Apical membrane permeability of mdck cells.
Rivers, Rickey L., James A. McAteer, Jeffrey L. Clendenon, Bret A.
Connors, Andrew P. Evan, James C. Williams, Jr.
Department of Cell Biology and Anatomy, Medical University of South
Carolina, Charleston, SC 29425-2204 and Department of Anatomy,
Indiana University School of Medicine, Indianapolis, IN 46202
-5120
APStracts 3:0133C, 1996.
The osmotic water permeability (Pf) and permeability to non
-electrolytes were determined for the apical membrane of clonal strain
MDCK C-12 cells cultured as cysts with the apical membrane facing the
surrounding medium. Pf and solute permeabilities were calculated from
the rate of volume change of cysts by digitizing images at 1s
intervals following instantaneous osmotic challenge. Image
measurement was fully automated using a program that separated the
image of the cyst from the background by using adaptive intensity
thresholding and shape analysis. Pf, calculated by curve fitting to
the volume loss data, averaged 2.4 +/- 0.1[mu]m/s, and was increased
by addition of amphotericin B. The energy of activation for Pf was
high (16.3 kcal/mole), and forskolin (50?[mu]M) had no effect on Pf .
Two populations of MDCK cysts were studied: those with 2-3 cells, and
those that appeared to be composed of only one cell. The Pf of multi
-cell cysts was the same as single-cell cysts suggesting that
paracellular water flow is not significant. Solute permeability was
measured using paired osmotic challenges (sucrose and test solute) on
the same cyst. Urea permeability was not different from zero, while
the permeabilities of acetamide and formamide were consistent with
their relative oil-water partition coefficients. Our data are similar
to values from studies on the permeability properties of vesicles of
water-tight epithelial apical membrane. The combination of the unique
model of MDCK apical-out cysts and fully automated data analysis
enabled determination of apical membrane permeability in intact
epithelial cells with high precision.
Received 3 April 1995; accepted in final form 5 April 1996.
APS Manuscript Number C191-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 8 May 96