Role of Ca2+-independent phospholipase A2 in the regulation of inducible nitric oxide synthase in cardiac myocytes

Abstract

We have previously shown that the regulation by interleukin-1β (IL-1β) of inducible nitric oxide synthase (iNOS) involves phospholipase A2 (PLA2) metabolites in neonatal ventricular myocytes. Based on studies in which ONO- RS-082 is used to inhibit secretory PLA2 and methyl arachidonyl fluorophosphonate is used to inhibit cytosolic PLA2, our data suggest that a secretory PLA2 metabolite was involved in the regulation by IL-1β of iNOS. In addition, a third PLA2 isoform, which is Ca2+ independent (iPLA2), has also been detected in cardiac myocytes and shown to be regulated by cytokines. We tested whether iPLA2 metabolites are involved in the regulation by IL-1β of iNOS with the use of bromoenol lactone (BEL), a specific and irreversible inhibitor of iPLA2. For this, we measured IL-1β- stimulated nitrite (NOx) production with use of the Griess reagent, prostaglandin E2 (PGE2) production with use of an enzyme immunoassay, and arachidonic acid release in the presence and absence of BEL. We also detected iNOS and iPLA2 proteins by Western blotting. Treatment with IL-1β (5 ng/mL) for 24 hours stimulated NOx production by 8-fold and iNOS protein levels by at least 10-fold. In addition, arachidonic acid release was increased by 1.6- fold and PGE2 production was increased by 300-fold. When neonatal ventricular myocytes were treated with 10 μmol/L BEL, both IL-1β-stimulated PGE2 production and arachidonic acid release were inhibited. BEL inhibited IL-1β-stimulated NOx production and iNOS protein by 88% and 93%, respectively. Lysophosphatidic acid, but not arachidonic acid or lysophosphatidylcholine, stimulated iNOS expression. Our results indicate that an iPLA2 metabolite, perhaps lysophosphatidic acid, may be involved in the IL-1β-signaling pathway, regulating the synthesis of iNOS.