C.J. Kenyon

The role of glucocorticoid activity in the inheritance of hypertension: Studies in the rat

Young (3-week-old) spontaneously hypertensive rats (SHR) had significantly higher basal plasma corticosterone levels than WKY rats and maximum responses to ACTH were also higher. In isolated adrenocortical cells from these rats, corticosterone production was also more responsive to ACTH in SHR. There was no significant difference in aldosterone production. Mononuclear leucocytes from older (10-week-old) SHR had a higher affinity for dexamethasone but a smaller number of binding sites per cell. The SHR therefore has higher circulating glucocorticoid levels and the target cells have a higher apparent affinity for this agonist. However, the target cells also have a smaller binding capacity. The precise resultant effect of these changes on glucocorticoid activity will require additional studies on specific glucocorticoid-dependent variables.

Regulation of adrenocortical steroidogenesis by benzodiazepines

Benzodiazepines affect steroidogenesis in at least four ways depending on concentration and adrenocortical cell type. Firstly, at micromolar concentrations, they inhibit steroidogenic enzymes. Competition for microsomal 17- and 21-hydroxylase activity explains the inhibition of ACTH-stimulated aldosterone and cortisol synthesis by diazepam and midazolam. At slightly higher concentrations, we have evidence that 11 beta-hydroxylase activity is also inhibited. Secondly, at sub-micromolar concentrations, calcium influx is inhibited. T-type and L-type calcium channels appear to be blocked, this impairs signal response coupling and, in particular, decreases angiotensin- and K(+)-stimulated aldosterone synthesis in zona glomerulosa cells. Thirdly, the mitochondrion of steroidogenic tissues is a sensitive site for the stimulatory effects of benzodiazepines. Aldosterone synthesis from added HDL-cholesterol by cultured bovine zona glomerulosa cells is stimulated by diazepam, RO5-4864 and PK11195. The fourth site of benzodiazepines effect on steroidogenesis is particular to zona glomerulosa cells. In addition to cholesterol side chain cleavage, the final part of the aldosterone biosynthetic pathway, the conversion from deoxycorticosterone is controlled. Although high micromolar concentrations of diazepam appear to be inhibitory, lower nanomolar concentrations stimulate the synthesis of aldosterone from added deoxycorticosterone. In vivo, a fifth site of benzodiazepine activity may influence plasma steroid concentrations. Competition between steroids and benzodiazepines for hepatic clearance enzymes may affect half lives of both drugs and hormones.

Dexamethasone-suppressible hyperaldosteronism. Adrenal transition cell hyperplasia?

Dexamethasone-suppressible hyperaldosteronism is a rare familial syndrome in which hypokalemia, suppression of plasma renin concentration, and elevated aldosterone secretion are corrected by treatment with glucocorticoids. Regulation of adrenocortical function and body electrolytes was studied in two affected brothers. Both were hypertensive (210/128 and 160/106 mm Hg) with hypokalemia (3.3 and 3.5 mM) and low plasma renin concentrations. Aldosterone was elevated intermittently with levels as high as 45 ng/dl (normal range, 4-16 ng/dl). Cortisol concentrations were normal but were correlated with aldosterone levels (r = 0.9 and 0.7). Concentrations of 11-deoxycorticosterone (19 and 21 ng/dl; normal range, 4-16 ng/dl) and 18-hydroxycortisol (1000 and 950 ng/dl; normal range, 34-150 ng/dl) were elevated, and diurnal changes in both were the same as those seen with aldosterone. Infusion of adrenocorticotropic hormone (ACTH) caused exaggerated increases of aldosterone, 11-deoxycorticosterone, and 18-hydroxycortisol; cortisol response was normal. A 4-week trial of dexamethasone normalized blood pressure and caused a natriuresis, a fall in aldosterone, and a rise in plasma renin. Administration of ACTH after dexamethasone treatment again caused exaggerated increases of aldosterone. Aldosterone did not respond to angiotensin II before dexamethasone therapy (r = 0.01), but it showed a normal response after therapy (r = 0.8, p less than 0.01). Neither administration of dopamine (1 microgram/kg/min) nor long-term therapy with bromocriptine (2.5 mg t.i.d. for 4 weeks) affected aldosterone biosynthesis. Thus, loss of dopaminergic inhibition of mineralocorticoid biosynthesis does not account for hyperaldosteronism in this condition.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the benzodiazepines diazepam, des-N-methyldiazepam and midazolam on corticosteroid biosynthesis in bovine adrenocortical cells in vitro; location of site of action

