Androgens inhibit the formation of catechol estrogens by estrogen 2-hydroxylase present in rat liver microsomal preparations

The inhibition of estrogen 2-hydroxylase by androgens was demonstrated in screening assays and has been further investigated under initial velocity conditions. The ability of testosterone, 5 alpha-dihydrotestosterone, and dehydroepiandrosterone to block the conversion of estradiol to 2-hydroxyestradiol by male rat liver microsomal preparations was determined using two radiotracer methods--the conversion of [4-14C]estradiol to [4-14C]2-hydroxyestradiol and the release of 3H2O from [2-3H]estradiol. The apparent Ki's for the androgens ranged from 12.0 to 14.0 microM, with the apparent Km for the substrate estradiol in these assays of 2.08 microM. Multiple inhibition studies with the androgens and 2-bromoestradiol, an effective estrogen inhibitor, in male rat liver microsomes resulted in Dixon plots consisting of a series of nonparallel, intersecting lines. Thus, the androgens and 2-bromoestradiol are non-exclusive inhibitors, i.e. the binding of one compound to the enzyme does not interfere with the binding of the other. These interactions of androgens suggest that the steroid hormonal environment be considered in the examination of the physiological role(s) of the estrogen 2-hydroxylase and the catechol estrogen products.     

Specific nuclear uptake of intracellularly-produced estrogen by rat granulosa cells

Granulosa cells of the ovarian follicle are unique in that they both synthesize steroid hormones and respond to exogenously-administered steroids. Isolated granulosa cells from ovaries of gonadotropin-primed rats were incubated in the presence of [3H]testosterone, which the cells convert to [3H]estradiol. Nuclear extracts of these cells were analyzed by high-performance liquid chromatography in a system of 40% acetonitrile. When cells were incubated in the presence of [3H]testosterone alone, a significant portion of the radioactivity present in nuclei co-eluted with authentic estradiol. The nuclear binding was considered to be specific, since 50-75% of total binding was suppressed when the incubation medium contained excess unlabeled estrogen. Moreover, when an antibody to estradiol was included in the medium, specific nuclear uptake of [3H]estradiol was not abolished, but rather was increased. Granulosa cells may, therefore, directly utilize endogenously-produced estradiol, a mechanism which may play a role in the regulation of ovarian cells.     

Aromatase and testosterone fatty acid esters: the search for a cryptic biosynthetic pathway to estradiol esters.

The estradiol fatty acid esters (lipoidal derivatives, LE2) are extremely potent estrogens that accumulate in fat, including fat of menopausal women. These steroidal esters are protected from metabolism and are converted to the free, biologically active steroid through the action of esterases. Previous studies have shown that biosynthetic pathways in the adrenal gland exist in which steroid fatty acid esters are substrates. This led us to determine whether a cryptic aromatase pathway exists in which testosterone esters could be converted directly into LE2. We tested a representative fatty acid ester, testosterone stearate, both as an inhibitor and as a substrate for the aromatase enzyme from human placental microsomes. This ester had neither activity. In addition, we tested [1 beta-3H]testosterone acetate as a substrate for this enzyme complex, measuring the production of 3H2O as evidence of aromatization. Although the rate of reaction was considerably slower than that of testosterone, 3H2O was produced. However, when [2, 4, 6, 7-3H]testosterone acetate was incubated and the steroidal products isolated, we found that hydrolysis of the substrate had occurred. Both [3H]-labeled testosterone and estradiol were found, and very little if any [3H]estradiol acetate was formed. Thus, we conclude that an aromatase pathway involving testosterone esters does not exist and that the sole source of LE2 is through direct esterification of estradiol.     

Binding characteristics of [3H]delta(5)-androstene-3beta,17beta-diol to a nuclear protein in the rabbit vagina

