A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells

Journal Article · Wed Aug 01 04:00:00 EDT 1990 · American Journal of Physiology; (USA)

OSTI ID:6532362

Venglarik, C J; Bridges, R J; Frizzell, R A ^[1]

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1. Univ. of Alabama, Birmingham (USA)

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.

• OSTI ID: 6532362
• Journal Information: American Journal of Physiology; (USA), Journal Name: American Journal of Physiology; (USA) Vol. 259:2 Pt 1; ISSN 0002-9513; ISSN AJPHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALKALI METAL COMPOUNDS
ALKALI METAL ISOTOPES
AMP
ANIMAL CELLS
ANIMAL TISSUES
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOASSAY
BIOLOGICAL PATHWAYS
BODY
CHLORINE COMPOUNDS
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ELECTRON CAPTURE RADIOISOTOPES
EPITHELIUM
GASTROINTESTINAL TRACT
HALOGEN COMPOUNDS
INTERMEDIATE MASS NUCLEI
INTESTINES
IODINE 125
IODINE ISOTOPES
ISOMERIC TRANSITION ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
LARGE INTESTINE
MAMMALS
MAN
MEMBRANE PROTEINS
MEMBRANE TRANSPORT
MINUTES LIVING RADIOISOTOPES
NUCLEI
NUCLEOTIDES
ODD-EVEN NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANS
PORINS
POTASSIUM COMPOUNDS
PRIMATES
PROSTAGLANDINS
PROTEINS
RADIOISOTOPES
RUBIDIUM 86
RUBIDIUM ISOTOPES
TISSUES
TRACER TECHNIQUES
TUMOR CELLS
VERTEBRATES