Immunoprecipitation Assay of Alpha-fetoprotein Synthesis by Cultured Mouse Hepatoma Cells Treated with Estrogens and Glucocorticords

This investigation was to study the biosynthesis of ³H-labeled alpha-fetoprotein (AFP) by cultured mouse hepatoma (HEPA-2) cells. Both the function and regulation of this oncodevelopmental gene are unknown. However, evidence indicates that mechanisms controlling the expression of AFP involve aspects of both normal embryonic development and neoplastic transformation. The secretion of AFP was analyzed during different phases of the growth cycle to provide information on AFP production using standard culture conditions. The highest rate of secretion occurred during the stationary phase, followed by the late logarithmic and early logarithmic phases of growth, respectively. The production of AFP was then determined following the addition of glucocorticords and estrogens in an attempt to understand hormonal factors that may be involved. Studies utilizing estradiol-17β indicated that the secretion of AFP did not appear to be sensitive to this steroid even though sucrose density gradient analysis of HEPA-2 cytosol, for estrogenic receptors, revealed competitive binding moieties in the 8S and 4S regions of the gradient. In contrast, the secretion of the total complement of proteins, including AFP, was significantly stimulated by the glucocorticords, dexamethasone and corticosterone. Analysis of HEPA-2 cytosol for glucocorticord receptors revealed binding components in the 7S and 3–4S regions of the gradient. The ³H-dexamethasone binding appeared to be stereospecific since nonlabeled dexamethasone, but not nonlabeled estradiol-17β, effectively displaced the bound radioactivity. The glucocorticoid-binding component in HEPA-2 therefore displayed characteristics reported for glucocorticord receptors in normal liver and other hepatomas.     

Binding of 2-hydroxyestradiol and 4-hydroxyestradiol to estrogen receptors from human breast cancers

The binding of catechol estrogens, epoxyenones and methoxyestrogens was evaluated using estrogen receptors in cytosol prepared from human breast cancers. The relative affinity of 2-hydroxyestradiol, a metabolite formed in vitro from estradiol-17 beta by breast cancer cells, was indistinguishable from that of estradiol-17 beta. 4-Hydroxyestradiol, which is also a metabolite of estradiol-17 beta, associated with the estrogen receptor with a relative affinity approximately 1.5-fold greater than that of estradiol-17 beta. Epoxyenones and methoxyestrogens were weak competitors compared to the binding of estradiol-17 beta, exhibiting relative affinities 3% or less than the affinity of estradiol-17 beta. Sucrose density gradient centrifugation revealed that both 2- and 4-hydroxyestradiol inhibited the binding of estradiol-17 beta to both the 4S and 8S isoforms of the estrogen receptor in a competitive manner, with a Ki = 0.94 nM for 2-hydroxyestradiol and a Ki = 0.48 nM for 4-hydroxyestradiol. It can be concluded that these data demonstrate a specific receptor-mediated estrogenic action for both of these catechol estrogens.     

Stimulation of IgG production by glucocorticoids in human myeloma lymphoblasts

LICR-LON-HMy2 cells (HMy2 cells), an established line of human myeloma lymphoblasts, produce and secrete IgG, and have been used for production of human-human hybridomas. We have previously shown that HMy2 cells are growth-inhibited by glucocorticoids and contain high affinity, saturable, steroid-specific glucocorticoid receptors. Here we report that treatment for 0-4 days with the synthetic glucocorticoid dexamethasone (1,4-pregnadien-9-fluoro-16 alpha-methyl-11 beta,17 alpha,21-triol-3,20-dione) leads to time-dependent increases in IgG secretion rates as measured by goat anti-human IgG antibodies in an enzyme-linked immunosorbent assay. Stimulation of IgG secretion is dependent on the concentration of dexamethasone employed, with half-maximal stimulation occurring between 1.10(-9) and 1.10(-8) M, and maximal stimulation occurring at 1.10(-7) M. Stimulation of IgG secretion is specific for active glucocorticoids such as cortisol and dexamethasone; treatment of cells with 17 beta-estradiol, progesterone, dihydrotestosterone, and aldosterone has little, if any, effect on IgG secretion. Finally, dexamethasone markedly stimulates both secreted and newly synthesized IgG, as determined by continuous and pulse labeling of extracellular and intracellular proteins, respectively, followed by binding to protein A-Sepharose, gel electrophoresis, and autoradiography. Thus, although dexamethasone effects on post-translational or secretory processes have not been ruled out, our data indicate that increased biosynthesis of IgG accounts for most, if not all, of the observed increase in IgG secretion rates. In summary our results demonstrate that despite the known immunosuppressive effects of glucocorticoids, these hormones can stimulate IgG biosynthesis and secretion in human myeloma lymphoblasts in vitro.     

Inhibition of synthesis of alpha-fetoprotein by glucocorticoids in cultured hepatoma cells

alpha-Fetoprotein (AFP) synthesis was studied in the presence and absence of glucocorticoids in rat hepatoma Mc-A-RH-7777 cells. Radioimmunoassay of media from cell cultures grown in the presence of glucocorticoid (dexamethasone or cortisol) showed a reduction in AFP, an increase in albumin, and no significant change in transferrin accumulation, as compared to controls. Labeling experiments with L-[35S]methionine indicated that in both cells and media of dexamethasone-treated cultures there was a 50--80% reduction in polypeptide precipitated by anti-AFP serum, as compared with controls; no change was seen in polypeptide precipitated by anti-transferrin serum. Pulse and pulse-chase experiments demonstrated that dexamethasone inhibited the synthesis of AFP but not its secretion. The half-time for secretion of AFP in the presence and absence of dexamethasone was 43 min.     

Glucocorticoids Increase Tyrosine Hydroxylase Activity in Cultured Murine Neuroblastoma

Cyclic AMP and glucocorticoids appear to have a role in regulating the activity of tyrosine hydroxylase (TH), as well as the expression of "morphological differentiation" in murine neuroblastoma. Monolayer cultures of C-1300 murine neuroblastoma (clone NBP2) were treated with the following compounds in ethanol: dexamethasone, triamcinolone acetonide, hydrocortisone, cortexolone, androstenedione, testosterone, estradiol-17 beta; or with the phosphodiesterase inhibitor Ro20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone]. Treatment with either 200 micrograms/ml Ro20-1724 or 50 micrograms/ml dexamethasone produced significant increases in TH activity compared to alcohol controls (1.44 vs. 0.82 nmol 14CO2/mg protein/hr compared to 0.095). Triamcinolone acetonide or hydrocortisone also produced smaller, but significant, increases in TH activity compared to dexamethasone. When steroid activities were compared at 25 microM concentration and after 60 min of incubation (to maximize TH activity), triamcinolone acetonide was not as effective (62%) as dexamethasone. The relatively inactive glucocorticoid cortexolone produced a slight but significant increase, while the androgens androstenedione and testosterone and the estrogen estradiol-17 beta were without effect.     

A study of the cytoplasmic receptors for glucocorticoids in intestine of pre- and postweanling rats.

Glucocorticoids cause both enzymic and morphologic changes in the rat intestine during the time of weaning. To obtain information regarding the mechanism of these actions, we examined the cytoplasmic fraction of intestines from 18-day-old rats for the presence of specific glucocorticoid-binding proteins which are characteristics of target tissues. Incubation of slices of intestine with [3H]dexamethasone in a physiological medium at 2 degrees showed the presence of a cytoplasmic binding macromolecule with high specificity for steroids having glucocorticoid activity. The binding reaction was saturable (concentration of binding sites equals 0.24 pmol per mg of protein) and of high affinity (dissociation constant equals 9.3 nM). Binding was reversible on addition of nonlabeled dexamethasone (t 1/2 equals 5.2 hours), indicating that the usual assay procedure measured both corticosterone-filled and unoccupied binding sites. Sucrose density gradient centrifugation showed that the receptor-dexamethasone complex from intestinal cytosol sedimented at the same rate as that from liver (8.2 S). The receptor-dexamethasone complex was stable at 2 degrees for at least 24 hours in intestinal slices, but in isolated cytosol fractions there was considerable loss of binding even in the presence of high concentrations of [3H]dexamethasone. Furthermore, mixing experiments showed that the presence of cytosol from intestinal mucosa (but not from the muscle layers) caused a dissociation of dexamethasone from receptors of liver cytosol. This suggested the presence of some interfering factor in isolated mucosal cytosol and meant that quantitative studies had to be confined to intact slices. Although the reasons for the instability of steroid-receptor complexes in the presence of isolated intestinal cytosol are not understood, the instability is believed to be associated with homogenization and, therefore, is believed to have no physiological significance. Finally, the ontogenesis of cytoplasmic glucocorticoid receptors in intestinal slices was examined and the pattern compared with that in liver and lung. Receptor activity was present in intestine from late fetal life through adulthood, but concentrations were significantly higher during the first two postnatal weeks than at all other times. By contrast, receptor activity detected in cytosol prepared from rat lung was high around the time of birth, while that in liver rose steadily during the first postnatal week and remained at high levels. Thus specific receptors for glucocorticoids are present in the rat intestine during periods of both responsiveness and unresponsiveness. This suggests that although corticosteroids exert their effects through the cytoplasmic receptors, this early event in glucocorticoid action may not be a controlling step for changes in responsiveness during development.     

The inhibition of commitment of mouse erythroleukemia cells by steroids involves a glucocorticoid-receptor mediated process(es) acting at the nuclear level

Dexamethasone has been shown to inhibit dimethylsulfoxide (DMSO)-induced differentiation of mouse erythroleukemia (or Friend) cells by blocking commitment to terminal erythroid maturation. In this study, we confirmed previous reports indicating the presence of glucocorticoid receptors in murine erythroleukemia cells and examined the mechanism(s) by which steroids block commitment. Untreated murine erythroleukemia cells contain dexamethasone receptors which decrease in number during DMSO-induced cell differentiation. When steroids of different classes (estrogenic, androgenic, glucocorticoid) were tested for inhibition of commitment and for displacement of [3H]dexamethasone from its receptors in DMSO-treated cells, we observed that the glucocorticoids dexamethasone, prednisolone and hydrocortisone, all blocked commitment and substantially displaced [3H]dexamethasone. In contrast, steroids other than glucocorticoids failed to inhibit commitment or displace [3H]dexamethasone. Analysis of kinetics of dexamethasone binding to chromatin revealed that dexamethasone binds to the nucleus via the receptor and preferentially interacts with active chromatin. Inhibition of commitment by dexamethasone persisted in cells released from this agent and reincubated with DMSO in the presence of another glucocorticoid of similar affinity to steroid receptors; inhibition of commitment, however, was not obtained when cells removed from dexamethasone were incubated in the presence of beta-estradiol, progesterone and testosterone. These data indicate that inhibition of commitment of mouse erythroleukemia cells by steroids is associated with binding to glucocorticoid receptors and may involve interactions of steroids and their receptors with regions of chromatin.     

Depletion of [3H]methyltrienolone cytosol binding in glucocorticoid-induced muscle atrophy

The present study was undertaken to determine cytosol binding properties of [3H]methyltrienolone, a synthetic androgen, in comparison with [3H]dexamethasone, a synthetic glucocorticoid, under conditions of glucocorticoid excess in skeletal muscle. Male hypophysectomized rats received either seven daily subcutaneous injections of cortisone acetate (CA) (100 mg X kg-1 body wt) or the vehicle, 1% carboxymethyl cellulose. Following treatment, both [3H]dexamethasone and [3H]methyltrienolone-receptor concentrations were decreased from those in vehicle-treated rats by more than 90 and 80%, respectively, in CA-treated animals. Scatchard analysis of [3H]methyltrienolone binding in muscles of vehicle-treated animals became nonlinear at high concentrations of labeled ligand and were reanalyzed by a two-component binding model. The lower affinity, higher capacity component, which was attributed to binding of methyltrienolone to a "dexamethasone" component, disappeared in muscles of CA-treated rats and Scatchard plots were linear. Receptor concentrations of the higher affinity lower capacity "methyltrienolone" component were similar in muscles of vehicle-treated and CA-treated groups. From competition studies, the high relative specificities of glucocorticoids for [3H]methyltrienolone binding in muscles of vehicle-treated animals were markedly reduced by CA treatment. In addition, the binding specificity data also showed strong competition by progesterone and methyltrienolone for [3H]dexamethasone binding and estradiol-17 beta for [3H]methyltrienolone binding. These results demonstrate that most of the [3H]methyltrienolone binding is eliminated under in vivo conditions of glucocorticoid excess. Furthermore, the competitiveness of various steroids for receptor binding suggests that rat muscle may not contain classic (ligand-specific) glucocorticoid and androgen receptors.     

Glucocorticoid receptors and glucocorticoid-sensitive secretion of neutral proteinases in a macrophage line.

A continuous line of mouse macrophages (P388D1) has been shown to secrete elastase, collagenase, and plasminogen activator at activities comparable to those of macrophages elicited by an inflammatory stimulus in vivo. At physiologic concentrations anti-inflammatory glucocorticoids selectively and reversibly inhibited secretion of the three proteinases but did not inhibit secretion of lysozyme, a constitutive enzyme produced by the P388D1 cells. The secretion of the neutral proteinases was inhibited 50% by 2 to 10 nM dexamethasone. Proliferation of the macrophages was also glucocorticoid sensitive. The P388D1 macrophages contained about 4000 saturable glucocorticoid-binding sites per cell. Concentrations of hormone saturating the high affinity receptor site (for dexamethasone the dissociation constant for steroid-receptor binding, Kd, was 4 nM) correlated well with concentrations inhibiting secretion of the proteinases. Only glucocorticoids and progesterone competed for binding to the specific receptors. Temperature-sensitive translocation of hormone-receptor complexes from "cytoplasm" to nucleus similar to that found with rat thymocytes was demonstrated. Thus, the interaction between glucocorticoids and the P388D1 cell line provides a model for the regulation of macrophage secretion of neutral proteinases under normal and stress conditions.     

A new affinity matrix for mineralocorticoid receptors

The behavior of mineralocorticoid and glucocorticoid receptors of rabbit kidney cytosol was investigated on two affinity gels: a new affinity matrix prepared with a 3-O-derivative of carboxymethyloxime deoxycorticosterone (deoxycorticosterone gel) and a gel linked to a 17 beta-dexamethasone derivative (dexamethasone gel). Deoxycorticosterone gel was highly specific, since it retained mineralocorticoid but not glucocorticoid receptors, and dexamethasone gel exhibited high selectivity for glucocorticoid receptors since it did not bind mineralocorticoid receptors. The use of these two matrices allowed separation of mineralocorticoid and glucocorticoid receptors and further characterization of each type of cytosolic receptors after its isolation. Cytosolic mineralocorticoid and glucocorticoid receptors stabilized by tungstate were found to have a Stokes radius of approximately 6 nm, as determined by high performance size exclusion chromatography and a sedimentation coefficient of approximately 9 S, determined on a glycerol density gradient containing tungstate, under either high or low salt conditions. The hydrodynamic parameters, binding characteristics, and specificity of mineralocorticoid receptors were the same in the untreated and dexamethasone gel-treated cytosol. Similarly glucocorticoid receptor characteristics remained unchanged after deoxycorticosterone gel treatment, indicating biochemical independence of cytosolic mineralocorticoid and glucocorticoid receptors. The [3H]aldosterone receptor complex eluted from deoxycorticosterone gel was recovered with a 30-40% yield and a purification factor of about 1000. Purified mineralocorticoid receptors had the same sedimentation coefficient as cytosolic mineralocorticoid receptors (9 S) but a different Stokes radius (4 versus 6 nm). The decrease in the Stokes radius of the purified mineralocorticoid receptors was probably due to the gel filtration method. These results indicate that the newly synthesized matrix specific for mineralocorticoid receptors constitutes a powerful tool for their extensive purification.     

Glucocorticoid-receptor interaction and induction of murine mammary tumor virus.

The relationship between the cellular uptake of glucocorticoid hormones, the binding of these hormones to specific in vitro receptors, and the induction of mouse mammary tumor viruses in an established mouse mammary tumor cell line was highly correlated. These results suggest that the induction of mouse mammary tumor virus by glucocorticoid hormones is a physiological process acting through a mechanism of high affinity, saturable steroid-receptors. A temperature-sensitive or salt-dependent step following glucocorticoid-receptor interaction was required for nuclear uptake of the steroid. Induction studies with different adrenocorticoids indicate that the synthetic glucocorticoid, dexamethasone (1,4-pregnadiene-9-fluor-16alpha-methyl-11beta,17alpha,21-triol-3,20-dione), is the most potent inducer of mouse mammary tumor viruses and all steroids which caused significant induction were glucocorticoids. Other glucocorticoids appear to stimulate murine mammary tumor virus production by a mechanism similar to that of dexamethasone; for example, corticosterone competes with dexamethasone for binding to the glucocorticoid receptor and blocks the uptake of dexamethasone into cells. Progesterone also blocks the cellular uptake of dexamethasone and can bind to the glucocorticoid receptor at low concentrations (10-7 to 10-8 M) but progesterone does not consistently induce virus at hormone concentrations even as high as 10-4 M. Thus, in this system, binding to a cytoplasmic receptor is necessary but not sufficient for induction by glucocorticoids. Estrogens and androgens interfere with receptor binding and cellular uptake of dexamethasone but only at much higher concentration (10-4 M) than progesterone, and do not induce mammary tumor virus production. Although there was a positive correlation between steroid structure, binding, and biologic induction, other factors clearly affect the physiological manifestations of steroid actions. Mouse cells with comparable cytoplasmic receptor levels and comparable nuclear uptake differed absolutely in their degree of murine mammary tumor virus induction following hormone treatment. Although all mouse cells examined contain comparable levels of murine mammary tumor virus DNA, only cells producing constitutive levels of murine mammary tumor virus RNA could be induced to higher levels by a variety of glucocorticoids.     

Cortisol 17 beta acid, transcortin, and the heterogeneity of rat brain glucocorticoid receptors

Binding of tracer or competing steroids to transcortin can compromise specificity studies on receptors for adrenal steroids. Recently Alexis et al. have used cortisol 17 beta acid at high concentrations to prevent steroid binding to any transcortin possibly contaminating rat brain cytosol preparations. On the basis of limited specificity studies of [3H]dexamethasone and [3H]corticosterone binding under such conditions, it was claimed that binding sites for the two steroids are indistinguishable, and it is thus unnecessary to invoke distinct binding sites for each glucocorticoid. We have extended these competition studies in the presence of cortisol 17 beta acid, and shown that in rat hippocampus Type I, corticosterone-preferring glucocorticoid receptors can be clearly distinguished both from transcortin and from Type II, dexamethasone-binding glucocorticoid receptors.     

Glucocorticoids increase cholecystokinin receptors and amylase secretion in pancreatic acinar AR42J cells

We recently reported in AR42J pancreatic acinar cells that glucocorticoids increased the synthesis, cell content, and mRNA levels for amylase (Logsdon, C.D., Moessner, A., Williams, J.A., and Goldfine, I.D. (1985) J. Cell Biol. 100, 1200-1208). In addition, in these cells glucocorticoids increased the volume density of secretory granules and rough endoplasmic reticulum. In the present study we investigate the effects of glucocorticoids on the receptor binding and biological effects of cholecystokinin (CCK) on AR42J cells. Treatment with 10 nM dexamethasone for 48 h increased the specific binding of 125I-CCK. This increase in binding was time-dependent, with maximal effects occurring after 48 h, and dose-dependent, with a one-half maximal effect elicited by 1 nM dexamethasone. Other steroid analogs were also effective and their potencies paralleled their relative effectiveness as glucocorticoids. Analyses of competitive binding experiments conducted at 4 degrees C to minimize hormone internalization and degradation revealed the presence of a single class of CCK binding sites with a Kd of approximately 6 nM and indicated that dexamethasone treatment nearly tripled the number of CCK receptors/cell with little change in receptor affinity. Treatment with 10 nM dexamethasone increased both basal amylase secretion and the amylase released in response to CCK stimulation. In addition, dexamethasone increased the sensitivity of the cells to CCK. The glucocorticoid decreased the concentration of CCK required for one half-maximal stimulation of amylase secretion from 35 +/- 6 to 8 +/- 1 pM. These data indicate, therefore, that glucocorticoids induce an increase in the number of CCK receptors in AR42J cells, and this increase leads to enhanced sensitivity to CCK.     

In vitro mycotoxin binding to bovine uterine steroid hormone receptors

The mycotoxins, aflatoxin B(1), aflatoxin M(1), aflatoxicol and zearalenone were tested for binding to bovine endometrial estrogen and progestin receptors. Radioinert estradiol-17beta, estrone, testosterone, and cholesterol were evaluated for binding to the estrogen receptor. Zearalenone and aflatoxicol but not aflatoxins B(1) and M(1) competed with estradiol-17beta for the estrogen receptor. The order of binding affinities for the estrogen receptor were zearalenone > estradiol-17beta > estrone > aflatoxicol. The affinity of zearalenone for the estrogen receptor was 2-3 times that of estradiol-17beta. Progesterone, cortisol, radioinert R 5020, and cholesterol were evaluated for binding to the progestin receptor. None of the tested compounds except R 5020 and progesterone competed for the progestin receptor. The significance of aflatoxicol binding to the estrogen receptor is unclear. It is proposed that aflatoxicol binding to the receptor may alter gene expression in target tissues or act at the level of the hypothalamus to inhibit gonadotropin secretion and ovulation. These effects could explain reports of reduced fertility in domestic animals following ingestion of aflatoxin contaminated feedstuffs. It is also suggested that the mechanism of adverse effects on fertility of chronic aflatoxin ingestion in cattle and other livestock should be more thoroughly investigated.     

Dexamethasone decreases phospholipase C beta1 isozyme expression in human vascular smooth muscle cells

The molecular characterization of the human PLC beta1 gene was just reported by Peruzzi et al. [Biochim. Biophys. Acta 1582 (2002) 46]. This prompted us to investigate the effects of dexamethasone on PLC beta1 expression in two types of human vascular smooth muscle cells--coronary artery smooth muscle cells (hCASMC) and aortic smooth muscle cells (hAoSMC), since glucocorticoids are known to affect the signaling pathways of Gprotein coupled receptors. Semi-quantitative RT-PCR was used to analyze mRNA expression and Western-blot for protein expression. Dexamethasone treatment in the two types of cells studied decreased (mRNA and protein) PLC beta1 isozyme expression. A rapid (2 h) fall in mRNA occurred in hCASMC after treatment, and hCASMC were more sensitive to dexamethasone (1 nM versus 100 nM) than hAoSMC. The major reduction (80%) was observed after 48 h of exposure in both VSMC. Treatment with mifeprisone, an antagonist of glucocorticoid receptors, blunted the dexamethasone effect on PLC beta1 mRNA and showed that this effect was mediated by glucocorticoids receptors.     

Glucocorticoids Enhance Histamine-Evoked Catecholamine Secretion from Bovine Chromaffin Cells

Abstract: The synthetic glucocorticoid dexamethasone enhanced histamine-evoked catecholamine secretion from cultured bovine chromaffin cells. Dexamethasone enhanced the effects of histamine on both adrenergic (epinephrine-rich) and noradrenergic (norepinephrine-rich) chromaffin cells but had a more dramatic effect on noradrenergic cells. Histamine-evoked secretion in noradrenergic cells appeared to become rapidly inactivated, whereas the rate of secretion in adrenergic cells was nearly constant for up to 2 h; dexamethasone treatment attenuated the inactivation seen in noradrenergic cells. The effect of dexamethasone appeared after a lag of several hours and was maximal by 24 h. The EC₅₀ for dexamethasone was ∼1 n M . The effect of dexamethasone was mimicked by the glucocorticoid agonist RU 28362 and was blocked by the antagonist RU 38486, indicating that the effects of these steroids were mediated by the glucocorticoid or type II corticosteroid receptor. Histamine-evoked catecholamine secretion in both dexamethasone-treated and untreated cells was blocked by the H₁ histamine receptor antagonist mepyramine but was not affected by the H₂ antagonist cimetidine; thus, dexamethasone appeared to enhance an H₁ receptor-mediated process. In the absence of glucocorticoids, H₁ receptor mRNA levels were higher in adrenergic than in noradrenergic cells. Dexamethasone increased H₁ receptor mRNA levels in both cell types. The increased expression of H₁ receptors presumably contributes to the enhancement of histamine-evoked catecholamine secretion by glucocorticoids. Glucocorticoids may play a physiological role in modulating the responsiveness of chromaffin cells to histamine and other stimuli.     

Glucocorticoid hormone receptors in rat pancreas

Although glucocorticoiods influence pancreatic function, it has not been established whether they act directly at the level of the pancreas, or indirectly by causing metabolic changes in other target tissues. As a step in elucidating the actions of glucocorticoids on the pancreas, a search was conducted for glucocorticoid hormone receptors in this tissue. Uptake and binding studies indicated that there were glucocorticoid hormone receptors in the high-speed cytosolic extract of rat pancreas. These receptors appear to be similar to other rat glucocorticoid receptors: they bind glucocorticoids rapidly in a reversible manner at 0°C, competitive binding analysis studies show that they have a preference for glucocorticoids and, like receptors, bind the synthetic steroids triamcinolone acetonide and dexamethasone with a higher affinity than corticosterone. Scatchard analysis demonstrated that there are 1.37 · 10^?13 mol glucocorticoid-binding sites/mg cytosolic protein. This demonstration of a glucocorticoid hormone receptor in pancreatic cytosol suggests that some of the effects glucocorticoids exert on pancreatic function are a consequence of their direct actions on this target tissue.     

Melatonin prevents glucocorticoid inhibition of cell proliferation and toxicity in hippocampal cells by reducing glucocorticoid receptor nuclear translocation

Glucocorticoids are the main product of the adrenal cortex and participate in multiple cell functions as immunosupressors and modulators of neural function. Within the brain, glucocorticoid activity is mediated by high-affinity mineralocorticoid and low-affinity glucocorticoid receptors. Among brain cells, hippocampal cells are rich in glucocorticoid receptors where they regulate excitability and morphology. Also, elevated glucocorticoid levels suppress hippocampal neurogenesis in adults. The pineal neuroindole, melatonin, reduces the affinity of glucocorticoid receptors in rat brain and prevents glucocorticoid-induced apoptosis. Here, the ability of melatonin to prevent glucocorticoid-induced cell death in hippocampal HT22 cells was investigated in the presence of neurotoxins. Results showed that glucocorticoids reduce cellular growth and also enhance sensitivity to neurotoxins. We found a G(1) cell cycle arrest mediated by an increase of cyclin/cyclin-dependent kinase inhibitor p21(WAF1/CIP1) protein after dexamethasone treatment and incremental change in amyloid beta protein and glutamate toxicity. Melatonin prevents glucocorticoids inhibition of cell proliferation and reduces the toxicity caused by glucocorticoids when cells were treated with dexamethasone in combination with neurotoxins. Although, melatonin does not reduce glucocorticoid receptor mRNA or protein levels, it decreases receptor translocation to nuclei in these cells.     

RU486 inhibits induction of aromatase by dexamethasone via glucocorticoid receptor in cultured human skin fibroblasts

Effects of RU486 on the induction of aromatase by dexamethasone via glucocorticoid receptor were determined using cultured human skin fibroblasts. Competition of [3H]dexamethasone binding to the cytosol receptor was 7 times stronger with RU486 than with dexamethasone. The order of the strength of competition was RU486 greater than dexamethasone greater than betamethasone greater than prednisolone greater than hydrocortisone. RU486 abolished a specific 8.6 S [3H]dexamethasone binding peak in the cytosol, determined using a sucrose density gradient analysis. Dexamethasone markedly induced aromatase and this event was strongly suppressed by RU486, in a dose-dependent manner, in the cultured skin fibroblasts. A linear correlation between the strength of competition and the induction of aromatase of various glucocorticoids was observed. RU486 non-competitively inhibited aromatase induction by dexamethasone determined from a double reciprocal plot of aromatase activity, with respect to [3H]androstenedione concentration in the presence of RU486. These results show that RU486 is a peripheral noncompetitive antiglucocorticoid on aromatase induction by glucocorticoid in human skin fibroblasts and that aromatase induction is a good marker for the biological function of glucocorticoid receptor in human skin fibroblasts.