The Binding of Dexamethasone and Triamcinolone Acetonide to Glucocorticoid Receptors in Rat Skeletal Muscle *

Specific binding of the synthetic glucocorticoids [l ,2,43H]dexamethasone (Qa-fluoro-llp,17a,Zl-trihydroxy-16amethyl-1,4-pregna-1,4-diene-3,20-dione) and [1,~2,4-~H]triamcinolone acetonide (9cu-fluoro-ll~,l6ol,l7~,Zl-tetrahydroxy-pregna-l,4-diene-3,20-dione-16,17-acetonide) by the cytoplasmic fraction of rat gastrocnemius muscle was studied. The cytosol binding reaction displayed stereospecificity and high affinity of binding. Biologically active glucocorticoids, administered to adrenalectomized rats or present during the in vitro binding reaction markedly depressed the binding of [3H]dexamethasone and [“H]triamcinolone acetonide. The biologically inactive stereoisomer, epicortisol, had no effect on the binding of the labeled hormones. The binding component displayed high affinity for [ aH]dexamethasone and [ 3H]triamcinolone acetonide (& = 1.9 and 1.2 X lo-* M, respectively). The number of binding sites was limited (0.1 pmoles per mg of cytosol protein), and Scatchard analysis suggests that only a single class of binding sites exists for both [3H]dexamethasone and [3H]triamcinolone acetonide. Competition studies indicated that the two glucocorticoids interact with the same binding site. The binding macromolecule appears to be a protein since binding is prevented by nagarse treatment and is dependent on the integrity of -SH groups. The glucocorticoid-protein complexes were characterized on 5 to 20% sucrose gradients. Both complexes sedimented at n 4 S in 0.3 M KCl, but only the [3H]triamcinolone acetonide-receptor complex sedimented at 7 S in the absence of salt. Since the specific binding component has the properties of a physiological glucocorticoid receptor, a direct effect of these hormones on skeletal muscle is suggested.

The effects of steroid hormones is the intracellular binding of the hormone to specific receptor molecules (1). Such protein receptors are found in the cytosol fraction of glucocorticoidresponsive tissues, and display selective and high affinity binding of these hormones (2). Their physicochemical properties as well as their implication in the biological response of various target tissues, e.g. liver, thymus, and certain cell lines growing in culture, were thoroughly described in several recent papers (3)(4)(5)(6)(7).
Exposure of muscles in ~iuo to glucocorticoid hormones results in a significant depression of DNA, RNA, and protein synthesis (8,9), and was recently found to cause activation of myofibrillar proteolytic activity (10). However, information on the sequence of events and mechanism of the antianabolic and catabolic effects of these hormones on muscle tissue is still limited.
It is not known whether pronounced muscle wasting brought about by glucocorticoid administration is a result of a direct interaction of the hormone with muscle cells, or a secondary consequence of hormonal action on visceral organs.
Such secondary effects could be mediated via changes in the hepatic demand for gluconeogenic amino acids, causing increased mobilization of these substrates from muscle proteins (11,12).
Since specific glucocorticoid receptors appear to be necessary for a direct physiological response to these hormones, the presence of such binding components in skeletal muscle might indicate a primary effect of the hormone on that tissue.
Although no binding of cortisol or corticosterone to subcellular fractions of rat skeletal muscle could be demonstrated by De-Venuto et al. (13) using a novel "equilibrium fractionation" technique, a recent paper by Simpson and White (14) reported on the binding of some biologically potent steroids in vivo to rat muscle, and Roth (15) examined the age-related changes in the binding of glucocorticoids to several rat tissues, including muscle. However, in these studies total rather than glucocorticoidspecific binding was measured and no attempt was made to characterize the binding component or the binding reaction itself.
In the present study we examined the specific binding of biologically potent glucocorticoid hormones to rat muscle cytosol.
[ Measurement of 3H-Labeled Steroid Binding-Two milliliters of the 27,999 X g muscle supernatant fraction were incubated with tritiated steroids (4 to 9 X lo-* M) at 0" for 2 hours or as indicated. [3H]triamcinolone acetonide for specific receptors but might bind to sites which do not play a role in mediating the biological activity of glucocorticoids.
On the other hand, an excess of biologically potent steroids should compete with the labeled hormone for both glucocorticoid-specific and nonspecific binding sites. For this reason the binding of the 3H-labeled steroids in the presence of nonlabeled epicortisol, minus the binding in the presence of nonlabeled biologically active steroids is taken as an indication of the relative amount of hormone-specific binding components. Fig. 1  A saturation type binding curve is obtained.
To estimate the number of binding sites and the binding affinity, the results were analyzed by the Scatchard technique (21). A plot of the amount of steroid bound against the ratio of bound to free steroid (Fig. 2, inset) yielded a straight line for both glucocorticoids.
From the slopes of the Scatchard plots the apparent dissociation constants for the reaction: hormone + binding component e bound hormone complex was calculated and found to be 1.9 X 10m8 M and 1.2 X 10e8 M for the binding of [aH]dexamethasone and [3H]triamcinolone acetonide, respectively.
The concentration of the binding sites, assuming that each site binds one molecule of steroid and that only a single class of specific binding sites exists under the conditions of the assay, is about 0.1 pmole per mg of cytoplasmic protein for each of these steroids.
The possibility that the two steroid hormones interact with the same class of binding sites in muscle cytosol was further tested in the experiment described in Fig. 3  were performed to determine the sedimentation profiles of the glucocorticoid binding components of rat muscle cytosol.
The 27,000 X g muscle supernatant fraction was incubated with [3H]dexamethasone or [aH]triamcinolone acetonide at 0", the unbound steroid was removed by charcoal adsorption and the bound complexes were subjected to 5 to 20% sucrose density gradient centrifugation (Fig. 4) For details see "Materials and Methods." The arrow indicates the sedimentation peak of bovine serum albumin (B&4). Gradients with KCl, --; gradients without KCl, ---. The biological potency of these hormones on muscle is reflected by their effect on the reduction of muscle mass (22,23). Under the conditions of the experiment, however, it is difficult to determine whether the reduction in the specific binding of [aH]dexamethasone is a reflection of the increased degree of saturation of the available binding sites by the in vivo administered steroid or an actual reduction in the number of binding sites. DISCUSSION The binding reaction described in this work was followed in vitro by incubating the 27,000 x g supernatant fraction of rat skeletal muscle with the labeled steroids at 0". Such a cell-free system has several advantages over the assay system utilized by Simpson and White for the determination of glucocorticoid binding to skeletal muscle (14), since the direct interaction of the hormone with the binding component is measured.
The in vitro assay at 0" minimizes possible bio-transformations of the hormone and eliminates changes in binding which might be a result of differences in cellular metabolism, cellular uptake, or binding by extracellular sites. By utilizing the cell-free system, evidence was obtained for the existence of specific binding sites for glucocorticoid hormones in rat skeletal muscle. The binding reaction displayed saturation kinetics and high affinity binding of [aH]dexamethasone and [aH]triamcinolone acetonide to a cytosol protein.
The formed complexes sedimented at about 4 S in sucrose density gradients containing 0.3 M KCl.
These properties, as well as the reduction of binding following treatment of the rats with potent glucocorticoid hormones, resemble those described for steroid-specific "receptor" proteins present in the cytosol fraction of various target tissues, including rat liver (3), thymus (4), as well as certain murine lymphoid tumors (24). Other studies* have indicated that in addition to the binding component described in this paper, muscle cytosol contains an [aH]cortisol-binding protein similar to the corticosteroid-binding globulin of rat plasma (25). However, binding of [aH]dexamethasone or ['Hltriamcinolone acetonide cannot be attributed to the presence of the corticosteroid-binding globulin in muscle cytosol, since that globulin does not bind these synthetic glucocorticoids (5,25).
In spite of the similarities in the kinetics and specificity of the ['Hldexamethasone-and [aH]triamcinolone acetonide-binding reactions, they differed with respect to their sedimentation profiles.
No sedimentation peak could be found for the t8H]dexamethasone protein complex in sucrose gradients at low ionic strength.
This difference might be a result of the interaction of the hormones with different, preexisting or induced, conformational forms of a single binding protein resulting in different stabilities of the formed complexes. Such a possibility was recently suggested by Giannopoulos el al. (26) to explain differences in the binding of cortisol and dexamethasone to nuclear binding sites of rabbit fetal lung.
It is likely that the glucocorticoid-protein complex, as a result of the rather specific binding of glucocorticoid hormones to skeletal muscle cytosol, is involved in the catabolic actions of these hormones on skeletal muscle.
The existence of specific intracellular receptors suggests that the interaction of steroids with muscle is of a "primary" type, in which the hormones directly interact with the muscle tissue, and the binding subsequently triggers the catabolic biological response.
If this is the mechanism of action one should expect a correlation between hormone binding and catabolic response. The finding of [3H]dexamethasone-and [aH]triamcinolone-specific binding to muscle cytosol is in accord with their high potency in inducing muscle protein catabolism and weight loss (22,23). Along with the findings on the reduction in binding following in viva administration of potent glucocorticoids, these resuhs can be taken as an indication for a direct rather than secondary effect of glucocorticoids on muscle. The observations on glucocorticoid hormones binding to skeletal muscle further imply that a basic common sequence of events mediates the biological response of different tissues to steroid hormones.
Since in tissues in which glucocorticoids mediate an essentially anabolic response (liver) a similar binding is known to occur, it is probable that the binding is the initial reaction occurring before any subsequent tissue-specific response, either anabolic or catabolic, is induced by the glucocorticoid hormones.