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Nitric oxide is a short-lived biologic mediator for diverse cell types. Synthesis of an inducible nitric oxide synthase (NOS) in murine macrophages is stimulated by lipopolysaccharide (LPS) and interferon gamma. In human hepatocytes, NOS activity is induced by treatment with a combination of tumor necrosis factor, interleukin 1, interferon gamma, and LPS. We now report the molecular cloning and expression of an inducible human hepatocyte NOS (hep-NOS) cDNA. hep-NOS has 80% amino acid sequence homology to macrophage NOS (mac-NOS). Like other NOS isoforms, recognition sites for FMN, FAD, and NADPH are present, as well as a consensus calmodulin binding site. NOS activity in human 293 kidney cells transfected with hep-NOS cDNA is diminished by Ca2+ chelation and a calmodulin antagonist, reflecting a Ca2+ dependence not evident for mac-NOS. Northern blot analysis with hep-NOS cDNA reveals a 4.5-kb mRNA in both human hepatocytes and aortic smooth muscle cells following stimulation with LPS and cytokines. Human genomic Southern blots probed with human hep-NOS and human endothelial NOS cDNA clones display different genomic restriction enzyme fragments, suggesting distinct gene products for these NOS isoforms. hep-NOS appears to be an inducible form of NOS that is distinct from mac-NOS as well as brain and endothelial NOS isozymes.

Nitric oxide (NO.) is a short-lived mediator which can be induced in a variety of cell types and produces many physiologic and metabolic changes in target cells. The inducible or high-output NO. synthase (NOS) pathway was first characterized in macrophages activated by lipopolysaccharide (LPS) and interferon gamma (IFN-gamma). Hepatocytes also express an inducible NOS following exposure to the combination of endotoxin (LPS) and tumor necrosis factor (TNF), interleukin 1 (IL-1), and IFN-gamma. In this study, to identify which of these cytokines, if any, was acting to induce the gene expression for hepatocyte NOS, we measured the levels of rat hepatocyte NOS mRNA by Northern blot analysis after stimulation by various combinations of endotoxin and cytokines in vitro. We found the mRNA for hepatocyte NOS to be a single band at approximately 4.5 kilobases which was maximally up-regulated (approximately 70-fold) by the combination of TNF, IL-1, IFN-gamma, and LPS. Abundance of NOS mRNA peaked 6-8 hr after stimulation and then declined by 25% at 24 hr. Unstimulated hepatocytes in vitro showed only a trace mRNA band after prolonged autoradiographic exposure. As single agents, TNF and IL-1 were the most effective inducers of hepatocyte NOS mRNA. Combinations of two or three stimuli revealed strong synergy between TNF, IL-1, and IFN-gamma. The increased mRNA levels correlated with elevated nitrogen oxide release and cGMP levels in the culture supernatants. Dexamethasone and cycloheximide inhibited induction of mRNA for hepatocyte NOS in a dose-dependent fashion. The addition of NG-monomethyl-L-arginine had no effect on mRNA levels but effectively blocked NO. formation. The inducible hepatocyte NOS mRNA was also detected in rat hepatocytes following chronic hepatic inflammation triggered by Corynebacterium parvum injection in vivo. These data demonstrate that the inducible NOS is functional in rat hepatocytes both in vitro and in vivo and that this pathway is under complex control. Endotoxin and inflammatory cytokines act synergistically to up-regulate gene expression for hepatocyte NOS, whereas glucocorticoids down-regulate the mRNA.