Polarized expression of cyclic amp-activated chloride channels in
isolated epithelial cells.
Torres, Rub[acute]en J., Guillermo A. Altenberg, Jonathan A. Cohn, and
Luis Reuss.
Department of Physiology and Biophysics, University of Texas
Medical Branch, Galveston, Texas 77555-0641, and Department of
Medicine, Duke University and Veterans Administration Medical
Centers, Durham, North Carolina 27710
APStracts 3:0208C, 1996.
We have described a preparation of Necturus gallbladder (NGB)
epithelium yielding isolated cells that retain structural and
functional polarity ("figure-eight" cells). These cells have
a normal membrane voltage and remain polarized for several hours
following isolation. Apical- and basolateral-membrane domains are
differentially labeled with hydrophobic fluorescent dyes; freeze
-fracture electron microscopy reveals two distinct membrane domains
separated by tight junctions; ZO-1, NHE3 and Na+,K+-ATPase are
present in the junctional, apical and basolateral region,
respectively; and cell-attached patch-clamp experiments reveal
different K+ currents in the two membrane domains (Torres et al., Am.
J. Physiol. (Cell), in press). Here, we show that NGB epithelial
cells express a protein cross-reactive with an antibody against human
cystic fibrosis transmembrane conductance regulator (CFTR). In
figure-eight cells, immunoreactivity was restricted to the apical
-membrane domain. Using intracellular microelectrodes and a novel
method of regional superfusion, we found that control cells have high
K+ conductances in both membranes, and a small basolateral Cl-
conductance, similar to findings in the epithelium. Activation of
adenylate cyclase with forskolin elicited a large apical-membrane Cl-
conductance and membrane depolarization. Whole-cell patch-clamp
studies yielded a forskolin-activated linear Cl- current, with high
Cl-/aspartate selectivity. In conclusion, a) figure-eight cells
maintain the conductive membrane properties present in the
epithelium, including polarized expression of cAMP-activated Cl-
channels, and b) the cAMP-activated Cl- conductance is underlied by a
CFTR homologue.
Received 16 January 1996; accepted in final form 6 June 1996.
APS Manuscript Number C23-6.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 4 July 96