Potentiation by steroids of the β-adrenergic agent-induced stimulation of cyclic AMP in isolated mouse thymocytes

There is an increasing amount of evidence suggesting that glucocorticoids may modulate the responsiveness of various cell types to β-adrenergic agents. In some systems, it has been shown, in addition, that steroids potentiate the elevation of cAMP induced by catecholamines. Little is known however of the mechanism underlying steroid action. We have studied this ‘permissive action’ in isolated thymocytes which have specific receptor sites for both glucocorticoids and β-adrenergic agents. The glucocorticoid compound dexamethasone did not alter intracellular cAMP level but markedly enhanced the stimulation produced by isoproterenol. This effect was instantaneous and was still measurable at 10^?7 M dexamethasone. A similar potentiating action was observed in the presence of corticosterone but also in the presence of sex steroids. Determination of β-receptors after cell preincubation in the presence of dexamethasone showed that rapid alterations in β-receptors are not involved in this permissive action. Experiments done in the presence of the calcium chelator, ethyleneglycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid, suggest that dexamethasone action could be related to a modification of calcium mobilization.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[³H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90 000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [³H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [³H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37°C. SDS-polyacrylamide gel electrophoresis of [³H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37°C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei aas well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.     

31P NMR characterization of cellular metabolism during dexamethasone induced apoptosis in human leukemic cell lines

³¹P NMR has been used to study the effects of dexamethasone on phosphorus metabolism in one dexamethasone (dex)-sensitive (CEM-C7) and three different dex-resistant (CEM-C1, CEM-4R4, and CEM-ICR27) human leukemic cell lines. The use of these cell lines, containing widely varying amounts of glucocorticoid receptors, made it possible to evaluate the receptor-mediated contributions to the modes of action of dexamethasone in these cells. To evaluate the effects of dexamethasone without any significant contribution from experimental conditions, all the experiments were done with parallel controls. Results obtained showed: (1) significantly different levels of phosphorylethanolamine (PE) and phosphorylcholine (PC) among cell lines, suggesting significant differences in phospholipid metabolism; (2) the dexamethasone induced reduction of phosphomonoester (PE + PC), ATP, and metabolic rates probably through glucocorticoid receptor mediated mechanisms; (3) the dexamethasone induced stimulation of cellular metabolism in a process which seems to be independent of glucocorticoid receptors; and (4) the dexamethasone induced alkaline shift of intracellular pH in all the cell lines except ICR27. The reduction in PME levels seems to be an earlier step in dexamethasone-induced apoptosis than the reduction in ATP. The degree of alkaline shift was found to correlate with the number of glucocorticoid receptors present. The possible involvement of phospholipid metabolites as second messengers in dexamethasone-induced apoptosis is discussed. © 1994 Wiley-Liss, Inc.     

Effects of dexamethasone on spermidine N1-acetyltransferase and ornithine decarboxylase activities in rat spleen

Treatment of rats with the glucocorticoid dexamethasone causes an increase in the activity of cytosolic spermidine N¹-acetyltransferase both in the spleen and thymus, but not, however, in liver, kidney or lung. The induced spermidine N¹-acetyltransferase activity in the spleen catalyses acetylation of spermidine as well as spermine and sym-norspermidine, but not of diamines and histones. The enzyme induction depends on the dose of dexamethasone, and is suppressed by cycloheximide, which suggests that de novo protein synthesis is required for the action of this glucocorticoid. N¹-acetylspermidine accumulates in the spleen after dexamethasone treatment, while spermidine progressively decreases and is partly converted into putrescine, the content of which transiently increases. In accordance with previous reports, dexamethasone was found to cause a rapid and large fall in the activity of spleen ornithine decarboxylase which was effected via the appearance of an inhibitor of the enzyme. Glucocorticoids exert large catabolic effects on lymphoid tissues, and further selectively affect the activities of spermidine N¹-acetyltransferase and ornithine decarboxylase in the thymus and spleen. These latter selective responses may represent an important early event in lymphoid tissue response to glucocorticoid hormones.     

Maintenance of quantal size and immediately releasable granules in rat chromaffin cells by glucocorticoid

Glucocorticoid is reported to regulate catecholamine synthesis and storage. However, it is not clear whether the actual amount of catecholamine released from individual granules (quantal size, Q) in mature chromaffin cells is affected by glucocorticoid. Using carbon fiber amperometry, we found that dexamethasone did not affect mean cellular Q or the proportional release from different populations of granules in rat chromaffin cells cultured for 1 day in a serum-free defined medium. After two extra days of culture in the defined medium, there was a rundown in mean cellular Q, and it was associated with a shift in the proportional release from the different granule populations. This phenomenon could not be rescued by serum supplementation but could be prevented by dexamethasone via an action that was independent of changes in voltage-gated Ca²⁺ channel (VGCC) density. Using simultaneous measurements of membrane capacitance and cytosolic Ca²⁺ concentration, we found that for cells cultured in defined medium dexamethasone enhanced the exocytotic response triggered by a brief depolarization (50 ms) without affecting the VGCC density or the fast exocytotic response triggered via flash photolysis of caged Ca²⁺. Thus glucocorticoid may regulate the number of immediately releasable granules that are in close proximity to a subset of VGCC. Because chromaffin cells in vivo are exposed to high concentrations of glucocorticoid, our findings suggest that the paracrine actions of glucocorticoid maintain the mean catecholamine content in chromaffin cell granules as well as the colocalization of releasable granules with VGCCs. catecholamines; paracrine action; exocytosis; calcium channels     

HPAC,a new human glucocorticoid-sensitive pancreatic ductal adenocarcinoma cell line

Summary A new human pancreatic cancer (HPAC) cell line was established from a nude mouse xenograft (CAP) of a primary human pancreatic ductal adenocarcinoma. In culture, HPAC cells form monolayers of morphologically heterogenous, polar epithelial cells, which synthesize carcinoembryonic antigen, CA 19-9, CA-125, cytokeratins, antigens for DU-PAN-2, HMFG1, and AUA1, but do not express chromogranin A or vimentin indicative of their pancreatic ductal epithelial cell character. In the presence of serum, HPAC cell DNA synthesis was stimulated by insulin, insulin growth factor-I, epidermal growth factor, and TGF-α but inhibited by physiologic concentrations of hydrocortisone and dexamethasone. Dose-dependent inhibition of DNA synthesis was limited to steroids with glucocorticoid activity. The inhibitory effect of dexamethasone was abolished by the glucocorticoid antagonist RU 38486. Binding of [³H]dexamethasone to cytosolic proteins was specific and saturable at 4° C. Scatchard analysis of binding data demonstrated a single class of high-affinity binding sites (K_d=3.8±0.9 nM; B_max=523±128 fmol/mg protein). Western blot analysis revealed a major protein band that migrated at a M_r of 96 kDa. Northern blot analysis identified an mRNA of approximately 7 kilobases which hybridized with a specific glucocorticoid receptor complementary DNA probe (OB7). These findings support a role for glucocorticoids in the regulation of human malignant pancreatic cell function.     

Regulation of liver metallothionein and plasma zinc by the glucocorticoid dexamethasone.

Administration of the glucocorticoid dexamethasone to adrenalectomized rats significantly decreased the serum zinc concentration within 14 hr. Dexamethasone did not detectably alter the liver zinc content, but markedly increased the proportion of zinc associated with liver metallothionein. The rate of incorporation of ³⁵S-cystine into this protein was stimulated to a maximal extent 7 hr after administration of the glucocorticoid. Poly(A)⁺ mRNA from liver polysomes was isolated and translated in a cell-free protein synthesizing system. Nearly twice as much polysomal metallothionein mRNA was found 7 hr following treatment with dexamethasone. These results suggest that glucocorticoids can regulate the plasma zinc concentration by a process that is related to the biosynthesis of the hepatic zinc-binding protein, metallothionein.     

Localization of the glucocorticoid receptor in rat liver cells: Evidence for plasma membrane bound receptor

• 1.1. The cytoplasmic glucocorticoid receptor of rat liver cells is in part recovered in the plasma membrane fraction.
• 2.2. After in vivo administration of [³H]dexamethasone, 0.35% of the radioactivity recovered is bound on plasma membranes.
• 3.3. Dexamethasone also binds in vitro specifically to plasma membranes. Expressed as fmol/mg protein, binding of dexamethasone to plasma membranes is comparable to binding to the soluble cytoplasmic fraction (cytosol).
• 4.4. Using polyclonal antibody to the glucocorticoid receptor and the indirect immunofluorescence technic, an intense decoration of the plasma membranes is observed, denoting a high concentration of glucocorticoid receptor on plasma membranes.
• 5.5. The localization of the receptor on plasma membranes could be of potential importance for its interaction with agents (mitogens, growth factors) initially acting on the cell membrane, regulating subsequent cell proliferation and growth at the level of the cell nucleus.

