It is unclear whether anabolic steroids act on skeletal muscle via the androgen receptor (AR) in this tissue, or whether there is a separate anabolic receptor. When several anabolic steroids were tested as competitors for the binding of [3H]methyltrienolone (MT; 17 beta-hydroxy-17 alpha-methyl-4,9,11-estratrien-3-one) to the AR in rat and rabbit skeletal muscle and rat prostate, respectively, MT itself was the most efficient competitor. 1 alpha-Methyl-5 alpha-dihydrotestosterone (1 alpha-methyl-DHT; mesterolone) bound most avidly to sex hormone-binding globulin (SHBG) [relative binding affinity (RBA) about 4 times that of DHT]. Some anabolic-androgenic steroids bound strongly to the AR in skeletal muscle and prostate [ RBAs relative to that of MT: MT greater than 19-nortestosterone ( NorT ; nandrolone) greater than methenolone (17 beta-hydroxy-1-methyl-5 alpha-androst-1-en-3-one) greater than testosterone (T) greater than 1 alpha-methyl-DHT]. In other cases, AR binding was weak (RBA values less than 0.05): stanozolol (17 alpha-methyl-5 alpha- androstano [3,2-c]pyrazol-17 beta-ol), methanedienone (17 beta-hydroxy-17 alpha-methyl-1,4-androstadien-3-one), and fluoxymesterolone (9 alpha-fluoro-11 beta-hydroxy-17 alpha-methyl-T). Other compounds had RBAs too low to be determined (e.g. oxymetholone (17 beta-hydroxy-2-hydroxymethylene-17 alpha-methyl-5 alpha-androstan-3-one) and ethylestrenol (17 alpha-ethyl-4- estren -17 beta-ol). The competition pattern was similar in muscle and prostate, except for a higher RBA of DHT in the prostate. The low RBA of DHT in muscle was probably due to the previously reported rapid reduction of its 3-keto function to metabolites, which did not bind to the AR [5 alpha-androstane-3 alpha, 17 beta-diol and its 3 beta-isomer (3 alpha- and 3 beta-adiol, respectively)]. Some anabolic-androgenic steroids (only a few synthetic) bound to SHBG (1 alpha-methyl-DHT much greater than DHT greater than T greater than 3 beta-adiol greater than 3 alpha-adiol = 17 alpha-methyl-T greater than methenolone greater than methanedienone greater than stanozolol). The ratio of the RBA in rat muscle to that in the prostate (an estimate of the myotrophic potency of the compounds) was close to unity, varying only between about 0.4 and 1.7 in most cases.(ABSTRACT TRUNCATED AT 400 WORDS)

A major obstacle to a better understanding of the fundamentals of stress corrosion cracking (SCC) has been the almost total lack of information about the nature of the corrodent within a growing stress corrosion crack, particularly near the advancing edge. While it is generally conceded that the local chemistry within the stress corrosion crack differs considerably from that of the bulk solution, the demonstration of this difference has been deterred by inaccessibility and the problems of obtaining representative samples of solution. The present communication describes methods by which the stress corrosion process is interrupted in such fashion as to permit opening the crack without undue mixing of the corrodent within it, and to enable one to analyze the small amount of corrodent present without undue chemical changes in the interval between termination of the SCC test and the completion of the analysis.The specimens used were for the most part rectangular and about 3 mm thick, with a saw cut at the middle of the long dimension. A metal wedge was pressed into the saw cut using a vise, thus stressing the root of the saw cut in tension. The specimen was then placed in either distilled water or 31/2% NaC1 solution (pH 6.5), with the wedge either paraffin coated or kept above the waterline to prevent galvanic effects. Some of the aluminum alloy specimens were long bars slotted at one end (parallel to the rolling plane) and stressed by opening the slot elastically with a set screw.After the stress corrosion crack had propagated a suitable distance (a centimeter or so), the specimen was removed from the solution and immersed in liquid nitrogen to freeze the corrodent in the crack in place and also to minimize any side reactions which might alter the composition of the corrodent until the analytical operation could be effected. The specimen was subsequently removed from the liquid nitrogen and broken apart in a vise or with pliers to expose the frozen solution on the SCC surfaces for analysis immediately on thawing. Some of the early experiments were conducted in a glove box filled with dry nitrogen (1), but it was found that in general they can be conducted satisfactorily in the open air.The inherent difficulties of microanalysis on the microvolume of solution available at the crack tip, the rugged fracture surface, and the obvious requirement of rapid analysis to prevent solution changes suggested a simple in situ approach to the problems of determining the solution chemistry of interest, i.e., the acidity and the soluble metallic constituents. (A listing of the acid and ion indicators used for this purpose is given in Tables I and II.) The test method involved the use of indicator-impregnated filter paper or indicator-coated silica gel (60-80 mesh) prepared by saturating the paper or gel with dilute aqueous solutions of indicator, and drying completely. The congored and alkacid papers were obtained commercially; the others were all prepared as described above. The silica gel particles were sprinkled on the chilled f...

Using a charcoal technique, we determined the relative binding affinity of some anabolic compounds for the androgen and glucocorticoid receptors in cytosol from rat skeletal muscle. Only a few of the compounds analyzed competed for the receptor-binding sites. The androgen and glucocorticoid receptors were analyzed in rat and mouse skeletal muscle cytosols by Scatchard analysis. In rats grouped according to sex and age, the cytosolic protein content was about the same in all groups, but the DNA content decreased with increased weight of the animal regardless of sex (male, female, or castrated male). The glucocorticoid receptor did not differ in concentration (2-3 pmol/g tissue) or ligand affinity (Kd, 10-40 nM) among the groups, but the androgen receptor concentration decreased with increased weight and age of the animals, more in the case of males than in the case of females or castrates. The Kd for the androgen receptor increased with age in males but was constantly about 0.2 nM for castrates or females. In adult intact rats, the androgen and glucocorticoid receptor concentrations in muscle cytosol from females were about 100 and 3000 fmol/g tissue, respectively, the corresponding values for males being about 50 and 2000 fmol/g tissue, respectively. Short term castration or adrenalectomy increased the concentration of and ligand affinity for the androgen and glucocorticoid receptors, respectively. After long term castration of male rats, the concentration of both receptors increased during 5 weeks to about the female level, only to decrease later. Neonatally castrated male rats had about the same androgen receptor concentrations and Kd values as female rats. Female mice had higher androgen receptor concentrations (approximately 700 fmol/g tissue) than rats. Intact male mice had about 200 fmol androgen receptor-binding sites/g tissue, and the same amount was found in mice bearing the testicular feminization (Tfm) mutant gene. In summary, the concentrations of androgen and glucocorticoid receptors in rat skeletal muscle are regulated at least by the testes. The presence of androgen receptors in skeletal muscle from Tfm mice is surprising and may motivate a reinvestigation of the regulation of androgen receptors in Tfm animals.

The binding of the radioactive synthetic hormonal steroids [3H]dexamethasone (9a-fluoro-1 ID, 17a,21-trihydroxy-l6a-methyl-l,4-pregnadiene-3,20-dione) and [3H]methyltrienolone (1 7P-hydroxy17a-methyl-4,9,1l-estratrien-3-one) to cytosol from rat skeletal muscle was studied using dextrancoated 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 "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-' under equilibrium conditions at 0 "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 colurnns, 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 (3-150. 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.Although it has long been known that glucocorti- [6,7], but the characterization of these receptors hasTriviul Numes. Dexamethasone. 9~-f l u o r o -l 1 /j. 17x.21 -trihydroxy-I 6%-meth yl-l ,4-pregnadiene-3,20-dione ; methyltrienolone, 17fl-hydroxy-17a-methyl-4,9,ll-estratrien-3-one; 5a-dihydrotestosterone, 17~-hydroxy-5a-androstan-3-one; triamcinolone acetonide, 9a-fluoro-11~,21-dihydroxy-l6a,l7a-[(methylethylidene)bis-(oxy)]-I ,4-pregnadiene-3,20-dione; 19-nortestosterone decanoate, 1 Y-nor-4-androsten-3-one-I 7fl-yl decanoake. not been extensive. Indeed, the presence of androgen receptors in skeletal muscle has been questioned by several authors [8 -101, and the anabolic action...

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