Time-dependent regulation by aldosterone of the amiloride-sensitive Na+ channel in rabbit kidney

The epithelial Na+ channel (ENaC) functions as the rate-limiting factor in aldosterone-regulated transcellular Na+ transport. In the study described here, the effect of aldosterone on ENaC mRNA levels, protein synthesis and benzamil-sensitive Na+ transport was investigated using primary cultures of immunodissected rabbit kidney connecting tubule and cortical collecting duct cells (CNT and CCD, respectively). After a lag time of 3 h, aldosterone caused transepithelial Na+ transport to increase, reaching maximal level of 260+/-44% after 16 h of incubation. The alpha, beta and gamma rabbit ENaC (rbENaC) mRNA levels, measured by semi-quantitative reverse transcriptase-polymerase chain reaction, were not changed by aldosterone during the first 3 h, but a twofold increase was apparent after 6 h; levels remained elevated for up to 16 h of incubation. Immunoprecipitation of [35S]methionine-labeled rbENaC revealed a rise in protein levels of the alpha and beta subunits, but the protein level of the gamma subunit remained constant. In conclusion, our data suggest that in rabbit CNT and CCD the early increase in Na+ transport caused by aldosterone is due to the activation or insertion of existing Na+ channels into the apical membrane, and that the late response is mediated by increased synthesis of the alpha and beta rbENaC subunits.