0013-7227/78/1035-1629$02.00/0 Endocrinology Copyright © 1978 by The Endocrine Society
Vol. 103, No. 5 Printed in U.S.A.
Estrogen Receptor in Adult Male Rat Liver* RAYMOND F. ATEN, ROBERT B. DICKSON, AND ARNOLD J. EISENFELD Reproductive Biology Section, Departments of Obstetrics and Gynecology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 ABSTRACT. A 30% ammonium sulfate precipitation has previously been utilized to partially purify the estrogen receptor(s) of female rat liver cytosol. This procedure has now been used to fractionate the estradiolbinding sites of adult male rat liver cytosol. The 30% ammonium sulfate precipitation partially purifies a group of estradiol-binding sites which have properties quite di$tinct from the large number of sites present in male liver cytosol. The partially purified male sites seem to have the same properties as the partially purified female estradiol-binding sites. They seem to be proteins
T
HE LIVER cytosol of adult female mammals contains estradiol-binding sites which have been shown to be estrogen specific and of high affinity (1-6). The properties of these sites are similar to those of the presumed estrogen receptor in uterus and suggest that the liver sites are estrogen receptors (1, 2). A substantial portion of the female rat liver estradiol-binding sites is precipitated by ammonium sulfate at 30% of saturation (7, 8). Evidence obtained in vivo indicates that these estrogen-binding sites, which are partially purified by ammonium sulfate precipitation, are estrogen receptors which can translocate to the nucleus (8). Ethinylestradiol was administered in vivo, and the partially purified estradiol-specific binding sites of liver cytosol and the estradiol-specific binding sites of purified nuclei were measured by exchange assay. A dose-dependent increase in the number of estradiol-specific binding sites of nuclei as well as a concomitant decrease in the number of partially purified cytosolic sites were observed.
that are estrogen specific and have a high estradiol affinity (KH = 1 X 10"'" M) and a low estrogen capacity (2.3 fmol/mg liver). The estradiol-binding sites of prepubescent male rat liver cytosol have also been fractionated by 30% ammonium sulfate precipitation. The redissolved ammonium sulfate precipitates from prepubescent male rat liver cytosol contain fewer estradiolbinding sites then those from adult male or female rats. It seems that adult male as well as adult female rat liver contains estrogen receptors. (Endocrinology 103: 1629, 1978)
Adult male rat liver cytosol also contains estradiol-binding sites. In contrast to the cytosol of the female, male rat liver cytosol contains a very high concentration of sites. The properties of these male rat liver cytosolic sites are different from those of the female and are discussed in the accompanying paper (9). In this paper, the estradiol-binding sites of male rat liver cytosol have been fractionated by 30% ammonium sulfate precipitation, and the properties of the redissolved ammonium sulfate-precipitated estradiol-binding sites have been compared to those of the partially purified female rat liver estradiol-binding sites. Materials and Methods Animals and materials
The rats were obtained from Charles River (CD strain). The intact adult females weighed 200-250 g; the intact males weighed 250-300 g. The immature males were 26 days old when sacrificed. The rats were anesthetized with ether and perfused with 20 ml normal saline through the left ventricle after incising the right atrium. The livers were then Received December 12, 1977. Address all correspondence and requests for reprints quickly excised and retained on ice. to: Raymond F. Aten, Department of Obstetrics and [2,4,6,7-3H]Estradiol (102 Ci/mmol) was obGynecology, Yale University School of Medicine, 333 tained from New England Nuclear. Before each Cedar Street, New Haven, Connecticut 06510. a portion of the tritiated estradiol * This work was supported by NIH Grants HD-8280 experiment, ([3H]E2) stock was evaporated to dryness under and CA-08341. 1629
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Kndo 1978 Vol 103 , No 5
ATEN, DICKSON, AND EISENFELD
nitrogen and redissolved in distilled H2O to give the required concentration. Radioactivity was determined in a Packard 3380 liquid scintillation spectrometer at 50% efficiency. All nonradioactive steroids were of highest purity available from commercial sources. Protease was obtained from Worthington Biochemical Corp. Parachloromercuriphenyl sulfonic acid was obtained from Sigma.
ing silica gel. The elution solvent was 80% chloroform-20% ethylacetate. Unlike liver cytosol, the redissolved ammonium sulfate-precipitated cytosols did not metabolize [3H]E2. Upon completion of the 2 nM or equilibrium-binding incubations, greater than 90% of the toluene-soluble radioactivity remained as unmetabolized [3H]E2.
Cytosol preparation
The concentration of protein in the fractions was determined by the method of Lowry et al. (10). The liver cytosols contained 10-15 mg/ml protein. Five to seven percent of the cytosol protein was recovered in the redissolved 30% ammonium sulfateprecipitated fractions. All of the results have been replicated. The SEMS (triplicate values) are indicated on the figures by brackets. If a bracket is not depicted, the SEM was smaller than could be clearly shown in the figure.
