Carbachol-induced cytosolic free Ca2+ increases in T84 colonic cells seen by microfluorimetry.
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.
Reinlib
L
L
Department of Medicine, Tufts University School of Medicine, Boston, Massachusetts.
Mikkelsen
R
R
Zahniser
D
D
Dharmsathaphorn
K
K
Donowitz
M
M
D001285:Atropine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D003110:Colonic Neoplasms; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D003620:Dantrolene / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D014700:Verapamil / Q000494:pharmacology
D004533:Egtazic Acid; D001285:Atropine; D002217:Carbachol; D014700:Verapamil; D003620:Dantrolene; D002118:Calcium