Relative Binding Affinity of Antiprogestins Zk 98.299 And Zk 98.734 For Progesterone Receptors in the Endometrium and Myometrium of Bonnet Monkeys

Abstract

Progesterone bound with high affinity to the endometrial and myometrial cytosol of ovariectomized bonnet monkeys pretreated with estradiol benzoate and progesterone. The equilibrium dissociation constant (K_d) of ³H-progesterone was 4.5 nM and 5.5 nM and the binding capacity was 1.7 nM and 1.4 nM for the endometrial and myometrial receptors, respectively. This experimental ‘model’ was used to assess the relative binding affinity (RBA) of progesterone, ZK 98.299 and ZK 98.734. The tested compounds showed competitive binding to cytoplasmic progesterone receptors. The RBF of progesterone in the endometrium (100) was more than that of ZK 98.299 (25.1) and ZK 98.734 (17.8). A similar RBA pattern was observed in the myometrial cytosol. Both ZK 98.299 and ZK 98.734, like progesterone, displaced the ³H-progesterone bound to the receptors. The administration of ZK 98.299 or ZK 98.734, during the mid-luteal phase, has been reported to shorten the cycle length in bonnet monkeys and marmosets, respectively. These compounds, therefore, appear to intercept the progesterone action by blocking progesterone binding sites in the target tissue. Since ZK 98.299 has higher binding affinity than ZK 98.734, it may be a more potent progesterone antagonist.     

Progesterone receptor quantification with radiolabeled promegestone (R 5020) in frozen sections of endometrium and breast cancer tissue

A technique for the determination of the progesterone receptor content at sections was developed. Series of coverglass-mounted unfixed frozen sections were incubated with [3H]R5020 only, to determine total binding, or with excess unlabeled R5020, to determine non-specific binding. Ligand binding in the tissue sections was measured by liquid scintillation counting after repeated washing of the coverslips. Elution of ligand binding proteins into the incubation buffer was quantitated with the dextran-coated charcoal method. Specific ligand binding was related to the total tissue protein content which was determined on parallel, unmounted sections. Scatchard analysis showed specific saturable and high affinity (Kd = 0.01-2 nM) section-bound and soluble binding sites in cryostat sections of calf uterus, human endometrium and breast cancer samples. Ligand specificity was studied by competition of [3H]R5020 with a 100-fold excess of various steroid receptor ligands. The competition was excellent for R5020 and progesterone, negligible for estrogens and slight for androgens and corticosteroids. These binding characteristics provide evidence that with this assay progesterone receptors are determined. Exchange experiments showed that with this method total, free as well as occupied, progesterone receptors can be measured. A highly significant linear correlation, and agreement in PR status classification between assay on cytosol and sections was obtained for a series of 21 breast cancer samples. Finally, progesterone receptor analysis using cryostat sections results in the recovery of 2-3 times more PR from the same amount of tissue as compared to the use of cytosol. These results indicate that progesterone receptors can be reliably assayed with Scatchard analysis using cryostat sections, which requires less tissue than the cytosol assay. This method, which is simple and easy to perform could be of practical importance, particularly when only small tissue samples (which also have to be analyzed morphologically or histochemically) are available and when quantitative radiochemical progesterone receptor data are required for direct comparison with (immuno-) histochemical information.     

Decreased concentrations and affinities of oestrogen and progesterone receptors of intrauterine tissue in human pregnancy

Cytoplasmic and nuclear receptors for oestrogen and progesterone were measured in non-pregnant myometrium and endometrium and compared to concentrations found in decidua of ectopic pregnancy (6-8 weeks gestation) and therapeutic abortions (8-16 weeks). Amnion, chorion, placenta, decidua and myometrium at full term pregnancy were also assayed for the same receptors. High affinity binding was confirmed in the non-pregnant tissue; in early pregnancy, decreases in concentrations of cytoplasmic receptors were demonstrated, these decreases becoming more marked as pregnancy progressed in the 1st trimester. Nuclear receptor concentrations were not significantly different. Significant decreases in the occurrence of positive receptors with the progression of pregnancy were also demonstrated for cytoplasmic and nuclear oestrogen and nuclear progesterone receptors. Tissue at full term pregnancy had no detectable receptors, irrespective of whether the patients were in labour or not. Increasing the range of the labelled steroids failed to demonstrate any low affinity binding sites and pre-assay removal of endogenous hormones also had no effect on receptor status. When endogenous hormones were removed, displaceable binding was demonstrated in the presence of excess unlabelled ligand. However, this binding did not conform with receptor dynamics on Scatchard analysis. Heating the cytosol prior to assay or failure to remove endogenous steroid hormones eliminated this binding. Cytosolic oestrogen and progesterone levels increased significantly in the decidua of therapeutic abortions, whilst term pregnant tissue had the highest concentration of endogenous hormones.     

