Aldosterone and corticosterone binding and effects on Na+ transport in cultured kidney cells

Abstract

A continuous line of cells (A6) derived from toad kidney has been shown to form epithelia in culture that manifest aldosterone-stimulatable transepithelial sodium transport. In this study an efficient filtration assay for nuclear binding of [3H]aldosterone was validated. Specific high-affinity aldosterone and corticosterone binding sites in the particulate (nuclear-enriched) fraction were characterized in intact epithelia. Despite metabolism of both steroids, two high-affinity binding sites for each were demonstrable: aldosterone, K'd1 = 0.85 (+/- 0.19) X 10(-10) and K'd2 = 1.6 (+/- 0.42) X 10(-8) M; corticosterone, K'd1 = 0.5 (+/- 0.31) X 10(-10) and K'd2 = 0.32 (+/- 0.19) X 10(-8) M. Analogue competition-binding studies indicated a qualitative difference in the two sites and co-occupancy of both sites by the two steroids. The sodium transport response to aldosterone and corticosterone approximated a linear function of occupancy of the lower affinity sites. Although the lower affinity sites resemble mammalian glucocorticoid receptors in terms of relative binding affinities for analogues, we conclude that they are the receptors which mediate the aldosterone and corticosterone stimulation of Na+ transport in these epithelia.

Aldosterone increases the apical Na+ permeability of toad bladder by two different mechanisms.

“…In these cell lines, however, the relative increase of I,, closely resembled occupancy of the type II receptor (28,29). These discrepancies may be accounted for by assuming that the early and late apical processes postulated in this study are mediated by type I and type II receptors, respectively.…”

“…It has been reported (15) that both the initial activation of Na+ channels (measured as a decrease of the tissue electrical resistance) and the enhanced induction of Na+/K+-ATPase correlate with the occupancy ofa type I receptor whose Kd kidney (A6) and toad bladder (TB6-C) cells as well (28,29).…”

Regulation of epithelial Na+channels by adrenal steroids: mineralocorticoid and glucocorticoid effects

“…In the toad urinary bladder, ϳ30% of the response to a high dose of aldosterone in vitro was attributable to occupancy of GR (20). The A6 cell line from the Xenopus laevis kidney responds to steroids mainly through GR (32,36). However, transfection of the cells with MR can rescue the effects of low doses of aldosterone (7).…”

Epithelial sodium channel regulated by aldosterone-induced protein sgk

“…Methods) was used to generate a library of partial-length cDNAs representing rapidly induced mRNAs in A6 cells (12). After an initial screen for false positives (see Materials and Methods), selected library cDNAs were sequenced, and one of the clones, A83, had 92% amino acid identity (96% similarity) with rat sgk (serum and glucocorticoid-regulated kinase), a recently cloned member of the serine-threonine protein kinase family (16).…”

“…We, therefore, wanted to identify aldosterone-induced genes whose products might stimulate ENaC activity. Toward this end, we used a PCRbased technique to generate a cDNA library representing rapidly induced genes in A6 cells, a CD-like cell line derived from Xenopus laevis kidney (12). We then characterized the effect of selected clones on ENaC activity in a coexpression assay.…”