All subsequent steps were performed at 0-4 C unless otherwise stated. The livers were weighed, minced, and homogenized in 6 vol (wt/vol) TEN buffer (0.01 M Tris-HCl, pH 7.4; 0.0015 M disodium EDTA; and 0.001 M NaN3) using conical glass homogenizers. The liver homogenates were centrifuged at 25,000 X g for 10 min. The homogenate supernates were then centrifuged at 105,000 X g for 60 min, and the clear supernates (cytosols) were retained. Ammonium sulfate precipitation A neutralized saturated ammonium sulfate solution was added to the liver cytosols to a final concentration of 30% (vol/vol); the suspensions were stirred for 45 min and then centrifuged at 25,000 X g for 10 min. The supernates were discarded, and the precipitates were redissolved in TEN buffer to a final volume equal to that of the original cytosol. Binding assays and thin layer chromatography Unless otherwise stated, 25-jul portions of the aqueous [3H]E2 solutions were transferred to assay tubes. When competition with nonradioactive steroids was examined, the competitors (dissolved in 5 jul ethanol) were added before the [3H]E2. The binding assays (at least in triplicate) were commenced by the addition of 200 ju,l fractions to the assay tubes. The samples were incubated in ice for 1 h (2 nM [3H]E2-binding incubations) or 20 h (equilibrium [3H]E2-binding incubations), and then the macromolecular bound radioactivity was determined using small polyacrylamide gel filtration columns (Biogel P10; 20,000 exclusion mol wt; 1.1 X 10 cm) maintained in a 4 C cold room. Tris-HCl (0.01 M, pH 7.4) was used for column equilibration and sample elution (1). Upon completion of the assay incubations, the identity of the radioactivity in the incubation mixtures and in the macromolecular bound fractions was determined by thin layer chromatography us-
Protein determination and data analysis
Results Partial purification by ammonium sulfate precipitation The macromolecular binding of tritiated 17/?-estradiol ([3H]E2) by the cytosols of adult female and male rat liver after incubation with 2 nM [3H]E2 is presented in Fig. 1. Male rat liver cytosol binds 7 times as much [3H]E2 as the female cytosol. These [3H]E2-binding conditions are not optimal for measuring the binding of [3H]E2 by adult male cytosol. As described in the accompanying paper, only a small fraction of the binding of [3H]E2 by adult male liver cytosol is measured after a 2 nM [3H]E2 incubation (9). While the binding of [3H]E2 by female liver cytosol is reduced by a 50-fold excess of nonradioactive diethylstilbestrol (DES) or 170-estradiol (E2) but not by 5adihydrotestosterone (DHT), the binding of [3H]E2 by male liver cytosol is not reduced. Unlike female rat liver cytosol, male liver cytosol binds [3H]E2 with an apparent moderate affinity, a high capacity, and a different steroid specificity. This binding component is described in the accompanying paper (9). When female liver cytosol is partially purified by precipitation with 30% ammonium sulfate and the binding of [3H]E2 by the redissolved precipitate is measured, a level of binding of [3H]E2 similar to that present in the
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MALE RAT LIVER ESTROGEN RECEPTOR CYTOSOLS
4.0 Control
3.0 2.0
E H DES (S3 E 2 ^ DHT
1.0
3 0 % ( N H 4 ) 2 S 0 4 FRACTION
1.0 O O
or DHT for 1 h at 0 C, and then the macromolecular bound radioactivity was determined by gel filtration. *, P < 0.05 less than control.
cytosol is observed (Fig. 1). The binding continues to be reduced by DES and E2 but not by DHT. Male liver cytosol has also been precipitated with 30% ammonium sulfate, and the binding of [3H]E2 by the redissolved precipitate has been determined. The redissolved male liver cytosol precipitate binds [3H]E2 at a level similar to that of partially purified female liver cytosol. In addition, the binding of [3H]E2 by the redissolved male liver cytosol precipitate is reduced by DES and E2 to the same extent as that of the partially purified female liver cytosol. When the binding of [3H]E2 by the female and male liver cytosols and their redissolved
1631
precipitates is expressed per milligram of protein, the relative binding levels are unchanged. For female liver cytosol, the binding is 5.5 ± 0.2 fmol/mg protein; for male liver cytosol, it is 38 ± 4 fmol/mg protein. For redissolved precipitated female liver cytosol, binding is 260 ± 10 fmol/mg protein; for redissolved precipitated male liver cytosol, it is 270 ± 10 fmol/mg protein. Comparison of the estradiol- binding properties of partially purified adult female and male liver cytosol In addition to the data presented in Fig. 1, the binding of [3H]E2 by the redissolved 30% ammonium sulfate-precipitated female and male cytosol has been determined in the presence of other nonradioactive steroids. The results are presented in Table 1. The binding of both is reduced by estrone and ethinylestradiol but not by corticosterone or progesterone. In addition, the results in Table 1 indicate that the binding of [3H]E2 by the redissolved female and male precipitates is reduced by proteolytic enzyme digestion and by parachloromercuriphenyl sulfonic acid or heat treatment. TABLE 1. Specificity and sensitivity of the binding of [3H]E2 by redissolved 30% ammonium sulfate-precipitated cytosol of adult female and male liver % Control binding Treatment Female
Male
12 ± 1° 12 ± 1" 115 ± 3 114 ± 3
13 ± 1" 9 ± 1" 110 ± 5 112 ± 7
27 ± 3 "
19 ± 1"
PCMS (5 X 10~ M)
11 ± 1"
9 ± 1"
Protease (0.25 mg/ml)
10 ±2"
8 ± 1"
Steroid competition 10~7 M Estrone 10"7 M Ethinylestradiol 10~5 M Corticosterone 10~r> M Progesterone Heat (50 C for 10 min) 3
The partially purified liver cytosols were incubated with 2 nM [3H]E2 in the absence or presence of the nonradioactive steroids for 1 h at 0 C ("Steroid competition"), with 2 nM [3H]E2 for 1 h at 0 C and then for 10 min at 50 C ("Heat"), with PCMS for 30 min at 0 C and then with 2 nM [nH]E2 for 1 h at 0 C ("PCMS"), or with 2 nM [3H]E2 and protease for 1 h at 25 C ("Protease"). Then the macromolecular bound radioactivity was determined by gel filtration. " P < 0.05 less than control.
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ATEN, DICKSON, AND EISENFELD
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The binding of [3H]E2 at equilibrium by the redissolved female and male liver cytosol precipitates has been determined. The fractions were incubated with various [3H]E2 concentrations, ranging from 0.03-5 nM for 20 h. An 8-h incubation with 2 nM [3H]E2 was sufficient to achieve greater than 95% of the binding measured at equilibrium. The results are presented as a Scatchard plot (Fig. 2). Equilibrium dissociation constants and binding capacities have been calculated from least squares fits of the data. When all data points are included (correlation coefficients >0.92), the equilibrium dissociation constants of the partially purified female and male liver cytosols are L2 X 1O"10 and 1.4 X 10"10 M, respectively. The binding capacities were the same for each (2.3 fmol/mg liver and 26 fmol/mg original cytosol protein). Since the data seem to deviate from linearity at the lower and at the highest concentration, a least squares fit of the data has also been calculated utilizing the data obtained with incubations containing an initial [3H]E2 concentration of 0.2-2 nM. Using these five data points (correlation coefficients >0.93), the calculated equilibrium dissociation constants were the same for female
Kndo Vol 103
1978 No 5
and male (0.8 X 10 10 M) as well as the capacities (2.0 fmol/mg liver or 23 fmol/mg original cytosol protein). The partially purified female and male liver cytosols seem to have similar equilibrium dissociation constants and binding capacities. Estradiol binding of prepubescent male liver The binding of [3H]E2 by prepubescent female rat liver has previously been described (7). The liver cytosol and redissolved 30% ammonium sulfate-precipitated cytosols of immature (25-29 days old) females had a low level of binding which was reduced by DES and E2. The binding of [3H]E2 by 26-day-old male rat liver cytosol and of the redissolved 30% ammonium sulfate precipitate of the cytosol has been examined after incubation with 2 nM [3H]E2 (Fig. 3). Prepubescent male rat liver cytosol and its redissolved ammonium sulfate CYTOSOL
.40
-£-,
J.30
k
£.20
P
.10
|
3
e »
2
i
I I
WI
m
I Control
CD DES
£353 PCMS 2
DHT
E 3 HEAT
30%(NH 4 ) 2 S0 4 FRACTION
1.0
2.0
3.0
BOUND M (x 10'°)
FIG. 2. Scatchard plot of the binding of [3H]E2 by partially purified female and male liver cytosol at equilibrium. Redissolved 30% ammonium sulfate-precipitated female (•, ) and male (O, ) liver cytosols were incubated with nine concentrations of [nH]E2) ranging from 0.03-5 nM for 20 h at 4 C, and then the macromolecular bound radioactivity was determined by gel filtration. The lines are a least squares fit of all the data.
