Identification of the phospholipase A₂ isoforms that contribute to arachidonic acid release in hypoxic endothelial cells: Limits of phospholipase A₂ inhibitors

Changes in endothelium functions during ischemia are thought to be of importance in numerous pathological conditions, with, for instance, an increase in the release of inflammatory mediators like prostaglandins. Here, we showed that hypoxia increases phospholipase A₂ (PLA₂) activity in human umbilical vein endothelial cells. Both basal PLA₂ activity and PG synthesis are sensitive to BEL and AACOCF3, respectively, inhibitors of calcium-independent PLA₂ (iPLA₂) and cytosolic PLA₂ (cPLA₂), while OPC, an inhibitor of soluble PLA₂ (sPLA₂) only inhibited the hypoxia-induced AA release and PGF_2α synthesis. Hypoxia does not alter expression of iPLA₂, sPLA₂ and cPLA₂ and cycloheximide did not inhibit PLA₂ activation, indicating that hypoxia-induced increase in PLA₂ activity is due to activation rather than induction. However, mRNA levels for sPLA₂ displayed a 2-fold increase after 2hr incubation under hypoxia. BAPTA, an intracellular calcium chelator, partially inhibited the AA release in normoxia and in hypoxia. Direct assays of specific PLA₂ activity showed an increase in sPLA₂ activity but not in cPLA₂ activity after 2 hr hypoxia. Taken together, these results indicate that the hypoxia-induced increase in PLA₂ activity is mostly due to the activation of sPLA₂. © 2002 Elsevier Science Inc. All rights reserved.