AJP - Cell Physiology, Vol 257, Issue 5 C976-C985, Copyright © 1989 by American Physiological Society

ARTICLES

Role of calcium in mediating action of carbachol in T84 cells

S. M. Wong, R. P. Lindeman, S. Parangi and H. S. Chase Jr
Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York 10032.

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.

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