Establishment and characterization of a human T-lymphocyte cell line immortalized by SV40 and with abnormal expression of TCR/CD3

OBJECTIVE: Elderly women with proximal femur fracture show a prolonged increase in plasma cortisol, which could have undesirable catabolic effects. Suppression of cortisol by dexamethasone is impaired, suggesting resistance to glucocorticoid effects at feedback inhibitory sites. We therefore wished to find out whether peripheral glucocorticoid sensitivity is normal. DESIGN: Peripheral blood mononuclear leucocytes were used as a model tissue. Blood samples were taken from elderly women about 2 weeks after hip fracture and from elderly control women. Each patient was then given 1 mg dexamethasone at 2300 h followed by further sampling at 0800 and 1600 h the next day. METHODS: Glucocorticoid-receptor binding parameters were measured by incubating whole cells with [3H]dexamethasone for 2 h at 37 degrees C. Inhibition of cell proliferation by dexamethasone was assessed by addition of [3H]thymidine to cells cultured for 65 h with concanavalin A. Cortisol and dexamethasone concentrations were measured in the dexamethasone suppression test. RESULTS: As expected, the hip-fracture patients had raised morning cortisol concentrations and impaired suppression by dexamethasone. The cells of the patients had similar numbers of glucocorticoid receptors to those of the control subjects but higher values for Kd (i.e. a lower binding affinity). The cells of the patients incorporated less [3H]thymidine than the control cells in the absence of dexamethasone. The percentage inhibition by a saturating concentration of dexamethasone was unchanged but the concentration giving half-maximal inhibition was decreased (sensitivity was increased) at the higher of the two concanavalin A concentrations used. CONCLUSIONS: These experiments in mononuclear leucocytes give no evidence of peripheral resistance to glucocorticoids in hip-fracture patients with impaired suppression of cortisol by dexamethasone.     

Impaired cortisol binding to glucocorticoid receptors in hypertensive patients

We compared glucocorticoid receptor binding characteristics and glucocorticoid responsiveness of human mononuclear leukocytes (HML) from hypertensive patients and matched normotensive volunteers. We also considered associations of these variables with plasma renin activity, aldosterone, cortisol, corticotropin, and electrolyte concentrations. We calculated binding affinity (Kd; nmol/L) and capacity (Bmax; sites/cell) for dexamethasone and cortisol from homologous and heterologous competition curves for specific [3H]dexamethasone binding sites on HML isolated from the blood of normotensive volunteers and subjects with essential hypertension. Glucocorticoid responsiveness of HML was evaluated as IC50 values (nmol/L) for dexamethasone and cortisol for the inhibition of lysozyme release. We measured plasma hormones by radioimmunoassay. Kd values (mean+/-SE) for cortisol in HML of hypertensive patients were higher than in control subjects (24.6+/-2.4 versus 17.5+/-1.7 nmol/L, P<.04). Binding capacity (4978+/-391 versus 4131+/-321 sites/cell), Kd values for dexamethasone (6.7+/-0.5 versus 5.7+/-0.3 nmol/L), and IC50 values for dexamethasone (3.4+/-0.3 versus 3.1+/-0.2 nmol/L) and cortisol (12.2+/-1.6 versus 9.5+/-0.3 nmol/L) were not significantly different. Patients with renin values less than 0.13 ng angiotensin I/L per second were markedly less sensitive to cortisol than those with higher values. Both Kd (30.3+/-2.5 versus 19.2+/-2.4 nmol/L) and IC50 values (15.5+/-1.8 versus 8.9+/-1.2 nmol/L) for cortisol were significantly higher in patients with lower renin values (P<.03). Other variables, including plasma hormone and electrolyte values and binding characteristics for dexamethasone, were not different. These data suggest that cortisol binding to glucocorticoid receptor is slightly impaired in patients with essential hypertension. In vivo, this could lead to inappropriate binding of cortisol to mineralocorticoid receptors. Hence, decreased sensitivity to cortisol is associated with renin suppression. This hypothesis is supported by evidence of hypertension and low renin activity, which others have described in patients with primary glucocorticoid resistance due to mutations of the glucocorticoid receptor.     

Effectors of cyclic adenosine 5'-monophosphate up-regulating-oxytocin receptors in rabbit amnion cells: isoproterenol, parathyroid hormone-related protein, and potentiation by cortisol

Forskolin (FSK; an activator of adenylyl cyclase) and cortisol synergistically increase the concentration of oxytocin receptors (OTRs) in rabbit amnion cells. The aims of this study were to characterize potential physiological regulators of OTR concentrations acting through adenylyl cyclase and to clarify the mechanisms of potentiation by cAMP and cortisol. Both isoproterenol (ISO) and parathyroid hormone-related protein (PTHrP) elevated amnion cell cAMP levels and OTR concentrations. The effects of ISO and PTHrP on OTR were potentiated by cortisol. Cortisol had no effect on the ability of ISO or PTHrP to stimulate adenylyl cyclase activity, and cAMP did not affect the number or affinity of glucocorticoid receptors in whole cells or in cytosol. Adenylyl cyclase activation, however, caused conversion of mifepristone (RU486) from a glucocorticoid antagonist to agonist. Thus, mifepristone elevated OTR receptor concentrations in the presence of FSK. In contrast, a structurally related glucocorticoid antagonist, onapristone (ZK98 299), was unaffected by cAMP. Because glucocorticoid receptors bound to mifepristone are capable of interacting with DNA, whereas onapristone-occupied receptors are not, we conclude that cAMP affects glucocoticoid receptor-DNA interactions, accounting for the synergistic effects of cAMP and cortisol on OTRs.     

Specific glucocorticoid binding at different levels of human motor activity

We studied the number of glucocorticoid receptors and dissociation constant in isolated human lymphocytes as well as blood concentrations of hormones produced by the hypothalamic-hypophyseal-adrenocortical system in three experimental series: at normal (17 subjects), decreased (10 subjects, a 360-d head-down bed rest) and increased (8 subjects, physical exercise on bicycle ergometer) levels of motor activity. In the first series we found that the number of glucocorticoid receptors and dissociation constant did not depend on the season, on the age of subjects nor on cortisol concentrations in blood. In the second series we observed the following: at the end of the first month of bed rest the number of glucocorticoid receptors and receptor affinity significantly increased; at the beginning of the third month of bed rest specific glucocorticoid binding significantly decreased and circadian rhythms of adrenocorticotropin and cortisol in blood varied markedly; at the end of the sixth month of bed rest the number of glucocorticoid receptors returned to prebed rest levels and dissociation constant decreased. In the third series physical exercises that induced an activation of the hypothalamic-hypophyseal-adrenocortical system (maximal physical exercises and prolonged submaximal exercises at 70% of maximal oxygen uptake) led to a significant increase in the number of glucocorticoid receptors without changes of dissociation constant. These results indicate that both a decrease and an increase of human motor activity resulted in significant changes of specific glucocorticoid binding which were not influenced by changes of circulating hormone concentrations in blood but by some other factors affected by physical activity.     

Cortisol inhibits apoptosis in carp neutrophilic granulocytes

The direct effect of cortisol treatment on carp neutrophil viability was examined in vitro. Cortisol treatment caused an inhibition of neutrophil apoptosis. The effect was blocked by glucocorticoid receptor blocker RU486, showing that rescue from apoptosis was receptor mediated. Using binding studies with radioactive cortisol, a single class of glucocorticoid receptors was detected with high affinity (Kd = 2.6 nM) and low capacity (497 receptors/cell) for cortisol binding. Both in vitro and in vivo cortisol treatment did not affect neutrophil respiratory burst activity. These data indicate that cortisol can augment the supply of functional neutrophilic granulocytes in conditions of acute stress, which may be essential for survival, since phagocytes form the first line of defence against micro-organisms.     

Structure and function of the hepatic form of 11 beta-hydroxysteroid dehydrogenase in the squirrel monkey, an animal model of glucocorticoid resistance

Both cortisol and aldosterone bind to and activate the mineralocorticoid receptor. Cortisol concentrations are generally 100- to 200-fold higher than aldosterone concentrations, yet mineralocorticoids clearly exert effects different from glucocorticoids. One hypothesis is that 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), which converts cortisol to biologically inactive cortisone, protects the mineralocorticoid receptor from cortisol. The circulating concentrations of cortisol in the squirrel monkey are 20- to 50-fold higher than human cortisol concentrations, yet this animal has no evidence of glucocorticoid or mineralocorticoid excess. We used this experiment of nature to test the hypotheses that the known (hepatic) form of 11 beta-HSD protects renal mineralocorticoid receptors from the action of cortisol and that it modulates glucocorticoid concentrations in target tissues. Using a long oligonucleotide based on the rat sequence, we cloned the squirrel monkey 11 beta-HSD complementary DNA and gene. The encoded monkey amino acid sequence is 75% and 91% identical to the corresponding rat and human sequences, respectively. The tissue abundance of the messenger RNA for the monkey enzyme was similar to or less than that seen for the rat and human enzymes. Both the monkey and human 11 beta-HSD complementary DNAs were cloned into an expression vector and used to transfect cultures of Chinese hamster ovary cells. Both vectors were transcribed and translated into equivalent amounts of 11 beta-HSD enzyme. The monkey enzyme was slightly more efficient than the human enzyme in converting [3H]cortisol to cortisone, and estimates of the Michaelis-Menten constant and maximum velocity of both enzymes are similar. These data indicate that the abundance and activity of the hepatic form of 11 beta-HSD are insufficient to inactivate the very high concentrations of cortisol in the squirrel monkey, suggesting that this form of 11 beta-HSD does not defend the mineralocorticoid receptor or protect tissues from high cortisol concentrations. Rather, this enzyme appears to favor conversion of cortisone to cortisol, thus maximizing tissue concentrations of cortisol to overcome glucocorticoid resistance associated with a 50% reduction in glucococorticoid receptors.     

Neonatal vitamin E treatment induces long term glucocorticoid receptor changes: an unusual hormonal imprinting effect

Single neonatal vitamin E treatment significantly altered the affinity (Kd) of thymic glucocorticoid receptors in male adolescent and adult rats. In six weeks old animals the affinity increased (and there is a tendency for an increase in receptor density), in twelve weeks old animals the affinity decreased. The thymic glucocorticoid receptors and uterine estrogen receptors of female animals were not influenced at all. Thousandfold tocopherol did not compete with labeled dexamethasone for their receptors, suggesting that neonatal vitamin E imprinting effect was not done at direct receptorial level.     

Glucocorticoid binding in mammary tissue slices of cattle in various reproductive states

Unlabeled cortisol and dexamethasone reduced tritiated cortisol and tritiated dexamethasone binding to 700 X g supernatant and precipitate fractions of mammary tissue slices from virgin heifers and from multiparous cows that were 1-mo prepartum (nonlactating), lactating (non-pregnant), or dry (nonpregnant, nonlactating). Unlabeled progesterone, testosterone, and 17beta-estradiol had no effect on tritiated glucocorticoid binding in 700 X g supernatant and precipitate fractions from these mammary tissue slices. The 700 X g fractions in mammary tissue slices from all cattle bound cortisol and dexamethasone with high affinity (Kd 10-10M). There were 1263 and 1955 molecules of cortisol and dexamethasone bound per mammary cell, respectively, in mammary tissue slices from lactating non-pregnant cows; in comparison virgin heifers bound 413 and 651 molecules of cortisol and dexamethasone; dry, non-pregnant cows bound 336 and 536 molecules of cortisol and dexamethasone; and 1-mo prepartum nonlactating cows bound 532 molecules of cortisol. Mammary tissue slices from cattle in reproductive states examined contained a major non-specific component which bound cortisol in both 700 X g tissue fractions. Since mammary tissue slices from lactating cattle bound more molecules of glucocorticoids than mammary slices from cattle in other reproductive states, we speculate specific glucorticoid binding may be associated with lactation.     

A chimeric Cre recombinase inducible by synthetic,but not by natural ligands of the glucocorticoid receptor

We have developed a new ligand-dependent chimeric recombinase (Cre-GRdex) by fusing the site-specific Cre recombinase to the ligand binding domain (LBD) of a mutant human glucocorticoid receptor (GRdex). The synthetic glucocorticoid receptor (GR) ligands dexamethasone, triamcinolone acetonide and RU38486efficiently induce recombinase activity in F9 murine embryonal carcinoma cells expressing constitutively Cre-GRdex. In contrast, no recombinase activity was detected in the absence of ligand or in the presence of the natural GR ligands corticosterone, cortisol or aldosterone. Moreover, physiological concentrations of these natural GR ligands do not affect Cre-GRdexrecombinase activity induced by dexamethasone. Thus, as previously shown using Cre-oestrogen receptor (ER) fusion proteins, Cre-GRdexmight be useful for achieving loxP site-directed mutagenesis in cultured cells and spatio-temporally controlled somatic cell mutagenesis in transgenic mice.     

A biomechanical evaluation of cyclic and static stretch in human skeletal muscle

Hexarelin (HEX) is a synthetic GHRP which acts on specific receptors at both the pituitary and the hypothalamic level to stimulate GH release both in animals and in humans. Like other GHRPs, HEX possesses also acute ACTH and cortisol-releasing activity similar to that of hCRH. The mechanisms underlying the stimulatory effect of GHRPs on hypothalamo-pituitary-adrenal (HPA) axis are still unclear, although a CNS-mediated action has been demonstrated. In 6 normal healthy young women (26-34 years) we studied the effects on ACTH and cortisol secretion of HEX (2.0 microg/kg i.v. at 0 min) alone and preceded by dexamethasone (DEXA, 1 mg p.o. at 23.00 h on the previous night) or alprazolam (ALP, 0.02 mg/kg p.o. at -90 min), a benzodiazepine which binds to GABA receptors and possesses CRH-mediated inhibitory activity on HPA axis. ACTH and cortisol secretion after saline administration as well as the GH response to HEX alone and preceded by DEXA or ALP were also studied. HEX administration elicited an increase in ACTH (peak vs. baseline, mean +/- SEM: 28.0 +/- 6.7 vs. 11.7 +/- 2.2 pg/ml, p < 0.05) and cortisol secretion (162.6 +/- 15.0 vs. 137.7 +/- 12.6 microg/l, p < 0.05). DEXA pretreatment strongly inhibited basal ACTH (3.2 +/- 0.7 pg/ml, p < 0.01) and cortisol levels (11.3 +/- 2.5 microg/l, p < 0.001) and abolished the ACTH and cortisol responses to HEX (3.6 +/- 0.9 pg/ml, p < 0.01 and 10.7 +/- 2.0 microg/l, p < 0.001), respectively. On the other hand, ALP pretreatment did not significantly modify basal ACTH (7.9 +/- 2.0 pg/ml) and cortisol levels (127.6 +/- 14.5 microg/l) but abolished the HEX-induced ACTH and cortisol secretions (8.6 +/- 2.4 pg/ml, p < 0.05 and 111.0 +/- 6.0 microg/l, p < 0.05), respectively. ACTH and cortisol levels after HEX when preceded by ALP overlapped with those recorded during saline. HEX induced a clear GH response (peak at 15 min vs. baseline: 65.5 +/- 20.5 vs. 2.2 +/- 0.7 microg/l, p < 0.03) which was blunted by ALP (peak at 15 min: 21.5 +/- 5.5 microg/l, p < 0.05) while it was not modified by DEXA pretreatment (78.7 +/- 7.6 microg/l). In conclusion, our present data demonstrate that the ACTH- and cortisol-releasing effect of HEX is abolished by either dexamethasone or alprazolam, a benzodiazepine, which is even able to blunt the GH-releasing activity of the hexapeptide. These findings suggest that, in physiological conditions, the stimulatory effect of GHRPs on HPA axis is sensitive to the negative glucocorticoid feedback and could be mediated by GABAergic mechanisms; the latter seem also involved in the GH-releasing activity of GHRPs.     

Increased glucocorticoid activity in men with cardiovascular risk factors

The association between hypertension and insulin resistance might be explained by increased activity of the principal glucocorticoid, cortisol. Recent data show that the intensity of dermal vasoconstriction after topical application of glucocorticoids is increased in patients with essential hypertension. In this report, we examine whether increased glucocorticoid sensitivity or secretion is associated with insulin resistance and is a cause or consequence of hypertension. We studied 32 men (aged 47 to 56 years) from a cross-sectional study and 105 men (aged 23 to 33 years) in whom predisposition to high blood pressure has been defined by their own blood pressure and the blood pressures of their parents. In both populations, increased dermal glucocorticoid sensitivity was associated with relative hypertension, insulin resistance, and hyperglycemia. In young men with higher blood pressure whose parents also had high blood pressure, enhanced glucocorticoid sensitivity was accompanied by enhanced secretion of cortisol, enhanced ligand-binding affinities for dexamethasone in leukocytes, and impaired conversion of cortisol to inactive metabolites (cortisone and 5beta-dihydrocortisol). Increased tissue sensitivity to cortisol, amplified by enhanced secretion of cortisol, is a feature of the familial predisposition to high blood pressure rather than a secondary effect of high blood pressure. It may be mediated by an abnormal glucocorticoid receptor, and it may contribute to the association between hypertension and insulin resistance.     

Patients with rheumatoid arthritis: study of the correlation between density of glucocorticoid receptors in synovial cells and clinical response to steroidal treatment

BACKGROUND: The target cellular response to glucocorticoids is proportional to the concentrations or affinity of specific receptors to these substances. AIM: To look for a correlation between glucocorticoid receptor concentrations in synovial wall cells and the clinical response to steroidal treatment in patients with rheumatoid arthritis. PATIENTS AND METHODS: Twenty eight patients with rheumatoid arthritis were studied. Each subject was subjected to a synovial biopsy, in which a dry radioautographic technique for diffusible compounds was used. Patients were treated afterwards with three 500 mg intravenous pulses of methilprednisolone. RESULTS: A mean of 44.8% of synovial cells (range 30.1-62.8%) had binding sites for 3H dexamethasone. All patients had a significant clinical improvement after methylprednisolone. Multiple regression analysis did not show a correlation between clinical response and glucocorticoid receptor concentration. CONCLUSIONS: The lack of association between glucocorticoid receptor concentrations and clinical response could be due to the large steroid dose used, that saturated all available receptors.     

Central nervous system mediates an antihypertensive property in glucocorticoid hypertension in dogs

OBJECTIVE: To determine whether the central nervous system has a pressor or a depressor role in glucocorticoid-induced hypertension. METHODS: Intracerebroventricular dexamethasone or its receptor antagonist, RU 38486, was administered in 20 trained conscious dogs. In addition, intracerebroventricular RU 38486 was administered in dogs treated with oral dexamethasone. RESULTS: Intracerebroventricular dexamethasone induced a dose-related reduction in blood pressure accompanied by decreased heart rate and cardiac output. In contrast, intracerebroventricular RU 38486 caused a slight but not significant elevation in blood pressure. Total peripheral resistance showed no significant change throughout the treatment with dexamethasone or RU 38486. In contrast, oral dexamethasone caused significant elevation of blood pressure associated with increased total peripheral resistance and reduced heart rate. In hypertensive dogs treated with oral dexamethasone, intracerebroventricular RU 38486 elicited a more severe form of hypertension accompanied by an attenuation of the heart rate and a reduction in cardiac output. Intracerebroventricular dexamethasone induced a significant reduction in plasma levels of adrenocorticotrophic hormone, cortisol, arginine vasopressin and noradrenaline. In addition, simultaneous central administration of RU 38486 with intracerebroventricular dexamethasone blocked the reduction in blood pressure and heart rate completely. CONCLUSION: The present data strongly suggest that endogenous glucocorticoid in the central nervous system may not have a role in the regulation of systemic haemodynamics and hormones under resting conditions, but does play an important part during the glucocorticoid excess state, for example glucocorticoid hypertension caused by oral treatment with dexamethasone. The glucocorticoid in the central nervous system opposed the elevation of blood pressure in glucocorticoid-induced hypertension by attenuating the reduction in heart rate and cardiac output via direct stimulation of glucocorticoid receptors in the brain.     

Specific glucocorticoid receptor in the iris--ciliary body of the rabbit

The cytoplasm of the iris--ciliary body of the rabbit contains a receptor capable of specifically binding dexamethasone. This binding protein has a high affinity for dexamethasone (average KD = 2.0 X 10(-8) M), a low capacity (average 4.8 X 10(-13) mol of steroid bound per milligram of protein), and extreme heat sensitivity; it exhibits a pattern of competition virtually identical to that obtained with glucocorticoid receptors from other tissues and shows characteristic physicochemical behavior in various salt concentrations. The demonstration of a specific dexamethasone receptor in the iris--ciliary body provides the first direct biochemical evidence that these tissues may function as a target organ for glucocorticoids.     

Uptake of cortisol by isolated rat liver cells. A phenomenon indicative of carrier-mediation and simple diffusion

