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Action of Micrurus dumerilii carinicauda coral snake venom on the mammalian neuromuscular junction
Toxicon 40 (2002) 167±174
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Action of Micrurus dumerilii carinicauda coral snake venom on the mammalian neuromuscular junction Francine G. Sera®m a, Marielga Reali a, Maria Alice Cruz-Hoȯing b, Marcos D. Fontana a,* a
Departamento de Farmacologia, Faculdade de CieÃncias MeÂdicas, Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6111, CEP 13083-970, Campinas, SP, Brazil b Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6109, 13083-970, Campinas, SP, Brazil Received 4 April 2001; accepted 18 August 2001
Abstract The venoms of coral snakes (mainly Micrurus species) have pre- and/or postsynaptic actions, but only a few of these have been studied in detail. We have investigated the effects of Micrurus dumerilii carinicauda coral snake venom on neurotransmission in rat isolated phrenic nerve-diaphragm muscle and chick biventer cervicis preparations stimulated directly or indirectly. M. d. carinicauda venom (5 or 10 mg/ml) produced neuromuscular blockade in rat (85±90% in 291.8 ^ 7.3 min and 108.3 ^ 13.8, respectively; n 5) and avian (95.0 ^ 2.0 min; 5 mg/ml, n 5) preparations. Neostigmine (5.8 mM) and 3,4diaminopyridine (230 mM) partially reversed the venom-induced neuromuscular blockade in rat nerve-muscle preparations. In neither preparation did the venom depress the twitch response elicited by direct muscle stimulation. The contractures induced by acetylcholine in chick preparations were inhibited by the venom (95±100%; n 4; p , 0.05). In rat preparations, the venom produced a progressive decrease in the amplitude of miniature end-plate potentials (m.e.p.ps control frequency 69.3 ^ 5.0/ min and control amplitude 0.4 ^ 0.2 mV) until these were abolished. Neostigmine (5.8 mM) and 3,4-diaminopyridine (230 mM) partially antagonized this blockade of m.e.p.ps. The resting membrane potential was not altered with the venom (10 mg/ml). M. d. carinicauda venom produced dose-dependent morphological changes in indirectly stimulated mammal preparations. Twenty-®ve per cent of muscle ®bers were affected by a venom concentration of 5 mg/ml, whilst 60.7% were damaged by 10 mg of venom/ml. In biventer cervicis preparations, the morphological changes were slower in onset and were generally characterized by undulating ®bers and, to a lesser extent, by zones of disintegrating myo®brils. A venom concentration of 5 mg/ml damaged 52.2% of the ®bers. These ®ndings indicate that M. d. carinicauda venom has neurotoxic and myotoxic effects and that the neuromuscular blockade involves mainly a postsynaptic action. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Blockade; Coral snake; M. d. carinicauda; Neuromuscular junction; Histological changes; Venom
1. Introduction The coral snakes comprise a group of about 120 species and subspecies of New World elapid snakes belonging primarily to the genus Micrurus (Roze, 1982, 1989; Roze and Bernal-Carlo, 1987). Envenoming by coral snakes results in progressive neuromuscular blockade, with death * Corresponding author. Tel.: 155-19-3788-7482; fax: 155-193289-2968. E-mail address: [email protected] (M.D. Fontana).
resulting from respiratory arrest through the action of preand postsynapatic neurotoxins at the neuromuscular junction (Kellaway et al., 1932; Lee et al., 1972; Vital Brazil et al., 1976/77; Vital Brazil and Fontana, 1983/84). Several studies have also shown that coral snake venoms are myotoxic (Arroyo et al., 1987; GutieÂrrez et al., 1983; Goularte et al., 1995). Micrurus dumerilii carinicauda is a coral snake distributed from the Norte de Santander region of northeastern Colombia through the Maracaibo Basin and eastward to Caracas and the state of Miranda in Venezuela (Roze, 1982;
0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0041-010 1(01)00217-3
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Campbell and Lamar, 1989). Since a knowledge of the mechanisms of action of this venom may be helpful in establishing protocols for the treatment of persons envenomed by this species, we have investigated the effects of M. d. carinicauda venom on neuromuscular transmission and muscle contractility in mammalian and avian neuromuscular preparations, and also evaluated the morphological changes caused by the venom.
2. Materials and methods 2.1. Reagents and venom Acetylcholine iodide, a-bungarotoxin, 3,4-diaminopyridine (3,4-DAP), M. d. carinicauda venom were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Neostigmine methylsulfate (Prostigmine) was from Roche and d-tubocurarine chloride was from Abbot. 2.2. Lethality (LD50) The intravenous lethality of M. d. carinicauda venom was determined in male Swiss white mice (18±22 g). Six animals per dose were used at four dose levels with a ratio of 1.25 between doses. The LD50 and 95% con®dence intervals, calculated as described by Weil (1952), were 0.764 mg/kg and 0.511±1.124, respectively. 2.3. Rat isolated phrenic nerve-diaphragm preparation Left hemi-diaphragms with a portion of the phrenic nerve were obtained from male Wistar rats (230±250 g) anesthetized with chloral hydrate (240 mg/kg, i.p.; Pentofarma Ltda, Brazil). The preparations were suspended in 40 ml of Tyrode solution (composition, in mM: NaCl 136.8; KCl 2.7; CaCl2 1.8; NaHCO3 11.9; MgCl2 0.25; NaH2PO4 0.3; and glucose 11.0) maintained at 378C and oxygenated with 95% O2 1 5% CO2 (BuÈlbring, 1946). The nerve was stimulated with supramaximal pulses of 0.2 ms duration at a frequency of 0.1 Hz. The stimuli were delivered by a Grass S48 stimulator and the muscle contractions were recorded isometrically using a Load Cell BG 50 GMS transducer coupled to a Gould RS 3400 physiograph. The tissues were allowed to stabilize for 10 min after which venom was added to ®nal concentrations of 5 or 10 mg/ml and the muscle contractions then recorded until complete blockade (5 mg/ml) or for up to 90 min with 10 mg/ml. In some experiments, the effect of neostigmine methylsulfate (5.8 mM) or 3,4-DAP (230 mM) on the neuromuscular responses was investigated. Direct stimulation (maximal voltage, 2.0 ms and 0.1 Hz) was used in preparations blocked with d-tubocurarine (d-Tc, 15 mM) or a-bungarotoxin (a-BTx, 13 mM).
2.4. Bioelectrical potentials Rat hemi-diaphragms were obtained as described above and mounted horizontally in a 40 ml Perspex organ bath with their thoracic surface facing upwards. The preparations were bathed in warm, oxygenated Tyrode solution (see above). The organ bath was placed on a Zeiss stereo microscope stage and the preparation was viewed under 40 £ magni®cation. Intracellular bioelectrical potentials were recorded in the end-plate region using conventional glass microelectrodes ®lled with 3 M KCl (resistance, 5±20 MV) (Fatt and Katz, 1951). The transmembrane potentials of ®ve ®bers in each preparation (n 5) were measured at 5 min intervals in each ®ber (total number of measurements 450) using a Bimos operational ampli®er (model CA 3140) with a typical input impedance of 1.5 TV. In some preparations, neostigmine (5.8 mM, n 3) or 3,4-diaminopyridine (230 mM, n 3) was added to the bath to evaluate the action of the venom on postsynaptic nicotinic receptors. Oscilloscope-stored records of miniature end-plate potentials (m.e.p.ps) were photographed before and at various times after the addition of venom to the bath. The recordings were displayed on a Tektronix 5103 N storage oscilloscope ®tted with differential and dual time base modules. Permanent records were obtained by photographing the potentials with a Polaroid C-5A camera. Changes in the m.e.p.ps were measured before and 30, 60 and 90 min post-venom (10 mg/ml). 2.5. Chick biventer cervicis nerve-muscle preparation Biventer cervicis muscles from chicks (4±10 days old) were obtained and mounted as described by Ginsborg and Warriner (1960). The preparations were suspended under a resting tension of 0.5 g in 5 ml of bathing solution (composition, in mM: NaCl 136.0; KCl 5.0, CaCl2 2.5; NaHCO3 23.8; MgSO4 1.2; KH2PO4 1.2; and glucose 11.0) maintained at 378C and oxygenated with a mixture of 95% O2 1 5% CO2. The preparations were stimulated indirectly with supramaximal pulses (7 V, 0.2 ms and 0.1 Hz) using a Grass S88 stimulator. Direct stimulation (20 V, 0.2 ms, 0.1 Hz) was done in preparations blocked with d-Tc (15 mM) or a-bungarotoxin (a-BTx, 13 mM). The muscle responsiveness to exogenous acetylcholine (ACh, 37 mM) and KCl (134 mM) in the absence of electrical stimulation was assessed before and after exposure to venom (5 mg/ml). In all cases, the muscle twitches and contractures were recorded using a Myograph F60 transducer coupled to a Narcotrace 40 physiograph. 2.6. Light microscopy After a 90 min incubation with venom, fragments of muscle from indirectly stimulated preparations (n 3 for venom concentrations of 5 and 10 mg/ml in rat diaphragm and 5 mg/ml in chick biventer cervicis) were taken at
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Fig. 2. Ineffectiveness of washing in reversing the neuromuscular blockade produced by M. d. carinicauda venom in the rat phrenic nerve-diaphragm preparation. a, Control; b, addition of venom (5 mg/ml); c, neuromuscular blockade 300 min after addition of venom; and d±n, repeated washing of the preparation with Tyrode solution.
compared using Student's t-test, with p , 0.05 indicating signi®cance. 3. Results Fig. 1. Neuromuscular blockade produced by M. d. carinicauda venom (5 mg/ml) in the phrenic nerve-diaphragm preparation: antagonistic effect of neostigmine methylsulfate (Neo) 1I and 3,4diaminopyridine (3,4-DAP) 1II. 1I: a, control; b, addition of venom; and c, addition of Neo (5.8 mM) 285 min after the venom. 1II: a, control; b, addition of venom; and c, addition of 3,4-DAP (230 mM) 295 min after the venom.
3.1. Phrenic nerve-diaphragm preparation M. d. carinicauda venom dose-dependently inhibited indirectly evoked twitches in diaphragm muscle. At a concentration of 5 mg/ml, the venom caused blockade in 291.8 ^ 7.3 min (n 5), whereas at 10 mg/ml, blockade occurred in 108.3 ^ 13.8 min (n 5). In six experiments, neostigmine (5.8 mM) partially reversed (28.8%) the neuromuscular blockade when added to the organ bath after 85% blockade by a venom concentration of 5 mg/ml (Fig. 1). 3,4-DAP (230 mM, n 3) produced similar reversal of the venom-induced blockade (Fig. 1II). In contrast, repeated washing of the preparations was ineffective in reversing the venom-induced blockade (Fig. 2). The venom had no effect on the twitch tension of directly stimulated muscle in curarized (14.6 mM d-tubocurarine) preparations or in the presence of a-bungarotoxin (13 mM) (Fig. 3).
random and ®xed in Bouin solution for 24 h after which the samples were washed three times with a solution of water and ammonia. Following dehydration through a graded ethanol series, the tissues were embedded in LKB Historesin and incubated at 608C overnight. Sections, 2 mm thick, were cut using a Leica RM 2035 microtome (Vienna, Austria) and stained with 0.5% methylene blue for examination by light microscopy. Photomicrographs (Fugi-Film, 100 ASA) were obtained using a Nikon Optiphot (Nippon, Tokyo, Japan). Indirectly stimulated preparations incubated with Tyrode solution (n 3) or fresh preparations without prior incubation (0 h, n 3) were used as controls. The percentage of intact and damaged ®bers was determined in three sections from three different tissue blocks for each animal (n 3). Approximately 480±750 cells were counted per section, except for biventer cervicis for which 250 cells per sample were counted.
