ABSTRACT
The neuromuscular activity of Bbil-TX, a PLA2 with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5-10 µg/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87 ± 7, 41 ± 7 and 19 ± 2 min (mean ± SEM; n = 4-6) for 1, 5 and 10 µg/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10 µg/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10 µg/ml) BC preparations. However, Bbil-TX (10 µg/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116 ± 17 IU/ml (basal) to 710 ± 91 IU/ml after 45 min). Bbil-TX (5 µg/ml)-induced blockade was markedly inhibited at 22-24 °C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3-30 µg/ml, n = 4-6) caused partial time- and concentration-dependent blockade in PND preparations (52 ± 2% at the highest concentration). Bbil-TX (30 µg/ml) also markedly reduced the MEPPs frequency [from 26 ± 2.5 (basal) to 10 ± 1 after 60 min; n = 5; p < 0.05] and the quantal content [from 94 ± 14 (basal) to 24 ± 3 after 60 min; n = 5; p < 0.05] of PND preparations, but caused only minor depolarization of the membrane resting potential [from -80 ± 1 mV (basal) to -66 ± 2 mV after 120 min; n = 5; p < 0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA2 that contributes to the neuromuscular blockade caused by B. b. smargadina venom.
Subject(s)
Neuromuscular Blockade , Phospholipases A2, Secretory/pharmacology , Reptilian Proteins/pharmacology , Viper Venoms/chemistry , Acetophenones/metabolism , Animals , Bothrops , Carbachol/pharmacology , Chickens , Diaphragm/drug effects , Diaphragm/metabolism , Male , Mice , Miniature Postsynaptic Potentials/drug effects , Muscle Contraction/drug effects , Muscle, Skeletal/drug effects , Neuromuscular Junction/drug effects , Phrenic Nerve/drug effects , Phrenic Nerve/metabolismABSTRACT
In this work, we studied the neuromuscular blockade caused by Micrurus altirostris venom (0.1-10 microg/mL) in indirect stimulated chick biventer cervicis and mouse phrenic nerve-diaphragm preparations and the ability of commercial antivenom (Instituto Butantan) and antiserum raised in rabbits to neutralize neurotoxicity and lethality in chicks and mice (LD(50) 0.042 and 0.255 mg/kg), injected i.m. and i.p., respectively, with venom (5 LD(50)):antivenom or antiserum mixtures (n = 6) of 1:1-1:2.5-1:5-1:10-1:20. The venom caused a complete and irreversible neuromuscular blockade in both preparations, inhibited the acetylcholine and carbachol contractures, without interfering on KCl response. The neuromuscular blockade was not Ca(2+) or temperature-dependent and did not affect the response to direct stimulation. Only a venom:antivenom or antiserum ratio of 1:20 neutralized the neuromuscular blockade in vitro and protected chicks and mice against 5 LD(50) of venom. Our results indicated that Micrurus altirostris venom interferes with postsynaptic neurotransmission and that commercial antivenom and rabbit antiserum have low efficacy in neutralizing the neurotoxicity and lethality of this venom.
