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The effect of crotoxin on the longitudinal muscle-myenteric plexus preparation of the guinea pig ileum
NeuropharmacologyVol. 28, No. I, pp. 741-747, 1989
0028-3908/89 S3.00 + 0.00
Copyright8 1989PergamonPressplc
Printed in Great Britain. All rights reserved
THE EFFECT OF CROTOXIN ON THE LONGITUDINAL MUSCLE-MYENTERIC PLEXUS PREPARATION OF THE GUINEA PIG ILEUM ZILDA M. MUNIZ and C. R. DINIZ Departamento de Bioquimica e Imunologia, Instituto de Ciencias Biolbgicas, Universidade Federal de Minas Gerais, Av. AntBnio Carlos, 6627~CP2486, 30160-Belo Horizonte-MG, Brasil (Accepted 2 October 1988)
Summary-The effects of crotoxin, the neurotoxin of the venom of the South American rattlesnake (Croralus durissus terrificus), was studied by using the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. Crotoxin (0.024.0 PM) caused depression of the twitch response of the electrically stimulated preparation. This transitory depression depended on the concentration of crotoxin; since crotoxin diminished the output of acetylcholine, this depression may be due to the inhibition of the release of acetylcholine from the plexus. Crotoxin also induced an early contraction, followed by relaxation; as the contraction was inhibited by aspirin and indomethacin, it may have resulted from the release of prostaglandin. In addition, a late persistent contracture was observed after the early contraction. The contracture was resistant to blockage by muscarinic, histamine and serotonin antagonists, to hexamethonium, a non-depolarizing ganglionic blocking substance and to tetrodotoxin, a sodium channel blocker. The contracture was blocked by an elevated concentration of calcium (10 mM) and by verapamil, a calcium channel blocker. Ke)l words-acetylcholine,
crotoxin, myenteric muscle, neurotoxin, smooth muscle, snake venom.
Crotoxin, the major toxic component of the venom of the South American rattlesnake, Crotalus durissus terrificus, was the first snake neurotoxin to be isolated (Slotta and Fraenkel-Conrat, 1938). It is a complex of two different subunits (Riibsamen, Breithaupt and Habermann, 1971; Hendon and Fraenkel-Conrat, 1971), a basic protein which carries the phospholipase A, activity, component B (12.000D) and an acidic component, component A or crotapotin (lO.OOOD), which has no known catalytic properties and no toxicity by itself, but enhances the toxicity of component B (Riibsamen et al., 1971; Hendon and Fraenkel-Conrat, 1971; Breithaupt, Riibsamen and Habermann, 1974; Breithaupt, Omori-Satoh and Lang, 1975; Breithaupt, 1976a; Eaker, 1978; Fraenkel-Conrat, 1982; 1983). In the case of the vertebrate neuromuscular junction, crotoxin acts presynaptically as it interferes with the release of acetylcholine (ACh) from the motor nerve terminals (Vital Brazil and Excell, 1971; Hawgood and Smith, 1977; Chang and Lee, 1977). This mechanism underlines the blockade of the skeletal neuromuscular transmission and the resulting peripheral paralysis (Vital Brazil, 1966; Vital Brazil, Franceschi and Waisbich, 1966; Vital Brazil and Excell, 1971; Chang and Lee, 1977; Hawgood and Smith, 1977). The present study concerned the effect of crotoxin in the myenteric plexus-longitudinal muscle preparation of the guinea pig. It was undertaken to clarify the action of crotoxin on smooth muscle and relate it, if possible, to its action on the skeletal neuro-
muscular junction. Slotta (1956) showed that crotoxin induces a transient contraction of the guinea pig ileum, which is antagonized by indomethacin (Saihara and Mendes, 1982). Muniz and Diniz (1983) confirmed these results and observed, in addition, a partial inhibition of the twitch responses in the electrically-stimulated myenteric plexus-longitudinal muscle preparation, caused by crotoxin. Similar results were also observed by Anad6n and MartinezLarrafiagua (1985). Evidence of a possible direct action of crotoxin upon the smooth muscle cells of the guinea pig ileum are shown here. In addition, further indications of presynaptic effects of crotoxin on this preparation are presented.
741
METHODS Drugs
Acetylcholine chloride, atropine sulphate, choline chloride, hexamethonium sulphate, morphine hydrochloride, yohimbine, (Merck); aspirin, eserine sulphate, indomethacin, mepyramine maleate, tetrodotoxin (Sigma); methysergide (Sandoz); verapamil (Knoll); Sephadex G-75 and G-25 (Pharmacia); carboxymethyl cellulose CM-52 (Whatman). Venom
The Crotalus durissus terrificus venom, used in this work, was obtained from snakes kept at the University Serpentarium, collected under ice, centrifuged and lyophyllized.
742
ZILDAM. MUNIZand C. R. DINIZ
Crotoxin
Both natural and reconstituted crotoxin was used in this work. Natural crotoxin was precipitated from Crotalus durissus terrificus venom, as described by Slotta and Fraenkel-Conrat (1938). Crotoxin was further purified by gel filtration in Sephadex G-75. Components A and B were separated by cationic chromatography from purified crotoxin and the complex (component A and B) was reconstituted as described by Riibsamen et al. (1971). The activity of phospholipase was assayed by the method of Vidal and Stoppani (1971). The toxicity of Crotalus durissus terrzjicus venom, crotoxin, component B and component A was determined by intraperitoneal injection of 0.1 ml of test solution per 10 g body weight into 20 g albino mice. All solutions were made in saline. Mortality during the first 24 hr was used for calculation. At least 30 mice were needed for each determination of the median lethal dose (LD,,), by the method of Klrber (1937). The values found were (in mg/kg): 0.164 f 0.016 for Crotalus durissus terrzjicus; 0.094 f 0.019 for crotoxin; 1.8 1 f 0.23 for component B; more than 10.0 for component A. The values for reconstituted crotoxin (component B + component A) was 0.087 Ifr 0.019 mg/kg. The results are in accordance with those of other authors (Hendon and Fraenkel-Conrat, 1971; Nakazone, Roger0 and Goncalves, 1984). Experimental procedure
The myenteric plexus-longitudinal muscle preparation of guinea pig (either sex) (30&500 g) was used in these experiments. After discarding 1Ocm of the terminal portion of the ileum (Munro, 1953), a 25 cm length of the remaining ileum was cut and its longitudinal muscle was dissected, as described by Paton and Zar (1968). The preparations (78 f 8 mg of tissue) were folded in order to provide for measurable release of ACh into the organ bath (3 ml). The length of the folded preparation was about 4-5 cm. For field stimulation, square-wave pulses of 0.5 msec duration, at a frequency of 0.08 or 10 Hz, were delivered through two platinum electrodes placed at the top and the bottom of the organ-bath by a stimulator made in this laboratory (Muniz, 1985). The strength of the stimulus was sufficient to produce a maximum contraction of the strip at 0.08 Hz. The contractions of the strips were recorded on a smoked kymograph with an auxotonic level (Paton, 1957; Paton, 1961), magnification 1 x 8, load 0.1 g/cm deflection on the kymograph. An initial load of about 0.5 g was used, and the heights of contractions were measured from the base line so obtained. The preparation was bathed in a modified Krebs-Ringer solution of the following composition (mM); NaCl 113, KC1 4.7, CaCl, 2.5, KH,PO, 1.2, MgSO, 1.2, NaHCO, 25, glucose 11 and choline chloride 0.02; it was kept at 37°C and bubbled with 95% O2 and 5% COz. For the study of the effect of
ions on the action of crotoxin, the composition of the bathing fluid was modified as shown in “Results”. To avoid precipitation when the concentration of CaCl; was raised to 10 mM., KH,PO., was omitted from the solution and the concentration of NaHCO, was reduced to 12.5mM. In these circumstances the pH measured was maintained constant at pH 7.4 f 0.2. The preparations were allowed to equilibrate in the bathing fluid for approximately 60 min before the initiation of the experiment. The ACh released by electrical stimulation was assayed on segments of the guinea pig ileum (Paton and Zar, 1968; Paton and Vizi, 1969). Calculation and statistics: the height of the twitch responses and the shift of the base line of the myenteric plexus-longitudinal muscle preparation, after the addition of crotoxin and other substances, were used to assess quantitatively their effects in regard to the controls. The height of twitch responses of the preparation measured in mm before the addition of toxin or drugs, was used as the control (100%). The results were reported as the mean f SD. Statistical differences were evaluated by NewmannKeuls test (Snedecor and Cochran, 1980). The level for significance was set at P < 0.05.
