TY - JOUR
T1 - Isolation and Characterization of Rat Cholangiocyte Vesicles Enriched in Apical or Basolateral Plasma Membrane Domains
AU - Tietz, Pamela S.
AU - Holman, Ralph T.
AU - Miller, Laurence J.
AU - LaRusso, Nicholas F.
N1 - Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 1995/11
Y1 - 1995/11
N2 - 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.
AB - 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.
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U2 - 10.1021/bi00047a007
DO - 10.1021/bi00047a007
M3 - Article
C2 - 7492544
AN - SCOPUS:0028835048
SN - 0006-2960
VL - 34
SP - 15436
EP - 15443
JO - Biochemistry
JF - Biochemistry
IS - 47
ER -