Regulation of phenobarbital induction of the cytochrome P450 2b9/10 genes in primary mouse hepatocyte culture. Involvement of calcium- and cAMP-dependent pathways.
Phenobarbital (PB) has long been known as an inducer of drug-metabolizing enzymes in liver, but the molecular mechanism underlying this induction is still poorly understood. Using primary mouse hepatocyte culture, we have investigated the possible involvement of different regulatory pathways in PB action, by exposing PB-treated cells to various protein kinase/phosphatase modulators. Our results showed a negative role of the cAMP-dependent pathway, as treatment with cAMP-dependent protein kinase (PKA) activators (10 microM dibutyryl-cAMP and 50 microM forskolin) dramatically inhibited PB-induced Cyp2b9/10 mRNA accumulation, whereas PKA inhibitor potentiated the PB responsiveness of this gene. The cGMP-dependent protein kinase (PKG) seems to play a positive role as PKG inhibitor reduced the PB-induced level of Cyp2b9/10 mRNA. We also obtained two lines of evidence for the involvement of Ca2+ in modulating PB action. Firstly, measurements of intracellular Fura-2 fluorescence ratio in murine hepatocytes showed that long-term PB incubation (24 and 48 h) led to a significant increase of [Ca2+]i. Secondly, treatment with an intracellular Ca2+ chelator (BAPTA-AM) nearly completely abolished PB-induced Cyp2b9/10 expression. Ca2+ thus appeared to mediate PB action likely via Ca2+/calmodulin-dependent protein kinase II, as KN62, a specific inhibitor of this enzyme, also dramatically inhibited PB induction of the Cyp2b9/10 genes.
Marc
N
N
INSERM U456, Faculté de Pharmacie, Rennes, France.
Galisteo
M
M
Lagadic-Gossmann
D
D
Fautrel
A
A
Joannard
F
F
Guillouzo
A
A
Corcos
L
L
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D003994:Bucladesine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000378:metabolism / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072467:Cytochrome P450 Family 2; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010634:Phenobarbital / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013250:Steroid Hydroxylases; D015533:Transcriptional Activation / Q000187:drug effects*
D002614:Chelating Agents; D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; D012333:RNA, Messenger; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D005576:Colforsin; D004533:Egtazic Acid; D003994:Bucladesine; D003577:Cytochrome P-450 Enzyme System; D000242:Cyclic AMP; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C121503:Cyp2b9 protein, mouse; D000072467:Cytochrome P450 Family 2; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D054729:Calcium-Calmodulin-Dependent Protein Kinase Type 1; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C517598:Camk1 protein, mouse; C517681:Pnck protein, mouse; D002118:Calcium; D016257:Fura-2; D010634:Phenobarbital