Tytgat Institute for Liver and Intestinal Research, Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Hepatology. 2012 Jan;55(1):173-83. doi: 10.1002/hep.24691.
Human cholangiocytes are continuously exposed to millimolar levels of hydrophobic bile salt monomers. We recently hypothesized that an apical biliary HCO3- umbrella might prevent the protonation of biliary glycine-conjugated bile salts and uncontrolled cell entry of the corresponding bile acids, and that defects in this biliary HCO3- umbrella might predispose to chronic cholangiopathies. Here, we tested in vitro whether human cholangiocyte integrity in the presence of millimolar bile salt monomers is dependent on (1) pH, (2) adequate expression of the key HCO3- exporter, anion exchanger 2 (AE2), and (3) an intact cholangiocyte glycocalyx. To address these questions, human immortalized cholangiocytes and cholangiocarcinoma cells were exposed to chenodeoxycholate and its glycine/taurine conjugates at different pH levels. Bile acid uptake was determined radiochemically. Cell viability and apoptosis were measured enzymatically. AE2 was knocked down by lentiviral short hairpin RNA. A cholangiocyte glycocalyx was identified by electron microscopy, was enzymatically desialylated, and sialylation was quantified by flow cytometry. We found that bile acid uptake and toxicity in human immortalized cholangiocytes and cholangiocarcinoma cell lines in vitro were pH and AE2 dependent, with the highest rates at low pH and when AE2 expression was defective. An apical glycocalyx was identified on cholangiocytes in vitro by electron microscopic techniques. Desialylation of this protective layer increased cholangiocellular vulnerability in a pH-dependent manner.
A biliary HCO3- umbrella protects human cholangiocytes against damage by bile acid monomers. An intact glycocalyx and adequate AE2 expression are crucial in this process. Defects of the biliary HCO3- umbrella may lead to the development of chronic cholangiopathies.
人类胆管细胞持续暴露于毫摩尔水平的疏水性胆盐单体中。我们最近提出假设，即胆管顶端的 HCO3-伞可能防止胆盐水解结合物胆汁盐的质子化和相应胆汁酸的不受控制的细胞内进入，并且该胆管 HCO3-伞的缺陷可能导致慢性胆管疾病。在这里，我们在存在毫摩尔胆盐单体的情况下，在体外测试了人胆管细胞的完整性是否取决于 (1) pH 值，(2) 关键 HCO3- 外排体阴离子交换器 2 (AE2) 的充分表达，以及 (3) 完整的胆管细胞糖萼。为了解决这些问题，我们用 chenodeoxycholate 及其甘氨酸/牛磺酸缀合物在不同 pH 值下处理人永生化胆管细胞和胆管癌细胞。通过放射性化学法测定胆汁酸摄取量。通过酶法测定细胞活力和细胞凋亡。用慢病毒短发夹 RNA 敲低 AE2。通过电子显微镜鉴定胆管细胞糖萼，用酶法脱唾液酸化，并通过流式细胞术定量唾液酸化。我们发现，在体外人永生化胆管细胞和胆管癌细胞系中，胆汁酸摄取和毒性取决于 pH 值和 AE2，在低 pH 值和 AE2 表达缺陷时速率最高。通过电子显微镜技术在体外鉴定到胆管细胞的顶端糖萼。该保护层的脱唾液酸化以 pH 依赖性方式增加胆管细胞的易损性。
胆管 HCO3- 伞可保护人胆管细胞免受胆汁酸单体的损害。完整的糖萼和足够的 AE2 表达在这个过程中至关重要。胆管 HCO3- 伞的缺陷可能导致慢性胆管疾病的发展。
