NITRIC-OXIDE DOWN-REGULATES HEPATOCYTE-INDUCIBLE NITRIC-OXIDE SYNTHASE GENE-EXPRESSION

Background: The expression of inducible nitric oxide synthase (iNOS) c ontributes to the systemic manifestations of sepsis. Objective: To det ermine whether nitric oxide (NO) can exert negative feedback regulatio n on iNOS gene expression. Setting: Molecular biology research laborat ory of the department of surgery. Study Design: Isolated rat hepatocyt es were cultured with a cytokine mix consisting of tumor necrosis fact or alpha, interleukin 1 beta, and interferon gamma in the presence or absence of the NO donor S-nitroso-N-acetyl-D,L-penicillamine. Main Out come Measures: Nitrite and nitrate (NO2- and NO3-) levels were assayed . Hepatocyte iNOS messenger RNA and protein levels were assessed. Elec tromobility shift assays were performed for NF-kappa B DNA binding act ivity. Finally, iNOS enzyme activity was determined using high-perform ance liquid chromatography. Results: Cytokine mix-induced hepatocyte i NOS mRNA and protein production and the addition of the NO donor S-nit roso-N-acetyl-D,L-penicillamine markedly attenuated iNOS mRNA and prot ein levels. Gel shift assays of the nuclear extracts disclosed that de creased cytokine mix-induced DNA binding activity for NF-kappa B in a concentration-dependent manner. Finally, NO failed to significantly in hibit iNOS enzyme activity. Conclusions: These data indicate that NO d ownregulates iNOS gene transcription, and that the effect is mediated in part by inhibiting NF-kappa B activity. These results identify a no vel negative feedback mechanism whereby NO down-regulates iNOS gene ex pression, possibly to limit overproduction during the septic response.