Hydrogen peroxide-induced arachidonic acid release in L929 cells; roles of Src, protein kinase C and cytosolic phospholipase A2α

Abstract

Hydrogen peroxide (H2O2) stimulates the release of arachidonic acid from cells, but the signaling mechanism(s) involved remains to be elucidated. We investigated the roles of α-type cytosolic phospholipase A2 (cPLA2α), Src family kinases (Src) and protein kinase C (PKC) in the release of arachidonic acid from L929 cells (a murine fibroblast cell line), C12 cells (a variant of L929 that lacks cPLA2α) and a stable clone of C12 cells expressing cPLA2α (C12–cPLA2α cells). In the presence of 10 μM A23187, 100 nM phorbol myristate acetate (PMA) and 1 mM H2O2 synergistically stimulated arachidonic acid release from L929 cells and C12–cPLA2α cells, and to a much lesser extent from C12 cells. The reagents alone and co-treatment with PMA and H2O2 without A23187 had marginal effects. No arachidonic acid was released by PMA/A23187 or H2O2/A23187 in CaCl2-free buffer and the release was inhibited by a selective cPLA2α inhibitor (3 μM pyrrophenone). Addition of 10 μM H2O2, which did not stimulate arachidonic acid release with A23187, enhanced the response to PMA/A23187. The release induced by PMA/A23187 and by H2O2/A23187 was significantly inhibited by a PKC inhibitor (10 μM GF109203X) and in PKC-depleted cells, and by a Src inhibitor (2 μM PP2). The phosphorylation of extracellular signal-regulated kinase 1/2 induced by PMA/A23187 and H2O2/A23187 was significantly decreased by inhibitors of PKC and Src. These findings suggest that H2O2 with Ca2+ stimulates arachidonic acid release via cPLA2α in a Src- and PKC-dependent manner in L929 cells. The role of cross-talk between Src and PKC in arachidonic acid release is discussed.