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Postsynaptic blockade of neuromuscular transmission by toxin II from the venom of the South African ringhals cobra (Hemachatus haemachatus)
Toxicon. 1976, Vol. 14, DP. 167-173 . >
n Pree. Printed 1n Great Britain.
POSTSYNAPTIC BLOCKADE OF NEUROMUSCULAR TRANSMISSION BY TOXIN II FROM THE VENOM OF THE SOUTH AFRICAN RINGHALS COBRA (HEMACHATUS HAEMACHATUS) RoY G. BENGIS and Dnvm F. Nosl.l: Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi 39216, U.S.A. (.lccepted for publication 30 October 1975)
R. G. Bexols and D. F. Nosl.s . Postsynapti c blockade of neuromuscular transmission by toxin II from the venom of the South African ringhals cobra (Hemachatus hae»raclratus) . Toxicon 14, 167-173, 1976 .-Purified toxin II (2"5-25 lig per ml) from the venom of the South African ringhals cobra (Hemachatus haemachatus) produces a slow decline in twitch tension of toad sciatic nerve-gastrocnemius preparations . These studies established that toxin II is a neurotoxin that eliminates the sensitivity of the postyunctional membrane to acetylcholine, indicating a primary postsynaptic site of action . Pretreatment with gallamine or curare aff orded no protection against this toxin. Any effect toxin II may have on acetylcholinesterase is probably minor, sincethe effects of toxinII are not enhanced or reversed by acetylcholine, nor does acholinesterase inhibitor reverse theeffects of the toxin.The postsynaptic blockade is nondepolarizing and is irreversible by conventional methods. INTRODUCTION
Toxnv under study is one of the six fractions isolated from South African ringhals (STRxDOM and Btu, 1971). When injected into mice it produces symptoms suggestive of neurotoxic poisoning. Its molecular weight of 6838 suggests, according to L13B'S (1971) classification of neurotoxins, that it may belong to the class of neurotoxins which block transmission by acting at postsynaptic receptor sites. This study, using the isolated sciatic nerve-gastrocnemius muscle of the American toad, investigates whether the toxin is in fact a neurotoxin, which synaptic component it affects most, whether it depolarizes the muscle membrane, and whether its effects can readily be reversed. Neurotoxins have become useful tools in the study of biological processes, and it is important to study the biological properties of new pure toxins as they become available. T11H
(Hemachatus haemachatus) venom
MATERIALS AND METHODS On hundred mg of purified ringhals toxin II was obtained from the National Chemical Research Laboratory, Council for Scientific and Industral Research, Pretoria, Republic of South Africa (compliments of Drs. F. J. Joubert, A. J. C. Strydom, and D. P. Botes). The effects of the toxin were investigated using the isolated gastrocnemius muscle-sciatic nerve preparation from the American toad (Br~jo americonus) . The muscle and nerve were bathed at room temperature (20-22 °C) in fresh frog Ringer's solution of the following composition (mM) : NaCI 114" 65 ; KCl 2"5; CaCI, dihyd. 1"8 ; MgC1,tíH,O 1 "0; NaH,PO,H,O 0"85 ; Na,HPO, 2"15 ; and glucose 2"2. The final pH of the solution ranged from 7"25 to 7"35 . For most of the experiments, the nerve-muscle preparation was placed in an organ bath with a capacity of 30 ml. A partition placed transversely within this bath had a slit for lxssage of the sciatic nerve. Packing 167 TOálCON 1976 Nat. !t
168
ROY G. BENGLS and DAVID F. NOBLE
vaseline about the slit effectively isolated the solutions in the two compartments of the organ bath. 95 ~ Op per 5 % COs was bubbled through the Ringer's solution in an upper reservoir, and the pH during an experiment rarely dropped below 7~0. The oxygenated solution dripped continually from the upper reservoir into the organ bath containing the muscle, and the resulting overflow was recycled to the upper reservoir at a rate of 2 ml per min using a Beckman solution metering pump . The total capacity of the entire system was 200 ml. Square wave stimulation pulses with an amplitude of 4 V and duration of 0~2 cosec were delivered to the sciatic nerve or muscle membrane using a Grass model S.D .S physiological stimulator. The Achilles tendon was attached to a Grass Model F.T. lOC strain gauge tension transducer with 4/0 silk and tension was recorded on a Grass polygraph. Micrcelectrodes for intracellular recording contained 3M KCl in combination with an agar bridge to minimize any functional potential. These micrcelectrodes were selected with resistances ranging from 15 to 40 Mfï and with tip potentials less than 10 mV. The intracellular potentials were amplified with a Transidyne General type MTA-6 differential input d.c . preamplifier and photographed from a Tektronix 5103N storage oscilloscope with a Polaroid camera. As detailed in the Results section, various drugs were added to the organ bath at various times for comparative and reversal studies, and for the attempted acetylcholine bioassay . The following are the usual drug doses used with the concentration referring to the salt of the drug : Ballamine triethiodide, 100 ug per ml ; tubceurarine chloride, 15 kg per ml ; succinylcholine chloride, 100 ug ; hyaluronidase, 0075 units per ml ; physostigmine salicylate, 2 ug per ml ; neostigmine, 1'66 pg per ml . A few procedures required different doses. Any variations are mentioned in the more detailed discussion of the Results section. The attempted acetylcholine assay experiments followed the procedure of McIrrrosrt and PeRísv (1950) using isolated bullfrog (Rmra catesbiana) rectos abdominus muscles. The muscle strip is placed for 30 min in 5 ml of frog Ringer's solution, and for another 30 min in the same solution containing physostigmine salicylate . The response to standard concentrations of acetylcholine were then tested . Gastrocnemius muscles from the same toad stood for 30 min in frog Ringer's solution also containing physostigmine salicylate . Toxin II was added to the solution containing one of these test muscles and 30 min later, both muscles were stimulated tetanically for 20 min with 20 imp. per sec stimuli. The bathing solution was then poured into a 5 ml bath containing the frog rectos muscle . The tension, when compared with the tension produced by the standard acetylcholine solutions, provided a rough measure of the acetylcholine released from the poisoned and control toad gastrocnemius muscle. RFSULTS
