Glucocorticoids decrease endothelin-A- and -B-receptor expression in the kidney.

Glucocorticoids play an important role in circulatory homeostasis and in excess they cause hypertension. These corticosteroids affect the expression of many genes involved in blood pressure control including preproendothelin-1 (PPET-1), the precursor of endothelin-1 (ET-1), a potent vasoactive peptide. We have previously shown that glucocorticoids increase PPET-1 mRNA levels in rat aorta. Moreover, they also affect ET(A)- and ET(B)-receptor expression in various in vitro and in vivo situations. Both ET-1 and glucocorticoids exert direct effects in the kidney and are involved in vascular resistance and sodium balance. We therefore sought to determine the effects of glucocorticoids on renal PPET-1, ET(A)- and ET(B)-receptor expression in an animal model of glucocorticoid-induced hypertension. Wistar rats were given 2.5 mg/l of dexamethasone, a glucocorticoid agonist, in their drinking water for 1 or 5 days. Our data reveal that dexamethasone administration increases systolic blood pressure (SBP) in rats. SBP rose from 120 +/- 3 to 139 +/- 4 and 150 +/- 5 mmHg after 1 and 5 days treatments, respectively (p < 0.05). Furthermore, dexamethasone administration decreased ET(A) and ET(B)-receptor expression in the rat kidney. This effect was observed after 1 day of dexamethasone treatment with ET(A) and ET(B)-receptor mRNA levels decreasing to 83 +/- 2% and 80 +/- 5% of control values. respectively (p < 0.01). Both ET(A)- and ET(B)-receptor mRNA levels further declined to 67 +/- 3 and 65 +/- 6% of control values after 5 days of dexamethasone treatment, respectively (p < 0.001). Interestingly, kidney PPET-1 expression was not affected by dexamethasone administration. Our results suggest a contribution of renal ET receptors in glucocorticoid actions on blood pressure control.

Transcriptional regulation of pre-pro-endothelin-1 gene by glucocorticoids in vascular smooth muscle cells.

Endothelin is a potent vasoactive peptide involved in the maintenance of vascular tone and in pathophysiological states. Endothelin-1 synthesis is controlled at the transcriptional level. We report that glucocorticoids increase the pre-pro-endothelin-1 gene transcription rate in vascular smooth muscle cells. The effect of glucocorticoids is dose-dependent (EC50 approximately 2-3 nM) and completely blocked by co-incubation with the glucocorticoid antagonist RU 38486. The rise in pp-Et-1 steady state mRNA levels is rapid and transient with a maximal three-fold stimulation within one hour of glucocorticoid administration. Glucocorticoid treatment does not affect the half-life of pre-pro-endothelin-1 mRNA as shown by actinomycin D studies. Furthermore, cycloheximide treatment concomitantly with RU 28362 did not reverse the stimulatory effect of glucocorticoids on pre-pro-endothelin-1 mRNA levels. Nuclear run-on analysis shows that glucocorticoids increase the transcription rate of pre-pro-endothelin-1. Our results suggest a role for glucocorticoids in the regulation of biosynthesis and action of this important vasoactive peptide in vascular smooth muscle cells.

Glucocorticoids increase preproendothelin-1 expression in rat aorta.

Glucocorticoids play an important role in circulatory homeostasis. They modulate peripheral resistance by their direct action on blood vessels. We have previously shown that glucocorticoids increase preproendothelin-1 gene transcription rate in vascular smooth muscle cells (Morin C et al. 1998, BBRC 244:583). Endothelin-1 is a potent vasoactive peptide involved in the maintenance of vascular tone as well as in pathophysiological states. In this report we show that glucocorticoids increase preproendothelin-1 expression in rat aorta. The rise in preproendothelin-1 steady state mRNA level is dose-dependent with a maximal 2.3-fold stimulation 8 hours following a 0.35 and a 2.5 mg/kg dexamethasone injection. A lower dose of dexamethasone (0.13 mg/kg) also increased preproendothelin-1 expression in rat aorta. Since endothelin-1 levels are regulated at the transcriptional step, the increase in mRNA is likely to be paralleled by increased protein synthesis and vasoconstrictor action. Moreover, systolic blood pressure was significantly elevated in rats who received the 0.35 and 2.5 mg/kg doses of dexamethasone. Together, our results strongly suggest that the increase in preproendothelin-1 expression by glucocorticoids contributes to the regulatory action of these corticosteroids on blood pressure in health and disease.

Glucocorticoids but not mineralocorticoids modulate endothelin-1 and angiotensin II binding in SHR vascular smooth muscle cells.

Both glucocorticoids and mineralocorticoids are involved in circulatory homoeostasis and blood pressure control. In recent years direct effects of both steroid classes on vascular smooth muscle cells (VSMC) have been reported. We have thus examined the effects of RU 28362, a pure glucocorticoid agonist, and aldosterone, the physiologic mineralocorticoid, on the binding to VSMC from spontaneously hypertensive rats (SHR) of two key vasoactive peptides, endothelin-1 and angiotensin II. Binding of angiotensin II rose, and that of endothelin-1 declined, in a time- and dose-dependent fashion with maximal effects observed at 24 h and half-maximal effects for each at 2-3 nM RU 28362. Scatchard analysis showed that for both endothelin-1 and angiotensin II, RU 28362 alters receptor number but not affinity; competition studies with receptor-selective ligands (BQ123, S6C, DuP753 and PD123319) show that glucocorticoids specifically elevate (X2) AT-1 receptors and specifically lower (to approximately 30%) levels of ETA receptors. Treatment of VSMC with the antiglucocorticoid RU 38486 reversed the effect of glucocorticoids on endothelin-1 and angiotensin II binding, confirming the Type II (glucocorticoid) receptor mediated effect of the glucocorticoids. Aldosterone (100 nM) also lowers endothelin-1 binding and increases angiotensin II binding in VSMC; that this effect reflects aldosterone occupancy of classical glucocorticoid receptors is shown by the blockade of the aldosterone effect by an equal concentration (100 nM) of RU 38486--i.e. there is no evidence for an action of aldosterone via mineralocorticoid receptors. We interpret our results as evidence for a complex modulation of receptors for vasoactive peptides in VSMC by glucocorticoid but not mineralocorticoid hormones.

Regulation of 21-hydroxylase activity by steroids.

In this study, we investigated the effect of steroids on guinea pig and bovine adrenal steroidogenesis, especially 21-hydroxylase activity. Analysis of guinea pig adrenal steroids indicated the presence of high concentrations of androstenedione in the guinea pig adrenal; furthermore, in vitro studies using guinea pig adrenal cortex cells in primary culture confirmed that androstenedione is one of the major C19 steroids produced and secreted. The direct action of steroids on steroid production by adrenal cells was investigated. Our data indicate that steroids themselves increase C19 steroid synthesis and inhibit glucocorticoid production by guinea pig adrenal cells without affecting gene expression for steroidogenic enzymes. Incubation of a series of C19 steroids, namely, androstenedione, with guinea pig adrenal cell cultures demonstrated that the decrease in 21-hydroxylase activity is largely independent of the androgenic activity of C19 steroids. RU38486, a synthetic C18 steroid possessing a 4-ene-3-ketosteroid with an aryl group at position 11 and a very low affinity for the androgen receptor, also irreversibly altered 21-hydroxylase activity. An effect of RU38486 on 21-hydroxylase activity was also demonstrated in bovine adrenal cells. Further studies with bovine adrenal cells showed that the decrease in 21-hydroxylase activity induced by RU38486 was accompanied by a small but significant inhibition of P450c21 protein levels at both basal and ACTH-stimulated levels. In summary, our data indicate that alteration of 21-hydroxylase activity by steroids is likely due to a direct action on P540c21 protein, and the levels of androstenedione in the adrenal are high enough to inhibit 21-hydroxylase activity.

Isozymes of 11 beta-hydroxysteroid dehydrogenase: which enzyme endows mineralocorticoid specificity?

Intracellular enzymes which interconvert circulating hormones between active and inactive forms aid in regulating the biological activity of the ligand in a cell-specific manner. This is particularly important in mineralocorticoid target tissues where glucocorticoids and mineralocorticoids have equivalent affinity for the mineralocorticoid receptor. Inactivation of glucocorticoids at the 11-hydroxyl position by the action of 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) permits the occupation of the mineralocorticoid receptor by aldosterone in the presence of much higher levels of circulating cortisol. The suppression of dehydrogenase activity allows glucocorticoids to activate the mineralocorticoid receptor, leading to classical mineralocorticoid type effects such as sodium retention and potassium excretion. A number of 11 beta-OHSDs are currently candidate protectors of the mineralocorticoid receptor. This review examines the attributes of these 11 beta-hydroxysteroid dehydrogenase isozymes and suggests reasons why a high affinity, NAD-dependent enzyme appears to be the most likely candidate to endow specificity on the mineralocorticoid receptor.

Studies of adrenal steroidogenic enzymes in guinea pigs.

In the present study we found that 3 beta-hydroxysteroid dehydrogenase 4-ene-5-ene-isomerase (3 beta-HSD), 17-hydroxylase and 17,20-lyase (P-450c17), and 21-hydroxylase (P-450c21) activities in a suspension of cells from guinea pig zona reticularis (RE) were 10- to 15-fold less than those measured in cells from zona fasciculata-glomerulosa (FG). Whereas the secretion of cortisol and C-19 steroids was remarkably increased during treatment of FG cells with adrenocorticotropic hormone (ACTH), no response could be detected when using cells from zona RE. By contrast, the measurement of a series of C-21 and C-19 steroids shows that the concentrations of several steroids were greater in the zona RE than in the zona FG. In addition, using Northern blot analysis, we have observed that the basal steady-state levels of mRNA for cholesterol side-chain cleavage (P-450scc), 3 beta-HSD, P-450c21, P-450c17, and P-450c11 were in the same range in the two zones and an administration of ACTH caused, in both zona FG and zona RE, a two- to threefold decrease in P-450c17 and P-450c21 steady-state mRNA levels, whereas P-450c11, 3 beta-HSD, and P-450scc steady-state mRNA levels remained unchanged. Our data suggest the presence of some factor(s) capable of rapidly deactivating the steroidogenic enzymes in the zona RE.

The heterogeneity of 11-beta-hydroxysteroid dehydrogenase activities in the rat hippocampus implies a complex regulation of steroid hormone action.

1. The rat hippocampus 11-beta-hydroxysteroid dehydrogenase (11-HSD) displays a different substrate specificity to that of other tissues. S1 nuclease analysis was used to determine whether the hippocampal messenger RNA is different from that found in other tissues. 2. S1 nuclease analysis using probes spanning the full length cDNA demonstrated that there were no differences in sequence between the hippocampal 11-HSD and the enzyme originally cloned from the liver. 3. These results suggest that there may be multiple 11-HSD isoforms in the hippocampus with different substrate specificities.

Pregnenolone fatty acid esters incorporated into lipoproteins: substrates in adrenal steroidogenesis.

The presence of fatty acid ester derivatives of pregnenolone (PFA) has been recently reported in guinea pig plasma and adrenals. Moreover, it has equally been demonstrated that PFA is present in plasma lipoproteins, including high density lipoproteins (HDL) and low density lipoproteins (LDL). We report here the conversion of LDL- and HDL-incorporated [3H]PFA in guinea pig and bovine adrenocortical cells into nonconjugated steroids, thus indicating that PFA can be used as substrate for steroid production. When guinea pig glomerulosa-fasciculata (FG) cells were incubated for 48 h in the presence of either LDL-incorporated [3H]PFA or HDL-incorporated [3H]PFA, 66 +/- 2% and 47 +/- 1% of the added radioactivity were converted to nonconjugated steroids, respectively. The transformation of LDL-incorporated [3H]PFA into nonconjugated steroids was further increased in FG cells when 10 nmol/liter ACTH were present in the culture medium. This ACTH-induced liberation of nonconjugated steroids from LDL-incorporated [3H]PFA was strongly inhibited by a 10-fold excess of unlabeled LDL, while no competitive effect of unlabeled HDL was observed on the generation of tritiated nonconjugated steroids from HDL-incorporated [3H]PFA. Moreover, nonconjugated metabolites formed during incubation of FG cells with LDL-incorporated [3H]PFA and HDL-incorporated [3H]PFA resembled those observed when FG cells were incubated with tritiated pregnenolone. The formation of nonconjugated steroids was similarly observed when lipoprotein-incorporated [3H]PFA was incubated with bovine adrenocortical cells. Our data suggest that [3H]PFA incorporated into lipoproteins can be internalized into adrenal cells, after which the [3H]PFA is hydrolyzed into nonconjugated pregnenolone, which is readily used as substrate for adrenal steroidogenesis.

Effects of C19 steroids on adrenal steroidogenic enzyme activities and their mRNA levels in guinea-pig fasciculata-glomerulosa cells in primary culture.

The present study examined the effects of steroids on steroidogenic enzyme activity in adrenal glands. Guinea-pig fasciculata-glomerulosa (FG) cells maintained in primary culture were exposed to steroids for 48 h. Although the treatment with androstenedione alone had no effect on 3 beta-hydroxysteroid dehydrogenase 4-ene-5-ene-isomerase (3 beta-HSD), 17-hydroxylase and 17,20-lyase activities, there was inhibition of 11-hydroxylase and 21-hydroxylase activities. When FG cells were exposed to 10 nmol ACTH/l for the last 24 h of incubation, ACTH alone had no effect on steroidogenic enzymes but, while combined with androstenedione, it further decreased 21-hydroxylase activity and stimulated 17-hydroxylase and 17,20-lyase activities. Cortisol, corticosterone, oestradiol and 11 beta-hydroxy androstenedione had no effect on steroidogenic enzyme activities while the inhibitory effect on 21-hydroxylase activity was only observed with androstenedione, testosterone and dihydrotestosterone. Addition of hydroxyflutamide, a pure antiandrogen, did not block the inhibitory effect of androstenedione on 21-hydroxylase and 11-hydroxylase activities. The reduction in oxygen tension from 19 to 2% which was aimed at examining the oxygen-mediated effects on steroidogenic enzymes, revealed that the reduction in 21-hydroxylase activity induced by androstenedione could not be prevented by low oxygen tension. An interaction of C19 steroids at the level of the enzymes is also suggested by our finding that androstenedione had no effect on basal and ACTH-stimulated steady-state 11-hydroxylase, 17-hydroxylase, 17,20-lyase and 21-hydroxylase mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

Effect of chronic ACTH treatment on guinea-pig adrenal steroidogenesis: steroid plasma levels, steroid adrenal levels, activity of steroidogenic enzymes and their steady-state mRNA levels.

We report here the effects of a 7-day treatment of guinea-pigs with ACTH on adrenal mRNA levels for steroid-transforming enzymes. Adrenal 3 beta-hydroxysteroid dehydrogenase 4-ene-5-ene-isomerase (3 beta-HSD), 17-hydroxylase, 17,20-lyase, 21-hydroxylase and 11-hydroxylase activities were also examined as well as plasma and adrenal steroid levels. Our data reveal that chronic ACTH-treatment stimulated all post-pregnenolone enzyme activities in glomerulosa-fasciculata cells. Plasma steroid levels increased 8 h after the last injection of ACTH and returned to the control levels 24 h later whereas, in the adrenal, the content in steroids in the group sacrificed 8 h after the last injection of ACTH were similar to the values of the control group and decreased markedly 24 h later. It is suggested that the steroid turn-over in the adrenal may be affected by the chronic ACTH-treatment. On the other hand, despite the significant stimulation in steroid-transforming enzyme activities, our data reveal that chronic ACTH administration caused a decrease in mRNA levels for P450c21 and P450c17 while P450scc, 3 beta-HSD and P450c11 remained unchanged. Taken together, these results suggest that in vivo chronic ACTH-treatment of guinea-pigs increases adrenal steroidogenic capacity by increasing steroid secretion and steroid enzyme activity. Moreover, the chronic treatment with ACTH may have a post-transcriptional effect on steroidogenic enzymes gene expression by affecting the half-life of their mRNAs.

Effect of ACTH on steroidogenic enzymes in guinea pig fasciculata-glomerulosa cells: changes in activity and mRNA levels.

