TRANSCRIPTIONAL REGULATION OF NA/K-ATPASE BY CORTICOSTEROIDS, GLYCYRRHETINIC ACID AND 2ND-MESSENGER PATHWAYS IN RAT-KIDNEY EPITHELIAL-CELLS

Corticosteroid regulation of Na/K-ATPase is of key importance in the modulation of Na+ transport across renal tubular epithelia. In amphibian renal cells, aldosterone induction of Na/K-ATPase alpha(1) and beta(1) subunit gene transcription is mediated by an indirect mechanism dependent on the synthesis of a labile protein. In mammalian target cells, while both mineralo- and glucocorticoids increase the levels of Na/K-ATPase alpha(1) and beta(1) subunit mRNA and enzyme activity, they are diminished by glycyrrhetinic acid (GE), the active ingredient of licorice. To investigate the mechanisms underlying the regulation of mammalian renal Na/K-ATPase, levels of alpha(1) and beta(1) mRNA were measured in rat kidney epithelial (NRK-52E) cells treated with a range of concentrations of aldosterone, corticosterone and GE in the presence of a specific inhibitor of mRNA synthesis, dichlororibofuranosylbenzimidazole (DRB), an inhibitor of total RNA synthesis, actinomycin D (ActD), and the protein synthesis inhibitor cycloheximide (CHX). In addition, GE was co-incubated with the sodium channel antagonist benzamiloride (BZ). The increase in both alpha(1) and beta(1) mRNA levels following aldosterone and corticosterone was completely abolished by treatment with ActD and DRB, while CHX did not affect this response. Similarly, the GE-induced decrease in alpha(1) and beta(1) mRNA was also completely abolished by ActD and DRB, but not by CHX or by BZ. The half-lives of alpha(1) and beta(1) mRNA in these cells 1 (means +/- S.E.M., n=4), estimated from the rate of mRNA decay in the presence of DRB, were 6.8 +/- 0.3 and 4.8 +/- 0.2 h respectively. This was unaffected by GE. The inhibitory action of GE on alpha(1) and beta(1) mRNA levels was accompanied by a dose-dependent decrease in levels of intracellular cAMP (means +/- S.E.M., n=4) from 395 +/- 28 fmol cAMP/mu g total cell protein to between 275 +/- 19 fmol/mu g total cell protein (0.1 mu M GE) and 78 +/- 11 fmol/mu g total cell protein (10 mu M GE). This was abolished following down-regulation of protein kinase C by prolonged treatment with the phorbol ester tetradecanoylphorbol-13-acetate (TPA), and by pertussis toxin (PT), but not by cholera toxin (CT). Indeed, subunit mRNA levels were increased by 8-bromo-cAMP (2.2-fold) and stimulators of adenylate cyclase activity, i.e. forskolin (2.1-fold), PT (2.1-fold) and CT (1.9-fold), but not by TPA. In keeping with their effects on GE inhibition of cAMP synthesis, TPA and PT (but not CT) abolished the GE-induced decrease in subunit mRNA. In conclusion, corticosteroid induction and GE inhibition of Na/K-ATPase subunit gene expression in rat kidney epithelial cells occur at the transcriptional level and do not require de novo synthesis of an intermediary protein. Furthermore, GE attenuation of subunit gene transcription may be mediated by both cAMP-dependent protein kinase A and diacylglycerol-protein kinase C pathways via interaction with a PT-sensitive G(i) protein.