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Mode of neuromuscular blocking action of ceruleotoxin
21, No . 2, pp. 301 -307, 1983 . Printed in Grat Britain.
0041 -0101/83/020301 -07503.00/0 7 1983 Pergamon Press Ltd.
Taxiroh, Vol.
MODE OF NEUROMUSCULAR BLOCKING ACTION OF CERULEOTOXIN C. L. Ho' and C. Y . LEE~ " 'Institute of Biological Chemistry, Academia Sinica and 'Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (Amepted Jor publication 23 September 1982) C . L . Ho and C . Y . Lee . Mode of neuromuscular blocking action of ceruleotoxin . Toxicon 21, 301- 307, 1983 . - Cenileotoxin is an acidic toxin protein from a Bungarus venom which has recently been clarified as B. fauiatus venom . The toxin at a low concentration (10~g/ml) abolished the twitch response of the indirectly stimulated biventer cervicis muscle of the chick, without affecting the response to acetylcholine or carbamylcholine . At a higher concentration (10'`g/ml), the toxin, in addition to inhibition of the twitch response, caused contracture in the chick biventer cervicis muscle and reduced the response to acetylcholine or carbamylcholine in both the innervated chick cervical and denervated rat diaphragm muscles . Electrophysiological studies on the rat phrenic nerve-diaphragm preparation showed that ceruleotoxin at a low concentration caused an initial inhibition followed by recovery of the quantal content of endplate potentials and then a second phase of inhibition leading to complete failure. At a higher concentration, the toxin gradually reduced the resting membrane potential of the diaphragm muscle to a level lower than SO mV within 2 hr . In addition, enzyme assay showed that the toxin possessed phospholipase A activity comparable to that of other basic phospholipases A, of snake venom origin . It is concluded that ceruleotoxin is a phospholipase A, with presynaptic and myotropic actions on vertebrate nerve-muscle system similar to those of notexin from Notechir scutatus scutatus venom . INTRODUCTION CERULEOTOXIN (CeTX), an acidic toxin protein originally reported to be isolated from a batch of Bungarus caeruleus venom (BON and CHANOEUx, 1975, 1977a), was recently found to be actually a component of Bungarus fasciatus venom (BON and SALIOU, 1982) . This toxin blocks the depolarisation caused by carbamylcholine on the isolated electroplaque from Electrophorus electricus and the increase of "Na* and "K* efflux caused by cholinergic agonists on excitable receptor-rich microsacs prepared from Torpedo marmorata electric organ . However, at variance with typical postsynaptic snake toxins, this toxin does not interfere with the binding of ['H]acetylcholine or of a-['H]toxin from Naja nigricollis to the cholinergic receptor site . Based on these findings, BoN and CHANGEUX (19776) have proposed that CeTX is a new type of snake toxin which blocks selectively the electrogenic action of acetylcholine on the postsynaptic membrane without binding to the cholinergic receptor site and that it therefore might serve as a useful tool to characterise the cholinergic ionophore . On the other hand, they have also found that CeTX possesses phospholipase activity (BoN and CHANaEUx, 1977a) . Since several presynaptic neurotoxins from snake venoms possess phospholipase A, activity (EAICER, 1978; KARLSSON, 1979; LEE and Ho, 1980, 1982) and some of them also have " To whom correspondence should be addressed .
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C. L. HO AND C. Y. LEE
more or less postsynaptic and/or myotoxic action (HARRIS et al., 1975, 1980; LEE et al., 1976), it was deemed necessary to investigate the effect of CeTX on vertebrate skeletal muscle to clarify the mode of action of the toxin. A preliminary account of this work has already been published (LEE and Ho, 1978).
MATERIALS AND METHODS Acidic phospholipase A, from Ngja ngja atra and basic phospholipase A, from Ngja nigricollis venoms were purified by chromatography on CM-Sephadex C-23 columns and gel filtration on Sephadex G-S0, as described by Lo et al. (1972) and by Wu et al. (1977), respectively. ß-Bungarotoxin was isolated from the venom of Bungarus multicinctus according to the method of LeE et al. (1972) . Ceruleotoxin was a gift from Dr. C. Bon, Institut Pasteur, Paris. Notexin was a gift from Dr . E. Karlsson, Institute of Biochemistry, Uppsala. Acetylcholine chloride, carbachol (carbamylcholine) and phosphatidylcholine (dipalmitol) were purchased from Sigma Chemical Co ., U.S .A . All common chemicals used were of reagent grade. Biventer cervicis nerve-muscle preparation of the chick
The biventer cervicis muscle of Leghorn chicks (3-7 days old) was prepared according to the method of GINSBORG and WARRINER (1960) . The isolated preparation was suspended in 10 ml of Krebs solution (composition in mM: NaCI, 126; KCI, 5.0; CaCI,, 2.5 ; MgSO,, 1 .2; KH,PO,, 1.2; NaHCO,, 23 .8 ; dextrose, 11 .2) maintained at 3710 .3°C and oxygenated with a mixture of 95% O, and S% CO,. The preparation was stimulated indirectly with supramaximal square pulses of 0.3 msec duration at a frequency of 0.2 Hz . Isometric contraction was recorded with a Grass FT 03 force- displacement transducer attached to a Grass 7P polygraph. Acetylcholine and carbachol responses of the muscle were tested by adding each agent to the resting muscle and washing with fresh Krebs solution after the evoked contraction reached the maximum. Phrenic nerve-diaphragm preparation oJthe Long Evans rat
The phrenic nerve-diaphragm preparation of the rat was prepared aaording to the method Of BÜLHRING (1946) . Denervated preparations were prepared from rats in which one side of the phrenic nerves had been cut at the cervical level for at least 7 days . For recording muscle contractions, the preparation was suspended in an organ bath containing 20 ml of Tyrode solution (composition in mM : NaCI, 136; KCI, 2.8 ; CaCI,, 1 .8 ; MgCI,, 1.1 ; NaH,PO,, 0.33; NaHCO,, 11 .9 ; dextrose, 5 .6) maintained at 3710 .3°C and oxygenated with a mixture of 95% O, and S% CO,. Muscle contractions were elicited either by stimulation of the nerve with supramaximal square pulses of 0.05 msec duration, or by direct stimulation of the muscle with pulses of 1 msec duration at a frequency of 0.03 or 0.1 Hz, and recorded as described above for the chick muscle . For measuring the electrical events, the muscle was mounted horizontally in an organ bath containing 15 ml Tyrode solution maintained at 3210.5°C . In order to record the endplate potentials (e .p .p .s), the concentration of MgCI, in Tyrode solution was increased to 10-13 mM and that of CaC6 was reduced to 0 .9 mM . The e.p .p .s were elicited by stimulating the phrenic nerve with square pulses of 0 .05 msec at a frequency of 1 Hz. Measurement of the resting membrane potential and the quanta! content of e.p.p.s
For intracellular recording, a microelectrode made of a glass capillary having a thin filament attached on the inside wall (W-P 1B-150) was used . The resistance of the microelectrode was in the range 5-20 M4 . Intracellular recordings were made only on the superficial muscle layers . The quanta/ content of e.p .p .s was measured from the ratio of the mean amplitude of e.p .p .s to the mean amplitude of miniature e.p .p .s (m .e.p .p .s) (D8L CA5IILLD 8nd KATZ, 1954). Recording of the compound action potentials of rat phrenic nerve
The phrenic nerve-diaphragm preparation was prepared as described above . The phrenic nerve was dissected free of fat and connective tissue and bathed in oxygenated Tyrode solution maintained at 3210 .5°C . Compound nerve action potentials were evoked antidromically with square pulses (0.05 cosec, 0.2 Hz) by a bipolar platinum electrode plead at the muscle end of the nave . The evoked action potentials were picked up from the cut end of the nerve and amplified through a Grass P16 micrcelectrode DC/AC amplifier and displayed on a Tetronix 5103-D13 storage oscilloscope . Assay of phaspholipase activity
Enzyme activity was determined by measuring the amount of NaOH needed to titrate the fatty acid liberated from the emulsified phosphatidylcholine at 37°C, pH 7.4 . The method is essentially the same as that described by $TROtJO et al. (1976).
Mode of Action of Cenileotoxin
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RESULTS
Evidence for presynaptic effects of CeTX In the chick biventer cervicis muscle preparation, a low concentration (10'°g/ml) of CeTX gradually depressed the twitch response to indirect stimulation, leading to a complete transmission block in about S hr, whereas the responses to both acetylcholine (ACh) and carbamylcholine (Carb) after the neuromuscular block were not reduced or were even increased (Fig . 1). The average time for complete transmission block was 29819 min (meatitS .E ., n=3) and the responses to ACh and Carb, tested after block, were 11St11 % (n = 3) and 250129% (n = 3) of the control, respectively . In the rat phrenic nerve-diaphragm preparation, the same concentration of CeTX blocked neuromuscular transmission in about 6 hr (37217 min, n = 2) without affecting the muscle contraction elicited by direct stimulation. Control preparations survived for more than 10 hr. Electrophysiological studies on the rat phrenic nerve - diaphragm preparation revealed that CeTX (2 x 10-° g/ml) caused a triphasic change in the quantal content of e.p.p.s: an immediate depression followed by incomplete recovery of the quantal content and then by a complete failure of e.p.p.s. (Fig. 2). The amplitude of m.e.p.p.s was not significantly changed even after complete failure of e.p.p.s, indicating that the sensitivity of postsynaptic ACh receptors remains unaffected by CeTX up to this concentration. A two to three-fold increase in the frequency of m.e.p.p. was observed during the period between the recovery phase and about 1 hr after the failure of e.p.p.s. A similar triphasic effect on the release of neurotransmitter in skeletal muscle has been described in other snake presynaptic toxins (CHANG and LEE, 1977; Ho and LEE, 1981 ; LEE et al., 1982). Postsynaptic and musculotropic effects of CeTX At a higher concentration (10-° g/ml) of CeTX, the tone of the chick biventer cervicis muscle was progressively increased, the twitch height decreased and the responses to both ACh and Carb were greatly depressed (Fig . 3A). The average time for complete block was 13419 min (n = 3) and the responses to ACh and Carb tested at complete or nearly complete block were 1212% (n = 3) and 2914% (n = 3) of the control, respectively . In the denervated diaphragm preparation of the rat, the same concentration of CeTX gradually depressed the muscle contraction evoked by direct stimulation with a slight increase of the tone. The response to ACh was greatly reduced and that to Carb was abolished (Fig. 3B). The resting membrane potentials of the rat diaphragm muscle were gradually decreased to less than 50 mV in 2 hr and to less than 40 mV in 4 hr by the same concentration of CeTX (Fig. 4).
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300
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EFFECT OF LOW CONCENTRATIONS OF CERULP.OTOXIN ON THE CHICK HIVEN7ER CERVICIS NERVE-MUSCLE PREPARATION .
Two muscle preparations were isolated from the same chick, one treated with CeTX (B) and another serving as control (A). The preparation was stimulated indirectly at a rate of 0 .2 Hz. Arrows indicate the application of acetylcholine (ACh, S x l0i g/atl), carbachol (Cart, 5 x 10'' g/ml) and the ta~xin (10~ g/ml) . Horizontal bars indicate the duration of drug action . Figures under the tracing denote min after toxin application .
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C . L. HO AND C. Y. LEE
,2
~0
FIG . Z . EFFECTS OF CERULEOTOXIN ON THE QUANTAL CONTENT OF DIAPHRAGM .
e. p . p .
AND AMPLITUDE OF
m.e .p .p .
OF THE RAT
CeTX (2 x 10-° g/ml) was added at 0 time . For quantal content of e.p .p . ( ~ - - - ~ ), each point represents mearLtS .E . of 3-S calculated values from 3 preparations . For m.e .p.p. amplitude (O - - - O), each point represents meantS.E . of 150-250 observations from 3 preparations.
L
ACh
t ACh FIG .
3.
L~
Carb .
t
5
L
t Carà
L
ACh C~b
CeTX
t CeTX
25
89 ~ AiCh
109t, Carb
EFFECT OF A HIGH CONCENTRATION OF CERULEOTOXIN ON THE CHICK BIVENTER CERVICIS NERVE-MUSCLE (A) AND THE CHRONICALLY DENERVATED RAT DIAPHRAGM PREPARATION (B).
Arrows indicate the application of acetylcholine (ACh, lß-" g/ml), carbachol (Carb, S x 10-' g/ml) and the toxin (10'` g/ml). Horizontal bars indicate the duration of drug action and the figures under each tracing denote min after toxin application.
Mode of Action of Ceruleotoxin
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-30
o
so
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305
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Tim¢ (min) FIG . 4 . EFFECT OF CERULEOTOXIN ON RESTING MEMBRANE POTENTIALS OF THE RAT DIAPHRAGM . Toxin (10-' g/ml) was added at 0 time . Each point represents 15 observations from 3 preparations .
Fffect on the compound action potential of the rat phrenic nerve The action potential of the rat phrenic nerve was not appreciably affected by a low concentration (10-° g/ml) of CeTX. Even at a concentration of 10-° g/ml, it required 360 min to cause a 25% decrease of the action potential. Therefore, it is unlikely that the neuromuscular block by CeTX is due to axonal conduction block. Phospholipase A activity of CeTX The catalytic activity of CeTX on phosphatidylcholine was compared with phospholipase As preparations isolated from other snake venoms . As shown in Table 1, the enzyme activity of CeTX is comparable to that of notexin, a toxic phospholipase A, from Notechis scutatus scutatus venom.
TAHLE 1 . COMPARISON OF THE ENZYME ACTIVITY OF PHOSPHOLIPASES As (PLA,) PURIFIED FROM VARIOUS SNAKE VENOMS
Toxin
Hydrolysis of phosphatidylcholine (pmole fatty acid/min/mg)
Ngja ngja afro PLA,
418 t IO (S)
Ngja nigricollir basic PLA, ß-Bungarotoxin
268 t 5 (S) 238 t 15 (S)
Notexin Ceruleotoxin
173 t 6 (S) 195 t 16 (S)
Data shown are mean t S .E . The number in the parentheses denotes the number of experiments .
306
C. L. HO AND C. Y . LEE DISCUSSION
The present study on the effect of CeTX on vertebrate nerve - muscle preparations has shown that the toxin blocks neuromuscular transmission at lower concentrations without depressing the ACh response of the chick biventer cervicis muscle and abolishes the indirectly evoked contractions without depressing those evoked by direct stimulation of the rat diaphragm muscle . These results suggest a presynaptic site of action of CeTX at low concentrations . In addition, the toxin also caused an immediate initial depression and a late phase of inhibition of the e.p .p .s in rat diaphragm similar to those caused by other presynaptically acting toxins, such as ß-bungarotoxin, crotoxin, Mojave toxin and caudoxin (CHANG and LEE, 1977 ; Ho and LEE, 1981 ; LEE et al., 1982 ; LEE and Ho, 1980, 1982) . At higher concentrations, CeTX caused contracture, diminished the ACh and Carb responses of the chick and denervated rat skeletal muscles and also depolarized the membrane of the rat diaphragm muscle, as did notexin, a presynaptically acting toxin (LEE et al., 1976 ; HARRIS et al ., 1980). The enzyme activity assayed by titration of the fatty acid liberated from phospholipid was also about the same as that of notexin. From these results we conclude that CeTX is not a novel-type toxin, but a phospholipase A, with both presynaptically neurotoxic and myotoxic activities, analogous to other phospholipase A, toxins such as notexin (HARRIS et al., 1973, 1975 ; LEE et al., 1976 ; LEE and Ho, 1980, 1982) . The difference in site of action of CeTX found in fish and mammalian tissues may be due to the high sensitivity of Torpedo postsynaptic membrane, possibly the cholinergic ionophore, to phospholipase A, activity . However, there appears to be no parallelism between this postsynaptic effect and the neurotoxicity in toxins with phospholipase A, activity . For example, a relatively nontoxic phospholipase A, isolated from Naja naja siamensis venom has been shown to inhibit the sodium efflux of Torpedo membrane vesicles at a concentration 10 -15 times lower than that required by a postsynaptic toxin from the same venom (ANDREASEN and MCNAMEE, 1977) . The recent report that crotoxin also caused a postsynaptic block on membrane vesicles of electroplaque (BON et al ., 1979) at concentrations lower than those used by CHANG and LEE (1977) to demonstrate the presynaptic effect of crotoxin in birds and mammals seems to support this contention . Acknowledgements - We thank Dr. C. Box for his generous gift of ceruleotoxin . We are also grateful to Dr . KARLSSON for his generous gift of notexin.
E.