RESULTS Exposure of the preparation to crotoxin, at concentrations of O.O6--4pM, caused an early transient contraction of the muscle, observed about 1 min after the addition of crotoxin (Figs 1B and D), followed by a long lasting contracture of 2-4 hr. Field stimulation at a low frequency (0.08 Hz) produced twitch responses of the preparation (Fig. lA), that were constant for 4 hr or more. Exposure of stimulated strips to crotoxin produced depression of the twitch, within 2-5 min, that recovered slowly even in the course of continued exposure to the toxin (Fig. 1B). These results were also observed in the presence of Crotalus durissus terrtjicus venom (20 pg/ml, 3 experiments) and component B. The contractile effects (early transient contraction and long lasting contraction), induced by these substances, could also be observed in non stimulated preparations (Fig. 1D). Crotoxin-induced early transient contraction and twitch depression were concentration-dependent (Table 1). There was no dose-effect relationship in the case of the long lasting contraction (Table 1). It was necessary to increase the concentration of component B (l-20 PM) in the bath to obtain twitch depression and long lasting contraction of the same magnitude as that reached with crotoxin; however equal concentration of crotoxin and component B induced an early transient contraction response of the same magnitude. Component A, employed in concentrations of l-8 p M (6 experiments), showed no effects in this preparation. Reconstituted crotoxin behaved like natural crotoxin (Table 1).
Crotoxin and the myenteric preparation
I60
Ii0
$0
6
TIME (mint
743
so
Ii0
LTOX,N
TIME(minl
D
C
$0
6
Ii0 TIME
(minj
d0
I20 TIME I min J
iOTO,lN
Fig. 1. Typical response of an innervated longitudinal muscle strip from the guinea pig ileum preparation in the absence and presence of crotoxin. In (A) and (B), the preparations were stimulated electrically and
in (C) and (D) they were not stimulated electrically. The twitch responses were generated at every 12set following field stimulation (single pulses, 0.5 msec duration, 0.08 Hz, supramaximal intensity). Crotoxin (1.0 PM) was added as indicated (B and D). The experiment shown in (B) is representative of the data of Table 1.
Once stimulated by the toxin (l-2 PM), the muscle became insensitive to further additions of crotoxin (l-4 PM) and remained so, even after the washing out of the preparation for 1 hr in normal bathing fluid, although the response to BaCI, (0.1 mg, 2 experiments), ACh (S-50 ng, 5 experiments) and histamine (10-100 ng, 4 experiments) were not changed. The crotoxin-induced twitch depression seemed to be of a presynaptic nature, since ACh, histamine and BaCl,-evoked contraction remained unaffected by crotoxin. The output of ACh evoked by a low rate of stimulation (0.08 Hz) was partially depressed by crotoxin; at 1 PM, the inhibition amounted to 18.5 + 12% (mean f SD, 6 experiments), during the initial 30min of mcubation with crotoxin. At high rate of stimulation (10 Hz), the output of ACh was markedly depressed by crotoxin used in a concentration of 1 to 4pM (Fig. 2). The ability of various drugs, which interfere with neuromuscular transmission or muscle contractility, to alter the effects of crotoxin was determined. PreTable 1. Dose-effect of crotoxin on the guinea pig ileum, stimulated as in Figure l(C). Values in the Table represent the percentage of the control twitch response Concentration Crotoxin l&M) 0.06 0.12 0.25 0.50 1.oo l.OO(‘) 2.00 4.00
n
EC TD LC (percentage of control twitch height)
3 3 3 4 7 3 5 4
10 f 5 17k4 20 + 7 22 f 5 25 k 9 28 + 8 37 * 9 41*9
n-number of experiments. (*)-reconstituted crotoxin. Values are mean + SD. EC = early transient contraction; LC = long-lasting contraction.
9*5 17*4 24 + 5 26 f 8 29 f 7 32 + 6 33 k 8 4159
TD = depression
44 f 27 47+ 17 49k 16 55& 11 42 & 21 45+ 14 51 * 17 50+ 14
of twitch;
treatment (30-60min) of the preparation with the a-adrenoreceptor antagonist, yohimbine (0.1-2 PM), the A, receptor antagonist, mepyramine (0.01-2 PM), the serotonin receptor antagonist, methysergide (0.03-l PM) and a non-depolarizing ganglionic blocking agent, hexamethonium (3-30 p M), did not significantly change the early transient contraction,
2 F 2
60 40-
I ’
1
20
1
60
100 Crotolln
140
I80
I
220
TIME (min)
Fig. 2. Inhibition by crotoxin of the output of acetylcholine evoked by electrical stimulation. Longitudinal muscle strip of the guinea pig ileum in eserinized Krebs solution. The rates of release of acetylcholine, in the absence and presence of crotoxin, have been expressed as a percentage of the initial rate of the output of acetylcholine. The preparation was stimulated for periods of 1 min at 10 Hz, at intervals of 10 min. Crotoxin was present as indycateci. O---0, Control; l -e crotoxin 1 PM; A--A 2 PM; A--A 4pM. The values of the output of acetylcholine (%) are mean f SD of 3 experiments, except at crotoxin 4 pM, which represents only 1 experiment.
