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 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.