Skip to main content
PMC home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1992 Oct;90(4):1608–1613. doi: 10.1172/JCI116030

Microfilament-dependent activation of Na+/K+/2Cl- cotransport by cAMP in intestinal epithelial monolayers.

J B Matthews 1, C S Awtrey 1, J L Madara 1
PMCID: PMC443209  PMID: 1328303

Abstract

cAMP-mediated stimulation of Cl- secretion in the human intestinal cell line T84 is accompanied by significant remodeling of F-actin, and both the secretory and cytoskeletal responses may be largely ablated by previous cell loading with phalloidin derivatives, reagents that prevent dynamic reordering of microfilaments (1991. J. Clin. Invest. 87:1903-1909). In this study, we examined the effect of phalloidin loading on the cAMP-elicited activity of the individual membrane-associated transport proteins involved in electrogenic Cl- secretion. Efflux of 125I and 86Rb was used to assay forskolin-stimulated Cl- and K+ conductances, respectively, and no inhibitory effect of phalloidin could be detected. Na+/K(+)-ATPase pump activity, assessed as bumetanide-insensitive 86Rb uptake and the ability of monolayers to generate a Na+ absorptive current in response to apical addition of a Na+ ionophore, was not different between control and phalloidin-loaded monolayers. Forskolin was found to stimulate Na+/K+/2Cl- cotransport (bumetanide-sensitive 86Rb uptake) in time-dependent fashion. In the absence of any agonist, cotransporter activity was markedly decreased in phalloidin-loaded monolayers. Furthermore, under phalloidin-loaded conditions, the forskolin-elicited increase in bumetanide-sensitive 86Rb uptake was markedly attenuated. These findings suggest that cAMP-induced activity of Cl- channels, K+ channels, and the Na+/K(+)-ATPase are not influenced by F-actin stabilization. However, cAMP-induced activation of the Na+/K+/2Cl- cotransporter appears to be microfilament-dependent, and ablation of this event is likely to account for the inhibition of cAMP-elicited Cl- secretion seen in the phalloidin-loaded state. Such findings suggest that Na+/K+/2Cl- cotransporter is functionally linked to the cytoskeleton and is a regulated site of cAMP-elicited electrogenic Cl- secretion.

