Biochemical and biological evaluation of gyroxin isolated from Crotalus durissus terrificus venom

Gyroxin, a thrombin-like enzyme isolated from Crotalus durissus terrificus venom and capable of converting fibrinogen into fibrin, presents coagulant and neurotoxic activities. The aim of the present study was to evaluate such coagulant and toxic properties. Gyroxin was isolated using only two chromatographic steps - namely gel filtration (Sephadex G-75) and affinity (Benzamidine Sepharose 6B) - resulting in a sample of high purity, as evaluated by RP-HPLC C2/C18 and electrophoretic analysis that showed a molecular mass of 30 kDa. Gyroxin hydrolyzed specific chromogenic substrates, which caused it to be classified as a serine proteinase and thrombin-like enzyme. It was stable from pH 5.5 to 8.5 and inhibited by Mn²+, Cu²+, PMSF and benzamidine. Human plasma coagulation was more efficient at pH 6.0. An in vivo toxicity test showed that only behavioral alterations occurred, with no barrel rotation. Gyroxin was not able to block neuromuscular contraction in vitro, which suggests that its action, at the studied concentrations, has no effect on the peripheral nervous system.

Understanding the in vitro neuromuscular activity of snake venom Lys49 phospholipase A2 homologues.

Phospholipases A(2) (PLA(2)s) with a lysine substituting for the highly conserved aspartate 49, Lys49 PLA(2) homologues, are important myotoxic components in venoms from snakes of Viperidae family. These proteins induce conspicuous myonecrosis by a catalytically-independent mechanism. Traditionally, the Lys49 PLA(2) homologues are classified as non-neurotoxic myotoxins given their inability to cause lethality or paralytic effects when injected in vivo, even at relatively high doses. However, a series of in vitro studies has shown that several Lys49 PLA(2) homologues from Bothrops snake venoms induce neuromuscular blocking activity on nerve-muscle preparations in vitro. The interpretation of these findings has created some confusion in the literature, raising the question whether the Lys49 PLA(2) homologues present some neurotoxic activity. The present article reviews the in vitro neuromuscular effects of Lys49 PLA(2) homologues and discusses their possible mechanisms of action. It was concluded that the neuromuscular blockade induced by Lys49 PLA(2) homologues in isolated preparations is mainly a consequence of the general membrane-destabilizing effect of these toxins.

Effects of nandrolone decanoate on the neuromuscular junction of rats submitted to swimming.

This study addressed the effects of nandrolone decanoate (ND) on contractile properties and muscle fiber characteristics of rats submitted to swimming. Male Wistar rats were grouped in sedentary (S), swimming (Sw), sedentary+ND (SND), and swimming+ND (SwND), six animals per group. ND (3 mg/kg) was injected (subcutaneously) 5 days/week, for 4 weeks. Swimming consisted of 60-min sessions (load 2%), 5 days/week, for 4 weeks. After this period, the sciatic nerve extensor digitorum longus (EDL) muscle was isolated for myographic recordings. Fatigue resistance was assessed by the percent (%) decline of 180 direct tetanic contractions (30 Hz). Safety margin of synaptic transmission was determined from the resistance to the blockade of indirectly evoked twitches (0.5 Hz) induced by pancuronium (5 to 9x10(-7) M). EDL muscles were also submitted to histological and histochemical analysis (haematoxylin-eosin (HE); nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR)). Significant differences were detected by two-way ANOVA (p<0.05). ND did not change body mass, fatigue resistance or kinetic properties of indirect twitches in either sedentary or swimming rats. In contrast, ND reduced the safety margin of synaptic transmission in sedentary animals (SND=53.3+/-4.7% vs. S=75.7+/-2.0%), but did not affect the safety margin in the swimming rats (SwND=75.81+/-3.1% vs. Sw=71.0+/-4.0%). No significant difference in fiber type proportions or diameters was observed in EDL muscle of any experimental group. These results indicate that ND does not act as an ergogenic reinforcement in rats submitted to 4 weeks of swimming. On the other hand, this study revealed an important toxic effect of ND, that it reduces the safety margin of synaptic transmission in sedentary animals. Such an effect is masked when associated with physical exercise.

Influence of temperature upon effects of crotoxin and gamma-irradiated crotoxin at rat neuromuscular transmission.

