Bode Hans-Peter, Wang LiFu, Cassio Doris, Leite M Fatima, St-Pierre Marie V, Hirata Keiji, Okazaki Keisuke, Sears Marvin L, Meda Paolo, Nathanson Michael H, Dufour Jean-François
Department of Gastroenterology, University of Bern, Bern, Switzerland.
Hepatology. 2002 Sep;36(3):631-40. doi: 10.1053/jhep.2002.35274.
Hepatocytes and other digestive epithelia exchange second messengers and coordinate their functions by communicating through gap junctions. However, little is known about intercellular communication in cholangiocytes. The aim of this study was to examine expression and regulation of gap junctions in cholangiocytes. Connexin expression was determined by confocal immunofluorescence in rat bile ducts and in normal rat cholangiocyte (NRC) cells, a polarized cholangiocyte cell line. Intercellular Ca(2+) signaling was monitored by fluorescent microscopy. Microinjection studies assessed regulation of gap junction permeability in NRC cells and in SKHep1 cells, a liver-derived cell line engineered to express connexin 43. Immunochemistry showed that cholangiocytes from normal rat liver as well as the NRC cells express connexin 43. Localization of apical, basolateral, and tight junction proteins confirmed that NRC cells are well polarized. Apical exposure to ATP induced Ca(2+) oscillations that were coordinated among neighboring NRC cells, and inhibition of gap junction conductance desynchronized the Ca(2+) oscillations. NRC cells transfected with a connexin 43 antisense were significantly less coupled. Transcellular dye spreading was inhibited by activation of protein kinase A or protein kinase C. The same was observed in transfected SKHep1 cells, which expressed only connexin 43. Rat cholangiocytes and NRC cells express connexin 43, which permits synchronization of Ca(2+) signals among cells. Permeability of connexin 43-gap junctions is negatively regulated by protein kinases A and C. In conclusion, cholangiocytes have the capacity for intercellular communication of second messenger signals via gap junctions in a fashion that is under hormonal control.
肝细胞和其他消化上皮细胞通过间隙连接进行通讯，交换第二信使并协调它们的功能。然而，关于胆管细胞间通讯的了解却很少。本研究的目的是检测胆管细胞中间隙连接的表达和调控。通过共聚焦免疫荧光法测定大鼠胆管和正常大鼠胆管细胞（NRC）系（一种极化的胆管细胞系）中连接蛋白的表达。通过荧光显微镜监测细胞间Ca(2+)信号。显微注射研究评估了NRC细胞和SKHep1细胞（一种经基因工程改造以表达连接蛋白43的肝源性细胞系）中间隙连接通透性的调控。免疫化学显示，正常大鼠肝脏的胆管细胞以及NRC细胞均表达连接蛋白43。顶端、基底外侧和紧密连接蛋白的定位证实NRC细胞具有良好的极化状态。顶端暴露于ATP可诱导相邻NRC细胞间协调的Ca(2+)振荡，而抑制间隙连接电导会使Ca(2+)振荡失同步。用连接蛋白43反义核酸转染的NRC细胞耦合性明显降低。蛋白激酶A或蛋白激酶C的激活可抑制跨细胞染料扩散。在仅表达连接蛋白43的转染SKHep1细胞中也观察到同样的现象。大鼠胆管细胞和NRC细胞表达连接蛋白43，这使得细胞间的Ca(2+)信号能够同步。连接蛋白43间隙连接的通透性受到蛋白激酶A和C的负调控。总之，胆管细胞有能力通过间隙连接以一种受激素控制的方式进行第二信使信号的细胞间通讯。
