cAMP-associated inhibition of phenobarbital-inducible cytochrome P450 gene expression in primary rat hepatocyte cultures.
The effects of elevated intracellular cyclic adenosine monophosphate (cAMP) in regulating phenobarbital (PB)-inducible gene expression in primary rat hepatocyte cultures were investigated. Cells were exposed to various concentrations (0.1-100 microM) of cAMP analogs and/or activators of intracellular cAMP-dependent pathways. Effects of these treatments were assessed either using a 1-h pulse prior to PB (100 microM) exposure or in conjunction with PB during a 24-h exposure period. PB-inducible responses were measured in hepatocytes by hybridization to cytochrome P450 (CYP) CYP2B1, CYP2B2, and CYP3A1 mRNAs. The cAMP analogs, 8-bromo-cAMP, 8-(4-chlorophenylthio)-cAMP, dibutyryl cAMP, and (Sp)-5,6-DCl-cBiMPS ((Sp)-5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3', 5'-monophosphorothioate), and the activators of adenylate cyclase, forskolin and glucagon, dramatically inhibited PB-mediated induction of CYP2B1 and CYP2B2 in a concentration-dependent manner. A similar inhibition of PB-induced CYP3A1 mRNA levels was effected by the cAMP analogs and glucagon. The phosphodiesterase inhibitors isobutylmethylxanthine and RO 201724 potentiated the cAMP responses. Increasing the concentration of PB (0.05-1.00 mM) did not alleviate the cAMP-mediated repression. A requirement for protein kinase A (PKA) was demonstrated by the use of (Sp)-cAMPS, a highly specific activator of PKA, whereas the inactive diastereoisomer, (Rp)-cAMPS, was ineffective in modulating PB induction. The response to cAMP was specific since elevated intracellular cAMP levels did not perturb beta-naphtholflavone-mediated induction of CYP1A1, CYP1A2, microsomal epoxide hydrolase, or dexamethasone-mediated induction of CYP3A1 gene expression. Nor did elevated intracellular cAMP modulate the liver-selective albumin gene expression levels. The results of the present study demonstrated striking inhibition of PB-mediated CYP gene induction by cAMP and PKA activators, indicating a negative regulatory role for the cAMP signal transduction pathway on PB gene induction.
Sidhu
J S
JS
Department of Environmental Health, University of Washington, Seattle, 98195, USA.
Omiecinski
C J
CJ
D000418:Albumins / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D001571:Benzoflavones / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000819:agonists / Q000037:antagonists & inhibitors; D006152:Cyclic GMP / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D003907:Dexamethasone / Q000494:pharmacology; D004004:Dichlororibofuranosylbenzimidazole / Q000031:analogs & derivatives / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D005934:Glucagon / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010634:Phenobarbital / Q000494:pharmacology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D051381:Rats; D013237:Stereoisomerism; D013250:Steroid Hydroxylases / Q000096:biosynthesis*; D013873:Thionucleotides / Q000494:pharmacology; D015533:Transcriptional Activation; D019324:beta-Naphthoflavone
D000418:Albumins; D001571:Benzoflavones; D010726:Phosphodiesterase Inhibitors; D013873:Thionucleotides; C071644:5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole-3',5'-monophosphorothioate; D005576:Colforsin; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D004004:Dichlororibofuranosylbenzimidazole; D019324:beta-Naphthoflavone; D003994:Bucladesine; D003907:Dexamethasone; D005934:Glucagon; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C104464:CYP3A protein, human; C465138:Cyp3a23-3a1 protein, rat; D051544:Cytochrome P-450 CYP3A; C025619:steroid 16-beta-hydroxylase; D017868:Cyclic AMP-Dependent Protein Kinases; D006152:Cyclic GMP; D010634:Phenobarbital