- Email: [email protected]
The effect of cobra venom cardiotoxin on locust skeletal muscle
JOURNAL
OF INVERTEBRATE
PATHOLOGY
30,
232-236 (1977)
The Effect of Cobra Venom Cardiotoxin
on Locust Skeletal
Muscle’
J. W. DEITMER,~ N. PRIMOR, AND E. ZLOTKIN Department
of Entomology and Venomous Animals, The Hebrew University
of
Jerusalem, Jerusalem, Israel
Received September 21, 1976 The strong paralytic and Iethai action of the venom of the cobra Naja mossambica mossambica on locusts is mainly due to its cardiotoxic components. When cardiotoxin was applied to the locust extensor tibiae nerve-muscle preparation, it caused a gradual and irreversible decrease in the membrane resting potential of the muscle fibers. The time course of this potential drop was dose dependent. The electrical responses of the muscle fiber, caused by stim~ation of the motor nerve, progressively decreased due to the depolarization of the membrane. The membranedepolarizing action of cardiotoxin could be prevented by high calcium (10 mM) and by lanthanum (1 mM) in the bathing solution. In nerve-muscle preparations obtained from cardiotoxinparalyzed locusts, normal resting and action potentials were recorded for at least 2 hr after cardiotoxin was injected into the animals.
INTRODUCTION Cobra venoms are known to contain several groups of pharmacologically active c~diotoxins and proteins: neurotoxins, phospholipases (Zlotkin, 1973). It has previously been found that the paralysis and lethality of fly larvae and isopods (Crustacae), as induced by the venom of the South African cobra Naja mossambica mossambica, is mainly due to its phospholipatic components. The neurotoxins, which comprise the main lethal and paralytic factor to vertebrates in cobra venoms, were found to be inactive to the above arthropods (Zlotkin et al., 1975). ~elimin~y essays performed recently indicate that the main paralytic and lethal factors to locusts in the above cobra venom are actually its cardiotoxic components. The effect of cardiotoxin on locusts served as the object of the present work. 1 Supported by Grant 730 from the United StatesIsrael Binational Science Foundation (BSF), Jerusalem, Israel. z Present address: Department of Physiology, Medical School, University of Bristol, England.
MATERIALS
AND METHODS
Venom and Toxins Crude venom. The crude venom of N. m. mossam~ica was supplied by D. Miller, Johannesburg, South Africa. biologically active fractions. The phospholipatic, cardiotoxic, and neurotoxic fractions were obtained by Sephadex G-50 column chromatography of the above venom according to the method previously described (Zlotkin et al., 1975). Curdiotoxin. One of the several cardiotoxins present in the N. m. mossambica venom (Louw, 1974) was obtained in a purified form by using ion-exchange column chromato~aphy (Fraction De, Frimor and Zlotkin, 1977). In order to obtain fraction Dq, 700 mg of cardiotoxic material obtained by Sephadex G-50 gel filtration (Zlotkin et al., 1975) were applied on an 80 x 2-cm column filled with Biorex 70 (Bio-rad) and equilibrated by 0.4 M ammonium acetate buffer, PH 7.0, with a flow rate of 12 ml/ hr. The initial stage of elution under equilibrium conditions was followed by a series of stepwise increases in molarity (0.55 M for fraction D). This product was applied on the nerve-muscle preparation. 232
Copyright 6 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-201 I
EFFECTS OF COBRA CARDIOTOXIN TABLE THE
1
LETHAL POTENCY TO LOCUSTS OF THE CRUDE VENOM OF NAJA MOSSA.MBKA MOSSAMEUCA AND ITS DERIVED TOXINS LDw
Toxic material
wld
Crude venom= Phospholipatic fraction” Cardiotoxic fraction* Neurotoxic fraction0 Purified cardiotoxin 4’ Purified cardiotoxin D4E
8.0 25.0 3.9 40.0 4.0 2.6
Percentage of total toxicityd
loo 10 89 1 28 48
o Obtained from D. Miiller, Johannesburg, South Africa. L Obtained by Sephadex G-50 column chromatography of the crude venom. The phospholipatic, cardiotoxic, and neurotoxic fractions correspond to fractions I and II, III, and IV, respectively, as presented by Zlotkin et al., 1975. e Obtained by Biorex 70 ion-exchange chromatography of the Sephadex G-50 fraction III (Primor and Zlotkin, 1977). d Calculated according to the content in the crude venom of the different fractions (Zlotkin et ai., 1975; Primor and Zlotkin, 1977) and their specific toxicities.