ZILDA
744
M. MUNIZand C. R.
Table 2. Effects of ions and drugs on the action of crotoxin (1 PM) on the myenteric plexus longitudinal muscle preparations. All strips were stimulated as in Figure I(C). The values in the Table represent the percentage of the control twitch response
Treatment Crotoxin (1 PM) + 1. Krebs 2. 1OmM Ca*+ 3. 0.62 mM Cal+ 4. IOmM Mg*+ 5. Indomethacin (30pM) 6. Aspirin (0.2 mM) 7. ‘l-fX (1.6 PM) 8. Atropine (0.17 PM) 9. Yohimbine (O.lOpM) 10. Mepyramine (0.02 PM) 1I. Methysergide (0.03 pM) 12. Hexamethonium (30 FM)
n
EC (pe*centagTeDf contr? twitch height)
I
25 * 9
29 If 7
42k21
6 3 3 5 3 3 3 3 3 3 3
0.0’ 85k14’ I1 f8 0.0. 0.0. o.o* 28k3 27k 12 26514 2159 22 f 8
39f2 3559 39 f 20 28k 17 32k 12
0.01 7a+11* 43 f 23 43* 12 39k 18 42 f 6 62+21 56+21 40+11 30 * 7 55i 15
23k4 25klO 19 + 13 26k9
n-number of experiments. Values are mean k SD. *Significantly different from treatment 1 (Newman”, Kells-test, P < 0.05) TTX = tetrodotoxin; other abbreviations as in Table I.
the long-lasting contraction or the twitch depression (Table 2). Although atropine (0.01-l PM), the muscarinic receptor antagonist, blocked the electrically evoked twitch, it did not significantly alter the early transient contraction or the long-lasting contraction induced by crotoxin (Fig. 3A, Table 2). Tetrodotoxin (0.05-0.5 PM), the sodium channel blocker, prevented the twitch responses induced by electrical stimulation and the early transient contraction elicited by crotoxin. However, the long-lasting contraction was not affected (Fig. 3B, Table 2). The cyclooxygenase inhibitors indomethacin (5-30 PM) and aspirin (0.1-0.3 mM) prevented the early transient contraction, but did not affect the longlasting contraction or the twitch depression (Fig. 3C, Table 2). Verapamil (0.5-4 PM), a calcium channel blocker, prevented the early transient contraction and the long-lasting contraction in addition to abolishing the twitch response (Fig. 4A). The influence of calcium and magnesium on the effects of crotoxin (1 p M) on the longitudinal muscle preparation was also tested. In the presence of 10 mM calcium, both the early transient contraction and the long-lasting contraction evoked by crotoxin were blocked (Fig. 4B, Table 2). In the preparation equilibrated with 10 mM of magnesium (Table 2), crotoxin caused the usual response. When the concentration of calcium was reduced to 0.62 mM, the early transient contraction and the long-lasting contraction were significantly increased (Table 2). Crotoxin did not produce the early transient contraction and the long-lasting contraction in preparations equilibrated with a physiological solution, which did not contain calcium; upon addition of 0.62 mM calcium, crotoxin elicited the long-lasting contraction (Fig. 4C). Crotoxin also produced the long lasting contraction in the preparation, equilibrated in a depolarizing solution, in which all the NaCl of the Krebs solution was replaced by an equimolar concentration of KCl.
DINIZ
In the myenteric plexus-longitudinal muscle preparation, crotoxin caused an inhibition of the electrically-induced response of the preparation. The inhibition started rapidly and was spontaneously reversible, in spite of the continued presence of the toxin. Anadon and Martinez-Larrafiagua (1985) and Muniz (1985) found similar results using this preparation. Crotoxin did not act by reducing the excitability or the contractility of the smooth muscle, since no reduction in the response of the tissue to several spasmogens, was observed. In this work it was also observed that the output of ACh, evoked by high frequency stimulation, was markedly depressed by crotoxin, indicating that inhibition of electricallyinduced twitch was presynaptic in nature. Crotoxin also blocked the output of ACh induced by tityustoxin (De Lima and Diniz, 1985), a scorpion venom toxin, which acts presynaptically, through a
A
. t ATrow!E CROrnXlN
. f
INDOYtt
Y
ca0mx1*
Ii0
cb
nut (mia)
i
Ii0 nut 1-d
Fig. 3. (A) Effect of atropine on the action of crotoxin on the innervated longitudinal muscle preparation, stimulated as in Figure l(C). The preparation was exposed to atropine (1 PM) and, 15 min later, crotoxin (1 PM) was added to the bath as indicated. (B) Effect of tetrodotoxin (TTX) on the action of crotoxin on the innervated longitudinal muscle preparation stimulated as in Figure l(C). The preparation was exposed to tetrodotoxin (0.5 PM) and, 15 min later, crotoxin (2pM) was added to the bath as indicated. (C) Effect of indomethacin on the early contraction of the innervated longitudinal muscle preparation, stimulated as in Figure l(C). The preparation was exposed to indomethacin (30 PM) throughout the experiment. Crotoxin (1 .OPM) was added to the bath as indicated.
Crotoxin and the myenteric preparation A
M.
t s0
VERAPAMIL CROTOXIN
6
T1t.e (mid
B
. ;
CRORJXIN
‘.
0 t CC 0,82mM
si,
Ii0 t 6 CRodxlw Cal’ 0,62mM
60
i0
TIME [min)
tie
2io 4 tlwlmln)
Fig. 4. (A) Effect of verapamil on the contracture, induced by crotoxin, in an isolated innervated longitudinal muscle strip from the guinea pig ileum preparation, stimulated as in Figure l(C). The preparation was exposed to crotoxin (1 FM) and, 20 min later, verapamil (4.4 FM) was added to the bath as indicated. The complete inhibition of the twitch contraction and the contracture induced by crotoxin can be seen. (B) Effect of calcium 10 mM, on the early contraction and contracture, induced by crotoxin in an isolated innervated longitudinal muscle strip from the guinea pig ileum preparation, stimulated as in Figure l(C). The preparation was exposed to 10 mM calcium-containing Krebs solution throughout the experiment. Crotoxin (1 PM) was added to the bath as indicated. (C) Requirement of calcium for the long-lasting contraction, induced by crotoxin on isolated innervated longitudinal muscle strips from the guinea pig ileum preparation, After pre-incubation for 60 min in Caz+-free physiological solution, Ca*+ to reach a concentration of 0.62 mM was added (7). A contraction that was abolished after the preparation was washed with Ca*+-free physiological solution (1) can be seen. Crotoxin (1 PM) was added to the bath as indicated (0). Addition of Ca*+ to reach a concentration of 0.62 mM was again added (t ). The sustained contraction, was produced.
sodium channel mechanism. Hexamethonium did not alter the depression of the twitch induced by crotoxin, which indicates that its site of action is probably the postganglionic cholinergic nerve terminal. This inhibition cannot be explained by invoking the participation of catecholamines, prostaglandins or serotonin. The possibility that other autacoids might be the mediator of this effect has not been excluded.
