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


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 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Barthelson, R.A., Jacoby, D.B., Widdicombe, J.H. 1987. Regulation of chloride secretion in dog tracheal epithelium by protein kinase C.Am. J. Physiol. 53:C802-C808Google Scholar
  2. Chao, A.C., Dix, J.A., Sellers, M.C., Verkman, A.S. 1989. Fluorescence measurement of chloride transport in monolayer cultured cells: Mechanisms of chloride transport in fibroblasts.Biophys. J. 56:1071–1081PubMedGoogle Scholar
  3. Chen, P.-Y., Illsley, N.P., Verkman, A.S. 1988. Renal brush border chloride transport mechanisms characterized using a fluorescent indicator.Am. J. Physiol. 254:F114-F120PubMedGoogle Scholar
  4. Chen, P.-Y., Verkman, A.S. 1988. Sodium-dependent chloride transport in basolateral membrane vesicles isolated from rabbit proximal tubule.Biochemistry 27:655–660PubMedGoogle Scholar
  5. Coleman, D.L., Tuet, I.K., Widdicombe, J.H. 1984. Electrical properties of dog tracheal cells grown in monolayer culture.Am. J. Physiol. 246:C355-C359PubMedGoogle Scholar
  6. Durand, J., Durand-Arczynska, W., Schoenenweid, F. 1986. Oxygen consumption and active sodium and chloride transport in bovine tracheal epithelium.J. Physiol. (London) 372:51–62Google Scholar
  7. Fong, P., Illsley, N.P., Widdicombe, J.H., Verkman, A.S. 1988. Chloride transport in apical membrane vesicles from bovine tracheal epithelium: Characterization using a fluorescent indicator.J. Membrane Biol. 104:233–239Google Scholar
  8. Fong, P., Widdicombe, J.H. 1989. Potassium dependence of the basolateral chloride uptake mechanism in primary cultures of canine tracheal epithelium.Biophys. J. 55:606a Google Scholar
  9. Frizzell, R.A. 1987. Cystic fibrosis: A disease of ion channels.TINS 10:190–193Google Scholar
  10. Frizzell, R.A., Rechkemmer, G., Shoemaker, R.L. 1986. Altered regulation of airway epithelial cell chloride channels in cystic fibrosis.Science 233:558–560PubMedGoogle Scholar
  11. Geck, P., Heinz, E. 1986. The Na−K−2Cl cotransport system.J. Membrane Biol. 91:97–105Google Scholar
  12. Greger, R., Schlatter, E. 1981. Presence of luminal K+, a prerequisite for active NaCl transport in the cortical thick ascending limb of Henle's loop of rabbit kidney.Pfluegers Arch. 392:92–94Google Scholar
  13. Gruenert, D.C., Basbaum, B.C., Welsh, M.J., Li, M., Finkbeiner, W.E., Nadel, J.A. 1988. Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.Proc. Natl. Acad. Sci. USA 85:5951–5955PubMedGoogle Scholar
  14. Illsley, N.P., Verkman, A.S. 1987. Membrane chloride transport measured using a chloride-sensitive fluorescent indicator.Biochemistry 26:1215–1219PubMedGoogle Scholar
  15. Krapf, R., Berry, C.A., Verkman, A.S. 1988a. Estimation of intracellular chloride activity in isolated perfused rabbit proximal tubules using a fluorescent probe.Biophys. J. 53:955–962PubMedGoogle Scholar
  16. Krapf, R., Illsley, N.P., Tseng, H.C., Verkman, A.S. 1988b. Structure-activity relationships of chloride-sensitive fluorescent indicators for biological application.Anal. Biochem. 169:142–150PubMedGoogle Scholar
  17. Pearce, D., Verkman, A.S. 1989. NaCl reflection coefficients in proximal tubule apical and basolateral membrane vesicles: Measurement by induced osmosis and solvent drag.Biophys. J. 55:1251–1259PubMedGoogle Scholar
