Peroxynitrite provides the peroxide tone for PGHS-2-dependent prostacyclin synthesis in vascular smooth muscle cells

Abstract

Endotoxin-treated vascular smooth muscle cells (VSMCs) were recently shown to release high amounts of prostacyclin (PGI2) dependent on the induction of prostaglandin endoperoxide synthase-2 (PGHS-2). In contrast to endothelial PGI2-synthase, for which nitration and inhibition by peroxynitrite was reported, addition of SIN-1 as a peroxynitrite-generating system did not cause inhibition but rather doubled PGI2 release by VSMC. The hypothesis of peroxynitrite supplementing an unsaturated peroxide tone for PGHS-2 was supported by H2O2 exerting the same effect. Studies performed with purified PGHS-2 revealed maximal elevation of enzyme activity in the presence of equimolar concentrations of *NO and *O2-, which together form peroxynitrite, while excessive production of either one radical was inhibitory. Most importantly, 6-keto-PGF1alpha formation by intact VSMC depended on a nearly equimolar generation of *NO and *O2- for providing the endogenous peroxide tone. These findings, together with the observation that an excess of exogenously added *NO, as well as uric acid as a scavenger of peroxynitrite potently reduced PGI2 release, underlined the role of peroxynitrite as the dominating and rate-limiting intracellular mediator of peroxide tone in VSMC. The results allow us to postulate a new cross-talk between the *NO and the prostanoid pathways with a crucial role for peroxynitrite in providing the peroxide tone for a continuous activation of PGHS-2.