Botulinum Toxin Inhibits Arachidonic Acid Release Associated with AcetylcholineRelease from PC12 Cells

摘要

The molecular mechanisms of depolarization-induced calcium-dependentacetylcholine (ACh) release and its inhibition by botulinum neurotoxin type A (BoTx) are not clear. We studied these mechanisms in an in vitro cholinergic neuronal pheochromocytoma PC12 cell line model. Cultured monolayer PC12 cells were differentiated by treatment with 50 ng/ml nerve growth factor (NGF) for 4 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 (three to four times) in (3H)Ach release in a Ca2+-dependent manner. K+-stimulated (3H)ACh release was totally inhibited by pretreatment of cells with BoTx (2 nm) for 2 h. High K+ also stimulated the release of arachidonic acid (3H)AA from the cell membrane, which was inhibited by BoTx (2 nm). Addition of phospholipase A2 (PLA2) inhibitors (quinacrine, 4-bromophenacyl bromide, manoalide) to the perfusion