Nitric Oxide Down-regulates Hepatocyte–Inducible Nitric Oxide Synthase Gene Expression

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Abstract
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 donorS-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 donorS-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 overproduction during the septic response. Arch Surg. 1997;132:1177-1183