Roelofsen H, Bakker C T, Schoemaker B, Heijn M, Jansen P L, Elferink R P
Department of Gastrointestinal and Liver Diseases, Academic Medical Centre, Amsterdam, The Netherlands.
Hepatology. 1995 Jun;21(6):1649-57.
The hepatocanalicular transport of a large number of organic anions, such as bilirubin glucuronides and glutathione conjugates in the rat, is mediated by an adenosine triphosphate (ATP)-dependent transport system, which is termed canalicular multispecific organic anion transporter (cMOAT). This system is mainly defined by its deficiency in mutant TR rats. We have previously reported that in cultured hepatocytes the fluorescent organic anion glutathione-bimane (GS-B) accumulates in intracellular vesicles and that this transport is mediated by cMOAT. We now show that this intracellular accumulation of fluorescent organic anion is largely absent in freshly isolated hepatocytes but appears when cells are incubated in suspension at 37 degrees C or cultured for periods of 1 to 24 hours. The appearance of intracellular cMOAT activity coincides with the disappearance of 70% of cMOAT activity from the plasma membrane as measured by the transport activity of the cells for the organic anion dinitrophenyl-glutathione (GS-DNP). Both the appearance of intracellular cMOAT and the disappearance of transport activity from the plasma membrane were completely inhibited at temperatures below 20 degrees C. Residual cMOAT activity in 24-hour cultured hepatocytes could be further diminished by incubation of the cells with 1 mumol/L monensin or 10 mmol/L methylamine. We conclude that after disruption of the cell polarity by collagenase isolation of the hepatocytes, remnants of apical membrane containing cMOAT are rapidly endocytosed when the cells are kept at 37 degrees C. Evidence suggests that at least part of the transporters may recycle back to the plasma membrane after endocytosis. These observations may be relevant for the understanding of regulation of canalicular transport.
在大鼠体内，大量有机阴离子（如胆红素葡萄糖醛酸苷和谷胱甘肽共轭物）的肝小管转运由一种三磷酸腺苷（ATP）依赖性转运系统介导，该系统被称为小管多特异性有机阴离子转运体（cMOAT）。该系统主要通过其在突变TR大鼠中的缺陷来定义。我们之前报道过，在培养的肝细胞中，荧光有机阴离子谷胱甘肽-双硫腙（GS-B）积聚在细胞内囊泡中，且这种转运由cMOAT介导。我们现在表明，这种荧光有机阴离子在新鲜分离的肝细胞中基本不存在细胞内积聚现象，但当细胞在37℃悬浮培养或培养1至24小时时会出现。细胞内cMOAT活性的出现与通过细胞对有机阴离子二硝基苯基-谷胱甘肽（GS-DNP）的转运活性所测得的质膜上70%的cMOAT活性的消失相一致。在低于20℃的温度下，细胞内cMOAT的出现以及质膜转运活性的消失均被完全抑制。24小时培养的肝细胞中残余的cMOAT活性可通过将细胞与1μmol/L莫能菌素或10mmol/L甲胺孵育而进一步降低。我们得出结论，在用胶原酶分离肝细胞破坏细胞极性后，当细胞保持在37℃时，含有cMOAT的顶端膜残余物会迅速被内吞。有证据表明，至少部分转运体在内吞后可能会循环回到质膜。这些观察结果可能与理解肝小管转运的调节有关。
