Skip to main content
NCBI home page
The Journal of Clinical Investigation logoLink to The Journal of Clinical Investigation
. 1994 May;93(5):1900–1910. doi: 10.1172/JCI117181

Regulated Cl transport, K and Cl permeability, and exocytosis in T84 cells.

M E Huflejt 1, R A Blum 1, S G Miller 1, H P Moore 1, T E Machen 1
PMCID: PMC294297  PMID: 8182123

Abstract

We measured stimulant-induced changes of exocytosis that are associated with increases in Cl secretion (i.e., short circuit current, ISC, in microA/cm2) and apical (ap) Cl permeability (PCl) and basolateral (bl) K permeability (PK) (both in cm/s) in T84 monolayers. PCl and PK were measured by permeabilizing the bl or ap membrane with nystatin. PCl was also measured with a fluorescent dye 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). A noninvasive and sensitive method (release of 35SO4-labeled glycosaminoglycan [GAG], a fluid-phase marker of Golgi-derived vesicles) was used to measure exocytosis at both ap and bl membranes. At rest, ISC = 3.6, PK = 0.8 x 10(-6), PCl = 2.1 x 10(-6) with SPQ and 2.4 x 10(-6) electrically, and there was constitutive GAG secretion (i.e., exocytosis) to both ap and bl sides (bl > 2 x ap). Carbachol (C) increased: ISC (delta = 18.6), PK (6.5x), PCl (1.8-2.9x), and exocytosis to both ap (2.2-3.5x) and bl (2.0-3.0x) membranes. Forskolin (F) increased ISC (delta = 29), PCl (5.5-11x) and ap exocytosis (1.5-2x), but had no effect on PK or bl exocytosis. Synergistic effects on ISC occurred when C was added to F-treated cells but not vice versa, even though the characteristic effects of F+C on PCl, PK, and/or GAG secretion were identical to those exhibited when stimulants were added individually. Cl secretion results from coordinated activation of channels at ap and bl membranes, and exocytosis may play a role in these events.