Tests of lethality Tests of lethality were performed on female adults of Locusta migratoria migrutorioides (1.8-2.0 g body weight) by injection of the toxic material through the intersegmental membrane between the fifth and the sixth abdominal pleurites. The amount of toxic material causing the lethality of 50% of test animals, defined as a unit of toxicity (LD&, was determined according to the method of Reed and Muench (1938). Increasing doses (in geometric series and with proper corrections for the small differences in body weight) of toxic material were administered to groups of 7-10 test animals (Zlotkin et al., 1971). The determination of death was based on the inability of the locusts to move 48 hr after the injection. Nerve -Muscle
Preparation
The extensor tibiae nerve-muscle preparation of adult locust as well as its dis-
ON THE LOCUST
233
section have been described in detail by Hoyle (1955). The muscle fibers were stimulated indirectly by applying short-current pulses to the motor nerve which was sucked into a polyethylene tube. Membrane potentials were recorded intracellularly from superficial fibers of different muscle bundles using a conventional glass microelectrode technique. The locust saline had the following composition: 154 rnM NaCl; 10 mM KCI; 2 mM CaCl, (altered after Hoyle, 1955). In highCa saline, iso-osmolar amounts of NaCl were replaced with CaCl,. In some experiments, LaCl, (1 mM) was added to the normal bathing solution; the change in the osmolarity was considered as negligible. All solutions were buffered with 0.2 M Trismaleate-NaOH to pH 6.8 (20.1). RESULTS
Lethal Potency and Symptomatology The lethal effect of the crude venom of N. m. mossambica and its derived toxic fractions, when injected into locusts, is shown in Table 1. A dose of 3-5 LDsO units causes a complete and irreversible paralysis of the locust 15-25 min after the injections. Locusts lying on their sides with extended hind legs can be indicated as a characteristic symptom. With sublethal doses test animals do not lie, but they appear phlegmatic in their mobility. Such a partial paralysis is generally followed by recovery. Effect on the Muscle Membrane When cardiotoxin was added to the bathing solution, the membrane resting potential of the muscle fiber decreased with a dose-dependent time course (Fig. 1). During the drop in the resting potential, the electrical responses elicited by stimulating the motor nerve were reduced in amplitude until only small excitatory junction potentials (ejps) could be evoked (Fig. 1, inset).
DEITMER,
234
PRIMOR, AND ZLOTKIN ,70 3 .60 f 5 50 9. ‘D ?i 40 2 a 30 : 20 j k 10 zj5
OJ
, 0
I
5
,
10
I
t (mid
0
‘5
FIG. 1. Time course of cardiotoxin effect on the membrane resting potential of the muscle fiber (4 &ml: open circles; 0.8 &ml: filled circles) and on the amplitude of the postsynaptic response (0.8 pgi ml: squares). Filled symbols represent same experiment and same muscle fiber. The arrow indicates application of cardiotoxin. Inset: Intracellular recordings: (a) control, and (b) 3, (c) 4, (d) 5, and (e) 10 min after application of 0.8 pg/rnl of cardiotoxin, (a,b,c) “Slow” and “fast” ejps; (d,e) only “fast” ejps. The postsynaptic response above a 40-mV amplitude (at a membrane potential of about 40 mV, 4 min after application of the toxin) consists of the ejp and the electrically excitable response; below a 40-mV amplitude, only ejps are evoked by nerve stimulation. Figures in inset are original registrations which are partially retouched for clarity.
