Carbachol-induced cytosolic free Ca2+ increases in T84 colonic cells seen by microfluorimetry

L. Reinlib, R. Mikkelsen, D. Zahniser, K. Dharmsathaphorn, Mark Donowitz

Research output: Contribution to journalArticle

Abstract

Changes in cytosolic free Ca2+ ([Ca2+](i)) in response to the secretagogue carbachol have been characterized in the human color cancer cell line T84, a model Cl- secretory cell. In this study, [Ca2+](i) was determined with the fluorescence indicator fura-2 at the single-cell level with a fluorescent microscope-imaging system. Basal [Ca2+](i) in T84 cells in Ringer-HCO3 solution was 76 ± 4 nM and was decreased by exposure to Ca2+ free solution or 25 μM verapamil. The cholinergic agonist carbachol caused a concentration-dependent rise in [Ca2+](i) with a K(m) of 4 μM and a peak increase in [Ca2+](i) of ~ 50 nM. The onset of the [Ca2+](i) increase was within 3 s, occurred uniformly among cells, and peaked at 10-15 s. The increase in [Ca2+](i) was heterogenous in the length of time the [Ca2+](i) remained elevated above basal, and cell responses could be divided into at least two groups on that basis. Blocking the contributions of intracellular Ca2+ with dantrolene inhibited the increase in [Ca2+](i) as early as could be determined, whereas blocking the extracellular contribution with ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), verapamil, or nifedipine inhibited a slightly later increase in [Ca2+](i). In conclusion, the initial detectable increase in [Ca2+](i) caused by carbachol is due to the release of Ca2+ from internal stores, whereas the contribution of extracellular Ca2+ occurs later and at least partially involves a nifedipine- and verapamil-sensitive process.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume257
Issue number6
StatePublished - 1989
Externally publishedYes

Fingerprint

Cytophotometry
Carbachol
Verapamil
Nifedipine
Dantrolene
Cholinergic Agonists
Ethylene Glycol
Fura-2
Egtazic Acid
Ether
Color
Fluorescence
Cell Line
Acids
Neoplasms

Keywords

  • fura-2
  • intracellular free calcium

ASJC Scopus subject areas

  • Physiology
  • Gastroenterology

Cite this

Carbachol-induced cytosolic free Ca2+ increases in T84 colonic cells seen by microfluorimetry. / Reinlib, L.; Mikkelsen, R.; Zahniser, D.; Dharmsathaphorn, K.; Donowitz, Mark.

In: American Journal of Physiology - Gastrointestinal and Liver Physiology, Vol. 257, No. 6, 1989.

Research output: Contribution to journalArticle

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AU - Donowitz, Mark

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