel-filtration procedures and other tech- niqiies, it may he possiMe to piirify many active components. We hope that fiirther research will he planned in future to give much clearer results than ihose which wcre possihle hefore this International Symposium ou Animal Venoms. Heferences 1. SUZUKI, T., HAGIWARA, H., and TAKAGI, Y., J. Pharmacol. Soc. Jap., 74, 167, 1954. 2. SUGINO, Y., J. Amer. Chem. Soc., 7», 5Ü74. 1957. 3. OKAZAKI, R., Biocheni. Biaphys. Res. Conim., 1, 34, 1959. 4. SUZUKI, T., J. Biol. Chem., 237, 1393, 1962. 5. YOSHIKAWA, H., Bioche7n. Biophys. Res. Connn., 5, 71, 1961. 6. SUZUKI, T., and IWANAGA, S., J. Phnrmacol. Soc. Jap., 78, 354, 1958. 7. SUZUKI, T., and IWANAGA, S., J. Pharmacol. Soc. Jap., 78, 368, 1958. 8. SUZUKI, T., IWANAGA, S., and SATAKE, M., ,/. Pharmacol. Soc. Jap., 80, 857, 1960. 9. SUZUKI, T., IWANAGA, S., and SATAKE, M., J. Pharmacol. Soc. Jap., 80, 861, 1960. 10. WAKUI, K.. and KAWACHI, S., J. Phanimcol. Soc. Jap., 81, 1394, 1961. 11. SATAKE, M., MURATA, Y., and SUZUKI, T., J. Biochem., .53, 438, 1963. 12. SATO, T., IWANAGA, S., -WIZUSHIMA, Y., and SUZUKI, T., ./. Bioche^n., 57, 380, 1965. 13. SUZUKI, T., IIZUKA, K., and MURATA, Y., J. Plumiiacol. Soc. Jap., 80, 868, 1960. 14. IWANAGA, S.. OMORI, T., OSHIMA, G., and SUZUKI, T., J. Biochem., 57, 392, 1965. 15. KONDO, H., KONDO, S., IKEZAWA, H., MURATA, R., and OHSAKA, A., Jap. J. med. Sei. Biol., 13, 43, 1960. 16. IWANAGA, S., SATO, T., MIZUSHIMA, Y., and SUZUKI, T., J. Bioche^n., 58, 123, 1965. 17. KATO, H., IWANAGA, S., and SUZUKI, T., Experientia, 22, 49, 1966. cm 2 3 z 5 6 11 12 13 14 15 Mem. Inst. Butantan Internac. 33(2):411-42-4, 1966 W. B. ELLIOTT, JOAN M. AUGUSTYN, and CARL GANS 411 11. S()!\IE ACTIO.NS OF SNAKE VENOM ON iMlTOCHONDHIA W. B. ELLIOTT, JOAN M. AUGUSTYN, and CARL GANS Department of Biochemistry and Bioloçjy, State University of New York at Buffalo, Buffalo, New York, U.S.A. l\TKOI)UCTION I lie fiiPt indicalion of lipolytic aclivity of Piiake veiioni came froni the work df Eüdecke (1) wlio fhowed lhat vciioni rcacted w ith lecilliin of lilood jdasma •o iHodiico a lipiiiolyíic agont. TIk> first dcmonslration of lhe sirong inhihition df eleetion tians])ort caine from lhe work of Ghosh and Chalterjee (2) wlio showed dii inhihilion willi very diliite snake venoms aeling on jrigeotehrain eells over a 11/^ hoiir jreriod prior to testing for electron transport ea|)ahilit\'. Nygaard and Siiinner (3) showed lhat inhil)ition hy leeilhinase A nnist he oecurring al more than one site heeause itdiil)ition inereased with snhstrates eiiler- 'iig al leveis furlher away from oxygen. Edwards and Hall (4) showed thal ex])osiire of a suecinoxidase |)re])aralioti •o l\aja naja venom produced an inhihition of snceinale oxidation whieh was proportional lo lhe fally aeid released hv the venom and thal nnsalurated fatty ‘toid added as lhe sodium sall caiised a similar inhihition. However. apparently heeause of lhe inhihition produced hy CJostridiu.m ucicliii toxin (whieh conlains d phosj)holipase lhal splits phos|)horyl( holine from lecithÍTi) lhey eoiu luded lhal phospholipid was required for eleciron lrans|)orl. They showed lhat added Ivso- locilhins did noi inhihil. Aravindakshan and Hragançal,5) showed thal inlra|)eril()neal injections of í^iija naja venom led to the isolation of parliallv uneouj)led ral liver and hrain niitochondria. Peirushka, Quastel aiid Scholefield (6) showed uncoupling aclivity of hoiled i^aja naja venom was reversihie hy |)hospholii)id. Since Marsden and Heid (7) fiad re|)orted the app('arance of myoglohin in lhe urine of jjeople envení)mated l'y hnhydrina sclii.stosa, we decided lo Iry lo release lhe cylochromes from milo- 1'hondria hy use of A', schislosa venom (8). Allhough a slight release oecurred, 've hecame more inierested in lhe actions on mitochondrial swelling and energy liansformation. In a study made wilh Húngaras jasciutus venom (9), we de- nionslraled 1| thal low leveis of the venom (5-10 /ig/mg protein) juoduced "iiconpling of oxidalive phos])horylation after 1-2 minutes ex])osure and 2) that lhe reduclion of ADH*/() ratios (an index of lhe efficiency of the transformalion of the energy in reduced coenzymes inlo terminal i)hosphate hond energy of ATI’) paralleled lhe increase in venom (1-3 /tg/mg protein) used lo Ireal rat liver niilochondria. cm SciELO 10 11 12 13 14 15 412 SOME ACTIONS OF SNAKE VENOM ON MITOCHONDRIA Jf larger amoiiiils of venoin vvcrf used, lhe aíided ADP iidiihited respiralion rather lhan stimulated res|)iralion. A similar inhihitory a(-tioii of ADI’ in aged mitochoiidrial pre|)arations has hecn termed reversc acceptor control l)y Leiiitiger and Gregg(lO). The itdiil>ition of DPNH linked suhstrates was stronger lhan lhat for succinate oxidalion (9). However, reversal of eleclron transpori was possilile in the presence of reversc arce|)lor control (9). B. fasciatus venom hoiled for 20 minutes at pH 5.9 to produce a phosplio- lipase A j)reparation (11) caiised imcoupling hiit not reverse acceptor control (9). Since senim alhumin (1% final concentration) reversed the imcoiipling activity (9) and .seriim alhumin had shovvn a similar reversal of the iincou])ling activity caused hy fatty acâds present in mitochrome prej)arations (12), it was considered prohahie thal the release of fatty acids was responsihle for the venom action. In fact, the addition of neutral triglycerides to mitochondria during venom treatment greatly eidianced the inhihition of mitochondrial energy transformations, while addition of triglyceride to control mitochondria did not affect tlie energy transformations. Several workers have reported thal fatty acids cause either inhihition of respiralion (13) or uncoupling of oxidative phos])horylation (12, 14). A study of the venoms from a numher of elapids showed that Buiigarus fa.s- ciatus, Micrurus fiãvius. Naja naja and Walterinncsia aegyptia venoms all showed uncoupling and reverse acceptor control activities, while Uenisonia pallidiceps venom did not sliow either activity. None of three VIPEIIINAE tested had either activity. Of ten CHOTALINAE tested, Agkislrodon piscivorus was highly active in holh activities sludied and A. bilincatiis and Botiirops jararaca produced moderate uncoupling of mitochondria (15). At lhe jtresenl time we are attempting to isolale the factor(s) in Bungariis jasclutus venom responsihle for lhe jjroduction of lhe uncoupling and/or reverse acceptor (control activity ou incuhalion with mitochondria. Mktiiods highest ])urily foi lo ws: .Ml reagents were ohtained cornmercially and were of the availahle. B. jasciatus venom. collected in our lahoratory, was slored as unpooled, freshiy collected venom was frozcn iinmediately in an alcohol-dry ice mixture and either stored in a de.ssicator at —20"C (hereafter designated as frozen venom) or lyophilized in a Virtis Centrifugai Bio-Dryer and stored in a dessicator at —20'’C (hereafter designated as lyophilized venom). Allernatively, freshiy collected venom was stored in a dessicator at room ternperature (here¬ after designated as dessicated venom). Eresh venom (hereafter designated as fresh venom) was collected and used immediately. Commercial krail venom (Pure Toxin, liereafter designated as commercial venom) purchased as a lyo- j)hilized powder from Miami Ser])entarium was stored at —20"C and reconstituted lo a 10% solulion in 0.05 M potassium phos])hate, |)H 7.4. Horizontal starch gel electro])horcsis was carried oiit according lo the ()ro- cedure of Smithies(16) using the. discontinuous Iris-citrate huffer system of Poulik(17). Gels were stained with Amidoschwarz lOB (National Biological Slain Color Index 20470). Concenlraled staining solulion conlained 0.5 grams cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Slmp. Internac. ■ 33 ( 2 ): 411 - 424 , 1966 W. B. ELLIOTT, JOAN M. AUGUSTYN, and CARL GANS 413 Amidoschwarz in 100 ml of aii acetic acid-ethanoi-water solulion 1:4:5. Diliite staiiiing solution consisled of one part concentrated staining solulion diluled willi two parts acetic acid-ethanol-water solution 1 :4:5. In fractionation experiments, niilking was carried out at 4'’C. Elution of the components of fresh B. jasciatus vcnom after starcli gel electroplioresis was according to the method of Master and Hao (18) wilh the following modification : after electrophoresis the starch gel was cut lengthwise into two halves. One half was stained with Amidoschwarz to determine the degree of migration of each of the components. A record of each pattern was made wilh a Polariod MP-3 camera. Photographs were takcn ou Polariod Land Film Packets, Type 55 P/N (Polariod Corp., Camhridge, Mass.) using a Tiffen Photar Filler (No. 8, Yel- low 2). The second half of the starch gel was then cut into strips according to the pattern indicated in the stained half. Isolated fractions were tested for uncoup- ling and reverse acceptor control activities with rat liver homogenates pref)ared in a mannitol-sucrose-EDTA médium as descrihed hy Ziegler et al. (19). Oxygen consumption was measiired with a sealed oxygen electrode (20). Polarization of the Clark electrode and recording of the changes in oxygen tension was provided hy a polarograph (Gilson Medicai Electronics, Middleton, Wisc., Mo- ■dei K). Protease activity was determined hy the degree of clot lysis in a system consisting of 0.15 ml Bovine fihrinogen, 5 mg/ml (Armour), 0.05 ml of the venom fraction to he tested and 0.05 ml thromhin (citrate thromhin approximate- ly 1.2 units). Protein concentration was determined using Haas’ modification (21) of Lowry’s method (22). Kesui.ts and discussion In earlier work with Butigarus fasciatus venom (23), we had exj)erienced difficulty with the stahility of lyophilized venom, reconstituted with distilled water, when held at 2‘’C or when held in the frozen State at —ló^C. Loss of production of reverse acceptor control activity occurred more ra])idly than loss of uncou[)ling activity. Forrnation of a white preci()itate accompanied the loss of activity (24). Because of the instahility of the factors heing studied in whole venom, we have avoided pooling of venom, preferring to use the venom from a single snake in ■ a given experiment and/or storage ])rocedure. We have repeated each experimeni and/or storage ])rocedure a numher of times using venom from different snakes to insiire that the ty])ical results presented are not the result of individual variation. In the initial studies of the separation of B. ju.sciatius venom hy starch gel ’electro|)horesis, either frozen or lyo|)hilized venom was used to test the procedures for electro[)horcsis and staining. Since the patterns for the two storage forms differed, a systematic stiidy of the effect of storage conditions on the electro- phoretic patterns was undertaken. When cornpared to fresh venom, frozen venom consistently showed a loss of all or a major portion of the fourth hand from the cathode end atui the |)resence of a lightly staining hand ahead of the first hand from the cathode end (the latter hand is not always visihle in photographs of lhe |)atterns). Venom dessicated over silica gel ,al rooni temperalure gave a pattern in which the protein hands jtresent are shifted in relation to the patterns of fresh 'venom (Fig. 1). cm 2 3 z 5 6 11 12 13 14 15 414 SOME ACTIONS OF SNAKE VENOM ON MITOCHONDRIA The nallerii of lyo])hilize(l vciiorn ajipears to he closely n'late(l lo lhat of fresh venom exeept for lhe ahsence of tlie firsl hand migraliiig tovvards lhe ealhodc (Fig. 1). 12 3 4 IA I A 12 3 4 B I ^ I _A. 0 time 2 hours 16 hours Frozen Lyophilized Dessicated Fresh Flg, 1 —■ Varlation In starch gel electrophoresis pattorn oT Biinriaruíi fuschitun venom with different methods of storage. Eleclrophoresls was carried out at 4oC, for 9 hours, 300 volts. Starch gel was stained with concentrated Amldoschwarz lOB, prepared as described in Metiiods, and washed in 951» ethanol-.'5% acetic aciates talamancae 11’anama), Phyl- lohales pratti (Panama), Pliyllohales lagahris (Panama), Dendrohatcs minutas (Panama), Dendrohates aiiralus (Tahoga, Panama). Of these specics, oídy Den¬ drohales auralus, which is known as lhe poison arrow frog of Panama, the liny Dendrohates minutas, and some of the eolor varielies of Dendrohates pumilio had a])|)reciahle toxicity in skin cxtracts. None of these conld, however, he com])ared lo lhe toxicity of lhe extremely poisonous Kokoi’ of Colomhia. No halracholoxin could he delecled in any of these other frogs so lhat the Kokoi’ of Colomhia ap|)ears at present to he quite nnique in this respect. 'riiese studies on Panamanian frogs are lhe result of a slimnlating collahora- tion wilh (Tiarles W. Myers of lhe Gorgas Memorial Lahoralory, Panama. He has discovered thal Dendrohates pumilio, a small red and hlack frog in large SciELO 10 11 12 13 14 15 16 cm 2 3 5 6 430 THE VENOMS OF AMPHIBIANS areas of Cenlral America explodes into a great iiumher of varioiisly colored island pojjulations iti the Bocas regioii of Baiuima. Tlie coloralioii lielweeii |)opulations is extremely varied l)olh in lhe dorsal and veiitral aspects. Whal factors liave caused this diversification are at presenl iinkiiowii. Since warning coloration iii ami)hihians is often assiimed lo he associaled wilh venoinoiis secrelions, a com- [larisori of the toxicities aj various poputalions wilh hrightness of coloration. was carried oiil (J. W. Daly and C. W. Myers. In preparalion) . No correlation heiween color and toxicity was foiind alllioiigli holh faclors varied widely heiween [) 0 |)nlalions. Cerlain hrighlly colored írogs were almosl nonloxic while one darkd)lne frog, very proleclively colored, conlained large araonnls of venoin. The loxicily was measnred hy snhcnlaneous injeclion in mice and also hy visnalizalion of loxic princi[)les A and R on thindayer chromalo- ])lales. The Iwo loxic princi]des were lhen isolated hy alumina colnmn chroinalo- graj)hy and silica gel thindayer chromalography. These Iwo loxic principies were found hy high resolution mass speclromelry lo have empirical formidae of CioH.isNO^ and ChiHusNO^, respectively. The nltraviolel ahsorption speclra showed only end ahsorption. The infrared sjrectra showed no carhonyl, donhle hond or oxazolidine ring. The mass speclra provided evidence for 4 rings, a carhinol- amine, and one or Iwo hydroxyl groups. Both compound A and B formed 0-methyl elhers on Irealment wilh melhanolic hydrochloric acid. Compound A forms a 0,N-Diacetyl derivalive. The foregoing data siiggest that eompounds A and B are related in strnclnre lo the salamander alkaloids. These stndies were carried oiit on only ahout 1 mg of each comjiound and the final strnctural ehicidalion wili require additional material. A greal variety of olher amphihians are known to contain loxic suhslances iti their skin secrelions, and il apiiears ihal lheir invesligation shonld he qnile profilahle in terms of discovery of novel chemical slrnclures and com])ounds of high pharmacological activily. Uekekences 1. ERSPAMER, V., VITALI, T., ROSEGHIN, M., and CEI, J. M., Experientia, 21, 504, 1965. 2. MÃRKI, F., AXELROD, J., and WITKOP, B., Biochim. Biopinjs. Acta. .58, 367, 1962. 3. ERSPAMER, V., ROSEGHIN, M., and CEI, J. M., Biochem. Pharmacol. 13, 1083, 1964. 4. ERSPAMER, V., ANASTASI, A., BERTACCINI, G., and CEI, J. M., Experien- tia, 2», 489, 1964. 5. ERSPAMER, V., Abstrncts International Syniposiuni on Hypotensive PoJypep- tiden, October 25-29, 1965, Florence, Italy. 6. MOSHER, H. S., FUHRMAN, F. A., BUCHWALD, H. D., and FISCHER, H. G., Science, 144, 1100, 1964. 7. SCHÕPF, C., Experientia, 17, 285, 1961. 8. HABERMEHL, G., Z. Naturforsch., 20, 1129, 1965. 9. MaRKI, F., and WITKOP, B., Experientia, 19, 329, 1963. 10. DALY, J. W., WITKOP, B., BOMMER, P., and BIEMANN, K., J. Amer. Chem. Soc., 87, 124, 1965. SciELO Mem. Inst. Butantan Simp. Internac. 33(2):425-432, 1966 JOHN W. DALY and BERNARD WITKOP 431 Discussion F. E. Russell: “Dr. Daly, in the slide showing the effects of the toxin on a Bulbring nerve-muscle preparation you noted that this demonstrated the neuro- muscular blocking activity of the toxin. Did not the slide also show a reduction in the directiy ilicited contractions which would eertainly make it difficult to evaluate the neuromuscular blocking effect; and secondly, how can you be sure, in this preparation, that the principal effect is not on the nerve, rather than on the muscle?” J. Daly: “I was not clear enough in explaining that in sciatic-nerve-Sartorius muscle preparation in Bufo rruirinus, that the nerve action potential was unaffected and the muscle action potential decreased. The muscle at this point stili responded to direct stimulation thus indicating a block in neuromuscular transmission.” cm SciELO 10 11 12 13 14 15 16 KUNO MKYER Mem. Inst. Butant.an Simp. Internac. 33(2):433-440, 1966 433 16. CAHDIOTOXIC STEROIDS EKOM TOADS KUNO MEYER Pharmazeutisches Institut der Universitãt, Basel, Switzerland Knowledge of tlie poisonoiisness of llie load goes liack lo antiquity. Fhysiciaiis of lhe classic ])('riod mentioned in tlieir writings medicines prepared froin loads, and described their effecl on lhe liearl and respiralion. In lhe sevenleenlh and eighleenlli cenluries dried loads vvere lemporarily admilled as “hnfones exsiecali” in lhe official pharniaco|)oeias and were iised as diurelies againsl dro|)sy and olher diseases, even hefore digilalis was inlroduced l)y Williering. In China and Jajjan llie dried venomous secrelion of lhe Cliinese load, fonned inlo roíind, smoolh, dark hrown dises and knovvn as Ch’an Sii or Senso, is slill iised lo-day againsl various diseases. The skin glands of elli|)lical form, lhe so-called jraraloids, eonlain lhe hnlk of lhe venom and are localed in lhe rear of lhe ear. A iniich snialler ainonnl of venom is also .secreled l)y lhe small verriieose skin glands lhal cover lhe whole hack of lhe animal. In ihis secrelion tvvo groiqrs of loxic suhslanees are foimd logelher wilh olher compoiinds. I . lhe heart-aclive principies, represenling sleroid derivalives, nsnally kiiown as biijogenins (hufagins), or syslemalically as hiifadienolides, and bujotoxins (eonjugales of lhe ImfogeTiins wilh suherylarginine), which are |)rimarily res- |)onsihIe for lhe |)harmacologicaI effecl of lhe poisonous secrelion, and 2. the basic componenls of lhe hufolenine lype as well as lhe hormones adrenaline and noradrenaline. Klueidalion of lhe chemieal slruelnre of lhe hnfogenins which are lhe suh- ject of ihis i)aj)er has heen slarled hy H. Wieland and his associales iti (Jermany, hy Jensen and Chen in America, and hy several grouj)S in Japan siich as Kolake and Kuvvada, and Kondo and Olmo. Ilut oídy dnring lhe lasl two decades mi- eqnivoeal j)roof of lhe sleroid nalure of lhe hnfogenins has heen ])resenled and lhe delails of lheir slruelnre com])Ielely eincidaled. On lhe following lahie all load species are lisled whose venomons secrelion has heen snhjecled lo a closer chemieal invesligalion. The load ])oisons can he ohlained from lhe following sonrces: froin lhe dried skins of lhe animais, from sccrelions of lhe skin glaiuls which are slimnialed hy cleclrical irrilalion of lhe living animais, or hesl, from dried paraloid secrelion. I In ihis conneclion il shonld he menlioned lhal lhe poisonous suhslanees have also heen detecled and isolaled in olher paris of lhe hody, e.g. in lhe hlood, and lhe ovaries.) The eollection of lhe venomons secrelion from lhe living loads can he effecled as follows: the animais are firmly held wilh one hand and jiressed down on a hoard that is covered wilh a glass plate. The raised, ohiong gland ac- cm SciELO 10 11 12 13 14 15 16 434 CARDIOTOXIC STEROIDS FROM TOADS ('umiilations locatecl Iiehiiid lhe ear are lield near lheir hase, l)etweeii ihuml) and forefinger and sqneezed firmiy, so lhal lhe milky secrelion squirls onlo lhe uiider- side of lhe glass jílate. The glass plales cdiarged wilh lhe venom secrelion are kept in a horizontal position (secrelion on toj)) at room teinperalure wilhoiit exj)osnre to direct snnlight until lhe secrelion is dry and can easily he scraped off. This slow drying prohahly permits lhe enzyine contained in lhe raw secre- tion to split lhe hufotoxins into hnfogenins and snherylarginine. The ahove ])rocednre can l)e repeated after a rest period of 30-40 minutes and yields again a consideral)le quantity of secrelion. Afterwards lhe venom glands are complelely empty and regeneration lakes place afler a period of 4 lo 6 weeks. An excellent soiirce for ohtaining a series of hnfogenins is Ch’an Sn or Senso, menlioned al)ove. This is still ohlainahle in large quanlities from lhe pharmacies, for instance in Hong Kong and Tokyo. SPECIES SOURCE Length of animais Approx. amount of venom (drled) in mg per animal Bufo alvarius GIRARD Southern Arizona and Southern Cali¬ fórnia to México 80-165 mm 400 Bufo america7tus HOLBROOK Eastern part oí North America from the Hudson Bay southwarti 54-110 mm 16 Bufo arejiarum HENSEL (are7iarius LUTZ) Uruguay, Northern Argentina, South¬ ern Brazii 75 mm 87 Bufo asper Indonésia, Siam, Malayan Peninsuia 260 mm — Bufo blombergi MYERS & FUNKHOUSER Columbia 200 mm 1200 Bufo bufo bufo LINNAEUS = Bufo vulparis LAURENTI Europe, not including the Mediter- ranian, temperate zone of Asia 60-80 mm 13 Bufo bufo ijartjarizans = Bufo (jarrjarizans CANTOR = Bufo asiaticus China 75-114 mm 19 Bufo críicifer WIED 1821 Costline of Brazii and Argentina up to 75 mm 18 B7ifo for7nos7is BOULEN- GER Japan 125 mm 75 Bufo woodhaiisi fowleri IimKLEY New England and New York South to Geórgia and westward along the Great Lakes to Michigan. Along the gulf coast to central Texas 51-82 mm 14 Bufo granulosus SPIX subsp. fernandeze GALLARDO East-north-eastern part of the South American contlnent from Panama to the Southern part of Buenos Ai¬ res in Argentina 50-55 mm Bufo ictericus SPIX 1824 = Bufo marinus BOULENGER, 1882, part. Brazii up to 140 mm 190 SciELO Mem. Inst. Butantan Simp. Internac. 33(2):433-440, 19eB KUNO MEYER 435 SPECIES SOURCE Length of animais Approx. amount of venom (dried) in mg per animal Bufo marinus (LINNAEUS 1758) = Bufo marinus West Inclies, México, Central and South America up to 200 mm 580 (L.) SCHNEIDER Bufo mauritanicus SCHLEGEL Morocco, Algeria and Tunisia 122 mm 190 Bufo melanosiictus Southeast Asía, Indonésia 116 mm 90 Bufo paracnemis LUTZ Guiana, Eastern Brazil 130-220 mm 240 Bufo peltocephalus TSCHUDI Cuba 130 mm 120 Bufo quercícus HOLBROOK North Carolina to Florida vvest from Louisiana 19-32 mm 2 Bufo regularis REUSS África, wide spread up to 136 mm 180 Bufo fipinulosus WIEGMANN i=B. chi- Chile, Peru up to 100 mm — lensifi (TSCHUDI) Bufo vaUiceps WIEGMANN Louisiana, East and South Texas to New México and Costa Rica 53-125 mm 18 B ufo virid ifi v irid 1 s LAURENTI Europe, not including Iberian penín¬ sula, North África, Near East, east- ward to MongoIia, Tibet and Hima- laya area 80-140 mm 27 For the isolalion of tlic liiirogoniiis from lhe dried j)aratoid sccrelion (e.g. from Cli’aii Su) lhe íinely j)idverized (povvdered) inalerial is niixed wilh an e(]iial volume (or more) of sand and exlraeted iu a Soxhiel a|)[)araliis or in a jjercolalor witii cldoroform. Tlie yellowisli-hrown eoloiired extracts, containing lhe slerois and lhe hnfogenins, are snlmiilled to an inilial pnrificalion in order lo se[)arale lhese Iwo classes of com])onnds. 7'he hnfogenins so olilained are ehromalogra[)hed on alumina or silica Gel. Thus, relatively easily and in a short time, the major hnfogenins can he ohlained in a crystalline State. However, tho.se snhstances ])resent in small or miniile cpianlities may reqnire fnrlher ])nri- fiealion hy ehromalographic melhods, for example parlilion eliromalography, which may he nsed direclly for se|)aration of lhe crnde venom, instead of ahsorj)tion eliromalography on alnmina or silica Gel. Mixlures which are difficnll lo separale may he resolved hy jirejiarative jiaper eliromalography or dispersion on long colnmns of silica Gel. Wilh lhe aid of Pajier Chroniatography or Thin Layer Chromalogra])hy it is possihle in most cases to ohtain an nneqnivocal identificalion of the hnfogenins, thus enahling the chemisl to analyse ininnle amonnts of the venom. Fnrther- more, the.se melhods are most reliahle and easy for delermining the hornogeneity of the crystalline materiais. The chromalograms are dried and the migrated snhstances can he detected hy sjiraying wilh a .solnlion of ShCh, in cldoroform (20 gr/lOO ml) and heating lo ahont 80‘’-120'’ for several minutes. The dif- 436 CARDIOTOXIC STEROIDS FROM TOADS ferenl colours ihus appcariiig in day- or iillraviolel liglit lielj) furthcr lo charac- lerize tlieso suljstances. Since tlie l)iifogenins have a sirong ultraviolet al)sorpüoii at al)oiil 290-300 iim lhey can lic located direclly oii lho j)aper hy a pliolocopy with filtred ultraviolel light, or eveti })etter wilh a nionochromator. By this way 0.005 mg of a hufogeniii can hc detected. The different coloralions or lhe change of lhe colours which occur when lhe Inifogeniiis are trealed wilh strong acids are very usefui in idenlificalion. ll should he emjdiasized here lhat reliahie residis are only ohlained if pure cryslals are iised and at lhe same time aullientic sulistances are lested as well. Witli concentrated sulphuric acid or 84% sniphnrie acid it is liest lo use white spot plates, Inil colour reaclions wilh a solulion of SliCls iu chloroform (20 gr/100 ml) are jierformed on filler paper. Only minute amounls of suhstances are needed (0.05 mg). The coloured sjiots should also he examined uuder ultraviolel light. The fãehermann colour reacliou and its rnodifications have hecome ohsolele. The chemistry of the rufogem.xs In contrast lo lhe C 23 -sleroids of the digilaiis and strophanlhus tyjie, the hufogeuins are C 24 -steroids. Their sleroid nalure was first deducted from de- hydrogenalion experiments: chrysene was ohlained from hufotalin, and -melhyl- cyclojienteno-phenanthrene from cinohufagin, cinohufolalin and marinolmfagin. Direcl transformalion of a hufogenin inlo a sleroid of kuown struclure has not heen achieved iinlil 1939. 1) The Laclone groitp An inlegrant jiart of all hufogenins is lhe laclone group. Wieland and Wey- land first formulalcd ihis laclone grouji as five-memhered on lhe hasis of experi¬ ments with ozone fformation of formic acid). Later a slructure with only one douhle hond in a six-rnemhered laclone was jirojiosed. In 1936 Wieland and associales correctly inter|)reled lhe ultraviolel spectrum [X 290-300 um (log e = 3.75) I of the hufogenins as derived from an «-jiyrone structure. The same s|)eclrum is found wilh coumalic acid rnelhyl ester as well as wilh the scylla glycosides and their aglycones. The struclure of lhe laclone ring is unamhigous- ly jiroved hy the givcii .spectral datas, hy degradation ex|)erimenls and also hy synthesis. 2) 77m Stcroid ring skelefon Slereochciniatry. The stereochemislry of the ring junctures at C-5, C-8, C-9, C-10, C-13, is the same for all naturally occurring hufogenins and corresponds lo lhe ring junctures of the hile acids series, wilh lhe only exception of the C/D ring juncture, which is cis instead of trans. 3) The Degradutwn Can he effecled hy different ways. By hydrogenolysis the hufogenins can he transforrned into a hile acid. As relalively large amounls of suhstauce are needed, this method has heen giveu iip. It has heen shown lhat the hest method cm 2 3 z 5 6 11 12 13 14 15 16 438 CARDIOTOXIC STEROIDS FROM TOADS of degrailation is lhe permangaiiatc oxidation of Üu- peracelylalcd liufogeniiis. I?y this melhod llic corrcsponding clianic acids caii hc ol)laitu‘d iii a relalively high yield (iij) lo 50%). 'p-Hydroxycholanic acid COOH 5P-Hydroxy-5p-etlanic acid Fig. 3 4) Thr Oxy^eti fiinclions Tlie htifogenins diffor primarily iti lhe mindicr and posilion of llie hydroxyl groiij>s, wliicli an‘ scatlercd all ovor lhe .skelelon. Eacli hufogenin has al leasl a secondary hydroxyl groiip al C-8 (reeenlly 8-kelol)iifogenins have heen isolaled in miniile cpianlilies) and niosl hufogenins have as an inlegranl paii a leiiiary liydroxyl grou|) al C-14 (similar lo lhe eardio-aclive aglyeones of llie digilalis- slrophanlhiis group). Those hufogenins carrying an oxido ^roítp al C-14/C-15 helong lo a separale gronp. Addilional hydroxyl gronps are fonnd al C-5, C-11, C-12, C-10 (moslly in aeelylaled forni) and al C-19. A keto group was deleeled al C-12 and an rddehyde, group al C-10. R„ R,, R„ R, = II = Bufalin R„ R„ R, = H; R.. = IIO = (lamabufotalin R„ R., R, = H; R., = OCOCH., = Bufotalln R,, R,„ R, = H; R, = HO = Telocinobufagin R„ R, = 11; = HO = Hellebrigenol cm SciELO LO 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):433-440, 1966 KUNO MEYER 439 R,, Rj, Rj = H = Reslbufogenln Rj, Rj = H; R, = HO = Marinobufagln R,, Rj = H; Rj = OCOCHj = Cinobufagin R, = H: R, = OCOCH.,: R, = HO = Cinobufaginol R, = HO; R, = OCOCH^; R, = H = Cinobufotalin The bufotoxins Toad venoms coiitain hesido the l)ufogeidns a fiiillier lype of cardio-active suhslaiices, lhe so-called hiifotoxiiis. These represciil conjugales ot hufogenins witli suherylarginitie and wcrc isolalc-d and investigaled in a numlier of lahora- tories dnring lhe I930’s. With lhe exceplion of Wielainrs hufotoxin, all are rather poorly cliaracterized dne lo lhe fact lhal lhese snhslances are exlremely difficnil lo ohlain in honiogeneous iryslals. This is even lhe case when modern chromalographic melhods are applied. No doul)l llie hiifoloxins so far descrihed are niixlures, and il seems very prol)ahle lhal WielaiKhs Inifoloxin also was ini- pure. Oiir assum])lion lhal lhe snherylarginine resl (in Wieland’s Biifoloxin) is linked to lhe hytiroxy groiip at C-14, seems, as Fieser aiready suggested, very iiidikely. New investigations made in onr lahoratory tenlatively confirm lhal lhe linkage poinl is al C-3. Enzyme ,NH 2 Bufotoxin->■ Bufogenin + O COOU SUBERYEARGININE The BlOGEiNESlS OF THE TOAI) POISONS Very liltle is knowfn ahonl lhe hiosynlhelic pathway of lhe formation of lhe toad j)oisons. Tfie close relalionship in lhe slrueliire of lhe hnfadienolides and lhe hile acids indicate that these tvvo lypes of sleroids are formed froin tlie saine cm SciELO 10 11 12 13 14 15 16 440 CARDIOTOXIC STEROIDS FROM TÜADS hasic suhslancf. rscliesclic and Korte assiinied llial llic laclonn ling nf tlu’ liiifadicnolidcs is liuill iip frorn oxalaceloacclic acid. Tlu- invesligaüons so far lindcitaken wilh lahedled siibslancc-s, vvliitli coidd serve as l)uilding slones for lhe hiosvnlliesis of lhe hiifadieiiolides, have only shovvn, that radioaclive ear- l)onale, or sodiuni aeetate-l-(i''*, and sodiurn acelale-2-C'‘‘ are nol incorporated. ()n Üie olher hatid, l)y feeding fi. marinus wilh C “-eonlaitdng algae or hy pa- renteral adniinistralion of eholeslerol-d-C'** it was jiossihle lo ohtain radioaclive marinolnifagin and rnarinohufoloxin, res|)eelively. These residis show lhal eholesterol is a precursor in lhe synlhesis of Üie liufogenins and hiifoloxins. This ohservalion wonld siiggesl lhal eholeslerol or a elosely relaled eonipound is lhe major soiirce of lhe eardiotonie slerols in lhe load. Tlli: SYMHKSIS OK lUlFADIEXOiaDES While lhe synlhesis of a eardenolide, i.e. Digiloxigenin was |)repared oídy reeenlly, lhe synlhesis of a Co, sleroid of lhe hufogenin lype has lieen aehieved in 1961 l)y llerlin and eo-workers. Ihil lliis laelone possessed neilher lhe 14/3- configiiralion nor llie hydroxy groiip al C-14, whieli means lhal no “irue” Inifa- dienolide eould lie synlhesized. IIkferences 1. MICHL, H., and KAISER, E., Toxicon, I, 175, 1963. DiscnssioN E. A. Zteller: “In order lo find out lhe hydroxyl grouj) to which lhe suberyl- arginine is attached, enzymic studies wilh simple model substances and the enzymo liresent in crude venom prejiaration eould be carried out. Did anybody try this way?” K. Meyer: "The isolation of the enzyme was not yet tried.” cm SciELO, LO 11 12 13 14 15 16 JOHN J. PISANO Mem. Inst, Butantan Simp. Internar. 3;i(2):441-446, 19(16 441 47. WASP KININ JOHN ,1. PISANO Ldborcttory of Cliniatl Biochemistry, Natiomil Heart Institute, Natioiuil Institutex of Hedlth. Bethesâa, Mítrylwnd, U.S.A. ) Diiritig aii ('xaminalion for liistaiiiiiic-releasÍMy Irypsin. Ilornet venom {Vespa crabro) like thal of the wasp conlains large amoimts of histamine and serolonin Init has in addition very high leveis of acetyleholine and a single kinin wilh the charaeterislie kinin activity hiit whieh conld he clearly distingnished from wasj) kinin hy chromalographic and enzymalic tests(6). Hornel kinin, like hradykinin, was fonnd to he re.sislani lo inaclivation hy Irypsin. However, relative lo hradykinin it was only al)onl one lenlh as aclive in con- tracling lhe gninea-pig ilenm as in causing relaxation of lhe ral dnodenum or lowering rahhil arterial hlood [)ressiire. Uniike was]) or hornet venom, hee venom {Apis tnellijica) contains lillle or no serolonin or kinin (2). It does, however, produce a delayed, slow conlraction of lhe mepyramine-treated gninea-|)ig ileuni. This activity is characterized hy a rapid desensilization and may he due lo an enzyme. The inosl ihorough sludy of the pharmacologically aclive suhslances in hee venom, s[)anning 16 years, has come from lhe Ihiiversity of Wurzhnrg (7). Histamine, hyaluronidase, phos|(holi[)ase A and an active hasic fraclion were re- ported in earlier sludies. Recent work on lhe fiirlher characterization of hasic fraction has cniminated in lhe isolation of two poly|)e))tides, meliltin and apamin (8). Meliltin forms rnicells in solnlion and this ])ro])erty may conlrihiite lo an imderslanding of ils polency in lowering the snrface tension of acjueous Solutions and ils powerfnl hemolylic action (9). The amino acid sequence of mcdiltin, a 2 3 4 .5 6 7 b !) 10 major comiionenl of hee venom is (X)-(lly-lleii-(íly-Ala-Val-Leii-Lys-Val-Leu-Thr- Another minor meliltin has aiso heen characterized and differs from lhe major peplide hy an unknown amino acid at the N-lermituis hefore glycine and after 2'i 21 22 2.0 24 25 20 the idenlical secpience of lhe firsl 19 residues, Ileu-Ser-Arg-Lys-Lys-Arg-dhi (NHj)- -CÍnlNH,)., (10). Apamin is [iresenl in miich lower leveis lhan meliltin and is jiliarmacological- ly characterized hy a long-lasling excitation of lhe central nervous syslem of mice. It has lhe following amino acid composition: Ala,), Argj, Asp,, l/^Cys 4 , Glu:), HiS], LeUa, Lysj, PrO], Thr, (8). Uniike ihose of the hee, wasp and hornet ])ej)tides have jiharmacological jiroperlies like the known kinins (Tahle 1). These imjiortant actions as well as their jiotency make it of inleresl to determine lheir structures. The present reporl conlains hoth a snnimary of earlier work (11,12,18) and lhe mosl recent findings which permil a proposal for lhe slrnclnre of lhe major kinin in Pa- I i s I e. s venom. cm 2 3 z 5 6 11 12 13 14 15 16 JOHN J. PISANO Mcm. Inst. Butantan Simp. Internac. 33(2):441-44H, 1966 443 STÜDIES ON POLISTES MaTKIíIALS AM) MKTHODS Ifasjjs riirec s|)i‘cies of wasp Polistes annularis LiiiTiaeiis, P. juscatus Lepcletier, and P. exclanutns Vicreck were delivi-red alive to lhe lalioratory. These were niixed aiid wlien nol iininediately used they were slored al 3-5" for several days or uj) to Iwo weeks al room lem|)eraliire wlien fed eanned or fresh frnit. Other foods siicli as cooked, strained ineat or live silk works were rejecled. Most of lhe was])s, nj) to 16,(K)0 were immediately frozen at —10". Their alidomens were disseeled and the terminal 3 segments which eontained lhe venom glands, saes, dnels, and lanceis were slored iip lo 6 nionlhs at —10". Exiraction of venom and isolation of kinins Several exiraction jirocednres were coin|)ared in order lo find lhe most convenient procedure for ohtaining snitahle qiiantities of material. An electric shock techniqne (14) was nsed to ohtain piire venom which served as a standard. Extraction of venom ajiparatuses with glacial acetic acid as jirevioiisly descrihed (4) and homogenization of lhe disseeled ahdomen in 6% trichloroacetic acid were lhe other procedures employed. The essential ste[)s in the latler procedure are; laj hornogenizalion of lhe disseeled ahdomens in 6% trichloroacetic acid, (h) removal of trichloroacelic acid frorn the extract with ether, (c) adsorption of the kinins on a jirejiarative cohimn of CM-Sephadex and elution with ammoniiim formate, (d) fractionation on Sejihadex G-10. (e) fractionation on an analytical cohimn of CM-Sephadex hy gradient elntion with ammoninm formate, (f) repeat of stej) 5 nsing a flalter gradient. Hioassays Kinins were measiired on lhe eslriis rat nlerns siispended in de Jalon’s sohi- tion (12). Whenever scrolonin may have heen presenl, 1 íig/ml lysergic acid diethylamide was added lo the halhing fluid. Anlihislaminics were not added hecanse lhe nterus was insensitive lo the leveis of histamine enconntered. Rio- a.ssays nsing the gninea-])ig ileum and rat duodeiuim were performed in the nsual manner(15). Vahies were hased on the responses to single and douhle doses of lhe nnknown sample and slandards. Hat hlood pressiire was recorded with a Stathain Strain gange and Sandhorn Hecorder. Results Essentiallv the same results, i.e., kinin activity equiactive to 1.2-1.5 /rg hradykinin in the rat nteriis assay, were ohtained when venom was collected hy electric shock, or extracted from ajiparatuses hy glacial acetic acid, or extracted from ahdomens with trichloroacetic acid. Hecanse the latler procedure was more convenient. it was nsed lo extract kinins from 6000 wasps. After the jireparative CM-Sephadex slep. kinin activity was ohtained ecpiiactive to 6.4 mg hradykinin. Chromalography on tlu' analytieal CM-Sephadex cohimn resnlled in the isolation cm SciELO 10 11 12 13 14 15 16 444 WASP KININ of 3 ])eaks of kiiiiti activity. The sanie resiills were olilainccl wheii glacial acelic aeicl exlracls of venoni apparaluses were ehromalographed on CM-eelliilose (12). The firsl Iwo ])eaks of activity. Polistes kiiiitis 1 and 2, accoiinted for less than 10% of the total kinin activity and Polistes kinin 3 approxiniately 90%. Keeovery of kinins from the cohitnns vvas essentially (|iiantilative. In preliminary tests einploying the estnis ral uterus, duodemim and hlood pressure tests and the guinea-i)ig ilenrn, all three kinins shovved activities similar to hradykinin and kallidin iTahle I); however, they eould he distinguished from the known kinins in j)reliminary stiidies. For example when hradykinin was iised as the standard Polistes kinin 3 was found to he ahoiit 7 times more active on the guinea-pig ilenrn than rat dnodennm, 1 times more active in lower- ing ral hlood pressnre than on the ilenrn, 3 times more active on the ilenrn than on lhe nlerus and 2 limes more active on the nlerns than dnodemim. Thns relalive to hradykinin Polistes kitiin 3 was most aclive in lowering hlood |)ressure, the order of activity hcing hlood pressnre > ilenm > ntenis > dno¬ dennm. The structnre of Polistes kinin 3 is cnrrently nnder invesligalion. In- cnhation of the kiidn with chymolry|)sin deslroyed hiological activity hnt lryi>sin, [)epsin or collagena.se did not. The action of tryjjsin was particniarly inlerest- ing becanse it cleaved lhe pejitide into a fragment which was slighlly more aclive on lhe nlerus and which was readily isolated on a coinmn of CM-Se])hadex. In a typical experimenl, 1.0 mg of Polistes kinin 3 (recovered from CM-Sephadex and conlaining some sall and moislnre) was incnhated in 0.5 ml of 0.05 M ammonium hicarhonate pH 7.0 and 0.02 ml of 0.1% Irypsin for 3 hours al 37°. This digestion cansed an increase in hradykinin-like activity from 300 lo 400 jjLg when lested on lhe ral nlerus. When analyzed on a CM-Sephadex colnmn no activity was recovered where Polistes kinin 3 is normally foimd. Inslead activity equivalent to 390 pg hradykinin was fonnd as a single ])eak mnch earlier than lhe position of Polistes kinin 3. Annno acid analysis of the new aclive jicplide revealed lhat it had lhe same composition as hradykitiiti hnt, in addilion, also contained glycine. Comi)ari.son hy TLC of lhe dansyl derivative of the active lry])lic pe|)lide and synlhelic glycylhradykiiun showcd they were indistingnishahie (13). Dansyl glycine was identified in an acid hydro- lysale of the pe[)tide derivative showing lhal glycine was al lhe N-lerminns. Additional evitience for the idenlity of lhe active Iryplic fragment of Polistes kinin 3 as glycylhradykinin was the identical potencies on the rat ntenis of the nalnral and synthetic pejilides. Acid hydrolysales of lhe wasp kinin showed thal in addilion to lhe amino acids in glycylhradykinin lhere are 3 residnes of lysine and 1 each of glntamie acid, threonine, aspartic acid, lencine and arginine. Fxperiments performed in our lahoralory hy Dr. T. Nakajima have indicated lhat lhe N-lerminus is hlocked, as the jieptide did nol reacl with lencinaminopeptidase nor in lhe kàlman jiro- cedure. Furlhermore only r-dansyl-lysine was ohserved in hydrolysales of the dansylated peptide. Upon Ireatment with lry|)sin lhe following were ohserved: free lysine, lencylarginine, glycylhradykinin and an N-terrninal hlocked telrapejitide which afler acid hydrolysis was fonnd lo contain glntamie acid, threonine, as- parlic acid and lysine. Heaction of the wasp kinin with carhoxy|)ejilidase B yielded only free arginine which was also ohserved afler Ireatment with chymolrypsin. Horse nrinary kal- likrein, like tryjrsin, also s])lit glycylhradykinin from the wasp kinin hnt only cm 2 3 z 5 6 11 12 13 14 15 16 446 WASP KININ oiie addilioiial |)ej)li(]e was foimd which u])on acid hydrolysis yielded 3 residiies of lysitie and onc nach of gliilainic acid, llirconinc, asi)artic acid, leiicine and arginine. Thesc residts arc in agrecnicnl vvilli prcscnl knowlcdge of the spccificily of liorse nrinary kallikrcin which is known lo hc an endope])lidase which splils oídy arginyl liondsilG). If lhe vvasp peplide is firsl dansylalcd, in which caso all lhe f-amino groups of lysinc rcaci, lhen Irypsin and horse nrinary kallikrcin gave lhe satne jirodiicts, i.e., glycylliradykinin and a second peplide conlaining all lhe reinaining aniino acids, jiliis f-dansyl lysine. The data iip lo ihis |)oint indicate lhe following slrnclnre: ((dn, Thr, As|), Lysj) Lys-Leu-Arg-ídy-hrady- kinin. Enzymatic digeslion of P o I i s I e x kinin 3 wilh a coinhinalion of l^ro- nase, carhoxype|)lidases A and 15, Irypsin, chyrnotry|)sin (5 and prolidase yiehUal as])aragine, |)yroglutanne acid atui pyroglnlarnylthrconine. Asjtarlie or glntaniic acid was nol ohserved and ihreonine was lowered hy an ainonnl cxpecled froni the |)yroglutamylthreonine foimd. Other data ohtained wilh Iryjisin and chymo- trypsin C indieale lhal lhe 3 lysine residnes are .se(|iienlial. Ilence lhe follow¬ ing tenlative slrnclnre for /* o I i s I e s kinin 3 is: l’yr-Thr-Asn-I,ys-Lys-Lys-Len- -Arg-Gly-Arg-Pro-Pro-(51y-Phe-Ser-Pro-l’he-Arg. Idenlificalion of lhe hradykinin slrnctnre in was]) venom, logelher wilh an earlier re[)orl on its presenee in amphihian skin (17) indieale lhe widespread ocenrrenee of lhe kinin in nalnre and .snjiport accninnlating evidence for lhe significance of kinins in rnan. Certainly, lheir presenee in snch a highly s])ecial- ized finid as venom which aiso contains well-known pharmacologically active snhstances, inclnding hislamine, serolonin, acetylcholine, phospholypase A. hyaln- ronidase, snggesls lhal kinins and jiarlienlarly lhe hradykinin moleenle are nniipiely active snhslances of physiologieal imjiorlance. Kekekencks 1. JAQUES, R., and SCHACHTER, M., Brit. J. Phttrmacoh, 3, 53, 1954. 2. SCHACHTER, M., and THAIN, E. M., Brit. J. Pharmacol., 9, 352, 1954. 3. SCHACHTER, M., Anu. Rev. Phimnacol., 4, 281, 1964. 4. HOLDSTOCK, D. J., MATHIAS, A. P., and SCHACHTER, M., Brit. J. Phar- mdcol., 12, 149, 1957. 5. MATHIAS, A. P., and SCHACHTER, M., Brit. .]. Pharvuicol., 13, 326, 1958. 6. BHOOLA, K. D., CALLE, J., and SCHACHTER, M., .7. Physiol.. 1.59, 167, 1961. 7. BEARD, R. L., Ann. Rev. Entomoí... 8, 1, 1963. 8. HABERMANN, E., and REIZ, K. G., Biochem. Z.. 341, 451, 1965. 9. HABERMANN, E., and REIZ, K. G., Biochem. Z., 343, 192, 1965. 10. HABERMANN, E., and .lENTSCH, ,J., Arch. exp. Path. Piumnakol., 253, 40, 1966. 11. PRADO, J. L., TAMURA, Z., FURANO, E., PISANO, J. J., and UDENFRIEND, S., in E. G. ERDOS, N. BACK, and F. SICUTERI (Editor.s), Hypotenifive Peptulex, Springer Verlag, N.Y., 1966, p. 93. 12. PISANO, J. J., PRADO, J. L., FURANO, E., and UDENFRIEND, S., Bio¬ chem. Pharmacol., in press. 13. TAMURA, Z., PISANO, J. .1., and UDENFRIEND, S., Biochem, Pharmacol., in press. 14. 0’C0NN0R, R., ROSENBROOK, W., JR., and ERICKSON, R., Science, 139. 420, 1963. 15. PRADO, E. S., PRADO, .1. L., and BRANDI, C. M. W., Arch. int. Pharmaco- dyn.. 137, 3.58, 1962. 16. PRADO, E. S., Personal communication. 17. ANASTASI, A., ERSPAMER, V., and BERTACCINI, G., Comp. Biochem. Phy.siol.. 14, 43. 1965. cm 2 3 z 5 6 11 12 13 14 15 16 Mcm. Inst. Butantan Slmp. Internac. 33(2):447-432, 19(i6 CATIIERINE ROCHAT, HERVÉ ROCHAT, FRANCOIS MIRANDA et SERGE LISSITZKY 447 KS. PUKIFÍCATION DES NEEHOTOXINES DU SCOHPION ANDROCTONUS AUSTRALIS CATHERINE ROCHAT, HERVÉ ROCHAT, FRANÇOIS MIRANDA et SERGE LISSITZKY Luhomtoire de Biocliimie Médicale, Fuculté de Médecine, Bd. d’Alès, MarseiUe 5e, Frunce Des locherc lies aiilérieiiros (l-.S) oii moiilré qiu> les veiiiiis de deiix espèees de scorjtions Nord-Afrieaiiis coiileiiaieiil chaeuii deiix iieiiiotoxiiies dont la jtiirifi- calioii a élé réaiisée par réteiilioii réversilile stir Sephadex G-25 et ])ar ehronia- logra])liie irécliaiige (Pioiis siir Anil)erlite IIÍC-50. Les jtrotéiiies Itasitpies ohte- mies élaient honiogènes en idlraeentrifiigation, eit éleclrophorèse de zone sur gel (ramidon et eii ehromatograpliie d’éqiiililire sur Amherlite IHC-50. Un poids moléculaire de 11.000 à 18.000 avait été ohtenti |)ar idtracentrifugation. Des Iravaux idtérieiirs oiit niontré que le traitement des toxiues à des ])H éloigiiés de la neutralité eonduisait à leiir dissoeiatiou eu luoiionières. late nouvellc métliode de |)urifieation a été niise au |)oiut, jtermettaut d’ol)te- nir les toxiues moiionières avee uii reiidemeut élevé. Le uiatériel de dé|)art était le venin lirut proveiiaiit oxyinéllivlcysléiiie. Le poids niolécidaire délerniiné par éqiiilil)re de sédimentatioii selou Sved- mouoiodoaeétique qui oiit fourni 7,10 (toxiiie I) H 7,52 résidus (loxiiie II) de minations oiit été réalisées dans une ullraeenlrifugeuse Spinco E à 15.220 el 15.410 rev./iiiin (loxines 1 et 11) pendant 88,5 el 116 li à 20". Les loxines étaienl dissoiites dans Tacélate d’animoninni 0.20 ,1/ [iH 6,90 à une eoncentration de 0,897. Ees valeurs olitenues soul três proelies du jxiids luoléeulaire mininuim ealeulé (faprès la eoniposition en aeides aininés (6.822 el 7.249 pour 1 et II). Dans Taeide aeétique 0,5 N, les loxines I et II pré.sentenl des niaxinuini (ral)Sor|)tion 275 el à 276 mp res|)eetivemenl. Pour ees longueurs (Ponde les coeffieients tPextinetion moléeulaire soul 10,71X10" (loxine 1) et 18,08X10' (toxitie 11). TABLEAU II COMPOSITION EN ACIDES AMINÉS DES NEUROTOXINES D'ANDROCTONUS AUSTRALIS 1 à 2 mg (le toxine pure ont cté hydroly.sés pendant 20 et 70 h dans IICI (íN ã IIC» .selon MOORE et STEIN (7) et analysê.s par chromalographie sur colonne selon PIEZ et MOR¬ RIS (8) avec un Autoanalyzer Technicon. Chaque valeur repré.sente la moyenne de deux analyses pour chacun des temps d’hydrolyse. Les valeurs de la sérine, de la thrííonine et de la tyrosine ont été calculées par extrapolation au temps d’hydroIyse 0. Acide aminc Toxine I (rapport molalrc ♦) Toxine II (rappoiT molaire *) A. asiiartique 9,04 (9) 8,13 (8) Thréonine 2,00 (2) 3,07 (3) Scrine 5,70 (0) 2,12 (2) A. glutamlque 0,0 (01 4,13 (4) Prolinc 5,90 (0) 2,80 (3) Glyclne 0,04 ((>) 7,02 (7) Alanine 1,00 (1) 3,12 (3) Cystlne (1/2) 7,55 («) 7,92 (8) Valinc 4,29** (4) 4,08 (4) Méthlonlne 0,0 (0) 0,0 (0) Isolcucino 2,40** (3) 0,98 (1) Leurlne 4,01** (4) 1,75 (21 Tyrosine 2,S3 (3) 7,04 (7) Phcnylalaninc 1,01 (1) 0,99 (1) Lysine 5,S7 (0) 5,00 (5) Ilislidine 0,99 (1) 1,90 (2) Arginine 2,03 (2) 2,99 (3) NIIj amklé (0) (9) Tryptophane (1) (1) Total (i3 04 Poids moléeulaire minimum 0.822 7.249 En prenant phénylalanlne = 1,0. Entre parenthèses 1'entler le plus proehe. Valeur obtenue après 200 h d'hy(lroly.se. Détermination spectrophotométrlque selon BEAVEN et IIOLIDAY (6). cm SciELO 10 11 12 13 14 15 450 PURIFICATION DES NEURÜTOXINES DU SCORPION ANDROCTONUS AUSTRALIS Lcs acides amiiiés N-tcrminaux oni clé détcrminés |)ar a/iiciisi.s. Their venoms produce on inanimal.s similar symplonialoloiíies Inil lhe degree of loxieily is tiol lhe sanie (1, 2). Allempls lo fraelionale lhese venoms, ol)lained l)y eleelrieal slimulalion, led Diniz and (/Onçalves (4, 1) lo demonslrale lhal lhey are mixlures of proleins, moslly of l)asic naliire; lhe main melliod used was ])a|)er and slarefi zone eleelro- jjhoresis. The following eoraponenls were fonnd: a) toxic h) smoolh miisele slimidaling e) inereasing eaiiillary permeahilily atid d) hvíihironidase; a and h were inaelivated hy ])roleolylic enzymes. From several enzymes assayed. only d showcd measiirahie aelivily. Kecenlly Gomez and Diniz suhmilled /’. scrrulalus venorn lo a fraelionalion proeednre nsing a eomhination of |)reeipilalion and colmnn chromalography on Se])luidex (T25 and CjVT-eelhdose; lhey have eonfirmed llie prolein com|)lexity of lhe venorn and, in addilion, isolaled a higtdy |)iirified loxic eomponenl which migraled as a single eom|)onenl on eellnlose aeelale pa|)er eleelroplioresis ( Fig. 1). f Titpus serrulaliis toxin CelluIo.se acetate paper electrophoresl.s Acctate buffer pll 5.5, 0.05, 7 hours Fig. 1 — Cellulose acetate paper eiectrophore.sis ot the toxic component obtalned after chromatography of the component in a CM cellulo.se column. Acetate buffer pll 5.5 lí = 0.05. A single component was obtalned. .Mkcha.msim or .vctid.x of T i t v u s vknom As was |)oinled onl in previoiis paragraplis, lhe venomons secrelions are complex mixlures. When llie wliole venorn or exlraels of lhe venorn ap|)aralus aro used lo sludy llie venorn meehanism of aelion, diffieullies lo inlerprel resulls cm 2 3 z 5 6 11 12 13 14 15 454 CHEMICAL PROPERTIES AND BIOLOGICAL ACTIVITY OF T I T YU S VENOM arisc. In lhe case of scorpion venoms, use has l)een niade in general eilher of lelsons exlracts or of whole venom ohtained l)y electrical or manual sliinulalion. In most cases, following injeelions into whole animais, lhe effects ohserved on hlood pressure, liearl rate, respiration, glandular secrelions, etc., are used lo at- lcmj)t an underslanding of lhe mechanism of action. In spite of lhe difficulties involved several authors as Houssay (5), Magalhães (1), Carvalho (6), Ramos and Corrado(7), Freire-Maia and Ferreira (8) have reached tlie conchision that lhe T ityus venom contains neurotoxic sul)slanees. The sludy of lhe effeets of T it y u s venom on isolated organs disclosed some effects that point to lhe parasympalhetic nerves as lhe mediators of the loxic action (3,9). T ityus venom contracts the smooth musele of the guinea- pig ileum. This effect is iidiihited hy atropine and jiotentiated hy eserine; ganglion l)locking agents such as hexamethonium have no effect and rnorphine jiartially antagonizes the aclivity of the venom on this preparation. These re- sults (Fig. 2) led lo the supposition that T ityus venom contained at least HAT 0 3 6 12 atf ooif'? Flg. 2 — Gulnea-plg ileum preparation suspended In 10 ml Tyrode solutlon. 