Neuromuscular blockage by toxin II Submerging the muscle in a bath containing 5 ßíg toxin II per ml produces progressive neuromuscular blockade. If the muscle is indirectly stimulated continuously once per sec, twitch tension falls to 50~ of the control level in an average of 7 min (12 preparations) after introducing toxin II (Fig. la) whereas in control muscle, 7 min of such stimulation reduces twitch tension only l 0~ (Fig. 1 b). The contribution of twitch tension reduction made by muscle fatigue is entirely eliminated if the muscle is stimulated only once every 5 min with a 15 sec train of 1 per sec stimulation . With such a stimulation schedule there is no twitch tension reduction in the control muscles, not even after 3 hr of stimulation . Muscles bathed in toxin II showed a 50~ reduction after 45 min when stimulated in this manner. The slow rate at which tension declines suggests that the toxin concentration may be too low or that the diffusion rate of the polypeptide toxin to its site of action is the limiting factor. Changing the toxin concentration to various doses between 2~5 and 25 1íg per ml did not affect the rate of tension decline. Further, curare (1 S Fíg per ml) or gallamine (1001íg per ml) blocked tension development at a rate very similar to that of toxin II (Fig. 2), indicating that it is probably a diffusion factor . Site of action Two simple procedures demonstrated that toxin II affects neither nerve action potential conduction nor muscle excitation--contraction coupling . The solution bathing the muscle may be isolated from that bathing its efferent nerve by sealing the connection slit between TOXICON 1976 Vol. 14
Toxin II of Ringhals Cobra la)
169
Toxin added
Time,
min
FIG. 1 . EFFECT OF TOXIN II ON TOAD GASTROCNEMIUS TENSION PRODUCED BY SCIATIC NERVE STIMULATION .
Continual stimulation with square wave pulses of 4 V amplitude, 0~2 wsec duration at 1 per sec. Muscles in Ringer's solution . A. Toxin II added at arrow. Concentration of toxin is 2" 5 Rg per ml. B. Contralateral muscle used as control. No toxin added.
Time,
FIG.
i.
min
COMPARLSON OF THE EFFECTS OF CURARE ( ~), GAt?~MiNF TENSION DEVELOPMENT.
(Q)
AND TOXIN II (~
On
Note similar time course for curare, gallawine and toxin induced paralysis during the first 15 min. A control preparation (") with no drugs or toxin added shows a 70 ~ decrease in tension development in 1 hr, due to the fatigue factor . However, this preparation never totally lost its responsiveness even after 4 hr of continual stimulation .
the two compartments in the organ bath with vaseline. Since adding the toxin only to the nerve compartment had no effect on tension development, the toxin could not be blocking action potential conduction in the nerve. After toxin II was added to the muscle compartment, tension development to nerve stimulation decreased in the usual manner . However, since the muscle still contracted vigorously when stimulated directly, its membrane and contractile proteins must have retained their integrity. Since toxin II is a neurotoxin which affects neither action potential conduction in the nerve nor muscle excitation-contraction TOXICON 1976 Vol. If
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ROY G. BENGIS and DAVID F. NOBLE
coupling, it must act at the synapse. In general, a neurotoxin may block synaptic transmission by interfering with transmitter synthesis, storage or release, transmitter reception, or acetylcholinesterase, and it may have multiple effects. The additional procedures to be described attempted to determine which of these actions the toxin actually has. If a neuromuscular blocker prevents acetylcholine, added directly to the organ bath, from eliciting a muscle contraction, it must have postsynaptic action . Toxin II interferes with postsynaptic reception, because doses of acetylcholine as high as 25 4rg per ml failed to reverse the induced paralysis . One-hundredth of this dose produces contraction in an unpoisoned muscle. At the synapse there are two possible reasons why acetylcholine added directly to the bathing solution may fail to cause muscle contraction. One possibility is that the postsynaptic receptors are blocked or occupied (with or without depolarization of the muscle membranes) . The second possibility is that cholinesterase activity is enhanced, thus reducing the efficacy of exogenous or endogenous acetylcholine. This second possibility can be eliminated in the case of toxin II induced paralysis because neither cholinesterase inhibitors (0166 Fig neostigmine per ml nor 25 !Ag acetylcholine per ml) or even a combination of the two caused any reversal of paralysis. Conversely, if toxin II had a cholinesterase inhibiting effect, then the nerve-muscle preparation would become sensitized to