745
A slow, and not sustained, contraction of the longitudinal muscle is observed after exposure of the preparation to crotoxin. This effect of crotoxin, first observed by Slotta (1956), is inhibited by indomethacin (Saihara and Mendes, 1982; Anadon and Martinez-Larrafiagua, 1985; Muniz, 1985). These results are confirmed in the present paper. Since this contraction was affected by indomethacin and aspirin, which are inhibitors of cyclooxygenase, it may be mediated by a prostaglandin. This response was also sensitive to tetrodotoxin, an alkaloidal toxin, which abolishes neuronal activity and to verapamil, a potent calcium channel blocker (Rosenberger, Ticku and Triggle, 1979; Janis and Scriabine, 1983); however it was not significantly sensitive to atropine as has already been shown by Saihara and Mendes (1982), Muniz (1985) and Anadon and MartinezLarrafiagua (1985). Notexin (5 pg/ml), also a snake venom phospholipase A, neurotoxin, induces this type of contraction in this preparation (Harris and Zar, 1978). However, /I-bungarotoxin, another snake venom phospholipase A, neurotoxin, in concentrations as large as 20 pg/ml had no effect on the twitch response of the guinea pig ileum, stimulated coaxially, or on the contractility of the smooth muscle after 4 hr of treatment (Chang and Lee, 1963; Kato, Pinto, Glavinovic and Collier, 1977). Finally, the data presented in this paper show that crotoxin induced contracture of the guinea pig ileal longitudinal smooth muscle. This contracture occurred in the presence or absence of field stimulation of the preparation. It was often reversible and lasted for 224 hr. After recovery the responsiveness of the preparation to field stimulation, BaCl,, ACh and histamine was not changed. Crotoxin-induced contracture was blocked by a high level of calcium and by verapamil and was enhanced by a low level of calcium. The contracture was insensitive to atropine, suggesting that ACh is presumably not involved in this effect. It was also not significantly affected either by mepyramine or by methysergide, which suggests that the release of histamine and serotonin is not involved in this process. This effect of crotoxin, in contrast to the early contraction, was not inhibited either by indomethacin or by aspirin, thus suggesting that a prostaglandin is not envolved. The possibility that bradykinin, vasointestinal peptide (VIP), substance P or other related autacoids might be mediators of this effect, has not been excluded. However, the lack of effect of tetrodotoxin, which abolishes nerve-mediated responses in the ileum without suppressing the excitability of the muscle (Gershon, 1967), would argue against the release of neurotransmitter, mediated by neural activity. The requirement of calcium for the crotoxin-induced contracture suggests a Ca-dependent mechanism, as is also suggested by the finding that verapamil inhibited the contracture. It thus appears that crotoxin affects the smooth muscle cell directly. However crotoxin caused no apparent damage to the preparation, since the
ZILDA
746
M. MUNIZ and C. R.
responsiveness of the preparation, as indicated before, was not changed and the rates of release of lactate dehydrogenase, a soluble cytoplasmic constituent, were not affected by this toxin (data not shown). Although the direct effect of crotoxin on the skeletal muscle, at the time of neuromuscular block, is insignificant (Chang and Lee, 1977), large concentrations of and prolonged treatment with crotoxin affect the skeletal muscle directly, inducing the contracture (Breithaupt, 1976b). In the chick biventer cervices muscle, crotoxin and Component B evoked a slow progressive contracture, even in the presence of (+)-tubocurarine (Chang and Su, 1981). This crotoxin-induced contracture in the skeletal muscle, like the crotoxin-induced contracture in the longitudinal muscle-myenteric plexus, was abolished by increasing the concentration of calcium in the bath fluid, even after the contracture had developed.
The present studies demonstrate that the effects of crotoxin on smooth muscle, seen as depression of the twitch and contracture, were qualitatively similar to the action of this toxin upon the skeletal muscle and were probably caused by the same mechanisms. Acknowledgements-This work was supported by CNPq (Conselho National de Desenvolvimento Cientifico e Tecnologico), FINEP (Financiadora de Estudos e Projetos) and a fellowship from CAPEYCoordenacBo de Aperfeicoamento de Pessoal de Nivel Superior) (Muniz, Z. M.). We gratefully acknowledge the encouragement of, and helpful discussions with Professors Paulo S. BeirHo and Dalton L. Ferreira-Alves.
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p. 41. Programas e resumos Pirassununna. Slotta K. (i956) Further experiments on Erotoxin. In: Venoms (Buckley E. and Pornes N.. Eds). DD. 253-257. American Association for the Advancement* of Science Publications, Washington. Slotta K. and Fraenkel-Conrat H. L. (1938). Schlangen-
Crotoxin and the myenteric preparation gifte, III: Mitteihmg: Reinigung und Krystallisation des Klapperschlangen-giftes. Ber. Drch. Chem. Ges. 71: 107&1081.
Snedecor G. W. and Cochran W. G. (1980) Statistical Methoak 7th edn. The Iowa State University Press, Ames. Vidal J. C. and Stoppani A. 0. M. (1971) Isolation and purification of two phospholipases A from Bothrops Venoms, Arch. biochem. Biophys. 145: 543-556. Vital Brazil 0. (1966) Pharmacology of crystalline crotoxin.
747
II. Neuromuscular blocking action. Mem. Inst. Buranran 33: 98 l-992. Vital Brazil 0. and Excel1 B. J. (1971) Action of crotoxin and crotaction from the venom of Crotalus durissus terrificus (South American rattlesnake) on the frog neuromuscular junction, J. Physiol. 212: 3435P. Vital Brazil O., Franceschi J. P., Waisbich E. (1966) Pharmacology of crystalline crotoxin. I. Toxicity. Mem. Inst. Butantan 33: 973-980.
0028-3908/89 S3.00 + 0.00
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THE EFFECT OF CROTOXIN ON THE LONGITUDINAL MUSCLE-MYENTERIC PLEXUS PREPARATION OF THE GUINEA PIG ILEUM ZILDA M. MUNIZ and C. R. DINIZ Departamento de Bioquimica e Imunologia, Instituto de Ciencias Biolbgicas, Universidade Federal de Minas Gerais, Av. AntBnio Carlos, 6627~CP2486, 30160-Belo Horizonte-MG, Brasil (Accepted 2 October 1988)
Summary-The effects of crotoxin, the neurotoxin of the venom of the South American rattlesnake (Croralus durissus terrificus), was studied by using the myenteric plexus-longitudinal muscle preparation of the guinea pig ileum. Crotoxin (0.024.0 PM) caused depression of the twitch response of the electrically stimulated preparation. This transitory depression depended on the concentration of crotoxin; since crotoxin diminished the output of acetylcholine, this depression may be due to the inhibition of the release of acetylcholine from the plexus. Crotoxin also induced an early contraction, followed by relaxation; as the contraction was inhibited by aspirin and indomethacin, it may have resulted from the release of prostaglandin. In addition, a late persistent contracture was observed after the early contraction. The contracture was resistant to blockage by muscarinic, histamine and serotonin antagonists, to hexamethonium, a non-depolarizing ganglionic blocking substance and to tetrodotoxin, a sodium channel blocker. The contracture was blocked by an elevated concentration of calcium (10 mM) and by verapamil, a calcium channel blocker. Ke)l words-acetylcholine,
crotoxin, myenteric muscle, neurotoxin, smooth muscle, snake venom.