0.4 Ag of atropine sulphate left 2 minutes in contact wlth the preparation Inhlblts the action of subsequent additlons of 5 tig of toxin preparation (T) or 0.08 /íU oí acetyl- choline chloride (A); 0.04 iig of histamlne (H) is not affected. The recovery time is about the same for the toxin and acetylcholine. (Aceording to Diniz and Valerl, 1959). One component acting on jiarasympathetic jiostganglionic fil)ers(9). This effect migfit he either a direct one or mediated ihrough an autojiharmacological stili- slance jiresenl in lhe nervous endings of guinea-pig ileum plexuses. Recently, in collahoration with Torres (10), we incuhaled fragments of guinea-])ig ileum wilh T i ty u s venom and lesled the release of acetylcholine aceording lo Pa- lon(ll); in the flasks containing venom, a smooth musele conlracling suhslance, with |)harmacological and some chemical projierties similar lo acetylcholine, ac- cumulaled. We concluded that the conlracting effect of the venom on the guinea- pig ileum is indireet and mediated through acetylcliolinc. Since lhe venom does not inhihit cholineslerase (4) lhe release of acetylcholine mnst he due to an effect of lhe venom in some otlier place of lhe melaholic pathway of lliis suhslance. Many symptoms of the scor|)ion venom intoxicalion could fie interpreted hy ad- milting lhe release of acetylcholine in the nervous slrucTures. These resiilts were, however, in a|)parenl discordance with lhe findings of Ramos and Corrado(7); Freire-Maia and Ferreira (8) wlio found that in some condilions lhe venom produced hyperlension, lachycardia, hyjjerglycemia, relaxed lhe airojiinized rahhit duodenum and jirodnced olher symptoms siiggeslive of sympalhelic medialion. We decided ihus lo investigale the action of lhe venom iising the perfused guinea-jiig heari which offered lhe possihilily to analyse in lhe same slruclure effects mediated hy acetylcholine or adrenaline, wilhoul the inlerferencc of lhe eomjilex slructures of lhe whole animal. cm SciELO 0 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):453-456, 1966 455 CARLOS R. DINIZ, MARCUS V. GOMEZ, ABÍLIO ANTÔNIO, and ALEXANDRE P. CORRADO tC 'O c Cd cm 456 CHEMICAL PROPERTIES AND BIOLOGICAL ACTIVITY OF T I T Y U S VENOM We were ahle to sliovv thal 20 /xg of veiiom pcrfiised throiigh tlie heart jirodiiced first liradycardia simullaneousiy willi a reduclion of slrenghl of hearl beals. These exjieriments suggested a participation of cdiolinergic, simultaiieously or followed liy adrenergic mechaiiisnis in lhe actiou of llie venom (Fig. 3). Tliis hypothesis was confirmed liy use of driigs whicli interfere vvitli holli me- chanisms. The negative chronolrojiic and inotropic effects were hloeked hv atropine and hemicholinium anc! |)otentiated hy jirosligniine. Hexamethoiuuin had no effecl; heta sympatholitics, as Inderal, ir.hihiled lhe ])ositive effects. Previous treatment of lhe giiinea-pig with reser|)ine, a sid)Stance knovvn to deplete catecholamines, prevented the positive inotropic and chronotropic effects, allovv- ing however lhe negative effects lo sliow. The imiiortanl poinl now is to decide if these effects are prodiiced liy dif- ferent components or l)y the same suhstance. We cannot decide at lhe moment. hiil the toxin ohtained in the various steps of the piirificalion procedure, jiresenled hoth effects on the heart. Based on these resnlts, we can specniate ahoiil llie site of action of lhe venom. A liypothesis which sàlisfies lhe resnlts ohtained in onr experiments is lhat lhe venom is a postganglionic fiher nenrotoxin. If we admit with l]nrn and Rand tliat even noradrenaline is liherated hy a previous release of acetylcholine in lhe adrenergic nerves, only one toxic snhstance, that may acl always liy releasing acetylcholine from the anlonomous nervons syslem, can ex|)lain all lhe effects hitherto ohserved in lhe isolated organs. We may conclnde these remarks hy pointing ont the interesting jiossihilities offered hy the stndy of interaction of T i t y ii s toxin with the strnctures of the nervons syslem. Acknowledgement — Part of this work became possible through a grant re- ceived from the Fundação de Amparo à Pesquisa do Estado de São Paulo. One of us (C. R. D.) wish to thank the Faculdade de Medicina de Ribeirão Prêto, the opportunity to visit the University of Minas Gerais during the realization of this work. HkKEHEiNCES 1. MAGALHÃES, O., Mem. Inst. Osw. Cruz, 31, 5, 1928. 2. BÜCHERL, W., Mem. InM. BuUintan, 25, 53, 1953. 3. DINIZ. C. R., and GONÇALVES, J. M., in E. E. BUCKLEY, and N. PORGES (Editors), Venonis, Amer. Ass. Advanc. Sei., Washington, 1956, p. 131. 4. DINIZ, C. R., and GONÇALVES, J. M., Biochim. Biophys. AcUi, 41, 470, 1960. 5. HOUSSAY, B., J. Physioh Patliol. Gen., 18, 305, 1919. 6. CARVALHO, P., Arch. Inst. Benj. Bnpt., 3, 101, 1937. 7. RAMOS, A., and ÇORRADO, A. P., An. Fac. Med. Univ. São Pauto, 28, 81, 1954. 8. FREIRE-MAIA, L., and FERREIRA, M. C., Mem. Inst. Osw. Cruz, 59, 11, 1961. 9. DINIZ, C. R., and VALERI, U., Arch. Int. Pt\urmacodyn., 121, 1, 1959. 10. TORRES, J. M., and DINIZ, C. R., Ciência e Cultura, 16, 197, 1964. 11. PATON, W. D. M., Brit. J. Pharmacol., 12, 119, 1957. cm 2 3 z 5 6 11 12 13 14 15 16 FARMACOLOGIA P HARMACOLOGY Mem. Inst. Butantan Simp. Internac. 33(2):457-460, 1966 M. ROCHA E SILVA 457 50. PHAKMACOLOGY OF VENOMS — INTKODUCTOHY REMAHKS M. ROCHA E SILVA Departamento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, São Paulo, Brasil The present Iiitroduclory Hemarks on lhe Pliaimacology of Venoms should cover a large area treated in this Symposiiim, since most of lhe suhjects which are going to lie discussed here have a heariíig on lhe Pharmacology of Venoms. Especially all papers on lhe hiochemical aspeets of lhe action of venoms are of lhe litmost importanee to iinderstand their ])harmacological aclions. Olivionsly, I have to limit my talk to a fevv personal recollections al)ont onr past and present work involving animal venomo. In the heginning, 1 was mainly interested in the mechanism of anaphylaxis and relatcd jdienomena, from the [)oinl of view of a release of histamine. Con- sequenlly, 1 hecame ehiefly interested in the work hy Feldherg and Kellaway (1937-38), in Australia, ahonl lhe release of histamine hy the Australian and Indian cohra, the Denisouiu sitpcrha and the Naja naja. Both venoms, when perfused throngh the gninea-pig hmg, |)rodnced a sharp release of histamine, leading almost to exhanstion of the stock of this amine in that organ of the gninea-pig. However, cther experiments hy the Australian workers, on the cir- cnlalory effects of the venoms, as well as on lhe effects npon lhe isolated smooth mnsele of the gninea-pig ilenm, vvere eonsistent wilh lhe idea that hesides his¬ tamine, other endogenonsly released ])rinci|)les might jiarticipate in sneh enveno- mations. An im|)ortant onteome of these experiments was lhe eonclnsion that, nnder certain conditions, a slow-reacling sid)stance (SHS) was released and eould exj)lain some of the featnres of lhe shoeks hy animal venoms. These experiments showing the release of histamine and of a slow-reacting snhstance, hy snake venoms, estahlished an ohvions ])arallelism helween lhe symp- toms of envenoniation and ihose of anaphylaxis, as had heen descrihed in the düg, hy Kichet and Porlier (1902), in lhe guinea-|)ig, hy Theohald Smilh (1906) and in lhe rahhil, hy Arthns and Breton (1903-5). This analogy was the most striking having in view the way hy which ana- ])hylaxis was discovered hy Kichet and Portier. in 1902. It is well known that the Erench physiologists were stndying the venom of lhe sea anémona, of the genns P h y .s a I i a- and A c I i ti i a, using glycerinaled extracts of their tentacles. When injecled wilh a lelhal dose of these extracts the animais showed a [nctnre similar to that lhey conld have iindcr the ana])hylaclic crisis: diarrhea, abdominal cramps, fali in hlood jiressnre, coma and dealh. By section, lhe animais showed profnse hemorrhages and slagnalion of hlood in lhe portal region. 1 SciELO INTKODUCTORY REMARKS 458 PHARMACOI.OGY OP' VENOMS The new |)lieiiomenon, ohserved hy líichel and Porlier and lo vvhieh lltey "ave lhe name of Anaphylaxis, vvas lhat a similar ])ictiire eonld he ohlained vvilh a small fraclion of lhe lelhal dose, if such a small amounl of lhe exlraels was given 15 lo 20 days afler lhe animal had heen suhmilled lo a previoiis injeclion of a non-lelhal dose of lhe extraels. Therefore, lhey have coneluded lhal inslead of heeoming |)roleeled (imnumized) hy a previoiis admitiislralioa of lhe loxin, lhe animais ])resenled an increased sensilivily lo il. fo denole ihis ahsenee of ])roleclion or pliylaxis, Hichel and Forlier eoined lhe name which hecame famoiis of Anuj)liylaxis. This slory is well known lo all ihose who have worked in ana|ihylaxis, hui lhe ])oint I wish lo raisc, is lhe im[)licalion eonlained in lhal ?iame of Anaphy¬ laxis, lhal lhe |)rimary agenl should he lhe loxic malerial and lhal lhe symploms arising al lhe second injeclion were oídy exacerhalion of lhe primary loxic ef- fecls of lhe iililized malerial. Whal Hichel and Forlier tried lo im|)ly wilh lhe name of ana])hylaxis was lhe rediiclion of protcction lo lhe loxic effects of lhe glycerinaled exlracls of P h y s a I i a or Actinia lenlacles. Laler on. lhe name was widely ulilized lo indicale lhe developmenl of loxic effecls hy malerials. such as seruni, ovalhumiu and so forlh, which are prirnarily non-loxic and he- come so afler lhe repelilion of lhe Irealmenl. In 1904, Arihus discovered sero-anaphylaxis, i.e., lhe developmenl of loxic effecls in lhe rahhil hy rejrealed injeclions of horse serum, and in 1906, lhe immiinologisls wilh Theohald Smilh, descrihed in guinea-|)igs lhe ana|ihylaclic reaclion lo lhe reinjeclion of sera lo which lhe animal is normally insensilive. We know whal greal success had ihis name of Ana])hylaxis lo indicale a greal (leal of palhological |)henomena in animais and in humans, where von Firquel and Schick descrihed serum disease and allergy. liul, why should Hichel he fooled in his ex])erimenls lo lhe poinl lo ihink lhal whal he was ohserving al lhe .second injeclion was in realily lhe ahsenee aj prol.ecíion lowards lhe |)rimary loxic effecls of his malerial exlracled from lhe lenlacles of lhe Acliniae? Il is easy now lo answer such a rpieslion. In facl. lhe sym|)lomalology [)roduced hy many venorns from animal origin, re- semliles or ruimies lhe effecls lhal we know lo he lypical of Anaphylaxis. In olher words. whal he look as ati inlensifiealion of lhe primary effecls of his loxin. lhal he used lo call aelinoeongeslin. were aclually lhe similar .symploms j)roduced hy Anaphylaxis, which oceur as a conscípience of an endogenous in- loxicalion. hy release of aclive suhslances, among which hislamine is cerlaiidy one of lhe mosi imporlanl. al leasl in dogs and guinea-|)i"s. Hui, of course. lhe aelinoeongeslin when acling as a loxic malerial releases hislamine and/or olher medialors direcllv from lheir endogenous slores, ihough when aclÍTig in lhe sensilized animal lo produce anaphylaxis. lhe symplomalology develo])s ihroiigh an enlirelv differeni mechatiism, namely hy comhinalion wilh anlihodies formed afler lhe firsl itijeclion. Il is ihis profound analogy helween envenomalion hv animal poisons and lhe symplomalology of Ana[)hylaxis lhal was so nicely explored hv Arlhus in his hook “De TAnaplivlaxie à rimmunilé”. ])uhlished in 1921. in Faris. Hui, in lhe meanlime. lhe imporlanl conlrihulion hy Sir Henry Dale. in Kngland. sludying lhe effecls of hislamine and poslulaling ils parlici|)alion in lhe mechanism of Anaj)hylaxis, should he considered. Iti 1919, Dale pro|)osed lhe name of Aniopliarniaeology lo denole ihis class of |)henomena ilevelo|)ing in lhe aniiTial hody hy lhe release of endogenous aclive malerials, especially hislamine. cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan M. ROCHA E SILVA 459 Simp. Internac. 83(2):457-460, 1966 Aiifl then, lhe excelleiil work which followeck hy Feldherg atui Kelhnvay (1937-38) atui others to shovv lhe importaiice of lhe release of histamiiie atui slow-reacting siihstances (SRS) iii lhe mechatiism of productioii of shocks hy suake veiionis. Anolher kiiid of shock in wliieh histamiiie ajtpears lo paitici|)ale lo a very imporlaiit exletil is lhe shock jtrodiiced hy A s c a r i s, iii guinea-pigs and dogs. 4’liis kiiid of shock was stiidied hy myself, with Grafia, Porto atui Andrade (in 1915-16), froni lhe point of view of a release of histaniine. It is enoiigh lo take a portion of a single worni, lo niacerate it in saline and inject inio a 10 kg dog lo prodnce an extreniely severe shock, reseinhiing in all details anaphylaxis in ihis sjiecies. For lhal reason vve have called this shock an Anaphylaxis-Uke reaction, instcad of nsing lhe coniinon expression of Anaphylactoid. Ry lhal time. I thonght lhal lhe phenonienon conld he explained as Irur anajthvlaxis. assnmitig lhal lhe dogs were [trohahly sensitized to lhe Ascuris material. In agreement with this point of view, Heraltlo and his colleagnes, in 1961, have presented evidences thal lhe effecl of A s c a r i s in the gninea-pig a]i]jears lo depend npon a real State of sensitizalion, prohahiy as a conseqnence of infesla- lion hy NEMATODA parasites, which are common in snch animais. I cannol dwell any longer npon this interesling asjtect of the jthenomenon. that once more estahiished so dose connections helween Anaphylaxis and the envenomation hy animal venoms. In lhe ]ueceding discussion we have mentioned mainly histamine as the ])rinciple released hy snake venoms and Ascuris extracts. It was. indeed. with this idea in mind lhal, in 1918, we slarled doing ex- perimenls to decide whelher the venom of liollirops jararaca prodnces its sym|)- lomatülogy in dogs, hy releasing histamine from lhe liver. \X’e were doing. with Rerahio. liver perfnsions and have assayed lhe venom, hronght lo ns hy Hosen- fehl. to .^ee wlu-ther it releases histamine from dog’s liver. Since we had show n, in onr previons experiments with Ascuris extracts, that the hlood was im- jtorlanl for lhe relea.se of histamine, we perfnseti lhe liver with defihrinated hlood to which lhe venom was added al lhe momeni of lhe |)erfnsion. The ontpnt was nol histamine, hnl hradykinin and it hecame clear that the venom released this material from lhe glohnlin fraction and that hesides lhe venom, aiso Irvpsin wonld release hradykinin. Hui. whal was mosl imporlant, lhe liver itself had nothing lo do with lhe release of lhe new snhstance, and if we added lhe venom direclly lo the defihrinated hlood or to the ])seudo-glohulin prepared from it. lhe same activily or even more, was released and conld he demonstraled npon lhe isolaled gninea-pig ileuni, made insensitive to lhe venom hy repealed |)revious addilions of the same. Hnl here, again, we had a sirong analogy hetween lhe action of lryi)sin. which was fonnd lo [)roduce very sirong anaphylactoid symptoms when given to dogs and rahhils, and lhe envenomation hy snake venoms. Those who are presenl to this section of Pharmacology know very well, and some hetter lhan I, lhe conditions in which hradykinin is released hy venoms and toxins. I wili nse lhe few minnles stili Icft lo discnss some of lhe ])harmacological actions of hradykinin: as a vaso-dilalor ageni not only npon lhe systemic hnt aIso npon lhe coronary circnlation, as a powerfnl slimulating sid)stance npon the smooth mnscle of the intestinal Iract in certain animal s|)ecies and lhe isolaled nlerns of many sjiecies lesled. as lhe mosl jiowfrfnl agenl lo jirodnce increased cajiil- cm 2 3 z 5 6 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33 ( 2 ): 461 - 466 , 1966 E. KAISER anci W. RAAB 461 51. LIBERATION OF PHARMACOLOGICALLY ACTIVE SUBSTANCES FROM MAST CELLS BY ANIMAL VENOMS E. KAISER and W. RAAB Department of Medicai Chemistry, University of Vienna, Vienna, Áustria In many inslances, reactions following contacl or ])arenteral adminislration of animal venoms are indislinguishahle from anaphylactoid reactions. For the first lime lhe analogy helween antigen adminislration in a sensitized animal and lhe action of a snake venom (Crotalus adamanleus) was clearly demonstrated hy Arthiis (1). It is now generally accepted that diiring anaphylaxis several Itiologically active suhstances are liherated and activated from precursors (histamine, 5-hydro- xytryptamine, hejjarine, slow reacting suhstances, kinins) (2,8,4). On the hasis of the similarity helween the effects of snake venoms and histamine (5, 6, 7), lhe liheration of histamine from perfused lissues hy animal venoms was investigated hy Feldherg and Kellaway (8). The release of histamine was first attrihuted to the jihospholipase activity of the venom and to the lyso- ])hosphatides formed (9). Rocha e Silva and coworkers (10, 11) demonstrated lhe liheration of histamine from isolated organs following perfnsion with ])roteo- lylic enzymes and snake venoms. Resides histamine, slow reacting suhstance a|)pears in lhe perfnsion finid of isolated Inng n?Kler the infhience of snake and Itee venoms ( 12). Sdiydroxylryptamine is liherated hy animal venoms from platelets (18). Bradykinin can he formed from lhe glohnlin fraction of normal plasma (hradykininogen) nnder lhe infhience of snake venoms or trypsin (14, 15). In anajihylactic and anaphylactoid reactions (administralion of animal ve¬ noms) identical mediators are formed and liherated (16). In hoth types of reactions, tissne mast cells play an important role. Certain chemical snhslances as well as enzymes prodnce anaphylactoid reactions hy degrannlating mast cells and hy liheraling hiologically active snhslances; concomitanlly kinins are activated from precursors. In Tahle I, a snmmary is given of direcl and indirect mediators of ana- phylaclic shock, which are found in animal venoms. Histamine has heen identified in the venoms of HYMENOPTEBA (17. 18), DIPTEIÍA ( 19 ), LEPIDOPTEUA (20), HEMIPTEUA (21 ) , S c o / O p e ;i á r o. (22) and OCTOPODA (28). S-Hydroxytryptumine is present in lhe venoms of ACTINAKIA (24, 25), GASTIIOPODA (26), OEPHALOPODA (27,28) HVMENOPTERA (29),AIIACH- NIDA (30,81) and in skin secretions of A5IPHIBIA (32,88,84). 1 SciELO 462 LIBERATION OF PIIARMACOLOGICALLY ACTIVE SUBSTANCES FROM MAST CELLS BY ANIMAL VENOMS TABLE I — ANAPHYLACTOID SUBSTANCES IN ANIMAL VENOMS DIRECT EFFECTORS: Illstamine 5-hy(lroxytryptamine (5-HT, serotonin) klnins INDIRECT EFFECTORS: (Ilistamlne, 5-IIT, kinins) Enzymes (proteases, phospholipase, esterases, hyaluronkiase) Kinin-proiiucing fraction Anaphylatoxin-producing fraction Iligh molecular mast cell dcpletors of unknovvn structure I,ovv molecular mast cell clepletors of knovvn structure Surface active agents A characteristic kinin was foiiiid iii wasp vt-noni (26,35,36,37). In lhe venom of Bolhrops jararaca a l)ra(lykiniii potcntiatiiig factor lias heen foiind (38). Il sliould he mentioiied lhal liistaniine (39, dO), 5-hydroxytryplaniine (41. 39, 10) and kinins (42) exert a mast-cell depleling effect; for this reason. these siihslances are eonsidered lo lie indireet effectors of anaphylaetoid shoek, too. Several animal venorns eonlain mast cell depleling (13) enzymes (44) (proleases, phospholipase, esterases. hyahironidase). The kinin-prodiicing fraclions are mainiy related to lhe enzymes of'lhe veiionis. An anaphylaloxin-proãucing fraction was found in Cohra venom (45); il is knovvn lhal anaphylatoxin degrannlales mast etdls (46,47). Iligh inoiccalar mast cell dcpletors have heen exlracled from lhe jeily fish 148, 19. 50, 51), from lhe eelworm of swine (52) and from lhe skin seerelions of A >l 1* 111 B IA (53,31, 54, 55, 56). Spcrininc whieh is knovvn to liherale histamine (57) is fonnd in spider venom (.58). Ilolotiiarin, a sa|)onin4ike snhslanee, was isolaled from Uolothuria vagabunda (59). It has heen shown lhat surface active agenls are potent mast eell deplelors (60). In Kig. 1, a simplified siimmary is given on llie tneelianisni wliicli |)rovokes anaphviaetoid shoek, with s|)eeial referenee to lhe aetion of animal venorns. Morphologieally, masl eell degramilalion hy animal venorns ean ea.sily he ohserved. liiopsies taken at various iniervals afler injeelions of animal venorns (Agkistrodon piscivorns, skin secrelion of Honibina variegata) demonslrate lhe differenl stages of mast eell degraniilalion. 3 hoiirs afler injeelion, praelicallv all mast eells are found (l(>granulale(l. The severe vascular ehanges at sites of injeelions seem to result from liherated masl eell suhstauees as vvell as from direct loxie effeets of lhe vmioms (55. 61 ). Mast eell degranulation hy animal venorns ean also he demonstraled in isolated eells hy mieroseopie exarninalion or hy hioehemieal analysis of lhe siis|)etision fluid ( 50 ). cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):461-466, 1966 E. KAISER and W. RAAB 463 Fig. 1 — Anaphylactoid shock provoked by animal venoms. Fig. 2 — Inhibitory effect of antihistamine-, anliserotonin- and antikinin- substances on mortality íollovving snake venom. Aiia[)hylactoi(l reactioiis caii In- [ireveiitod by admiiiistralion of aiilaniiiie sulistances: antihislamiiies, aiitiserotoiiins and antikiiiins. Aiitiliistainines did iiot exerl any sigiiificaiil influence oii loxicity of siiake venoms (62, 6ií), experi- cm SciELO 10 11 12 13 14 15 4fi4 LIBERATION OF PHARMACOLOGICALLY ACTIVE SUBSTANCES FROM MAST CELLS BY ANIMAL VENOMS ments with aiitiserolonins and antikinins have iiol yel l)cen rcporled. In a series of preliminary experimenls, we invesligated lhe prolective action oí dif- ferent antamines on lhe loxicity of the venom of Agkistrodon piscivorus in mice. Pretreatment with antihistamines or antiserotonins did not show any signifieant influence on survival time following injection of a lethal dose of Agkistrodon piscivorus venom (20 mg/kg However after administralion of a comhined antihistamine-antiserotonin suhstance (cyprohej)tadine), a longer survival time was foimd. In several animais rapid death due to anaphylactoid shock was prevented. Optimal resnits were ohtained with a eomhined antihistamine-anti- serotonin-antikinin suhstance (WA-335 Dr. Karl Thomae, Germany). A pre¬ treatment with this suhstance (15 minutes hefore injection of the venom) did not only ehange survival lime, hui the lethal dose of the venom was tolerated liy a numher of mice. The high antikinin aclivity might exert a direct antitoxic effect hy neulralizing toxic fractions of lhe venom (Fig. 2). The prevention of shock l)y hroad spectrum anli-anaphylactoid suhstances might he of therapeutic value in man. References 1. ARTHUS, M., De Vanaphylaxie à Vimmunité, Masson, Paris, 1921. 2. MONGAR, J. L., and SCHILD, H. O., Physiol. Rev., 42, 226, 1962. 3. RAAB, W., and KAISER, E., Klin. Wschr., 43, 345, 1965. 4. UNGAR, G., and HAYASHI, H., Ann. Allergy, 16, 542, 1958. 5. ESSEX, H. E., and MARKOWITZ, J., Amer. J. Physiol., 92, 698, 705, 1930. 6. KELLAWAY, C. H., Brit. J. exp. Pathol., 10, 281, 1929. 7. KELLAWAY, C. H., and LE MESSURIER, D. H., Aiísí. J. exp. Biol. med. Sei., 14, 57, 1936. 8. FELDBERG, W., and KELLAWAY, C. H., J. Physiol. (Lond.), 90, 257, 1937. 9. FELDBERG, W., and KELLAWAY, C. H., J. Physiol. (Lond.), 94, 187, 1938. 10. ROCHA E SILVA, M., C. R. Soc. Biol., 130, 186, 1939. 11. ROCHA E SILVA, M., and ANDRADE, S., J. biol. Chem., 149, 9, 1943. 12. FELDBERG, W., HOLDEN, H., and KELLAWAY, C., J. Physiol. (Lond.), 94, 232, 1938. 13. MARKWARDT, F., BARTHEL, W., GLUSA, E., and HOFFMANN, A., Arch. exp. Pathol. Pharmacol., 252, 297, 1966. 14. HAMBERG, U., and ROCHA E SILVA, M., Arch. intern. Pharmacodyn., 110, 222, 1957. 15. ROCHA E SILVA, M., BERALDO, W. T., and ROSENFELD, G., Amer. J. Physiol., 156, 261, 1949. 16. KAISER, E., and RAAB, W., Z. angew. Zool., 52, 1, 1965. 17. HABERMANN, E., in Recent Advances in the Pharmacology of Toxins, Per- gamon Press, Oxford, 1965. 18. SLOTTA, K., and BORCHERT, P., Mem. Inst. Butantan, 26, 279, 1954. 19. ECKERT, D., PAASONEN, N., and VARITIANEN, A., Acta Pharmacol., 7, 16, 1951. 20. VALLE, J. R., PICARELLI, Z. P., and PRADO, J. L., Arch. intern. Pharmaco¬ dyn., 98, 324, 1954. cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Slmp. Internac. 33(2):461-466, 1966 E. KAISER and W. RAAB 465 21. YAMASAKI, M., J. Orient. MecL, 34, 401, 1941; Chem. Abstr., 35, 8124, 1941. 22. KONISI, M., Okayama Igakkai Zasshi, 48, 1309, 1936; Ber. ges. Physiol., 95, 528, 1936. 23. BETAZZI, P., Arch. int. Physiol., 18, 313, 1921. 24. MATHIAS, A. P., ROSS, D. M., and SCHACHTER, M., J. Physiol. (London.) 151, 269, 1960. 25, WELSH, J. H., and MOORHEAD, M., J. Neurochem., 6, 146, 1960. 26. BHOOLA, K. D., CALLE, J. D., and SCHACHTER, M., J. Physiol., 159, 167, 1961. 27. ERSPAMER, V., Arzneimittel-Forsch., 2, 253, 1952. 28. VIALLI, M., and ERSPAMER, V., Mikrochemie, 24, 253, 1938. 29, JAQUES, R., and SCHACHTER, M., Brit. J. Pharmacol., 9, 53, 1954. 30. ADAM, K. R., and WEISS, C., Nature, 178, 421, 1956. 31. ADAM, K. R., and WEISS, C., Nature, 183, 1398, 1959. 32. ERSPAMER, V., and ASERE, B., J. biol. Chem., 200, 311, 1953. 33. ERSPAMER, V., and VIALLI, M., Ricerca scient., 22, 1420, 1952. 34. KISS, G., and MICHL, H., Toxicou, 1, 33, 1962. 35. HERXHEIMER, A., and SCHACHTER, M., NaUire, 183, 1510, 1959. 36. HOLDENSTECK, D. J., MATHIAS, A. P., and SCHACHTER, M., Brit. J. Pharmacol., 12, 149, 1957. 37. SCHACHTER, M., and THAIN, E. M., Brit. J. Pharmacol., 9, 352, 1954. 38. FERREIRA, S. H., Brit. J. Pharmacol., 24, 163, 1965. 39. KALZ, F., and FEKETE, Z., J. invest. Derm., 35, 135, 1960. 40. KALZ, F., and FEKETE, Z., J. invest. Derm., 36, 37, 1961. 41. FELDBERG, W., and SMITH, A. N., Brit. J. Pharmacol., 8, 406, 1953. 42. STERN, P., NIKULIN, A., and FERLUGA, J., Arch. intern. Pharmacodyn., 141, 528, 1962. 43. SELYE, H., The Mast Cells, Butterworth, London, 1965. 44. KAISER, E., and MICHL, H., F. Deuticke, Wien, 1958. 45. VOGT, W., and SCHMIDT, G., Experientia, 20, 207, 1964. 46. MOTA, I., Immunology, 2, 403, 1959. 47. ROCHA E SILVA. M., and ARONSON, M., Brit. J. exp. Path., 33, 577, 1952. 48. HõGBERG, B., SUDOW, G., THON, I. L., and UVNÃS, B., Acta physiol, scand., 38, 265, 1957. 49. HÕGBERG, B., THUFVESSON, G„ and UVNÂS, B., Acta physiol. scand., 38, 135, 1957. 2 3 4 5 6 SCÍELO;LO 11 12 13 14 15 r.IBERATrON OF PIIARMACOLOGICALLY ACTIVE SUBSTANCES FROM MAST CELLS BY ANIMAL VENOMS 50. UVNAS, B., Ann. N.Y. Acad. Sei., SO. 751, 1960. 51. UVNÂS, B., J. med. pluirrn. Chem., 4, 511, 1961. 52. DIAMANT, B., HOGBERG, B., THON, I. L., and UVNÃS, B., Acta pliysiol. scand., 42, Suppl. 145, 1957. 53. KAISER, E., and KRAMAR, R., Toxicon, in press. 54. MICHL, H., and BACHMAYER, H., Mschr. Chemie (Wien), 94, 814, 1963. 55. RAAB, W., and KAISER, E., Arcii. klhi. exp. Derm., 220, 374, 1964. 56. BACHMAYER, H., MICHL, H., and ROSE, B., Toxicon, in press, 1966. 57. KUTTNER, K., HUBER, G., and JAQUES, R., Experientki, 18, 130, 1962. 58. FISCHER, F. G., and BOHN, H., Ann. Chemie, 603, 232, 1957. 59. YAMANOUCHI, T., Publ. Sete Marine Biol. Lab., 4, 183, 1955; Chem. Abstr., .50, 3660, 1956. 60. GROSSBERG, A. L., and GARCIA-AROCHA, M., Science, 120, 762, 1954. 61. RAAB, W., and KAISER, E., Toxicon, 3, 19, 1965. 62. PARRISH, H. M., SCATTERDAY, J. E., and MOORE, W., J. Am. vet. med. Ass., 129, 522, 1956. 63. SCHõTTLER, W. H. A., Amer. J. trop. Med., 3, 1083, 1954. Discüssion E. A. Zeller: “If serotonin, histamine, and other biogenic amines play an es- sential role in the action of certain venoms, then it should be possible to change the response to venom by changing the metabolism of those biogenic amines. Since amine oxidases, e.g. diamine oxidases (histaminase) and monoamine oxidases can be blocked in vivo, I wonder whether anybody has pretreated laboratory animais with inhibitors of the enzym.es before the venoms were administered?” E. Kaiser: “As far as I know substances blocking monoamine oxidase have not been used until now. Preliminary experiments in our laboratory have shown that pretreatment with compound 48/80 (synthetic mast cell depletor) significantly diminishes anaphylactoid reactions following snake venoms.” II. Edery: “Would you please tell us more details about this anti-kinin substance you mentioned. It is specific, it acts in other organ system reactive to kinins?” E. Kaiser: "I am sorry to say that only very limited Information is available on this substance (WA-335, Dr. Thomae, Western Germany). At the present mo- ment I can only tell you that the substance has a high antihistamine, antisero- tonin and antikinin activity. I am not informed about pharmacological tests i)er- formed by Dr. Thomae.” E. R. Trethewie: "Do you think adenosine and related enzymes released are significant in that cardiac effects? One can select a venom that is simpler in effect, e.g., Pseudechis porphyrincus (Australian Black Snake), antihistamine with lieparine does reduce its mortality. Polyvinylpyrrolidone will i)rolong life with lhe hemotoxic Tiger Snake venom.” E. Kaiser: “Our experiments were restricted to the venom of A. jji.scivorus until now. We have no personal experience on the cardioto.xic effects of the venom.” cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):467-476, 196(5 ADOLFO MAX ROTHSCIllLD 467 52. MECI1AN4SM OF HISTAMINE HELEASE HY ANIMAL VENOMS ADOLFO MAX ROTHSCHILD Depdrtumento de Farmacologia, Faculdade de Medicina de Ribeirão Preto, São Paulo, Brasil Tlif aldlity lo rploase histaniine from lissues is a pliainiacodynaniic projjerty of many animal venoms. Allliougli not gtmerally considered as a major cause o[ lhe lethal actions of siich venoms, lhe hypolensioii and increased peripheral vascular j)ermeahilily which are j)roduced hy hislamine, can eidiance lhe loxic aelioiis of olher venom componenls. as vvell as of endogenous faclors released hy lhe venom in lhe hillen suhjeel. In ihis presetilalion I shall limit myself lo a discussiou of lhe hiochemical aspecls of lhe release of hislamine indueed hy Iwo venoms; lhat of lhe Hrazilian ralllesnake Crotalu.s diiri.s.sii.s terriliciís and lhal of lhe honey hee Api.'! nieilijcra. The resulls lo he presenled are moslly ihose of our own sludies, performed in eollahoralion wilh Dr. Mercedes P. de Oliveira, A. Caslania, Vera Porlieri and Suzana Hiheiro da Cosia. ft has heen known siuee 1956 lhal crotamine, a hasic jnotein foimd in lhe venom of CROTALIDAE of soulhern Brazil (1), is capahle of releasing hislamine from ral lissues. This resull was firsl ohlained hy Moura Gonçalves and Rocha e Silva (2) using lhe perfused hind leg pre|)aralion of lhe ral; lhe aulhors concluded lhal crolamine is lhe cause of lhe well-known hislamine releasing ahilily of ihis crotalic venom. There are hovvever, cerlain varielies of Hrazilian ralllesnakes, more ahimdanl in lhe norlhern and cenlral regions of lhe country, which do nol conlain crolamine in lheir venoms. In a sludy of lhe jdiarmaco- dvnamic properlies of such venoms, vve noled lhal lhey were highly aclive hislamine releasing agents, having a ])olency e(|ual or higher lhan lhal of crolamine ilself. Eig. 1 shovvs lhe release of hislamine from isolaled ral masl cells hy crolamine and crolamine-free whole ralllesnake venom. In ihis, as vvell as in most suhsequenl sludies, hislamine release was assayed on lhe washed masl cells isolaled from lhe periloneal cavily of lhe lal. Such cells are good re- pre.senlalives of lhe lissuediound forms of lhese cells which, as il is well-known, are hislamine slorage siles in many s|)ecies. Il is hy lhe slimulalion of lheir granular secrelion or hy unspecific cylolylic damage, lhal hislamine release is hroughl ahoul in vivo. Il is known lhal lhe so-called liemolylic snake venoms are oflen ])owerful hislamine releasing agenls in vivo or in perfused lissues. Trelhewie in Auslralia, has aclually suggesled (3) lhal lhese Iwo aclivilies e.g. hemolysis and amine releasing aclion are due lo lhe same chemical enlily. We have Iried lo verify whelher in crolamine-free ralllesnake venom, hislamine-releasing aclivily was as- socialed wilh liemolylic or ralher, lo use a more precise lerm, ])hos|)holipase A Supportcd, in part, by grant No. DA-ARO-49-()92-66-Gini U.S. Army Re.search Office. cm 2 3 z 5 6 11 12 13 14 15 468 MECHANISM OF IIISTAMINE RELEASE BY ANIMAR VENOMS aclivity. To ihis ciid, wc made a chromalograiihic analysis of crotamino-frec ralllesnake veiiom oii a And)erlite lrc-5() ion-excliaTige columii accordiiig to lhe techiii(]iie descrihed l)y Hahermaiiti (4). Fig. 2 shows lhe residis. Il ean he Flg. 1 — Histamine release from Isolated rat mast cells by crotamine and crotamine-free vvhole rattle- snake venom. ml effluent Flg. 2 — Chromatographlc analysis oI crotamine- free crotalie venom on Amberlite XE-64, CG-50 lonic resin. seeii that hemolytic aetivity, measiired as phospholipase A liy lhe egg-yolk eoagu- lation lest, coidd he clearly differenliated from hislamine releasing aclivily as measiired hy lhe isolated mast cell teclmique. This aclivity was eluted sliortiy after, hut definilely iiot together willi crolacliii I tall peak), lhe major protein of lliis ralllesnake venom, which aiso seems lo he lhe major factor res])on.sihle for lhe lelhality of snch venom (4). It thiis hecomes clear lhal lhe histamine re¬ leasing factor of erolalic venom, which vve have lentalively called enzyme fraclion I, is neither crolaclin nor phospholipase A. By nsing lhe comhined fractions showing highest histamine releasing aclivity, we jiroceeded wilh an analysis of ils projierties. These are shown in Tahie I, which aIso comjiares them wilh TABI.E I — HISTAMINE RELEASING ACTIVITY OF CROTAMINE AND OF ENZYME FACTOR I FROM RATTLESNAKE VENOM Treatment Histamine releasing aetivity ♦ Enzyme factor I Crotamine Ileat (5’, lOCPC) Lost Retained Dialysis Retained I-ost Digestion by trypsin Lost 9 Electrophoretlc moblllty (pH 7.7) Slow, anionlc Fast, cationic Assayed on rat isolated peritoneal fluid mast oell.s. cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan ADOLFO MAX ROTHSCHILD 46Í) Simp. Internac. 33(2):467-476, 1966 ihose of crolamine. Il can he seeii lhat \ve are most prolialily tlealiiig with an enzyme as the lhermal instaliilily, |)rotein natiire and high molecular vveight data indicale. \Ve suspect lhal enzyme factor I is a proteolytic enzyme having chymo- trypsindike properties and base this assumption on three facts: a) chymotryptic activity has lieen demonslrated in crotalic venom by Deutsch & Diniz (5) in 1955; 1») cbymolrypsin is a hislamine-releasing agenl (6,7) in contrast to trypsin which will not act on the isolaled mast cell; c) our product was able to destroy ])radykinin, a pro])erty of cbymolrypsin, and which is an extremely sensitive, although not specific test, for this enzyme. The absence of a direct histamine releasing effect of crotalic phospholipase A was a somevvbat unexpected finding, even more so because of reports in the literature (Uvniis and collaborators (8)) showing that snake or bee venom phospho¬ lipase A was indeed a potent histamine releasing agent on isolated mast cells of the rat. It seemed possible to us that the preparations used by Uvnas could have been contaminated with other venom factors capable of releasing histamine. In order to investigate this problem, we decided to study the histamine releasing properties of a highly piirified and very active jíreparalion of phospholipase A írom bee venom, obtained through the courtesy of Prof. Habermann (9) of Germany. It turned out, that this enzyme was only slightly active on the isolated mast cell, even when assayed in relatively high doses as shown in Table II. Not TABLE II — EFFECT OF PHOSPHOLIPASE A TREATED EGG YOLK ON THE RELEASE OF FIISTAMINE FROM ISOLATED RAT MAST CELLS Releasing agent * Percent histamine released Phospholipase A, 20 íig/ml Egg yolk (4í7) pretreated with phospholipase A 7.3 (37°, 40') 91.0 Egg yolk (4%) 0.0 * Allovveci to act for 20 min, 37°C on the mast cell .suspension. imexpectedly, |)hospholi[)ase A was also inactive as a hemolytic agent, an obvious concliision from the known fact that lhe red ceirs membrane phospholipids are shielded from attack by this enzyme (10). They are however highly sensitive to the action of the product of this enzyme’s action on a susceptible substrate like egg yolk’s fresb lecithin. The data on Table 11 indicate that rat mast cells readily loose lheir histamine when treated with lhe producls of the action of jihospholijiase A on egg yolk, which we know to conlain lysolecithin. Thus, in vitro release of histamine bv phospbolipase A can only take place in an in- direct way. The question which now arose in our minds was: would the enzyme bchave in the same manner in an in vivo test for hislamine-releasing activity? Would lherefore the numerous results about lhe histamine releasing activity of hemolytic vcnoms in vivo or in perfused tissues demonslrated by other authors, have to bc ascribed enlirely lo lhe presence of non-phos|)holipase A components? In order lo answer lhese questions, we jierformed a very simjile test for the deteclion of histamine releasing activity in vivo. This is lhe well-known Trypan blue skin 1 SciELO 470 MECHANISM OF HISTAMINE RELEASE BY ANIMAL VENOMS capillary [lermealnlily k>sl, which consists of lhe followiiig: a ral or a giiinea-pig is iiitravenousiy iiijected wilh a dye like Trypaii or Evans hhie, which nonnally does nol leave lhe circtdalioti lo enler lissiie fluid spaces l)ecause of ils iiialiilily lo cross capillary wall ])crmeahilily harriers. If sucli harriers are liovvever allered hy aii experimenlal procedure siich as lhe local release of hislamine. a suhslance highly aclive on ca|)illary permeahilily, diffusion of lhe dye across lhe vascular Wall can lake |)lace and a hhie si)Ot will ap|)ear al lhe sile of hislamine release. l'ig. 3 shovvs lhe resulls of inlradernial injeclion of hee venoni j)hospholipase A B'ig. 3 — Effect of bee venom phosphollpase A on capillary per- meabllity In rat skln. Dark areas correspond to the injection of respectively 0.01, 0.1, 1,0 and 5.0 jig of enzyme. Contra-lateral injectlons of saline failed to produce comparable respon.ses. inlo ral skin doses ranging frorn 10 /rg to ,5 /<,g. ll can he seen lhal the enzyrne was highly effeclive in hnvering ca|)illary diffusion harriers and lhal ihis effect hore relation to lhe dose iised. This effecl was nol a direcl one; it is prohahly medialed hy the release of skin hislamine and serolonin since it coiild he shovvn (13) lhal the [lermeahility effects of phospholi|)ase A coiild he completely siippressed in animais |)revionsly Irealed wilh a mixlure of an inhihitor of the vascular effects of hislamine I diphentiydramine) and an inhihilor of the vascular effects of serolouiu |H()L-M8, hromiysergic acid dielhylamide). This laller had Io he iised hecatise as it is well-kiiovvu, wheuever mast cell damage occurs iu the ral. uol only hislamine luit serolouiu as well, is released. Serolouiu is even more jioteul on rat capillary |)ermeahility than hislamine. The ohviotis couchision from lhese resulls was lheu, lhal jihosjiholijiase A was ahle to release mast cell amines in vivo, eveu ihough it proved iucapahie of doiug the same on the isolated mast cell in vitro. Talile 11 had shown lhat phosjiholipase A can iuduce copioiis liistamiue release from isolated mast cells in vitro hui only iu au iudirect way, namely, lliroiigh lhe formaliou of lysolecilhiu. As it is well-knowu, this suhslance, a highly cytolytic. agent, is lhe resnit of the spliltiug of an acid radical from lecithin Ijy cm SciELO 10 11 12 13 14 15 16 SciELO i'o 2 3 5 6 11 12 13 14 15 L cm MECHANISM OF HISTAMINE RELEASE BY ANIMAL VENOMS 472 Fig. 4b ^..11 I Fig. 4c SciELO cm Mem. Inst. Butantan Simp. Internac. 33(2):467-476, 1966 ADOLFO MAX ROTHSCHILD 473 Fig. 4d histamiiu' releasiiig stini llial histamiiie liroaks 1 pliarinacological actioiis lhe effect of conipound ig stiniiili and il is likely, lliat il is oiily iii llie slied granules reaks loose froni ils intracellular lies, hecoinitig free lo exerl utions iii lhe hody. Fig. Id shows, for lhe sake of comiiarisoit, npoiind 48/80, lhe inost potent chemical histamine releaser iii of another sani])le of shows lhe kiiown, on lhe niasl mesenlerv. rat SciELO 474 MECHANISM OF IIISTAMINE RELEASE BY ANIMAL VENOMS llie effects ot ineliltin; ihis is a polype])li(le siil)statKc, isolated fiom l)ee veiiom liy Hahermann (14), which has an iiitense and a[)part‘iitly unspecific cytolytic effect. It induces 100 per cent release of histamine froni isolated mast cells (13). As it can be seen, its effects are not so imich a degranulation hiit rather a dis- solution of the celFs houiidaries. This as[)ect is lypical of unspecific cell damage, and is, in its mechanisni of aclion quite different from granule secretion as evoked by compound 48/80 or the antigen-antibody reaction. The action of melittin is however similar to tbat of lysolecithin. Table III sbows that tbere is a ineans TABLE III — MODES OF ACTION OF IIISTAMINE RELEASING AGENTS Group I: Stimulants of mast cell secretory activlty (effects inhlblted by metabolic in- hibltors (anoxla, DNP, NaCN) Antigen-antibody reactions Epinephrine Compound 48/80 Curares Chymotrypsin Enzyme factor I from crotalic venom Crotamine Group II: Non-specifie cytolytic agents (effects not Inhlblted by metabolic Inhlbitors) Surfactants (octylamlne, Tween 20, etc.) Phosphollpase A Lysolecithin (egg yolk) Melittin Crotamine of distinguisbing belween lhe niecbanisin of action of histamine releasing agents. 1 consider those agents wiiose action is inhihited by metabolic inhibitors like oxygen lack, dinitrophenol or cyanide, lo be Irue stimulants of the secretory macbinery of the mast cell. In contrast, the group of histamine releasers wbose action is not blocked by metabolic inhibitors, are to be considered unspecific cytolytic agents, capable of riiiiluring lhe mast cell in lhe same way as lhey would injure most any cell in lhe mammaliaii organism. It can be seen that among histamine releasing agents conlained in animal venoms, phospholipase A, melittin and, to a partial extent, crotamine, are included in this group. Kefeuences 1. MOURA GONÇALVES, J., in E. E, BUCKLEY, and N. PORGES (Editors), Venoms, Amer. Ass. Advanc. Sei., Washington, D.C., 1965, p. 261. 2. MOURA GONÇALVES, J., and ROÇHA E SILVA, M., Ciência e Cultura, Brasil, 1(), 163, 1958. 1 SciELO Mem. Inst. Hutantan ADOLFO MAX ROTHSCUILD 475 Simp. Internac. 33(2):467-47fi, 1966 3. TRETHEWIE, E. R., Aust. J. exp. biol. med. Sei., 17, 145, 1939. 4. HABERMANN, E., Biochern. Z., 329, 405, 1957. 5. DEUTSCH, H. F., and DINIZ, C. R., J. biol. Chem., 216, 17, 1955. 6. UVNÂS, B., Ann. N.Y. Acud. Sei., 166, 880, 1963. 7. SAEKI, K., Jap. J. Pharmacol, 14, 375, 1964. 8. UVNÃS, B., Cliemotherupiu, 3, 137, 1961. 9. HABERMANN, E., Biochern. Pharmacol., Suppl., 12, 187, 1963. 10. CONDREA, E., MAMMON, Z., ALOOF, S., and DE VRIES, A., Biochhn. Bio- phys. Acta, 84, 365, 1964. 11. KELLER, R., Helv. Physiol. Pharmacol. Acta, 22, C76, 1964. 12. FELDBERG, W., and KELLAWAY, C. H., Aust. J. exp. biol. med. Sei., 15, 461, 1938. 13. ROTHSCHILD, A. M., Brit. J. Pharmacol., 25, 59, 1965. 14. NEUMANN, W., and HABERMANN, E., Arch. exp. Pathol. Pharmacol., 22, 367, 1954. Discussion F. Kornalik: “How do you explain the fact, that ELAPIDAE venoms, especial- ly the venom of Naja naja which are known to have big amounts o£ phospholipase A are not able to liberate any histamine from the tissue? Have you te.sted the phospholipase A from Prof. Habermann in another way than the skin test for the presence of spreading factor?” A. M. Rothschild: “I have no personal experience with such venoms. How- ever the statement that the venom of Naja naja does not release histamine from tissues contradicts observations reported by a considerable number of authors wbo have found this venom to be quite powerful as a histamine releasing agent. We have not done a characterization of this enzyme because Prof. Habermann (9) has presented convincing proof that it is essentially free of enzymatic contaminants including hyaluronidase. Furthermore, our skin lests definitely implicate histamine and serotonin as mediators; hyaluronidase is known not to release these amines.” J. L. Prado: “The bradykinin destroying effect of enzyme I fraction of snake venom seemed too slow to me; would it really be a chymotryptic enzyme?” A. M. Rothschild: “There is, I believe, fair evidence that the histamine releas¬ ing activity of Fraction I is of an enzymic nature. Aithough our results do not conclusively characterize this activity as being chymotrypsin-like, they point in this direction. Slugishness of action does not necessarily rule out this possibility since even whole crotalic venom is slow in attacking a typical chymotryptic sub- strate like ATEE.” E. A. Zeller: “Have you tested the substrate pattern of your enzyme factor which you suggested appears to be a chymotrypsin-like proteinase?” A. M. Rothschild: “No.” cm 2 3 L. 5 6 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):477-50K, 1968 PHILIP ROSENBERG 477 53. l’SE OF VENOMS IN STUDIES ON NEHVE EXClTATIOiX PHILIP ROSENBERG Departinent of Neurology, College of Physicians and Surgeons Columbia University, New York, N.Y. Introduction Various techniques have Iteeii utilized over the past several decades in at- tcmpliiig to understand hioeleclrogenesis, lhe jirimary eveiil in neiiral aclivdty. Ulilizing eleetrophysiological techniques it was ]) 0 ssil)le lo analyze inany aspecls of electrical activity of conducting lissues (1,21. However, for elucidaling the Chemical and molecular hasis of nerve activity it was necessary that techniques of modem hiochemislry and pharmacology he used. Over lhe pasl 25 years the sludies of Nachmansohn and coworkers have [irovided us with lhe essential steps involved in lhe generation of hioelectricity allhough many details require further investigation (3-5). The impetus which led to our studying of the effecls which slruclure and organizalion of hiological memhranes and their environment have on the function- ing of lhe ACh syslem, and on lhe action of compounds apjilied externally lo l)iological lissue, will he hetter understood if we hriefly review the role allrihuted to acetylcholine (ACh) in the excitahie memhrane during nerve conduction (Fig. 1). ACh in resting condition is jiresent in a hound form. Any stimulus reach- ing lhe memhrane leads to release of the ester which comhines with a receptor. In the reaction of ACh with the rece])lor a change in conformalion of lhe receptor is thought to occur which leads lo a shifl of charge initiating lhe permeahility changes of lhe mend)rane associated with the passage of a nerve impulse, i.e. sodium ions enter lhe axon and potassium ions leave. The rapid inactivation of the ester hy acelylcholinesterase (AChE) permits the recejilor to return to its resting condition. A ])rerequisite for this lheory was the demonslralion of lhe presence in all conducting tissue of choline acelylasc (ChAc) lhe enzyme which synthesizes ACh and AChE the enzyme which hydrolyzes ACh. Of crucial im- |)ortance to lhe development of the theory has heen, however, the evidence that hlock of eilher AChE or ChAc hy jiotent or comjictitive inhihitors leads inevitahly to hlock of electrical activity. Thls Work was supported by the Divlsion of Research Grants and Fellowships, U.S. Public Health Service, Grants NB-03304, NB-04367 and B-400 and training grant 2B-5216; by the National Science Foundation Grants 12901 and 1913 and by the Muscular Dys- trophy Associatlons of America Inc. Recipient of a Public Health Service Research Career Development Award, 5-K3- NB-21, 862. cm 2 3 L 5 6 11 12 13 14 15 478 USE OF VENOMS IN STUDIES ON NERVE EXCITATION ELEMENTARY PROCESS O-r " 2 A T P 1 s! 2 PYRUVIC ♦ 02 VIA CITRIC ACID CYCLE 30 ATP 1 fCHOLINE ACETATE j . _) L PHOSPHOCREATINE Co A t ACETATE :holIN E \ cetylaseJ ' -ACETVL-Coâ a M p + PP Fig. 1 Some invesligators liave, hovvever, not accepted lhe lheory lhat ACh lias an essential role in axonal conduction, and liave inainlained that ACh is essential for transmission of impulses only al certain jimclional regions. One of their major ohjections to Nachmansolm’s theory was the failiire of ACh, and other quaternary nilrogen derivatives lo affect conduction in nerve axons in coutrasl to their povverfui aclion on junctions. The excilahie and conducting axonal memliranes, in coutrasl to those al synaptic junctions, are surrounded hy slructures rich in liiiid material, the Schvvann cells. which form a iiermeahilily harrier jirevenling li[)id insoluhle compounds from reaching the memhrane. Exjierimenlal evidence for the existence of liarriers was ohtaincd hy the demonstration that lioth ACh and lhe quaternary cholinesterase inhihitor neosligmine, failed lo jie- netrate into the axo])lasm of lhe squid giant axon afler their a])|)licaliou to lhe fiher in the externai solution, whereas lipid solulile lertiary nilrogen derivalivE's, •sucli as physosligmine, that lilocks electrical aclivity, were fouud in the axo- plasm (6,7). lii pre|)aratious such as the ralihit vagus and axons from lhe lohsler walking leg in which the permeahility liarriers are apparenlly incomplele, direct effecls on conducliou have heen ohtained with ACh, and related comjiouuds, such as d-tuliocuraririe (curare) knowii lo compele with ACh for recejilor sites at junctions (8-11). If A(ih and curare are inactive on lhe squid giant axon and other axonal prejiarations hecause of permeahility liarriers it appeared possihle that hy (hemical Ireatment one could decrea.se lhese liarriers and iheii demonslrate actions of lijiid insoluhle com])ouuds which iuleract with lhe ACh syslem. After exjiosure of the frog scialic nerve to a detergent. axonal conduction was reversihly hlocked hy ACh, curare, neostigmine and other quaternary nitrogen derivatives (12). In our sludies we elected to use the squid giaut axou (Fig. 2) hecause it is a large single fiher and is non-myelinated, i.e. is surrounded hy a relatively ihin Schwann cell. In such a prejiaration chemical treatment migliL he ex|)ected to he more effective and hetter conlrollahle lhau in a mulliíihred [ireparation. This preparation also has lhe uniipie advaulage lhat its axoplasm lan he readily extruded in suf- íicient amounts lo allow penetralion sludies to he carried oul. In addition it was usefui lo sludy a preparation for which a considerahle amounl of data are cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33{2):477-508, 1966 PHILIP ROSENBERG 479 already availahie, and willi which oiir residis coidd lie correlaled. Tliis review shall presenl and snmmarize some of llie findings we have made over lhe past 5 years (14-25). Fig. 2 Effects of tertiary and quaternary mtrogen dekivates on conduction While a few phannacologieal agenis have heen tesled on eonduclion of lhe sqnid giant axon, no systenialic eornjiarison had heen made hetween terliary and qualernary nilrogen conlaining eomponnds. If a permeahility harrier ]irevenls ]i])id insolnhle qualernary nilrogen comjjounds from reaehing lhe conducting niemlirane. lhen we shoidd find lhese compoimds lo he inactive on lhe squid axon even if lhey interacl wilh lhe ACh syslem at junclions where lhe jiermealiility harriers are mueh w'eaker. \\ e should also find lhal lijnd solnhle compoimds which affecl jnnctional Iransmission shonld also affecl axonal conduction. This is whal we did find on conirol squid axons in Tahle 1 where we have also cornpared lhe |)otency of various compoimds on lhe squid axon wilh lhal ohserved at lhe synaplic junclions of lhe isolaled single eleclroplax where harriers are either miuimal or ahsent, and where approximale affinily lo rece[)tors jireseut 1 SciELO 480 USE OF VENOMS IN STUDIES ON NERVE EXCITATION inay Ix' delermineíl (28,29). The terliary iiitrogeii derivatives are effective in verv similar eoncentrations oii lliese two [jreparations. One couid not, however, expect exaet agreement, even if lhe |)ermealiility harriers were tom])letely ahsent, l)ecause of species differences, sornewhat differciil experimental eondilions and most likely other faelors involved. In eonlrast all cjuaternary nilrogen eontaining componnds except noracelylcholine and jiyridine aldoxime dodecyliodide (PAD) are ineffeetive on lhe sqnid axon in eonlrast to their high ])olency al lhe synapse. Noracetylelioline and PAD are li])id solulile derivatives of acetyleholine and jjyridine aldoxime methiodide (PAM) having a dodecyl group vvhieh replaces a methyl group on the nitrogen. We shall disciiss lhe resulls ohlained on the venom Ireated sqiiid axon in seetion 8. ACh and curare were equally inert ou control sqnid axous which were very carefully dissected free of all adhering sniall nerve filiers as well as axons iu which not all of the small nerve fihers were removed(28). Physosligmine was also found to he almost equally potent in hoth pre|)aralions. These resulls indicate lhal lhe adhering small nerve fihers and assoeiated connective tissue do not constitule a strong ])ermeaf)ility harrier. The major harrier is a|)parently the Schwann cell surrounding the giant axon. For convenience we have lherefore usually used j)reparations in which no S])ecial effort was made to remove all adjacent fihers, although as will he diseussed in seclions 8, 4 and 8 venom effects and penetration of ACh were comjiared wilh hoth ly])es of prejiarations. These results show lhat lipid soluhle com|)ounds expected to interact wilh lhe ACh syslem affecl conduction along lhe axon as well as at the junction, whereas lipid insoluhle componnds hlock junctional Iransmission hul not axonal conduction. Venom pretreatment of squid giant axon In attempling lo reduce the {)ermeahility harriers surrounding the squid giant axon, various enzymes, detergenis and other componnds in the highesl eoncentra¬ tions which had no effect on conduction were a|)|)lied to the axon for 80 minutes. After removal of lhe pretreatmenl ageiil and rinsing in normal sea waler for 15 to 80 minutes, 1.4X10~’‘ M enrare was applied for 80 minutes. Curare was used as a lesi compound for iudiealing disruplioti of permeahilily harriers siuce il is highly ])oteut al synapses and is not readily melaholized hy enzymes of hiologieal tissue sueh as for examjtle ACh is hy AChK. Curare had no effect on conduction following ex|)osure of lhe axon lo digitonin, hyaluronidase, Irypsin, chymotry|)sin, hydrolase mixture, lij)ase, lysozyme, pa])ain, alkaline ])hosphatase, sodium desoxycholale, Span 20, Tween 20, neuraminidase, saponin, arsenite, dim- clhyl formarnide, dimelhyl sulfoxide or hislamine (14,21). Following prelreat- ment with Naja naja I hooded cohra) snake venom, however, curare was found lo reversihly hlock conduction (14). An axon rendered sensilive to lhe action of curare remained so over a long ])eriod of lime. For example curare was equally effective whelher [)relrealment was followed hy |)lacing in sea waler for 10 min. or 95 min. (Fig. 8). This figure shows effects ohlained wilh a com- hination of hooded cohra venom plus a delergent, however, similar resulls were ohtaiued with venom alone. Since snake venoms apj)eared lo render the axons sensitive to curare a .syslematic sludy of lhe effects of curare following venom jjrelrealmenl of lhe axon was underlaken. Of a series of 21 venoms lesled Agkis- trodon p. piscivoriis (Collonmoulh moccasiu) venom was most effective in render- ing the axon sensitive to curare (Tahle II). Fxamples of the effects of curare cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Inlernac. 