Full text

PDF
1900

Images in this article

1906Image
on p.1906

Selected References

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

  1. Anderson M. P., Welsh M. J. Calcium and cAMP activate different chloride channels in the apical membrane of normal and cystic fibrosis epithelia. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6003–6007. doi: 10.1073/pnas.88.14.6003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bacskai B. J., Friedman P. A. Activation of latent Ca2+ channels in renal epithelial cells by parathyroid hormone. Nature. 1990 Sep 27;347(6291):388–391. doi: 10.1038/347388a0. [DOI] [PubMed] [Google Scholar]
  3. Bae H. R., Verkman A. S. Protein kinase A regulates chloride conductance in endocytic vesicles from proximal tubule. Nature. 1990 Dec 13;348(6302):637–639. doi: 10.1038/348637a0. [DOI] [PubMed] [Google Scholar]
  4. Bradbury N. A., Jilling T., Berta G., Sorscher E. J., Bridges R. J., Kirk K. L. Regulation of plasma membrane recycling by CFTR. Science. 1992 Apr 24;256(5056):530–532. doi: 10.1126/science.1373908. [DOI] [PubMed] [Google Scholar]
  5. Bradbury N. A., Jilling T., Kirk K. L., Bridges R. J. Regulated endocytosis in a chloride secretory epithelial cell line. Am J Physiol. 1992 Mar;262(3 Pt 1):C752–C759. doi: 10.1152/ajpcell.1992.262.3.C752. [DOI] [PubMed] [Google Scholar]
  6. Brown D., Hirsch S., Gluck S. An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations. Nature. 1988 Feb 18;331(6157):622–624. doi: 10.1038/331622a0. [DOI] [PubMed] [Google Scholar]
  7. Brown D. Vesicle recycling and cell-specific function in kidney epithelial cells. Annu Rev Physiol. 1989;51:771–784. doi: 10.1146/annurev.ph.51.030189.004011. [DOI] [PubMed] [Google Scholar]
  8. Buanes T., Grotmol T., Landsverk T., Raeder M. G. Ultrastructure of pancreatic duct cells at secretory rest and during secretin-dependent NaHCO3 secretion. Acta Physiol Scand. 1987 Sep;131(1):55–62. doi: 10.1111/j.1748-1716.1987.tb08205.x. [DOI] [PubMed] [Google Scholar]
  9. Cartwright C. A., McRoberts J. A., Mandel K. G., Dharmsathaphorn K. Synergistic action of cyclic adenosine monophosphate- and calcium-mediated chloride secretion in a colonic epithelial cell line. J Clin Invest. 1985 Nov;76(5):1837–1842. doi: 10.1172/JCI112176. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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 Membr Biol. 1990 Feb;113(3):193–202. doi: 10.1007/BF01870071. [DOI] [PubMed] [Google Scholar]
  11. Cliff W. H., Frizzell R. A. Separate Cl- conductances activated by cAMP and Ca2+ in Cl(-)-secreting epithelial cells. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4956–4960. doi: 10.1073/pnas.87.13.4956. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Denning G. M., Ostedgaard L. S., Cheng S. H., Smith A. E., Welsh M. J. Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia. J Clin Invest. 1992 Jan;89(1):339–349. doi: 10.1172/JCI115582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Devor D. C., Simasko S. M., Duffey M. E. Carbachol induces oscillations of membrane potassium conductance in a colonic cell line, T84. Am J Physiol. 1990 Feb;258(2 Pt 1):C318–C326. doi: 10.1152/ajpcell.1990.258.2.C318. [DOI] [PubMed] [Google Scholar]
  14. Dharmsathaphorn K., Mandel K. G., Masui H., McRoberts J. A. Vasoactive intestinal polypeptide-induced chloride secretion by a colonic epithelial cell line. Direct participation of a basolaterally localized Na+,K+,Cl- cotransport system. J Clin Invest. 1985 Feb;75(2):462–471. doi: 10.1172/JCI111721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Dharmsathaphorn K., Pandol S. J. Mechanism of chloride secretion induced by carbachol in a colonic epithelial cell line. J Clin Invest. 1986 Feb;77(2):348–354. doi: 10.1172/JCI112311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Forstner G., Zhang Y., McCool D., Forstner J. Mucin secretion by T84 cells: stimulation by PKC, Ca2+, and a protein kinase activated by Ca2+ ionophore. Am J Physiol. 1993 Jun;264(6 Pt 1):G1096–G1102. doi: 10.1152/ajpgi.1993.264.6.G1096. [DOI] [PubMed] [Google Scholar]
  18. Forte T. M., Machen T. E., Forte J. G. Ultrastructural changes in oxyntic cells associated with secretory function: a membrane-recycling hypothesis. Gastroenterology. 1977 Oct;73(4 Pt 2):941–955. [PubMed] [Google Scholar]
  19. Gluck S., Cannon C., Al-Awqati Q. Exocytosis regulates urinary acidification in turtle bladder by rapid insertion of H+ pumps into the luminal membrane. Proc Natl Acad Sci U S A. 1982 Jul;79(14):4327–4331. doi: 10.1073/pnas.79.14.4327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kimura J. H., Lohmander L. S., Hascall V. C. Studies on the biosynthesis of cartilage proteoglycan in a model system of cultured chondrocytes from the Swarm rat chondrosarcoma. J Cell Biochem. 1984;26(4):261–278. doi: 10.1002/jcb.240260406. [DOI] [PubMed] [Google Scholar]
