Endothelial cell activation by endotoxin involves superoxide/NO-mediated nitration of prostacyclin synthase and thromboxane receptor stimulation

In bovine coronary artery segments, peroxynitrite inhibits prostacyclin (PGI(2)) synthase by tyrosine nitration. Using this pharmacological model, we show that a 1 h exposure of bovine coronary artery segments to endotoxin (lipopolysaccharide [LPS]) inhibits the relaxation phase following angiotensin II (Ang II) stimulation and causes a vasospasm that can be suppressed by a thromboxane A(2) (TxA(2)) receptor blocker. In parallel, PGI(2) synthesis decreases in favor of prostaglandin E-2 formation. Immunoprecipitation and costaining with an anti-nitrotyrosine antibody identified PGI(2) synthase as the main nitrated protein in the endothelium. All effects of LPS could be prevented in the presence of the nitric oxide (NO) synthase inhibitor N-omega-monomethyl-L-arginine and polyethylene-glycolated Cu/Zn- superoxide dismutase. Thus, the early phase of endothelial cell activation in bovine coronary arteries by inflammatory agents proceeds by a protein synthesis-independent priming process for a source of superoxide that we tentatively attribute to xanthine oxidase. Upon receptor activation, Ang II stimulates NO and superoxide production, resulting in a peroxynitrite-mediated nitration and inhibition of PGI(2) synthase. The remaining 15-hydroxy-prostaglandin 9,11-endoperoxide (PGH(2)) first activates the TxA(2)/PGH(2) receptor and then is converted to prostaglandin E-2 (PGE(2)) by smooth muscle cells. PGE(2) together with a lack of NO and PGI(2) is known to promote the adhesion of white blood cells and their immigration to the inflammatory locus.