Binding of 2-Hydroxyestradiol to Rat Anterior Membranes* Pituitary Cell

THEJOURNALOF BIOLOGICAL CHEMISTRY Vol. 255,No. 20, Issue of October 25. pp. 983&9&w, 1980 Printed in U S A .

Binding of 2-Hydroxyestradiol to Rat Anterior Pituitary Cell Membranes* (Received for publication, April 7,1980)

James M. Schaefferlfj, Sandy Stevensl, Roy G. Smithll, and Aaron J. W. Hsueh$ From the$Department of Reproductive Medicine, ”025, University of California, San Diego, La Jolla, California 92093, the !Department of Obstetrics a n d Gynecology, Baylor College of Medicine, Houston, Texas 77025, and thellDepartment of Biochemistry, University of Texas Schoolof Medicine, Houston, Texas 77025

We have studied the specific binding of 1,3,6(10)-estratrien-2,3,17j”ol (2-hydroxyestradiol) toan enriched membrane fraction isolated from the rat anterior pituitary gland. Specific [6,7-3H]2-hydroxyestradiol-17B ([SH12-hydroxyestradiol)binding is saturable and displays a high and low affinity binding component. The apparent dissociation constants, KO,are 4 f 2 X 10”’ and 2 X lo-‘ M with 13 fmol and 2.6 pmol bound/mg of protein, respectively. The specific high affinity binding increases linearly with increasing amounts of tissue protein. The binding is both temperature-and pH-dependent, with maximal binding at 37°C and pH 7.4. The 2-hydroxyestradiol binding is shown to be stereospecific. Dopamineand spiroperidol (a dopamine antagonist) competitively inhibit the specific binding of [3€€12-hydroxyestradiolto thehigh a n i t y binding site of 1X lo-’ M and 2 X lo-‘ with inhibition constants, KI, M, respectively. Related catecholestrogens (2-hydroxyestrone and 2-hydroxyestriol) are also competitive inhibitors of [3H]2-hydroxyestradiol binding with KI values of 1.5 X lo-’ M and 1.9 X lo-’ M, respectively. None of the estrogens (estrone, estradiol, estriol) or 2methoxyestrogen derivatives (2-methoxyestrone, 2methoxyestradiol, 2-methoxyestriol) inhibits [3H]2-hydroxyestradiol binding at concentrations upto M. Norepinephrine, epinephrine, and serotonin are also ineffective as inhibitors at concentrations of lo-’ M and M. inhibited less than 20% at Centrifugation through a stepwise discontinuous sucrose density gradient is used to separate subcellular components of the anterior pituitary cells. Approximately 90 per cent of the specific [3H]2-hydroxyestradiol binding is associated with the material at the 47.4 to 52.9% sucrose interface, the layer most highly enriched for 6’”nucleotidase (an enzyme marker for plasma membranes). Studies of tissue specificity indicate that specific 2-hydroxyestradiol binding sites are heterogeneously distributed in nervous tissue with the highest concentration of binding sites in the anterior pituitary, cerebralcortex, and hypothalamus and lower levels found in the thalamus and striatum.Low levels of 2-hydroxyestradiol binding sites are also identified in liver and uterus. The present demonstration of specific 2-hydroxyestradiol binding to the anterior pituitary membrane provides information on the mechanism of catecholestrogen action. * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 5 To whom reprint requests are tobe sent.

Catecholestrogens are 2-hydroxylated estrogen derivatives endogenously present in various target tissues including the central nervous system and anterior pituitary gland (1-3). These compounds are of interest because of their unique structural resemblance to both estrogens and biologically active catecholamines. The conversion of estrogens to catecholestrogens is enzymatically mediated by a specific estrogen 2hydroxylaseenzyme present inhigh concentration in the anterior pituitary and othertissues (4-6).Although the physiological role of catecholestrogens is unclear, it has been demonstrated that catecholestrogens have both estrogenic and dopaminergic properties. Catecholestrogens have been shown to bind to uterine estrogen receptors (7,8) and initiate uterotrophic responses similar to those elicited by estrogens (7).In the cultured anterior pituitarycells, 2-hydroxyestradiol’ was shown to directly sensitize the pituitary cells to gonadotropin-releasing hormone similar to thatinduced by estradiol (9). In addition, catecholestrogens are competitive inhibitors of the dopamine-metabolizing enzymes, catechol-0-methyltransferase (10, 11) and tyrosine hydroxylase (12), suggesting their role in regulating catecholamine metabolism. Recently, we reported that 2-hydroxyestradiolis a competitive inhibitor of the dopamine receptor in the rat anterior pituitary membrane suggesting an interaction of 2-hydroxyestradiol with a membrane receptor (13). Although steroid hormones are thought to initiate nuclear events in target cells by interaction with intracellular receptors (14, E),there are several lines of evidence suggestingthe existence of steroid hormone receptors in the cell membrane (16-23). Estradiol interaction with uterine and liver plasma membrane binding sites has been reported (16, 17), although the possibility exists that some of these receptor sites are due to cytoplasmic contamination of the membrane preparation (24).

