Search Results

You are looking at 1 - 2 of 2 items for

  • Author: C B Whorwood x
  • Refine by Access: All content x
Clear All Modify Search
Restricted access

C B Whorwood and P M Stewart


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 α1 and β1 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 α1 and β1 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 α1 and β1 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 α1 and β1 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 α1 and β1 mRNA was also completely abolished by ActD and DRB, but not by CHX or by BZ. The half-lives of α1 and β1 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 α1 and β1 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/μg total cell protein to between 275 ± 19 fmol/μg total cell protein (0·1 μm GE) and 78±11 fmol/μg total cell protein (10 μm 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 Gi protein.

Restricted access

M Shimojo, C B Whorwood, and P M Stewart


11β-Hydroxysteroid dehydrogenase (11β-HSD) catalyses the interconversion of biologically active cortisol to inactive cortisone in man, and corticosterone to 11-dehydrocorticosterone in rodents. As such, this enzyme has been shown to confer aldosterone-selectivity on the mineralocorticoid receptor and to modulate cortisol/corticosterone access to the glucocorticoid receptor (GR). Two kinetically distinct isoforms of this enzyme have been characterized in both rodents and man; a low-affinity NADP(H)-dependent enzyme (11β-HSD1) which predominantly acts as an oxo-reductase and, more recently, a high-affinity NAD-dependent uni-directional dehydrogenase (11β-HSD2). In this study we have analysed the expression of both 11β-HSD1 and 11β-HSD2 isoforms in rat adrenal cortex and medulla and have investigated their possible roles with respect to glucocorticoid-regulated enzymes mediating catecholamine biosynthesis in adrenal medullary chromaffin cells.

Using a rat 11β-HSD1 probe and a recently cloned in-house mouse 11β-HSD2 cDNA probe, Northern blot analyses revealed expression of mRNA species encoding both 11β-HSD1 (1·4kb) and 11β-HSD2 (1·9kb) in the whole adrenal. Consistent with this, 11β-dehydrogenase activity (pmol 11-dehydrocorticosterone formed/mg protein per h, mean ± s.e.m.) in adrenal homogenates, when incubated with 50 nm corticosterone in the presence of 200 μm NAD, was 97·0 ± 9·0 and with 500 nm corticosterone in the presence of 200 μm NADP, was 98·0 ± 1·4 11-Oxoreductase activity (pmol corticosterone formed/mg protein per h) with 500 nm 11-dehydrocorticosterone in the presence of 200 μm NADPH, was 187·7 ± 31·2. In situ hybridization studies of rat adrenal cortex and medulla using 35S-labelled antisense 11β-HSD1 cRNA probe revealed specific localization of 11β-HSD1 mRNA expression predominantly to cells at the corticomedullary junction, most likely within the inner cortex. In contrast, 11β-HSD2 mRNA was more abundant in cortex versus medulla, and was more uniformly distributed over the adrenal gland. Negligible staining was detected using control sense probes.

Ingestion of the 11β-HSD inhibitor, glycyrrhizic acid (>100mg/kg body weight per day for 4 days) resulted in significant inhibition of adrenal NADP-dependent (98·0 ± 1·4 vs 42·5 ± 0·4) and NAD-dependent (97·0 ± 9·0 vs 73·2 ± 6·7) 11β-dehydrogenase activity and 11-oxoreductase activity (187·7 ± 31·2 vs 67·7 ± 15·3). However, while levels of 11β-HSD1 mRNA were similarly reduced (0·85 ± 0·07 vs 0·50 ± 0·05 arbitrary units), those for 11β-HSD2 remained unchanged (0·44 ± 0·03 vs 0·38 ± 0·01). Levels of mRNA encoding the glucocorticoid-dependent enzyme phenylethanolamine N-methyltransferase which catalyses the conversion of noradrenaline to adrenaline, were also significantly reduced in those rats given glycyrrhizic acid (1·12 ± 0·04 vs 0·78 ± 0·04), while those for the glucocorticoid-independent enzyme tyrosine hydroxylase (1·9 kb), which catalyses the conversion of tyrosine to DOPA, were unchanged (0·64 ± 0·04 vs 0·61 ± 0·04).

In conclusion, the rat adrenal gland expresses both 11β-HSD1 and 11β-HSD2 isoforms. 11β-HSDl gene expression is localized to the adrenal cortico-medullary junction, where it is ideally placed to regulate the supply of cortex-derived corticosterone to the medullary chromaffin cells. This, together with our in vivo studies, suggests that 11β-HSD1 may play an important role with respect to adrenocorticosteroid regulation of adrenaline biosynthesis. The role of 11β-HSD2 in the adrenal remains to be elucidated.