Preservation of structural and functional polarity in isolated
epithelial cells.
Torres, Rub[acute]en J., Guillermo A. Altenberg, Julio A. Copello,
Guido Zampighi, and Luis Reuss.
Department of Physiology and Biophysics, University of Texas
Medical Branch, Galveston, Texas; Department of Neurobiology,
University of California, Los Angeles, California
APStracts 2:0452C, 1995.
We describe a method to isolate epithelial cells from gallbladders of
Necturus maculosus with preserved structural and functional polarity.
Isolation was carried out with a mixture of collagenase and protease,
with only a brief exposure to a divalent-cation-free medium. About
40% of the isolated epithelial cells had a "figure-eight"
shape and retained metabolic and cell-membrane integrity. Figure
-eight cells display features consistent with preserved polarity for
several hours, including: a) the "apical" and
"basolateral" membrane domains were differentially labelled
by a hydrophobic fluorescent dye, b) freeze-fracture electron
microscopy verified two plasma-membrane domains differing in the
presence of microvilli and folds, and separated by tight junctions,
c) proteins such as ZO-1, NHE3 and Na+,K+-ATPase, remained localized
in the junctional, apical and basolateral region, respectively, d)
following apical-surface exposure to wheat-germ agglutinin, the label
remained in the apical membrane after cell isolation, and e) patch
-clamp experiments demonstrated polarized expression of K+ channels.
Polarity was rapidly lost following removal of extracellular Ca2+,
exposure to trypsin, or ATP depletion. Therefore, this preparation
allows for structural and functional studies of epithelial transport
in single cells retaining the essential features present in the
assembled epithelium.
Received 14 August 1995; accepted in final form 12 December 1995.
APS Manuscript Number C502-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 23 December 95