Simultaneous Measur of Cell (Ca) and glr Explore Calcium's Role

To explore the role of calcium in mediating the action of carbachol in chloride-secreting epithelia, we simultaneously measured intracellular free (Ca) ((Ca).) and the potassium conductance (gx) of the basolateral membrane in T84 cells grown on collagen-coated filters. (Call was measured with fura-2 and fluores- cence microscopy and expressed as a relative value ((Ca)() normalized to control. To assess changes in basolateral gK, we measured the short circuit current (I~ in the presence of luminal amphotericin and a transepithelial mucosa-to-serosa K + gradient (Germann, W.J., M. E. Lowy, S. A. Ernst, and D. C. Dawson. 1986.J. Gen. Physiol. 88:237-251). Treatment of the monolayers with carbachol resulted in a parallel increase and then decrease in (Ca)~ and gx. The carbachol-induced changes in gg appeared to be dependent on the increase in (Ca)i because stimulation ofg x was significantly diminished when the hormone-induced increase in (Ca)~ was blunted, either by loading the cells with BAPTA or by reducing the extracellular (Ca). The carbachol-stimulated increase in gK appeared to be the direct result of the increase in steady-state (Ca)~. The changes in gK and (C,a)~ after stimulation with carbachol were correlated and ionomycin also increased gK and (Ca)~ in a parallel manner. The carbachol-induced Agi~ per A(Ca)~, however, was greater than that after ionomycin. Because ionomycin and carbachol appear to open the same channel, a conclusion based on inhibitor and selectivity experiments, carbachol may have a second action that amplifies the effect of calcium on gx.