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Induction by phenobarbital of the mRNA for a specific variant of rat liver microsomal cytochrome P-450.

ABSTRACT: The treatment of rats for 4 days with phenobarbital causes an apparent 3-fold increase in the amount of total liver cytochrome P-450. By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, metyrapone binding and immunoprecipitation, this increase was found to be due to a much larger increase in a restricted number of specific cytochrome P-450 variants. A radioimmunoassay technique demonstrated that the major phenobarbital-inducible variant, of molecular weight 52 000, is induced 24-fold by phenobarbital. Immunoprecipitation analysis of products of translation in vitro with an antibody specific to the 52 000-mol.wt. cytochrome P-450 showed that phenobarbital induces the mRNA in polyribosomes for this variant 20-fold. Evidence is presented for the action of phenobarbital at the transcriptional and translational levels.

SUBMITTER: Phillips IR

PROVIDER: S-EPMC1163106 | biostudies-other | 1981 Jun

REPOSITORIES: biostudies-other

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Project description:The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones.

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Induction by phenobarbital of the mRNA for a specific variant of rat liver microsomal cytochrome P-450.

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