the effects of exogenous acetylcholine and just the opposite was noted . Hence modifications of cholinesterase activity is an unlikely or possibly minor effect of toxin II which, if present, is masked by the overwhelming receptor blockade effect, already shown by these data to be a major mechanism of action of this toxin. It is unknown whether toxin II acts at the same postsynaptic site as curare or gallamine. These three drugs must block the same receptors if gallamine or curare can protect the receptors from toxin II. LeE (1971) reported that pretreatment with curare protects against some cobra toxins, and SxYnnt et al. (1973) obtained partial protection with curare against a neurotoxin fraction of coral snake venom. The procedure to determine whether curare or gallamine can protect the muscle from toxin II began by completely blocking neuromuscular transmission with gallamine (100 pg per ml) or curare (IS pg per ml), respectively . Then the muscle was bathed for an hour in a new solution containing toxin II (5 Ng per ml) in addition to the gallamine or curare . Next it was washed with a third solution containing curare or gallamine but no toxin. Following this lengthy preparation, we attempted to reverse paralysis with acetylcholine or neostigmine. In these experiments pretreatment with gallamine or curare failed to alter the irreversibility of paralysis caused by toxin II. This inability to protect indicates either that toxin II acts at a somewhat different site from curare and gallamine, or that it displaces these other neuromuscular blockers from the receptors. The demonstrated postsynaptic effect of toxin II cannot a priori preclude a dual presynaptic effect. Such a dual action is not expected because in all literature surveyed, toxins with molecular weights in the same range as toxin II acted solely on the postjunctional membrane, and because the authors are unaware of any literature report of a toxin with a dual mechanism. Nevertheless, experiments to measure any pocsynaptic effect by toxin II were conducted. The nerves to a poisoned muscle and its unpoisoned contralateral muscle were stimulated identically for 20 min at 20 imp. per sec, and the quantity of acetylcholine released by the two preparations were compared. These measurements were inconclusive because of variable bioassay sensitivity. Acetylcholine diffusion out of the muscle was facilitated by stripping the muscle membrane and adding 15 units of hyaluronidase to the collecting bath. Only two experiments out of twelve yielded usable data . In both of these experiments, more acetylcholine was collected from the poisoned muscle than from the TOXICON 1976 Vol. 1~
Toxin II of Ringhals Cobra
17 1
unpoisoned contralateral control muscles. These results, though inconclusive, do suggest toxin II lacks a presynaptic effect . Pharmacological studies
These studies determined that toxin II blocks postsynaptic receptors without depolarizing the muscle, and that toxin II induced paralysis is irreversible by such common procedures as adding acetylcholine and neostigmine, or by prolonged washout of the preparation. Membrane potential was recorded continuously for 45 min after adding toxin II, and only slight gradual fluctuations were observed as follows. Initially the cell hyperpolarized somewhat, possibly because toxin II blocks the steady depolarization maintained by back ground spontaneous transmitter release. A later slight membrane depolarization occurred which may be attributed to a sodium influx around the microelectrode puncture. Eight different preparations were used and the resting membrane potential ranged between -60 and -80 mV, with a mean of -72 mV. The slight fluctuations mentioned during the 45 min monitoring did not exceed 8 mV in either direction. Intracellular recordings obtained after the addition of either curare or toxin II were identical . However, the addition of a known depolarizer, succinylcholine, produced immediate but transient depolarization . After recording from each cell, a second cell in the nearby vicinity was punctured, and in all cases its resting membrane potential was within 5 mV of the original cell's resting membrane potential. Muscle paralysis by toxin II was irreversible by the usual procedures . In particular, it was found that: (a) Doses of acetylcholine as high as 25 pg per ml had no effect. Such a dose is a hundred times as great as the concentration which reverses neuromuscular block by gallamine or curare . (b) Doses of neostigmine as high as 2"5 pg per ml failed to reverse toxin II induced paraylsis. This dose is ten times the dose which can reverse paralysis by curare or gallamine. (c) Washing the preparation for 7 hr at a rate of 5 ml per min did not even partially reverse paralysis by toxin II. Apparently the affinity between toxin II and its postsynaptic binding site is much greater than the affinity of curare or gallamine for their binding sites. If the binding sites for these various compounds are the same, such relatively permanent attachment of toxin II may explain why gallamine or curare fail to protect the muscle from paralysis by toxin II . DISCUSSION