Crotoxin, the major toxic component of the venom of the South American rattlesnake, Crotalus durissus terrificus, was the first snake neurotoxin to be isolated (Slotta and Fraenkel-Conrat, 1938). It is a complex of two different subunits (Riibsamen, Breithaupt and Habermann, 1971; Hendon and Fraenkel-Conrat, 1971), a basic protein which carries the phospholipase A, activity, component B (12.000D) and an acidic component, component A or crotapotin (lO.OOOD), which has no known catalytic properties and no toxicity by itself, but enhances the toxicity of component B (Riibsamen et al., 1971; Hendon and Fraenkel-Conrat, 1971; Breithaupt, Riibsamen and Habermann, 1974; Breithaupt, Omori-Satoh and Lang, 1975; Breithaupt, 1976a; Eaker, 1978; Fraenkel-Conrat, 1982; 1983). In the case of the vertebrate neuromuscular junction, crotoxin acts presynaptically as it interferes with the release of acetylcholine (ACh) from the motor nerve terminals (Vital Brazil and Excell, 1971; Hawgood and Smith, 1977; Chang and Lee, 1977). This mechanism underlines the blockade of the skeletal neuromuscular transmission and the resulting peripheral paralysis (Vital Brazil, 1966; Vital Brazil, Franceschi and Waisbich, 1966; Vital Brazil and Excell, 1971; Chang and Lee, 1977; Hawgood and Smith, 1977). The present study concerned the effect of crotoxin in the myenteric plexus-longitudinal muscle preparation of the guinea pig. It was undertaken to clarify the action of crotoxin on smooth muscle and relate it, if possible, to its action on the skeletal neuro-
muscular junction. Slotta (1956) showed that crotoxin induces a transient contraction of the guinea pig ileum, which is antagonized by indomethacin (Saihara and Mendes, 1982). Muniz and Diniz (1983) confirmed these results and observed, in addition, a partial inhibition of the twitch responses in the electrically-stimulated myenteric plexus-longitudinal muscle preparation, caused by crotoxin. Similar results were also observed by Anad6n and MartinezLarrafiagua (1985). Evidence of a possible direct action of crotoxin upon the smooth muscle cells of the guinea pig ileum are shown here. In addition, further indications of presynaptic effects of crotoxin on this preparation are presented.
741
METHODS Drugs
Acetylcholine chloride, atropine sulphate, choline chloride, hexamethonium sulphate, morphine hydrochloride, yohimbine, (Merck); aspirin, eserine sulphate, indomethacin, mepyramine maleate, tetrodotoxin (Sigma); methysergide (Sandoz); verapamil (Knoll); Sephadex G-75 and G-25 (Pharmacia); carboxymethyl cellulose CM-52 (Whatman). Venom
The Crotalus durissus terrificus venom, used in this work, was obtained from snakes kept at the University Serpentarium, collected under ice, centrifuged and lyophyllized.
742
ZILDAM. MUNIZand C. R. DINIZ
Crotoxin
Both natural and reconstituted crotoxin was used in this work. Natural crotoxin was precipitated from Crotalus durissus terrificus venom, as described by Slotta and Fraenkel-Conrat (1938). Crotoxin was further purified by gel filtration in Sephadex G-75. Components A and B were separated by cationic chromatography from purified crotoxin and the complex (component A and B) was reconstituted as described by Riibsamen et al. (1971). The activity of phospholipase was assayed by the method of Vidal and Stoppani (1971). The toxicity of Crotalus durissus terrzjicus venom, crotoxin, component B and component A was determined by intraperitoneal injection of 0.1 ml of test solution per 10 g body weight into 20 g albino mice. All solutions were made in saline. Mortality during the first 24 hr was used for calculation. At least 30 mice were needed for each determination of the median lethal dose (LD,,), by the method of Klrber (1937). The values found were (in mg/kg): 0.164 f 0.016 for Crotalus durissus terrzjicus; 0.094 f 0.019 for crotoxin; 1.8 1 f 0.23 for component B; more than 10.0 for component A. The values for reconstituted crotoxin (component B + component A) was 0.087 Ifr 0.019 mg/kg. The results are in accordance with those of other authors (Hendon and Fraenkel-Conrat, 1971; Nakazone, Roger0 and Goncalves, 1984). Experimental procedure
The myenteric plexus-longitudinal muscle preparation of guinea pig (either sex) (30&500 g) was used in these experiments. After discarding 1Ocm of the terminal portion of the ileum (Munro, 1953), a 25 cm length of the remaining ileum was cut and its longitudinal muscle was dissected, as described by Paton and Zar (1968). The preparations (78 f 8 mg of tissue) were folded in order to provide for measurable release of ACh into the organ bath (3 ml). The length of the folded preparation was about 4-5 cm. For field stimulation, square-wave pulses of 0.5 msec duration, at a frequency of 0.08 or 10 Hz, were delivered through two platinum electrodes placed at the top and the bottom of the organ-bath by a stimulator made in this laboratory (Muniz, 1985). The strength of the stimulus was sufficient to produce a maximum contraction of the strip at 0.08 Hz. The contractions of the strips were recorded on a smoked kymograph with an auxotonic level (Paton, 1957; Paton, 1961), magnification 1 x 8, load 0.1 g/cm deflection on the kymograph. An initial load of about 0.5 g was used, and the heights of contractions were measured from the base line so obtained. The preparation was bathed in a modified Krebs-Ringer solution of the following composition (mM); NaCl 113, KC1 4.7, CaCl, 2.5, KH,PO, 1.2, MgSO, 1.2, NaHCO, 25, glucose 11 and choline chloride 0.02; it was kept at 37°C and bubbled with 95% O2 and 5% COz. For the study of the effect of
ions on the action of crotoxin, the composition of the bathing fluid was modified as shown in “Results”. To avoid precipitation when the concentration of CaCl; was raised to 10 mM., KH,PO., was omitted from the solution and the concentration of NaHCO, was reduced to 12.5mM. In these circumstances the pH measured was maintained constant at pH 7.4 f 0.2. The preparations were allowed to equilibrate in the bathing fluid for approximately 60 min before the initiation of the experiment. The ACh released by electrical stimulation was assayed on segments of the guinea pig ileum (Paton and Zar, 1968; Paton and Vizi, 1969). Calculation and statistics: the height of the twitch responses and the shift of the base line of the myenteric plexus-longitudinal muscle preparation, after the addition of crotoxin and other substances, were used to assess quantitatively their effects in regard to the controls. The height of twitch responses of the preparation measured in mm before the addition of toxin or drugs, was used as the control (100%). The results were reported as the mean f SD. Statistical differences were evaluated by NewmannKeuls test (Snedecor and Cochran, 1980). The level for significance was set at P < 0.05.