M. d. carinicauda venom (5 mg/ml) blocked the twitches elicited by indirect stimulation in this preparation. In all ®ve experiments, the contractures produced by ACh (37 mM), but not by KCl (134 mM), were inhibited after exposure to venom (Fig. 4).
2.7. Statistical analysis
3.3. Action of M. d. carinicauda venom on bioelectrical potentials
The twitch tension measurements were expressed as the mean ^ standard error of the mean (S.E.M.), where appropriate. Differences between groups or treatments were
M. d. carinicauda venom (10 mg/ml) did not alter the membrane resting potential of phrenic nerve-diaphragm preparations after an incubation of up to 90 min (®ve ®bers,
3.2. Chick biventer cervicis preparation
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Fig. 3. Effect of M. d. carinicauda venom (10 mg/ml) on phrenic nerve-diaphragm blockade produced by a-bungarotoxin (a-BTx, 13 mM). a, Indirect electrical stimulation and addition of a-BTx; b, neuromuscular blockade 50 min after a-BTx; c, direct electrical stimulation and addition of M. d. carinicauda venom (10 mg/ml); and d±f, 30, 60 and 90 min after venom addition, respectively.
with 25 measurements from each preparation) (Figs. 5 and 6). However, the venom decreased the m.e.p.ps' amplitude and frequency, eventually abolishing them. This inhibition of m.e.p.ps was reverted by neostigmine (5.8 mM, n 3) (Fig. 5A), although the m.e.p.ps reappeared at a lower frequency and amplitude. In contrast, after the addition of 3,4-DAP (230 mM), the m.e.p.ps always returned with a frequency and amplitude equal to or only slightly smaller than those recorded before venom addition (Fig. 5B).
Fig. 5. Effects of M. d. carinicauda venom (10 mg/ml) on miniature end-plate potentials (m.e.p.ps) in the rat diaphragm: antagonistic effect of neostigmine methylsulfate (Neo) and 3,4-diaminopyridine (3,4-DAP). A. I. Control; II. Blockade 90 min after the addition of venom; III. 5 min after the addition of Neo (5.8 mM). B. I. Control; II. Blockade 97 min after the addition of venom; III. 5 min after the addition of 3,4-DAP (230 mM).
3.4. Light microscopy
Fig. 4. Effect of M. d. carinicauda venom (5 mg/ml) on the chick biventer cervicis preparation. a, Control, indirect electrical stimulation and addition of acetylcholine (ACh, 37 mM) and potassium chloride (KCl, 134 mM); b, addition of venom; and c, neuromuscular blockade 90 min after venom, and addition of ACh and KCl.
Histological examination of control hemi-diaphragms showed that the muscle tissue was normal in appearance, with the muscle ®bers presenting regular light and dark bands. Cross sections of the tissue showed the normal polygonal outline of cells grouped in well-de®ned fascicles (Fig. 7). In contrast, rat hemi-diaphragms incubated with M. d. carinicauda venom showed areas with various stages of myo®bril degeneration, depending on the venom concentration. At a concentration of 5 mg/ml, the venom produced small vacuolations in 25% of the muscle ®bers (Fig. 8A, B), along with the presence of undulations and zones of ®ber hypercontraction (Fig. 8C). A higher venom concentration (10 mg/ml) produced foci of highly damaged ®bers with areas of extensive cell disintegration (Fig. 8D, E)
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Fig. 6. Membrane potential of rat diaphragm muscle ®bers (n 5) incubated with M. d. carinicauda venom (5 mg/ml, B and 10 mg/ml, X). The membrane potential was determined as described in Section 2. Each point represents the mean ^ S.E.M. of ®ve experiments.
intermingled with normal or only slightly affected areas of tissue. Approximately 61% of the ®bers showed some damage. Control chick biventer cervicis muscle showed ®bers with a normal morphology (Fig. 9A, B). Incubation with venom (5 mg/ml) in the absence (Fig. 9C) or presence (Fig. 9D) of indirect stimulation for 90 min produced hypercontracted ®bers without visible striations, although large areas of apparently undamaged ®bers were also seen.
4. Discussion The venoms of M. fulvius and M. nigrocinctus, coral snakes from North and Central America, respectively, depress the responses elicited by direct muscle stimulation and depolarize the muscle ®ber membrane (Weis and McIsaac, 1971; Goularte et al., 1995). In contrast, the venoms of the South American coral snakes M. lemniscatus, M. frontalis and M. corallinus do not alter the twitch tension produced by direct muscle stimulation (Vital Brazil, 1965) or the membrane resting potential (Vital Brazil et al., 1976/ 77; Vital Brazil and Fontana, 1983/84). As shown here, M. dumerilii carinicauda venom (5 or 10 mg/ml) also did not depress the responses to direct muscle stimulation and did not depolarize the muscle ®ber membrane. Thus, like other South American coral snake venoms, M. d. carinicauda venom does not alter skeletal
Fig. 7. Morphological appearance of control rat hemi-diaphragm preparations incubated with Tyrode solution. A and B, Longitudinal and cross sections of muscle ®bers showing well-de®ned striations, peripherally-located nuclei and normal polygonal appearance. Bars 30 mm.