REFERENCES T. J. and McNAMEE, M. G. (1977) Phospholipase A inhibition of acetylcholine receptor function in Torpedo California membrane vesicles. Biochem. biophys. Res. Commun . 79, 958. BoN, C . and CHANGEUx, J.-P. (1975) Ceruleotoxin : an acidic neurotoxin from the venom of Bungarus rneruleus which blocks the response to a cholinergic agonist without binding to the cholinergic receptor site. FEBS Lett . 59, 212. BoN, C. and CHANGEUX, J.-P. (1977a) Chemical and pharmacological characterization of toxic polypcptide from the venom of Bungarus caeruleus. Eur. J. Biochem. 74, 31 . BoN, C. and CHANGEUx, J.-P. (1977b) Ceruleotoxin: a possible marker of the cholinergic ionophore Eur. J. Biochem. 74, 43 . Box, C., C~tANCeux, J .-P., JENC, T. W. and F~rnteL-CoNRAT, H. (1979) Postsynaptic effects of crotoxin and its isolated subunit. Eur. J. Biochem. 99, 471. Born, C. and SAL.IOU, B. (1982) Isolation of "ceruleotoxin" from Bungarus fasciatus venom. Toxirnn 211, 111. Bü~eetrrc, E. (1946) Observation on the isolated phrenic nerve diaphragm preparation of the rat. Br. J. Pharmac. 1, 38 . ADREASEN,
Mode of Action of Ceruleotoxin
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CHANG, C. C. and Lee, J. D. (1977) Crotoxin, the neurotoxin of South American rattlesnake venom, is a presynaptic toxin acting like ~-bungarotoxin . Naunyn-Schmiedebergss Archs Pharmac. 296, 139. DEL CASTILLO, J. and KwTZ, B. (1954) Quantal component of the endplate potential . J. PhysioL, Load. 124, 360. EAKER, D. (1978) Studies of presynaptically active neurotoxic and myotoxic phospholipase A, . In : Versatility of Proteins, p. 413 (Li, C. H., Ed .) . New York : Academic Press. GINSBORG, B. L. and WnRRiNER, L. (1960) The isolated chick biventer cervicis nerve- muscle preparation. Br. J. Pharmac. 15, 410. HARRIS, J. B., JOHNSON, M. A. and KARLSSON, E. (1973) Pathological responses of rat skeletal muscle to a single subcutaneous injection of a toxin isolated from the venom of Australian tiger snake, Notenhis scutatus scutatus. Clin . ezp. Pharmac. Physiol. 2, 383. HARRIS, J . B., JOHNSON, M. A. 8nd MACDONELL, C. A. (1980) Muscle necrosis induced by some presynaptic neurotoxin. In : Natural Toxins, p. 569 (EAxeR, D. end WADSTRÖM, T., Eds) . Oxford : Pergamon Press. HARR~s, J . B., KARLSSON, E. Snd THESLEFF, S. (1973) Effects of an isolated toxin from Australian tiger snake (Notenhis scutatus scutatus) venom at mammalian neuromuscular junction. Br. J. Pharmac. 47, 141. Ho, C. L. and LEE, C. Y. (1981) Presynaptic action of Mojave toxin isolated from the Mojave rattlesnake (Crotalus scutulatus) venom. Toxicon 19, 889. KARLSSON, E. (1979) Chemistry of protein toxins in snake venoms . In: Handbook of Experimental Pharmacology, Vol. 52, Snake Venom, p. 159 (LEE, C. Y., Ed.) . Berlin : Springer . LEE, C. Y. and Ho, C. L. (1978) Cenileotoxin : a phospholipase A with presynaptic and myotropic effects . In : Abstracts of the 7th International Congress on Pharmacology, Paris, p. 571 . Oxford: Pergamon Press. LEE, C. Y. and Ho, C. L. (1980) Pharmacology of presynaptic neurotoxins from snake venoms . In: Natural Toxins, p. 539 (FAKER, D. and WAnsrROM, T., Eds.) . Oxford : Pergamon Press. LEE, C. Y. and Ho, C. L. (1982) The pharmacology of phospholipase A, isolated from snake venoms, with particular reference to their effects on neuromuscular transmission . In : Advances in Pharmacology and Therapeutics II, Vol. 4, Biochemical -Immunologirnl Pharmacology, p. 37 (YOSHIDA, H., HAGnaARA, Y. and EeASH~, S., Eds.) . Oxford : Pergamon Press. LEE, C. Y., CHANG, S. L., KAU, S. T, and LuH, S. H. (1972) Chromatographic separation of the venom of Buregarus multicinctus and characterization of its components . J. Chromat. 72, 71 . LEE, C. Y., CttEN, Y. M. and KARLSSON, E. (1976) Postsynaptic and musculotropic effects of aotexin, a presynaptic neurotoxin from the venom of Notenhis scutatus scutatus (Australian tiger snake) . Taxirnn 14, 493 . LEE, C. Y., Ho, C. L. and BOTES, D. P. (1982) Site of action of caudoxin, a neurotoxic phospholipase A, from the horned puff adder (Bitis caudalis) venom. Taxicon 20, 637. Lo, T. B., CHANO, W. C. and CFIANG, C. S. (1972) Chemical studies on phospholipase A from Formosan cobra (Ngja ngja atm) venom. J. Formosan med. Ass. 71, 318. STRONG, P. N., GOERKE, L, OHERG, S. G. 8nd KELLY, R. B. (1976) ~-BUIIgarOtOXln, a presynaptic toxin with enzymatic activity . Pros, natn . Aced. Sci. U.S.A . 73, 178. Wu, S. H., CHANG, W. C. and Lo, T. B. (1977) Isolation and enzymatic characterization of phospholipase A from several snake venoms . J. Chin, biochem. Soc. 6, 9.
