Phospholipase A(2) and arachidonic acid-mediated mechanism of neuroexocytosis: A possible target of botulinum neurotoxin a other than SNAP-25

The vesicular neuroexocytosis process consists of two important steps: fusi on of transmitter-loaded vesicles at release sites on the presynaptic nerve terminal membrane; followed by the release of transmitter molecules into t he synaptic cleft, We previously reported that in nerve growth factor (NGF) -differentiated PC12 cells, arachidonic acid (AA) release is associated wit h acetylcholine (ACh) release, botulinum neurotoxin A (BoNT/A) inhibits bot h processes and AA itself or a phospholipase A(2) (PLA(2)) activator can ca use ACh release in BoNT/A-poisoned cells in which SNAP-25 has supposedly be en hydrolyzed, In the present study, we examined the roles of two endogenou s intraterminal components in neuroexocytosis: the membrane fusogenic agent AA; and the vesicle fusion protein SNAP-25, A PLA(2) activator, mastoparan , was used to induce the release of AA and ACh from NGF-differentiated PC12 cells. Release depended upon the mastoparan concentration, as well as Ca2 influx via the neuronal-type voltage-sensitive Ca2+ channels. Release of A Ch followed a rise in intracellular free Ca2+ concentration; the increased Ca2+ activated PLA, and, thereby, increased the AA level. Scanning and tran smission electron microscopy confirmed that mastoparan-induced ACh and AA r elease were not due to simple diffusion through damaged plasma membranes, T reatment of PC12 cells with appropriate antisense oligonucleotides blocked SNAP-25 expression, as judged by Western blot protein analysis with a speci fic monoclonal antibody. Despite apparent elimination of SNAP-25, treatment of differentiated PC12 cells with mastoparan and high (80 mM) K+ induced A Ch exocytosis. The results support the conclusion that PLA(2) and AA have i mportant roles in neuroexocytosis that are independent of SNAP-25, Both PLA (2) and AA have been shown to be involved in actin cytoskeletal organizatio n related to vesicle fusion and exocytosis. This mechanism may be an altern ative target of BoNT/A other than SNAP-25.