A Novel Insight into the Mechanism of Action of Botulinum Toxin in a Cholinergic Neuronal Cell Culture Model.

Botulinum neurotoxin type A BoTx, which is an identified biological threat agent acts by blocking the Ca 2 -dependent release of the neurotransmitter acetylcholine ACh from presynaptic nerve terminals at neuromuscular junctions. There is no effective antidote against this deadly poison primarily due to a lack of understanding of the exact sequence of events leading to the neuromuscular blockade. We studied this mechanism in an in vitro cholinergic neuronal pheochromocytoma PC 1 2 cell line model. Cultured monolayer PC12 cells were differentiated by treatment with 50 ngml nerve growth factor NGF for 4 to 5 days to enhance cellular ACh synthesis and release. Stimulation of these cells with high K 80 mM in the perfusion medium caused a marked increase 3-4X in 3Hach release in a Ca 2 -dependent manner. K-stimulated 3HACh release was totally inhibited by pretreatment of cells with BoTx 2 nM for 2 h. High K also stimulated the release of arachidonic acid 3HACh from the cell membrane, which was inhibited by BoTx 2 nM. Addition of quinacrine, a phospholipase A2 PLA2 inhibitor, to the perfusion medium inhibited Kstimulated 3HACh and 3HAA release in a dose-dependent manner. Inclusion of exogenous AA, the PLA2, activator mellitin or PLA, itself prevented the effect of BoTx. These results demonstrate that in NGF differentiated PC12 cells, AA release is associated with ACh release, BoTx inhibits both processes, and increased AA can protect against BoTx. Drugs that can modulate PLA2 activity or the level of intracellular AA or its metabolites may therefore be prospective countermeasures against BoTx poisoning.