Calcium-independent phospholipase A2 is regulated by a novel protein kinase C in human coronary artery endothelial cells.
We demonstrated previously that thrombin stimulation of endothelial cells activates a membrane-associated, Ca(2+)-independent phospholipase A2 (iPLA2) that selectively hydrolyzes arachidonylated plasmalogen phospholipids. We report that incubation of human coronary artery endothelial cells (HCAEC) with phorbol 12-myristate 13-acetate (PMA) to activate protein kinase C (PKC) resulted in hydrolysis of cellular phospholipids similar to that observed with thrombin stimulation (0.05 IU/ml; 10 min). Thrombin stimulation resulted in a decrease in arachidonylated plasmenylcholine (2.7 +/- 0.1 vs. 5.3 +/- 0.4 nmol PO4/mg of protein) and plasmenylethanolamine (7.5 +/- 1.0 vs. 12.0 +/- 0.9 nmol PO4/mg of protein). Incubation with PMA resulted in decreases in arachidonylated plasmenylcholine (3.2 +/- 0.3 nmol PO4/mg of protein) and plasmenylethanolamine (6.0 +/- 1.0 nmol PO4/mg of protein). Incubation of HCAEC with the selective iPLA2 inhibitor bromoenol lactone (5 mM; 10 min) inhibited accelerated plasmalogen phospholipid hydrolysis in response to both PMA and thrombin stimulation. Incubation of HCAEC with PMA (100 nM; 5 min) resulted in increased arachidonic acid release (7.1 +/- 0.3 vs. 1.1 +/- 0.1%) and increased production of lysoplasmenylcholine (1.4 +/- 0.2 vs. 0.6 +/- 0.1 nmol PO4/mg of protein), similar to the responses observed with thrombin stimulation. Downregulation of PKC by prolonged exposure to PMA (100 nM; 24 h) completely inhibited thrombin-stimulated increases in arachidonic acid release (7.1 +/- 0.6 to 0.5 +/- 0.1%) and lysoplasmenylcholine production (2.0 +/- 0.1 to 0.2 +/- 0.1 nmol PO4/mg of protein). These data suggest that PKC activates iPLA2 in HCAEC, leading to accelerated plasmalogen phospholipid hydrolysis and increased phospholipid metabolite production.