Cytochrome P450IIIA activity and cytokine-mediated synthesis of nitric oxide.
BACKGROUND
Although nitric oxide synthase (NOS) is a cytochrome P450-like hemoprotein with additional sequence homology to cytochrome P450 reductase, the role of the cytochrome P450 system in cytokine-mediated NO synthesis is unknown.


METHODS
To characterize the role of the P450 system in the synthesis of NO, NO production, NOS enzyme activity, and steady state NOS mRNA and protein expression were characterized in the setting of P450 isoform activity inhibition by using a model of isolated rat hepatocytes in primary culture. Cimetidine (0 to 10 mmol/L) was chosen as a specific inhibitor of P450IIIA activity. NO production was induced by interleukin-1 (50 ng/ml) and tumor necrosis factor (500 units/ml) and quantified by measurement of its metabolite, nitrite, in the culture medium. Steady state NOS mRNA and protein expression were determined by reverse-transcriptase polymerase chain reaction and immunoblot analysis, respectively. NOS enzyme activity was measured by the conversion of tritiated-L-arginine to tritiated-L-citrulline.


RESULTS
Inhibition of P450IIIA activity was associated with a concentration-dependent decrease in cytokine-mediated NO production. Levels of NOS mRNA and protein were not altered. The NOS enzyme assay was notable for stable concentrations of intermediate, N-OH-L-arginine, and decreased production of the final end product, L-citrulline. Dixon plot kinetic analysis of cimetidine-mediated inhibition of NOS yielded an inhibition constant Ki = 1.76 mmol/L.


CONCLUSIONS
These results indicate that cytochrome P450IIIA isoform may play a posttranslational role in cytokine-mediated NO synthesis in this model of isolated rat hepatocytes in primary culture.
Kuo
P C
PC
Department of Surgery, Stanford University Medical Center, Calif 94305, USA.
Abe
K Y
KY
Dafoe
D C
DC
D000594:Amino Acid Oxidoreductases / Q000235:genetics; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000096:biosynthesis; D001483:Base Sequence; D002927:Cimetidine / Q000494:pharmacology; D002956:Citrulline / Q000096:biosynthesis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D016207:Cytokines / Q000502:physiology*; D007536:Isomerism; D008297:Male; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D014158:Transcription, Genetic
D016207:Cytokines; D015335:Molecular Probes; D012333:RNA, Messenger; D002956:Citrulline; D009569:Nitric Oxide; C068309:N(omega)-hydroxyarginine; D002927:Cimetidine; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases