J.C. Cogo

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.

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 Duvernoys 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 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 Duvernoys 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 Duvernoys secretion.

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.