Phospholipase A(2) activity of beta-bungarotoxin is essential for induction of cytotoxicity on cerebellar granule neurons.
The aim of this study was to investigate the mechanism of the cytotoxic effect of beta-bungarotoxin (beta-BuTX), a presynaptic neurotoxin, on rat cerebellar granule neurons (CGNs). The maturation of CGNs is characterized by the prominent dense neurite networks that became fragmented after treatment with beta-BuTX, and this cytotoxic effect of beta-BuTX on CGNs was in a dose- and time-dependant manner. The cytotoxic effect of beta-BuTX was found to be more potent than other toxins, such as alpha-BuTX, cardiotoxin, melittin, and Naja naja atra venom phospholipase A(2). Meanwhile, undifferentiated neuroblastoma neuronal cell lines, IMR-32 and SK-N-MC, and astrocytes were found to be resistant to beta-BuTX. These results indicated that only the mature CGNs were sensitive to beta-BuTX insults. None of the following chemicals: antioxidants, K(+)-channel activator, K(+)-channel antagonists, intracellular Ca(2+) chelator, Ca(2+)-channel blockers, NMDA receptor antagonists, and nitric oxide synthase inhibitor tested, were able to reduce beta-BuTX-induced cytotoxicity. However, secretory type phospholipase A(2) inhibitors (glycyrrhizin and aristolochic acid) and a free radical scavenger (5,5-dimethyl pyrroline N-oxide, DMPO) could attenuate not only beta-BuTX-induced cytotoxicity but also ROS production and caspase-3 activation. These data suggest that phospholipase A(2) activity of beta-BuTX may be responsible for free radical generation and caspase-3 activation that accounts for the observed cytotoxic effect. It is proposed that the CGNs can be a useful tool for studying interactions of the molecules on neuronal plasma membrane with beta-BuTX that mediates the specific cytotoxicity.
Chen
Yu-Jen
YJ
Department of Medical Technology and Institute of Biotechnology, Yuanpei University of Science and Technology, Hsinchu, Taiwan. yjchen@mail.yust.edu.tw
D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D002038:Bungarotoxins / Q000494:pharmacology*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology; D009498:Neurotoxins / Q000494:pharmacology*; D010741:Phospholipases A / Q000378:metabolism*; D011796:Quinacrine / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors
D002038:Bungarotoxins; D004791:Enzyme Inhibitors; D005609:Free Radicals; D009498:Neurotoxins; D010741:Phospholipases A; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D011796:Quinacrine