Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors.

Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca(2+) and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca(2+)], derived from intracellular stores, followed, only in myotubes, by a large Ca(2+) influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca(2+) permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca(2+) mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death.

Antibothropic action of Casearia sylvestris Sw. (Flacourtiaceae) extracts.

Casearia sylvestris Sw., popularly known in Brazil as 'guaçatonga', has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C. sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects.

Antineurotoxic activity of Galactia glaucescens against Crotalus durissus terrificus venom.

Ethanolic extract of leaves of Galactia glauscescens (GGE) at concentration of 100 and 500 microg/ml prevented the neuromuscular paralysis induced by Crotalus durissus terrificus venom on mouse phrenic nerve-diaphragm preparation.

Determination of primary structure of two isoforms 6-1 and 6-2 PLA2 D49 from Bothrops jararacussu snake venom and neurotoxic characterization using in vitro neuromuscular preparation.

In this paper we reported the purification, the biological characterization and the amino acid sequence of two new isoforms basic 6-1 (Bj-IV) and 6-2 (Bj-V) PLA(2) D49 purified from the Bothrops jararacussu venom. The isoforms 6-1 and 6-2 had a sequence of amino acids of 121 amino acid residues 6-1: DLFEWGQMIL KETGKNPFPY YGAYGCYCGW GGRGKPKDKD TDRCCYVHDC CYKKLTGCPK TDDRYSYSWL DLTIVCGEDD PCKELCECDK AIAVCFRENL GTYNKKYRYH LKPCKKADKP C and pI value 7.83 and 6-2: DLWQFGQMIL KETGKIPFPY YGAYGCYCGW GGRGGKPKDG TDRCCYVHDC CYKKLTGCPK TDDRYSYSWL DLTIVCGEDD PCKELCECDK AIAVCFRENL GTYNKKYRYH LKPCKKADKP C with a pI value of 7.99. Skeletal muscle preparations from the young chicken have been used previously in order to study the effects of toxins on neuromuscular transmission, providing an important opportunity to study the differentiated behavior of a toxin before more than one model, because it shows differences in its sensibilities. Both isoforms have produced neuromuscular blockade in young chicken biventer cervicis nerve-muscle preparations in presence or absence of crotapotin crotalic (F3 and F4) indicating that catalytic activity was not essential for neuromuscular action in this preparation.

The effects of Bothrops jararacussu venom and its components on frog nerve-muscle preparation.

The effect of Bothrops jararacussu venom was studied in cutaneous pectoris nerve muscle preparations and in the desheathed sciatic nerve of the frog. The venom rapidly inhibited muscle twitch--tension, evoked either directly or indirectly through the motor nerve and abolished the compound action potential of the muscle and of the sciatic nerve. After fractionation of the venom by Sephadex G-50 column chromatography, all the activity was recovered in a fraction containing 30% of the total venom protein and highly enriched in two polypeptides with apparent Mr of 13-15,000, as revealed by two-dimensional polyacrylamide gel electrophoresis. The concentration of active subfraction required to obtain 50% paralysis in 1 h was 8 micrograms protein/ml. The active subfraction contained low levels of phospholipase A activity, whereas no proteolytic activity was detected. The paralyzing activity of the active subfraction on nerve-muscle preparations was not dependent on the presence of Ca2+, suggesting that phospholipase A activity is not required for the toxic effect. The active subfraction was found to cause an initial spontaneous contracture and fasciculation of the nerve-muscle preparation, and a rapid depolarization of the muscle membrane. The frequency of miniature endplate potentials was normal throughout the period of exposure to the active subfraction, although occasionally initial transient bursts were observed. At the end of the incubation, nerve endings still responded to high [K+] and to black widow spider venom. The exposure (1-2 h) to blocking concentrations of venom active subfraction provoked different degrees of morphological alteration of the muscle fibers. In contrast, no ultrastructural alterations were observed in nerve terminals, giving further support to the idea that terminals are not a prime site of the venom action. In addition to its effect on the nerve muscle-preparation, the active subfraction at higher concentrations, showed a Ca2+-dependent hemolytic activity. In the light of these results, the properties of the active subfraction of B. jararacussu venom are compared with those of other known membrane-active toxins.

Histopathological changes in avian kidney caused by Bothrops insularis (jararaca ilh a) venom and a phospholipase A2-containing fraction.

The histopathological changes induced in avian kidney by the intramuscular injection of Bothrops insularis (jararaca ilh a) venom and its phospholipase A2 (PLA2)-containing fraction were examined. Acute experiments (3 h and 24 h) with B. insularis crude venom (20 microg and 80 microg) or its PLA2-contaning fraction (10 microg and 40 microg) resulted in significant structural damage to the kidneys of 5-12-day-old chicks. Histopathological analysis indicated that the venom and its fraction acted on the renal tubules and glomeruli. The morphological changes, although widespread, varied in intensity from cell to cell, and from tubule to tubule in venom-injected chicks. The tubular and glomerular changes produced by the venom and its PLA2-containing fraction may be the result of a direct cytotoxic effect potentiated by ischemia-related disturbances in the regional hemodynamics. The venom and its fraction affected more segments along reptilian-type nephrons than along mammalian ones. This divergent sensitivity to the venom and its fraction may reflect the species-specific characteristics of B. insularis snake, an example of geographical isolation influencing its diet which is almost exclusively avian.

Properties of the early phases of crotoxin poisoning at frog neuromuscular junctions.

Addition of crotoxin to the frog neuromuscular junction in either 0.9 mM Ca2+ plus tubocurarine or 0.5 mM Ca2+ Ringer solution produced a triphasic change in the amplitude of nerve-evoked endplate potentials (e.p.p.s) and, with 0.5 mM Ca2+, a biphasic change in miniature endplate potential (m.e.p.p.) frequency. The secondary phase of rising e.p.p. amplitude was associated with an increase in facilitation of e.p.p. amplitude with closely spaced twin impulses; the increase in spontaneous release lagged behind that of evoked release. When a calcium chelator, BAPTA, was loaded into presynaptic nerve terminals to buffer cytosolic free Ca2+, both e.p.p. amplitude and twin-impulse facilitation were increased by crotoxin to a similar extent relative to that in the control without BAPTA. The duration of the increase in twin-impulse facilitation was reduced but the duration of the increase in e.p.p. amplitude was unaffected by BAPTA loading. The presence of BAPTA did not alter the characteristic changes in spontaneous release in response to crotoxin. These results suggest that the augmentation of evoked and spontaneous transmitter release by crotoxin is not primarily due to changes in cytosolic Ca2+. The response time between stimulus and e.p.p. peak was lengthened in all phases due to prolongation of the interval between stimulus and e.p.p. onset and, in the secondary and tertiary phases, slowing of e.p.p. rise-time. The protein kinase C inhibitor, H-7, produced complex changes in e.p.p.s. under control conditions but did not alter the triphasic response characteristic of intoxication. These results suggest that crotoxin initiates a primary disturbance in the phasic release process leading to a series of time-gated changes which include transient facilitation then uncoupling of phasic release and generalized acceleration of spontaneous release.

Fractionation of Bothrops jararacussu snake venom: partial chemical characterization and biological activity of bothropstoxin.

A myotoxin, bothropstoxin (BthTX), showing no detectable phospholipase A2 activity, was purified to homogeneity from the venom of the Brazilian snake Bothrops jararacussu by a combination of gel filtration on Sephadex G-75 and ion-exchange chromatography on SP-Sephadex C-25. Four phospholipases (Sm-SP1 to Sm-SPIV) were also isolated, the latter showing, similarly to BthTX (Sm-SPv) myonecrotic activity. Approximate mol. wts, as determined by SDS-PAGE, and pI of Sm-SPI to Sm-SPIV are: 22,400-4.2; 15,500-4.8; 13,800-6.9; and 13,200-7.7, respectively. BthTX is a single chain protein, approximate mol. wt 13,000, with 16 half-cystine residues, pI = 8.2 and LD50 = 7.5 mg/kg (i.p.) and 4.8 mg/kg (i.v.) for 20 g mice. The ten first N-terminal amino acid residues show a significant homology to other toxins with phospholipase structure. BthTX is specifically myotoxic, contrary to crude B. jararacussu venom which, although also myotoxic, affects intramuscular arteries and veins leading to thrombosis. BthTX and Sm-SPIV also differ from toxins isolated from the venom of other Brazilian snakes which are strongly hemorrhagic.

Effect of Bothrops insularis venom on the mouse and chick nerve-muscle preparation.

The effects of Bothrops insularis venom were examined in vivo in mice and chicks and in vitro using the mouse phrenic nerve diaphragm and chick biventer cervicis muscle preparations. Incubation of the indirectly or directly stimulated mouse preparation with B. insularis venom (20-80 micrograms/ml) produced an initial increase in twitch tension followed by irreversible blockade. With direct stimulation in the presence of D-tubocurarine, no increase in twitch tension was observed prior to the onset of blockade. A venom-induced effect on presynaptic activity was suggested by the marked increase in the frequency of the mepps recorded in vitro 5-15 min after venom addition. A direct muscular effect was shown by the dose- and time-dependent reduction in the resting membrane potential of the diaphragm. Chick preparations were more sensitive than those of the mouse. In the isolated chick biventer cervicis muscle preparation, B. insularis venom induced a contracture and a dose-dependent block of responses to indirect stimulation. At low venom concentrations (1-5 micrograms/ml), no significant release of creatine kinase (CK) was observed from this preparation. However, a dose-dependent release of CK was detected at higher doses (10-80 micrograms/ml). For morphological studies, B. insularis venom was injected into the chick left pectoralis muscle. At low doses (0.4 microgram), only an inflammatory reaction was present, while at high doses (20-80 micrograms) increasing numbers of necrotic fibers were observed as well as occlusive thrombosis and hemorrhage. The muscular effect, also observed on the incubated muscle, points to a direct myolytic action of the whole venom.

Effects induced by bothropstoxin, a component from Bothrops jararacussu snake venom, on mouse and chick muscle preparations.

Bothropstoxin, a 13,700 mol. wt myotoxic phospholipase homologue isolated from the venom of Bothrops jararacussu and devoid of PLA2, proteolytic or hemolytic activities, inhibited muscle twitch tension, evoked either directly or indirectly through stimulation of the motor nerve in the mouse phrenic-diaphragm preparations. The compound action potential of the muscle was also abolished with a similar time course. In addition, the toxin (0.7 mM) evoked membrane depolarization which was inhibited in the presence of 10 mM Ca2+. In chick biventer cervicis muscle, the toxin (2 mM) induced a contracture that reached its maximum amplitude in 44.8 +/- 15.6 min (n = 6) and was not blocked by either d-tubocurarine or tetrodotoxin. The time to maximum amplitude was reduced to 5.5 +/- 1.0 min (n = 4) in nominally Ca(2+)-free Krebs solution and was completely abolished in Ca(2+)-free Krebs solution containing 1 mM EGTA.

The ability of specific antivenom and low temperature to inhibit the myotoxicity and neuromuscular block induced by Micrurus nigrocinctus venom.

In the isolated mouse diaphragm preparation, Micrurus nigrocinctus venom produced a dose-dependent contracture and blockade of the contractile response to direct and indirect electrical stimulation of the muscle. This effect could not be completely reversed by repeated washing of the preparation nor by the addition of neostigmine or 3, 4-diaminopyridine. The observation that the direct blockade had to be preceded by indirect blockade together with the capacity for venom to prevent the ACh- but not the KCl-induced contractures in biventer cervicis and chronically denervated preparations strongly suggests a curarimimetic action for the venom. The temperature at which the experiment was performed greatly influenced the neuromuscular blocking and myotoxic actions of the venom and suggests that the venom component responsible for these effects is thermolabile. Both the neuromuscular blocking action and the myotoxicity of the venom could be prevented by a specific M. nigrocinctus antivenom regardless of whether this was added together with or after the venom. The muscle morphological changes induced by the venom were accompanied by a corresponding increase in the release of creatine kinase (CK) into the incubation medium. This release was, however, submaximal (35%) when compared to that induced by the detergent Triton X-100. In contrast to what has been demonstrated for other Micrurus venoms (M. frontalis, M. corallinus, M. lemniscatus and M. spixii), our results show that the myotoxic effect induced by M. nigrocinctus venom is important for the development of blockade of the muscle contractile response.

Muscle necrosis and regeneration after envenomation by Bothrops jararacussu snake venom.