Diazepam and midazolam inhibited cortisol and aldosterone synthesis in bovine adrenal cells in vitro. The biologically active metabolite des-N-methyldiazepam did not. Midazolam was a more potent inhibitor (IC50: 6 micrograms/ml) than diazepam (IC50: 13 micrograms/ml) in ACTH-stimulated cells. Both compounds inhibited steroidogenesis at several points in the biosynthetic chain; the greatest effects were on 17 alpha hydroxylation and 21 hydroxylation. Diazepam had a relatively greater effect on 17 alpha hydroxylation; midazolam on 21 hydroxylation. Both were less potent inhibitors of 11 beta hydroxylation and had little apparent effect on side chain cleavage. Thus microsomal hydroxylation is more vulnerable to benzodiazepines than mitochondrial hydroxylation. It is suggested that the drugs act by competing with steroid mixed function oxidases for cytochrome P-450. The plasma concentrations required for these effects are high in relation to therapeutic levels but may be achieved, for example, during acute infusions or when they are used in combination with imidazole drugs such as cimetidine.

In vivo and in vitro effects of carbenoxolone on glucocorticoid receptor binding and glucocorticoid activity.

Carbenoxolone potentiates the mineralocorticoid activity of endogenous glucocorticoid hormones by inhibiting the enzyme 11 beta-hydroxysteroid dehydrogenase, which converts cortisol and corticosterone to inactive 11-oxo-derivatives. We addressed the question of whether glucocorticoid activity is also affected by carbenoxolone. Using a rat model involving low dose corticosterone treatment, we found that carbenoxolone neither potentiated nor inhibited the modest increases in blood pressure or reductions in weight gain caused by steroid treatment. Other indices of glucocorticoid activity including white blood cell number, thymus weight, and down regulation of the glucocorticoid receptor were unaffected. In vitro studies with liver and kidney cytosol preparations indicated that carbenoxolone did compete for 3H-dexamethasone binding sites. Carbenoxolone was 5-10 times more effective than glycyrrhetinic acid, 20-30 thousand times less effective than dexamethasone, and is therefore, approximately 1000 times less effective than corticosterone. Analysis of dexamethasone-binding curves indicated a single class of receptor. We conclude that carbenoxolone at the dose tested does not have intrinsic glucocorticoid activity in vivo, nor does it modulate the activities of corticosterone. Carbenoxolone binds weakly to the glucocorticoid receptor. It is not clear whether this weak affinity accounts for some or any of the direct in vitro effects of high concentrations of carbenoxolone that others have described.

The role of dopamine in the control of corticosteroid secretion and metabolism

The relation between aldosterone and its trophins is altered by electrolyte status and in some hypertensive conditions in man by a mechanism or mechanisms not understood. Dopamine has been suggested as the agent for the altered sensitivity of plasma aldosterone to angiotensin II based on the results of studies with dopamine itself, both in vivo and in vitro, and with pharmacological agonists and antagonists. The evidence derived from these studies is presented and discussed. Questionable specificity of the agents used makes interpretation difficult. Similarly, dopamine infusion rates used in man and animals have resulted in plasma concentrations far in excess of those found normally and these pharmacological concentrations have been shown to alter both the clearance rate of exogenous angiotensin II, and the pattern of steroid response to ACTH. Direct study of adrenal tissue has provided more promising results. The adrenal cortex possesses specific dopamine receptors and dopamine has been shown to modify aldosterone biosynthesis in vitro. Moreover, dopamine is present in adrenocortical tissue in concentrations in the range calculated to operate the receptors. However, there is, as yet, no evidence that dopamine concentrations change in a physiological meaningful way, for example, during changes in sodium status.

The role of calcium ions in the mechanism of acth stimulation of cortisol synthesis

Removal of free calcium ions from the incubation medium of isolated bovine adrenocortical cells with EGTA reduced basal cortisol synthesis and blocked the effects of ACTH; additional calcium restored normal steroid synthesis. Calcium channel blockers, verapamil and nitrendipine and the calmodulin antagonist, trifluoperazine inhibited ACTH-stimulated cortisol synthesis in a dose-dependent manner (IC50s of 6.2, 10 and 5.2 microM, respectively). Steroidogenic effects of dibutyryl cyclic AMP were prevented with 50 microM verapamil or trifluoperazine. Calcium ionophore A23187 at 1 microM increased cortisol synthesis 2-3 fold which was less than the normal response to ACTH. Stimulatory effects of ionophore and cyclic AMP or ACTH were not additive. ACTH-stimulation of cortisol synthesis appears to involve cyclic AMP-dependent uptake of extracellular calcium ions, possibly by a mechanism requiring calmodulin. Increases in intracellular calcium ions cannot wholly mimic ACTH actions.

