Br. J. Phrmc. (1979), 67, 199-25 EFFECTS OF THE VENOM OF THE GREEN ngusticeps ON SKELETAL MUSCLE AND TRANSMISSION J.C. BARRETT1 & A.L. HARVEY Deprtment of Physiology nd Phrmcology, University of Strthclyde, George Street, Glsgow, GI IXW MAMBA, Dendrospis NEUROMUSCULAR 1 The venom of the green mmb, Dendrospis ngusticeps, ws tested for effects on neuromusculr trnsmission nd skeletl muscle contrctility in isolted phrenic nerve-hemidiphrgm preprtions of the rt nd mouse, chick biventer cervicis muscle preprtions nd in neurl cultures of embryonic chick skeletl muscle. 2 The venom (1 to 4 pg/ml) ugmented the responses to indirect but not direct stimultion. As the venom did not hve nticholinesterse ctivity nd did not increse receptor sensitivity, it is likely tht the venom enhnced relese of cetylcholine. 3 Higher concentrtions of venom (4 to 8 jg/ml) inhibited cetylcholine receptor sensitivity. 4 Prolonged exposure to the higher concentrtions of venom produced filure of muscle contrctility. Signs of muscle degenertion were seen in skeletl muscle cultures. Introduction Investigtion of venoms -from elpid nd hydrophid snkes hs led to the discovery of severl polypeptides tht hve specific phrmcologicl ctions nd re useful tools for study of neuromusculr processes. The toxins include - nd,b-bungrotoxin from the Tiwn krit Bungrus multicinctus, cobrtoxins from vrious species of Nj, nd erbutoxins from the sesnke Lticud semifscit (for reviews, see Lee, 1972; Tu, 1977). Among the elpid fmily, cobr nd krit venoms hve been studied extensively but reltively little ttention hs been given to the phrmcologicl properties of venoms from the Africn mmbs. Previously, neurotoxins hve been found in the venoms of the western green mmb, Dendrospis viridis (Bnks, Miledi & Shipolini, 1974) nd of the blck mmb, Dendrospis polylepis (Strydom, 1972). In the present study the ctions of venom from the green mmb, Dendrospis ngusticeps, were exmined on skeletl muscle nd neuromusculr trnsmission. Some of these results were presented to the 3rd Symposium of the Interntionl Society on Toxinology Europen Section, London, September, 1978. Methods Rt phrenic nerve-hemidiphrgm preprtion The rt phrenic nerve-hemidiphrgm preprtion (Bilbring, 1946) ws mounted in Krebs-Henseleit solu- 1 Present ddress: Tropicl Products Institute, 56-62 Gry's Inn Rd., London. 7-1188/79/1199-7 SO 1. tion of the following composition (mm); NCl 118.4, KCI 4.7, MgSO4.7H2 1.2, CC12 2.5, NHCO3 25, nd glucose 11.1; t 37 C nd bubbled with 2 contining 5% CO2. The phrenic nerve ws stimulted t frequency of.1 Hz with rectngulr pulses of.2 ms durtion nd of strength greter thn tht required to elicit mximl twitches of the hemidiphrgm. For direct muscle stimultion, neuromusculr trnsmission ws bolished by the irreversible cholinoceptor blocking gent, erbutoxin b (5 jg/ml) (Tmiy & Ari, 1966). The hemidiphrgm ws then stimulted directly through hook electrode inserted into the rib tissue, with rectngulr pulses of 2 ms durtion t frequency of.1 Hz nd suprmximl strength. Pired preprtions were set up from ech rt, one hemidiphrgm for direct nd the other for indirect stimultion. Chick biventer cervicis nerve-muscle preprtion The chick biventer cervicis nerve-muscle preprtion (Ginsborg & Wrriner, 196) ws mounted nd stimulted under identicl conditions to those described for the rt hemidiphrgm. Indirect stimultion ws vi the motor nerve in the tendon while for direct stimultion of preprtions irreversibly blocked with erbutoxin b (1 gg/ml), the stimulting electrode ws moved into contct with the belly of the muscle. Periodiclly, in the bsence of nerve stimultion, responses were obtined to cetylcholine or crbchol. Mcmilln Journls Ltd 1979
2 J.C. BARRETT & A.L. HARVEY Anticholinesterse ssy The nticholinesterse ctivity of the venom ws ssessed by mesurement of the cholinesterse ctivity of intct chick biventer cervicis muscles in the presence of the venom, by the colorimetric method of Ellmn, Courtney, Andres & Fetherstone (1961). Two biventer cervicis muscles from 2 dy old chicks were used for ech ssy. The rection flsk contined 5.8 ml of.1 M phosphte buffer (ph 8.) nd ws mintined t 32 C. The rection ws begun by ddition of.2 ml cetylthiocholine iodide (.75 M, freshly mde up). At 15 min intervls for 1 h,.2 ml ws removed nd dded to cuvette contining 2.9 ml.1 M phosphte buffer (ph 8.) nd.1 ml 5,5-dithiobis-2-nitrobenzoic cid (.1 M). A blnk determintion ws performed on flsk contining tissue nd venom (8 jg/ml) but no substrte. Cholinesterse ctivity of the venom itself ws ssessed in four flsks contining ll the regents but no tissue. The bsorbnce chnge t 412 nm, per min, in four control preprtions ws compred to tht in the presence of the venom t two concentrtions (2 nd 8 jig/ml; four preprtions ech). All flsks were incubted for 3 min before ddition of cetylthiocholine. Isolted tissue electrophysiology Mouse phrenic nerve-hemidiphrgm preprtions were pinned to the bse of 1 ml bth. The bth ws perfused t rte of 2.5 to 5 ml/min with Krebs- Henseleit solution previously erted with 2 contining 5% CO2. The muscle ws mintined t temperture of 28 to 3 C. Endpltes were locted by inspection under 4 x mgnifiction using stndrd binoculr microscope fitted with Leitz UM 2/.33 long-working-distnce objective. Intrcellulr recordings of membrne potentil nd endplte potentils (e.p.ps) were mde with conventionl glss microelectrodes filled with 2 M potssium cette (8 to 1 megohm resistnce). Potentils were mplified with WPI 71 electrometer (bnd width to 3 khz), monitored on one oscilloscope nd lso recorded on 35 mm film from slve oscilloscope. Neuromusculr trnsmission ws depressed by equilibrtion with physiologicl slt solution to which (+ )-tubocurrine (3 to 5 gm) ws dded. To determine the ction of the venom on the e.p.p., the nerve ws stimulted t frequency of.5 Hz with rectngulr pulses of.2 ms durtion nd suprmximl voltge. Effects of the venom were investigted by perfusion of the preprtion with Krebs-Henseleit solution to which venom (2 to 2 jg/ml) hd previously been dded. This voided disturbnce of the bth, so tht potentils could be mesured from the sme cell before nd during exposure to the venom. Cell cultures of embryonic chick skeletl muscle Suspensions of myoblsts were obtined from the leg muscles of 1 to 11 dy chick embryos by the method of Konigsberg, McElvin, Tootle & Hermn (196). Two ml of medium contining 5 x 11 cells/ml ws dded to 35 mm plstic Petri dish tht hd previously been coted with collgen. The growth medium ws Egle's Minimum Essentil Medium (M.E.M.; Egle, 1959) contining 5% of 5% v/> whole embryo extrct nd 15% horse serum. In order to prevent overgrowth of replicting fibroblst cells, the medium ws replced on the second dy of culture, fter the beginning of myoblst fusion, by one contining 1 pm cytosine rbinoside, DNA synthesis inhibitor. Therefter, the medium ws replced on every third dy with fresh medium. Cultures were incubted t 37 C in wter-sturted tmosphere of 5% CO2 in ir. Myotube formtion occurred t 2 to 3 dys, nd cultures were 7 to 1 dys old when used (Hrvey & Dryden, 1974). Cultures were mounted on n inverted phse-contrst microscope nd mintined t 37 C on heted stge. Membrne potentils were recorded with n intrcellulr glss microelectrode filled with 3 M potssium chloride (electrode resistnce 1 to 2 megohms) nd mounted obliquely on Leitz micromnipultor. Potentil differences between the penetrting microelectrode nd chlorided silver wire bth electrode were monitored vi d.c.-premplifier on n oscilloscope nd on pen recorder. Responses were obtined to iontophoretic ppliction of cetylcholine. Microelectrodes for iontophoresis were filled with.5 M cetylcholine chloride nd connected to stimultor through one chnnel of WPI 75 electrometer. The verge resistnce of the cetylcholine electrode ws 1 megohms. Cre ws tken to select electrodes tht did not lek drug spontneously in the bsence of bcking current. The dose of cetylcholine is expressed s nnocoulombs (nc) of chrge pssed through the electrode. Responses, verged from t lest 1 cells, were clculted s depolriztion s percentge of the resting potentil. All recordings were mde in Egle's M.E.M. which ws mintined t ph 7.4 by bubbling with CO2. When control mesurements of membrne potentil nd dose-response curves to cetylcholine hd been obtined, the culture medium ws replced with fresh medium contining the crude venom t different concentrtions. In some experiments, mesurements were mde immeditely following exposure of the culture to venom; in other experiments the culture ws returned to the incubtor before membrne potentils nd dose-response curves were recorded. In those experiments where cultures were incubted with venom overnight, the venom-contining medium ws sterilized by membrne filtrtion.
