ACTIVATION OF CYTOSOLIC PHOSPHOLIPASE A2[alpha] THROUGH NITRIC OXIDE‐INDUCED S‐NITROSYLATION: INVOLVEMENT OF INDUCIBLE NITRIC OXIDE SYNTHASE AND CYCLOOXYGENASE‐2

Abstract

Cytosolic phospholipase A2α (cPLA2α) is the rate‐limiting key enzyme that releases arachidonic acid (AA) for eicosanoid production. This study describes that cPLA2α protein is S‐nitrosylated and its activity is enhanced by nitric oxide (NO). Forced expression of inducible nitric oxide synthase (iNOS) in human epithelial cells induced cPLA2α S‐nitrosylation, enhanced its catalytic activity and increased the release of AA. The iNOS‐induced cPLA2α activation is blocked by the specific iNOS inhibitor, 1400W. Addition of the NO donor, GSNO, to isolated cell lysates induced S‐nitrosylation of cPLA2α, in vitro. Incubation of cultured cells with the iNOS substrate L‐Arginine and NO donor significantly increased cPLA2α activity and AA release. Furthermore, COX‐2 induction or expression markedly enhanced iNOS‐induced cPLA2α S‐nitrosylation and activation, leading to 9, 23, 20 fold increase of AA release and 100, 38, 88 fold of PGE2 production in A549, SG231, and HEK293 cells, respectively, whereas COX‐2 alone leads to less than 2 fold change. These results indicate that COX‐2 has the ability to enhance iNOS‐induced cPLA2α S‐nitrosylation and that maximal PG synthesis is achieved by the synergistic interaction among iNOS, cPLA2α and COX‐2. These findings disclose a novel link among cPLA2α, iNOS and COX‐2 which form a multiprotein complex leading to cPLA2α S‐nitrosylation and activation.