Carbachol Increases Basolateral K + Conductance in T 84 Cells

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 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.