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The aim of this study was to elucidate the mechanism of the neurotoxic effect of β-bungarotoxin (β-BuTX, a snake presynaptic neurotoxin isolated from the venom of Bungarus multicinctus) on cultured cerebellar granule neurons. β-BuTX exerted a potent, time-dependent, neurotoxic effect on mature granule neurons. Mature neurons, with an abundance of neurite outgrowths, were obtained after 7-8 days in culture. By means of microspectrofluorimetry and fura-2, we measured the intracellular Ca2+ concentration ([Ca2+]i) and found it to be increased markedly. BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tertrakis(acetoxymethyl ester)], EGTA, MK801 (dizocilpine maleate), and diltiazem prevented not only the elevation of [Ca2+]i, but also the β-BuTX-induced neurotoxic effect. The signaling pathway involved in the elevation of [Ca2+]i in β-BuTX-induced neurotoxicity was studied. The results obtained indicated that β-BuTX initially increased the production of reactive oxygen species and subsequently reduced mitochondrial membrane potential and depleted ATP. All of these events in the signaling pathway were blocked by MK801, diltiazem, EGTA, and BAPTA-AM. These findings suggest that the neurotoxic effect of β-BuTX is mediated, at least in part, by a cascade of events that include the direct or indirect activation of N-methyl-d-aspartate (NMDA) receptors and L-type calcium channels that, in turn, lead to Ca2+ influx, oxidative stress, mitochondrial dysfunction, and ATP depletion. Therefore, we suggest that this polypeptide neurotoxin, as a result of its high potency and irreversible properties, is a useful tool to elucidate the mechanisms of neurodegenerative diseases.