POSSIBLE INVOLVEMENT OF PHOSPHATIDYLINOSITOL 3-KINASE IN REGULATED EXOCYTOSIS - STUDIES IN CHROMAFFIN CELLS WITH INHIBITOR LY294002

Several lines of evidence suggest that phosphorylated products of phos phatidylinositol play critical functions in the regulation of membrane trafficking along the secretory pathway. To probe the possible involv ement of phosphatidylinositol 3-kinase (PI 3-kinase) in regulated exoc ytosis, we have examined its subcellular distribution in cultured chro maffin cells by immunoreplica analysis and confocal immunofluorescence . We found that the PI 3-kinase heterodimer consisting of the regulato ry and catalytic subunits was associated essentially with the subplasm alemmal cytoskeleton in both resting and nicotine-stimulated chromaffi n cells. Attempts to immunoprecipitate PI 3-kinase with anti-phosphoty rosine antibodies failed, suggesting that the activity of PI 3-kinase was not modulated by tyrosine phosphorylation and/or physical interact ion with SH2-containing proteins in stimulated chromaffin cells. LY294 002 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a potent inhibi tor of PI 3-kinase, produced a dose-dependent inhibition of catecholam ine secretion evoked by various secretagogues. Furthermore, cytochemic al experiments with rhodamine-labeled phalloidin revealed that LY29400 2 blocked the disassembly of cortical actin in chromaffin cells stimul ated by a depolarizing concentration of potassium. Our results suggest that PI 3-kinase may be one of the important regulatory exocytotic co mponents involved in the signaling cascade controlling actin rearrange ments required for catecholamine secretion.