This demonstrates, that action potentials were still conducted in the motor axons and were transmitted to the muscle, although the muscle fiber membrane was not able to generate action potentials due to the refractory effect of the depolarization. Continuous washing of the preparation with toxin-free saline for 30-60 min did not recover the membrane-depolarizing effect of the cardiotoxin. When the application of cardiotoxin was preceded by an increase in the external calcium concentration from 2 to IO mM, the depolarizing effect of the cardiotoxin was prevented (Fig. 2). Addition of 1 mM lanthanum to the external solution before application of the cardiotoxin also prevented depolarization of the membrane (Fig. 2). In the La-containing solution the electrical responses elicited by motor nerve stimulation rapidly decreased within 2 min. This was probably due to blockage of the neuromuscular transmission caused by lanthanum. Nerve-muscle preparations were dissected from two locusts which had been completely paralyzed by the injection of 5
and 10 pg of cardiotoxin, respectively. Measurements of the membrane resting potential as well as of the indirectly evoked electrical responses of different fibers revealed no effect of the injected toxin on the periphery: The resting and action potentials of the muscle fibers were found to be unchanged as compared with those of untreated animals. DISCUSSION
The cardiotoxins are a group of low molecular weight strongly basic proteins isolated from snake venoms devoid of enzymatic activity. They possess a wide range of pharmacological activities such as inducing contracture in vertebrate skeletal, smooth, and heart muscles, block of axonal conduction, local irritation, weak direct hemolytic activity and cytophatic action to tumor cell cultures (Lee et al., 1968, 1972, Chang et al., 1972, Condrea, 1974). To these various actions one may now add the high lethal potency of cardiotoxins to locusts. The lethal effect of the cobra venom and its cardiotoxic components is even stronger
EFFECTS OF COBRA CA~IOTOXIN - -704
0’
r 0
5
10 t fminf
i5
FIG. 2. The effect of lanthanum ions (1 mM: triangles) and of high calcium concentration ( 10 mM: filled circles) on the cardiotoxin-induced muscle membrane depolarization. After changing the calcium-rich saline to a normal calcium solution concentration (2 mM: open circles), card~otoxin (U~gjmi) caused a quick and drastic drop in the muscle membrane resting potential. in the same preparation and experiment. The arrow indicates application of cardiotoxin.
than that of potent scorpion venoms, which are known for their high specific toxicity to insects (Kamon and Shulov, 1963; Zlotkin et al., 1971). On the basis of the protein contents of the different Sephadex G-50 venom fractions (Zlotkin et al., 1975), it could have been calculated that the cardiotoxic fraction is responsible for about 90% of the crude venom’s lethality to the locusts (Table I). The cardiotoxin is about 6-7 times more potent to locusts than the phospholipatic fraction (Table l), which was the main lethal factor for fly larvae and isopods (Zlotkin et al., 1975). This may indicate the physiological-pharmacological diversity of arthropods. On the other hand, the neurotoxic fraction, which was inactive to fly larvae and isopods, was equally inactive to locusts. Cobra venom neurotoxins are known to paralyze vertebrates through a highly specific competitive blockage of the cholinergic receptors at the postsynaptic membrane at the neuromuscular junction (Changeux et al., 1970). The tolerance of arthropods to cobra neurotoxins is certainly due to the fact that their neuromuscular transmitters are noncholinergic (Gerschenfeld, 1973). The strong and relatively quick lethal and
ON THE LOCUST
235
paralytic action of cardiotoxin has actually suggested the investigation of its effect on the present nerve-muscle preparation. It was shown in the present investigation that cardiotoxin had a strong depolarizing effect on the fiber membrane of locust skeletal muscle. This was also demonstrated in frog skeletal muscles, in which this depolarization could have been antagonized by a high Ca concentration (Lee et al., 1968, 1972). Calcium ions have equally antagonized the axonal, hemolytic, and cytotoxic effects of cardiotoxin (Condrea, 1974). It was suggested that cardiotoxin may bind to the same sites on the membrane surface as do Ca ions. In locust muscle the membrane depolarization caused by cardiotoxin was prevented by a high Ca-concentration and also by 1 mM lanthanum. Recently it was shown that 1 mM La3+ blocked the Ca-dependent action potentials in insect skeletal muscle (Deitmer and Rathmayer, 1976), as was demonstrated for barnacle muscle fibers (Hagiwara and Takahashi, 1967). It was suggested there that La ions have a high affinity to “Ca binding sites” of the membrane surface. Electron microscopy on barnacle muscle fibers revealed that, indeed, La ions compete with Ca ions for the same binding sites (Henkart and Hagiwara, 1976). The ability of La ions to prevent the membrane-depolarizing effect of cardiotoxin, as shown in the present study, strongly supports the hypothesis that the cardiotoxin attaches to the “Ca binding sites” of the membrane. The question which we encountered was to what extent is the muscle membranedepolarizing effect due to ~ardiotoxin responsible for its paralyzing and lethal action to the whole animal. The experiments on preparations obtained from cardiotoxinenvenomated locusts demonstrate that the paralysis of the locust cannot be attributed to the muscle membrane depolarization. It is suggested, therefore, that the cardiotoxin lethal-paralytic action to the locusts may be due to effects on the central nervous system,
236
DEITMER,
PRIMOR, AND ZLOTKIN
This hypothesis, however, must be investigated by further experimentation. REFERENCES C. C., WEI, J. W., CHUANG, S. T., AND LEE, C. Y. 1972. Are the blockades of nerve conduction and depolarization of skeletal muscle induced by cobra venom due to phosphofi~se A, neurotoxin or cardiotoxin?J. Formosaa Med. Assoc., 71,323-327. CHANGEUX, J. P., KASAI, M., AND LEE, C. Y. 1970. Use of snake venom toxin to characterize the chofingeric receptor protein. Froc. Nat. Acad. Sci. USA, 67, 1241-1247. CONDREA. E. 1974. Membrane active polypeptides from snake venom: Cardiotoxins and haemocytotoxins. Experientia, 30, 121- 129. DEITMER, J. W., AND RATHMAYER, W. 1976. Calcium action potentials in larval muscle fibers of the moth Ephestia k~ha~el~a Z. (Lepidoptera). J. Camp.
CHANG,
Physiol. 112, GERSCHENFELD,
123-
132.
H. M. 1973. Chemical transmission in invertebrate central nervous system and neuromuscular junctions. Physiol. Rev., 53, 2-92. HAGIWARA, S., AND TAKAHASHI, K. 1967. Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane. J. Gen. Physiol., 50, 583-601. HENKART, M., AND HAGIWARA, S. 1976. Localization of the Ca binding sites associated with the Ca spike in bernacle muscle. j. Membrane Bioi., 27, l-20. HOYLE. G. 1955. The anatomy and innervation of focust skeletal muscle. Proc. Roy Sot. London Ser. B. 143,281-292.
KAMON, E., AND SHULOV, A. 1963. Estimation of locust resistance to scorpion venom. J. Insect Physiol., 5, 206-214. LEE, J.
G. Y., CHANG. C. C., CHIU, T. H., S., TSENG, T. C., AND LEE, S.
Pharmacological from Formosan bergs Arch. LEE. C. Y.,
CHIU, P. Y. 1968.
properties of cardiotoxin isolated cobra venom. Naunyn Schmiede-
Pharmakol
Exp.
Pathol.,
259,360-374.
LIN, J. S., AND WEY. J. W. 1972. Identification of cardiotoxin with cobramine B, DLF. Toxin o! and cobra venom cytotoxin. in “Toxins of Animal and Plant Origins” (A. de Vries and E. Kochwa, eds.), Vol. 2, p. 307. Gordon and Breach, New York. Louw, A. I., 1974. The purification and properties of five neurotoxic polypeptides from Naja mossambica mossambica venom. Biochim. Biophys. Actu, 336, 470-480. PRIMOR, N., AND ZLOTKIN, E. 1977. Per OS toxicity of venoms and toxins to blotiies.In “Proceedings, 5th International Symposium on Toxinology, San Jose, Costa Rica, August 1976.” In press. REED, I,. J., AND MUENCH. H. 1938. A simple method for estimating fifty per cent end points. Amer. J. Hyg.. 27, 493-497. ZLOTKIN. E., FRAENKEL, LISSITZKY, S. 1971. The
G.. MIRANDA, F.. AND effect of scorpion venom on blowfly larvae: A new method for the evaluation of scorpion venom potency. Toxicon. 9, 1-8. ZLOTKIN, E. 1973. Chemistry of animal venoms. Experientia. 29, 1453ZLOTKIN, E., MENASH& F., AND L~SSITZKY.
arthropods Physiol.,
1466.
M., ROCHAT, H., MIRANDA, S. 1975. Protein toxic to in the venom of efapid snakes. J. Insect
21, 1605-1611.
