Sidhu J S, Omiecinski C J
Department of Environmental Health, University of Washington, Seattle, 98195, USA.
J Biol Chem. 1995 May 26;270(21):12762-73. doi: 10.1074/jbc.270.21.12762.
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.
研究了细胞内环状单磷酸腺苷（cAMP）升高对原代大鼠肝细胞培养物中苯巴比妥（PB）诱导基因表达的调节作用。将细胞暴露于不同浓度（0.1 - 100 microM）的cAMP类似物和/或细胞内cAMP依赖性途径的激活剂。这些处理的效果通过在PB（100 microM）暴露前进行1小时脉冲处理或在24小时暴露期内与PB联合使用来评估。通过与细胞色素P450（CYP）CYP2B1、CYP2B2和CYP3A1 mRNA杂交来测量肝细胞中PB诱导的反应。cAMP类似物8 - 溴 - cAMP、8 - （4 - 氯苯基硫代） - cAMP、二丁酰cAMP和（Sp） - 5,6 - DCl - cBiMPS（（Sp） - 5,6 - 二氯 - 1 - β - D - 呋喃核糖基苯并咪唑 - 3'，5' - 单磷酸硫酯）以及腺苷酸环化酶激活剂福斯可林和胰高血糖素，以浓度依赖性方式显著抑制PB介导的CYP2B1和CYP2B2诱导。cAMP类似物和胰高血糖素对PB诱导的CYP3A1 mRNA水平有类似的抑制作用。磷酸二酯酶抑制剂异丁基甲基黄嘌呤和RO 201724增强了cAMP反应。增加PB的浓度（0.05 - 1.00 mM）并不能减轻cAMP介导的抑制作用。使用（Sp） - cAMPS（一种高度特异性的蛋白激酶A（PKA）激活剂）证明了对PKA的需求，而无活性的非对映异构体（Rp） - cAMPS在调节PB诱导方面无效。对cAMP的反应具有特异性，因为细胞内cAMP水平升高不会干扰β - 萘黄酮介导的CYP1A1、CYP1A2、微粒体环氧化物水解酶诱导或地塞米松介导的CYP3A1基因表达诱导。细胞内cAMP升高也不会调节肝脏选择性白蛋白基因表达水平。本研究结果表明cAMP和PKA激活剂对PB介导的CYP基因诱导有显著抑制作用，表明cAMP信号转导途径对PB基因诱导具有负调节作用。
