A novel fluorescence-based method to evaluate ileal apical sodium-dependent bile acid transporter ASBT.
A novel fluorescence-based method to evaluate ileal apical sodium-dependent bile acid transporter ASBT.
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DOI：
10.1016/j.xphs.2020.11.030
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发表时间：
2020-12
影响因子：
3.8
通讯作者：
Qiunan Zhu;Hisakazu Komori;Rikako Imamura;I. Tamai
中科院分区：
文献类型：
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作者：
Qiunan Zhu;Hisakazu Komori;Rikako Imamura;I. Tamai
本研究旨在证明荧光团标记的胆汁酸衍生物 N-(24-[7-(4-N,N-二甲基氨基磺酰基-2,1,3-苯并恶二唑)]氨基-3α,7α,12α-三羟基-27-去甲-5β-胆汁斯坦-26-酰基)-2'-氨基乙磺酸盐 (tauro-nor-THCA-24-DBD) 作为底物的有用性顶端钠依赖性胆汁酸转运蛋白（ASBT，SLC10A2）的研究，该转运蛋白在回肠远端表达，用于胆汁酸的重吸收，并寻找一种新的基于荧光的方法来评估 ASBT 活性。在HPLC分析中，tauro-nor-THCA-24-DBD的色谱图显示双峰：该化合物的R-和S-异构体。当 ASBT 在非洲爪蟾母细胞中表达时，其摄取量高于对照卵母细胞，表明两者均由 ASBT 转运。因此，结果分别作为峰1、峰2和它们的总和进行分析。表达 ASBT 的卵母细胞中牛磺去甲 THCA-24-DBD 的浓度依赖性摄取达到饱和，峰 1 的 Km 122 μM 和 Vmax 1.49 pmol/卵母细胞/30 分钟，峰 2 的 Km 122 μM 和 Vmax 1.49 pmol/卵母细胞/30 分钟，峰 2 30.7 μM 和 1.34 pmol/卵母细胞/30 分钟，40.6 μM 和 2.36 pmol/卵母细胞/30 分钟分别为总和。在牛磺胆酸存在和无Na + 条件下，这些摄取减少。此外，在 Caco-2 细胞中，tauro-nor-THCA-24-DBD 的摄取也是 Na+ 依赖性且可饱和的。此外，选择性 ASBT 抑制剂 eobixibat 降低了这些摄取。因此，得出的结论是，tauro-nor-THCA-24-DBD是ASBT的底物，可用于评估肠道ASBT转运活性。
This study aimed to demonstrate usefulness of the fluorophore-labeled bile acid derivative, N-(24-[7-(4-N,N-dimethylaminosulfonyl-2,1,3-benzoxadiazole)]amino-3α,7α,12α-trihydroxy-27-nor-5β-cholestan-26-oyl)-2′-aminoethane sulfonate (tauro-nor-THCA-24-DBD) as a substrate of apical sodium-dependent bile acid transporter (ASBT,SLC10A2), which is expressed at distal ileum for reabsorption of bile acids and to find a novel fluorescence-based method to evaluate ASBT activity. In HPLC analysis, chromatogram of tauro-nor-THCA-24-DBD showed double peaks: R- and S-isomers of the compound. When ASBT was expressed inXenopus laevisoocytes, their uptakes were higher than those by control oocytes, demonstrating both are transported by ASBT. Therefore, results were analyzed separately as peak 1, peak 2 and sum of them. Concentration dependent uptake of tauro-nor-THCA-24-DBD in ASBT-expressing oocytes was saturable with Km 122 μM and Vmax 1.49 pmol/oocyte/30 min for peak 1, 30.7 μM and 1.34 pmol/oocyte/30 min for peak 2, and 40.6 μM and 2.36 pmol/oocyte/30 min for sum, respectively. These uptakes were decreased in the presence of taurocholic acid and in the Na+free condition. Furthermore, in Caco-2 cells, tauro-nor-THCA-24-DBD uptake was also Na+-dependent and saturable. Additionally, these uptakes were decreased by elobixibat, a selective ASBT inhibitor. Accordingly, it was concluded that tauro-nor-THCA-24-DBD is a substrate of ASBT and useful to evaluate the intestinal ASBT transport activity.