In this study, we investigated the binding characteristics of [3H]Delta(5)-androstene-3beta,17beta-diol to rabbit vaginal cytosolic and nuclear extracts and in freshly excised intact tissue strips. [3H]delta(5)-Androstene-3beta,17beta-diol bound to a protein(s) in the vaginal nuclear extract with high affinity (K(d)=3-5 nM) and limited capacity (50-100 fmol/mg protein). No specific binding was detected in the cytoplasmic extracts. Competitive binding studies showed that binding of [3H]delta(5)-androstene-3beta,17beta-diol was effectively displaced with unlabeled delta(5)-androstene-3beta,17beta-diol but not with dehydroepiandrosterone, testosterone, dihydrotestosterone, triamcinolone acetonide, or progesterone. However, estradiol at high concentrations partially displaced bound [3H]delta(5)-androstene-3beta,17beta-diol. Incubation of freshly excised vaginal tissue strips with [3H]delta(5)-androstene-3beta,17beta-diol in the absence or presence of excess unlabeled delta(5)-androstene-3beta,17beta-diol for 1h at 37 degrees C resulted in specific binding to a soluble macromolecule in the nuclear KCl extracts. In addition, quantitative measurement of estrogen receptor, androgen receptor and delta(5)-androstene-3beta,17beta-diol binding protein was performed by equilibrium ligand binding assays using extracts of distal vaginal tissue from intact animals or ovariectomized animals treated for 2 weeks with vehicle, estradiol, testosterone, or estradiol plus testosterone. These changes in steroid hormone levels resulted in opposing trends between the estrogen receptor and delta(5)-androstene-3beta,17beta-diol binding protein, suggesting that delta(5)-androstene-3beta,17beta-diol binding protein is regulated differently by the hormonal milieu than the estrogen receptor. These data suggest that rabbit vaginal tissue expresses a novel binding protein which specifically binds delta(5)-androstene-3beta,17beta-diol and is distinct from the androgen and estrogen receptors.     

Interaction of sex steroids with proteins from the soluble fraction of swine and cattle liver (a preliminary analysis)]

The interactions of [3H]estradiol, [3H]testosterone and [3H]progesterone with soluble proteins from porcine and calf liver were studied. The specific binding of [3H]progesterone and [3H]testosterone in calf liver cytosol seems to be due to serum transcortin or its intracellular precursor (analog). Contrariwise, the specific binding of [3H]progesterone observed in porcine liver cytosol was absent in the serum. This binding was characterized by slow association and dissociation dynamics, moderate affinity for the [3H]-ligand and a high binding capacity. The structural determinants of the ligands were studied by competitive inhibition of the [3H]-ligand binding. The delta 4-3-keto group in the steroid A-ring was found to be the most important determinant. An intensive metabolism of [3H]progesterone was observed during its incubation with cytosol (data from thin-layer chromatography). A 3H-metabolite (presumably, 20 beta-dihydroprogesterone) was predominant in the bound ligand fraction. The data obtained suggest that proteins of a steromodulin type are widely distributed in the mammalian liver.     

The effects of various steroids on testosterone metabolism by the sexually mature rabbit epididymis

We examined the influences of steroids present in the epididymis on androgen metabolism by epididymal tissue and on the binding of androgen metabolites to the epididymal androgen receptor in castrated adult rabbit epididymides under in vitro conditions. The conversion of [3H]testosterone to [3H]17 beta-hydroxy-5 alpha-androstan-3-one (5 alpha-DHT) and to [3H]5 alpha-androstane-3 alpha (beta), 17 beta-diol was inhibited by unlabeled steroids in the following manner progesterone greater than testosterone greater than estradiol. Unlabeled 5 alpha-DHT did not inhibit [3H]testosterone metabolism indicating that product inhibition is not an important regulatory event. The antiandrogen cyproterone acetate did not inhibit the formation of 5 alpha-reduced metabolites of [3H]testosterone. All of the compounds used inhibited androgen binding to the classically defined cytoplasmic and nuclear androgen receptor.     

Multiple estrogen binding sites in the uterus: stereochemistry of receptor and non-receptor binding of diethylstilbestrol and its metabolites

Indenestrol A (IA), an oxidative metabolite of the synthetic estrogen diethylstilbestrol (DES), has high binding affinity for estrogen receptor in mouse uterine cytosol but possesses weak biological activity. Racemic mixture of optically active [3H]indenestrol A (IA-Rac) was separated and purified into individual enantiomers on a semi-preparative scale by HPLC with a Chiralpak OP(+) column. The structure-activity relationship was investigated among the [3H]IA enantiomers (IA-R and IA-S) and [3H]DES through direct saturation binding assays using mouse uterine cytosol. Specific binding curves and Scatchard plots were obtained for each [3H]ligand; DES, IA-Rac, IA-R and IA-S. IA-S enantiomer (Kd = 0.67) binds to the estrogen receptor with the same affinity as DES (Kd = 0.71) and four times higher affinity than IA-R (Kd = 2.56). The number of binding sites for IA-S is approximately the same as estradiol, DES and IA-Rac while IA-R binds far fewer sites than the other ligands. Saturation binding assays indicated that [3H]DES and [3H]IA enantiomers exhibited a higher level of non-specific binding to the cytosol receptor compared to estradiol which has a low level of non-specific binding. These binding studies led to the detection of an additional binding component for the stilbestrol compounds in estrogen target tissue cytosol preparations. Sucrose density gradient separation assays under low salt conditions showed that both [3H]DES and [3H]IA compounds bound to the 8S form of the receptor, the same as E2. But, in addition both DES and IA bound to another binding component in 4S region. The binding to the 4S component were partially displaced by the addition of excess unlabeled E2 and DES. Further characterization of the 4S component is described.     

Binding properties of androgen receptors. Evidence for identical receptors in rat testis, epididymis, and prostate.

Androgen receptors in crude and partially purified 105,000 X g supernatant fractions from rat testis, epididymis, and prostate were studied in vitro using a charcoal adsorption assay and sucrose gradient centrifugation. Androgen metabolism was eliminated during receptor purification allowing determination of the kinetics of [3H]-androgen-receptor complex formation. In all three tissues, receptors were found to have essentially identical capabilities to bind androgen, with the affinity for [3H] dihydrotestosterone being somewhat higher than for [3H] testosterone. Equilibrium dissociation constants for [3H] dihydrotestosterone and [3H] testosterone (KD = 2 to 5 X 10(-10) M) were estimated from independently determined rates of association (ka congruent to 6 X 10(7) M-1 h-1 for [3H] dihydrotestosterone and 2 X 10(8) M-1 h-1 for [3H] testosterone) and dissociation (t 1/2 congruent to 40 hr for [3H] dihydrotestosterone and 15 h [3H] testosterone). Evaluation of the effect of temperature on androgen receptor binding of [3H]testosterone allowed estimation of several thermodynamic parameters, including activation energies of association and dissociation (delta H congruent to 14 kcal/mol), the apparent free energy (delta G congruent to -12 kcal/mol), enthalpy (delta H congruent to -2.5 kcal/mol), and entropy (delta S congruent to 35 cal col-1 K-1). Optimum receptor binding occurred at a pH of 8. Receptor stability was greatly enhanced when bound with androgen. Receptor specificity for testosterone and dihydrotestosterone was demonstrated by competitive binding assays. The potent synthetic androgen, 7 alpha, 17 alpha-dimethyl-19-nortestosterone, inhibited binding of [3H] testosterone or [3H] dihydrotesterone nearly as well as testosterone and dihydrotestosterone while larger amounts of 5 alpha-androstane-3alpha, 17 beta-diol and nonandrogenic steroids were required. Sedimentation coefficients of androgen receptors in all unfractionated supernatants were 4 and 5 to 8 S. Differences in sedimentation coefficients were observed following (NH4)2SO4 precipitation which did not influence the binding properties of the receptors. These results, together with measurements of3alpha/beta-hydroxysteroid oxidoreductase activity in vitro, suggest that organ differences in receptor binding of [3H] dihydrotestosterone and [3H] testosterone in vivo result from relative differences in intracellular concentrations of these androgens rather than from differences in receptor affinities.     

Estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase from rabbit liver--II. Mechanisms of enzyme-steroid interaction

Binding of [3H]estradiol, [3H]testosterone and [3H]progesterone to purified NADP-dependent estrophilic 3 alpha,3 beta,17 beta,20 alpha-hydroxysteroid dehydrogenase (EHSD) from rabbit liver cytosol has been examined. The three steroids bind to the enzyme with moderate [corrected] affinity (Ka congruent to 10(7) [corrected] M-1 at 4 degrees C) and equal binding capacity. High-rates were shown for both association and dissociation processes. The steroids competitively inhibited the binding of each other to EHSD. At the same time, their relative binding affinities (RBA) were dependent on the nature of [3H]ligand. The results of RBA determinations for 72 steroids and their analogues by inhibition of [3H]progesterone binding to EHSD suggest that androgens and gestagens bind preferentially to the same site on EHSD molecule, while estrogens (at least by their D-ring) bind to another site. The assumption that EHSD molecule has more than one binding site for steroids is corroborated by (i) substrate inhibition revealed for a number of steroids; (ii) the estrogen ability to potentiate 20 alpha-reduction of progesterone; (iii) stimulatory effect of 5 alpha (beta)-androstane-3 alpha (beta), 17 beta-diols on [3H]testosterone and progesterone binding; and (iv) reciprocal effect of NADP on [3H]estradiol and [3H]testosterone binding to EHSD. Significant differences in sensitivity to pH and changes in NaCl concentration upon metabolism and binding of various steroids have been found. At concentrations of 16 mM dithiothreitol potentiated catalytic conversion of some steroids and had no effect on metabolism of others. Both the affinity for steroids and binding capacity of EHSD are found to be cofactor-dependent. It is speculated that EHSD has a complex active center including at least two mutually influencing steroid-binding sites tightly related with cofactor-binding site. The polyfunctionality of EHSD may be due to both the excess of functional protein groups that form individual constellations upon binding of any steroid and also to conformational lability of EHSD molecule implying alternative orientations of steroids at the binding site.     

Interaction of medroxyprogesterone acetate with cytosol androgen receptors in the rat hypothalamus and pituitary

The binding of medroxyprogesterone acetate (MPA) with cytosol androgen receptors from rat pituitary and hypothalamus was studied. The pituitary and hypothalamic cytosol androgen receptors from adult castrated female rats were in vitro labeled using 3H natural (testosterone (T) and 5 alpha-dihydrotestosterone (DHT] and [3H]synthetic (methyltrienolone) androgens as radioligands. The [3H]androgen-receptor complexes sedimented with a coefficient of 8S in linear sucrose gradients. When incubated with an excess of radioinert MPA, specific binding was abolished indicating interaction of MPA with androgen receptors. Furthermore specific [3H]MPA-androgen cytosol receptor complexes could be identified in these neuroendocrine tissues when a post-gradient receptor labeling technique was used in the absence or presence of radioinert MPA, DHT, and triamcinolone acetonide. A study of binding kinetics disclosed that the equilibrium dissociation constant and saturation binding capacity for the MPA binder, were similar to those exhibited by DHT binding to androgen receptors in both studied tissues under identical experimental conditions. The overall results were interpreted as demonstrating that MPA interacts with cytosol steroid receptors other than those of progesterone in the rat hypothalamus and anterior pituitary. The data are consistent with MPA binding to androgen receptors.     

Synthesis and aryl hydrocarbon receptor binding properties of radiolabeled polychlorinated dibenzofuran congeners

Microchlorination of 1,4,9[3H]dibenzofuran gave several polychlorinated dibenzofuran (PCDF) products and 2,3,7,8-[3H]tetrachlorodibenzofuran (TCDF), 1,2,3,7,8-[3H]pentachlorodibenzofuran (PeCDF), and 1,2,3,6,7,8-/1,2,3,4,7,8-hexachlorodibenzofuran (HCDF) of high specific activity (57, 34, and 32.5 Ci/mmol, respectively) were purified by preparative high-pressure liquid chromatography. These compounds were investigated as radioligands for the rat liver cytosolic aryl hydrocarbon (Ah) receptor protein. Like 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD), the radiolabeled PCDF congeners exhibited saturable binding with the receptor protein and sucrose density gradient analysis of the radiolabeled ligand-receptor complexes gave specific binding peaks with comparable sedimentation profiles. The rank order of radioligand binding affinities (Kd values) was 2,3,7,8-TCDD greater than 2,3,7,8-TCDF greater than 1,2,3,6,7,8-HCDF greater than 1,2,3,7,8-PeCDF and the maximum difference in Kd values for the four radioligands was less than 13-fold (0.44-5.9 nM). The interactions of the PCDF radioligands with the cytosolic receptor all exhibited saturable binding curves and linear Scatchard plots and the slopes of their Hill plots were in the range 1.0-1.1, thus indicating that cooperativity was not a factor in these binding interactions. The relative stabilities and dissociation kinetics of the radioligand-receptor complexes were highly dependent on the structure of the radioligand. The dissociation curves of the 2,3,7,8-[3H]TCDD and PCDF receptor complexes were biphasic and this suggests that there may be a temporal shift in ligand binding affinities. However, the rates of dissociation did not correlate with the rank order of ligand binding affinities. The stabilities of the radioligand-receptor complexes were also dependent on the structures of the radioligands; for example, the 2,3,7,8-[3H]TCDD-receptor complex degraded more rapidly than the PCDF-receptor complex and these relative stabilities were clearly not related to the Kd values or the relative in vivo or in vitro biologic potencies of these halogenated aryl hydrocarbons.     