muscle ®ber membrane excitability. Rather, the primary cause of the neuromuscular blockade induced by this venom was a postsynaptic action. This hypothesis is supported by the observations that the venom decreased the amplitude of the m.e.p.ps before abolishing them and that the response of the biventer cervicis preparation to ACh (37 mM) was inhibited when neuromuscular transmission was blocked. Severe myonecrosis has been reported for M. d. carinicauda venom in mice (GutieÂrrez et al., 1983), but at concentrations greater than those used here for pharmacological studies (15 and 30 mg/ml versus 5 and 10 mg/ml). At a venom concentration of 5 mg/ml, the changes seen in the chick and rat preparations were similar, and in both cases the sarcolemma was hardly affected. At the higher concentration (10 mg/ml) tested in phrenic nerve-diaphragm preparations, the myonecrosis was of the myolytic type (Homma and Tu, 1971). The observation that the venom did not affect the contractile responses to direct muscle stimulation suggested
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Fig. 8. Myonecrotic changes in ®bers of rat diaphragm incubated with M. d. carinicauda venom for 90 min. A, B and C: 5 mg/ml, D and E: 10 mg/ml. In A and B, cross sections of normal polygonal-shaped cells showing microvacuoles (small arrows). In C, D, and E, longitudinal sections showing dark muscle ®bers in various stages of necrosis, including dispersed myo®laments and hypercontracted areas (h) alternating with regions of cytoplasmic disintegration ( p ). Some ®bers exhibit a lace-like appearance (l), probably caused by recurrent hypercontracted discontinuities of sarcomeres. Bars 20 mm (B) and 30 mm (A, C, E).
that the morphological changes did not adversely in¯uence muscle contractility and, therefore, did not contribute to the venom-induced neuromuscular blockade or to the changes in membrane resting potential. The lack of correlation between the extent of ®ber damage and the changes in membrane resting potential probably re¯ected the fact that the latter was measured close to the end-plate regions of the ®bers, whereas for the morphological studies, the ®bers examined included also those distant from these areas. It is probable that the number of damaged ®bers in the endplate regions would be less than in the muscle in general.
These ®ndings indicate that M. d. carinicauda venom produces neuromuscular blockade by a postsynaptic action that is apparently independent of possible myotoxic effects of the venom. As shown above, the contractions to direct muscle stimulation in rat preparations were unaffected by incubation with M. d. carinicauda venom. This lack of effect was similar to that reported for the venoms of M. corallinus (Vital Brazil and Fontana, 1983/84) and M. spixii (Vital Brazil et al., 1995) which also do not affect the responses to direct muscle stimulation and not depolarize the muscle ®ber membrane.
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Fig. 9. Chick biventer cervicis preparation incubated with Tyrode solution and M. d. carinicauda venom (5 mg/ml) for 90 min. In A and B, control cross sections and longitudinal sections showing well-delineated muscle fascicles and cells with peripheral nuclei and dark and light bands. In C and D, longitudinal sections of muscle ®bers incubated with venom with or without indirect electrical stimulation, respectively. Electrical stimulation did not affect the hypercontraction of the muscle ®bers produced by venom. Note the constricted areas (arrow) along the hypercontracted ®ber (h). Bars 30 mm.
The effect of neostigmine on the blocked twitches in rat diaphragm was small and transient, but allowed the reappearance of m.e.p.ps that had been abolished by the venom. However, the very low frequency and amplitude of the restored m.e.p.ps suggested that most receptors in the end-plate were still blocked by the venom. The observation that the restored m.e.p.ps had a low frequency and amplitude suggests that only a few receptors were blocked competitively by the venom; most were probably blocked non-competitively by the venom since a higher concentration of neostigmine did not improve the
restoration of the m.e.p.ps. This action is similar to that proposed for M. spixii venom.
Acknowledgements This work was supported by CAPES (Brazil). The authors thank Antonio Vilson dos Santos and Gustavo Henrique da Silva for technical assistance and Dr Stephen Hyslop for correcting the English of the manuscript.
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References Arroyo, O., Rosso, J.P., Vargas, O., GutieÂrrez, J.M., Cerdas, L., 1987. Skeletal muscle necrosis induced by a phospholipase A2 isolated from the venom of coral snake Micrurus nigrocinctus nigrocinctus. Comp. Biochem. Physiol. 87, 949±952. BuÈlbring, E., 1946. Observations on the isolated phrenic nerve diaphragm preparation of the rat. Br. J. Pharmacol. 1, 38±61. Campbell, J.A., Lamar, W.W., 1989. The Venomous Reptiles of Latin America. Comstock Publ. Assoc, New York. Fatt, P., Katz, B., 1951. An analysis of the end-plate potential recorded with an intra-cellular electrode. J. Physiol. 115, 320±370. Ginsborg, B.L., Warriner, J.N., 1960. The isolated chick biventer cervicis nerve muscle preparation. Br. J. Pharmacol. 15, 410±415. Goularte, F.C.L., Cogo, J.C., GutieÂrrez, J.M., Rodrigues-Simioni, L., 1995. Effects of Micrurus nigrocinctus snake venom on mouse and chick neuromuscular preparations. Toxicon 31, 135±136. GutieÂrrez, J.M., Lomonte, B., Portilla, E., Cerdas, L., Rojas, E., 1983. Local effects induced by coral snake venoms: evidence of myonecrosis after experimental inoculation of venoms from ®ve species. Toxicon 21, 777±783. Homma, M., Tu, A.T., 1971. Morphology of local tissue damage in experimental snake envenomation. Br. J. Exp. Pathol. 52, 538±542. Kellaway, S.H., Cherry, R.O., Williams, F.E., 1932. The peripheral action of the Australian snake venoms. II. The curare-like action in mammals. Austral. J. Exp. Biol. Med. Sci. 10, 181±194.
