Glucocorticoid Binding Capacity Research Articles

Glucocorticoid receptor expression and binding capacity in patients with burn injury.

Burn injuries are associated with strong inflammation and risk of secondary sepsis which both may affect the function of the glucocorticoid receptor (GR). The aim of this study was to determine GR expression and binding capacity in leucocytes from patients admitted to a tertiary burn center. Blood was sampled from 13 patients on admission and days 7, 14 and 21, and once from 16 healthy subjects. Patients were grouped according to the extent of burn and to any sepsis on day 7. Expression and binding capacity of GR were determined as arbitrary units using flow cytometry. GR expression and binding capacity were increased compared to healthy subjects in most circulating leucocyte subsets on admission irrespective of burn size. Patients with sepsis on day 7 displayed increased GR expression in T lymphocytes (51.8%, P < 0.01) compared to admission. There was a negative correlation between GR binding capacity in neutrophils and burn size after 14 days (P < 0.05). GR expression and binding capacity are increased in most types of circulating leucocytes of severely burned patients on their admission to specialized burn care. If sepsis is present after 1 week, it is associated with higher GR expression in T lymphocytes and NK cells.

Five ways to skin a cat: An unexpected diversity of stress profiles in five species of ground squirrels

Event Abstract Back to Event Five ways to skin a cat: An unexpected diversity of stress profiles in five species of ground squirrels Brendan Delehanty1* and Rudy Boonstra1 1 University of Toronto at Scarborough, Biological Sciences, Canada Comparative physiologists often hypothesize that animals facing similar problems will have similar solutions; that is, we expect general patterns to emerge. The alternative hypothesis is that few, if any, patterns will emerge because each species is able to so finely manipulate its physiology that there are any number of ways to solve the same problem. We studied stress axis (hypothalamic-pituitary-adrenal axis) function over the course of the breeding season in a group of closely related ground squirrel species. Because the stress axis is highly conserved among vertebrates in terms of its basic functioning, and because all of the ground squirrel species we studied face similar reproductive challenges (all are hibernators that compete for mating opportunities shortly upon emerging from hibernation), we expected that species sharing similar life history strategies would have similar stress profiles. For each species we obtained pre- and post-breeding stress profiles of males that included stress-induced glucocorticoid (GC) levels, GC levels in response to a hormone challenge protocol (dexamethasone suppression and ACTH stimulation), blood glucose levels, free fatty acids, and complete blood counts. We found that life history did not predict stress profile: each species had a unique stress profile, with no two species sharing even basic trends in GC levels and binding capacity. Trends in total GC concentrations, GC binding capacity, and free GC concentrations over the course of the breeding season varied between all 5 species studied. Moreover, the downstream measures of stress (glucose, free fatty acids, hematocrit, complete blood counts) showed similar variability and could not be predicted from simple measures of GC concentrations. We conclude that many of our simplifying assumptions about how the stress axis supports or constrains reproduction need to be revisited. Acknowledgements Supported by NSERC grant to R.B. and NSTP Grant to B.D. Keywords: downstream effects, Glucocorticoids, ground squirrels, Reproduction, stress profiles Conference: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology, Ann Arbor, United States, 13 Jul - 16 Jul, 2011. Presentation Type: Poster Topic: Stress hormones Citation: Delehanty B and Boonstra R (2011). Five ways to skin a cat: An unexpected diversity of stress profiles in five species of ground squirrels. Front. Endocrinol. Conference Abstract: NASCE 2011: The inaugural meeting of the North American Society for Comparative Endocrinology. doi: 10.3389/conf.fendo.2011.04.00125 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 20 Jul 2011; Published Online: 09 Aug 2011. * Correspondence: Mr. Brendan Delehanty, University of Toronto at Scarborough, Biological Sciences, Toronto, ON, M1C 1A4, Canada, brendan.delehanty@utoronto.ca Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Brendan Delehanty Rudy Boonstra Google Brendan Delehanty Rudy Boonstra Google Scholar Brendan Delehanty Rudy Boonstra PubMed Brendan Delehanty Rudy Boonstra Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Developmental changes in neural corticosteroid receptor binding capacity in altricial nestlings

