Abstract
Neuromuscular preparations exposed to B. marajoensis venom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposed in vitro to BmjeTX-I and BmjeTX-II toxins from B. marajoensis venom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.
Highlights
In Brazil, the accidents caused by Bothrops snakes are close to 74% according to the National System of Accidents Notification (SINAN) [1]; they constitute a major public health problem in Brazil
Synaptophysin, an integral protein of the synaptic vesicle, binds to synaptobrevin and may act to prevent vesicle docking at the axolemma of the terminal bouton; SNAP25 is a plasma membrane protein (t-SNARE), which in association with synaptobrevin forms a stable complex responsible for vesicle docking, priming, and fusion [17]. Alterations in this complex can affect the neurotransmitter release and induce a presynaptic-blocking effect. In this communication we describe our observations on the cellular pathology of the motor nerve terminal and skeletal muscle fibres exposed in vitro to two major toxins isolated from the venom of B. marajoensis
In order to support the twitch tension findings which showed that BmjeTX-I and BmjeTX-II phospholipase A2 (PLA2) caused a presynaptic neuromuscular blockade, we further investigated the neuromuscular junction (NMJ) ultrastructure by Transmission Electron Microscopy (TEM)
Summary
In Brazil, the accidents caused by Bothrops snakes are close to 74% according to the National System of Accidents Notification (SINAN) [1]; they constitute a major public health problem in Brazil. The envenomation picture is characterized by pronounced myonecrosis [3,4,5] and systemic effects [6]. Such effects result from proteolytic, myotoxic, blood-clotting, and hemorrhagic activities attributed to the phospholipases A2 contained in their venoms. It is likely that a direct action of phospholipase A2 (PLA2) myotoxins plus an indirect action caused by tissue anoxia has key roles
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