FIG. 3. The binding of [3H]E2 per mg liver or protein of prepubescent (26 days old) male rat liver cytosol and of redissolved 30% ammonium sulfate-precipitated liver cytosol. The fractions were incubated with 2 nM ['1H]E2 in the absence or presence of a 50-fold excess of DES, E2, or DHT for 1 h at 0 C, with 5 x 10~3 M PCMS for 30 min at 0 C and then with 2 HIM [3H]E2 for 1 h at 0 C, or with 2 nM [3H]E2 for 1 h at 0 C, then for 10 min at 50 C. Then the macromolecular bound radioactivity was determined by gel filtration. *, P < 0.05 less than control.
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MALE RAT LIVER ESTROGEN RECEPTOR precipitate contain a low level of binding which is reduced by DES and E 2 but not by DHT. The ammonium sulfate fractionation precipitates 25% of the cytosol [ 3 H]E 2 -binding sites and increases [ 3 H]E 2 binding per mg protein by 8-fold. The binding of [ 3 H]E 2 by the redissolved ammonium sulfate-precipitated cytosol is reduced by parachloromercuriphenyl sulfonic acid (PCMS) but not by heat treatment (Fig. 3). The binding of [ 3 H]E 2 by cytosol is not reduced by either treatment. Discussion The estradiol-binding sites of female and male rat liver cytosol have been fractionated by ammonium sulfate precipitation. The redissolved 30% ammonium sulfate precipitates contain 5-7% of the cytosol protein and have substantially reduced levels of estradiol-metabolizing activity. This metabolic activity is present in unfractionated liver cytosol and has made the examination of the estradiol-binding sites of liver cytosol difficult (2). The partially purified female and male liver cytosols contain tritiated estradiol-binding sites which are estrogen specific and appear to be protein(s) which are heat sensitive and contain sulfhydryl groups. In addition, these sites have an apparent high affinity (Kd = 1 X 10"10 M) and low capacity (2.0-2.3 fmol/mg liver) for tritiated estradiol. These parameters were determined from Scatchard plots including and excluding data points which deviated from linearity at low estrogen concentrations. Such deviations have also been observed in Scatchard plots of uterine estrogen receptors (11). For the uterine estrogen receptor, this deviation was suggested as an indication of cooperativity. The basis for this deviation by the partially purified estradiol-binding sites of liver cytosol is unknown. The properties of these partially purified estradiol-binding sites from female and male liver cytosol are the same as those that have been reported for adult female rat liver cytosol sites (1, 2). It has been proposed that the female cytosol sites are estrogen receptors (7, 8). It would seem that male liver also contains estrogen receptors.
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The estradiol-binding sites of female rat liver cytosol have also been fractionated by 40% ammonium sulfate precipitation (6). In that report, a larger portion (20%) of the cytosol protein was recovered, and the precipitated fraction contained estradiol-metabolizing activity. A substantial portion of the estradiol-binding sites of adult female rat liver cytosol are recovered in the 30% ammonium sulfate fraction. A similar quantity of estradiol-binding sites are recovered in the 30% ammonium sulfate-precipitated male liver cytosol. These recovered sites represent only a small fraction of the sites found in male liver cytosol. There is a large quantity of estradiol-binding sites in adult male liver cytosol which have a moderate estradiol affinity and a steroid specificity quite distinct from the ammonium sulfate-precipitated sites. The male liver cytosol sites are discussed in the accompanying paper (9). Prepubescent male rat liver cytosol also contains estradiol-binding sites and has been fractionated by 30% ammonium sulfate precipitation. The level of sites in the redissolved ammonium sulfate precipitates of prepubescent male rat liver cytosol is a tenth of that found in partially purified adult male liver cytosol. This low level of sites, compared to the adult, is also found in female rats (7). The lack of heat sensitivity suggests that the estradiol-binding sites of prepubescent male rat liver cytosol may be different from those of the adult. In female rats, the difference in levels of liver estrogen-binding sites between prepubescent and adults was shown to be correlated with the ability of ethinylestradiol administration to increase the levels of plasma renin substrate (7). Renin substrate is synthetized in the liver, and results with perfused liver indicate that the increased levels are a direct effect of estrogens on liver (12). This developmental correlation would also seem to be so in male rats as 1) the present results demonstrate that there is also a substantial difference between the levels of estradiol-binding sites in partially purified liver cytosol of prepubescent and adult male rats; 2) it has already been demonstrated that the plasma renin substrate
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ATEN, DICKSON, AND EISENFELD
levels are not substantially elevated after ethinylestradiol administration to prepubescent males (13); and 3) plasma renin substrate levels are elevated after ethinylestradiol administration to adult male rats (14). It would seem that male as well as female rat liver contains estrogen-binding sites, which may be estrogen receptors. These receptors might modulate at least one liver function, the synthesis of renin substrate. A major reason for examining mammalian liver estrogen receptor(s) has been their potential role in modulating plasma protein levels and other aspects of liver function (15-18). This modulation may contribute to the side effects which have been reported for women taking estrogens alone or in oral contraceptives (15, 17, 19-21). It is possible that a liver estrogen receptor may also modulate plasma protein levels and other aspects of liver function in men receiving estrogens; estrogen treatment of men has been observed to change the level of several plasma proteins in a dosedependent fashion (22). This modulation might also contribute to side-effects which have been reported for men receiving estrogens [men receiving 5-mg doses of diethylstilbestrol for prostatic carcinoma had an increased risk of fatal and nonfatal cardiovascular disease (23), and a therapeutic trial of estrogen administration to attempt to diminish recurrence of myocardial infarction in men was discontinued when an increase in thromboembolism was observed (24)]. The presence and function of an estrogen receptor in the liver of men remains to be demonstrated. Acknowledgments The authors thank Ms. Irene Visintin for her invaluable assistance and Ms. Susan Losacco for typing the manuscript.
