AJP - Gastrointestinal and Liver Physiology, Vol 257, Issue 6 950-G960, Copyright © 1989 by American Physiological Society

ARTICLES

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

L. Reinlib, R. Mikkelsen, D. Zahniser, K. Dharmsathaphorn and M. Donowitz
Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts.

Changes in cytosolic free Ca2+ ([Ca2+]i) in response to the secretagogue carbachol have been characterized in the human colon 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 microM verapamil. The cholinergic agonist carbachol caused a concentration-dependent rise in [Ca2+]i with a Km of 4 microM and a peak increase in [Ca2+]i of approximately 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(beta-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.

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