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

B. S. Taylor, Y. M. Kim, Q. Wang, R. A. Shapiro, T. R. Billiar and D. A. Geller
Department of Surgery, University of Pittsburgh, Pa., USA.

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.