The Effect of Hydrogen Peroxide on inducible Nitric Oxide Synthase Expression in Murine Macrophage RAW264.7 Cells

Ahn, Joong Hyun; Song, Jeong Sup

Abstract

BACKGROUND: Nitric oxide is a short-lived effector molecule derived from L-arginine by the nitric oxide synthase(NOS). Nitric oxide plays a role in a number of physiologic and pathophysiologic functions including host defense, edema formation, and regulation of smooth muscle tone. Some kinds of cells including macrophage are known to produce large quantities of nitric oxide in response to inflammatory stimuli such as interleukin-1beta(IL-1beta), tumor necrosis factor-alpha(TNF-alpha), interferon-gamma(IFN-gamma) and lipopolysaccharide(LPS). Reactive oxygen species are also known to be important in the pathogenesis of acute cell and tissue injury such as acute lung injury model.
METHODS
Using the RAW264.7 cells, we have examined the ability of oxidant hydrogen peroxide(H2O2) to stimulate nitric oxide production and inducible NOS mRNA expression. Also, we have examined the effects of NOS inhibitors and antioxidants on H2O2 induced nitric oxide production.
RESULTS
Stimulation of RAW264.7 cells with combinations of 100 ng/ml IL-1beta, 100 ng/ml TNF-alpha, and 100 U/ml IFN-gamma or 100 U/ml IFN-gamma and 1 microg/ml LPS induced the synthesis of nitric oxide as measured by the oxidation products nitrite(NO2-) and nitrate(NO3-). Addition of 250 microM- 2 mM H2O2 to the cytokines significantly augmented the synthesis of NO2- and NO3-(p<0.05). When cells were incubated with increasing concentrations of H2O2 in the presence of IL-1beta, TNF-alpha and IFN-gamma at constant level, the synthesis of NO2- and NO3- was dose-dependently increased(p<0.05). 3. NG-nitro-L-arginine methyl ester(L-NAME), dose dependently, significantly inhibited the formation of NO2- and NO3- in cells stimulated with LPS, IFN-gamma and H2O2 at constant level(p<0.05). 4. Catalase significantly inhibited the H2 O2-induced augmentation of cytokine- induced NO2- and NO3- formation(p<0.05). But, boiled catalase did not produce a significant inhibition in comparison with the native enzyme. Another antioxidant 2-mercaptoethanol and orthophenanthroline dose-dependently suppressed NO2- and NO3- synthesis(p<0.05). Northern blotting demonstrated that H2O2 synergistically stimulated the cytokine-induced iNOS mRNA expression in RAW264.7.
CONCLUSION
These results suggest that H2O2 contributes to inflammatory process by augmenting the iNOS expression and nitric oxide synthesis induced by cytokines.