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). Stimulation of rat pleural mesothelial cells with combinations of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and LPS induced the synthesis of nitric oxide as measured by the oxidation products nitrite (NO2-) and nitrate (NO3-). Addition of 25-50 microM H2O2 to the cytokines significantly augmented the synthesis of NO2- and NO3-. Stimulation 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 3.8- and 3.5-fold, respectively. These effects were inhibited by NG-nitro-L-arginine methyl ester and cycloheximide as well as by catalase. Immunoblotting demonstrated that H2O2 augmented cytokine-induced synthesis of iNOS protein. These effects were inhibited by certain antioxidants and metal chelators, suggesting that the hydroxyl radical may mediate the oxidant-induced effect. Northern blotting demonstrated that H2O2 greatly augmented steady-state levels of iNOS mRNA, suggesting that H2O2 acted in part at the transcriptional level.