33(2):477-508, 1966 PHILIP ROSENBERG 481 Flg. 3 TABLE I — MINIMAL CONCENTRATIONS OF SEVERAL COMPOUNDS REQUIRED TO BLOCK CONDUCTION OF CONTROL AND VENOM TREATED SQUID GIANT AXONS AND SYNAPTIC TRANSMISSION IN THE ISOLATED SINGLE ELECTROPLAX Compounds were appliecl for 30 minutes. Venom treated axons vvere pretreated for 30 minutes vvith 15 ng/ml A()kif:trodon p. piscivonis venom. > indicates that at concentra- tion shown no effect on conduction was observed. Noracetyicholine = ;S-acetoxyethyl- dimethyidodecyl ammonium lodide; PAD = Pyridine aidoxime dodecyiiodide; PAM = 2- Pyridine aidoxime methiodide. COMPOUND [M] Squid TO BLOCK CONDUCTION Axon * Control Venom Treated Synapse *• Tertiary Niirogen Derwativefi Atroi)ine 2 X lO--' 3X10-' 3 X10-' Methantheiine 2 X 10-^ — 5X10-1 Physostigmine 7X10-3 1 X 10-3 7 X 10-' Procaine 3 X 10--’ — 1X10-= Dibucaine 3 X 10-'> — 3X10-= Diphenhydramine 4X10--* — 2 X 10-' Chiorpromazine 1 X 10-« — 1 X 10-1 Qnaternary Nitrogen DerivaCives Acetylcholine > 10-' 2 X 10-1 3X10-0 Curare > 10-= 3X10-3 3X10-» Decamethonium > 10-' 2 X 10-' 3X10-0 Benzoylcholine > 10-' 2 X 10-= 1X10-= Chlorisondamine > 10-' 1X10-= 2 X 10-1 PAM > 10-1 1X10-= — PAD 5 X 10-' — 5 X 10-3 Noracetyicholine 2X10--Í — 2 X 10-' Choline > 10-1 > 10-' > 10-' Data from Ro.senberg et al. (14-16). Data from Ro.senberg et al. (26, 27). cm SciELO 10 11 12 13 14 15 482 USE OF VENOMS IN STUDIES ON NERVE EXCITATION íollovviiig coltonmoulh moccasiii veiiom are sliown in Figs. 4 and 5. lii lhe conceiitralions used for prelreatmenl tlie venoms had iio effect on the aclion polenlial. Hooded cobra venom depolarizes the lobster axoii (30), biit il is not apparent whether depolarizalion is the cause of the conduction hlock or occiirs suhsequenl lo the hlock. On the squid axon lhe resting and action potentials SCA WATIR I- 3.8v COTTONMOUTH VENOM 8.Sv SE A WATER -1 I 50m V 1.4x10-3 M CURARE Fig. 5 S E A WATER F 1 SciELO Mem. Inst. Butantan Simp. Internac. 33(2):477-508, 1966 PHILIP ROSENBERG 483 vvere foiind to decline simidtaneously (Fig. 6), whereas after coltoninoiith moc- casiii venom, depolarization occurred only after the action potential was niarkedlv reduced (Fig. 1). There was no difficulty of deterinining wliether hlock of conduction was diie to curare or venom since the effects of the venoms on the action potential were always irreversihle whereas conduction hlock hy curare was usually readily reversihle. Curare had little or no effect on the resting potential after venom pretreatment (Fig. 5) which agrees with its effects at junctions where mV Venom cm SciELO 10 11 12 13 14 15 484 USE OF VENOMS IN STUDIES ON NERVE EXCITATION íL also Iilocks comliictioii luit does iiol depolarize. It ai)|)arenlly ialeraels wilh the receptor lo lilock the effecls of ACli. Iiut does not cause llie chaiige in con- formatioii of lhe receptor responsil)le for altered conduclance. IL may be called a receptor inhihitor iii contrast to comj)ounds siich as ACh whicli depolarize and may lie classified as receptor activators. The ahility of the snake venoms to render curare active ahnost exactiy cor¬ relates with their direct potencies on electrical activity of the squid giant axon (Tahle II) indicating that the same venom component may he resjjonsihie for TABLE II — CONCENTRATIONS OF VENOM PRETREATMENT WHICH RENDERED THE SQUID GIANT AXON SENSITIVE TO CURARE, AND CONCENTRATIONS REQUIRED TO BLOCK DIRECTLY THE CONDUCTED ACTION POTENTIAL (AP). VENOMS WERE APPLIED FOR ABOUT 30 MINUTES > incllcates that at concentration shown no effect on AP was observeU. -I-+-h, -h+, -h, and - indica te respectively a 80 - 100 %, 50-80%, 20-50% and 0-20% decrease in the AP produced in 30 minutes or iess by 1.4 X 10-^ M curare alter venom pretreatment. The eflect of curare was readiiy reversible. Results taken from Refs 14-16, 24. VENOM íig/mi Direct block of AP Pretreat. Curare effect Afjkistrodon p. piscivoriis 50 15 + + -h Notechis scíitatus 100 30 + + 4- Acanthophis antarcticns 100 30 + + Naja naja 100 30 -f -1- Heloderma horridum 100 30 + -1- HeJoderma suftpectuni 100 30 + -1- Enhydrlna schistosa 100 20 — Dendroaspis poJi/Iepin 100 30 -1- Bunffarus coeruleus 250 50 -I--H Ophloph agu .s han n ah 400 100 -1- -l Crotalufi atrox 500 150 -t- Affkistrodon c. mokeson 1000 200 + fíothrops atrox 1000 200 + Bitis arietans 2000 500 + + Vipera russeUii > 1000 500 + Centruroides scuipturatufi > 100 100 — Vespula arenaria > 100 100 — Latrodectus f/eomeiricus > 100 100 — La trodeet ua varidn s > 250 250 — CrotaluH h. horridus > 500 500 — Cr o t a 1 UM a da m a n te us > 2000 2000 — holh effects. Stiidies hy others of venom effects at lhe neuromuscular junction (31, .12) have also shown collonmouth venom to he more ])Otenl lhan Crolaliis ada- niaiitcus (Lastern diamondhack ralllesnake) venom, which in our sltidies was one of the leasl effeclive of the snake venoms. Likevvise lhe relalive effeclive- ness of hooded cobra and ralllesnake venoms in our studies and in cansing demyelinaling changes in the central nervons system (33, 34) are similar. In contrast ralllesnake venom is more loxic lhan cottonmouth moccasin venom (35, 1 SciELO Mem. Inst. Butantan PHILIP ROSENBERG 485 Simp. Internai'. 33 ( 2 ):- 477 - 508 , 1966 riG). It is t>s])i‘ciallv inlfiesting lhal lhe heitiolylie and heniagghilinin aclivities of .«everal siiake veiiorns as foiiiid liy Miiilon (351 is iii excelleiit agreemenl witli their ahilities lo render eiirare aclive, hor example lie reporls lhal Kastern diamondhack ralllesnake venoni has lillle or no hemolytie aelivily whereas Cro- Niliis atro.x (Vieslern dianiondhaek ralllesnake) venom is more |iotenl whieli also agrees wilh onr findings. He also fonnd Agkisirodon c. mukcson (eojiperhead moecasin) lo he Icss jrolenl llian eollonmoulli moceasin. The marked similarity lietvveen his and oiir residis may indieale lhal llie same venom eom])onent is resjronsihie for holh effeels measnred. Sinee eollonmonlli moceasiti venom apjteared mosl effeclive in rendering llie axon sensilive lo eiirare it was selected for additional slndies. The venom Ireated axon not only lieeame sensilive lo curare Inil as shown in Tahie I. .4Ch. deca- melhoniiim and several olher qnalernary nilrogen derivalives l)Ioeked condnclion. Examjiles of lhe ACh effeels on collonmoiith venom Ireated axons are showii in Figs. 8 and 9. As can l)e seen in Fig. 9 llie effeels of ACh were nol aiways reversihle. In a large series ahoiil 1 onl of !■ experiments wilh ACh were reversihle. In experiments wilh intraeellnlar eleelrodes, 1 X 10“" M .4Ch follow- ing venom jirelreatmenl hloeked eondnelion and deereased lhe resting irolential ahonl in 30 min. while in eonlrol axons ACh had no effeel on lhe resting or aetion potenlial. However. 4X10“^ 41 ACh following venom prelrealment had a marked effeel on lhe aelion jiolenlial hiil no ap|)arenl effeel on lhe resling ])olenlial. This is in conlrasl lo olher ])re|)arations where lhe effeels of ACh on lhe aelion potenlial are paralleled hy eonciirrenl effeels on lhe resling po- lenlial 19-11). The eoneentralions of qnalernary nilrogen derivalives shown as heing effeclive in venom Irealed axons iTahle I) are llie minimal eoneenlralions whieh in al leasl a few exjierimenls hloeked eondnelion. For fairly eonsistenl effeels it was neeessarv to nse eoneentralions ,5 lo 10 fold higher. The eoncentration of t(nalernary nilrogen derivalives lisled in Tahie I as heing effeclive on lhe venom Ireated axon are 10 to 1000 fold grealer than ihose reqnired al lhe svnajise. However, al leasl pari of this differenee is ipiile ohvionsly dne to lhe ineomplete rednction of permeahilily harriers snrronnding lhe axon, .so lhal ordv a relatively small fraelion of lhe exlernally ajiplied ACh. enrare ele. is ahie to |)enelrale Isee .seetion 4). The inaetivity of eholine in venom Ireated axons, despite lhe fael lhal il penetrales nnder lhese eonditions tsee seetion 4) show lhal lhe effeels of ACh are sjieeifie. At those synapses where ACh is highly ))olent. sneh as for example lhe eleelroplax synapse. il has also heen fonnd lhal eholine is inert (Tahie I). In conlrasl to lhe (jnalernary eomponnds shown in Tahie I whieh were effeclive on venom Irealed axons earhamvleholine and neo- sligmine even in 5 X 10~- 41 eoneentralions were inaetive. These eomponnds are apparenlly even more lipid insoinhie than ACh. enrare, or eholine sinee neo- stigmine does not jienelrale inlo lhe sqnid axon nnder eonditions where lhe venom markedly inereased lhe ])enetration of lhe olher eomponnds (seetion 4). In these exjieriments we ohserved only hloeking effeels of ACh and nol anv eleetrogenic aelion. Wilh lhe melhod of appiieation nsed, one eannot expeel to mimie lhe snggested physiological aelion of ACh. Physiologieally ACh wonld he liheraled wilhin a memhrane of ahonl 80 A" thiekness. Il wonld lhen ael, in mieroseeonds, on a iirotein reeeplor in elose proximily, prohahiy only a few A" away. W hereas ihis aelion may he very effieient and rajiid. ACh when ap|)lied exlernally miisl pcnelrate throngh a relatively large amonnt of lissne hefore reaehing lhe reie[)lor of lhe active axonal memhrane. Thns. lhe eondilions are nol eomparahle. cm 2 3 z 5 6 11 12 13 14 15 SciELOiio 2 3 5 6 11 12 13 14 15 16 L. cm Mem. Inst. Butantan Simp. Internac. 33 ( 2 ): 477 - 508 , 1986 PHILIP ROSENBERG 487 It was foiind (Talile [) thal lhe potency of atropine and physostigmine, tertiary nitrogen derivatives, was also increased hy venom pretrealmenl so that lhe concentrations effective on lhe axon hecome very dose to those effective at the electro])lax synapse. These findings are in agreement with the idea that venoms are disriipting permeahility harriers, since even relatively lipid soluhie materiais prohahly cannot freely diffuse through the memhrane surroiinding lhe giant axon. The findings with atropine, physostigmine and the other compounds shown in Tahle I appears to estahlish that a fiinctional ACh receptor and AChK are present in the sqiiid axon. Effect of ve.xoms on perme.ability of squid giant axon We interpreted the demonstration that ACh and curare affect electrical activity after exposiire of squid giant axons to coltonmoiilli venom as heing due to a rediiction of the permeahility harrier which prevents lipid insoluhle compounds from reaching the conducting memhrane. In view of lhe crucial question in- volved, viz. whether ACh and curare act on the receptor in the axonal memhrane, this iuterpretation was suhmilted to a direcl tesl; i.e., whether the venom which has heen shown to allow ACh and curare to affect electrical activity also allows them to penetrate. Axons were exposed to Solutions of venom or in the case of control axons to normal sea water for 30 min., followed hy a 10 min. washiug and hy a 60 min. exjjosure to the C'^ lahelled compound heing studied. The axoplasm was then extruded and C’^ assayed. A summary of resulls ohtained are shown in Fig. 10. These data clearly estahlished that ACh and curare do penetrate after trealment with lhe low concentrations of cotlonmouth moccasin venom which renders these compounds active. In contrast after exposiire to even high concentrations of Eastern diamondhack ratllesnake venom, curare neither penetrates nor does it affect electrical activity. In control axons lhe penelration of ACh, choline and dimelhylcurare is less lhan 1 per cent of that exjiected if no harrier were present. Even these low values however may not rejiresent aclnal penelration hut may he dne to conlamination during lhe process of extrusion, or the presence of trace amounts of C’‘-tertiary nitrogen containing compounds or trace im|)nrities in the radioactive samples of the qnaternary compounds. This suggestion is snpjiorted hy the finding that the penelration of ACh in venom trealed axons appears to increase with increasing lime of ACh incuhation, whereas in axons not pretreated with venom the apparent ACh penelration after 5 min. incuhation is as great as after 60 min. The amonnt of contaminalion occurring during extrusion would he expected lo remain approximalely constant regardiess of whether the ACh incuhation time was 5 or 60 min. We fonnd lhe penetration of ACh to he equally low hoth in axons dissected free of all adhering small nerve fihers as well as in axons containing mnch con- nective tissue and surrounding small nerve fihers. This agrees with our findings that ACh had no effect on conduction in hoth lypes of prejiaralions (23), hut is in disagreement with a report that ACh rajndly enlers lhe axoplasm of finely dis¬ sected axons (37). In that study, moreover, the electrical activity of the axons was not checked, which makes the meaning of the ex[)erimenls questionahie. In contrast to the qnaternary compounds lhe li[)id soluhie tertiary nitrogen derivativo Irimethylamine readily penelrated (Fig. 10) even without venom treatment which confirms an earlier report (6). ,1 1 SciELO 488 USE OF VENOMS IN STUDIES ON NERVE EXCITATION ACh Choline Dimethyl Trimelhy! Curare omine Fig. 10 [n lhe studies (lescril)ed radioactivity has l)eeti iiieasiired iii lhe axoplasm, althoiigh our iiiterest is iii knovviiig lhe cortcenlraliou of lhe compounds reaching lhe aclive siles of lhe mendirane which are surroimdÍTig lhe axoplasin. Allhoiigli only aliout 80 A'’ ihick (38,89), lhe ineinhraiie is eomplex and lhe exact localioii of lhe ACli syslein is nol kiiown. ll is impossible lo kiiow whal lhe concenlralion of ACh or curare may he iti lhe meml)rane compared lo llial foimd iu lhe axo- plasm. Even llie meaning of concenlralion in such a memhrane is nncertain. Considering lhe wide variely of compoimds lo which nerve axons diiring lheir enlire lenglh may he exposed in lhe liody. one woidd ex])ecled llial lhe receplor areas of lhe excilahie memhrane may he especially well prolecled againsi lhe aclion of externai comjioimds. Measuring lhe concenlralion of lhe compoimds lesled in lhe whole axon would he of qiieslionahie significance, since lhere is non specific hinding of charged molecnies such as ACh and curare wilh mauy macromolecules in lhe Schvvann cell, i.e., exlernal lo lhe aclive neuronal memhrane )23 ). 1 SciELO Mem. Inst. Butantan Slmp. Internac. 33(2):‘477-50S, 1966 PHILIP ROSENBERG 489 Since \ve tiad foiiiul tlial lhe ]) 0 \veiful CliE inhihilor iieostigniiiie does not affeel eoiiductioii eveii after venom pretieatnieiit. \ve nieasiired its per ceiit ])e- iielralioii iiito lhe axoplasin iising a magnetic diver deviee(21l. Oii coiitrol axons or afler trealmeiit of lhe axon wilh 25 p-g/nil cotloiinioiilh moccasiii venom only 0.1 per cenl penelration of neo.sligniine vvas ohserved. vvhile afler 100 ,u.g/rnl of venom, a concenlralion whicli hloeked eonduclion. 1 |)er cenl petielralion vvas ohserved. The poor penelralion of neosligmine lherefore appears lo l)e in good agreemeiit vvith ils ineffecliveness in Idoeking eleclrical aclivily. Neosligmine thus is apparenlly even more lijvid insoluhle lhan ACh or curare. The increased jvermealiilily prodiiced hy llie venoms agrees wilh snggeslions made many years ago. even hefore any experimenlal evidence vvas availalde, lhat niany effecls of venoms are dne lo increased permealiilily of hiological memlvranes. More recently il was foimd lhal moecasin venom increases llie passage of per- fnsion fliiid ihrough frog alria(40) and lhe penelralion of jvrocaine into õog seiatie nerve(41). Cohra venom releases ACh from hinding particles of lhe eenlral nervous system ( 421 vvhile collonmoulh moecasin venom causes lhe release of gliilamine oxalacelic transarninase from cerlain rat lissue(4). Easlern dia- mondhack ralllesnake venom vvhich is ineffeclive in our sludíes vvas akso imahle lo disrnpl rahhil liver microsomes in conlrasl lo anolher venom vvhich vvas foimd effective (44). The ahilily of many venoms lo hemolyze red hiood cells is vvell known (45-47). Ralllesnake venom hovvever has lillle or no aclivily as a hemo- lysin or hemagglulinin vvhereas coltonmoulh moecasin is very aclive (35). Il is significanl lhal in many of lhe sludies moecasin or cohra venom is more [lolent lhan ralllesnake venom in disrnpling memhranes vvhich agrees wilh our residis. TABLE III — PENETRATION OF VARIOUS C* LABELLED CO.MPOUNDS INTO THE AXOPLASM OF CONTROL AND AGKISTRODON P. PISCIVORUS VENOM TREATED SQUID AXONS AU compouncls vvere appiied for one hour in a concenlralion of 4.5 X 10-“ M excepl for 1 X 10-“ M indoleacelic acid, 1.9 X lo-'> M dieldrin and 2.5 X 10-‘ M diphenyl- hydanloin, corlisol and dehydroepiandroslerone sulfale. AgkÍKtrodon p. piscivoriis venom applied for 30 minules in a concenlralion of 100 ,/ig/ml blocked eonduclion. None of lhe olher compounds had any effecl on eonduclion during lhe 1 hour appiicaliun. GABA = 7 Amino bulyiic acid, DOPA = 3,4 dihydroxyphenyialamine, DOPA mine = 3,4 dihydroxyphenylelhylamine. Dala from Iloskln and Ro.senberg (22). COMPOUND Per Conlrol cenl penelralion Venom prelrealed Glucose 20 Mannitol 1 _ Sucrose 1 40 Glulamale 1 35 Glutamine 2 35 Aspartate 2 _ GABA 4 25 DOPA 2 35 DOPA mine 3 70 Serotonin 6 50 Indoleacetic acid 25 _ Acelylsalicylic acid 35 Diphenylhydanloin 60 _ Corli.sol 100 _ Dieldrin 100 _ Dehyd roepland roslerone .sui fale 4 — cm SciELO 10 11 12 13 14 15 490 USE OF VENOMS IN STUDIES ON NERVE EXCITATION The stiulies of the |)erineahility jjroperties of the squid exlentlcd hy ineasiiring Üie penetration iiito the axojjlasm of a wide variety of compoiinds (Tahle III). Excepl for glucose, ])enetration of the siil)stances studied ap])ears to depend largely on the exteiit of the non-polar, lipo[)hilic charaeter of the eompoimd. This conclusion is hased on experiments with squid axou, how- ever it appears possihle thal cell memhranes from widely different sources may have sufficient properties iu eommon to give validity to such generalization. The glucose penetration appears to he metaholically mediated, indeed studies with specifieally laheled glucose indicated the participation of the jientose phos])hate pathway as a metaholic route in axonal memhrane and associated cell wall material and partial or complete ahsence of the oxidative system in the axoplasm (22). Clutamate, glutamine, GABA, aspatate, DOPA, DOPA mine and serotonin, all of which penetrated poorly have two or more iotnzahle groups each, selected from carhoxyl, amine and j)henolic hydroxyl groups, and exist almost always in a charged form. In contrast aspirin, indolacetic acid and diphenylhydantoin which have only one ionizahle group, and which would he in equilihrium with a significant amount of the dissociated lipophilic form, penetrated the untreated squid axon much more rapidly. Dieldrin is an extremely water-insoluhle in- secticide, and cortisol is a lipid-soluhle steroid. In contrast dehydroe])iandro- Fig. 11 cm SciELO Mem. Inst. Butantan Slmp. Internac. 33(2):477-508, 1966 PHILIP ROSENBERG 491 slerone sulfate is a water-solulile com]jletely ioiiized derivative of an olhervvise water-insoluhle steroid. The steroid and steroid sulfate thus constitute a striking exaniple of the effect on iiermealúlity of introducing a highly polar coni])letely dissociated substituent into a lipophilic moleeule. Moccasin venom treatment seems to iucrease the |)enetration of many poorly penetrating compouiids in a not too specific way. The permeahility harrier of the squid axon may he the axolemma, Schwann cell or connective tissue surroimding the axon. According to as yet unpid)lished ohservations of Dr. David Rohertson with electron microscojiy of venom pretreated axons, the venom produced marked alteration and disintegration of the Schwann cell; the axonal plasma memhrane appeared to he not affected (Fig. 11). Ville- gas and Villegas (38) have shown that Schwann cells are traversed by relatively large channels, however their tortuous nature may still make penetration difficult. Higher concentrations of venom may hlock electrical activity hy a gross disru|)tion of the axonal memhrane or its receptor areas. The venoms might also increase the concentration of curare and ACh which reaches the active sites hy disrupting nonspecific hinding sites with which charged molecules such as curare and ACh might he ex])ected to interact (e.g. nucleic acid, chondroitin sulfate etc.). Ai.tekatiox oe acetylcholi.ne penetration into, and effects on VENOM-TREATED SQUID AXONS An ap])arently surprising result in our studies with venom pretreated squid axons was the finding that low concentrations of physostigmine applied in com- hination with ACh did not enhauce its action as would have heen expected with a |)otent ChE inhihitor, hut markedly antagonized its action. Curare in contrast did not antagonize the action of ACh (Tahie IV). Similar unexpected ohserva¬ tions had previously heen made on the vagiis and lohster nerves (10,48). Floth of these findings are opposite to that observed at junctional sites where low con¬ centrations of physostigmine enhauce the action of ACh and curare antagonizes TABLE IV — PENETRATION INTO AXOPLASM AND EFFECTS ON ACTION POTENTIAL (AP) OF VENOM TREATED SQUID GIANT AXONS BY C“ ACETYLCHOLINE (ACh) ALONE AND COMBINED WITH OTHER COMPOUNDS Axons were exposed to 4.5 X 10-' M ACh for one hour. AH axons were pretreated with 25 /ig/ml Apkistroilon p. piscivorvs which had no effect on the AP. Data taken from Hoskin and Rosenberg (19), and Rosenberg and Podieskl (16). COMPOUNDS Decrease Per AP cent Penetration ACh 90 4 ACh-FchoIine 2.7X10-" M 70 3 ACh + oholine 5.4X10-3 M 40 — ACh-l-choline 2.7X10-'' M 20 1 ACh-|-neostigmine 1.7X10-2 M 30 1 ACh + physostigmine 5X10-* M 30 1 ACh + physostigmine 5X10-“ M 40 1 ACh + curare 1X10-“ M 90 — ACh-F curare 7X10-' M 100 15 cm SciELO 10 11 12 13 14 15 492 USE OF VENOMS IN STUDIES ON NERVE EXCITATION ils acüon. Botii clioliiie aiul iifosligmine. vvhicli cvcii iii liigli coiifentralions havc 110 cffect on coiuliiction of lhe venoni trealed squid axon aiso inarkediy antagonize lhe effeel of ACh on lhe action potential (Tahle IV). Nachmansohn had suggesled lhal there may he a competition helvveen lhese (:oin|)onnds for tlie palliways of penetration in tlie structnres surrounding lhe eondueting memhrane; i.e., externai to the reee|)tor areas of lhe nieinhrane. We llierefore undertook slndies wilh radioactive eoinpoiinds to see hovv well the effects on electrieal activity eorrelated with the iienetration of ACh. Choline. neoptigmine and physostigmine decreased the [lenetration of ACh in venom treated axons froin ahoiil 4 per cent to ahoiit 1 per cent whieh is lhe levei of penetration ohserved in eontrol axons not treated wilh venom (Fig. 10 ). In conlrasl curare did nol decrease hui actnally increased the jienetration of ACh (Tahle IV). The resuits indieate that physostigmine. neostigmine and choline decrease the effects of ACh on conduction hy eompeting wilh ACh for ])enetration jiathways. The competition ajipears lo he concentralion dependent for choline, and choline seems to compete with ACh for penetration on a mole for mole hasis since con- centrations of choline ahont eipial to that of ACh are needed to dernonslrate anlagonism. In contrast the antagonistic action of physostigmine is mueh more potent than that of choline sinee 5X 10“' M physostigmine decreased lhe pe- netralion of ACh as mnch as 5 X lü“" M agreeing wilh lhe ohservations on electrieal activity. Since neostigmine, choline and lhe low concentralion of jihyso- stigmine have no direct effects on conduction il would he difficull to assume lhal lhey are com|)eling with ACh for a speeific site in lhe memhrane essenlial for electrieal activity. This conelusion is further su|)[)orted hy the ohservalion that it is no longer [jossihie to demonstrate anlagonism of ACh |)enelration hy choline when the jiermeahility harriers lo ACh are no longer the limiting faclor when the axons are jiretreated with 100 /xg/ml eotlonmoulh moccasin venom, a concenlration whieh hlocks electrieal activity and inarkedly increases the |)enelra- lion of ACh (Kig. 10). The.se resnits eonfirm and exlend the previous ohservations on the importance of structural harriers snrroimding lhe axonal eondueting memhranes in determin- ing effects of Chemical compounds on electrieal activity. ACdi. choline, neostigmine and curare are all qiiaternary nitrogen derivatives while physostigmine al the pH used is ])artly in lhe eharged fonn. They may all reacl with negatively charged gronps in the rednced hui slill persisting harrier and may modify lhe effeels ohserved when two of lhem are ap|died simultaneousiy. A|)parenlly, howev(>r. lhey do not react with the same gronps in lhe sqnid axon preparation since curare facilitates lhe passage of ACh while choline and neostigmine decrease ils passage. In conlrasl curare markediy decreases lhe penetration of a (piaternary nitrogen compound in skeletal miiscle (49) emphasizing lhe variahility of inler- action helween compounds whieh may he ohserved in differeni |)reparations. Il was of inieresi to invesligate whether there was a degree of siiecificity ahont this comjielilion phenomenon. The |)enetralion of ACh was lherefore lesled in the presence of sucro.“e. a non charged jiharmacologically indifferent com|)ound. Sucrose had no significanl effeel on ACh jienelralion (28). indicating lhal charged molecules may more effeclively compete wilh A(.'h for jienetration sites than un- charged molecules. Our findings on lhe axon are due to the enlirely different environrnent from that al the jimclion where for exam[)le neostigmine and phy.so- sligmine at low eoncenlralions increase the action of ACh hy lhe inhihition of AChK and neostigmine in addilion hy activating the reee[)tor. 'lhe exacl sites at cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan PHILIP ROSENBERG 493 Simp. Internac. 33(2):477-5Ü8, 19H6 vvliifh tliese toin|joiin(ls aiitapoiiizt' lhe |K‘iielialion uf ACh is iiol known. hiit it must he kepl iii mind, lhat llie Schwatiii eell siirroumhng lhe squid gianl axon is alioul 2000 A thick eompared to lhe 80 A thickness of lhe excilahle niemhiaiie itself. Externally added ACh decreases lhe action |)oteiilial and de|)olarizes lhe rahl»it vagus after renioval of llie sheath (48). Hovvever. lhe likelihood of lliere l)eing a physiological significaiice to this effect was eoiisidered remote hecause neostigmine and olher conij)ounds wliicli had no effecl on conduction antagonized lhe action of ACh (50). As in llie sqnid axon lhe condneting niemhrane in lhe vagus pre])aration is surrounded l)y a large layer of otlier structures. and thus il appears lo us likely lhal an effect on penetralion similar to lhat descrilied here is lhe exjdanation for their findings. InCREASEI) CnOl.INESTERASE ACTIVITY GE IXTACT CEI.LS CAESEI) BY VE.XOMS To determine vvhether snake venom increa.sed permeahilily to ACh in jjie- paralions olher than lhe squid giant axon it was necessary to use techniques nol requiring lhe extrusion of lhe cells contents. It lias heen shown thal lhe ratio of Chf] activity in an intact and homogenized preparation, to vvhich we shall refer to as the jiermeahility constant (PC), serves as an index of lhe iiermeahility harrier (51). If the CliE of the tissue were completely accessihle to ACh, which is used as the suhstrate for measiiring the ChE activity, then the PC wonld he 1, whereas complete inaccessihilily would give a PC ratio of 0. The PC ratios in control |)reparalions of squid stellar nerve, eel eleclrojilax, walking leg nerve of lohster, frog sartorius muscle and rahhit cerehral córtex indicated lhat the eel eleclrojilax has the strongest harrier to ACh whereas the squid stellar nerve has lhe weakesl (Tahle V). The squid stellar nerve is composeil of lhe gianl axon plus small nerve fihers. Since gianl axons contain only of lhe total ChE activity of the squid stellar nerve (52) these figures would no) indicate lhe harrier iiresenl in lhe giant axon. Hecause of lhe relalively low euzyme activity of the giant axon it is nol possihie to measure small changes in its activity. The grealest fraction of ChE of lhe gianl axon is located in the envelojie. The surface arca of lhe giant axon is niuch le.ss than lhe total surface area of lhe small nerve fihers. Therefore, it is nol surprising lhat lhe majorily of lhe (diE is in lhe small fihers. Agkistrodo/i p. piscivorus and lo a lesser exlent Crotalus adainanletis venom caiised an increase in the ChE activity oí intact axons of lhe sqnid stellar nerve. lhe fihers of the lohster walking leg nerve and lhe isolated eel electroplax (Tahle V). The grealer effectiveness of cottonmouth lhan ratllesnake venom in increas- ing |)ermeahilily agrees wilh ])revious findings in olher systems lhal ratllesnake venom is less effective in its ahilily lo disrupl memhranes. Since lhe venoms used have no ChE activity themselves (20,53,54), their effects must he altrihuted lo a greater accessihilily of ChE in the intact preparations as previousiy disciissed. In contrasl to the complete ineffecliveness of Eastern diamondhack ratllesnake venom on lhe squid gianl axon (Tahle 11 and Eig. 10), il increased the permeahil- ity of the sqnid stellar nerve ailhongh less lhan the collonmonlh moceasin venom. The increased permeahility cau.sed hy lhe venoms in lohster nerves agrees wilh lhe findings lhat collonmonlh venom decreases the concenIralioTi of ACli required to affect electrical activity 111 ). cm 2 3 z 5 6 11 12 13 14 15 494 USE OF VENOMS IN STUDIES ON NERVE EXCITATION TABLE V — EFFECT OF VENOMS ON PERMEABILITY OF VARIOUS PREPARATIONS AS JUDGED BY THE ABILITY OF 5 X 10-» M ACETYLCHOLINE (ACh) TO PENETRATE AND ASSAY ALL OF THE AVAILABLE CHOLINESTERASE (ChE) IN INTACT BIO- LOGICAL PREPARATIONS The permeabillty constant (PC) is the ratlo oI ChE activity of intact and homogenized tissue. AP = Agkistrodon piscivorns venom; CA = Crotalus adamanteus venom. AIl ChE activities are expressed as ACh hyd/g/hr except for the electroplax results whlch are presented as iiM ACh hyd/cell/hr (Average cell vveight is 40 mg). Data taken from Rosenberg and Dettbarn (20, 25). TISSUE Venom /ig/ml ChE PC Homog. Intact Squid Stellar Nerve 57 42 0.74 AP 15 57 44 0.77 AP 50 56 54 0.96 CA 50 58 46 0.79 CA 200 55 50 0.91 Eel Electroplax 18 3 0.17 AP 50 20 5 0.25 AP 400 29 10 0.34 AP 1000 32 20 0.63 CA 50 20 4 0.20 CA 1000 32 9 0.28 Walking Leg Nerve of Lobster — — 925 337 0.36 AP 100 900 442 0.49 AP 1000 886 698 0.79 CA 1000 900 475 0.53 Frog Sartorius Muscle 32 13 0.41 AP 1000 32 27 0.84 Rabbit Cerebral Córtex 260 138 0.53 AP 1000 285 152 0.53 CA 1000 256 156 0.61 Permealiility l>ariiers are j)re.çent iii the intact ral)l)it cereliral córtex pliees as is sliown hy the a])|)roximately doiihling of ChE activity oh.served ii])on homo- genization, hut neither venom vvas ahle to significantly increase the accessihility of the enzyme to ACh in tliese slices. This is in agreement with earlier stndies showing that Naja naja venom cannot hydrolyze ])hos|)holipids in hrain slices (55). As we shall discnss in Section 8. s])litting of phospholipids might he essential for increasing permeahility. An interesting and imexpected finding was the demons- tration in homogenized electrojilax of strong permeahility harriers. This was the only |)reparation in which the venoms increased the ChE activity even of homo¬ genized tissne. The harriers in the homogenized tissiie conld he ])artially eliminated hy longer jieriods of homogenization or hy freezing and thawing of cells; how- ever, the use of venoms appeared to yield the highesl enzyme activity. Jn the homogenized electroplax hoth venoms were ahout eqnally effective in contrast to cm SciELO 10 11 12 13 14 15 16 Mem, Inst. }5utantan PHILIP ROSENBERG 495 Slmp. Internac. 33(2):477-50í<, 1966 llie weaker effects of Crotulus venoin iii iiilact jireparations. This niay iiulicate an inahility of rattlesnake veiiom comjionents to ])enelrate lo siilistrate sites in the inemhraiie of intact preparatioiis (see section 8). Ueraiise, as disciisscd ahove, suhstrates siich as ACh do nol jienelrate to all of the ChE it is diffieiilt to determine the total enzyme aclivitv in intact nerve fihers. In studies aimed at measuring ChE activity of a tissiie following exposnre lo an “irreversil)le” organophosphale ChE inhihitor, it is essential lo measnre activity in intact preparations, since excess inhihition diie to tlie jiresence of lincomhined inhihitor inay occiir at the time of homogenization. Several ])ro- cediires have heen nsed in attempts to measiire the total activity in the intact preparation. Hovvever none have heen conipletely satisfactory (56, 57). Our re¬ sidis indicate that venoms may he useful in future studies where it will he essential to ohtain some indication of lhe total ChE activity in intact jireparatious. IJSE OF VENOMS IN TESTlNG FOK ESSENTIALITY OF CHOLlNESTERASE IN CONDLCTION The ahility of venoms to decrease permeahility harriers in the sqiud axon and ex])ose ChE to ACh (Fig. 10, Tahle V) offered lhe jiossihility of designing experinients to test for the essentiality in nerve conduction of ChE, the enzyme vvhich hydrolyzes ACh. Organophos])hates, such as diisopropyl jihosphorofluoridate (DEP) or diethyl-p-nitro]ihenyl phosphate (Paraoxon) jihosphorylale and therehy irreversihly inhihil ChE and hlock electrical activity (3-51. It is possihle to re- activale the phos|)horylated enzyme wilh certain nucleophilic compounds among which pyridine-2-aldoxime methiodide IPAM) is extremely potent and has a high degree of specificily for ])hos[)horylated ChE (58). ff hlock of conduction hy organophosphates is specificallv due lo hlock of ChE and if ChE is essential for conduction as postulated hy Nachmansohn (3-5) it might he jiossihle hy using PAM lo specifically restore conduction. Experinients of this lyjie are extremely difficult to carry ont however, hecause PAM, like ACh and curare heing a lijiid insoluhle quaternary nilrogen compound cannot penetrale lhe conducting mem- hranes of nerve and nurscle. In the lohster axon where lhe permeahility harrier is less as evidenced hy the ahility of ACh to directly hlock conduction (10,11). it was possihle to overcome conduction hlock hy Paraoxon with PAM (59). It was lherefore worthvvhilc' lo lesl on lhe venom Ireated squid axon, where the permeahility harriers are retluced and the aclive sites of ChE exposed, whelher PAM could restore electrical activity which had heen liloeked hy DEP or Paraoxon. It was found that in axons not venom pretreated lhe hlock of conduction hy Paraoxon, iu the concenlrations used and lhe lime of exjiosure appiied, was reversihle whereas in axons ireated wilh 25 jiig/ml coltoumoulh moccasin venom lhe hlock was irreversihie (25). At lhe lime of reversihle hlock of conduction ChE activity is stili presenl and in no case was conduction found in the ahsence of enzyme activity (3-5,60,61 ). This indicates that Paraoxon may he reacting with some component of lhe memhraue other than ChE, possihly with the active site of lhe receptor, to cause reversihle hlock. The irreversihie conduction hlock hy Paraoxon after venom may he either hecause of the greater exposnre of ChE to Paraoxon or hecause venom prelreatrnent inactivales the enzyme which hydro¬ lyzes organophos|)hates. It was found that lhe squid axon has eonsiderahle amounts of this enzymatie activity, holh in the envelope and in the axoplasm (62). Conduction iu 11 out of 16 venom Ireated axons was irreversilily hlocked hy cm 2 3 z 5 6 11 12 13 14 15 496 USE OF VENOMS IN STUDIES ON NERVE EXCITATION Paraoxon, whereas iii axons exposed lo PAM followiTig Paraoxoti llu> lilock of conduction l)v l^araoxoii was irreversilile iii onlv 6 of 17 axons (25). An aclnal ex|)erjment wilh Paraoxon and PAM is sliown in Fig. 12. I’AM aiso restored electrical activity which had I)een lilocked hy DFP|25). These exjicrinienls confirmed lhe inseparahle associalion helween eleclrical and CliF aclivily. B _ í Fig. 12 MeCHAMSM of VENOM ACTIO.N ON THE SQUII) GIANT AXON In addilion lo non-enzyniic íoni|)onenls venonis conlain inany enzymes in- clnding plios])holi|)ase A, hyaliironidase, jiroleolylic enzymes, 1-ainino acid oxidase, phosjdiodieslerase, ele. (15, 16,61-66). Venoms oí lhe ELAPIDAE such as Naja naja venom liave FhF aclivily (55,51). ll is ohviously of greal inlerest lo de- lermine lhe comiMinent (or coinjionenls) of lhe venonis responsihie for increasing llie jierrnealiilily of lhe sqnid giani axon. The effecls of varions venom fraclions, enzymes and jihospholipids are shovvn in Talile VI. Thosí' componnds which hlocked lhe aclion |)olenliaI did so irre- versihly. The concenlralions of lhese agenls nsed as |)relrealmenl had no effecls on lhe aclion polenlial. The effeci of curare following phospholipase D was ir- reversihle whereas all olher curare effecls were reversihle. A non dialyzahie fraclion of Naja. naja venom rich in |)hos])holipase A (17, 67) was more polenl lhan Naja naja venom (Tahie II) holh in ils direcl effecls on lhe aclion polenlial and as a prelrealmenl ageni (Tahie VI). ACh aIso affecled condnclion afler lhe |)hos|)holi|)ase rich fraclion. In conirasl a dia¬ lyzahie fraclion of cohra venom poor in ])hospholi])ase A (17,67), recpiired relalively high concenlralions lo hlock condnclion, and following jirelrealmenl of lhe axon wilh il curare was com|)lelely inaclive (17). The comjiaralive phos|)ho- li|)ase A aclivilies of lhe rich and poor fraclions shown in Fig. 15, indicale lhal lhe rich fraclion is al leasl 200 limes as aclive as lhe poor fraclion. cm SciELO 10 11 12 13 14 15 16 Simp. Internac. Mem. Inst. Butantan 33(2):477-508, 1966 PHILIP ROSENBERG 497 TABLE VI — CONCENTRATIONS OF SEV^ERAL VENOM FRACTIONS, ENZYMES AND PHOSPIIOLIPIDS USED AS PRETREATMENTS TO RENDER THE SQUID AXON SEN- SITIVE TO CURARE, AND CONCENTRATIONS REQUIRED TO BLOCK THE CONDUCTED ACTION POTENTIAL (AP) Compouncis were applled for about .30 minutes. > indicates that at the eoncentration shown no effect on AP was observed. + + +, ++, and — indicate respectlvely a 80- 100%, 50-80% and 0-20% decrease in the AP produced in 30 minutes or less by 1.4 X 10-» M curare after pretreatment ot the axon. OH— and H-h indicate.s that venom solutlon was bolled for 15 minutes at pH 8.5 or 5.5 after which the solutlon was cooled, pH readjusted and then tested. Data taken from Rosenberg and Podleski (15) and Rosenberg and Ng (17). COMPOUND tig/m\ Curare effecl AP block Pretreat Phospholipase A rlch fractlon 20 2 + -H- Phospholipase A poor fractlon 200 100 - - Agkistrodon j). piscivorus OH- > 1000 200 — Agkistroãon p. piscivorus H4- 200 50 -n- -t- Naja naja OII- > 400 200 — Naja 7iaja H-t- 100 50 + + Phospholipase C > 500 500 — Phospholipase D > 100 10 -I- + L-Amino acid o.xidase > 100 100 — Hyaluronldase > 100 100 — Cobroxin 500 200 — Cobra venom Neurotoxin > 250 250 — Lysoleclthln 500 100 — Egg lecithin > 1000 Beef heart lecithin > 1000 Pliospliolijiase A in venoins is rtisislanl lo lioiling al acid pH wherras it is destroyed liv hoiling at aii alkaiine ])H (68-70). The otlier enzynies known lo lie present in venom are destroyed l)y lioiling al acid or alkaiine ])H. Some cm SciELO 10 11 12 13 14 15 498 USE OF VENOMS IN STUDIES ON NERVE EXCITATION investigators tlierefore use acid heated veiiom Solutions as their source of plios|)ho- li])ase A. As seen iti Tahle VI alcaliuc heated Solutions of A. piscivorus and Naja naja did not render curare active while acid heated Solutions vvere effective. An acid heated solution of cobra venom has also been found to de])olarize and hlock conduction in lohsler axons(30). The comparative phospholipase activities of lhe acid and alkaline heated Solutions are shown in Fig. 14. These results also indicated that ])hospholipase A is res])onsihle for decreasing the ])ermeahility bar- rier in the squid giant axon. Neither ]-amino acid oxidase nor hyaluronidase, both of which are ])resent in snake venoms, had any effecl on the squid axon (Tahle VI). A preparation of phos[)hodiesterase frotn venom ajijieared to be highly |)otent, however \ve found it to contain eonsiderable phospholipase A activity (17). Two different ])repara- lions of nenrotoxin from N. naja venom (Cobroxin and cobra venom neurotoxin), although higbly toxic to animais were also inactive on the squid axon. No relalionshij) has been found between venom toxicity and any of the enzymatic fractions in venom (64, 71-74). As noted in Tahle VI three |)hospholi])ids were inactive as also were ])ho.s[)hatidylelhanolamine, [)hos|)balidylserine, L-a-cephalin and L-«-lecithin. Phospholipases C and D are not presenl in snake venoms and we shall discuss their actions laler. The results shown in Tahle VI indicate that phospholijiase A of venoms is responsible for their ability lo iticrease lhe jiermeability of the squid axon to normally impenetrahle lipid-insoluble com|)ounds. Also in suj)j)ort of this su])- posilion is the finding that Nolrchis scutata.s a venom highly potent in our sludies (Tahle 11) also contains high eoncentralions of ])hospholipase A (75). In contrasl Centruroide.s scalpturatas (scorpion) venom which contains no ])hos])holipase A (76) was inactive in onr test syslem (Tahle II), although it produced repetitive firing of the sqidd axon (24). As shown in Fig. 15 ])hospholi])ase A hydrolyzes a íally acid ester from the (i ])osition of eertain glycerophosjíhatides yielding the cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Slmp. Internac. 3S(ü):477-508, 1966 PHILIP ROSENBERG 499 O CH,—o—X, I ,D / R—C—O—CH o i t CHj—O—P—O—X,.—N(+)—Xj / I O(-) X. Xj X, Phosphatidylcholine (lecithin) ü II C—R (CH.,)., (CH,); Phosphatidylcthanolamine O II C—R (CH.,), H 3 Phosphatidylserine O II C—R CH.^—CH 1 H, Phosphatidalchoiine CH=CH—R - COÜ(-> (CH,), (CH,); (choline plasmalogen) Fig. 15 corres])oiiding lysopliosphatide. 1’revious studies hy many workers have showii lhat phospholipase A is caj)alile of disrupling memhranes (33, 34,69, 77,78). The soiirce of ])hosj)liolipase A in mosl of these sliidies was acid heated solulions of venom. Since we ol>served such a great variahilily in venom j)olency (Tal)le II) we decided lo compare the |)liospholipase A activities of cerlain venoms. In most of the nieasiireinents of ])hos]diolipase A activities we used egg or heef heart lecithin rather than ])urified sul)slrates l)ecause hydrolysis was slower and in- complete wdth piirified snhstrates (17). Snake venom phospliolipase A has l)cen reported to hydrolyze egg yolk and seriim ])hos])holipids at a rale 10 to 20 fold greater than that olilained with juirified ovolecitliin ])reparations, and it was sug- gested lhat this may he diie to the specific natnre of the comhination of phospho- lipid and protein in lhe crude snhslrate ])rcparation (79). The sample of heef heart lecithin iised contained ahoul 60% phospliatidalcholine (choline j)lasma- logen) and 40% phosphatidylcholine (lecithin). It was re])orled hy Gottfried and Kapport (80) lhat cohra venom hydrolyzes egg lecithin at only a slighlly faster rate than heef lecithin whereas AVeslern diamondhack ratllesnake venom hydrolyzes egg lecithin considerahly helter than heef lecithin. In addition when thcy ohtained purified plasmalogen from heef lecithin the difference in hydrolysis of lecithin and jilasmalogen hy ratllesnake venom was even more marked. Since Kastern diamondhack ratllesnake venom did not render curare active whereas cohra and coltonmoiith moccasin venom did it appeared lhat an action of the venom iijion j)lasmalogen might he responsihle for their ahility lo render curare active. As seen in Fig. 16 we found that collonmouth venom hydrolyzed the two snhstrates at similar rates, whereas rattlesnake venom is somewhat more ef- fective in hydrolyzing egg than heef lecithin. Quantitatively however this dif¬ ference in reaction rale does not a|)pear snfficient to aceonnt for the marketl differences in potency of these two venoms on lhe sqnid axon. When the hydro¬ lysis of heef lecithin hy various venoms was com])arcd it was fonnd that two of the venoms most cffeclive in rendering curare active also hydrolyzed heef lecithin at lhe highest rates, hnt hvdrolysis hy lhe other three venoms did not correlate with their ahility lo render curare active (Fig. 17). It will he necessary to actnally cm SciELO 10 11 12 13 14 15 500 USE OF VENOMS TN STUDIES ON NERVE EXCITATION mcasure the phosjjholipids in tlie squid axoii as wcdl as lhe changes ])roduced hy cotlonniouth and rattlesnake venoms to see if there is a correlaüoii hetween s])lit- tiiig of any j)articular phospholipid and potency of the venoms. The nervous syslem of course eontains many ])hospholi])ids in addition to lecithin and choline j)lasinalogeti ([)hosphatidalcholine) ; for example, phos[)hatidylethanolainine, phospha- tidylserine, |)hosj)hatidalethanolamine, phospfialidalserine, inositol pliosphatides and sphingomyelins (81-87). The concenlralion of phosphalidaleholine is actnally nnich less than thal of phosphatidalelhanolainine (87, 88). Lecithin is nol even a coin- ])arcnt of the myelin lipids whereas j)lasmalogens, inositol pliosphatides and s])hingo- inyelins are (81,89,901. Quantitalively plasinalogens forni one of the largest jiart of lhe jihosjihalides fonnd in while niatler 183, 91). In addition lo lhe possihilily thal lhe phospholipase A in coltoiimoulh and rattlesnake venoms have different siieeificities tow'ard phos|)holi|)id suhstrates it is also possihle thal there are isozymes of phospholipase A vvhieh have differenees in lheir molecular structnre, whieh may explain the marked differenees in lheir jiotencies. ll has heen reported that there are differenees in lhe electro|)horetic migration of lhe ])hos|)holipase A fraetions from different venoms (92, 93). Il has also heen jioinled onl thal it can no longer he assnmed that a single enzyme in each venom is responsihie for the hydrolysis of all phosiiholipid snhslrates (80). In cerlain venoms there liavi^ heen foimd more than one |diospholi|)ase A, each wilh different |)hysical properties (75,91), and different sjiecifie activities when tesled on varions phospholi|)ids. It is of s|)ecial pertinence to our data thal the j)hospholi|)ase A from llinghals and Naja naja venom are ahle lo split phospho- lipids in certain memhranes whereas lhe phos])holi[)ase A from V. palf.slinac is imahle to do so (55,95). Similar differenees conld he res|)onsihle for the marked difference in |)olency hetween rattlesnake and colloninoiilh venom. In addition to the possihilily that the phospholi|)ases from rattlesnake and cottonmonlh venoms have different snhstrale specificities or different molecnle strnclnre there is also lhe possihilily that there is anolher faclor in lhe active venoms whieh |)repares the memhrane for attack hy venom pliospliolipai^c. Such a factor, the ilirect lytie faclor (DLF) has heen isolated from cohra and hee cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 3.S(2):477-50S, 196fi PHILIP ROSENBERG 501 venoms. liiil is nol ])iest>iil iii lerlaiii vijier venonis ( 7H. 93. 96-98). For i'xaiii|)le waslied erytlirocytes are resistani lo lhe aelion of pliospholiiiase A ohlaioed from varioiis veiioms. whereas lhe addilioii of DLF, whieh ilself has no |)hospholi])ase A aclivily rendeis lhe red cell phospliolipids snsceplihle lo atlack liy jiliospliolipase A of lhese venoms (96). Df.F mij 2 ;hl allow venoni jihospholipase A lo slijil llie phospholijiids in lhe inlael sqnid axon. If this faclor were ahsenl in raltlesnake hiit ])resenl in eollonnioiilh venoms this conld exjdain our results. Assnming thal phosjiholipase A is lhe aclive eomjionent of tlie venoms it is of inleresl to knovv whether lhe venom effects are due to a direct aelion of phospholipase A on phos]dioli])ids or dne to lhe liheration, hy lhe aelion of phospholipase A on pliosjdialides, of lysophosphalides in lhe memhrane. For example lysolecilhin a eompoimd wilh delergenl projierlies is formed hy lhe hydro- lysis of lhe fatly aeid esler al lhe 13 posilion of leeithin hy phospholipase A (Fig. 15). It is ihoiighl lhal indirect hemolysis of red hlood cells hy venoms is due lo lhe aelion of lhe lysophosphalides formed from lhe aelion of jdiosjiholipase A on lhe mernhranal phos])holipids (99). Bolh j)hos|)holi])ase A and lysoleeilhin cause demyelinaliug ehanges iu lhe ceulral nervous syslem (83), while lysolecilhiu and venom eause a release of ginlamie-oxaloaeelie Irausamiuase from vvhole eell preparalions (13). Il was aiso suggesled lhal hloek of eondueliou in lohsler nerves hy healed venom solulious may he due lo lhe formalion of lyso|)hos])ha- lides(3()l. Olher invesligalors (77, 78, 100, 101), for various reasons, ihiuk lhal lhe effects they have ohserved are due to a direct aelion of phosjiholipase A and nol due to a liheration of lysojihosphatides. For example, in two of lhese sludies (78,100), ])hospholipase C had a similar effecl as healed venom Solutions which was takeii as evidence lhal a lyso|)hosphatide is nol imporlant in lhe aelion of phosidiolipase A. The aelion of ])hos|)holipase C on ])hos|)hatides does nol give rise to lysophosphalides (Fig. 15). Iu our sludies however phospholi|)ase C was inactive (Tahie VI). Iu altemptiug to decide whether phos[iholipase A is actiug direclly or whether lyso[)hos|)hatides are responsihie for increasiug permealiility an ohservalion made on squid giant axons after removal of olher fihers is of relevante. Afler having found thal ACh was inactive on conduction and unahie lo penelrate hoth on cleaned and less earefully dissecled axons (see section 2 and 3) we have foi- lhe sake of eonvenience used gianl axons wilh only partial removal of surrouud- ing fihers. The apparentiy sur|)rising ohservalion was later made thal collon- nioiith venom is |)ractically inerl on axons earefully dissecled, lhal is, wilh all sinall iierve fihers aud as much conuective tissue as possihie removed(15). Fol- lowing ap|)licatiou of even high coucenlrations of cottonmouth venom on finely dissecled axons, curare and ACh were inactive and peuetraled no hetter lhan iu axons nol prelrealed wilh venom (23). These results are iu marked conlrasl to effects of lhe delergenl celyltrimelhviammonium hromide whith is known lo direcl¬ ly disru|)t mcmhraues, and which was even more polent ou lhe fiuely dissecled axon lhan on lhe preparation wilhout removal of small fihers (23). A likely explanation for lhe ohservalion wilh venom on cleaned axons is thal lhe venom is actiug through lhe formalion in or near lhe memhrane of a secoudary |)roduct which iu luru affects electrical aclivily hy disru|)ling liarriers eilher withiu or externai lo lhe memhrane. Iu lhe closely dissecled axon there would he less suhslrate on which phospholipase A conld act aud therefore less lysojihosjihatides formed. The lyso comjiouuds may aIso increase lhe aetivity of |)hos|)holipase A (102). Attempts were made to demonstrale lhe produetiou hy coltoumouth venom of a eompound wilh polenl effeels on electrical aetivity. The venom was ineuhated cm SciELO 10 11 12 13 14 15 502 USE OF VENOMS IN STUDIES ON NERVE EXCITATION vvith sqiiicl axons and with t'gg and lieef lecilhin, hiit no coni|)onnd was ohlained froni the incuhation mixture with potent effects on electrical activity. Tliese negative results ohviously do nol exclnde lhe possiliility that such cornpoimds may he íormed since adding a lyso[)hosphatide extcrnally may not give results eqnivalent to its formation within lhe preparation. Although phospholi])ase C and D are not present in venoms, the effects of lhese two comj)oiinds on the squid axon are interesting. While neither of them had any direct effects on the action ])otential, ])hospholipase D even in relatively low amounts rendered curare active (Tahie VI). The hlock of conduction hy curare after phospholipase D could however not lie reversed. Hydrolysis of phosphatides hy {)hospholipase D removes a cationic nitrogen 11'ig. 15). One may specidate that the cationic nitrogens of curare may he attracted to the negatively charged phosphates. The curare molecules may pass from one ])hos])hate lo another and finally penetrate to the active sites of tlie neuronal memhrane and affect electrical activity. The concentration gradient of curare and its hinding might he such that with washing it would he difficult to remove enough of lhe curare to reverse its effects on electrical activity. Hydrolysis of phosphatides hy phospholij)ase C removes the j)hosphate grou[)ing as well as the cationic nitrogen leaving an uncharged diglyceride which would not he expected to hind curare. SUMMARY It has been possible with the aid of venoms to analyze some of the effects which the structure and organization of biological membranes and their environ- ment have on the physiological functioning of the acetylcholine (ACh) system and on the action of compounds when appiied externally to biological tissues. 1) Lipid soluble tertiary nitrogen containing compounds such as atropine and physostigmine which are known to interact with the ACh system at junctions also block conduction in lhe squid giant axon, indicating the presence of the ACh system in this preparation. In contrast lipid in.soluble quaternary nitrogen con¬ taining compounds such as ACh and curare do not affect conduction of the a.xon. 2) ACh and curare reversibly block conduction after pretreatment of the squid axon with concentrations of certain snake venoms which by themselves have no affect on conduction. Of 21 venoms tested Agkistrodon p. piscivoriís (cotton- mouth moccasln) venom was the most effective in rendering the axon sensitive to curare whereas Crotulus acUivwmteus (Eastern diamondback rattlesnake) venom was completely inactive. 3) ACh and curare do not penetrate through the structural barrier surround- ing the excitable membrane of the giant axon in contrast to lipid soluble tertiary nitrogen derivatives which can penetrate. Cottonmouth moccasin venom markedly increased the permeability of this structural barrier as evidenced by increased penetration of radioactive ACh, curare and choline. In contrast Eastern diamond¬ back rattlesnake venom did not increase permeability. Cottonmouth venom also increased the permeability of the barrier to various sugars, amines and amino acids. The barrier is formed presumably to a large extent by the Schwann cell. Pre- liminary electron microscopic studies indicate that fragmentation of the Schwann cell surrounding the giant axon by venom may be responsible for the increased permeability. 4) Choline, neostigmine and physostigmine when appiied in combination with ACh to venom treated axons markedly decreased the effect of ACh. This action was shown to be due to a competilion for sites of penetration, thereby allowing less ACh to reach the excitable membrane!. cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Slmp. Internac. 33(2):477-508, 1966 PHILIP ROSENBERG 503 5) In addition to increasing penetration of ACh in the squid giant axon, cottonmouth venom aiso increased its penetration in the squid stellar nerve, isolated single eei electroplax, walking leg nerve of lobster and frog sartorius muscle. 6) It was possible to demonstrate the essentiality of cholinesterase for con- duction on the venom treated squid giant axon. The organophosphate choline¬ sterase inhibitor Paraoxon caused a block of axonal conduction which could not be reversed by washing with sea water but which was readily reversed by pyridine aldoxime methiodide, a specific reactivator of organophosphate inhibited choline¬ sterase. 7) It appears that phospholipase A is at least one of the components res- ponsible for the action of venoms on the squid giant axon. Acknowledyements — For encouragement and advice as well as assistance in preparation of this manuscript, I am grateful to Professor David Nachmansohn. I am pleased to acknowledge the collaboration in many of these studies of Drs. W-D. Dettbarn, F.C.G. Hoskin and T. R. Podleski, all of whom I would like to thank. The Marine Biological Lab., Woods Hole, Mass. where many of the studies were carried out deserves special thanks for the facilities which they made available to us. I also would like to express my appreciation to all those who provided samples of several of the compounds used in these studies, as acknowledged in the original publications. Legends Fig. 1 — Schematlc presentation of the elementary process controlling membrane permeabllity during electrlcal activity and integration of ACh into the metabolic path- ways. The role of ACh In the permeabllity cycle may be pietured as follows: 1) In rest- Ing condltion ACh is bound to a storage proteln (S). The membrane is polarized. 2) ACh is released by current flow; the free ester combines with the receptor protein (R). A conformational change of the receptor (symbolized by dotted llne) leads to a shift of charge; this process increases permeabllity; it is the trlgger action by which the ionic concentration gradient becomes effective. 3) The ester receptor complex is in dynamic equilibrium with free ester and receptor. The free ester is attacked by ACh-esterase (E). 4) The hydrolysls of the ester permits the receptor to return to its original condltion. The permeability barrier is reestabllshed and the membrane is repolarlzed (From Nach¬ mansohn, 3). F\g. 2 — Cross section of the giant axon of squid (above) compared to that of rabbit sclatlc nerve (below) at the same magnlficatlon (From Young, 13). Fig. S — Effect of 1.4X10-'’ M curare on squid axon rinsed in normal sea water CW) for 10 min. (left) and for 95 min. (rlght) alter the application, per ml, of 10 fig of cobra venom (CV) plus 20 jig of cetyltrimethylammonium chloride (CTA) (From Rosenberg and Ehrenpreis, 14). Fig. 1) — Effect of d-tubocurarine chloride (curare) on the giant axon of squid fol- lowlng pretreatmcnt with 15 /jg/ml cottonmouth moccasin venom. S. W. indicates return to sea water (From Rosenberg and Podleski, 15). Fig. 5 — Effect of curare on the restlng and action potential of the squid giant axon following exposure to cottonmouth venom. A, control; B, after exposure to 15 /rg/ml venom for 15 min.; C, 15 min. after return to sea water (stlmulus voltage remained constant for this period); D, E, 4 and 8 min. after exposure to 1.4 mM curare; F, 22 min. after return to sea water (From Rosenberg and Podleski, 16). Fig. 6 — Effect of 67 íig/ml of hoodcd cobra venom on the resting potential (. —.) and action potential (o — o) of the giant axon of squid (From Rosenberg and Podleski, 15). Fig. 7 — Effect of 50 /íg/ml of cottonmouth moccasin venom on the restlng potential (. —.) and action potential (o—o) of the giant axon of squid (From Rosenberg and Podleski, 15). 1 SciELO 504 USE OF VENOMS IN STUDIES ON NERVE EXCITATION Fi(i. S — Effect of acetylcholine (ACh) on the electrical activity of the giant axon üf squkl following exposure to 15 /Jg/ml o£ cottonmouth moccasln venom. S. W. indicates return to sea vvater (From Rosenberg and Podieski, 15). Fi;i. !> — Effect of acethylcholine on the re.sting and action potential of the squid giant axon following e.xposure to cottonmouth venom. A, controi; B, after exposure to 15 íig/mi venom for 30 min.; C, 15 min. after return to sea vvater; D, E, 15 and 25 min. after exposure to 4.4X10-= M ACh; F, 30 min. after return to sea vvatei (From Rosen¬ berg and Podieski, 16). Fif/. 10 — Penetration of radioactivcly (C") labeied ACh, choline, dimethylcurare and trimethylamlne into the axoplasm of squid giant axon with and vvithout exposure to cottonmouth moccasin venom. The percentage indicates the radioactivity of the axoplasm compared to that In the outside fluld. The figures belovv the columns Indlcate the /tg of venom/ml. In contrast to the moccasin venom, that of the Eastern dlamondback rattlesnake (R. S.), even In 1000 /ig/ml had no signlflcant effect on the penetration of dimethylcurare. Trimethylamlne readily penetrates with and vvithout exposure to venom (accnrding to data of Rosenberg and Iloskln, IS). Fi;/. 11 — Electron micrographs of squid giant axons A, controi and B, after treat- ment with 100 /ig/ml Nuju naja venom. KMnO,-Pb stalns used. Magnificatlon of A = 17,000, B = 25,000. AX = a.xoplasm; SC = Schwann cell; CT = connective tlssue. The a.xolemma of the squid giant axon is between Schwann cell and axoplasm. Vesiculatlon and fragmentatlon of venom treated Schwann cell is scen. (By courte.sy of Dr. David Robertson unpubllshed observations). Fíd. 12 — Restoratlon by pyridine-2-aldoxime methiodide (PAM) of electrical activity blocked by Paraoxon in venom treated squid giant axon. A, controi; B, after exposure to 25 vug/ml cottonmouth moccasin venom for 30 min.; C, D after exposure to 0.01 M Paraoxon for 2 and 5 min.; E, 30 min. after return to sea vvater; F, G, after e.xposure to 0.05 M PAM for 5 and 20 min. Time slgnal is 750 cycles per second (From Rosen¬ berg and Dettbarn, 25). Fiíi. 13 — Phospholipase A activities of 5 /ig of phospholipase A rich (Rich) and 100 /ig of phospholipase A poor (Poor) fraetlons of Naja naja venom and activity of 5 /ig of Naja naja venom (Venom). 1 mg of egg (-) or beef (— — —) lecithin vvas the substrate. Results are shovvn as means ± S. E. of the mean (From Rosenberg and Ng, 17). Fiil. Ht — Phospholipase A activity of cottonmouth moccasin venom heated at pll 5.5 (11+) and pll S.5 (OH — ). Results are shovvn as means ± S. E. of the mean. Egg leci- thln (1 mg) vvas the substrate (From Ro.senberg and Ng, 17). Faj. 15 — Structure of severai glycerophosphatides. A, C and D indicate the points of hydrolysis by pho.spholipa.se A, C and D, respectively. R = hydrocarbon Chain (From Rosenberg and Ng, 17). Fii/. 16 — Phospholipase A activity of cottonmouth moccasin (C.M.) and Eastern dlamondback rattlesnake (E.R.) venom. 1 mg of egg (-) or beef (— — —) lecithin vvas the substrate. Results are shovvn as means ± S. E. of the mean (From Rosenberg and Ng, 17). Fiii. n — Phospholipase A activities of 5() /ig of cottonmouth moccasin (C.M.), hooded cobra (II.C.), Russells vlper (R.V.), Eastern diamondback rattlesnake (E.R.) and king cobra (K.C.) venoms. 1 mg of beef lecithin was used as substrate. Results are shovvn as means ± S. E. of the mean (From Ro.senberg and Ng, 17). Hkieke.nces 1. HODGKIN, A. L., The Conduction of the Nerve Impulse, C. C. Thomas, Springfield, Illinois, 1964. 2. ECCLES, J. C., The Physiolo(jy of Syndp.ses, Academic Press, Inc., New York, 1964. cm SciELO 10 11 12 13 14 15 16 Mem. Simp. 33(2): Inst. Butantan Internac. 477-50.S. 1966 PHILIP ROSENBERG 505 21 . NACHMANSOHN, D., Chemical and Molecular Busis of Nerve Activity, Aca- demic Press, Inc., New York, 1959. NACHMANSOHN, D., in M. SELA (Editor), New Perspectives in Biology, Vol. IV, Elsevier Publ. Co., B. B. A. Library, New York, 1964. NACHMANSOHN, D., Chemical Control of the Permeability Cycle in Excitable Membranes During Electrical Activity, Ann. N.Y. Acad. Sei., in press. ROTHENBERG, M. A., SPRINSON, D. B., and NACHMANSOHN, D., J. Neiirophysiol., 11, 111, 1948. BULLOCK, T. H., NACHMANSOHN, D., and ROTHENBERG, M. A., J. Neuro- physiol., 9, 9, 1946. DETTBARN, W-D., Nature, 186, 891, 1960. ARMETT, C. J., and RITCHIE, J. M., J. Physiol. (Lond.), 15.5, 372, 1961. DETTBARN, W-D., and DAVIS, F. A., Biochem. biophys. Acta (Amst.), 66, 397, 1963. DETTBARN, W-D., Life Sciences, 12, 910, 1963. WALSH, R. R., and DEAL, S. E., Amer. J. Physiol., 19", 547, 1959. YOUNG. J. Z., Doubt and Certainty in Science, Oxford Univ. Press, New York, 1952. ROSENBERG, P., and EHRENPREIS, S., Biochem. Pharmacol., 8, 192, 1961. ROSENBERG, P., and PODLESKI, T. R., J. Pharmacol. exp. Ther., 1.37, 249, 1962. ROSENBERG, P., and PODLESKI, T. R., Biochim. biophys. Acta (Amst.), 75, 104, 1963. ROSENBERG, P., and NG, K. Y., Biochim. biophys. Acta (Amst.), 75, 116, 1963. ROSENBERG, P., and HOSKIN, F. C. G., J. gen. Physiol., 46, 1065, 1963. HOSKIN, F. C. G., and ROSENBERG, P., J. gen. Physiol., 47, 1117, 1964. ROSENBERG. P., and DETTBARN, W-D., Biochem. Pharmacol., 13, 1157, 1964. BRZIN, M., DETTBARN, W-D., and ROSENBERG, P., Biochem. Pharmacol,, 14, 919, 1965. HOSKIN, F. C. G., and ROSENBERG, P., J. gen. Physiol., 49, 47, 1965. ROSENBERG, P., and HOSKIN, F. C. G., Biochem. Pharmacol., 14, 1765, 1965. ROSENBERG, P., Toxicou, 3, 125, 1965. ROSENBERG, P., and DETTBARN, W-D., Proc. of the International Sym- posium on Animal Veno7ns, Pergamon Pub. Co., London, in press. ROSENBERG, P., HIGMAN, H., and NACHMANSOHN, D., Biochim. bio¬ phys. Acta (Amst.), 44, 151, 1960. ROSENBERG, P., and HIGMAN, H., Biochim. biophys. Acta (Amst.), 45, 348, 1960. HIGMAN, H., PODLESKI, T. R., and BARTELS, E., Biochim. biophys. Acta (Amst.), 75, 187, 1963. BARTELS. E., Biochim. biophys. Acta (Amst.), 109, 194, 1965. TOBIAS. J. M., J. cell. comp. Physiol., 46, 183, 1955. cm SciELO 10 11 12 13 14 15 506 USE OF VENOMS IN STUDIES ON NERVE EXCITATION 31. RUSSELL, F. E., and LONG, T. E., in H. R. VIETS (Editor), Second int. aymy. on Myasthenia gravis, Vol. III, C. C. Thomas, Springfield, 1961, p. 101. 32. KELLAWAY, C. H., and HOLDEN, H. F., Aust. J. exp. Biol. mecl. Sei., 10, 167, 1932. 33. MORRISON, L. R., and ZAMECNIK, P. C., Arch. Neurol. Psychiat. (Chie.), 63, 367, 1950. 34. BIRKMAYER, W., and NEUMAYER, E., Dtsch. Z. Nervenheilk., 17 1957. 50. 117, 35. MINTON JR., S. A., in E. E. BUCKLEY, and N. PORGES (Editors), Venoms, Amer. Ass. Advanc. Sei., Washington, 1956, p. 145. 36. RUSSELL, F. E., J. Amer. med. Ass., 177, 903, 1961. 37. MITCHELL, R., TRUANT, A. P., and CLARK, B. B., Proc. Soc. exp. Biol., 81, 5, 1952. 38. VILLEGAS, R., and VILLEGAS, G. M., J. gen. Pliysioh, 43 (suppl. 5), 73, 1960. 39. VILLEGAS, G. M., and VILLEGAS, R., J. Ultrastruct. Res., 3, 362, 1960. 40. BROWN, R. V., Amer. J. Physiol., 130, 613, 1940. 41. HADIDIAN, Z., in E. E. BUCKLEY, and N. PORGES (Editors), Venoms, Amer. Ass. Advanc. Sei., Washington. 1956, p. 205. 42. WHITTAKER, V. P., Biochem. Pharmacol., 5, 392, 1961. 43. MeARDLE, B., THOMPSON, R. H. S., and WEBSTER, G. R., J. Neicro- chem., 5, 135, 1960. 44. ISSELBACHER, K. J., Biochem. biophys. Res. Commun., .5. 243, 1961. 45. ZELLER, E. A., in J. B. Summer, and K. Myrbaek (Editors), The En- zymes, Vol. I, Aead. Press, N.Y., 1951, p. 986. 46. BUCKLEY, E. E., and PORGES, N. (Editors), Venoms, Amer. Ass. Advane. Sei., Washington, 1956. 47. GHOSH, B. N., and SARKAR, N. K., in E. E. BUCKLEY, and N. PORGES (Editors), Venoms, Amer. Ass. Ad\'anc. Sei., Washington, 1956, p. 189. 48. ARMETT, C. J., and RITCHIE, J. M., J. Physiol. (Lond.), 1.52, 141 ,1960. 49. CREESE, R., TAYLOR, D. B., and TILTON, B., J. Pharmacol. exp. Ther., 139, 8, 1963. RITCHIE, J. M., and ARMETT, C. J., J. Pharmacol. exp. Ther., 139, 201, 1963. 51. ROSENBERG, P., and DETTBARN, W-D., Biochim. biophys. Actn (Amst.), 69, 103, 1963. 52. BRZIN, M., DETTBARN, W-D., ROSENBERG, P., and NACHMANSOHN, D., J. Cell Biol., 26, 353, 1965. 53. ZELLER, E. A., Experientia, 3, 375, 1947. 54. ZELLER, E. A., Helv. chim. Acta, 32, 94, 1949. 55. KLIBANSKY, C., SHILOAH, J., and DE VRIES, A., Biochem. Pharmacol., 13, 1107, 1964. 56. DETTBARN, W-D., and HOSKIN, F. C. G., Biochim. biophys. Acta (Amst.), 62, 566, 1962. 57. DETTBARN, W-D., and ROSENBERG, P., Biochem. Pharmacol., 11, 1025, 1962. cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Slmp. Internac. ;<3(2):477-508, 1966 PHILIP ROSENBERtJ 507 58. 59. 60. 61. 62. WILSON, I. B., and GINSBURG, S., Biochim. biophijs. Acta (Amst.), 18, 168, 1955. DETTBARN, W-D., ROSENBERG, P., and NACHMANSOHN, D., Life Sciences, 3, 55, 1964. BULLOCK, T. H., GRUNDFEST, H., NACHMANSOHN, D., and ROTHEN- BERG, M. J., J. Neurophysiol, 10, 63, 1947. GRUNDFEST, H., NACHMANSOHN, D., and ROTHENBERG, M. A., J. Neu¬ rophysiol., 10, 155, 1947. HOSKIN, F. C. G., ROSENBERG, P., and BRZIN, M., Proc. nat. Acad. Sei. (Wash.), 55, 1231, 1966. ZELLER, E. A., Adviinc. Enzymol., 8, 458, 194S. YANG, C. C., CHEN, C. J., and SU, C. C., J. Biochem., 46, 1201, 1959. BRAGANÇA, B. M., and ARAVINDAKSHAN, 1., in K. A. C. ELLIOTT, I. H. PAGE, and J. H. QUASTEL (Editor.s), Neurochemistry, C. C. Thomas, Spring- field, 1962, p. 840. 66 . 67. GHOSH, B. N., DUTT, P. K., and CHOWDHURY. D. K., Soc., 16, 75, 1939. J. Indian chem. 77. 78. 79. LAL, H., SUMYK, G., and SHEFNER, A., Arch. int. Pharmacodyn, 159, 452, 1966. HUGHES, A., Biochem. J., 29, 437, 1935. BRAGANÇA, B. M., and QUASTEL, J. H., Biochem. J., 53, 88, 1953. MAGEE, W. L., and THOMPSON, R. H. S., Biochem. J., 77, 526, 1960. YANG, C. C., CHIU, W. C., and KAO, K. C., .7. Biochem,, 48, 706, 1960. YANG, C. C., KAO, K. C., and CHIU, W. C., J. Biochem., 48, 714, 1960. NEUMANN, W., and HABERMANN, E., in E. E. BUCKLEY, and N. PORGES (Editors), Venams, Amer. Ass. Advanc. Sei., Washington, 1956, p. 171. MASTER, R. W. P., and RAO, S. S., J. biol, Chem., 236, 1986, 1961. DOERY, H. M., and PEARSON, .1. E., Biochem. J., 78, 820, 1961. MASTER, R. W. P., RAO, S. S., and SOMAN, P. D., Biochim. biophys. Acta (Am.st.), 71, 422, 1963. NYGAARD, A. P., DIANZANI, M. U., and BAHR, G. F., Exp. Cell. Res., 6, 453, 1954. PETRUSHKA, E., QUASTEL, J. H., and SCHOLEFIELD, P. G., Catiad. J. Biochem., 37, 975, 1959. CONDREA, E., DE VRIES, A., and MAGER, J., Biochim. biophys. Acta (Amst.), .58, 389, 1962. GOTTFRIED, E. L., and RAPPORT, M. M., J. biol. Chem., 237. 329, 1962. ROSSITER, R. J., in K. A. C. ELLIOTT, I. H. PAGE, and J. H. QUASTEL (Editors), Neurochemistry, Vol. Hl, C. C. Thomas, Springfield, 1962, p. 10. RAPPORT, M. M., and NORTON, W. T., Ann. Rev. Biochem., 31, 103, 1962. WEBSTER, G. R., Biochim. biophys. Acta (Amst.), 44, 109, 1960. BRANTE, G., Acta physiol. scand., 18 (suppl. 63), 1, 1949. JOHNSON, A. C., McNABB. A. R., and ROSSITER, R. J., Biochem. J., 45, 500, 1949. cm SciELO 10 11 12 13 14 15 508 USE OF VENOMS IN STUDIES ON NERVE EXCITATION 86. McCAMEN, R. E., and ROBINS, E., J. Neiirochem., 5, 18, 1959. 87. HACK, M. H., GUSSIN, A. E., and LOWE, M. E., Comp. Biocliem. Physiol., r., 217, 1962. 88. RAPPORT, M. M., and ALONZO, N. F., J. biol Chem., 235, 1953, 1960. 89. JOHNSON, A. C., McNABB, A. R., and ROSSITER, R. J., Arch. Neuroh Psychkit. (Chie.), 64, 105, 1950. 90. ROSSITER, R. J., in K. A. C. ELLIOTT, I. H. PAGE, and J. H. QUASTEL (Editors), Neurocliemistry, Vol. III, Thomas, C. C., Springfield, 1962, p. 870. 91. RAPPORT, M. M., and LERNER, B., Biochhn. biopltys. Actn (Amst.), 33, 319, 1959. 92. WAKIU, K., and KAWACHI, S., J. PJutrmacol. Soc. (Japan), 79, 1177, 1959. 93. CONDREA, E., DE VRIES, A., and MAGER, J., Biochim. biophys. Acta (Amst.), 84, 60, 1964. 94. SAITO, K., and HANAHAN, D. J., Biochemistry. 1. 521, 1962. 95. KIRSCHMANN, C., CONDREA, E., MOAV, N., ALOOF, S., and DE VRIES, A.. Arch. int. PlKtrmacoclyn., 150, 372, 1964. -V 96. CONDREA, E., MAMMON, Z., ALOOF, S., and DE VRIES, A., Biochim. bio¬ phys. Acta (Amst.), 84, 365, 1964. 97. GENNARO, J. F., and RAMSEY, H. W., Amer. J. trop. Mecl.. 8, 546, 1959. 98. CONDREA, E., and DE VRIES, A., Toxicon, 2, 261, 1965. 99. ROY, A. C., Nature, 1.55, 696, 1945. 100. MELDRUM, B. S., and THOMPSON, R. H. S., Ginfs Hosp. Rep., 111, 87, 1962. 101. ARAVINDAKSHAN, I., and BRAGANÇA, B. M., Biochem. •/., 79, 84, 1961. 102. ROHOLT, O. A., and SCHLAMOWITZ, M.. A) f/í. Biochem.. 94, 364, 1961. Disclssion C. Y. Lee: “I would like to ask your comment on the paper by Feng & Hsieh who demonstrated that the nerve action potential is unaffected after complete in- activation of the acetylcholinesterase by TEPP.” P. Rosenberg: “The difficulty in studies atlempting to correlate cholinesterase activity and electrical activity is the method used to measure enzyme activity. The tissue cannot be homogenized since excess inhibition may oceur al the time of homogenization due to presence of e.xcess uncombined inhibitor. If one uses intact tissue with acetylcholine as substrate then one is only measuring the readily available or “externai” cholinesterase since acetylcholine cannot penetrate to all of the enzyme. We have discussed all of these problems in a publication a few years ago in “Biochemical Pbarmacology”. Feng and Hsieh did not take into consideration all of the sources of error in this type of study.” F. Kornalik: “Have you got all the snake venoms from one source only?” P. Rosenberg: “Most of lhe venoms were obtained from the Ross Allen Reptile Institute, Silver Springs, Florida, except for some of the Australian venoms which were obtained from Light & Co., England, and a few which were kind gifts of investigators from various parts of the U.S.A. and which are acknowledged in my publications.” cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):509-518, 1966 J. H. WELSH 509 54. SEHOTO.MN AND KELATED THYPTAMINE DEHIVATIVES LN SXAKE VENOMS J. H. WELSH BioloçjicaJ Laboratories, Hurvurd Vniversity, Cambrklge, Mass., U.S.A. IXTRODLXTION Cerlain liiogenic amÍTies siich as acptylcholiiie, serotoiiin aiul histamine often are foiind iii veiioms in verv large aniounts compared with other natural sources (Talile ll. Since tliese suhstances are known lo produce ])ain wlien a])|)lied to Elister areas it has Eeen siiggested tlial lliey ])lay a defensive role rather thaii conlriliute iii any importaiit degree to lhe toxieity of the venom (1,2). TABLE I — SOME EXCEPTIONALLY HIGH LEVELS FOR CERTAIN OF THE I.OVV -MOLECULAR WEIGIIT COMPONENTS OF VENOMS Species Common name Value Acetylcholine Reference Vespa crahro (hornet) 10-100 mg/g dry venom (11) 10-50 mg/g dry venom (12) Zyoaena spp. (moths) 1.6-60 mg/g (Accessory se.x glands and ducts. Organs of defense?) (13) Deiulrodfipis 13 species) (mambas) 7-30 mg/g dry venom (3) 5-Hydroxytryptamine (Serotonin) Leiurus quinquestriatus (scorpion) up to 4 mg/g dry venom (14) Phoneutria fera 1 Brazllian spider) 1.5-2.7 mg/g dry venom (15) Vespa, crahro (hornet) up to 19/mg/g dry venom sacs (12) Synoeca sur\y\ama (wasp) 13 mg/g dry sting apparatus (15) Rana plpiens (írog) 0.3-1.0 mg/g wet skin (2) Other AMPHII3IA ímany species) up to 4.5 mg/g dry skin (16) Histamine Ap\s melUfera (honey bee) 10 mg/g dry venom (17) Vespa- vulqaris (wasp) 10 mg/g dry venom sacs (18) Vespa, crahro (hornet) 14-30 mg/g dry venom sacs (12) Supported hy Grant NB-00623 from the Instltute of Neurological Diseases anU Blind- ness, USPHS. cm SciELO 10 11 12 13 14 15 510 SEROTONIN AND líELATED TRYPTAMINE DERIVATIVES IN SNAKE VENOMS Until recenlly, liüle allenlion lias lieen paid lo lhe possihie occurrence of such siilistances in snake vonoms. Acelylclioline has now lieen fourid in large amounts in the venoms of three speeies of D e n d r o a s p i s {'ò). However, the occurrence of serotonin and catecholamines in snake venoms is in disjinte. Zara- fonetis and Kaias (4) report lheir presence in venoms of Crotalus atrox, C. ada- matUcus and Agkistrodon pLmvoriis, luit Anlon and Gennaro (5) failed to find serotonin or norejiinejihrine in the venoms of the two latter s|)ecies. In a further stndy of this ])rohlem the venoms of 20 speeies of elapid, vijierid and crotalid snakes have heen examined hy hioassay, sjieetroflnorometry, and chromalography for the possihie presence of serotonin and related indolealkyl- amines. The results wili he presented in this jiaper. Materiai.s and methods The speeies of snakes whose venoms have heen studied are listed in 'Pahle II. Venoms from seven speeies of Ilrazilian Bothrops were ohtained from the lintantan Institnle throngh the kiiulness of Dr. Hoge. Venom samjiles of the remaining speeies were ])urchased from the Miami (Florida) Serpentarinm. In most instances only one pooled sam[)le of venom from a given s|)ecies was studied. Three methods were employed to iletect the presence of serotonin or related substances. However, some venoms were examined hy only one or two of the following methods: 1) Hioassay. The isolated heart of the ciam. Mercenária [Venus) merce¬ nária. which detects small amonnts of serotonin and related componnds (6), was used aceording to the method descrihed hy Welsh and Twarog{7). 2) Spectrojluorometry. Venoms were extracted of Hogdanski ct al. (8) and finoresiamce and excitation with a Farrand speetroflnorometer in the [iresence of ?>N aceording to the method sjiectra were recorded HCI. 8) Chrornatopraphy. Venoms were extracted with 50 (ler cent acetone or 70 per cent ethanol and the eoncentrate suhjeeted to paper and silica gel chromato- gra[)hy. Several developing systems suitahle for separation indolc componnds were used, as well as several standard methods for visualizing snch snhstances. lIlCStILTS The cedures, follow: results are snmmarized in Tahie II. Certain details eoneerning jiro- and the significance of the results ohtained with eaeh method of assay BIOASSAY — No hioassays were done with venoms of lhe Brazilian speeies of Bothrops. Whole venoms of all other .speeies were tested on one or more isolated Mercenaria hearts. After dissolving in distilled water, from 0.01 mg to 0.25 mg of venom per ml of ])erfusion finid was added to the hath. Tests were made hefore and after treatment of the hearts with henzoquinoninm iMyto- lon) in order to hloek the jiossihle inhihilor action of acetylcholine which is presenl in some venoms (e.g. Dend roas pis in very large amounts (.8)). cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):509-518, 1966 J. H. WELSH 511 TABLE II — SUMMARY OF RESULTS OF THE SEROTONIN THREE METHODS FOR DETECTING 5-HT 5-HT Indole-reacting SPECIES (Spectrofluoro- spots (Bioassay) metry) (Chromatography) ELAPIDAE Elapinae Bungarus fasciatns + ? None detected None Hemachatus haemachatus + ? None detected 1 Naja naja — None cietected 1 Pseudechis mortoneiisis + ? None detected None Dendroaspinae De^idroaspis polylepls — None detected 1 Dendroaspis ancjusticeps — Not run VIPERIDAE VIPERINAE Vipera russeUii + ? 1.9 íig/g 2 Bitis gabonica + 30 /ig/g 4 ClíOTALINAE Crotalus horrulus — None detected 2 or 3 Sistrurus m. harbouri + 6.3 /ig/g 4 Agkistrodo7i contortrix + Not run 4 Agkistrodon piscivorus + 1.7 íig/g 5 Boihrops utrox + None detected 3 B 0 th r o p s (7 Brazilian .specles) Not run None detected Not run Serotoniii and certain rtdaled conipoimds cause an increasc in ampliUide and freqnency of l)eat of tlie Mercenária heart, wilh little increase in toniis lintil relatively liigh concentralions are reached. The catecholamines and histamine also have an excilor action hut the M e r c e n a. r i a heart is far less sensitive to lhese llian to serotonin. Also, lhey cause an increase in tonus resulting in a niarked rise in baseline. Methysergide (UML 491) is an cffective hlocking agent for serotonin on tlie M e r c e n a r i a lieart hut not for catecholamines and hista- inine (9). When a venom was scen to excite the heart it was tested again after the addition of methysergide to the hath. A sample recording of the actions of liitis gabonica and Sistrurus m. hur- boiiri venoms hefore and after methysergide is seen in Fig. 1. The exciter action of liitis venom is completely aholished hy methysergide and that of Sistru¬ rus venom is much reduced. This suggcsts that serotonin or a dose relativo is j)resent in these venoms. The failure of methysergide to hlock completely the exciter action of S i s t r ii r u s venom, at the given dose levei, might he due to the presence of a catecholamine or histamine in this venom. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan J. H. WELSH 513 Simp. Internac. 33(2):5()9-51S, 1966 Heferring lo Tahle II. it iiiay I)i' .seeii lliat. of lhe six elapid venoms tested. aii exeiter aetion on lhe Mercenária heai l was found in B u n u r ii s. II ew achatas and Bseudechis venoms hnl lhese were nol ly|)ically sero- lonindike. Of lhe seven viperid and crolalid venoms lested on lhe Merce¬ nária heari, all hnl lhal from Crotalas hórridas had some serotonindike exciler aetion. In some venoms. notahiy ihose of Bseudechis, S i s I r u r n s, A. pis- civorus and Bolhrops alrox, a calecholatnine or histamine may he ])resent hnl \ve have made no aUem])l lo look for lhese compoimds. The residis from the hioassays are only indicalive of lhe presente of sero- tonin or relaled snhstanees and ihey hecome significant only when vievved in lhe light of the residis from speelrofiiiorometric and chromatogra])hic examinalion of lhe same venoms. SPECTROFLUOROMETRY — In niosi instances. only one samjile of venom of a given species was exlraeted and ils fliiorescence and excitalion s[)eelra reeorded wilh a speelrofluoromeler. Wilh the iiistriiment lhal was nsed, the imcorreeted íliioreseence jieak for serolonin in 8 N HCI ajipears al 510 mp and the excitalion ])eak at 805 mp. (Fig. 2). Venoms of 14 species so examined showed no peaks corresponding wilh ihose for serolonin. Sample records of reeorded specira of ihree of lhese species are reprodiiced in Fig. 2. Extracts of venoms of foiir of the VIPERIDAE showed excitalion peaks or shoulders at 305 m/x hiil a strong fluorescence al 450 m/x ohsciired lhe characterislic 540 m/x peak for serolonin (see, for example, records for Bilis gabonica, Fig. 2). In addilion to the excitalion jieak or shoulder al 805 m/i. Bilis and Sisiru- r u s venoms gave a second excitalion peak near 280 m/x. This might he dne to try|)tamine or one of ils melaholites lhal carne throngh the extraction procedure (see residis from chromalography). Only 10-25 mg of each ernde venom were exlraeted for the sjiectrofhioro- melric analysis. If some venoms contained low' lewels of serolonin lhe characteiistic excitalion jieak might nol have heen detected. Cerlain viperid and crolalid ve¬ noms. however, do give evidente for lhe presente of serolonin hy ihis melhotl. CHROMATOGRAPHY — The residis of oídv one cliromalograjihic jnocednre which was nsetl for all of the venoms (excejtl B o t h r o p s venoms from Bn- tanlan) will he re|)ortetI. This involvetl the nse of ascentiing paper chromato- graphy wilh 10-honrs developmenl in hntanol acelic acid:H20 (60:15:25). Sero- tonin anti olher inilole-reacting spots were matie visihie hy sjnaying wilh |)-di- melhylaminocinnanialdehytle (10). This DM(iA reagent is more sensitive hiil less seleclive lhan EhrlichV reagenl. The nimihers of inilole-reacting s|)ots, made visihie hy ihis procetinre. are given in Tahle II. Of lhe six ela|)id venoms. three gave one s])ot each; two gave none; one was ilonhtfid. Chromalograms of seven viperid anil crotaliil venoms showeti from two to five sjiots made visihie hy the DMCA reagent. In an allempi to iilentify lhe components of the venoms responsihie for lhese several spots, serolonin anil cerlain relaled conijionnds were spotted wilh lhe venoms. The following known snhslances were nsetl: tryjitophan. 5-hydroxy- Iryjitophan. Iryptamine. 5-hydroxytry])tamine (serolonin). hnfolenine. N-methyl- tryplaniine. !\.N’-dimelhyllryptamine. indole. indolyI-8-acetic aciti. and melatonin. cm 2 3 z 5 6 11 12 13 14 15 514 SEROTONIN AND RELATED TRYPTAMINE DERIVATIVES IN SNAKE VENOMS FLUORESCENCE EXCITATION I. Pse u de c hl s i 0 0 50 0 6 0 0 30 0 4 0 0 MU Flg. 2 — Examples of fluorescence and excitation spectra oí a serotonin (5-KT) standard of three venoms shovving no detectable 5-IIT; and of Bitis gaboiüca venom with a characteristic excitation peak at 305 mii. See text for further detail. An examjile of a cliromatograin of S i s t r u r u s venoin rim with six of the standards is reproduct'd in Fig. d. Tlie venom liad heen exiracted with 50 per cent acetone and volumes eqnivalent to 10 and 20 mg of venom sjiotted. Tlie venom may lie seen to have vielded two jnominent and two faint s])ots made visihie hy lhe DMCA reagenl. cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Butantan Slmp. Internac. 33(2):509-518, 1966 J. n. WELSII 515 w Melat. Venom T-phan Venom T-mine 20 mg 10 mg 5-HT Fro n t Venom Bufo. 20 mg Venom 10 ma N-CH^-T Fig. 3 — Chrnmatogram of S i s t r u r u s venom (10 and 20 mg) run in butanol; acetlc acid: vvater with 5 /ig amounts of each of the followlng standards; melatonln (Melat); tryplophan (T-phan); tryptamine (T-mine); .serotonin (5-HT); bufotenlne (Bufo) and N-melhyllryptamine (N-CII^-T). Spraycd with DMCA rtagent. This ehromatogram w:)s dcvelopcd for 12 hrs and the Hf value.s are higher than those for a run of optimal duration (8-10 hrs). riie lowt'sl venom s|)()l lias an lif corrcsjionding closely with thal for sero- lonin (5-HT). The iiexl higher, and niost jirominent, venom s])ot is at a levei near lhal of hiifotenine. There is a faint venom sjiol at the same Hf as N- methyltry|)tamine. Ilowever, this solvent system fails to sejiarate this uinitie from N,I\'’-dimethyltry|)tamine. A fourth faint venom spot is seen near the front. Melatonin rims to this jiosition hiil so do indole and indolyl-5-aeetie aeid. Chromatograms of the venom of Agkistrodon jHscirorus (water moeeasin) gave fonr indole-reaeting sjiots that eorrespond with those from Sistruriis cm SciELO 11 12 13 14 15 16 17 516 SEROTONIN AND HELATKD TRYPTAMINE DERIVATIVES IN SNAKE VENOMS vciioin aiul, iii addilion, a fiflli, latc-apijcariíi" s|)()l willi aii Hf .10 and 40. Tlii.s is dose lo wlicre S-liydroxylryploplian runs in luilanol laeetie aeid:vvaler. Wilh A. piscivorus venotn (20 mp) tlie fast-ninninjí sjiol al Hf 95 was mneli more olivioiis lhan vvilii S i s ! r ii r u x venom (20 rng) : il gave a purple eolor vvith tlie DM(fA reagenl wlueli is aiso Irne for indolyl-,l-aeetie acid. Agkixtro- dofi conlortrix (eopperficad) venom (20 mg) gave no spol al Hf 95. The ollier fonr s|)ols corresponded eiosely willi lliose from A. piscirorns venom. An insnffieient variely of developing syslems liave l)een Iried, tliiis far, lo permil tlie eerlain idenlifiealion of any of lhe indole-reaeling suhslanees in snake venoms invesligaled in ihis sliidy. Il wili he neeessary lo use addilional solvenl syslems and a wider variely of slandard iudole derivalives hefore lhe idenlily of serolonin and relaled snhslauees in any given snake venom is more cerlainly delermined. Discussion Keele and Armslrong (I ). in lheir hook Siihslimcrs Producinf’ Pain and Itch, diseiiss lhe eonslilnenls of snake venoms lhal eonlrihule lo jiroduelion of pain. From lheir eilalious, pain jiroduelion al lhe sile of lhe hile would ajijiear to he more eommou aniong lhe vijierids and erolalids lhan among lhe elajiids. Rradykinin releasing faetors eonslilute oue ela.ss of agenls resjionsihle for jiaiii Jiroduelion in snake venoms. They slale (ji. 217) “Snake venoms do nol eonlain ACh, 5-HT or hislamine, ihongh many of them release hislamine from lissues.” We novv know lhal venoms of lliree sjieeies of l) e n d r o a s p i s eonlain large arnoiinls of aeetyleholine (1). and from lhe residis of lhe jireseul sliidv il is aji- jiarenl lhal some snake venoms eonlain a variely of indole-reaeling eomjionnds, one of whieh is jiroliahly serolonin. This is a highly jiolenl jiain-jirodncer. However, Armslrong has found lhal Iryjilamine and eerlain of ils derivativos other lhan serolonin are aIso very effeelive jirodueers of jiain vvhen ajijilied to enlaneous nerve endings. Aniong lhese are l-hydroxylryjilamine, I\'-melhyl- Iryjilamine, N.N’-dimethyl-5-hydroxylryiitannne ( hnfolenine), and N-melhyl-5-hydro- xylryjilamine. The last is even more effeelive lhan .serolonin (5-hydroxylryjitamine). From lhe ehromalograjihie residis of lhe jireseni sltidy il ajijiears lhal some snake venoms may eonlain al least ihree of lhe.se aetive Iryjilamine derivalives, namely serolonin, liiifolenine, and N-melhyllryjilamine. If serolonin and hufolenine are jiresent in some venoms, then lhe inlermediale. N-melhyl-5-hydroxylryjilamine, may aiso he jiresenl. Il vvill he of mueh inleresl if fiirlher sindy reveals lhal some snake venoms do, in faet, eonlain an assemhly of Iryjilamine derivalives knovvn to he aniong lhe mosi aelive in jirodueing entaneoiis jiain in man. AckHoivledgements — Mytolon was supplied by lhe Sterling-Winthrop Research Institute and methysergide by Sandoz Pharmaceuticals. Carolyn S. Batty. Joyce B. Zipf and Lois D. Williams assisted in Ihis sludy. Hkeehknces 1. KEELE, C. A., and ARMSTRONG, D., Subutances Pro(tucin/i(>lii)asc A aclivily One ml of lenfold saline dihiled egg yolk was incnhaled al 87" for incnxis- ing [teriods of lime wilh 0.05 ml venom or phospholipase A fraclion conlaining 0.5 p.g prolein. lhe mieslerified fally acids (UFA) were del('t'mined hy lilralion using lhe melhod of Dole (16). 'Plie ticlivily was calcnialed as m-ctpiiv. of free cm SciELO 10 11 12 13 14 15 16 Mem. Inst. Hutanlan Simp. Inlernac. 33{2):523-5^0, 196(5 II. I. BICHEIl 525 ari(] n-leased por enzynio fraolioti por niiiiulo. For ox|)orimonls only llioso plios])lio- lij)ase A proparalioiis wore usod wliioh lihoralod al loasl 1.6 ni-ocpiiv. lIFA/rnin por 1 /ig proloiii. Metiious Expcrimrnls on mico Inlraporiloiioal mouso-LD.-,ii of wliole voiioni and ils separate fraclions wero ilolorniinod on looally hrod Swiss all)ino inioo, using 5 animal-s por doso. Fal- rulation of LD.-,„ was mado aooording lo Ilood and Miioncli (17). Exiicrimcnls on cats Tlio oxporiinonts vvoro oarriod oul on anao.slholizod inlaot oals and s[)inal cais, woiglnng 2-d.,5 kg. Anaoslliosia was induood liy ollior and rnainlainod ihrongliont lho ex])oriment hy ropoatod injoolion inlo a fenioral voin of 0.5-1 nd of a mixtiiro oonlaining 10 ing lliioponlono sodiiim and dO /xg alropino sulfalo por nd. Illood clolling was provonlod hy inlravonous adndidstralion of hoparin (5,000 iiidts/nd/kg hody woighl). All aclivilios stiidied wero rooordod on a Grass modol 5 |)olygra])h. Blood pressiiro was rogistered from lhe eannidated feinoral arlery using a Stalham Iransdueer. Hespiralion was moasurod l)y nioans of a iherniooouplo inlroduced inlo llie canniilaled Iraeliea. The oleelrooardiograni was ohlained using needie eloelrodes insorlod inlo lho oxlrondtios. lVri])heral eiroidalion was sliidied l)y [)holo])lelhysniography, using a Hoffman 55-e silicone ])holocell wilh high sen- silivily in lhe infrared range, |daced around llie oaFs |)aw. Variations in inlensily of lho liglil reaohing llio scnsilivo area of lhe ])holooell on Iransillutninalion of lhe oxposod oxlrendly are a measure of varialions in lhe ainounl of hlood in llio liglil |)alh and ihus indioalo va.sooonslriclion (deoroasing volunio) or vaso- dilalion (iticreasing volunio) (1ré|iaration iFélail pas curariséo, mais dans un 2.*' tonips los 2 substancos agis- sont |)our donnor une itdiibilion complèto avant rotour des mouvomonts (Fig. 1). Fig. 1 -- RAT; Contractions du muscle tibial antérieur in sitii. Stimulation alternee du nerf (Indlrecte) et du muscle (directe) toutes les 10 secondes (nerf sclatique: 0,U msec, 3 V — muscle 0,1 msec, KO V). En dT: d-tubo- curarlne — 40 meg/kg l/v: seule la contraction indlrecte est déprlmée. En C: Crotamine — 500 meg/kg i/v: contraclure typique avec reprise fugace des contractions indirectes puis Inhibition complete des deux contractions. Ce muinlien de la contraclure après curarisation est un argumenl de plus pour une uetion niusculaire. La orotamino diminuo la sousibilité à tiiio 2.'' injeolion tillérioure do d-lubo- oiirariuo. décarnéthoniuni — Après Cm, la oonlracturo est maiuteuue mais est moins forte que sur une pré|)aration non parliollemont inhibéo. La crotamine aiigmenle la sousibilité à uno 2.'^ injoction do Cm. AnTACONISME des lONS Antagonisme des ions L'«++ — Comino poiii la véralrino Finjoctiou de CaCb. aiuiule la coutracluro orotaminiquo ot la jtréviont si CaCL est injocté préala- blomont. cm SciELO LO 11 12 13 14 15 16 544 ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES CROTALinAE, ELAPIDAE ET HYDROPHIIDAE Aniagonismc des ions + — L’aclion i-st dii mêmo lype (]u’avec' (Ài"'' ils soiil nioins toxiques mais leiir effet est ])lus fugarc. Tacuyimiylaxie ckoisÉE — Nous avons moiilré une lacliypliyiaxie eroisée. entre crotamiue et venin à crotamine el vice-versa. Par coiitre le veniu saus crotaminc et la crotamiue administrés run a|)rès Tautre et vice-versa n’onl en- traíné aucuue altéuuation de la coiitracture crotaminique. La tachyphyla.xie se- rait donc duc. à ce.ltc dernière siibstance. PRÉPARATION NERF PHRÊNIQUE — DIAPHRAGME ISOLÉE DE RAT Action |'kopI!E — Crotamine (7 mcg/ml) cntraine une aclion contractu- rante avec élévation du niveau de hase et décontraction difficile, la contracture est généralement immédiate ou peu différée. Klle décroil saus lavage, progres- sivement mais reparail par lavage. Puis des conlractures ai)])araissent s|)onta- ucment à intervalles lougs imjjrévisililes. Actiom des ions — Ca++ [)révient la contracture (CaCL 2()() mcg/ml) celle-ci ap])araít aii lavage. Ca++ sup])rime la contracture cu cours. Mg+^-d"- mais aholit plus difficilcmeni une contracture en cours. iNTElíACTION AVEC EES 1’AHAI.YSANTS NEUKOMESCULAllíES d-tuhocurarine (dose enlraínant une faihle paralysie). La crotamine entraí- iie une faihle contracture qui ap[)aralt nettemcut au lavage. décaméllwriiiun, après Irès faihle contracture par crotamiue et lavage u'eu- traiiie pas de contracture importante. Taciiyphyi.axie CHOiSÉE ohicnue sur ce lest jiar venin :i crotamiue ct cro- tamine ou vice-versa. RECTUS ABDOMINIS DE GRENOUILLE Action pkopke — Aucun effet contracturanl sur ce tesl. 2 à 5 mcg/ml de crotaminc seusihilise considérahiemeni le rectas au K + douhlanl ou triplant les effets du K"*" (30 à '15 miii. a|)rès), cette sensihilisation persiste après lavage et se prolonge 12 hcures et plus. Les ious Ca"'"“’“ (250 mcg/ml) supprimeiit les effets de la crotamine vis à vis du 1n + . 10 mcg/ml de crotamine douhlent les effets de racétylcholine, mais cette action disparait après lavage. ILÉON DE COBAYE I.a crotamine (2,5 mcg/ml) provoque la contracture des fihres lisses avec apparilion des rnouvements spontaués de la fihre lisse, disjiaraissant juir lavages répétés. Peu ou ])as d’action sensilulisante à racétylcholine. cm SciELO LO 11 12 13 14 15 16 Mem. Inst. líutantan Simp. Internac-, JEAN CIIEYMOL, FRANCOIS BOURILEET, MONIQUE HOCII 545 33(2):541-554, 1966 VENIN DE CROTALUS DURISSUS TERRIFICUS SANS CHOTAMINE TOXICITÉ — 250 nicg/kg i/v chez la Souris jirovoqueiit une gênc respi- raloire de loutes les souris après 5 minutes. SOÇf meurent en 24 heures. Ce venin est donc plus toxique que celui à crotainine, coticliision donnée égaleinent |)ar Vital-Brazil qui [)ense que Ia toxieité du venin dii C. d. terrijicus est due à la crotoxine. PRÊPARATION NEUROMUSCULAIRE IN SITU DE RAT — Après une lé- gère augmenlalion de l’aniplitude des eontraclions au nioment de Einjeetion, ou ohserve une j)aralysie de la préparation três lente à s’étal)lir et irréversiblc, les myogramnies sont identiques sur le tibial aiitérieur excite directement comine sur le tiliial excite indirectement par son nerf. II scmhle donc que le venin agisse direciement sur le niuscle. Très léger et fugace antagonisme par Ca++ et clilorhydrat de Clioliue. Après venin sans crotamiue le venin à crotainine exerce son effet contractiirant liahitiiel si la paralysie n’est pas trop avancée, mais Eeffet est atténué. Le venin sans crotainine sensihilise aiix inhihiteurs neuromusculaires tant d-tuhocurarine que siiccinyldicholine. Si sur une j)ré|)aration neuromusculaire de Chat, on injecte dans Eartère tihiale de racétylclioline, on a une contraction ])lus grande ou égale à celle don¬ née jiar Eexcitation indirecte [lar le nerf, ajirès le venin Eeffet acétylcliolinique est diminué. Donc les récepleurs de la plaque nioirice paraissent également toiicliés. Sur le diapliragme isolé de Hat on note une jiaralysie |)rogressive lente et irréversihie. RECTUS ABDOMINIS DE GRENOUILLE — Ajirès un certain teinjis (do¬ ses > à 10 mcg/ml) on note des contractions de grande amplitude mais de courte diirée atténiiahles ou su])|)rinialiles jiar les ions Ca"'" + . Pas des niodifica- tions des seusihilités au K+ ou à EA/C. L’action contraclurante de la crotainine est-elle vératrinique? Ce rapproclie- luent a été présenté par Moussatché et Conçalves en 1956. Quatre points sont comimuis entre les effets de ces deiix substances: 1 — Mêiiies symptònies d'intoxication chez la Souris. 2 — Sensibiliseut le reclas abdoniinis de Grenouille aiix K+ (c’est Eeffet veratriniqiie de llACri). o — Cênent la décontraction du muscle. 4 — Ont leurs effets antagonisés par les ions Ca++ et Mg++. Par contre lEaiitres effets sont nettement différents. Entre autres: 1 — Sur une préparation neuromusculaire non stimulé, Ia crotainine jirovo- (|ue une contracture, la vératrine ne le fait pas. 2 — Sur une préparation neuromusculaire stimiilée directement ou indirecte- ment, isolée ou in sita, la vératrine provoque une augmentation de Eamplitude des contractions sans élévation de la ligue de base alors que la crotamiue ])ro- voipie une élévation de la ligue de base et diminution jiendant la contracture de Eamplitude des contractions. cm 2 3 z 5 6 11 12 13 14 15 546 ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES CROTALIDAE, ELAPIUAE ET HYDROPIIIIDAE 3 — La crotamiiH’ presente iine paralysie seeoiulaire jamais ol)servée avec la vcralrine. 1 — La crolainine inésente le ])liénomèiie cie lacliyphylaxie et non la veratrine. 5 — Lc's effels cie la vératrine varient en fonclions cie la frécpience cie sli- nnilation, alors cpie ceux cie la erotamine sonl iclenlic^ues, quelle cpie soil la fre- ejuenee cie stimulalion. 6 — A|)rès cl-liil)ociirarine (dose entrainanl une paralysie parlielle), la cro- tamine ])rocliiil ime coiitraeture ]mis la paralysie soiivcmt lolale. La vératrine entraine la rejerise cies contraetions et rangmentation crani[)litncle aii-delà clii nivean crorigine. VENINS DE NAJA {N. naja, N. haje, N. nigricullis) (Venins provenant de Elnstitut Pastenr (annexe de Garches — Dr. Bocpiet, Paris) Les actions (jiialilalivcs sont cin mêine type pour les trois, noiis déerirons |)liis particniièrenient eelles cin N. naja indiqnani à la fin les différenees snrtout dor- clre qnantitalif entre les trois. TOXICITÉ — L’aspect qualitatif est tonjonrs seinhlalile mais la valenr quan¬ titativo assez varialile cTiin échantillon à raiilre liien (|ne eenx-ci soient eonservés à Fctat see en tuhes liien lioncliés, |)arfois scellés et en glacic're. Cliiffres varia- Ides. Souris hlanche (DL.-.n i/v allant de 390 meg/kg à 850 meg/kg). I.es Solutions sont tonjonrs préjcarées extem])oranément. Paelivité diminiiant en 21 à 48 lienres de façon sensihle. La mori [)résente tonjonrs le mêrne aspect. Apirs nn tem|)s de latenee ])lns on moins long, gêne respiratoire qiii s’aeeentne jiiscpFà Taspliyxie, le coenr eon- timiant à liattre après Tarrêt respiratoire. Même tahlean, ehez le Poussin de 8 jonrs, hien qne ceini-ci soit [)lns sensi- lile que la Souris, la DL.-,» i/v se situant vers 100 meg/kg. PREPARATION NEUROMUSCULAIRE IN SITU DE RAT Action PiíonHK — A|)rès injection par voie i/v ou i/artérielle, la jiaralysie s’installe a|)rès un ternps de latenec' jilus ou moiiis loug, seloti la dose adminis- trée, elle est lent (|)lusieurs lieures), progre.ssive et irréversilile clans les 7 à 8 lienres des expcriencos. Si Ton enregistre rexcitation clirecte et indirecte du musele, on constate qne le musele reste eontractile jusqiFà Ia fin, même (|uancl Texcitation indirecte ])ar le nerf est inefficaee. Cet aspect est comparahle à une curarisation classiíjue, la Iransniission ner- veuse est annulée alors que la contraetion directe par excilaíion du musele persiste. Ess.ai i)'.\>'TACt)NTSTES — La néostigmine, récirophcmium sur une paralysie jiar venin de Naja encore à moitié des contraetions uormales, assure une re¬ prise nelte mais fugace. cm SciELO LO 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):541-554, 1966 JKAN CHEYMOL, FRANCOIS BOURILLET, MONIQUE ROCH 547 Sur une même |)réparation eu cours de paralysie par veiiin de N a j a, le ve- niu de Crotaliis durissus lerrijicus crotaminicus, donne sa contracture hahituelle, puis une reprise des mouvements, mais la j)aralysie progressive et déjinitive ré- apparait après. IntERACTION AVEC INHIBITEUKS NEEKOMUSCULAIRES, D-TUBOCURARINE - Alors que Ton ])eul injecter j)ar voie i/v jusqidà 6 fois 20 mcg/kg de d-tul)ocurarine sans curarisation, les mêmes iiijections aj)rès une seide injection de 100 mcg/kg de venin de N. naja soul toujours progressivement efficaces. Avec la succinylcholiue les résultats sont conlradictoires. Chose peu êlon- nante Ic Rat nc réagissant pas de jaçon piire aux acétylcholinomimétiques, la pre- mière injection agit selon ce type, mais par répétition des doses 1' évolution se fait vers le type compétitij. CHUTE DE LA TÊTE DU LAPIN Sur des la])ins étalomiés par rapporl à la d-tidiocurarine ou à la succinyldi- choline, nous avons rnontré qu’une injection de venin de N. naja (80 mcg/kg) diminuait de 50% les doses de d-tuhocurarine nécessaires pour ohtenir la chute de la tête et ceci, sensihle 9 heures après Finjection du venin se maintient en s’atténuant jusqiFau 9.® joiir. Rour les raisons indiquées ci-avanl, les résultats avec la succinyldicholine sont contradictoires. DIAPHRAGME ISOLÉ DE RAT — Le venin de N. naja entraíne après un certain temps de latence une contracture (élevation du niveau de hase) dispa- raissant par lavage. En même temps s’installe la paralysie progressive, lente, ir- réversihle. Néostigmine, édroplionium antagonisent nettement mais passagèrement la ])aralysle. Les ions Ca++ et Mg++ agissent de même. Le venin de C. d. terrijicus crotaminicus donne sa contracture haliituelle, mais la paralysie réappa- rait inexorahle. Commc pour la pré])aration in sita, le venin de N. naja sensihilise le dia- phragme à la d-tuhocurarine. Avec la succinyldicholine ])as de sensihilisation mais parfois antagonisme. Ceci augmente la parenté de Taction du venin de N. naja avec les acétylcholinocompétitifs. DIAPHRAGME DÉNERVÊ ISOLÉ DE RAT — Sur une telle pré|)aration les mouvements provoqués })ar excitation directe du muscle étani enregistrés si on ar- rête les mouvements et ajoute de TA/C durant Farrêl, on a une contraction d’am- plitude normale. Après addilion de venin on voit Faction de FA/C disparaítre (Fig. 2). On retrouve ces effets sur une préparation in situ de Chat par in¬ jection d’A/C dans Fartère tihiale avant et après venin de N. naja. On peut donc admettre (jiie le venin de Naja diminue la sensihilité des plaques motrices à Facétylcholine. RECTUS ABDOMINIS DE GRENOUILLE — A doses faihles le venin de N. naja n’a aucun effet. A doses fortes on ohtieiit des contractures, ne dispa- raissant ])as par lavage. A la dose de 0,5 mcg/kg, on a siqjpression des effets de Facétylcholine, mais aucune action sur les effets du K + . 1 SciELO 548 ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES CROTALIDAE, ELAPIDAE ET HYDROPHIIDAE Fig. 2 — RAT; Hémidiaphragme chroniquement dénervé et isolé: Stimulatlon du muscle (0,1 c/s — 0,1 à 0,2 msec — (50-80 V). La stimulatlon électrique est interiompue pour permettre 1'additlon au bain de 1’acétylcholine (30 sec) et le lavage eontlnu de la préparation (9,5 min). La stimulatlon électrique est reprise 5 min avant une nouvelle additlon d’acétylcholine. En Ac, Tacé- tylchollne (0,75 mcg/ml) est ajoutée avant et 30-45 et 60 min après Taddition n répéléi" cl lente de faililes doses (50 à 100 nicg/k") nous a [lerinis (rexpérirtieuler. Noloiis égalenieut que sur le diajiliragme isolé de Hal, raiilagouisme par la néostigmine esl plus niarqué sur le veniu de N. nigricoilis. VEMNS D’lf]I)ROPHIII)AE: I.upeinis hurdwickii, Uydrophis cyanocinctus Enliydrinu .schisíosa (Venins [irovenant de rinstitut Pasteur de Saigon, Dr. Bariue) Les Irois venins expériinenlés ont des aetions qualilatives eomnnuies, nous pouvons donc traiter leur aetion neuronniseulaire en niêine leinjis. TOXICITÊ — V. Eapemis harduickii: DL.,,, i/v Souris 110 nieg/kg voisine de celle du Naja. haje, le jiliis loxitpie des 5 Naja que nous avons éludiés. On eonslate une (*xcitalion légère après rinjeclion, après un temps de latenee de cm SciELO LO 11 12 13 14 15 16 Simp. Internac. 33(l):45-54. 1966 JEAN CIIEYMOL, FRANCOIS BOURILLET, MONIQUE ROCII 549 33(2):541-554, 1966 10 à 15 mimiles, une gêne res|)iratoire s’installe, suivie d’une paralysie progres¬ sivo généralement morlelle. Après une dose non mortelle, il faut au nioins 5 heures pour qne les trouhles res|)iratoires disparaissent. PRÉPARATION NEUROMUSCULAIRE DE RAT — L’excitation électrique iu- direcle par le nerf devient inefficace, 1’excitalion électrique directe par le muscle reste efficace (dose paralysante senil 75-100 nieg/kg). II y a une grande res- semhlance avec Teífel du venin de Naja, mais ici pas d'effet hypotenseur con- joinl gênanl. Donc typc curarisalion classique, mais la paralysie est irréversible. Néostig- niine et édro[)honium ont une actiou antagoniste fugace. 11 y a sensiOilisation uette aux doses non ])aralysantes de d-tnhocurariue. SuR LA PRÉPARATION' isolÉe diaphragme DE Rat, OU note une paralysie len- tement irréversiltle sMnstallant après nn temps de latence plus ou moius long selou la dose (2 mcg/ml — la paralysie a[)parait après 20 à 80 min., devient totale, malgré les lavages, 80 à 40 minutes après). Là encore, Texcitatiou électri- qne indirecto ])ar nerf devient inefficace, rexcitalion électrique directe du muscle reste efficace. Mêines antagonismes fugaces jiar néostigmine et édropliouium. Même sensiliilisatiou uette aux doses non paralysantes de d-tuhocurariue. Actiox de l’acétylcholine exogène a) Sur un diajihragme clironique dénervé, isolé 10 jours ajuès. rexcitalion directe dn muscle étanl inlerrompne, Taddition d’A/C nVntraine aucnne con- traction après venin d’ HYDROPHIIDAE. li) Sur une ])réparation in silit de Chat. Texcitation indirecte étanl inter- rompue, rinjection dans Tartère tihiale d’A/C. nVntraine aucnne contraction après venin de L a p e m i s (l’ig. 8). RECTUS ABDOMINIS ISOLÉ DE GRENOUILLE Sur ce lest venius tl’ HYDROPHIIDAE: pas (radiou pro|)re, diminulion con- sidérahle des effets de rA/C, ]ias de sensihilisalion aux ions K'*'. Kn conclnsion adiou nenromusculaire du même type que celle des Naja, mais peut-êtrc plus dépouillée. car il n’y a pas de fadenr cardiovasculaire donc radiou hy[)olensive surajoulée rend ])arfois pour ceux-ci rexpérimenlalion diffi- cile sur les ])ré])aralions in siln. Mécanisme daclion péripliériquc nenromusculaire des venins de Naja et d’ HYDROPHIIDAE étudiés. Le trois venins de Naja (N. naja, N. haje, N. nigricollis) d les trois ve¬ nins d’ HYDROPHIIDAE {Lupemis hurdwickii, Jíydrop/iis cyanocinclus. Enhydri- na. schistosa) éludies ont une adiou neuromnscidaire du même Ivjie. Celle adiou ressemhle ndtement à une curarisation typiqne. On conslale après lenr admi- nislralion; Argnmcnls ])our lype curarisalion classique (ly|)e Claiide Bernardi 1 — IVxcilalion éledri(|ne indirecte ])ar le nerf devient inefficace, rexcitalion éledritjne dn muscle reste efficace. cm 2 3 z 5 6 11 12 13 14 15 550 ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES CROTALIDAE, ELAPIDAE ET HYDROPHIIDAE jl mín Fig. 3 — CHAT: Contractlons du muscle tlbial antérieur in situ. Stimulatlon du nerf sciatique (0,3 msec — 3 V — 0,1 c/sec) interrompue pendant 2 mín pour permettre ITnjectlon rapide d’acétylcholine en Ac (2,5 mcg) dans l’ar- tère tibiale (portlon dlstale à contre courant). A: contracture lémoin en V Lap, injection i/v de 50 mcg/kg de venln de Lapemis (doíe paralysante seull). La contracture e.st presque totalement inhibée au bout de 12 min: clle récupére ensuite progressivement au bout de 30 min (B), 45 min (C) et 1 h (D), mais sans jamais pouvoir atteindre Tamplitude initiale. 2 — iiéosligmhie, édroplionium, antagonisme, nc’t mais fugace. 3 — synergic avec les acétylclioliiiocompíílitifs, antagonisme (?) avec acétylclio- rmcmiméti(|ues. 4 — injection directe dans Tartère (Prep. nerf-muscle in silu) inefficace addi- tion d’A/C diapliragme isolé innervé ou énervé (dejniis 15 joiirs) inefficace. Ceei montre que TA/C exogène n’agit |)lus sur les récepteurs s])écifiques et laisse supposer (juMI en est de même pour PA/C eudogène. S’il y a diminutiou de la syntlièse de TA/C (?l, cela ne tient pas à uu déficit en clioline, Tadini- nistration de celle-ci ne modifiant pas la paralysie. Aux doses supraliminaires de venin, raugmentation de fréquence des excita- tions (augmentant la consommatiou d’A/C endogène) parait accélérer la paralysie. 5 — diminutiou ou sujipression des effets de TA/C sur le rccíus ahdominis de Grenouille. Arj^umenta contre une ciirarisaíion. clu.saique: 1 — ternps de lateuce plus ou moins jírolougé et jiaralysie irréversihle, 2 — pas (Tantagonismes vrais, jniisque fugaces. cm SciELO LO 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. JEAN CHEYMOL, FRANCOIS BOURILLET, MONIQUE ROCH 551 33(2):541-554, 1966 Bieii qu’il senihle y avoir avec ces veniiis une actioii siir les récepteiirs spé- cifiqiies à racétylcholine de Ia ])laqiie niotrice, on ne peul doiic Fassimiler à la paralysie réversil)le par les acétylcliolinoconipétitifs (curares historiqiies ou subs- taiices de synlhèse). Le blocage des réceplcurs |)arail ici irréversible. CONCLUSIONS D’inie façon uii peu sinqdisle, ou peul opposer: L’actiou musciilaire des veuins de CROTALIDAE: contracturaule dans le cas des veuins à crotamine, paralysanle pour les veniiis sans crolaniine. L’a(lioii curarisante de lype compétitif des veuins de Naja et tl’ HYDRO- PHIIDAE. SuMMARY I. The nenronuiscidar aclions of several cobra (common cobra. Naja naja, Egyplian cobra, N. haje, and spitling cobra. N. nigricollis) and sea-snake (Lapc- mis hardivickii, Jlydrophis cyariocinctas and En/iydrina schistosa) venonis were studied as well as ihose of both varieties of ibe South American ratllesnake iCroUdiis durissus lerrijicas) venoni. lhe crolamin conlaining onc and lhe variely devoid of lhal substance. The aclions of crolamin itself, a basic ])rolein isolated l)y Moura Gonçalves, was also invesligaled. II. All tbree cobra venoms studied are neuromuscidar blocking agenls: tbey exert a perij)beral, slovvly indiiced and irreversible action. The jiaralysis of neuromuscular transmission a])[)ears concurrenlly wilh a cardiovascular depression on iti .‘iilii |)reparations or wilb a conlraclure on isolated ])reparations. N. haje. venom is a more potcnl ueuromuscniar blocking agenl tban a cardiovascular de- pressing or conlraclure inducing substance vvbile N. nigricoilis venom is more aclive in producing cardiovascular depression or skelelal muscle conlraclure tban in cliciting neuromuscular blockade. The venom of N. naja occu])ies, from tbis poinl of view, an inlermediate position. 1. The skelelal muscle paralysis is not due lo a direct action of lhe cobra venoms on ibe muscle fibre; lhe conlraclure, on lhe conlrary, is ])roduced at tbis levei. 2. l'bc inlerruption of nenromuscular transmission produced by tbe Naja venoms is, at least in some measnre, exj)lainable by a slow and irreversilile inhibition of end-plale recej)tors: therefore ibeir neuromuscular blocking action resemble a curarization in Cl. Bernard sense. It is wilh tbe venom of N. naja ibat tbis itdiibilion of recejilors cau be beller evidenccc oh- served on inlact and conscious animais as well as on in sitii or isolated prc|)aralions. This effect is jjroduced hy a direcl action of crotamin on mnscle fihres. A secondary paralysis is inconstantiy prodnced. fn s])ite of some similarilies with veratrin, lhe action of crotamin and cro- taminic venom can not he compared with a veratrinie one. V. The non crotaminic ralllesnake venom shows a weak and slowly indueed paralytic effect whicli can nol he easily evidenced on accoimt of its slrong cardio¬ vascular depressing properlies. This |)aralytic effect is dne lo a ilirecl action on lhe mnscle fihre without inliihition of lhe end-plate recei)tors. Rekereaces VENIN DE CROTALE ^ CROTAMINE 1. BARRIO, A., et VITAL BRAZIL, O., Rev. Inst. Malbnin, 16, 22-40, 1954, 2. ESSEX, H. E., et MARKOWITZ, J., Arner. J. Physiol., 92, 317, 348, 695-706, 1930. 3. FRAENKEL-CONRAT, H., et SINGER, B., Arch. Biochem., 60, 64-73, 1956. 4. GRALEN, N., et SVEDBERG, T., Biochem. J., .32, 1375-77, 1938. 5. HABERMANN, E., et NEUMANN, W. P., Biochem. Z., 327, 170-85, 1955. 6. HABERMANN, E., et NEUMANN, W. P., Biochem. Z., 329, 405-415, 1957. 7. HOUSSAY, B. A., et coll., C. R. Soc. Biol., 87, 821-824, 1922. 8. HOUSSAY, B. A., et coll., C. R. Soc. Biol., 88, 367-68, 1923. 9. KLAUBER, Rattlesnakes, Berkeley University Press, 1956. 10. MOURA GONÇALVES, J., Venoms, 261-274, 1956. 11. MOUSSATCHÉ, H., et VIEIRA, G. D., An. Acud. brasil. Cienc., 25, 249-58, 1953. 12. MOUSSATCHÉ, H., GONÇALVES, J. M., VIEIRA, G. D., et HASSON, A„ Venoms, 275-79, 1956. 13. SCHENBERG, S., Science. 129, 1361-63, 1959. 14. SLOTTA, K. H., et FRAENKEL-CONRAT, H. L., Nuture, 142, 213, 1938. 15. VELLARD, J., C. R. Soc. Biol., 130, 463-64, 1939. cm SciELO LO 11 12 13 14 15 16 Mem. Inst. Butantan -Slmp. Intcrnac. 33(2):541-554, 1966 JEAN CHEYMOL, FRANCOIS BOURILLET, MONIQUE ROCII 553 VENINS DE NAJA 16. ARTHUS, M., Arch. int. Physiol., 10, 161-191, 1910. 17. BOQUET, P., Toxicon, 3, 243-79, 1986. 18. BRUNTON, T. L., et FAYRER, J., Proc. roy. Soc., 31, 358-374, 1872. 19. BRUNTON, T. L., et FAYRER, J., Proc. roy. Soc., 32, 68-133, 1873 . 20. CICARDO, V. H., C. R. Soc. Biol., 120, 732-733, 1935. 21. SU, C., J. Formosan med. Ass., 59, 1083-1091, 1960. 22. DETRAIT, J., IZARD, Y., et BOQUET, P., C. R. Soc. BioL, 153, 1722-24, 1959. 23. ELLIOT, R. H., PhiU. trans. roy. Soc., 197, 361-406, 1955. 24. GAUTRELET, J., HALPERN, N., et CORTEGGIANI, E., Arch. int., 38, 293- 352, 1934. 25. KELLAWAY, C. H., BuH. John’s Hopk. Hosp., (50, 18-39, 1937. 26. KELLAWAY, C. H., CHERRY, R. O., et WILLIAMS, F. E., Aiist. J. exp. Biol. med. Sei., 10, 181-194, 1932. 27. LEE, C. Y., et PENG, M. T., Arch. int. PhurmacoL, 133, 180-192, 1961. 28. MELDRUM, B. S., Brit. J. Phurmacol., 25, 197-205, 1965. 29. MELDRUM, B. S., Physiol. Rev., 17, 393-445, 1965. 30. RAGOTZI, V., Arch. Pathol. Aniitom. Physiol., 123, 201-234, 1890. 31. SARKAR, N. K., et MAITRE, S. R., Amer. J. Physiol., 163, 209-211, 1950. 32. VICK, J. A. et coll., Arch. int. Phurmacol., 153, 424-429, 1965. 33. VITAL BRAZIL, O., Thèse fac. med. São Paulo. 1983. 34. VITAL BRAZIL, O., et BARRIO, A., Rev. Inst. Mulbrán. 16, 1-18, 1954. 35. WALL, A. J., Proc. roy. Soc., 32, 333-362, 1881. VENINS D'HYDROPHIJDAE 36. BARME, M., Ven. and pois. animais and noxious plants of the Pacific region, Pergamon Press, 1963, pp. 373-378. 37. BARME, M., et DETRAIT, J., C. R. Acad. Sc:., 218, 312-315, 1959. 38. CARREY, J. E., et WRIGHT, E. A., Trans. roy. Soc. trop. Med. Hyg., 54, 50- 67, 1960. 39. CARREY, J. E., et WRIGHT, E. A., Trans. roy. Soc. trop. Med. Hyg., 55, 153, 1961. 40. FRASER, T. R., et ELLIOT, R. H., Proc. roy. Soc., 74, 104-109, 1904. 41. FRASER, T. R., et ELLIOT, R. H., Pliilul. Trans. roy. Soc., 107, 249-279, 1905. 42. HALSTEAD, B. W., Dangerous marine animais, 1959, pp. 93-99. cm SciELO 10 11 12 13 14 15 554 ACTION NEUROMUSCULAIRE DES VENINS DE QUELQUES CROTALIDAE, ELAPIDAE ET HYDROPHIIDAE 43. MARSDEN, A. T. H., et REID, H. A., Brit. med. J., 1290-93, 1961. 44. REID, H. A., Brit. med. J., 2, 73-78, 1956. 45. REID, H. A., Brit. med. J., 2, 1284-89, 1961. 46. REID, H. A., Symyosium sur an. et yl. ven. du Pacif., Pergamon Press, 1963, pp. 355-362. 47. ROGERS, L., Proc. roy. Soc., 71, 481-496, 1903. 48. ROGERS, L., Proc. roy. Soc., 72, 305-319, 1903. 49. TAUB, A. M., et ELLIOTT, W. B., Toxicon, 2, 87-92, 1964. 50. WERLER, J. E., et KEEGAN, H. L., SymyosUim sur an. et yl. ven. du Pacif., Pergamon Press, 1963, pp. 219-325. Discussion A. Barrio comments: “Nos cupo a Oswaldo Vital Brazil y a mi seiialar por primera vez la existência de dos tipos de veneno de Crotalus durissus terrificus en cuanto a su acción neuromuscular: uno (tipo I) que producia paralisis y otro (tipo II) que en forma muy llamativa prov'ocaba espasmos. Posteriormente pude demonstrar que este cuadro era producido por la crotamina, substancia aislada por Moura Gonçalves, dei veneno de C. díirissus terrificus procedente de la Argentina. Comparamos la acción dei veneno tipo II con la veratrina. Aclaro que denomina¬ mos a esta ponzofia “v'eratrine-like”, nunca quisimos senalar identidad absoluta y total entre estas dos substancias.” cm SciELO LO 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2);555-572, 1966 C. Y. LEE anti C. C. CHANG 555 58. MODES OF ACTJONS OF P15FIF1ED TOXINS FROM ELAPID VEXOMS ON NEUROMISCULAR TKAXSMISSIOX C. Y. LEE and C. C. CHANG PJiarmacological Institute, Colíege of Medicine, National Taiwan University, Taipei. Taiwan, China The actions of venoms from snakes lielonging lo tlie faniily of ELAPIDAE 011 neuroiTuisciilar traiisniission liave recenlly lieen reviewed liy Meldnini ( 1 ). The view lhat peripheral jiaralysis of res])iratory imiscles is lhe principal cause of dealh from elapid venoms aj)])ears lo he well eslahlislied (2, 8, T)- The ciirare- like aclion of elapid venoms has iiecn exlensively sliidied and several aulhors havo presenled evidence lhat neiiromiiscular lilocking venoms have a ])ostsyna])tic site of aclion (for references see Meldrum (1 ) I. Altliough a “non-depolarizing’' lilock of neuromusciilar transmission. like lhal of curare, has heen |)OStulated as lhe mode of aclion for Formosan cohra {Naja naja atra) venom (5) and Iianded krait (Bungarus inalticinctus) veuorn (6). some differences lielvveen actions of lhese venoms and ihose of curare were also noled. Thus, lhe neuromuscular hlock was not effectively relieved hy anticholin-eslerases or reversed hy washiiig- the muscle paralysed liy lhese venoms at higher concentrations; and acetvlcholiiie release from llie presynaptic terminais was more or less imjiaired. Resides, lhe aclion of cohra venom is complicaled wilh a direcl musculolropic effecl (5). and a depolarizing aclivity on skelelal muscle has heen found in ihis venom (7). Since snake venom could he regarded as a mixiure of proleins or jioly- pcjilides, il was considered lhal lhe complexily of lhe venom ailions mighl he due lo comhined effecls of differeni componenis contained in lhe same venom. Using zone electrojihoresis on slarch at pH 5.0, lhe venom of B. multicinctus was separated inlo four fractions (8). One lacks neuromuscular hlocking jiro- perties Init contains cholinesterase. One called “n-Rungaroloxin'’ jiroduces a neuromuscular hlock of relalively ra])id ousei in vitro and in vivo. Il does nol aller lhe acelylcholine outpul from lhe ral phrenic nerve endiugs hui il aholishes lhe respon.se of lhe chick hivenler cervicis muscle lo acelylcholine. The Iwo most eleclroposillve fraclions, called ‘‘yS- and y-Bungaroloxin” respeclively. juoduce neuromuscular hlock and a severe reduction in acelylcholine ouljnil in lhe ral dia|ihragm afler a laleni jieriod of ahoul one hour and this jieriod is not shorlened hy increasing lhe dose. Xeuromuscular hlock produced hy lhese two fraclions in lhe chick hivenler cervicis muscle is nol associaled wilh anv diminulion of sensilivily lo acelylcholine. Mice given large doses of lhese fractions show hyperirrilahilily al firsl and, afler a lalenl period of ahoul one hour. die suddenly wilh dysjmea and convulsions. Aidetl by thc U.S. Army Meti, Re.s. Develop. Command Research Grant DA-MD-19- 193-6d-G10S and by the National Councll on Science Development, Republic of China. cm 2 3 z 5 6 11 12 13 14 15 MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS ON NEUROMUSCULAR TRANSMISSION Frotn Naja naja a/ra vciiom two loxic fractions were sejiaralod uiuler the sainc cleclrophoretic ('011(11110118(9,10). One callcd Najaloxin or cobra nciiro- toxin produces non-depolarizing Idock of neiiromii.sciilar transniission liiil, iinlike lhe vvliole veiiom, doe.s iiol diminisli lhe acetylclioliiic oul]nil froni lhe rat phrenic iierve, nor has aiiy direcl actioii oii lhe miisele fihres. This neiiroloxiii is also free frotii phospliolipase A, histaniiiie releasing and local irritanl aclivilies conlaiiied in lhe vvhole veiiom. Anolher fractioii, which is lhe mosl eleclroposilivc and called carclioloxin, produces a neuroniusciilar Idock vvilli coiitracinre, cardiac arrest in syslolic .«tale and niany ollier |)harmacological effects(ll). From lhe ahove-mentioiied studies, il is ohvious lliat al lea.®! Iwo tyfies of effect.s are oh.servah]e al the neuromiiscular junclion. A curare-Iike iion-depolariz- ing lilock is produced liy colira neurotoxin and «-Bungaroloxin. Tlie olher type of effeci is impairment of acelylcholiiie release from the iiresynaptic terminais, which is produced hy /?- and y-Hungaroloxiiis. The [iresent pajier descrihes lhe resuits of our recent eleclrophysiological study of lhese purified loxins on the iieuromuscular Iransmission. A preliniinary accounl of some of lhe residis has hcen givcii in a coinmunicalion al lhe XXIII Internalional Congress of Physiological Sciences (12). Materials and methods Parijicalion of toxin.i: — «- and y8-l?ungaroloxins were isolaled from lhe venom of Hiinganis nuillicinclus accordirig lo lhe mcthod descrilied hy Chang & Lee (8). Cohra neurotoxin and cardioloxiu were juirified from the venom of Naja naja atra hy CM-Scphadex column chromalograjihy as descrilied hy Lo, Chen & Lee (13). Potencies of lhese purified loxins were checked hy their toxicities in mice and effecis on the chick hivenler cervicis muscle. I niraccilular inicrocleclrodc rccordtng: — The conventional microelectrode rccording techniipie (11) was adopicd using glass rnicroelectrodi^s filled wilh 3M KLl and having 6-10 Mí2 resislance. No ca|iacily compensation for lhe micro¬ electrode was incorporaled. (Irass model P6 DL jireamplifier wilh ils calhode- follower prohe and leklronix 5()2A oscilloscojie were used. For the ral jdirenic iierve-diajiliragm preparaliou, Tyrode solution oxygenaled wilh ÇSÇF Oo + 5% C ()2 was em|)loye(l. The temperalnre was kept al 32-35 ± ().5'’C. For the frog nerve-saiTorius mnscle lhe jireparalion was susjiended in Kinger solution, conlaining NaCl 117 mM. KCI 2.0 mM. CaCl. 1.8 niM and NallCO., 6 niM, at rooni temperalnre (20-21'’C). 'I’he end-plale foeus was localized wilh lhe aid of lhe lime-coiirsc of lhe niinialure end-plale ]iolenlial (FPP) or evoked FPP. Indirecl sliniulalion was ajiplied ihrough a pair of electrodes on lhe nerve wilh snpramaximal reclangular ])ulses of 0.2 msec duralion, and direcl sliniulalion ihrough electrodes. one on the niuscle-tendon junclion and lhe olher in lhe halh fluid, also wilh 0.2 msec rectangnlar ]ndses. 'I crniinal nerve .spikc: — Lxiracellnlar reeording of lhe terminal nerve sjiike wilh a microelectrode having resislance of ahoul 5 MU was ])erformed on lhe frog sarlorius muscle, aceording lo lhe techniijue descrilied hy Kaiz & Milcdi (15). The niusde was imniohilized hy adding 11 niM MgLL lo the Hingir solution. Lhidor such condilion, lhe lerminal nerve sjiike potenlial could he rccorded to- gcther wilh an EPP. cm SciELO LO 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):555-572, 1966 C. Y. LKE and C. C. CIIANG 557 Antidronüc activily: — The nicthofl clescriliod liy Katulic & Straugliaii 1161 for the recording of anlidromic aclivity of the isolated ral j)hrenic iierve was followed. To llie niodified Tyrode solution, conlaining 3.6 mM CaCl 2 and 0.12 niM MgCU, iieostigmiiie melhylsid|)liate was addcd to give a final concenlralion of 0.3 /ig/nil. Tlie lemperaliiro was ke])l al 22 dz O.S^C. linder ihis condition it was |)ossilile to rocord the antidromic repetitivo discharges of lhe nerve follow- ing single nerve volleys for more lhen 2 honrs if the stimulns frequency was kej)t at 0.05/see or less. Bivcnter cervicis iierve-muscle preparalion oj lhe chick: — The isolated hi- venter cervicis nerve-muscle j)re[)aration (17) was siispended in 20 ml of Krehs soliilion which was maintained at 37 ± 0.5"C and l)id)hled with 9S^/c IT and 5% CO 2 . The preparalion was stimnlated indirectly with snpramaximal reet- angular pulses of 0.5 msec duralion at a rale of 6 per min. Hesui.ts and discussion Efjecl on resting membranc poleritlals: — As shown in Tahle I, all of lhe three purified neiirctoxins, cohra neurotoxin, a- and /3-Bimgarotoxins, did not cause any changes in the resling memhrane |)olentials of mustle fihres at eithcr end-plate or non-end-plate zone of the rat diaphragm at a concentration as high as 10 /ig/rn], In eontrast, cardiotoxin as well as erude cohra venom caused a ])rogressive reduction of the resting potentials. Our resuits are al variance with ihose of Meldrum (7) who reported that lhe neurotoxic fraction isolated from Indian cohra {Naja naja) venom depolarized the frog sartorius muscle. It is likely that ^Ieldrum’s neurotoxic fraction mighl he co}Uaminaled hy lhe cardio- loxic comjronent. TABI.E I — EFFECT ON RESTING MEMBRANE POTENTIALS Membrane potential.s were recordcd from both the end-plate and non end-plate zone.s of muscle fibers of the rat diaphragm at the Indicated periods after addition of 10 /ig/ml of each agent. n = Number of observations. TOXIN Membrane potentials (mV ± S.D.) Control 0-5 min 5-10 min 10-15 min 15-20 min (»-Bungarotoxln 75.3 ± 3.7 77.3 ± 6.5 79.3 ± 3.2 81.6 ± G.ü 74.7 ± 4.1 (n = 33) (n = 9) (n = 9) (n = 11) (n = 6) ^-Bungarotoxin 76.5 ± 5.0 _ _ — 72.9 ± 8.2 (n = 27) (n = 23) Cobra neurotoxin 78.1 ± 4.4 81.4 ± 4.4 77.0 ± 3.9 78.4 ± 4.2 76.0 ± 4.8 (n = 28) (n = 11) (n = 5) (n = 10) (n = 7) Crucie cobra venom 83.0 ± 3.7 49.3 ± 8.3 .34.0 ± 17.2 29.0 ± 6.2 23.0 ± 6.8 (n = 30) (n = 9) (n = 11) (n = 9) (n = 8) Cardiotoxin 81.4 ± 4.1 73.7 ± 8.5 54.4 ± 9.7 45.3 ± 14.2 28.9 ± 12.8 (n = 30) (n = 11) (n = 10) (n = 9) (n = 12) cm SciELO 10 11 12 13 14 15 558 MODES OF ACTIONS OF FURIFIED TOXINS FROM ELAPID VENOMS ON NEUROMUSCULAR TRANSMISSION Kflccl ou aclion potentiuls: — As sliown in Fig. 1, no a|)i)rec'ial)le clianges liolh in lhe am|)Iitiule and lime course were fonnd in lhe aclion polcnlials elicited hy direct stimnialion of llie mnscle fihres paralysed l)y a high concenlration (10 /xg/ml) of eilher a-Bungarotoxin or colira neuroloxin. Tliis is in agreement willi our previous findings lhal lliese nenroloxins do not affect lhe ninscle fihre itself. «-BUNGAROTOXIN (I X lO'5)' COBRA NEUROTOXIN (I X 10-5) 2 msec 50 mV Fig. 1 — Effect on action potentials. Action potentials recorded from rat diai)hragm preparation.s are shown. The flrst traoing of each pair (a & c) shows control action potential evoked by indirect stimulatlon. The second tracings are action potentials evoked by direct .stlmulatlon after the neuromuscular transmls.slon was blocked by 10 íig/ml of (v-Bungarotoxin (b) and 10 /tg/ml of cobra neuroto.xln (d), iespectlvcly. Ay/ccf on rnd-platc polcnlials (EPI\s): — EPFs were easily recorded from lhe mnscle fil)res of ifie ral diaphragm, immedialely afler llieir mechanical res- jionses on nerve slimnialion had heen aliolished l>y /Idhmgaroloxin al a con- cenlralion of 1 /ig/ml. üy conlrasl, in lhe cases of «dhmgaroloxin and cohra neuroloxin, sn|)erficial mnscle fihres were innch more rapidiv paralysed lhan lhe deeper ones and no KPPs conld he recorded from lhe superficial mn.scle fihres when lhe mnscle was paralysed. In order lo record KI’Ps from lhe diaphragm [laralysed hy cohra neuroloxin, lhe prejiaralions was firsl immersed in a con- eentralion of 1 /xg/ml for ahonl one honr lo hlock lhe ncnromnscniar Iransmission, lhen washed for one honi', and finally swilclied lo a lowcr conceniralion, snch as ().0.'l-0.0,5 ;ng/ml, which was jnsl enongh lo ])revenl reappearance of mechanical response on nerve slimnialion. Uniike cohra neuroloxin. lhe effecl of «-Piiingaro- loxin was progressive and irreversihie and hence no sleatly slale conld he al- lained in lhe pre.«ence of lhe loxin. In lliis case, lower concenlralions, snch as 0.4-0.,S /j,g/ml, were a|)plied for ahonl 2-8 honrs anil then, lhe preparalion was washed wilh fresh Tyrode solnlion for recording of KPPs. The lirne-conrses of llie KI’Ps ihns ohiained were compared wilh ihose ohlained in llie preparalions jiaralysed hy olher nenromnsctdar hlocking agenls, snch as dimelhyllnhocnrarine (DMTfi), decamelhoninm iCinl and MgCK. As shown in Fig. 2 and Tahie II, lhe KPPs ohlained in lhe diaphragm Irealed wilh each neuroloxin, whellicr acling |)resynaplically or poslsyna|)lically. invariahiy showed a shorler lime-conrse lhan ihosc ohlained in lhe mnscle Irealed wilh cm SciELO LO 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):555-572. 1966 C. Y. LEE and C. C. CHANG 559 a-BUNGAROTOXIN ^-BUNGAROTOXIN DECAMETHONIUM COBRA NEUROTOXIN c 1 MAGNE51UM f -ju-Í 2 msec 2 mV Fig. 2 — EPPs as aftected by toxlns and ofher neuromuscular blocking agents. EPPs reeorded from the rat phreníc nerve-diaphragm preparation vvhich was treated wlth the respective neuromuscflar blocking agent as descrlbed in Table II. TABLE II — TIME-COURSES OF THE EPPs AS AFFECTED BY NEUROTOXINS AND OTHER NEUROMUSCULAR BLOCKING AGENTS The concentration of each agent shDvvn in the table is the final one except that of a-Bungarotoxin and cobra neurotoxin; foi details see the text. * For spontaneous miniature EPPs only those vvhich had amplitudes comparable vvith the EPPs vvere selected. n = Number of observations. AGENT Concentration n Amplitude (mV±S.D.) Time from onset to peak (msec+S.D.) Time from peak to 14 decay (msec±S.D.) a-Bungarotoxln 0.4 /ig/ml for 3 hrs., then wash-out 19 2.35 ± 1.28 0.71 ± 0.14 0.62 ± 0.18 /3-Bungarotoxln 1.0 Hg/ml 17 3.04 4 1.12 0.57 ± 0.11 0.97 ± 0.21 Cobra neurotoxin 1.0 /ig/ml for 1 hr., then reduced to 0.05 /ig/ml 68 3.14 i 1.29 0.59 ± 0.13 1.02 ± 0.29 Dimethyltubocurarine 0.8 íig/ml 31 3.43 ± 0.94 0.82 ± 0.20 1.54 ± 0.12 Decamethonium 40.0 ^g/ml 71 3.41 ± 1.16 0.95 ± 0.28 2.38 ± 0.78 Mg + + 12 mM 45 3.04 ± 0.60 0.41 ± 0.03 0.71 ± 0.09 Miniature EPP* — 21 2.27 ± 0.46 — 0.78 ± 0.16 1 SciELO 560 MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS ON NEUROMUSCULAR TRANSMISSION eitlier DMTC or Cn, and vvere ralher dose to ihose of lhe magnesiiim-paralysed muscle. These results suggest that lhe more prolonged EPP ohtained in lhe muscle treated either vvith DMTC or Cm as compared vvith that ohtained in the magnesium- or neurotoxin-paralysed muscle may he due to some jjharmacological effecl exerted hy DMTC or C,,, on the end-i)late. Ejjecl on spontaneous miniature EEPs: — Brooks (18) has ohserved a decline in the frequency of miniature EPPs in a jjreparation treated with holulinum- loxin. Neuromuscular hlock occurred either hefore or after the complete dis- appearance of miniature EPPs. This result led him to conclude that hotulinnm- toxin blocks the neuromuscular transmission at the nerve terminais rather than at the arhorization as postulated hefore hy himself (19). The effect of /?-Bun- garotoxin was sludied in the conjunction since it also hlocks neuromuscular transmission at presyna|)tic site (8). The frequency of a|)pearance of miniature EPPs was first increased 2-8 times hy /8-Bungarotoxin (0.3-3 p.g/ml) during 1-2 hours after the application, then gradually decreased and finally no miniature EPPs conld he fonnd (Figs. 3 & 4). Stimulation of the j)hrenic nerve revealed that EPPs were al)oli.shed hefore lhe com|)lete disappearance of miniature EPPs. It was occasionally found that a hurst of miniature EPPs ap|)eared for several minutes hefore their complete disappearance (Eig. 5 ). In contrast. hoth a-Bungaroloxin and cohra nenrotoxin reduced the amplitude of miniature EPPs progressively without affecting the rate of discharge, and final¬ ly the miniature EPPs had disappeared hefore neuromuscular hlock took place. 200 mse^ I mV Flg. .3 — Effect of fi-Rungarotoxin on tlie spontaneou.s miniature EPPs. Continuou.? record- ing on moving film, 2 .sec per svveep. (a) Control recordeii 45 min after .setling up of the preparation; (I)) 80 min after addition of 0,3 /i-ttungarotoxin. Note the increase in lhe frequency of miniature EPPs. cm SciELO LO 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):555-572, 1966 C. Y. LEE and C. C. CHANG 561 Flg. 4 — Effect of /3-Bungarotoxin on the trequency of miniature EPPs. A dif- ferent preparation from that shown in Fig. 3. Control was taken 30-60 min atter setting up ot the preparation and shown as the filled cirele in the leít side. /?-Bun- garotoxin (3 Mg/ml) was added at 0 time. At 240 min, none ot muscle fibres contracted on nerve stimulation. Each elrcle represents the mean trequency ot miniature EPPs obtained trom 20 sec observatlon of one end-plate. Fig. 5 — Burst ot miniature EPPs induced tay ^-Bungarotoxin. The same preparation as described in Fig. 3. A burst ot miniature EPPs from the same end-plate is shown. It was recorded at about 200 min atter addition of 0.3 (Ug/ml /t-Bungarotoxin. There was about 1 min interval between (a) and (b). This end-plate did not respond to nerve stimulation when the record was taken. Antuí^onism ivitli neostig,mim’: — As shown in Mg. 6 (a & ii), in the ral (liaphragni, treated wilh eillier «-lUingarotoxin or eohra neuroloxin. tlie EPF was increased in ils size and jnolongpd in its linie-conrse l)y neosliginine (1 /j.g/nd). Some of lhe Kl’l’s hecame large enongh to generale action potentials. Neo- stigmine aiso exerted a similar effect on llie miniature KPPs recorded in lhe diaphragm afler the evoked EPPs liad lieen aholished hy /3-I]imgarotoxin (Eig. 6, c & (1). It mnsl l)e staled here. however, lhal • 10 34 2.09 0.85 46.1 ± 7.6 25.1 ± 6.4 24.7 ± 5.8 5 7 2.10 0.97 47.6 ± 6.3 22.4 ± 7.4 16.4 4.5 £2 in c -C o 50 76 2.36 1.22 74.1 + 15.7 o o u rH O o S \ fcí a. 20 72 2.28 ± 1.10 84.2 21.2 50.6 ± 18.7 c E a 00 o 10 75 2.34 1.38 89.2 26.8 56.9 ± 25.1 39,1 ± 22.5 rt M C ^ 0) ò o CO o 5 48 2.22 0.97 100.9 24.9 61.6 25.6 41.8 ± 18.7 U H *-> d 29 3.86 ± 1.72 84.4 12.9 26 3.17 1.32 93.7 16.0 71.4 19.3 29 3.76 1.19 106.0 23.9 70.7 ±: 24.8 47.6 ± 16.2 11 3.56 ± 0.70 98.4 ± 16.9 77.5 ±_ 11.6 57.6 ± 17.3 pulse intervals in llie J3MTC-treate(! miiscle were similar to lliose ohlaincd hy Huhliard (23). To our siirprise, however, Cm elicited a mucli more raiiid decline of EPPs in tlie rat diapliragm pre|)aration and eansed a marked \Wdensky inhiliition, eonlrary to lhe finding lliat C,,, eansed a snslained contraction in the eal lihialis mnsele(24). The FiPPs in cohra nenroloxin-lreated ])reparation deelined almost as rajiidly as ihose in I)l\1TC-lrealed one. On the other hand, the EPPs in lhe jirejiaralion Irealed wilh n-l>ungaroloxin shovved mneh slower decline lhan did lhe EPPs in preparations Ireated with other hloeking agents descrihed ahove. Actnally in some jnnetions lhe EPPs maintained a conslant levei I Eig. 7. a). cm SciELO 10 11 12 13 14 15 564 MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS ON NEUROMUSCULAR TRANSMISSION I I I I I Flg. 7 — Patterns of trains of EPPs. The phrenic nerve was stimulateci vvith a train of 10 pulses at the frequency of 100/sec. The experimental condltlons vvere the same as clescribeci In Tahle II. (a) n-Bungarotoxin; (b) fi-Bungarotoxin; (c) cobra neurotoxin; (d) Dlmethyltubocurarlnc; (e) Decamethonlum; (f) 12 mM MgCl,^. In fontrast to lliese agenls, all of wliicli are helieved to act on the post- synajitic memlirane, ^-Hiingarotoxin eauseti the amplitude of KPPs to fliietuate irregiilarly (Kig. 7, h), and there was oeeasional conduction hlock so thal some EPPs were left ont. Analysis of the EPP sequence revealed thal no decline in its amjiliinde oecurred dtiring the train of 10 pnises (Tahle IV). As shown in Tahle IV and Fig. 7 f, trealmeni with high magnesiiim ion also eansed a fhicluation of EPPs and facilitalion as rejiorled hy dei Castillo & Katz (25) and Hiihhard 125) hiiL no eondnclion hlock was ohserved. Il is inleresling in this regard that hoth /8-Bnngaroloxin and magnesinm ion hlock lhe nenromiiscnlar transmission hy inhihition of acetylcholine release. TAHLE IV — THE TRAIN OF EPPs AS AFFECTED BY PRESYNAPTIC BLOCKING AGENTS Phrenic nerve was stimulated with a traih of suprama.ximal pulses for 100 msec at 10 msec pulse interval. Amplitude of the EPP was shown as the mean (mV + S.D.) since the EPP fluctuated irrcgularly (Fig. 7-b) and had no definite correlation with the first one. E.P.P. (mV ± S.D.) AGENT n (mV Ist ± S.D.) (mV 2nd ± S.D.) (mV 5th ± S.D.) (mV lOth ± S.D.) ^-Bungarotoxin 1X 10-“ for 3 hours, lhen wash-out 13 1.47 ± 0.74 1.(52 ± 0.80 1.48 ± 0.71 1.35 ± 0.72 Mg, 12 mM 8 2.44 ± l.lü 3.14 ± 0.83 3.38 ± 0.97 3.70 ± 1.16 cm SciELO LO 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):555-572, 1966 C. Y. LEE and C. C. CHANG 565 However, condiiction hlock, occiirred in lhc> /3-Hiingarotoxiii-treaU-d nuiscle duriíig repetitiva stiimdatioii, lieeame more marked and the average amplitude of EPPs vvas reduced as the slimidation was prolonged. As sliown in Fig. 8 and Tahie V, the conduetion faihire inereased to more than twiee during 500 msec and the average amplitude of EPP decreased hy ahout 66%. Jn eoiUrast. neither conduetion hlock nor decrease in the amplitude of E!PP was ohserved iu the magnesium-paralysed musele during ])rolonged stimulation. These results may ex])lain why the musele treated with /S-Rungarotoxin tends to shovv Wedeiisky itdiihition. ...iiiiiiÉwiiiiuiinüilillliivi Fig. 8 — EfCect of /i-Bungarotoxln and Mg ion on the train of EPPs. The phrenic nerve vvas stimulated with a train of 50 puises at the frequency of 100/sec. (a) Treated with 1 /íg/ml /3-Bungaroto.xin for 185 min and then washed. Note the faiiure in re.sponse to the stimuiation and decrease in the ampiitude of EPP. (b) 12 mM MgCL. TABLE V — COMPARISON OF THE RATE OF FIRING AND THE AMPLITUDE OF FIRED EPP ON REPETITIVE STIMULATION BETWEEN PREPARATIONS TREATED WITH /5-BUNGAROTOXIN AND THOSE WITH Mg ION The diaphragm vvas treated either with 1 /vg/ml /i-Bungarotoxin or with 12 mM Mg- Tyrode’s soiution. A train of 50 puises at the frequency of lüO/.sec vvas given. The number of responses to the first 10 pulses and that to the last 10 pulses of the train vvere counted and the average amplitude of EPPs vvas compared. AGENT No. of junctions fired (mean ± S.D.) First 10 stimuli Last 10 stimuli Amplitude of EPP (mV ± S.D.) First 10 stimuli Last 10 stimuli + + Mg /S-Bungarotoxin 3.66 ± 1.06 1.10 ± 0.76 3.35 ± 1.08 0.37 ± 0.32 Klfect on aniidroniic aclivitirs: — li has heen shown lhat eurare aholishes repelilive anlidromie discharges of molor nerve fihres, evoked hy orthodromic nerv(> impul.se under the influenee of anticholineslerase agenls, at a dose levei mueh lovver than that required for hloeking neuromuseular transmission (26,27). As shown in l'ig. 9, the repetitive discharges of the rat phrenic nerve evoked cm SciELO 10 11 12 13 14 15 Calibration 40;jV MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS ON NEUROMUSCULAR THANSMISSION hy a single volley in the presence of neosligmine (0.3 p,g/nil) were abolished hy all of lhe ihree neuroloxins hefore lhe oceiirrence of complele neiironuiscular hloek. oi-BUNGAROTOXIN ^-BUNGAROTOXIN GOBRA NEUROTOXIN (IXIO'^) (lxlO~^) lOO mm 130 min 60 min 20 msec Fig. 9 — Eífect on antUiromic activities. Lovver traclng In each figure shovvs the antiíiromlc activities of the rat phrenic nerve prepaiation in the presence of neo- stigmine (0.3 /;g/mi) as (iescribeci in the Method. Upper tracing shovvs the muscle action potential monitoreci with an extraceiiuiar microeiectrocie. Upper figures (a, b & c) Show the control activities and lovver figures, (ci) 100 min after «-Bungarotoxin (1 /ig/ml), (e) 130 min after /?-Bungarotoxin (1 íig/ml) and (f) 60 min after cobra neurotoxin (1 /jg/mi), respectiveiy. Ejfcci on Icnninal nerve sjnke: — While the lerminal nerve s|)ike recorded with extraceiiuiar mieroelectrode in lhe frog sartoriu.s was aholished hy eardio- loxin (Fig. 10), il remained unaffeeted after lhe F.IM’. simullaneonsly recorded with the satne eleelrode, had heen aholished hy eaeh of lhe ihree neuroloxins (Fig. 11). These findings indieate lhat none of the.se neurotoxins interferes with the eouduetion in nerve axons up lo the nerve terminais, whereas eardioloxin hloeks nerve eouduetion juoliahly hy ils dejiolariziug effeet. This effcel of eardio¬ loxin inay explain lhe inhihilion of aeetyleholine out|)ut froni the nerve endings hy the ernde eohra venoinllO). On the other hand, sinee lhe eouduetion in nerve axons is unaffeeted hy j8-Bungarotoxin up lo lhe nerve terminais, lhe inhihition of aeetyleholine release hy this loxin is jirohahly due to its action ou the exeilation-seeretion eou|)liug systeni. Kjject oj stimnliis jreqnency on the lime-conrse oj neurotniiscnlnr hloek hy P-Bun^urotoxin: — Hughes and Whaler (2Í)) have reeentiy shown lhat nerve stimnialion with higher frecpieneies canses a marked enhaneemeni of neuro- mtisenlar hloek hy holnlinum-toxin. This lihenomenon was inlerpreled as jirodueed hy an inerease of the permeahility lo lhe hotidinnm toxin in consetjuenee of a sustained depolarizalion of lhe nerve endings eaiised hy nerve stimnialion al cm SciELO LO 11 12 13 14 15 16 SciELOiio 2 3 5 6 11 12 13 14 15 L cm 568 MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS ON NEUROMUSCULAR TRANSMISSION scarcely affccted liy increasing the rate of stimiilation. Siiice’ lhe iiplake of /3-Bungarotoxin appears to Ite a rather ra])i(l ])rocess(8), it is iinlikely that the enhancement of neiiromiiscular hlock is due to an increase in penneahility to /8-Bungarotoxin, caused hy stimuli of liigh freqiiencics as postulated for hotulinum- toxin hy Hughes and Whaler (28). This resiilt rather indicates that the action of /5-Biingarotoxin oii the nerve endings is infhienced hy the activity of the nerve endings. Pulse intervals Flg. 12 — Etfect oT stlmulus frequency on the time-course of neuromuscular block. The phrenic nerve was stimulated with single pulses at frequencles varylng from 0.75/mln to 48/mln, Each polnt represents the mean of 4-15 experlments. Ejject of calcium and magnrsiiim ions on the time-course of neuromuscular hlock hy /3-Ihingaroloxin: — One heinidiaphragm prejiaration was immersed in modified Tyrode sohition containing either low calcium (0.45 tnM) or high magncsiiim (12 niM). The pre|)aralion usually failed to respond to nerve stimula- tion williin 20 min. and j^-Bungaroloxin (1 /tg/ml) was llien added. Inleresling enough, the resjionse to nerve stimulalion was [lartially re.slored on addition of jS-Biingaroloxin and llien decreased jirogrcssively (Big. 15). The conlralalcral hernidiaphragm was immersed in normal Tyrode sohition and similarly treated with /3-Bungarotoxin to serve as the eontrol. When j8-Bungarotoxin induced a com[)lete neuromuscular hlock of the eontrol jireparation, liolh of lhe jireparalions were washed oul wilh normal Tyrode sohition. While no recovery of respon.se occiirred in lhe eonirol preparalion, lhe response of lhe test pre|)aratioii was restored after rejieated washing, and it took anolher 100-150 min. to eaiise iieiiro- mirscular liloek again. Thiis, low ealeiiim as well as high rnagnesium can mark- edly [irolong the time for neuromuscular hlock hy /3-Bungarotoxiii (Tahle VI). Sueli antagonism helweeii j8-Bungaroloxin and ealeitim or magnesiiim ion may suggesl that lhey share a eommoii site of action at the nerve endings. cm SciELO LO 11 12 13 14 15 16 Mem. Inst. Butantan Simp. Internac. 33(2):555-572, 1966 C. Y. LEE and C. C. CHANG 569 Effect of low calcium on the N-M blocking action of ^-Bunga^otoxin CaCf20.45mM yS-Bungarotoxin I x IO' r6 140 min t 240min CaCl 2 I 8mM Fig. 13 — Effect of calcium and ^-Bungarotoxln on neuromuscular transmisslon. Rat diaphragm prepara- tion; for detail see the text. Note the restoration of neuromuscular transm.ission on addltlon of ;8-Bungaro- toxin 1 jug/ml to the paralysed preparatlon. TABLE VI — EFFECT OF CALCIUM AND MAGNESIUM ON THE NEUROMUSCULAR BLOCKING ACTION OF /1-BUNGAROTOXIN For the experimental detail see the text. The time taken for the test preparatlon to cause neuromuscular block was that from the addition of ^-Bungarotoxln (1 /jg/ml) to complete arrest after washlng. n = no. of experiments. Pretreatment Concentration Time for N-M block by j3-Bungarotoxin (Min. ± S.D.) None — 153 ± 12.8 (n = 15) MgCl, 12.00 mM 266 ± 63.3 (n = 6) CaCL 0.45 mM 313 ± 45.0 (n = 4) Ejfect of d-liibocururine pretreulnient on the neuromuscular blocking action of a-Bungarotoxin: — It has recently heeii demonstrated that pretreatment with fl-tul)OCiirarine protecls the chick hiveiiter cervicis miiscle from the neuromuscular hiccking action of cohra neurotoxin (10). In order to see whether d-tulio- curarine also can ])rotect lhe muscle from the paralytic effect of /3-llungarotoxin, one of lhe Ivvo hivenler cervicis muscles from lhe same chick was pretreated with d-tuhocurarine (10 /rg/ml) for 10 min. and the other was immersed in Krelis solution to serve as tlie control. ív-Hungarotoxin (0.3 p,g/ml) was added to lioth prejjarations simiiltaneously. When the resjionse of the control prejiara- ticn to nerve stimulation was aholished, hoth pre])arations were washed with fresh Krehs solution once every 5 min. As shown in Fig. 14, while no recovery was cm SciELO 570 MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS ON NEUROMUSCULAR TRANSMISSION a éOmin CHICK BIVENTER CERVICIS Fig. 14 — Effect of ti-tubocurarlne on the neuromuscular blocking actlon oí «-Bungarotoxin. Chick biventer cervicis muscles. a: control treateci only vvith «-Bungarotoxln, 0.3 ;ig/ml. b: contralateral preparatlon pre- treated vvith 10 /jg/ml of d-tubocurarine. Note the remarkable restoratlon of response after repetitlve vvashlng in the d-tubocurarlne-pretreated preparatlon In eontrast to the control. found in the cotUrol |)rc|)aralioii despile re|)(‘ated vvashiiigs for d lioiirs, lhe response of llie ])reparalion ])retrealed wilh d-tuliociirarine recovered sleadily lo ahout 70% of the original height after d hoiirs’ washing. In eontrast. no pro- tection ])y d-luhociirarine was found against the neuromuscular hlocking aetioii of ^-Hungaroloxin. These findings, logether with our ])revious ohservatiou vvith eohra neuroloxin, strongly suggest lhat «-Ifuiigaroloxin as vvell as eohra neuro- toxin l)locks neuromusenlar transrnission hy eoml)ining vvith the S])ecific reee[)lor of acethylcholine al the end-plate, just like d-tuhocurarine does. SuMM.VHY AND CONCl.USIÜNS Cobra neuroloxin: — Cohra neuroloxin de|)resses EPF vvilhout affecling rest- ing nienihrane potential, niusele aelion ])otential. and terminal nerve s|)ike. The amiilitude of lhe EPP is increased and ils time-eourse prolonged liy neostigmine. Antidromic aetivities of the phrenic nerve in the |)resenee of neostigmine are aholished. The amjililude of successive EPPs on repetitive stimulation deelines markediy as in lhe curarized miisele. Erom these findings it is eoncluded lhat cm SciELO 11 12 13 14 15 16 17 Mem. Inst. Butantan Simp. Internac. 