Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells

• Published in: The Journal of membrane biology 1995-05, Vol.145 (2), p.129-141
• Main Authors: Fine, D M, Lo, C F, Aguillar, L, Blackmon, D L, Montrose, M H
• Format: Article
• Language: English
• Subjects: Anions - metabolism; Biological Transport; Bumetanide - pharmacology; Carrier Proteins - antagonists & inhibitors; Carrier Proteins - metabolism; Cations - metabolism; Cell Membrane Permeability - drug effects; Cell Polarity; Cell Size; Chloride Channels - drug effects; Chloride Channels - metabolism; Chlorides - metabolism; Colforsin - pharmacology; Colonic Neoplasms - pathology; Cyclic AMP - antagonists & inhibitors; Cyclic AMP - physiology; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Electrophysiology; Furosemide - pharmacology; Gluconates - pharmacology; Humans; Intestinal Mucosa - drug effects; Intestinal Mucosa - metabolism; Intracellular Fluid - metabolism; Ionomycin - metabolism; Nitrates - pharmacology; Organ Specificity; Quinolinium Compounds - metabolism; Sodium-Potassium-Chloride Symporters; Tumor Cells, Cultured; Valinomycin - pharmacology
• ISSN: 0022-2631; 1432-1424
• DOI: 10.1007/BF00237371

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.