References 1. Eisenfeld, A. J., R. Aten, M. Weinberger, G. Haselbacker, K. Halpern, and L. Krakoff, Estrogen receptor in the mammalian liver, Science 191: 862, 1976. 2. Eisenfeld, A. J., R. F. Aten, G. K. Haselbacher, and K. Halpern, Specific macromolecular binding of estradiol in the mammalian liver supernate, Biochem Pharmacol 26: 919, 1977. 3. Viladiu, P., C. Delgado, J. Pensky, and 0. H. Pearson,
Kndo • 1978 Vol 103 • No 5
Estrogen binding protein of rat liver, Endocrine Res Commun 2: 273, 1975. 4. Chamness, G. C, M. E. Costlow, and W. L. McGuire, Estrogen receptor in rat liver and its dependence on prolactin, Steroids 26: 363, 1975. 5. Powell-Jones, W., P. Davies, and K. Griffiths, Specific binding of [3H]oestradiol by cytoplasmic protein components of female rat liver, J Endocrinol 69: 167, 1976. 6. Beers, P. C, and W. Rosner, The binding of estrogens in the liver of the rat: demonstration and endocrine influences, J Steroid Biochem 8: 251, 1977. 7. Eisenfeld, A. J., L. R. Krakoff, and R. F. Aten, Developmental correlation of higher levels of estrogen binding by macromolecules in rat liver supernate and of increases in plasma renin substrate levels following estrogen administration, Biochem Pharmacol 26: 923, 1977. 8. Aten, R. F., M. J. Weinberger, and A. J. Eisenfeld, Estrogen receptor in rat liver: translocation to the nucelus in vivo, Endocrinology 102: 433, 1978. 9. Dickson, R. B., R. F. Aten, and A. J. Eisenfeld, An unusual sex steroid binding protein in mature male rat liver cytosol, Endocrinology, 103: 1636, 1978. 10. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin-phenol reagent, J Biol Chem 193: 265, 1951. 11. Ellis, D. J., and H. J. Ringold, The uterine estrogen receptor: a physicochemical study, In McKerns, K. W. (ed.), The Sex Steroids, Appleton-Century-Crofts, New York, 1971, p. 73. 12. Nasjletti, A., and G. M. C. Masson, Studies on angiotensinogen formation in a liver perfusion system, Circ Res (Suppl 2) 30, 31: 187, 1972. 13. Krakoff, L. R., and A. J. Eisenfeld, Hormonal control of plasma renin substrate, Circ Res (Suppl 2) 41: 43, 1977. 14. Menard, J., P. Corvol, A. Foliot, and J. P. Raynaud, Effects of estrogens on renin substrate and uterine weights in rats, Endocrinology 93: 747, 1973. 15. Spellacy, W. N., Metabolic effects of oral contraceptives, Clin Obstet Gynecol 17: 53, 1974. 16. Conrad, J., M. Samama, and Y. Salomon, Antithrombin III and the oestrogen content of combined oestrogen-progestagen contraceptives, Lancet 2: 1148,1972. 17. Laragh, J. H., L. Baer, H. R. Brunner, F. R. Buhler, J. E. Sealey, and E. D. Vaughn, Jr., Renin, angiotensin, and aldosterone system in pathogenesis and management of hypertensive vascular disease, Am J Med 52: 633, 1972. 18. Fletcher, A. P., N. Alkjaersig, and R. Burstein, Effects of contraceptives on vascular system, In Hafez, E. S. E., and T. N. Evans (eds.), Human Reproduction, Harper and Row, New York, 1973, p. 539. 19. Vessey, M. P., Thromboembolism, cancer, and oral contraceptives, Clin Obstet Gynecol 17: 65, 1974. 20. Doar, J. W. H., Metabolic side-effects of oral contraceptives, Clin Endocrinol Metabol 2: 503, 1973. 21. Edmondson, H. A., B. Henderson, and B. Benton, Liver-cell adenomas associated with use of oral contraceptives, N EnglJMed 294: 470, 1976.
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MALE RAT LIVER ESTROGEN RECEPTOR 22. Musa, B. U., U. S. Seal, and R. P. Doe, Elevation of certain plasma proteins in man following estrogen administration: a dose-response relationship, J Clin Endocr(nol Metab 25: 1163, 1965. 23. Blackard, C. E., R. P. Doe, G. T. Mellinger, and D. P. Byar, Incidence of cardiovascular disease and death
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in patients receiving diethylstilbestrol for carcinoma of the prostrate, Cancer 26: 249, 1970. 24. The Coronary Drug Project, Findings leading to discontinuation of the 2.5 mg/day estrogen group, JAMA 226: 652, 1973.
Erratum In the article, "Antimineralocorticoid Action of Progesterone in the Rat: Correlation of the Effect on Electrolyte Excretion and Interaction with Renal Mineralocorticoid Receptors," by G. Wambach and J. R. Higgins, references 26 and 27 should have been given as below (Endocrinology 102: 1686, 1978). 26. Porter, G. A., R. Bogoroch, and I. S. Edelman, On the mechanism of action of aldosterone on sodium transport: the role of RNA synthesis, Proc Natl Acad Sci 52: 1326, 1964. 27. Funder, J. W., J. A. Robinson, D. Feldman, K. N. Wynne, and W. R. Adam, 16/?-Hydroxydehydroepiandrosterone: the dichotomy between renal receptor binding and urinary electrolyte activity, Endocrinology 99: 619, 1976.