RESULTS Exposure of the preparation to crotoxin, at concentrations of O.O6--4pM, caused an early transient contraction of the muscle, observed about 1 min after the addition of crotoxin (Figs 1B and D), followed by a long lasting contracture of 2-4 hr. Field stimulation at a low frequency (0.08 Hz) produced twitch responses of the preparation (Fig. lA), that were constant for 4 hr or more. Exposure of stimulated strips to crotoxin produced depression of the twitch, within 2-5 min, that recovered slowly even in the course of continued exposure to the toxin (Fig. 1B). These results were also observed in the presence of Crotalus durissus terrtjicus venom (20 pg/ml, 3 experiments) and component B. The contractile effects (early transient contraction and long lasting contraction), induced by these substances, could also be observed in non stimulated preparations (Fig. 1D). Crotoxin-induced early transient contraction and twitch depression were concentration-dependent (Table 1). There was no dose-effect relationship in the case of the long lasting contraction (Table 1). It was necessary to increase the concentration of component B (l-20 PM) in the bath to obtain twitch depression and long lasting contraction of the same magnitude as that reached with crotoxin; however equal concentration of crotoxin and component B induced an early transient contraction response of the same magnitude. Component A, employed in concentrations of l-8 p M (6 experiments), showed no effects in this preparation. Reconstituted crotoxin behaved like natural crotoxin (Table 1).
Crotoxin and the myenteric preparation
I60
Ii0
$0
6
TIME (mint
743
so
Ii0
LTOX,N
TIME(minl
D
C
$0
6
Ii0 TIME
(minj
d0
I20 TIME I min J
iOTO,lN
Fig. 1. Typical response of an innervated longitudinal muscle strip from the guinea pig ileum preparation in the absence and presence of crotoxin. In (A) and (B), the preparations were stimulated electrically and
in (C) and (D) they were not stimulated electrically. The twitch responses were generated at every 12set following field stimulation (single pulses, 0.5 msec duration, 0.08 Hz, supramaximal intensity). Crotoxin (1.0 PM) was added as indicated (B and D). The experiment shown in (B) is representative of the data of Table 1.
Once stimulated by the toxin (l-2 PM), the muscle became insensitive to further additions of crotoxin (l-4 PM) and remained so, even after the washing out of the preparation for 1 hr in normal bathing fluid, although the response to BaCI, (0.1 mg, 2 experiments), ACh (S-50 ng, 5 experiments) and histamine (10-100 ng, 4 experiments) were not changed. The crotoxin-induced twitch depression seemed to be of a presynaptic nature, since ACh, histamine and BaCl,-evoked contraction remained unaffected by crotoxin. The output of ACh evoked by a low rate of stimulation (0.08 Hz) was partially depressed by crotoxin; at 1 PM, the inhibition amounted to 18.5 + 12% (mean f SD, 6 experiments), during the initial 30min of mcubation with crotoxin. At high rate of stimulation (10 Hz), the output of ACh was markedly depressed by crotoxin used in a concentration of 1 to 4pM (Fig. 2). The ability of various drugs, which interfere with neuromuscular transmission or muscle contractility, to alter the effects of crotoxin was determined. PreTable 1. Dose-effect of crotoxin on the guinea pig ileum, stimulated as in Figure l(C). Values in the Table represent the percentage of the control twitch response Concentration Crotoxin l&M) 0.06 0.12 0.25 0.50 1.oo l.OO(‘) 2.00 4.00
n
EC TD LC (percentage of control twitch height)
3 3 3 4 7 3 5 4
10 f 5 17k4 20 + 7 22 f 5 25 k 9 28 + 8 37 * 9 41*9
n-number of experiments. (*)-reconstituted crotoxin. Values are mean + SD. EC = early transient contraction; LC = long-lasting contraction.
9*5 17*4 24 + 5 26 f 8 29 f 7 32 + 6 33 k 8 4159
TD = depression
44 f 27 47+ 17 49k 16 55& 11 42 & 21 45+ 14 51 * 17 50+ 14
of twitch;
treatment (30-60min) of the preparation with the a-adrenoreceptor antagonist, yohimbine (0.1-2 PM), the A, receptor antagonist, mepyramine (0.01-2 PM), the serotonin receptor antagonist, methysergide (0.03-l PM) and a non-depolarizing ganglionic blocking agent, hexamethonium (3-30 p M), did not significantly change the early transient contraction,
2 F 2
60 40-
I ’
1
20
1
60
100 Crotolln
140
I80
I
220
TIME (min)
Fig. 2. Inhibition by crotoxin of the output of acetylcholine evoked by electrical stimulation. Longitudinal muscle strip of the guinea pig ileum in eserinized Krebs solution. The rates of release of acetylcholine, in the absence and presence of crotoxin, have been expressed as a percentage of the initial rate of the output of acetylcholine. The preparation was stimulated for periods of 1 min at 10 Hz, at intervals of 10 min. Crotoxin was present as indycateci. O---0, Control; l -e crotoxin 1 PM; A--A 2 PM; A--A 4pM. The values of the output of acetylcholine (%) are mean f SD of 3 experiments, except at crotoxin 4 pM, which represents only 1 experiment.