Lee, C.Y., Chang, C.C., Chen, Y.M., 1972. Reversibility of neuromuscular blockade by neurotoxins from elapid and sea snake venoms. J. Formosan Med. Assoc. 71, 344±350. Roze, J.A., 1982. New World coral snakes (Elapidae): a taxonomic and biological summary. Mem. Inst. Butantan 46, 305±338. Roze, J.A., 1989. New species and subspecies of coral snakes, genus Micrurus (Elapidae), with notes on type specimens of several species. Am. Mus. Novitates 2932, 1±15. Roze, J.A., Bernal-Carlo, A., 1987. Las serpientes corales venenosas del generoÐLeptomicrurus (Serpentes, Elapidae) de SurameÂrica com descripcioÂn de una nueva subespeÂcie. Boll. Mus. Reg. Sci. Nat. Torino 5, 573±608. Vital Brazil, O., 1965. AcaÅo neuromuscular da pecËonha de Micrurus. O Hospital 68, (4), 183±224. Vital Brazil, O., Fontana, M.D., 1983/84. AcËoÄes preÂ-juncionais e poÂs-juncionais da pecËonha da cobra coral Micrurus corallinus na juncËaÄo neuromuscular. Mem. Inst. Butantan 47/48, 13±26. Vital Brazil, O., Fontana, M.D., Pellegrini Filho, A., 1976/77. Physiologie et theÂrapeutique de l'envenomation causee par le venin de Micrurus frontalis. Mem. Inst. Butantan 40/41, 221±240. Vital Brazil, O., Fontana, M.D., Heluany, N.F., 1995. Mode of action of the coral snake Micrurus spixii venom at the neuromuscular junction. J. Nat. Toxins 4, 19±33. Weil, C.S., 1952. Tables for convenient calculation of median-effective dose (DL50 and ED50) and instruction in their use. Biometrics 8, 249±263. Weis, R., McIsaac, R.J., 1971. Cardiovascular and muscular effects of venom from coral snake Micrurus fulvius. Toxicon 9, 219±228.
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Action of Micrurus dumerilii carinicauda coral snake venom on the mammalian neuromuscular junction Francine G. Sera®m a, Marielga Reali a, Maria Alice Cruz-Hoȯing b, Marcos D. Fontana a,* a
Departamento de Farmacologia, Faculdade de CieÃncias MeÂdicas, Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6111, CEP 13083-970, Campinas, SP, Brazil b Departamento de Histologia e Embriologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6109, 13083-970, Campinas, SP, Brazil Received 4 April 2001; accepted 18 August 2001
Abstract The venoms of coral snakes (mainly Micrurus species) have pre- and/or postsynaptic actions, but only a few of these have been studied in detail. We have investigated the effects of Micrurus dumerilii carinicauda coral snake venom on neurotransmission in rat isolated phrenic nerve-diaphragm muscle and chick biventer cervicis preparations stimulated directly or indirectly. M. d. carinicauda venom (5 or 10 mg/ml) produced neuromuscular blockade in rat (85±90% in 291.8 ^ 7.3 min and 108.3 ^ 13.8, respectively; n 5) and avian (95.0 ^ 2.0 min; 5 mg/ml, n 5) preparations. Neostigmine (5.8 mM) and 3,4diaminopyridine (230 mM) partially reversed the venom-induced neuromuscular blockade in rat nerve-muscle preparations. In neither preparation did the venom depress the twitch response elicited by direct muscle stimulation. The contractures induced by acetylcholine in chick preparations were inhibited by the venom (95±100%; n 4; p , 0.05). In rat preparations, the venom produced a progressive decrease in the amplitude of miniature end-plate potentials (m.e.p.ps control frequency 69.3 ^ 5.0/ min and control amplitude 0.4 ^ 0.2 mV) until these were abolished. Neostigmine (5.8 mM) and 3,4-diaminopyridine (230 mM) partially antagonized this blockade of m.e.p.ps. The resting membrane potential was not altered with the venom (10 mg/ml). M. d. carinicauda venom produced dose-dependent morphological changes in indirectly stimulated mammal preparations. Twenty-®ve per cent of muscle ®bers were affected by a venom concentration of 5 mg/ml, whilst 60.7% were damaged by 10 mg of venom/ml. In biventer cervicis preparations, the morphological changes were slower in onset and were generally characterized by undulating ®bers and, to a lesser extent, by zones of disintegrating myo®brils. A venom concentration of 5 mg/ml damaged 52.2% of the ®bers. These ®ndings indicate that M. d. carinicauda venom has neurotoxic and myotoxic effects and that the neuromuscular blockade involves mainly a postsynaptic action. q 2001 Elsevier Science Ltd. All rights reserved. Keywords: Blockade; Coral snake; M. d. carinicauda; Neuromuscular junction; Histological changes; Venom
1. Introduction The coral snakes comprise a group of about 120 species and subspecies of New World elapid snakes belonging primarily to the genus Micrurus (Roze, 1982, 1989; Roze and Bernal-Carlo, 1987). Envenoming by coral snakes results in progressive neuromuscular blockade, with death * Corresponding author. Tel.: 155-19-3788-7482; fax: 155-193289-2968. E-mail address: [email protected] (M.D. Fontana).
resulting from respiratory arrest through the action of preand postsynapatic neurotoxins at the neuromuscular junction (Kellaway et al., 1932; Lee et al., 1972; Vital Brazil et al., 1976/77; Vital Brazil and Fontana, 1983/84). Several studies have also shown that coral snake venoms are myotoxic (Arroyo et al., 1987; GutieÂrrez et al., 1983; Goularte et al., 1995). Micrurus dumerilii carinicauda is a coral snake distributed from the Norte de Santander region of northeastern Colombia through the Maracaibo Basin and eastward to Caracas and the state of Miranda in Venezuela (Roze, 1982;
0041-0101/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved. PII: S 0041-010 1(01)00217-3
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Campbell and Lamar, 1989). Since a knowledge of the mechanisms of action of this venom may be helpful in establishing protocols for the treatment of persons envenomed by this species, we have investigated the effects of M. d. carinicauda venom on neuromuscular transmission and muscle contractility in mammalian and avian neuromuscular preparations, and also evaluated the morphological changes caused by the venom.
