Steroid antagonism of the hypertensinogenic activity of adrenocortical steroids

Previous studies in sheep have provided evidence for a separate "hypertensinogenic" class of adrenocortical steroid activity which is not simply related to their classical mineralocorticoid (MC) and/or glucocorticoid (GC) actions. This study investigated the structure-activity relationships of the effects of structural analogues of prednisolone on mean arterial pressure (MAP), and MC and GC actions in sheep. Infusions of these synthetic GC at 0.6 and 24 mg/day produced variable pressor effects which were dissociated from their MC and GC actions. In other experiments, the minimum adrenocortical steroid requirement to reproduce the onset of ACTH-dependent hypertension was determined. Infusion of cortisol, aldosterone, 17 alpha-hydroxy progesterone and 17 alpha,20 alpha-dihydroxy-4-pregnene-3-one was found to be sufficient to reproduce the hypertensive response to ACTH administration in sheep. A subsequent experiment showed that substitution of cortisol by the more potent synthetic GC, prednisolone had no effect on MAP. Therefore, cortisol appears to exert an essential action in ACTH hypertension which is not dependent on its GC activity. Other studies have found that prednisolone (100 mg/day) antagonized 9 alpha-fluoro-prednisolone (0.6 mg/day) induced hypertension but not its MC effects. The effect of progesterone (500 mg/day) and the progesterone analogues, norethisterone, medroxy-progesterone and 16 alpha-methyl progesterone on ACTH (5 micrograms/kg per day) hypertension was investigated. Progesterone completely blocked the hypertension and MC effects of ACTH infusion, while medroxy-progesterone partially blocked the increase in MAP. These data support our concept of a "hypertensinogenic" class of steroid activity.     

Steroid antagonism of the ‘hypertensinogenic’ activity of 9α-fluoroprednisolone

We have previously reported that adrenocortical steroids raise blood pressure by a ‘hypertensinogenic’ mechanism of action which is not simply related to their classical ‘mineralocorticoid’ or ‘glucocorticoid’ actions. This study presents evidence for specific antagonism of this ‘hypertensinogenic’ activity. The effects of separate IV infusions of prednisolone (P) 100 mg/d and 9α-fluoro-prednisolone (9αF-P) 0.6 mg/d on mean arterial pressure (MAP), plasma [K], plasma [glucose] and urinary NA excretion (UNaV) after 2 days were studied in sheep. In the same group of sheep which received P alone for 2 days, 9αF-P was given for a further 2 days while continuing the P infusion (P + 9αF-P). P alone had no effect on MAP or plasma [K] or U_NaV but increased plasma [glucose], effects which are characteristic of ‘glucocorticoid’ activity. 9αF-P alone increased MAP by 14 mmHg (P<0.001) and reduced plasma [K] and U_NaV but had no effect on plasma [glucose]. Thus 9αF-P exhibited both ‘hypertensinogenic’ and ‘mineralocorticoid’ activity. In the sheep which received the combined P + 9αF-P infusion, the increase in MAP normally produced by 9αF-P was blocked. Although pretreatment with P blocked the pressor effect of 9αF-P, it did not alter the ‘mineralocorticoid’ effects, namely hypokalaemia and urinary Na retention, produced when 9αF-P was infused alone. These results provide further evidence for our concept of a ‘hypertensinogenic’ class of steroid activity and are the first demonstration of specific antagonism of steroid induced hypertension.     

Paradoxical involvement of glucocorticoid receptors in the aldosterone-induced impairment of ACTH secretion from perifused pituitary glands

The present investigation was aimed at examining whether interaction of aldosterone with specific mineralocorticoid receptors at the level of the pituitary gland may account for the inhibitory effect of that steroid on ACTH secretion. By using pituitaries from neonatal rats, which we show to completely lack specific mineralocorticoid receptors but to contain a functional glucocorticoid receptor system, we demonstrated the persistence of aldosterone-induced inhibition of ACTH release from perifused glands. Conversely, when the glucocorticoid receptors sites were blocked in pituitaries from mature rats by means of a potent antiglucocorticoid (RU 38486), thus leaving unaltered mineralocorticoid binder, aldosterone no longer dampened hormonal output. We conclude that the latter steroid affected corticotropic activity by interacting not with its proper and specific receptor, but rather with the glucocorticoid binding sites.     

Tritiated 9α-fluorocortisol metabolism and binding in rat kidney

Metabolism of 9α-fluorocortisol (9α-F) has been studied in rat kidney slices, homogenate, and in the isolated perfused kidney. These studies show that the rate of 9α-F metabolism varies depending upon the experimental conditions. The major metabolite formed, identified by mass spectrometry, is 20(ξ)-dihydro-9α-fluorocortisol. The kidney slice experiments show that only ³H-9α-F and none of the metabolites bind to cytosolic receptors. In competition experiments performed with tritiated and unlabeled 9α-fluorocortisol, aldosterone (A), dexamethasone (DM) and triamcinolone acetonide (TA), 9α-F was found to bind to mineralocorticoid receptors with a lower affinity than A but also to glucocorticoid receptors with a higher affinity than A. The Scatchard plot analysis indicated that ³H-9α-F is characterized by K_D: 8.6 × 10^?9M and N: 1.9 × 10^?13moles/mg of protein. In conclusion it is felt that 9α-F would not be a better “marker” than aldosterone for the renal mineralocorticoid receptors.     

Induction of 5-aminolevulinate synthase by activators of steroid biosynthesis

Different cytochromes P450 are involved in steroid biosynthesis. These cytochromes have heme as the prosthetic group. We previously reported that ACTH, an activator of glucocorticoid biosynthesis in adrenal, requires heme biosynthesis for a maximal response. In the present study, we investigated the effect of ACTH, and the effect of two activators of the adrenal mineralocorticoid synthesis, endothelin-1 and low sodium diet on 5-aminolevulinate-synthase (ALA-s) mRNA. ALA-s is the rate-limiting enzyme in heme biosynthesis. It was found that infusion of rats with ACTH for 1 h caused an increase of adrenal ALA-s mRNA and activity accompanied by an increase in plasma corticosterone. CYP21, a cytochrome involved in the synthesis of both corticosterone and aldosterone, was not modified at the RNA level in adrenal glands by 1 h of ACTH infusion. Consistently, infusion of endothelin-1 for 1 h increased ALA-s mRNA and aldosterone content in adrenal gland without modifying CYP21 mRNA levels. To study if ALA-s is also regulated by the main physiological stimuli that increase adrenal mineralocorticoid secretion, we fed rats with low salt diet for 2 or 15 days. Low salt diet treatment increased adrenal gland ALA-s mRNA levels. On the other hand, the rapid stimulation of ALA-s mRNA by ACTH which acts through cyclic AMP was confirmed in H295R human adrenocortical cells, the only human adrenal cell line that has a steroid secretion pattern and regulation similar to primary cultures of adrenal cells. Our findings suggest that the acute activation of adrenal steroidogenic cytochromes by trophic hormones involves an increase in heme biosynthesis which will favor the production of active cytochromes.     

Effects of ACTH on plasma levels of adrenal steroids in essential hypertension.

Plasma concentrations of progesterone (P), deoxycorticosterone (DOC), 17-hydroxyprogesterone (17-OH P), corticosterone (B), deoxycortisol (S), cortisol (F) and aldosterone (A) in 8 control subjects (mean age: 40.5 years) and 10 patients with essential hypertension (EH) (mean age: 48.5 years) were determined before, 4 and 8 hours after an infusion of ACTH at a rate of 25 units per 8 hours. Secretion rates (SR) of 18-hydroxy-11-deoxycorticosterone (18-OH DOC) were measured 24 hours before and again on the day of ACTH infusion. All subjects were studied on the fourth day of a diet containing 135 mEq of sodium and 90 mEq of potassium. There was no statistically significant difference between 8 control subjects and 10 patients with EH in the 7 plasma steroid levels and the SR of 18-OH DOC before ACTH infusion. The mean plasma P response to ACTH was slightly lower in controls than in patients with EH, while that of 17-OH P (in male subjects) was slightly higher. The mean plasma B response was significantly lower after 4 hours of ACTH infusion (p less than 0.01), while that of DOC was significantly higher after 8 hours of ACTH infusion (p less than 0.05) in patients with EH. The mean plasma S rose significantly more in patients with EH (p less than 0.025) at 4 and 8 hours after ACTH infusion. The mean plasma F response to ACTH infusion was slightly lower in patients with EH than in controls. The mean response of 18-OH DOC SR to ACTH infusion was slightly higher in patients with EH than in controls. The mean plasma A response was significantly higher in patients with EH than in controls 4 (p less than 0.05) and 8 hour (p less than 0.001) after an ACTH infusion. These results could be explained in part by abnormalities in the 17- and 11-hydroxylase systems, and that the abnormality in 11-hydroxylation was more pronounced than that in the 17-position. Furthermore, we suspect that the sensitivity of adrenal aldosterone to ACTH might be increased or another accelerated pathway to aldosterone biosynthesis might exist in patients with EH.     

Effect of 3beta-hydroxysteroid dehydrogenase inhibition by trilostane on blood pressure in the Dahl salt-sensitive rat

The brains of rats and humans express the enzymes required for the synthesis of aldosterone from cholesterol, including the 3beta-steroid dehydrogenase that catalyzes the conversion of pregnenolone to progesterone in the pathway of adrenal steroid synthesis. Salt-induced hypertension in the Dahl inbred salt-sensitive (SS/jr) rat is associated with normal to low levels of circulating aldosterone, yet it is abrogated by the central infusion of mineralocorticoid receptor antagonists. To test the hypothesis that de novo synthesis of aldosterone in the brain has a pathophysiological role in the salt-induced hypertension of the SS rat, the 3beta-steroid dehydrogenase antagonist trilostane was infused continuously intracerebroventricularly or subcutaneously in two different cohorts of Dahl SS/jr rats, one female, the other male, during and after the development of salt-induced hypertension. The doses of trilostane used had no effect on blood pressure when infused subcutaneously. Animals receiving vehicle intracerebroventricularly experienced a 30- to 45-mmHg increase in systolic blood pressure measured by tail cuff. The intracerebroventricular, but not subcutaneous, infusion of 0.3 microg/h trilostane effectively blocked the increase in systolic blood pressure and reversed the hypertension produced by drinking 0.9% saline. Trilostane was equally effective in female and male rats. Weight gain, serum aldosterone and corticosterone concentrations, and behavior assessed subjectively and by elevated plus maze were unchanged by the trilostane treatment. These studies suggest that the synthesis in the brain of a mineralocorticoid receptor agonist, probably aldosterone, is responsible in part for the salt-induced hypertension of the inbred Dahl SS/jr rat.     

Mineralocorticoid receptors and hypertension

Mineralocorticoid receptors (MR) have equal affinity for the mineralocorticoid aldosterone, and the physiological glucocorticoids cortisol and corticosterone. In epithelial tissues in vivo, MR are protected against glucocorticoid occupancy by the enzyme 11β-hydroxysteroid dehydrogenase, allowing access by the lower circulating levels of the physiological mineralocorticoid aldosterone. In non-epithelial tissues, including the heart and most areas of the central nervous system, MR are not so protected, and their physiological ligand is cortisol/corticosterone. Intracerebroventricular infusion studies have shown that aldosterone occupancy of such unprotected circumventricular MR is necessary for mineralocorticoid hypertension, and the hypertensinogenic effects of peripherally infused aldosterone can be blocked by intracerebroventricular infusion of the MR antagonist RU28318. Prolonged (8 weeks) administration of mineralocorticoids to salt-loaded rats has been shown to be followed by hypertension, cardiac hypertrophy and cardiac fibrosis. Whether the hypertrophy and fibrosis reflect primary effects of aldosterone via cardiac MR, or effects secondary to occupancy of protected, epithelial MR, remains to be determined, as does the mechanism of action of salt loading in this model of mineralocorticoid hypertension.     

