Torchia E C, Shapiro R J, Agellon L B
Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
Hepatology. 1996 Jul;24(1):206-11. doi: 10.1002/hep.510240133.
The liver recovers bile acids from the portal circulation primarily via an active process that is dependent on sodium ions. Hepatocytes lose the ability to transport bile acids in culture, and, in liver-derived permanent cell lines, this ability is severely reduced or absent. To study the importance of bile acids in regulating liver-specific functions (e.g., cellular bile acid and cholesterol metabolism), we have re-established active bile acid transport in cultured cells. The complementary DNA (cDNA) encoding the rat sodium/taurocholate cotransporting polypeptide (ntcp) was placed under the control of a cytomegalovirus promoter and transfected into the rat hepatoma cell line, McArdle RH-7777. Transfected cells were screened for the ability to take up [3H]-taurocholate. Clones that displayed the ability to take up taurocholate were expanded (designated McNtcp) and further characterized. The apparent Michaelis constant (Km) for taurocholate uptake was similar among the different clones. The observed maximum velocity (Vmax), however, differed and was positively correlated with the abundance of recombinant ntcp messenger RNA (mRNA). The highest level of taurocholate uptake activity observed in McNtcp cells was comparable with that of freshly isolated hepatocytes. Efflux of accumulated taurocholate from McNtcp cells proceeded in a manner similar to primary hepatocytes, indicating that McArdle RH-7777 cells have retained the ability to secrete bile acids. Moreover, taurocholate uptake in McNtcp cells was inhibited by other bile acid species. Based on the observed kinetic parameters, the reconstituted McArdle RH-7777 cells mimic the ability of primary hepatocytes to transport bile acids.
肝脏主要通过依赖钠离子的主动过程从门静脉循环中回收胆汁酸。肝细胞在培养过程中会丧失转运胆汁酸的能力，并且在源自肝脏的永久细胞系中，这种能力会严重降低或缺失。为了研究胆汁酸在调节肝脏特异性功能（如细胞胆汁酸和胆固醇代谢）中的重要性，我们在培养细胞中重新建立了活跃的胆汁酸转运。将编码大鼠钠/牛磺胆酸盐共转运多肽（ntcp）的互补DNA（cDNA）置于巨细胞病毒启动子的控制下，并转染到大鼠肝癌细胞系McArdle RH - 7777中。筛选转染细胞摄取[3H] - 牛磺胆酸盐的能力。表现出摄取牛磺胆酸盐能力的克隆被扩增（命名为McNtcp）并进一步表征。不同克隆之间牛磺胆酸盐摄取的表观米氏常数（Km）相似。然而，观察到的最大速度（Vmax）不同，并且与重组ntcp信使RNA（mRNA）的丰度呈正相关。在McNtcp细胞中观察到的最高水平的牛磺胆酸盐摄取活性与新鲜分离的肝细胞相当。McNtcp细胞中积累的牛磺胆酸盐的流出方式与原代肝细胞相似，表明McArdle RH - 7777细胞保留了分泌胆汁酸的能力。此外，McNtcp细胞中牛磺胆酸盐的摄取受到其他胆汁酸种类的抑制。基于观察到的动力学参数，重建的McArdle RH - 7777细胞模拟了原代肝细胞转运胆汁酸的能力。
