The central goal of this project is to characterize the mechanisms of regulation of the four cellular transport processes involved in net Cl movement across secretory epithelia. At the basolateral membrane, these include NaCl co-transport mechanisms, Na/K pumps, and K channels; and, at the apical membrane, Cl channels. The effects of cAMP- and Ca-dependent agonists will be evaluated by monitoring ionic currents and transepithelial fluxes across polyene-treated tissues and by application of conventional and ion-selective microelectrode and patch clamp techniques to isolated tracheal epithelium, primary tracheal cell cultures and colonic tumor cell cultures. The effects of cAMP- and Ca-dependent intracellular regulatory systems (e.g. protein kinases) on Cl and K conductances and channel kinetics will also be examined. Secretagogue-induced cell volume alterations and the role of volume regulation in the concerted response to secretory agonists will be determined. Finally, we will evaluate the cellular basis for dual regulation of NaCl co-transport and K conductance in two absorptive epithelia whose transport processes resemble those found in secretory tissues. Our studies will provide a complete characterization of the mechanisms underlyng cAMP- and Ca-dependent regulation and permit construction of a dynamic model of the events involved in stimulation of Cl-secreting epthelia.
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