Binding of glucocorticoid antagonists to androgen and glucocorticoid hormone receptors in rat skeletal muscle

The binding of ten steroids possessing antiglucocorticoid activity has been studied in rat skeletal muscle cytosol. The affinity of these steroids for both the androgen and the glucocorticoid receptors was determined by competition with radioactive R1881 (methyltrienolone, metribolone) and dexamethasone, respectively. The antiglucocorticoid activity of these compounds was assessed in rat hepatoma (HTC) cells by measuring their inhibitory effect on the glucocorticoid-induced tyrosine aminotransferase activity. This led to identification of five novel in vitro glucocorticoid antagonists. All the steroids tested bound to both the glucocorticoid and the androgen receptors in muscle. Four steroids had an affinity for the glucocorticoid receptor higher than for the androgen receptor. The assumption is made that the steroids tested also behave as antagonists when binding to the glucocorticoid receptor in muscle and behave as agonists when binding to the androgen receptor. On this basis, the data allow one to compute a potential anticatabolic (PAG) and a potential anabolic (PAA) index for each compound. These indices might be of predictive value to determine whether these steroids exert their anabolic action in muscle through the glucocorticoid receptor or through the androgen receptor. The data also make it unlikely that satellite cells are a preferential target for anabolic steroids in muscle.     

Some biochemical effects of triamcinolone acetonide on rat liver and muscle

1. The powerful anti-inflammatory glucocorticoid triamcinolone acetonide, administered to rats at 20 and 2.5mg/kg, leads to a decrease in the incorporation in vivo of [(3)H]uridine and [(32)P]orthophosphate into hind-limb skeletal muscle. 2. At the higher dose, this decrease in the rate of incorporation of precursors into RNA precedes a decrease in the incorporating ability of muscle ribosomes, which commences about 4-5h after drug administration, but is unaccompanied by any changes in the concentration of tissue ATP or free amino acids. 3. The ribosomal dysfunction extends to polyribosomes, which can only be successfully isolated from the muscle of triamcinolone-treated animals after the addition of alpha-amylase to the tissue homogenate to remove glycogen. 4. The specific radioactivity of muscle protein labelled in vivo with (14)C-labelled amino acids does not decrease progressively after triamcinolone administration. After 2h there is an apparent stimulation of incorporation which leads to an overall discrepancy between measurements of protein-synthetic activity made in vivo and in vitro. 5. There is a significant increase in muscle-glycogen concentration between 8 and 12h after the administration of triamcinolone acetonide (20mg/kg), although a significant decrease occurs after 4h. The fall in glycogen concentration may be due to a decrease in the rate of synthesis of protein essential for glucose uptake into the tissues. 6. As judged by (a) incorporation of (14)C-labelled amino acids into protein, (b) [(3)H]uridine and [(32)P]-orthophosphate incorporation into RNA, (c) the rate of induction of tryptophan pyrrolase and (d) changes in the pool sizes of taurine and tryptophan, the responses in liver followed the same time-course as those in muscle after administration of the drug.     

Specific binding of dexamethasone to plasma membranes from skeletal muscle

A procedure was developed to isolate plasma membranes from rabbit skeletal muscle. K⁺-dependent phosphatase activity was used as marker enzyme for plasma membranes and was determined in the presence of CHAPS (3-((3-cholamidopropyl)dimethylammonio)-1-propanesulfonate), a zwitterionic detergent. Ca²⁺-ATPase and succinate dehydrogenase activities were used as marker enzymes for sarcoplasmic reticulum and mitochondria, respectively. Electron-microscopy revealed that plasma membranes were in the form of vesicles. Significant proteolysis of membrane proteins was observed during extraction, which was inhibited by EGTA and 20 mM molybdate. SDS-polyacrylamide gel electrophoresis revealed the disappearance of an intense 96 kDa protein band when membranes were purified in the absence of EGTA and molybdate. Specific binding sites for [³H]dexamethasone were identified in plasma membranes after freezing and incubation with CHAPS. Dithiothreitol was essential for steroid binding and ATP increased it. Under standardized assay conditions, binding was complete with 50 min à 37°C. No binding occurred at 0°C, nor if EGTA and molybdate were absent from the extraction medium.     

