Calcium-independent phospholipase A₂ is regulated by a novel protein kinase C in human coronary artery endothelial cells

Abstract

. Calcium-independent phospholipase A 2 is regulated by a novel protein kinase C in human coronary artery endothelial cells. Am J Physiol Cell Physiol 288: C475-C482, 2005; doi:10.1152/ ajpcell.00306.2004.-We demonstrated previously that thrombin stimulation of endothelial cells activates a membrane-associated, Ca 2ϩ -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 Ϯ 0.1 vs. 5.3 Ϯ 0.4 nmol PO4/mg of protein) and plasmenylethanolamine (7.5 Ϯ 1.0 vs. 12.0 Ϯ 0.9 nmol PO4/mg of protein). Incubation with PMA resulted in decreases in arachidonylated plasmenylcholine (3.2 Ϯ 0.3 nmol PO4/mg of protein) and plasmenylethanolamine (6.0 Ϯ 1.0 nmol PO 4/mg of protein). Incubation of HCAEC with the selective iPLA 2 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 Ϯ 0.3 vs. 1.1 Ϯ 0.1%) and increased production of lysoplasmenylcholine (1.4 Ϯ 0.2 vs. 0.6 Ϯ 0.1 nmol PO 4/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 Ϯ 0.6 to 0.5 Ϯ 0.1%) and lysoplasmenylcholine production (2.0 Ϯ 0.1 to 0.2 Ϯ 0.1 nmol PO 4/mg of protein). These data suggest that PKC activates iPLA 2 in HCAEC, leading to accelerated plasmalogen phospholipid hydrolysis and increased phospholipid metabolite production. lysophospholipids; cell signaling; phospholipid metabolism; arachidonic acid WE DEMONSTRATED PREVIOUSLY that thrombin stimulation of human coronary artery endothelial cells (HCAEC) activates a membrane-associated, Ca 2ϩ -independent phospholipase A 2 (iPLA 2 ). This enzyme selectively hydrolyzes membrane plasmalogen phospholipids, leading to an increase in the production of lysoplasmalogens and arachidonic acid. Activation of endothelial cell (EC) membrane-associated iPLA 2 is also observed when EC are incubated for short intervals with phorbol 12-myristate 13-acetate (PMA), suggesting that the increase in iPLA 2 activity is mediated by protein kinase C (PKC). In addition, downregulation of PKC activity by prolonged incubation with PMA also results in complete inhibition of thrombin-stimulated iPLA 2 activity (6).

Mechanisms of Hypotonicity-Induced Calcium Signaling and Integrin Activation by Arachidonic Acid-Derived Inflammatory Mediators in B Cells

“…2C). Moreover, it has been demonstrated that DAG can directly activate PLA 2 (18). Although we did not obtain evidence for physiological activation of PLA 2 by hypotonicity in B cells, it is possible that pharmacological concentrations of DAG (i.e., 200 M OAG) used in previous experiments and in the AA release assay above do so.…”

“…We previously identified TRPV4 mRNA and TRPV4-like cation currents in primary B cells (8). Therefore, we tested the sensitivity of hypotonicity-activated Ca 2ϩ signals to the TRPV4 blocker ruthenium red (RR) (18). RR did not inhibit hypotonicity-induced Ca 2ϩ release from stores, but did attenuate Ca 2ϩ influx (Fig.…”

Phospholipase A2-catalyzed hydrolysis of plasmalogen phospholipids in thrombin-stimulated human platelets

“…This suggests that a Ca 2+ -dependent step may exist in the signaling process between PAR and iPLA 2 activation. In previous studies, we have demonstrated that iPLA 2 in ventricular myocytes and endothelial cells is dependent upon protein kinase C (PKC) activity (32,33). If a Ca 2+ -dependent PKC phosphorylates iPLA 2 γ in platelets, this may explain our observation of the lack of iPLA 2 activation by thrombin-stimulated platelets suspended in a Ca 2+ -free buffer.…”

Recovery of PEX1-Gly843Asp peroxisome dysfunction by small-molecule compounds

“…In addition, PKC phosphorylation activates phospholipase A2 to generate arachadonic acid and 1-alkyl-or 1-acyl-lysophospholipids from glycerophospholipids. Inhibition of PKC results in the inhibition of phospholipid hydrolysis (34) and could possibly result in the accumulation of plasmalogens as a secondary effect.…”