Role of calcium in mediating action of carbachol in T84 cells.

To examine the role of calcium in mediating carbachol's action in secretory epithelia, we simultaneously measured intracellular free [Ca] [( Ca]i) and transepithelial chloride transport in T84 cells grown on collagen-coated filters. [Ca]i was measured with fura-2 and fluorescence microscopy and expressed as a relative value [( Ca]'i) normalized to control. Chloride transport was measured as the short-circuit current (Isc) with a voltage clamp. Monolayers were pretreated with cyclic AMP to augment the response of Isc to carbachol, a procedure that did not qualitatively change the response of the monolayer to carbachol. The carbachol-induced changes in Isc appeared to be dependent on the increase in [Ca]i. First, carbachol caused both Isc and [Ca]'i to increase in parallel. Isc increased from 32 +/- 5 to 70 +/- 9 microA and then declined to 57 +/- 16 microA while [Ca]'i increased from 72 +/- 14 to 156 +/- 22 nM and then declined to 133 +/- 45 nM. Second, the carbachol-induced increases in Isc and [Ca]'i were correlated. The greater the hormone-stimulated rise in [Ca]'i, the higher the increase in Isc. Third, carbachol's stimulation of Isc was blunted by preventing the calcium spike with the cellular calcium buffer 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid (BAPTA). Although the carbachol-induced increase in [Ca]'i appeared necessary for the increase in Isc, it was not clear if carbachol's action was solely the result of an increase in [Ca]'i. Increasing [Ca]'i with ionomycin, although causing Isc and [Ca]'i to increase in parallel, failed to increase Isc to the levels observed with carbachol. These experiments suggest that although the carbachol-induced increase in Isc is dependent on the increase in [Ca]i, the hormone may activate a second process that increases the sensitivity of the calcium-activated transport process to changes in [Ca]i.