GREEN MAMBA VENOM 21 +o 1 L) I-.rv.m 25 F 2 F 15 1! C7 ^ O o Figure 1 3 6 9 12 Time (min) Effect of Dendrospis ngusticeps venom on the rt phrenic nerve-hemidiphrgm preprtion. () Indirectly stimulted preprtions; (b) directly stimulted preprtions. (U) Venom 1 jg/ml; (V) 2 jig/ml; () 4 pg/ml; (A) 8 pg/mi. Drugs Dendrospis ngusticeps venom ws obtined from Sigm Chemicl Co. (Lot No. 12C-73). Other drugs used were: cetylcholine chloride, cetylthiocholine iodide, crbchol chloride, cytosine rbinoside nd (+)-tubocurrine chloride (ll from Sigm Chemicl Co.). Tissue culture medi were purchsed from Flow Lbortories, nd erbutoxin b ws generously supplied by Professor N. Tmiy, Tohoku University, Jpn. Results Rt phrenic nerve-hemidiphrgm At concentrtions of 1 to 4 jg/ml of venom there ws n immedite increse in the height of responses to indirect stimultion (Figure 1). With 1 jg/ml the twitch height incresed to lmost 25% of control, stbilizing t this ugmented level fter bout 6 min. With 2 nd 4 gg/ml the responses to indirect stimultion decresed fter the initil ugmenttion. At higher concentrtion of venom (8 jg/ml) there ws short-lived increse in the height of indirectly elicited contrctions tht ws followed by progressive muscle prlysis (Figure 1). In contrst, there ws never ny increse in twitch height in preprtions tht were stimulted directly. Concentrtions of venom between 1 to 8 gg/ml cused slow, progressive decrese in muscle contrctions (Figure 1). The development of blockde ws fster t the higher venom concentrtions, lthough it ws slower thn tht observed in indirectly stimulted preprtions exposed to 4 or 8 gg/ml venom. The decline in twitch height could not be reversed by wshing. A second ddition of venom to indirectly stimulted preprtions during the ugmenttion produced by 1 jg/ml venom did not cuse further increse in twitch height, but rther hstened the onset of block. Chick biventer cervicis nerve-muscle preprtion Qulittively similr results were obtined on the chick biventer cervicis nerve-muscle preprtion. The venom (1 to 4 gg/ml) mrkedly ugmented the responses of indirectly but not directly stimulted preprtions. At higher concentrtions of venom (4 to 16 gig/ml) the ugmenttion ws followed by slowly developing, irreversible blockde of the twitch response. A similr blockde ws observed on directly stimulted preprtions but with much longer time course. In some preprtions trnsient contrcture occurred on ddition of venom. During the development of the ugmenttion of responses to indirect stimultion, there ws no mrked increse in the responses to cetylcholine nd crbchol (see Figure 2 for typicl experiment with cetylcholine contrctures nd 2 gg/ml venom). When tested t the time of mximum ugmenttion of indirectly elicited contrctions, responses to both cetylcholine nd crbchol were depressed (Figure 3). Cholinesterse ctivity Venom (8 gg/ml) hd no bility to hydrolyse cetylthiocholine within 6 min incubtion t 32 C, indicting tht the venom itself did not hve significnt cholinesterse ctivity. With intct chick biventer cervicis muscles, control cholinesterse ctivity per muscle ws.66 (±.4) x 1-3 bsorbnce units/min (men + s.e. men of 4 determintions), while in the presence of 2 nd 8
22 J.C. BARRETT & A.L. HARVEY A t K! LI..J 5min Figure 2 Effect of Dendrospis ngusticeps venom (2 pg/ml) on responses of the chick biventer cervicis nervemuscle preprtion to indirect stimultion (.1 Hz) nd cetylcholine (2 mm). Acetylcholine ws dded t the dots nd wshed out fter 3 s. Venom ws dded t the rrow, nd gin fter ech cetylcholine wshout. 