Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes

David A. Geller*, Charles J. Lowenstein, Richard A. Shapiro, Andreas K. Nussler, Mauricio Di Silvio, Stewart C. Wang, Don K. Nakayama, Richard L. Simmons, Solomon H. Snyder, Timothy R. Billiar

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

838 Scopus citations

Abstract

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 γ. In human hepatocytes, NOS activity is induced by treatment with a combination of tumor necrosis factor, interleukin 1, interferon γ, 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.

Original languageEnglish
Pages (from-to)3491-3495
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume90
Issue number8
DOIs
StatePublished - 15 Apr 1993
Externally publishedYes

Keywords

  • Endotoxin
  • Interferon γ
  • Interleukin
  • Lipopolysaccharide
  • Tumor necrosis factor

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