cPLA2α-evoked formation of arachidonic acid and lysophospholipids is required for exocytosis in mouse pancreatic β-cells

Abstract

Using capacitance measurements, we investigated the effects of intracellularly applied recombinant human cytosolic phospholipase A2 (cPLA2α) and its lipolytic products arachidonic acid and lysophosphatidylcholine on Ca2+-dependent exocytosis in single mouse pancreatic β-cells. cPLA2α dose dependently (EC50 = 86 nM) stimulated depolarization-evoked exocytosis by 450% without affecting the whole cell Ca2+ current or cytoplasmic Ca2+ levels. The stimulatory effect involved priming of secretory granules as reflected by an increase in the size of the readily releasable pool of granules from 70–80 to 280–300. cPLA2α-stimulated exocytosis was antagonized by the specific cPLA2 inhibitor AACOCF3. Ca2+-evoked exocytosis was reduced by 40% in cells treated with AACOCF3 or an antisense oligonucleotide against cPLA2α. The action of cPLA2α was mimicked by a combination of arachidonic acid and lysophosphatidylcholine (470% stimulation) in which each compound alone doubled the exocytotic response. Priming of insulin-containing secretory granules has been reported to involve Cl- uptake through ClC-3 Cl- channels. Accordingly, the stimulatory action of cPLA2α was inhibited by the Cl- channel inhibitor DIDS and in cells pretreated with ClC-3 Cl- channel antisense oligonucleotides. We propose that cPLA2α has an important role in controlling the rate of exocytosis in β-cells. This effect of cPLA2α reflects an enhanced transgranular Cl- flux, leading to an increase in the number of granules available for release, and requires the combined actions of arachidonic acid and lysophosphatidylcholine.