Pardaxin Stimulation of Phospholipases A₂and Their Involvement in Exocytosis in PC-12 Cells

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 2'-amino-3'-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.

The Birth of Neurochemical Maps

“…The observation that arachidonic acid had a stimulatory effect on exocytosis is also consistent with the results of previous studies, which showed increase in exocytosis in PC12 cells after PLA 2 activation (28,29). In addition, cPLA 2 alphaevoked formation of arachidonic acid and lysophospholipids was found to be required for exocytosis in mouse pancreatic beta-cells (30).…”

“…In this context, it is possible that eicosapentaenoic acid and arachidonic acid could present the ideal combination of acyl length/unsaturation necessary for optimal fluidity of the cell membrane and vesicle fusion. It has been reported that the fusion of synaptic vesicles with presynaptic membrane induced by arachidonic acid or PLA 2 activity could occur independently of calcium ions (33) or a key protein of the SNARE complex, SNAP-25 (29).…”

Cellular Function of Calcium-Independent Phospholipase A2

“…Paradaxin, a voltage-dependent ionophore that releases catecholamine from PC-12 pheochromocytoma cells, was revealed to activate iPLA 2 to stimulate AA release and eicosanoid production, which facilitate catecholamine release through an increase in fusion of vesicle to membranes. 38) In rat mesenteric resistance arteries, acetylcholinemediated vascular relaxation was attenuated by BEL, and external addition of AA to BEL-treated cells restored the relaxation completely. 39) These results suggest that AA released by iPLA 2 plays an important role in acetylcholine-induced relaxation in the arteries.…”

Translationally Controlled Tumor Protein Stimulates Dopamine Release from PC12 Cells via Ca2+-Independent Phospholipase A2 Pathways

“…Arachidonic acid and its metabolites are likely candidates in Ca 2+ -independent neurotransmitter release [17,34]. PLA 2 s are involved in a variety of cellular functions, including signal transduction and exocytosis [19,35].…”

“…PLA 2 s are involved in a variety of cellular functions, including signal transduction and exocytosis [19,35]. In a number of studies with PC12 cells, PLA 2 s are suggested to be involved in both Ca 2+ -dependent exocytosis [36,37,38,39] and in Ca 2+ -independent [34] exocytosis. All major PLA 2 groups (cPLA 2 s, sPLA 2 s, and Ca 2+ -independent iPLA 2 s) are present in the central nervous system [40,41], and iPLA 2 was also suggested to be the major PLA 2 in the cytosolic fraction of developing and adult rat brains [42].…”