M2 muscarinic ([3H]N-methyl scopolamine) binding in micropunches of rat ventricular myocardium: characterization and modification by progesterone.

A new technique is outlined for the characterization and quantification of M2 muscarinic binding sites (receptors) in micropunches (1 mm diam.), cut from slices (350 microns), of fresh cardiac tissue using the hydrophilic antagonist [3H]N-methyl scopolamine. The use of this water-soluble ligand allows us to label, and quantify, M2 receptors on the cell surface of intact cells contained within the micropunch. We believe that cardiac micropunches offer a simple but powerful approach to the investigation of membrane receptor regulation in tissue that largely retains the in vivo cytoarchitecture. Specific binding is reversible, stereospecific, saturable, of high affinity, and has the drug specificity typical of an M2 muscarinic receptor. In rat left ventricle, Bmax was 151.2 +/- 10.3 fmol/mg protein while KD was 1.0 +/- 0.1 nM. Nonspecific binding of the ligand was very low, varying from 2.8% (at 0.27 nM) to 7.7% (at 3.58 nM). This micropunch assay was used to determine that progesterone can compete with the muscarinic ligand for the M2 receptor in vitro (IC50 = 50 x 10(-6) M). The steroids estradiol and testosterone, as well as ouabain, were without effect. Progesterone inhibited [3H]N-methyl scopolamine binding competitively (KD reduced from 1.9 to 4.3 nM) without affecting the rate of association of the ligand. However, progesterone induced a rapid dissociation of the ligand from its receptor. We conclude that the micropunch assay described here is suitable for the continued study of sex hormone effects on cardiac function.     

ZK98299, a novel antiprogesterone that does not interact with chicken oviduct progesterone receptor

Steroid antagonists, at receptor level, are valuable tools for elucidating the mechanism of steroid hormone action. We have examined and compared the interaction of avian and mammalian progesterone receptors with progestins; progesterone and R5020, and a newly synthesized antiprogesterone ZK98299. In the chicken oviduct cytosol, [3H]R5020 binding to macromolecule(s) could be eliminated with prior incubation of cytosol with excess radioinert steroids progesterone or R5020 but not ZK98299. Alternatively, [3H]ZK98299 binding in the chicken oviduct was not abolished in the presence of excess progesterone, R5020, or ZK98299. In the calf uterine cytosol, [3H]R5020 or [3H]ZK98299 binding was competeable with progesterone, R5020 and ZK98299 but not estradiol, DHT or cortisol. Furthermore, immunoprecipitation and protein A-Sepharose adsorption analysis revealed that in the calf uterine cytosol, the [3H]R5020-receptor complexes were recognized by anti-progesterone receptor monoclonal antibody PR6. This antibody, however, did not recognize [3H]ZK98299-receptor complexes. When phosphorylation of progesterone receptor was attempted in the chicken oviduct mince, presence of progesterone resulted in an increased phosphorylation of the known components A (79 kDa) and B (110 kDa) receptor proteins. Presence of ZK98299 neither enhanced the extent of phosphorylation of A and B proteins nor did it reverse the progesterone-dependent increase in the phosphorylation. The avian progesterone receptor, therefore, has unique steroid binding site(s) that exclude(s) interaction with ZK98299. The lack of immunorecognition of calf uterine [3H]ZK98299-receptor complexes, suggests that ZK98299 is either interacting with macromolecule(s) other than the progesterone receptor or with another site on the same protein. Alternatively, the antisteroid binds to the R5020 binding site but the complex adopts a conformation that is not recognized by the PRG antibodies.     