     

The effect of thymosin on glucocorticoid receptors in lymphoid cells

The present study investigates the effect of a partially purified thymus factor, thymosin Fraction 5, and an homogeneous polypeptide component of Fraction 5, thymosin α₁, on glucocorticoid resistance and glucocorticoid receptors in mouse thymocytes. Treatment of thymocytes in vitro with thymosin Fraction 5 or α₁, results in an increase in the percentage of glucocorticoid-resistant cells. Studies on the specific whole-cell binding of [³H]dexamethasone and steroid competition experiments demonstrate the existence of high-affinity (K_d = 1.0 × 10^?8M) specific glucocorticoid receptors in mouse thymocytes. Preincubation with thymosin Fraction 5 or α₁ appears to cause a reduction in the specific [³H]dexamethasone binding to intact thymocytes.     

Effect of dexamethasone,ammonium lons,and serum-deprivation on intracellular proteolysis in cultured muscle cells

Intracellular proteolysis was measured in primary cultures of newborn rat skeletal (gastrocnemius) and cardiac muscle cells by release to the medium of trichloroacetic acid-soluble label from cells grown in the presence of ¹⁴C-labeled phenylalanine. Exposure of the cultured cells to 10^?7M dexamethasone for 5 days starting at day 0 of culture resulted in an enhancement of proteolysis in skeletal muscle but not in cardiac muscle cells. Dexamethasone did not affect cell viability measured by release of label from cells preloaded with Na₂ ⁵¹CrO₄, release of creatine phosphokinase, and release of lactic dehydrogenase into the culture medium. Incorporation of ³H-thymidine into the cells increased during the first 3 to 4 days of culture and subsequently decreased, indicating that cell proliferation ceases at that time. When the exposure to dexamethasone was started on day 4 of culture, i.e., at a postmitotic stage, and continued for 4 days, proteolysis was again found to increase in skeletal but not cardiac cells, thereby suggesting that the response to the hormone is independent of the proliferative state of the culture. Ammonium chloride at a concentration of 10 mM produced a 50% reduction of the basal proteolysis in cultures of skeletal muscle cells and did not affect proteolysis in cardiac muscle cells. Exposure to ammonium chloride did not prevent the dexamethasone-induced increase of proteolysis in skeletal muscle cells. Serum-deprivation induced enhanced proteolysis which was not affected by NH₄Cl in both cell types. It is concluded that the differential responses of the two cultures to dexamethasone reflects the sparing of heart proteins and concomitant wasting of skeletal muscle proteins by glucocorticoid hormones in vivo, and that the enhancement of proteolysis by the glucocorticoid hormone or by serum-deprivation is not sensitive to the lysosomotropic agent NH₄Cl. Thus, while a lysosomal-autophagic enzyme system is responsible for almost half of the basal proteolysis, the accelerated proteolysis occurs via ammonium chloride-insensitive enzymes.     

Δ1-11-oxa-11-deoxycortisol: A new antiglucocorticoid with activity in vivo

A new steroid-like compound, Δ¹-11-oxa-11-deoxycortisol, was tested in a one-week growth suppression, thymus suppression and adrenal weight suppression bioassay for possible glucocorticoid antagonist activity in $\text{vivo}$. We hypothesized that this compound would have antiglucocorticoid activity based on previous studies of 11-deoxycortisol and Δ^1,9(11)-11-deoxycortisol, which were optimal glucocorticoid antagonists $\text{in vivo}$ in adrenalectomized rats, but which lost antiglucocorticoid activity in intact animals, apparently due to adrenal 11β-hydroxylation. Thus, Δ¹-11-oxa-11-deoxycortisol, a compound which cannot undergo llβ-hydroxylation, was synthesized and tested as an antiglucocorticoid. This analog had an affinity for the rat thymus glucocorticoid receptor similar to that of its parent compounds (Ki 0.9-3.1×10^?7M). A dose of 1 $\text{mg}\text{rat}$ antagonized the effect of 15μg of dexamethasone in the growth suppression assay (p<0.05) and in the thymus suppression assay (p<0.06), but did not antagonize dexamethasone-induced adrenal weight suppression. Δ¹-11-oxa-11-deoxycortisol did not exhibit glucocorticoid activity in any of the three assays. These data suggest that Δ¹-11-oxa-11-deoxycortisol may be a pure competitive antagonist of dexamethasone.     

Regulation of neurotensin secretion in a mammalian C cell line: effect of dexamethasone

We established in culture two colony clones of rMTC 44-2 cells, rMTC 44-2B and 44-2C which secrete substantially greater quantities of neurotensin (NT) than the parent cell line. We describe here the effects of the synthetic glucocorticoid, dexamethasone, on NT and cAMP release. Medium and intracellular levels of NT and cAMP were measured by specific RIAs. Long-term release experiments were performed in Dulbecco's Modified Eagle's Medium supplemented with 15% horse serum (DMEM). Short-term release experiments were performed in Krebs-Ringer-bicarbonate-glucose buffer (KRBG) supplemented with 1.0 mm Ca²⁺. Dexamethasone stimulated NT release and increased intracellular NT levels. The ED₅₀ values for stimulation of NT release following 24 or 48 h incubation of cells in DMEM with dexamethasone were 5 · 10^?9 and 7 · 10^?9 M, respectively. Dexamethasone markedly enhanced intracellular levels of NT in rMTC 44-2 cells while it decreased cell growth. Cells pretreated with dexamethasone for 48 h released greater amounts of NT in response to Ca²⁺ (1.0 mM) with or without K⁺ (50 mM) or NE (10^?6 M) following a 10 min incubation with these substances in KRBG. This experimental paradigm was also used to measure the efflux of cAMP following a brief (10 min) exposure of cells to NE. We conclude that the rMTC 44-2B and 44-2C cells are useful tools for studying the effects of dexamethasone on the regulation of cell growth, as well as the secretion of NT and cAMP.     

Lack of Correlation Between the Glucocorticoid Receptor Density and the in Vitro Growth-Inhibitory Effect of Dexamethasone in Human Leukemia Cell Lines

Abstract

We have developed a whole cell binding assay with [³H] dexamethasone as the ligand for the measurement of the glucocorticoid receptor (GR) content of normal and malignant human leukocytes. A panel of eleven phenotypically well-defined human leukemia cell lines were investigated for their GR expression and in vitro sensitivity to glucocorticoids.

There were great variations in the GR contents of different cell lines (2200–18100 sites/cell) while no marked differences in the binding affinities of the GRs were seen. No obvious correlation was found between the GR content and the phenotype of the cell line nor between the GR content and the in vitro growth inhibition by glucocorticoids.     

Dexamethasone regulation of marrow stromal-derived osteoblastic cells

The clonal subtypes of cells in the osteogenic family represented by fibroblastoid MBA-15.33, preosteoblast MBA-15.4, and mature osteoblastic MBA-15.6 cells were used to study the effects of glucocorticoid (dexamethasone). The role of dexamethasone was monitored on cell attachment when plated on various protein substrata (BSA, collagen I, and Matrigel). A 24 h exposure of the cells to 10^-6 M or 10^-7 M dexamethasone differential affects their attachment preference. MBA-15.33 and MBA-15.4 cells increased their attachment capability on collagen I, while MBA-15.6 cells' attachment was inhibited. Pretreatment with (10^-6 M) dexamethasone caused an increase in attachment on Matrigel by MBA-15.33 cells and to less extent by MBA-15.4 cells. Additionally, measurements of two enzymatic activities were monitored; one is alkaline phosphatase (ALK-P), and the second is neutral endopeptidase (CD10/NEP). MBA-15.33, MBA-15.4, and MBA-15.6 cells were exposed to dexamethasone or to various growth factors (bone morphogenic protein (BMP-2 and BMP-3), TGFβ, and IGF-I). In some experiments, pretreatment of cells by dexamethasone was followed by exposure to the growth factors. The cells' challenged cellular responses were not uniform and revealed a differential pattern when their ALK-P and CD10/NEP enzymatic activities were measured. © 1996 Wiley-Liss, Inc.     

Neutrophils Are Not Less Sensitive Than Other Blood Leukocytes to the Genomic Effects of Glucocorticoids

BackgroundNeutrophils are generally considered less responsive to glucocorticoids compared to other inflammatory cells. The reported increase in human neutrophil survival mediated by these drugs partly supports this assertion. However, it was recently shown that dexamethasone exerts potent anti-inflammatory effects in equine peripheral blood neutrophils. Few comparative studies of glucocorticoid effects in neutrophils and other leukocytes have been reported and a relative insensitivity of neutrophils to these drugs could not be ruled out.ObjectiveWe assessed glucocorticoid-responsiveness in equine and human peripheral blood neutrophils and neutrophil-depleted leukocytes.MethodsBlood neutrophils and neutrophil-depleted leukocytes were isolated from 6 healthy horses and 4 human healthy subjects. Cells were incubated for 5 h with or without LPS (100 ng/mL) alone or combined with hydrocortisone, prednisolone or dexamethasone (10⁻⁸ M and 10⁻⁶ M). IL-1β, TNF-α, IL-8, glutamine synthetase and GR-α mRNA expression was quantified by qPCR. Equine neutrophils were also incubated for 20 h with or without the three glucocorticoids and cell survival was assessed by flow cytometry and light microscopy on cytospin preparations.ResultsWe found that glucocorticoids down-regulated LPS-induced pro-inflammatory mRNA expression in both cell populations and species. These drugs also significantly increased glutamine synthetase gene expression in both equine cell populations. The magnitude of glucocorticoid response between cell populations was generally similar in both species. We also showed that dexamethasone had a comparable inhibitory effect on pro-inflammatory gene expression in both human and equine neutrophils. As reported in other species, glucocorticoids significantly increase the survival in equine neutrophils.ConclusionsGlucocorticoids exert genomic effects of similar magnitude on neutrophils and on other blood leukocytes. We speculate that the poor response to glucocorticoids observed in some chronic neutrophilic diseases such as severe asthma or COPD is not explained by a relative lack of inhibition of these drugs on pro-inflammatory cytokines expression in neutrophils.     

Differential effects of glucocorticoids on insulin-like growth factor I action in cultured human fibroblasts

Glucocorticoids act synergistically with insulin-like growth factor I (IGF-I) to stimulate DNA synthesis and replication of cultured human fibroblasts. In the present study, we further define glucocorticoid and IGF-I interactive effects on human fibroblast metabolism and growth. IGF-I stimulated dose-dependent increases in early metabolic events. Half-maximal effectiveness was seen at 5–8 ng/ml IGF-I, with mean maximal responses of 1.5-, 2-, and 6-fold for [³H]2-deoxyglucose uptake, [¹⁴C]glucose incorporation, and [¹⁴C]aminoisobutyric acid (AIB) uptake, respectively. A 48-hour preincubation with 10^?7 M dexamethasone markedly enhanced both the sensitivity and maximal effectiveness of IGF-I stimulation of AIB uptake. In contrast, dexamethasone had no effect on IGF-I-stimulated glucose uptake and utilization. Maximum specific binding of [125I]IGF-I to fibroblast monolayers was identical in ethanol control and glucocorticoid-treated cells, with 50% displacement at ～5 ng/ml IGF-I. In addition to its synergism with IGF-I, preincubation with dexamethasone augmented insulin and epidermal growth factor (EGF) stimulation of [³H]thymidine incorporation; dexamethasone had no effect on platelet-derived growth factor or fibroblast growth factor action. Two-dimensional gel electrophoresis identified two specific glucocorticoid-induced proteins in human fibroblast cell extracts with molecular weights of 45K and 53K and pls of 6.8 and 6.3, respectively. These data indicate that IGF-I receptor-mediated actions in human fibroblasts are differentially modulated by glucocorticoids. Glucocorticoids are synergistic with IGF-I in stimulating mitogenesis and amino acid uptake, without having any apparent effect on IGF-I-stimulated glucose metabolism. Glucocorticoid enhancement of growth factor bioactivity may involve modulation of a regulatory event in the mitogenic signaling pathway subsequent to cell surface receptor activation. © 1995 Wiley-Liss, Inc.