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 alpha, interleukin 1 beta, and interferon gamma 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-kappa B 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-kappa B 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-kappa B activity. These results identify a novel negative feedback mechanism whereby NO down-regulates iNOS gene expression, possibly to limit overproduction during the septic response.