The toxin investigated in this study is a fraction of the venom of the ringhals (Hemachatus haemachatus) . The biochemistry of ringhals venom has been studied by Ci-ntlsrErrsEx (195, $TRYDOM and BOTES (1971), MsBS (1969), KuhInR et al. (1973), and others . Christensen reported that the venom contains a hemolysin, a proteolysin, a coagulin, a phosphodiesterase, phosphomonoesterase, ophio-z-amino oxidase, and a polypeptide neurotoxin . $TRYDOM and BOTES (1971) isolated six toxins from ringhals venom using gradient chromatography on Amberlite CG-50. Toxins II and IV were further purified by gel filtration on 5ephadex G-50 . Both were found, by virtue of the symptoms produced, to be neurotoxins with median lethal dose values in mice of 0" 11 pg per g and 0"09 pg per g for toxins II and IV, respectively. They found these two toxins to contain 61 amino acid residues and to have molecular weights of 6838 and 6831, respectively . Both toxins fall into Lee's group i (LEg, 1971), comprising neurotoxins with molecular weights between 710XICON 7976 YoJ. 14
172
ROY G. BENGIS and DAV1I) F. NOBLE
6000 and 8000, and accordingly would be expected to have a postsynaptic action . This study confirms STRYDOM and BOTE'S (1971) conclusion that toxin II is a neurotoxin, and because it shows that the toxin acts postsynaptically, suggests the general validity of Lee's classification. Like other toxins studied, it appears to have only a single physiologically significant action (e.g. LES'rER, 1973) . CHRISTENSEN (195 observed that 100 N~g of whole ringhals venom given intravenously to mice caused death in 133-950 min, and higher doses were fatal in less than 1 min . The question arises as to why purified toxin II takes so long (average 45 min) to produce total paralysis in the preparations used. There are several possible reasons . (a) Because the venom was introduced directly into the blood stream in Christensen's work, it was conveyed directly to its site of action . In these in vitro isolated preparations, the toxin had to diffuse from the bathing medium to its site of action . (b) Christensen used whole venom, with all its constituent enzymes for facilitating absorption, dispersal, and diffusion of the toxin components . The in vitro studies used purified toxin II. (c) Christensen used mammals (mice) whereas these studies used toads. It was reasoned that since toads form a major part of the ringhals' diet, they should be highly sensitive to toxin II. The effect of toxin II appears to be irreversible by conventional methods such as adding exogenous acetylcholine and cholinesterase inhibitors, or by washout. Such irreversibility has been observed in other toxins extracted from venoms . For example, the toxin fractions of many elapid snake venoms block the acetylcholine receptors of the postjunctional membrane relatively irreversibly (CHRISTENSEN, 1955 ; CHAPMAN, 1968 ; LEE, 1971). However, the effects of elapid snake venoms are not irreversible over extended periods oftime. There are many reports in the literature of clinical cases of neurotoxin envenomation that have recovered totally after several days of intensive care. PEARN (1971) described a case of a 28-yr-old man who recovered totally from the bite of a kraft after the administration of massive doses of antiserum, followed by 8 days of positive pressure ventilation and 15 additional days of intensive medical and physical therapy . HuxwiTZ and Htri,h (1971) reported a case of Berg-adder bite in a teenage boy. The overt symptoms of total opthalmoplegia, anosmia, loss oftaste, ptosis, and loss of reflexes regressed after 6 days of therapy. Thus over long periods of time the effects of neurotoxic snake venoms are reversible. The antiserum combining with the toxin antigen may somehow reduce affinity between receptor and toxin and effectively `pry' it loose . Alternate explanations for this long-term reversiblity are that tissue peptidases hydrolize the toxin in situ or that the end plate forms new acetylcholine receptors . AcknowJer~ements-The authors thank Dr. HucH L. KEecnx for his help and interest. The authors also thank Dr . F. J. JOUBERT, A. J. C. STxYUOM, and D. P. Booms of the National Chemical Research Laboratory, Council for Scientific and Industrial Research, Pretoria, Republic of South Africa, for supplying 100 mg of purified ringhals toxin II . REFERENCES CxnrFtax, D. S. (1968) The symptomatology, pathology and treatment of the bites of venomous snakes of Central and South Africa . In : Venomous Animals and Their Venoms, Vol. I, pp. 463-527, (HUCHERL, W., Buc~a.ev, E. E. and DEUIAFEY, V., Eds.) . New York : Academic Press. C~rst~vsex, P. A. (1955) South African Snake Venoms and Anttvenoms . Publ. S. Afr. Inst . med. Res., p. 130. Huxwrrz, B. J. and Hui.t., P. R. (1971) Berg-adder bite . S. Afr. med. J. 42, 969. Kurux, V., RacErrr, T. A. and Eta.toT-r, W. B. (1973) Anticholinesterase activity of elapid venoms . Toricon 11, 131 . LEE, C. Y. (1971) Elapid neurotoxine and their mode of action . In : Snake Yenoms and Envenomation, pp . 111-126, (Mnv~rox, S. A., Ed .) . New York : Marcel Dekker. TOXICON 1976 Yot. l~
Toxin II of Ringhals Cobra
17 3
LesrcR, H. A. (1970) Postsynaptic action of cobra toxin at the myoneural junction . Nature, Lond. 227, 727. M~cIrrrosx, F. C. and PeRxY, W. L. M. (1950) Biological estimation of acetylcholine. In : Methods in Medical Research, Vol. 3, pp . 78-88. London : Bailliere. Mess, D. (1969) Comparison of biochemical properties of elapid snake venoms. Toxicon 6, 267. Penxrr, J. H. (1971) Survival after snake-bite with prolonged neurotoxic envenomation . Med. J. Aust . 2, 259. SNYDER, G. K., R .~,tKSEV, H. W., TAYLOR, W. J. and CHiou, C. Y. (1973) Newomuscular blockade of chick biventer cervicis nerve-muscle preparations by a fraction from coral snake venom. Toxicon 11, 505. SrttYnoM, A. J. C. and Bores, D. P. (1971) Purification, properties, and complete amino acid sequence of two toxins from ringhals (Hemachatus haemachatus) venom. J. biol. Chem . 246, 1131 .
