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

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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 mm medium potassium + 10 μm valinomycin), cAMP activated electrogenic chloride uptake permselective for Cl−≈Br−>NO −3 >I−. The electrogenic transport pathway was inhibited in chloridedepleted cells. Results suggest that chloride depletion limits activation of electrogenic chloride flux.

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References

  1. Anderson, M.P., Berger, H.A., Rich, D.P., Gregory, R.J., Smith, A.E., Welsh, M.J. 1991. Nucleoside triphosphates are required to open the CFTR chloride channel. Cell 67:775–784

    Google Scholar 

  2. Anderson, M.P., Gregory, R.J., Thompson, S., Souza, D.W., Paul, S., Mulligan, R.C., Smith, A.E., Welsh, M.J. 1991. Demonstration that the CFTR is a chloride channel by alteration of its anion selectivity. Science 253:202–205

    CAS  PubMed  Google Scholar 

  3. Anderson, M.P., Sheppard, D.N., Berger, H.A., Welsh, M.J. 1992. Chloride channels in the apical membrane of normal and cystic fibrosis airway and intestinal epithelia. Am. J. Physiol. 263:L1-L14

    Google Scholar 

  4. Anderson, M.P., Welsh, M.J. 1990. Fatty acids inhibit apical membrane chloride channels in airway epithelia. Proc. Natl. Acad. Sci. 87:7334–7338

    Google Scholar 

  5. Anderson, M.P., Welsh, M.J. 1991. Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia. Proc. Natl. Acad. Sci. USA 88:6003–6007

    Google Scholar 

  6. Bajnath, R.B., Groot, J.A., de Jonge, H.R., Kansen, M., Bijman, J. 1993. Synergistic activation of nonrectifying small conductance chloride channels by forskolin and phorbol esters in cell-attached patches of the human colon carcinoma cell line HT-29cl,19A. Pfluegers Arch. 425:100–108

    Google Scholar 

  7. Bear, C.E., Li, C., Kartner, N., Bridges, R., Jensen, T., Ramjeesingh, M., Riordan, J.R. 1992. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell 68:809–818

    Article  CAS  PubMed  Google Scholar 

  8. Bear, C.E., Reyes, E.F. 1992. cAMP activated chloride conductance in the colonic cell line, Caco-2. Am. J. Physiol. 262:C251-C256

    Google Scholar 

  9. Berger, H.A., Travis, S.M., Welsh, M.J. 1993. Regulation of the cystic fibrosis transmembrane conductance regulatory Cl− channel by specific protein kinases and protein phosphatases. J. Biol. Chem. 268:2037–2047

    Google Scholar 

  10. Berschneider, H.M., Knowles, M.R., Asiskhan, R.G., Boucher, R.C., Tobey, N.A., Orlando, R.C., Powell, D.W. 1988. Altered intestinal chloride transport in cystic fibrosis. FASEB J. 2:2625–2629

    Google Scholar 

  11. Bradbury, N.A., Bridges, R.J. 1994. Role of membrane trafficking in plasma membrane solute transport. Am. J. Physiol. 267:C1-C24

    Google Scholar 

  12. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254

    Article  CAS  PubMed  Google Scholar 

  13. Cliff, W.H., Frizzell, R.A. 1990. Separate Cl−1 conductances activated by cAMP and Ca2+ in Cl−-secreting epithelial cells. Proc. Natl. Acad. Sci. USA 87:4956–4960

    Google Scholar 

  14. Dechecchi, M.C., Tamanini, A., Berton, G., Cabrini, G. 1993. Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene. J. Biol. Chem. 268(15):11321–11325

    Google Scholar 

  15. Donowitz, M., Welsh, M.J. 1987. Regulation of mammalian small intestinal electrolyte secretion. In: Physiology of the Gastrointestinal tract. 2nd edition. L.R. Johnson, editor, pp. 1351–1388, Raven, New York

    Google Scholar 

  16. Egan, M., Flotte, T., Aflone, S., Solow, R., Zeitlin, P.L., Carter, B.J., Guggino, W.B. 1992. Defective regulation of outwardly rectifying Cl− channels by protein kinase A corrected by insertion of CFTR. Nature 358:581–584

    Google Scholar 

  17. Fittschen, C. Henson, P.M. 1994. Linkage of Azurophil granule secretion in neutrophils to chloride ion transport and endosomal transcytosis. J. Clin. Invest. 93:247–255

