A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells.
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.
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
C J
CJ
Venglarik
R J
RJ
Bridges
R A
RA
Frizzell
D001464:Barium / Q000494:pharmacology; D002034:Bumetanide / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D018999:Charybdotoxin; D018118:Chloride Channels; D002712:Chlorides / Q000502:physiology*; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms; D015232:Dinoprostone / Q000494:pharmacology; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D006801:Humans; D007454:Iodides / Q000378:metabolism; D007457:Iodine Radioisotopes; D007473:Ion Channels / Q000187:drug effects / Q000502:physiology*; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D008565:Membrane Proteins / Q000502:physiology*; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D011865:Radioisotope Dilution Technique; D012413:Rubidium / Q000378:metabolism; D012414:Rubidium Radioisotopes; D012604:Scorpion Venoms / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000502:physiology*; D062367:ortho-Aminobenzoates / Q000494:pharmacology
D018118:Chloride Channels; D002712:Chlorides; D007454:Iodides; D007457:Iodine Radioisotopes; D007473:Ion Channels; D008565:Membrane Proteins; D015221:Potassium Channels; D012414:Rubidium Radioisotopes; D012604:Scorpion Venoms; D062367:ortho-Aminobenzoates; D002034:Bumetanide; D018999:Charybdotoxin; D005576:Colforsin; D001464:Barium; D008353:Mannitol; D015759:Ionomycin; D002217:Carbachol; C001209:fenamic acid; D012964:Sodium; D015232:Dinoprostone; D012413:Rubidium