Nitric oxide-associated regulation of hepatocyte glutathione synthesis is a guanylyl cyclase-independent event.
BACKGROUND
In a system of rat hepatocytes in primary culture, inhibition of cytokine-mediated nitric oxide (NO) production has been shown to be protective in states of oxidative stress. In the absence of oxidative injury, inhibition of NO synthesis has been associated with decreased intracellular levels of reduced glutathione.


METHODS
To further characterize the role of NO in hepatocyte glutathione metabolism, cytokine-mediated NO synthesis was inhibited by addition of a competitive substrate inhibitor. Reduced glutathione, NO metabolites, and enzyme activity and steady-state mRNA levels of the rate-limiting enzyme for reduced glutathione (GSH) synthesis, gamma-glutamylcysteine synthetase, were determined in the presence and absence of the substrate inhibitor. A diffusible cyclic guanosine monophosphate (cGMP) analog, 8-bromo-cGMP, was added in selected instances to determine the potential role of soluble guanylyl cyclase in glutathione metabolism.


RESULTS
Inhibition of cytokine-induced NO synthesis was associated with depletion of glutathione. These levels were restored in the presence of pharmacologic concentrations of a NO donor. Along with decreased glutathione levels, gamma-glutamylcysteine synthetase enzyme activity and steady state mRNA levels were also decreased with inhibition of NO synthesis. Addition of 8-bromo-cGMP did not alter glutathione content or gamma-glutamylcysteine synthetase enzyme activity and steady-state mRNA levels.


CONCLUSIONS
In this system of cultured rat hepatocytes, cytokine-mediated NO synthesis may be protective in states of oxidative stress through regulation of glutathione synthesis.
Kuo
P C
PC
Department of Surgery, University of Maryland Medical Systems, Baltimore, USA.
Abe
K Y
KY
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005721:Glutamate-Cysteine Ligase / Q000235:genetics; D005978:Glutathione / Q000096:biosynthesis*; D006162:Guanylate Cyclase / Q000378:metabolism*; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology; D008099:Liver / Q000166:cytology* / Q000201:enzymology; D008233:Lymphotoxin-alpha / Q000494:pharmacology; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology
D007375:Interleukin-1; D008233:Lymphotoxin-alpha; D012333:RNA, Messenger; D009569:Nitric Oxide; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D006162:Guanylate Cyclase; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione