MOLECULAR-CLONING AND EXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE FROM HUMAN HEPATOCYTES

Nitric oxide is a short-lived biologic mediator for diverse cell types . Synthesis of an inducible nitric oxide synthase (NOS) in murine macr ophages is stimulated by lipopolysaccharide (LPS) and interferon gamma . In human hepatocytes, NOS activity is induced by treatment with a co mbination of tumor necrosis factor, interleukin 1, interferon gamma, a nd LPS. We now report the molecular cloning and expression of an induc ible human hepatocyte NOS (hep-NOS) cDNA. hep-NOS has 80% amino acid s equence homology to macrophage NOS (mac-NOS). Like other NOS isoforms, recognition sites for FMN, FAD, and NADPH are present, as well as a c onsensus calmodulin binding site. NOS activity in human 293 kidney cel ls transfected with hep-NOS cDNA is diminished by Ca2+ chelation and a calmodulin antagonist, reflecting a Ca2+ dependence not evident for m ac-NOS. Northern blot analysis with hep-NOS cDNA reveals a 4.5-kb mRNA in both human hepatocytes and aortic smooth muscle cells following st imulation with LPS and cytokines. Human genomic Southern blots probed with human hep-NOS and human endothelial NOS cDNA clones display diffe rent genomic restriction enzyme fragments, suggesting distinct gene pr oducts 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.