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.     

Pharmacological properties of the enhanced-affinity glucocorticoid fluticasone furoate in vitro and in an in vivo model of respiratory inflammatory disease

Fluticasone furoate (FF) is a novel enhanced-affinity glucocorticoid that has been developed as topical therapy for allergic rhinitis. The pharmacological properties of FF have been investigated using a number of in vitro experimental systems. FF demonstrated very potent glucocorticoid activity in several key pathways downstream of the glucocorticoid receptor (GR) as follows: the transrepression nuclear factor-kappaB (NF-kappaB) pathway, the transactivation glucocorticoid response element pathway, and inhibition of the proinflammatory cytokine tumor necrosis factor-alpha. Furthermore, FF showed the greatest potency compared with other glucocorticoids for preserving epithelial integrity and reducing epithelial permeability in response to protease- and mechanical-induced cell damage. FF showed a 30- to >330,000-fold selectivity for GR-mediated inhibition of NF-kappaB vs. the other steroid hormone receptors, substantially better than a number of other clinically used glucocorticoids. In studies examining the respiratory tissue binding properties of glucocorticoids, FF had the largest cellular accumulation and slowest rate of efflux compared with other clinically used glucocorticoids, consistent with greater tissue retention. The in vivo anti-inflammatory activity of FF was assessed in the Brown Norway rat ovalbumin-induced lung eosinophilial model of allergic lung inflammation. At a dose of only 30 microg, FF achieved almost total inhibition of eosinophil influx in the lung, an inhibition that was greater than that seen with the same dose of fluticasone propionate. In conclusion, the potent and selective pharmacological profile of FF described here could deliver an effective, safe, and sustained topical treatment of respiratory inflammatory diseases such as allergic rhinitis and asthma.     

Glucocorticoid receptors in purified subpopulations of human peripheral blood lymphocytes.

On the premise that the differential effects of glucocorticoids on various aspects of the immune response may be mediated by differences in the glucocorticoid receptors in the effector cells, subpopulations of human peripheral blood lymphocytes were examined for these receptors as well as for glucocorticoid responsiveness. Purified T and non-T lymphocytes, when studied by a sensitive whole cell assay technique, contained equivalent amounts of specific glucocorticoid receptor, which, by binding affinity and specificity measurements, were indistinguishable from each other. Furthermore, under in vitro incubation conditions, macromolecular synthesis in both of these cell populations was inhibited by glucocorticoid at concentrations which saturated the receptor sites. It is concluded that the putative differential effects of glucocorticoids on T and non-T lymphocyte-associated functions are probably not mediated by differences in the glucocorticoid receptors in these cell populations.     

Ketoconazole inhibition and glucocorticoid action in the human lymphoblastic leukemia cell line CEM-C7

The antifungal drug, ketoconazole, was reported to antagonize the induction of the enzyme tyrosine aminotransferase (TAT) by glucocorticoids in hepatoma tissue culture (HTC) cells, and to compete with glucocorticoids for binding to the glucocorticoid receptor. Since glucocorticoids inhibit the growth of the human leukemia cell line CEM-C7, ketoconazole might be expected to reverse this inhibition. Unexpectedly, ketoconazole inhibited CEM-C7 cell growth without utilizing glucocorticoid receptors. This was confirmed by ketoconazole inhibition of the growth of a receptor-less subline of CEM-C7 cells which are insensitive to glucocorticoids. Ketoconazole competed with triamcinolone acetonide (TA) for binding to the glucocorticoid receptor in cell-free supernatant prepared from CEM-C7 cells, but this was greatly reduced if ketoconazole and TA were incubated with intact cells prior to preparation of the cell-free supernatant. Ketoconazole inhibited induction by TA of the enzyme glutamine synthetase only at concentrations of 45-90 microM. We conclude that ketoconazole antagonism of glucocorticoid activity in CEM-C7 cells is probably not of pharmacologic significance due to the large concentrations required, and its reduced interaction with receptors in intact cells. The growth inhibitory activity of ketoconazole may be of interest in cancer chemotherapy.     

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 potential mechanism in medroxyprogesterone acetate teratogenesis.

MPA (medroxyprogeste)rone acetate) has been shown to be te)ratogenic in rabbits but not in rats or mice (Andrew and Staples 1977). Since normal steroid action appears to be mediated, in large part, through interaction with specific steroid receptors, it was postulated that the species difference in teratogenicity might be due to a difference in the interaction of MPA with target cells. A primary event in steroid-cell interaction is the binding of a steroid to intracellular receptors. Studies were initiated to measure the specific nature of MPA binding to glucocorticoid and progestin receptors in appropriate rat and rabbit target tissues. The competition of MPA with 3H-dexamethasone binding in liver cytosol (glucocorticoid receptor) and with 3H-progesterone binding in uterine cytosol (progesterone receptor) was determined. In rabbit liver cytosol, MPA was as effective at competing for specific dexamethasone binding as the natural glucocorticoids and considerably more effective than the nonspecific steroids. In rat liver cytosol MPA was only 10% as effective as the natural glucocorticoids and the competition could not be distinguished from that of nonspecific steroids. A similar species difference was not seen in uterine cytosol; MPA competed with progesterone in a similar fashion in both rat and rabbit. These data demonstrate a distinct species difference in the competitive nature of MPA for the glucocorticoid receptor but not for the progestin receptor. The results suggest that MPA, or possibly a metabolite, may be teratogenic in rabbits by binding with specific glucocorticoid receptors to inhibit or alter normal steroidal function in embryo-fetal development.     

Pharmacological characterization of type II glucocorticoid binding sites in AtT20 pituitary cell culture

Recent evidence indicates that at least two functional glucocorticoid receptors (Type I and Type II) are present in many tissues. It has also become increasingly recognized that, as in other systems, stimulus-response relationships for steroid hormones are often nonlinear. Thus, precise pharmacological parameters are required to establish a functional relationship(s) between binding site and response characteristics. We therefore pharmacologically characterized a glucocorticoid binding site present in AtT20 mouse pituitary cells, a cell line extensively used in studying Type II glucocorticoid receptor function. By several different criteria, glucocorticoids were shown to bind to a single class of binding sites, which, in comparison to available literature, correspond to classical Type II glucocorticoid receptors. No evidence for Type I adrenal steroid binding sites was observed, under the experimental conditions used. Unambiguous Kb values for both glucocorticoid agonists and antagonists were therefore calculated. These parameters should prove of use in elucidating the relationships between glucocorticoid receptor activation and different responses in both AtT20 cells and other glucocorticoid responsive tissues.     

Cyclic AMP specific, calcium independent phosphodiesterase from a malignant murine mast cell tumor.

Certain pyrazolo-steroids are extremely potent anti-inflammatory agents but are predicted to be inactive glucocorticoids on the basis of their structure. However, one representative compound is found to possess a high affinity for cytoplasmic glucocorticoid receptors. The biological activity of this steroid is greater than that predicted from its affinity for receptors. This may be due to an exceptionally slow rate of dissociation of the receptorsteroid complex, which would prevent an accurate determination of the equilibrium affinity constant.     

Glucocorticoid binding during the differentiation of 3T3-F442A fibroblasts into adipocytes. A possible regulatory effect of insulin

Until recently, few studies had been carried out on receptors for glucocorticoids in adipocytes, although the role of these steroids is considerable. In the present studies, we chose the pre-adipocyte line 3T3-F442A, which constitutes an excellent model for investigating the differentiation and function of adipocytes. Using a whole cell assay system, we showed the existence of a homogenous class of sites with the characteristics of glucocorticoid receptors, that is, high-affinity binding which is reversible, specific and saturable. Whatever the state of cellular differentiation, the affinity of the receptor for dexamethasone did not vary, although we observed an increase in the number of sites during differentiation. When cells were differentiated in the presence of insulin, there was a further increase in the binding capacity; moreover, insulin deprivation of such adipocytes caused a decrease in the number of sites. Our results therefore suggest that factors other than the glucocorticoids themselves influence dexamethasone binding. It is suggested that insulin plays a role in the regulation of the number of glucocorticoid receptors.     

Crosstalk between TNF and glucocorticoid receptor signaling pathways

TNF is a Janus-faced protein. It possesses impressive anti-tumor activities, but it is also one of the strongest known pro-inflammatory cytokines, which hampers its use as a systemic anti-cancer agent. TNF has been shown to play a detrimental role in inflammatory diseases such as rheumatoid arthritis and inflammatory bowel disease. Glucocorticoids are strongly anti-inflammatory and exert their therapeutic effects through binding to their receptor, the glucocorticoid receptor. Therefore, glucocorticoids have been used for over half a century for the treatment of inflammatory diseases. However, many patients are or become resistant to the therapeutic effects of glucocorticoids. Inflammatory cytokines have been suggested to play an important role in this steroid insensitivity or glucocorticoid resistance. This review aims to highlight the mechanisms of mutual inhibition between TNF and GR signaling pathways.     

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.     

Fetal and adult sheep adrenal cortical cells contain glucocorticoid receptors

Specific receptors for glucocorticoids were identified in the fetal and adult sheep adrenal cortex by a whole-cell binding assay using [3H]triamcinolone acetonide ([3H]TA) as the radiolabelled ligand. [3H]TA binding sites were saturable and of high affinity, with dissociation constant (Kd) of 2-3nM. Scatchard analysis revealed a single class of binding sites with a binding capacity (Bmax) of 207 and 5 fmol/10(6) cells for d100 fetuses and adults, respectively. By single point analyses at saturating [3H]TA concentration, we found that glucocorticoid receptors (GR) were present in the fetal adrenal cortex as early as d60. Highest concentrations were found at d100-110. GR decreased to d125, then increased to term (approx. d145) before declining again in newborn and adult animals. This demonstration of glucocorticoid receptors in ovine fetal adrenal cortical cells provides a mechanistic basis for the concept that glucocorticoids may act, perhaps in a paracrine or autocrine fashion, to influence adrenocorticotropin (ACTH)-induced activation of fetal adrenal function and the events leading to parturition.     

Glucocorticoid receptors in mouse mammary tumors: Specific binding to nuclear components.

The specific interaction of glucocorticoids with nuclei of mouse mammary tumor was studied in vitro by incubation of the tissue with [3H]dexamethasone at 25 degrees. It was demonstrated that the mammary tumors contain a limited number of specific nuclear binding sites which were saturated with low hormone concentrations (10-8 M)9 The concentrations of specific binding sites in the nuclei were related to the concentration of cytoplasmic binding sites of unincubated tissues and varied between individual tumors. The binding component in the nuclei appeared to be a protein and was easily solubilized with 0.4 M KCl containing buffers. The ability of various corticoids to block the nuclear localization of the steroid correlated well with their glucocorticoid potency. Estradiol and progesterone at concentrations of 10-6 M were also effective in competing for the glucocorticoid receptor binding sites. However, while the glucocorticoids such as hydrocortisone and corticosterone translocated to nuclear sites also specific for dexamethasone, estradiol and progesterone competed for the cytoplasmic binding sites and did not translocate to the nucleus. The possible significance of the interaction of various steroids with the glucocorticoid receptors in mammary tumors is discussed.