Characterization and quantification of the androgen and glucocorticoid receptors in cytosol from rat skeletal muscle

The binding of the radioactive synthetic hormonal steroids [3H]dexamethasone (9 alpha-fluoro-11 beta, 17 alpha, 21-trihydroxy-16 alpha-methyl-1,4-pregnadiene-3,20-dione) and [3H]methyltrienolone (17 beta-hydroxy-17 alpha-methyl-4,9,11-estratien-3-one) to cytosol from rat skeletal muscle was studied using dextran-coated charcoal to separate unbound and receptor-bound steroid. The rates of association, dissociation, and degradation of the complexes of dexamethasone and methyltrienolone with receptor were highly dependent on temperature. The temperature dependence of association was greater for dexamethasone, and that of degradation was greater for methyltrienolone. Dissociation rates were insignificant for both steroid-receptor complexes compared to association and degradation rates. The apparent equilibrium dissociation constants for the binding of dexamethasone and methyltrienolone to their receptor binding sites were about 7 and 0.3 nM, respectively, regardless of temperature (0. 15 or 23 degrees C). The lack of influence of temperature on the equilibrium constants indicate that the binding was of hydrophobic character, and the corresponding free energy changes upon binding of dexamethasone and methyltrienolone to their respective binding sites were -41 and -49 kJ mol-1 under equilibrium conditions at 0 degrees C. The apparent maximum number of binding sites determined from Scatchard plots under these conditions was about 1900 fmol/g of tissue, 3500 fmol/mg of DNA or 30 fmol/mg of protein in the case of the dexamethasone receptor, and the corresponding figures for the methyltrienolone were about 100 fmol/g of tissue, 200 fmol/mg of DNA or 2 fmol/mg of protein. The ligand specificities of the binding sites for dexamethasone and methyltrienolone were typical of a glucocorticoid and an androgen receptor, respectively. Both steroid-receptor complexes were retained on DNA-cellulose columns, and were eluted by NaCl at an ionic strength of 0.1. The DNA-cellulose step purified about 20 times, and was used to allow gel exclusion chromatography and electrofocusing. Both steroid-receptor complexes were excluded from a column of Sephadex G-150. Electrofocusing in preparative columns gave reproducible patterns consisting of three peaks for each receptor. The apparent isoelectric points were 5.4, 5.6 and 6.2 for the glucocorticoid receptor, and 5.9, 6.2 and 8.5 for the androgen receptor.     

Expression of glucocorticoid receptors in the regenerating human skeletal muscle

Many stress conditions are accompanied by skeletal muscle dysfunction and regeneration, which is essentially a recapitulation of the embryonic development. However, regeneration usually occurs under conditions of hypothalamus-pituitary-adrenal gland axis activation and therefore increased glucocorticoid (GC) levels. Glucocorticoid receptor (GR), the main determinant of cellular responsiveness to GCs, exists in two isoforms (GRalpha and GRbeta) in humans. While the role of GRalpha is well characterized, GRbeta remains an elusive player in GC signalling. To elucidate basic characteristics of GC signalling in the regenerating human skeletal muscle we assessed GRalpha and GRbeta expression pattern in cultured human myoblasts and myotubes and their response to 24-hour dexamethasone (DEX) treatment. There was no difference in GRalpha mRNA and protein expression or DEX-mediated GRalpha down-regulation in myoblasts and myotubes. GRbeta mRNA level was very low in myoblasts and remained unaffected by differentiation and/or DEX. GRbeta protein could not be detected. These results indicate that response to GCs is established very early during human skeletal muscle regeneration and that it remains practically unchanged before innervation is established. Very low GRbeta mRNA expression and inability to detect GRbeta protein suggests that GRbeta is not a major player in the early stages of human skeletal muscle regeneration.     

Cortisol binding in rat skeletal muscle.

Studies of the reversible binding of [3H]cortisol by rat gastrocnemius muscle cytoplasm in vitro reveal specific binding in the 27,000 times g supernatant fraction at 0 degrees. The [3H]cortisol-binding molecule had an apparant Kd value of 1.7 times 10-7 M and the number of binding sites was 0.99 pmol per mg of cytosol protein. Only a single class of [3H]cortisol-binding sites could be detected, whose protein nature was suggested by its susceptibility to nagarse. The [3H]cortisol-protein complex sedimented at similar to 4 S in a 5 to 20% sucrose gradient either in the presence or absence of 0.3 M KCl. Binding increased more than 2-fold in adrenalectomized rats and was markedly reduced in the muscle of rats pretreated with cortisol. In contrast to the binding of [3H]dexamethasone and [3H]triamcinolone acetonide to receptor proteins in muscle, no correlation was found between the ability of various steroids to complete wtth [3H]cortisol binding and their glucocorticoid potency: [3H]cortisol binding was inhibited by a 1000-fold higher concentration of unlabeled cortisol and progesterone but not by dexamethasone or triamcinolone acetonide. It is therefore suggested that the [3H]cortisol-binding reaction is not directly involved in the biological effects of all potent glucocorticoids in skeletal muscle. The [3H]cortisol-binding protein in muscle cytosol could not be unequivocally distinguished from rat plasma corticosteroid-binding globulin, because both had similar steroid specificity and temperature stability, were not markedly affected by--SH reagents, and displayed similar sedimentation properties.     

Characterization of rat skeletal muscle sarcolemmal insulin receptors and a sarcolemmal insulin binding inhibitor

When insulin receptors of rat skeletal muscle sarcolemmal vesicles were solubilized with Triton X-100, the specific binding of 125I-labeled insulin increased by more than 10-fold over that seen in the intact vesicles. Partial purification of the skeletal muscle insulin receptors on wheat germ agglutinin affinity columns increased the total insulin binding activity by 7-fold and reduced the Kd for insulin binding from 1.92 to 0.20 nM, suggesting that an inhibitor of insulin binding was removed by this purification step. This was confirmed when the unbound fractions of the affinity column were dialyzed and reconstituted with the insulin receptors. The inhibitory activity in the sarcolemmal extract could not be accounted for by the presence of Triton X-100. The skeletal muscle inhibitor was more potent in inhibiting insulin binding to skeletal muscle insulin receptors than to liver or adipose receptors. The inhibitor was very effective in inhibiting insulin binding to wheat germ agglutinin-purified IM-9 receptors, but had negligible effects on insulin binding to intact IM-9 cells. The properties of the alpha and beta subunits of the skeletal muscle insulin receptors appear to be the same as those of insulin receptors of other tissues: cross-linking of 125I-labeled insulin to the receptor revealed a band of 130,000 daltons, and insulin stimulated the phosphorylation of bands of 90,000 and 95,000 daltons in the receptor preparation. The skeletal muscle insulin binding inhibitor elutes from molecular sieves in a major 160,000-dalton peak and minor 75,000-dalton peak. The binding inhibitor is not inactivated by heat, by mercaptoethanol, or by trypsin, pepsin, or proteinase K. Collectively, these data suggest that the inhibitor may be a small molecule that aggregates with itself, with larger proteins, or with detergent micelles.     

Nuclear glucocorticoid receptor binding in L6 skeletal muscle cells in culture

Mechanisms underlying glucocorticoid hormone actions on skeletal muscle remain incompletely understood. This problem may be amenable to solution with a simple cell culture system in which the hormonal environment can be controlled. In this report, we demonstrate that the L6 muscle cell line may provide such a system. These cells, which possess many morphological and functional characteristics of skeletal muscle, originate as mononuclear myoblasts, which fuse to form multinucleated myotubes. L6 myoblasts and myotubes contain an intracellular glucocorticoid receptor that has binding parameters and ligand specificity similar to those of glucocorticoid receptors of classical glucocorticoid target tissues. A major advantage of the use of cultured cells is ease of isolation of myonuclei that display specific glucocorticoid receptor binding. L6 muscle cells should provide a valuable model system for further studies of the mechanisms of glucocorticoid hormone actions on muscle.     

Specific cytosol binding of diethylstilbestrol in rat skeletal muscle

Rat skeletal muscle cytosol proteins bound ³H-diethylstilbestrol (³H-DES). More than 90% of this binding was high capacity and low affinity. Serum albumin accounted for roughly 50–60% of the binding, as evidenced by its precipitation with anti-rat albumin IgG. About half of the binding was distinguishable from albumin and other serum proteins by its precipitation in 40% saturated ammonium sulfate. This material sedimented at 4–5S in high-salt sucrose gradients, and resolved into two components (8S and 4–5S) in low-salt. Following incubation at 23–27°C for one hour, 2% of the bound ³H-DES in whole cytosol (approximately 2 fmole/mg cytosol protein) was retained by DNA-cellulose, and was eluted with 0.6 M KCl. This small fraction of the total binding was inhibited by estrogens and DES analogues: estradiol-17β, DES, dienestrol, and hexestrol were strong inhibitors; isodienestrol, dimethylstilbestrol, estradiol-17α, estrone, tamoxifen, MER-25, CI-628, and nafoxidine were weak inhibitors; dihydrotestosterone, testosterone, and prednisone did not compete. These observations indicate that specific estrogen-binding sites exist in rat skeletal muscle.     

Comparative teratogenicity of triamcinolone acetonide and dexamethasone in the rhesus monkey (Macaca mulatta)

Pregnant rhesus macaques were treated with 0.5 or 2.5 mg/kg triamcinolone acetonide (TAC) or 1.0 or 10.0 mg/kg dexamethasone sodium phosphate (DEX) between 20 and 50 gestational days (GD). Treatment with TAC at 2.5 mg/kg resulted in a fetal loss of 71%; 3/5 recovered fetuses displayed an encephalocele or meningocele. All other treatment groups displayed minor cranial skeletal abnormalities consistent with glucocorticoid-mediated teratogenesis. DEX was shown to have a lower teratogenic potential than TAC in this species.     

Ouabain binding of the rat's heart muscle cells after neonatal glucocorticoid (triamcinolone) treatment

In adult rats the ouabain-sensitive ATP-ase activity of the heart muscle as well as the amount of specifically bound 3H-ouabain decreased by 1/3 as a result of a single triamcinolone treatment undertaken in the neonatal period. Neonatal ouabain treatment did not alter the triamcinolone binding of the thymus of adult animals. The experiments again call attention to the phenomenon that the steroid hormones, even though specific for their target cells, are able to induce "alien" imprinting when administered in the neonatal period. They are also able to bind to steroid receptors of other cells altering, thereby, the responsiveness of these cells in adulthood.     

Submicromolar free calcium modulates dexamethasone binding to the glucocorticoid receptor

The relative distribution of bound cis- and trans-(NH₃)₂PtCl₂ at specific sites in SV40 DNA is evaluated by monitoring the extent to which five restriction endonucleases, each of which cleave at a single, unique site, are inhibited as a result of the DNA modification. The order of cleavage inhibition is Bgl 1 ? Bam HI > Hpa II, Kpn I > Eco RI. Both isomers produce a comparable effect for any particular endonuclease. Inhibition correlates with the % (G+C) content within and about the recognition sequences. That modified sequences immediately adjacent to the recognition sequence influence cleavage is further supported by differential cleavage observed with the multicut Hind III endonuclease. The binding of cis-(NH₃)₂PtCl₂ at the hyper-reactive Bgl 1 site may well be directly responsible for inhibiting SV40 replication.     

Kinetics of dexamethasone binding to the glucocorticoid receptor of intact erythroid cells from rat fetal liver

The erythroid cells from the rat fetal liver have been shown to possess a receptor for glucocorticoids. In the present work, the characteristics of [3H]dexamethasone binding have been studied on intact cells, in order to minimize receptor degradation, and at 4 degrees C, in order to prevent the activation of the hormone-receptor complex. Dissociation kinetics were those of a first-order reaction and the value of the rate constant of dissociation was similar to the values available in the literature. When studied at low concentrations of the ligand and using short-term incubations, association kinetics were apparently those of a simple bimolecular reaction. But at high ligand concentrations and/or using long-term incubations, association kinetics indicated a more complex reaction. Our results were compatible with the model proposed by Pratt W.B., Kaine J.L. and Pratt V.D. (J. Biol. Chem. 250 (1975) 4584-4591) for cytosolic preparations. This model implies the rapid formation of a transient unstable form of the complex, further converted into a stable form with slower kinetics. Equilibrium dissociation constant of the first (rapid) reaction was 80 microM and the rate constant of 'stabilization' was of the order of 70 X 10(-3) min-1. These values agree with the results of Pratt et al. relative to a cytosolic preparation from rat thymocytes.     

Purification and autophosphorylation of insulin receptors from rat skeletal muscle

Insulin receptors of rat skeletal muscle were purified by first extracting a plasma membrane-enriched pellet obtained from a muscle homogenate with Triton X-100, followed by WGA-Sepharose and insulin-Sepharose affinity chromatography. Routinely, 4-5 micrograms of purified receptor were obtained from 15 g of tissue. The purified receptors are composed of two major polypeptides with molecular weights of 130,000 and 95,000, respectively. The binding of [125I]insulin by the purified receptors was analyzed by a Scatchard plot. There are at least two binding components. The high-affinity component, with an apparent association constant (Ka) of 2.0 X 10(9) M-1, comprises 10% of the total insulin binding sites; while the low-affinity component, with a Ka value of 1.4 X 10(8) M-1, represents 90% of the binding sites. Assuming the insulin receptor to have a molecular weight of 300,000, the receptor binds 1.7 mol of insulin per mol at saturation. Insulin is capable of stimulating the autophosphorylation of the beta-subunit of the muscle insulin receptor (Mr 95,000) by 5-10-fold. The stoichiometry of this phosphorylation reaction was determined as 0.8 phosphate per insulin binding site after a 10 min incubation with 100 nM insulin. In a previous report, I showed that the insulin stimulation of glucose transport in diaphragms from neonatal rats was small, even although the diaphragms had normal levels of insulin receptors and glucose transporters (Wang, C. (1985). Proc. Natl. Acad. Sci. USA 82, 3621-3625). To determine whether or not receptor autophosphorylation might be related to this insensitivity to insulin, the level of receptor phosphorylation was quantitated in diaphragms from rats at different stages of development. Autophosphorylation remains unchanged from birth to 21 days of age, suggesting that the lower insulin-stimulated glucose uptake by diaphragms at early stages of postnatal development as compared to that by diaphragms of older rats, is not due to a difference in receptor kinase.     

β-adrenergic receptors in rat skeletal muscle effects of thyroidectomy

Thyroid hormones may participate in the regulation of β-adrenergic receptors in skeletal muscle sarcolemmal membrane. Since skeletal muscles are not innervated by sympathetic nerve endings, the biochemical mechanism involved in the control of β-adrenergic receptors by thyroid hormones appears to be mediated by thyroid-induced regulation of serum levels of catecholamines.     

Progestin-induced enhancement of dexamethasone dissociation from glucocorticoid hormone receptors

Several groups have reported that progesterone accelerates the rate of steroid dissociation from the agonist site of the glucocorticoid receptor. It has been proposed that this enhancement reflects the binding of progestins to a second steroid-binding site. Since progestins are frequently antagonists of glucocorticoid hormone action, we decided to characterize this site more fully. In particular, in this study, we investigated whether the cytosolic preparations of four separate glucocorticoid target tissues from the same species all contained this second site and whether it was similar in each case. Cytosolic extracts of rat heart, liver, kidney, and pancreas were examined. In each case it was found that the rate at which prebound tritiated dexamethasone dissociated from the glucocorticoid receptor was faster in the presence of nonradioactive progesterone. The magnitude of this effect was essentially the same in each case. These results indicated that the second site was present in each preparation. To determine if the site was similar in each extract, we studied the steroid specificity of the enhancement of dissociation. This was determined by quantitating the degree to which each of a series of test steroids could cause augmentation of dissociation. Progesterone, R-5020, medroxyprogesterone, deoxycorticosterone, 17-OH-progesterone, and cortexolone were evaluated. The results for all four cytosolic preparations showed that either progesterone or R-5020 was the most potent steroid while both cortexolone and 17-OH-progesterone were essentially without effect. Medroxyprogesterone and deoxycorticosterone were usually of intermediate potency. These results suggest that the cytosolic extracts of all glucocorticoid target tissues have a similar second steroid-binding site which demonstrates a preference for progestins and that interaction with this site causes the glucocorticoid receptor to decrease the affinity with which it binds agonists.