Differences in temperature-sensitive receptor binding of glucocorticoids in spontaneously hypertensive and normotensive Wistar-Kyoto rats

Glucocorticoid receptor binding was compared in liver cytosol preparations from spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats using homologous displacement of [3H]dexamethasone. At 5 degrees C, there was no difference in receptor binding affinity or concentration between strains for dexamethasone, corticosterone or aldosterone. At 37 degrees C, affinity for dexamethasone was lower than at 5 degrees C for both rat strains and decreased with time. However, at this higher temperature, binding affinity in the SHR preparation was consistently higher than in the WKY preparation. The WKY preparation had a higher receptor concentration. The rate of dissociation of the [3H]dexamethasone-receptor complex prepared at 5 degrees C and then incubated at 37 degrees C was rapid but not different between strains. A possible explanation of these results is that the relationship of the heat shock proteins to the receptor heterocomplex is different between strains. Evidence exists of a genetic difference in Hsp 70 between SHR and WKY rats, although its cosegregation with blood pressure has not been established.

Stimulation of aldosterone secretion by benzodiazepines in bovine adrenocortical cells

Abstract— Previous studies have indicated that peripheral benzodiazepine receptor (PBR) ligands inhibit aldosterone secretion in isolated adrenal zona glomerulosa cells although positive responses have been demonstrated in other steroidogenic tissues. In the present study, aldosterone secretion was measured in bovine cells after 6 days of primary culture. At this time, basal aldosterone secretion was very low and cells appeared less sensitive to the steroidogenic effects of extracellular [K+] (maximal response required K+ concentration > 32 mmol/L) but were sensitised to angiotensin II (maximal response achieved with 3 nM) when compared with previous studies with freshly isolated cells. Diazepam concentration in the range 0.1 nM to 1 μM increased basal aldosterone secretion, an effect which was not enhanced by pre‐treatment with diazepam. The effects were small compared with those of angiotensin II or K+. Over the same concentration range, diazepam also potentiated the stimulatory effects of sub‐maximally effective concentrations of angiotensin II. When cells were treated with high‐density lipoprotein (HDL‐3) as a source of cholesterol, diazepam and the PBR ligands Ro5‐4864 and PK11195 also stimulated aldosterone secretion at nanomolar concentrations. In addition, the conversion of added 11‐deoxycorticosterone (DOC) to aldosterone was increased by nanomolar concentrations of diazepam and Ro5‐4864 but inhibited by high micromolar concentrations of these drugs (100 μM). We conclude that adrenocortical responses to PBR ligands are complex. At high concentrations, inhibitory effects involving competition for steroidogenic enzymes and calcium channel blockage predominate. At low concentrations, an enhancement of basal, angiotensin‐II and cholesterol‐dependent aldosterone synthesis is revealed which may involve a PBR‐mediated mitochondrial uptake of cholesterol and DOC.

Dantrolene inhibits adrenal steroidogenesis by a mechanism independent of effects on stored calcium release

The muscle relaxant dantrolene has been widely used in signal transduction studies as an inhibitor of intracellular calcium release. However, in vivo studies have shown that the drug may inhibit steroidogenesis by a mechanism which is distinct from its effects on calcium mobilization. Using freshly isolated cells and mitochondria from the outermost regions of bovine adrenal cortex we have shown that dantrolene (0.2 mM) significantly inhibits steroid synthesis stimulated by either angiotensin II (AII) or by addition of various precursors. Our results suggest that dantrolene inhibits the rate-limiting steps of adrenocortical steroidogenesis, i.e. the intramitochondrial conversion of cholesterol to pregnenolone (for both aldosterone and cortisol) and the conversion of corticosterone to aldosterone (for aldosterone), by a mechanism independent from its known effects on calcium release. A possible alternative mechanism may involve direct inhibition of cytochrome P450-dependent hydroxylation reactions.