Fetal renal and blood pressure responses to steroid infusion after early prenatal treatment with dexamethasone

Maternal infusion of dexamethasone for 48 h early in gestation results in upregulation of mRNA for mineralocorticoid and glucocorticoid (MR and GR) receptors and angiotensin II receptors in ovine fetal kidneys late in gestation. This study sought to determine whether dexamethasone exposure results in changes in renal function and blood pressure responsiveness to infused cortisol or aldosterone in the late-gestation fetus. Merino ewes carrying single fetuses were infused with isotonic saline (Sal; n = 9) or dexamethasone (Dex, 0.48 mg/h; n = 10) for 48 h between days 26 and 28 of gestation (term = 150 days). At 115-122 days, renal function and blood pressure were measured in fetuses during a 4-h infusion of saline, cortisol (100 microg/h), or aldosterone (5 microg/h). Infusions were given in random order at least 2 days apart. Basal blood pressure and renal function were similar in Sal and Dex groups and did not change over the course of saline infusion. Cortisol infusion caused similar increases in blood pressure, urine flow, and glomerular filtration rate (GFR) in the groups. Aldosterone infusion caused a significantly different GFR response between the groups [P(treatment x time) < 0.05], but increase in K excretion and decrease in Na-to-K ratio were similar in the groups. The similar results obtained with cortisol and aldosterone infusion suggest no increased renal functional maturity to those hormones after early prenatal dexamethasone exposure. This suggests that changes in mRNA for MR and GR in kidneys of dexamethasone-exposed fetuses do not result in functional differences and highlights the renin-angiotensin system, as reported previously, as more important in this model.     

Corticosteroids mediate fast feedback of the rat hypothalamic-pituitary-adrenal axis via the mineralocorticoid receptor

The aim of this study was to investigate fast corticosteroid feedback of the hypothalamic-pituitary-adrenal (HPA) axis under basal conditions, in particular the role of the mineralocorticoid receptor. Blood samples were collected every 5 min from conscious rats at the diurnal peak, using an automated blood sampling system, and assayed for corticosterone. Feedback inhibition by rapidly increasing concentrations of ligand was achieved with an intravenous bolus of exogenous corticosteroid. This resulted in a significant reduction in plasma corticosterone concentrations within 23 min of the aldosterone bolus and 28 min of methylprednisolone. Evaluation of the pulsatile secretion of corticosterone revealed that the secretory event in progress at the time of administration of exogenous steroid was unaffected, whereas the next secretory event was inhibited by both aldosterone and methylprednisolone. The inhibitory effect of aldosterone was limited in duration (1 secretory event only), whereas that of methylprednisolone persisted for 4-5 h. Intravenous administration of canrenoate (a mineralocorticoid receptor antagonist) also had rapid effects on the HPA axis, with an elevation of ACTH within 10 min and corticosterone within 20 min. The inhibitory effect of aldosterone was unaffected by pretreatment with the glucocorticoid receptor antagonist RU-38486 but blocked by the canrenoate. These data imply an important role for the mineralocorticoid receptor in fast feedback of basal HPA activity and suggest that mineralocorticoids can dynamically regulate basal corticosterone concentrations during the diurnal peak, a time of day when there is already a high level of occupancy of the cytoplasmic mineralocorticoid receptor.     

An adrenocortical tumor secreting weak mineralocorticoids

An adrenocortical carcinoma (15.5 g) secreting excessive amounts of steroids with weak mineralocorticoid activity in a 25-year-old woman was studied with particular reference to its in vivo and in vitro secretions of steroids. Severe hypertension, occasional low serum potassium and suppressed PRA were the major clinical findings, and were improved with removal of the tumor. In the preoperative stage, plasma levels of 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were all increased. However, the plasma level of aldosterone was repeatedly normal. Although plasma levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone were very high, those of other late step steroids, i.e. 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione and testosterone were almost normal. From these findings, a major etiological role of weak mineralocorticoids such as 11-deoxycorticosterone, 18-hydroxycorticosterone and corticosterone in her hypertension was suggested. Pregnenolone and 17-hydroxypregnenolone in tumor tissue were increased, but 11-deoxycorticosterone, corticosterone, aldosterone, cortisol and adrenal androgens such as dehydroepiandrosterone, androstenedione and testosterone were below normal or low normal. In vitro production of 11-deoxycorticosterone, aldosterone or cortisol by the tumor tissue slices was very low and scarcely responded to synthetic ACTH.     

Mineralocorticoids act centrally to regulate blood-borne tumor necrosis factor-alpha in normal rats

Excessive mineralocorticoid receptor (MR) stimulation induces neurohumoral excitation and cardiac and vascular fibrosis. In heart failure (HF) rats, with excessive neurohumoral drive, central infusion of the MR antagonist spironolactone (SL) decreases blood-borne TNF-alpha. This study aimed to determine whether DOCA, a precursor of aldosterone, acts centrally to stimulate TNF-alpha production in normal rats. DOCA (5 mg sc daily for 8 days) induced a progressive increase in TNF-alpha beginning on day 3 and increased tissue TNF-alpha in hypothalamus, pituitary, and heart but not in other brain and peripheral tissues harvested on day 9. A continuous intracerebroventricular infusion of SL (100 ng/h) blocked the plasma TNF-alpha response. Oral SL (1 mg/kg) blocked the plasma and tissue TNF-alpha responses. Thus DOCA increases TNF-alpha in brain, heart, and blood in normal rats. Activation of brain MR appears to account for the increase in plasma TNF-alpha. These findings have important implications for the understanding of pathophysiological states (e.g., HF, hypertension) characterized by high circulating levels of aldosterone.     

The influence of Aspalathus linearis (Rooibos) and dihydrochalcones on adrenal steroidogenesis: quantification of steroid intermediates and end products in H295R cells

The steroid hormone output of the adrenal gland is crucial in the maintenance of hormonal homeostasis, with hormonal imbalances being associated with numerous clinical conditions which include, amongst others, hypertension, metabolic syndrome, cardiovascular disease, insulin resistance and type 2 diabetes. Aspalathus linearis (Rooibos), which has been reported to aid stress-related symptoms linked to metabolic diseases, contains a wide spectrum of bioactive phenolic compounds of which aspalathin is unique. In this study the inhibitory effects of Rooibos and the dihydrochalcones, aspalathin and nothofagin, were investigated on adrenal steroidogenesis. The activities of both cytochrome P450 17α-hydroxylase/17,20 lyase and cytochrome P450 21-hydroxylase were significantly inhibited in COS-1 cells. In order to study the effect of these compounds in H295R cells, a human adrenal carcinoma cell line, a novel UPLC-MS/MS method was developed for the detection and quantification of twenty-one steroid metabolites using a single chromatographic separation. Under both basal and forskolin-stimulated conditions, the total amount of steroids produced in H295R cells significantly decreased in the presence of Rooibos, aspalathin and nothofagin. Under stimulated conditions, Rooibos decreased the total steroid output 4-fold and resulted in a significant reduction of aldosterone and cortisol precursors. Dehydroepiandrosterone-sulfate levels were unchanged, while the levels of androstenedione (A4) and 11β-hydroxyandrostenedione (11βOH-A4) were inhibited 5.5 and 2.3-fold, respectively. Quantification of 11βOH-A4 showed this metabolite to be a major product of steroidogenesis in H295R cells and we confirm, for the first time, that this steroid metabolite is the product of the hydroxylation of A4 by human cytochrome P450 11β-hydroxylase. Taken together our results demonstrate that Rooibos, aspalathin and nothofagin influence steroid hormone biosynthesis and the flux through the mineralocorticoid, glucocorticoid and androgen pathways, thus possibly contributing to the alleviation of negative effects arising from elevated glucocorticoid levels.     

Selectivity and potency of the retroprogesterone dydrogesterone in vitro

Dydrogesterone is widely used for menstrual disorders, endometriosis, threatened and habitual abortion and postmenopausal hormone replacement therapy. Although progestins have a promiscuous nature, dydrogesterone does not have clinically relevant androgenic, estrogenic, glucocorticoid or mineralocorticoid activities. To date, systematic biochemical characterization of this progestin and its active main metabolite, 20α-dihydrodydrogesterone, has not been performed in comparison to progesterone. The objective of this study was to evaluate the selectivity and potential androgenic/antiandrogenic effects of dydrogesterone and its metabolite in comparison to progesterone and medroxyprogesterone acetate by analyzing their interference with AR signaling in vitro. We characterized dydrogesterone and its metabolite for their binding and transactivation of androgen and other steroid hormone receptors and for their potential inhibitory effects against androgen biosynthetic enzymes, 17β-hydroxysteroid dehydrogenase types 3 and 5 and 5α-reductase types 1 and 2. We found that dydrogesterone resembled progesterone mainly in its progestogenic effects and less in its androgenic, anti-androgenic, glucocorticoid and antiglucocorticoid effects; whereas, 20α-dihydrodydrogesterone showed reduced progestogenic potency with no androgenic, glucocorticoid and mineralocorticoid effects. Effects on the androgen and glucocorticoid receptor differed depending on the technology used to investigate transactivation. Progesterone, but not dydrogesterone and 20α-dihydrodydrogesterone, exerted anti-androgenic effects at the pre-receptor level by inhibiting 5α-reductase type 2. Dydrogesterone, 20α-dihydrodydrogesterone and progesterone inhibited the biosynthesis of testosterone catalyzed by 17β-hydroxysteroid dehydrogenase types 3 and 5; however, due to their micromolar K_i values, these activities appeared to be not of relevance at therapeutic levels. Overall, our data show that the anti-androgenic potential of dydrogesterone and 20α-dihydrodydrogesterone is less pronounced compared to progesterone.     

Influence of morphine treatment in pregnant rats on the mineralocorticoid activity of the adrenals in their neonates

Exposure of pregnant rats to morphine, from day 11 to day 18 of gestation, was previously reported to induce both an adrenal atrophy and hypoactivity of the glucocorticoid function in newborns at term, but did not affect, in vitro, the responsiveness of those glands to adrenocorticotrophin hormone (ACTH) concerning corticosterone release. Moreover, these effects were mediated by maternal hormones from the adrenal glands. In the present work, we investigated the effects of a prenatal morphine exposure on the mineralocorticoid activity of the adrenals in neonates. The first aim of the present study was to determine in these newborns 1) the adrenal and plasma aldosterone concentrations at birth time and during the early postnatal period 2) the plasma levels of Na+ and K+ at birth time, 3) the in vitro responsiveness of the newborn adrenals to angiotensin II (A(II)) and ACTH. The second aim of our study was to investigate the mineralocorticoid activity of the adrenals in newborns from adrenalectomized mothers treated with morphine during gestation. According to present data morphine given to intact mothers induced in newborns a severe adrenal atrophy but increased adrenal aldosterone content and plasma aldosterone level. However, prenatal morphine was unable to affect significantly Na+/K+ ratio in both mothers and newborns. In vitro, the adrenals of neonates from morphine-treated mothers were unresponsive to An and ACTH for promoting aldosterone release; in contrast, aldosterone secretion was significantly stimulated by high potassium levels (55 mEq). Maternal adrenalectomy performed one day before the beginning of morphine treatment prevented morphine-induced adrenal atrophy but was unable to affect significantly the adrenal mineralocorticoid function of the offspring. Such data suggest that a prenatal morphine exposure stimulated both aldosterone synthesis and release in neonates. However, this basal hyperfunction did not appear to be coupled with an enhanced adrenal responsivity to AII or ACTH. Prenatal morphine-induced hyperactivity of the mineralocorticoid function of the newborn adrenals, which drastically contrast with hypoactivity of the glucocorticoid one, was independent of adrenal factors from maternal origin.     

The steroid metabolome in lamotrigine-treated women with epilepsy

Background

Epilepsy in women may be associated with reproductive disorders and alterations in serum steroid levels. Some steroids can be induced by epilepsy and/or treatment with antiepileptic drugs; however, there are still limited data available concerning this effect on the levels of other neuroactive steroid metabolites such as 3a-hydroxy-5a/b-reduced androstanes.

Aim

To evaluate steroid alterations in women with epilepsy (WWE) on lamotrigine monotherapy.

Subjects and methods

Eleven WWE and 11 age-matched healthy women underwent blood sampling in both phases of their menstrual cycles (MCs). The steroid metabolome, which included 30 unconjugated steroids, 17 steroid polar conjugates, gonadotropins, and sex hormone-binding globulin (SHBG), was measured using gas chromatography–mass spectrometry (GC–MS) and radioimmunoassay (RIA).

Results

WWE had lower cortisol levels (status p < 0.001), but elevated levels of unconjugated 17-hydroxypregnenolone (status p < 0.001). Progesterone was higher in the follicular menstrual phase (FP) in WWE than in the controls (status × menstrual phase p < 0.05, Bonferroni multiple comparisons p < 0.05), whereas 17-hydroxyprogesterone was higher in WWE in both menstrual phases (status p < 0.001). The steroid conjugates were mostly elevated in WWE. The levels of 5α/β-reduced androstanes in WWE that were significantly higher than the controls were etiocholanolone (status p < 0.001), 5α-androstane-3α,17β-diol (status p < 0.001), and the 5α/β-reduced androstane polar conjugates (status p < 0.001).