The lesions caused by sublethal doses of Bothrops jararacussu venom injected into tibialis anterior (tib. ant.) muscles of mice were studied with paraffin sections. Doses of 5 and 20 micrograms produced a large area of necrosis in tib. ant., but hardly affected neighbouring muscles. Phagocytosis of necrotic remnants was followed by marked regeneration of the muscle fibres. Within two weeks of the 5 micrograms dose there was recovery to near normal appearance and slight fibrosis. With 20 micrograms, a circumscribed scar and stronger interstitial fibrosis developed in the tib. ant. Most regenerated muscle fibres were small, but varied in diameter, retained central nuclei for three months (the longest survival) and were surrounded by collagen. Doses of 80 and 200 micrograms produced widespread coagulative necrosis of tib. ant., though neighbouring leg muscles were relatively spared. Myonecrosis was evident microscopically at 10 min, and over the next week the necrotic muscle remained acellular and devoid of inflammatory reaction except at the very edge. Blood vessels within and outside tib. ant. often became hyalinized and thrombosed. Phagocytosis of debris proceeded from the periphery, and after two weeks the muscle was replaced by fibro-adipose tissue. There was little if any muscle fibre regeneration. Abscesses developed in the vicinity of the injection site in several mice receiving high venom doses, but never after low doses or saline. Muscle necrosis after B. jararacussu venom seems due primarily to direct action of the venom, though vascular thrombosis and ischaemia may contribute. The venom can cause fibrosis of muscle and hinder or prevent muscle fibre regeneration.

Effects of Bothrops pirajai venom on the mouse extensor digitorum longus (EDL) muscle preparation.

The effects of Bothrops pirajai snake venom on the mouse extensor digitorum longus (EDL) preparation were examined using myographic, histopathological and biochemical approaches. B. pirajai venom (10, 25 or 50 microg/ml) dose dependently and irreversibly blocked the contractile response of indirectly stimulated EDL muscle. Histopathological analysis of EDL muscle incubated with venom showed dose-dependent damage with a loss of the normal tissue structure and the appearance of highly dark, edematous fibers together with myofibrils in various stages of condensation. At high doses of venom (50 microg/ml), loss of muscle cells was observed. In non-stimulated EDL, B. pirajai venom (10 and 50 microg/ml) caused a time-dependent release of CK which was maximal after 120 min. These results suggest that a component(s) present in the B. pirajai venom has a direct myolytic action on the skeletal muscle.

Neutralizing capacity of antisera raised in horses and rabbits against Crotalus durissus terrificus (South American rattlesnake) venom and its main toxin, crotoxin.

Crotalus durissus terrificus (South American rattlesnake) venom possesses myotoxic and neurotoxic activities, both of which are also expressed by crotoxin, the principal toxin of this venom. We have investigated the ability of commercial equine antivenom and antivenoms raised in rabbits against C. d. terrificus venom and crotoxin to neutralize the physiological and morphological changes induced by this venom and crotoxin in electrically-stimulated phrenic nerve-diaphragm (PND) and extensor digitorum longus (EDL) preparations of mice. The time required to produce 50% neuromuscular blockade in the PND and EDL preparations was, respectively, 103+/-9 and 59+/-6 min for C. d. terrificus venom (10 microg/ml) and 75+/-9 and 110+/-7 min for crotoxin (10 microg/ml). The antivenoms dose-dependently inhibited this neuromuscular activity of the venom and crotoxin. At a venom:antivenom ratio of 1:3, the rabbit antivenoms were as effective as the commercial equine antivenom. The creatine kinase (CK) concentrations in the organ bath containing EDL muscle were 290 and 1020 U/l following a 120 min exposure to C. d. terrificus venom and crotoxin, respectively. All of the antivenoms neutralized the release of CK by crotoxin, but were ineffective against C. d. terrificus venom. Histological analysis of the two preparations showed that rabbit anticrotoxin antivenom protected against the myotoxic action of C. d. terrificus venom and crotoxin better than the other antivenoms. We conclude that antisera raised in rabbits are better than equine antiserum in neutralizing the neurotoxic and myotoxic activities of C. d. terrificus venom and crotoxin.

Neutralization of the pharmacological effects of bothropstoxin-I from Bothrops jararacussu (jararacuçu) venom by crotoxin antiserum and heparin.

Bothropstoxin-I (BthTX-I), the principal myotoxin of Bothrops jararacussu venom, is devoid of phospholipase A(2) (PLA(2)) activity but capable of blocking neuromuscular transmission in mouse nerve-muscle preparations. In this study, the ability of crotoxin antiserum and heparin in preventing the neurotoxic and myotoxic effects of BthTX-I was investigated. Phrenic nerve-diaphragm preparations (PND) stimulated indirectly with supramaximal stimuli (0.2 ms, 0.1 Hz) were incubated with BthTX-I (20 microg/ml) alone or with BthTX-I preincubated with antiserum or heparin for 30 min at 37 degrees C prior to testing. Control preparations were incubated with Tyrode solution, antiserum or heparin alone. BthTX-I (20 microg/ml) produced 50% neuromuscular blockade in the PND preparations in 31+/-4min, with complete blockade occurring in 120 min. The antiserum and heparin significantly prevented the neuromuscular blockade caused by BthTX-I (84 +/- 4% and 100% protection, respectively). Light microscopy examination of the muscles at the end of the 120 min incubation showed that BthTX-I damaged 48 +/- 6% of the fibers. Preincubating the toxin with antivenom significantly reduced the extent of this damage (only 15 +/- 4% of fibers affected, corresponding to 69% protection, P<0.01) whereas heparin offered no protection (34 +/- 7% of fibers affected, not significantly different from that seen with toxin alone). These results show that the antivenom was more effective in neutralizing the myotoxic effects of BthTX-I than was heparin.

The effects of Duvernoy's gland secretion from the xenodontine colubrid Philodryas olfersii on striated muscle and the neuromuscular junction: partial characterization of a neuromuscular fraction.

The effect of Philodryas olfersii Duvernoy's secretion was studied in vivo in mice and chicks as well as in the mouse phrenic nerve-diaphragm and the chick biventer cervicis preparations. The whole secretion (20-40 micrograms/ml) increased the creatine kinase (CK) levels in mice but had no effect on the mouse phrenic nerve-diaphragm preparation. In the chick, the secretion caused head drop and paresia as well as irreversible blockade of the twitch-tension evoked by indirect stimulation in the chick biventer cervicis preparation (50% paralysis in 34.5 +/- 2.7 min, n = 4). The secretion also caused muscle contracture (30% of the maximal twitch-tension generated) after a latency of nearly 9 min. Following fractionation on a Superose 12 FPLC column, the neuromuscular activity was recovered in the high mol. wt fraction (Peak I). At a concentration of 10 micrograms/ml in the chick biventer cervicis preparation, Peak I caused 50% paralysis within 18.5 +/- 3.0 min (n = 4), and evoked a strong contracture (70% of the maximal twitch-tension generated). The contractile responses of the chick preparation to ACh and KCL were partially blocked (90%) by the whole secretion and totally blocked by Peak I. CK release was increased by the whole secretion but not by Peak I. The whole secretion also produced various degrees of muscle cell lysis and extensive widening of the intercellular spaces. The latter showed a loosely arranged membranous network. In general, Peak I caused only minor morphological alterations compared with the whole secretion, although these were still significantly different from those observed in the control preparations. The changes principally involved hypercontraction of the muscle fibers. Based on the above results, we conclude that Peak I contains the factor(s) responsible for the in vitro effects on neuromuscular transmission, whereas the direct myotoxic effect is apparently caused by at least one other component of the Duvernoy's secretion.

No role for enzymatic activity or dantrolene-sensitive Ca2+ stores in the muscular effects of bothropstoxin, a Lys49 phospholipase A2 myotoxin.

The role of low levels of phospholipase A2 (PLA2) activity and intracellular Ca2+ stores in the pharmacological action of bothropstoxin (BthTX), a myotoxic Lys49 PLA2 homologue isolated from the venom of Bothrops jararacussu, was investigated. We examined the muscular effects of BthTX in the mouse diaphragm and its PLA2 activity in radiolabeled human and rat primary cultures of skeletal muscle. Although it is a Lys49 PLA2 homologue, BthTX had a low, but easily detectable, level of enzymatic activity relative to two Asp49 PLA2 enzymes from Naja naja kaouthia and Naja naja atra venoms, and this activity was reduced by about 85% in the presence of Sr2+ (4.0 mM). However, the replacement of 1.8 mM Ca2+ by 4 mM Sr2+ did not alter the BthTX-induced contracture and blockade of the muscle twitch tension. In addition, Sr2+ decreased by 50% the time required to cause 50% paralysis, and evoked approximately a four-fold increase in the number of spontaneous spikes. In isolated sarcoplasmic reticulum preparations, BthTX opened the intracellular Ca2+ release channel (ryanodine receptor) and lowered the threshold of Ca(2+)-induced Ca2+ release by a second, as yet unidentified, mechanism. However, in intact muscle, dantrolene, an antagonist of some forms of intracellular Ca2+ release, had no effect on the actions of BthTX. These findings do not support any role for the low levels of PLA2 activity, or dantrolene-sensitive intracellular Ca2+ stores, in the action of BthTX. The mechanism whereby Sr2+ stimulates the pharmacological activity of BthTX remains to be clarified.

