Interleukin 1 beta decreases prostacyclin synthase activity in rat mesangial cells via endogenous peroxynitrite formation

We have reported that peroxynitrite (PON) selectively inactivated prostacyc lin synthase (PGIS) by a mechanism of tyrosine nitration at the active site [Zou, Martin and Ullrich (1997) Biol. Chem. Hoppe-Seyler 378, 707-713]. We have now extended our studies on rat mesangial cells (RMC) and show that n itration can occur under the influence of cytokines. Pretreatment of RMC wi th interleukin 1 beta (IL-1 beta), which up-regulated cyclo-oxygenase 2 and inducible nitric oxide synthase (NOS-2), significantly attenuated the conv ersion of [C-14]prostaglandin H-2 (PGH(2)) into the stable prostacyclin (PG I(2)) metabolite 6-oxo-prostaglandin F-1 alpha (6-oxo-PGF(1 alpha)). The pr esence of superoxide dismutase (SOD, 100 units/ml) or the NOS synthase inhi bitor N-omega-monomethyl-L-arginine (100 mu M) as well as cycloheximide (10 mu M) plus actinomycin(10 mu M) abolished IL-1 beta-mediated down-regulati on of 6-oxo-PGF(1 alpha) from PGH(2). At the same time, 6-oxo-PGF(1 alpha) production from arachidonate (AA) increased at the expense of prostaglandin E-2 (PGE(2)). Neither NO alone generated from different NO donors nor supe roxide from xanthine/xanthine oxidase (1-100 m-units/ml) inhibited PGI(2) s ynthesis, either from PGH(2) or from AA. Bolus additions of chemically synt hesized PON or the PON generator 3-morpholinosydnonimine N-ethyl-carbamide (SIN-1) exhibited a potent inhibition of 6-oxo-PGF(1 alpha) release from bo th PGH(2) and AA. In addition, immunoprecipitation of nitrotyrosine-contain ing proteins from PON- and SIN-1-treated RMC yielded distinct nitrated PGIS bands but also from IL-1 beta-pretreated cells alone, compared with a lack of nitrated PGIS in control cells. Taken together, our results strongly su ggest that IL-1 beta pretreatment of RMC via NOS-2 leads to the production of PON with the consequence of a partial nitration and inhibition of PGIS.