Number Of Glucocorticoid Binding Sites Research Articles

Lymphocyte glucocorticoid receptor expression level and hormone-binding properties differ between war trauma-exposed men with and without PTSD

Posttraumatic stress disorder (PTSD) has been shown to be associated with altered glucocorticoid receptor (GR) activity. We studied the expression and functional properties of the receptor in peripheral blood mononuclear cells (PBMCs) from non-traumatized healthy individuals (healthy controls; n=85), and war trauma-exposed individuals with current PTSD (n=113), with life-time PTSD (n=61) and without PTSD (trauma controls; n=88). The aim of the study was to distinguish the receptor alterations related to PTSD from those related to trauma itself or to resilience to PTSD. Functional status of the receptor was assessed by radioligand binding and lysozyme synthesis inhibition assays. The level of GR gene expression was measured by quantitative PCR and immunoblotting. Current PTSD patients had the lowest, while trauma controls had the highest number of glucocorticoid binding sites (Bmax) in PBMCs. Hormone-binding potential (Bmax/KD ratio) of the receptor was diminished in the current PTSD group in comparison to all other study groups. Correlation between Bmax and KD that normally exists in healthy individuals was decreased in the current PTSD group. Contrasting Bmax data, GR protein level was lower in trauma controls than in participants with current or life-time PTSD. Current PTSD is characterized by reduced lymphocyte GR hormone-binding potential and by disturbed compensation between Bmax and hormone-binding affinity. Resilience to PTSD is associated with enlarged fraction of the receptor molecules capable of hormone binding, within the total receptor molecule population in PBMCs.

Effects of the glucocorticoid antagonist RU486 in spontaneously hypertensive and Sprague Dawley rats

The effect of a low dose of the gluco-corticoid receptor antagonist, RU486, was tested in spontaneously hypertensive (SHR) and in Sprague Dawley (SD) rats. In SD rats, RU486 (50 micrograms/day, 18 days) significantly increased growth rate and thymus weight, probably by antagonising the actions of endogenous glucocorticoid hormones. Blood pressure and plasma corticosterone levels were not affected by RU486 at this dose. In leucocytes, RU486 treatment in vivo reduced the number of glucocorticoid binding sites but did not affect binding affinity. In contrast, growth rate and thymus weight were not altered in SHR when treated with the same dose of RU486 for a longer period (60 days). Blood pressure was unaffected as were leucocyte glucocorticoid receptor characteristic. Glucocorticoid receptor binding characteristics for RU486 were similar for liver cytosol of SD and SHR. We conclude that the apparent difference in sensitivity to RU486 between strains is probably not due to differences in interaction of the antagonist with the glucocorticoid receptor but may be caused by differences in pharmacokinetics of RU486 between strains.

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.

Glucocorticoid receptors in mononuclear blood cells and their correlation to endogenous and exogenous corticoids in healthy and asthmatic children.

The number and affinity of glucocorticoid binding sites in peripheral mononuclear cells (MNC) of asthmatic and healthy children were determined by a whole cell (3H)dexamethasone binding assay at 37 degrees C. Using HPLC determination, corresponding serum levels of non-protein-bound (free) cortisol, whole cortisol and cortisone as well as urine excretion of free cortisone and cortisol were assessed. The average number of binding sites (BS) per cell and the dissociation constant (KD) respectively, in atopic asthmatics (7768 +/- 666 BS/MNC resp. KD = 17.2 +/- 2 nM) did not differ from the values measured in our control group (8333 +/- 691 BS/MNC resp. 25.4 +/- 4.8 nM). Within the age range 1 month-15.8 years neither age-dependent changes nor sex-related differences in the number of binding sites or the KD values could be detected. Active or currently inactive asthmatics, and patients under different antiasthmatic drug regimes, had similar binding sites on MNC. No differences in serum levels of cortisol, cortisone and free cortisol or in free cortisol and free cortisone of 24-h urine samples were found between healthy children and asthmatics. After a short course of prednisolone therapy for an acute severe asthmatic attack the number of glucocorticoid binding sites in peripheral MNC decreased to an average of 4632 +/- 421 BS/MNC, whereas the dissociation constant did not change significantly (14.5 +/- 3.6 nM). The corticoid-hormone pattern in the serum, 24-h urine excretion, and the normal number and affinity of glucocorticoid receptors on peripheral MNC suggest that there is no primary, general impairment of glucocorticoid metabolism in asthmatic children.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro evidence that hypothyroidism modifies glucocorticoid receptors

We have previously reported that hypothyroidism is accompanied by modifications of the cytosolic rat liver glucocorticoid receptors. In the present study we have used molybdate, which stabilizes the glucocorticoid receptor from hypothyroid rats, allowing its characterization. We confirm a decrease in the number of glucocorticoid binding sites in the liver of hypothyroid rats as compared to that in normal rats (8000 per cell versus 23000 per cell for normal rats). At 22°C and in the absence of molybdate, thermal activation shows the same kinetics in both complexes from normal and hypothyroid rats, though activated hormone-receptor complexes from hypothyroid rats are progressively degraded ( t 1 2 = 230 min). At the same temperature the dissociation rate of native complexes from hypothyroid rats is slower ( K −1 = 8 × 10 −3 min −1) than normal ( k −1 = 16 × 10 −3 min −1). Competitive dissociation studies using [ 3H]dexamethasone indicate that native complexes from hypothyroid rats show altered affinities for agonist and antagonist. Hypothyroidism decreases the number of rat liver glucocorticoid receptors and alters their properties, as evidenced by their greater instability and differences in steroid binding. These effects may be due to regulating factors and/or slight, probably post-transcriptional, modifications of the binding protein.

Glucocorticoid receptors in Epstein-Barr virus-transformed human lymphocytes.

Glucocorticoid receptors were studied in cultured human lymphocytes from normal donors after transformation with the Epstein-Barr Virus (EBV) and compared to those of circulating human mononuclear leukocytes. Both whole cell and cytosol fractions were examined for [3H]-dexamethasone binding. The concentration and absolute number of glucocorticoid binding sites were increased five-fold in the transformed cells when compared to the non-transformed human mononuclear leukocytes. However, the affinity (Kd) of the glucocorticoid receptor for dexamethasone was the same in both types of cells. The denatured glucocorticoid receptor, covalently labelled with [3H]-dexamethasone-21-mesylate, was identified by SDS polyacrylamide gel electrophoresis as a protein moiety with Mr approximately equal to 92,000, similar to that obtained from human non-transformed mononuclear leukocytes. The pattern of the activation of the hormone-receptor complexes, analyzed by phosphocellulose chromatography, was similar in both types of cells, and also the time-courses of loss of specific binding during thermal activation were similar. These results suggest that viral transformation is associated with increases in the concentration and the absolute number of glucocorticoid receptors whereas other qualitative receptor characteristics remain similar. Thus, transformation of cells with EB virus can provide a constant source of glucocorticoid receptors for study.

Glucocorticoid receptors in sheep brain tissues during development.

In this study we describe changes in the number of glucocorticoid binding sites that occur in cytosols of pituitary, hypothalamic, and hippocampal tissue obtained from fetal, newborn, and adult sheep. We observed specific, saturable binding of [3H]triamcinolone acetonide by cytosols of all three tissues. There were both age-related and tissue-related differences in the number of binding sites observed. By midgestation (70 days) significant quantities of binding sites were present in pituitary and brain tissues, and the number remained relatively constant throughout gestation and in the neonatal period. The number of binding sites declined markedly in cytosols from adult tissues. At all ages there were more binding sites present in pituitary cytosols than in cytosols of hypothalamus and hippocampus. The sucrose density gradient profile of the radioactive ligand-binding site complex is characteristic of a glucocorticoid receptor. Thus, by midgestation at least a portion of the biochemical machinery mediating glucocorticoid effects is present in the fetal pituitary and in specific brain tissues considered important in the modulation of ACTH secretion by corticosteroids.

Low levels of glucocorticoid binding sites in circulating lymphocytes of premature infants suffering from hyaline membrane disease

The number and affinity of glucocorticoid binding sites in lymphocytes of newboms and prematures were determined by a whole cell [ 3H]dexamethasone binding assay. The average binding capacities were as follows: 1758 ±245 binding sites/cell in cord blood, 2758 ± 307 binding sites/cell in mature newborns ( K d : 6,23 × 10 −1M), and 2031 ± 330 binding sites/cell in prematures. No specific binding was measurable in several cases. All the prematures, who did not display a measurable glucocorticoid binding capacity, suffered from a serious hyaline membrane disease (HMD), they died on 3.5–4 days of life. HMD diagnosis was established in 12 cases in toto. The number of glucocorticoid binding sites was significantly smaller in this group (639 ± 216 per cell) than in the case of the rest of prematures (2959 ± 404 per cell, P < 0.001). Our results suggest that lack of glucocorticoid receptors may be one of the causes of the HMD.

Age-Related Changes in the in vitro Glucocorticoid Responsiveness of Rat Spleen and Splenic Leukocytes

Specific in vitro glucocorticoid uptake and binding activity of spleen and splenic leukocytes were examined in adult (2- to 3-month-old) and senescent (24- to 28-month-old) male, adrenalectomized Sprague-Dawley and Wistar rats. No significant differences in these parameters were evident between young and old animals of either strain. Moreover, cortisol inhibition of [5-3H]-uridine uptake and the total number of glucocorticoid binding sites of spleen and its leukocytes were found to be similar in these two age groups. It was concluded that age has no effect on the glucocorticoid responsiveness of the splenic tissues of the two strains of male rats studied.

Binding of glucocorticoids in fetal tissues

Glucocorticoids enhance the activity of a number of enzyme systems in fetal tissues. In the rabbit fetal lung [ 3H]-dexamethasone binds to cytosol and nuclear receptors with a high degree of specificity which is not competed for by other hormones or metabolites. The cytosol [ 3H]-dexamethasone-receptor complex sediments at 4 s in sucrose density gradients in the presence of 0.6 M KCl and at approximately 7–8 s in the absence of salt, whereas the nuclear complex sediments at approximately 4–5 s in the presence or absence of 0.4 M KCl. At 37°C the saturation of nuclear binding sites is reached with 5 × 10 −8−1 × 10 −7 M [ 3H]-cortisol or with 1 × 10 −8 M [ 3H]-dexamethasone. There is an increase in the uptake of [ 3H]-cortisol by lung nuclei (c.p.m./mg DNA) with advancing gestation, beginning on day 20 and reaching a maximum on day 28–30. A lower uptake is observed in lung nuclei of the newborn, the one-month-old and the adult rabbit. The maximum uptake of [ 3H]-cortisol by fetal lung nuclei correlates well with the concentration of surface-active phospholipids extracted from lungs of rabbit fetuses. [ 3H]-Dexamethasone binds to macromolecules in the cytosol and nuclei of fetal rabbit small intestine. The binding is specific for steroids with glucocorticoid potency. There is a parallelism between the increase in the total number of glucocorticoid binding sites in the cytosol and nuclei and the increase in tissue weight up to term. The concentration of binding sites (pmol/mg protein or DNA) is maximum at day 25 to 26 of gestation, followed by a decrease to adult levels within a few days of birth. Alkaline phosphatase activity is first detectable on day 25 of gestation and increases rapidly thereafter. There seems to be a temporal relationship between the development of the fetal rabbit small intestine and the level of glucocorticoid receptors in the cytosol and nuclei.

Reduced glucocorticoid binding site concentration in cortical neuronal perikarya from senescent rats

Isolated neuronal perikarya from cerebral cortices of senescent (24-26-month-old) male rats exhibit a 55-65% reduction in the concentration of specific cytosol glucocorticoid binding sites when compared to mature counterparts (12-13 months). Neuronal preparations from the two age groups are essentially identical with respect to yield, protein content, physiochemical integrity and purity as judged by enrichment for free glycine and reduction in carbonic anhydrase specific activity (relative to unfractionated cortical cell suspensions). Adrenalectomy does not alter the number of glucocorticoid binding sites in either group, and age differences do not appear due to differential partitioning of sites between nucleus and cytoplasm. It is suggested that loss of receptors from the cortical neuron population represents a possible mechanism by which glucocorticoid responsiveness may be decreased during aging.