We demonstrated that
beta-bungarotoxin (beta-BuTX), a snake presynaptic
neurotoxin, exhibited a potent cytotoxic effect on cultured cerebellar granule neurons. The mechanism of action of beta-BuTX and the cytoprotective agents against beta-BuTX were studied. The neuronal death of cerebellar granule neurons induced by beta-BuTX was manifested with apoptosis and
necrosis processes as revealed by neurite fragmentation, morphological alterations, and staining apoptotic bodies with the
fluorescent dye Hoechst 33258. By means of microspectrofluorimetry and
fura-2, we measured intracellular Ca2+ concentration, [Ca2+]i and found that [Ca2+]i was increased markedly prior to the morphological changes and cytotoxicity. The downstream pathway of the increased [Ca2+]i was investigated: there was increased production of
free radicals, decreased mitochondrial membrane potential, and depleted cellular
ATP content.
MK801 and
suramin effectively suppressed these detrimental effects of beta-BuTX. Furthermore, the [3H]
MK801 binding was reduced by unlabeled
MK801, beta-BuTX, and
suramin. Thus, activation of
N-methyl-D-aspartate (
NMDA) receptors appeared to play a crucial role in the cytotoxic effects following betaBuTX exposure. In conclusion, the novel finding of this study was that a
polypeptide beta-BuTX exerted a potent cytotoxic effect through sequential events, including activating
NMDA receptors followed by increasing [Ca2+]i, ROS production, and impaired mitochondrial energy metabolism.
Suramin, clinically used as a trypanocidal agent, was an effective antagonist against beta-BuTX. Data suggest that
suramin might have value to detect the possible pathway of certain neuropathological disorders.