OF INVERTEBRATE
PATHOLOGY
30,
232-236 (1977)
The Effect of Cobra Venom Cardiotoxin
on Locust Skeletal
Muscle’
J. W. DEITMER,~ N. PRIMOR, AND E. ZLOTKIN Department
of Entomology and Venomous Animals, The Hebrew University
of
Jerusalem, Jerusalem, Israel
Received September 21, 1976 The strong paralytic and Iethai action of the venom of the cobra Naja mossambica mossambica on locusts is mainly due to its cardiotoxic components. When cardiotoxin was applied to the locust extensor tibiae nerve-muscle preparation, it caused a gradual and irreversible decrease in the membrane resting potential of the muscle fibers. The time course of this potential drop was dose dependent. The electrical responses of the muscle fiber, caused by stim~ation of the motor nerve, progressively decreased due to the depolarization of the membrane. The membranedepolarizing action of cardiotoxin could be prevented by high calcium (10 mM) and by lanthanum (1 mM) in the bathing solution. In nerve-muscle preparations obtained from cardiotoxinparalyzed locusts, normal resting and action potentials were recorded for at least 2 hr after cardiotoxin was injected into the animals.
INTRODUCTION Cobra venoms are known to contain several groups of pharmacologically active c~diotoxins and proteins: neurotoxins, phospholipases (Zlotkin, 1973). It has previously been found that the paralysis and lethality of fly larvae and isopods (Crustacae), as induced by the venom of the South African cobra Naja mossambica mossambica, is mainly due to its phospholipatic components. The neurotoxins, which comprise the main lethal and paralytic factor to vertebrates in cobra venoms, were found to be inactive to the above arthropods (Zlotkin et al., 1975). ~elimin~y essays performed recently indicate that the main paralytic and lethal factors to locusts in the above cobra venom are actually its cardiotoxic components. The effect of cardiotoxin on locusts served as the object of the present work. 1 Supported by Grant 730 from the United StatesIsrael Binational Science Foundation (BSF), Jerusalem, Israel. z Present address: Department of Physiology, Medical School, University of Bristol, England.
MATERIALS
AND METHODS
Venom and Toxins Crude venom. The crude venom of N. m. mossam~ica was supplied by D. Miller, Johannesburg, South Africa. biologically active fractions. The phospholipatic, cardiotoxic, and neurotoxic fractions were obtained by Sephadex G-50 column chromatography of the above venom according to the method previously described (Zlotkin et al., 1975). Curdiotoxin. One of the several cardiotoxins present in the N. m. mossambica venom (Louw, 1974) was obtained in a purified form by using ion-exchange column chromato~aphy (Fraction De, Frimor and Zlotkin, 1977). In order to obtain fraction Dq, 700 mg of cardiotoxic material obtained by Sephadex G-50 gel filtration (Zlotkin et al., 1975) were applied on an 80 x 2-cm column filled with Biorex 70 (Bio-rad) and equilibrated by 0.4 M ammonium acetate buffer, PH 7.0, with a flow rate of 12 ml/ hr. The initial stage of elution under equilibrium conditions was followed by a series of stepwise increases in molarity (0.55 M for fraction D). This product was applied on the nerve-muscle preparation. 232
Copyright 6 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.
ISSN 0022-201 I
EFFECTS OF COBRA CARDIOTOXIN TABLE THE
1
LETHAL POTENCY TO LOCUSTS OF THE CRUDE VENOM OF NAJA MOSSA.MBKA MOSSAMEUCA AND ITS DERIVED TOXINS LDw
Toxic material
wld
Crude venom= Phospholipatic fraction” Cardiotoxic fraction* Neurotoxic fraction0 Purified cardiotoxin 4’ Purified cardiotoxin D4E
8.0 25.0 3.9 40.0 4.0 2.6
Percentage of total toxicityd
loo 10 89 1 28 48
o Obtained from D. Miiller, Johannesburg, South Africa. L Obtained by Sephadex G-50 column chromatography of the crude venom. The phospholipatic, cardiotoxic, and neurotoxic fractions correspond to fractions I and II, III, and IV, respectively, as presented by Zlotkin et al., 1975. e Obtained by Biorex 70 ion-exchange chromatography of the Sephadex G-50 fraction III (Primor and Zlotkin, 1977). d Calculated according to the content in the crude venom of the different fractions (Zlotkin et ai., 1975; Primor and Zlotkin, 1977) and their specific toxicities.
