FURTHER CHARACTERIZATION AND COMPARISON OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN MOUSE, RAT, AND HUMAN HEPATOCYTES

Marked differences in induced nitric oxide (NO) synthesis occur between species. We have previously shown that both human and rat hepatocytes express an inducible NO synthase in response to cytokines and lipopolysaccharide. In this study, we compare the expression and regulation of cytokine-induced NO synthase in hepatocytes isolated from three species, human, rat, and mouse. On stimulation with tumor necrosis factor alpha (TNF alpha), interleukin-1 beta (IL-1 beta), interferon gamma (IFN gamma), and lipopolysaccharide (LPS), it was found that hepatocytes from all three species produce high levels of NO with levels of production exhibiting the following hierarchy: rat hepatocytes > mouse hepatocytes > human hepatocytes, Whereas rat and mouse hepatocytes express inducible NO synthase messenger RNA (mRNA) in response to TNF alpha, IL-1 beta, or IFN gamma as a single stimulus, human hepatocytes respond to LPS alone. Inhibition of NO generation through transforming growth factor (TGF-beta(1)) was seen in mouse (77% +/- 5.9) and rat hepatocytes (17% +/- 2.6) whereas only about 10% was seen in human hepatocytes. Epidermal growth factor (EGF) was shown to inhibit NO synthesis in human and mouse hepatocytes but not rat. A marked NO-dependent inhibition of total protein synthesis was seen in rat and human hepatocytes, whereas mouse hepatocytes showed almost no inhibition in protein synthesis when stimulated. NO-dependent cyclic guanosine monophosphate (cGMP) release was found in all three species. Comparative studies on cytosol for inducible NO synthase enzyme activity showed that mouse and rat hepatocyte cytosol needed only L-arginine and reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH) to exhibit NO formation, whereas cytosol from human hepatocytes required the addition of 5,6,7,8-tetrahydrobiopterin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) to exhibit maximal NO synthase activity. Our results show even though hepatocytes from all three species can express considerable inducible NO synthase activity, important differences exist in the characteristics and effects of the NO synthesis.