Transcriptional regulation of Na/K-ATPase by corticosteroids, glycyrrhetinic acid and second 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 (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/microgram total cell protein to between 275 +/- 19 fmol/micrograms total cell protein (0.1 microM GE) and 78 +/- 11 fmol/micrograms total cell protein (10 microM 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.(ABSTRACT TRUNCATED AT 400 WORDS)
University of Birmingham Department of Medicine, Queen Elizabeth Hospital, Edgbaston, UK.
C B
CB
Whorwood
P M
PM
Stewart
D000305:Adrenal Cortex Hormones / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D005786:Gene Expression Regulation; D006034:Glycyrrhetinic Acid / Q000494:pharmacology*; D007668:Kidney / Q000166:cytology / Q000201:enzymology*; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015290:Second Messenger Systems; D015398:Signal Transduction / Q000187:drug effects; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*
D000305:Adrenal Cortex Hormones; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D000242:Cyclic AMP; D000254:Sodium-Potassium-Exchanging ATPase; D006034:Glycyrrhetinic Acid