Abstract
Multidrug resistance-associated protein (MRP) 3/ABCC3 and MRP4/ABCC4 are ATP-binding cassette (ABC) transporters expressed in the sinusoidal membrane of hepatocytes. The purpose of the present study was to establish organic anion-transporting polypeptide (OATP) 1B1/MRP2/MRP3 and OATP1B1/MRP2/MRP4 triple transfectants as in vitro model of the hepatobiliary transport of anionic drugs. To find in vivo relevant Mrp3 probes, wild-type and Mrp3(−/−) mice were given gemfibrozil, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridymethyl)benzothiazole (E3040), troglitazone, bisphenol A, and 4-methylumbelliferone orally. Plasma concentrations of the glucuronide conjugates were significantly lower in Mrp3(−/−) mice than in wild-type mice. The systemic exposure of gemfibrozil, E3040, and troglitazone were similar in wild-type and Mrp3(−/−) mice. 4-Methylumbelliferone and bisphenol A were undetectable in the plasma. In MRP3-expressing membrane vesicles, ATP-dependent uptakes of the glucuronide conjugates of estradiol, gemfibrozil, E3040, and troglitazone were markedly greater than those in controls, whereas MRP4-expressing membrane vesicles exhibited significant ATP-dependent uptake of gemfibrozil glucuronide and estradiol glucuronide. MRP3 or MRP4 was expressed in the OATP1B1/MRP2 double transfectants using adenovirus. The expression levels of OATP1B1 and MRP2 proteins were maintained both in the OATP1B1/MRP2/MRP3 and OATP1B1/MRP2/MRP4 triple transfectants, whereas MRP3 and MRP4 were localized in the basal membrane. Significant reductions in the basal-to-apical flux of the glucuronide conjugates of estradiol, gemfibrozil, E3040, and troglitazone were observed in the OATP1B1/MRP2/MRP3 triple transfectants compared with those in the double transfectants, whereas significant reduction was observed only for gemfibrozil glucuronide and estradiol glucuronide in the OATP1B1/MRP2/MRP4 triple transfectants. These results suggest that MRP3- or MRP4-triple transfectants provide a simple and useful in vitro system for evaluating their importance in the hepatobiliary transport of drugs.
- MRP, multidrug resistance-associated protein
- MDCK, Madin-Darby canine kidney
- OATP, organic anion-transporting polypeptide
- ABC, ATP-binding cassette
- Gem-G, gemfibrozil 1-O-β-glucuronide
- E3040, 6-hydroxy-5,7-dimethyl-2-methylamino-4-(3-pyridymethyl)benzothiazole
- E3040G, E3040 glucuronide
- TGZ-G, troglitazone glucuronide
- Bis-AG, bisphenol A glucuronide
- E217βG, 17β-estradiol-17β-d-glucuronide
- LC, liquid chromatography
- MS, mass spectrometry
- HEK, human embryonic kidney
- MOI, multiplicity of infection
- 4-MUG, 4-methylumbelliferone glucuronide.
Footnotes
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This study was supported in part by a grant-in-aid for Scientific Research (A) (20249008) from the Ministry of Education, Culture, Sports, Science and Technology; and in part by the Industrial Technology Research Grant Program in 2006 from New Energy and Industrial Technology Development Organization of Japan.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
- Received February 17, 2009.
- Accepted July 21, 2009.
- Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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