3 2 11 L-ll I T AC T AC TA C TA C Control 1pg/ml 2pg/ml 4(g/ml Figure 3 Effects of Dendrospis ngusticeps venom on responses of the chick biventer cervicis preprtion to indirect stimultion (T), cetylcholine (A, 2mM) nd crbchol (C, 2 gm). Responses to cetylcholine nd crbchol were determined t pek twitch ugmenttion. Ech column represents the men of t lest 6 preprtions; verticl lines show s.e. men. gig/ml venom the ctivity per muscle ws.55 ( ±.5) x 1-3 nd.8 (±.6) x 1-3 bsorbnce units/min, respectively. Effect on endplte potentils Mouse phrenic nerve-hemidiphrgm preprtions were prtilly prlysed by perfusion with physiologicl sline contining (+)-tubocurrine (3 to 5 gm). After the preprtion stopped contrcting in response to indirect stimultion, subthreshold endplte potentils could be recorded. Subsequent exposure to venom (t 2 nd 1 ig/ml) cused such drmtic increse in the mplitude of the endplte potentils tht the preprtions begn to twitch in response to stimultion, mking intrcellulr recording impossible. Typicl records of endplte potentils before nd 5 min fter ppliction of 2 tg/ml venom re shown in Figure 4. The time course of the endplte potentils did not chnge pprecibly in the presence of the venom nd there ws no chnge in muscle resting membrne potentil throughout the experiment. Effect on neurl muscle cultures Postjunctionl blocking ctivity of the venom ws ssessed on neurl cultures of chick embryonic skeletl muscle. Effects on resting membrne potentil nd cell morphology were lso tested. At concentrtions of 5 to 4 tg/ml the venom depressed responses to iontophoretic ppliction of cetylcholine (Figure 5). Lower concentrtions (.1 to 2 tg/ml) hd no effect, even fter overnight incubtion. Three hours incubtion of cultures with 5 or 1 pg/ml of venom did not significntly depress the verge resting membrne potentil, but 25 to 5%/ reduction ws found following 2 h in 2 or 4 )tg/ml. The depolriztion ws generl ccompnied by signs of cellulr dmge, such s extensive vcuole formtion, extrusion of cytoplsm nd peeling of myotubes from the surfce of the culture dish (Figure 6). It
GREEN MAMBA VENOM 23 2mV[ t;.ib 2ms ; Figure 4 Typicl endplte potentils (e.p.p.) recorded in mouse phrenic nerve-hemidiphrgm preprtion prlysed by (+ )tubocurrine (5 pm). () Control e.p.p.; (b) e.p.p. fter 5 min exposure to 2 pg/ml Dendrospis ngusticeps venom. should be noted tht reduction or bolition of cetylcholine responses ws found with concentrtions of the venom tht cused neither depolriztion nor obvious cellulr dmge, lthough it is not known if fibre input resistnce ws ltered by venom. Additionlly, in cultures in which lrge res hd been destroyed, cells could be found with membrne potentils more negtive thn -2 mv but the cetylcholine responses of these cells were lmost completely blocked. Discussion The venom of Dendrospis ngusticeps hd three concentrtion-dependent ctions on skeletl muscle: t low concentrtions it ugmented the contrctions of indirectly stimulted preprtions; t higher concentrtions it inhibited responses to cetylcholine; nd t still higher concentrtions it cused filure of muscle contrctility. Perhps the most interesting effect of the venom ws the mrked ugmenttion of twitch responses of indirectly stimulted rt phrenic nerve-hemidiphrgm nd chick biventer cervicis preprtions. In n erlier report tht concentrted on the crdiovsculr ctions of green mmb venom, Osmn, Ismil & El Asmr (1973) described similr effect on the rt hemidiphrgm. These uthors suggested tht the ugmenttion might be due to supersensitiztion of the cholinoceptors, lthough no evidence ws presented to support such hypothesis. Since the venom did not ugment the contrctions of directly stimulted preprtions, the site of ction would pper to be t the neuromusculr junction rther thn on the muscle fibres themselves. It ws, thus, possible tht the venom hd nticholinesterse ctivity, incresed postsynptic receptor sensitivity or incresed cetylcholine relese. In the chick biventer cervicis nerve-muscle preprtion responses to cetylcholine nd crbchol were unffected, or depressed, t times when responses to indirect stimultion were incresed three fold. This supports the erlier conclusion tht there is no increse in muscle contrctility nd lso mkes it unlikely tht there hs been supersensitiztion of cholinoceptors, s suggested by Osmn et l. (1973). Tht there ws little difference in the effect of the venom on responses to crbchol nd exogenous cetylcholine suggests tht the venom does not ct s n nticholinesterse. This conclusion ws confirmed by the cholinesterse ssy, where biochemicl tests indicted tht the venom t concentrtions up to 8 pg/ml hs no significnt nticholinesterse ctivity. By process of elimintion, it my be concluded tht the ugmenttion of twitch height produced by venom is result of presynptic ction to increse trnsmitter relese. More direct evidence in fvour of this conclusion ws obtined from electrophysiologicl experiments on the mouse phrenic nerve-hemidiphrgm preprtion where the venom incresed the mplitude of the endplte potentil without the concomitnt prolongtion tht would be ssocited with n nticholinesterse effect. At concentrtions of venom higher thn those cusing ugmenttion, there ws slow irreversible blockde of the twitch responses. Although similr effect ws observed on directly stimulted preprtions, it ws fster in indirectly stimulted preprtions, suggesting tht the ntgonism is lrgely t the neuromusculr junction. Since responses of the chick biventer cervicis preprtion to cetylcholine, crbchol nd indirect stimultion were depressed simultneously, the ntgonism would pper to be postjunctionl rther thn by filure of trnsmitter relese. The presence of neurotoxins tht cuse nondepolrizing blockde by binding to postjunctionl cholinoceptors is common feture mong elpid snke venoms (see Lee, 1972); such toxins hve lredy been reported in Dendrospis viridis venom (Bnks et l., 1974) nd in Dendrospis polylepis venom (Strydom, 1972). Electrophysiologicl studies on neurl cultures of chick embryo skeletl muscle llowed investigtion of effects of the venom on the postjunctionl cholinoceptors in the bsence of interference due to effects of the venom on the presynptic nerve terminl. Receptors on cultured muscle hve previously been shown to be similr phrmcologiclly to receptors on more conventionl skeletl muscle preprtions (for review, see Hrvey & Dryden, 1977). At concentrtions which hd no effect on the verge resting
24 J.C. BARRETT & A.L. HARVEY E -i._ C CL E o 1 [ 2 3 56 1t 1 I t t l I t I.5 1 8 32 64nC ACh.5 1 8 32 64 loonc ACh -i r- 6. 5 CL _- Co 4.6 4 o 3 o c 2 N 1.5 Q..5 1. 1 Acetylcholine (nc) 1 Figure 5 () Effects of Dendrospis ngusticeps venom on responses of cultured chick embryonic muscle fibres to iontophoretic ppliction of cetylcholine. Left pnel: typicl control dose-response curve; right pnel: dose-response curve in myotube fter 8 min exposure to 2 jg/ml venom. (b) Men dose-response curves to cetylcholine before nd fter venom: (@) control responses; () responses fter 8 min exposure to 5 pg/ml venom; (V) responses fter 8 min exposure to 2 ig/ml venom. Ech point represents the men of t lest 1 determintions; verticl lines show s.e. men. membrne potentil, the venom mrkedly depressed the depolriztion elicited by iontophoreticlly pplied cetylcholine, suggesting tht one or more components in the venom hve postjunctionl receptor blocking ctions. At higher concentrtions the venom hd cytotoxic effect on the muscle fibres in tissue culture which resulted in cell destruction. This direct ction of the venom on skeletl muscle my be the cuse of the slow decrese in twitch height of directly stimulted rt hemidiphrgm nd chick biventer cervicis preprtions tht ws observed t higher concentrtions of venom. Thus, the venom of Dendrospis ngusticeps hs severl ctions on skeletl muscle nd on neuromusculr trnsmission, including n unusul fcilittory effect. Further studies on isolted venom components re in progress in order to elucidte more precisely the mechnisms of ction of the venom. Previous studies on purifiction of polypeptides from green mmb venom filed to isolte single component tht pproched the ctivity of crude venom when injected into mice (Viljoen & Botes, 1973; 1974), but it is hoped tht more extensive phrmcologicl testing will be successful. We thnk the Science Reserch Council for Studentship (to J.C.B.).
GREEN MAMBA VENOM 25 Figure 6 Cytotoxic ction of Dendrospis ngusticeps venom on cultured chick embryonic muscle fibres. Upper pnel: phse-contrst photomicrogrph of culture before ddition of venom. Lower pnel: photomicrogrph of the sme culture fter 6 min exposure to 2 1ig/ml venom. Br indictes 5 gm. References BANKS, B.E.C., MILEDI, R. & SHIPOLINI, R.A. (1974). The primry sequences nd neuromusculr effects of three neurotoxic polypeptides from the venom of Dendrospis viridis. Eur. J. Biochem., 45, 457-468. BUJLBRING, E. (1946). Observtions on the isolted phrenic nerve-diphrgm preprtion of the rt. Br. J. Phrmc. Chemother. 1, 38-61. 5O~ EAGLE, H. (1959). Amino cid metbolism in mmmlin cell cultures. Science, N.Y 13, 432-437. ELLMAN, G.L., COURTNEY, K.D., ANDRES, V. & FEATHER- STONE, R.M. (1961). A new nd rpid colorimetric determintion of cholinesterse ctivity. Biochem. Phrmc., 7, 88-95. GINSBORG, B.L. & WARRINER, J. (196). The isolted chick biventer cervicis nerve-muscle preprtion. Br. J. Phrmc. Chemother., 15, 41-411. HARVEy, A.L. & DRYDEN, W.F. (1974). Depolriztion, desensitiztion nd the effects of tubocurrine nd neostigmine in cultured skeletl muscle Eur. J. Phrnc., 27, 5-13. HARvEY, A.L. & DRYDEN, W.F. (1977). Electrophysiologicl nd phrmcologicl properties of skeletl muscle in culture. J. Phrm. Sci., 66, 913-922. KONIGSBERG, I.R., McELVAIN, N., TOOTLE, M. & HERR- MANN, H. (196). The dissocibility of deoxyribonucleic cid synthesis from the development of multinuclerity of muscle cells in culture. J. biophys. biochem. Cytol., 8, 333-343. LEE, C.Y. (1972). Chemistry nd phrmcology of polypeptide toxins in snke venoms. A. Rev. Phrmc., 12, 265-286. OSMAN, O.H., ISMAIL, M. & EL-AsMAR, M.F. (1973). Phrmcologicl studies of snke (Dendrospis ngusticeps) venom. Toxicon, 11, 185-192. STRYDOM, D.J. (1972). Snke venom toxins: the mino cid sequences of two toxins from Dendrospis polylepis polylepis (blck mmb) venom. J. biol. Chem., 247, 429-442. TAMIYA, N. & ARAI, H. (1966). Studies on se snke venoms: crystlliztion of erbutoxins nd b from Lticud semifscit venom, Biochem. J., 99, 624-63. Tu, A.T. (1977). Venoms: Chemistry nd Moleculr Biology. New York: John Wiley. VILJOEN, C.C. & BoTEs, D.P. (1973). Snke venom toxins. The purifiction nd mino cid sequence of toxin Fvll from Dendrospis ngusticeps venom. J. biol. Chem., 248, 4915-4919. VILJOEN, C.C. & BoTEs, D.P. (1974). Snke venom toxins. The purifiction nd mino cid sequence of toxin TA2 from Dendrospis ngusticeps venom. J. biol. Chem., 249, 366-372. (Received November 3, 1978. Revised Februry 19, 1979.)