Role of Ca2+-independent phospholipase A(2) in the regulation of induciblenitric oxide synthase in cardiac myocytes

We have previously shown that the regulation by interleukin-1 beta (IL-1 be ta) of inducible nitric oxide synthase (iNOS) involves phospholipase A(2) ( PLA(2)) metabolites in neonatal ventricular myocytes. Based on studies in w hich ONO-RS-082 is used to inhibit secretory PLA(2) and methyl arachidonyl fluorophosphonate is used to inhibit cytosolic PLA(2), our data suggest tha t a secretory PLA(2) metabolite was involved in the regulation by IL-1 beta of iNOS. In addition, a third PLA(2) isoform, which is Ca2+ independent (i PLA(2)), has also been detected in cardiac myocytes and shown to be regulat ed by cytokines. We tested whether iPLA(2) metabolites are involved in the regulation by IL-1 beta of iNOS with the use of bromoenol lactone (BEL), a specific and irreversible inhibitor of iPLA(2). For this, we measured IL-1 beta-stimulated nitrite (NOx) production with use of the Griess reagent, pr ostaglandin E-2 (PGE(2)) production with use of an enzyme immunoassay, and arachidonic acid release in the presence and absence of BEL. We also detect ed iNOS and iPLA(2) proteins by Western blotting. Treatment with IL-1 beta (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 incr eased by 1.6-fold and PGE(2) production was increased by 300-fold. When neo natal ventricular myocytes were treated with 10 mu mol/L BEL, both IL-1 bet a-stimulated PGE(2) production and arachidonic acid release were inhibited. BEL inhibited IL-1 beta-stimulated NOx production and iNOS protein by 88% and 93%, respectively. Lysophosphatidic acid, but not arachidonic acid or l ysophosphatidylcholine, stimulated iNOS expression. Our results indicate th at an iPLA(2) metabolite, perhaps lysophosphatidic acid, may be involved in the IL-1 beta-signaling pathway, regulating the synthesis of iNOS.