Androgen and estrogen binding in rat skeletal and perineal muscles
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Texas A&M University on 11/12/14 For personal use only.
JEAN Y. DUBIS,R E N ~LESAGE, E AND ROLANDR. TWEMBLAY kaboratoire cf'EarAocrinologie-Me'tabolisme, kc Centre Hospitalier de k' &Inhersite'kaval, Quebec, Qut. GI Y 4G2 Received July 14,1975 DubC, J. Y.,Lesage, R. & Tremblay, R. R. (1976) Androgen and estrogen binding in rat skeletal and perineal rnuscBes. Can. 9.Biochem. 54, 0-55 Specific ira v i m binding sf E3H]testosterone (T), Sac-[3HHldihydrotestostepone(DHT), and [3H]estradiol (E2)was demonstrated in the 30 OMI x g supernatant (cytosol) of thigh muscles (TM) and of the levator ani - bulbocavernosus muscle complex (LA-BC) by gel filtration through Sephadex G-25 columns. Bn T M cytosol, T and E2 [are bound with high affinity (BY, = 1.1 x 109M-I and 2.3 x lo9 M - l , respectively) whereas DHT binding is of lower affinity (Ka = 5.0 x 1Q7M-I).] In LA-BC cytssol, T, E2, and D H T are bound with high affinity (Ka 1.9 x lo9 M-', 0.3 x B09M-', and 0.5 x lo9 M - I , respectively). Competition experiments suggest that the binding of the three hormones (T, El,and DHT) is due to different proteins. In addition to TM and LA-BC, T and E2binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.
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Dube, J. Y . , Lesage, R. kk Trernblay, R. R. (1976) Androgen and estrogen binding in rat skeletal and perineal muscles. Can. 9.Blochem. 54, 58-54 Nous dimontrons in vitrs la liaison spkcifique de la [3H]testostkrone (T), de la Su-[%IHihydrotestosterone (DHT) et du [3H]oestradiol (E2) dans le sumageant (cytosol) 30 000 x g des muscles fdmoraaax (TM) et du cornplexe rnusculaire levatsr ani - bulbocavernosus (LA-BC) par gel filtration sur colonnes de Sephadex 6-25.Dans le cytosol TM, les hormones T et Ea sont likes avec grande affinite (Ka = 1.1 x lo9 M-' et 2.3 x 10' M - ' , respectivement) tandis que pour Ba liaison de DHT, 19affixlitCest moins [ClevCe (K, = 5.0 x lo7 M - I ) . Dans le cytosol LA-BC, T, El et DHT] ssnt aussi Biees avec grande affinitk (K, = 1.9 x 10' '44-I, 0.3 x lo9 M-I et 0.5 x IQW-', respectivement). Les exxpCriences de compktition swgg&rentque la Iiaison des trois hormones (T, E, et DWT) est due B differentes protkines. En plus des muscles T M et LA-BC, la liaison des hormones T et E2 p u t aussi se faire dans d'autres muscles de rats miiles et fernelles, done %egastrocnemien, le pectoralis, Be diaphragme eb le coeur. [Traduit par le journal]
Intra~ductiow hormone receptors Since the discovery of in androgen a nd estrogen target tissues, there has been an uncertainty as to whether the muscles would contain such receptors. In fact many groups of workers failed to detect androgen receptors in muscks. Moreover, in several in vivo studies dealing with known androgen target tissues such as the Prostate and the seminal vesicles, the skeletal muscles were used as a control tissue which supposedly contained no receptors. However, recent studies (1-3) have shown that rrat and guinea-pig Ievator ani muscle cytosol possesses high affinity androgen receptors. TWOrecent reports (4, 5) indicate that similar receptors are also present in the rat thigh muscle. Our own studies have shown that there is a specific binding of Sa-[3H]dihydr~testosterone(DHVa in cock's breast muscles cytossl in virvo (6) while during in vivs experiments, with [aM]testosterone (T), T is the main steroid bound to cytosol proteins (7). Hn 'Abbreviations: DHT, dihydrotestosterone; T. testosterone; E,, estradiol; TM, thigh muscles; LA-BC, levator ani - bulbocavernosus.
the present work, we have studied some of the characteristics of androgen binding proteins in rat levator - bulb~cavernosusmuscle complex ( L A - w ) and in thigh m ~ s c l e(TM). Furthermore7 the Present study rePofls for the first time the Presence of specific binding of [3~]estradiol(E,) in the toso sol of both m ~ s c l etypes.
Materials and Methods Adult male (275-325 g) and female rats (240-275 g) were obtained from Ee Centre de Biomedecine, Qutbec. Qui. Each animal was castrated, under ether anesthesia. 24 h before the experiment, the males through a scrota1 incision and the femdes through small laterd incisions. The various muscle tissues were qraickly dissected out and kept on ice untif homogenization in 0.01 M Tris-HCii buffer pH 7.4 containing 1.5 ITN EDTA. The homogenization was performed with a Polytron homogenizer as previously described (6) with 18 volumes of buffer for LA-BC1 muscles and five volumes of buffer for the other muscles. The homogenates were then centrifuged at 330000 x g for 364 wain in a Sorvall centrifuge. Essentially similar results were obtained when the hasmogenates were centrifuged at BOB000 x g for 1 h. The supernatant (cytosol) was used as the source of binding proteins.
DUB& ET AL.
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Texas A&M University on 11/12/14 For personal use only.
The cytosol was incubated for 2-4 h at 0 "e with [3M]steroid alone or in the presence of 3.4 x 1W6M concentration sf the corresponding unlabeled steroid. [I ,2-3H]Testosterone (4Q Cilmmol), [6,7-WH]estrradiol (49.3 Cilmmol), and 5a-[l.2-3H]dihydrotestosterone (44 Ci/mrnol)were obtained from New England Nuclear Cop. These radioactive steroids were purified by paper chromatography before use. The bound and free steroids were separated by filtration through Sephadex G-25 columns. The column length was 28 em and the diameter 1.2 cm. The fractions corresponding to the macromolecular peak were collected and counted in 10 rnl of standard toluene PPO-POPOP scintillation mixture in a Mark II (Nuclear Chicago) scintillator. Resdts Preliminary experiments showed that specific binding of T , DHT, and E, were present in TM and LA-BC. We observed higher levels of binding in 24-h castrated rats as compared to intact rats. We then decided to use castrated rats for all subsequent experiments. The maximum binding was observed after short incubation times (30 min) at 0 "C and remained constant for at least 8 h. We have also tested three different homogenization media on the binding of T and DHT in TM and LA-BC: Tris-HC1 buffer, pH 7.4' Tris-HC' '." M' pH 7.47 containing EDTA; and sucr0se9 0.25 M' The Tris-HC1-EDTA buffer gave higher and more consistant results than the two other media. Under similar experimental conditions, we could not demonstrate any specific binding of T, DHT, and E, in the blood plasma. In a second series of experiments, we have determined the association constants (K.) of T, DHT, and E, in the cytosol of thigh muscles and levator ani - bulbocavernosus muscle complex- Figure 1 shows a representative experiment of the binding of increasing concentrations of in thigh muscle
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Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Texas A&M University on 11/12/14 For personal use only.
JEAN Y. DUBIS,R E N ~LESAGE, E AND ROLANDR. TWEMBLAY kaboratoire cf'EarAocrinologie-Me'tabolisme, kc Centre Hospitalier de k' &Inhersite'kaval, Quebec, Qut. GI Y 4G2 Received July 14,1975 DubC, J. Y.,Lesage, R. & Tremblay, R. R. (1976) Androgen and estrogen binding in rat skeletal and perineal rnuscBes. Can. 9.Biochem. 54, 0-55 Specific ira v i m binding sf E3H]testosterone (T), Sac-[3HHldihydrotestostepone(DHT), and [3H]estradiol (E2)was demonstrated in the 30 OMI x g supernatant (cytosol) of thigh muscles (TM) and of the levator ani - bulbocavernosus muscle complex (LA-BC) by gel filtration through Sephadex G-25 columns. Bn T M cytosol, T and E2 [are bound with high affinity (BY, = 1.1 x 109M-I and 2.3 x lo9 M - l , respectively) whereas DHT binding is of lower affinity (Ka = 5.0 x 1Q7M-I).] In LA-BC cytssol, T, E2, and D H T are bound with high affinity (Ka 1.9 x lo9 M-', 0.3 x B09M-', and 0.5 x lo9 M - I , respectively). Competition experiments suggest that the binding of the three hormones (T, El,and DHT) is due to different proteins. In addition to TM and LA-BC, T and E2binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm, and heart.
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Dube, J. Y . , Lesage, R. kk Trernblay, R. R. (1976) Androgen and estrogen binding in rat skeletal and perineal muscles. Can. 9.Blochem. 54, 58-54 Nous dimontrons in vitrs la liaison spkcifique de la [3H]testostkrone (T), de la Su-[%IHihydrotestosterone (DHT) et du [3H]oestradiol (E2) dans le sumageant (cytosol) 30 000 x g des muscles fdmoraaax (TM) et du cornplexe rnusculaire levatsr ani - bulbocavernosus (LA-BC) par gel filtration sur colonnes de Sephadex 6-25.Dans le cytosol TM, les hormones T et Ea sont likes avec grande affinite (Ka = 1.1 x lo9 M-' et 2.3 x 10' M - ' , respectivement) tandis que pour Ba liaison de DHT, 19affixlitCest moins [ClevCe (K, = 5.0 x lo7 M - I ) . Dans le cytosol LA-BC, T, El et DHT] ssnt aussi Biees avec grande affinitk (K, = 1.9 x 10' '44-I, 0.3 x lo9 M-I et 0.5 x IQW-', respectivement). Les exxpCriences de compktition swgg&rentque la Iiaison des trois hormones (T, E, et DWT) est due B differentes protkines. En plus des muscles T M et LA-BC, la liaison des hormones T et E2 p u t aussi se faire dans d'autres muscles de rats miiles et fernelles, done %egastrocnemien, le pectoralis, Be diaphragme eb le coeur. [Traduit par le journal]
Intra~ductiow hormone receptors Since the discovery of in androgen a nd estrogen target tissues, there has been an uncertainty as to whether the muscles would contain such receptors. In fact many groups of workers failed to detect androgen receptors in muscks. Moreover, in several in vivo studies dealing with known androgen target tissues such as the Prostate and the seminal vesicles, the skeletal muscles were used as a control tissue which supposedly contained no receptors. However, recent studies (1-3) have shown that rrat and guinea-pig Ievator ani muscle cytosol possesses high affinity androgen receptors. TWOrecent reports (4, 5) indicate that similar receptors are also present in the rat thigh muscle. Our own studies have shown that there is a specific binding of Sa-[3H]dihydr~testosterone(DHVa in cock's breast muscles cytossl in virvo (6) while during in vivs experiments, with [aM]testosterone (T), T is the main steroid bound to cytosol proteins (7). Hn 'Abbreviations: DHT, dihydrotestosterone; T. testosterone; E,, estradiol; TM, thigh muscles; LA-BC, levator ani - bulbocavernosus.
the present work, we have studied some of the characteristics of androgen binding proteins in rat levator - bulb~cavernosusmuscle complex ( L A - w ) and in thigh m ~ s c l e(TM). Furthermore7 the Present study rePofls for the first time the Presence of specific binding of [3~]estradiol(E,) in the toso sol of both m ~ s c l etypes.
Materials and Methods Adult male (275-325 g) and female rats (240-275 g) were obtained from Ee Centre de Biomedecine, Qutbec. Qui. Each animal was castrated, under ether anesthesia. 24 h before the experiment, the males through a scrota1 incision and the femdes through small laterd incisions. The various muscle tissues were qraickly dissected out and kept on ice untif homogenization in 0.01 M Tris-HCii buffer pH 7.4 containing 1.5 ITN EDTA. The homogenization was performed with a Polytron homogenizer as previously described (6) with 18 volumes of buffer for LA-BC1 muscles and five volumes of buffer for the other muscles. The homogenates were then centrifuged at 330000 x g for 364 wain in a Sorvall centrifuge. Essentially similar results were obtained when the hasmogenates were centrifuged at BOB000 x g for 1 h. The supernatant (cytosol) was used as the source of binding proteins.
DUB& ET AL.
Can. J. Biochem. Downloaded from www.nrcresearchpress.com by Texas A&M University on 11/12/14 For personal use only.
The cytosol was incubated for 2-4 h at 0 "e with [3M]steroid alone or in the presence of 3.4 x 1W6M concentration sf the corresponding unlabeled steroid. [I ,2-3H]Testosterone (4Q Cilmmol), [6,7-WH]estrradiol (49.3 Cilmmol), and 5a-[l.2-3H]dihydrotestosterone (44 Ci/mrnol)were obtained from New England Nuclear Cop. These radioactive steroids were purified by paper chromatography before use. The bound and free steroids were separated by filtration through Sephadex G-25 columns. The column length was 28 em and the diameter 1.2 cm. The fractions corresponding to the macromolecular peak were collected and counted in 10 rnl of standard toluene PPO-POPOP scintillation mixture in a Mark II (Nuclear Chicago) scintillator. Resdts Preliminary experiments showed that specific binding of T , DHT, and E, were present in TM and LA-BC. We observed higher levels of binding in 24-h castrated rats as compared to intact rats. We then decided to use castrated rats for all subsequent experiments. The maximum binding was observed after short incubation times (30 min) at 0 "C and remained constant for at least 8 h. We have also tested three different homogenization media on the binding of T and DHT in TM and LA-BC: Tris-HC1 buffer, pH 7.4' Tris-HC' '." M' pH 7.47 containing EDTA; and sucr0se9 0.25 M' The Tris-HC1-EDTA buffer gave higher and more consistant results than the two other media. Under similar experimental conditions, we could not demonstrate any specific binding of T, DHT, and E, in the blood plasma. In a second series of experiments, we have determined the association constants (K.) of T, DHT, and E, in the cytosol of thigh muscles and levator ani - bulbocavernosus muscle complex- Figure 1 shows a representative experiment of the binding of increasing concentrations of in thigh muscle
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