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PREDOMINANT CONTRIBUTION OF ORGANIC ANION TRANSPORTING POLYPEPTIDE OATP-B (OATP2B1) TO APICAL UPTAKE OF ESTRONE-3-SULFATE BY HUMAN INTESTINAL CACO-2 CELLS

Division of Pharmaceutical Sciences, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Japan (Y.S., Yo.K., S.I., K.M., Yu.K., A.T.); Department of Molecular Biopharmaceutics, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Yamasaki, Noda, Japan (I.T.); and Department of Pharmacy, Uppsala University, Uppsala, Sweden (P.A.)

Human organic anion transporting polypeptide OATP-B (OATP2B1) is a pH-sensitive transporter expressed in the apical membranes of small intestinal epithelial cells. In this study, we have examined the contribution of OATP-B to the uptake of [³H]estrone-3-sulfate in Caco-2 cells in comparison with those of its homologs OATP-D (OATP3A1) and OATP-E (OATP4A1). Immunocytochemical study revealed that OATP-B is expressed in the apical membranes of Caco-2 cells. The uptake of [³H]estrone-3-sulfate by Caco-2 cells was Na⁺-independent and inhibited by several organic anions. It showed biphasic saturation kinetics with K_m values of 1.81 µM and 1.40 mM. The uptake of [³H]estrone-3-sulfate by human embryonic kidney (HEK) 293 cells stably expressing OATP-B (HEK293/OATP-B) was also Na⁺-independent and inhibited by several organic anions. The K_m value for estrone-3-sulfate uptake by OATP-B (1.56 µM) was close to that for the high-affinity component observed in Caco-2 cells. The mRNA expression level of OATP-B was higher than that of OATP-D or OATP-E in Caco-2 cells and in human jejunum biopsies from healthy volunteers. The values of [³H]estrone-3-sulfate uptake normalized to OATP-B mRNA expression were similar in Caco-2 cells and HEK293/OATP-B cells. The specific activity of OATP-B per mRNA expression was much higher than that of OATP-D and OATP-E. [³H]Estrone-3-sulfate uptake by membrane vesicles prepared from HEK293/OATP-B cells exhibited an overshoot phenomenon in the presence of an inwardly directed H⁺ gradient, suggesting that an H⁺ gradient is the driving force of estrone-3-sulfate transport by OATP-B. These results suggest that OATP-B is predominantly responsible for the apical uptake of estrone-3-sulfate in Caco-2 cells.

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