  21. Lewis S. A., Eaton D. C., Clausen C., Diamond J. M. Nystatin as a probe for investigating the electrical properties of a tight epithelium. J Gen Physiol. 1977 Oct;70(4):427–440. doi: 10.1085/jgp.70.4.427. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lindeman R. P., Chase H. S., Jr Protein kinase C does not participate in carbachol's secretory action in T84 cells. Am J Physiol. 1992 Jul;263(1 Pt 1):C140–C146. doi: 10.1152/ajpcell.1992.263.1.C140. [DOI] [PubMed] [Google Scholar]
  23. Luikart S. D., Sackrison J. L., Thomas C. V. Altered glycosaminoglycan production by HL-60 cells treated with 4-methylumbelliferyl-beta-D-xyloside. Blood. 1985 Oct;66(4):866–872. [PubMed] [Google Scholar]
  24. 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]
  25. Mandel K. G., McRoberts J. A., Beuerlein G., Foster E. S., Dharmsathaphorn K. Ba2+ inhibition of VIP- and A23187-stimulated Cl- secretion by T84 cell monolayers. Am J Physiol. 1986 Mar;250(3 Pt 1):C486–C494. doi: 10.1152/ajpcell.1986.250.3.C486. [DOI] [PubMed] [Google Scholar]
  26. Marcon M. A., McCool D., Forstner J., Forstner G. Inhibition of mucin secretion in a colonic adenocarcinoma cell line by DIDS and potassium channel blockers. Biochim Biophys Acta. 1990 Apr 9;1052(1):17–23. doi: 10.1016/0167-4889(90)90051-e. [DOI] [PubMed] [Google Scholar]
  27. McCool D. J., Marcon M. A., Forstner J. F., Forstner G. G. The T84 human colonic adenocarcinoma cell line produces mucin in culture and releases it in response to various secretagogues. Biochem J. 1990 Apr 15;267(2):491–500. doi: 10.1042/bj2670491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Miller S. G., Moore H. P. Movement from trans-Golgi network to cell surface in semiintact cells. Methods Enzymol. 1992;219:234–248. doi: 10.1016/0076-6879(92)19025-2. [DOI] [PubMed] [Google Scholar]
  29. Negulescu P. A., Machen T. E. Intracellular ion activities and membrane transport in parietal cells measured with fluorescent dyes. Methods Enzymol. 1990;192:38–81. doi: 10.1016/0076-6879(90)92062-i. [DOI] [PubMed] [Google Scholar]
  30. Paul A., Mergey M., Veissière D., Hermelin B., Cherqui G., Picard J., Basbaum C. B. Regulation of secretion in cultured tracheal serous cells by protein kinases A and C. Am J Physiol. 1991 Aug;261(2 Pt 1):L172–L177. doi: 10.1152/ajplung.1991.261.2.L172. [DOI] [PubMed] [Google Scholar]
  31. Phillips T. E., Huet C., Bilbo P. R., Podolsky D. K., Louvard D., Neutra M. R. Human intestinal goblet cells in monolayer culture: characterization of a mucus-secreting subclone derived from the HT29 colon adenocarcinoma cell line. Gastroenterology. 1988 Jun;94(6):1390–1403. doi: 10.1016/0016-5085(88)90678-6. [DOI] [PubMed] [Google Scholar]
  32. Prince L. S., Tousson A., Marchase R. B. Cell surface labeling of CFTR in T84 cells. Am J Physiol. 1993 Feb;264(2 Pt 1):C491–C498. doi: 10.1152/ajpcell.1993.264.2.C491. [DOI] [PubMed] [Google Scholar]
  33. Reinlib L., Mikkelsen R., Zahniser D., Dharmsathaphorn K., Donowitz M. Carbachol-induced cytosolic free Ca2+ increases in T84 colonic cells seen by microfluorimetry. Am J Physiol. 1989 Dec;257(6 Pt 1):G950–G960. doi: 10.1152/ajpgi.1989.257.6.G950. [DOI] [PubMed] [Google Scholar]
  34. Schwartz N. B., Galligani L., Ho P. L., Dorfman A. Stimulation of synthesis of free chondroitin sulfate chains by beta-D-xylosides in cultured cells. Proc Natl Acad Sci U S A. 1974 Oct;71(10):4047–4051. doi: 10.1073/pnas.71.10.4047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shorofsky S. R., Field M., Fozzard H. A. Mechanism of Cl secretion in canine trachea: changes in intracellular chloride activity with secretion. J Membr Biol. 1984;81(1):1–8. doi: 10.1007/BF01868804. [DOI] [PubMed] [Google Scholar]
  36. Smith J. J., McCann J. D., Welsh M. J. Bradykinin stimulates airway epithelial Cl- secretion via two second messenger pathways. Am J Physiol. 1990 Jun;258(6 Pt 1):L369–L377. doi: 10.1152/ajplung.1990.258.6.L369. [DOI] [PubMed] [Google Scholar]
  37. Velasco A., Hidalgo J., Pérez-Vilar J., García-Herdugo G., Navas P. Detection of glycosaminoglycans in the Golgi complex of chondrocytes. Eur J Cell Biol. 1988 Dec;47(2):241–250. [PubMed] [Google Scholar]
  38. 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]
  39. Wong S. M., Lindeman R. P., Parangi S., Chase H. S., Jr Role of calcium in mediating action of carbachol in T84 cells. Am J Physiol. 1989 Nov;257(5 Pt 1):C976–C985. doi: 10.1152/ajpcell.1989.257.5.C976. [DOI] [PubMed] [Google Scholar]
  40. Wong S. M., Tesfaye A., DeBell M. C., Chase H. S., Jr Carbachol increases basolateral K+ conductance in T84 cells. Simultaneous measurements of cell [Ca] and gK explore calcium's role. J Gen Physiol. 1990 Dec;96(6):1271–1285. doi: 10.1085/jgp.96.6.1271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yiu S. C., Lambert R. W., Bradley M. E., Ingham C. E., Hales K. L., Wood R. L., Mircheff A. K. Stimulation-associated redistribution of Na,K-ATPase in rat lacrimal gland. J Membr Biol. 1988 Jun;102(3):185–194. doi: 10.1007/BF01925712. [DOI] [PubMed] [Google Scholar]
  42. Yiu S. C., Lambert R. W., Tortoriello P. J., Mircheff A. K. Secretagogue-induced redistributions of Na,K-ATPase in rat lacrimal acini. Invest Ophthalmol Vis Sci. 1991 Oct;32(11):2976–2984. [PubMed] [Google Scholar]

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

RESOURCES