Effects of 8-cpt-cAMP on the epithelial sodium channel expressed in Xenopus oocytes

Vasopressin stimulates the activity of the epithelial Na channel (ENaC) thr ough the cAMP/PKA pathway in the cortical collecting tubule, or in similar amphibian epithelia, but the mechanism of this regulation is not yet unders tood. This stimulation by cAMP could not be reproduced with the rat or Xeno pus ENaC expressed in Xenopus oocyte. Recently, it was shown that the alpha -subunit cloned from the guinea-pig colon (agp) could confer the ability t o be activated by the membrane-permeant cAMP analogue 8-chlorophenylthio-cA MP (cpt-cAMP) to channels produced by expression of alpha gp, beta rat and gamma rat ENaC subunits. In this study we investigate the mechanism of this activation. Forskolin treatment. endogenous production of cAMP by activati on of coexpressed beta adrenergic receptors., or intracellular perfusion wi th cAMP did not increase the amiloride-sensitive Na current, even though th ese maneuvers stimulated CFTR (cystic fibrosis transmembrane conductance re gulator)-mediated Cl currents. In contrast, extracellular 8-cpt-cAMP increa sed alpha gp, beta rat and gamma rat ENaC activity but had no effect on CFT R. Swapping intracellular domains between the cpt-cAMP-sensitive alpha gp a nd the cpt-cAMP-resistant alpha rat-subunit showed that neither the N-termi nal nor the C-terminal of at ENaC was responsible for the effect of cpt-cAM P. The mechanisms of activation of ENaC by cpt-cAMP and of CFTR by the cAMP /PKA pathway are clearly different. cpt-cAMP seems to increase the activity of ENaC formed by alpha gp and beta gamma rat by interacting with the extr acellular part of the protein.