Benedetti A, Di Sario A, Marucci L, Svegliati-Baroni G, Schteingart C D, Ton-Nu H T, Hofmann A F
Department of Gastroenterology, University of Ancona, School of Medicine, Italy.
Am J Physiol. 1997 Jun;272(6 Pt 1):G1416-24. doi: 10.1152/ajpgi.1997.272.6.G1416.
When secreted into bile, unconjugated dihydroxy bile acids are absorbed passively by cholangiocytes according to the cholehepatic circulation hypothesis. A fraction of these are likely to be conjugated during transcellular transport. Experiments were performed using fluorescent conjugated bile acids to test whether carrier-mediated transport of conjugated bile acids is present in the basolateral domains of polarized cholangiocytes of intrahepatic bile ductules isolated from rat liver. The time course of the cellular localization of cholyl-NBDAB-Gly and chenodeoxycholyl-NBDAB-Gly, which are anionic fluorescent derivatives of the corresponding glycine-conjugated bile acids, was characterized using an image-analysis system. With 0.3-3 microM solutions, fluorescence was present at 1 and 3 min in the basolateral area of cholangiocytes. Staining in the apical region occurred later, with a peak after 15 min of incubation. The basolateral uptake of the two fluorescent bile acids was temperature dependent and Na+ independent, and was not influenced by the addition of amiloride, by lowering of the medium pH to 6.0, or by preincubation with valinomycin. Uptake was partially inhibited by the absence of Cl- or HCO3- in the perfusate, by preincubation with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), and by the presence of different organic anions or unconjugated and conjugated bile acids in the medium. When cells were preloaded with an ethyl ester of chenodeoxycholyl-NBDAB-Gly, which is hydrolyzed by intracellular esterases, the decrease of cell fluorescence was partly inhibited by H2DIDS, whereas it was stimulated by the presence of 20 microM cholyltaurine in the medium. It is concluded that transport of conjugated bile acid anions across the basolateral membrane of the polarized rat cholangiocyte is carrier mediated. The conjugated bile acid transporter is likely to be an anion exchanger and is likely to be involved in bile secretion whenever conjugated bile acids or other organic anions are transported from the base of the biliary ductular epithelial cells into the plasma of the periductular capillary plexus.
根据胆肝循环假说，未结合的二羟基胆汁酸分泌入胆汁后，胆管细胞会被动吸收。其中一部分在跨细胞转运过程中可能会发生结合。实验使用荧光结合胆汁酸来测试从大鼠肝脏分离的肝内胆小管极化胆管细胞的基底外侧区域是否存在载体介导的结合胆汁酸转运。使用图像分析系统对相应甘氨酸结合胆汁酸的阴离子荧光衍生物胆酰-NBDAB-甘氨酸和鹅去氧胆酰-NBDAB-甘氨酸的细胞定位时间进程进行了表征。在0.3-3 microM溶液中，胆管细胞基底外侧区域在1分钟和3分钟时出现荧光。顶端区域的染色出现较晚，孵育15分钟后达到峰值。两种荧光胆汁酸的基底外侧摄取是温度依赖性的且不依赖于Na+，不受amiloride的添加、培养基pH降至6.0或与缬氨霉素预孵育的影响。灌注液中缺乏Cl-或HCO3-、与4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）预孵育以及培养基中存在不同的有机阴离子或未结合和结合的胆汁酸会部分抑制摄取。当细胞预先加载经细胞内酯酶水解的鹅去氧胆酰-NBDAB-甘氨酸乙酯时，细胞荧光的降低部分受到H2DIDS的抑制，而培养基中存在20 microM胆酰牛磺酸则会刺激荧光降低。结论是，结合胆汁酸阴离子跨极化大鼠胆管细胞基底外侧膜的转运是由载体介导的。结合胆汁酸转运体可能是一种阴离子交换器，并且每当结合胆汁酸或其他有机阴离子从胆小管上皮细胞底部转运到小叶间毛细血管丛血浆中时，可能参与胆汁分泌。