Structure and function of the Ah receptor: sulfhydryl groups required for binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to cytosolic receptor from rodent livers

Cytosol from rodent liver was exposed to a variety of sulfhydryl-modifying reagents to determine if the cytosolic Ah receptor contained reactive sulfhydryl groups that were essential for preservation of the receptor's ligand binding function. At a 2 mM concentration in rat liver cytosol, all sulfhydryl-modifying reagents tested (except iodoacetamide) both blocked binding of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to unoccupied receptor and caused release of [3H]TCDD from receptor sites that had been labeled with [3H]TCDD before exposure to the sulfhydryl-modifying reagent. Exposure of cytosol to iodoacetamide before labeling with [3H]TCDD prevented subsequent specific binding of [3H]TCDD, but iodoacetamide was not effective at displacing previously bound [3H]TCDD from the Ah receptor. The mercurial reagents, mersalyl, mercuric chloride, and p-hydroxymercuribenzoate, were more effective at releasing bound [3H]TCDD from previously labeled sites than were alkylating agents (iodoacetamide, N-ethylmaleimide) or the disulfide compound 5,5'-dithiobis(2-nitrobenzoate). Presence of bound [3H]TCDD substantially protected the Ah receptor against loss of ligand binding function when the cytosol was exposed to sulfhydryl-modifying reagents. This may indicate that the critical sulfhydryl groups lie in or near the ligand binding site on the receptor. Subtle differences exist between the Ah receptor and the receptors for steroid hormones in response to a spectrum of sulfhydryl-modifying reagents, but the Ah receptor clearly contains a sulfhydryl group (or groups) essential for maintaining the receptor in a state in which it can bind ligands specifically and with high affinity.     

Identification of an androgen receptor in the adult chicken oviduct

The chicken oviduct androgen receptor was characterized by sucrose density gradient centrifugation, Scatchard analysis, competition studies, and affinity labeled with dihydrotestosterone 17 beta-bromoacetate. A specific 8.5 S peak was seen on 0.01 M KCl sucrose density gradients when the receptor was labeled with [3H]5 alpha-dihydrotestosterone. Specific 4.6 S peaks were seen when receptor labeled with [3H]5 alpha-dihydrotestosterone or [3H]dihydrotestosterone 17 beta-bromoacetate was analyzed on 0.3 M KCl sucrose density gradients. Scatchard analysis of [3H]5 alpha-dihydrotestosterone binding by oviduct cytosol was consistent with two binding sites. A Kd of 0.13 nM was found for the high affinity androgen receptor. Competition studies showed the following order of ligand affinity: 5 alpha-dihydrotestosterone greater than dihydrotestosterone 17 beta-bromoacetate greater than progesterone greater than estradiol. A 61.2 kDa protein was specifically covalently labeled with [3H]dihydrotestosterone 17 beta-bromoacetate. The chicken oviduct androgen receptor possesses characteristics similar to other androgen receptors, and provides a good source of androgen receptor for physicochemical studies of the native receptor protein.     

High affinity progesterone binding sites of human uterine microsomal membranes

Microsomal membranes sedimented at 40 000 g were prepared from human myometrium samples. The progesterone binding properties of microsomal suspensions were determined by incubating microsomes and [3H]progesterone at 4 degrees C. Dextran-coated charcoal was used for the separation of bound and free steroids. Membrane-associated progesterone binding sites of high affinity were identified in microsomes prepared from pregnant and nonpregnant uteri. The binding was saturable (Kd approximately 4 X 10(-9) M, concentration of binding sites 400-900 fmol/mg microsomal protein) and specific for natural progesterone. Of 21 steroids tested only 21-hydroxy-4-pregnene-3,20-dione, 17 alpha-hydroxyprogesterone and testosterone showed moderate competition against progesterone with relative affinities between 7.0-20.0% (R.A. of progesterone 100%). 5 alpha-Dihydroprogesterone and 5 alpha-dihydrotestosterone showed weak cross reaction (relative affinities 2.5 and 2.0%, respectively). Corticosteroids, estrogens and the 5 synthetic progestins tested showed only weak competition with relative affinities lower than 1.0%. These microsomal progesterone binding sites of high affinity and limited capacity resemble steroid hormone receptors but they are different from the soluble cytosolic progesterone receptor of human uterus in terms of steroid specificity. The physiological function of this microsomal progesterone receptor is unknown.     

Autoradiographic visualization of alpha 1-adrenergic receptors in cervix of early pregnant rat

alpha 1-Adrenergic receptors were identified, characterized, and localized in rat cervix on Day 6 of pregnancy by autoradiography. Autoradiographic study was performed in slide-mounted rat cervix sections using [3H]-prazosin ([3H]-PRAZ) as ligand. Binding was time dependent and specific. Pharmacological study indicated that specific [3H]-PRAZ binding was inhibited with high affinity by prazosin and phenylephrine and low affinity by yohimbine and clonidine. In cervix, the alpha 1-adrenergic receptors were localized mainly to the inner circular layer of the myometrium. Binding to the outer longitudinal layer of myometrium was moderate, and binding was absent in the endometrium. The regional distribution of alpha 1-adrenergic receptors strongly suggests that the circular layer of myometrium may function as an important modulator of contractile response of the cervix, probably involved in the retention of blastocysts at the utero-cervical end of the horn.     

Immunologically distinct binding molecules for progesterone and RU38486 in the chick oviduct cytosol

[3H]Progesterone and [3H]RU38486 binding in the chick oviduct cytosol is associated with macromolecules which sediment as 8 S and 4 S moieties, respectively, in molybdate-containing 5-20% sucrose gradients. The [3H]progesterone binding could be displaced by excess progesterone, but not by RU38486. Conversely, the [3H]RU38486 binding was able to compete with RU38486 but not by excess progesterone. A preparation containing antibodies against chick oviduct progesterone receptor recognized only the [3H]progesterone-receptor complex but not the 4 S, [3H]RU38486 binding component of the chick cytosol. In the calf uterus cytosol, [3H]R5020 (a synthetic progestin) and [3H]RU38486 were associated with 8 S molecules and the peaks of radioactivity were displaceable upon preincubation with radionert steroids. In addition, the complexes were recognized by antibodies to chick oviduct progesterone receptor. Our data suggest that in the chick oviduct cytosol, RU38486 does not bind to progesterone receptor, but interacts with an immunologically distinct macromolecule.     

Purification of a progesterone receptor from rabbit uterus

A progesterone receptor has been purified to homogeneity from rabbit uterus by steroid affinity chromatography. The receptor was obtained in 5% yield, with a specific activity for [³H]progesterone binding of 14,580 pmol/mg protein. The pure receptor migrated as a single band on SDS-polyacrylamide electrophoresis, with a MW of 70,000. Progesterone binding to the receptor was heat labile and was displaced by an excess of R5020. Photoaffinity labeling of the pure receptor with [³H]R5020 corresponded to the major photoaffinity labeled species in crude cytosol.     

Identification of three muscarinic receptor subtypes in rat lung using binding studies with selective antagonists

Heterogeneity of the muscarinic receptor population in the rat central and peripheral lung was found in competition binding experiments against [3H]quinuclidinyl benzilate [( 3H]QNB) using the selective antagonists pirenzepine, AF-DX 116 and hexahydrosiladifenidol (HHSiD). Pirenzepine displaced [3H]QNB with low affinity from preparations of central airways indicating the absence of M1 receptors in the trachea and bronchi. Muscarinic receptors in the central airways are comprised of both M2 and M3 receptors since AF-DX 116, an M2-selective antagonist, bound with high affinity to 70% of the available sites while HHSiD, an M3-selective antagonist bound with high affinity to the remaining binding sites. In the peripheral lung, pirenzepine bound with high affinity to 14% of the receptor population, AF-DX 116 bound with high affinity to 79% of the binding sites while HHSiD bound with high affinity to 18% of the binding sites. The presence of M1 receptors in the peripheral airways but not in the central airways was confirmed using [3H]telenzepine, an M1 receptor ligand. [3H]Telenzepine showed specific saturable binding to 8% of [3H]QNB labeled binding sites in homogenates of rat peripheral lung, while there was no detectable specific binding in homogenates of rat trachea or heart. The results presented here demonstrate that there are three muscarinic receptor subtypes in rat lungs, and that the distribution of the different subtypes varies within the lungs. Throughout the airways, the dominant muscarinic receptor subtype is M2. In the trachea and bronchi the remaining receptors are M3, while in the peripheral lungs, the remaining receptors are both M1 and M3.     

Specific glucocorticoid binding in human uterine tissues, placenta and fetal membranes

The binding of [3H]dexamethasone to cytosol fractions of human myometrium, endometrium, decidua, chorion, amnion and placenta has been studied. All tissues examined contained high affinity, low capacity binding sites with high specificity for glucocorticoids. Maximum specific binding of [3H]dexamethasone was reached after about 10 h at 0-4 degrees C and remained stable for at least the next 12 h. Sucrose density gradient analysis showed that the binding macromolecules sedimented at 7.9 S in hypotonic solutions and at 4.35 in solutions containing 0.4 M KCl. In the presence of sodium molybdate, the sedimentation coefficients shifted both in the absence and presence of 0.4 M KCl to 8.9 and 5.7 S, respectively. The apparent equilibrium dissociation constants (Kd) of the glucocorticoid binding sites were similar in most tissues, ranging between 1 and 6 nM, with the exception of the placenta in which the binding sites showed a higher Kd (13-22 nM). In all tissues studied, the binding affinities were similar in nonpregnant and pregnant patients and in patients at different stages of pregnancy or in labor. The concentration of the binding sites in the different tissues ranged from 11 to 268 fmol/mg protein, higher concentrations being found in myometrium, placenta and amnion and lower concentrations found in endometrium, chorion and decidua. The number of binding sites was higher in the myometrium of nonpregnant than pregnant women, but was similar in the myometrium of women at term pregnancy before or during labor. In the placenta, the number of binding sites increased significantly from early pregnancy to midpregnancy, while in chorion, amnion and decidua the number of binding sites did not change during pregnancy. It is concluded that human uterine tissues, placenta and fetal membranes contain specific binding sites with properties characteristic of glucocorticoid receptors suggesting that these tissues may respond directly to glucocorticoids.     

Effects of progestin antagonists, glucocorticoids and estrogen on progesterone-induced protein secreted by rabbit endometrial stromal cells in culture

Progesterone enhances the synthesis of a 42 kDa protein secreted by rabbit endometrial stromal cells in primary culture. The duration of that response, the effects of estrogen and the inhibitory ability of antiprogestin steroid analogs, RU486, ZK98.299 and ZK98.734, were tested. Although there was a progressive decrease in the amount of the 42 kDa protein synthesized during a 6-day culture period, progesterone stimulated its rate of synthesis greater than 2-fold throughout that period. The addition of estrogen did not prevent the progressive decrease in the amount of the protein synthesized, nor did it enhance the progesterone effect when the culture medium contained phenol red. Estrogen alone did slightly induce 42 kDa protein synthesis by cells grown in phenol red-free medium, and the progesterone response was accentuated to the same degree. When present in a concentration that was 100-fold that of the progesterone, RU486, ZK98.299 and ZK98.734 each abolished stimulation. This antagonistic effect was overcome by addition of an equimolar concentration of progesterone. Deoxycorticosterone (DOC) also stimulated 42 kDa protein synthesis. The antiprogestins blocked this stimulatory effect, even when both steroids were in equimolar concentrations. There was no difference in the ability of ZK98.299 or ZK98.734 to block DOC stimulation, even though ZK98.734 exhibits no antiglucocorticoid activity [J. Steroid Biochem. 25 (1986) 835]. Therefore, it is likely that the DOC effect is mediated by the progesterone receptor system. These studies indicate that enhanced synthesis of the 42 kDa protein represents a progesterone receptor mediated event and that the cell culture system described can be used as a bioassay for determination of antiprogestin activity.     

Progesterone and oestrogen receptors in the female genital tract throughout pregnancy in tammar wallabies

