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[Induction and kinetic characterization of nitric oxide synthase in hepatocytes]
1Faculty of Laboratory Medicine, Chongqing University of Medical Sciences, Chongqing 410046, China.
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.