Role of Ca 2+ -Independent Phospholipase A 2 in the Regulation of Inducible Nitric Oxide Synthase in Cardiac Myocytes

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Abstract
—We have previously shown that the regulation by interleukin-1β (IL-1β) of inducible nitric oxide synthase (iNOS) involves phospholipase A 2 (PLA 2 ) metabolites in neonatal ventricular myocytes. Based on studies in which ONO-RS-082 is used to inhibit secretory PLA 2 and methyl arachidonyl fluorophosphonate is used to inhibit cytosolic PLA 2 , our data suggest that a secretory PLA 2 metabolite was involved in the regulation by IL-1β of iNOS. In addition, a third PLA 2 isoform, which is Ca 2+ independent (iPLA 2 ), has also been detected in cardiac myocytes and shown to be regulated by cytokines. We tested whether iPLA 2 metabolites are involved in the regulation by IL-1β of iNOS with the use of bromoenol lactone (BEL), a specific and irreversible inhibitor of iPLA 2 . For this, we measured IL-1β–stimulated nitrite (NOx) production with use of the Griess reagent, prostaglandin E 2 (PGE 2 ) production with use of an enzyme immunoassay, and arachidonic acid release in the presence and absence of BEL. We also detected iNOS and iPLA 2 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 PGE 2 production was increased by 300-fold. When neonatal ventricular myocytes were treated with 10 μmol/L BEL, both IL-1β–stimulated PGE 2 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 iPLA 2 metabolite, perhaps lysophosphatidic acid, may be involved in the IL-1β–signaling pathway, regulating the synthesis of iNOS.

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