The tammar, Macropus eugenii, is a monovular macropodid marsupial which has a post-partum oestrus and an 11 month embryonic diapause. Progesterone and oestradiol cytosol receptors were measured by Scatchard analyses and single point analysis in the lateral vagina, endometrium and myometrium of the gravid and contralateral non-gravid uterus throughout pregnancy, immediately after parturition and during seasonal reproductive quiescence. In endometrial tissues, both progesterone and oestradiol receptors doubled in concentration in both gravid and non-gravid uteri between day 0 and day 5 of pregnancy, coinciding with previously described peak values in peripheral plasma progesterone and oestrogen. Receptor concentrations in endometrial tissue during seasonal quiescence were not significantly different from those immediately after reactivation. After day 12 of pregnancy, downregulation of both progesterone and oestradiol cytosolic receptors occurred concomitant with the increase in progesterone in the peripheral plasma. However, there was a unilateral increase in oestradiol receptor concentrations in endometrium obtained from the non-gravid uterus between day 25 of the 26.5 day gestation and immediately after parturition. Myometrial receptor concentrations mirrored those of the endometrium but were lower. Concentrations of progesterone receptor in the lateral vaginae were at the lower limit of detection, while the oestradiol cytosol receptor concentrations were even lower in this tissue. Thus, the steroid receptor concentrations provide another example of local unilateral endocrine responses in the reproductive tract of the tammar. These results also indicate that the downregulation of progesterone and oestradiol receptors that occurs in both uteri in mid- and late-pregnancy is selectively and locally reversed before parturition in the non-gravid endometrium in response to the local effects of follicular oestradiol from the ipsilateral ovary.     

Characterization of the progesterone receptor of rabbit uterus with the synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione

The synthetic progestin 16α-ethyl-21-hydroxy-19-norpregn-4-ene-3,20-dione (Org 2058) was used to characterize the progesterone receptor in the uterine cytosol of the rabbit. [³H] Org 2058 binds to a homogeneous population of protin binding sites with an apparent association equilibrium constant of 7.7· 10⁸ M⁻¹ at 0°C. The concentration of protein-bound steroid at saturation is 2.3 pmol per mg of cytosol protein. [³H] Progesterone binds to the same set of binding sites but exhibits a 4–5 fold lower apparent association constant. The difference in affinity is mainly due to a 13-fold slower rate of dissociation of the synthetic progestin compared with progesterone. Org 2058 competes very efficiently for the binding of [³H] progesterone to the uterine cytosol, and progesterone also competes, although less efficiently, for the binding of [³H]-Org 2058. There is a good correlation between the progestational activity of various steroids and their ability to compete with [³H] Org 2058 binding to the cytosol. At 0°C, there is no metabolic transformation of either Org 2058 or progesterone in the uterine cytosol.When filled with the steroid, the progesterone receptor is stable, but in the absence of the steroid the receptor binding sites are thermolabile and show a rapid decay at 20°C . Org 2058 is more effective than progesterone in protecting the receptor against thermal inactivation. The rate constant of association and dissociation of [³H] Org 2058 and the cytosol receptor are strongly dependent on temperature and the activation energy of the dissociation reaction is 17.8 kcal/mol. The equilibrium association constant is less dependent on temperature and exhibits ΔH° of −4.7 kcal/mol. The binding reaction shows a positive entropy change of 23 cal · K⁻¹ · mol⁻¹.At low ionic strength the complex of Org 2058 and the progesterone receptor tends ot aggregate. It sediments as a broad peak on sucrose gradients (4–6 S), and is excluded from columns of Sephadex G-100 and G-200. At concentrations of NaCl above 0.15 M, the receptor sediments in sucrose gradients as an homogeneous peak at 3.6 S, but upon gel filtration it aggregates and a complex elution pattern is observed, that prevents a precise estimation of the molecular weight.     

Characterization of an antiestrogen-binding protein in high salt extracts of human breast cancer tissue

An antiestrogen binding protein which binds [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)-phenyl]1,2-diphenylbut-1(Z)-ene) with high affinity (Kd = 1.1 X 10(-9) M) is present in high salt (0.6 M KCl) extracts of washed breast cancer tissue pellets. Its concentration in high salt extract is higher than its concentration in cytosol. The characteristics of the antiestrogen binding protein from cytosol and salt extract of breast cancer tissue are indistinguishable. It specifically binds triphenylethylene and other nonsteroidal antiestrogens and displays little or no binding affinity for estrogens, progesterone, dihydrotestosterone and cortisol. The antiestrogen binding protein is of unusually large size as judged by gel filtration on agarose 0.5 m and sedimentation analysis on 5-20% sucrose density gradients. Differential centrifugation studies indicate that it is not principally microsomal in origin. This protein is more thermostable than the estrogen receptor from which it can also be distinguished by ion exchange chromatography. The antiestrogen binding protein was eluted from DEAE-Sephacel by 0.05 M KCl indicating that it is less negatively charged than the estrogen receptor which was eluted by 0.1 M KCl. Lipoprotein fractionation of breast cancer cytosol using potassium bromide density gradients did not reveal specific antiestrogen binding activity associated with any recognized class of lipoprotein. Specific [3H]tamoxifen binding sites were pelleted in potassium bromide gradients consistent with the apparent large size of this protein. The physical characteristics of the antiestrogen binding protein in normal human tissue (myometrium) and neoplastic tissue (breast cancer) are remarkably similar, possibly reflecting a highly conserved structure.     

