CHARACTERIZATION OF EARLY ALDOSTERONE-INDUCED RNAS IDENTIFIED IN A6 KIDNEY EPITHELIA

The early aldosterone-induced increase in Na reabsorption across tight epithelia is characterized by a transcription-dependent activation of epithelial Na channels (ENaC) and pumps (Na,K-ATPase). In order to co ntribute towards the identification of transcriptionally regulated med iators of this process, we first tested mRNAs of proteins previously s uggested to be involved. Epithelia were treated for 1 h with 10(-6) M aldosterone, a concentration which produces a maximal transport respon se and at which both mineralo- and glucocorticoid receptors are occupi ed. Northern blot analysis showed no change in mRNAs of trimeric G pro tein alpha subunits, calmodulin, and mitochondrial energy metabolism p roteins, whereas Na,K-ATPase alpha 1 and beta 1 subunit mRNAs were sli ghtly increased (1.2- to 1.4-fold). In a second approach, we visualize d 5000 cDNA bands generated from A6 RNAs by differential display polym erase chain reaction (PCR). After 1 h of aldosterone treatment, approx imate to 0.5% of these appeared to be regulated. Four cDNA fragments c orresponding to early adrenal-steroid-upregulated RNAs (ASURs) were cl oned and for two of them cDNAs containing entire coding sequences were isolated by library screening. ASUR4 is the Xenopus laevis homologue of human E16 and rat TA1, a membrane protein structurally related to y east and prokaryotic permeases, and ASUR5 is the A transcript of Xenop us K-ras2. The rapid inductions of the four ASURs correspond to direct transcriptional effects since they were not inhibited by cycloheximid e but were blocked by actinomycin D. The K-1/2 values were similar or slightly below those reported for stimulation of Na transport. These c haracteristics of RNA accumulation and their time courses suggest a po ssible role of one of these induced RNAs in the mediation of the early effect of aldosterone on Na transport.