Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Feb 15;89(4):1438–1442. doi: 10.1073/pnas.89.4.1438

Carbachol-activated calcium entry into HT-29 cells is regulated by both membrane potential and cell volume.

H Fischer 1, B Illek 1, P A Negulescu 1, W Clauss 1, T E Machen 1
PMCID: PMC48466  PMID: 1311099

Abstract

Intracellular Ca2+ ([Ca2+]i) was measured in single Cl(-)-secretory HT-29/B6 colonic carcinoma cells with the Ca2+ probe fura-2 and digital imaging microscopy. Resting [Ca2+]i was 63 +/- 3 nM (n = 62). During treatment with the muscarinic agonist carbachol, [Ca2+]i rapidly increased to 901 +/- 119 nM and subsequently reached a stable level of 309 +/- 23 nM, which depended on Ca2+ entry into the cells from the extracellular solution. The goal of this study was to characterize the Ca2+ entry pathway across the cell membrane with respect to its dependence on membrane potential and cell volume. Under resting conditions [Ca2+]i showed no apparent dependence on either potential or cell volume. After stimulating Ca2+ entry with carbachol (100 microM), [Ca2+]i increased with hyperpolarization (low-K+ or valinomycin treatment) and decreased with depolarization (high-K+ or gramicidin treatment) of the cell, as expected from changes in driving force for Ca2+ entry. In stimulated cells, hypotonic solutions caused [Ca2+]i to increase, whereas hypertonic solutions blocked Ca2+ entry. The shrinkage-induced decreases in [Ca2+]i were only slightly affected when the membrane potential was increased with valinomycin, suggesting that shrinkage directly affects the carbachol-activated Ca2+ conductance. In contrast, the swelling-induced increase in [Ca2+]i was significantly reduced in valinomycin-treated cells, suggesting an indirect dependence on a swelling-activated K+ conductance. Thus, carbachol-stimulated Ca2+ entry is under the dual control of membrane potential and cell volume. This mechanism may serve as a regulatory influence that determines the extent of Ca2+ influx during cholinergic stimulation.

Full text

PDF
1438

Selected References

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

  1. Bolívar J. J., Cereijido M. Voltage and Ca2+-activated K+ channel in cultured epithelial cells (MDCK). J Membr Biol. 1987;97(1):43–51. doi: 10.1007/BF01869613. [DOI] [PubMed] [Google Scholar]
  2. Christensen O. Mediation of cell volume regulation by Ca2+ influx through stretch-activated channels. Nature. 1987 Nov 5;330(6143):66–68. doi: 10.1038/330066a0. [DOI] [PubMed] [Google Scholar]
  3. Christensen O., Zeuthen T. Maxi K+ channels in leaky epithelia are regulated by intracellular Ca2+, pH and membrane potential. Pflugers Arch. 1987 Mar;408(3):249–259. doi: 10.1007/BF02181467. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Foskett J. K., Melvin J. E. Activation of salivary secretion: coupling of cell volume and [Ca2+]i in single cells. Science. 1989 Jun 30;244(4912):1582–1585. doi: 10.1126/science.2500708. [DOI] [PubMed] [Google Scholar]
  7. Germann W. J., Ernst S. A., Dawson D. C. Resting and osmotically induced basolateral K conductances in turtle colon. J Gen Physiol. 1986 Aug;88(2):253–274. doi: 10.1085/jgp.88.2.253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  9. Hosey M. M., Lazdunski M. Calcium channels: molecular pharmacology, structure and regulation. J Membr Biol. 1988 Sep;104(2):81–105. doi: 10.1007/BF01870922. [DOI] [PubMed] [Google Scholar]
  10. Kreusel K. M., Fromm M., Schulzke J. D., Hegel U. Cl- secretion in epithelial monolayers of mucus-forming human colon cells (HT-29/B6). Am J Physiol. 1991 Oct;261(4 Pt 1):C574–C582. doi: 10.1152/ajpcell.1991.261.4.C574. [DOI] [PubMed] [Google Scholar]
  11. Lewis S. A., Wills N. K. Use of ionophores in epithelia: characterizing membrane properties. Methods Enzymol. 1989;171:715–736. doi: 10.1016/s0076-6879(89)71039-9. [DOI] [PubMed] [Google Scholar]
  12. Matthews G., Neher E., Penner R. Second messenger-activated calcium influx in rat peritoneal mast cells. J Physiol. 1989 Nov;418:105–130. doi: 10.1113/jphysiol.1989.sp017830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Merritt J. E., Rink T. J. Regulation of cytosolic free calcium in fura-2-loaded rat parotid acinar cells. J Biol Chem. 1987 Dec 25;262(36):17362–17369. [PubMed] [Google Scholar]
  14. Negulescu P. A., Machen T. E. Intracellular Ca regulation during secretagogue stimulation of the parietal cell. Am J Physiol. 1988 Jan;254(1 Pt 1):C130–C140. doi: 10.1152/ajpcell.1988.254.1.C130. [DOI] [PubMed] [Google Scholar]
  15. Parod R. J., Putney J. W., Jr The role of calcium in the receptor mediated control of potassium permeability in the rat lacrimal gland. J Physiol. 1978 Aug;281:371–381. doi: 10.1113/jphysiol.1978.sp012428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Penner R., Matthews G., Neher E. Regulation of calcium influx by second messengers in rat mast cells. Nature. 1988 Aug 11;334(6182):499–504. doi: 10.1038/334499a0. [DOI] [PubMed] [Google Scholar]
  17. Taniguchi J., Guggino W. B. Membrane stretch: a physiological stimulator of Ca2+-activated K+ channels in thick ascending limb. Am J Physiol. 1989 Sep;257(3 Pt 2):F347–F352. doi: 10.1152/ajprenal.1989.257.3.F347. [DOI] [PubMed] [Google Scholar]
  18. Worrell R. T., Butt A. G., Cliff W. H., Frizzell R. A. A volume-sensitive chloride conductance in human colonic cell line T84. Am J Physiol. 1989 Jun;256(6 Pt 1):C1111–C1119. doi: 10.1152/ajpcell.1989.256.6.C1111. [DOI] [PubMed] [Google Scholar]
  19. Yada T., Oiki S., Ueda S., Okada Y. Intestinal secretagogues increase cytosolic free Ca2+ concentration and K+ conductance in a human intestinal epithelial cell line. J Membr Biol. 1989 Dec;112(2):159–167. doi: 10.1007/BF01871277. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES