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1.
Immunocytochemical study of the glucocorticoid receptor in rat liver nuclei after hyperthermic stress 总被引:1,自引:0,他引:1
The presence of glucocorticoid receptors (GR) in rat liver nuclei over a 24 h time period following hyperthermic stress at 41 degrees C was immunocytologically studied using unfixed nuclear smears. Liver nuclei in unstressed animals were found to be immunonegative for GR. However, intense GR immunopositivity followed by a subsequent gradual decrease in receptor levels was observed in the nuclei of test animals during the first 2 h after stress. This stress-related increase in the receptor nuclear level was greater than the increase seen after dexamethasone administration. These results suggest that hyperthermic stress could potentiate the hormonal stimulation of receptor nuclear translocation. 相似文献
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Stress-induced changes of glucocorticoid receptor in rat liver. 总被引:4,自引:0,他引:4
M Alexandrová P Farkas 《The Journal of steroid biochemistry and molecular biology》1992,42(5):493-498
The effect of corticosterone injection and of acute and repeated stress on rat liver cytosol glucocorticoid receptor was studied to ascertain whether corticosterone-induced glucocorticoid receptor (GR) regulation also takes place in intact animals as it does in adrenalectomized ones. Adult male rats were exposed to six different stressors (swimming, 10 mg/kg histamine i.p., 500 mU/kg vasopressin s.c., heat, immobilization and cold) acutely or three times daily for 18 days (repeated stress). Each of the stressors applied acutely provoked a pronounced increase of plasma corticosterone with subsequent induction of hepatic tyrosine aminotransferase activity. Depletion of cytosol receptor was however only noticed after swimming and histamine injection. On the other hand, sustained hypersecretion of corticosterone evoked by repeated stress significantly reduced the number of GR in rat liver cytosol without any change in Kd. It is concluded that in the presence of intact adrenal glands cytosol receptors are more resistant to corticosterone-induced depletion than in their absence. Further, repeated stress causes down-regulation of GR in the liver, most probably by sustained corticosterone secretion, yet the effect of other stress factors cannot be excluded. 相似文献
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Glucocorticoid receptor (GR) hormone-binding activity, its physical characteristics, and GR mRNA levels were studied in the liver, brain and muscle of normal (saline-injected) and hypermetabolic septic rats 24 h after the subcutaneous injections of E. coli. The GR levels (hormone-binding activity) declined by about 40%, 56%, and 40% in septic liver, brain, and muscle cytosol, respectively. The mechanism of the decrease in the GR levels in sepsis was studied in liver. The GR levels remained low (45% of control hormone-binding) even after 48 h of E. coli administration. The decrease in the liver GR occurred in the 9S untransformed GR. The 9S GR from septic liver transformed to the 4S form in proportions comparable to the control liver GR. In addition, the 4S GR from control and septic liver was capable of binding to DNA-cellulose to a similar extent. The GR mRNA level in septic liver declined by about 30%. Thus, a decrease in GR hormone-binding activity in sepsis appears to be due to a decline in the steady-state GR mRNA level and not from a change in the qualitative properties of the GR protein. 相似文献
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A Yoshida T Noguchi S Taniguchi Y Mitani M Ueda K Urabe T Adachi Y Okamura C Shigemasa K Abe 《Endocrinologia japonica》1986,33(6):769-775
The changes in the cytosol glucocorticoid receptor (GR) content during a long-term administration of a glucocorticoid were studied to examine the mechanism of the development of steroid hormone resistance. Dexamethasone (Dex) (0.2 microgram/ml and 2.0 micrograms/ml) was given to adrenalectomized rats, and the GR content was determined using the exchange assay 1, 10, 20 and 50 days after the start of administration. The activity of tyrosine aminotransferase (TAT) in the cytosol was also assayed as a measure of the biological responsiveness of these animals to the administered glucocorticoid. The dissociation constant (Kd) was elevated and the Bmaxs of the GR in the cytosol were decreased by the lower concentration of Dex. The Bmaxs decreased to 30% of the untreated controls within 24 h and this lower level was maintained as long as the hormone treatment continued. On the other hand, the cytosol obtained from animals treated with 2.0 micrograms/ml of Dex for 20-24 days did not show any measurable amount of binding to 3H-Dex. The activity of TAT was elevated 24 h after the administration of Dex but decreased gradually and steadily with time during the experimental period. To examine the biological potency of remaining GR in the liver cytosol, 2.0 micrograms/ml Dex was again administered after a long-term treatment. This treatment eliminated the remaining GR completely and induced TAT at almost the same rate as observed in the untreated control animals.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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A. Pekki J. Koistinaho T. Ylikomi P. Vilja H. Westphal P. Touhimaa 《The Journal of steroid biochemistry and molecular biology》1992,41(3-8):753-756
Recent immunohistochemical studies suggest that the unoccupied glucocorticoid receptor (GR) is cytoplasmic and that the ligand causes its translocation into the target cell nucleus. The subcellular location of GR is especially interesting in that other members of the steroid receptor superfamily appear to be nuclear. The intracellular distribution of GR was studied immunohistochemically using a new freeze-drying and vapor fixation method which eliminates the protein diffusion and redistribution possibly caused by liquid fixation techniques. We used two monoclonal antibodies against rat liver GR. Dried samples of the adrenalectomized rat brain and uterus were fixed in p-benzoquinone vapor for 3 h at 60°C and embedded in paraffin. Sections were stained with a biotinylated mouse monoclonal GR antibody using the avidin-biotin-peroxidase complex. Both unoccupied and occupied GR were found in the nucleus of the target cells, fibroblasts in the uterus and nerve cells in the cortex of the brain. The staining was saturated with the cytosol of cos cellls transfected with GR. No cytoplasmic staining was seen even 2 days after adrenalectomy. In conclusion we propose that GR is also located in the nucleus independently of occupation. 相似文献
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S Okret 《Journal of steroid biochemistry》1983,19(3):1241-1248
Seven antisera against the glucocorticoid receptor (GR), raised in different rabbits immunized with highly purified (in case of five rabbits apparently homogeneous) preparation of GR from rat liver cytosol, were compared concerning titer and cross-reactivity. The titers of protein A-purified antisera (10 mg/ml) were in the range 1:100-1:320 as measured by enzyme-linked immunosorbent assay, ELISA, (defined as the dilution giving 50% of maximum absorbance). All seven antisera bound to the rat GR with a Stokes radius of 6.1 nm, but no antiserum reacted with the proteolytically induced steroid binding domain with Stokes radius 3 nm. However, the antigenic determinant(s) of the non-ligand-binding domain(s), split off from the steroid binding domain, is preserved following digestion with alpha-chymotrypsin or trypsin, respectively, since immunoactivity is still detectable by ELISA. Only two of four antisera tested cross-reacted with the GR from human lymphocytes. The same two antisera cross-reacted with chick embryo liver GR. Four out of four antisera tested cross-reacted with mouse liver GR as well as with rabbit lung GR. For these antisera, antibody binding to the GR prior to steroid- or DNA-binding did not influence the ability of the GR to interact with the ligand or DNA-cellulose, respectively. No difference regarding avidity of the antisera for activated or non-activated GR was observed. Furthermore, none of the antisera tested cross-reacted with the estrogen, progestin, androgen or mineralocorticoid receptors in rat. These findings indicate that the antisera from different rabbits raised against the same antigen all react with a certain domain of the rat GR, but show species differences as well as receptor class specificity. 相似文献
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Filipović D Gavrilović L Dronjak S Demajo M Radojcić MB 《Physiological research / Academia Scientiarum Bohemoslovaca》2008,57(2):205-213
The aim of the present study was to define the stress-induced pattern of cytosolic glucocorticoid receptor (GR) and Hsp70 protein in the liver of male Wistar rats exposed to different stress models: acute (2 h/day) immobilization or cold (4 degrees C); chronic (21 days) isolation, crowding, swimming or isolation plus swimming and combined (chronic plus acute stress). Changes in plasma levels of corticosterone were studied by radioimmunoassay (RIA). The results obtained by Western immunoblotting showed that both acute stressors led to a significant decrease in cytosolic GR and Hsp70 levels. Compared to acute stress effects, only a weak decrease in the levels of GR and Hsp70 was demonstrated in chronic stress models. Chronically stressed rats, which were subsequently exposed to novel acute stressors (immobilization or cold), showed a lower extent of GR down-regulation when compared to acute stress. The exception was swimming, which partially restores this down-regulation. The observed changes in the levels of these major stress-related cellular proteins in liver cytosol lead to the conclusion that chronic stressors compromise intracellular GR down-regulation in the liver. 相似文献
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V K Moudgil L Vandenheede C Hurd N Eliezer G Lombardo 《The Journal of biological chemistry》1987,262(11):5180-5187
We have examined the influence of urea on the properties of the rat liver glucocorticoid receptor (GR). A 1-h incubation of hepatic cytosol with 1-3 M urea at 0 or at 23 degrees C caused a progressive decrease in the steroid binding efficiency of GR. Urea treatment of cytosol incubated with 20 nM [3H]triamcinolone acetonide caused transformation of glucocorticoid-receptor complexes (GRc) and resulted in an increase in the binding of GRc to DNA-cellulose and ATP-Sepharose. The transforming effect was maximal with 2.5 M urea at 0 degrees C for 1 h, and it caused a shift in the rate of sedimentation of the 9 S untransformed GRc to a 4 S form, similar to that observed upon incubation of the cytosol GRc at 23 degrees C. This 9 to 4 S transformation could also be observed in the presence of Na2MoO4. The Stokes radii of the GRc eluted from a Bio-Gel-A-0.5m agarose column were determined to be 5.9 and 4.9 nm in the absence and presence of 2.5 M urea. The aqueous two-phase partitioning analysis revealed a significant change in surface properties of GR following urea treatment; the observed partition coefficient values (cpm upper phase/bottom phase) were 0.022, 0.208, and 0.60 for GRc, GRc + 23 degrees C, and GRc + 2.5 M urea, respectively. Furthermore, the urea treatment rendered the GRc less negatively charged, forcing their appearance in the flow-through fractions of a DEAE-Sephacel column. These results suggest that urea is a potent in vitro modulator of the physicochemical behavior of GR, influencing both the steroid binding and the process of receptor transformation. 相似文献
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Glucocorticoid hormone receptor exists in the cytoplasm of target cells in the form of dynamic multiprotein heterocomplexes with heat shock proteins Hsp90 and Hsp70, and additional components of the molecular chaperone machinery. Whole body hyperthermic stress was previously shown to induce alterations in protein composition of these complexes increasing the share of Hsp70, but participation of individual Hsp70 family members was not investigated. In the present study the association of glucocorticoid receptor with constitutive and inducible forms of Hsp70 in the liver cytosol of rats exposed to 41 degrees C whole body hyperthermic stress was examined. Immunoprecipitation of glucocorticoid receptor heterocomplexes by monoclonal anti-receptor antibody (BuGR2) followed by quantitative immunoblotting revealed the presence of both nucleocytoplasmic Hsp70 family members, constitutive--Hsc70 and inducible--Hsp72, within the complexes. Immediately after the stress only Hsc70 was found in association with glucocorticoid receptor. However, after the induction of Hsp72 by stress, its appearance within the glucocorticoid receptor heterocomplexes was also recorded and the presence of both Hsp70 forms within the heterocomplexes was evident by the end of examined 24h period after the stress. This study confirms that heat stress affects protein composition of rat liver glucocorticoid receptor heterocomplexes increasing the share of Hsp70 and shows that this increase could be equally ascribed to constitutive and inducible forms of Hsp70. 相似文献
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Brkljacić J Milutinović DV Dundjerski J Matić G 《Journal of biochemical and molecular toxicology》2004,18(5):257-260
The subject of the present study is the influence of mercury on association of rat liver glucocorticoid receptor (GR) with heat shock proteins Hsp90 and Hsp70. The glucocorticoid receptor heterocomplexes with Hsp90 and Hsp70 were immunopurified from the liver cytosol of rats administered with different doses of mercury. The amounts of co-immunopurified apo-receptor, Hsp90 and Hsp70 were then determined by quantitative Western blotting. The ratio between the amount of heat shock protein Hsp90 or Hsp70 and the amount of apo-receptor within immunopurified heterocomplexes was found to increase in response to mercury administration. On the other hand, the levels of Hsp90 and Hsp70 in hepatic cytosol remained unaltered. The finding that mercury stimulates association of the two heat shock proteins with the glucocorticoid receptor, rendering the cytosolic heat shock protein levels unchanged, suggests that mercury affects the mechanisms controlling the assembly of the receptor heterocomplexes. 相似文献
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The glucocorticoid receptor (GR) was partially characterized in mouse renal cytosol. A sensitive and reproducible [3H]dexamethasone binding assay suitable for use with small quantities of cytosolic protein, was developed. Studies defined the optimal equilibrium binding conditions, metabolism of [3H]dexamethasone in adult renal cytosol, specificity of binding of the GR, and molecular weight of the GR-[3H]dexamethasone complex by gel filtration chromatography. The assay was subsequently used to measure the renal GR during different stages of foetal and postnatal development, as well as in glomerular and renal tubular preparations from adult mice. An almost linear increase in GR occurred from day 13 to day 18 of gestation with levels rising from 100 to 201 fmol/mg cytosol protein; this was followed by a sharp rise in receptor concentration just after birth to 343 fmol/mg cytosol protein. Adult levels, 410-433 fmol/mg cytosol protein, were reached by 2 weeks after birth. The equilibrium dissociation constants (Kd) of the [3H]dexamethasone-receptor complex were similar in adult and in embryonic cytosols (range, 2.8-11.8 nM; mean +/- SD = 6.5 +/- 2.9 nM). Specific binding was assessed to be 3- to 5-fold greater in tubular than in glomerular preparations. These data on the localization and ontogeny of GR during murine metanephric development provide a basis for study of glucocorticoid-mediated effects on various models of congenital and acquired renal disease. 相似文献
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This investigation used cytosol fraction of rat liver to examine the effects of insulin (INS) on functional properties of glucocorticoid receptor (GR). Male Wistar rats (220-250 g b.wt.) were injected with INS (50 microg/200 g b.wt, i.p.) and 18 h after INS administration used for experiments. INS-stimulated dissociation of G-R complexes was significantly increased by 133% compared to control level. However, INS treatment significantly stimulated stability of GR protein by 138% above control value. Furthermore, results show that INS stimulated activation of formed cytosol [3H] TA-R complexes by 143% in respect to control. [3H]TA-R complexes from INS treated animals could be activated and accumulated at higher rate in cell nuclei of control animals. The physiological relevance of the data was confirmed by INS-related stimulation of Tryptophan oxigenase (TO) activity. It was observed that INS stimulated TO activity while INS injected to adrenalectomized rats, exhibited less effects compared to control. The results indicate that a glucocorticoid hormone (CORT) enhances INS induced stimulation of TO activity, as evidenced by enhanced enzyme activity. Presented data suggest: that INS treatment leads to modifications of the GR protein and the nuclear components and that INS activates the rat liver CORT signaling pathway which mediates, in part, the activity of TO. 相似文献
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Anatomical resolution of two types of corticosterone receptor sites in rat brain with in vitro autoradiography and computerized image analysis 总被引:4,自引:0,他引:4
The rat brain contains two receptor systems for corticosterone (CORT): the glucocorticoid (GR) and corticosterone or mineralocorticoid-like (CR) receptor sites. We have studied the localization of these receptors by in vitro autoradiography and by in vitro cytosol binding assays in microdissected brain areas. In vitro autoradiography revealed that CR receptor sites are almost entirely restricted to the septal-hippocampal complex, whereas the presence of GR extends throughout the brain. Highest levels of GR are present in the lateral septum, hippocampal, cortical and thalamic regions and the paraventricular nucleus. In vitro determination of binding of 3H-labelled steroids to CR and GR in cytosol of "punched out" brain tissue revealed a similar neuroanatomical distribution as observed with the autoradiographic analysis. In addition, it was found that CORT binds to CR (KD approximately 0.5 nM) with 5-10-fold higher affinity than to GR (KD approximately 2.5-5 nM). 相似文献
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《Journal of receptor and signal transduction research》2013,33(5):761-774
AbstractRecently developed “exchange assays” have been used to measure total cytosolic glucocorticoid receptor (GR) binding activity as compared to standard GR assays which measure unoccupied receptor. In the current study we modified these methods and extended the applications of such assays. Experiments defined the conditions whereby two sulfhydryl-binding agents, p-hydroxymercuribenzoate (PHMB) and mersalyl, completely inhibited binding of the glucocorticoid receptor to ligand in mouse renal cytosol. Reactivation of steroid-binding activity was restored by addition of dithiothreitol. The present study demonstrates 12% higher GR binding activity when this exchange assay is performed using saturated glucocorticoid-receptor complexes, rather than standard cytosol. Combining the data from the standard and exchange mouse renal cytosolic GR assays, it was determined that, at physiologic tissue corticosterone levels, the respective mean concentrations of unoccupied, occupied, and total GR were 467, 89, and 556 fmol/mg cytosol protein. Measurement of receptor concentrations by the use of these methods permits precise experimental differentiation of factors which affect total, as well as unoccupied GR. 相似文献
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The influence of cadmium on the rat liver glucocorticoid receptor (GR) binding capacity, on the cytosolic level of 90 kDa
heat shock protein (Hsp90), and on the association of the two proteins was investigated. The results showed that the mode
of metal application led to diverse alterations in hormone binding to the GR. Reduction of the GR binding capacity observed
afterin vitro treatment was proportional to the applied metal concentrations. In animals administered different doses of cadmium, GR binding
capacity was not reduced, except in those that received the highest dose. A concomitant elevation of Hsp90 level was detected
both in the cytosol and within the GR untransformed heterocomplexes. The results suggest that cadmium-induced reduction of
the GR binding capacity seenin vitro was prevented in intact animals by the elevated level of Hsp90 within the GR heterocomplexes.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献