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 phosphatidylinositol play critical functions in the regulation of membrane trafficking along the secretory pathway. To probe the possible involvement of phosphatidylinositol 3-kinase (PI 3-kinase) in regulated exocytosis, we have examined its subcellular distribution in cultured chromaffin cells by immunoreplica analysis and confocal immunofluorescence. We found that the PI 3-kinase heterodimer consisting of the regulatory and catalytic subunits was associated essentially with the subplasmalemmal cytoskeleton in both resting and nicotine-stimulated chromaffin cells. Attempts to immunoprecipitate PI 3-kinase with anti-phosphotyrosine antibodies failed, suggesting that the activity of PI 3-kinase was not modulated by tyrosine phosphorylation and/or physical interaction with SH2-containing proteins in stimulated chromaffin cells. LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], a potent inhibitor of PI 3-kinase, produced a dose-dependent inhibition of catecholamine secretion evoked by various secretagogues. Furthermore, cytochemical experiments with rhodamine-labeled phalloidin revealed that LY294002 blocked the disassembly of cortical actin in chromaffin cells stimulated by a depolarizing concentration of potassium. Our results suggest that PI 3-kinase may be one of the important regulatory exocytotic components involved in the signaling cascade controlling actin rearrangements required for catecholamine secretion.
Chasserot-Golaz
S
S
Institut National de la Santé et de la Recherche Médicale U-338, Biologie de la Communication Cellulaire, Strasbourg, France.
Hubert
P
P
Thiersé
D
D
Dirrig
S
S
Vlahos
C J
CJ
Aunis
D
D
Bader
M F
MF
D000199:Actins / Q000648:ultrastructure; D000311:Adrenal Glands / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002458:Cell Fractionation; D019439:Chromaffin Cells / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D002867:Chromones / Q000494:pharmacology*; D003599:Cytoskeleton / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005089:Exocytosis / Q000187:drug effects*; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D018613:Microscopy, Confocal; D009025:Morpholines / Q000494:pharmacology*; D009638:Norepinephrine / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000648:ultrastructure; D000081082:Phosphoinositide-3 Kinase Inhibitors
D000199:Actins; D002118:Calcium; D002867:Chromones; D004791:Enzyme Inhibitors; D009025:Morpholines; D009638:Norepinephrine; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one