Altricial nestlings typically do not show an adrenocortical response during the early post-hatch period. This may be a result of an immature hypothalamic-pituitary-adrenal axis, or an enhanced control of the axis by negative feedback. To examine whether the dampened adrenocortical response is due to higher receptor densities in hypothalamus and hippocampus, the major sites for negative feedback and tonic inhibition, we explored the ontogenetic changes in glucocorticoid (GR) and mineralocorticoid receptor (MR) binding capacities in the brain of white-crowned sparrow nestlings. During the 10-day nestling period, MR binding capacity decreased with age, whereas GR capacity was not affected. In addition, this overall decline in MR levels was driven entirely by a decline in cerebellar MR. No age-related changes were observed in hippocampal or hypothalamic areas. Our findings suggest that enhanced negative feedback does not play a major role in the attenuated adrenocortical responses seen in white-crowned sparrow nestlings.

Binding constants and stability of dexamethasone-binding protein from rat heart.

A pronounced decrease in specific dexamethasone binding capacity of heart cytosol was observed. The inactivation of unbound glucocorticoid receptor was not due to the action of proteases since the rat of albumin digestion was very slow in the particulate preparations. The rate of inactivation of glucocorticoid binding capacity depends on cytosol protein concentration and temperature. Addition of hormone and glycerol to cytosol markedly slow binding capacity inactivation. The rapid loss of binding activity, especially in the cytosol with high protein concentration, makes difficult to perform equilibrium studies for simultaneous determination of binding affinity and a number of binding sites. Under various conditions the apparent dissociation constants were measured and compared with the dissociation constants, calculated from the rate of constants of association and dissociation, in order to find out optimal conditions for simultaneous determination of KD and [R0].

Maintenance of glucocorticoid receptor function following severe heat-shock of heat-conditioned cells

The competence of the glucocorticoid receptor to regulate gene expression is thought to depend on Hsp70-driven continuous reactivation following spontaneous inactivation of its hormone-binding state. We show here that the glucocorticoid-binding capacity of HeLa cells fell with increasing temperature in the range 43–45 °C in a manner that closely paralleled the loss of soluble receptor protein. Receptor activity was maintained during moderate (43 °C) but not severe (45 °C) heat shock. Hsp70 was rapidly rendered insoluble and was replenished by soluble chaperone at 43 but not 45 °C. In heat-conditioned cells expressing different levels of Hsp70, we observed a positive correlation between the concentration of active receptor and the amount of Hsp70 rendered insoluble by heat shock. Much higher amounts of Hsp70 were rendered insoluble and receptor competence to regulate gene expression was preserved after severe heat shock of appropriately heat-conditioned cells. An excess of Hsp90 was found associated with resolubilized heat-inactivated receptor from severely heat-shocked cells. The data indicate that GR activity is maintained, provided that denaturation and/or aggregation of the receptor is prevented by Hsp70; and that the concentration of the chaperone is the limiting determinant of receptor activity in heat-shocked HeLa cells.

Blunted pituitary-adrenocortical stress response in adult rats following neonatal dexamethasone treatment.

Glucocorticoids have a prominent impact on the maturation of the stress-related neuroendocrine system and on the postnatal establishment of adaptive behaviour. The present study aimed at investigating the stress responsiveness of the hypothalamo-pituitary-adrenocortical (HPA) axis in young and adult rats after neonatal treatment with the synthetic glucocorticoid agonist, dexamethasone. Newborn male Wistar rats were injected s.c. with 1 microg/g dexamethasone on postnatal days 1, 3 and 5. Circulating adrenocorticotropic hormone (ACTH) and corticosterone concentrations were measured in the resting state and following a 30-min cold stress at the age of 10 days, as well as after a 30-min restraint stress at the age of 14 weeks. Also in adults, pituitary and adrenocortical hormone responsiveness was evaluated after i.v. administration of 2 microg/kg corticotropin releasing hormone (CRH). In addition, glucocorticoid (GR) and mineralocorticoid receptor (MR) binding capacities were assessed in the pituitaries of adult rats. The results showed that at day 10 basal ACTH concentration was elevated while the cold stress-evoked ACTH response was attenuated in the dexamethasone-treated rats. As adults, treated rats showed a suppressed elevation of both ACTH and corticosterone plasma concentrations in response to restraint, while basal hormonal concentrations were not altered. There was no difference in the magnitude of the CRH-induced elevation of ACTH and corticosterone concentrations initially; however, the dexamethasone-treated animals showed a prolonged secretion of both hormones. These animals also showed a selective decrease in pituitary GR binding capacity. Neonatal dexamethasone treatment strongly suppressed body weight gain, and adrenal and thymus weights in the early phase of postnatal development. By adulthood, the body and adrenal weights were normalized while thymus weight was greater than in controls. These findings indicate that neonatal dexamethasone treatment permanently alters HPA axis activity by reducing stress responses to cold and restraint probably through supra-pituitary actions, and by decreasing the effectiveness of feedback through a diminished GR binding in the pituitary.

Asymmetrical Distribution of Hippocampal Mineralocorticoid Receptors Depends on Lateralization in Mice

Previous experiments showed that the activation of the hypothalamic-pituitary-adrenal (HPA) axis during stress was associated with behavioral lateralization used as a marker of population heterogeneity in mice. Furthermore, brain asymmetries have been demonstrated in neurotransmitter metabolism and neuroendocrine modulation. As the hippocampus modulates the activity of the HPA axis in stress and basal conditions, we postulated that hippocampal corticoid receptors may be asymmetrically distributed and that asymmetry may differ according to behavioral lateralization of animals. In order to answer these questions, binding capacity of mineralocorticoid (MR) and glucocorticoid (GR) receptors was determined in right and left hippocampi of mice previously selected for paw preference. The results show that regardless of behavioral lateralization, there was a tendency for a right dominance in MR binding capacity in the hippocampus but interestingly, the percentage of right/total MR binding capacity was inversely correlated with individual paw preference scores. The affinity of MRs did not depend on behavioral lateralization. GR binding capacity was similar in each hemisphere and no relationship was found between GR binding capacity and paw preference scores. These results suggest that hippocampal receptors for corticoids may play an important role in the asymmetrical brain control of immune reactivity.

Temporary loss of glucocorticoid receptor-mediated regulation of gene expression in heat-shocked cells

The effect of heat shock on the transcriptional activity of glucocorticoid receptor was assessed using HeLa cells stably transfected with the chloramphenicol acetyltransferase (CAT) gene the transcription of which is controlled by two glucocorticoid-responsive elements placed directly upstream of a core promoter. Heat shock inactivated the high-affinity glucocorticoid binding capacity of the cells and nullified the rate of accumulation of CAT mRNA in the presence of hormone. Hormonal responsiveness was restored on return to normal temperature concomitantly with recovery of high-affinity glucocorticoid binding capacity. Heat inactivation of the receptor was coincident with loss of its solubility and apparently unrelated to receptor degradation.

Effects of adrenalectomy on glucocorticoid and mineralcorticoid receptor binding capacity and mRNA expression in the rat hippocampus and spinal cord

Quantitative densitometry has been used to measure cholinesterase bands in amniotic fluids after polyacrylamide gel electrophoresis. The ratio of acetylcholinesterase (AChE) to pseudocholinesterase (PChE) could be used both for diagnosis and for differential diagnosis of fetal abnormalities. Normal amniotic fluids had AChE/PChE density ratios of zero, while neural tube defect fluids were all above 0.15. Ventral wall defect samples had ratios below 0.15, as did false positives due to contamination with human fetal serum. In samples which had been accidentally contaminated with fetal calf serum, density ratios were above 0.15. Similar results were obtained with both slab gel and rod gel electrophoresis.

Short-Term Adrenalectomy Increases Glucocorticoid and Mineralocorticoid Receptor mRNA in Selective Areas of the Developing Hippocampus

In the central nervous system, adrenal steroids bind to two different types of corticosteroid receptors: glucocorticoid (GR) or mineralocorticoid (MR). In vitro biochemical and autoradiographic techniques have been used to infer GR and MR protein abundance in the hippocampus. Adrenalectomy (ADX) is routinely performed to measure the normal receptor number in absence of corticosterone (B), which would otherwise interfere with the binding reaction, The developing rodent has low basal B levels until the third week of life. We were interested in whether removal of circulating B may have a greater impact in the developing hippocampus than in the adult animal. In this study we examined the effect of a 14-h ADX on hippocampal GR and MR binding capacity (B max) by standard binding techniques and on gene expression by in situ hybridization. ADX was performed on Day 6, 10, 14, 18, 22, 28, 35, and 45 and on adult animals. GR B max increased from Day 6 to adult levels by Day 22 (d6 = 159.0 ± 27; d22 = 369.1 ± 43; a = 344.8 ± 23, fmol/mg protein ± SE). In contrast, MR B max had adult levels on Day 6 and increased above these until Day 45, when it decreased and approached adult concentrations (d6 = 83.2 ± 22; d45 = 123 ± 23; a = 76.9 ± 13, fmol/mg protein ± SE). The greatest absolute increase for both receptors occurred between Days 22 and 45 and correlated with increases in GR and MR gene expression. Moreover, age- and region-specific changes were evident in the developing hippocampus. In addition, the adult animal also exhibited an MR mRNA upregulation after 14 h of adrenalectomy. We propose extreme caution when interpreting GR and MR B max values obtained after short-term adrenalectomy in both adult and developing animals since upregulation of these genes is evident in this short time frame.

Effect of 6-hydroxydopamine and 5,7-dihydroxytryptamine on tissue uptake and cell nuclear retention of corticosterone in the rat hypothalamus

Previous studies have shown that norepinephrine and serotonin can modulate the glucocorticoid (GC) binding capacity in the hippocampus. The aim of the present study was to evaluate the role of these neurotransmitters in regulating GC receptors in the hypothalamus. Injection of the neurotoxin 6-hydroxydopamine (6-OHDA) into the ventral noradrenergic bundle (VNAB) and 5,7-dihydroxytryptamine (5,7-DHT) into the raphe nuclei caused a marked depletion in norepinephrine and serotonin, respectively, in the paraventricular nucleus (PVN) and mediobasal hypothalamus (MBH). The injection of these neurotoxins did not change the basal levels of ACTH and corticosterone. Injection of 6-OHDA into the VNAB caused a significant reduction in the cell nuclear binding of corticosterone in the PVN but not in the MBH. Conversely, injection of 5,7-DHT into the raphe nuclei caused a significant reduction in cell nuclear binding of corticosterone in the MBH but did not affect binding in the PVN. These results demonstrate that at least part of the nuclear corticosteroid receptors in the PVN and MBH are differentially regulated by the noradrenergic and serotonergic systems.

Characterization of Glucocorticoid Binding Capacity in Human Mononuclear Lymphocytes: Increase by Metyrapone is Prevented by Dexamethasone Pretreatment

Abstract Autoregulation of receptor systems by their own ligands is a well established biological phenomenon. While down-regulation of the glucocorticoid binding capacity by glucocorticoids has been shown in animals and humans, data on up-regulation processes in humans are lacking. To further explore glucocorticoid receptor plasticity in relation to endogenous ligands, glucocorticoid binding parameters were assessed in 15 healthy controls before and after oral administration of 1.5 g metyrapone with and without dexamethasone pretreatment. Administration of metyrapone resulted in blockade of the feedback of the hypothalamic-pituitary-adrenal system as shown by the rise in adrenocorticotropin levels, while pretreatment with 1 mg dexamethasone completely suppressed adrenocorticotropin concentrations. Glucocorticoid binding sites per lymphocyte exhibited an increase of 63% following metyrapone administration, which was prevented by dexamethasone pretreatment. Comparison of morning and afternoon glucocorticoid binding sites per cell in 11 healthy volunteers further revealed a diurnal rhythm of glucocorticoid receptor sites. These data suggest that human lymphocyte glucocorticoid receptors are under autoregulatory control.

Hippocampal glucocorticoid receptor and behavior: A correlative study in rats and mice

A correlation has been demonstrated between binding capacity for [ 3H]corticosterone in the hippocampus and the performance in passive and active avoidance in the rat, and impaired behavior in adrenalectomized rats by exogenous corticosterone is restored. On this basis we have studied the possible correlation between strain-dependent behavioral differences and the glucocorticoid binding capacity in the hippocampus in mice and rats. In Naples high- (NHE) and low-excitability (NLE) rat strains, genetically selected on the basis of divergent locomotor activity upon forced exposure to a spatial novelty situation, no differences were found in glucocorticoid maximal binding capacity while both strains had a lower capacity than Naples random-breed (NRB) control rats. However, the intra-strain correlative analysis of hippocampal glucocorticoid receptors number and behavioral scores demonstrated that motor and emotional indexes of arousal to novelty were positively correlated in NLE-and negatively in NHE-, while no correlation was present in NRB rats. Using two inbred strains of mice, C57BL/6 and Balb/c, extensively investigated for learning abilities, the lower active avoidance score of C57BL/6 was associated with a lower binding capacity for [ 3H]corticosterone in the hippocampus. Altogether the above results support the involvement of the hippocampal glucocorticoid receptor in the modulation of some adaptive behavioral responses, while do not prove that genetic differences in behavior rest on parallel differences in binding capacity for glucocorticoid hormone.

Human glucocorticoid receptor cDNA contains sequences sufficient for receptor down-regulation.

Glucocorticoid receptors are ligand-dependent transcription factors that are subject to down-regulation by their cognate ligand; however, the mechanisms mediating this physiological response are not completely understood. Since analysis of the human glucocorticoid receptor (hGR) cDNA sequence revealed the presence of sequences with homology to both positive and negative glucocorticoid regulatory elements, we have examined the potential of hGR to bind to the hGR cDNA by Southwestern blot analysis. The data revealed that glucocorticoid receptors exhibited specific binding to their own cDNA. To determine whether this binding was of functional significance in the down-regulation of glucocorticoid receptors, we analyzed the effect of glucocorticoids on hGR protein levels from COS 1 cells transfected with an hGR cDNA expression vector. These transfected cells produced intact hGR that were capable of ligand-dependent regulation of a co-transfected glucocorticoid-responsive reporter gene. Glucocorticoid treatment of hGR-transfected cells resulted in down-regulation of hGR (assayed by both glucocorticoid binding capacity and hGR protein levels) within 24 h of steroid administration. To determine if the glucocorticoid-induced down-regulation of transfected hGR was compatible with effects at the levels of receptor gene expression and RNA stability, we examined hGR mRNA steady state levels. Reductions from 2- to 6-fold were observed in hGR mRNA levels following glucocorticoid treatment of transfected COS 1 cells. This down-regulation of transfected hGR mRNA could not be attributed to either the Rous sarcoma virus promoter, which drives hGR expression, or to other sequences present in the vector plasmid since transcription of a related plasmid containing a chloramphenicol acetyltransferase gene in place of the hGR cDNA was not regulated by glucocorticoids. Down-regulation of hGR mRNA by glucocorticoids in transfected cells occurred in a time- and dose-dependent manner that is consistent with a glucocorticoid receptor-mediated process. Glucocorticoid-induced down-regulation of hGR mRNa steady state levels was not observed in COS 1 cells transfected with cDNAs encoding mutant hGR (defective in either steroid or DNA binding), which indicates that functional steroid and DNA binding domains of the expressed hGR were required for down-regulation. Interestingly, treatment of transfected COS 1 cells with the glucocorticoid antagonist RU486 also resulted in down-regulation of transfected hGR mRNA. Deletion analysis revealed that the region of the hGR cDNA that was responsible in part for the observed down-regulation in response to glucocorticoid was contained within a 1-kilobase restriction fragment (from base pair +527 to +1526).(ABSTRACT TRUNCATED AT 400 WORDS)

Aluminum fluoride inhibition of glucocorticoid receptor inactivation and transformation

Fluoride, in the presence of aluminum ions, reversibly inhibits the temperature-mediated inactivation of unoccupied glucocorticoid receptors in cytosol preparations from mouse L cells. The effect is concentration-dependent, with virtually complete stabilization of specific glucocorticoid-binding capacity at 2 mM fluoride and 100 microM aluminum. These concentrations of aluminum and fluoride are ineffective when used separately. Aluminum fluoride also stabilizes receptors toward inactivation by gel filtration and ammonium sulfate precipitation. Aluminum fluoride prevents temperature-dependent transformation of steroid-receptor complexes to the DNA-binding state. Aluminum fluoride does not inhibit calf intestine alkaline phosphatase, and unoccupied receptors inactivated by this enzyme in the presence of aluminum fluoride can be completely reactivated by dithiothreitol. The effects of aluminum fluoride are due to stabilization of the complex between the glucocorticoid receptor and the 90-kDa mammalian heat-shock protein hsp90, which suggests that aluminum fluoride interacts directly with the receptor. Endogenous thermal inactivation of receptors in cytosol is not accompanied by receptor dephosphorylation. However, inactivation is correlated with dissociation of hsp90 from the unoccupied receptor. These results support the proposal that hsp90 is required for the receptor to bind steroid and dissociation of hsp90 is sufficient to inactivate the unoccupied receptor.

Strain-dependent differences in hippocampal glucocorticoid binding capacity and active avoidance in the mouse

Maximal individual [ 3H]corticosterone binding capacity in the hippocampus was lower in C57BL/6 mice than in BALB/c mice, and positively correlated with active avoidance learning in the two strains. Moreover, a parallel difference in the activity of hypothalamo-pituitary adrenocortical axis (HPAA) was found, consisting in a level of plasma corticosterone in C57BL/6 higher than in BALB/c mice. These results confirm the genetically determined differences in behavior of C57BL/6 and BALB/c mice, and demonstrate their association with differences in hippocampal corticosterone binding capacity, pointing to a functional relationship between the behavioral and neuroendocrine parameters.

Isolation and characterization of a mouse L cell variant deficient in glucocorticoid receptors

The growth of mouse L cell fibroblasts is inhibited by glucocorticoids, and we have selected spontaneous glucocorticoid-resistant L cells in culture. One cloned variant exhibits a stable phenotype in the absence of selective conditions. This variant contains no specific glucocorticoid-binding capacity, no immunoreactive glucocorticoid receptor protein, and no detectable glucocorticoid receptor messenger RNA. A glucocorticoid-dependent reporter gene requires exogenous glucocorticoid receptor cDNA and steroid in order to be expressed in this variant. Genomic DNA analysis of the variant cell line indicates that there has been no gross alteration in receptor gene structure. These results suggest that the variant may be deficient in transcription of the glucocorticoid receptor gene.

Regulation of Rat Pancreatic Glucocorticoid Receptors by Thyroxine during Development*

The effect of T4 on the development of pancreatic glucocorticoid receptors was studied in normal and adrenalectomized rat pups. Daily injection of T4 (0.1 microgram/g BW) to intact pups starting 3 days before death at 10, 15, and 20 days of age resulted in a precocious increase in pancreatic glucocorticoid-binding capacities. Intact pups made hypothyroid by propylthiouracil feeding exhibited lower glucocorticoid-binding capacities in their pancreata. Scatchard analysis demonstrated an increase in the number of glucocorticoid-binding sites in the pancreata of T4-treated intact rats compared to that in normal intact rats. In hypothyroid groups the number of glucocorticoid-binding sites was much lower than that in normal intact rats. The Kd values, however, were unchanged in hypothyroid, hyperthyroid, and control groups. Rat pups who underwent adrenalectomy at 12 days of age had undetectable plasma corticosterone levels and showed an increase in their pancreatic glucocorticoid-binding capacity 3 days after operation. Replacement of corticosterone resulted in a binding level similar to that in the sham-operated group. However, injection of T4 alone to adrenalectomized pups led to a further increase in pancreatic glucocorticoid-binding capacity above that due to adrenalectomy alone. When both T4 and corticosterone were given together to adrenalectomized pups their pancreatic glucocorticoid-binding capacities increased to levels above those in the adrenalectomized group, but lower than those in pups receiving T4 alone. Our results suggest that T4 modulates the development of rat pancreatic glucocorticoid receptors and, at least in part, acts via pathways independent of adrenal function.

Corticosteroid receptors in liver cytosol of the clawed toad, Xenopus laevis: Daily and seasonal variations

In postmetamorphic Xenopus laevis liver cytosol the glucocorticoid binding capacity R o and the dissociation constant K d were determined. The receptor assay included an incubation period of 24 hr at 0–4° with sodium molybdate to stabilize the receptor. Dexamethasone, triamcinolone acetonide, and corticosterone as tritiated ligands were compared regarding the R 0 (67.6, 57.2, and 30.7 fmol/mg protein), the K d (3.54, 0.56, and 9.03 n M), and the rate of dissociation in young specimens of X. laevis. In adult toads the [ 3H]dexamethasone receptor binding capacity was threefold higher in females than in males (153.86 ± 12.19, 54.29 ± 4.5 fmol/mg protein)—with about the same K d (3.97 ± 0.57, 4.08 ± 0.28 n M). Young toads were kept under an artificial light regime (light from 600 to 1800 hr) and dexamethasone binding was measured every 3 hr. Unlike K d , R 0 showed a significant diurnal variation with maximal values at 600 and 1800 hr, which occurred about 9 hr after a maximal level of corticosterone in serum was reached (900, 2100). Seasonal variations of the [ 3H]dexamethasone and [ 3H]corticosterone binding capacity were different in both sexes of adult X. laevis. Maximal values in males were found in June/July and October/November. In females, the R 0 was increased in the second half of the year with the maximum in August (275.5 ± 45.02 fmol/mg protein). No correlation between R 0 and the concentration of corticosterone or aldosterone existed.

The biopotency of dexamethasone at causing hepatic glucocorticoid receptor down-regulation in the intact mouse

The effect of dexamethasone administered intraperitoneally on hepatic glucocorticoid receptor binding capacity was measured in adrenalectomized male Swiss Webster mice. The liver content of dexamethasone was also measured. Within 30 min of a 5 μg injection, the hepatic content of dexamethasone reached a maximum and fell quickly thereafter. By 6 h the hepatic content of dexamethasone had decreased to 25% of maximum and by 24 h the liver did not contain detectable dexamethasone. At this 24 h point, the glucocorticoid binding capacity was reduced to 50% of control. This decrease reflected down-regulation. Other studies revealed that only glucocorticoids caused this effect and doses of dexamethasone as low as 0.5–5 ng caused a clear down-regulation in binding capacity. Doses that cause receptor down-regulation are also effective at inducing tyrosine aminotransferase, suggesting that dexamethasone down-regulates its own receptors over a physiologically meaningful dosage range. It is concluded that dexamethasone causes a dose-dependent down-regulation of the glucocorticoid receptor in mouse liver.