AUGMENTATION OF CYTOKINE-INDUCED NITRIC-OXIDE SYNTHESIS BY HYDROGEN-PEROXIDE

The inducible isoform of nitric oxide synthase (iNOS) is induced upon stimulation of cells with cytokines and lipopolysaccharide (LPS). Stim ulation of rat pleural. mesothelial cells with combinations of interle ukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), inte rferon-gamma (IFN-gamma), and LPS induced the synthesis of nitric oxid e as measured by the oxidation products nitrite (NO2-) and nitrate (NO 3-). Addition of 25-50 mu M H2O2 to the cytokines significantly augmen ted the synthesis of NO2- and NO3-. Simulation with IL-1 beta and TNF- alpha plus H2O2 or IL-1 beta and LPS plus H2O2 increased the synthesis of NO2- and NO3- by N-G-nitro-L-arginine methyl ester and cycloheximi de as well as by catalase. Immunoblotting demonstrated that H2O2 augme nted cytokine-induced synthesis of iNOS protein. These effects were in hibited by certain antioxidants and metal chelators, suggesting that t he hydroxyl radical may mediate the oxidant-induced effect. Northern b lotting demonstrated that H2O2 greatly augmented steady-state levels o f iNOS mRNA, suggesting that H2O2 acted in part at the transcriptional level.