title: Stimulation-dependent recruitment of cytosolic phospholipase A2-alpha to EA.hy.926 endothelial cell membranes leads to calcium-independent association.
abstract: Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme involved in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid is predominantly converted into prostacyclin, a potent vasodilator and inhibitor of platelet activation. As the rate-limiting step in prostacyclin production is the generation of free arachidonic acid by cPLA2-alpha, this enzyme has become an attractive pharmacological target and the focus of many studies. Following stimulation with calcium-mobilizing agonists, cPLA2-alpha translocates to intracellular phospholipid membranes via its C2 domain. In this study, the calcium-induced association of cPLA2-alpha with EA.hy.926 endothelial cell membranes was investigated. Subcellular fractionation and immunofluorescence studies showed that following stimulation with histamine, thrombin or the calcium ionophore A23187, cPLA2-alpha relocated to intracellular membranes. Treatment of A23187-stimulated cells with EGTA or BAPTA-AM demonstrated that a substantial pool of cPLA2-alpha remained associated with membrane fractions in a calcium-independent manner. Furthermore, immunofluorescence microscopy studies revealed that cells stimulated for periods of greater than 10 min showed a high proportion of calcium-independent membrane-associated cPLA2-alpha. Calcium-independent membrane association of cPLA2-alpha was not due to hydrophobic or cytoskeletal interactions. Finally, the recombinant C2 domain of cPLA2-alpha exhibited calcium-independent membrane binding to membranes isolated from A23187-stimulated cells but not those isolated from nonstimulated cells. These findings suggest that novel mechanisms involving accessory proteins at the target membrane play a role in the regulation of cPLA2-alpha. Such regulatory associations could enable the cell to discriminate between the varying levels of cytosolic calcium induced by different stimuli.
authors.affiliation: School of Biochemistry and Molecular Biology, University of Leeds, UK.
authors.forename: Seema
authors.initials: S
authors.lastname: Grewal
authors.forename: Jennifer
authors.initials: J
authors.lastname: Smith
authors.forename: Sreenivasan
authors.initials: S
authors.lastname: Ponnambalam
authors.forename: John
authors.initials: J
authors.lastname: Walker
mesh_terms: D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology*; D004533:Egtazic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000201:enzymology*; D054513:Group IV Phospholipases A2; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D018613:Microscopy, Confocal; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D021381:Protein Transport; D014471:Umbilical Veins
chemical_list: D007527:Isoenzymes; D000001:Calcimycin; D004533:Egtazic Acid; D010741:Phospholipases A; D054513:Group IV Phospholipases A2; D054467:Phospholipases A2; D002118:Calcium