Abstract
The expression of three cytochromes P450 (CYP3A4, CYP2C9, and CYP2B6) was investigated in primary human hepatocyte cultures following treatment with four calcium channel modulators (CCM) of the dihydropyridine family, three antagonists (nifedipine, nicardipine, and isradipine), and one agonist (BK8644). Induction of CYP3A4 was studied by Northern blot, Western blot, and enzymatic activity. Induction began between 1 and 10 μM CCM and was dependent on the presence of dexamethasone (100 nM) in the medium. CYP3A4 mRNA accumulation started only after 16 h of treatment because pregnane X receptor (hPXR) synthesis was needed. Cotransfection experiments showed that the proximal and the distal PXR response elements of the CYP3A4 promoter and hPXR (HepG2 cells) or dexamethasone-induced hPXR (primary hepatocytes) were necessary to obtain full induction. Furthermore, glutathione S-transferase pull-down assays demonstrated that the CCM tested can act as hPXR ligands. In addition, cotransfection experiments in CV1 cells showed that these compounds failed to reverse CAR (constitutively activated receptor) inactivation by androstenol. Finally, 10 μM CCM induced both CYP2C9 and CYP2B6, strengthening the evidence that hPXR is involved in the regulation of these genes. All together, these results widen the field of hPXR activators to a new class of ligand, namely the CCM of the dihydropyridine family.
Footnotes
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This work was supported in part by GlaxoWellcome (L.D.) and la Ligue Nationale contre le Cancer (J.-M.P.).
- Abbreviations used are::
- CCM
- calcium channel modulators
- P450
- cytochrome P450
- PXR
- pregnane X receptor
- hPXR
- human PXR
- CAR
- constitutively activated receptor
- mCAR
- mouse CAR
- DMSO
- dimethyl sulfoxide
- TCPOBOP
- 1,4-bis[2-(3,5-dichloropyridyloxy)]-benzene
- PCR
- polymerase chain reaction
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- GST
- glutathione S-transferase
- PAGE
- polyacrylamide gel electrophoresis
- CSA
- cyclosporin A
- NIF
- nifedipine
- BK
- BK8644
- ISRA
- isradipine, RIF, rifampicin
- NIC
- nicardipine
- SRC
- steroid receptor coactivator
- PB
- phenobarbital
- DEX
- dexamethasone
- CARLA
- coactivator receptor ligand assay
- ER6
- everted repeat 6
- Received March 26, 2001.
- Accepted July 9, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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