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J. Biol. Chem., Vol. 275, Issue 39, 30176-30185, September 29, 2000
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From the Laboratory of Epithelial Cell Biology, Renal-Electrolyte
Division, Department of Medicine, University of Pittsburgh,
Pittsburgh, Pennsylvania 15261
To define aspects of lipid composition and
bilayer asymmetry critical to barrier function, we examined the
permeabilities of liposomes that model individual leaflets of the
apical membrane of a barrier epithelium, Madin-Darby canine kidney type
1 cells. Using published lipid compositions we prepared exofacial
liposomes containing phosphatidylcholine, sphingomyelin,
glycosphingolipids, and cholesterol; and cytoplasmic liposomes
containing phosphatidylethanolamine, phosphatidylserine, and
cholesterol. The osmotic permeability of cytoplasmic liposomes to water
(Pf), solutes, and NH3 was
18-90-fold higher than for the exofacial liposomes
(Pf(ex) = 2.4 ± 0.4 × 10
Reconstituting the Barrier Properties of a Water-tight Epithelial
Membrane by Design of Leaflet-specific Liposomes*,
4 cm/s, Pf(cy) = 4.4 ± 0.3 × 103 cm/s;
Pglycerol(ex) = 2.5 ± 0.3 × 108 cm/s,
Pglycerol(cy) = 2.2 ± 0.02 × 106 cm/s; PNH3(ex) = 0.13 ± 0.4 × 104 cm/s,
PNH3(cy) = 7.9 ± 1.0 × 103
cm/s). By contrast, the apparent proton permeability of exofacial liposomes was 4-fold higher than cytoplasmic liposomes
(PH+(ex) = 1.1 ± 0.1 × 102 cm/s, PH+(cy) = 2.7 ± 0.6 × 103 cm/s). By adding
single leaflet permeabilities, we calculated a theoretical
Pf for a Madin-Darby canine kidney apical
membrane of 4.6 × 104 cm/s, which
compares favorably with experimentally determined values. In exofacial
liposomes lacking glycosphingolipids or sphingomyelin, permeabilities
were 2-7-fold higher, indicating that both species play a role in
barrier function. Removal of cholesterol resulted in 40-280-fold
increases in permeability. We conclude: 1) that we have reconstituted
the biophysical properties of a barrier membrane, 2) that the barrier
resides in the exofacial leaflet, 3) that both sphingomyelin and
glycosphingolipids play a role in reducing membrane permeability but
that there is an absolute requirement for cholesterol to mediate this
effect, 4) that these results further validate the hypothesis that each
leaflet offers an independent resistance to permeation, and 5) that
proton permeation was enhanced by sphingolipid/cholesterol interactions.
*
This work was supported by a research fellowship from the
National Kidney Foundation and by National Institutes of Health Grant
DK43955.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The on-line version of this article (available at
http://www.jbc.org) contains a table with the complete acyl chain
composition of our artificial membranes.
To whom correspondence should be addressed: Laboratory of
Epithelial Cell Biology, Renal-Electrolyte Div., 1218 Scaife Hall, 3550 Terrace St., University of Pittsburgh, Pittsburgh, PA 15261. Tel.:
412-648-9636; Fax: 412-648-2117; E-mail:
zeidel@msx.dept-med.pitt.edu.
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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