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Vol. 103, No. 5 Printed in U.S.A.
Estrogen Receptor in Adult Male Rat Liver* RAYMOND F. ATEN, ROBERT B. DICKSON, AND ARNOLD J. EISENFELD Reproductive Biology Section, Departments of Obstetrics and Gynecology and Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 ABSTRACT. A 30% ammonium sulfate precipitation has previously been utilized to partially purify the estrogen receptor(s) of female rat liver cytosol. This procedure has now been used to fractionate the estradiolbinding sites of adult male rat liver cytosol. The 30% ammonium sulfate precipitation partially purifies a group of estradiol-binding sites which have properties quite di$tinct from the large number of sites present in male liver cytosol. The partially purified male sites seem to have the same properties as the partially purified female estradiol-binding sites. They seem to be proteins
T
HE LIVER cytosol of adult female mammals contains estradiol-binding sites which have been shown to be estrogen specific and of high affinity (1-6). The properties of these sites are similar to those of the presumed estrogen receptor in uterus and suggest that the liver sites are estrogen receptors (1, 2). A substantial portion of the female rat liver estradiol-binding sites is precipitated by ammonium sulfate at 30% of saturation (7, 8). Evidence obtained in vivo indicates that these estrogen-binding sites, which are partially purified by ammonium sulfate precipitation, are estrogen receptors which can translocate to the nucleus (8). Ethinylestradiol was administered in vivo, and the partially purified estradiol-specific binding sites of liver cytosol and the estradiol-specific binding sites of purified nuclei were measured by exchange assay. A dose-dependent increase in the number of estradiol-specific binding sites of nuclei as well as a concomitant decrease in the number of partially purified cytosolic sites were observed.
that are estrogen specific and have a high estradiol affinity (KH = 1 X 10"'" M) and a low estrogen capacity (2.3 fmol/mg liver). The estradiol-binding sites of prepubescent male rat liver cytosol have also been fractionated by 30% ammonium sulfate precipitation. The redissolved ammonium sulfate precipitates from prepubescent male rat liver cytosol contain fewer estradiolbinding sites then those from adult male or female rats. It seems that adult male as well as adult female rat liver contains estrogen receptors. (Endocrinology 103: 1629, 1978)
Adult male rat liver cytosol also contains estradiol-binding sites. In contrast to the cytosol of the female, male rat liver cytosol contains a very high concentration of sites. The properties of these male rat liver cytosolic sites are different from those of the female and are discussed in the accompanying paper (9). In this paper, the estradiol-binding sites of male rat liver cytosol have been fractionated by 30% ammonium sulfate precipitation, and the properties of the redissolved ammonium sulfate-precipitated estradiol-binding sites have been compared to those of the partially purified female rat liver estradiol-binding sites. Materials and Methods Animals and materials
The rats were obtained from Charles River (CD strain). The intact adult females weighed 200-250 g; the intact males weighed 250-300 g. The immature males were 26 days old when sacrificed. The rats were anesthetized with ether and perfused with 20 ml normal saline through the left ventricle after incising the right atrium. The livers were then Received December 12, 1977. Address all correspondence and requests for reprints quickly excised and retained on ice. to: Raymond F. Aten, Department of Obstetrics and [2,4,6,7-3H]Estradiol (102 Ci/mmol) was obGynecology, Yale University School of Medicine, 333 tained from New England Nuclear. Before each Cedar Street, New Haven, Connecticut 06510. a portion of the tritiated estradiol * This work was supported by NIH Grants HD-8280 experiment, ([3H]E2) stock was evaporated to dryness under and CA-08341. 1629
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Kndo 1978 Vol 103 , No 5
ATEN, DICKSON, AND EISENFELD
nitrogen and redissolved in distilled H2O to give the required concentration. Radioactivity was determined in a Packard 3380 liquid scintillation spectrometer at 50% efficiency. All nonradioactive steroids were of highest purity available from commercial sources. Protease was obtained from Worthington Biochemical Corp. Parachloromercuriphenyl sulfonic acid was obtained from Sigma.
ing silica gel. The elution solvent was 80% chloroform-20% ethylacetate. Unlike liver cytosol, the redissolved ammonium sulfate-precipitated cytosols did not metabolize [3H]E2. Upon completion of the 2 nM or equilibrium-binding incubations, greater than 90% of the toluene-soluble radioactivity remained as unmetabolized [3H]E2.
Cytosol preparation
The concentration of protein in the fractions was determined by the method of Lowry et al. (10). The liver cytosols contained 10-15 mg/ml protein. Five to seven percent of the cytosol protein was recovered in the redissolved 30% ammonium sulfateprecipitated fractions. All of the results have been replicated. The SEMS (triplicate values) are indicated on the figures by brackets. If a bracket is not depicted, the SEM was smaller than could be clearly shown in the figure.
All subsequent steps were performed at 0-4 C unless otherwise stated. The livers were weighed, minced, and homogenized in 6 vol (wt/vol) TEN buffer (0.01 M Tris-HCl, pH 7.4; 0.0015 M disodium EDTA; and 0.001 M NaN3) using conical glass homogenizers. The liver homogenates were centrifuged at 25,000 X g for 10 min. The homogenate supernates were then centrifuged at 105,000 X g for 60 min, and the clear supernates (cytosols) were retained. Ammonium sulfate precipitation A neutralized saturated ammonium sulfate solution was added to the liver cytosols to a final concentration of 30% (vol/vol); the suspensions were stirred for 45 min and then centrifuged at 25,000 X g for 10 min. The supernates were discarded, and the precipitates were redissolved in TEN buffer to a final volume equal to that of the original cytosol. Binding assays and thin layer chromatography Unless otherwise stated, 25-jul portions of the aqueous [3H]E2 solutions were transferred to assay tubes. When competition with nonradioactive steroids was examined, the competitors (dissolved in 5 jul ethanol) were added before the [3H]E2. The binding assays (at least in triplicate) were commenced by the addition of 200 ju,l fractions to the assay tubes. The samples were incubated in ice for 1 h (2 nM [3H]E2-binding incubations) or 20 h (equilibrium [3H]E2-binding incubations), and then the macromolecular bound radioactivity was determined using small polyacrylamide gel filtration columns (Biogel P10; 20,000 exclusion mol wt; 1.1 X 10 cm) maintained in a 4 C cold room. Tris-HCl (0.01 M, pH 7.4) was used for column equilibration and sample elution (1). Upon completion of the assay incubations, the identity of the radioactivity in the incubation mixtures and in the macromolecular bound fractions was determined by thin layer chromatography us-
Protein determination and data analysis
Results Partial purification by ammonium sulfate precipitation The macromolecular binding of tritiated 17/?-estradiol ([3H]E2) by the cytosols of adult female and male rat liver after incubation with 2 nM [3H]E2 is presented in Fig. 1. Male rat liver cytosol binds 7 times as much [3H]E2 as the female cytosol. These [3H]E2-binding conditions are not optimal for measuring the binding of [3H]E2 by adult male cytosol. As described in the accompanying paper, only a small fraction of the binding of [3H]E2 by adult male liver cytosol is measured after a 2 nM [3H]E2 incubation (9). While the binding of [3H]E2 by female liver cytosol is reduced by a 50-fold excess of nonradioactive diethylstilbestrol (DES) or 170-estradiol (E2) but not by 5adihydrotestosterone (DHT), the binding of [3H]E2 by male liver cytosol is not reduced. Unlike female rat liver cytosol, male liver cytosol binds [3H]E2 with an apparent moderate affinity, a high capacity, and a different steroid specificity. This binding component is described in the accompanying paper (9). When female liver cytosol is partially purified by precipitation with 30% ammonium sulfate and the binding of [3H]E2 by the redissolved precipitate is measured, a level of binding of [3H]E2 similar to that present in the
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MALE RAT LIVER ESTROGEN RECEPTOR CYTOSOLS
4.0 Control
3.0 2.0
E H DES (S3 E 2 ^ DHT
1.0
3 0 % ( N H 4 ) 2 S 0 4 FRACTION
1.0 O O
or DHT for 1 h at 0 C, and then the macromolecular bound radioactivity was determined by gel filtration. *, P < 0.05 less than control.
cytosol is observed (Fig. 1). The binding continues to be reduced by DES and E2 but not by DHT. Male liver cytosol has also been precipitated with 30% ammonium sulfate, and the binding of [3H]E2 by the redissolved precipitate has been determined. The redissolved male liver cytosol precipitate binds [3H]E2 at a level similar to that of partially purified female liver cytosol. In addition, the binding of [3H]E2 by the redissolved male liver cytosol precipitate is reduced by DES and E2 to the same extent as that of the partially purified female liver cytosol. When the binding of [3H]E2 by the female and male liver cytosols and their redissolved
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precipitates is expressed per milligram of protein, the relative binding levels are unchanged. For female liver cytosol, the binding is 5.5 ± 0.2 fmol/mg protein; for male liver cytosol, it is 38 ± 4 fmol/mg protein. For redissolved precipitated female liver cytosol, binding is 260 ± 10 fmol/mg protein; for redissolved precipitated male liver cytosol, it is 270 ± 10 fmol/mg protein. Comparison of the estradiol- binding properties of partially purified adult female and male liver cytosol In addition to the data presented in Fig. 1, the binding of [3H]E2 by the redissolved 30% ammonium sulfate-precipitated female and male cytosol has been determined in the presence of other nonradioactive steroids. The results are presented in Table 1. The binding of both is reduced by estrone and ethinylestradiol but not by corticosterone or progesterone. In addition, the results in Table 1 indicate that the binding of [3H]E2 by the redissolved female and male precipitates is reduced by proteolytic enzyme digestion and by parachloromercuriphenyl sulfonic acid or heat treatment. TABLE 1. Specificity and sensitivity of the binding of [3H]E2 by redissolved 30% ammonium sulfate-precipitated cytosol of adult female and male liver % Control binding Treatment Female
Male
12 ± 1° 12 ± 1" 115 ± 3 114 ± 3
13 ± 1" 9 ± 1" 110 ± 5 112 ± 7
27 ± 3 "
19 ± 1"
PCMS (5 X 10~ M)
11 ± 1"
9 ± 1"
Protease (0.25 mg/ml)
10 ±2"
8 ± 1"
Steroid competition 10~7 M Estrone 10"7 M Ethinylestradiol 10~5 M Corticosterone 10~r> M Progesterone Heat (50 C for 10 min) 3
The partially purified liver cytosols were incubated with 2 nM [3H]E2 in the absence or presence of the nonradioactive steroids for 1 h at 0 C ("Steroid competition"), with 2 nM [3H]E2 for 1 h at 0 C and then for 10 min at 50 C ("Heat"), with PCMS for 30 min at 0 C and then with 2 nM [nH]E2 for 1 h at 0 C ("PCMS"), or with 2 nM [3H]E2 and protease for 1 h at 25 C ("Protease"). Then the macromolecular bound radioactivity was determined by gel filtration. " P < 0.05 less than control.
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ATEN, DICKSON, AND EISENFELD
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The binding of [3H]E2 at equilibrium by the redissolved female and male liver cytosol precipitates has been determined. The fractions were incubated with various [3H]E2 concentrations, ranging from 0.03-5 nM for 20 h. An 8-h incubation with 2 nM [3H]E2 was sufficient to achieve greater than 95% of the binding measured at equilibrium. The results are presented as a Scatchard plot (Fig. 2). Equilibrium dissociation constants and binding capacities have been calculated from least squares fits of the data. When all data points are included (correlation coefficients >0.92), the equilibrium dissociation constants of the partially purified female and male liver cytosols are L2 X 1O"10 and 1.4 X 10"10 M, respectively. The binding capacities were the same for each (2.3 fmol/mg liver and 26 fmol/mg original cytosol protein). Since the data seem to deviate from linearity at the lower and at the highest concentration, a least squares fit of the data has also been calculated utilizing the data obtained with incubations containing an initial [3H]E2 concentration of 0.2-2 nM. Using these five data points (correlation coefficients >0.93), the calculated equilibrium dissociation constants were the same for female
Kndo Vol 103
1978 No 5
and male (0.8 X 10 10 M) as well as the capacities (2.0 fmol/mg liver or 23 fmol/mg original cytosol protein). The partially purified female and male liver cytosols seem to have similar equilibrium dissociation constants and binding capacities. Estradiol binding of prepubescent male liver The binding of [3H]E2 by prepubescent female rat liver has previously been described (7). The liver cytosol and redissolved 30% ammonium sulfate-precipitated cytosols of immature (25-29 days old) females had a low level of binding which was reduced by DES and E2. The binding of [3H]E2 by 26-day-old male rat liver cytosol and of the redissolved 30% ammonium sulfate precipitate of the cytosol has been examined after incubation with 2 nM [3H]E2 (Fig. 3). Prepubescent male rat liver cytosol and its redissolved ammonium sulfate CYTOSOL
.40
-£-,
J.30
k
£.20
P
.10
|
3
e »
2
i
I I
WI
m
I Control
CD DES
£353 PCMS 2
DHT
E 3 HEAT
30%(NH 4 ) 2 S0 4 FRACTION
1.0
2.0
3.0
BOUND M (x 10'°)
FIG. 2. Scatchard plot of the binding of [3H]E2 by partially purified female and male liver cytosol at equilibrium. Redissolved 30% ammonium sulfate-precipitated female (•, ) and male (O, ) liver cytosols were incubated with nine concentrations of [nH]E2) ranging from 0.03-5 nM for 20 h at 4 C, and then the macromolecular bound radioactivity was determined by gel filtration. The lines are a least squares fit of all the data.
FIG. 3. The binding of [3H]E2 per mg liver or protein of prepubescent (26 days old) male rat liver cytosol and of redissolved 30% ammonium sulfate-precipitated liver cytosol. The fractions were incubated with 2 nM ['1H]E2 in the absence or presence of a 50-fold excess of DES, E2, or DHT for 1 h at 0 C, with 5 x 10~3 M PCMS for 30 min at 0 C and then with 2 HIM [3H]E2 for 1 h at 0 C, or with 2 nM [3H]E2 for 1 h at 0 C, then for 10 min at 50 C. Then the macromolecular bound radioactivity was determined by gel filtration. *, P < 0.05 less than control.
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MALE RAT LIVER ESTROGEN RECEPTOR precipitate contain a low level of binding which is reduced by DES and E 2 but not by DHT. The ammonium sulfate fractionation precipitates 25% of the cytosol [ 3 H]E 2 -binding sites and increases [ 3 H]E 2 binding per mg protein by 8-fold. The binding of [ 3 H]E 2 by the redissolved ammonium sulfate-precipitated cytosol is reduced by parachloromercuriphenyl sulfonic acid (PCMS) but not by heat treatment (Fig. 3). The binding of [ 3 H]E 2 by cytosol is not reduced by either treatment. Discussion The estradiol-binding sites of female and male rat liver cytosol have been fractionated by ammonium sulfate precipitation. The redissolved 30% ammonium sulfate precipitates contain 5-7% of the cytosol protein and have substantially reduced levels of estradiol-metabolizing activity. This metabolic activity is present in unfractionated liver cytosol and has made the examination of the estradiol-binding sites of liver cytosol difficult (2). The partially purified female and male liver cytosols contain tritiated estradiol-binding sites which are estrogen specific and appear to be protein(s) which are heat sensitive and contain sulfhydryl groups. In addition, these sites have an apparent high affinity (Kd = 1 X 10"10 M) and low capacity (2.0-2.3 fmol/mg liver) for tritiated estradiol. These parameters were determined from Scatchard plots including and excluding data points which deviated from linearity at low estrogen concentrations. Such deviations have also been observed in Scatchard plots of uterine estrogen receptors (11). For the uterine estrogen receptor, this deviation was suggested as an indication of cooperativity. The basis for this deviation by the partially purified estradiol-binding sites of liver cytosol is unknown. The properties of these partially purified estradiol-binding sites from female and male liver cytosol are the same as those that have been reported for adult female rat liver cytosol sites (1, 2). It has been proposed that the female cytosol sites are estrogen receptors (7, 8). It would seem that male liver also contains estrogen receptors.
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The estradiol-binding sites of female rat liver cytosol have also been fractionated by 40% ammonium sulfate precipitation (6). In that report, a larger portion (20%) of the cytosol protein was recovered, and the precipitated fraction contained estradiol-metabolizing activity. A substantial portion of the estradiol-binding sites of adult female rat liver cytosol are recovered in the 30% ammonium sulfate fraction. A similar quantity of estradiol-binding sites are recovered in the 30% ammonium sulfate-precipitated male liver cytosol. These recovered sites represent only a small fraction of the sites found in male liver cytosol. There is a large quantity of estradiol-binding sites in adult male liver cytosol which have a moderate estradiol affinity and a steroid specificity quite distinct from the ammonium sulfate-precipitated sites. The male liver cytosol sites are discussed in the accompanying paper (9). Prepubescent male rat liver cytosol also contains estradiol-binding sites and has been fractionated by 30% ammonium sulfate precipitation. The level of sites in the redissolved ammonium sulfate precipitates of prepubescent male rat liver cytosol is a tenth of that found in partially purified adult male liver cytosol. This low level of sites, compared to the adult, is also found in female rats (7). The lack of heat sensitivity suggests that the estradiol-binding sites of prepubescent male rat liver cytosol may be different from those of the adult. In female rats, the difference in levels of liver estrogen-binding sites between prepubescent and adults was shown to be correlated with the ability of ethinylestradiol administration to increase the levels of plasma renin substrate (7). Renin substrate is synthetized in the liver, and results with perfused liver indicate that the increased levels are a direct effect of estrogens on liver (12). This developmental correlation would also seem to be so in male rats as 1) the present results demonstrate that there is also a substantial difference between the levels of estradiol-binding sites in partially purified liver cytosol of prepubescent and adult male rats; 2) it has already been demonstrated that the plasma renin substrate
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1634
ATEN, DICKSON, AND EISENFELD
levels are not substantially elevated after ethinylestradiol administration to prepubescent males (13); and 3) plasma renin substrate levels are elevated after ethinylestradiol administration to adult male rats (14). It would seem that male as well as female rat liver contains estrogen-binding sites, which may be estrogen receptors. These receptors might modulate at least one liver function, the synthesis of renin substrate. A major reason for examining mammalian liver estrogen receptor(s) has been their potential role in modulating plasma protein levels and other aspects of liver function (15-18). This modulation may contribute to the side effects which have been reported for women taking estrogens alone or in oral contraceptives (15, 17, 19-21). It is possible that a liver estrogen receptor may also modulate plasma protein levels and other aspects of liver function in men receiving estrogens; estrogen treatment of men has been observed to change the level of several plasma proteins in a dosedependent fashion (22). This modulation might also contribute to side-effects which have been reported for men receiving estrogens [men receiving 5-mg doses of diethylstilbestrol for prostatic carcinoma had an increased risk of fatal and nonfatal cardiovascular disease (23), and a therapeutic trial of estrogen administration to attempt to diminish recurrence of myocardial infarction in men was discontinued when an increase in thromboembolism was observed (24)]. The presence and function of an estrogen receptor in the liver of men remains to be demonstrated. Acknowledgments The authors thank Ms. Irene Visintin for her invaluable assistance and Ms. Susan Losacco for typing the manuscript.
