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

D. M. Fine, C. F. Lo, L. Aguillar, D. L. Blackmon, M. H. Montrose

Research output: Contribution to journalArticle

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) (μm) increased furosemideinsensitive 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 bumetanideinsensitive 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 chloridedepleted cells. When cells with normal chloride content were depolarized (135 m m medium potassium + 10 μm valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl-≈Br->NO3->I-. The electrogenic transport pathway was inhibited in chloridedepleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.

Original languageEnglish (US)
Pages (from-to)129-141
Number of pages13
JournalThe Journal of Membrane Biology
Volume145
Issue number2
DOIs
StatePublished - May 1995

Keywords

  • Cell volume
  • Cellular chloride content
  • Cyclic AMP
  • Intestine
  • Secretion

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

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