2. Materials and methods 2.1. Reagents and venom Acetylcholine iodide, a-bungarotoxin, 3,4-diaminopyridine (3,4-DAP), M. d. carinicauda venom were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Neostigmine methylsulfate (Prostigmine) was from Roche and d-tubocurarine chloride was from Abbot. 2.2. Lethality (LD50) The intravenous lethality of M. d. carinicauda venom was determined in male Swiss white mice (18±22 g). Six animals per dose were used at four dose levels with a ratio of 1.25 between doses. The LD50 and 95% con®dence intervals, calculated as described by Weil (1952), were 0.764 mg/kg and 0.511±1.124, respectively. 2.3. Rat isolated phrenic nerve-diaphragm preparation Left hemi-diaphragms with a portion of the phrenic nerve were obtained from male Wistar rats (230±250 g) anesthetized with chloral hydrate (240 mg/kg, i.p.; Pentofarma Ltda, Brazil). The preparations were suspended in 40 ml of Tyrode solution (composition, in mM: NaCl 136.8; KCl 2.7; CaCl2 1.8; NaHCO3 11.9; MgCl2 0.25; NaH2PO4 0.3; and glucose 11.0) maintained at 378C and oxygenated with 95% O2 1 5% CO2 (BuÈlbring, 1946). The nerve was stimulated with supramaximal pulses of 0.2 ms duration at a frequency of 0.1 Hz. The stimuli were delivered by a Grass S48 stimulator and the muscle contractions were recorded isometrically using a Load Cell BG 50 GMS transducer coupled to a Gould RS 3400 physiograph. The tissues were allowed to stabilize for 10 min after which venom was added to ®nal concentrations of 5 or 10 mg/ml and the muscle contractions then recorded until complete blockade (5 mg/ml) or for up to 90 min with 10 mg/ml. In some experiments, the effect of neostigmine methylsulfate (5.8 mM) or 3,4-DAP (230 mM) on the neuromuscular responses was investigated. Direct stimulation (maximal voltage, 2.0 ms and 0.1 Hz) was used in preparations blocked with d-tubocurarine (d-Tc, 15 mM) or a-bungarotoxin (a-BTx, 13 mM).
2.4. Bioelectrical potentials Rat hemi-diaphragms were obtained as described above and mounted horizontally in a 40 ml Perspex organ bath with their thoracic surface facing upwards. The preparations were bathed in warm, oxygenated Tyrode solution (see above). The organ bath was placed on a Zeiss stereo microscope stage and the preparation was viewed under 40 £ magni®cation. Intracellular bioelectrical potentials were recorded in the end-plate region using conventional glass microelectrodes ®lled with 3 M KCl (resistance, 5±20 MV) (Fatt and Katz, 1951). The transmembrane potentials of ®ve ®bers in each preparation (n 5) were measured at 5 min intervals in each ®ber (total number of measurements 450) using a Bimos operational ampli®er (model CA 3140) with a typical input impedance of 1.5 TV. In some preparations, neostigmine (5.8 mM, n 3) or 3,4-diaminopyridine (230 mM, n 3) was added to the bath to evaluate the action of the venom on postsynaptic nicotinic receptors. Oscilloscope-stored records of miniature end-plate potentials (m.e.p.ps) were photographed before and at various times after the addition of venom to the bath. The recordings were displayed on a Tektronix 5103 N storage oscilloscope ®tted with differential and dual time base modules. Permanent records were obtained by photographing the potentials with a Polaroid C-5A camera. Changes in the m.e.p.ps were measured before and 30, 60 and 90 min post-venom (10 mg/ml). 2.5. Chick biventer cervicis nerve-muscle preparation Biventer cervicis muscles from chicks (4±10 days old) were obtained and mounted as described by Ginsborg and Warriner (1960). The preparations were suspended under a resting tension of 0.5 g in 5 ml of bathing solution (composition, in mM: NaCl 136.0; KCl 5.0, CaCl2 2.5; NaHCO3 23.8; MgSO4 1.2; KH2PO4 1.2; and glucose 11.0) maintained at 378C and oxygenated with a mixture of 95% O2 1 5% CO2. The preparations were stimulated indirectly with supramaximal pulses (7 V, 0.2 ms and 0.1 Hz) using a Grass S88 stimulator. Direct stimulation (20 V, 0.2 ms, 0.1 Hz) was done in preparations blocked with d-Tc (15 mM) or a-bungarotoxin (a-BTx, 13 mM). The muscle responsiveness to exogenous acetylcholine (ACh, 37 mM) and KCl (134 mM) in the absence of electrical stimulation was assessed before and after exposure to venom (5 mg/ml). In all cases, the muscle twitches and contractures were recorded using a Myograph F60 transducer coupled to a Narcotrace 40 physiograph. 2.6. Light microscopy After a 90 min incubation with venom, fragments of muscle from indirectly stimulated preparations (n 3 for venom concentrations of 5 and 10 mg/ml in rat diaphragm and 5 mg/ml in chick biventer cervicis) were taken at
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Fig. 2. Ineffectiveness of washing in reversing the neuromuscular blockade produced by M. d. carinicauda venom in the rat phrenic nerve-diaphragm preparation. a, Control; b, addition of venom (5 mg/ml); c, neuromuscular blockade 300 min after addition of venom; and d±n, repeated washing of the preparation with Tyrode solution.
compared using Student's t-test, with p , 0.05 indicating signi®cance. 3. Results Fig. 1. Neuromuscular blockade produced by M. d. carinicauda venom (5 mg/ml) in the phrenic nerve-diaphragm preparation: antagonistic effect of neostigmine methylsulfate (Neo) 1I and 3,4diaminopyridine (3,4-DAP) 1II. 1I: a, control; b, addition of venom; and c, addition of Neo (5.8 mM) 285 min after the venom. 1II: a, control; b, addition of venom; and c, addition of 3,4-DAP (230 mM) 295 min after the venom.
