Neuromuscular activity of Micrurus laticollaris (Squamata: Elapidae) venom in vitro

Abstract

In this work, we have examined the neuromuscular activity of Micrurus laticollaris (Mexican coral snake) venom (MLV) in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations, the MLV induced an irreversible concentration- and time-dependent (1-30 µg/mL) neuromuscular blockade, with 50% blockade occurring between 8 and 30 min. Muscle contractures evoked by exogenous acetylcholine were completely abolished by MLV, whereas those of KCl were also significantly altered (86% ± 11%, 53% ± 11%, 89% ± 5% and 89% ± 7% for one, three, 10 and 30 µg of venom/mL, respectively; n = 4; p < 0.05). In mouse phrenic nerve-diaphragm preparations, MLV (1-10 µg/mL) promoted a slight increase in the amplitude of twitch-tension (3 µg/mL), followed by neuromuscular blockade (n = 4); the highest concentration caused complete inhibition of the twitches (time for 50% blockade = 26 ± 3 min), without exhibiting a previous neuromuscular facilitation. The venom (3 µg/mL) induced a biphasic modulation in the frequency of miniature end-plate potentials (MEPPs)/min, causing a significant increase after 15 min, followed by a decrease after 60 min (from 17 ± 1.4 (basal) to 28 ± 2.5 (t15) and 12 ± 2 (t60)). The membrane resting potential of mouse diaphragm preparations pre-exposed or not to d-tubocurarine (5 µg/mL) was also significantly less negative with MLV (10 µg/mL). Together, these results indicate that M. laticollaris venom induces neuromuscular blockade by a combination of pre- and post-synaptic activities.

Figure 1

Neuromuscular blockade caused by M. laticollaris venom (one, three, 10 and 30 µg/mL) in biventer cervicis (BC) preparations. (A) Graphic of the concentration- and time-dependent blockade; (B) Columns representing muscle contractures to exogenous ACh (110 µM) and KCl (40 mM) after 120 min of incubation or complete neuromuscular blockade are expressed as a percentage of the responses observed in control preparations (Krebs solution alone). The points in A and the columns in B are the mean ± SEM (n = 4); *p < 0.05 compared to control preparations in both A and B panels.

Figure 2

Concentration- and time-dependent neuromuscular blockade caused by M. laticollaris venom (one, three and 10 µg/mL) in phrenic-nerve diaphragm (PND) preparations. Note that a 3 µg/mL venom concentration caused a minor neuromuscular facilitation after 10 min of incubation. The points are the mean ± SEM (n = 4); * p < 0.05 compared to control preparations.

Figure 3

Neuromuscular blockade caused by M. laticollaris venom in BC and PND preparations. (A) Representative recordings showing the blockade caused by M. laticollaris venom (10 µg/mL) in a BC preparation indirectly stimulated at 37 °C. Note that responses to exogenous ACh (▲, 110 µM) were completely abolished by venom, whereas those to KCl (■, 40 mM) had a minor decrease; (B) Representative recordings showing the blockade caused by M. laticollaris venom (10 µg/mL) in a PND preparation indirectly stimulated at 37 °C. This venom concentration caused contracture in the baseline from 20 min of incubation.

Figure 4

Miniature end-plate potential (MEPP) measurements in PND preparations treated with M. laticollaris venom. The columns are the mean ± SEM of four muscles in which three different end-plates were measured per interval; * p < 0.05 compared to t₀ (control) values.

Figure 5

Membrane resting potential measurements in curarized and uncurarized PND preparations treated with M. laticollaris venom. (A) The depolarizing effect caused by venom (10 µg/mL) in uncurarized preparations; (B) The lack of depolarization by d-tubocurarine (d-Tc, 5 µg/mL) after 120 min of incubation; (C) The depolarizing effect caused by venom (10 µg/mL) in preparations previously treated with d-Tc (5 µg/mL) for 10 min. Note that pretreatment with d-Tc did not prevent the depolarizing effect caused by venom. The columns are the mean ± SEM of four muscles in which six different end-plates were measured per interval; * p < 0.05 compared to t₀ (control) values in all panels.