ABSTRACT 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 A 2 inhibitors we show that PX stimulation of phospholipase A 2 (PLA 2 ) enzymes is coupled with induction of exocytosis. We investigated the relationship between PX-induced PLA 2 activity and neurotransmitter release by measuring the levels of arachidonic acid (AA), prostaglandin E 2 (PGE 2 ), and dopamine release. In the presence of extracellular calcium, the cytosolic PLA 2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF 3 ) inhibited by 100, 70, and 73%, respectively, the release of AA, PGE 2 , and dopamine induced by PX. The mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor 2Ј-amino-3Ј-methoxyflavone (PD98059) reduced by 100 and 82%, respectively, the release of AA and PGE 2 induced by PX. In the absence of extracellular calcium, the calcium-independent PLA 2 (iPLA 2 ) inhibitors methyl arachidonyl fluorophosphonate, AACOCF 3 , and bromoenol lactone (BEL) inhibited by 80 to 90% PX stimulation of AA release, by 65 to 85% PX stimulation of PGE 2 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, PGE 2 , and dopamine induced by PX. Our data suggest that PX stimulates exocytosis by activating cystolic PLA 2 and iPLA 2 , leading to the generation of AA and eicosanoids, which, in turn, stimulate vesicle competence for fusion and neurotransmitter release. Hormones and neurotransmitters are usually released from cells by exocytosis, when a rise in cytosolic calcium triggers fusion of the secretory vesicle membrane with the plasma membrane SNAREs are targets for the botulinum and tetanus toxins Aside from toxins that inhibit neurotransmitter release, there are others that cause a massive release of neurotrans-E.B.-S. and S.A.-R. contributed equally to this work