In this paper, we report the synthesis of [3H]2-hydroxyestradiol and have employed this labeled ligand to study the possible presence of cell membrane receptors for catecholestrogens. We demonstrate, for the first time, the presence of high affinity, stereospecific 2-hydroxyestradiol binding sites associated with rat anterior pituitary membranes. MATERIALSANDMETHODS

Chemicals-Steroids were purchased from Research Plus Steroid Labs (Wilton, NH). In some cases, the steroids were further purified by thin layer chromatography on silica gel plates in a solvent system of chloroform, cyclohexane, and acetic acid (22:l). Spiroperidol was a gift from Dr. Ian Creese (San Diego, CA). Potassium nitrodisdfonate (Fremy’s salt) was purchased from Ventron COT. (Danvem

’ The abbreviations used are: 2-Hydroxyestradiol, 1,3,5(10)-estratrien-2,3-17P-triol; [3H]2-hydroxyestradiol, [6,7-’H]2-hydroxyestradiol-17/?.

9838

2-Hydroxyestradiol Binding

to Anterior Pituitary

MA). AU other chemicals were obtained from Sigma co. (st. Louis, MO). [3H]Spiroperidol (24 Ci/mmol) was purchased from New England Nuclear (Boston, MA). [6,7-3H]Estradiol (52 Ci/mmol) was bought from Amersham Searle (Desplaines, IL). Animals-Random cycling female rats (175 to 200 g, body weight) purchased from Simonson Laboratories (Escondido, CA) were used in all experiments. Preparation of [6, 7-3H]2-Hydroxyestradwl-l 7p (fH]2-Hydroxyestradiol)-The [3H]2-hydroxyestradiolwas synthesized by a modification of the method of Gelbke et al. (25). One hundred microliters of [6,7-3H]estradiol-17Pwas evaporated in a 5-ml vial under a stream of nitrogen. The estradiol was redissolved in 500 pl of acetone and 800 p1 of 10%acetic acid in water (w/v). Following the addition of 5 mg of potassium nitrosodisulfonate, the vial was capped and the mixture was stirred rapidly for 20 min at room temperature. An additional 5 mg of potassium nitrosodisulfonate was added and stirring was continued for 30 min. The quinone products (a mixture of 2,3- and 3,4quinones) were extracted with 3- X 1-ml portions of chloroform. The combined chloroform extracts were washed with 1 N HCI (2 X 1 m l ) and saturated NaCl in water (2 X 1 ml). The quinone solution was then transferred to a clean 5-ml microvial using a small quantity of chloroform, and, after the addition of 100 pl of glacial acetic acid, the quinones were reduced to catechols by rapid stirring with 3 mg of solid potassium iodide. After stirring 2min, 100 plof 5% sodium bisulfite was added to remove iodine formed during the reaction. One milliliter of distilled water was then added, and the chloroform layer was removed using a Pasteur pipette. The aqueous phase was extracted twice with 1 ml of dichloromethane: ethyl acetate (LI, v/v), and the combined organic extracts were washed with 1 N HC1 (2 X 1 m l ) and saturated NaCl solution (2 X 1 ml). One milliliter of methanol containing 40 pg of ascorbic acid and 10 p1 of glacial acetic acid were added to theproduct solution to retard oxidation. The solvent was then evaporated in vacuo. Residual water was removed by the addition of 1 ml of benzene:ethanol (l:l, v/v) followed by evaporation in vacuo. [3H]2-Hydroxyestradiol was separated from [3H]4-hydroxyestradiol and [3H]estradiol by paper chromatography using the procedure described by Gelbke et al.(25) (Fig. 1). A strip of Whatman No. 3MM paper (5 X 57 cm) was saturated with a freshly prepared solution of formamide:methanol (l:l, v/v) containing 1.9% (w/v) ascorbic acid. The paper was blotted and allowed to air dry for 1 h. The product mixture was dissolved in 500 pl of methanol and a 225-4 aliquot was applied to the paper and dried under a stream of nitrogen. Elution was carried out with chlorobenzene:ethyl acetate (3:1, v/v) saturated with formamide and ascorbic acid. The paper was allowed to air dry overnight and then the spots were identified using a Packard model 7201 radiochromatogram scanner. Baseline separations were obtained with R,values of 0.21 for 2-hydroxyestradiol, 0.33 for 4-hydroxyestradiol, and 0.71 for estradiol (Fig. 1). These R,values were identical to those obtained using unlabeled estradiol, 4-hydroxyestradiol, and 2hydroxyestradiol (Fig. 1). Of the total radioactivity applied to the paper, 24% was associated with 2-hydroxyestradiol, 10% with 4-hydroxyestradiol, and 32% with unreacted estradiol. A second preparation differing only in the length of treatment with potassium nitrosodisulfonate (15 min) resulted in associated radioactivity of 22% for 2-hydroxyestradiol, 13%for 4-hydroxyestradiol, and 38% for unreacted estradiol. The remainder of the radioactivity was associated with the origin, the solvent front, and a minor peak near the origin. 2-Hydroxyestradiol was recovered from the paper by the method of Gelbke and Knuppen (26). 2-Hydroxyestradiol was stable on the paper at room temperature for at least 1 week, although the paper slowly turned pink due to ascorbic acid oxidation. The pink color was not present in the purified product. Proof of identity was obtained by thin layer chromatography using a solvent system of chloroform: cyc1ohexane:acetic acid (2:2:1), and greater than 99% of the radioactivity co-migrated with the authentic 2-hydroxyestradiol (R,= 0.46). Purity of the labeled material was verified by thin layer chromatography every 2 weeks and no degradation was noted after 10 weeks. Preparation of the Rat Anterior Pituitary Membranes-The animals were decapitated and the anterior pituitaries were quickly removed, homogenized in Krebs’ bicarbonate buffer (124 mM NaCl, 5 mM KCl, 26 IIlM NaHC03, 1.2 mM KH~POI,1.3 nm MgS04, and 0.75 mM CaCld, pH 7.4, at 4°C (100 mg of tissue/lO ml of buffer) with a glass-glass Kontes homogenizer, and centrifuged 10 min a t 1,OOO x g. The supernatant was then centrifuged 20 min at 28,000 x g. The resulting pellet was resuspended by homogenization in Krebs’ bicarbonate buffer (approximately 0.5 to 1.0 mg of protein/ml) with 0.1% ascorbate, aliquoted into I-ml fractions, and used immediately. Some

Membranes

9839

of the membrane preparations were frozen at -20°C and used at a later time with no apparent loss of activity (up to 2 months) if ascorbate was added after thawing the membrane preparation. Binding Assays-The membrane preparations were incubated with [3H]2-hydroxyestradiol a t 37°C for 45 min in the presence (nonspecific binding) or absence (total binding) of a 100-fold molar excess of unlabeled 2-hydroxyestradiol. Specific binding was calculated by subtracting nonspecific from total binding. The incubation was terminated by rapid filtration over Whatman GF/B filters and rinsed with 15 ml (3 X 5 m l ) of ice-cold Krebs’ bicarbonate buffer. The filters were placed into glass vials containing 5 ml of toluenebased scintillation fluid containing 5% Concifluor (Beckman, Fullerton, CA). The radioactivity on the filter was determined by liquid scintillation spectrometry at 53% efficiency. Purification of Plasma Membranes-Purified plasma membranes were prepared by a modification of the procedure described by Neville (27). Fifteen anterior pituitaries were homogenized in 3 ml of 1 mM NaHC03, 5mM P-mercaptoethanol buffer using a glass-glass homogenizer and centrifuged at 2,000 X g for 30 min. The pellet was resuspended in 4 ml of buffer and centrifuged 20 min at 1,220 X g. The supernatant was saved and thepellet was resuspended in 4 ml of buffer and centrifuged a t 2,000 X g for 20 min. The supernatants were pooled and centrifuged 30 min a t 30,000 X g; the resulting pellet was then resuspended in sucrose to a final density of 1.18 (47.4% sucrose) and layered onto a discontinuous sucrose gradient. The gradient (35 m l ) was composed of equal volume layers of sucrose with densities of 1.14 (36.8% sucrose), 1.16 (42.1% sucrose), 1.18 (47.4% sucrose), 1.20 (52.9% sucrose), and 1.22(53.8% sucrose). The gradient was centrifuged at 115,000 X g for 2 h in a Beckman SW 27 rotor. The material sedimenting at the various interfaces was removed with a Pasteur pipette, diluted 5-fold in Krebs’ buffer, and centrifuged 20 min at 28,000 X g. Thepellet was resuspended in 2.5 ml of Krebs’ buffer and used in [3H]2-hydroxyestradiol binding studies or organelle marker enzyme assays. Enzyme Assays-5’-Nucleotidase (plasma membrane marker) and glucose-6-phosphatase (microsome marker) activities were measured according to the method of Aronson and Touster (28). Glucose-6phosphate dehydrogenase (cell cytoplasm marker) activity was determined by the method of Cohen and Rosemeyer (29). Protein concentrations were measured by the method of Lowry et al. (30) using bovine serum albumin as thestandard. RESULTS

Optimal Conditions f0r[~H]2-Hydroxyestradiol BindingSpecific [3H]2-hydroxyestradiolbinding increased linearly as a function of tissue protein concentration between 0.1 and 2.4 m g / d of protein (Fig. 2). The optimal temperature for [’H]2hydroxyestradiol binding was between 32 and 37°C. At 22°C there was a 30% decrease in specific [3H]2-hydroxyestradiol binding, and at 4 and 60°C there was greater than 90% decrease in specific binding (Fig. 3A). At 37”C, the optimal pH for [3H]2-hydroxyestradiolbinding was7.4, although there is a very broad range of maximal bindingbetween pH 6.5 and pH 8.0 (Fig. 3B). Metabolism of [3H]2-hydroxyestradiolduring the 45-min incubation period was assessedby ethanol extraction of the steroid followed by thin layer chromatography (chloroform, cyclohexane, acetic acid, 2:2:1) as described under “Materials and Methods.” No detectable metabolism of [’H]2-hydroxyestradiol was observed. Equilibrium Binding Parameters-Anterior pituitary membrane fractions were prepared as described under “Materials and Methods,” and 0.9-ml aliquots (approximately 0.7 mgof protein) were incubated 45 min at 37°C with 0.6 nM [’H]2-hydroxyestradiol in the presence or absence of increasing concentrations of unlabeled 2-hydroxyestradiol (Fig. 4). [’H]2-Hydroxyestradiol binding to membrane fractions was inhibited by increasing concentrations of unlabeled 2-hydroxyestradiol in a biphasic manner, suggesting the presence of two binding sites. Binding parameters of the low affinity binding site were further examined by incubating aliquots of the membrane preparation with increasing concentrations of [’H]2-hydroxyestradiol (0.1 to 10 x M) in the presence or


9840


absence of a 100-fold molar excess of unlabeled 2-hydroxyestradiol to determine specific 2-hydroxyestradiol binding as described under “Materials and Methods” (Fig. 5A). The binding was saturable with a KO of 2 X M and maximum capacity of 2.6 pmol bound/mg of protein. Because of the relatively low specific activity of the labeled ligand and the low endogenous concentration of 2-hydroxyestradiol in the M),(2,3) we concentrated on characterization pituitary

1-

Front

Membranes

of the high affinity binding sites using equilibrium binding studies. To examine the high affinity 2-hydroxyestradiol binding site, anterior pituitary membranes were incubated for 45 min at 37°C in the presence of increasing concentrations of [’H]2hydroxyestradiol (0.025 to 2 nM) with (nonspecific binding) or without (total binding) a 100-foldmolar excess of unlabeled 2hydroxyestradiol. Specific binding of [3H]2-hydroxyestradiol to membrane fractions was saturable with increasing concentrations of [3H]2-hydroxyestradiol(Fig. 5B). The dissociation constant, KO, was calculated to be 4 2 X 10”’ M with 13 fmol bound/mg of protein. Nonspecific bindingincreased linearly and represented between 15 and 40% of the total [3H]2hydroxyestradiol binding. Binding Kinetics-The rate constant of association, k l , for [3H]2-hydroxyestradiol binding to anterior pituitary membranes was determined by incubating the membranes with 2.0 nM [3H]2-hydroxyestradiol at 37°C for varying lengths of time. [3H]2-Hydroxyestradiolbinding was half-maximal at 12 min and plateaued by 45 min (Fig.6A).The pseudo-first order rate

*

3 FIG. 1. Isolation of synthesized[sH12-hydroxyestradiol. After the conversion of estradiol to itsdihydroxylated derivatives, the methanol extract (225 pl) containing [3H]2-hydroxyestradiol, r3H]4hydroxyestradiol, and unreacted [3H]estradiol was applied to a strip of formamide-impregnated Whatman No. 3MM paper, dried under nitrogen, and chromatographed using a solvent system of chlorobenzene:ethyl acetate (3:1, v/v) as described under “Materialsand Methods.’’ The distribution of radioactivity was determined using a Packard model 7201 radiochromatogram scanner. Authentic samples of 2hydroxyestradiol, 4-hydroxyestradiol, and estradiol were used as standards.

Protein, mg

FIG. 2. Specific [SH]2-hydroxyestradiol binding as a function of tissue concentration. Various amounts of anteriorpituitary membrane preparations were incubated in the presence of 2 nM [3H]2-hydroxyestradiol.The amount of [3H]2-hydroxyestradiol specifically bound to themembranes was determined as described under “Materials and Methods.” Each point is the average of three determinations from the same membrane preparation. The standard error of the mean is less than 15%.

Temperature, “C

FIG. 3 (left and center). Specific [3H]2-hydroxyestradiol binding as a function of temperature and pH. A, anterior pituitary membranes (-1.0 mg of protein) were incubated a t various temperatures for 45 min with 2 nM [3H]2-hydroxyestradiol,and theamount of specific [3H]2-hydroxyestradiolbinding was determined as described under “Materials andMethods.” The amount of [3H]2-hydroxyestradiol bound is expressed as the per cent of the maximal specific binding, which is 13 fmol/mg of protein a t 32 and 37°C. B, anterior pituitary membranes were incubated in 50 mM 4-(2-hydroxethyl)-lpiperazineethanesulfonicacid (Hepes) buffer a t various pH values for 45 min at 37°C with 2 nM [3H]2-hydroxyestradiol,and the amount of specific binding was determined. The amount of [3H]2-hydroxyestradiol bound is expressed as theper cent of the maximal specific binding at pH 7.4 which is 13 fmol/mg. Each point is the average of three

determinations from the same membrane preparation. The standard error of the mean for each point is less than 10%. FIG. 4 (right). Displacement of [3H]2-hydroxyestradiol binding torat anterior pituitary membranes by 2-hydroxyestradiol (2-OH-E$). The membranes were incubated with 0.6 nM r3H]2-hydroxyestradiol in the presence or absence of increasing concentrations of 2-hydroxyestradiol. The amount of [”H]2-hydroxyestradiolbinding in the presence of M 2-hydroxyestradiol represented nonspecific binding and was subtracted from all tubes. The amount of binding is expressed as a per cent of the [3H]2-hydroxyestradiol bound in the absence of unlabeled 2-hydroxyestradiol. Eachpoint is the average of three determinations. This experiment was repeated three times with consistently similar results.

'i

2-Hydroxyestradiol Binding Anterior to Pituitary Membranes

I5t

9841

B

H 4

F

f

N

Tlms, Mlnutso

T l m r . Y,"",..

FIG. 5 (left). Equilibrium binding of [3H]2-hydroxyestradiol to rat anterior pituitary membranes. Increasing concentrations of ["H]2-hydroxyestradiol (2-OH-E2)were incubated with anterior pituitary membranes in the presence (nonspecificbinding) or absence (total binding) of a 100-fold molar excess of 2-hydroxyestradiol. Specific binding was determined by subtracting nonspecific from total binding. A, the low affinity binding site was examined using 0.1 to 10.0 X M [3H]2-hydroxyestradiol;B, the high affinity binding was examined using 0.025 to 2.0 nM [3H]2-hydroxyestradiol.Eachpoint is the average of three determinations from the same membrane preparation. The standard error of the mean was less than 10%.Scatchard analysis (inset) of the saturation binding data is shown. FIG. 6 (right). Rate of association of specific C3H]2-hydroxyestradiol binding torat anterior pituitary membranes. Specific binding was measured as a function of time by incubating anterior

pituitary membranes with 2 nM ['H]2-hydroxyestradiol at 37°C as described in the text. A , the rate constant of association, k l , was derived by dividing the initial slope of the binding of r3H]2-hydroxyestradiol to the membranes by the concentration of [3H]2-hydroxyestradiol and the concentration of binding sites. Each point represents the average of four determinations. The standard error of the mean was less than 15%. B, the rate of dissociation was determined by adding a 500-fold molar excess of unlabeled 2-hydroxyestradiol at 60 min after the initiation of the incubation and measuring the displacement of [3H]2-hydroxyestradiol from the membranes as described in the text. A logarithmic analysis of data is shown in the inset; the y axis represents In [(binding a t time t)/(binding at time zero)]. The negative slope represents the dissociation rate constant, k-1.

constant of association is 0.055 nM-' min-'. The rate constant of dissociation of the [3H]2-hydroxyestradiolreceptor complex was determined by incubating the membranes for 60 min at 37°C in the presence of 2.0 nM [3H]2-hydroxyestradiol. UnlaM) was then added to the beled 2-hydroxyestradiol incubation media and the rateof decline of specifically bound [3H]2-hydroxyestradiol was measured as a function of time (Fig. 6B). Thehalf-life of the [3H]2-hydroxyestradiol receptor of dissociation, k - l , complex was 1.8 min and the rate constant was 0.06 min". The dissociation constant k - I / k + l ,was calculated to be 11 X 10"' M in close agreement with the value of 4 k 2 X 10"' M obtained from the equilibrium data. Stereospecificity of fHJ2-Hydroxyestradiol Binding-To assess the specificity of [3H]2-hydroxyestradiol binding to anterior pituitarymembranes, the ability of various compounds to displace [3H]2-hydroxyestradiol binding was examined. Because of the known estrogenic and dopaminergic properties of the catecholestrogens, various estrogens and catecholaminergic compounds were tested. As shown in Table I, the most potent inhibitors of [3H]2-hydroxyestradiolbinding were 2-hydroxyestradiol > dopamine > 2-hydroxyestrone > 2-hydroxyestriol > spiroperidol. The estrogens (estrone, estradiol, and estriol) and 2-methoxyestrogens (2-methoxyestrone, 2-methoxyestradiol, and 2-methoxyestriol) were ineffective at M. Other catecholamines (epinephrine and norepinephM and rine)and serotonin had no inhibitory effect at M. The inhibitionconstants, Kr, inhibit less than 20%at for spiroperidol, dopamine, 2-hydroxyestrone, and 2-hydrox1X 1.5 X and 1.9 X lop5M, yestriol were 2 X respectively. The phenothiazines (chlorpromazine, promethazine, and fluphenazine) and bromoergocriptine had a slight inhibitory effect (less than 20% inhibition of ['H]B-hydroxyesM; data notshown). tradiol binding at Incubation of anterior pituitary membranes withincreasing M to 2.0 concentrations of [3H]2-hydroxyestradiol (0.05 X X M) in the presence or absence of 1.0 X lo-" M inhibitor (dopamine, spiroperidol, 2-hydroxyestrone, or P-hydroxyestriol) was performed (Fig. 7, A and B). The double reciprocal plots of this data demonstrate that the inhibition of r3H]2hydroxyestradiol binding by these inhibitors is of a competitive nature (Fig. 7, A and B). Tissue Specificity of fH]2-HydroxyestradioI Binding-

TABLE I The effect of estrogens, estrogen derivatives, and catecholaminerelated compounds on the specific binding of L3H]2hydroxyestradiol to anterior pituitarymembranes Anterior pituitary membranes were incubated with 0.6 X IO-' M [JH]2-hydroxyestradiol in the absence or presence of varying concentrations of the different inhibitors. KI was calculated using the formula: KI = ZCm/(I+ C/KO)where the ZC, is the concentration of the compound required to inhibit 50% ofthe specific binding (determined by log probit plots), cis the concentration of [3H]2-hydroxyestradiol, and KD is the dissociation constant (31). Inhibitor KI M

2-Hydroxyestrone 2-Hydroxyestriol Estrone Estradiol Estriol 2-Methoxyestrone 2-Methoxyestradiol 2-Methoxyestriol Dopamine Norepinephrine Spiroperidol Serotonin

1.5 X 1 0 - ~ 1.9 X 1 0 - ~

>lo-'

1.0 x

>lo-'

2.0 x

TABLE I1 Regional distribution of [3H]2-hydroxyestradwl binding sites Membranes from various tissues were obtained using the techniques described for preparation of anterior pituitary membranes. Specific binding was determined by incubation of the membranes with 2.0 nM [3H]2-hydroxyestradiol as described under "Materials and Methods." Region

2-Hydroxyestradiol bound fmoNmg

Pituitary Hypothalamus Cerebral cortex Striatum Cerebellum Thalamus Brain stem Uterus Liver Retina

13 & 2.1 9 14

5 7 3 4

2 5 2

9842

2-Hydroxyestradiol Bindingto Anterior Pituitary Membranes 1

1

1

8

/

n

I

l

l

,

,

1~2~U~mxvestradiol],nM

FIG. 7. Lineweaver-Burke analysis of the inhibition of specific binding of [3HJ2-hydroxyestradiol. A, in the presence of dopamine (H,M), spiroperidol (A-A, M), or no inhibitor (M control). , Specific binding was determined by incubating the anterior pituitary membranesin the presence of C3H]2hydroxyestradiol (0.2 to 2.0 X 10"' M) as described in the text. The lines were plotted using linear regression analysis. Eachpoint represents the mean of three determinations: standard error of the mean

- values were less than 10%.The inhibition constants were calculated

using the formula: y intercept = -'/KO (1+ ( n ) / K , .B, the anterior pituitary membranes were incubated in the presenceof [3H]2-hydroxyestradiol (0.05 to 2.0 X lo-' M) with no inhibitor (v control), 2-hydroxyestrone(2-OH-El)(A-A, 1X M),or2-hydroxyestriol (2-OH-E3)(t"., 1X M). Specificbinding was determined as described in A.

TABLE111 nucleotidase, an enzyme marker for plasma membranes (29). Subcellular distributionof 2-hydroxyestradwl binding activity and Glucose-6-phosphate dehydrogenase activity was used as a enzyme markers after separation through a sucrose density cytoplasmic marker (29) and is found only invery low concengradient trations at the 42.1 to 47.4% sucrose interface. Glucose-6Anteriorpituitarymembraneswerepreparedandcentrifuged phosphatase activity was used as a microsomal marker and through a discontinuoussucrosedensitygradient.Thesubcellular found to present at the 42.1 to 47.4%, 47.4 to 52.9%, and 52.9 components collected atthesucroseinterfaceswereassayedfor specific 2-hydroxyestradiol binding, 5'-nucleotidase, glucose-6-phos- to 53.8% sucrose interfaces. These results suggest the associphatase,andglucose-6-phosphatedehydrogenaseactivity as de- ation of the binding sites with the plasma membrane fraction devoid of cytoplasmic contamination. However, one cannot scribedunder"Materials and Methods."Theexperimentwasrepeated with two separate membrane preparations and the distribution rule out the possible binding of [3H]2-hydroxyestradiol to of binding capacity and enzyme activity was similar. microsomes. 2-Hy&oxye+ tradiol binding

5"Nuc-eotidase

6-phosphatase

Glucose'-phosphate dehydrogenase

DISCUSSION

Specific binding of 2-hydroxyestradiol to pituitary membrane receptor sites has been demonstrated in the ratanterior pituitary gland. Using [3H]2-hydroxyestradiolas the ligand, homogenate Whole 6.7 5.8 5.90 two separate binding sites were observed, a high affinity site, membranes Crude 13 10.5 10.7 0.40 KO = 4 f 2 X 10"' M, and a lower affinity site, KO = 2 X % sucrose M (Figs. 4 and 5). The high affinity binding site was shown to 3.9 0.28 4.3 36.8-42.1% interface be stereospecific and highly localizedin the plasma membrane 80 48.5 17.0 0.05 42.1-47.4% interface fraction. Since endogenous levels of 2-hydroxyestradiol have 10 30.8 28.1 47.4-52.9% interface 22.4 14.7 52.9-53.8% interface M (2, 3), the high been reported to be approximately affinity site may be of physiological significance. Units expressed as micromolesP, released/30 min. Stereospecificity of the 2-hydroxyestradiol binding site was * Units expressed as 100 X A d 2 . 0 7 . determined by examiningthe ability of related compounds to ['HH]2-Hydroxyestradiolbinding sites in various regions of the inhibit [3H]2-hydroxyestradiolbinding. The closely related 2brain and other non-neuronal tissues (Table 11). The 2-hy- hydroxyestrone and 2-hydroxyestriol were both competitive droxyestradiol binding sites were heterogeneously distributed inhibitors of [3H]2-hydroxyestradiolbinding with KI values of M and 1.9 X M, respectively (Fig. 7B). In throughout the brain, although all regionsexamined had 1.5 X measurable amounts of binding sites. Anterior pituitary, cer- contrast, estrone, estradiol, estriol, and their 2-methoxylated ebral cortex, and hypothalamus had the highest concentration derivatives (2-methoxyestrone, 2-methoxyestradiol, and 2of binding and lower levels were foundin the striatum, cere- methoxyestriol) were ineffective inhibitors of ['H]2-hydroxM (Table I). bellum, brain stem, and thalamus. Low concentrations of 2- yestradiol binding at concentrations up to hydroxyestradiol binding sites were also observedin liver, The observation that the parent estrogens do not inhibit specific [3H]2-hydroxyestradiol binding provides evidence uterus, and retina. Subcellular Distribution of [3H/2-HydroxyestradwL Bind- that the observed binding is not due to an interaction of 2ing-Binding of [3H]2-hydroxyestradiolto rat anterior pitui- hydroxyestradiol with the classical intracellular estrogen retary membranes was studied in various fractions prepared by ceptors in the anteriorpituitary (7,8). Enzymatic methylation stepwise sucrose density gradient centrifugation. The binding of 2-hydroxyestradiol has been shown to be a major catabolic activity present at the interface of 42.1 to 47.4% sucrose pathway in vivo (l),and therefore the ineffectiveness of the constituted 85 to 95% of the total activity in all fractions. The methoxylated derivatives to inhibit ['H]2-hydroxyestradiol specific binding of [3H]2-hydroxyestradiolper mg of protein binding suggests that enzymatic methylation may serve to in this fraction was increased by -6-fold over the starting terminate 2-hydroxyestradiol action at the level of its memmaterial from 13 fmol/mg of protein to 80 fmol bound/mg of brane receptor. Related catecholamines, indolamines, and doprotein (Table 111).The distribution of ['H]Z-hydroxyestradiol pamine antagonists were also examined as to their ability to binding in these fractions paralleled the distribution of 5'- inhibit specific ['H]2-hydroxyestradiol binding.None of these fmol/mg units/mg protein protein"

units/m protein .f



compounds were effective at concentrations up to M (Table I). We previously reported that 2-hydroxyestradiol specifically inhibits [3H]spiroperidolbinding to thedopamine receptor in the anterior pituitary (13). The stereospecificity of the high affinity 2-hydroxyestradiol receptor described in this paper is distinct from that of the dopamine receptor. Spiroperidol competes for the [3H]2-hydroxyestradiolbinding with a KI of 2 x M (Fig. 7A), whereas the [3H]2-hydroxyestradiol binds toits high affinity binding site with a dissociation constant, KD,of 4 f 2 X 10"' M (Fig. 5B). Conversely, [3H]spiroperidol binds to theanterior pituitarydopamine receptor with a K Dof 1 X 10"' M and 2-hydroxyestradiol competes for M (13). In the spiroperidol binding site with a KI of 9 X addition, the concentration of the spiroperidol binding site (213 fmol/mg of protein) reported in our earlier paper (13) is 15-foldhigher than thatof the high affinity 2-hydroxyestradiol binding site (13 fmol/mg of protein) reported in this paper. Although the characteristics of the low affinity 2-hydroxyestradiol binding site are unknown, the present data suggest that thehigh affiiity 2-hydroxyestradiol binding site and the dopamine receptor are separate entities. To demonstrate that the [3H]2-hydroxyestradiol binding was associated with a membrane-bound receptor and not a cytoplasmic binding protein, anterior pituitaries were homogenized and various subcellular components were separated by centrifugation through a discontinuous sucrose gradient as described by Neville (27).Fractions enriched for plasma membranes, microsomes, and cytoplasm were identified by the presence of 5'-nucleotidase, glucose-6-phosphatase, and glucose-6-phosphate dehydrogenase, respectively. Eighty-five to 95% of the 2-hydroxyestradiol binding capacity was localized at the42.1 to 47.4% sucrose interface (Table 111).This fraction has -6-fold enrichment of plasma membrane enzyme markers and less than 2% contamination of cytoplasmic enzyme markers, demonstrating that the[3H]2-hydroxyestradiolbinding site is not associated with a cytoplasmic component. The observation that specific 2-hydroxyestradiol binding is associated with the plasma membranes and not due to cytoplasmic contamination is consistent with the observed differences in stereospecificity between the 2-hydroxyestradiol binding site and the classical intracellular estrogen receptor. In addition, the difference in optimal temperature of binding between cytoplasmic estrogen receptors (4OC) (8) and the membranebound 2-hydroxyestradiol binding site (37°C) (Fig. 3A)further supports the notion that the 2-hydroxyestradiol binding site is distinct from the intracellular estrogen receptor. Membranes isolated from other brain regionswere also examined for the presence of 2-hydroxyestradiol binding sites. The highest concentration was observed in the anterior pituitary, cerebral cortex, and hypothalamus, regions reported to have high concentrations of estradiol 2-hydroxylase, the enzyme whichcatalyzes the conversion of estradiol to 2-hydroxyestradiol (4, 5). The striatum, a region highly enriched with dopamine receptors (32, 33), has a low level of 2-hydroxyestradiol receptors, further suggesting that the2-hydroxyestradiol binding site is distinct from the dopamine receptor. Although it is well known that steroid hormones mediate their action via interacting with specific intracellular receptors (14, 15), recent data from several laboratories suggest additional mechanisms of steroid action. The observation that progesterone covalently linked to polyethylene oxide (molecular weight, 20,000) induces oocyte maturation demonstrates that progesterone stimulates oocyte maturation without entering the cells (19).However, specific binding of progesterone