ZILDA
744
M. MUNIZand C. R.
Table 2. Effects of ions and drugs on the action of crotoxin (1 PM) on the myenteric plexus longitudinal muscle preparations. All strips were stimulated as in Figure I(C). The values in the Table represent the percentage of the control twitch response
Treatment Crotoxin (1 PM) + 1. Krebs 2. 1OmM Ca*+ 3. 0.62 mM Cal+ 4. IOmM Mg*+ 5. Indomethacin (30pM) 6. Aspirin (0.2 mM) 7. ‘l-fX (1.6 PM) 8. Atropine (0.17 PM) 9. Yohimbine (O.lOpM) 10. Mepyramine (0.02 PM) 1I. Methysergide (0.03 pM) 12. Hexamethonium (30 FM)
n
EC (pe*centagTeDf contr? twitch height)
I
25 * 9
29 If 7
42k21
6 3 3 5 3 3 3 3 3 3 3
0.0’ 85k14’ I1 f8 0.0. 0.0. o.o* 28k3 27k 12 26514 2159 22 f 8
39f2 3559 39 f 20 28k 17 32k 12
0.01 7a+11* 43 f 23 43* 12 39k 18 42 f 6 62+21 56+21 40+11 30 * 7 55i 15
23k4 25klO 19 + 13 26k9
n-number of experiments. Values are mean k SD. *Significantly different from treatment 1 (Newman”, Kells-test, P < 0.05) TTX = tetrodotoxin; other abbreviations as in Table I.
the long-lasting contraction or the twitch depression (Table 2). Although atropine (0.01-l PM), the muscarinic receptor antagonist, blocked the electrically evoked twitch, it did not significantly alter the early transient contraction or the long-lasting contraction induced by crotoxin (Fig. 3A, Table 2). Tetrodotoxin (0.05-0.5 PM), the sodium channel blocker, prevented the twitch responses induced by electrical stimulation and the early transient contraction elicited by crotoxin. However, the long-lasting contraction was not affected (Fig. 3B, Table 2). The cyclooxygenase inhibitors indomethacin (5-30 PM) and aspirin (0.1-0.3 mM) prevented the early transient contraction, but did not affect the longlasting contraction or the twitch depression (Fig. 3C, Table 2). Verapamil (0.5-4 PM), a calcium channel blocker, prevented the early transient contraction and the long-lasting contraction in addition to abolishing the twitch response (Fig. 4A). The influence of calcium and magnesium on the effects of crotoxin (1 p M) on the longitudinal muscle preparation was also tested. In the presence of 10 mM calcium, both the early transient contraction and the long-lasting contraction evoked by crotoxin were blocked (Fig. 4B, Table 2). In the preparation equilibrated with 10 mM of magnesium (Table 2), crotoxin caused the usual response. When the concentration of calcium was reduced to 0.62 mM, the early transient contraction and the long-lasting contraction were significantly increased (Table 2). Crotoxin did not produce the early transient contraction and the long-lasting contraction in preparations equilibrated with a physiological solution, which did not contain calcium; upon addition of 0.62 mM calcium, crotoxin elicited the long-lasting contraction (Fig. 4C). Crotoxin also produced the long lasting contraction in the preparation, equilibrated in a depolarizing solution, in which all the NaCl of the Krebs solution was replaced by an equimolar concentration of KCl.
DINIZ
In the myenteric plexus-longitudinal muscle preparation, crotoxin caused an inhibition of the electrically-induced response of the preparation. The inhibition started rapidly and was spontaneously reversible, in spite of the continued presence of the toxin. Anadon and Martinez-Larrafiagua (1985) and Muniz (1985) found similar results using this preparation. Crotoxin did not act by reducing the excitability or the contractility of the smooth muscle, since no reduction in the response of the tissue to several spasmogens, was observed. In this work it was also observed that the output of ACh, evoked by high frequency stimulation, was markedly depressed by crotoxin, indicating that inhibition of electricallyinduced twitch was presynaptic in nature. Crotoxin also blocked the output of ACh induced by tityustoxin (De Lima and Diniz, 1985), a scorpion venom toxin, which acts presynaptically, through a
A
. t ATrow!E CROrnXlN
. f
INDOYtt
Y
ca0mx1*
Ii0
cb
nut (mia)
i
Ii0 nut 1-d
Fig. 3. (A) Effect of atropine on the action of crotoxin on the innervated longitudinal muscle preparation, stimulated as in Figure l(C). The preparation was exposed to atropine (1 PM) and, 15 min later, crotoxin (1 PM) was added to the bath as indicated. (B) Effect of tetrodotoxin (TTX) on the action of crotoxin on the innervated longitudinal muscle preparation stimulated as in Figure l(C). The preparation was exposed to tetrodotoxin (0.5 PM) and, 15 min later, crotoxin (2pM) was added to the bath as indicated. (C) Effect of indomethacin on the early contraction of the innervated longitudinal muscle preparation, stimulated as in Figure l(C). The preparation was exposed to indomethacin (30 PM) throughout the experiment. Crotoxin (1 .OPM) was added to the bath as indicated.
Crotoxin and the myenteric preparation A
M.
t s0
VERAPAMIL CROTOXIN
6
T1t.e (mid
B
. ;
CRORJXIN
‘.
0 t CC 0,82mM
si,
Ii0 t 6 CRodxlw Cal’ 0,62mM
60
i0
TIME [min)
tie
2io 4 tlwlmln)
Fig. 4. (A) Effect of verapamil on the contracture, induced by crotoxin, in an isolated innervated longitudinal muscle strip from the guinea pig ileum preparation, stimulated as in Figure l(C). The preparation was exposed to crotoxin (1 FM) and, 20 min later, verapamil (4.4 FM) was added to the bath as indicated. The complete inhibition of the twitch contraction and the contracture induced by crotoxin can be seen. (B) Effect of calcium 10 mM, on the early contraction and contracture, induced by crotoxin in an isolated innervated longitudinal muscle strip from the guinea pig ileum preparation, stimulated as in Figure l(C). The preparation was exposed to 10 mM calcium-containing Krebs solution throughout the experiment. Crotoxin (1 PM) was added to the bath as indicated. (C) Requirement of calcium for the long-lasting contraction, induced by crotoxin on isolated innervated longitudinal muscle strips from the guinea pig ileum preparation, After pre-incubation for 60 min in Caz+-free physiological solution, Ca*+ to reach a concentration of 0.62 mM was added (7). A contraction that was abolished after the preparation was washed with Ca*+-free physiological solution (1) can be seen. Crotoxin (1 PM) was added to the bath as indicated (0). Addition of Ca*+ to reach a concentration of 0.62 mM was again added (t ). The sustained contraction, was produced.
sodium channel mechanism. Hexamethonium did not alter the depression of the twitch induced by crotoxin, which indicates that its site of action is probably the postganglionic cholinergic nerve terminal. This inhibition cannot be explained by invoking the participation of catecholamines, prostaglandins or serotonin. The possibility that other autacoids might be the mediator of this effect has not been excluded.
