title: [Induction and kinetic characterization of nitric oxide synthase in hepatocytes].
abstract: OBJECTIVE
  To study the synergistic responses of nitric oxide synthase (NOS) induction in rat hepatocytes to LPS and various cytokines in vitro and the kinetic characteristics of iNOS.
  
  
  METHODS
  The hepatocytes were isolated by in-situ pre-perfusion and collagenase circulatory perfusion of rat livers. The effects of LPS associated with IFN-r, TNF-alpha and IL-1beta or IL-6 on NOS activity, cGMP, and NO(2)(-)+NO(3)(-) were observed in hepatocytes, respectively. Also the kinetic characteristics of this enzyme and dose response of corticosteroids on the induction of iNOS were analyzed.
  
  
  RESULTS
  The maximum induction of NOS activity was observed in hepatocytes treated by LPS in combination with IFN-r, TNF-alpha and IL-1beta or IL-6. The kinetic analysis of this iNOS demonstrated specific constants of Km=10.8 micromol/L, Vmax=263.2 pmol/min/mg protein(for L-Arg), and Ki of 0.56, 0.94 micromol/L for competitive inhibitor, L-NMMA and NNA, respectively. The time course of induction showed that iNOS activity peaked at 9 h; however, significant increase in release of NO(2)(-)+NO(3)(-) and cGMP sustained for at least 18 h. Dexamethasone and hydrocortisone dramatically inhibited the NOS induction in hepatocytes in vitro with IC50 of 3.5+/-10(-8)mol/L and 2.6+/-10(-6)mol/L, respectively.
  
  
  CONCLUSIONS
  The expression of inductive NOS in hepatocytes requires specific synergetic action of cytokines, and the inducible characteristics may play an important pathogenesis in endotoxemia and septic shock.
authors: Faculty of Laboratory Medicine, Chongqing University of Medical Sciences, Chongqing 410046, China.
authors: X
authors: X
authors: Tang
authors: D
authors: D
authors: Zhou
authors: G
authors: G
authors: Kang
mesh_terms: D000818:Animals; D016207:Cytokines / Q000494:pharmacology; D004790:Enzyme Induction; D022781:Hepatocytes / Q000201:enzymology*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008297:Male; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D051381:Rats; D017208:Rats, Wistar
chemical_list: D016207:Cytokines; D008070:Lipopolysaccharides; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat