Characterization of hepatic nitric oxide synthase: identification as the cytokine-inducible form primarily regulated...

Induction of hepatic nitric oxide synthase (NOS) by tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interferon-gamma (IFN gamma), interleukin-6 (IL-6), and lipopolysaccharide was assessed as activity and immunoreactive protein. Hepatic NOS activity was cytosolic and had cofactor requirements consistent with inducible nitric oxide synthase (NOS2). NOS induction by TNF alpha was dose dependent from concentrations of 0.06 to 60 nM and was increased 2-3-fold by IFN gamma. NOS induction was reflective of total TNF alpha binding to hepatocyte receptors. Hepatocyte TNF alpha binding fit a biphasic curve with high affinity (K(d) = 1.4 nM, Bmax = 3157 sites) and low affinity (K(d) = 157 nM, Bmax = 204,948 sites) elements. NOS2 activity was induced by lipopolysaccharide, IL-1 beta, TNF alpha, and IFN gamma but not by IL-6. All cytokine stimuli were inhibited by antioxidants. Oxygen radical generation was directly measured as dichlorofluoroscein fluorescence in isolated mitochondria. Mitochondria from TNF alpha-treated hepatocytes generated more oxygen radicals than did controls. Antioxidants reduced mitochondrial generation of oxygen radicals. Activation of the transcription factor nuclear factor-kappa B by TNF alpha, IFN gamma, and IL-1 beta was assessed by gel shift analysis. Cytokine treatment increased nuclear factor-kappa B binding, and the addition of antioxidants or rotenone inhibited cytokine activation. Taken together, these data suggest that oxygen radicals, possibly generated by mitochondria, play a major role in NOS2 induction by cytokines.