Song J Y, Van Marle J, Van Noorden C J, Frederiks W F
Laboratory of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, The Netherlands.
Eur J Cell Biol. 1996 Nov;71(3):277-85.
Ca2+, Mg2+-ATPase is a membrane-bound enzyme localized at the bile canalicular membranes of hepatocytes. Cytoskeleton and tight junctions are important for maintenance of the polar distribution of plasma membrane proteins. In order to understand the mechanisms involved in the redistribution of Ca2+, Mg2+-ATPase due to cholestasis, the relationship between Ca2+, Mg2+-ATPase, microfilaments and tight junctions was examined. Cholestasis was induced in rat liver by common bile duct ligation (CBDL) for 2 weeks. Localization of Ca2+, Mg2+-ATPase activity was studied at the light and electron microscopic level. Double-staining of the enzyme and F-actin was performed using phase-contrast and fluorescence microscopy of 7-nitrobenzene-2-oxa-1,3-diazole phallacidin (NBD-ph), respectively. Immunofluorescence microscopy of ZO-1 was applied for the observation of tight junctions. Furthermore, cytoskeleton and junctional complexes were investigated electron microscopically in saponin-extracted tissues. The results showed that CBDL induced redistribution of Ca2+, Mg2+-ATPase activity from the apical to the entire plasma membrane of hepatocytes, which seemed to occur independently of F-actin. F-actin was present at all membrane domains of hepatocytes in control liver, whereas CBDL increased the amounts of F-actin mainly at the bile canalicular membranes. An inverse distribution pattern of Ca2+, Mg2+-ATPase activity and F-actin was found in epithelial cells of bile ducts in control and cholestatic livers. Marked alterations in microfilaments were observed at the bile canaliculi, which were defined as hypertrophy and atrophy and were in association with changes in tight junctions. Structural impairment of the tight junctions was proven by disordered immunofluorescence of ZO-1. It is concluded that changes in the distribution of Ca2+, Mg2+-ATPase and F-actin due to CBDL are independent of each other. CBDL-induced disorders of microfilaments are related to impairment of structural integrity of tight junctions that is suggested to be responsible for the redistribution of Ca2+, Mg2+-ATPase in hepatocytes.
钙镁 -ATP酶是一种位于肝细胞胆小管膜上的膜结合酶。细胞骨架和紧密连接对于维持质膜蛋白的极性分布很重要。为了了解胆汁淤积导致钙镁 -ATP酶重新分布的机制，研究了钙镁 -ATP酶、微丝和紧密连接之间的关系。通过胆总管结扎（CBDL）诱导大鼠肝脏胆汁淤积2周。在光学和电子显微镜水平研究钙镁 -ATP酶活性的定位。分别使用7-硝基苯-2-恶唑-1,3-二氮杂萘鬼笔环肽（NBD-ph）的相差显微镜和荧光显微镜对该酶和F-肌动蛋白进行双重染色。应用ZO-1的免疫荧光显微镜观察紧密连接。此外，在皂素提取的组织中通过电子显微镜研究细胞骨架和连接复合体。结果表明，CBDL诱导钙镁 -ATP酶活性从肝细胞顶端质膜重新分布到整个质膜，这似乎独立于F-肌动蛋白发生。在对照肝脏中，F-肌动蛋白存在于肝细胞的所有膜结构域，而CBDL主要增加了胆小管膜处F-肌动蛋白的量。在对照和胆汁淤积肝脏的胆管上皮细胞中发现钙镁 -ATP酶活性和F-肌动蛋白呈反向分布模式。在胆小管处观察到微丝有明显改变，表现为肥大和萎缩，并与紧密连接的变化相关。ZO-1免疫荧光紊乱证明了紧密连接的结构损伤。结论是，CBDL导致的钙镁 -ATP酶和F-肌动蛋白分布变化相互独立。CBDL诱导的微丝紊乱与紧密连接结构完整性受损有关，这被认为是肝细胞中钙镁 -ATP酶重新分布的原因。
