Oxidant-mediated AA release from astrocytes involves cPLA2 and iPLA2

Excessive generation of reactive oxygen species (ROS) in the central nervous system (CNS) is a leading cause of neuronal injury. Despite yet unknown mechanisms, oxidant compounds such as H2O2 have been shown to stimulate the release of arachidonic acid (AA) in a number of cell systems. In this study, H2O2 and menadione, a compound known to release H2O2 intracellularly, were used to examine the phospholipases A2 (PLA2) responsible for AA release from primary murine astrocytes. Both H2O2 and menadione dose-dependently stimulated AA release, and the release mediated by H2O2 was completely inhibited by catalase. H2O2 also stimulated phosphorylation of extracellular signal-regulated kinases (ERK1/2) and cytosolic phospholipase A2 (cPLA2). However, complete inhibition of cPLA2 phosphorylation by U0126, an inhibitor for mitogen-activated protein kinase kinase (MEK) and GF109203x, a nonselective PKC inhibitor preferring the conventional and novel isoforms, only reduced H2O2-stimulated AA release by 50%. MAFP, a selective, active, site-directed, irreversible inhibitor of both cPLA2 and the Ca2+-independent iPLA2, nearly completely inhibited H2O2-mediated AA release; but, HELSS, a potent irreversible inhibitor of iPLA2, only inhibited H2O2-mediated AA release by 40%. Along with the observation that H2O2-mediated AA release was only partially inhibited upon chelating intracellular Ca2+ by BAPTA, these results indicate the involvement of both cPLA2 and iPLA2 in H2O2-mediated AA release in murine astrocytes.