References 1. Eisenfeld, A. J., R. Aten, M. Weinberger, G. Haselbacker, K. Halpern, and L. Krakoff, Estrogen receptor in the mammalian liver, Science 191: 862, 1976. 2. Eisenfeld, A. J., R. F. Aten, G. K. Haselbacher, and K. Halpern, Specific macromolecular binding of estradiol in the mammalian liver supernate, Biochem Pharmacol 26: 919, 1977. 3. Viladiu, P., C. Delgado, J. Pensky, and 0. H. Pearson,
Kndo • 1978 Vol 103 • No 5
Estrogen binding protein of rat liver, Endocrine Res Commun 2: 273, 1975. 4. Chamness, G. C, M. E. Costlow, and W. L. McGuire, Estrogen receptor in rat liver and its dependence on prolactin, Steroids 26: 363, 1975. 5. Powell-Jones, W., P. Davies, and K. Griffiths, Specific binding of [3H]oestradiol by cytoplasmic protein components of female rat liver, J Endocrinol 69: 167, 1976. 6. Beers, P. C, and W. Rosner, The binding of estrogens in the liver of the rat: demonstration and endocrine influences, J Steroid Biochem 8: 251, 1977. 7. Eisenfeld, A. J., L. R. Krakoff, and R. F. Aten, Developmental correlation of higher levels of estrogen binding by macromolecules in rat liver supernate and of increases in plasma renin substrate levels following estrogen administration, Biochem Pharmacol 26: 923, 1977. 8. Aten, R. F., M. J. Weinberger, and A. J. Eisenfeld, Estrogen receptor in rat liver: translocation to the nucelus in vivo, Endocrinology 102: 433, 1978. 9. Dickson, R. B., R. F. Aten, and A. J. Eisenfeld, An unusual sex steroid binding protein in mature male rat liver cytosol, Endocrinology, 103: 1636, 1978. 10. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, Protein measurement with the Folin-phenol reagent, J Biol Chem 193: 265, 1951. 11. Ellis, D. J., and H. J. Ringold, The uterine estrogen receptor: a physicochemical study, In McKerns, K. W. (ed.), The Sex Steroids, Appleton-Century-Crofts, New York, 1971, p. 73. 12. Nasjletti, A., and G. M. C. Masson, Studies on angiotensinogen formation in a liver perfusion system, Circ Res (Suppl 2) 30, 31: 187, 1972. 13. Krakoff, L. R., and A. J. Eisenfeld, Hormonal control of plasma renin substrate, Circ Res (Suppl 2) 41: 43, 1977. 14. Menard, J., P. Corvol, A. Foliot, and J. P. Raynaud, Effects of estrogens on renin substrate and uterine weights in rats, Endocrinology 93: 747, 1973. 15. Spellacy, W. N., Metabolic effects of oral contraceptives, Clin Obstet Gynecol 17: 53, 1974. 16. Conrad, J., M. Samama, and Y. Salomon, Antithrombin III and the oestrogen content of combined oestrogen-progestagen contraceptives, Lancet 2: 1148,1972. 17. Laragh, J. H., L. Baer, H. R. Brunner, F. R. Buhler, J. E. Sealey, and E. D. Vaughn, Jr., Renin, angiotensin, and aldosterone system in pathogenesis and management of hypertensive vascular disease, Am J Med 52: 633, 1972. 18. Fletcher, A. P., N. Alkjaersig, and R. Burstein, Effects of contraceptives on vascular system, In Hafez, E. S. E., and T. N. Evans (eds.), Human Reproduction, Harper and Row, New York, 1973, p. 539. 19. Vessey, M. P., Thromboembolism, cancer, and oral contraceptives, Clin Obstet Gynecol 17: 65, 1974. 20. Doar, J. W. H., Metabolic side-effects of oral contraceptives, Clin Endocrinol Metabol 2: 503, 1973. 21. Edmondson, H. A., B. Henderson, and B. Benton, Liver-cell adenomas associated with use of oral contraceptives, N EnglJMed 294: 470, 1976.
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MALE RAT LIVER ESTROGEN RECEPTOR 22. Musa, B. U., U. S. Seal, and R. P. Doe, Elevation of certain plasma proteins in man following estrogen administration: a dose-response relationship, J Clin Endocr(nol Metab 25: 1163, 1965. 23. Blackard, C. E., R. P. Doe, G. T. Mellinger, and D. P. Byar, Incidence of cardiovascular disease and death
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in patients receiving diethylstilbestrol for carcinoma of the prostrate, Cancer 26: 249, 1970. 24. The Coronary Drug Project, Findings leading to discontinuation of the 2.5 mg/day estrogen group, JAMA 226: 652, 1973.
Erratum In the article, "Antimineralocorticoid Action of Progesterone in the Rat: Correlation of the Effect on Electrolyte Excretion and Interaction with Renal Mineralocorticoid Receptors," by G. Wambach and J. R. Higgins, references 26 and 27 should have been given as below (Endocrinology 102: 1686, 1978). 26. Porter, G. A., R. Bogoroch, and I. S. Edelman, On the mechanism of action of aldosterone on sodium transport: the role of RNA synthesis, Proc Natl Acad Sci 52: 1326, 1964. 27. Funder, J. W., J. A. Robinson, D. Feldman, K. N. Wynne, and W. R. Adam, 16/?-Hydroxydehydroepiandrosterone: the dichotomy between renal receptor binding and urinary electrolyte activity, Endocrinology 99: 619, 1976.
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