beta-Bungarotoxin (beta-BuTx), the presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide subunits. A phospholipase A2 subunit named A chain, and a non-phospholipase A2 subunits named B chain. The A chain and B chain are covalently linked by one disulfide bridge. Although it has been widely accepted that the toxic effect of beta-BuTx is attributed to the disturbance of presynaptic transmitter release, however the inhibition of transmitter release by beta-BuTx is still obscure. Here we investigate the mechanism that mediates facilitation of transmitter release at the neuromuscular junction induced by beta-BuTx, using Xenopus nerve-muscle coculture. Application of beta-BuTx and isotoxins BM12, BM13 led to a marked increase in the frequency of spontaneous synaptic currents (SSCs) after a short period (12~18 min) of latency. The synaptic potentiation induced by these toxins was abolished when Ca2+ in the medium is substituted by Ba2+ (a potent phospholipase A2 inhibitor). Application of PLP-BM12 and PLP-BM13, which have been chemical-modification to lose their PLA2 activity from BM12 and BM13, failed to potentiate the transmitter release.

Phenylarsine oxide (PAO) is a membrane-permeable trivalent arsenic compounds, which interfere the biochemical activity of intracellular enzymes or proteins through reacting specifically with sulfhydryl and vicinal dithiol groups in the protein structure. Although the deleterious effects of arsenic compounds in bioorganisms have been extensively studied, however its role in the synaptogenesis is still obscure. Here we test the role of PAO on the synaptic activity at developing Xenopus neuromuscular synapse by using whole-cell patch clamp recording. Bath application of PAO dose-dependently increases the frequency of spontaneous synaptic currents (SSC frequency) and reaches its maximal effect at 10 £gM. The SSC frequency is robustly facilitated in 10~15 minutes after PAO application and then the release of neurotransmitter were abruptly ceased due to the degenerative collapse of the presynaptic motoneuron. Pretreatment of the culture with Ca2+ chelator BAPTA-AM significantly blunted the SSC frequency facilitation induced by PAO, suggesting a rise in Ca2+ in presynaptic motoneuron is a prerequisite. The PAO-induced SSC frequency facilitation is unaffected even that Ca2+ is eliminated from culture medium or addition of pharmacological Ca2+ channel inhibitor cadmium, indicating the influx of extracellular Ca2+ is not needed for the rise of [Ca2+]i. Depletion of endoplasmic reticulum Ca2+ pool with thapsigargin effectively hampered the PAO-induced SSC frequency facilitation. Pretreatment of ryanodine receptor inhibitor TMB-8 but not IP3 receptor inhibitor XeC significantly occluded the increase of SSC frequency elicited by PAO. Furthermore, bath application of the culture with either mitochondria oxidative phosphorylation uncoupler FCCP or mitochondrial permeability transition pore inhibitor cyclosporin A significantly abolished the SSC facilitating effect of PAO. Pretreatment the culture with TMB-8 and cyclosporin A have no addictive effects on the occlusion of PAO-induced SSC frequency facilitation, suggesting a consecutively released Ca2+ from internal store through ryanodine receptor and mitochondria is responsible for PAO-induced SSC frequency facilitation. The synaptic facilitating effect of PAO is eliminated while incubated with free radical scavenger n-acetylcysteine. Furthermore, treating cultures with complex III of electron transport chain (ETC) inhibitor antimycin A, but not complex I inhibitor rotenone, abolished PAO-induced facilitation of synaptic transmission. PAO elicited no facilitation effects on SSC frequency when pretreatment the culture with either thiol-modifying agent NEM or thiol-reducing agent DTT. Overall, results from our current study provide evidences that reactive oxygen species derived from PAO inhibition on complex III of ETC induce the open of MPT pore in mitochondria, the accompanied Ca2+ leak from mitochondria and Ca2+-induced Ca2+ release from endoplasmic reticulum resulted in a robustly release of neurotransmitter and a destructive damage on the neuron.