ISOLATION AND CHARACTERIZATION OF RAT CHOLANGIOCYTE VESICLES ENRICHED IN APICAL OR BASOLATERAL PLASMA-MEMBRANE DOMAINS
ISOLATION AND CHARACTERIZATION OF RAT CHOLANGIOCYTE VESICLES ENRICHED IN APICAL OR BASOLATERAL PLASMA-MEMBRANE DOMAINS
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DOI：
10.1021/bi00047a007
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发表时间：
1995-11-28
期刊：
影响因子：
2.9
通讯作者：
LARUSSO, NF
中科院分区：
文献类型：
--
作者：
TIETZ, PS;HOLMAN, RT;LARUSSO, NF
胆管细胞是排列在肝内胆管上的上皮细胞，由具有离散的顶端（内腔）和基底外侧域的质膜组成。虽然这些结构域被认为含有不同的转运蛋白、交换剂、通道和受体，但目前还没有用于分离这些不同膜隔室的方法。因此，我们的目的是开发一种技术，以分离质膜从胆管细胞丰富的顶端或基底侧域。我们从胆管结扎（BDL）后3周的大鼠中分离出富含胆管细胞的细胞群，BDL是一种刺激选择性胆管细胞增殖的策略。使用等密度离心线性蔗糖梯度，我们准备了一个混合的胆管细胞质膜（MCPM）的馏分，我们进一步产生单独的顶端和基底侧胆管细胞质膜（ACPM和BCPM，分别）。我们通过特异性标记酶测定、透射电子显微镜（TEM）、脂质分析、各向异性测量、一维和二维凝胶电泳以及囊性纤维化跨膜传导调节因子（CFTR）的定量免疫印迹来表征这些组分。标记酶测定和透射电镜显示，MCPM部分基本上没有其他细胞器，但富含约70倍的磷酸二酯酶I，一般质膜标记; ACPM和BCPM适当地富集在各自的顶端和基底侧标记。ACPM和BCPM的TEM显示囊泡膜的均匀制剂，没有其他细胞器的污染。脂质分析，一维和二维凝胶电泳，CFTR免疫印迹，和各向异性测量显示明确的差异，脂质和蛋白质的组成和流动性之间的ACPM和BCPM域。这种方法提供了一个初步的尝试，开发高度纯化的顶端和基底膜结构域的胆管细胞，这为进一步的研究提供了一个起点。
Cholangiocytes, the epithelial cells that line intrahepatic bile ducts, are composed of plasma membranes with discrete apical (lumenal) and basolateral domains. While these domains are thought to contain different transporters, exchangers, channels, and receptors, no methodology currently exists for the isolation of these different membrane compartments. Thus, our aim was to develop a technique to isolate plasma membranes from cholangiocytes enriched in apical or basolateral domains. We isolated a cholangiocyte-enriched population of cells from rats 3 weeks after bile duct ligation (BDL), a maneuver which stimulates selective cholangiocyte proliferation. Using isopycnic centrifugation on linear sucrose gradients, we prepared a mixed cholangiocyte plasma membrane (MCPM) fraction from which we further generated separate apical and basolateral cholangiocyte plasma membranes (ACPM and BCPM, respectively). We characterized these fractions by specific marker enzyme assays, transmission electron microscopy (TEM), lipid analysis, anisotropy measurements, one- and two-dimensional gel electrophoresis, and quantitative immunoblots of the cystic fibrosis transmembrane conductance regulator (CFTR). Marker enzyme assays and TEM revealed that the MCPM fraction was essentially devoid of other organelles but was enriched approximately 70-fold in phosphodiesterase I, a general plasma membrane marker; the ACPM and BCPM were appropriately enriched in the respective apical and basolateral markers. TEM of ACPM and BCPM revealed homogeneous preparations of vesiculated membranes without contamination by other organelles. Lipid analysis, one- and two-dimensional gel electrophoresis, CFTR immunoblots, and anisotropy measurements showed unequivocal differences in lipid and protein composition and in fluidity between the ACPM and BCPM domains. This methodology provides an initial attempt to develop highly purified apical and basolateral membrane domains from cholangiocytes, which provides a starting point for further studies.