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

DL Duval, DR Miller, J Collier and RE Billings

Department of Environmental Health, College of Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins 80523, USA.

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.

Volume 50, Issue 2, pp. 277-284, 08/01/1996
Copyright © 1996 by American Society for Pharmacology and Experimental Therapeutics

This article has been cited by other articles:

				J. E. Talmadge Pathways Mediating the Expansion and Immunosuppressive Activity of Myeloid-Derived Suppressor Cells and Their Relevance to Cancer Therapy Clin. Cancer Res., September 15, 2007; 13(18): 5243 - 5248. [Abstract] [Full Text] [PDF]

				C. Zhang, L. M. Walker, J. A. Hinson, and P. R. Mayeux Oxidant Stress in Rat Liver after Lipopolysaccharide Administration: Effect of Inducible Nitric-Oxide Synthase Inhibition J. Pharmacol. Exp. Ther., June 1, 2000; 293(3): 968 - 972. [Abstract] [Full Text]

				A Akbay, K Cinar, O Uzunalimoglu, S Eranil, C Yurdaydin, H Bozkaya, and M Bozdayi Serum cytotoxin and oxidant stress markers in N-acetylcysteine treated thioacetamide hepatotoxicity of rats Human and Experimental Toxicology, November 1, 1999; 18(11): 669 - 676. [Abstract] [PDF]

				Y. Morel and R. Barouki Down-regulation of Cytochrome P450 1A1 Gene Promoter by Oxidative Stress. CRITICAL CONTRIBUTION OF NUCLEAR FACTOR 1 J. Biol. Chem., October 9, 1998; 273(41): 26969 - 26976. [Abstract] [Full Text] [PDF]

				H. Jaeschke, M. A. Fisher, J. A. Lawson, C. A. Simmons, A. Farhood, and D. A. Jones Activation of Caspase 3 (CPP32)-Like Proteases Is Essential for TNF-{alpha}-Induced Hepatic Parenchymal Cell Apoptosis and Neutrophil-Mediated Necrosis in a Murine Endotoxin Shock Model J. Immunol., April 1, 1998; 160(7): 3480 - 3486. [Abstract] [Full Text] [PDF]

				M.-L. Kuo, Y.-P. Chau, J.-H. Wang, and P.-J. Lin The Role of Src Kinase in the Potentiation by Ethanol of Cytokine- and Endotoxin-Mediated Nitric Oxide Synthase Expression in Rat Hepatocytes Mol. Pharmacol., September 1, 1997; 52(3): 535 - 541. [Abstract] [Full Text]