0041 -0101/83/020301 -07503.00/0 7 1983 Pergamon Press Ltd.
Taxiroh, Vol.
MODE OF NEUROMUSCULAR BLOCKING ACTION OF CERULEOTOXIN C. L. Ho' and C. Y . LEE~ " 'Institute of Biological Chemistry, Academia Sinica and 'Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China (Amepted Jor publication 23 September 1982) C . L . Ho and C . Y . Lee . Mode of neuromuscular blocking action of ceruleotoxin . Toxicon 21, 301- 307, 1983 . - Cenileotoxin is an acidic toxin protein from a Bungarus venom which has recently been clarified as B. fauiatus venom . The toxin at a low concentration (10~g/ml) abolished the twitch response of the indirectly stimulated biventer cervicis muscle of the chick, without affecting the response to acetylcholine or carbamylcholine . At a higher concentration (10'`g/ml), the toxin, in addition to inhibition of the twitch response, caused contracture in the chick biventer cervicis muscle and reduced the response to acetylcholine or carbamylcholine in both the innervated chick cervical and denervated rat diaphragm muscles . Electrophysiological studies on the rat phrenic nerve-diaphragm preparation showed that ceruleotoxin at a low concentration caused an initial inhibition followed by recovery of the quantal content of endplate potentials and then a second phase of inhibition leading to complete failure. At a higher concentration, the toxin gradually reduced the resting membrane potential of the diaphragm muscle to a level lower than SO mV within 2 hr . In addition, enzyme assay showed that the toxin possessed phospholipase A activity comparable to that of other basic phospholipases A, of snake venom origin . It is concluded that ceruleotoxin is a phospholipase A, with presynaptic and myotropic actions on vertebrate nerve-muscle system similar to those of notexin from Notechir scutatus scutatus venom . INTRODUCTION CERULEOTOXIN (CeTX), an acidic toxin protein originally reported to be isolated from a batch of Bungarus caeruleus venom (BON and CHANOEUx, 1975, 1977a), was recently found to be actually a component of Bungarus fasciatus venom (BON and SALIOU, 1982) . This toxin blocks the depolarisation caused by carbamylcholine on the isolated electroplaque from Electrophorus electricus and the increase of "Na* and "K* efflux caused by cholinergic agonists on excitable receptor-rich microsacs prepared from Torpedo marmorata electric organ . However, at variance with typical postsynaptic snake toxins, this toxin does not interfere with the binding of ['H]acetylcholine or of a-['H]toxin from Naja nigricollis to the cholinergic receptor site . Based on these findings, BoN and CHANGEUX (19776) have proposed that CeTX is a new type of snake toxin which blocks selectively the electrogenic action of acetylcholine on the postsynaptic membrane without binding to the cholinergic receptor site and that it therefore might serve as a useful tool to characterise the cholinergic ionophore . On the other hand, they have also found that CeTX possesses phospholipase activity (BoN and CHANaEUx, 1977a) . Since several presynaptic neurotoxins from snake venoms possess phospholipase A, activity (EAICER, 1978; KARLSSON, 1979; LEE and Ho, 1980, 1982) and some of them also have " To whom correspondence should be addressed .
301
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C. L. HO AND C. Y. LEE
more or less postsynaptic and/or myotoxic action (HARRIS et al., 1975, 1980; LEE et al., 1976), it was deemed necessary to investigate the effect of CeTX on vertebrate skeletal muscle to clarify the mode of action of the toxin. A preliminary account of this work has already been published (LEE and Ho, 1978).
MATERIALS AND METHODS Acidic phospholipase A, from Ngja ngja atra and basic phospholipase A, from Ngja nigricollis venoms were purified by chromatography on CM-Sephadex C-23 columns and gel filtration on Sephadex G-S0, as described by Lo et al. (1972) and by Wu et al. (1977), respectively. ß-Bungarotoxin was isolated from the venom of Bungarus multicinctus according to the method of LeE et al. (1972) . Ceruleotoxin was a gift from Dr. C. Bon, Institut Pasteur, Paris. Notexin was a gift from Dr . E. Karlsson, Institute of Biochemistry, Uppsala. Acetylcholine chloride, carbachol (carbamylcholine) and phosphatidylcholine (dipalmitol) were purchased from Sigma Chemical Co ., U.S .A . All common chemicals used were of reagent grade. Biventer cervicis nerve-muscle preparation of the chick
The biventer cervicis muscle of Leghorn chicks (3-7 days old) was prepared according to the method of GINSBORG and WARRINER (1960) . The isolated preparation was suspended in 10 ml of Krebs solution (composition in mM: NaCI, 126; KCI, 5.0; CaCI,, 2.5 ; MgSO,, 1 .2; KH,PO,, 1.2; NaHCO,, 23 .8 ; dextrose, 11 .2) maintained at 3710 .3°C and oxygenated with a mixture of 95% O, and S% CO,. The preparation was stimulated indirectly with supramaximal square pulses of 0.3 msec duration at a frequency of 0.2 Hz . Isometric contraction was recorded with a Grass FT 03 force- displacement transducer attached to a Grass 7P polygraph. Acetylcholine and carbachol responses of the muscle were tested by adding each agent to the resting muscle and washing with fresh Krebs solution after the evoked contraction reached the maximum. Phrenic nerve-diaphragm preparation oJthe Long Evans rat
