AJP - Cell Physiology, Vol 259, Issue 2 C358-C364, Copyright © 1990 by American Physiological Society
A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells
C. J. Venglarik, R. J. Bridges and R. A. Frizzell
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
We developed a convenient flux assay that permits simultaneous measurement
of Cl and K conductance pathways in Cl-secreting epithelial cells.
Monolayers of the colonic tumor cell line T84 were preloaded with 125I and
86Rb, and isotope effluxes were monitored by a sample-replace procedure.
The adenosine 3',5'-cyclic monophosphate (cAMP)-mediated agonists forskolin
and prostaglandin E2 increased I efflux with little effect on Rb efflux,
whereas the Ca-mediated agonists ionomycin, A23187, and carbachol increased
both I and Rb effluxes. Simultaneous determinations of I and Cl or Rb and K
effluxes indicated that I and Rb provide good measures of the effluxes of
Cl and K, respectively. Forskolin- and ionomycin-stimulated I effluxes were
inhibited by the Cl-channel blockers diphenylamine-2-dicarboxylate (DPC),
5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB), and
2-[cyclopentyl-6,7-dichloro-2,3-dihydro-2-methyl-1-oxo-1H-
inden-5-yl)oxy]acetic acid (IAA-94) and by high external K. The Rb efflux
evoked by ionomycin was inhibited by the K-channel blockers Ba and
charybdotoxin. These findings suggest that I and Rb effluxes provide
qualitative estimates of agonist-stimulated Cl and K conductance pathways.
Thus this method can provide a simple and relatively inexpensive screening
assay for Cl and K conductances in cultured cells to assess the effects of
agonist, blockers, or genetic manipulations.