  18. Shorofsky, S.R., Field, M., Fozzard, H.A. 1984. Mechanism of Cl secretion in canine trachea: Changes in intracellular chloride activity with secretion.J. Membrane Biol. 81:1–8Google Scholar
  19. Smith, P.L., Welsh, M.J., Stoff, J.S., Frizzell, R.A. 1982. Chloride secretion by canine tracheal epithelium: I. Role of intracellular cAMP levels.J. Membrane Biol. 70:215–226Google Scholar
  20. Sun, A.M., Hebert, S.C. 1989. ADH alters the K+ requirement for the luminal furosemide-sensitive NaCl symporter in mouse medullary thick limbs (MTAL)Kidney Int. 35:489 (Abstr.)Google Scholar
  21. Verkman, A.S., Takla, R., Sefton, B., Basbaum, C., Widdicombe, J.H. 1989. Fluorescence assay of chloride transport in liposomes reconstituted with chloride transporters.Biochemistry 28:4240–4244PubMedGoogle Scholar
  22. Wangemann, P., Wittner, M., Di Stefano, A., Englert, H.C., Lang, H.J., Schlatter, E., Greger, R. 1986. Cl channel blockers in the thick ascending limb of the loop of Henle. Structure activity relationship.Pfluegers Arch. 407 (Suppl. 2):S128-S141Google Scholar
  23. Weinman, S.A., Reuss, L. 1984. Na+−H+ exchange and Na+ entry across the apical membrane ofNecturus gallbladder.J. Gen. Physiol. 83:57–74PubMedGoogle Scholar
  24. Welsh, M.J. 1983a. Evidence for a basolateral membrane potassium conductance in canine tracheal epithelium.Am. J. Physiol. 244:C377-C384PubMedGoogle Scholar
  25. Welsh, M.J. 1983b. Intracellular chloride activities in canine tracheal epithelium. Direct evidence for sodium-coupled intracellular chloride accumulation in a chloride-secreting epithelium.J. Clin. Invest. 71:1392–1401PubMedGoogle Scholar
  26. Welsh, M.J. 1984. Energetics of chloride secretion in canine tracheal epithelium: Comparison of the metabolic cost of chloride transport with the metabolic cost of sodium transport.J. Clin. Invest. 74:262–268PubMedGoogle Scholar
  27. Welsh, M.J. 1985. Ion transport by primary cultures of canine tracheal epithelium: Methodology, morphology, and electrophysiology.J. Membrane Biol. 88:149–163Google Scholar
  28. Welsh, M.J. 1986. Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: Cellular electrophysiology.J. Membrane Biol. 91:121–128Google Scholar
  29. Welsh, M.J. 1987. Electrolyte transport by airway epithelia.Physiol. Rev. 67:1143–1184Google Scholar
  30. Welsh, M.J., Liedtke, C.M. 1986. Chloride and potassium channels in cystic fibrosis airway epithelia.Nature (London) 322:467–470Google Scholar
  31. Widdicombe, J.H. 1986. Cystic fibrosis and ‘beta’-adrenergic response of airway epithelial cell cultures.Am. J. Physiol. 251:R818-R822PubMedGoogle Scholar
  32. Widdicombe, J.H., Barthelson, R.A. 1988. Altered chloride transport across primary cultures of tracheal epithelium in cystic fibrosis.In: Cellular and Molecular Basis of Cystic Fibrosis. G. Mastella and P. Quinton, editors. pp. 471–478. San Francisco PressGoogle Scholar
  33. Widdicombe, J.H., Coleman, D.L., Finkbeiner, W.E., Friend, D.S. 1987. Primary cultures of the dog's tracheal epithelium: Fine structure, fluid and electrolyte transport.Cell Tissue Res. 247:95–103PubMedGoogle Scholar
  34. Widdicombe, J.H., Nathanson, I.T., Highland, E. 1983. Effects of “loop” diuretics on ion transport by dog tracheal epithelium.Am. J. Physiol. 245:C388-C396PubMedGoogle Scholar

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

Personalised recommendations