Inducible nitric oxide synthase mediates prostaglandin h2 synthase nitration and suppresses eicosanoid production.

Nitric oxide (NO) modulates the biological levels of arachidonate-derived cell signaling molecules by either enhancing or suppressing the activity of prostaglandin H(2) isoforms (PGHS-1 and PGHS-2). Whether NO activates or suppresses PGHS activity is determined by alternative protein modifications mediated by NO and NO-derived species. Here, we show that inducible NO synthase (iNOS) and PGHS-1 co-localize in atherosclerotic lesions of ApoE(-/-) mouse aortae. Immunoblotting and immunohistochemistry revealed Tyr nitration in PGHS-1 in aortic lesions but markedly less in adjacent nonlesion tissue. PGHS-2 was also found in lesions, but 3-nitrotyrosine incorporation was not detected. 3-Nitrotyrosine formation in proteins is considered a hallmark reaction of peroxynitrite, which can form via NO-superoxide reactions in an inflammatory setting. That iNOS-derived NO is essential for 3-nitrotyrosine modification of PGHS-1 was confirmed by the absence of 3-nitrotyrosine in lesions from ApoE(-/-)iNOS(-/-) mice. Mass spectrometric studies specifically identified the active site residue Tyr385 as a 3-nitrotyrosine modification site in purified PGHS-1 exposed to peroxynitrite. PGHS-mediated eicosanoid (PGE(2)) synthesis was more than fivefold accelerated in cultured iNOS(-/-) versus iNOS-expressing mouse aortic smooth muscle cells, suggesting that iNOS-derived NO markedly suppresses PGHS activity in vascular cells. These results further suggest a regulatory role of iNOS in eicosanoid biosynthesis in human atherosclerotic lesions.