745
A slow, and not sustained, contraction of the longitudinal muscle is observed after exposure of the preparation to crotoxin. This effect of crotoxin, first observed by Slotta (1956), is inhibited by indomethacin (Saihara and Mendes, 1982; Anadon and Martinez-Larrafiagua, 1985; Muniz, 1985). These results are confirmed in the present paper. Since this contraction was affected by indomethacin and aspirin, which are inhibitors of cyclooxygenase, it may be mediated by a prostaglandin. This response was also sensitive to tetrodotoxin, an alkaloidal toxin, which abolishes neuronal activity and to verapamil, a potent calcium channel blocker (Rosenberger, Ticku and Triggle, 1979; Janis and Scriabine, 1983); however it was not significantly sensitive to atropine as has already been shown by Saihara and Mendes (1982), Muniz (1985) and Anadon and MartinezLarrafiagua (1985). Notexin (5 pg/ml), also a snake venom phospholipase A, neurotoxin, induces this type of contraction in this preparation (Harris and Zar, 1978). However, /I-bungarotoxin, another snake venom phospholipase A, neurotoxin, in concentrations as large as 20 pg/ml had no effect on the twitch response of the guinea pig ileum, stimulated coaxially, or on the contractility of the smooth muscle after 4 hr of treatment (Chang and Lee, 1963; Kato, Pinto, Glavinovic and Collier, 1977). Finally, the data presented in this paper show that crotoxin induced contracture of the guinea pig ileal longitudinal smooth muscle. This contracture occurred in the presence or absence of field stimulation of the preparation. It was often reversible and lasted for 224 hr. After recovery the responsiveness of the preparation to field stimulation, BaCl,, ACh and histamine was not changed. Crotoxin-induced contracture was blocked by a high level of calcium and by verapamil and was enhanced by a low level of calcium. The contracture was insensitive to atropine, suggesting that ACh is presumably not involved in this effect. It was also not significantly affected either by mepyramine or by methysergide, which suggests that the release of histamine and serotonin is not involved in this process. This effect of crotoxin, in contrast to the early contraction, was not inhibited either by indomethacin or by aspirin, thus suggesting that a prostaglandin is not envolved. The possibility that bradykinin, vasointestinal peptide (VIP), substance P or other related autacoids might be mediators of this effect, has not been excluded. However, the lack of effect of tetrodotoxin, which abolishes nerve-mediated responses in the ileum without suppressing the excitability of the muscle (Gershon, 1967), would argue against the release of neurotransmitter, mediated by neural activity. The requirement of calcium for the crotoxin-induced contracture suggests a Ca-dependent mechanism, as is also suggested by the finding that verapamil inhibited the contracture. It thus appears that crotoxin affects the smooth muscle cell directly. However crotoxin caused no apparent damage to the preparation, since the
ZILDA
746
M. MUNIZ and C. R.
responsiveness of the preparation, as indicated before, was not changed and the rates of release of lactate dehydrogenase, a soluble cytoplasmic constituent, were not affected by this toxin (data not shown). Although the direct effect of crotoxin on the skeletal muscle, at the time of neuromuscular block, is insignificant (Chang and Lee, 1977), large concentrations of and prolonged treatment with crotoxin affect the skeletal muscle directly, inducing the contracture (Breithaupt, 1976b). In the chick biventer cervices muscle, crotoxin and Component B evoked a slow progressive contracture, even in the presence of (+)-tubocurarine (Chang and Su, 1981). This crotoxin-induced contracture in the skeletal muscle, like the crotoxin-induced contracture in the longitudinal muscle-myenteric plexus, was abolished by increasing the concentration of calcium in the bath fluid, even after the contracture had developed.
The present studies demonstrate that the effects of crotoxin on smooth muscle, seen as depression of the twitch and contracture, were qualitatively similar to the action of this toxin upon the skeletal muscle and were probably caused by the same mechanisms. Acknowledgements-This work was supported by CNPq (Conselho National de Desenvolvimento Cientifico e Tecnologico), FINEP (Financiadora de Estudos e Projetos) and a fellowship from CAPEYCoordenacBo de Aperfeicoamento de Pessoal de Nivel Superior) (Muniz, Z. M.). We gratefully acknowledge the encouragement of, and helpful discussions with Professors Paulo S. BeirHo and Dalton L. Ferreira-Alves.
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the release of acetylcholine induced by Tityus serrulatus scorpion venom. Toxicon 23: 588. Eaker D. (1978) Studies of presynaptically neurotoxic and myotoxic phospholipase A,. In: Versatility ofproteins (Li C. H., Ed.), pp. 413431. Academic Press, New York. Fraenkel-Conrat H. (1982-83) Snake venom neurotoxins related to phospholipase A,. J. Toxic. Toxin Rev. 1: 205-22 1. Gershon M. D. (1967) Effects of tetrodotoxin on innervated smooth muscle preparations. J Pharmac. Chemother. 29: 259-279.
Harris J. B. and Zar M. A. (1978) The effects of a toxin isolated from Australian tiger snake (Notechis scutufus scutatus) venom on autonomic neuromuscular transmission Br. J. Phnrmac. 62: 349-358. Hawgood B. J. and Smith J. W. (1977) The mode of action at the mouse neuromuscular junction of the phospholipase A crotapotin complex isolated from venom of the South American rattlesnake. Br. J. Pharmac. 61: 607-614.
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Muniz 2. M. (1985) Efeito da crotoxina sobre a resposta contratil do musculo longitudinal de ileo de cobaia. MSc Thesis, Belo Horizonte, Instituto de Ciincias Biologicas da UFMG. 144~. Muniz Z. M. and Diniz C. R. (1983) Crotoxina afeta a resposta contratil do mlisculo liso induzida por estimulo eletrico de campo. Arq. Biol. Tech. 26: 279. Munro A. F. (1953) Effect of autonomic drugs on the responses of isolated preparations from the guinea pig intestine to electrical stimulation. J. Physiol. 120: 41-52. Nakazone A. K., Roger0 J. R., Gonpalves (1984) Crotoxin I. Immunology and interaction of the subunits. Braz. J. med. Biol. Res. 17: 119-128. Paton W. D. M. (1957) A pendulum auxotonic lever. J. Physiol. 137: 35-36P.
Paton W. D. M. (1961) A theory of drug action based on the rate of drug-receptor combination. Proc. R. Sot. BIW 21-69. Paton W. D. M. and Vizi E. S. (1969) The inhibitory action of noradrenaline and adrenaline on acetylcholine output by guinea-pig ileum longitudinal muscle strip. Br. J. Pharmac. 35: 10-28.
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Breithaupt H., Omori-Satoh T., Lang J. (1975) Isolation and characterization of three phospholipases A from the crotoxin complex. Biochim. biophys. Acta 403: 355-369. Breithaupt H., Rtlbsamen K., Habermann E. (1974) Biochemistry and pharmacology of the crotoxin complex. Eur. J. Biochem. 49: 333-345.
Chang C. C. and Lee C. Y. (1963) Isolation of neurotoxins from the venom of Bungarus mulGcinctus and their modes of neuromuscular blocking action. Arch. inc. Pharmacodyn. 144: 241-257. Chang C. C. and Lee J. D. (1977) Crotoxin, the neurotoxin of South American rattlesnake venom, is a presynaptic toxin acting like B-Bungarotoxin. Naunyn -Schmiedebergs Arch. Pharmac. 2%: 159-168.
Chang C. C. and Su M. J. (1981) A study on the interaction of crotapotin with crotoxin phospholipase A,, notexin and other presynaptic neurotoxins. Br. J. Pharmac. 73: 495-503. De Lima
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