Tests of lethality Tests of lethality were performed on female adults of Locusta migratoria migrutorioides (1.8-2.0 g body weight) by injection of the toxic material through the intersegmental membrane between the fifth and the sixth abdominal pleurites. The amount of toxic material causing the lethality of 50% of test animals, defined as a unit of toxicity (LD&, was determined according to the method of Reed and Muench (1938). Increasing doses (in geometric series and with proper corrections for the small differences in body weight) of toxic material were administered to groups of 7-10 test animals (Zlotkin et al., 1971). The determination of death was based on the inability of the locusts to move 48 hr after the injection. Nerve -Muscle
Preparation
The extensor tibiae nerve-muscle preparation of adult locust as well as its dis-
ON THE LOCUST
233
section have been described in detail by Hoyle (1955). The muscle fibers were stimulated indirectly by applying short-current pulses to the motor nerve which was sucked into a polyethylene tube. Membrane potentials were recorded intracellularly from superficial fibers of different muscle bundles using a conventional glass microelectrode technique. The locust saline had the following composition: 154 rnM NaCl; 10 mM KCI; 2 mM CaCl, (altered after Hoyle, 1955). In highCa saline, iso-osmolar amounts of NaCl were replaced with CaCl,. In some experiments, LaCl, (1 mM) was added to the normal bathing solution; the change in the osmolarity was considered as negligible. All solutions were buffered with 0.2 M Trismaleate-NaOH to pH 6.8 (20.1). RESULTS
Lethal Potency and Symptomatology The lethal effect of the crude venom of N. m. mossambica and its derived toxic fractions, when injected into locusts, is shown in Table 1. A dose of 3-5 LDsO units causes a complete and irreversible paralysis of the locust 15-25 min after the injections. Locusts lying on their sides with extended hind legs can be indicated as a characteristic symptom. With sublethal doses test animals do not lie, but they appear phlegmatic in their mobility. Such a partial paralysis is generally followed by recovery. Effect on the Muscle Membrane When cardiotoxin was added to the bathing solution, the membrane resting potential of the muscle fiber decreased with a dose-dependent time course (Fig. 1). During the drop in the resting potential, the electrical responses elicited by stimulating the motor nerve were reduced in amplitude until only small excitatory junction potentials (ejps) could be evoked (Fig. 1, inset).
DEITMER,
234
PRIMOR, AND ZLOTKIN ,70 3 .60 f 5 50 9. ‘D ?i 40 2 a 30 : 20 j k 10 zj5
OJ
, 0
I
5
,
10
I
t (mid
0
‘5
FIG. 1. Time course of cardiotoxin effect on the membrane resting potential of the muscle fiber (4 &ml: open circles; 0.8 &ml: filled circles) and on the amplitude of the postsynaptic response (0.8 pgi ml: squares). Filled symbols represent same experiment and same muscle fiber. The arrow indicates application of cardiotoxin. Inset: Intracellular recordings: (a) control, and (b) 3, (c) 4, (d) 5, and (e) 10 min after application of 0.8 pg/rnl of cardiotoxin, (a,b,c) “Slow” and “fast” ejps; (d,e) only “fast” ejps. The postsynaptic response above a 40-mV amplitude (at a membrane potential of about 40 mV, 4 min after application of the toxin) consists of the ejp and the electrically excitable response; below a 40-mV amplitude, only ejps are evoked by nerve stimulation. Figures in inset are original registrations which are partially retouched for clarity.
This demonstrates, that action potentials were still conducted in the motor axons and were transmitted to the muscle, although the muscle fiber membrane was not able to generate action potentials due to the refractory effect of the depolarization. Continuous washing of the preparation with toxin-free saline for 30-60 min did not recover the membrane-depolarizing effect of the cardiotoxin. When the application of cardiotoxin was preceded by an increase in the external calcium concentration from 2 to IO mM, the depolarizing effect of the cardiotoxin was prevented (Fig. 2). Addition of 1 mM lanthanum to the external solution before application of the cardiotoxin also prevented depolarization of the membrane (Fig. 2). In the La-containing solution the electrical responses elicited by motor nerve stimulation rapidly decreased within 2 min. This was probably due to blockage of the neuromuscular transmission caused by lanthanum. Nerve-muscle preparations were dissected from two locusts which had been completely paralyzed by the injection of 5
and 10 pg of cardiotoxin, respectively. Measurements of the membrane resting potential as well as of the indirectly evoked electrical responses of different fibers revealed no effect of the injected toxin on the periphery: The resting and action potentials of the muscle fibers were found to be unchanged as compared with those of untreated animals. DISCUSSION
The cardiotoxins are a group of low molecular weight strongly basic proteins isolated from snake venoms devoid of enzymatic activity. They possess a wide range of pharmacological activities such as inducing contracture in vertebrate skeletal, smooth, and heart muscles, block of axonal conduction, local irritation, weak direct hemolytic activity and cytophatic action to tumor cell cultures (Lee et al., 1968, 1972, Chang et al., 1972, Condrea, 1974). To these various actions one may now add the high lethal potency of cardiotoxins to locusts. The lethal effect of the cobra venom and its cardiotoxic components is even stronger
EFFECTS OF COBRA CA~IOTOXIN - -704
0’
r 0
5
10 t fminf
i5
FIG. 2. The effect of lanthanum ions (1 mM: triangles) and of high calcium concentration ( 10 mM: filled circles) on the cardiotoxin-induced muscle membrane depolarization. After changing the calcium-rich saline to a normal calcium solution concentration (2 mM: open circles), card~otoxin (U~gjmi) caused a quick and drastic drop in the muscle membrane resting potential. in the same preparation and experiment. The arrow indicates application of cardiotoxin.
than that of potent scorpion venoms, which are known for their high specific toxicity to insects (Kamon and Shulov, 1963; Zlotkin et al., 1971). On the basis of the protein contents of the different Sephadex G-50 venom fractions (Zlotkin et al., 1975), it could have been calculated that the cardiotoxic fraction is responsible for about 90% of the crude venom’s lethality to the locusts (Table I). The cardiotoxin is about 6-7 times more potent to locusts than the phospholipatic fraction (Table l), which was the main lethal factor for fly larvae and isopods (Zlotkin et al., 1975). This may indicate the physiological-pharmacological diversity of arthropods. On the other hand, the neurotoxic fraction, which was inactive to fly larvae and isopods, was equally inactive to locusts. Cobra venom neurotoxins are known to paralyze vertebrates through a highly specific competitive blockage of the cholinergic receptors at the postsynaptic membrane at the neuromuscular junction (Changeux et al., 1970). The tolerance of arthropods to cobra neurotoxins is certainly due to the fact that their neuromuscular transmitters are noncholinergic (Gerschenfeld, 1973). The strong and relatively quick lethal and
ON THE LOCUST
235