The influence of temperature upon the effects of crotoxin (CTX), from Crotalus durissus terrificus venom, and gamma-irradiated (60Co, 2000 Gy) crotoxin (iCTX) was studied in rat neuromuscular transmission 'in vitro'. Indirect twitches were evoked in the phrenic-diaphragm preparation by supramaximal strength pulses with a duration of 0.5 ms and frequency of 0.5 Hz. The phospholipase A(2) (PLA(2)) enzymatic activity of CTX and iCTX was assayed against phosphadityl choline in Triton X-100. At 27 degrees C, CTX (14 microg/ml) did not affect the amplitude of indirectly evoked twitches. However, at 37 degrees C, CTX induced a time-dependent blockade of the neuromuscular transmission that started at 90 min and was completed within 240 min. iCTX (14 microg/ml) was inneffective on the neuromuscular transmission either at 27 or 37 degrees C. The PLA(2) enzymatic activity of CTX at 37 degrees C was 84 and that at 27 degrees C was 27 micromol fatty acid released/min/mg protein, and that of the iCTX at 37 degrees C was 39 micromol fatty acid released/min/mg protein. Thus, it was concluded that the mechanism of detoxification of CTX by gamma radiation at the neuromuscular level relies on the loss of its PLA(2) enzymatic activity.

Reduction of crotoxin-induced neuromuscular blockade by gamma radiation.

A comparative study between crotoxin and gamma irradiated crotoxin was performed on the indirectly evoked twitches and tetani of sciatic nerve-extensor digitorum longus muscle of rats. Crotoxin (3 to 14 microg/ml) decreased the amplitude of twitches and induced a slight tetanic fade, and irradiated crotoxin did not significantly affect either twitch amplitude or tetanic tension. Since gamma radiation reduced the neurotoxicity of crotoxin it may be useful for the production of anticrotalic serum.

Pre- and postsynaptic mechanisms involved in tetanic fade induced by pancuronium in the isolated rat muscle.

The mechanisms underlying the fade of the tetanic contraction induced by pancuronium were studied in vitro by means of myographical and electrophysiological techniques in the extensor digitorum longus muscle of the rat. Pancuronium (0.5 mumol/l) induced a complete fade of the tetanic contraction while leaving the twitch unaffected. At the same concentration it decreased the amplitude and increased the tetanic rundown of trains of endplate potentials (e.p.ps) evoked in the frequency of 50 Hz. The electrophysiological changes induced by pancuronium were due to decreases in both quantal sizes and quantal contents of the e.p.ps. The former effect was the result of a postsynaptic competitive action and the latter of a presynaptic inhibitory action of that compound. The decrease in quantal content affected the e.p.ps starting from the first in the train and became larger during the generation of the sequence of e.p.ps. This intensified their tetanic rundown. It is concluded that the fade of the tetanic contraction induced by pancuronium is due to a summation of pre- and postsynaptic actions and, therefore, not only to an increase in the tetanic rundown of e.p.ps. Possible explanations for the distinct abilities of neuromuscular blockers in affecting tetani and twitches in a differential manner are also discussed.

Mechanisms of hexamethonium-induced tetanic fade in the isolated rat muscle.

The action of hexamethonium on neuromuscular transmission was investigated on the rat extensor digitorum longus muscle in vitro. Hexamethonium (5 x 10(-4) M) induced a complete fade of the tetanic contraction while leaving the twitch unaffected. At the same concentration, hexamethonium induced a significant decrease in the amplitude of the endplate potentials evoked at 50 and 100 Hz. Additionally, hexamethonium (5 x 10(-4) M) significantly increased the tetanic rundown of the endplate potential trains evoked at 100 Hz. The former effect was mainly the result of a frequency-independent decrease in the quantal size of the endplate potentials. This decrease seemed to be due to a postsynaptic blocking action of hexamethonium. The increase in tetanic rundown was due to a presynaptic action of hexamethonium. Such an action led to a frequency-dependent decrease in the quantal release of transmitter during repetitive stimulation of the motor nerve. It is concluded that both pre- and postsynaptic actions are necessary for hexamethonium to induce tetanic fade without affecting the twitch; and that, if it is accepted that the presynaptic action of hexamethonium is exerted on presynaptic receptors, these are not of the ganglionic type since the presynaptic effect of hexamethonium was less pronounced than the postsynaptic one.