Oude Elferink R P, Bakker C T, Roelofsen H, Middelkoop E, Ottenhoff R, Heijn M, Jansen P L
Department of Gastrointestinal and Liver Diseases, Academic Medical Center, Amsterdam, The Netherlands.
Hepatology. 1993 Mar;17(3):434-44.
Transport of organic anions within hepatocytes and the possible involvement of intracellular vesicles were studied with fluorescence microscopy. For this purpose monochlorobimane, a nonfluorescent hydrophobic compound that readily permeates into cells and is conjugated with glutathione to form the fluorescent glutathione bimane, was used. In the isolated perfused livers of normal rats, glutathione bimane is rapidly secreted into bile. In contrast, in our study of livers from mutant TR- rats, a 100-fold reduction in glutathione bimane secretion into bile occurred. Mutant TR- rats have an inherited defect in the canalicular multispecific organic anion transporter, which mediates the ATP-dependent secretion of a wide range of organic anions over the canalicular membrane into bile. When cultured Wistar and TR- hepatocytes were loaded with glutathione bimane, both cell types displayed a strong cytosolic fluorescence. Wistar cells completely lost this cytosolic fluorescence at incubation on monochlorobimane-free medium because of secretion of glutathione bimane. A clear punctate fluorescence remained, however, which was scattered through the cell with some perinuclear concentration. In some cells vesicular fluorescence was also concentrated around a canaliculus. In contrast, TR- cells lost their cytosolic fluorescence more slowly and completely lacked the vesicular fluorescence. Making cells selectively permeable with digitonin directly after loading them with glutathione bimane to remove cytosolic fluorescence again revealed the presence of fluorescent vesicles in Wistar cells and their absence in TR- cells. In Wistar cells vesicular fluorescence could be increased by preincubation with monensin or methylamine, compounds that have been shown to interfere with plasma membrane recycling. In conclusion, these results suggest that apart from secretion over the plasma membrane, the canalicular multispecific organic anion transporter may be involved in accumulation of organic anion in intracellular vesicles. It is hypothesized that this intracellular localization of the canalicular multispecific organic anion transporter is caused by recycling of the transporter between the plasma membrane and intracellular membranes.
利用荧光显微镜研究了肝细胞内有机阴离子的转运以及细胞内囊泡可能发挥的作用。为此，使用了单氯双硫腙，它是一种非荧光疏水性化合物，很容易渗透到细胞内，并与谷胱甘肽结合形成荧光性的谷胱甘肽双硫腙。在正常大鼠的离体灌注肝脏中，谷胱甘肽双硫腙会迅速分泌到胆汁中。相比之下，在我们对突变型TR -大鼠肝脏的研究中，谷胱甘肽双硫腙分泌到胆汁中的量减少了100倍。突变型TR -大鼠在胆小管多特异性有机阴离子转运体上存在遗传性缺陷，该转运体介导多种有机阴离子通过ATP依赖的方式经胆小管膜分泌到胆汁中。当用谷胱甘肽双硫腙加载培养的Wistar和TR -肝细胞时，两种细胞类型均显示出强烈的胞质荧光。Wistar细胞在不含单氯双硫腙的培养基中孵育时，由于谷胱甘肽双硫腙的分泌，胞质荧光完全消失。然而，仍有明显的点状荧光残留，其分散在细胞中，在核周有一定浓度。在一些细胞中，囊泡荧光也集中在胆小管周围。相比之下，TR -细胞胞质荧光消失得更慢，并且完全没有囊泡荧光。在用谷胱甘肽双硫腙加载细胞后，立即用洋地黄皂苷使细胞选择性通透以再次去除胞质荧光，结果显示Wistar细胞中存在荧光囊泡，而TR -细胞中不存在。在Wistar细胞中，用莫能菌素或甲胺预孵育可增加囊泡荧光，这两种化合物已被证明会干扰质膜循环。总之，这些结果表明，除了通过质膜分泌外，胆小管多特异性有机阴离子转运体可能参与了细胞内囊泡中有机阴离子的积累。据推测，胆小管多特异性有机阴离子转运体的这种细胞内定位是由该转运体在质膜和细胞内膜之间的循环所致。