Membranes

9843

to the oocyte membrane has not been demonstrated. Membrane-associated specific estrogen and glucocorticoid binding sites have been reported in rat liver (16, 18). Furthermore, specific estrogen binding sites have been observed in membrane fractions isolated from rat uterine cells (17). The observation of membrane-bound estrogen receptors in therat uterus is in apparent contradiction with the results of Muller et al. (24) who suggest that the observed specific estradiol binding may be accounted for by cytoplasmic contamination. Although the physiologicalsignificance of our findings is unclear, this studymay provide a model system for the study of catecholestrogen action and membrane-associated steroid hormone receptors. REFERENCES 1. Fishman, J. (1976) Neuroendocrinology 22,363-374 2. Paul, S., and Axelrod, J. (1977) Science 197,657-659 3. Ball, P., Emons, G., Haupt, O., Hoppen, H.-O., Knuppen, R. (1978) Steroids 31,249-258 4. Paul, S. M., Axelrod, J., and Diliberto, E. J., Jr. (1977) Endocrinology 101,1604-1610 5. Barbieri, R. L., Canick, J. A., and Ryan, K. J. (1978) Steroids 32, 529-538 6. Ball, P., and Knuppen, R. (1978) Endocrinology 47, 732-737 7. Martucci, C., and Fishman, J. (1979) Endocrinology 105, 12881292 8. Davies, I. J., Naftolin, F., Ryan, K. J., Fishman, J., and Siu, J. (1975) Endocrinology 97,554-557 9. Hsueh, A. J. W., Erickson, G. F., and Yen, S. C.C. (1979) Endocrinology 97,554-557 10. Ball, P., Knuppen, R., Haryst, M., and Breuer, H. (1972) J. Clin. Endocrinol. Metab. 34, 736-746 11. Lloyd, T., Weisz, J., and Breakefield, X.0.(1978) J. Neurochem. 31, 245-250 12. Lloyd, T., and Weisz, J. (1978) J. Biol. Chem. 253,4841-4843 13. Schaeffer, J. M., and Hsueh, A. J. W. (1979) J. Biol. Chem. 254, 5606-5608 14. O'Malley, B. W., and Means, A. R. (1974) Science 183,610-620 15. Jensen, E. V., and Jacobson, H. I. (1962) Recent Prog. Horm. Res. 18,387-408 16. Pietras, R. J., and Szego, C. M. (1979) J. Steroid Biochem. 11, 1471-1483 17. Pietras, R. J., and Szego, C. M. (1979) J. Cell. Physiol. 98, 145160 18. Suyemitsu, T., and Terayama, H. (1975) Endocrinology 96,14991508 19. Godeau, J. F., Schorderet-Slakine, S., Hubert, P., and Baulieu, E.-E. (1978) Proc. Nut. Acad.Sci. U. S. A. 75,2353-2357 20. Ishikawa, K., Hanaoka, Y., Kondo, Y., and Imai, K. (1977) Mol. Cell. Endow. 9,91-100 21. Dufy, B., Vincent, J.-D., Fleury, H., DuPasquier, P., Gourdji, D., and Tixier-Vidal, A. (1979) Science 204,509-511 22. Yagi, K. (1973) Brain Res. 53,343-352 23. Kelly, M. J., Moss, R. L., Dudley, C. A,, and Fawcett, C. A. (1977) Exp. Brain Res. 30.43-52 24. Muller, R. E., Johnston, T. C., and Wotiz, H. H. (1979) J. Biol. Chem. 254,7895-7900 25. Gelbke, H. P., Haupt, O., and Knuppen, R. (1973) Steroids 21, 205-218 26. Gelbke, H. P., and Knuppen, R. (1972) J. Chromatogr. 71, 465471 27. Neville, D. M. (1960) J. Biophys. Biochem. Cytol. 8,413-422 28. Aronson, N. N., and Touster, 0. (1974) Methods Enzymol. 31, 90-102 29. Cohen, P., and Rosemeyer, M. A. (1969) Eur. J. Biochem. 8, 1-7 30. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193,265-275 31. Cheng, Y.-C., and Prusoff, W. H. (1973) Biochem. Pharmacol. 22, 3099-3108 32. Seeman, P., Chan-Wong, M., Tedesco, J., and Wong, K. (1975) Proc. Nut. Acad.Sci. U. S. A . 72,4376-4380 33. Creese, I., Burt, D. R., and Snyder, S. H. (1975) Life Sci. 17,9931002