paralytic action of cardiotoxin has actually suggested the investigation of its effect on the present nerve-muscle preparation. It was shown in the present investigation that cardiotoxin had a strong depolarizing effect on the fiber membrane of locust skeletal muscle. This was also demonstrated in frog skeletal muscles, in which this depolarization could have been antagonized by a high Ca concentration (Lee et al., 1968, 1972). Calcium ions have equally antagonized the axonal, hemolytic, and cytotoxic effects of cardiotoxin (Condrea, 1974). It was suggested that cardiotoxin may bind to the same sites on the membrane surface as do Ca ions. In locust muscle the membrane depolarization caused by cardiotoxin was prevented by a high Ca-concentration and also by 1 mM lanthanum. Recently it was shown that 1 mM La3+ blocked the Ca-dependent action potentials in insect skeletal muscle (Deitmer and Rathmayer, 1976), as was demonstrated for barnacle muscle fibers (Hagiwara and Takahashi, 1967). It was suggested there that La ions have a high affinity to “Ca binding sites” of the membrane surface. Electron microscopy on barnacle muscle fibers revealed that, indeed, La ions compete with Ca ions for the same binding sites (Henkart and Hagiwara, 1976). The ability of La ions to prevent the membrane-depolarizing effect of cardiotoxin, as shown in the present study, strongly supports the hypothesis that the cardiotoxin attaches to the “Ca binding sites” of the membrane. The question which we encountered was to what extent is the muscle membranedepolarizing effect due to ~ardiotoxin responsible for its paralyzing and lethal action to the whole animal. The experiments on preparations obtained from cardiotoxinenvenomated locusts demonstrate that the paralysis of the locust cannot be attributed to the muscle membrane depolarization. It is suggested, therefore, that the cardiotoxin lethal-paralytic action to the locusts may be due to effects on the central nervous system,
236
DEITMER,
PRIMOR, AND ZLOTKIN
This hypothesis, however, must be investigated by further experimentation. REFERENCES C. C., WEI, J. W., CHUANG, S. T., AND LEE, C. Y. 1972. Are the blockades of nerve conduction and depolarization of skeletal muscle induced by cobra venom due to phosphofi~se A, neurotoxin or cardiotoxin?J. Formosaa Med. Assoc., 71,323-327. CHANGEUX, J. P., KASAI, M., AND LEE, C. Y. 1970. Use of snake venom toxin to characterize the chofingeric receptor protein. Froc. Nat. Acad. Sci. USA, 67, 1241-1247. CONDREA. E. 1974. Membrane active polypeptides from snake venom: Cardiotoxins and haemocytotoxins. Experientia, 30, 121- 129. DEITMER, J. W., AND RATHMAYER, W. 1976. Calcium action potentials in larval muscle fibers of the moth Ephestia k~ha~el~a Z. (Lepidoptera). J. Camp.
CHANG,
Physiol. 112, GERSCHENFELD,
123-
132.
H. M. 1973. Chemical transmission in invertebrate central nervous system and neuromuscular junctions. Physiol. Rev., 53, 2-92. HAGIWARA, S., AND TAKAHASHI, K. 1967. Surface density of calcium ions and calcium spikes in the barnacle muscle fiber membrane. J. Gen. Physiol., 50, 583-601. HENKART, M., AND HAGIWARA, S. 1976. Localization of the Ca binding sites associated with the Ca spike in bernacle muscle. j. Membrane Bioi., 27, l-20. HOYLE. G. 1955. The anatomy and innervation of focust skeletal muscle. Proc. Roy Sot. London Ser. B. 143,281-292.
KAMON, E., AND SHULOV, A. 1963. Estimation of locust resistance to scorpion venom. J. Insect Physiol., 5, 206-214. LEE, J.
G. Y., CHANG. C. C., CHIU, T. H., S., TSENG, T. C., AND LEE, S.
Pharmacological from Formosan bergs Arch. LEE. C. Y.,
CHIU, P. Y. 1968.
properties of cardiotoxin isolated cobra venom. Naunyn Schmiede-
Pharmakol
Exp.
Pathol.,
259,360-374.
LIN, J. S., AND WEY. J. W. 1972. Identification of cardiotoxin with cobramine B, DLF. Toxin o! and cobra venom cytotoxin. in “Toxins of Animal and Plant Origins” (A. de Vries and E. Kochwa, eds.), Vol. 2, p. 307. Gordon and Breach, New York. Louw, A. I., 1974. The purification and properties of five neurotoxic polypeptides from Naja mossambica mossambica venom. Biochim. Biophys. Actu, 336, 470-480. PRIMOR, N., AND ZLOTKIN, E. 1977. Per OS toxicity of venoms and toxins to blotiies.In “Proceedings, 5th International Symposium on Toxinology, San Jose, Costa Rica, August 1976.” In press. REED, I,. J., AND MUENCH. H. 1938. A simple method for estimating fifty per cent end points. Amer. J. Hyg.. 27, 493-497. ZLOTKIN. E., FRAENKEL, LISSITZKY, S. 1971. The
G.. MIRANDA, F.. AND effect of scorpion venom on blowfly larvae: A new method for the evaluation of scorpion venom potency. Toxicon. 9, 1-8. ZLOTKIN, E. 1973. Chemistry of animal venoms. Experientia. 29, 1453ZLOTKIN, E., MENASH& F., AND L~SSITZKY.
arthropods Physiol.,
1466.
M., ROCHAT, H., MIRANDA, S. 1975. Protein toxic to in the venom of efapid snakes. J. Insect
21, 1605-1611.