    Google Scholar 

  18. Gabriel, S.E., Clarke, L.L., Boucher, R.C., Stutts, M.J. 1993. CFTR and outward rectifying chloride channels are distinct proteins with a regulatory relationship. Nature 363:263–268

    Google Scholar 

  19. Hardcastle, J., Hardcastle, P.T., Taylor, C.J., Goldhill, J. 1991. Failure of cholinergic stimulation to induce a secretory response from the rectal mucosa in cystic fibrosis. Gut 32:1035–1039

    Google Scholar 

  20. Huet, C., Sahuquillo-Marino, C., Coudrier, E., Louvard, D. 1987. Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT29) provide new models for cell polarity and terminal differentiation. J. Cell Biol. 105:345–357

    Google Scholar 

  21. Illsley, N.P., Verkman, A.S. 1987. Membrane chloride transport measured using a chloride-sensitive fluorescent probe. Biochemistry 26:1215–1219

    Google Scholar 

  22. Jefferson, D.M., Valentich, J.D., Marini, F.C., Grubman, S.A., Iannuzzi, M.C., Dorkin, H.L., Li, M., Klinger, K.W., Welsh, M.J. 1990. Expression of normal and cystic fibrosis phenotypes by continuous airway epithelial cell lines. Am. J. Physiol. 259:L496-L505

    Google Scholar 

  23. Kim, H.D., Tsai, Y.-S., Franklin, C.C., Turner, J.T. 1988. Characterization of Na+/K+/2Cl− cotransport in cultured HT29 human colonic adenocarcinoma cells. Biochim. Biophys. Acta 946:397–404

    Google Scholar 

  24. Kunzelmann, K., Tilmann, M., Hansen, Ch.P., Greger, R. 1991. Inhibition of epithelial chloride channels by cytosol. Pfleugers Arch. 418:479–490

    Google Scholar 

  25. Li, M., McCann, J.D., Welsh, M.J. 1990. Apical membrane Cl− channels in airway epithelia: anion selectivity and effect of an inhibitor. Am. J. Physiol. 259:C295-C301

    Google Scholar 

  26. Liedtke, C.M. 1989. Alpha-adrenergic regulation of Na-Cl cotransport in human airway epithelium. Am. J. Physiol. 257:L125-L129

    Google Scholar 

  27. Lytle, C., Forbush III, B. 1992. The Na-K-Cl cotransporter protein of shark rectal gland. II. regulation by direct phosphorylation. J. Biol. Chem. 267:25438–25443

    Google Scholar 

  28. Matthews, J.B., Smith, J.A., Tally, K.J., Awtrey, C.S., Nguyen, H., Rich, J., Madara, J.L. 1994. Na-K-2Cl cotransport in intestinal epithelial cells: influence of chloride efflux and F-actin on regulation of cotransporter activity and bumetanide binding. J. Biol. Chem. 269:15703–15709

    Google Scholar 

  29. Montrose, M.H., Friedrich, T., Murer, H. 1987. Measurements of intracellular pH in single LLC-PK1 cells: recovery from an acid load via basolateral Na+/H+ exchange. J. Membrane Biol. 97:63–78

    Google Scholar 

  30. Montrose-Rafizadeh, C., Blackmon, D.L., Hamosh, A., Oliva, M.M., Hawkins, A.L., Curristin, S.M., Griffin, C.A., Yang, V.W., Guggino, W.B., Cutting, G.R., Montrose, M.H. 1992. Regulation of CFTR gene transcription and alternative RNA splicing in a model of developing intestinal epithelium. J. Biol. Chem. 267:19299–19305

    Google Scholar 

  31. Montrose-Rafizadeh, C., Guggino, W.B., Montrose, M.H. 1991. Cellular differentiation regulates expression of Cl− transport and cystic fibrosis transmembrane conductance regulator mRNA in human intestinal cells. J. Biol. Chem. 266:4495–4499

    Google Scholar 

  32. Morris, A.P., Cunningham, S.A., Benos, D.J., Frizzell, R.A. 1992. Cellular differentiation is required for cAMP but not Ca2+-dependent Cl− secretion in colonic epithelial cells expressing high levels of cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 267:5575–5583

    Google Scholar 

  33. Morris, A.P., Cunningham, S.A., Tousson, A., Benos, D.J., Frizzell, R.A. 1994. Polarization-dependent apical membrane CFTR targetting underlies cAMP-dependent Cl− secretion in epithelial cells. Am. J. Physiol. 266:C254-C268

    Google Scholar 

  34. Morris, A.P., Frizzell, R.A. 1993. Ca2+-dependent Cl− channels in undifferentiated human colonic cells (HT-29). II. regulation and rundown. Am. J. Physiol. 264:C977-C985

    Google Scholar 

  35. O'Loughlin, E.V., Hunt, D.M., Gaskin, K.J., Stiel, D., Bruzuszcak, I.M., Martin, H.C., Bambach, C., Smith, R. 1991. Abnormal epithelial transport in cystic fibrosis jejunum. Am. J. Physiol. 260:G758-G763

    Google Scholar 

  36. Prince, L.S., Workman, R.B. Jr., Marchase, R.B. 1994. Rapid endocytosis of the cystic fibrosis transmembrane conductance regulator chloride channel. Proc. Natl. Acad. Sci. 91:5192–5196

    Google Scholar 

  37. Robertson, M.A., Foskett, J.K. 1994. Na transport pathways in secretory acinar cells: membrane cross talk mediated by [Cl]i. Am. J. Physiol. 267:C146-C156

    Google Scholar 

  38. Rowe, W.A., Blackmon, D.L., Montrose, M.H. 1993. Propionate activates multiple ion transport mechanism in the HT29–18-C1 human colon cell line. Am. J. Physiol. 265:G564-G571

    Google Scholar 

  39. Rowe, W.A., Lesho, M.J., Montrose, M.H. 1994. Polarized Na/H exchange function is pliable in response to transepithelial gradients of propionate. Proc. Natl. Acad. Sci. 91:6166–6170

    Google Scholar 

  40. Solc, C.K., Wine, J.J. 1991. Swelling-induced and depolarizationinduced Cl− channels in normal and cystic fibrosis epithelial cells. Am. J. Physiol. 261:C658-C674

    Google Scholar 

  41. Thiemann, A., Grander, S., Pusch, M., Jentsch, T.J. 1992. A chloride channel widely expressed in epithelial and nonepithelial cells. Nature 356:57–60.

    Google Scholar 

  42. Tilly, B.C., Kansen, M., Van Gageldonk, P.G., van den Berghe, N., Galjaard, H., Bijman, J., deJonge, H.R. 1991. G-proteins mediate intestinal chloride channel activation. J. Biol. Chem. 266:2036–2040

    Google Scholar 

  43. Wagoner, K., Pallotta, B.S. 1988. Modulation of acetylcholine receptor desensitization by forskolin is independent of cAMP. Science 240:1655–1657

    Google Scholar 

  44. Wang, X., Marunaka, Y., Fedorko, L., Dho, S., Foskett, J.K., O'Brodovich, H. 1993. Activation of Cl− currents by intracellular chloride in fibroblasts stably expressing the human cystic fibrosis transmembrane conductance regulator. Can. J. Physiol. Pharmacol. 71:645–649

    Google Scholar 

  45. Willumsen, N.J., Boucher, R.C. 1989. Activation of an apical Cl− conductance by calcium ionophores in cystic fibrosis airway epithelia. Am. J. Physiol. 256:C226-C233

    Google Scholar 

  46. Winters, C.J., Reeves, W.B., Andreoli, T.E. 1990. Cl− channels in basolateral renal medullary membranes: Determinants of singlechannel activity. J. Membrane Biol. 118:269–278

    Google Scholar 

  47. Worrell, R.T., Butt, A.G., Cliff, W.H., Frizzell, R.A. 1989. A volume-sensitive chloride conductance in human colonic cell line T84. Am. J. Physiol. 256:C1111-C1119

    Google Scholar 

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Authors and Affiliations

  1. Departments of Medicine and Physiology, Johns Hopkins University School of Medicine, 720 Rutland Ave., 21205, Baltimore, MD

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

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  1. D. M. Fine
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  2. C. F. Lo
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  3. L. Aguillar
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  4. D. L. Blackmon
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  5. M. H. Montrose
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Additional information

The technical assistance of Dwight Derr is gratefully acknowledged. We also thank Dr. Chahrzad Montrose-Rafizadeh for help in performance of the chloride efflux experiments. This work was supported by National Institutes of Health grants RO1-DK42457 and PO1-DK44484.

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Fine, D.M., Lo, C.F., Aguillar, L. et al. Cellular chloride depletion inhibits cAMP-activated electrogenic chloride fluxes in HT29-18-C1 cells. J. Membarin Biol. 145, 129–141 (1995). https://doi.org/10.1007/BF00237371

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  • DOI: https://doi.org/10.1007/BF00237371

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