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The Journal of Membrane Biology

, Volume 113, Issue 3, pp 193–202 | Cite as

Chloride conductive and cotransport mechanisms in cultures of canine tracheal epithelial cells measured by an entrapped fluorescent indicator

  • A. C. Chao
  • J. H. Widdicombe
  • A. S. Verkman
Articles

Summary

To study Cl conductive and cotransport mechanisms, primary cultures of canine tracheal cells were grown to confluency on thin glass cover slips and on porous filters. Transepithelial resistance was >100 ω·cm2, and short circuit current (Isc=2–20 μA/cm2), representing active secretion of Cl, increased >threefold with addition of 10 μm isoproterenol to the serosal solution. Cells made transiently permeable in hypotonic solution were loaded with the Cl-sensitive fluorophore 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ) (5mm, 4 min, 150 mOsm). The electrical properties of the cell monolayers were not altered by the loading procedure. Intracellular SPQ fluorescence was monitored continuously by epifluorescence microscopy (excitation 360±5 nm, emission>410 nm). SPQ leakage from the cells was <10% in 60 min at 37°C. Intracellular calibration of SPQ fluorescencevs. [Cl] (0–90mm) was carried out using high-K buffers containing the ionophores nigericin (5 μm) and tributyltin (10 μm); SPQ fluorescence was quenched with a Stern-Volmer constant of 13m−1. Intracellular Cl activity was 43±4mm. Cl flux was measured in response to addition and removal of 114mm Cl from the bathing solution. Addition of 10 μm isoproterenol increased Cl efflux from 0.10 to 0.27mm/sec. The increase was inhibited by the Cl-channel blocker diphenylamine-2-carboxylic acid (1mm). In the absence of isoproterenol, removal of external Na or addition of 0.5mm furosemide, reduced Cl influx by >fourfold. In ouabain-treated monolayers, removal of external K in the presence of 5mm barium diminished Cl influx by >twofold, suggesting that Cl entry is in part K dependent. These results establish an accurate optical method for the realtime measurement of intracellular Cl activity in tracheal cells that does not require an electrically tight cell monolayer. The data demonstrate the presence of an isoproterenol-regulated Cl channel and a furosemide-sensitive cation-coupled transport mechanism.

Key Words

fluorescence SPQ cell culture chloride transport trachea isoproterenol furosemide Na−K−Cl transport 

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Copyright information

© Springer-Verlag New York Inc. 1990

Authors and Affiliations

  • A. C. Chao
    • 2
  • J. H. Widdicombe
    • 1
  • A. S. Verkman
    • 2
  1. 1.Departments of Medicine and Physiology Cardiovascular Research InstituteUniversity of CaliforniaSan Francisco
  2. 2.Departments of Medicine and Physiology Cystic Fibrosis Research CenterUniversity of CaliforniaSan Francisco

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