The phrenic nerve-diaphragm preparation of the rat was prepared aaording to the method Of BÜLHRING (1946) . Denervated preparations were prepared from rats in which one side of the phrenic nerves had been cut at the cervical level for at least 7 days . For recording muscle contractions, the preparation was suspended in an organ bath containing 20 ml of Tyrode solution (composition in mM : NaCI, 136; KCI, 2.8 ; CaCI,, 1 .8 ; MgCI,, 1.1 ; NaH,PO,, 0.33; NaHCO,, 11 .9 ; dextrose, 5 .6) maintained at 3710 .3°C and oxygenated with a mixture of 95% O, and S% CO,. Muscle contractions were elicited either by stimulation of the nerve with supramaximal square pulses of 0.05 msec duration, or by direct stimulation of the muscle with pulses of 1 msec duration at a frequency of 0.03 or 0.1 Hz, and recorded as described above for the chick muscle . For measuring the electrical events, the muscle was mounted horizontally in an organ bath containing 15 ml Tyrode solution maintained at 3210.5°C . In order to record the endplate potentials (e .p .p .s), the concentration of MgCI, in Tyrode solution was increased to 10-13 mM and that of CaC6 was reduced to 0 .9 mM . The e.p .p .s were elicited by stimulating the phrenic nerve with square pulses of 0 .05 msec at a frequency of 1 Hz. Measurement of the resting membrane potential and the quanta! content of e.p.p.s
For intracellular recording, a microelectrode made of a glass capillary having a thin filament attached on the inside wall (W-P 1B-150) was used . The resistance of the microelectrode was in the range 5-20 M4 . Intracellular recordings were made only on the superficial muscle layers . The quanta/ content of e.p .p .s was measured from the ratio of the mean amplitude of e.p .p .s to the mean amplitude of miniature e.p .p .s (m .e.p .p .s) (D8L CA5IILLD 8nd KATZ, 1954). Recording of the compound action potentials of rat phrenic nerve
The phrenic nerve-diaphragm preparation was prepared as described above . The phrenic nerve was dissected free of fat and connective tissue and bathed in oxygenated Tyrode solution maintained at 3210 .5°C . Compound nerve action potentials were evoked antidromically with square pulses (0.05 cosec, 0.2 Hz) by a bipolar platinum electrode plead at the muscle end of the nave . The evoked action potentials were picked up from the cut end of the nerve and amplified through a Grass P16 micrcelectrode DC/AC amplifier and displayed on a Tetronix 5103-D13 storage oscilloscope . Assay of phaspholipase activity
Enzyme activity was determined by measuring the amount of NaOH needed to titrate the fatty acid liberated from the emulsified phosphatidylcholine at 37°C, pH 7.4 . The method is essentially the same as that described by $TROtJO et al. (1976).
Mode of Action of Cenileotoxin
303
RESULTS
Evidence for presynaptic effects of CeTX In the chick biventer cervicis muscle preparation, a low concentration (10'°g/ml) of CeTX gradually depressed the twitch response to indirect stimulation, leading to a complete transmission block in about S hr, whereas the responses to both acetylcholine (ACh) and carbamylcholine (Carb) after the neuromuscular block were not reduced or were even increased (Fig . 1). The average time for complete transmission block was 29819 min (meatitS .E ., n=3) and the responses to ACh and Carb, tested after block, were 11St11 % (n = 3) and 250129% (n = 3) of the control, respectively . In the rat phrenic nerve-diaphragm preparation, the same concentration of CeTX blocked neuromuscular transmission in about 6 hr (37217 min, n = 2) without affecting the muscle contraction elicited by direct stimulation. Control preparations survived for more than 10 hr. Electrophysiological studies on the rat phrenic nerve - diaphragm preparation revealed that CeTX (2 x 10-° g/ml) caused a triphasic change in the quantal content of e.p.p.s: an immediate depression followed by incomplete recovery of the quantal content and then by a complete failure of e.p.p.s. (Fig. 2). The amplitude of m.e.p.p.s was not significantly changed even after complete failure of e.p.p.s, indicating that the sensitivity of postsynaptic ACh receptors remains unaffected by CeTX up to this concentration. A two to three-fold increase in the frequency of m.e.p.p. was observed during the period between the recovery phase and about 1 hr after the failure of e.p.p.s. A similar triphasic effect on the release of neurotransmitter in skeletal muscle has been described in other snake presynaptic toxins (CHANG and LEE, 1977; Ho and LEE, 1981 ; LEE et al., 1982). Postsynaptic and musculotropic effects of CeTX At a higher concentration (10-° g/ml) of CeTX, the tone of the chick biventer cervicis muscle was progressively increased, the twitch height decreased and the responses to both ACh and Carb were greatly depressed (Fig . 3A). The average time for complete block was 13419 min (n = 3) and the responses to ACh and Carb tested at complete or nearly complete block were 1212% (n = 3) and 2914% (n = 3) of the control, respectively . In the denervated diaphragm preparation of the rat, the same concentration of CeTX gradually depressed the muscle contraction evoked by direct stimulation with a slight increase of the tone. The response to ACh was greatly reduced and that to Carb was abolished (Fig. 3B). The resting membrane potentials of the rat diaphragm muscle were gradually decreased to less than 50 mV in 2 hr and to less than 40 mV in 4 hr by the same concentration of CeTX (Fig. 4).
orrx FIa . 1 .
300
.ar~
ää,
EFFECT OF LOW CONCENTRATIONS OF CERULP.OTOXIN ON THE CHICK HIVEN7ER CERVICIS NERVE-MUSCLE PREPARATION .
Two muscle preparations were isolated from the same chick, one treated with CeTX (B) and another serving as control (A). The preparation was stimulated indirectly at a rate of 0 .2 Hz. Arrows indicate the application of acetylcholine (ACh, S x l0i g/atl), carbachol (Cart, 5 x 10'' g/ml) and the ta~xin (10~ g/ml) . Horizontal bars indicate the duration of drug action . Figures under the tracing denote min after toxin application .
