Yano M, Marinelli R A, Roberts S K, Balan V, Pham L, Tarara J E, de Groen P C, LaRusso N F
Department of Internal Medicine, Center for Basic Research in Digestive Diseases, Mayo Medical School, Clinic and Foundation, Rochester, Minnesota 55905, USA.
J Biol Chem. 1996 Mar 22;271(12):6702-7. doi: 10.1074/jbc.271.12.6702.
During bile formation by the liver, large volumes of water are transported across two epithelial barriers consisting of hepatocytes and cholangiocytes (i.e. intrahepatic bile duct epithelial cells). We recently reported that a water channel, aquaporin-channel-forming integral protein of 28 kDa, is present in cholangiocytes and suggested that it plays a major role in water transport by these cells. Since the mechanisms of water transport across hepatocytes remain obscure, we performed physiological, molecular, and biochemical studies on hepatocytes to determine if they also contain water channels. Water permeability was studied by exposing isolated rat hepatocytes to buffers of different osmolarity and measuring cell volume by quantitative phase contrast, fluorescence and laser scanning confocal microscopy. Using this method, hepatocytes exposed to hypotonic buffers at 23 degrees C increased their cell volume in a time and osmolarity-dependent manner with an osmotic water permeability coefficient of 66.4 x 10(-4) cm/s. In studies done at 10 degrees C, the osmotic water permeability coefficient decreased by 55% (p < 0.001, at 23 degrees C; t test). The derived activation energy from these studies was 12.8 kcal/mol. After incubation of hepatocytes with amphotericin B at 10 degrees C, the osmotic water permeability coefficient increased by 198% (p < 0.001) and the activation energy value decreased to 3.6 kcal/mol, consistent with the insertion of artificial water channels into the hepatocyte plasma membrane. Reverse transcriptase polymerase chain reaction with hepatocyte RNA as template did not produce cDNAs for three of the known water channels. Both the cholesterol content and the cholesterol/phospholipid ratio of hepatocyte plasma membranes were significantly (p < 0.005) less than those of cholangiocytes; membrane fluidity of hepatocytes estimated by measuring steady-state anisotropy was higher than that of cholangiocytes. Our data suggests that the osmotic flow of water across hepatocyte membranes occurs mainly by diffusion via the lipid bilayer (not by permeation through water channels as in cholangiocytes).
在肝脏形成胆汁的过程中，大量的水通过由肝细胞和胆管细胞（即肝内胆管上皮细胞）组成的两个上皮屏障进行转运。我们最近报道，一种水通道，即28 kDa的水通道形成整合蛋白，存在于胆管细胞中，并表明它在这些细胞的水转运中起主要作用。由于水通过肝细胞的转运机制仍不清楚，我们对肝细胞进行了生理学、分子学和生物化学研究，以确定它们是否也含有水通道。通过将分离的大鼠肝细胞暴露于不同渗透压的缓冲液中，并通过定量相差显微镜、荧光显微镜和激光扫描共聚焦显微镜测量细胞体积，研究了水通透性。使用这种方法，在23℃下暴露于低渗缓冲液的肝细胞以时间和渗透压依赖性方式增加其细胞体积，渗透水通透性系数为66.4×10^(-4) cm/s。在10℃进行的研究中，渗透水通透性系数降低了55%（在23℃时，p < 0.001；t检验）。这些研究得出的活化能为12.8 kcal/mol。在10℃下用两性霉素B孵育肝细胞后，渗透水通透性系数增加了198%（p < 0.001），活化能值降至3.6 kcal/mol，这与人工水通道插入肝细胞质膜一致。以肝细胞RNA为模板的逆转录聚合酶链反应未产生三种已知水通道的cDNA。肝细胞质膜的胆固醇含量和胆固醇/磷脂比均显著低于胆管细胞（p < 0.005）；通过测量稳态各向异性估计的肝细胞膜流动性高于胆管细胞。我们的数据表明，水通过肝细胞膜的渗透流动主要是通过脂质双层的扩散发生的（不像胆管细胞那样通过水通道渗透）。
