Abstract
Several of the hepatic microsomal cytochromes P-450 (CYP) including CYP3A are inducible by phenobarbital (PB). However, the intracellular pathways involved in the action of PB on CYP3A remain poorly known. With the aim to unravel some of the main aspects of PB signaling, we first devised a simple model of mouse cultured primary hepatocytes in which CYP3A mRNA and protein were strongly induced by PB in the absence of dexamethasone and were at maximum levels after a 48-h treatment with a 2-mM dose of PB. Under these culture conditions, we studied the effects of inhibitors and activators of different protein kinases or phosphatases on CYP3A mRNA and protein induction by PB. CYP3A-induced expression was inhibited by activators of cyclic AMP-dependent protein kinase (PKA) (dibutyryl-cyclic AMP and forskolin) whereas inhibition of PKA by PKA inhibitor enhanced induction. 8-br-cGMP produced effects similar to the activators of PKA, and so did the specific inhibitor of cGMP-dependent protein kinase, β-phenyl-1,N2-etheno-8-bromoguanosine-3,5′-cyclic monophosphorothioate, Rp-isomer (Rp-8-Br-PET-cGMPS). Inhibition of Ca2+/calmodulin-dependent protein kinase by KN-62 or the intracellular Ca2+ chelator BAPTA-AM produced an inhibition of CYP3A induction by PB. Specific inhibitors of protein kinase C, mitogen-activated protein kinase kinase, phosphatidylinositol-3-kinase, or serine/threonine phosphatase did not produce any effect. Taken together, our results suggest that CYP3A induction by PB is regulated positively by calmodulin-dependent protein kinase and cGMP-dependent protein kinase, and negatively by PKA in mouse hepatocytes in primary culture.
Footnotes
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Send reprint requests to: Dr. D. Lagadic-Gossmann, INSERM U456, Détoxication et Réparation Tissulaire, Facultédes Sciences Pharmaceutiques et Biologiques, Université de Rennes I, 2, Avenue du Professeur Léon Bernard, 35043 Rennes cedex, France. E-mail: Dominique.Lagadic{at}rennes.inserm.fr
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↵1 This work was supported by the Institut National de la Sante et de la Recherche Medicale and European Economic Community contract EUROCYP: BMH4-CT96–0254. M.G. is a recipient of a postdoctoral fellowship from the “Universidad de Granada” (Spain). N.M. is a recipient of a fellowship from the “Ministère de la Recherche et de l’Enseignement Supérieur” (France).
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↵2 Present address: Institut National de la Sante et de la Recherche Medicale U517, Faculté de Médecine, 7 Boulevard Jeanne d’Arc, 21033 Dijon Cedex, France.
- Abbreviations:
- CYP
- cytochrome P-450
- PB
- phenobarbital
- cAMP
- cyclic AMP
- PKA
- cAMP-dependent protein kinase
- PKG
- cGMP-dependent protein kinase
- CaM PK
- Ca2+/calmodulin-dependent protein kinase
- PKC
- protein kinase C
- MAP
- mitogen-activated protein
- Rp-8-Br-PET-cGMPS
- β-phenyl-1,N2-etheno-8-bromoguanosine-3′,5′-cyclic monophosphorothioate, Rp-isomer
- db-cAMP
- N6,O2-dibutyryl-cAMP
- PKAI
- protein kinase A inhibitor
- ITS
- 5 mg/l insulin, 2.25 mg/l transferrin, 2.5 mg/l sodium selenite
- SSC
- standard sodium citrate
- TBS
- Tris-buffered saline
- CREB
- cAMP response element-binding protein
- Received March 31, 1999.
- Accepted May 18, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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