Department of Gastroenterology, Hepatology and Infectious Diseases, Medical Faculty, Heinrich-Heine University, Düsseldorf, Germany.
Hepatology. 2012 Dec;56(6):2387-97. doi: 10.1002/hep.25955.
The sodium taurocholate cotransporting polypeptide (Ntcp) is the major bile salt uptake transporter at the sinusoidal membrane of hepatocytes. Short-term feedback regulation of Ntcp by primary bile salts has not yet been investigated in vivo. Subcellular localization of Ntcp was analyzed in Ntcp-transfected HepG2-cells by flow cytometry and in immunofluorescence images from tissue sections by a new automated image analysis method. Net bile salt uptake was investigated in perfused rat liver by a pulse chase technique. In Flag-Ntcp-EGFP (enhanced green fluorescent protein) expressing HepG2-cells, taurochenodeoxycholate (TCDC), but not taurocholate (TC), induced endocytosis of Ntcp. TCDC, but not TC, caused significant internalization of Ntcp in perfused rat livers, as shown by an increase in intracellular Ntcp immunoreactivity, whereas Bsep distribution remained unchanged. These results correlate with functional studies. Rat livers were continuously perfused with 100 μmol/L of TC. 25 μmol/L of TCDC, taurodeoxycholate (TDC), tauroursodeoxycholate (TUDC), or TC were added for 30 minutes, washed out, followed by a pulse of (3) [H]-TC. TCDC, but not TDC, TUDC, or TC significantly increased the amount of (3) [H]-TC in the effluent, indicating a reduced sinusoidal net TC uptake. This effect was sensitive to chelerythrine (protein kinase C inhibitor) and cypermethrin (protein phosphatase 2B inhibitor). Phosphoinositide 3-kinase (PI3K) inhibitors had an additive effect, whereas Erk1/2 (extracellular signal activated kinase 1/2), p38MAPK, protein phosphatase 1/2A (PP1/2A), and reactive oxygen species (ROS) were not involved.
TCDC regulates bile salt transport at the sinusoidal membrane by protein kinase C- and protein phosphatase 2B-mediated retrieval of Ntcp from the plasma membrane. During increased portal bile salt load this mechanism may adjust bile salt uptake along the acinus and protect periportal hepatocytes from harmful bile salt concentrations.
牛磺胆酸钠共转运蛋白（Ntcp）是肝细胞窦状膜上主要的胆汁盐摄取转运体。初级胆汁盐对 Ntcp 的短期反馈调节尚未在体内进行研究。通过流式细胞术分析转染 HepG2 细胞的 Ntcp 的亚细胞定位，并通过新的自动化图像分析方法分析组织切片的免疫荧光图像。通过脉冲追踪技术研究灌注大鼠肝脏的净胆汁盐摄取。在表达 Flag-Ntcp-EGFP（增强型绿色荧光蛋白）的 HepG2 细胞中，牛磺胆酸钠（TCDC）而非牛磺胆酸钠（TC）诱导 Ntcp 的内吞作用。TCDC 而非 TC 导致灌注大鼠肝脏中 Ntcp 的显著内化，如细胞内 Ntcp 免疫反应性增加所示，而 Bsep 分布保持不变。这些结果与功能研究相关。用 100μmol/L 的 TC 连续灌注大鼠肝脏。用 25μmol/L 的 TCDC、牛磺脱氧胆酸钠（TDC）、牛磺熊脱氧胆酸钠（TUDC）或 TC 灌注 30 分钟，然后冲洗，再用脉冲（3）[H]-TC 进行脉冲。TCDC 而非 TDC、TUDC 或 TC 显著增加流出物中的（3）[H]-TC 量，表明窦状净 TC 摄取减少。这种作用对 Chelerythrine（蛋白激酶 C 抑制剂）和 Cypermethrin（蛋白磷酸酶 2B 抑制剂）敏感。磷酸肌醇 3-激酶（PI3K）抑制剂具有加性作用，而细胞外信号激活激酶 1/2（Erk1/2）、p38MAPK、蛋白磷酸酶 1/2A（PP1/2A）和活性氧（ROS）不参与。
TCDC 通过蛋白激酶 C 和蛋白磷酸酶 2B 介导的从质膜中回收 Ntcp 来调节窦状膜上的胆汁盐转运。在门静脉胆汁盐负荷增加时，该机制可以沿腺泡调节胆汁盐摄取，并防止周边肝细胞受到有害的胆汁盐浓度的影响。