Characterization of estrogen and progesterone receptors in monkey endometrium: methodology and effects of estradiol and/or progesterone on endometrium of castrate monkeys.

Appropriate methods for repeated surgical collection of endometrial tissue from rhesus monkeys, and characterization of cytosol and nuclear estrogen (E₂) and progesterone (P) receptors (R) are described. Tissue collection was made in the mid-luteal phase at abdominal fundal hysterotomy. Functional status of the ovaries was determined by visual inspection and RIA of E₂ and P in serum. Receptor assay procedures were devised permitting the measurement of total cytosol and nuclear receptor concentration. Sucrose density gradients of labelled cytosol were made and a 4S saturable binding component for ³H P and for ³H E₂ were found. Equilibrium dissociation constants of ³H E₂ and ³H R5020 were 2.1×10^?10M and 3.6 × 10^?9M, respectively. These binding characteristics are similar to those found in human endometrium and suggest that these surrogate primates have extensive utility in investigation of factors influencing E₂R and PR concentrations in endometrial tissue during the menstrual cycle and implantation. Simulated menstrual cycle were produced in 20 castrate monkeys by sequential treatment with estradiol and progesterone in silastic capsules. RIA of E₂ and P, and gonadotropins in peripheral serum provided assuredness of the hormonal status of each monkey under treatment. Cytosol and nuclear receptors for E₂ and P were measured in the endometrium after different intervals of the treatment. E₂ receptor (E₂R) levels were not changed during the estrogen sequence, but were lowered by progesterone therapy in both cytosol and nuclear components. Progesterone receptor (PR) synthesis in cytosol was induced by exogenous estrogen. The total concentration of PR decreased with the uptake of P by the cell; meanwhile, the ratio of cytosol to nuclear P receptors declined. These data suggest that this sequential estrogen-progesterone regimen induces the changes in E₂R and PR patterns in the endometrium of ovariectomized monkeys which occurs due to ovarian cyclicity in the normal menstrual cycle.     

Dissociation between adenosine receptors and adenylate cyclase in the smooth muscle of guinea pig myometrium

We have previously demonstrated that adenosine causes contraction of guinea-pig myometrium in a fashion consistent with the presence of a purinergic receptor of the A1 subtype. Incubation of guinea-pig uterine smooth muscle membranes with the stable adenosine analogue [3H]cyclohexyladenosine [( 3H]CHA) resulted in rapid, reversible association of radioligand to saturable sites. The affinity (KD) of the receptor for [3H]CHA determined from kinetic experiments (3.14 nM) is in good agreement with that determined in saturation experiments (KD = 4.5 nM). Scatchard analysis of specific [3H]CHA binding (Bmax = 79 fmol/mg protein) is consistent with a single class of binding sites for [3H]CHA. Computer analysis of competition of [3H]CHA binding by the stereoisomers of phenylisopropyl adenosine, R-PIA (KI = 5.3 nM) and S-PIA (KI = 69 nM), as well as the 5'-substituted analogue, ethylcarboxamide adenosine (NECA; KI = 4.2 nM) suggest that [3H]CHA binding occurs to a single class of receptors of the AI subtype. Contractile studies employing these agents reveal that the relative order of potency, based on ED50 values, correlates well with the relative order of competition of agonist binding, based on equilibrium binding constants. Direct assay of myometrial adenylate cyclase failed to show that adenosine receptors in this smooth muscle are coupled to adenylate cyclase. We conclude here that a smooth muscle adenosine receptor is not coupled to adenylate cyclase, yet subserves muscle contraction. These data are important in light of recent attempts to classify adenosine receptors as dual regulators of adenylate cyclase.