Adrenocorticotropin (ACTH) is known to exert an acute effect on adrenal steroidogenesis as well as long-term effects by regulation of gene expression. In order to further study the long-term action of ACTH, guinea pig fasciculata-glomerulosa (FG) cells in primary culture were treated for up to 72 h with ACTH. The effects of this treatment on steroid secretion, enzyme activity and mRNA levels for steroid enzymes were measured. While the rate of 17-deoxy C-21 steroid secretion decreased over the 72-h period of incubation with ACTH, the 17-hydroxy C-21 steroid secretion rate remained constant for the first 24 h of incubation and declined thereafter; the rate of 4-ene C-19 steroid secretion increased over the 72-h incubation period. ACTH treatment increased 17-hydroxylase and 17,20-lyase activities and the maximal stimulation was reached after 48 h. In contrast, the activity of 21-hydroxylase (P450c21) steadily declined over the 72-h incubation period. ACTH also caused an increase in mRNA levels for P450c21, 17-hydroxylase and 17,20-lyase (P450c17), 3 beta-hydroxysteroid dehydrogenase 4-ene-5-ene-isomerase (3 beta-HSD) and cholesterol side-chain cleavage enzyme (P450scc). The maximal stimulation for the four mRNAs was observed after 18 h of incubation with ACTH, decreasing afterwards except for P450c17 mRNA levels which remained elevated over the 72-h incubation period. Despite the increase in mRNA levels for 3 beta-HSD and P450c21, no increase in their respective enzyme activities was observed and 21-hydroxylase activity even declined over the 72-h incubation period with ACTH, thus suggesting that mechanism(s) other than gene expression alone regulate steroid secretion in FG cells. In conclusion ACTH caused major changes in steroid distribution due to increased 17-hydroxylase and 17,20-lyase activities and decreased 21-hydroxylase activity in FG cells in culture. Moreover, our data revealed major differences in the induction of mRNAs for steroidogenic enzymes and their activities following ACTH treatment.

Steroidogenesis in guinea pig adrenal cortex: effects of ACTH on steroid secretion and steroidogenic enzyme activities and expression.

In this study, we investigated guinea pig adrenal steroidogenesis, specially, C19 steroid production. Analysis of adrenal steroids by high performance liquid chromatography and gas chromatography indicated the presence of androstenedione and 11ß-hydroxyandrostenedione. Adrenal androstenedione and 11ß-hydroxyandrostenedione levels were stimulated by ACTH administration while only 11ß-hydroxyandrostenedione was increased in plasma. In vitro studies using adrenal cortex cells in primary culture confirmed that 11ß-hydroxyandrostenedione is the major C19 steroid secreted. The chronic treatment of guinea pig with ACTH stimulated all adrenal post-pregnenolone enzyme activities and decreased P 450c17 mRNA levels while P 450scc, 3ß-hydroxysteroid dehydrogenase and P450c11 mRNAs remained unaffected. Treatment of adrenal cells in primary culture with ACTH for 72 h changed the distribution of steroids secreted and decreased 21-hydroxylase activity while 17α-hydroxylase and 17,20-lyase activities were increased favoring C19 steroid production. In ACTH-treated cells, the mRNA levels for P450c21 and P450c17 increased and reached a peak at 18 h. Our data indicate that treatment with ACTH stimulates adrenal steroidogenic capacity by increasing steroid secretion and causes transcriptional and post-transcriptional effects on steroidogenic enzymes gene expression. Finally, the direct action of steroids on steroid production by adrenal cells in primary culture was investigated. Our data indicate that steroids themselves increase C19 steroid synthesis and inhibit glucocorticoid production without affecting gene expression for steroidogenic enzymes.

Effects of RU 486 on adrenal steroidogenesis in the guinea-pig.

Recent reports have shown that RU 486, a synthetic glucocorticoid and progestin antagonist, has direct effects on tissues secreting steroids. In order to characterize the effects of RU 486 on steroidogenesis further, guinea-pig fasciculata-glomerulosa (FG) cells in primary culture were treated for 48 h with RU 486. RU 486 caused an alteration of basal as well as ACTH-stimulated steroid secretion. Corticosterone and cortisol secretion decreased by 50% while the secretion of 17-hydroxyprogesterone and C19 steroids were increased. The activity of steroidogenic enzymes was measured using tritiated steroids. In RU 486-treated cells, the activity of 21-hydroxylase was dramatically inhibited while there was an increase in 17-hydroxylase and 17,20-desmolase activities. The effects of RU 486 on enzyme activities were dependent upon dose and time. The effects of the compound were not reversed by concomitant treatment of FG cells with R-5020 or dexamethasone, thus suggesting that RU 486 acted directly on steroidogenic enzymes to alter their activity.