3.1. Phrenic nerve-diaphragm preparation M. d. carinicauda venom dose-dependently inhibited indirectly evoked twitches in diaphragm muscle. At a concentration of 5 mg/ml, the venom caused blockade in 291.8 ^ 7.3 min (n 5), whereas at 10 mg/ml, blockade occurred in 108.3 ^ 13.8 min (n 5). In six experiments, neostigmine (5.8 mM) partially reversed (28.8%) the neuromuscular blockade when added to the organ bath after 85% blockade by a venom concentration of 5 mg/ml (Fig. 1). 3,4-DAP (230 mM, n 3) produced similar reversal of the venom-induced blockade (Fig. 1II). In contrast, repeated washing of the preparations was ineffective in reversing the venom-induced blockade (Fig. 2). The venom had no effect on the twitch tension of directly stimulated muscle in curarized (14.6 mM d-tubocurarine) preparations or in the presence of a-bungarotoxin (13 mM) (Fig. 3).
random and ®xed in Bouin solution for 24 h after which the samples were washed three times with a solution of water and ammonia. Following dehydration through a graded ethanol series, the tissues were embedded in LKB Historesin and incubated at 608C overnight. Sections, 2 mm thick, were cut using a Leica RM 2035 microtome (Vienna, Austria) and stained with 0.5% methylene blue for examination by light microscopy. Photomicrographs (Fugi-Film, 100 ASA) were obtained using a Nikon Optiphot (Nippon, Tokyo, Japan). Indirectly stimulated preparations incubated with Tyrode solution (n 3) or fresh preparations without prior incubation (0 h, n 3) were used as controls. The percentage of intact and damaged ®bers was determined in three sections from three different tissue blocks for each animal (n 3). Approximately 480±750 cells were counted per section, except for biventer cervicis for which 250 cells per sample were counted.
M. d. carinicauda venom (5 mg/ml) blocked the twitches elicited by indirect stimulation in this preparation. In all ®ve experiments, the contractures produced by ACh (37 mM), but not by KCl (134 mM), were inhibited after exposure to venom (Fig. 4).
2.7. Statistical analysis
3.3. Action of M. d. carinicauda venom on bioelectrical potentials
The twitch tension measurements were expressed as the mean ^ standard error of the mean (S.E.M.), where appropriate. Differences between groups or treatments were
M. d. carinicauda venom (10 mg/ml) did not alter the membrane resting potential of phrenic nerve-diaphragm preparations after an incubation of up to 90 min (®ve ®bers,
3.2. Chick biventer cervicis preparation
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Fig. 3. Effect of M. d. carinicauda venom (10 mg/ml) on phrenic nerve-diaphragm blockade produced by a-bungarotoxin (a-BTx, 13 mM). a, Indirect electrical stimulation and addition of a-BTx; b, neuromuscular blockade 50 min after a-BTx; c, direct electrical stimulation and addition of M. d. carinicauda venom (10 mg/ml); and d±f, 30, 60 and 90 min after venom addition, respectively.
with 25 measurements from each preparation) (Figs. 5 and 6). However, the venom decreased the m.e.p.ps' amplitude and frequency, eventually abolishing them. This inhibition of m.e.p.ps was reverted by neostigmine (5.8 mM, n 3) (Fig. 5A), although the m.e.p.ps reappeared at a lower frequency and amplitude. In contrast, after the addition of 3,4-DAP (230 mM), the m.e.p.ps always returned with a frequency and amplitude equal to or only slightly smaller than those recorded before venom addition (Fig. 5B).
Fig. 5. Effects of M. d. carinicauda venom (10 mg/ml) on miniature end-plate potentials (m.e.p.ps) in the rat diaphragm: antagonistic effect of neostigmine methylsulfate (Neo) and 3,4-diaminopyridine (3,4-DAP). A. I. Control; II. Blockade 90 min after the addition of venom; III. 5 min after the addition of Neo (5.8 mM). B. I. Control; II. Blockade 97 min after the addition of venom; III. 5 min after the addition of 3,4-DAP (230 mM).
3.4. Light microscopy
Fig. 4. Effect of M. d. carinicauda venom (5 mg/ml) on the chick biventer cervicis preparation. a, Control, indirect electrical stimulation and addition of acetylcholine (ACh, 37 mM) and potassium chloride (KCl, 134 mM); b, addition of venom; and c, neuromuscular blockade 90 min after venom, and addition of ACh and KCl.
Histological examination of control hemi-diaphragms showed that the muscle tissue was normal in appearance, with the muscle ®bers presenting regular light and dark bands. Cross sections of the tissue showed the normal polygonal outline of cells grouped in well-de®ned fascicles (Fig. 7). In contrast, rat hemi-diaphragms incubated with M. d. carinicauda venom showed areas with various stages of myo®bril degeneration, depending on the venom concentration. At a concentration of 5 mg/ml, the venom produced small vacuolations in 25% of the muscle ®bers (Fig. 8A, B), along with the presence of undulations and zones of ®ber hypercontraction (Fig. 8C). A higher venom concentration (10 mg/ml) produced foci of highly damaged ®bers with areas of extensive cell disintegration (Fig. 8D, E)
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Fig. 6. Membrane potential of rat diaphragm muscle ®bers (n 5) incubated with M. d. carinicauda venom (5 mg/ml, B and 10 mg/ml, X). The membrane potential was determined as described in Section 2. Each point represents the mean ^ S.E.M. of ®ve experiments.
intermingled with normal or only slightly affected areas of tissue. Approximately 61% of the ®bers showed some damage. Control chick biventer cervicis muscle showed ®bers with a normal morphology (Fig. 9A, B). Incubation with venom (5 mg/ml) in the absence (Fig. 9C) or presence (Fig. 9D) of indirect stimulation for 90 min produced hypercontracted ®bers without visible striations, although large areas of apparently undamaged ®bers were also seen.