Full text

PDF
1610

Images in this article

1611Image
on p.1611
1611Image
on p.1611

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bennett V. Proteins involved in membrane--cytoskeleton association in human erythrocytes: spectrin, ankyrin, and band 3. Methods Enzymol. 1983;96:313–324. doi: 10.1016/s0076-6879(83)96029-9. [DOI] [PubMed] [Google Scholar]
  2. Breitwieser G. E., Altamirano A. A., Russell J. M. Osmotic stimulation of Na(+)-K(+)-Cl- cotransport in squid giant axon is [Cl-]i dependent. Am J Physiol. 1990 Apr;258(4 Pt 1):C749–C753. doi: 10.1152/ajpcell.1990.258.4.C749. [DOI] [PubMed] [Google Scholar]
  3. Cantiello H. F., Stow J. L., Prat A. G., Ausiello D. A. Actin filaments regulate epithelial Na+ channel activity. Am J Physiol. 1991 Nov;261(5 Pt 1):C882–C888. doi: 10.1152/ajpcell.1991.261.5.C882. [DOI] [PubMed] [Google Scholar]
  4. Dawson D. C., Richards N. W. Basolateral K conductance: role in regulation of NaCl absorption and secretion. Am J Physiol. 1990 Aug;259(2 Pt 1):C181–C195. doi: 10.1152/ajpcell.1990.259.2.C181. [DOI] [PubMed] [Google Scholar]
  5. Dharmsathaphorn K., Madara J. L. Established intestinal cell lines as model systems for electrolyte transport studies. Methods Enzymol. 1990;192:354–389. doi: 10.1016/0076-6879(90)92082-o. [DOI] [PubMed] [Google Scholar]
  6. Dharmsathaphorn K., McRoberts J. A., Mandel K. G., Tisdale L. D., Masui H. A human colonic tumor cell line that maintains vectorial electrolyte transport. Am J Physiol. 1984 Feb;246(2 Pt 1):G204–G208. doi: 10.1152/ajpgi.1984.246.2.G204. [DOI] [PubMed] [Google Scholar]
  7. Duffey M. E., Hainau B., Ho S., Bentzel C. J. Regulation of epithelial tight junction permeability by cyclic AMP. Nature. 1981 Dec 3;294(5840):451–453. doi: 10.1038/294451a0. [DOI] [PubMed] [Google Scholar]
  8. Haas M., Forbush B., 3rd Photolabeling of a 150-kDa (Na + K + Cl) cotransport protein from dog kidney with a bumetanide analogue. Am J Physiol. 1987 Aug;253(2 Pt 1):C243–C252. doi: 10.1152/ajpcell.1987.253.2.C243. [DOI] [PubMed] [Google Scholar]
  9. Haas M., Forbush B., 3rd [3H]bumetanide binding to duck red cells. Correlation with inhibition of (Na + K + 2Cl) co-transport. J Biol Chem. 1986 Jun 25;261(18):8434–8441. [PubMed] [Google Scholar]
  10. Haas M., Johnson L. G., Boucher R. C. Regulation of Na-K-Cl cotransport in cultured canine airway epithelia: a [3H]bumetanide binding study. Am J Physiol. 1990 Oct;259(4 Pt 1):C557–C569. doi: 10.1152/ajpcell.1990.259.4.C557. [DOI] [PubMed] [Google Scholar]
  11. Halm D. R., Rechkemmer G. R., Schoumacher R. A., Frizzell R. A. Apical membrane chloride channels in a colonic cell line activated by secretory agonists. Am J Physiol. 1988 Apr;254(4 Pt 1):C505–C511. doi: 10.1152/ajpcell.1988.254.4.C505. [DOI] [PubMed] [Google Scholar]
  12. Hardy M. A., DiBona D. R. Microfilaments and the hydrosmotic action of vasopressin in toad urinary bladder. Am J Physiol. 1982 Sep;243(3):C200–C204. doi: 10.1152/ajpcell.1982.243.3.C200. [DOI] [PubMed] [Google Scholar]
  13. Kim H. D., Tsai Y. S., Franklin C. C., Turner J. T. Characterization of Na+/K+/Cl- cotransport in cultured HT29 human colonic adenocarcinoma cells. Biochim Biophys Acta. 1988 Dec 22;946(2):397–404. doi: 10.1016/0005-2736(88)90415-4. [DOI] [PubMed] [Google Scholar]
  14. Kirk K. L., Halm D. R., Dawson D. C. Active sodium transport by turtle colon via an electrogenic Na-K exchange pump. Nature. 1980 Sep 18;287(5779):237–239. doi: 10.1038/287237a0. [DOI] [PubMed] [Google Scholar]
  15. Madara J. L., Stafford J., Barenberg D., Carlson S. Functional coupling of tight junctions and microfilaments in T84 monolayers. Am J Physiol. 1988 Mar;254(3 Pt 1):G416–G423. doi: 10.1152/ajpgi.1988.254.3.G416. [DOI] [PubMed] [Google Scholar]
  16. Mandel K. G., Dharmsathaphorn K., McRoberts J. A. Characterization of a cyclic AMP-activated Cl-transport pathway in the apical membrane of a human colonic epithelial cell line. J Biol Chem. 1986 Jan 15;261(2):704–712. [PubMed] [Google Scholar]
  17. McRoberts J. A., Beuerlein G., Dharmsathaphorn K. Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line. J Biol Chem. 1985 Nov 15;260(26):14163–14172. [PubMed] [Google Scholar]
  18. McRoberts J. A., Erlinger S., Rindler M. J., Saier M. H., Jr Furosemide-sensitive salt transport in the Madin-Darby canine kidney cell line. Evidence for the cotransport of Na+, K+, and Cl-. J Biol Chem. 1982 Mar 10;257(5):2260–2266. [PubMed] [Google Scholar]
  19. Molitoris B. A., Nelson W. J. Alterations in the establishment and maintenance of epithelial cell polarity as a basis for disease processes. J Clin Invest. 1990 Jan;85(1):3–9. doi: 10.1172/JCI114427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Nash S., Parkos C., Nusrat A., Delp C., Madara J. L. In vitro model of intestinal crypt abscess. A novel neutrophil-derived secretagogue activity. J Clin Invest. 1991 Apr;87(4):1474–1477. doi: 10.1172/JCI115156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nelson W. J., Hammerton R. W. A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity. J Cell Biol. 1989 Mar;108(3):893–902. doi: 10.1083/jcb.108.3.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. O'Donnell M. E. [3H]bumetanide binding in vascular endothelial cells. Quantitation of Na-K-Cl cotransporters. J Biol Chem. 1989 Dec 5;264(34):20326–20330. [PubMed] [Google Scholar]
  23. O'Grady S. M., Palfrey H. C., Field M. Characteristics and functions of Na-K-Cl cotransport in epithelial tissues. Am J Physiol. 1987 Aug;253(2 Pt 1):C177–C192. doi: 10.1152/ajpcell.1987.253.2.C177. [DOI] [PubMed] [Google Scholar]
  24. O'Neill W. C., Klein J. D. Regulation of vascular endothelial cell volume by Na-K-2Cl cotransport. Am J Physiol. 1992 Feb;262(2 Pt 1):C436–C444. doi: 10.1152/ajpcell.1992.262.2.C436. [DOI] [PubMed] [Google Scholar]
  25. Owen N. E., Prastein M. L. Na/K/Cl cotransport in cultured human fibroblasts. J Biol Chem. 1985 Feb 10;260(3):1445–1451. [PubMed] [Google Scholar]
  26. Paris S., Pouysségur J. Growth factors activate the bumetanide-sensitive Na+/K+/Cl-cotransport in hamster fibroblasts. J Biol Chem. 1986 May 15;261(14):6177–6183. [PubMed] [Google Scholar]
  27. Shapiro M., Matthews J., Hecht G., Delp C., Madara J. L. Stabilization of F-actin prevents cAMP-elicited Cl- secretion in T84 cells. J Clin Invest. 1991 Jun;87(6):1903–1909. doi: 10.1172/JCI115215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Venglarik C. J., Bridges R. J., Frizzell R. A. A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells. Am J Physiol. 1990 Aug;259(2 Pt 1):C358–C364. doi: 10.1152/ajpcell.1990.259.2.C358. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Investigation are provided here courtesy of American Society for Clinical Investigation

RESOURCES