Predominant contribution of organic anion transporting polypeptide OATP-B (OATP2B1) to apical uptake of estrone-3-sulfate by human intestinal Caco-2 cells
Predominant contribution of organic anion transporting polypeptide OATP-B (OATP2B1) to apical uptake of estrone-3-sulfate by human intestinal Caco-2 cells
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DOI：
10.1124/dmd.106.009530
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发表时间：
2006-08-01
影响因子：
3.9
通讯作者：
Tsuji, Akira
中科院分区：
文献类型：
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作者：
Sai, Yoshimichi;Kaneko, Yosuke;Tsuji, Akira
人有机阴离子转运多肽OATP-B（OATP 2B 1）是一种表达于小肠上皮细胞顶端膜的pH敏感性转运蛋白。在本研究中，我们检查了OATP-B与其同系物OATP-D（OATP 3A 1）和OATP-E（OATP 4A 1）相比对Caco-2细胞摄取[H-3]雌酮-3-硫酸酯的贡献。免疫细胞化学研究显示OATP-B表达于Caco-2细胞的顶膜。Caco-2细胞对[H-3]雌酮-3-硫酸酯的摄取不依赖于Na+，并受多种有机阴离子的抑制。稳定表达OATP-B的人胚肾（HEK）293细胞（HEK 293/OATP-B）对[H-3]雌酮-3-硫酸酯的摄取也不依赖于Na+，并受多种有机阴离子的抑制。OATP-B（1.56 μ M）对雌酮-3-硫酸盐摄取的Km值接近Caco-2细胞中观察到的高亲和力组分。在Caco-2细胞和健康志愿者空肠活检组织中，OATP-B的mRNA表达水平高于OATP-D或OATP-E。在Caco-2细胞和HEK 293/OATP-B细胞中，标准化为OATP-B mRNA表达的[H-3]雌酮-3-硫酸盐摄取值相似。OATP- B的单位mRNA表达比活性远高于OATP-D和OATP- E。[H-3]由HEK 293/ OATP-B细胞制备的膜囊泡对雌酮-3-硫酸酯的摄取在向内定向的H+梯度存在下表现出过冲现象，表明H+梯度是OATP-B转运雌酮-3-硫酸酯的驱动力。这些结果表明，OATP-B主要负责Caco-2细胞中雌酮-3-硫酸酯的顶端摄取。
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 [H-3]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 [H-3] 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 mu M and 1.40 mM. The uptake of [H-3] 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 Km value for estrone-3-sulfate uptake by OATP-B (1.56 mu M) 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 [H-3] 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. [H-3] 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.