TY - JOUR
T1 - Characterization of apical and basolateral plasma membrane domains derived from cultured rat cholangiocytes
AU - Tietz, Pamela
AU - Levine, Susan
AU - Holman, Ralph
AU - Fretham, Chris
AU - Larusso, Nicholas F.
N1 - Funding Information:
1This work was supported by NIH Grant DK 24031 and by the Mayo Foundation, Rochester, MN.
PY - 1997/12/15
Y1 - 1997/12/15
N2 - Cholangiocytes, the epithelial cells that line intrahepatic bile ducts, are composed of plasma membranes with discrete apical (lumenal) and basolateral domains that contain different channels, transporters, and receptors. In recent work, we developed a long-term, primary culture system of normal rat cholangiocytes (NRC). Our aims here were to prepare and characterize apical and basolateral plasma membrane vesicles from NRC. Using serial isopycnic centrifugation on sucrose gradients, we generated separate apical and basolateral plasma membrane vesicles. We characterized these vesicles by transmission electron microscopy, specific marker enzyme assays, and immunoblotting; we also determined the percentage of sealed vesicles and their intravesicular volume and sidedness using biochemical approaches. Our results showed that vesicles derived from the apical and basolateral plasma membrane domains of NRC were highly purified, predominately sealed, right- side-out vesicles that differed in size and lipid and protein composition. This experimental model represents a novel tool that will be useful for additional functional studies on the channels, transporters, and receptors differentially distributed in the plasma membrane of biliary epithelia.
AB - Cholangiocytes, the epithelial cells that line intrahepatic bile ducts, are composed of plasma membranes with discrete apical (lumenal) and basolateral domains that contain different channels, transporters, and receptors. In recent work, we developed a long-term, primary culture system of normal rat cholangiocytes (NRC). Our aims here were to prepare and characterize apical and basolateral plasma membrane vesicles from NRC. Using serial isopycnic centrifugation on sucrose gradients, we generated separate apical and basolateral plasma membrane vesicles. We characterized these vesicles by transmission electron microscopy, specific marker enzyme assays, and immunoblotting; we also determined the percentage of sealed vesicles and their intravesicular volume and sidedness using biochemical approaches. Our results showed that vesicles derived from the apical and basolateral plasma membrane domains of NRC were highly purified, predominately sealed, right- side-out vesicles that differed in size and lipid and protein composition. This experimental model represents a novel tool that will be useful for additional functional studies on the channels, transporters, and receptors differentially distributed in the plasma membrane of biliary epithelia.
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U2 - 10.1006/abio.1997.2431
DO - 10.1006/abio.1997.2431
M3 - Article
C2 - 9417776
AN - SCOPUS:0031574367
SN - 0003-2697
VL - 254
SP - 192
EP - 199
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -