Skip to main content
SpringerLink
Log in

The outwardly rectifying Cl− channel is not involved in cAMP-mediated Cl− secretion in HT-29 cells: evidence for a very-low-conductance Cl− channel

  • Published:
Pflügers Archiv Aims and scope Submit manuscript

Abstract

The patch-clamp technique and transepithelial current measurements in conjunction with analysis of transepithelial current noise were employed in order to clarify the role of the outwardly rectifying, depolarization-induced Cl− channel (ORDIC) during cAMP-mediated Cl− secretion in HT-29/B6 cells. Confluent monolayers growing on permeable supports were used in order to ensure the apical location of measured Cl− channels. The ORDIC needed to be activated by excision and/or depolarization, and was found in both cAMP-stimulated and non-stimulated cells. Both 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and 4,4′-dinitro-2,2′-stilbenedisulphonate (DNDS) induced fast flickery-type blocks of the ORDIC at low, micromolar blocker concentrations and were used as a probe for ORDIC. However, these substances were ineffective in blocking transepithelial forskolin-induced Cl− secretion of monolayers in Ussing chambers. No inhibitory effect at all was detected for DNDS up to 1 mmol/l. NPPB blocked the ORDIC at low concentrations (IC50=0.5±0.3 μmol/l) by reducing its open probability, but NPPB did not block forskolin-induced Cl− secretion unless high concentrations were used (IC50=240±10 μmol/l). In order to exclude effects of NPPB other than on the apical Cl− channel, trans-epithelial measurements were performed in basolaterally amphotericin-permeabilized, forskolin-stimulated preparations, and a serosal-to-mucosal Cl− gradient was applied as a driving force. Under these conditions, NPPB's inhibitory effects were also very small. Noise analysis of this gradient-driven Cl− current showed a very-low-frequency Lorentzian noise component (f c=1.4±0.2 Hz), which was not compatible with Lorentzians predicted from single-channel gating of ORDIC. As revealed from fura-2 fluorescence measurements, forskolin-stimulated Cl− secretion occurred in the absence of changes in intracellular Ca2+. Thus, we conclude that there is an apical Cl− channel in HT-29/B6 that is activated through the cAMP-mediated pathway and is insensitive to NPPB and DNDS, and the kinetics of which are incompatible with ORDIC kinetics. Therefore, despite its prevalence in isolated patches and even in cell-attached recordings, the ORDIC appears not to be involved in cAMP-mediated Cl− secretion by HT-29/B6 cells. From noise analysis, a very-small-conductance (probably below 1 pS), slow-gating Cl− channel was calculated as the conductive site in the apical membrane during forskolin stimulation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Breuer W, Skorecki KL (1989) Inhibition of PGE2 synthesis by a blocker of epithelial chloride channels. Biochem Biophys Res Commun 163:398–405

    Google Scholar 

  2. Bridges RJ, Worrell RT, Frizzell RA, Benos DJ (1989) Stilbene disulfonate blockade of colonic secretory Cl− channels in planar lipid bilayers. Am J Physiol 256:C902–912

    Google Scholar 

  3. Colquhoun D, Sigworth FJ (1983) Fitting and statistical analysis of single channel records. In: Sakmann B and Neher E (eds) Single channel recording. Plenum, New York, pp 191–263

    Google Scholar 

  4. Cunningham SW, Worrell RT, Benos DJ, Frizzell RA (1992) cAMP-stimulated ion currents in Xenopus oocytes expressing CFTR cRNA. Am J Physiol 262, C783-C788

    Google Scholar 

  5. Dalemans W, Barbry P, Champigny G, Jallat S, Dott K, Dreyer D, Crystal RG, Pavirani A, Lecocq J-P, Lazdunski M (1991) Altered chloride ion channel kinetics associated with the ΔF508 cystic fibrosis mutation. Nature 354:526–528

    Google Scholar 

  6. DeWolf I, Van Driessche W (1986) Voltage-dependent Ba2+ block of K+ channels in the apical membrane of frog skin. Am J Physiol 251:C696–706

    Google Scholar 

  7. Diener M, Rummel W (1989) Actions of the Cl− channel blocker NPPB on absorptive and secretory transport processes of Na+ and Cl− in rat descendent colon. Acta Physiol Scand 137:215–222

    Google Scholar 

  8. Dreinhöfer J, Gögelein H, Greger R (1988) Blocking kinetics of Cl− channels in colonic carcinoma cells (HT-29) as revealed by 5-nitro-2-(3-phenylpropylamino)bencoic acid (NPPB). Biochim Biophys Acta 946:135–142

    Google Scholar 

  9. Fischer H, Van Driessche W, Clauss W (1989) Evidence for apical sodium channels in frog lung epithelial cells. Am J Physiol 256:C764-C771

    Google Scholar 

  10. Fischer H, Kromer W, Clauss W (1991) Two types of chloride channels in hen colon epithelial cells identified by patch clamp experiments. J Comp Physiol B 161:333–338

    Google Scholar 

  11. Fischer H, Illek B, Negulescu PA, Clauss W, Machen TE (1992) Carbachol-activated calcium entry into HT-29 cells is regulated by both membrane potential and cell volume. Proc Natl Acad Sci USA 89:1438–1442

    Google Scholar 

  12. Frizzell RA, Rechkemmer G, Shoemaker RL (1986) Altered regulation of airway epithelial cell chloride channels in cystic fibrosis. Science 233:558–560

    Google Scholar 

  13. Gögelein H (1988) Chloride channels in epithelia. Biochim Biophys Acta 947:521–537

    Google Scholar 

  14. Gray MA, Harris A, Coleman L, Greenwell JR, Argent BE (1989) Two types of chloride channel on duct cells cultured from human fetal pancreas. Am J Physiol 257:C240-C251

    Google Scholar 

  15. Greger R, Schlatter E, Gögelein H (1985) Cl− channels in the apical membrane of the rectal gland “induced” by cAMP. Pflügers Arch 403:446–448

    Google Scholar 

  16. Greger R, Nitschke RB, Lohrmann E, Burhoff I, Hropot M, Englert HC, Lang HJ (1991) Effects of arylaminobenzoate-type chloride channel blockers on equivalent short-circuit current in rabbit colon. Pflügers Arch 419:190–196

    Google Scholar 

  17. Halm DR, Rechkemmer GR, Schoumacher RA, Frizzell RA (1988) Apical membrane chloride channels in a colonic cell line activated by secretory agonists. Am J Physiol 254:C505-C511

    Google Scholar 

  18. Hamill OP, Marty A, Neher E, Sakman B (1981) Improved patch clamp technique for high resolution current recordings from cells and cell-free membrane patches. Pflügers Arch 391:85–100

    Google Scholar 

  19. Hayslett JP, Gögelein H, Kunzelmann K, Greger R (1987) Characteristics of apical chloride channels in human colon cells (HT-29). Pflügers Arch 410:487–494

    Google Scholar 

  20. Illek B, Fischer H, Kreusel KM, Hegel U, Clauss W (1992) Volumesensitive K+ channels in the basolateral membrane of HT-29/B6 monolayers blocked by lidocaine, quinidine, NPPB and barium. Am J Physiol, in press

  21. Keeling DJ, Taylor AG, Smith PL (1991) Effects of NPPB on the chloride transport in intestinal tissues and the T84 cell line. Biochim Biophys Acta 1115:42–48

    Google Scholar 

  22. Kreusel KM, Fromm M, Schultzke JD, Hegel U (1991) Cl− secretion in epithelial monolayers of mucus-forming human colon cells (HT-29/B6). Am J Physiol 261:C754-C582

    Google Scholar 

  23. Kunzelmann K, Grolik M, Kubitz R, Greger R (1992) Conductance properties and cAMP dependent activation of small conductance Cl− channels in HT-29 colon carcinoma cells. Pflügers Arch, in press

  24. Li M, McCann JD, Liedtke CM, Nairn AC, Greengard P, Welsh MJ (1988) Cyclic-AMP-dependent protein kinase opens chloride channels in normal but not cystic fibrosis airway epithelium. Nature 331:358–360

    Google Scholar 

  25. Lukacs GL, Nanda A, Rotstein OD, Grinstein S (1991) The chloride channel blocker NPPB uncouples mitochondria and increases the proton permeability of the plasma membrane in phagocytic cells. FEBS Lett 288:17–20

    Google Scholar 

  26. Marty A, Tau YP, Trautwein A (1984) Three types of calcium-activated Cl− channels in rat lacrimal glands. J Physiol (Lond) 357:293–325

    Google Scholar 

  27. Reinach P, Schoen HF (1990) NPPB inhibits the basolateral membrane K+ conductance in the isolated bullfrog cornea. Biochim Biophys Acta 1026:13–20

    Google Scholar 

  28. Schoumacher RA, Shoemaker RL, Halm DR, Tallant EA, Wallace RW, Frizzel RA (1987) Phosphorylation fails to activate chloride channels from cystic fibrosis airway cells. Nature 330:752–754

    Google Scholar 

  29. Sigworth FJ, Sine S (1987) Data transformation for improved display and fitting of single channel dwell time histograms. Biophys J 52:1047–1054

    Google Scholar 

  30. Singh AK, Afink GB, Venglarik CJ, Wang R, Bridges RJ (1991) Colonic Cl channel blockade by three classes of compounds. Am J Physiol 260:C51-C63

    Google Scholar 

  31. Solc CK, Wine JJ (1991) Swelling-induced and depolarization-induced Cl− channels in normal and cystic fibrosis epithelial cells. Am J Physiol 261:C658-C674

    Google Scholar 

  32. Tabcharani JA, Hanrahan JW (1991) On the activation of outwardly rectifying anion channels in excised patches. Am J Physiol 261:G992-G999

    Google Scholar 

  33. Tabcharani JA, Chang XB, Riordan JR, Hanrahan JW (1991) Phosphorylation-regulated Cl− channel in CHO cells stably expressing the cystic fibrosis gene. Nature 352:628–631

    Google Scholar 

  34. Tilmann M, Kunzelmann K, Fröbe U, Cabanchik I, Lang HJ, Englert HC, Greger R (1991) Different types of blockers of the intermediate-conductance outwardly rectifying chloride channel in epithelia. Pflügers Arch 418:556–563

    Google Scholar 

  35. Wangemann P, Wittner M, DiStefano A, Englert HC, Lang HJ, Schlatter E, Greger R (1986) Cl−1 channel blockers in the thick ascending limb of the loop of henle. Structure-activity relationship. Pflügers Arch 407 [Suppl 2]:S128-S141

    Google Scholar 

Download references

Author information

Author notes

  1. Wolfgang Clauss

    Present address: Institut für Tierphysiologie, Wartweg 95, W-6300, Giessen, FRG

Authors and Affiliations

  1. Institut für Veterinärphysiologie, Klinikum Steglitz, Freie Universität Berlin, W-1000, Berlin 33, Federal Republic of Germany

    Horst Fischer, Beate Illek & Wolfgang Clauss

  2. Institut für Klinische Physiologie, Klinikum Steglitz, Freie Universität Berlin, W-1000, Berlin 33, Federal Republic of Germany

    Klaus -M. Kreusel & Ulrich Hegel

  3. 235 Life Science Addition, Division of Cell and Developmental Biology, Molecular and Cell Biology Department, University of California, 94720, Berkeley, CA, USA

    Horst Fischer, Beate Illek & Terry E. Machen

Authors
  1. Horst Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Klaus -M. Kreusel
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Beate Illek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Terry E. Machen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ulrich Hegel
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Wolfgang Clauss
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fischer, H., Kreusel, K.M., Illek, B. et al. The outwardly rectifying Cl− channel is not involved in cAMP-mediated Cl− secretion in HT-29 cells: evidence for a very-low-conductance Cl− channel. Pflügers Arch 422, 159–167 (1992). https://doi.org/10.1007/BF00370415

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00370415

Key words

Search

Navigation