CAMP-associated Inhibition of Phenobarbital-inducible Cytochrome P450 Gene Expression in Primary Rat Hepatocyte Cultures

Abstract

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.