Characterization of a pretranscriptional pathway for induction by phenobarbital of cytochrome P450 3A23 in primary cultures of adult rat hepatocytes.
Our laboratory has proposed that phenobarbital (PB), a typical lipophilic agent that induces some members of the supergene family of liver microsomal cytochromes P450 (e.g., CYP2B1/2 and CYP3A23), acts through a complex process inhibitable by the presence of growth hormone (GH), the absence of some components of the extracellular matrix, or a disrupted cytoskeleton. To verify that these manipulations of the culture environment block specific steps in the PB induction pathway rather than simply exerting nonspecific or toxic effects on CYP2B1/2 gene transcription, we have now examined PB induction of CYP3A23, a gene known to also be transcriptionally activated by dexamethasone (DEX) through a "nonclassical" pathway apparently involving the glucocorticoid receptor. We found that in primary cultures of adult rat hepatocytes treated with PB, induction of CYP3A23 mRNA, just as we reported for induction of CYP2B1/2 mRNA, required the use of Matrigel (a reconstituted basement membrane) and was blocked by the presence of cytoskeletal inhibitors (colchicine or cytochalasins) or of physiologic concentrations of GH in the culture medium. Moreover, PB induction of CYP3A23 and of CYP2B1/2 mRNAs was greatly diminished by inhibitors of cAMP-dependent protein kinase (PKA). In striking contrast, induction of CYP3A23 mRNA by DEX was unaffected by any of these alterations of the culture conditions that block its induction by PB. We conclude that the effects of extracellular matrix, GH, disruption of the cytoskeleton, and activation of cAMP-dependent protein kinase, pharmacologically define multiple, pretranscriptional steps in the pathway(s) for PB induction of liver cytochromes P450.
Brown
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SE
UCHSC, Hepatobiliary Research Center, Denver, Colorado 80262, USA.
Quattrochi
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LC
Guzelian
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PS
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000235:genetics; D003907:Dexamethasone / Q000008:administration & dosage; D004790:Enzyme Induction; D013006:Growth Hormone / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008297:Male; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis* / Q000235:genetics; D010634:Phenobarbital / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D014158:Transcription, Genetic
D012333:RNA, Messenger; D003907:Dexamethasone; D013006:Growth Hormone; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D001189:Aryl Hydrocarbon Hydroxylases; C465138:Cyp3a23-3a1 protein, rat; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D011494:Protein Kinases; D010634:Phenobarbital