Regulation of cAMP-sensitive colonic epithelial Na⁺ channel in oocyte expression system

Abstract.

In amphibian epithelia and in cortical collecting duct the antidiuretic peptide arginine-vasopressin (AVP) stimulates activity of epithelial Na⁺ channels (ENaCs). Generally, the AVP action upon Na⁺ (re)absorption is believed to be a cAMP/protein-kinase-A mediated mechanism. In the Xenopus oocyte expression system, however, a clear stimulation of ENaC activity by cAMP could not be reproduced with channel subunits cloned from A6 cells or rat colon. We have recently shown that membrane-permeant 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) stimulates activity of a hybrid ENaC in Xenopus oocytes, that consists of an α-subunit cloned from guinea-pig colon and the β- and γ-subunit originating from rat colon (gpαrβγENaC). In the present study, we have further investigated the mechanisms by which cpt-cAMP upregulates gpαrβγENaC activity. Interestingly, we found AVP to stimulate the gpαrβγENaC in oocytes. Also, treatment with GTP-γ-S largely activated this channel. In contrast, as a conflicting result, forskolin had no stimulatory effect on the cAMP-sensitive gpαrβγENaC. Experiments with Brefeldin A (BFA) or nocodazole suggested that only a minor part of cpt-cAMP-induced activation is probably due to an additional translocation of channel proteins into the oocyte membrane. In conclusion, the stimulatory effect of synthetic cpt-cAMP does not seem to be exclusively provided by classical cAMP/PKA-associated transduction mechanisms, i.e., as in A6 cells.