304
C . L. HO AND C. Y. LEE
,2
~0
FIG . Z . EFFECTS OF CERULEOTOXIN ON THE QUANTAL CONTENT OF DIAPHRAGM .
e. p . p .
AND AMPLITUDE OF
m.e .p .p .
OF THE RAT
CeTX (2 x 10-° g/ml) was added at 0 time . For quantal content of e.p .p . ( ~ - - - ~ ), each point represents mearLtS .E . of 3-S calculated values from 3 preparations . For m.e .p.p. amplitude (O - - - O), each point represents meantS.E . of 150-250 observations from 3 preparations.
L
ACh
t ACh FIG .
3.
L~
Carb .
t
5
L
t Carà
L
ACh C~b
CeTX
t CeTX
25
89 ~ AiCh
109t, Carb
EFFECT OF A HIGH CONCENTRATION OF CERULEOTOXIN ON THE CHICK BIVENTER CERVICIS NERVE-MUSCLE (A) AND THE CHRONICALLY DENERVATED RAT DIAPHRAGM PREPARATION (B).
Arrows indicate the application of acetylcholine (ACh, lß-" g/ml), carbachol (Carb, S x 10-' g/ml) and the toxin (10'` g/ml). Horizontal bars indicate the duration of drug action and the figures under each tracing denote min after toxin application.
Mode of Action of Ceruleotoxin
zo
-30
o
so
tzo
teo
305
aro
Tim¢ (min) FIG . 4 . EFFECT OF CERULEOTOXIN ON RESTING MEMBRANE POTENTIALS OF THE RAT DIAPHRAGM . Toxin (10-' g/ml) was added at 0 time . Each point represents 15 observations from 3 preparations .
Fffect on the compound action potential of the rat phrenic nerve The action potential of the rat phrenic nerve was not appreciably affected by a low concentration (10-° g/ml) of CeTX. Even at a concentration of 10-° g/ml, it required 360 min to cause a 25% decrease of the action potential. Therefore, it is unlikely that the neuromuscular block by CeTX is due to axonal conduction block. Phospholipase A activity of CeTX The catalytic activity of CeTX on phosphatidylcholine was compared with phospholipase As preparations isolated from other snake venoms . As shown in Table 1, the enzyme activity of CeTX is comparable to that of notexin, a toxic phospholipase A, from Notechis scutatus scutatus venom.
TAHLE 1 . COMPARISON OF THE ENZYME ACTIVITY OF PHOSPHOLIPASES As (PLA,) PURIFIED FROM VARIOUS SNAKE VENOMS
Toxin
Hydrolysis of phosphatidylcholine (pmole fatty acid/min/mg)
Ngja ngja afro PLA,
418 t IO (S)
Ngja nigricollir basic PLA, ß-Bungarotoxin
268 t 5 (S) 238 t 15 (S)
Notexin Ceruleotoxin
173 t 6 (S) 195 t 16 (S)
Data shown are mean t S .E . The number in the parentheses denotes the number of experiments .
306
C. L. HO AND C. Y . LEE DISCUSSION
The present study on the effect of CeTX on vertebrate nerve - muscle preparations has shown that the toxin blocks neuromuscular transmission at lower concentrations without depressing the ACh response of the chick biventer cervicis muscle and abolishes the indirectly evoked contractions without depressing those evoked by direct stimulation of the rat diaphragm muscle . These results suggest a presynaptic site of action of CeTX at low concentrations . In addition, the toxin also caused an immediate initial depression and a late phase of inhibition of the e.p .p .s in rat diaphragm similar to those caused by other presynaptically acting toxins, such as ß-bungarotoxin, crotoxin, Mojave toxin and caudoxin (CHANG and LEE, 1977 ; Ho and LEE, 1981 ; LEE et al., 1982 ; LEE and Ho, 1980, 1982) . At higher concentrations, CeTX caused contracture, diminished the ACh and Carb responses of the chick and denervated rat skeletal muscles and also depolarized the membrane of the rat diaphragm muscle, as did notexin, a presynaptically acting toxin (LEE et al., 1976 ; HARRIS et al ., 1980). The enzyme activity assayed by titration of the fatty acid liberated from phospholipid was also about the same as that of notexin. From these results we conclude that CeTX is not a novel-type toxin, but a phospholipase A, with both presynaptically neurotoxic and myotoxic activities, analogous to other phospholipase A, toxins such as notexin (HARRIS et al., 1973, 1975 ; LEE et al., 1976 ; LEE and Ho, 1980, 1982) . The difference in site of action of CeTX found in fish and mammalian tissues may be due to the high sensitivity of Torpedo postsynaptic membrane, possibly the cholinergic ionophore, to phospholipase A, activity . However, there appears to be no parallelism between this postsynaptic effect and the neurotoxicity in toxins with phospholipase A, activity . For example, a relatively nontoxic phospholipase A, isolated from Naja naja siamensis venom has been shown to inhibit the sodium efflux of Torpedo membrane vesicles at a concentration 10 -15 times lower than that required by a postsynaptic toxin from the same venom (ANDREASEN and MCNAMEE, 1977) . The recent report that crotoxin also caused a postsynaptic block on membrane vesicles of electroplaque (BON et al ., 1979) at concentrations lower than those used by CHANG and LEE (1977) to demonstrate the presynaptic effect of crotoxin in birds and mammals seems to support this contention . Acknowledgements - We thank Dr. C. Box for his generous gift of ceruleotoxin . We are also grateful to Dr . KARLSSON for his generous gift of notexin.
E.
REFERENCES T. J. and McNAMEE, M. G. (1977) Phospholipase A inhibition of acetylcholine receptor function in Torpedo California membrane vesicles. Biochem. biophys. Res. Commun . 79, 958. BoN, C . and CHANGEUx, J.-P. (1975) Ceruleotoxin : an acidic neurotoxin from the venom of Bungarus rneruleus which blocks the response to a cholinergic agonist without binding to the cholinergic receptor site. FEBS Lett . 59, 212. BoN, C. and CHANGEUX, J.-P. (1977a) Chemical and pharmacological characterization of toxic polypcptide from the venom of Bungarus caeruleus. Eur. J. Biochem. 74, 31 . BoN, C. and CHANGEUx, J.-P. (1977b) Ceruleotoxin: a possible marker of the cholinergic ionophore Eur. J. Biochem. 74, 43 . Box, C., C~tANCeux, J .-P., JENC, T. W. and F~rnteL-CoNRAT, H. (1979) Postsynaptic effects of crotoxin and its isolated subunit. Eur. J. Biochem. 99, 471. Born, C. and SAL.IOU, B. (1982) Isolation of "ceruleotoxin" from Bungarus fasciatus venom. Toxirnn 211, 111. Bü~eetrrc, E. (1946) Observation on the isolated phrenic nerve diaphragm preparation of the rat. Br. J. Pharmac. 1, 38 . ADREASEN,
Mode of Action of Ceruleotoxin
307
CHANG, C. C. and Lee, J. D. (1977) Crotoxin, the neurotoxin of South American rattlesnake venom, is a presynaptic toxin acting like ~-bungarotoxin . Naunyn-Schmiedebergss Archs Pharmac. 296, 139. DEL CASTILLO, J. and KwTZ, B. (1954) Quantal component of the endplate potential . J. PhysioL, Load. 124, 360. EAKER, D. (1978) Studies of presynaptically active neurotoxic and myotoxic phospholipase A, . In : Versatility of Proteins, p. 413 (Li, C. H., Ed .) . New York : Academic Press. GINSBORG, B. L. and WnRRiNER, L. (1960) The isolated chick biventer cervicis nerve- muscle preparation. Br. J. Pharmac. 15, 410. HARRIS, J. B., JOHNSON, M. A. and KARLSSON, E. (1973) Pathological responses of rat skeletal muscle to a single subcutaneous injection of a toxin isolated from the venom of Australian tiger snake, Notenhis scutatus scutatus. Clin . ezp. Pharmac. Physiol. 2, 383. HARRIS, J . B., JOHNSON, M. A. 8nd MACDONELL, C. A. (1980) Muscle necrosis induced by some presynaptic neurotoxin. In : Natural Toxins, p. 569 (EAxeR, D. end WADSTRÖM, T., Eds) . Oxford : Pergamon Press. HARR~s, J . B., KARLSSON, E. Snd THESLEFF, S. (1973) Effects of an isolated toxin from Australian tiger snake (Notenhis scutatus scutatus) venom at mammalian neuromuscular junction. Br. J. Pharmac. 47, 141. Ho, C. L. and LEE, C. Y. (1981) Presynaptic action of Mojave toxin isolated from the Mojave rattlesnake (Crotalus scutulatus) venom. Toxicon 19, 889. KARLSSON, E. (1979) Chemistry of protein toxins in snake venoms . In: Handbook of Experimental Pharmacology, Vol. 52, Snake Venom, p. 159 (LEE, C. Y., Ed.) . Berlin : Springer . LEE, C. Y. and Ho, C. L. (1978) Cenileotoxin : a phospholipase A with presynaptic and myotropic effects . In : Abstracts of the 7th International Congress on Pharmacology, Paris, p. 571 . Oxford: Pergamon Press. LEE, C. Y. and Ho, C. L. (1980) Pharmacology of presynaptic neurotoxins from snake venoms . In: Natural Toxins, p. 539 (FAKER, D. and WAnsrROM, T., Eds.) . Oxford : Pergamon Press. LEE, C. Y. and Ho, C. L. (1982) The pharmacology of phospholipase A, isolated from snake venoms, with particular reference to their effects on neuromuscular transmission . In : Advances in Pharmacology and Therapeutics II, Vol. 4, Biochemical -Immunologirnl Pharmacology, p. 37 (YOSHIDA, H., HAGnaARA, Y. and EeASH~, S., Eds.) . Oxford : Pergamon Press. LEE, C. Y., CHANG, S. L., KAU, S. T, and LuH, S. H. (1972) Chromatographic separation of the venom of Buregarus multicinctus and characterization of its components . J. Chromat. 72, 71 . LEE, C. Y., CttEN, Y. M. and KARLSSON, E. (1976) Postsynaptic and musculotropic effects of aotexin, a presynaptic neurotoxin from the venom of Notenhis scutatus scutatus (Australian tiger snake) . Taxirnn 14, 493 . LEE, C. Y., Ho, C. L. and BOTES, D. P. (1982) Site of action of caudoxin, a neurotoxic phospholipase A, from the horned puff adder (Bitis caudalis) venom. Taxicon 20, 637. Lo, T. B., CHANO, W. C. and CFIANG, C. S. (1972) Chemical studies on phospholipase A from Formosan cobra (Ngja ngja atm) venom. J. Formosan med. Ass. 71, 318. STRONG, P. N., GOERKE, L, OHERG, S. G. 8nd KELLY, R. B. (1976) ~-BUIIgarOtOXln, a presynaptic toxin with enzymatic activity . Pros, natn . Aced. Sci. U.S.A . 73, 178. Wu, S. H., CHANG, W. C. and Lo, T. B. (1977) Isolation and enzymatic characterization of phospholipase A from several snake venoms . J. Chin, biochem. Soc. 6, 9.