Pardaxin stimulation of phospholipases A₂ and their involvement in exocytosis in PC-12 cells

Pardaxin (PX) is a voltage-dependent ionophore that stimulates catecholamine exocytosis from PC-12 pheochromocytoma cells both in the presence and absence of extracellular calcium. Using a battery of phospholipase A₂ inhibitors we show that PX stimulation of phospholipase A₂ (PLA₂) enzymes is coupled with induction of exocytosis. We investigated the relationship between PX-induced PLA₂ activity and neurotransmitter release by measuring the levels of arachidonic acid (AA), prostaglandin E₂ (PGE₂), and dopamine release. In the presence of extracellular calcium, the cytosolic PLA₂ inhibitor arachidonyl trifluoromethyl ketone (AACOCF₃) inhibited by 100, 70, and 73%, respectively, the release of AA, PGE₂, and dopamine induced by PX. The mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor 2′-amino-3′-methoxyflavone (PD98059) reduced by 100 and 82%, respectively, the release of AA and PGE₂ induced by PX. In the absence of extracellular calcium, the calcium-independent PLA₂ (iPLA₂) inhibitors methyl arachidonyl fluorophosphonate, AACOCF₃, and bromoenol lactone (BEL) inhibited by 80 to 90% PX stimulation of AA release, by 65 to 85% PX stimulation of PGE₂ release, and by 80 to 90% PX-induced dopamine release. Using vesicle fusion-based enzyme-linked immunosorbent assay we found similar levels of inhibition of PX-induced exocytosis by these inhibitors. Also, PX induced the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes, an effect that was augmented by N-methylmaleimide. This complex formation was completely inhibited by BEL. Botulinum toxins type C1 and F significantly inhibited the release of AA, PGE₂, and dopamine induced by PX. Our data suggest that PX stimulates exocytosis by activating cystolic PLA₂ and iPLA₂, leading to the generation of AA and eicosanoids, which, in turn, stimulate vesicle competence for fusion and neurotransmitter release.