The uptake of cortisol by isolated rat liver cells was studied. Cortisol was taken up rapidly; the uptake increased with increasing temperature and reached a plateau after 45 s at 37 degrees, C, after 60 s at 27 degrees C, and after 90 s at 22 degrees C; at 5 degrees C the uptake increased linearly with time. The uptake was linear up to 1.5 mg of cell protein. Analysis of uptake as a function of increasing concentration of cortisol in the external medium indicated the presence of two saturable systems: a high-affinity system with an apparent Km value of 190 +/- 25 nM and a low-affinity system with an apparent Km value of 2200 +/- 180 nM. Above 600 nM, the rate of uptake of cortisol increased almost linearly with increasing cortisol concentration. Treatment of cells with KCN or 2,4-dinitrophenol inhibited the two saturable components, leaving the nonsaturable system unaffected. The affinity constants, Ka, were 6 X 10(6) M-1 and 0.6 X 10(6) M-1 for the high and low affinity components, respectively. These values increased approximately two-fold when uptake rates were corrected for diffusion. Cortisone and corticosterone inhibited the uptake of cortisol by liver cells competitively; dexamethasone inhibited cortisol uptake noncompetitively. Similarly, oestrone, oestradiol and testosterone decreased the uptake of cortisol, at a concentration of 2000 nM in the external medium, by 20, 49 and 35 percent, respectively; the inhibition was noncompetitive. p-Chloromercuribenzoate, N-ethylmaleimide and 1-fluoro-2,4-dinitrobenzene decreased the uptake of cortisol. Ouabain did not influence the uptake of cortisol; varying the external sodium concentration also did not affect uptake of cortisol. Cyclic-3',5'-adenosine monophosphate had a stimulatory effect. The results show that the first step, before cortisol is bound to intracellular binding proteins, is the uptake of cortisol by proteins in the plasma membrane. At lower concentrations of cortisol, uptake takes place by saturable processes; at higher concentrations saturation is not achieved, indicating that simple diffusion becomes the major route of transport into the cell. The proteins in the plasma membrane probably function as carriers to transport the glucocorticoid into the cell.     

Effect of dexamethasone on cell proliferation of neuroepithelial tumor cell lines

The effect of glucocorticoid on cell proliferation, the expression of glucocorticoid receptor, and the relationship between inhibition of cell growth and apoptosis were investigated in four established neuroepithelial tumor cell lines (KNS42, T98G, A172, and U251MG). Glucocorticoid receptor expression was located in the cytoplasm of untreated cells, but translocated into nuclei after treatment with dexamethasone in KNS42, T98G, and A172 cells. U251MG did not express glucocorticoid receptors. Dexamethasone significantly inhibited the growth of KNS42 and T98G cell lines, at high concentrations in contrast to growth stimulation at low concentration. Dexamethasone inhibited proliferation of A172 cell line at all concentrations from 10(-4) M to 10(-7) M. These were prevented by RU38486, a specific glucocorticoid antagonist. Apoptosis did not occur in any cell lines after dexamethasone treatment. There was no response to glucocorticoid by U251MG cells. Dexamethasone treatment of neuroepithelial tumor cells expressing glucocorticoid receptors causes translocation into the nucleus to modulate cell proliferation upon binding of different concentrations of dexamethasone in vitro. Dexamethasone inhibits proliferation of some neuroepithelial cell lines, not by glucocorticoid-induced apoptosis. The bimodal potential of glucocorticoid to stimulate or suppress proliferation of neuroepithelial tumor cells expressing glucocorticoid receptor must be considered in clinical trials.     

The role of mineralocorticoid receptors in hypothalamic-pituitary-adrenal axis regulation in humans

In rodents, two types of glucocorticoid receptors, the mineralocorticoid (MR; type I) and the glucocorticoid (type II) receptors, have been demonstrated to play a role in hypothalamic-pituitary-adrenal (HPA) axis regulation. Because MR shows a very high affinity for cortisol, it has been suggested that MR plays an important role in restraint of CRH and ACTH secretion during the nadir of the circadian rhythm. Although a number of studies have established the importance of MR in rodents, the functional role of MR in humans has not been determined. These studies evaluated whether spironolactone, an MR antagonist, had a detectable effect on HPA axis regulation in humans, and whether the effect was greatest during the evening, when plasma cortisol concentrations are in the MR range. Compared to the placebo day, after a single dose of spironolactone at either 0800 or 1600 h, there is a significant increase in plasma cortisol, which is preceded by a rise in ACTH and beta-endorphin. A significant effect of spironolactone on cortisol secretion was demonstrated with no differences between the morning and evening. Because the effect of spironolactone on cortisol was short lived, a second experiment was conducted using two doses of spironolactone, again sampling in the morning and evening. After two doses of spironolactone, plasma cortisol levels showed a significant and sustained spironolactone-induced elevation for the entire sampling period. However, neither plasma beta-endorphin nor ACTH was increased compared to levels on the placebo day. These data suggest that MR appear to play a clear role in HPA axis regulation during the time of the circadian peak as well as the trough. Furthermore, MR blockade may affect the sensitivity of the adrenal to ACTH.