title: Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells.
abstract: Cyclic AMP-activated chloride fluxes have been analyzed in HT29-18-C1 cells (a clonal cell line derived from a human colon carcinoma) using measurements of cell volume (electronic cell sizing), cell chloride content (chloride titrator) and intracellular chloride activity (6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ). HT29-18-C1 was shown to mediate polarized chloride transport. In unstimulated cells, the apical membrane was impermeable to chloride and net chloride flux was mediated by basolateral furosemide-sensitive transport. Forskolin (10 microM) increased furosemide-insensitive chloride permeability of the apical membrane, and decreased steady-state intracellular chloride concentration approximately 9%. Cellular chloride depletion (substitution of medium chloride by nitrate or gluconate), caused greater than fourfold reduction in cellular chloride concentration. When chloride-depleted cells were returned to normal medium, cells regained chloride and osmolytes via bumetanide-sensitive transport, but forskolin did not stimulate bumetanide-insensitive chloride uptake. The inhibition of cAMP-activated chloride reuptake was not explained by limiting cation conductance, cell shrinkage, choice of substitute anion, or decreased generation of cAMP in chloride-depleted cells. When cells with normal chloride content were depolarized (135 mM medium potassium + 10 microM valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl- approximately Br- > NO3- > I-. The electrogenic transport pathway was inhibited in chloride-depleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.
authors.affiliation: Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Fine
authors.forename: C F
authors.initials: CF
authors.lastname: Lo
authors.forename: L
authors.initials: L
authors.lastname: Aguillar
authors.forename: D L
authors.initials: DL
authors.lastname: Blackmon
authors.forename: M H
authors.initials: MH
authors.lastname: Montrose
mesh_terms: D000838:Anions / Q000378:metabolism; D001692:Biological Transport; D002034:Bumetanide / Q000494:pharmacology; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002412:Cations / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D016764:Cell Polarity; D048429:Cell Size; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000473:pathology; D000242:Cyclic AMP / Q000037:antagonists & inhibitors / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism; D004594:Electrophysiology; D005665:Furosemide / Q000494:pharmacology; D005942:Gluconates / Q000494:pharmacology; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D007424:Intracellular Fluid / Q000378:metabolism; D015759:Ionomycin / Q000378:metabolism; D009566:Nitrates / Q000494:pharmacology; D009928:Organ Specificity; D011806:Quinolinium Compounds / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters; D014407:Tumor Cells, Cultured; D014634:Valinomycin / Q000494:pharmacology
chemical_list: D000838:Anions; C505032:CFTR protein, human; D002352:Carrier Proteins; D002412:Cations; D018118:Chloride Channels; D002712:Chlorides; D005942:Gluconates; D009566:Nitrates; D011806:Quinolinium Compounds; D028021:Sodium-Potassium-Chloride Symporters; D002034:Bumetanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D014634:Valinomycin; D015759:Ionomycin; D005665:Furosemide; C052335:6-methoxy-N-(3-sulfopropyl)quinolinium; D000242:Cyclic AMP; C030691:gluconic acid