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 m odulation of Na+ transport across renal tubular epithelia. In amphibia n renal cells, aldosterone induction of Na/K-ATPase alpha(1) and beta( 1) subunit gene transcription is mediated by an indirect mechanism dep endent 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 ar e diminished by glycyrrhetinic acid (GE), the active ingredient of lic orice. To investigate the mechanisms underlying the regulation of mamm alian renal Na/K-ATPase, levels of alpha(1) and beta(1) mRNA were meas ured in rat kidney epithelial (NRK-52E) cells treated with a range of concentrations of aldosterone, corticosterone and GE in the presence o f a specific inhibitor of mRNA synthesis, dichlororibofuranosylbenzimi dazole (DRB), an inhibitor of total RNA synthesis, actinomycin D (ActD ), and the protein synthesis inhibitor cycloheximide (CHX). In additio n, GE was co-incubated with the sodium channel antagonist benzamilorid e (BZ). The increase in both alpha(1) and beta(1) mRNA levels followin g 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 complet ely 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 ac companied 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 pro tein 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 abol ished following down-regulation of protein kinase C by prolonged treat ment with the phorbol ester tetradecanoylphorbol-13-acetate (TPA), and by pertussis toxin (PT), but not by cholera toxin (CT). Indeed, subun it mRNA levels were increased by 8-bromo-cAMP (2.2-fold) and stimulato rs of adenylate cyclase activity, i.e. forskolin (2.1-fold), PT (2.1-f old) 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 p athways via interaction with a PT-sensitive G(i) protein.