Construction of Triple-Transfected Cells [Organic Anion-Transporting Polypeptide (OATP) 1B1/Multidrug Resistance-Associated Protein (MRP) 2/MRP3 and OATP1B1/MRP2/MRP4] for Analysis of the Sinusoidal Function of MRP3 and MRP4

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.