33(2):555-572, 1966 C. Y. LEE and C. C. CHANG 571 lhe inode of iieiiiomuscular l)lockiiig action of cohra iieuroloxiii is essciitially similar to tliat of curare, although lhe former acts mucli more slowly and less reversihlv than lhe latler. a-llimguroloxin: — The effects of a-l]imgaroloxin on lhe nenromiiseular Iransmission are similar lo ihose of cohra neurotoxin in lhe following aspects; (1) depression of EPP wilhout affecting resliiig memhrane ])olenlial, muscle action potenlial and terminal nerve spike; (2) enlargement and |)rolongation of EPP hy neostigmine; (3) iidiihilion of anlidromic discharges of lhe motor nerve; and 14) protection from ils ])aralytic effect hy d-luhocurarine. Hovvever. «- Bungaroloxin differs from cohra neurotoxin in lhat its jiaralylic. effect is irre- versihle and not restored hy neostigmine and thal the decline of successive EPPs is much less marked, so that sustained contraction is ohserved on repetitive stimulalion. ji-Hungaroloxin: — ;S-Bimgaroloxin increases lhe frequency of sponlaneons miniature EPPs in the early stage anil reslorcs ueuronmscnlar transmission im- jiaired hy eilher low calcinm or high magnesium, hnl in lhe later stage, lhe numher of miniature EPPs is diminished without change in size. Neuronmscidar hlock as well as aholilion of EPP takes place hefore the comjilete disapjiearance of miniature EPPs. All lhese findings are in good accordance wilh onr previous conclusion thal j8-Bimgarotoxin acts jifesynaptically, reducing the acethylcholine output from the nerve endings and leaving the sensitivity of the end-j)late to acetylcholine unaffected. Since the terminal nerve sjtike remains unaffecled, it ajipears that /3-Bungaroloxin acts on lhe motor nerve endings, prohahly affecting the excilalion-secretion conpling System. Cardiotoxin: — Cardiotoxin causes conlraclure of the muscle as well as neuromuscular hlock hy deiiolarizing hoth the muscle and nerve fihres; the terminal nerve s])ike is aholished and direct slimulation of lhe muscle íihre íails lo evoke an action [lotenlial. This effect of cardiotoxin on nerve tissue is prohahly resjionsihie for lhe rednction of acetylcholine output from the nerve endings caused J)y crude cohra venom. Hekehk.xcks 1. MELDRUM, B. S., Pharmacol. Rev., 17, 393-445, 1965a. 2. KELLAWAY, C. H., CHERRY, R. O., and WILLIAMS, F. E., Aiist. J. exp. Biol. med. Sei., 10, 181-194, 1932. 3. LEE, C. Y., and PENG, M. T., Arch. int. Pharmacodyn., 83, 180-192, 1961. 4. VICK, J. A., CIUCHTA, H. P., and POLLEY, E. H. — Arch. int. Pharmacodyn., 153, 424-429, 1965. 5. SU, C., J. Formosan med. Ass., 59, 1083-1091, 1960. 6. CHANG. C. C., J. Formosan med. Ass., 59, 315-322; 416-426, 1960. 7. MELDRUM, B. S., Brit. J. Pharmacot., 25, 197-205, 1965b. 8. CHANG, C. C., and LEE, C. Y., Arch. int. Pharmacodyn., 144, 241-257, 1963. 9. LEE, C. Y., J. Showa med. A.s.s., 23, 221-229, 1963. 10. SU, C., CHANG, C. C., and LEE, C. Y., Toxicon, in press. 11. LEE, C. Y., CHANG, C. C., CHIU, T. H., TSENG, T. C., and LEE, S. Y., Int. Symp. Animal Venoins, São Paulo, Pharmacological properties of cardio¬ toxin isolated from the venom of Naja naja atra, 1966. 12. LEE, C. Y., and CHANG, C. C., Abstracts of the XXIII Int. Congress Physiol. ScL, Tokyo, Modes of neuromuscular hlocking action of neurotoxins isolated from elapine venoms, 1965. cm SciELO 10 11 12 13 14 15 16 572 MODES OF ACTIONS OF PURIFIED TOXINS FROM ELAPID VENOMS ON NEUROMUSCULAR TRANSMISSION 13. LO, T. B., CHEN, Y. H., and LEE, C. Y., J. Chin. chem. Soc., II, 13, 25, 1966. 14. FATT, P., and KATZ, B., J. Phyftiol. (Lond.), 115, 320-370, 1951. 15. KATZ, B., and MILEDI, R., Proc. roy. Soc., B, 161, 453-482, 1935. 16. RANDIC, M., and STRAUGHAN, D. M., J. Physiol. (Lond.), 173, 130-148, 1964. 17. GINSBORG, B. L., and WARRINER, J., Brit. J. Pharmacoh, 15, 410-411, 1960. 18. BROOKS, V. B., J. Physiol. (London), 134, 264-277, 1956. 19. BROOKS, V. B., J. Physiol. (Lond.), 123, 501-515, 1954. 20. BURGEN, A. S. V., DICKENS, F., and ZATMAN, L. J., J. Physiol. (Lond.), 109, 10-24, 1949. 21. OTSUKA, M., ENDO, M., and NONOMURA, Y., Jap. J. Physiol., 12, 573-583, 1962. 22. THESLEFF, S., J. Physiol. (Lond.), 148, 659-664, 1959. 23. HUBBARD, J. I., J. Physiol. (Lond.), 169, 641-662, 1963. 24. PATON, W. D. M., and ZAIMIS, E. J., J. Physiol. (Lond.), 112, 311-331, 1951. 25. DEL CASTILLO, J., and KATZ, B., J. Physiol. (Lond.), 124, 560-573, 1954. 26. FENG, T. P., and LI, T. H., Chin. J. Physiol., 16, 37-50, 1941. 27. RIKER, W. F., WERNER, G., ROBERTS, J., and KUPERMAN, A., Ann. N.Y. Acad. Sei., 81, 328-344, 1959. 28. HUGHES, R., and WHALER, B. C., J. Physiol. (Lond.), 160, 221-233, 1962. Discussion P. E. Russell: “First, may I compliment you on a very interesting and definitive study. It is perhaps infortunate that curare was studied in depth before venoms, for we have a tendency to drift into the habit of calling things “curare-like”. In reality, many substances have a “curare-like” activily, and these substances are in no way related chemically, and indeed the difficulties may not be as closely associated with chemistry as they are with the physiological abuse of “curare-like”. Your Work indicates that the common acceptance of certain venoms and their fractions as post-synaptic agents .should be reexamined. I agree with you, and I would like to ask whether or not you care to discuss the specific site of action involved or if you have condueted studies in this direction?” C. Y. Lee: “Although cobra neurotoxin as well as alpha-Bungarotoxin has some presynaptic action (e.g. such of antidromic discharges of motor nerve), we believe, N-M block caused by these two neurotoxins is post-synaptic and lhe specific site of action is acetylcholine receptor on the end-plate, since d-tubocurarine pretreatment can protect the paralysing action of these neurotoxins. On the other hand, the action of beta-Bungarotoxin is exclusively presynaptic, leaving the sensitivity of end-plate to acetylcholine unaffected. The site of action is on motor nerve end- ings, probably on the excitation-secretion coupling system, since the terminal nerve spike remains unaffected.” E. Garcia Mendes: “How about the molecular weight of lhe alpha- and beta- Bungarotoxin?” C. Y. Lee: “The molecular weight of alpha-Bungarotoxin has been estimated to be about 8,000. Since beta-Bungarotoxin has not been obtained in pure State, the exact molecular weight is unknown, but we have evidence that it is higher than that of aipha-Bungaroto.xin.” D. Mebs: “Is your AT (t j (í-neurotoxin-preparation the same of Mr. Yang? Is it íree of enzymes?” C. Y. Lee: “Judging from the LD50 in mice, our cobra neuroto.xin is almost identical with Dr, Yang’s cobrato.xin and it is free from various enzymic activities, such as protease, cholinesterase, phospholipase A and hyaluronidase (unpublished observations).” cm 2 3 z 5 6 11 12 13 14 15 Mem. Inst. Butantan Slmp. Internac. 33(2):573-582, 1966 ANIMA DEVI and N. K. SARKAR 573 59. CAHDIOTOXIC AND CAKDIOSTIMULATING FACTORS IN COBRA VENOM ANIMA DEVI and N. K. SARKAR Ueyartment of Fhysiology, University of Calcutta, índia and Canada Department of Aíjriculture, Animal Research Institute, Central Experimental Farm, Ottawa, Canada The suhjecl of snakes and lheir venoms is of special interest lo people of all tropical coiR.tries, as lhe numlier of deaths lhat occiir every year all over lhe world runs, according to lhe World Health Organizalion is as high as 40,000. In Índia alone. some 12,000 lo 15,000 people die every year and in South America 3,000 to 4,000 deaths occur each year. The numher of deaths from snake hites is likewise considerahly high compared lo their total population; in Burma, Indo- China, Australia, África, West Indies, and the troj)ical helt of North and South America. The temperate parts of the glohc are far less severely affected. The venoms of snakes helonging lo ELAPIDAE are generally more toxic than the venoms of VIPERIDAE as is evident from Tahle I, where the toxicities of venoms of some snakes helonging to ELAPIDAE, VIPERIDAE and CROTALIDAE which are generally responsihle for causing most of lhe deaths, are recorded (1). The toxicity of a venom depcuds u[)on many faclors, snch as seasonal chauges. lenght (size) of the snake, amount of venom collecled, frequency of milking, whether or not fonud in the field (foresl) or takeu from snakes in captivily, etc. The toxicity of a venom also varies with the nature of the animal emjiloyed for lhe cxperiment (2). Venom is a mixlure of several |)roteins. Relatively fevv of them have lieeu separated until recenlly. 1’artial or complete sejiaraliou of them has bcen achieved liy emj)loying such procedures as Am;,S ()4 fractionalion, starch-gel eleclro|)horesis, jjaper electrophoresis and anion and cation cxchauge chromatography. Nearly all venoms depending npon lhe species of origin contain at leasl eight to ten (somclimes more) well characlerized com])onenls. Most of them if not all, are enzymes; each one is differeni from the other in their liehaviour lovvards suhslrales. Their activities vary from s])ecies lo s])ecies and even from venom lo venom, collected from lhe same snake, at different seasons of the year (3). The enzymes that are commoidy found in lhe venom of all s})ecies helong- iug lo ELAPIDAE, CROTALIDAE and VIPERIDAE, are phosidiolipase A, L-amino acid oxidase, deoxyrihonuclease, rihonuclease, phos|)hodiesterase, non-specific |)hos- jihomonoesterase, 5'-nuclcolidase, adenosine-5’-triphosphatase and are recorded in 'Pahle II. Proteases and amino acid eslerases are getierally found in lhe venoms of VIPERIDAE and CROTALIDAE. Colira venom exhiliits oídy feehie proteolylic activity, liut coulains several pei)tidases. Venoms of ELAPIDAE in contrasl lo lliose of VIPERIDAE contain enzymes capahie of hydrolyzing acelylcholine and i nou-cholinesters (4 ). The venom of Formosan cobra contains besides acelylcholin- eslerase, an irdiibilor of ihis enzyme too, which can be reversibly blocked by cm 2 3 z 5 6 11 12 13 14 15 TABLE I — RELATIVE TOXICITIES OF VENOMS OF SOME COMMON SNAKES OF ELAPIDAE, VIPERIDAE AND CROTALIDAE SNAKES Estlmated MLD for Man (150 Ibs. body welght) In mgs. ELAPIDAE Bungarus candidus (blue krait) 1.5 Nntechis scutatttn (tlger Gnake) 2.0 Naja naja (Intiian cobra) 20.0 Dendroaspis angusticeps (mamba) 20.0 CROTALIDAE Bothrops atrox (fer-de-]ance) 70.0 Crotalus atrox (Western dlamondback rattlesnake) 140.0 Crotalus adamanteus (Eastern dlamondback rattlesnake) 2S0.0 VIPERIDAE Vipera russellii (Indlan daboia) 50 Bitis arietans (African puff adder) 120 TABLE II Enzymes Cobra venom Vipera runsellü Venom C. adamanteus Venom C. atrox Venom 5'-Nucleotldase (AMPase) + + + + Dlphosphopyrldlne nucleo- tldase (DPNa.se) + not known + 4 - Adenoslne trlphosphatase (ATPase) + + + + Deokyrlbonuclease (DNa.se) + + + + Rlbonuclease (RNase) + + + + Phosphodlesterase + + + + Phosphomonoesterases (nonspeclflc) + + + Phosphollpase A (In some venoms In addltlon to thls Phosphollpase B or C Is found) + + + + Protease + + + + L-amlno acld oxídase + + + Acetylcholinesterase (and cholinesterase) + — ESTEROLYTIC Proteases ENZYMES IN SNAKE VENOMS Proteolytlc enzyme (pro- teln degradlng enzyme) + + + Synthetic amíno acid es- ters + + .+ Clotting enzymes — + + — Bradykinin — releasing enzyme not known + + Bradykinin — destroying enzyme _ not known + + Clotting Inhlbltor (Inhlblts clotting due to Its actlon on phophollplds) — — — cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan ANIMA DEVI and N. K. SARKAR 575 SImp. Internac. 33(2):573-582, 1966 Mg ioiis (5l. Colira venoni exhil)its adenosiiie (li|)lio?|)halase and pyrophospliatase aitivities in addilion lo it8 well known ATPase activity altlioiigli lhe activities of the?e tvvo enzyines are low (6). KiisselTs viper venom can iniliale coagulalion of lilood, whcreas colira venom acts as an iíduliilor. The presence of ])hospho- lipase I? in venonis of l’seinlcchis porphyriacm (Ansiralian snake), C. aduinan- teus and A. piscivorits (North Anieriean snakes) has heen recenlly reporled(7). Sorne venorns also exhil)it phos|)holipase (i activily. Althongh many of lhe enzymes fonnd in lhe venorns of ELAPIDAE, VIPERIDAE anil CROTALIDAE are eornnion hnl lhe eanse of dealh appears to he different in eaeh case, e.g. it is generally helieved lhal the priniary cause of doath in colira |)oisoning is the faihire of respiratory system lirouglit alioiit either liy the. paralysis of the central nervous system or hy the paralysis of diajdiragm mnscle (8-11). The heart is affected at a miich later stage. Tlie changes in hlood pressure following the intravenous injection of cohra venom into anesthetized animais, have heen recordei! hy many vvorkers; lhey all invariahly fonnd failnres of rcs|)iration and cirenlation 18-131. Many of these investigators helieved that the heart vvas primarily and direetly affected 18 , 9-13) . Feldherg and Kcll- awaylTl) pointed ont tliat the circnlatory faihire vvas secondary to the effects of the venom on other organs. Marked electrographic changes in the rhylhm and eondnction of the heart resnlting from an intravenous administration of C. d. terrijicus venom led Soaje-Eehaqüe to consider myocardial inefficiency as the jirimary canse of the faihire of circidation (13). The action of cohra venom on isolated frog heart has also heen extensively stndied hy many vvorkers (11, 15-19). They all foimd an initial increase in the amplitude of contraction follovved hy complete stoppage of all the movements of an isolated frog heart, when it vvas ])erfnsed vvith venom solntion. The effeet as noted hv most of the vvorkers dependei! largely on the concentration of the venom solntion nsed. These stndies, carried ont in silu, in vivo and in vilro, indicate that the ohserved effects of the venom may he ex|)lained on the hasis of its action on myocardinm. Whether or not it vvas actnally so, vvas investigated many years ago in onr lahoratory 119). The vvork to he jiresented here vvas hased on three different series of exjierirnents and vvas initiated to determine the seipiences and relationshi]) hetvveen the changes that follovv after the intravenous injection of venom into animais. MkTHOOS A.M) MATIClilAI.S as follovvs: For the stndy of the effeet of the venom on circnlation and resjiira- tion, ailiilt eats vveighing hetvveen 2.0 to 2.5 kg. vvere chosen. Urethane I 1.8 g/kg. of hodv vveight) vvas introdnced intramnsenlarly into the animal tvvo lionrs prior to dissection. Femoral vein vvas cannnlated for the administration of the venom Inenrotoxin or cardiotoxin) dissolved in 2 ml. of saline. Carotid artery vvas cannnlated for measnrement of the changes of hlood pressnre, and the changes in respiration vvhere recorded hy rneans of a pnenmograph. For the stndy of the effects of cohra venom on excised heart, toads of average vveight of 100 to 120 gms. vvere chosen. The heart vvas perfnsed vvith Solutions of cohra venom Inenrotoxin or cardiotoxin I of desired concentration. The eontractions of the heart vvhere recorded on a kymograph. The heart vvas cm 2 3 z 5 6 11 12 13 14 15 576 CARDIOTOXIC AND CARDIOSTIMULATING FACTORS IN COBRA VENOM first perfuscd witli lhe Hiiiger soliilion iiiilil it heeame |)liysiologic'ally normal; al lliis poini, lhe eaniuila was filled wiüi 2 ml. of llie l{inger-venom soliition of desired eoncenlration and lhe drip slarted and lhe eoncenlrations reeorded. Wlien all lhe venom soliUion was iised np, il was replaced liy Hinger solulion. The sludy of lhe effeets of lhe venom (neuroloxin or cardioloxin) on lhe transmission of nerve impulse were carried out wilh frog nervc-musele preiiarations kej)t immersed in venom solulion for differeni lenghls of time. The eonlraction of lhe muscle was elicited hy indireet (ihroiigh lhe nerve) and direci electrical stimulalion. The venoms were collected írom snakes of lhe same species {Naja naja) and lhe same size, as far as possihle, during lhe monlhs of Ajiril and Jime, freeze- dried, and kepl in an ice hox (2-l'’C) in a darkd)rown slojipered glass hollle imlil use. Hesui.ts The residis ohiained can hc summarized as follows: Kollowing lhe injeclion of a large do.se of cohra venom (2 mg/kg of hody weighl), a sharp fali in syslemic arterial jiressure is noted. The cnsiiing respiralion hecomes more and more shallow and rajiid, and tends lo he ahdominal in tyjie imlil finally ccases; lhe IkP. drops lo zero mm ílg and lhe animal dies, aiiparenlly hecause of lhe faihires of res])iration and circulalion. The a[)|)licalion of artificial respiralion can not jirevent lhe dealh of lhe animal. Even if a smaller dose of lhe venom (0..5 mg/kg of hody weighl) is administered, lhe changes in re.sjiiralion and hlood [iressnre follow lhe same pallerns. If artificial re.spiration is ajijilied, lhe hearl gradually improves, 15.P. slowly and |)rogressively rises imlil it is reslored lo normal levei. If lhe artificial respiralion is discontinned at ihis poinl, natural resjiiralion revives slowly and lhe animal survives for many hours. If now a larger quaniity of venom (1.5 mg/kg) is administered, hlood pre.ssure drops almosl inslantaneously lo zero anil respiralion ceases imrnedialely. The lelhal effecls of ihis second dose can nol he counieracled hy lhe ap|)licalion of aiiificial res|)iralion. If on lhe olher hand, a smaller do.se (0.2 mg/kg) of venom is inilially iniroduced, a very slight fali in arlerial hlood pressurc oflen accomjuinied wilh certain irregnlarilies in res])iralion, is ohserved. If no furlher venom is adminislered, lhe ll.P. slowly rises iinlil il reaches lhe normal levei and remains steady thereafter. Adminisiralion of ihree lo four doses increasing each time lhe arnonnl of venom iniroduced, (tolalling 2.25 mg/kg) al regular inlervals, does not affecl eilher 15.P. or res])iralion. Al lhe end of líie fotirlh adminisiralion. a large quaniily of venom can he iniroduced even in a single dose, wilhoiil af- fecling eilher lhe 15.P. or respiralion. Afler 1 lo 6 hours, 15.P. drops lo zero, res|iiralion ceases and lhe animal dies. If lhe hearl is examined jusl imrnedialely afler lhe dealh of lhe animal, il is found in syslolie coniraclure. If neuroloxin, [lurified from lhe same venom, is injecled inio aneslhelized cais, faihires of resjuralicn and circulalion are ohserved almosl al lhe same lime. The laller can he reslored lo normal levei if ariificial respiralion is ajiplied. wliich remains sleady lhereafler for many hours (6 lo 8 hours) uniil lhe animal dies hecause of olher secondary effecls. If lhe chest is opened and lhe hearl is examined, no syslolie arrest of lhe hearl like lhe one lhal has heen noied hefore wilh crude venom can he delecled (19). If lhe venom is healed ihrough various lempeialures lo ilestroy one or lhe olher aclive eonsliluenls of lhe venom and lesled lhereafler for ils aclion on cm 2 3 z 5 6 11 12 13 14 15 Mem. Inst. Butantan ANIMA DEVI and N. K. SARKAR 577 Simp. Internac. 33{2):573-5S2, 1966 resi)iratioii and circiilation, il heconies cvideni lhal il rclaiiis this |)iO]K‘rly iinlil il is heated lo 85‘’C for 30 minutes. At this temperature mosl of the enzynies are destroyed. Heinolysin wliieh is not destroyed at this temperature is inef- feelive in eausing res])iratory and cireulatory failures. Hecenlly Viek and his colleagues isolaled froni eol)ra venom three major [)hysiologically identifiahle eompouents (actually 12 coinpouents were separated). The first eomponent, whicli vvas a mixture of 3 to 4 sei)arate fraclions ])roduced a loss of cortical eleetrical activity when injecled intravenously into dogs. Tlie second compouenl (fraetions 5 to 8) eauses res])iratory ])aralysis and lhe ihiril component I fraction 12) affected cardiovascular system. On lhe hasis of tlie effect of fraction 12 on arterial hlood jiressure and heart rate, these workers coucluded thal colua venom has a definite action on cardiovascular system of the dog. Action of cohra venom on excised fhog heart If au isolaled frog heart is perfused vvith an exlremely diluled solulion of venom (1 in 100.000) no changes in lhe amjililude of conlraction could he noticed; no slimulating or de])ressing action of venom could either he detecTed if the heart is perfused with a less diluled venom solulion (1 in 10,000). An increase in the amjilitude of conlraction for a hrief jieriod is noticed when a venom sohition of 1 in 5,000 is used, which disa])pears on washings and lhe heart hecomes normal. If a more concentraled solulion is used, say 1 in 1,000. an increase iu am[)lilude is first ohserved and lhen certain irregularities and slow- ing of heart heats are noticed. The heart finallv slops heating. These effects of the venom can also he jiaiTially removed hy jirolonged washings with lhe Hiuger. If a more concentraled venom solulion (1 in 200) is used, no slimulal- ing action can he detected, instead, a ])rogressive decrease in lhe am|)litude of conlraction is ohserved. The heart finally sto])s iu syslolic coulracTure and can not he revived in this case eveu afler conlimious washings with Kiuger. The effects of venom on excised heart could uol he couuteracTed hy alropine and aulihistamine drugs. If the heart is perfused with a venom solulion )] in 200) previously heated ihrough varioiis temperatiires, il is found lhal venom loses its action at 85‘’C (20). Phospholipase A or neurotoxin has no such effect on excised heart. It is inleresting lo note that similar findings have heen recorded with dif- ferenl concenlralions of digilalis drugs (digilalis, slrophanlheine, etc.). In all cases where high concentralioiis ol these drugs have heen used. lhe heart slops in systolic contracture hut contrary lo lhe action of lhe venom, their effects could he easily washed oul with llinger. RecenI histo|)alhological sludies indicate lhal venom produces sjieclfic changes in lhe isolaled heaiTs of frogs and of guiuea- pigs which are quite differenl from those produced hy histamine, slro]ihan- iheine. etc. When an isolaled frog-nerve jireparaliou is ex]iosed lo a venom solulion, it loses its excitahilily in 4 lo 6 minutes depeudiiig upon lhe concenlralion of lhe venom solulion used (21,22). Neither direcl nor indirecl slimulalioa can produce conlraction. The ahility of lhe eleetrical stimulus lo eliciled conlraction when appiied through the uerve is lost earlier than if appiied direclly lo lhe muscle. The muscle contracts lo one third of its original lenghl. hecomes jiale and opaque. Nerves or mnscles ex])osed to oídy Hinger’s solulion maintain eleetrical |)ropeiTy cm 2 3 z 5 6 11 12 13 14 15 CARDIOTOXIC AND CARDIOSTIMULATING FACTORS IN COBRA VENOM for 4 lo 6 lioiirs. lí lhe ticrve-niuscle projiaralion is exjiosfd to tifuroloxiii, lhe iniiscle, retaiiis its excitahilily; iinder identical condilioiis oídy niuscle loses ils exritahilily if exposed lo cardiotoxin sohitioii. The ahilily of veiiom lo lilock contraetion of lhe imiscle is losl if il is healed at 55'’C for 10 miii. or 85"(i for 30 min., de|ieiiding iipon hovv llie eotilraclion is eliciled. i.e. whelher stimniated throiigh lhe nerve or ihrough lhe niuscle (21). At lhese lemperaliires acetyl- cholinesterase aclivily of tlie venom and ils cardiotoxic property are destroyed. The action of cardiotoxin can nol he prevenled or coimteracted hy K ions, anli- histainine driigs or liy EDTA, known lo inhiliil llie |)rotease action of tlie venom. Col)ra venom or cardiotoxin ihtis appears to have direct |)aralysing aclion on cardiac and skelelal niuscle hui hovv il acts is nol known. Neilher colira venom nor cardiotoxin can jiroduce any coniraction of aclomyosin filiers either extracled direclly from rahhil skelelal muscle or pre|iared synthelically hy mixing cryslailin myosin and adiu. Il has neilher any action either on myosin or aclin(23). Il ihns ajipears thal venom has no aclion on individual comiionenls lhal eonslilnle aclomyosin, lhe contraetile elemeni of lhe muscle. Ils aclion is visihie when only inlacl muscle cells are involved. The results olilained indicale lhal lhe ohserved changes in hlood pressure thal occurred following lhe injection of cobra venom, might he due lo its dirccl action on myocardium ralher than on central nervous system since nenrotoxin has no such effect. Il is possihlc lhal cohra venom conlains anolher factor resjionsihie for all lhe ohserved changes in lhe circnialion when lhe venom is injected into au aneslhetizeil cal and lhe changes in lhe movements of an isolated frog heart when il is jierfnsed wilh lhe venom sohition. This factor was isolated from lhe venom using lhe convcntional methods vvhich involved hcal denatnralion, fractional precijiilalion wilh AmoSO,, adsorplion and elnlion from Ca-()hos])hale- gel, iso-electric ])recipitation, elc. (21). The purified material was 20 times more aclive than lhe crnde venom, and lhe rnclecnlar weight as determined hy diffnsion melhod was fonnd lo he 30,()()() (25). Hecenlly il has heen purified using snch methods involving heal denalnralion, AnioSOj fraclionation, chromatography on DKAE-cohinm and starch-gel electro|)horesis. The molecular weight of ihis purified material as determined from sedimenlalion cisnslanl, is approxirnalely 13,()()() to 11,000. Il is 30-35 times more aclive than crnde venom. (inrionsly enongh, when a single dose of cardiotoxin (0.1 mg/kg) is ad- ministered intravenonsly into an anesthetized cat. an immediale sharj) fali in arterial hlood pressure to zero nnn Hg is ohserved; respiralorv failnre also occnrs at lhe same time. Artificial res|nralion if applied fails lo reslore thc hlood pressure lo normal levei. When lhe heart is exarnined, it is fonnd in sysiolic contraclnre. If an isolated toad heart is |)erfnsed wilh a cardiotoxin soinlion, there is at firsl some angmenlalion in lhe amplitude of lhe contraetion for a hrief period followed hy a gradual decrease in am])lilnde nnlil finally all lhe movements are slopped. The heart goes lo sysiolic contraclnre. The effect of cardiotoxin can nol he washed ont hy prolonged washing with Hinger. Il has no aclion oti nenromnscniar jimction nor on nerves hnt skelelal muscle kepl im- mersed in cardiotoxin solntion loses ils excilahilily. 'l he resnils so far ohtained. indicale lhal all lhe effecis recorded eilher wilh crnde cohra venom or cardiotoxin snggesi lhat lhese effects are nol possihly dne to lhe aclion of hislamine or histamine like componnds, released from tissnes hy the aclion of cohra venom or cardioloxin, as lhe laltcr has neilher any proleo- lylic activity nor any |)hospholipase A aclivily and can nol he implicaled in cm 2 3 z 5 6 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):573-582, 1960 ANIMA DEVI anel N. K. SARKAR 579 releasing liislamiiic or histaniiiie like compoiinds froni mast cells. There aro many rejioris in lhe literatiire siipporling lhe hy|)olhesis lhal llie effects of venom are (lue lo llie actioii of liistaniine liheraled. Tliey are niaiiily Iiased on reports of successfiil Irealmenl of snake I)iles wilh untihiiítamiiie drugs, ACTH (adreno- eorticolropic hormone) and corlisone (26-28). Tliese reports are no don!)l of some interesl to ns hnt are not as significant as imagined since a criticai analysis of the resnits reveals many short comings wliicli do not allow to draw any sla- tistically jiistified conclusions as lo eillier llierapenlic values. This is due to the lack of reliahle standard l)y vvhich the severity of snake hile ])oisoning can he judged and hecanse of lhe limiled niimher of cases Irealed snccessfnlly. The merits of lhese drngs have Iteen ifierefore reassayed in onr lahoratory hy more accuralely conlrolled animal experimenls. The antihistamine dnig used was 10-12()-dimelliyIaminoisopro]iyl I-])heno- ihiazine. The corlisone was a commercial preparation of 1 l-dehydro-17-hydroxy- corticoslerone-21-acelale olilained in lhe forni of a snsjicnsion in salinc. The doses of ACTH or corlisone adminislered were 25 lU per kg of hody weighl. The drngs were adminislered in all cases along with the venom. The resnits ohlained wilh lhese drugs clearly indicate their ineffectiveness to counteract the action of cohra venom on circnlalion and respiration; in other words, they can not |)revent the fali of arterial hlood jircssiire to zero mm Hg and counteract lhe resjiiralory faihire that oceur following the atiministralion of the mixture. None of the drugs can also prevent the slojipage of the movements of tlie excised frog heari when il is perfnsed with lhe venom or cardiotoxin solntion, they can not eilher counteract the action of cohra venom on nenro- muscular junction or miiscle fihers. Scholtler (29) did not also find any heneficial effect of antihistamine drngs in snake jioisoning in contrast to its a[)[)reciahle ciiralive effect againsl fatal dose of histamine. He did not also find any effeclive- ness of AOTH, corlisone, etc., againsl venom [loisoning. (ÍARI)IOSTIMUI.ATING PKINCI IM.i; IN COBRA VENOM If an isolaled (detached from lhe hody) frog heart is kept immersed in a venom solntion, il loses its ahilily lo prodiice rhythmic movements in 6 to 10 minutes depending upon the concentralion of the venom solntion used. If il is exposed to a very dilnted solntion say 1 in 5,000, it takes 2 to 3 honrs to stop heart heat. If on lhe other hand the heart is exposed to a venom solntion previons- ly heated at 98'’C for one honr, lhe heart continues to heat for as long as 12 to 14 honrs. The rhythmic' movements hecome more and more feehie wilh lime. Under identical conditions, the heart continues to heat for onlv 4 lo 6 honrs if kept immersed in Ringer solntion. Since at 98‘’C all the known components of the venom with exception of nenrotoxin are destroyed and the pnrified nenrotoxin has no snch effect on heart or skelelal mnscle, it may he possihle that the factor directly responsihle for maintaining the rhythmic movements of the heart for 12 to 14 honrs, has heen masked liy the presence of other factors, particnlarly hy cardiotoxin, which has just lhe op])osile effect on heart. This factor may he associated with cardio- loxin; and withonl the destrnetion of the latter, lhe stimnlaling effect pf venom conld not he delected. If instead of the heated venom solntion lhe isolaled heart is exj)osed to a mixtnre of trijihosphales of all the fonr nncleosides, ATI’, (ITP. CPT and UTP or TTP in eqnivalent proporlions, the heart continues to heat cm SciELO 10 11 12 13 14 15 580 CARDIOTOXIC AND CARDIOSTIMUDATING FACTORS IN COBRA VENOM for oiily Ct to í! liours. When siicli a inixime is rcplaced hy aiiy individual nucicolide say AMP, (dVlP, CMP, l^Ml^ or TMP otily a sliglil slimidaliiip; effect is ohserved wilh AMP. Since llic isolaled lioari íontinues lo l)cal for 12 lo 14 hours iu heated vcnoni solulioii and tlu> fact llial none of lhe foiir micleolides eillier individually or ('olicctively caii mainlain lhe rhylhniic niovemenls of llie lieart longer lhan 6 lo u liours, it is helieved llial lhe faclor responsihle for lhe sliniulating effecl of lhe venom is a proteiu, quite resistanl lo heal and has lovv molecular weight. Venom loses ihis properly if il is heated al 100‘’C for oue hour. This faclor is a eardioslimulaling faclor, as opposed to eardiotoxie faclor. Ils separalion and characlerizalion are novv under aclive iuvesligalion in our lahoratory. (iohra venom conlains hesides neurotoxin and enzymes, Iwo more faclors. 'lhe one, cardioloxin, is resjionsilile for lhe failure of circulalion and sloppage of all lhe niovemenls of an exci.scd heart. 'Plicse effecls can noi he aholished or reversed hy any prolonged washing or liy any antihistamine driigs, alro|)ine or corlicosteroids. Since lhe effecl of venom or cardioloxin is primarily dne lo either aclion ou cardiac or skeletal mnscle rallier lhan on central nervous sysiem, it is helieved cardioloxin should he lhe mosl ajipropriate name llial can he a.ssigned lo ihis faclor. The olhei faclor stimniales lhe hearl, ils effecl is only visihie when lhe olher faclor is destroyed. 'Plie presenl commiinicalion emphasizes lhe irnporlance of lhe isolalion of various components in relalively purcr form. and lhe sludy of lheir nature, hio- chemical, and pharmacological actions separalely as well as colleclively. Such comprehensive sindies are helieved lo he more usefnl and significanl in lhe under- slanding of lhe cause of dealh from snake hiles. l{i;i'EHE.\ci;s 1. MINTON, S. A., Sei. Amer., Illfi, 114, 1957. 2. KLAUBER, L. M., RutÚesnukes, tlieiv hdhitN, Vife historiex, (uul influenco on vuinkind, Press and Zoological Sociely of San Diego, Universily of Califórnia, 1956. 3. DEVI, A., in W. BÜCHERL, V. DELOFEU, and E. E. BUCKLEY (Editors), Venoms and Venomous Anrnials, Vol. II, Academic Press, Inc., New York, in press. 4. DEVI, A., and SARKAR, N. K., in W. BÜCHERL, V. DELOFEU, and E. E. BUCKLEY (Editors), Vetioms and Venomoua Animais, Vol. I. Acad. Press, Inc., New York, in press. 5. LEE. C. L., CHANG, C. C., and KAZUYA, K., in E. E. BUCKLEY, and N. PORGES (Editors), Venoms, Amer. Ass. Avanc. Sei., Washington, 1956 p. 197. B , . , 6. JOHNSON, M., KAYE, M. A. G., HEMS, R., and KREBS, H. A., Biochem. J., ãt, 625, 1953. 7. DOERY, H., and PEARSON, ,1. E.. Biochem. J., i)2, 599, 1964. 8. ELLIOT, R. H., Philad. Trans. roy. 8oc., B, 197, 361, 1905. 9. MACHT, D. I., Med. Rec., 1.53, 369, 1941. cm 2 3 z 5 6 11 12 13 14 15 Mem. Inst. Hutantan Simp. Intcrnac. 33(2):573-582, 1966 ANIMA DKVI and N. K. SARKAR 581 4 10 . 11 . 12 . 13. 14. 15. 16. 17. 18. 19 . 20 . 21 . 22 . 23. 24. 25. 26. 27. 28. 29. HOUSSAY, B. A., NEGRETE, .1., and MAZZOCCO, P., Rev. As. ynéd. argent., Sec. Soc. Biol., 35, 185, 1922. CUSHNY, A. R., and YAGI, S., Philad. Trans. roy. Soc., B, 308, 1, 1916. SARKAR, B. B., MAITRA, S. R., and GHOSH, B. N., Indian J. med. Res., 30. 453, 1942. SOAJE-ECHAQUÊ, E.. Rev. Soc. argent. Biol., Ifi. 475. 1940. FELDBERG, W., and KELLAWAY, C. H., Ausl. J. exp. Biol. med. Sei., 15. 441, 1937. KUSNETSOR, A. I., Byiãl. éksp. Biol. Med., 3, 295, 1936. GOTTDENKER, F., and WACHSTEIN, M., J. Pharmacol. exp. Tlier., 69, 117, 1940. GUNN, J. W. C.. and EPSTEIN, D., Quart. J. Pharmacol., 6, 182, 1933. BROWN, R. V., Amer. J. Physiol.. 134, 202, 1941. SARKAR, N. K., Ann. Biocheyn., 8, 11, 1948. SARKAR, N. K., MAITRA, S. R., and ROY, P. K., Toxicon, Ann. Biochem. Med., 6, 81, 1946. SARKAR, N. K., and MAITRA, S. R., Amer. J. Physiol., 163, 209, 1950. DEVI. A., MAITRA. S. R.. and SARKAR. N. K., Ann. Biochem. Med., 13, 23, 1954. SARKAR, N. K., Proc. Soc. exp. Biol. (N.Y.), 78, 469, 1951. SARKAR, N. K., J. Indian Chem. Soc., 34, 227, 1947. SARKAR, N. K., J. Indian Chem. Soc., 34, 61, 1947. MAIER, H. K., Vet. Med., 46, 463, 1951. CLUXTON, H. E., Proc. 2nd. Clin. Acth Conf., 3, 445, 1951. HOBACK, W. W., and GREEN, T. W., J. Amer. Med. Ass., 153, 236, 1953. SCHÕTTLER, W. H. A.. Amer. J. Trop. Med. Ilyg., 3, 1083, 1954. 10 . 11 . 12 . 13. 14. 15. 16. 17. 18. 19 . 20 . 21 . 22 . 23. 24. 25. 26. 27. 28. 29. cm 2 3 z 5 6 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):583-588, 1966 FINDLAY E. RUSSELL 583 60. CARDIOVASCULAR RESPONSES TO SNAKE VENOMS AND THEIR FRACTIONS FINDLAY E. RUSSELL University of Southern Califórnia, School of Medicine, Los Angeles, County General Hospital, Los Angeles, Califórnia, U.S.A. It has long heen kiiown lhal cerlain sitake venoms exert a deleterious effect ori the cardiovascular syslem. During more recenl years, altempts liave heen made to determine which fraction or fractions of snake venoms are res])onsil)le for these deleterious effects. Some attempt has also l)een made to define the relationships hetween the direct effects of the venom on the cardiovascular system and tliose which may he precipitated hy auto|)harmacological changes initially provoked l)y lhe venom. In the course of these various investigations, many imporlant data, which indicaled the complexity of the vascular response, have heen ohtained. The purpose of lhe j)resent paper is to present a shorl review of lhe compendium of knowledge on lhe cardiovascular effects produccd hy snake venoms, and to reflect on some experiences which, I feei, are imporlant as guide lines for future research on the pharmacology of these fascinaling toxins. First, I should like to discuss, in a very general vvay, some relalionshi|)s l)elween lhe j)hysio|)harmacological aclivities of snake venoms and lheir chemislry. We are wíell aware, as has heen pointcd oul hy numerous workers, thal snake venoms are complex mixlures, chiefly proteins, many of which have enzymalic aclivities. There was a time in our ihinking when all of lhe deleterious effects of venoms were linked wilh lhe enzymatie components of the loxin. We now know, of course, lhal ihis is nol tnie, and indeed, lhe more lelhal aclivities of most venoms — whelher lhey he snake, arthrojmds or fish — are nol generally associaled wilh one enzyme or evcn with several, although it does appear lhal certain enzymes may he closely hound with cerlain non-enzymalic |)roteins in a venom, and thal these condiinalions can he rpiite lelhal. On llie olher hand, lhe enzymes of snake venoms are cerlaitdv responsihie for some of lhe deleterious chauges provoked hy the crude toxin, and perhaps in particular with those changes which ajjjjear lo occur al lhe cell memhrane, whelher lhe cell hc one in the intima of a vessel or in suhcutaneous tissues, or even if it he a hlood cell. These unusual comhinations of mullijde enzymatie and non-enzymalic ])roleins, along with certain non-prolein suhstances, not only indicale very com[)lex or- gauization and |)hysio])harmacological activily, hui mighl apjiear to reflect upon a series of adaptive mechanisms during lhe evolulion of lhe venom. On lhe olher hand, snake venom enzymes may he involved in far more com|)Iex hiological functions, as has long heen pro])osed hy Professor Zeller in his many fine papers. I ani sure we are all hecoming impressed wilh lhe fact lhat. in general, there is a great deal of conformity in lhe organization of lhe chemislry of snake venoms, and while cerlain fractions are indeed quite differenl, chemically as 1 SciELO 584 CARDIOVASCULAR RESPONSES TO SNAKE VENOMS AND TIIEIR FRACTIONS vvell as physio[)harmac'ologic:ally, ])arlitiilai'!y al lhe Family levei, lhere appear to he more similarities lhan (liffereiiees tlum vvhal \ve luul llioughl oiie or Ivvo (leeades afio. Tliis same general im[)re.ssion is also ohtaimnl when one eonsiders tlie marine venoms and poisons, and indeed lo some cxteni even willi aiihropod toxins. There is a lendeney, ])erlia[)s forUinalely so, in lhe vvell-oiled seienliFie mind, to gather data inlo orderly groups For [)ur|)oscs oF elassiFieation. This is an admirahle Irait hnt it ean he Fraught wilh some dangers, and perhajis nowhere in hiology is this more evideni lhan in our ovvn Field. In lhe ahsenee oF reliahie dala there has heen a lendeney to arrange snake venoms into loose indeFinahIe elassiFicalions. These elas.siFiealions have, in many instanees, heen more eonFus- ing than hel[)Fnl. Even sueh widely iised lerms as luniroloxiii and cardiotoxin are inadeíjuale and misleading. For lhey are oFten ap[)lied lo lhe whole venom. and most venoms are eomplex rnixliires having several or many hiologieal pro- perties. Il has also heen shown thal neuroloxiiis ean. and oFten do, have cardio- loxic or hcmotoxic aetivity, or hoth; vurdiotoxins may have ncitroloxic or hemo- toxic aetivity, or hoth, and hcriioloxins may have the other aetivities. The laheling oF a venom as a neuroloxin is not oniy conFusing hnl dangerous. For it may lead a physieian to make unwise elinical decisions (1 ). No one has hrought this |)oint eloser to Foens lhan 11. Alistair Heid, vvho has reeenlly demonstraled that the prinei|)al clinieal Featnre oF eohra poi.soning in Malaya is local necrosis, and that ncuroloxic eFFeets in hiiman vietims are indeed rare(2). Furthermore, as Dr. Heid poinis oiit, and as has heen noted hy [jrevioiis workers, the cardiovaseular eFFeets oF lhese venoms are oFten times marked, and in many ea.ses are the eaiise oF death. It would seem, that imtil the Fraetions responsihie For the deleterioiis eFFeets oF a snake venom have heen isolated and studied individually and in eomhination, we need to exereise extreme eare in systematizing data vvhieh are hased parlly on hiologieal assay methods, ])artly on hioehemieal stndies, parlly on elinical ohservalions and |)arlly on in- Inilive himehes. There is anolher vveakness in our over-all approaeh lo lhe sliidy oF lhe physio|)harmaeologieal jiroperlies oF snake venoms. There is a lendeney to link .s])eeiFic ehemieal struetures wilh speeiFie hiologieal aetivities; i.e., thal suhstanee A |)roduees eFFeet A, and lhal suhstanee 15 produees eFFeet 15, ele. Il would appear lo me that it would he highly uniikely and unheeoming For nalure lo have developed venoms íti this manner. Partieularly, sinee perha|)S one oF lhe most irnporlant Faelors in the evolution oF a venom is lhe role |)layed hy lhe ada|)tion oF the prey or the oFFending animal. Bul whelher or not lliis eoneern is Founded, lhe very |)rinei|)le oF looking For a s|)eeiFie Fraetion lo exeri a speeiFie eFFeet is a dangerous one, For among olher things, il may oFten limit our eoneept and our experimental ap|)roaeh to determining the meehanism oF aelion oF a venom hy ihinking thal il neeessarily Follows thal one s])eeiFic Fraetion has one speeiFie Fimelion in or on one organ syslem, ailhough in some cases this may prove lo he true. Some oF lhe so ealled neuroloxins thal have heen seni lo us For Furlher evaluation in cardiovaseular preparalions during iluí ])asl Five years have had a more marked eFFeet on lhe cardiovaseular syslem than on lhe nervous system, and in some cases, in Fael, this eFFeet has Far oiil-shadoweil lhal on the laller syslem. In most oF lhese ea.ses lhe error in judgemeni has not weighed wilh lhe venom hut ralher wilh the experimenter, who, For example, having secn a mou.se in eonvulsions Following lhe injeelion oF lhe venom has presuined that iIkí cm SciELO 10 11 12 13 14 15 Mcm. Inst. Butantan FINDLAY E. RUSSKLL 585 Simp. Internac. 33(2):5,S3-58S, venoiii was a neuroloxin. wlicii iii rcalily llu- nioiisc is coiivulsing liecaiise' oí fcrcliral aiioxia caiiscd liy a niarkcdly raducad hlood supply lo ils hrain secoTidary lo syslcniic Iiy|)ol(‘nsion. 'Flieri' is onc olhar araa of cardiovascidar researcii oti wliicli I slioidd likc lo coninu-iil. '1’his involvas lhe ahoiaa of llia propar ax])arinianlal animal. Ona iniisl ha axaaadinply aarafid in applying dala darivad frorn sludies in ona gron|i of animais lo aonalnsions ahonl lha hiologiaal affaals of a vanom in anolhar gronp of animais or lo dala on lha dasign, usa and adaplion of a vanom. \Va ara all familiar vvith lha markad diffaranaas in lhe lelhality of vanonis for dif- faranl animais and hovv in soma aasas lha.sa ara ])ailieidarly ralatad lo s])aeifie aeologiaal prohlams. 1'nforlunalely, mueh of oiir informalioti on lha zooloxiao- logiaal ])roparlias of vanoms is hasad on sliidias vvilh niammals. vvhiah, of eoursa, liniils lhair a|)pliealion as far as oiir nndarslanding lha design of lhe loxin in lha animars armamanl. On lha olhar hand, il Is acpially dangarous lo appiy dala ohiainad on a fish narva-musala pra])aralion or lha frog haari, or llia aoak- roaah haarl, lo aonalnsions ahonl lha aalion of lha vanom on man, or avan on a rnammal. CerlaÍTdy lha mora divarsifiad onr sltidias llia mora imjiorlanl dala lhal vva vvill ohlain, hnl lha a|)pliealion of lhase dala nnisl ha gnardad zaalonsly. Kven in ap])lying dala ohiainad in mammals one mnsl ha exaaadingly aarafnl. For inslance, dala ohiainad from aardiovascniar slndies in dogs aannol he liherally ap|)liad lo hnmans. Il has haaome increasingly a])|)arpnl, jiarlicniarlv from slndies on shoek, lhal lhe dog may res|)ond quila diffarenlly lhan lhe human nnder similar experimenlal aondilions. The imporlanee of lhe porlal eiranlalion in lhe dog dnring varions hy|)olensive arises is far mora markad lhan in lhe hnman. In eeiiain cardiovascular condilions lhe dog mnsl he considered a porlal animal while man cerlainly is nol. Anolher difference is in lhe renal eiranlalion. In lhe rahhil and eal, as well as in lhe hnman, lhe renal vein is known lo dilala (piile raadily in responsa lo machanical and earlain chamiaal slimnii. It is far less rasponsiva in lhe dog. whila on lhe olher hand lhe s|)lenic vein in lha lallar animal dilalas (jnila easily vvilh s|)acifia drngs and slimnii. This jiiohalily raflaels lha high dagrae of sjilaen raservoir fnnalion in ihis s|)aaias. In lha eal. rahhil and hnman lhe S|)lenia reservoir is mnah less marked. In cerlain mammals, paiiienlarly lhe dog, aerlain venons valnes |)lay a more im[)orlanl role in lha vascular res|)onsa lhan lhay do in lha hnman. The cal, on lhe olher hand. ap- jiears lo res|)ond lo snake vanoms in a mannar mnch more lika lhe hnman lhan does lhe dog. In holh lhe cal and in lhe hnman lhe hypotensive crisis evoked hy C r o t a I tt s venom, for inslance. is associaled vvilh changes in lhe |mlmonary eiranlalion and perhaps in lhe larger ves.sels of lhe chesi (1,8, 1). These faw examples poini onl lhe greal care one mnsl lake in ap])lying dala from one gron|) of animais lo anolher, or lo hnmans. One niighl aonclnde lhal jierlnqis manv' of lhe differences noied in lhe literalnre helvveen resiion.ses lo varions venoms ara mora direclly ralalad lo lha choice of animal n.sed lhan lhe venom nsed or aven lo lhe leehniqne applied. Halher lhan deal vvilh speaific' dala on lhe eardiovasaniar effeels of snake venoms and lheir fraclion (and of lhase affaals mosi of ns are acqnainled), I shonld like lo revievv some of lha hasic eonea|)ls lhal one mnsl eonsidar if he ehooses lo measnre and inierprel lhe changes provokad hy vanoms in lhe de- pendenl variahies of lhe varions paramelers of lhe cardiovascular syslern. I■’ig. 1 illnslrales lhe ])rinciple of parallel cirenils and resislances in lhe aardiovascniar sysiam. Sinae lhe cardiovascular syslam aceomplishes ils hiological cm 2 3 z 5 6 11 12 13 14 15 586 CARDIOVASCULAR RESRONSES TO SNAKE VENOMS AND TIIEIR FRACTIONS fiincTions in a mechaiiical way, il can he seen lhat any chaiige in oiie of the paraineters will affect changes in tho resistance of oiu-, several or all other jiara- melcrs of lhe syslein. These changes will he reflected in lhe dependenl variahles, lhal is in the heart output, and the prcssures on lhe arterial and venous sides of the systemic and puhnonary circulations. The relationships can he piolled as HEAD NECK ARMS .UNGS TRUNK LIVER ^^1_ABDOMINAL ORGANS 1 .. .1 L SPLEEN KIDNEY PELVIC ORGANS LEGS Fig. 1 — Diagram of tho circulation showlng parameters, Inclufl- ing reslstances (moUitied from Wezler and Bõger, 1939). a matheniatical formulation, and this has heen done hy several workers. In 1954, hefore lhe days of eleclromagnetic flowmelers and other flow-jilessure nionitoring devices, Prof. Van Harreveld and I jiroposed a inodel of lhe eirculalion (Fig. 2), froni whieh eerlain equalions eould he developed and iised in defining more earefully llie changes jirovoked in lhe vascular parameters hy various venoms (5 ). cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):583-58S, 1966 FINDLAY E. RUSSELE 587 The relevant eqiialioiis descril)iiig lhe relations hetvveeii lhe parameters and lhe dependent variahies of lhe cireidation may lie sunimarized as follows: I P.si = V Pl: Csi (Rs fs) Csz Ts -j- Cin (Rl “1“ i‘l ) Ci,2 1‘l _V ( Rs + r.s )_ Csi (Rs + r,) + Cs2 rs + Cu (Rl + vl ) + Ci,2 r,, _ V n, Csi (Rs “h rs) “l” Cs2 rs - 1 - Cu ( Rl rn) -|- Cl 2 rL In these equalions, I is lhe hearl oiilpiil. P s, rejjresenls lhe syslemie arlerial [jressure. P l, indicales lhe lefl alrial i)ressure. V is relaled, ihough nol sy- tiüiiymous, wilh lhe lolal volume of lhe cireidaling l)lood. Kg and Kl are llie resislanees in lhe arlerioles and ea|)illaries of lhe syslemie and |)ulmonary eireula- lions respeelively (periplieral resislanee). The lolal resislanee in lhe venoiis syslem and all lhe faelors limiling lhe hlood flovv inlo lhe venlrieles are re])resenled hy lhe resislanees rg for lhe righl hearl and rj, for lhe lefl hearl. The largest faclor for lhe vahie r is lhe elaslieal resislanee of lhe venlrieular wall againsl filling. Cg^, C , ele...., are lhe eonslanls of eapacilanee (volume ehange per nnil ehange of pressiire) for lhe varioiis com|)onenls of lhe eireulalory ap|)aralns. These eornpoiuMils are an index of lhe lone of lhe larger vessels of lhe syslemic and |)ulmonary arlerial and venons syslems. íl ean lie seen lhal hy varying a parameler one can delerrnitie, ihrough meusuremenls in lhe dependenl variahies, lhe pressures and flows in lhe various eomponenls of lhe cardiovascular sy.slem. Today, ihis has heen made more simple hy lhe advenl of lhe eleclromagnelic flowmeter. Il is now ])ossihle, willi a lillle eare, lo measure hlood jjressnre and flow eoncomilanlly in a single vessel, and lo earry onl ihis ])roeednre in fonr or five vessels dnring a single experiment. The informalion ohlained from snch measnremenls nol only gives ns valnahle dala on lhe changes lhemselves and lheir relalionsliips lo olher vascular ])henomena, hui il gives us a ready insighl inlo lhe mechanism of aclion of lhe venom. This mighl he demonslraled hy several receul experimenls wilh C r o t a I ii s venoms. Using Iransducers lo measure cerlaiu arlerial and venons jjressures on ho Ih sides of lhe syslemic and jndmonary circnialions (some measnremenls heing cm SciELO 10 11 12 13 14 15 588 CARDIOVASCULAR RESPONSES TO SNAKE VENOMS AND TllEIR FRACTIONS takcii llirougli callieti-rs llireadcfl lliroiigh llio licari tindcr fliioroscopy), and hy taking simullaiieous Idood flow rccords willi ('l('clroinagiuTic ílowniotnrs fiom llic same and different vesscis, and liy concomilanl recording of llic eleclrocardio- grani and (‘Icctroenceiilialograni, and 1)V mcasnring ccrfhrosiiinal finid prcssnre and lhe rate and de|)th of respirations il [ias lieen ))Ossihli‘ lo delermine lhe seípience of evenis of lhe imniediale and |)reei|)ilons hypolensive eriíiiíi lhal oc- curs following lhe iniravenons injeetion of C r o ! a ! u s venoinld, I ). Il lias also lieen possihie, nsing lhese teehniqnes and cerlain isolaled organ teeliniqnes (d, 4), lo ohiain some insighl inio lhe meehanisni of aetion of ihis venoni. Frorn lhese sludies il has heen eoneinded lhal lhe hyqiolensive crisis is dne lo ehanges in lhe resislance vvilhin lhe puhnonary eirenil. Tliese elianges lead lo a decreased left heari oiil|nil ])ressnre and flow, wliieh in lurn jirovoke allera- lions in lhe flow and ])ressnre wilhin varions vesseis, ehanges in pei'i])heral resislance, ehanges in respiralion, eerehrospinal finid jiressnre, and in lhe eleelro- cardiogram and eleclroencephalogratn. Il was snggesled lhal lhe (hange in |iiilmonary resislance may he allrihuled lo lhe jiooling of hlood in lhe Inngs and larger vesseis of lhe chest, dne lo posl-cajiillary resislance froni eilher vascular conslriclion and/or lhe formalion of mnlliple ihromhi. It apjiears lhal each of lhese phenomena may |)lay a pari in lhe crisis. These ehanges are evideni in lhe hiiman, monkey and cal. They are much less conspicnoiis in lhe dog, where lhe mechanism for lhe hy[)olensive crisis may he quile differenl. The sludies previoiisly noted mighl .serve as a giiide for fulure work ou lhe cardiovascular effecls of venoms and venom fraclions, for lhe leihniques ])rovide a cotisideralile amounl of dala not olherwise ohtainahie, while also permilling a carcful l•heck and evalualion of inler|)retations. Bul even loday new lechniques are heing developed, and these may give us considerahiy more insighl inIo lhe mode of aclion of loxins lhan we aniicipale. Parlicnlarly encouraging is lhe possihilily of measnring definilive vascular ehanges vvilh lagged venom and hlood, ailhough even lhese lools have ohvious limils of usefniness. In all of lhese sludies, however, lhe crilical conlrihiilion will slill resi wilh lhe ex|)erimenler. As one sludies lhe progress in onr Science, as in all Sciences, il is ohvions lhal lhe mosi significani conirihulions are fnlly dejiendenl iqion lhe invesligalor and nol u|)on his eipiipmenl. The cardiovascular effecls of venoms and lheir fraclions are slowly heing unraveled hy scholars and not hy gadgeis, and we have seen in ihis meeling a demonsiralion of lhe kind of iniellecinal slimnialion and en- coiiragemenl lhal will greally enhance onr underslanding of lhe pro|)erlies of lhese mosl remarkahie loxins. Beitciíence.s 1. RUSSELL, F. E., in F. A. DAVIS (Editor), Cyclopedia of Medicine, Surgery and. tlw Specialtics, Vol. II, Philadolphia, 1962, p. 197. 2. REID, H. A., Brit. med. J., 2, 540, 1964. 3. RUSSELL, F. E., BUESS, F. W., and STRASSBERG, J., Toxicon, I, 5, 1962. 4. HALMAGYI, D. F. J., STARZECKI, B.. and HORNER, G. J., J. appl. Pliysiol. (U.S.A.), 20, 709, 1965. 5. RUSSELL, F. E., and VAN HARREVKLD, A. Árch. int. PJmiol.. 62, 322, 1954. cm 2 3 z 5 6 11 12 13 14 15 Mem. Inst. Bulíintan Simp. Internac. 33(2) :5S9-6n2, 1966 MASAIIIRO OKADA 589 61. PHAKMACOLOGY OF THE COMPONENTS OF TOAD VflNOM AND ALLIED SUBSTANCES MASAHIRO OKADA Department of Pharmacology, Faculty of Medicine, Tokyo, Japan The dried venom of lhe Chinese load has beeii used externally íroin aiitiquily as a home remedy called Senso iClFaii Su) for canker sores, looth-ache, and many local inflammatory conditions. Biil most pharmacologists have concenlrated them- selves on the sludy of ils cardiolonic action, and little is known on ils local actions. V7e have investigated lhe local aneslhetic aclion of each purified suhstance exlractcd from Senso (Tahie I). As descrihed in previoiis papers (1,2,3) lhe strong local aneslhelic properly was foiind generally in the steroid fraclions while the water-soluhie fraclions have local irritating aclion with very weak or almost no local aneslhetic jnoperlies. For example hufotenidine, a water-.solnhle derivalive of Iryptamine, has no aneslhetic action. For [jhartnacological lests, lhe snhstances insoinhie in waler were dis.solved in a mixtnre of waler and pro])ylene glycol. In ihose cases conlrol lests were made with lhe same concentralion of pro])ylene glycol. Snrface aiií^slhetic action on córnea was delennined with adnll inale rahhits. The conjunctival sac was filled with 0.2 ml of lhe solnlion of various concenlra- tions, and the lids were released after one minute. The wink reflex was elicited hy a horse-tail hair (0.17 mm in diameler, 0.7-0.75 g in jiressnre). The lesl of 6 jiricks was ajiplied on córnea every 5 minutes for 30 minnle‘S. The nnmhcr of limes lhe prick failed lo elicil a hlink reflex during the 30-minnle jieriod was added nj) and the sum gives an indicalion of lhe degree of aneslhesia. Therefore, 6 X 6 = 36 is the value thal indicales complete anesthesia for lhe 30-minnle period. Tahies If and III show .some resnlts. When lhese residis for each snhstance were jilotled on a log dose-response coordinale, the apiiroxiinalely slraighl lines so ohlained were not qiiile jiarallel to each otlier, which rnakes it difficnlt to slale exaclly the relative strenglh of anesthetics to cocaine (Fig. 1), hnt in roimd figures, when aneslhetic ])olency of cocaine hydrochloride is taken as 1, thal of jirocaine hydrochloride is 0.1, while that of Fraclion No. 200 is 30-60 I mean 40) as Tahie IV shows. This facl is remarkahle and what is more, No. 200 has almost no irritating aclion. cm SciELO 10 11 12 13 14 15 SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):589-602, 196{i MASAHIRO OKADA 591 TABLE II — NUMBER OF TIMES THE BLINK REFLEX IS ABSENT DURING A 30-MINUTE PERIOD (No. 200) Time (min) CONCENTRATION 0.011% 0.017% 5 4 2 3 2 2 2 3 2 5 4 5 5 4 2 3 4 10 5 5 6 5 4 5 5 5 6 6 6 6 6 6 6 6 15 4 2 4 5 1 3 4 5 6 6 6 6 6 6 6 6 20 2 0 2 3 0 0 2 3 6 4 6 6 5 6 5 2 25 0 0 0 1 0 0 0 0 4 3 4 4 4 4 4 0 30 COO 0 0 0 0 0 1 1 2 2 2 3 2 0 Totai 15 9 15 16 7 10 14 15 28 24 29 29 26 27 26 18 Time (min ) 0.025% 5 5 4 6 6 5 4 5 6 10 6 6 6 6 6 6 6 6 15 6 6 6 6 6 6 6 6 20 6 6 6 6 6 6 6 6 25 6 5 6 6 6 6 5 6 30 3 3 6 6 5 6 5 6 Tolai 32 30 36 36 34 34 33 36 TABLE III — NUMBER OF TIMES ' THE BLINK REFLEX IS ABSENT DURING A 30-MINUTE PERIOD. COCAINE-IICl Time (min) CONCENTRATION 0 . 2 % 0.4% 5 4 6 6 6 6 6 6 5 6 5 6 6 6 6 6 6 10 1 3 6 3 3 3 2 2 6 6 6 6 6 6 6 6 15 0 0 2 0 0 1 0 0 2 3 6 6 5 4 4 4 20 c 0 0 0 0 0 0 0 0 0 1 4 0 2 1 0 25 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 30 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Total 5 9 14 9 9 10 8 7 14 15 19 22 17 18 17 16 Time (min) CONCENTRATION 0 . 8 % 1 . 6 % 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 10 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 15 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 20 2 4 6 5 6 5 5 5 6 6 6 6 6 6 6 6 25 0 0 3 2 2 0 3 2 6 4 6 6 6 5 6 6 30 0 0 0 0 0 0 0 0 6 0 6 5 4 2 5 6 Totai 20 22 27 25 26 23 26 25 36 28 36 35 34 31 35 3ÍÍ cm SciELO 10 11 12 13 14 15 CSQ9 PIIARMACOLOGY OF THE COMPONENTS OF TOAD VENOM AND ALLIED SUBSTANCES Fig. la — Log Dose-Response curves oI Bufogenins and allied Compounds. TABLE IV — POTENCY IN TERMS OF COCAINE-IICl (=1.0). IN RABBIT CÓRNEA SURFACE ANESTHESIA Substance Number of animais Potency of surface anesthesia Rj R, R.^ Mean Cocalne-IICl 8 1 1 1 1 Procalne-IICl G 0.13 0.11 0.09 0.11 193 6 12.G 14.8 17.4 14.9 196 (i 13.8 16.2 19.1 16.4 194 () 9.6 11.2 13.5 11.4 197 (J 1.6 2.2 3.2 2.4 198 () 19.1 24.6 28.8 24.2 199 4 0 0 0 0 200 8 28.8 39.8 57.5 42.1 201 (í 5.0 4.7 Antllog (X,.-XO X !■; log dose of cocalne-IICl X»: log dose of substance R,: Response lO-levcl R,: Response 20-ievel R,: Response 30-level cm SciELO 10 11 12 13 14 15 Mem. Inst. Hutantan Simp. Internac. 33(2):589-6(>2, 1966 MASAIIIRO ÜKADA 593 The onset of local aiieslhcsia by No. 200 is soiiiewhat slovvcr lhan cocaiiie, l)iil its duralioii is mucli lotigcr. In infiltration ancslhesia test (wheal mclliod) on mati, lliis sulislance (No. 200) ])iodiices total local anesthesia in 5 minutes, whicli continues dnring 30 minutes to 1 liour in 0.0003% solntion and for 3 bonrs in 0.03% solntion, and no local tissne damage vvas leít. The fraction No. 200 corresponds to the snbstance of fraction niimber Fr. 52 in the repori hy Olmo and Komatsn (4). As was therein descrihed, this snbstance gave four spots with different Kf values by ])aper partition chromatography, biit repeated ])iirification finally afforded a snbstance giving only a single sj)Ot at Hf 0.61. Komatsn |3| examined the ehemieal and physical i)ro|)erties (melting point, ele- mentary analysis, Hf valiie, colour reaetion, ultraviolet and infrared S|)eelra) of this fraction and fonnd il to be identical with bnfalin isolated from toad venom by Knno Meyer. Local anesthetic activities of Meyer’s sample of bnfalin (No. 201!) and the sample (No. 209) isolated by Komatsn were eompared and we fonnd no difference between the tvvo, both qnalitatively and cpiantitatively. The assay method for local anesthetic activity was the same as previonsly descrihed, and the resnlts are shown in Tables V and VI. Samples No. 208 and No. 209 in 0.0025% solntion showed the maximnm hyjresthesia after abont 10 minutes and the drng action disap|)eared after abont 20 minutes. With 0.005% solntion, hypesthesia continned for abont 30 minutes, with 0.01% solntion for over 30 minutes, and an ahnost perfect anesthesia was exhibited. With 0.02%) solntion. complete anesthesia continned for over 30 minutes and there .seemed to be no irritation on the córnea. TABLK V — NUMBER OF TIMES THE BLINK REFI.EX IS ABSENT DURING A 30-MINUTE PERIOD — No. 208 Time (min) CONCENTRATION 0.0025 % 0.005 % 5 0 2 0 2 2 0 2 4 5 2 2 3 10 3 4 3 4 3 4 6 6 6 6 5 6 15 3 1 2 3 3 3 6 5 4 5 5 5 20 0 0 0 2 1 2 5 4 3 4 3 4 25 0 0 0 0 0 0 3 2 1 0 0 1 30 0 0 0 0 0 0 0 0 0 0 0 0 Total 6 7 5 11 9 9 22 21 19 17 15 19 Time (min) CONCENTRATION 0.01 % 0.02 % 5 5 5 5 4 5 3 6 6 6 6 6 6 10 6 6 6 6 6 6 6 () 6 6 6 6 15 6 6 6 6 6 6 6 (í 6 6 6 6 20 6 6 6 6 6 5 6 6 6 6 6 6 25 5 4 5 0 4 3 6 G 6 6 6 6 30 4 2 3 3 1 1 6 6 5 6 6 6 Total 32 29 31 31 28 24 36 36 35 36 36 36 cm SciELO 10 11 12 13 14 15 594 PlIARMACOLOGY OF THE COMPONENTS OF TOAD VENOM AND ALLIED SUBSTANCES TABDE VI — NUMBER OF TIMES THE BLINK REFLEX IS ABSENT DURING A 30-MINUTE PERIOD — No. ;209 Time (min) CONCENTRATION 0.0025 % 0.005% 5 2 0 0 2 2 2 4 4 3 3 3 3 10 4 3 0 4 4 4 6 6 5 6 5 5 15 2 3 í) 3 3 1 6 5 5 5 4 5 20 0 1 0 2 1 0 5 3 4 4 3 3 25 0 0 0 0 0 0 3 1 1 2 1 2 30 0 0 0 0 0 0 1 0 0 0 0 0 Total 8 7 0 11 10 7 25 19 18 20 16 18 CONCENTRATION Time (min) 0.01% 0.02% 5 5 5 5 5 4 4 6 G 6 6 6 6 10 6 6 6 6 6 6 6 6 6 6 6 6 15 6 6 G 6 6 6 6 6 6 G 6 6 20 5 6 5 G 4 5 6 6 6 6 6 6 25 4 5 5 5 3 4 6 6 6 6 6 6 30 1 3 3 4 1 2 6 6 6 G 4 6 Total 27 31 30 32 24 27 36 36 36 36 34 36 Do.«(‘-r(>S])oiisc curve is giveii in Fig. 2. Conijiarisoii of lhe values from this gra|)li wilh tho.se of eoeaine hyclroehloride aiul procaiue liydrochloride is giveii iii Talile VII. Fig. 2 — Log Dose-Re.sponse Curve.s. The local aiiesthelic adiou of samples No. 208 and 209 is over Iwice that of No. 200, and ahoul 90 times that of cocaine hydrochloride, and duration of lheir action is markedly lotiger, allhough it takes somewhat longer lime for the action lo ajipear. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):589-602, 1966 MASAHIRO OKADA 595 TABLE VII — POTENCY IN TERMS OF COCAINE HYDROCIILORIDE ( = 1.0) SURFACE ANESTIIESIA IN RABBIT CÓRNEA Compouncl No. of animais Poteney of surface aneslhesia R, R, R. Mean Cocalne-HCl 8 1.0 l.ü l.ü 1.0 Procalne-HCl 6 0.12 0.11 0.C9 0.11 No. 209 6 78.7 87.9 98.0 88.2 Antllog (X,-Xs) Xi-: log dose of cocalne-IiCI Xs: log-dose oí sub.stance R, Response 10-level R, Response 20-Ievel R.i Response 30-level riiis fact was also confirmed iii iiilradernial vvheal lesl on hiinian skin. lii lliis lest 0.1% soliition (iii 50% propylene glycol) of No. 209 was diliiled with physiological saline to niake 0.01% and 0.001% Solutions, 0.1 nil of eacli solution w'as injected intracutaneousiy in tlie forearm, a local |)atcli of anestliesia was jirodiiced and its degree and duration was tested l)y pin-pricks. With 0.001% solution, anestliesia lasted for ahout 30 minutes, while with 0.01% solution complete anestliesia lasted for several (3-7) hours, without leav- ing any tissue damage. These results are in marked contrast with the effect of cocaine hydrochloride and jirocaine hydrochioride, whose intracutaneous in- jection of 0.1 ml of 0.125% solution causes immediate apjiearauce of the action hut its durations are respectively 15 aud 6 miuutes. The local tissue-irritating jirojicrties of these fractions were estimated hy iutradcrmal injection of 0.3 ml of the following local anestheties into the clipped ahdominal skin of a rahhit hy Trypan Hlue test descrihed hy Hop|)e, Alexander, and Miller (5) and the magnitude and intensity of local hlue spots on the ahdominal skin caused hy the intravencus injection of trypan hlue (1%, 1 ml/kg) were measured after 30 miuutes, 1 hour, and 3 hours. With 2% or 5% jiro- caine-hydrochloride (5% propylene glycol solution) and 2% xylocaine-hydro- chloride (5% j)ro|)ylene glycol solution), large intensive hlue sjiots soou ap])eared, which could not he ohserved with 0.03% of hufalin (5% |no|)ylene glycol solution) and 0.01% of gamahufotalin (5% |)ropylene glycol solution). As has aiready heen descrihed (1) some local anesthetic activity has heen found in cinohufagin, cinohufatalin, gamahufotalin (Fig. la), etc., so that the local analgesic action of the Chinese crude drug, Ch’an Sn, the toad venom, cannot he accredited entirely lo hufalin. However, hufalin is the suhstance found until lhe present date that has the strongest local anesthetic action among hiifa- dienolides, and a way will he opened for the use of sleroid as a local analgesic hy com|)arative examination of such suhstances to lie j)re|)ared hy changing their side chain. Cinohufagin aiul citiohufalalin are ahout one-sixlh of hufalin in local anesthetic poteney, while resihufogenin has no such adiou. Digitoxigenin, a cardenolide jiossessing the same Chemical slructure as hufalin excejil for its 5-memhered lactone ring, has a very weak local anesthetic actioti, ahout one-fortieth that of hufaliu in surface anesthetic poteney. cm SciELO 10 11 12 13 14 15 596 PHARMACOI.OGY OF THE COMPONENTS OF TOAD VENOM AND ALLIED SUBSTANCES Ciliohufaginic acid, fonncd Iiy cleavage of the laclone ring through Iiydro- lysis, was found lo l)t' entircly dt-void of aiiestlietic adiou iu lhe eye. The sigiiifieaiue of lhe |)ref;etiee of uiiíaliiraled ladoiie ring, hesides lhal of hydroxyl iii Ca-posilion of sleroid ring, seenis lo l»; essenlial for lhe local aneslhdic adion. Hufaliii has, however, a very vveak anei-lhdic adion on llie nerve filicr as compared vvilh procaine (Tal)les VIII and IX). TABLE VIII Threshold anesthetic concentration Duration of anesthesia Toxicity (mouse) S| cu p .ií Ml Compound nerve ) B u s g § B C 3 ctí x: córnea (min.; o ,5 c . Cd — 3 Td O Cd _ c + ° « -S 2- bjo B M B s Sciatic (trog) cC OJ c ^ o i" U w 3 O ^ Cd Cd U Ot C > 1^- M r* Rabbit 1% sol. Human intracut 0.5 % SC Human intracut 0.5 9c SC lin l:õC S.C. LD I.V. LD. Procaine 0.250 4.0 0.04 0-2 16 171 750 45 Butacaine 0.125 0.31 0.02 30 45 200 50* 12* Dibucaine 0.004 0.003 0.0025 51 (0.1%) 100 221 10* 2.5-' Cocalne 0.125 0.32 0.02 22 12 200 110 11 Butalin _ 0.0025 0.001 30 (0.02%) ■ 30 — 4.0 0.35 (0.001% ) 150-480 (0.01 % ) • Rabbit TABLE IX Compound .Surface anes- thetic potency (rabbit córnea) ED,„ (rabbit córnea) LD,^ (mg/kg) (mouse) LD.„/ED,,„ mg 90 I.V. s.c. I.V. S.C. Cocalne 1.0 0.85 0.437 11 110 12.9 129 Procaine 0.11 8.50 4.37 45 750 5.3 88 Xylocaine 0.27 3.10 1.74 31.5 172 9.!) 54 Bufalln 88.2 0.01 0.ü(í 0.35 4.0 29.2 400 The effed of hiifalin and procaine on lhe eledrical excilahilily of nerve fiher wa.s examined, iising lhe scialic nerves of loads or hullfrogs. As descrihed hefore (6), hiifalin, in spile of ils very strong |)Olency in siirface aneslhesia, has otdy a vveak effed on lhe nerve fiher. Il was lherefore sujvposed lhal lhe sile of loeal aneslhdic adion of hufalin niight he lhe recejftor of sensory nerve ending. However, lhe sile of adion in queslion is now under delailed cxaminalion. cm SciELO 10 11 12 13 14 15 Mcm. Inst. Butantan Simp. Internac. 33(2):589-602, 1960 MASAHIRO OKADA 597 Hufadienolides as vvell as cardeiiolides induce a loiig-lastiiig contraclure iii llic isolated frog rectas ahdonünis inuscle, wliile cocaine, and otlier local anesthetics inliil)it llie acetylcholinc conlractiire of tlie imiscle. Tlie muscular contraclure curve consists of two stages: the stage of gradually increasing lonus, and tlie one inducing faster contracture follovving tlie foriner. The second stage is inhihited hy pretreatnient with d-tid)ocurarine, as well as hy letrodoloxin, while the firsl stage is not affected hy its juetrealment (presenled in Fig. 3). This contracture-inducing activity of hufadienolides and cardenolides runs parallel to the cardiotonic activity of these coinpounds. Arh Bufalin 1 : 2 000 001) 1 1 000 000 Arh Bafilin 1 : 100 000 .iher cl Tr. I : 10 000 0 Fig. 3 — Rufalin on Rectns Abdominis Muscie of the Frog. Method (i) Sciatic nerve trunk was arranged in a separating box, as inclicated in Fig. 4, slimulus was given to it at po.sitions a and b (S), action-current was led off from positions d and e (R) and recorded. Drug was applied to desheathed part c. (ii) Direct stimulus was given to a single nerve fibre. As shown in Fig. 5, single medultated nerve fibre was excised, internode of Ni-Nj-Nj was dried in the air, and Nj was stimulated directly after application of drug to the node. U. N Fig. 4 — Experimental nrrangement for nerve trunk. N: Nerve trunk. D.N: De¬ sheathed part. S: Stimulus. R: Reeord- Ing apparatus. a, h, c, d; Normal Ringer. C: Test solutlon. Fig. 5 — Experimental arrangement. N ij 3 Nodes exposed. N 13 : in 0.3% Coealne-Ringèr. N^: In test .solutlon. N,-Nj; Slimulatlng Circuit. Nj-Np Recordlng apparatus. cm SciELO 10 11 12 13 14 15 598 PHARMACOLOGY OF THE COMPONENTS OF TOAD VENOM AND ALLIED SUBSTANCES Action-current was conducted from space between Nj and N, by means of a low input resistance (IM ij ) D.C. amplifier combined with a cathode ray oscillo- scope, and recorded. Moreover, node Ni and Nj were previously anesthetized with 0.3% cocaine-Ringer solution. The stimulus was a square pulse of duration of 0.5 m sec. provided by a stimulator in (i) and (ü). Thus, the aspect of decreasing action-current at applied region, together with the elapse of time, was observed. Concentrations of the examined Solutions were 2x10~ 1x10 and 1 x 10 bufalin-Ringer’s solution and 0.05-1.0% procaine-Ringer’s solution, propylene glycol alone was examined at the same time.With the applied concentration of procaine- Ringer solution, action-current was found to decrease or disappear in nerve trunk and single nerve fibre. After the materiais were brought to Ringer solution, and thus fully recovered, they were applied with bufalin. With Ix 10 ~ 5 concentration there was noted almost no effect but with IXlO""* to 2x10“'* concentration there occurred reduction in action-current or inhibition on the conduction of excitation. However, almost the same effect appears in propylene glycol itself with the same concentration as of the solution used as solvent, so that the action of bufalin on nerve trunk and single medullated nerve fibre may be far weaker than its surface anesthetic action on córnea, and strong local anesthetic action may be presumed to be due to its specific effect on the receptor of sensory nerve ending. Its mechanism is now under investigation. As to the cardiotonic activity it has long heen l)elieved that the presence of OH groups in Cs- and Cj 4 -positions was a requisite in cardenolides. In resi- bufogenin there is no OH group in C^-position and there is a j 8 -epoxide («- epoxide is inactive) ring between C 14 - and Cis-positions. In spite of this Chemical structure, resihufogenin was found to have cardiotonic action nearly as strong as that of ouabain (Table X) hesides marked activities in respiratory excitation TABLE X — CONTRACTURE-INDUCING ACTIVITY OF THE VARIOUS CARDIO- ACTIVE STEROIDS AND THEIR DERIVATIVES (A) Digitoxigenin 4 - 4 + Bufalin 4 4 4 Oleandrigenin 4 4 Bufotalln 4 4 4 Digitoxin 4 4 Reslbufogenln 4 4 Dihydrodigitoxin 4 Cinobufagin 4 4 4 Ouabain Digitalinum verum mono¬ 4 4 Gamabufotalln 4 4 acetate 4 Strospeside 4 4 Lanato.slde C 4 4 17 ii-Dlgitoxlgentn /I-Anhyrirodlgitoxlgenin 14fv, 1.5ti-Epoxy- ( p ) -anhydrodlgitoxlgenln 17 (v-Digitalinum verum monoacetate /I-Anhydrogamabufotalin Deacetylclnobufaglnic acld (C) + 4 + + 4- 4- Tincture of dlgitalis purpurea leal (plgeon LD,'50 74,0 mg/kg) eftcctlve in 10-» cffective in doses of 1-3X10-* “ 1-3 X 10-“ “ IX10-5 “ IX10-“ 1 SciELO Mem. Inst. Butantan Slmp. Internac. 33(2):5S9-602, 1966 MASAHIRO OKADA 599 and elevation of Llood pressure tiotwilhstanding its very low toxicily. This facl is considered to play an importanl role in furlher sliidy of the cardiolonic action of these suhslances (Fig. 6, Tahles XI-XII). Cinobufagin Fig. 6 TABLE XI — LETHAL DO.SE OF BUFOGENINS AND AELIED COMPOUNDS IN THE CAT (HATCHER-MAGNUS METHOD) COMPOUND Bociy vveight (g) and sex- of animai Time of infusinn ímin.) M.L.D. (mg/kg) M.L.D. reported by ot.her workers (mg/kg) Bufalln 26S0 47 0.157 0.137 Gamabufotalin 2000 12 0.134 0.101 2400 30 0.137 Cinobufagin 2700 54 0.296 0.200 Resibufogenin 23.Õ0 110 3.2 5 Digitoxigenin 1900 49 0.411 0.450 Digitoxin 3450 130 0.365 0.325 Strophanthin 2400 64 0.118 0.116 cm SciELO 10 11 12 13 14 15 600 PIIARMACOLOGY OF THE COMPONENTS OF TOAD VENOM AND ALLIED SUBSTANCES TABEE XII — FROG SYSTOLIC STANDSTILL METHOD (hr.) COMPOUND ED„ (/ig/g) 95% Confidence limit (pígie) Bufalln 1.01 0.95-1.08 Gamabufotalin 1..15 1.26-1.44 Resibufogenln 7.11 5.29-8.93 Strophanthin 0.28 0.24-0.33 Digitoxin 1.75 1.62-1.89 Digitoxigenin fi.io 4.80-7.75 í)ii ihis mechanisni of respiralory excitatioii of resiliufogeniii lhe following expcriinent was made oii urethaiiized ral)l)il. Even afu-r intravenous itijeclion o[ 10 nig/kg of procaiiK-, wliich iidiihits llu' carolid sinus reflex, and causes com|)lete disa|)pearance of res])iratory-stiinulating action l)y 0.05 tng/kg nicotine tarlrale and O.d mg/kg loheline hvdrochioride, tlie resjiiratory stimiilaling action of resihiifogenin could lie ohserved, and it was foiind lo I)e alinost eqiial lo that induced hy single a 5 X 10“') and longer exposure, depolarization and spontaneous action potentials occiirred (Fig. 2). Perfusion of 80 O-* Q2 sec Fig. 2 — Spontaneous activity of a motor fibre supertused with Rlnger containing venom (5X10-’). Photograph taken after 18 minutes exposure to venom. the node with low-soditim Solutions aholished the effect of venom, and increasing the sodiiim to normal Hinger leveis restored the effect again. (lenerally, the action of venom on the resting potential was strongly dependent on the concentrations of sodiuin, the depolarization amplitude rising with increasing sodinni concentra- tion at a rate snggesting that the resting memhrane was rendered mnch more jicrmeahle to Na hy the venom (Fig. 3). The venom-indnced dejiolarizations and the spontaneous firing of impulses in the nerve fihres of a nerve-muscle jireparation wili, of cour.se, initiate Iwilch- ing of the muscle. Is there evidence for a direct action on miiscle as well? A contracture of isolated skeletal muscle can he ohtained with venom in the pre- sence of sufficient tuhocurarine to antagonize quite large qnantities of acetyl- choline (2). In Fig. 4, a rat diaphragm is heing stimulated directiy (suh- maximally) in the |)resencc of tuhocurarine. At lhe arrow 5 pg venom/ml was added, resulting in a contracture and increased amplitude of lhe twitches. Gradually. the effect diminishes as time goes on. and over the nexi 15-30 minutes cm SciELO 10 11 12 13 14 15 G06 SOME ASPECTS OF THE PHARMACOEOGY OF THE VENOMS OF AFRICAN SCORPIONS Flg. 3 — Relatlonship tietvveen Na concentration of the médium and membrane potential before and after exposure to venom (l()-“). Ordlnate, change of membrane potential In mV ídepolarizatlon positive). V = 0 is the normal resting potential before applicatlon of venom. Mcan values and standard errors of the means of 14 flbres. the twitch amplitiule decreases until lhe muscle hecoines almost iiiexcitahle. With higher concentrations of venom, violent spontaneoiis twitrlies are .«uperimposed initially. Il seems likely, therefore, lhal there is a direet effert on skelelal miiscle and, inkeeiiing witli lliis, n-eords resting memlirane jiotentials from Sartorius fihres show a slovv depolarization imder the inflnenee of venom tinlil an action ])Otential is elicited (Adam and Weiss, un])ul)lished oliservations). I’ossihly ihis, too, may l)e due to an effect on Na eonductanee throngh lhe memltrane hiit, as yet, there is no experimental evidence. Thiis lhe effeei olitained witii venom on an isolated or m situ nerve-musele prejtaration is [trohahiy dne to an aetion on lioth muscle and nerve, though it seems likely lhat lhe latler is affected more readily. CoMPAitisoN wiTH VEiiATHiNE: Since lhe early vvork of Hoiissay (10), the effects of several venoms on nerve and skeletal muscle have heen noted to re- semhle those of veralrine, or one of ils constitiienl alkaloids, veratridine. Fig. 5 shows the effect of dh /xg veralrine/ml on lhe direclly, std)maximally, slimulated rat diaphragm, inducing a coniracture and incn'ased Ivvitch am|)littide very similar to that shown in Fig. 4. Various differences, e.g. lhe smaller effect of cm SciELO 10 11 12 13 14 15 cm 2 3 4 5 6 SciELO ;lo 11 12 13 14 15 fiflQ SOME ASPECTS OF THE PHARMACOLOGY OF THE VENOMS OF AFRICAN SCORPIONS veratrine on muscle relaxalioii afler a twitcli, l)elwcen vcnom and veratriíie can 1)0 ficmonstrated (2,12) hiit oii tlio whole thoro is a strikiii" similarity. Vora- Irine has keen showii to liavo direot depolariziiiji effects on skoletal muscle and on nerve fihres, and tliese effoots have heen interpreted as duo lo increases in mcmhrane permeahility to Na (Id, 14, 15, 16). Tluis llie observed similarity in the actions of venom and of voralrine may havo its hasis in a oonirnon meclianism. Again, the effects of holh venom and veratrine resemhle tliose induced hy low- calcium Solutions and hy Ca-chelating agents, and can he al least partially an- lagonized l)y increasing lhe calcinm concentration of the médium (2, 16, 12). Such effects have heen demonstralcd with various venoms for many years(17). Displacement of (ia from memhranes was suggested as lhe meclianism of vera¬ trine aclion hy (lordon and Welsh (18) and it seems possihle that venoms could displace Ca in a similar way. One might sjieculate that the jiositively-charged venom molecules disfilay an affinity for the memhrane acidic jihospholipids in ])reference to (ia or lo Na, ihus interferinf Na-c System, hut it is prohahly not profilahle lo pursiie further any disciission of (ia-venom antagonism al the memhrane at presenl. However, it should he rememhered that various memhrane “slahilizers”, such as adrenaline or local anaesthetics, can he shown to hlock or reverse phases of venom action, and it has heen poslulated that their stahilizing effects may he mcdiated via lhe memhrane Ca (19, 20, 21). Pain production by VENOM: This is anolher nolahle effect of scorpion venom, and may well he related lo lhe actions on nerve fihres discussed previ- ously. In some cases it is possihle that lhe presence of large qnantilies of serotonin may conlrihute to the pain of a sting(22, 23) hut [irohahly the major jiain-producing suhslance is protein. Using lhe Armslrong-Keele cantharidin hlister lechnicpie (24), \ve found that venom could still cause jiain when its serotonin contenl was helovv ihreshold; that jiain conld he provoked hy lhe ajiplication of eluates from a protein hand after eleclrophoresis; and that it was usually |)ossihle to detect a cpialitative difference hetween lhe pain produced hy pure serotonin and that due lo venom (Adam, Smith & Weiss, unpuhlished ohservations). It .seems possihle that lhe same suhstanees which provoke neuro- muscular activity are resiionsihle for most of lhe jiain of a sting, and that this may he due to a direct stimulalion of sensory nerve fihres hy the meclianism already discus.sed. It is interesting to note that holh veratrine and citrale can induce pain al lhe hlister hase(25). PiiosPHOi.iPASE A ACTtviTY: Althougli /.. quinquestriaíiis venom has heen shown to have some haemolytic activity (26), it does not appear to contain jilios- pholipase A. Incuhation of venom with pnrified |ihospholi|)id suhsirales, human plasma, rat-hrain or rat-muscle homogenates resulls in a negligihie increa.se in free fatty acids, and a lack of degradation of di-acylphospholijiids ean he con- firmed chromatographieally (27). One wonders if lhere is a direct lylic faclor and whelher this might he the neuroloxic protein interfering with calion lrans|)oiT across lhe erylhrocyle memhrane. However. lhere is some evidence that the toxicities of various venoms do not parallel their haemolytic aclivities (28). To SUMMAHIZE: It seems likely that the main aclion of L. quínquestriatus venom is to interfere with the meclianism switching on and off lhe Na ])ermeahilily of cell memhranes. While this is a suilahle working hypolhesis for cnrrenl jiharmacological studic‘s, it is evident that lhe immediate recpiirement for further advances is lhe provision of pure venom fraclions in adequate amounts. cm SciELO 10 11 12 13 14 15 SciELO ^0 2 3 5 6 11 12 13 14 15 L. cm 610 SOME ASPECTS OF THE PHARMACOLOGY OF THE VENOMS OF AFRICAN SCORPIONS Discussion O. Vital Brazil (Department of Pharmacology, University of Campinas, Campi¬ nas, São Paulo: “Although it was known since the investigations of Maurano (1915), Vital Brazil (1918) and Houssay (1919) that the venom from the South American scorpions belonging to the genus Tityus was immunologically different from that of the African scorpions, their pharmacological actions were believed to be almost identical. However, by comparing the results obtained by Prof. Adam employing the venom of Leiuriis quinquestriatus and that of other Buthinae, with our own results using the venom of Tityus serrulatus, we can now appreciate that there are also important pharmacological differences between these venoms. In fact, the venom of T. serrulatus never produced, in our experiments, the contracture of the isolated rat diaphragm as described by Prof. Adam for the venoms of the African Buthinae. FIg. 1 — Isolated phrenlc nerve-dlaphragm preparatlon of the rat. The venom (10 mcg/ml) of Tityus serrulatus evoked contractlon.s of the diaphragm whlch were abollshed by d-tubocurarlne (10 mcg/ml). Instead, it produced intense twitchings of the muscle which were promptly abolished by d-tubocurarine (Fig. 1). Therefore, the venom of Tityus serrulatus seems to be devoid of the direct muscular action exhibited by the venoms of the African Buthinae. The twitchings as well as the increase caused by T. serrulatus venom in the amplitude of the response evoked by isolated supramaximal shocks applied to the nerve (Fig. 2), can be explained by acetylcholine release from the motor nerve terminais. Such a release of acetylcholine caused by the venom has recently been demonstrated in my laboratory at the University of Campinas. The isolated inervated, and sometimes the isolated chronically denervated hemi-diaphragm, were SciELO K. R. ADAM anti CH. WEISS Mem. Inst. Butantan Simp. Internac. 33(2):6n3-614, 1966 611 Fig. 2 — Isolated phrenic nerve-diaphragm preparation ot the rat. The venom of T. ser7'vlatus caused a great increase in the responses produced by nerve stimulation wlth supramaximal shocks delivered at a rate of 6 per minute. used in these experiments. They were suspended in Tyrode solution containing 0.2 per cent of glucose and 5 x oí neostigmine methylsulphate. The bath volume was 5 ml and its temperature, 37°C. The preparations were oxygenated by bub- bling a m.ixture of 95 per cent O; and 5 per cent COj. The fluids after bathing the diaphragm for 20 minutes were removed and immediately assayed for acetyl- choline by its depressor effect on the arterial blood pressure of anaesthetized small cats injected with hexamethonium and ephedrine. The results can be summarized as follows: 1. A very small spontaneous release of acetylcholine sometimes occurred, lhe acetylcholine content of the fluid being always less than 0.4 ng per 0.2 ml. There- fore, the spontaneous release of acetylcholine by the hemi-diaphragm was always less than 10 ng. 2. The venom promoted the release of acetylcholine from the inervated hemi- diaphragm. The acetylcholine content of the venom containing fluids which bathed the hemi-diaphragm for 20 minutes was seldom less than 2 ng per 0.2 ml; in most instances it varied from 2 to 4 ng per 0.2 ml (Fig. 3). Therefore, the acetylcholine chlorine released by the venom from the hemi-diaphragms could be estimated to be from 37.5 to 100 ng. 3. The v'nom did not release acetylcholine from chronically denervated hemi- diaphragms. * 4. Curarization ot the hemi-diaphragms with d-tubocurarine did not seem to reduce the release of acetylcholine by the venom. 5. The venom did not released acetylcholine when the fluid bathing the hemi- diaphragm contained procaine. G. The acetylcholine released by venom seemed to be calcium dependent. When Ihis ion was suppressed from the Tyrode solution no acetylcholine release could be demonstrated. When it was increased above the usual concentration in the Tyrode solution, an increase in the acetylcholine output was verified. The mechanism of acetylcholine release by lhe venom is unknown. Depolar- ization of the nerve fibres by the venom as Prof. Adam has verified, would explain it.”' 1 SciELO 612 SOME ASPECTS OF THE PHARMACOLOGY OF THE VENOMS OF AFRICAN SCORPIONS Flg. 3 — Arterial blooti pressure oí the cat. 1. and 2. — 0.2 ml of Tyrode solution vvlth neostigmine which bathed the diaphragm for 20 minutes (spontaneous release of acetylchollne); 3. — 0.2 ml of Tyrode solution vvlth neostigmine contalning 20 mcg/ml of T. serrulatus venom; 4. and 5. — 0.2 ml of Tyrode solution vvlth neostigmine contalning 20 mcg/ml of T. serrulatus venom after bathing the diaphragm for 20 minutes (acetylchollne released by the venom); 6. — 4 ng of acetylchollne chlorlne; 7. — the same as 4. and 5.; 8. — spontaneous release of acetylchollne after Tyrode solution vvlth neostigmine contalning the venom was removed and the diaphragm vvashed; 9. — the same 4. and 5; 10. and 11. — acetylchollne (4 ng) and acetylchollne relea.sed by the venom (0.2 ml) after the injection of 2 mg/kg of sulphate of atropine. Cat anaesthetlzed by pentobarbital (30 mg/kg, l.v.) and Injected vvlth hexamethonlum bromide and ephedrine sulphate. Heferences ÍIOUSSAY, B. A. — Action physiologique du venin des scorpions {Buthus quinquestrtatus et Tityus hahiensis). J. Physiol. Pathol. Generale, 18, 305-317, 1919. MAURANO. II. — Envenenamento escorpiônico e seu tratamento. Tese, 1915. RRAZIL, V. — Sôro anti-escorpiônico. Mem. Inst. Putantan, 1, 47-52, 1918. Disclission A. Slmlov: “1. Whether are there any differences in results obtained through use of fresh and lyophilised venom? 2. Whether any experiments were carried out with scorpion venom such as from Scorpio nmurus occurring in Sudan?” K. R. Adanis: “We have no evidence regarding the first question, but have this point very much in mind. We have not had the opportunity of investigating the venom of Scorpio maurus." P. Efruti: “I was deeply interesled in the observations presented by Prof. Adam. My experience concerns, unfortunately, human beings stung by Leiurus quinquestriatus. Besides of pains, observed predominantly in adults, we have observed SciELO SciELO ^0 2 3 5 6 11 12 13 14 15 L. cm Mem. Inst. Butantan E. C. DEE POZO 615 Slmp. Internac. 33(2):615-R26, 1966 68. PHAKMACOLOGY OF THE VENOMS OF MEXICAN CENTRUROIDES E. C. DEL POZO Instituto de Salubridad y Enfermedades Tropicales, México Hecent reports from electro])hysio]ogical aiui hiorhemical work seeni to show qualilative (liffereiices iii scor])ion venoins. Old descriptions of l)Oth diiiical and experimental poisoning are similar and only qnantitative discrepaneies are found in ol)servations eorresj)onding to a wide variety of speeies from distant parts of the world. Differenees in symptoms or signs of intoxieation seemed to be diie to variable amounts of venom or different content of aetive principies (1). The general actions; muscular Iwitchings, ptyalism, progressive respiratory irregularities reaeliing respiratory paralysis in some cases, pilo-ereetion, mydriasis, hlood pressure rise, signs of local pain, signs of laringeal constrielion, are to l)e found in aecounts of seorpion poisoning from any part of the world. Howevcr, the mechanism invoked to exj)lain such actions varied very much. At ])resent, fundamental work is heing done wilh seorpion venoms in many lahoratories and agreement seems to he reached ahout the effects on nervous System and neuromuscular junctions. Neverlheless new discrepaneies could be found in re[)orts from electrophysiological studies. Different teehniques are be- ing nsed and one shonld kee|) in mind lhal discre|)ancies are early and stimulat- ing aspecls of original research. In s])ite of the faet thal new |)aths of research in j)harmaeology of seorpion venoms may lead to the discovery of a common mechanism of action, it is es- sential to mention always the seorpion speeies nsed in each work. The descriplion that follows is limited to |)harmacological j)ro|)erties of venoni from Mexiean C c n I r ii r o i d c s, and it is based on works from my' laboratory. Onr comi)arative studies of the actions of venoms of C. siijjiisus siijjusus Pocock, C. noxiiis Hoffmann, C. Umpidus tecomanus Hoffmann and C. Umpidiis llmpidiis Karseh, grant a common consideralion. Only qnantitative differenees have heen found. l.arger doses of lhe less aclive venoms re])rodnce the effects of smaller doses of the more active ones. Muscui.ah KKKECTS — One of the niosi immediate effects of inlravenoiis injections of seorpion venom in cais, dogs, mice and rats is lhe aj)pearance of generalized muscular twitchings and fascicniar coniractions. This activity ori- ginates in lhe spinal cord. It persisls afler section of lhe brain slem, deaf- ferenlation or Iransection of the spinal cord. The fascicniar conlraclions helow lhe levei of lhe section are more marked than above, but they disa|)pear complctely when lhe s|)inal cord is destroyed or lhe motor nerves are ciit (Fig. 1). cm 2 3 z 5 6 11 12 13 14 15 616 PHARMACOLOGY OF THE VENOMS OF MEXICAN CENTRUR0IDE8 WBHi Fifí. 1 — A: Muscular efrects of an intravenous Injection of scorplon venom. B; No effecl is obtained when an equal amount of venom is injected after the muscular nerve was cut. (This and Figures 3 to 7 taken from dei Pozo and Anguiano (2)). Muscular twilchings and filirilialions are aiso ohlained if lhe venom is aj)- plied directly lo one mnscle or injeeted intrarlerially even with lhe corres])onding nerve cut. Tliis muscular aclivily does not oecur when lime has heen allowed for Wallerian degeneralion after denervation. Local ajipliealion of venom lo a muscular nerve trimk does noi |iroduee any effects on lhe museles 12). The local action of the venom lakes jilace on the muscidar end-plate region. This inference was eonfirmed when eleclrical records were taken from muscle and nerve. Kepelilive potentials were foimd following single shoeks a[)plied to lhe nerve cenlrally cut. This re[)etitive activity is conducled antidromically in lhe nerve and disappears when this nerve is disconnecled from lhe muscle. When records were taken from anterior and dorsal rools. the repelitive aclivily was seen in the former and not in the lalter (4) (Fig. 2). Big and longdasting conlradions are ohlained when single shoeks are aj)- plied lo museles under lhe adiou of scorpion venom. When suceessive resjionses are provoked al short intervals, the conlradions are |)rogressively smaller in amplitude and dnralion. Eledrical recordings show at the same lime a gradual ílirninution of the repetitive activity I, Figs. d and 4). cm SciELO 10 11 12 13 14 15 E. C. DEL POZO lOmseg. Flg. 2 — Eléctrica! recordlng from gastrocnemius muscle of the cat. Five superlmposecl responses to shocks applied to the nerve in each segment. A, betore; B to D, after successlve dosls of scorpion venom. (This and flg. 8 taken from dei Pozo, Salas and Pacheco, in press). SciELO cm Mem. Inst. Butantan Simp. Intcrnac. 33(2):615-626, 1966 According lo this evidence th(' grcat and long niiisctilar resjtonses to single shocks correspond to short letanic conlratlions consecnlive lo lhe repetitive aclivily jirovoked at lhe end-])Iate regions of jioisoned mnscles hy lhe arrival of single impulses. These effects on time and duralion of muscular contraction and the influencc of rejietilive stimulalion have heen extensively analyzed at differenl frequencies of stimulalion. The grajths of tension development of the tetanic contractions shows mechanograms resemhling those ohtained when higher frequencies of stimulalion are applied to normal mnscles 12) (Fig. 5). B Fig. 3 — Effect of scorpion venom on the amplitude and duration of muscular res¬ ponses. Maxlmal contractlons ot gastrocnemius of the cat, before (A) and atter the injeetlon of the venom (B). In successive contractions, the amplitude and duration are progressiveiy reduced (C). (D), 20 minutes later. Fig. 4 .— The length of the interval betvveen stimulations change the type ot responses In a muscle under the action ot scorpion venom. A, 3 minutes interval betvveen shocks. B, one second intervals. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):615-626, 1966 E. C. DEL POZO 619 Fig. 5 — Ettect of scorpion venom on muscular responses to high frequency stimulation. Pairs of responses before and after injectlon of the venom to stimulation to the following frequencies per second: A and D, 5; B and E, 13; C and F, 17; G and L, 25; H and M, 30; I and N, 60; J and O, 120; and K and P, 200. Gastrocnemius of the cat. The letaniziiig niinimal frequeiicy of lhe stiinuliis for one particular muscle is snialler tvheti poisotied than for a normal one, hiil after the initial complete letanus the individual responses lo each shock ajipear on the graph. This pecidiar develo])ment of lhe contraction is the oiijiosile of the normal graph ohtained at the minimal telanizing frequency, i.e., initial individual response lhal jirogressively i SciELO 620 PIIARMACOLOGY OF THE VENOMS OF MEXICAN CENTRUROWES fuse iii a complete lelaiuis. The long lasting initial coiitractions of the jioisoned miiscle and llie |)rogressive shorleniiig of lhe siiccessive res|)onses aceoimt for ihis phenomenoii (Fig. 6). Flg. 6 — Inversion of the stages of the responses to stimulation at a minimal tetaniz- ing frequcncy In a musrle under scorpion venom. A, 3 per second; B, 5 per seeond. When neiiromiiscular transmission has lieen liloeked l)y curare, scor|)ion venom given hy intravenons injecTion decnrarizes (2). Ilowever, large doses of lhe venom prodiice a complete hlock of neiiromnscular transmission. This hlock is not snjjpressed hy either |)rostigmÍ7i or curare. The miiscidar res|)onses to direct electrical or acetylcholine stimidalion are preserved (F igs. 7 and 8). Scorpion venom displays anli-cholinesterase aclivily as shown hy onr experi- mcnls tesling the fali of hlood pressure [)roduced l)y fixed arnonnts of aeelylcholine after hydrolysis wilh hlood senim with and withoul venom. This |)ropeiTy was also assayed com|)aratively with eserine on frog ahdominal mnscles (5). The anticholinesterase activity of differenl venoms was fonnd lo he plighl and withoul correlation wilh loxicity or lhe mnscle adivating jirojterlies. A stndy of lhe effects of venom (C. naxiiis Hoffmann ) on cholineslerases was examined on lhe isolated inteslines of guinea-i)igs and rahhils. Il was fonnd that the venom oídy inhiliit cholineslerases holh from hnman sernm (pseudo) and from the caudale nucleus of rahhits (specific) when present in very high concentralions (6). ín hrief, lhe muscular effects of scorpion |)OÍsoning are prodnced hy two actions of the venom: one, central, located on lhe s()inal cord, and lhe other, peripheral, on lhe neuromuseidar jnnclions. Both actions take jtlace at regions SciELO Fig. 7 — Decurarization by scorpion venom (B) anci by prostigmine (A). The first signal in both segments correspond to the injection o£ curare, The second In B to a second injection of curare (in A to atropine). The third signal in B maiks the time ot the Intravenous Injection of .scorpion venom. (In A, prostigmine). 2mvT 10 mse g Fig. 8 — Slmultaneous recording from sciatlc nerve (above) and gastrocne- mius muscle (below) to stimulations applied to the nerve. A, before, B, 1 minute after injection of scorpion venom, C, 5 minutes later. D, after curare. i SciELO PHARMACOLOGY OF THE VENOMS OF MEXICAN CENTRUROIDES of excilaMe ineinhranes, and il is rfasonaMe lo assume lhal lhe venom affects lhe [)ermeal)ilily of lliese niemhranes antl chaiige lhe ionic eqiiilihrium lielween holh sicles of lhe same. Macroeleetrode reeordings from lhe end-jilale regions of lhe sarloriíis musele of a cal showed a progressivo iiiversion of lhe loeal poleiilial imder lhe aelion of seorpion veiiorn (7). This is a direet evidenee of ehaiiges iti ioiiie dislrihiilion produced hy lhe seorpion loxiiis. Hesimkatory effects — The accidenlal or experimenlal |)oisoniiig wiüi seori)ion venom ])rodnee marked irregnlarily in differenl animais holh in frequency and amplilnde of lhe respiralory movemenls which in eases of slrong inloxiealion reaches a Cheyne-Slokes lype rhylhm and finally paralysis. The eenlral origin of lliis paralysis was altesled hy keeping lhe animais (eals) alive afler lhe para¬ lysis hy means of arlificial respiralion hroiight ahonl hy rhylhmie slimnlalion of holh |)hrenic nerves. In lhese exj)eriments, lo onr knowledge, was for lhe firsl lime appiied what aflerwards was inlrodneed under lhe name of e/ec/ro- phreiiic respiralion. The paper ])nhlished in 1945 refnled lhe hy])olhesis of enrarizalion as the origin of lhe respiralory j)aralysis (8). Bronehiolar ohstrnclions hy ahnndanl secrelions, laryngeal and hronchiolar mnseles eonlractions conlrihnte to lhe respiralory distress hut are not the canse of the asphixia. Same may he said of the fascicnlar eonlractions lhal appear in respiralory as well as in all skeletal mnseles. Periodic rhylhms of respiralion from central origin can he perceived among lhe hackground of irregular jerks uj) to the time of a{)pearance of Cheyne-Slokes ty|)e rhylhms. An addilional proof of lhe central aelion is lhal a minute amonnt of the venom injected into the cisterna magna prodnces immediale respiralory paralysis. Cardiovascui.ar effects — The inlravenous injection of seorpion venom prodnce in cais and other mammalians an initial increase of hlood pressnre and simnilaneons hradicardia. The j)ressnre then comes down slowly to normal values. During lhe periods of cyclic, respiralion, increases in hlood jiressiire aceompany the periods of apnea and hlood pressnre falis during hyj)ervenlilation. Finally. if the dose of venom was high the ])re.ssnre comes down gradually lo zero (9). The initial increase in hlood pressnre long time ascrihed lo a j)eripheral aclioti of the venom is dne lo effets on lhe S|)inal vaso-conslrictor |)resynaplic nenrons and lo the liheration of ej)inephrine aiso ihrongh slimnlalion of lhe adrenal activating |)re-gangliotdc nenrons of lhe spinal cord. Spinal animais afler total deslruction of the hrain, give equal or higher hlood pressnre increases wilh lhe venom, hnt lhe effecl does not aj)pear when the spinal cord is deslroyed even when lhe hlood ])ressnre levei is [)revionsly raised lo normal valnes. Denervated ])innae of white rahhits and cais showed vaso-constriction dne to liheraled epinephrine; it did not ocenr when lhe sym- ))athetic fihers lo lhe glands were cnl or the s|)inal cord was deslroyed (Figs. 9 and 10). Hradicardia corresponded also lo a central aelion. It disa|)peared when lhe medula was deslroyed or when the vagi were cnl. In lhese cases lhe heart was slightly acceleraled under lhe infinence of scorjiion venom. This effecl was also of Central origin hecanse il did not ocenr when the heart was denervated. The vascular reflexes produced hy lhe slimnlalion of the central end of lhe sciatic nerves peripherally cnl were not modifieil hy scor|)ion venom on enrarized cm 2 3 z 5 6 10 11 12 13 14 15 E. C. DEL POZO 623 Mem. Inst. Butantan Slmp. Internac. 33(2); 615-626, 1966 A 160- — 140- / 1 120- / _ h o o 80- 1; 60- 40- 20- li ■■ \)> '■t-J-.-A Fig. 9 — Increascs of blooci pressure produced by a first (A) and a second (B) intravenous Injection oí scorplon venom in a spinal cat by destructlon of the brain. (This and the following figure are taken from dei Pozo, Anguiano and Gonzãiez (9)). Fig. 10 — Scorplon vrnoin dce.s not produce increa.se of blood pressure when brain and spinal chord are destroyed. A, first in;ection of venom; B, injection of an equal volume of saiine; C, injection of 10 mcg of epincphrine; D, 5 mcg of epinephrine. cm SciELO 11 12 13 14 15 16 17 624 PHARMACOLOGY OF THE VENOMS OF MEXICAN CENTRUROIDES animais. This dissociation hetween lhe tonic vasopressor dircct effects and lhe reflex responses is an old physiological finding reporled hy Sherrington in 1906 (10), Porler in 1910(11) and Langley in 1924(12). Otiier effects — Other effecls of lhe scorj)ion venom siich as plyalism, mydriasis, j)iloereclion are also lo he cxplained l)y cenlral aciion on lhe syinpathelie ])re-ganglionic neurons heeaiise ihose effecls are greally rednced or disappear complelely hy denervation. Wc have seen thal lhe rich pharmacological aclions of scorpion venoms seem lo derive from a fundamental common mechanism present at different analomical regions. We have discovered already lhat ihis venom acls on lhe ly])ical })laces so-called centcrs and at neuromnscular junctions. We know lhat it works hy increasing lhe permeahilily of excilahle memhranes as shown hy lhe inversion of end-plale potential (7). Others investigators have reached similar results with finer tcchniqiies (13). Iti addition, valuahle hlochemical work is heing done in several lahoratories for lhe isolation of scorpion toxiris; we may he hopeful lhat now we are on lhe road to a hasic underslanding of lhe mechanism of aciion of these peculiar venoms. This first International Symposium has brought together the people working on the same problems in all parts of the earth. It will be an historical event. The importance of this meeting is increased because we meet at this famous Bu- tantan Institute, the leader of the venomological studies. We pay tribute to the illustrious Vital Brazil, inspired founder of the Institute and to all the group of workers that have followed his steps in this fascinating path of research. Kefekences 1. DEL POZO, E. C., in E. E. BUCKLEY, and N. PORGES (Editors), Venoms, Amer. Ass. Advanc. Sei., Washington, 1956. 2. DEL POZO, E. C., and ANGUIANO, G., Rev. Inst. Saliibr. Enferm. trop. (Mex.), 8, 231-263, 1947. 3. DEL POZO, E. C., Gac. méd. (Mex.), 78, 387-397, 1948. 4. DEL POZO, E. C., SALAS, M., and PACHECO, P., Extr. Comunic. VI Congr. Nac. Cienc. Fisiol., Mexlco, 1963, pp. 109-110. 5. DEL POZO, E. C., and DERBEZ, J., Rev. Inst. Salubr. Enferm. trop. (Mex.), 10 , 203-213, 1949. 6. DEL POZO, E. C., Brit. J. Pharmacol., 3, 219-222, 1948. 7. DEL POZO, E. C., SALAS, M., and PACHECO, P., Extr. Comunic. IX Congr. Nac. Cien. Fisiol., México, 1966, p. 141. 8. DEL POZO, E. C., GONZÁLEZ, Q. J., and MÊNDEZ, T., Rev. Inst. Salubr. Enferm. trop. (Mex.), e, 77-84, 1945. 9. DEL POZO. E. C., ANGUIANO, G., and GONZÁLEZ, Q. J., Rev. Inst. Salubr. Enferm. trop. (Mex.), 5, 227-240, 1944. 10. SHERRINGTON, C. S., The Integrative Action of the Nervous System, London, 1906, p. 242. SciELO Mem. Inst. Butantan E. C. DEL POZO 625 Simp. Internac. S3(2):615-626, 1966 11. PORTER, W. T., Amer. J. PInjsiol., 36, 418-422, 1910. 12. LANGLEY, J. N., J. Physiol., 59, 231-258, 1924. 13. ADAM, K. R., SCHMIDT, H., STÂMPFLI, R., and WEISS, C., Brit. J. Phar- macoL, 26, 666-677, 1966. Discussiox H. Edery: “AU these features you described on the neuromuscular transmission such as twitchings, and fasciculations are also produced by anticholinesterase organo- phosphorous compounds. I learnt from your paper that you tested the venom for anticholinesterase activity and it has a high concentration. I would like to ask if you would accept the idea that at least part of the effect on neuromuscular transmission were due to inhibition of cholinesterase at the end-plate levei. When you inject a substance intraarterially, you do get high levei concentrations. And thinking in this way of inhibition of cholinesterase, I would be keen to know the effects of the so-called reactivations of cholinesterase on the block produced by the venom.” E. dei Poso: “We did find anticholinesterase activity and we thought that this could be the explanation for the muscular effects of scorpion venom. However, when tested that possibility we found that the anticholinesterase effect was slight and did not keep relation or correlation with the muscle activating properties. For this last testing we compared venoms from different scorpion species with regards to anticholinesterase activity, to.xicity and muscle activating properties. One particular venom could have less anticholinesterase and more muscle activating properties than other.” P. Efrati: “Also my experience concerns mainly stings by Leiurus quinques- irxatus, I think, at least two clinicai signs, observed in general envenomation, could support the observation of E. C. dei Pozo about the influence of scorpion venom on the spinal cord: urinary retention and priapism, observed very often indeed.” cm 2 3 L. 5 6 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 3S(2):627-638, 1966 S. SCUENBERG and F. A. PEREIRA LIMA 627 61. I’HAHMAC()L()(;Y OF THE FOLYFEFTIDES FI{()M THE VENOM OF THE SPIDEK PHONELirRIA FERA S. SCHENBERG and F. A. PEREIRA LIMA Seção de Fisiologia, Instituto Butantan, São Paulo, Brasil Tliis jjaper resumes pharniaeo-hioehemical invesligations made witli lhe venom of tlie siiider Rhoneutria fera Perly, 183H iCteini.s jcrii,s, and C/em/.s nigrivenicr Keyserling, 18911, a speeies of lhe AKANEIDA order, LABIDOGNATA sidiorder, CTENIDAE Family and Cteninae suhfamily. Very dangerous hy its aggressivity and venom toxieity, this speeies is responsihie for lhe higher lethalily percenlage amongst olher venomous speeies from lhe Stale of São Paulo. The individual extraetions of the.se spiders may furnish a maximum of 1.8 mg dried venom in winter and 2.5 mg in sumnier (1, 2). After dried lhe venom has a grayish-white eoloiir. Notvvithslanding its toxieity heing eom|)arahle to that of C rol a I ii ,s and B o 1 It r o p s ophidian ve- noms, the small availahle venom arnoimts of individual spiders are generally only eai)ahle of attaiuing lelhal coucentrations in children. Venom efi ects on dogs In dogs lhe P. fera venom was preferentially admiuislered hy suheutaneous roule due to its high toxieity and lhe iniense hlood pressiire drop it induees wheu injeeled intravenously. Suheutaneous low lelhal doses (180-200 /ig/kg hody weight) provoke in dogs successively: intense loeal pain, violent sneezes, lacrimation, ahundant sialor- rhea, vomitiug, jiriapism, ])roslralion, semen elimination and in some eases death. Suhlethal doses generally are insuffieieul lo induee ])ria|)ism hy suheutaneous route. VVilh lhe exeeplion of priapism(8), all lhe aforementioned venom effeets were anteriorly reporled hy Vital Hrazil and VellardiT). Local pain — The venom suheutaneous injeetiou is excrueiatingly |)ainful, it makes dogs yelp for nearly an hour, foreiug lhem to maintain contraeted lhe injeeted hind leg for longer jieriods. The pain faelor is neiitralized hy the S])eeifie antivenin, whieh exeludes the possihility of this effeet heing eaused hy venom eontained hislamine or serotoidu. On the olher hand, the jtain faelor heing dialyzahle. its moleeule must he relatively small lo he eonsidered as a hradykiiun releasiug euzyme. Priapism onset may take plaee dnring the pain [)eriod. cm SciELO 10 11 12 13 14 15 628 PIIARMACOLOGY OF THE POLYPEPTIDES FROM THE VENOM (JF THE SPIDEU PHONKUTRtA FERA Sncezes — lii dogs siioezcs constilule one of llie firsl eiivonomatioii sigiis. 'I'licy are iiitermillent l)eing ohserved for more lhaii 24 h. The firsl attacks are very stroiig, lhe animal ihrows its head violently and nncontrollal)ly towards lhe floor, very often, liurting ils nose and lips. Lacrimalion and mydriasis — Dnring envenomalion lears drop conslanlly from lhe dog eyes, even in anesthetized animais. Mydriasis is indiiced early after venom injecTion, it ])ersists for many hours. The dogs ])resenl visual dislurhanees as a consequence of mydriasis. Siulorrhra — The venom ])rovoked sialorrhea is ahimdanl, pilocar|nne-like. IJnswallowed salive drops from lhe animal moulh continuousiy for hours. This effecl is hlocked hy alrojjine and doses of eserine and hexamelhoniurn. whieh inerease lo a small exlenl lhe poisoning signs, seem nol lo inlerfere in ils ma- nifeslation (3). Suheulaneous effeclive doses are ineffeetive in dogs under ehlo- ralosc, chioroform and harhiluries aneslhesia, hovvever, lhe same doses induee sialorrhea when appiied inlravenously. I‘riapism — The venom indueed priapism is aiso an intermillent and long- standing effecl I Hg. 1). It manifests re])ealedly for hours, very often exceed- iiig 24 h, in whieh cases edema is generally formed at lhe i)enis distai extremily correspondenl lo lhe glans. 1 — Priapi.sm inducctl by P. fcrct venom. The do" presents: adynamia, locomotion dirflculties and drow.siness. Priapism generally lakes ])lace at a more advanced envenomalion phase when lhe dog has alrinidy presented intoxicalion signs. Differiug from sialorrhea, venom j)riapism coidd nol he indueed in anesthetized dogs eveii when lhe venom was admiuislered inlravenously in larger doses than those whieh are effeclive hy sidi- culaneous route. Priapism is indueed in dogs having lhe medulla eul at DXII. cm SciELO Mem. Inst. Butantan Simp. Internac. 33(2);627-638, 1966 S. SCHENBERG and F. A. PEREIRA LIMA 629 indicating that it is independent froni the excitalioii of higher centers (5). Hiilike caiitharidin priapism, this of the arachnidan venom does not seem lo residt from reflexes caused l)y irritation of the iirinary tracts. In dogs, the venom provokes priapism hefore any micturition, as well as in animais with hoth urelers implanted into the skin; it is also not evoked hy perfusing venom Solutions ihrough the nrethra into the hladder. Senien eliminalion — In dogs lhe P. jera venom provokes eliminalion of semen during or after ])riapism onset. This effect seems to result from the excitation of other structiires than the seminal vesicles since dogs have not gol them (5 ). Toxicity — The venom intoxicalion pattern of dogs is characterized hy: adynamia, locomotion difficulties, prostration, drowsiness, vomiting, dyspnea, san- guinolent feces and dealh. By iising 200 jug/kg suhcutaneoiisly, lhe dog siirvives for several honrs hefore dealh occurs. Dogs also present sneezes, sialorrhea and priapism during lhe severe intoxicalion phase. Giiinea-pig ileum coiitractiori — C. Diniz separated from this venom Iwo polypeptides which contract the ileum of guinea-pigs; according lo his findings the fraction containing one of these polypeptides was also responsihle for the venom toxicity (6). Blood pressure jall — Endovenously, small venom doses ])rovoke a sharp hlood pressure fali. The loo small histamine ccntent of these venom doses have no comjiarahle effects on hlood pressure. Tachyphylaxis — Bafael H. L. Sampayo (7) reported that components of lhe hlack widow spider (Lalrodectiis rnaclana) venom provoke tachyphylaxis in dogs. Tachyphylaxis ])henomena vvere not ohserved for any one of the effeels eliciled hy the /h fera venom. The intermillenl actions would not have this characleristic in the case that the agenls res[)onsihles for these effects would have lach.yijhylaxis properties. Tachyphylaxis also shows that hoth these venoms dif- fer hy lhe molecular slructure of their components. Venom ekkects o\ mice C. Diniz was ahle to reproduce on alhino mice the jera venom ])riapism first ohserved in dogs (8). Lxcluding lacrimalion, mydriasis, sneezes, semen eliminalion and vomiting, which are difficull to he followed in mice, all the other P. jera venom effects can easily he tesled on these small rodents (5). Mice slrain and weighl are essential for reproduetive quantitative assays. The hest re.si)onses, for mice of lhe Instituto Butantan strain, are ohlained hy using animais of 22-25 gm. With less than 20 gm they are more sensihle lo loxic effects than to priapism. The method of Beed and Muench (9) shown lo he very suitahle lo eslimale statistically this venom actions on mice. Loca! pain — Not so noticiahle as in dogs, it is, however, exteriorized in mice hy contraction of the injected hind leg. The animais also hite lhe injectiou area in a scratching form. i SciELO 630 PHARMACOLOGY OF THE POLYPEPTIDES FROM THE VENOM OF THE SPIDER PHONICUTRIA FERA Sialorrhca — As Fig. 2 shows, ihis effecl can easilv l)e followed in inice. Al the Feginriitig small hiilihies of saliva acciimulale at lhe moulh. Later on, dcpending on the venom dosage, lhe anirnals inay have a large part or nearly all of its fur wel vvith saliva. Small doses of venom ])rovoke sialorrhea wilhoul any sign of toxicity. Wilh larger doses, sialorrhea a])|)ears hefore loxic mani- feslalions and eonlinues on its ap|)earance. The venom ED50 for sialorrhea is 0.43 mg per kg hody weight. Fig. 2 — Beginning of P. fera venom provoked sialorrhea in mire. The mouse is apparently normal, no sign of intoxication is evident. Priapism — Fig 3 shows clear manifeslalion of priapism in mice. The pria[)ism dose-effect relationshij) in mice is maintained until a maximum of respoirses in a gronp of animais are altained, thereafler, inereasing doses of venom jirovoke a deerease in lhe niimher of animais whieh presenl priajiism, and death oeenrs in some of ihern hefore ihis effeet a|)pears. In a similar way to sialorrhea, small doses of venom can induce ])riapism in mice wilhout any loxic manifestalions; in ihis point mice reacl differentiy from dogs. The jiriapism Fl)5() for mice is 0.25 mg per kg hody weight. 1 oxicity — The venom toxicity manifestalions in mice are: dyspnea, pros- tralion, distensive paralysis and death. The toxicity Ll)50 is 0.76 mg per kg hody weight. Distensive ixtnilysis — As can he seen in Fig. 4, in mice erude venom provokes a distensive jiaralysis of the hind legs and tail; this latter, diiring a certain period, remains hent in a distensive form npon the animal hack. The cm SciELO Fig. 3 — Priapism Induced In mouse by 10 iig ot P. fera venom. This dose is effective for priapism and sialorrhea but does not provoke intoxication signs. FIg. 4 — P. fera venom distensive paralysis. Characteristic position of hind legs and tail; the mouse died In a precocious rigor mortis. cm 632 PHARMACOLOGY OF THE POI.YPEPTIDES FROM THE VENOM OF THE SPIDER PHONEUTRIA FERA liiiul Ifgs paralysis lakcs place afler lhe induclion of sialorrhea and priapisni and remains iinlil dealh, which occurs with lhe animais aiready presenüng a precocious sorl of rigor mortis. VeNüM EFFECTS O.N OTHflR KODENTS (;uinea-])igs re|)rodiice ahnost all lhe venom actions provoked on dogs and miee, however. lhey have lieen less invesligaled lhan miee due to llieir larger venom eonsnm[)lion (3,5). Kals and rahhits are very resislant lo this venom actions, 500 /xg on rats of 150 gm only provoke secretion of lhe glands of Harder, and 1,000 /ig on rahbils of 2,000 gm only indncc small sialorrhea and lighl inloxicalion signs (3). HlIMAN BFING ACCIDENTS A ])aUern which resemhles lhat of dog envenomalion is also noticed in human heings siiffering from P. fera stings: local unhearahle ])ain, sialorrhea, visual stute Medicai Center at Syracuse, N.Y., U.S.A. There exists evideiice llial lhe silk glaiuls of lho spiifor Aranciis diademuliis Cl. Work iii olose ooiniection witii lhe central nervoiis system (CNSl. Tlie Ci\S a|)|)ears lo send signals to lhe glands, "teiling’’ ihtni to produce more or less silk; it peenis also “informed ” of lhe amoiiiit of silk availal)le in lhe ampullate glands at a given lime. Silk heing so importani in lhe s])ider’s life — for Ira])- huilding, moving aroimd, and eommimication — siich Iwo-way mechanisms a])- pears of greal survival value: enough material is made availahle, hut no energy is wasted on excess. The close interrelationship helween CNS and glands exjjlains lhat driigs which affect CNS fimction, the [)sychotro|)ics, influence silk gland activity and lhal wíehs hiiill afler these dnigs reflect lheir effects in weh-weighl and -])altern. The evidence vvill he reviewed. To estahlish the amoimt of silk which a s|)ider uses for one weh, the whole slruclure is ciil off from its siipports, rolled up on a j)iece of relalively nilrogen- free filter pa])er, and digested in selenium-siilfiiric acid. The amoimt of nitrogen determined Wíith an optical micromethod in lhe weh digesl is a measure of lhe lolal amoimt of polypejitide in the weh. Fig. 1 shows the residts of an experi- ment with 23 sjiiders which were treated wilh 1 mg/kg physostigmine hy mouth in sugar water and 23 control animais. The wehs were digested daily and on Tuesday hoth grou[)s showed lhe same mean of 39 microgram N per weh. 36 hoiirs afler lhe drug had heen given, on Wednesday morning. lhe ]diysostigmine- trealed animais had huilt wehs containing a mean of 48.9 ± 1-.4 microgram N per weh, while Controls huilt significanlly lighler wehs with 20-30 micrograms N. Tlie following day, h^riday, all animais, Controls and drugged sjiiders, huilt wehs with the same meau of 34.5 microgram N per weh. It was concluded that wehs afler physostigmine are heavier or contain more polypejilide ihread. In order lo lesi the hypolhesis tliat the cholinergic drug stimulates silk jiro- diiclion rather than promotes a more thorough scjueezing out of the ampullate silk glands, Peakall performed ex|)eriments using 3 kinds of methods (1, 2) : A: Glands were pulled emply of ihread at regular iniervals. f.e. every 6 hours, and lhe (jiiantity thread (udled was determined in lhe selenium-siilfiiric acid digesl. Such jirocedure lakes advaniage of lhe experience lhal a spider which sils on a rough surface leis oul thread onlo lhe rotaling axle of a motor imlil the glands are em|)ty. PeakalFs results wilh jihysostigminc and carhachol. 2 cholinergic drugs, and atro|)ine, an anticholinergic drug, show lhal with in- creasing dose of the drug, silk (juantily increased after lhe cholinergic and decreased after lhe anticholinergic drug. cm 2 3 z 5 6 11 12 13 14 15 640 SPIDER GLANDS AND PSYCHOTROPICS Fig. 1 — Nitrogen values in the digest of webs of 23 spiders treated with 1 mg/kg physostlgmlne on Tuesday nlght (12 hours before the Wednesday vveb-buUdlng time) and 23 Controls. Each polnt represents the mean ot all measurements, and lhe vertical Une the standard error ot the mean. The tvvo Thursday values are signiticantly ditterent belovv the 1% probability levei. Note the increase ot web nitrogen 36 hours atter the drug. li: Anollier procediirc usos laliclliiig of a silk preciiisor, alailinc, willl CM, iiijdcling il inlo the siiiders’ ahdomen, and measiiring lhe sjieed vvilh vvliich the lal)el liiriis iip in lhe silk. G hours afler alaiiine was given, signifieanily more lahel ajipeared in lhe silk afler |ihysosligmine, carhachol and jiaraoxon lhan in iinlrealed spiders (Tahie I). ll was aiso foiind lhal jiisl eni|)tying lhe glaiids proinoted incorporation of lhe lahel, and llial eniplying phis driig Irealnienl was iiol additive, hui ralher made the glands hehave like afler one Ireatmenl alone. We ean lherefore assume lhal tvvo mechanisms regulale sjieed of silk production: 1 ) a feed-hack from lhe lumen of lhe gland via lhe inner ejiilhelial memhrane and 2| a possihly neurohumoral meehanism via lhe outer epithelial memhrane. C: A ihird way of meastiring changes in silk production is the use of radio- aiilography: the lahelled silk precursor, again CM alanine, leaves hiack dois on a pholographic fihn which is sjiread ihinly over lhe sliced lissiie. By iising tliis metliod al different limes afler alanine injection, its progress from lhe hody fluids inlo lhe gland epitlielium and from there inlo lhe gland lumen can he followed. Tahie II shows lhal 4 hours afler injection of CM alanine, mosi of the lahel had left lhe intestine and hlood of lhe sjiiders and ajipeared in lhe gland lumen in jiulled or physostigmine Ireated animais, wliile atrojiine treated cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2) :639-644, 1966 PETEU N. WITT 641 TABLE I — AMOUNT OF INCORPORATION OF C-14-ALANINE INTO WEB PROTEIN DURING 6 hr. The labelled alanine was given at the start of the experlment. Threaci was pulled for determination 6 hr aftervvards. (From D. B. PEAKALL, Comp. Biochem. Physiol., 13, 467, 1964). TREATMENT No. of spiders Actlvity counts/min Standard Micrograms Standard error of nltrogen/ error of the mean 100 mg b.wt. the mean Unpulled, no drug 15 123 11.1 — — Unpulled, physostigmine 18 290 27.4 — — Unpulled, carbachol 14 301 25.4 — — Unpulled, paraoxon 6 329 60.7 — — Pulled, no drug 18 314 13.5 15.0 1.4 Pulled, phy.sostlgmlne 20 346 17.9 16.2 1.6 TABLE II — COUNTS FROM 7-DAY AUTORADIOGRAPIIS OF INTESTINE, BLOOD AND SILK GLAND EPITHELIUM AND LUMEN IN THE INTACT SPIDER Counts are per mm= with standard deviatlon, ground count (Bkd) were Sll, 1965). at magnlflcatlon from 50 count.s i 1-4/mm-. (From of 60. Each figure represents the average, [10 counts from each of five spiders). Back- D. B. PEAKALL, Coinp. Bioche^n. Physiol., 15, Time in hr Treatment Hind intestine Blood Epithelium of silk gland Lumen of silk gland 2 None 61.0 ± 8.0 29.7 4.3 4.4 ± 1.9 Bkd Re-pulled 55.1 ± 6.9 26.3 ± 5.1 8.8 ± 3.7 Bkd Physostigmine 54.9 ± 7.9 29.0 ± 2.9 6.1 ± 1.9 Bkd Atropine 64.1 ± 9.1 27.1 -± 5.0 Bkd Bkd 4 None 15.1 ± 2.5 17.8 ± 3.0 25.5 ± 4.1 4.9 ± 0.8 Re-pulled 10.3 ± 1.8 13.0 ± 5.2 54.3 ± 8.3 18.0 ± 4.2 Physostigmine 8.4 ± 2.1 8.9 ± 3.1 48.0 ± 7.5 12.5 ± 4.3 Atropine 16.7 ± 3.0 22.1 4.6 25.5 ± 4.8 6.1 ± 2.1 8 None Bkd 31.8 1.9 33.8 ± 4.8 38.4 ± 4.0 Re-pulled Bkd 4.7 ± 1.9 15.3 ± 2.8 55.6 ± 4.2 Physostigmine Bkd 5.6 ± 2.7 16.2 ± 3.5 62.8 ± 9.8 Atropine Bkd 14.0 + 7.4 44.4 ± 9.6 22.4 ± 7.5 cm SciELO 10 11 12 13 14 15 642 SPIDER GLANDS AND PSYCIIOTHOPICS or restiiig glands had iiol yot laken u[) lhe hulk of tlie lahel. Iii liislological slides lhe size and shapes of lhe amj)ullale glands and lhe position of lhe lahel ean he idenlified. If we assume thal aeelyleholine is lhe neurolransmiller snhslanee whieh is responsihle for carrying lhe signal for silk iirodnelion from lhe nerve lo lhe gland lissue, we mnsl look for places on lhe gland whieh eoidd hind aeelyleholine. Peakairs work shows auloradiographie ])roof thal lahelled aeelyleholine is ae- cnmnlaled on lhe gland e|)ithelium. This is possihly lhe area for reeeption ol lhe nenrohnmoral signal. Lei us now take a look al lhe whole animal and see lhe geographieal loeation and possihie interrelalionships of silk glands and Ci\S in Amneua diadcmutus Cl. F. Meier I personal eommnniealion) has idenlified nerves leading from lhe hig snheso|)hageal ganglion in lhe eej)halothorax lo lhe silk glands. Sneh nerves eould earry signals in hoth direetions, eoordinating leg movemenls of lhe weh- hnilding spider with silk suppiy in lhe gland. Behavioral experimenls wilh weh-huilding s])iders have shown thal there are 3 |)ossihle ways whieh ean lead lo wehs Iniill wilh a shorter ihread: 1. Atro[)ine sulfate, 1, 2 or 4 mg/kg, was given hy mouth to 19, 19 or 39 s|)iders 12 hours hefore weh-huilding time. The two lower doses eansed wehs whieh showed no change in size or regniarity hnl were huilt with wider meshes, eovering lhe same area with less ihread. The highesl dose eansed signifieanlly smaller and less regular wehs hiiill wilh less silk. This latler ehange lasled through lhe seeond day after drug a])])liealion. The inlerpretation assumes thal alro|)ine shows its effeet on polypeptide synlhesis in lhe spiderV glands as well as interferes with eentrally regulaled exaelness of movemenls. It is inleresting to note lhal not lhe size of lhe eatehing area in lhe weh was deereased wlien less ihread was availahle, hnl lhe shorter ihread was wider spaeed .so thal lhe Irap was full size and otdy losl lhe smallesl inseels. 2. S|)iders aiso hnilt wehs wilh shorter ihread afler a weighl had heen allaehed lo their haeks. These experimenls (3) were underlaken lo lesl lhe hy)) 0 - ihesis lhal ])siloeyhin, lhe hailueinatory mushroom poison, eansed in s|)iders similar effeets as an increase in hody weighl. This snhslanee is known to ehange in man pereeplion of one’s hody. Does a spider afler psiloeyhin “feel” heavier and lherefore huilds a weighl-weh? It eonld he shown thal 150 mg/kg |)silo- eyhin given to 9 and 23 s|)iders in Iwo independenl experimenls 12 hours he¬ fore weh-huilding lime, as well as a 30% increase in hody weighl of 15 s|)iders, deereased average thread lenglh signifieanlly hy ahonl 30%. However, when lhe wehs were digesled and N delerrnined, a signifieant differenee in lhe amounl of silk was found hetween lhe psiloeyhin and weight-wehs: lhe wehs after psilo¬ eyhin were huill wilh less silk, lhe shorter thread was as ihin as hefore; lhe heavier spiders, in eontrasl, huilt wehs wilh ihicker thread, using e(pial amoimts or even more prolein lhan hefore. The interprelalion for lhe results of lhe ex- periment wilh heavier s[)iders assumes thal lhey huilt a thieker thread lo hold lhemselves uj); as lhey had no more material lhan nsual in lheir glands, lhe thieker thread had to he shorler. The psiloeyhin wehs mnsl he inlerpreled in a differeni way, as will he seen laler. If lhe inlerpretation is eorreel, lhe s])iders’ CNS musl integrate information on ihread lenglh as well as silk cpianlily slored in lhe glands during weh eonstruclion. cm 2 3 z 5 6 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2)1639-644, 1966 PETER N. WITT 643 3. Experinieiits ])erfoi'me(l iiiider lhe iiifluence of llie Iraiiquilizer Valium (diazepani) may Help to inlerpret psilocybin effects (4). Wheii 100 nig/kg Valium were giveti lo 40 spiders 36 liours hefore web-building lime, all animais hiiilt smaller wehs wilh a shorler ihread and less material. This conld lie lhe result of a deerease in silk production in lhe glands ihrough lhe tranquilizei', or lhe glands were nol eompletely em|)tied at lhe end of weh construction. Experi- ments wilh ihread pulling afler Valium auswered lhe question in favour of lhe second inlerpretalion: when silk was |)ulled from 13 spiders one and Iwo days after 100 mg/kg Valium and from 9 eontrol spiders, no difference in quanlity could he found. The effeet of Valium is lherefore' interjireted as possihly af- fecting lhe spiders’ “drive” so lhat lhey huild smaller wehs usiug only part of lhe material. The glands stay jiarlly filled at lhe end of lhe eonstruetion jieriod. This, in lime, would slow down new silk synthesis hy lhe feed-hack mechanism which was shown hy Peakall. Thus, lhe three ways in which wehs wilh shorter thread were |)roduced ihrough drugs and weight changes, provide some evidenee for the close inter- eoimeclion helween silk glands and CNS. If the Iranquilized CNS “inslructs” lhe legs to huild a smaller weh, less silk is pulled from the glands. Eig. 2. Flg. 2 — Araneiis diaúematux CJ. sitting on a rough surface anel facing away from the camera. Note the posterior spin- nerets from which the 8th leg pulls two threads, and the anterior spinnerets from which a thread runs to the ground. illuslrates how the s]nder pulls silk from lhe spinnerets hy means of its hind leg. It can also lower itself on lhe ihread ihrough ils weighl and regulales S])eed and ])ossihly ihiekness wilh lhe eighlh leg. The funeition of WilsoiTs conirol valve (5,6) in this proeess and its interrelalionship wilh CNS and glands is a matter for ftilure investigalions which will we ho|)e furlher elarify |)syehotroj)ic drug effects on weh-huilding. 1 SciELO 644 SPIDER (3LANDS. AND PSYCIIOTROPICS Heferknces 1. PEAKALL, D. B., Comp. Biochem. Pliysiol., 12, 465, 1964. 2. PEAKALL, D, B., Comp. Biochem. Physiol., 15, 509, 1965. 3. CHRISTIANSEN, et al., J. PharmacoL, 1,S6, 31, 1962, 4. WITT, P. N., and REED, C. F., in press. 5. WILSON, Qiuirt. J. microhiol. Sei., 103, 549, 1962. 6. WILSON, Quart. J. microbiol. Sei., 104, 557, 1962. cm SciELO 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2):645-650, 1966 CHARLES F. REED 645 66. SPIDEH WEBS AND PSYCHOTROPICS CHARLES F. REED Temple University, PhíUideJphut, Pu., U.S.A. Perliaps, from the v'ie\v|)oint of lhe stiuleiit of venoms, llie spider wch is nothiiig more thaii an elahorate deviee for delivery of venoni lo lhe prey. (Jranling lhal inlerprelation, ihose of iis wlio are interesled iii iinderslaiiding animal hehavior are gralefid for lhe inlimacy required for tlie injeetion of venom: in lhe orh- weh we have a concrele segmeiit of hehavior es|)eeially susceplihle lo (jiianlilalive sludy. The weh is also a leslimony to the sensory and motor integrity of the •spider. While it may lie fnrther agreed lhal it is the result of innale hehavior patterns, we are not lherel)y relieved of the recpiirement to explain tlie s|)ider’s response to eontingencies of the moment (e.g. vvind and hroken strands), nor nltimately to explain the elahorate sensory, nervons and motor Systems whieh mediate ihose innate patterns. Eor it is the case that the lechnology of weh-hnihling is rather imperfectiy known. There have heen excellent naturalistic observations of lhe proeess and several attempts at motion piclures. There have also heen experimental inler- ferences in huihling: hy selectively injuring the animal (for instance hy removal of legs and hy the jirodnclion of lesions in the central nervons System), hy dis- rupting the ordinary luiilding eireumstances (as hy destroying threads or rotaling lhe frame dnring constrnetion ), and hy feeding lhe spiders drngs whieh affect lhe hehavior of rnany animais, (he psychotro|)ic drugs, and whieh in lhe sjrider have re|)roducihle and particular consequences for cerlain featnres of the weh. Drng effects on wehs have heen investigated exlensively for Iwo species. Zygiella x-nolula and Arancus diadematus. In these stndies, the ohject of measiire- ment has heen the compleled weh rather than the motions of the animal in the course or acl of huihling. There are many good rtasons for ihis choice, among olhers the reiuoducihilily of events whieh oceur at greal speed under eonditions of limited visihility, hut a prime consideration has heen the variahilily in the dimensions of the weh and lhe eonsequent necessity lo eniploy slatistieal com- j)arisons of many wehs. eilher of many animais under experimental and conlrol eonditions or of the saine aidmal treated afler a suitahiy lengthy weh-hnihling history had heen accnmnlated. Fig. 1. whieh shows lhe eom|)leted weh of .druncus diadematus, makes it ai)parenl lhal several different measnres or aspeets of the weh may he ehosen for evidenees of influenee of treatme^its. The eom|)nler, allowing as it does. many and rapid eonqnitations to he inade, diminished somewhat the limitations of lhe choice of parameters. It also |)ermitted ns lo pick nj) rather snhlle changes. apjjarenl in a series of wehs hnl perhajis not in lhe comparison of Iwo single represenlalives of treatment and control samples. (See for example Fig. 2, show- ing a represenlative drng effeel.) cm SciELO 10 11 12 13 14 15 Fig. 1 — Completed vveb of Araneus diudematus Cl. Structure in upper right comer is weight supported at 20 mm intervals by three thread.s. Fig. 2 — Radial and frame structure of Araneus vveb. The spider has been removed before beginning provisional spiral from hub tovvard periphery. (From BREED, A. L., LEVINE, V. D., PEAKALL, D. B. and WITT, P. N-, Ilehavtonr, 23. 43, 1964). Mem. Inst. Butantan Simp. Internac. 33 f2):645-650, 1966 CHARLES F. REED 647 The inaiiy jiaramelers of lhe wel) niay he reduced to ihree classes: the size. lhe shape, and llic regidarily of ihread placenieol in lhe wehs. Psycholropic drugs may he descrihed iii siniply emjcirieal leriiis of lhe effeets lhey wreak oii lhese classes. D-amphetamiiie, for example, in a dose of 600 mg/kg slimidates llie hnilding of smaller wehs than normal wilh irregular placeinent of certain ihreads (lhe spiral and radial ihreadsl. LSD 25, on lhe olher hand, can he adminislered in doses 10.3-0.5 nig/kg) which rednce lhe freqnency of vveh-hiiild- ing, luit have no effecl on the regularity of angles and spirals; in faci lhe regidarily of placement may he grealer than usual. Higher doses do have an adverse effect on angle regularity. These eni])irieal ohservalions, however, can only he lhe starling point for explanalion. ll is important of course lo note lhal lhe wehs change in ways sjiecific to lhe drug, hnl il is necessary to identify hasic elcments in lhe execution of lhe wel) struclure. There are four sources of alleralion which are now' apparcni. Dr. Witl has discussed the possihie role of thrcad supply in delermining weh dimensions. The inhihilion or stiniulation of protein-synthesis requires ad- juslmenls in the weh. This is nol an ohvious relalionship: it could he enteiiained as a possihilily lhat lhe s])ider comjiletes its weh wilh thread material slill availahle or that il runs short in lhe process. Ordinarily, however. the availahle material is rather com]iletely utilized, the sjiider coming to a slop which nol only completes lhe geometry of the weh hui empties lhe ampullate and aggregale glands. Diazepam, as Dr. Witl has shown. is an exce|)tion to ihis rule. An important point is lhal dearlh or ahiindance of material need nol imply loss of precision of placement of ihreads. However, if cholinergic action enlers inlo ihread produclion, il niiisl also he conlemiilated as one |)ossihle conlrihnlor lo the second source of weh change. se.nsory-molor disliirlxincc. Thal is. neural Iransmission may he affecled. Phvso- stigmine. however, does nol affecl jjrecision of jilacemenl ailhough it does slimnlate ihread-synthesis. Two olher suhstances potenlially involved in neural Iransmis¬ sion, on lhe olher hand. nor-epine])hrine and 5-hydroxylriplamine, affecl holh size and legularily of thread-|)lacing. Whatever lhe source of lhe dislurhance, il is clear lhal sensory-motor integralion is a second factor underlying weh alteralions. A relaled hnl conceplually inde])endenl source of disturhance may he called jailiire or clian^r in infonnalion-processins'. The ca])acily of lhe s])ider lo inle- grale sensory informalion can he affecled. lor inslance, hy central nervous system lesions. A simpler forni of interference is produced hy depriving lhe s[)ider of visual or laclual informaliou hv hlinding or hy removing legs. Pinally. il may he necessary lo invoke a higher-order adaptation on lhe part of lhe animal, reUridion of enprp;y output. If a weh is produced al all, it is smaller in area and in thread lenglh; il may or may not show thread irregularities. Slrychnine (30()-82() mg/kg) for inslance, diminishes frecpiency of hnilding and results in wehs of smaller spiral area and less oval shape. Caffeine (ahove 1 g/kg) also produces rounder wehs, disni|)lion in threads and long weh-huilding limes (several hours inslead of lhe usual 20 lo 30 minnles.) While each or several of lhese faclors may euler inlo lhe |)aiiicnlar dislurhances of weh conslrnclion, we have recently heen concenlraling on lhe malter of in¬ formalion. chiefly on lhe sensory informalion emjiloyed hy lhe s|)ider in ihread i SciELO SPIDER 8/64 t 4. p m 600 ^/g d-Amphetamine | fti' -' CONTROL DAY AREA 430.0 cm“ radii 35 AN6LES 1.29 DAY AFTER DRUG 149.6 cm* 27 5.96 Fig. 3 — Effect of d-amphetamine on web construction. The webs are separaled by one (lay, but the spider has been given 600 mg/kg 12 hours before beginning the second web. Reduction in area and in number of radii are observable, but it can also be demonstrated lhat central angles and spiral separation are more irregular than usual. This figure and the following illustrate rather than establish drug eífects; variation of webs requires statistical analysis of experimental treatment. íFrom WITT, P. N. BRETTSCIINEIDER, L., and BORIS, A. P., J. Pharmaeol. exp. Ther., iS2, 183, 1961). A B C Fig. 1 — Control, treatment and recovery webs, psllocybln. Drug vvas given 12 hours before web shown in B, dosage 150 mg/kg. Web A is web of previous day, C that of day following Web B. Reduction in area and number of radii in the treatment web are apparenl. (From Cl IRISTIANSEN, A., BAUM, RICARDA, and WITT, P. N., ■/. Phannarol. exp. Ther., 13H, 31, 1962). Á n (7 Area (cm'-') 293.6 227.3 31'1.5 Number of spirai turns 35 2S 29 Numl)er of radii 36 31 36 150 mg/kg psilocybin was given 12 hours before web B was built cm SciELO 10 11 12 13 14 15 Mem. Inst, Butantan Simp. Internac. 33(2); 645-650. 1966 CHARLES F. REED 649 jdacement. Bolh analysis of the coni|)lele(l webs and of ongoiiig huilding suggesl lhat lhere are stages in cüiistruclioii iii vvhich testing fimclions alteriiate witli actions taken iipoii tliose tests. A few examples will illustrate lhe ohservations. The firsl ihreads oí the weh are dependcnl U|)on lhe eonlingencies of localion : u])on the availahility of fastening points. F.ven in the lahoralory cages, the moor- ing ihreads differ froni day to day, perhaps heginning from lhe apparently random residues of lhe previoiis day. One means or anolher will he employed to set the firsl threads; if an air stream from a fan is directed across the cage, the s|)ider will let the thread he carried along it rather than lahorioiisly carrying the dragline from ])lace to place. Biit as constriiction proceeds, it is possihle to s])eak of typical structnres, where actions are giiided hy a narrower range of circiimstances than in the initial stages and where it is possihle to ohserve rather more invariahie events. It is interesting lhat while the spider may he easily distnrhcd dnring the selling of the initial threads, it is considerahly more difficnit to do so once the later stages of constrnction have hegnn. Assume lhat the sjiider has achieved a Y-structure of three threads witli a sparse perimeter of additional framing threads. Taking a station at the cenler of ihis plexus or huh, the animal appears to lest existing threads hy circling, lonching each of the lines with several legs; lhe necessity for an additional radial thread and/or siipporting stnictures is apparently estahiished during lhese move- ments, hui it is not clear what sensory data are heing utilized hy lhe animal. One jjossihility is lhat feedhack occurs from movement of a leg over a cerlain arc wilhoul contacting thread; anolher possihility is lhat lhe spider is utilizing some sensory data related to the degree of tension in lhe existing threads. Al any rate, if lhe first legs on one side of Araneus diadematus are removed, there are clear and irreversihle disturhances of lhe regularity of thread ]ilacement — ailhough such an experiment does not allow us lo dislinguish helween lhe Iwo foregoiug hy|)olhesis. Depending n|)on lhe results of lhe "tesl”, lhe spider exils from the huh along an existing thread, pulling a dragline vvhich is thereu])on atlached to the frame. The spider relurns to lhe huh along the new radial thread. If lhe new |)lacement is lo occur in the lower ((uadrauls of lhe weh. lhe animal may drop hy means of lhe dragline instead of crawling: lhere does seem to he an “ojrlion” and not a fixed aclion-])attern followiug lhe onlcome of lhe [nohing funclion. There is anolher o]vtion, a kind of suhslage which results not only in an additional radial line, hnl in an acce.ssory sup])oiiing or framing memher. In¬ stead of relurniug direclly lo lhe huh along lhe new radial line the s|)ider makes an allachmenl along lhe way. The altachmenl serves lo secure the radial line a second lime to the frame, and deviales it from its original direction. Bolh threads may receive additional reinforcement as lhe spider |)asses over them. The assemhling of lhe radial and frame elements jnoceeds hy this alternation of testing and placement until lhe sjjider succeeds in making a complete circuit of lhe hnh wilhoul receiving whatever signal it is lhat reí^uires additional su[)- ports. The inauguration of the nexl stage is so irnhedded in lhe preceding move- inenls thal it is dispntahle not only when it hegins hut whelher it is a separale stage at all. The se(|uence of movements thal have accompanied the radial-lest- ing continue, hut it is a|)parent lhat lhe s|)ider has also heen altaching thread to lhe radii as it circied, hridging the central angles. The resull of a com|)lele Circuit of the weh wilhout need for a radial strncture is lhe heginning of a light s])iral. Gradually the spider moves faiiher cul from the center; the pro- 1 SciELO 650 SPIDEU WEBS AND FSYCHOTROPICS visionai spiral lias hegim. This teniporary spiral ajipears only as a Iracp in the conijilelfd weli: it is (lovoiircd hy llu- s|)id(‘r as il losos ils iisefidncss as a giiide and su[)porl for the laying of the |)('rmanenl viscid s|)iral. The permanenl, tacky spiral is constructed in a elearly sejiarahle slage. First of all. the sjiider jiauses at the coinjiletion of the provisional spiral. The aggregate glands now ht‘gin to siipply material, and there seems to he a reipiireinent of time for this glandular shift-over. (It is possihie that the clear pause iu the center of the vveh, which oeeurs iu the radial hiulding is aiso a period in vvhieh the gland duct is filling vvith material.) The laying of the permanent spiral takes up hy far the greater |)roportion of wehdniilding time: the hasic structures are huild within the first ,5 minutes, the [lermaneut spiral oecujiies the remaining 15 to 20 minutes. The jirovisional sjiiral had proeeeded from within outwards and has hrought the spider to the periphery of the weh; now the work goes from out inwards. Thread-plaeing is halted short of the huh, leaving an o])en space or free zone hetvveen the inner- most turn and the huh. The S[)ider takes u|j its |)osition of vigilanee on the huh. It may remove some of the threads of the huh hy hiting thein out. Ks[)ecially elear movie se(]uences of these and other aetivities of A r a n c u s have reeentiy heen ohtaiued hy Bariium and VVitt. These films have revived the .speeulation that the ])ositioning of a |)artieular thread is determined hy the array of forces then e.xtant in the weh, that the spider tugs the threads and resjionds hy lay¬ ing a new thread if the resistanee to the tug falis helow a hy|K)thetieal hut unknown vahie. In order to test this eonjeetiire, we have heen attenijiting to make films of the seipienee of structures iu a single weh, with the ohjeetive of testing where the spider appears to test and iiredieting the jiosition of the next thread. A siiccessful emulation of the single event of new plaemnent should (lermit a state- ment of the range of tension to which the spider is sensitive in huilding, if indeed this is the teehnologieal hasis of its eonstrnetion at all. Discession yl. SJiulov: “Is there any correlation between the influence of drugs on the structure of the web and the changes in web’.s conslruction in time as a result of mutilation as cutting every one or two tarsi (Dr. Sehieps experiments) ?” P. N. Witt and C. F. ReecI: “The same structural change may be produced by different treatment.s. For instance, irregularities of central angles can be the con- sequenee of losing one first leg or 600 mg/kg d-amphetamine.” W. Bücherí: “1. In what kind of spiders you made your studies? 2. Have you used also other species of spiders or only orb-web-spiders?” P. N. Witt and C. F. Reed: “1. Araneus dUidematun and Zygielía-x-notata. 2. The orb-weavers have the distinct advantage of iiroducing webs with geometric fornis susceptible to mathematicat anatysis. Departures from ordinary variation can there- fore be specified in a way which is not possible with other webs.” D. Valente: “1. Há alguma correlação entre a feitura das teias e o sistema neuro-secretor? 2. O ciclo sexual e a idade têm influências na feitura das teias?” P. N. Witt and C. F. Reed: “1. Endocrine structures have been histologically identified in spiders, but their function is still unknown. 2. All of our drug experi¬ ments were done with adult female Araneus diadematus, so that the drug would be the main variabte. Males are irregular builders in later life.” cm 2 3 L 5 6 10 11 12 13 14 15 Mem. Inst. Butantan Simp. Internac. 33(2);651-658, 1966 WERNER RATHMAYER 651 67. THK EFFECT OF THE POISON OE SPIDEIi Ax\D DIGCEHWASPS ON THEIH PREY {HYMENOPrERA: POMPÍLÍDAE. SPIIECIDAE) WERNER RATHMAYER Zoologisches Institui. Frankfurt u.M., Germuny Ainong lhe HYJIENOPTKKA \ve find lhe most so])liistieate(l venonioiis aniinaP. With regard lo [jotencv I do iiot kiiovv of a slrongei poi.soii thaii thal oí l/ahro- bracoii jiiglundis, a iH acoiiid wasp, whieh ])aralyzes its ])rey, lhe G a 11 e r i a larva, cveii at a dilulioii of 1 pari veiiom in 200,000,000 ])arts of hemolymph (ll. AEo willi regard to duralioii of ])aralysis lhe ])redaceoiis vvas])s come first: a sj)ider sOmg hy lhe pom])ilid Cryptochilus uj/ini.s lived for foiir monlhs iii dee]) paralysis (2). Otie woiiders vvhy ])hysiologists and j)hannacologi?ts liave so far takeii liltie interest iii tlie solilary wasps. I wiil confine myself in lhe follovving lo lhe POMPILIDAE and SPHECIDAE. Wilh llieir sting ap])araliis llie females of lhese families inject venom into llieir ])rey whieh residis in paralysis of varying dnralion. The iiomjdlids hunl exclnsivelv for siiiders. On lhe lisl of prey of lhe SPHECIDAE we find all orders of inseels ano sjiiders loo. Imnioliilizalion of lhe prey is ilways related to eare of lhe off- S])ring. An egg is laid on lhe viclim and lhe haldiing larva feeds on lhe liv¬ ing ])rey. The niain snhjecl of ihis pajier is lhe ])hysiologieal effecl of lhe poisou. The dnralion of jiaralysis jirovides a firsl elneld). When we look al lhe pom- jdlids we find ihree calegories of paralysis: 1. In lhe mosl ]irimilive case re- ])resenled hy some memhers of lhe genns II o lu o n o I u s lhe females [laralyze s])iders, lay an egg on lhe viclim hnl hury lhem nol in lhe groimd. According to (4,51 all lhe sjiiders reeover a few miniiles aflei lhe sting and resume lheir regular aclivity. The larva lhal halehes a few days later wili feed on lhe sjiider and kill il. 2. females of lhe genus Anopliiis also suhdue spiders hy sliug- ing. 1 hey hury lhem aflerwards in chamhers in lhe ground and altaeh an egg. If one digs lhem oul one finds thal jiaralysis lasls much louger lhan wilh Hu- tu (> ll o t u .s, neveiiheless lhey usually reeover after a few hours. In lhe chamhers. however, lhey are. even after reeovering. imahle lo move hecause lhey are jiaeked so tighlly. Temporary paralysis is also re|)orled for N o t o c y p h ii s (6). 3. AIosl of lhe |)ompilids paralyse lhe s|)iders dee|ilv and permanently. The ])rey remains fresh, i.e. il is nol desieeated or eonlaminated for weeks. (ierlainly lhe vietims live dnring this lime. The striking facl is thal lhe prey slung hy cerlain jiompilids is aiways ahie lo reeover, lhal of others is pafalysed deejily and irreversihly. Since lhe stinging The recelpt of a travei grant by the Deutsche Forschungsgemeinschaft i.s gratefully acknouleilged. cm SciELO 10 11 12 13 14 15 652 THE EFFECT OF THE POISON OF SPIDER AND DIGGERWASPS ON TllEIR PREY (HYMENOPTERA: POMPILIDAE, SPHECIDAE) heliavior is very similar iii tlie whole faniily (7) oiie may assiinip tliat we liave to (leal willi (jiialitative or qiiaiililalive differetiees iii the eom|)osilioTi of the poisou or tlial differeiit pliysiological systems are affeeled. Kxjrerimeiits oii tliis poiiil are niiieh iieeded. 1’aralysis lasling only a fevv minutes eould lie caiised liy an instaliility of the active compound of the venom. The ohserved differeiices iii diiration of paralysis among memhers of the group that normally paralyse ir- reversihly ean he attrihuted to the amoimt of poisou iujeeted as ex])eriments vvilh a sphecid wasp, re|)orted later, show (8). How aud where the poisou of pompilids acts iu the orgainsm of the ])rey is eulirely uuknovvu. There is oue paper reportiug that the veuom gets iuto the hemolvmph of the prey(9). Teuetratiou of the stiug iuto the central uervous System therefore seems not to he a prere((uisite for jiaralysis aud prohahiy does iiot happeu iri lhe field. To sum up, the veuom of lhe POMPILIDAE has to he hroiighl iuto the hemolymph of a s])ider aud it paralyses the motor-system. There are iiilerestiiig geiius-sj)ecifie differeuces iu regard lo duraliou and completeuess of ])aralysis. Whelher lhey are due lo rpialitative or fuuclioual differeuces of lhe iujeeted veuom stili has to he eslahlished. Novv let us eousider lhe SPHECIDAE. In ihis family we also find differeuces iu duratiou aud com[)leteuess of paralysis. Molecrickets stimg hy L u r r u re- cover aiways .some minutes after stiiiging f 10, 11 I. Crickets slored hy Sphcx lobulus also recover 10 to 15 minutes after heiug stuug as well as the viclims of some species of Li ris and No togou ia. These examples, hovvever, and the a|)pareul killiug of the |uey hy the venom of uiany IloMHlCINl (12,15), .4 s í a I a sj)ecies ( 14, 15) and some Ckabkonim are exce|>lional. Delay hy mauy hours of onsel of ])aralysis is reported for crickets stung hy Liris nigra{\6) and for auts hunied hy Aphilanlhops jrigidus (17). The greal hulk of lhe sjihecoid wasps hovvever paralyse lheir prey dee|)ly and irreversihlv. I |)erformed ex])eriments with l'liilaulhiis Iriangiili/ui Wi) vvhich provided a first chie as lo the effeci of lhe poisou of ihis species ou its prey, lhe hon('y- hee. In mauy cases I eould walch lhe stinging direclly. The hee is alvvays slimg oídy onee iuto lhe ciitaneoiis memhrane hehiud lhe first jiair of legs I Fig. 1). Histological seclions demonsiraled that lhe stiug iu most of lhe cases in- vpstigaled did iiot pierce lhe veniral nerve, cord. Oue caii have hees stuug hy L li i I a u I li II s exjierimeulally al auy desired poiul hy cultiug a small vvindow iuto lhe cuticle aud placiug the stiug there. Il vvas shovvu that it is siifficieul for paralysis that lhe veuom luerely gets iuto lhe hemolymph. The furlher lhe poiul of [)unclnre is from lhe locomolor-sysiem lhe longer it takes uiilil lhe legs cea.se lo move (Fig. 2). The natural |)oiul of jmnclure hehiud lhe first pair of legs guaraulees the (piickest ousei of paralysis. Fvideuce vvas ohtaiued as to lhe mechauisrn of immohilizaliou. There are Ivvo íacts iudicatiug that [varalysis is not due lo a central adiou ou the uervous system hut ralher lo a peripheral hlocking. The immohilizatiou does noi stari inslaustaueously hy hlockiiig lhe movemeuts of all parts of the legs hui spreads progressively lovvard lhe periphery. The distai ])arls of lhe legs cau slill move al a lime vvhen the proximal oues are aiready immohilized. Siuce all muscles of a giveu leg are innervaled from lhe same maiu nerve truuk leaving lhe correspouding thoracic gangliou aud siuce lhe cell hodies of the moloneuroues lie closely logether al lhe periphery of lhe gatigliou, a central adiou of the veuom should had to a uearly simullaneous hlock or iuterruplion of the effereni iuuervatiou of lhe various leg muscles. This cm SciELO 10 11 12 13 14 15 WERNER RATHMAYER 653 Mem. Inst. Butantan Simp. Internac. 33(2):651-658, 1966 marked lime delay can lie ex|)lained hy lhe anatomy of lhe insect leg. The miiseles for movenieiit of lhe coxa and feimir lie in lhe hody cavity of lhe hee and lherefore wili he reached l)y lhe venom firsl. The muscles for lhe tihia are allaehed in lhe leg itself, lhe ones for lhe metatarsiis lie still further dislally. Jl will lake lime imlil via lhe hlood eirctdalion enoiigh poison has enlered lhe narrow leg and lherefore \ve encoimier lhe delay in onset of paralysis. Fig. 1 — Positlon of the dlggervvasp Phtliinthus triangulum whon attacking and stlnging, behind lhe firsl palr of legs of ils victim, the honeybee. A second ohservalion |)oinls in lhe same direclion. Wilh local adminislralion of small doses of venom il is possihle lo ])aralyse muscles vvhieh are innervaled hy lhe verv same ganglion as for example lhe muscr.lalure of the forevvings with- oiil affecling lhe muscles of the middlelegs. Again one comes to lhe conchision that ihis shonld he im|)ossihle hy an effecl of the venom on lhe CNS of the prey. It was shown further for lhe venom of I'h i I a n I h ii s thal il acis nol S])ecifically on the honeyhee hut ralher paralyses all tested insecls and s])iders loo (Tahle 1). The completeness of jiaralysis varies wilh lhe amoimt of poison injected. Wilh little doses lhe liees can comjilelely recover after initial im- cm SciELO fic;4 THE EFFECT OF THE EDISON OF SPIDER AND DIGGERWASPS ON THEIR PREY (IIYMENOPTERA: POMPILIDAE, SPHECIDAE) Flg. 2 — The variatlon of onset of paralysls of a honeybee’s legs when venom of Philanihus was injected at points 1, 2 or 3. mobilizalion. For a differetil spccii-s (19), demonstralcd lhat duralion and coni- plcteriess of paralysis varied willi llin niimlaT of .dings a])plied. I lliink in Uns case it is aiso an effecl of tlie amoiitil of venoin injected. Wliellier lhe resnlt llial lhe sling of F li i I u n I li ti s iisnally does noi reach lhe CNS may l)e generalized lo olher si)ecies of spliecids reqnires hislologic'al sludies of llie |(aralysed |)rey. The |)i'eeision willi which Liri.s iiig,ru ap|)lies four stirigs to lhe cricket (19) and lhe fact lhal lliey are placcd in dose topograi)hical rclalion lo lhe snhoesophagcal and lhe ihree thoracic ganglia leads lo lhe as- sumption lhal in this case lhe venom has to l>e hronght vcry dose lo lhe nervons centers responsilile for locornolion. A varialion of lhe mechanism of innnohilizalion and aIso in lhe effecl of llie poisou vvonld not hc snrprisitig when one ihinks of lhe greal nnmher of different insect families nsed as prcy. cm SciELO 10 11 12 13 14 15 Simp. Internac. Mem. Inst. Butantan 33(2);651-658, 1966 WERNER RATHMAYER 655 TABLE I EFFECT OF INJECTED VENOM OF PHILANTHUS TRIANGULVM ON VARIOUS ARTllROPODS ORDER Xo. of families tesled No. of genera tested No. of species tested Effect INSFXTA: HYMENOPTERA 5 26 36 P LEPIDOPTERA 2 4 4 P COLEOPTERA 1 1 1 P ORTHOPTERA 2 2 2 P HEMIPTERA 2 2 2 P ODONATA 1 1 1 p,