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 (I sc=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.
Similar content being viewed by others
References
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-C808
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. 56:1071–1081
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-F120
Chen, P.-Y., Verkman, A.S. 1988. Sodium-dependent chloride transport in basolateral membrane vesicles isolated from rabbit proximal tubule.Biochemistry 27:655–660
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-C359
Durand, J., Durand-Arczynska, W., Schoenenweid, F. 1986. Oxygen consumption and active sodium and chloride transport in bovine tracheal epithelium.J. Physiol. (London) 372:51–62
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. 104:233–239
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
Frizzell, R.A. 1987. Cystic fibrosis: A disease of ion channels.TINS 10:190–193
Frizzell, R.A., Rechkemmer, G., Shoemaker, R.L. 1986. Altered regulation of airway epithelial cell chloride channels in cystic fibrosis.Science 233:558–560
Geck, P., Heinz, E. 1986. The Na−K−2Cl cotransport system.J. Membrane Biol. 91:97–105
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. 392:92–94
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 85:5951–5955
Illsley, N.P., Verkman, A.S. 1987. Membrane chloride transport measured using a chloride-sensitive fluorescent indicator.Biochemistry 26:1215–1219
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. 53:955–962
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. 169:142–150
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. 55:1251–1259
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. 81:1–8
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. 70:215–226
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.)
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 28:4240–4244
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-S141
Weinman, S.A., Reuss, L. 1984. Na+−H+ exchange and Na+ entry across the apical membrane ofNecturus gallbladder.J. Gen. Physiol. 83:57–74
Welsh, M.J. 1983a. Evidence for a basolateral membrane potassium conductance in canine tracheal epithelium.Am. J. Physiol. 244:C377-C384
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. 71:1392–1401
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. 74:262–268
Welsh, M.J. 1985. Ion transport by primary cultures of canine tracheal epithelium: Methodology, morphology, and electrophysiology.J. Membrane Biol. 88:149–163
Welsh, M.J. 1986. Adrenergic regulation of ion transport by primary cultures of canine tracheal epithelium: Cellular electrophysiology.J. Membrane Biol. 91:121–128
Welsh, M.J. 1987. Electrolyte transport by airway epithelia.Physiol. Rev. 67:1143–1184
Welsh, M.J., Liedtke, C.M. 1986. Chloride and potassium channels in cystic fibrosis airway epithelia.Nature (London) 322:467–470
Widdicombe, J.H. 1986. Cystic fibrosis and ‘beta’-adrenergic response of airway epithelial cell cultures.Am. J. Physiol. 251:R818-R822
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 Press
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. 247:95–103
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-C396
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chao, A.C., Widdicombe, J.H. & Verkman, A.S. Chloride conductive and cotransport mechanisms in cultures of canine tracheal epithelial cells measured by an entrapped fluorescent indicator. J. Membrain Biol. 113, 193–202 (1990). https://doi.org/10.1007/BF01870071
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01870071