Demonstration of glucocorticoid receptors in the adrenal cortex: evidence for a direct dexamethasone suppressive effect on the rat adrenal gland

The adrenal cortex was evaluated for the presence of glucocorticoid receptors and functions. Substantial binding of [3H]dexamethasone was observed in aminoglutethimide-treated, hypophysectomized, and intact rats. Further studies demonstrated binding in cultured bovine adrenocortical cells and in Y-1 cells, a cloned murine cell line of adrenal cortical origin. Scatchard analysis of specific binding data in cytosol from hypophysectomized rats revealed an apparent Kd of approximately 15 nM and a receptor content (Nmax) of 123 fmol/mg cytosol protein. Analysis of Y-1 cell cytosol showed a Kd of approximately 17 nM and Nmax of 190 fmol/mg protein. The binding site in hypophysectomized rats had the following steroid specificities: high affinity for dexamethasone, corticosterone, and progesterone; moderate affinity for 11 beta-cortisol, and low affinity for testosterone, estradiol, pregnenolone, and 11 alpha-cortisol. Sedimentation in sucrose density gradients revealed 8S binding peaks in cytosols prepared from intact rat adrenal glands, Y-1 cells, and cultured bovine adrenocortical cells. Time- and temperature-dependent nuclear uptake of [3H]dexamethasone in Y-1 cells was demonstrated. In vivo treatment of hypophysectomized rats with dexamethasone significantly enhanced the rate of adrenal atrophy. ACTH stimulation tests in hypophysectomized rats showed a decreased corticosterone response in dexamethasone-treated rats compared to that in control animals. However, in vitro, there was no evidence for an effect of dexamethasone on ACTH-stimulated corticosterone production. The data indicate that the adrenal cortex possesses a high affinity binding site that fulfills the criteria for a glucocorticoid receptor. Glucocorticoid administration enhances adrenal atrophy and impairs adrenal function. We speculate that this action contributes to the suppressive effect of glucocorticoids on the pituitary-adrenal axis.     

Direct inhibitory effect of dexamethasone on steroidogenesis of human adrenal in vivo.

A rapid ACTH test was used to investigate the direct effect of glucocorticoid on the steroidogenesis by the adrenal cortex in man. The plasma cortisol response to 250 microgram iv synthetic alpha-ACTH-(1--24) (Cortrosyn) was determined in normal subjects pretreated with 2 and 12 mg dexamethasone. The 15- and 30-min increments in plasma cortisol levels in response to ACTH injection after pretreatment with 12 mg dexamethasone were significantly suppressed compared to values obtained after pretreatment with 2 mg dexamethasone. The present study suggests that there is a direct inhibitory effect of glucocorticoid on adrenocortical steroidogenesis in man.     

Two glucocorticoid binding sites on the human glucocorticoid receptor

Glucocorticoids are known to have a lytic effect in leukemic cells via interactions with the glucocorticoid receptor (GR). Cortisol and various synthetic glucocorticoids bind to the GR with one-site kinetics. Cortivazol (CVZ) is a unique, high potency synthetic glucocorticoid, which has a phenylpyrazol fused to the A-ring of the steroid nucleus and displays binding consistent with two or more sites in the cytosol from CEM C7 cells (a human acute lymphoblastic T-cell line). It has previously been shown that the lower affinity class of sites are similar in affinity and site molarity to those recognized by dexamethasone. The higher affinity sites bind CVZ with 20- to 50-fold greater affinity, consistent with CVZ's enhanced biological effects. In mutant leukemic cells resistant to the lytic effects of dexamethasone, CVZ both lyses the cells and recognizes a single class of sites similar to the high affinity site in CEM C7 cells. We have carried out experiments to define the nature of the higher affinity CVZ binding site. We now show that: 1) CVZ has more than one binding site in a second, independent, B-cell line, IM-9; 2) the antiglucocorticoid RU 38486 is able to block both CVZ's higher and lower affinity sites; 3) all of CVZ's binding sites are on a protein immunologically indistinguishable from the human GR; and 4) freshly isolated clones of CVZ-resistant cells have lost all binding sites for CVZ. These data indicate that CVZ is recognizing two glucocorticoid binding sites on the human GR or a protein very similar to it.     

Demonstration of specific high affinity receptors for aldosterone in cytosol of rat colon

Since both aldosterone and glucocorticoids increase cation transport in rat distal colon, and a specific glucocorticoid high affinity cytosolic receptor has been identified in this tissue, it was possible that the action of aldosterone was dependent on interaction with the glucocorticoid receptor. Studies were, therefore, performed to determine whether a specific high affinity receptor for aldosterone was present in rat distal colon. At 4 C, aldosterone binding was saturable and exhibited a high affinity site with an apparent Kd of 6.2 +/- 0.9 X 10(-10) M and a calculated number of binding sites of 57.2 +/- 10.8 fmol/mg cytosol protein. Scatchard plot analysis also revealed a low affinity site with a Kd of 5.9 +/- 1.1 X 10(-8) M and 961 +/- 191 fmol/mg cytosol protein-binding sites. Competitive binding studies demonstrated that the high affinity binding protein was specific for aldosterone, compared to either dexamethasone or RU-28362. Since a specific high affinity receptor protein for aldosterone is present in rat distal colon, these data are consistent with a direct action of aldosterone that is independent of the glucocorticoid receptor system.     

Glucocorticoid modulation of beta-adrenergic receptors of cultured rat arterial smooth muscle cells

Since both glucocorticoids and catecholamines are involved in the regulation of normal blood pressure, we investigated the modulation of beta-adrenergic receptors of cultured rat arterial smooth muscle cells by glucocorticoids. The synthetic glucocorticoids dexamethasone and RU 28362, at 10(-8) M concentration, increased maximum beta-adrenergic binding but had no effect on the dissociation constant (Kd). Each steroid caused an increase in maximum [3H]dihydroalprenolol binding over the concentration range of 10(-8) to 10(-6) M, but not at 10(-9) M. The glucocorticoid effect on beta-adrenergic receptors of arterial smooth muscle cells required a minimum of 20 hours of incubation in the presence of the steroid and was significantly inhibited by cycloheximide (10 micrograms/ml), indicating that the glucocorticoid effect required protein synthesis. The effect of dexamethasone on [3H]dihydroalprenolol binding was significantly inhibited by the glucocorticoid antagonist RU 38486. Basal and agonist-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) levels in arterial smooth muscle cells, before and after glucocorticoid treatment, were measured as an indicator of the physiological significance of the observed glucocorticoid-induced increase in beta-adrenergic receptor binding. While causing no change in the basal cAMP level, treatment of arterial smooth muscle cells with 10(-6) M dexamethasone for 24 hours increased the 10(-6) M isoproterenol-stimulated cAMP levels.     

Glucocorticoid enhancement of adrenocorticotropin-induced 3',5'-cyclic adenosine monophosphate production by cultured ovine adrenocortical cells

The present study examines the effect of chronic treatment of glucocorticoids on ACTH1-24- or forskolin-induced cAMP output of cultured sheep adrenocortical cells. Cells cultured for 2 days in the presence of 1 microM dexamethasone released more cAMP in response to ACTH1-24 than did untreated cells, both in the absence and presence of 0.5 mM 1-methyl-3-isobutylxanthine. Such an enhancing effect required greater than or equal to 21 h of treatment and was both concentration-dependent and steroid specific. The ED50 of dexamethasone was about 10 nM, while that of cortisol and corticosterone was about 1 microM; testosterone at concentrations less than or equal to 10(-5) M had no enhancing effect. Glucocorticoids enhanced the cAMP response to ACTH1-24 without altering its ED50. Treatment of cultures with aminoglutethimide or the antiglucocorticoid RU 38486 for 48 h resulted in a dose-dependent decrease in ACTH1-24-induced cAMP output. Moreover, RU 38486 antagonized the enhancing effect of dexamethasone. Glucocorticoids did not increase the cAMP response to forskolin. These results suggest that chronic exposure to glucocorticoids is necessary for the full expression of the cAMP response to ACTH1-24 of adrenocortical cells from adult sheep.     

Opposite effects of ACTH and glucocorticoids on adrenal DNA synthesis in vivo.

Administration of ACTH to rapidly growing weanling rats results in an increase of DNA synthesis in adrenal and a decrease in liver. Dexamethasone administration decreases both adrenal and liver DNA synthesis. When both hormones were administered to the same animals, the liver DNA synthesis was similar to that observed with dexamethasone alone, but the DNA synthesis in adrenal was lower than that obtained with ACTH alone, yet higher than that observed with dexamethasone. The plasma levels of corticosterone were similar in animals treated with ACTH or with ACTH plus dexamethasone. Aminoglutethimide stimulated adrenal DNA synthesis, but less than ACTH. This substance overcame partially the inhibitory effects of dexamethasone on liver DNA synthesis but did not in adrenal. When both ACTH and aminoglutethimide were given simultaneously, adrenal DNA synthesis was higher than that observed with each substance alone. In all experiments in which adrenal cytosol DNA polymerase was studied, the activity varied in the same direction as DNA synthesis. These results indicate opposing effects of ACTH and glucocorticoids on adrenal DNA synthesis. The finding of a glucocorticoid effect on the adrenal is supported by the demonstration of a glucocorticoid specific binding protein in adrenal cytosol. Cycloheximide blocks the stimulatory action of ACTH on both steroidogenesis and DNA synthesis. Actinomycin D, as well as dexamethasone, blocks only the DNA synthesis-promoting action of ACTH. This latter result suggests some differences in the metabolic pathways by which ACTH controls steroidogenesis and growth in the adrenal cell.     

Binding of cytosol receptor-glucocorticoid complexes by isolated nuclei of glucocorticoid-responsive and nonresponsive cultured cells.

Nuclear binding of the AtT-20 cytosol receptor-glucocorticoid complex was studied in a cell-free system using nuclei from steroid-responsive (AtT-20) and nonresponsive (EPO-G1) cell lines, both of which synthesize ACTH. The AtT-20 cell line was derived from a mouse pituitary adenocarcinoma, while the EPO cell line was established from a human malignant melanoma. The nonresponsive EPO cells lacked a cytosol receptor for glucocorticoids, and, when whole cells were incubated with labeled glucocorticoid, they were unable to concentrate the steroid in their nuclei. A cell-free system using AtT-20 cytosol preincubated with labeled glucocorticoid was used to study binding by isolated nuclei. Binding to isolated nuclei from both cell lines was indistinguishable, in terms of temperature sensitivity, binding capacity, and saturability. Sucrose density gradient analyses of KCl extracts of nuclei labeled under these cell-free conditions showed 3.2-3.6 S peaks. In contrast, a 4.0 S peak was observed consistently when unreacted cytosol was analyzed on high-salt gradients, suggesting that interaction with nuclei from both cell lines caused the receptor to alter its sedimentation characteristics. These findings suggest either that all cells contain nuclear acceptor sites and that target cell responsiveness is conferred solely by the presence or absence of the cytosol receptor, or that binding sites detected in isolated nuclei may be different from those observed in intact cells and may, in fact, obscure them.     

Perioperative management of patients treated with glucocorticoids.

HPA suppression is a common consequence of glucocorticoid therapy, whereas overt secondary adrenal insufficiency is a rare but life-threatening condition. Prolonged hypotension and a response to adequate doses of a glucocorticoid agent are not reliable ways to assess adrenocortical function. One must also demonstrate plasma cortisol levels that are inappropriately low for the clinical situation. Hypotension in patients previously treated with glucocorticoids is caused by loss of the permissive effect of glucocorticoids on vascular tone, which may be related in turn to enhanced PGI2 production in the absence of glucocorticoids. It is not caused by mineralocorticoid deficiency. Recurrent problems of study design and interpretation have plagued this area of investigation. Any patient who has received a glucocorticoid in doses equivalent to at least 20 mg a day of prednisone for more than 5 days is at risk for HPA suppression. If the doses are closer to but above the physiologic range, 1 month is probably the minimal interval. Recovery from prolonged exposure to high doses of glucocorticoids may take up to 1 year. Pituitary function returns before adrenocortical function. Recovery from short courses of treatment (e.g., 5 days) occurs more rapidly, in about 5 days. Recovery is time-dependent and spontaneous. The rate of recovery is a function of the dose and duration of therapy before tapering is started and while the dose is being reduced. ACTH therapy does not cause adrenocortical suppression but offers no advantage over glucocorticoids, has several disadvantages, and should no longer be used. Patients on alternate day glucocorticoid therapy have some suppression of basal cortisol levels but have normal or nearly normal responses to provocative tests of adrenocortical function. The standard short ACTH stimulation test is a reliable means of assessing adrenocortical function preoperatively. The low dose (1 microgram) short ACTH test is promising but has not been sufficiently well characterized, requires serial dilutions and cannot be recommended at this time. Studies of the physiologic adrenocortical response to surgical stress provide a basis for revised dose recommendations for perioperative coverage in the patient with known or suspected HPA suppression. Recommendations of a multidisciplinary group are presented.     

Sex differences in response to steroids in preterm sheep lungs are not explained by glucocorticoid receptor number or binding affinity.

We recently reported that prenatal glucocorticoid therapy is less effective at promoting an improvement in lung function in male than in female sheep. This observation, and the higher incidence of respiratory distress syndrome in human males, suggests that the male fetal lung may be less responsive to glucocorticoids than is the female fetal lung. Since glucocorticoids are known to exert their effects via specific cytoplasmic glucocorticoid receptors (GR), we hypothesized that there may be sexual dimorphism in either the number or binding affinity of lung GR. To test the hypothesis, binding of dexamethasone (a synthetic glucocorticoid, 0.5-40 nM) by cytosolic fractions of male (n = 16) and female (n = 16) fetal sheep lung was measured at 125 days gestation (term = 148 days). Scatchard analysis of dexamethasone binding showed that the total number of GR (Bmax) did not significantly differ between male (346 +/- 42 fmol/mg protein) and female (277 +/- 23 fmol/mg protein) fetuses. The measured binding affinity (Kd) in male fetal lungs (6.85 +/- 0.43 nM) was not significantly different from that in females (8.46 +/- 1.02 nM). In conclusion, this study suggests that sex differences in fetal sheep lung responses to glucocorticoid therapy are not due to differences in the number or binding affinity of lung GR.     

Thyroid function in adrenocortical insufficiency during withdrawal and re-administration of glucocorticoid substitution

The regulatory function of glucocorticoids on thyroid hormone concentrations was studied in patients with adrenocortical insufficiency (ACI, n = 8) and in healthy subjects (n = 6). In patients with ACI withdrawal of glucocorticoid substitution for 84 h led to an increase in serum concentrations of total triiodothyronine (TT3) from 110 +/- 20 to 133 +/- 22 ng/dl and a decrease of serum reverse-T3 (rT3) from 23 +/- 6 to 18 +/- 6 ng/dl, whereas subsequent administration of dexamethasone (0.5 mg po q.i.d. for 3 days) induced a fall in TT3 (129 +/- 22 to 88 +/- 16 ng/dl) and a rise in rT3 (17 +/- 5 to 37 +/- 11 ng/dl) concentrations. Serum levels of total thyroxine (TT4) were unchanged by either withdrawal or re-administration of glucocorticoids. Basal plasma thyrotrophin (TSH) concentrations were unchanged by glucocorticoid withdrawal and fell from 2.2 +/- 1.5 to 1.1 +/- 0.8 mU/l during subsequent dexamethasone therapy. In healthy subjects a decrease of TT3 (89 +/- 8 to 69 +/- 8 ng/dl) and an increase in rT3 (19 +/- 5 to 32 +/- 8 ng/dl) concentrations were seen after stimulation of endogenous cortisol production by prolonged infusion of ACTH1-24 (0.5 mg, t = 8 h on 2 consecutive days), whereas concentrations of TT4 remained unchanged. During subsequent administration of dexamethasone serum level of both. TT3 and rT3 returned to basal levels. Thus, changes in thyroid hormone concentrations are induced by alterations of substitution therapy in patients with ACI. In healthy subjects the application of 2 mg dexamethasone/day following prolonged maximal stimulation of endogenous cortisol by iv ACTH may represent a state of relative glucocorticoid deficiency, thus explaining the observed hormonal changes, which are inverse to those generally induced in healthy man by endogenous or exogenous glucocorticoids.     

Gonadotrophin-secretion in adrenocortical insufficiency: impact of glucocorticoid substitution

In patients with deficient endogenous glucocorticoid production due to primary adrenal insufficiency (n = 4) or bilateral adrenalectomy (n = 2) a rise in LRH-stimulated concentrations of LH was seen following withdrawal of substitution therapy for 84 h. Consecutive re-administration of glucocorticoids (dexamethasone 2.0 mg/day for 3 days) resulted in increased basal concentrations of LH and FSH and a diminished secretory response of LH upon iv LRH (100 micrograms). Five patients substituted with glucocorticoids because of adrenocortical insufficiency presented upon the administration of exogenous ACTH1-24 with unchanged basal and LRH-stimulated concentrations of LH and FSH as compared to a placebo experiment. These data suggest that the withdrawal and subsequent re-administration of gluco-corticoid substitution alters basal and stimulated concentrations of gonadotrophins in patients with adrenocortical insufficiency, thus providing evidence for the importance of adequate glucocorticoid supply in the regulation of gonadotrophin secretion.     

Adrenocortical and Pituitary Glucocorticoid Feedback in Abstinent Alcohol‐Dependent Women

Background: The long‐term ingestion of alcohol diminishes hypothalamic–pituitary–adrenal (HPA) axis reactivity in alcohol‐dependent men, potentially altering future relapse risk. Although sex differences in HPA axis functioning are apparent in healthy controls, disruptions in this system have received little attention in alcohol‐dependent women. In this study, we assessed the basal secretory profile of adrenocorticotropic hormone (ACTH) and cortisol, adrenocortical sensitivity in both the presence and absence of endogenous corticotropic pituitary activation, and feedback pituitary glucocorticoid sensitivity to dexamethasone. Methods: Seven women 4‐ to 8‐week abstinent alcohol‐only dependent subjects and 10 age‐matched female healthy controls were studied. All subjects were between 30 and 50 years old, not taking oral contraceptives, and were studied during the early follicular phase of their menstrual cycle. Circulating concentrations of ACTH and cortisol were measured in blood samples collected at frequent intervals from 2000 to 0800 hour. A submaximal dose of cosyntropin (0.01 μg/kg), a synthetic ACTH (1–24), was administered at 0800 hour to assess adrenocortical sensitivity. In a separate session, low‐dose cosyntropin was also administered following high‐dose dexamethasone (8 mg intravenous) to assess adrenocortical sensitivity in the relative absence of endogenous ACTH. In addition, the ACTH response to dexamethasone was measured to determine the pituitary glucocorticoid negative feedback. Sessions were 5 days apart, and blood draws were obtained every 5 to 10 minutes. Results: Mean concentrations and pulsatile characteristics of ACTH and cortisol over 12 hours were not statistically different between the 2 groups. Healthy controls had a somewhat higher (p < 0.08) net peak, but not net integrated, cortisol response to cosyntropin relative to the alcohol‐dependent women. There were no significant group differences in either the ACTH or cortisol response to dexamethasone nor in the net cortisol response to cosyntropin following dexamethasone. Conclusion: Significant differences in pituitary–adrenal function were not apparent between alcohol‐dependent women and matched controls. Despite the small n, it appears that alcohol‐dependent women do not show the same disruptions in HPA activity as alcohol‐dependent men. These findings may have relevance for gender‐specific treatment effectiveness.     

Androgen receptor specificity and growth response of a human cell line (NHIK 3025)

Growth of cultured NHIK 3025 cells stemming from a carcinoma of the human uterine cervix can be stimulated by the androgens 4-androstene-3β,17β-diol and testosterone, but is not influenced by oestradiol. In the cytosol fraction of these cells testosterone and 5α-dihydrotestosterone could be bound specifically to a steroid receptor with limited capacity (8 fmol/ mg cytosol protein), but no specific binding of oestradiol or of the synthetic progestagen R-5020 could be demonstrated. The specificity of the binding was studied by agar-gel electrophoresis and sucrose density gradient centrifugation. In the cytosol no specific binding of 4-androstene-3β,17β-diol could be demonstrated and addition of this steroid to cytosols containing [³H]testosterone did not interfere with [³H]testosterone binding. Receptor-bound steroid could be extracted from the nuclei after incubation of intact NHIK 3025 cells with ³H-labelled 4-androstene-3β,17β-diol, testosterone or 5α-dihydrotestosterone. However, considerable metabolism of 4-androstene-3β,17β-diol occurred and the radioactivity recovered from the bound fraction represented mainly testosterone and 5α-dihydrotestosterone. In addition to the androgen receptor the NHIK 3025 cells also contain a glucocorticoid receptor (190 fmol [³H]dexamethasone bound/mg cytosol protein).     

ACTH induces up-regulation of ACTH receptor mRNA in mouse and human adrenocortical cell lines

Corticotropin (ACTH) binds to specific receptors in the adrenal cortex and thereby regulates glucocorticoid and mineralocorticoid production. The number of ACTH binding sites on adrenocortical cells is increased by exposure of cells to activators of the cAMP pathway. The mechanism responsible for the increase in ACTH binding sites is not known. We therefore studied the levels of ACTH-R mRNA in mouse Y-l and human NCI-H295 (H295) adrenocortical carcinoma cell lines. ACTH induced an increase in mouse ACTH-R mRNA in Y-l cells that was time and dose dependent, increasing 6-fold over basal levels following exposure to 10⁻⁸ M ACTH for 19–24 h. The amount of human ACTH-R mRNA in H295 cells increased 2–4-fold following a 24 h exposure to 10⁻⁸ M ACTH, 1 mM dbcAMP, or 10⁻⁵ M Forskolin. Treatment of H295 cells with angiotensin II (A-II) was found to dramatically increase the level of ACTH-R mRNA. These data indicate that regulation of ACTH-R mRNA levels is at least one mechanism by which ACTH and A-II elevate the number of ACTH binding sites in the adrenocortical cell.     

A radioreceptor assay for evaluation of the plasma glucocorticoid activity of natural and synthetic steroids in man.

An assay for plasma glucocorticoid activity has been developed using specific glucocorticoid receptors. Unlike other assays for cortisol and certain synthetic corticosteroids, this radioreceptor assay measures the glucocorticoid activity of all natural an synthetic steroids. Steroids extracted from as little as 0.05 ml of plasma are incubated with 3H-dexamethasone and cytosol receptors from cultured rat hepatome cells. From 0.5 to 50 ng of cortisol are accurately detected. Glucocorticoid activities of adult determined by the assay correlate closely with corticoid levels obtained in the CBG-isotope and fluorometric assays. Other steroids are measured in proportion to both concentration and potency as glucocorticoids. Relative activities include: cortisol 100, dexamethasone 940, prednisolone 230, prednisone 3, estradiol 1 and androstenedione 1. A similar ranking of steroids was found using receptors from a human source (fetal lung). The assay has been useful in detecting glucocorticoid activity in unidentified medications and in measuring plasma glucocorticoid levels after administration of synthetic corticosteroids. A modification of the assay is described for also estimating the level of free (unbound) glucocorticoid activity in plasma. Assays are performed before and after removal by charcoal of steroids not bound to plasma transcortin. This consideration is shown to be particularly important with prednisolone where the unbound steroid level is much less than the total. Thus, the radioreceptor assay provides a relatively simple technique for measuring the total of free plasma of glucocorticoid activity in man due to any natural or synthetic steroid or combination of steroids.     

Glucocorticoid Receptors in Human Polymorphonuclear and Mononuclear Leucocytes

Both polymorphonuclear (PMN) and mononuclear (MN) leucocytes constitute targets for glucocorticoid hormones. In order to comparatively characterize the earliest steps of steroid action in these cell populations, we investigated the concentrations and specificities of glucocorticoid receptors in purified human PMNs and MNs by a whole-cell binding assay using (³H)dexamethasone as the ligand. PMNs and MNs were found to contain the same amounts of glucocorticoid receptors (4720 and 4900 receptor sites/cell, respectively). The equilibrium dissociation constant (K_d) of the interaction between the cellular receptor and (³H)dexamethasone was about the same (1 times 10^-8 M) in both cell populations. No significant differences in the specificities of the steroid binding sites in PMNs and MNs were found; competition studies revealed the following order of relative binding affinities for a number of compounds: betamethasone > dexamethasone > prednisolone > cortisol > deoxycortico-sterone = progesterone > aldosterone > testosterone > estradiol-17β. We conclude that known differences in the sensitivities of PMNs and MNs to glucocorticoids are apparently not caused by differences in the concentrations or characteristics of their glucocorticoid receptors.     

Glucocorticoid binding to adenohypophysis receptors and its physiological role.

The binding characteristics of glucocorticoids to cytoplasmic and nuclear fractions of the adenohypophysis were determined and an attempt was made to correlate steroid uptake with physiological action. Binding was of high affinity and exhibited a limited number of acceptor sites for dexamethasone (Dx). It was shown that the steroid-receptor complex dissociated rapidly at 37 degree C, but was stable at low temperatures. An inverse relationship was detected between the plasma corticosterone (B) titer and the capacity of receptors to combine 3h-Dx. Shortly after stress or injection of physiological doses of B,50 to 70% of the acceptor sites were saturated. Progressive desaturation of the occupied receptors appeared to depend upon time and the plasma B concentration. Some evidence was also provided supporting a possible correlation between the extent of Dx inhibition of in vitro ACTH secretion and the degree of binding sites' occupancy. These results suggest that glucocorticoid binding to pituitary receptors may be correlated with the physiological action of the steroids on ACTH release.     

Localization and role of transcortin-like molecules in the anterior pituitary

The uptake and binding of [³H]dexamethasone and [³H] corticosterone in the anterior pituitary has been studied with special reference to the transcortin-like protein and compared with the uptake and binding in the hippocampal region of the rat brain, which lacks this protein.Isolated pituitary cells of adrenalectomized rats contained both glucocorticoid receptors and transcortin-like molecules.A collagenase treatment was used for isolation of the intact pituitary cells. Exposure of cytosol from broken cells of the anterior pituitary to this enzyme destroyed nearly all binding activity for the corticosteroids. The results indicate an intracellular localization of the transcortin-like molecules in the anterior pituitary, although the possibility that plasma transcortin adheres to the cell surface cannot be excluded.Estradiol treatment increased the level of transcortin in the plasma and of transcortin-like molecules in the anterior pituitary. The concentration of the glucocorticoid receptors in the anterior pituitary and the hippocampus remained unaffected.Subcutaneous injection of doses up to 50 μg corticosterone per 100 g body weight did not compete for the binding in vitro of [³H]dexamethasone to glucocorticoid receptors in anterior pituitary cytosol. The same doses of corticosterone reduced binding of the synthetic steroid in cytosol of the hippocampus by 40%.Endogenous corticosterone competed poorly for the uptake of [³H]dexamethasone in cell nuclei of the anterior pituitary. In cytosol 71% of the receptor sites were still available for [³H] dexamethasone binding in vitro.It is proposed that transcortin-like molecules in the anterior pituitary, whether intra- or extracellularly, modulate the effect of corticosterone on pituitary-adrenal activity by mediating the extent of interaction of the steroid with the glucocorticoid receptor.     

Glucocorticoid dose dependent downregulation of glucocorticoid receptors in patients with rheumatic diseases

OBJECTIVE: The therapeutic success of low doses of glucocorticoids is mediated entirely by classical genomic effects, whereas that of high doses is also mediated to an as yet unknown extent by nongenomic effects. We assessed the relative therapeutic importance of these nongenomic effects in pulse therapy. METHODS: A [3H]dexamethasone radioligand binding assay was used to measure the number of glucocorticoid receptor sites (R, given as number of sites per cell) and glucocorticoid receptor binding affinity (Kd, given in nM) in peripheral blood mononuclear cells isolated from 26 healthy control blood donors and 27 patients with rheumatic diseases. Patients were divided into 4 groups on the basis of their glucocorticoid dose: 0 mg (Group A), < or = 0.25 mg (Group B), 0.25 to 1 mg (Group C), and > 1 mg (Group D) of prednisolone equivalent per kg per day. RESULTS: Sex independent normal values of 3605 +/- 1136 for R and 5.39 +/- 3.4 for Kd were found. At 5407 +/- 1968, the number of receptor sites in patients not receiving glucocorticoid therapy (Group A) was significantly higher than that of controls (p < 0.01). In patients receiving glucocorticoid therapy this value was reduced at 3855 +/- 866 (Group B), 3358 +/- 963 (Group C), and 2685 +/- 962 (Group D). The values in Groups C and D were significantly lower than those in untreated patients (p < 0.02). CONCLUSION: In pulse therapy doses of glucocorticoids that exceed receptor saturation are administered for several days, but in addition significant receptor downregulation occurs. Therefore, we assume an increase in the relative contribution of the nongenomic effects of glucocorticoids to the therapeutic success under these conditions.