An unusual presynaptic action of Bothrops insularis snake venom mediated by phospholipase A2 fraction.

A phospholipase A2-containing fraction was isolated from the venom of Bothrops insularis by a combination of gel filtration on Sephadex G-150 and ion exchange chromatography on DEAE-Sephadex. Peak IV of the latter chromatography containing all of the phospholipase A2 (PLA2) activity, was assayed on isolated neuromuscular preparations. In the mouse phrenic nerve-diaphragm incubated in Tyrode at 37 degrees C, the PLA2 fraction produced an initial increase in the twitch tension and in the frequency of the mepps, followed by a dose-dependent, irreversible blockade. The replacement of 1.8 mM Ca2+ by 4 mM Sr2 inhibited the neuromuscular blocking effect of the fraction. In the chick hiventer cervicis preparation incubated with Krebs solution at 37 degrees C, the PLA2 fraction induced blockade but did not affect the response to acetylcholine and K+, excluding the involvement of post-synaptic and direct muscular effects. A low temperature (18-22 degrees C) incubation prevented the neuromuscular effect from developing. These results suggest that the PLA2-containing fraction acts predominantly at presynaptic sites at the neuromuscular junction. This fraction also accounts for most of the pharmacological effects of the crude venom.

Characterization of a myotoxin from the Duvernoy's gland secretion of the xenodontine colubrid Philodryas olfersii (green snake): effects on striated muscle and the neuromuscular junction.

A myotoxin has been isolated from the Duvernoy's gland (DG) secretion of the xenodontine colubrid Philodrvas olfersii (green snake) by gel filtration on Sephadex G-100 SF. Under non-reducing and reducing conditions in SDS-PAGE, the myotoxin migrates as a single band with a mol. wt. of 20000. The toxin has 182 amino acid residues (approximately 20% acidic), a pI of 4.8 and a blocked N-terminal. In the chick biventer cervicis preparation, P. olfersii myotoxin partially blocks potassium-evoked contractures without affecting either the twitch-tension resulting from indirect stimulation or the contractures evoked by acetylcholine. Both the DG secretion and the myotoxin increase the serum creatine kinase (CK) levels of mice and stimulate the release of CK from the biventer cervicis preparation in a dose- and time-dependent manner. The varying degrees of muscle cell lysis and extensive widening of the intercellular spaces caused by the DG secretion are reproduced by the myotoxin, with the exception that in the latter the partial or total loss of transverse muscle striations is restricted to the muscle periphery. This myotoxin is the first such protein to be characterized from a DG secretion.

The pharmacological effect of Bothrops neuwiedii pauloensis (jararaca-pintada) snake venom on avian neuromuscular transmission.

The neuromuscular effects of Bothrops neuwiedii pauloensis (jararaca-pintada) venom were studied on isolated chick biventer cervicis nerve-muscle preparations. Venom concentrations of 5-50 micro g/ml produced an initial inhibition and a secondary increase of indirectly evoked twitches followed by a progressive concentration-dependent and irreversible neuromuscular blockade. At venom concentrations of 1-20 micro g/ml, the responses to 13.4 mM KCl were inhibited whereas those to 110 micro M acetylcholine alone and cumulative concentrations of 1 micro M to 10 mM were unaffected. At venom concentrations higher than 50 micro g/ml, there was pronounced muscle contracture with inhibition of the responses to acetylcholine, KCl and direct stimulation. At 20-24 degrees C, the venom (50 g/ml) produced only partial neuromuscular blockade (30.7 +/- 8.0%, N = 3) after 120 min and the initial inhibition and the secondary increase of the twitch responses caused by the venom were prolonged and pronounced and the response to KCl was unchanged. These results indicate that B.n. pauloensis venom is neurotoxic, acting primarily at presynaptic sites, and that enzyme activity may be involved in this pharmacological action.