Characterization of early aldosterone-induced RNAs identified in A6 kidney epithelia

Abstract

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 contribute towards the identification of transcriptionally regulated mediators of this process, we first tested mRNAs of proteins previously suggested to be involved. Epithelia were treated for 1 h with 10(-6 )M aldosterone, a concentration which produces a maximal transport response and at which both mineralo- and glucocorticoid receptors are occupied. Northern blot analysis showed no change in mRNAs of trimeric G protein alpha subunits, calmodulin, and mitochondrial energy metabolism proteins, whereas Na,K-ATPase alpha1 and beta1 subunit mRNAs were slightly increased (1.2- to 1.4-fold). In a second approach, we visualized 5000 cDNA bands generated from A6 RNAs by differential display polymerase chain reaction (PCR). After 1 h of aldosterone treatment, approximately 0.5% of these appeared to be regulated. Four cDNA fragments corresponding to early adrenal-steroid-upregulated RNAs (ASURs) were cloned 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 yeast and prokaryotic permeases, and ASUR5 is the A transcript of Xenopus K-ras2. The rapid inductions of the four ASURs correspond to direct transcriptional effects since they were not inhibited by cycloheximide but were blocked by actinomycin D. The K1/2 values were similar or slightly below those reported for stimulation of Na transport. These characteristics of RNA accumulation and their time courses suggest a possible role of one of these induced RNAs in the mediation of the early effect of aldosterone on Na transport.

Transcriptome of a mouse kidney cortical collecting duct cell line: Effects of aldosterone and vasopressin

“…Therefore, the 4-h time point represents a point of transition between the early and the late responses in which the early and the late sets of genes are potentially coexisting. In favor of this hypothesis, the mRNAs accumulation kinetics of several recently identified early genes reveal (i.e., Sgk) that the mRNA levels remain significantly induced at least 6 h after hormone stimulation (10,11). Second, the major transcriptional effects of vasopressin presumably occur after several hours of hormonal treatment as it was demonstrated for ENaC, CFTR, AQP2, and Na,K-ATPase (3,13,14).…”

“…The transcription-dependent aldosterone-signaling pathway (between receptor and membrane transport effectors) remains poorly understood. Only two gene products (Sgk and K-Ras) have recently been shown to be part of the pathway, but upstream and downstream mediators have not been identified at the molecular level (10,11).…”

Interactions of β and γENaC with Nedd4 Can Be Facilitated by an ERK-mediated Phosphorylation

“…Mutating ␥Thr-623 into an Ala residue significantly decreased incorporation of 32 P into this subunit without affecting labeling by [ 35 S]methionine. In three different experiments using ϳ40 oocytes each, the mutation of Thr-623 lowered the 32 P/ 35 S ratio of the immunoprecipitated subunit by 47 Ϯ 15%. Thus, Thr-623 is one of the ␥ residues phosphorylated in the oocyte system.…”

“…Oocytes labeled with 32 P or 35 S were washed and homogenized in buffer A ϩ protease inhibitors, and membranes were isolated by centrifugation through a sucrose cushion. Membranes were solubilized in 1% Triton X-100 in buffer A and centrifuged for 5 min at 11,000 ϫ g to remove insoluble material.…”

L‐type amino acid transporter 1 as a potential molecular target in human astrocytic tumors

“…3 The database search indicated that partial or incomplete sequences of LAT1 (E16, TA1 and ASUR4b) were already reported. 5,6,11 E16 and ASUR4b were identified to be up-regulated upon the mitogenic stimulation of lymphocytes and the stimulation of A6 epithelial cell line by aldosterone, respectively, 5,11 suggesting highly regulated nature of LAT1 gene expression. TA1 was identified as a tumor-associated sequence with the oncofetal pattern of expression in rat liver.…”