TOXICON 1976 Yol. 1 ~
n Pree. Printed 1n Great Britain.
POSTSYNAPTIC BLOCKADE OF NEUROMUSCULAR TRANSMISSION BY TOXIN II FROM THE VENOM OF THE SOUTH AFRICAN RINGHALS COBRA (HEMACHATUS HAEMACHATUS) RoY G. BENGIS and Dnvm F. Nosl.l: Department of Physiology and Biophysics, University of Mississippi Medical Center, Jackson, Mississippi 39216, U.S.A. (.lccepted for publication 30 October 1975)
R. G. Bexols and D. F. Nosl.s . Postsynapti c blockade of neuromuscular transmission by toxin II from the venom of the South African ringhals cobra (Hemachatus hae»raclratus) . Toxicon 14, 167-173, 1976 .-Purified toxin II (2"5-25 lig per ml) from the venom of the South African ringhals cobra (Hemachatus haemachatus) produces a slow decline in twitch tension of toad sciatic nerve-gastrocnemius preparations . These studies established that toxin II is a neurotoxin that eliminates the sensitivity of the postyunctional membrane to acetylcholine, indicating a primary postsynaptic site of action . Pretreatment with gallamine or curare aff orded no protection against this toxin. Any effect toxin II may have on acetylcholinesterase is probably minor, sincethe effects of toxinII are not enhanced or reversed by acetylcholine, nor does acholinesterase inhibitor reverse theeffects of the toxin.The postsynaptic blockade is nondepolarizing and is irreversible by conventional methods. INTRODUCTION
Toxnv under study is one of the six fractions isolated from South African ringhals (STRxDOM and Btu, 1971). When injected into mice it produces symptoms suggestive of neurotoxic poisoning. Its molecular weight of 6838 suggests, according to L13B'S (1971) classification of neurotoxins, that it may belong to the class of neurotoxins which block transmission by acting at postsynaptic receptor sites. This study, using the isolated sciatic nerve-gastrocnemius muscle of the American toad, investigates whether the toxin is in fact a neurotoxin, which synaptic component it affects most, whether it depolarizes the muscle membrane, and whether its effects can readily be reversed. Neurotoxins have become useful tools in the study of biological processes, and it is important to study the biological properties of new pure toxins as they become available. T11H
(Hemachatus haemachatus) venom
MATERIALS AND METHODS On hundred mg of purified ringhals toxin II was obtained from the National Chemical Research Laboratory, Council for Scientific and Industral Research, Pretoria, Republic of South Africa (compliments of Drs. F. J. Joubert, A. J. C. Strydom, and D. P. Botes). The effects of the toxin were investigated using the isolated gastrocnemius muscle-sciatic nerve preparation from the American toad (Br~jo americonus) . The muscle and nerve were bathed at room temperature (20-22 °C) in fresh frog Ringer's solution of the following composition (mM) : NaCI 114" 65 ; KCl 2"5; CaCI, dihyd. 1"8 ; MgC1,tíH,O 1 "0; NaH,PO,H,O 0"85 ; Na,HPO, 2"15 ; and glucose 2"2. The final pH of the solution ranged from 7"25 to 7"35 . For most of the experiments, the nerve-muscle preparation was placed in an organ bath with a capacity of 30 ml. A partition placed transversely within this bath had a slit for lxssage of the sciatic nerve. Packing 167 TOálCON 1976 Nat. !t
168
ROY G. BENGLS and DAVID F. NOBLE
vaseline about the slit effectively isolated the solutions in the two compartments of the organ bath. 95 ~ Op per 5 % COs was bubbled through the Ringer's solution in an upper reservoir, and the pH during an experiment rarely dropped below 7~0. The oxygenated solution dripped continually from the upper reservoir into the organ bath containing the muscle, and the resulting overflow was recycled to the upper reservoir at a rate of 2 ml per min using a Beckman solution metering pump . The total capacity of the entire system was 200 ml. Square wave stimulation pulses with an amplitude of 4 V and duration of 0~2 cosec were delivered to the sciatic nerve or muscle membrane using a Grass model S.D .S physiological stimulator. The Achilles tendon was attached to a Grass Model F.T. lOC strain gauge tension transducer with 4/0 silk and tension was recorded on a Grass polygraph. Micrcelectrodes for intracellular recording contained 3M KCl in combination with an agar bridge to minimize any functional potential. These micrcelectrodes were selected with resistances ranging from 15 to 40 Mfï and with tip potentials less than 10 mV. The intracellular potentials were amplified with a Transidyne General type MTA-6 differential input d.c . preamplifier and photographed from a Tektronix 5103N storage oscilloscope with a Polaroid camera. As detailed in the Results section, various drugs were added to the organ bath at various times for comparative and reversal studies, and for the attempted acetylcholine bioassay . The following are the usual drug doses used with the concentration referring to the salt of the drug : Ballamine triethiodide, 100 ug per ml ; tubceurarine chloride, 15 kg per ml ; succinylcholine chloride, 100 ug ; hyaluronidase, 0075 units per ml ; physostigmine salicylate, 2 ug per ml ; neostigmine, 1'66 pg per ml . A few procedures required different doses. Any variations are mentioned in the more detailed discussion of the Results section. The attempted acetylcholine assay experiments followed the procedure of McIrrrosrt and PeRísv (1950) using isolated bullfrog (Rmra catesbiana) rectos abdominus muscles. The muscle strip is placed for 30 min in 5 ml of frog Ringer's solution, and for another 30 min in the same solution containing physostigmine salicylate . The response to standard concentrations of acetylcholine were then tested . Gastrocnemius muscles from the same toad stood for 30 min in frog Ringer's solution also containing physostigmine salicylate . Toxin II was added to the solution containing one of these test muscles and 30 min later, both muscles were stimulated tetanically for 20 min with 20 imp. per sec stimuli. The bathing solution was then poured into a 5 ml bath containing the frog rectos muscle . The tension, when compared with the tension produced by the standard acetylcholine solutions, provided a rough measure of the acetylcholine released from the poisoned and control toad gastrocnemius muscle. RFSULTS