4. Discussion The venoms of M. fulvius and M. nigrocinctus, coral snakes from North and Central America, respectively, depress the responses elicited by direct muscle stimulation and depolarize the muscle ®ber membrane (Weis and McIsaac, 1971; Goularte et al., 1995). In contrast, the venoms of the South American coral snakes M. lemniscatus, M. frontalis and M. corallinus do not alter the twitch tension produced by direct muscle stimulation (Vital Brazil, 1965) or the membrane resting potential (Vital Brazil et al., 1976/ 77; Vital Brazil and Fontana, 1983/84). As shown here, M. dumerilii carinicauda venom (5 or 10 mg/ml) also did not depress the responses to direct muscle stimulation and did not depolarize the muscle ®ber membrane. Thus, like other South American coral snake venoms, M. d. carinicauda venom does not alter skeletal
Fig. 7. Morphological appearance of control rat hemi-diaphragm preparations incubated with Tyrode solution. A and B, Longitudinal and cross sections of muscle ®bers showing well-de®ned striations, peripherally-located nuclei and normal polygonal appearance. Bars 30 mm.
muscle ®ber membrane excitability. Rather, the primary cause of the neuromuscular blockade induced by this venom was a postsynaptic action. This hypothesis is supported by the observations that the venom decreased the amplitude of the m.e.p.ps before abolishing them and that the response of the biventer cervicis preparation to ACh (37 mM) was inhibited when neuromuscular transmission was blocked. Severe myonecrosis has been reported for M. d. carinicauda venom in mice (GutieÂrrez et al., 1983), but at concentrations greater than those used here for pharmacological studies (15 and 30 mg/ml versus 5 and 10 mg/ml). At a venom concentration of 5 mg/ml, the changes seen in the chick and rat preparations were similar, and in both cases the sarcolemma was hardly affected. At the higher concentration (10 mg/ml) tested in phrenic nerve-diaphragm preparations, the myonecrosis was of the myolytic type (Homma and Tu, 1971). The observation that the venom did not affect the contractile responses to direct muscle stimulation suggested
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Fig. 8. Myonecrotic changes in ®bers of rat diaphragm incubated with M. d. carinicauda venom for 90 min. A, B and C: 5 mg/ml, D and E: 10 mg/ml. In A and B, cross sections of normal polygonal-shaped cells showing microvacuoles (small arrows). In C, D, and E, longitudinal sections showing dark muscle ®bers in various stages of necrosis, including dispersed myo®laments and hypercontracted areas (h) alternating with regions of cytoplasmic disintegration ( p ). Some ®bers exhibit a lace-like appearance (l), probably caused by recurrent hypercontracted discontinuities of sarcomeres. Bars 20 mm (B) and 30 mm (A, C, E).
that the morphological changes did not adversely in¯uence muscle contractility and, therefore, did not contribute to the venom-induced neuromuscular blockade or to the changes in membrane resting potential. The lack of correlation between the extent of ®ber damage and the changes in membrane resting potential probably re¯ected the fact that the latter was measured close to the end-plate regions of the ®bers, whereas for the morphological studies, the ®bers examined included also those distant from these areas. It is probable that the number of damaged ®bers in the endplate regions would be less than in the muscle in general.
These ®ndings indicate that M. d. carinicauda venom produces neuromuscular blockade by a postsynaptic action that is apparently independent of possible myotoxic effects of the venom. As shown above, the contractions to direct muscle stimulation in rat preparations were unaffected by incubation with M. d. carinicauda venom. This lack of effect was similar to that reported for the venoms of M. corallinus (Vital Brazil and Fontana, 1983/84) and M. spixii (Vital Brazil et al., 1995) which also do not affect the responses to direct muscle stimulation and not depolarize the muscle ®ber membrane.
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Fig. 9. Chick biventer cervicis preparation incubated with Tyrode solution and M. d. carinicauda venom (5 mg/ml) for 90 min. In A and B, control cross sections and longitudinal sections showing well-delineated muscle fascicles and cells with peripheral nuclei and dark and light bands. In C and D, longitudinal sections of muscle ®bers incubated with venom with or without indirect electrical stimulation, respectively. Electrical stimulation did not affect the hypercontraction of the muscle ®bers produced by venom. Note the constricted areas (arrow) along the hypercontracted ®ber (h). Bars 30 mm.
The effect of neostigmine on the blocked twitches in rat diaphragm was small and transient, but allowed the reappearance of m.e.p.ps that had been abolished by the venom. However, the very low frequency and amplitude of the restored m.e.p.ps suggested that most receptors in the end-plate were still blocked by the venom. The observation that the restored m.e.p.ps had a low frequency and amplitude suggests that only a few receptors were blocked competitively by the venom; most were probably blocked non-competitively by the venom since a higher concentration of neostigmine did not improve the
restoration of the m.e.p.ps. This action is similar to that proposed for M. spixii venom.
Acknowledgements This work was supported by CAPES (Brazil). The authors thank Antonio Vilson dos Santos and Gustavo Henrique da Silva for technical assistance and Dr Stephen Hyslop for correcting the English of the manuscript.
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