Cyclic-3',5'-AMP (cAMP) plays a major role in regulating sodium transport across frog skin and other tight epithelia. This second messenger is thought to stimulate transepithelial Na+ movement by activating a cAMP-dependent kinase, which phosphorylates sites gating the apical Na+ channels. An increasing number of observations from this laboratory have suggested that the ubiquitous enzyme protein kinase C (PKC) may also be an important regulator of sodium transport across frog skin. The hypothesis has been developed that: (i) PKC phosphorylates sites identical with, or near, the regulatory sites phosphorylated by cAMP kinase, and (ii) that the natriferic action of insulin at the apical membrane is mediated by activation of PKC. The present proposal is focussed on this hypothesis, and aims at: (i) more rigorously examining whether PKC activation increases apical Na+ permeability (papNa), (ii) determining whether the apical natriferic action of insulin is mediated by PKC, (iii) examining whether translocation of PKC from cytosol to apical membrane is necessary for PKC activation to increase papNa, and (iv) determining whether the intracellular alkalinization (produced by both PKC activation and insulin in other cells) is important in the expression of this natriferic effect. Apical Na+ permeability, transepithelial Na+ transport and intracellular pH will be monitored with intracellular micropipets and pH-selective microelectrodes and transepithelial voltage clamping of intact isolated-epithelial and whole-thickness preparations of frog skin. Parallel studies of PKC activity and translocation will be conducted, using both enzymatic assays and monoclonal and polyclonal antibodies.