Neuromuscular blockage by toxin II Submerging the muscle in a bath containing 5 ßíg toxin II per ml produces progressive neuromuscular blockade. If the muscle is indirectly stimulated continuously once per sec, twitch tension falls to 50~ of the control level in an average of 7 min (12 preparations) after introducing toxin II (Fig. la) whereas in control muscle, 7 min of such stimulation reduces twitch tension only l 0~ (Fig. 1 b). The contribution of twitch tension reduction made by muscle fatigue is entirely eliminated if the muscle is stimulated only once every 5 min with a 15 sec train of 1 per sec stimulation . With such a stimulation schedule there is no twitch tension reduction in the control muscles, not even after 3 hr of stimulation . Muscles bathed in toxin II showed a 50~ reduction after 45 min when stimulated in this manner. The slow rate at which tension declines suggests that the toxin concentration may be too low or that the diffusion rate of the polypeptide toxin to its site of action is the limiting factor. Changing the toxin concentration to various doses between 2~5 and 25 1íg per ml did not affect the rate of tension decline. Further, curare (1 S Fíg per ml) or gallamine (1001íg per ml) blocked tension development at a rate very similar to that of toxin II (Fig. 2), indicating that it is probably a diffusion factor . Site of action Two simple procedures demonstrated that toxin II affects neither nerve action potential conduction nor muscle excitation--contraction coupling . The solution bathing the muscle may be isolated from that bathing its efferent nerve by sealing the connection slit between TOXICON 1976 Vol. 14
Toxin II of Ringhals Cobra la)
169
Toxin added
Time,
min
FIG. 1 . EFFECT OF TOXIN II ON TOAD GASTROCNEMIUS TENSION PRODUCED BY SCIATIC NERVE STIMULATION .
Continual stimulation with square wave pulses of 4 V amplitude, 0~2 wsec duration at 1 per sec. Muscles in Ringer's solution . A. Toxin II added at arrow. Concentration of toxin is 2" 5 Rg per ml. B. Contralateral muscle used as control. No toxin added.
Time,
FIG.
i.
min
COMPARLSON OF THE EFFECTS OF CURARE ( ~), GAt?~MiNF TENSION DEVELOPMENT.
(Q)
AND TOXIN II (~
On
Note similar time course for curare, gallawine and toxin induced paralysis during the first 15 min. A control preparation (") with no drugs or toxin added shows a 70 ~ decrease in tension development in 1 hr, due to the fatigue factor . However, this preparation never totally lost its responsiveness even after 4 hr of continual stimulation .
the two compartments in the organ bath with vaseline. Since adding the toxin only to the nerve compartment had no effect on tension development, the toxin could not be blocking action potential conduction in the nerve. After toxin II was added to the muscle compartment, tension development to nerve stimulation decreased in the usual manner . However, since the muscle still contracted vigorously when stimulated directly, its membrane and contractile proteins must have retained their integrity. Since toxin II is a neurotoxin which affects neither action potential conduction in the nerve nor muscle excitation-contraction TOXICON 1976 Vol. If
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ROY G. BENGIS and DAVID F. NOBLE
coupling, it must act at the synapse. In general, a neurotoxin may block synaptic transmission by interfering with transmitter synthesis, storage or release, transmitter reception, or acetylcholinesterase, and it may have multiple effects. The additional procedures to be described attempted to determine which of these actions the toxin actually has. If a neuromuscular blocker prevents acetylcholine, added directly to the organ bath, from eliciting a muscle contraction, it must have postsynaptic action . Toxin II interferes with postsynaptic reception, because doses of acetylcholine as high as 25 4rg per ml failed to reverse the induced paralysis . One-hundredth of this dose produces contraction in an unpoisoned muscle. At the synapse there are two possible reasons why acetylcholine added directly to the bathing solution may fail to cause muscle contraction. One possibility is that the postsynaptic receptors are blocked or occupied (with or without depolarization of the muscle membranes) . The second possibility is that cholinesterase activity is enhanced, thus reducing the efficacy of exogenous or endogenous acetylcholine. This second possibility can be eliminated in the case of toxin II induced paralysis because neither cholinesterase inhibitors (0166 Fig neostigmine per ml nor 25 !Ag acetylcholine per ml) or even a combination of the two caused any reversal of paralysis. Conversely, if toxin II had a cholinesterase inhibiting effect, then the nerve-muscle preparation would become sensitized to the effects of exogenous acetylcholine and just the opposite was noted . Hence modifications of cholinesterase activity is an unlikely or possibly minor effect of toxin II which, if present, is masked by the overwhelming receptor blockade effect, already shown by these data to be a major mechanism of action of this toxin. It is unknown whether toxin II acts at the same postsynaptic site as curare or gallamine. These three drugs must block the same receptors if gallamine or curare can protect the receptors from toxin II. LeE (1971) reported that pretreatment with curare protects against some cobra toxins, and SxYnnt et al. (1973) obtained partial protection with curare against a neurotoxin fraction of coral snake venom. The procedure to determine whether curare or gallamine can protect the muscle from toxin II began by completely blocking neuromuscular transmission with gallamine (100 pg per ml) or curare (IS pg per ml), respectively . Then the muscle was bathed for an hour in a new solution containing toxin II (5 Ng per ml) in addition to the gallamine or curare . Next it was washed with a third solution containing curare or gallamine but no toxin. Following this lengthy preparation, we attempted to reverse paralysis with acetylcholine or neostigmine. In these experiments pretreatment with gallamine or curare failed to alter the irreversibility of paralysis caused by toxin II. This inability to protect indicates either that toxin II acts at a somewhat different site from curare and gallamine, or that it displaces these other neuromuscular blockers from the receptors. The demonstrated postsynaptic effect of toxin II cannot a priori preclude a dual presynaptic effect. Such a dual action is not expected because in all literature surveyed, toxins with molecular weights in the same range as toxin II acted solely on the postjunctional membrane, and because the authors are unaware of any literature report of a toxin with a dual mechanism. Nevertheless, experiments to measure any pocsynaptic effect by toxin II were conducted. The nerves to a poisoned muscle and its unpoisoned contralateral muscle were stimulated identically for 20 min at 20 imp. per sec, and the quantity of acetylcholine released by the two preparations were compared. These measurements were inconclusive because of variable bioassay sensitivity. Acetylcholine diffusion out of the muscle was facilitated by stripping the muscle membrane and adding 15 units of hyaluronidase to the collecting bath. Only two experiments out of twelve yielded usable data . In both of these experiments, more acetylcholine was collected from the poisoned muscle than from the TOXICON 1976 Vol. 1~
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unpoisoned contralateral control muscles. These results, though inconclusive, do suggest toxin II lacks a presynaptic effect . Pharmacological studies
These studies determined that toxin II blocks postsynaptic receptors without depolarizing the muscle, and that toxin II induced paralysis is irreversible by such common procedures as adding acetylcholine and neostigmine, or by prolonged washout of the preparation. Membrane potential was recorded continuously for 45 min after adding toxin II, and only slight gradual fluctuations were observed as follows. Initially the cell hyperpolarized somewhat, possibly because toxin II blocks the steady depolarization maintained by back ground spontaneous transmitter release. A later slight membrane depolarization occurred which may be attributed to a sodium influx around the microelectrode puncture. Eight different preparations were used and the resting membrane potential ranged between -60 and -80 mV, with a mean of -72 mV. The slight fluctuations mentioned during the 45 min monitoring did not exceed 8 mV in either direction. Intracellular recordings obtained after the addition of either curare or toxin II were identical . However, the addition of a known depolarizer, succinylcholine, produced immediate but transient depolarization . After recording from each cell, a second cell in the nearby vicinity was punctured, and in all cases its resting membrane potential was within 5 mV of the original cell's resting membrane potential. Muscle paralysis by toxin II was irreversible by the usual procedures . In particular, it was found that: (a) Doses of acetylcholine as high as 25 pg per ml had no effect. Such a dose is a hundred times as great as the concentration which reverses neuromuscular block by gallamine or curare . (b) Doses of neostigmine as high as 2"5 pg per ml failed to reverse toxin II induced paraylsis. This dose is ten times the dose which can reverse paralysis by curare or gallamine. (c) Washing the preparation for 7 hr at a rate of 5 ml per min did not even partially reverse paralysis by toxin II. Apparently the affinity between toxin II and its postsynaptic binding site is much greater than the affinity of curare or gallamine for their binding sites. If the binding sites for these various compounds are the same, such relatively permanent attachment of toxin II may explain why gallamine or curare fail to protect the muscle from paralysis by toxin II . DISCUSSION
The toxin investigated in this study is a fraction of the venom of the ringhals (Hemachatus haemachatus) . The biochemistry of ringhals venom has been studied by Ci-ntlsrErrsEx (195, $TRYDOM and BOTES (1971), MsBS (1969), KuhInR et al. (1973), and others . Christensen reported that the venom contains a hemolysin, a proteolysin, a coagulin, a phosphodiesterase, phosphomonoesterase, ophio-z-amino oxidase, and a polypeptide neurotoxin . $TRYDOM and BOTES (1971) isolated six toxins from ringhals venom using gradient chromatography on Amberlite CG-50. Toxins II and IV were further purified by gel filtration on 5ephadex G-50 . Both were found, by virtue of the symptoms produced, to be neurotoxins with median lethal dose values in mice of 0" 11 pg per g and 0"09 pg per g for toxins II and IV, respectively. They found these two toxins to contain 61 amino acid residues and to have molecular weights of 6838 and 6831, respectively . Both toxins fall into Lee's group i (LEg, 1971), comprising neurotoxins with molecular weights between 710XICON 7976 YoJ. 14
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ROY G. BENGIS and DAV1I) F. NOBLE
6000 and 8000, and accordingly would be expected to have a postsynaptic action . This study confirms STRYDOM and BOTE'S (1971) conclusion that toxin II is a neurotoxin, and because it shows that the toxin acts postsynaptically, suggests the general validity of Lee's classification. Like other toxins studied, it appears to have only a single physiologically significant action (e.g. LES'rER, 1973) . CHRISTENSEN (195 observed that 100 N~g of whole ringhals venom given intravenously to mice caused death in 133-950 min, and higher doses were fatal in less than 1 min . The question arises as to why purified toxin II takes so long (average 45 min) to produce total paralysis in the preparations used. There are several possible reasons . (a) Because the venom was introduced directly into the blood stream in Christensen's work, it was conveyed directly to its site of action . In these in vitro isolated preparations, the toxin had to diffuse from the bathing medium to its site of action . (b) Christensen used whole venom, with all its constituent enzymes for facilitating absorption, dispersal, and diffusion of the toxin components . The in vitro studies used purified toxin II. (c) Christensen used mammals (mice) whereas these studies used toads. It was reasoned that since toads form a major part of the ringhals' diet, they should be highly sensitive to toxin II. The effect of toxin II appears to be irreversible by conventional methods such as adding exogenous acetylcholine and cholinesterase inhibitors, or by washout. Such irreversibility has been observed in other toxins extracted from venoms . For example, the toxin fractions of many elapid snake venoms block the acetylcholine receptors of the postjunctional membrane relatively irreversibly (CHRISTENSEN, 1955 ; CHAPMAN, 1968 ; LEE, 1971). However, the effects of elapid snake venoms are not irreversible over extended periods oftime. There are many reports in the literature of clinical cases of neurotoxin envenomation that have recovered totally after several days of intensive care. PEARN (1971) described a case of a 28-yr-old man who recovered totally from the bite of a kraft after the administration of massive doses of antiserum, followed by 8 days of positive pressure ventilation and 15 additional days of intensive medical and physical therapy . HuxwiTZ and Htri,h (1971) reported a case of Berg-adder bite in a teenage boy. The overt symptoms of total opthalmoplegia, anosmia, loss oftaste, ptosis, and loss of reflexes regressed after 6 days of therapy. Thus over long periods of time the effects of neurotoxic snake venoms are reversible. The antiserum combining with the toxin antigen may somehow reduce affinity between receptor and toxin and effectively `pry' it loose . Alternate explanations for this long-term reversiblity are that tissue peptidases hydrolize the toxin in situ or that the end plate forms new acetylcholine receptors . AcknowJer~ements-The authors thank Dr. HucH L. KEecnx for his help and interest. The authors also thank Dr . F. J. JOUBERT, A. J. C. STxYUOM, and D. P. Booms of the National Chemical Research Laboratory, Council for Scientific and Industrial Research, Pretoria, Republic of South Africa, for supplying 100 mg of purified ringhals toxin II . REFERENCES CxnrFtax, D. S. (1968) The symptomatology, pathology and treatment of the bites of venomous snakes of Central and South Africa . In : Venomous Animals and Their Venoms, Vol. I, pp. 463-527, (HUCHERL, W., Buc~a.ev, E. E. and DEUIAFEY, V., Eds.) . New York : Academic Press. C~rst~vsex, P. A. (1955) South African Snake Venoms and Anttvenoms . Publ. S. Afr. Inst . med. Res., p. 130. Huxwrrz, B. J. and Hui.t., P. R. (1971) Berg-adder bite . S. Afr. med. J. 42, 969. Kurux, V., RacErrr, T. A. and Eta.toT-r, W. B. (1973) Anticholinesterase activity of elapid venoms . Toricon 11, 131 . LEE, C. Y. (1971) Elapid neurotoxine and their mode of action . In : Snake Yenoms and Envenomation, pp . 111-126, (Mnv~rox, S. A., Ed .) . New York : Marcel Dekker. TOXICON 1976 Yot. l~
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LesrcR, H. A. (1970) Postsynaptic action of cobra toxin at the myoneural junction . Nature, Lond. 227, 727. M~cIrrrosx, F. C. and PeRxY, W. L. M. (1950) Biological estimation of acetylcholine. In : Methods in Medical Research, Vol. 3, pp . 78-88. London : Bailliere. Mess, D. (1969) Comparison of biochemical properties of elapid snake venoms. Toxicon 6, 267. Penxrr, J. H. (1971) Survival after snake-bite with prolonged neurotoxic envenomation . Med. J. Aust . 2, 259. SNYDER, G. K., R .~,tKSEV, H. W., TAYLOR, W. J. and CHiou, C. Y. (1973) Newomuscular blockade of chick biventer cervicis nerve-muscle preparations by a fraction from coral snake venom. Toxicon 11, 505. SrttYnoM, A. J. C. and Bores, D. P. (1971) Purification, properties, and complete amino acid sequence of two toxins from ringhals (Hemachatus haemachatus) venom. J. biol. Chem . 246, 1131 .
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