Arne Attramadal

Androgen receptors in the rat adrenal gland.

Abstract Cytosol fractions from rat adrenal glands contain specific androgen binding proteins showing high affinity ( K D ~ 3.9 × 10 9 M) and limited capacity (22 fmol/mg protein) for testosterone and with physicochemical properties identical to those of androgen receptors in the prostate, epididymis and testis. The androgen receptor complexes have a sedimentation coefficient of 7–8 S by sucrose density gradient centrifugation, and a mobility relative ( R F ) to bromophenol blue of 0.5 by electrophoresis in 3.25% polyacrylamide gels containing 0.5% agarose. Isoelectric focusing in agarose-polyacrylamide gels indicated a pI of 5.9. Androgen binding to these receptors is destroyed by heating at 45°C for 30min. by incubation with p -chloromercuriphenyl sulphonate (2 mM) or protease, but not by RNase. Testosterone (T) and 5α-dihydrotestosterone (DHT) were the most potent androgens in displacing [ 3 H]-T binding. However, 4-androsten-3,17-dione (A) and 5α-androstan-3α,17β-diol (Adiol) also significantly displaced [ 3 H]-T binding from the receptor. The reasons for this are the high activities of 17β-OH steroid dehydrogenase (converting A into T) and 3α-oxido-reductase (converting Adiol into DHT) in the cytosol fractions. These enzymes are active even at 0°C. Estradiol-17β in high concentrations (100-fold excess) also reduced the [ 3 H]-T binding, by approximately 60%, whereas corticosterone was without effect. The androgen receptors were found in similar concentrations in the adrenal cytosol of male and female rats, but were virtually absent in cytosol fractions obtained from rats with testicular feminization syndrome ( tfm rats). The presence of specific androgen receptors in the adrenal gland of normal rats indicates that androgens may affect adrenal steroid production by a direct action.

The uptake of 3H-oestradiol by the anterior hypophysis and hypothalamus of male and female rats

SummaryThe anterior hypophysis and hypothalamus of male and female rats given 3H-oestradiol were examined with regard to 1) the kinetics of the uptake of the radioactive material, 2) the chemical nature of the labelled material, and 3) the influence of non-labelled oestradiol-17β, oestradiol-17α and testosterone on the uptake of 3H-oestradiol. The anterior hypophysis was found to concentrate and retain oestradiol in basically the same manner in male and female rats. The pattern of the uptake was similar to that of the uterus and vagina, with a concentration peak 2 hours after the injection. Non-target tissues such as cerebral cortex, liver and blood attained their maximum uptake already 15 minutes after the injection. Thereafter the concentration gradually decreased. The ratio between the concentration of labelled material in the anterior hypophysis and brain cortex gradually increased until a peak was reached at 8 hours in both sexes. In the female, the concentration of labelled material in the anterior hypophysis was then 106.3 times greater than in the brain cortex, while in the male the ratio was 63.2.In the hypothalamus the uptake followed a pattern similar to that of the brain cortex. However, in the former the concentration of labelled material was consistently greater than in the latter. At maximum uptake, registered 4 hours after the injection, the concentration was about two times greater in the hypothalamus than in the cerebral cortex.The neurohypophysis contained, on an average, 1/6 of the amount of radioactive material registered in the anterior lobe one hour after the injection, but it was about two times greater than in the brain cortex.Isolation and identification of the radioactive material in the anterior hypophysis and hypothalamus showed that in both sexes nearly all of it was chemically unchanged oestradiol. Graded doses of non-labelled oestradiol-17β were found to decrease the uptake of 3H-oestradiol in the anterior hypophysis and hypothalamus almost linearly, while the concentration of labelled material in the brain was unaltered. Oestradiol-17α and testosterone were without significant effect on both the pituitary and hypothalamic accumulation of 3H-oestradiol. Therefore, a limited number of binding sites, with a high degree of specificity for oestradiol, appear to exist in both tissues. The results were essentially the same in male and female rats.

Receptors and binding of androgens in the prostate

Abstract The interaction between androgens and prostatic and other androgen-dependent tissues has been investigated in castrated male rats in vivo following administration of [1,2- 3 H] testosterone of high specific activity. Radioactive androgens were concentrated and retained in such tissues for a number of hours. The receptors in the ventral prostate appeared to have a limited capacity, as administration of 20 μg non-radioactive testosterone simultaneously with the tritiated testosterone significantly reduced the uptake of radioactivity, and 500 μg completely abolished it. Synthetic androgens, antiandrogens, progesterone and corticosterone also reduced the uptake. Autoradiography of the prostatic tissue revealed a selective labelling of the glandular epithelium, with most of the radioactivity associated with the nuclei. This conclusion was supported by subcellular fractionation of homogenized prostatic tissue. Isolation and identification of the radioactive steroids confirmed previous observations that 5α-dihydrotestosterone was the quantitatively most important radioactive compound in the various accessory sex organs. 5α-dihydrotestosterone accounted for 50–72 per cent of the radioactivity, whereas 3–17 per cent was recovered as unchanged testosterone. Following the administration of [1,2-3H]-androstenedione, accumulation of radioactivity was also observed in androgen-dependent tissues, and about 50 per cent of the radioactivity was identified as 5α-dihydrotestosterone. By Sephadex® G-100 gel filtration of a 105.000 g supernatant of a homogenate of the ventral prostate obtained one or two hours after the administration of [1,2-3H]-testosterone, the major fraction of the radioactivity was associated with macromolecules excluded from the gel. The radioactivity bound to the macromolecules was extractable with ether, suggesting a non-covalent binding, and 5α-dihydrotestosterone accounted for more than 90 per cent of the radioactivity. This cytosol androgen-macromolecule complex was destroyed by proteolytic enzymes and SH-reagents, but was unaffected by RNase and DNase. Incubations with [1,2-3H]-5α-dihydrotestosterone and a 105.000 g supernatant of the ventral prostate suggested the existence of two different species of cytosol macromolecules with binding affinity for 5α-dihydrotestosterone, one of which was excluded from the gel during Sephadex® G-100 gel chromatography, whereas the other was slightly retained. In the experiments with [1,2-3H]-testosterone administration in vivo the radioactive 5α-dihydrotestosterone was mostly bound to the former. Attempts to isolate [3H]-5α-dihydrotestosterone in muscle tissue following [1,2-3H]-testosterone administration were unsuccessful, suggesting that 5α-dihydrotestosterone may not be involved in the action of androgens on such tissue.

Quantitation of corticosteroid binding globulin (CBG) by steady state polyacrylamide gel electrophoresis (SS-PAGE)

Abstract A method suitable for quantitating corticosteroid binding globulin (CBG) in rat plasma has been developed using steady state polyacrylamide gel electrophoresis (SS-PAGE). The method is sensitive, specific and very precise. Using this technique CBG levels have been determined in immature and mature male and female plasma as well as in pregnant female plasma.

Androgen receptors in the rat epididymis do not disappear after castration

Abstract Binding of [ 3 H]-dihydrotestosterone to cytoplasmic androgen receptors in the rat epididymis was studied at varying times after castration (1, 5, 9, 33, 50 and 90 days), using two different methods: sucrose gradient centrifugation and polyacrylamide gel electrophoresis. Cytoplasmic androgen receptors were shown to be present in the epididymis at all times after castration. Earlier reports which describe the disappearance of cytoplasmic androgen receptors in the epididymis after castration are probably the result of methodological artifacts. Polyacrylamide gel electrophoresis was a more reliable technique than sucrose gradient centrifugation for demonstrating the presence of epididymal androgen receptors.

Catecholamine-responsive adenylate cyclase activity in human endomyocardial biopsies: Individual sensitivity to isoproterenol stimulation and propranolol inhibition

In the present study we have examined adenylate cyclase (AC) activity, the stimulation by isoproterenol and inhibition by propranolol, in endomyocardial biopsies from eleven patients with suspected cardiomyopathy. Biopsies were obtained by heart catheterization from the right endomyocardial surface of the interventricular septum. Three biopsies were taken from each patient (mean weight, 2.1 mg; range, 1.2-4.0 mg). One biopsy was studied by light microscopy. The two other biopsies were homogenized and AC activity in the homogenates was determined in the presence of different concentrations of isoproterenol and isoproterenol (5 micrograms/ml) combined with different concentrations of propranolol. Thus stimulation and inhibition curves were established for a pair of biopsies from each patient. Appropriate biopsy material was obtained in triplicate from only seven patients. In these patients the variance in maximal receptor stimulation (by isoproterenol) and inhibition (by propranolol) was significantly smaller in pairs of biopsies compared to the variance between all biopsies (p values from less than 0.05 to less than 0.025). Hence it is possible to determine AC activity, and adrenergic receptor function, in very small endomyocardial biopsies. New diagnostic possibilities could thereby be introduced.