Conclusions

WWE showed a trend toward higher circulating 3α-hydroxy-5α/β-reduced androstanes, increased activity of 17α-hydroxylase/17,20 lyase in the Δ⁵-steroid metabolic pathway, and increased levels of the steroid polar conjugates.     

Differential effects of mineralocorticoid blockade on the hypothalamo-pituitary-adrenal axis in pregnant and nonpregnant ewes

During pregnancy, plasma ACTH and cortisol are chronically increased; this appears to occur through a reset of hypothalamo-pituitary-adrenal (HPA) activity. We have hypothesized that differences in mineralocorticoid receptor activity in pregnancy may alter feedback inhibition of the HPA axis. We tested the effect of MR antagonism in pregnant and nonpregnant ewes infused for 4 h with saline or the MR antagonist canrenoate. Pregnancy significantly increased plasma ACTH, cortisol, angiotensin II, and aldosterone. Infusion of canrenoate increased plasma ACTH, cortisol, and aldosterone in both pregnant and nonpregnant ewes; however, the temporal pattern of these responses differed between these two reproductive states. In nonpregnant ewes, plasma ACTH and cortisol transiently increased at 1 h of infusion, whereas in pregnant ewes the levels gradually increased and were significantly elevated from 2 to 4 h of infusion. MR blockade increased plasma aldosterone from 2 to 4 h in the pregnant ewes but only at 4 h in the nonpregnant ewes. In both pregnant and nonpregnant ewes, the increase in plasma aldosterone was significantly related to the timing and magnitude of the increase in plasma potassium. The results indicate a differential effect of MR activity in pregnant and nonpregnant ewes and suggest that the slow changes in ACTH, cortisol, and aldosterone are likely to be related to blockade of MR effects in the kidney rather than to effects of MR blockade in hippocampus or hypothalamus.     

Studies on spirolactone steroid antagonists in ACTH-induced hypertension in sheep

This study investigated the anti-mineralocorticoid potency and haemodynamic effects of a series of mineralocorticoid antagonists of the spirolactone type (RU 28318, spironolactone, K-prorenoate, K-canrenoate and canrenone), for their ability to prevent the development of ACTH-induced hypertension in conscious sheep. In vivo bioassay, using aldosterone dependent changes in parotid salivary [Na+]/[K+] of sodium depleted adrenalectomized sheep, showed spironolactone was the most potent anti-mineralocorticoid tested. Infusions of the antagonists at equal doses alone for 4 days demonstrated that none affected mean arterial pressure, except for K-prorenoate which exhibited slight pressor activity. All the antagonists produced a natriuresis. Some of the steroid antagonists of the spirolactone group blocked the development of ACTH hypertension in sheep, spironolactone being the most effective. This study provides additional evidence for an essential mineralocorticoid component in ACTH-induced hypertension.     

NAD(P)H oxidase inhibitor prevents blood pressure elevation and cardiovascular hypertrophy in aldosterone-infused rats

Increased bioavailability of reactive oxygen species (ROS) has been implicated in the pathogenesis of mineralocorticoid hypertension. To find out the source of ROS, we evaluated the role of NAD(P)H oxidase in blood pressure (BP) elevation, cardiovascular hypertrophy, and fibrosis in aldosterone-salt rats. Aldosterone infusion (0.75 microg/h) significantly increased BP, which is attenuated by apocynin (1.5 mmol/L). Cardiac hypertrophy developed by aldosterone infusion was also normalized with apocynin. Greater mRNA for p22phox and NAD(P)H oxidase activity (more than twofold) in aorta of aldosterone-infused rats was reduced in apocynin-treated rats. Aldosterone infusion increased marginally procollagen I and III expression in LV compared to controls and apocynin decreased procollagen. Masson's Trichrome stain showed increased cardiac perivascular fibrosis, which was reduced by apocynin. These results suggest that NAD(P)H oxidase plays an important role in cardiovascular damage associated with mineralocorticoid hypertension.