The neuromuscular activity of Micrurus pyrrhocryptus venom and its neutralization by commercial and specific coral snake antivenoms

Abstract

The neuromuscular activity ofMicrurus pyrrochryptus venom was studied in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. The venom (0.5-50μg/ml) caused irreversible, time- and concentration-dependent blockade, with BC being more sensitive than PND (50% blockade with 10μg/ml in 22±;3min and 62±4min, respectively; mean±SEM, n=6; p<0.05). In BC preparations, venom (0.5μg/ml) progressively abolished ACh-induced contractures, whereas contractures to exogenous KCl and muscle twitches in curarized preparations were unaffected. The venom neither altered creatine kinase release (venom: 25.8±1.75IU/l vs control: 24.3±2.2IU/l, n=6, after 120min), nor it caused significant muscle damage (50μg of venom/ml vs control: 3.5±0.8% vs 1.1±0.7% for PND; 4.3±1.5% vs 1.2±0.5% for BC, n=5). The venom had low PLA(2) activity. Neurotoxicity was effectively neutralized by commercial Micrurus antivenom and specific antivenom. These findings indicate that M. pyrrhocryptus venom acts postsynaptically on nicotinic receptors, with no significant myotoxicity.

Keywords: Coral snake venom; neuromuscular blockade; neurotoxin; nicotinic receptor; postsynaptic.

Figure 1.

Concentration-dependent neuromuscular blockade caused by M. pyrrhocryptus venom in indirectly stimulated chick biventer cervicis (A) and mouse phrenic nerve-diaphragm (B) preparations. Each point represents the mean ±SEM of six experiments. C. Contractures of chick muscle to exogenous ACh, CCh and KCl after incubation with venom (1, 5, 10 and 50μg/ml). D. Recording of a chick biventer cervicis preparation incubated with M. pyrrhocryptus venom (5μg/ml, arrow, 0min) showing contractures to exogenous ACh (□ 110μM), CCh (▲ 8μM) and KCl (● 20mM) before and after incubation with venom. This recording is representative of six experiments, the mean values of which are shown in panels A-C. W = wash. *p<0.05 compared with control values.

Figure 2.

Comparison of the responses of mouse phrenic nerve-diaphragm preparations to indirect tetanic stimulation (T₁, T₂ and T₃; 70Hz, 0.2ms) in the presence of (A) α-bungarotoxin and (B) M. pyrrhocryptus venom (V). Note that the tetanic response after incubation with M. pyrrhocryptus venom was similar to that seen after exposure to α-bungarotoxin (n=6 each).

Figure 3.

Kinetics of the blockade of contractures to exogenous ACh in the presence of M. pyrrhocryptus venom (0.5μg/ml) in chick biventer cervicis preparations. Note the progressive decrease in the responses over time. Each bar represents the mean ±SEM of six experiments. *p<0.05 compared with the response before venom addition (0min) (n=5 for each interval).

Figure 4.

Myographic recording of mouse phrenic nerve-diaphragm contractions in response to M. pyrrhocryptus venom (V, 10μg/ml) under direct (D) and indirect (I) stimulations. The preparation was previously treated with 3μM d-tubocurarine (d-Tc) to inhibit any response of presynaptic origin (W = wash, n=6).

Figure 5.

Creatine kinase (CK) released from chick biventer cervicis preparations incubated with M. pyrrhocryptus venom (5μg/ml). The venom caused no significant release of CK at 120min incubation when compared with control preparations. The points are the mean ±SEM of six experiments.

Figure 6.

Neutralization by commercial antivenom (CAV) and specific antivenom (SAV) of the neuromuscular blockade caused by M. pyrrhocryptus venom (10μg/ml) in avian biventer cervicis (A) and mouse phrenic nerve-diaphragm (B) preparations. Negative control preparations were incubated with Krebs (A) or Tyrode (B) solution alone. The points are the mean ±SEM of six experiments (p<0.05).