Nitric oxide down-regulates hepatocyte-inducible nitric oxide synthase gene expression

Background: The expression of inducible nitric oxide synthase (iNOS) contributes to the systemic manifestations of sepsis. Objective: To determine whether nitric oxide (NO) can exert negative feedback regulation on iNOS gene expression. Setting: Molecular biology research laboratory of the department of surgery. Study Design: Isolated rat hepatocytes were cultured with a cytokine mix consisting of tumor necrosis factor α, interleukin 1β, and interferon γ in the presence or absence of the NO donor S-nitroso-N-acetyl- D,L-penicillamine. Main Outcome Measures: Nitrite and nitrate (NO₂^- and NO₃^-) levels were assayed. Hepatocyte iNOS messenger RNA and protein levels were assessed. Electromobility shift assays were performed for NF-κB DNA binding activity. Finally, iNOS enzyme activity was determined using high- performance liquid chromatography. Results: Cytokine mix-induced hepatocyte iNOS mRNA and protein production and the addition of the NO donor S-nitroso- N-acetyl-D,L-penicillamine markedly attenuated iNOS mRNA and protein levels. Gel shift assays of the nuclear extracts disclosed that decreased cytokine mix-induced DNA binding activity for NF-κB in a concentration-dependent manner. Finally, NO failed to significantly inhibit iNOS enzyme activity. Conclusions: These data indicate that NO down-regulates iNOS gene transcription, and that the effect is mediated in part by inhibiting NF-κB activity. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression, possibly to limit over production during the septic response.