Vol. 301, Issue 3, 953-962, June 2002

Pardaxin Stimulation of Phospholipases A₂ and Their Involvement in Exocytosis in PC-12 Cells

Eugenia Bloch-Shilderman, Saleh Abu-Raya¹ , Victoria Trembovler, Hassia Boschwitz, Arie Gruzman, Michal Linial and Philip Lazarovici

Department of Pharmacology and Experimental Therapeutics, School of Pharmacy, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel (E.B.-S., S.A.-R., V.T., A.G., P.L.); and Department of Biological Chemistry, Life Sciences Institute, The Hebrew University of Jerusalem, Israel (H.B., M.L.)

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.