Characterization of a [3H]methyltrienolone (R1881) binding protein in rat liver cytosol

The binding of radiolabelled methyltrienolone 17 beta-hydroxy-17 alpha-methyl-estra-4,9,11-trien-3-one (R1881) to adult male rat liver cytosol has been characterized in the presence of Na-molybdate to stabilize steroid-hormone receptors, and triamcinolone acetonide to block progestin receptors. Using sucrose density gradient analysis, male liver cytosol contains a [3H] R1881 macromolecular complex which sediments in the 8-9S region. 8S binding of R1881 to male rat serum, female liver cytosol or cytosol from a tfm rat cannot be demonstrated. Further metabolism of [3H] R1881 following 20h incubation with male rat liver cytosol was excluded: In the 8S region 97% of [3H] R1881 was recovered by thin layer chromatography. Characteristics of this [3H] R1881-8S binding protein include high affinity (Kd = 2.3 +/- 41 nM) and low binding capacity (18.8 +/- 3.3 fmol/mg cytosol protein), precipitability in 0-33% ammonium sulfate, and translocation to isolated nuclei following in vivo R1881 treatment. Whereas, the cytosol R1881-receptor is competed for by dihydrotestosterone, testosterone, and estradiol, [3H] estradiol binding in the 8S region is not competitive with androgens but does compete with diethylstilbestrol. The nuclear androgen binding site has a Kd = 2.8 nM for [3H] R1881, and is androgen specific (testosterone greater than 5 alpha-dihydrotestosterone greater than estradiol greater than progesterone greater than cyproterone acetate greater than diethylstilbestrol greater than dexamethasone greater than triamcinolone). Since a number of liver proteins including the drug and steroid metabolizing enzymes are, in part, influenced by the sex-hormone milieu, the presence of a specific androgen receptor in male rat liver may provide valuable insight into the regulation of these proteins.     

Xenoestrogen interaction with human sex hormone-binding globulin (hSHBG).

This study reports on some environmental chemicals with estrogenic activity (xenoestrogens) and their binding interaction for human plasma sex-hormone binding globulin (hSHBG). The binding affinity constant of these xenoestrogens was measured in equilibrium conditions by solid phase binding assay, and their ability to displace endogenous testosterone and estradiol from hSHBG binding sites was determined with an ammonium sulfate precipitation assay in native plasma from normal men and women. The data showed that some of these xenoestrogens bind hSHBG, with a reversible and competitive binding activity for both [3H]testosterone and [3H]17beta-estradiol and with no apparent decrease in the number of hSHBG binding sites. Their respective binding affinity constants were low, ranging from 0.02 to 7.8 10(5) 1 x mol(-1). However, in native plasma from normal men and women, they were able to dose-dependently increase concentrations of hSHBG-unbound testosterone and/or estradiol. In this study, 4-nonylphenol and 4-tertoctylphenol, two alkylphenols used as surfactants in many commercial products, and bisphenol A and O-hydroxybiphenyl, widely used in the plastics industry, were identified as potent hSHBG-ligands. Additionally, the flavonoid phytoestrogens genistein and naringenin were also identified as hSHBG ligands, whereas their glucoside derivatives, genistin and naringin, had no binding activity for hSHBG. From these data, it is suggested that hSHBG binding may transport some contaminant xenoestrogens into the plasma and modulate their bioavailability to cell tissues. On the other hand, xenoestrogens may also displace endogenous sex steroid hormones from hSHBG binding sites and disrupt the androgen-to-estrogen balance. Whether xenoestrogen SHBG ligands could reach high enough concentrations in the blood to expose humans to any such effect merits further investigation.     

Pretreatments with 5 alpha-dihydrotestosterone and the uptake of testosterone by cell nuclei in the brains of male rhesus monkeys

An in vivo competition method was used in adult male rhesus monkeys to determine if testosterone binds to high affinity binding agents, notably androgen receptors, in brain cell nuclei. Castrated males received 5 alpha-dihydrotestosterone propionate (DHTP, 20 mg, N = 6), testosterone propionate (TP, 100 mg, N = 3) or oil vehicle (controls, N = 6) followed 3 h later by 5 mCi [3H]testosterone [( 3H]T) as an intravenous bolus. Brain and peripheral tissue samples were removed after 60 min, homogenized and separated into supernatant and purified nuclear fractions. Radioactive metabolites of [3H]T [( 3H]estradiol, [3H]DHT) and unchanged [3H]T were identified by high performance liquid chromatography (HPLC). Androgen pretreatments reduced the nuclear uptake of [3H]T by 67-98% in hypothalamus (HYP), preoptic area (POA) and pituitary gland (PIT). This blockade was presumed to be due to prior occupation of nuclear androgen receptors by unlabeled androgens because pretreatments had no effects on levels of [3H]T in supernatants. Since [3H]T was the major radioactive androgen present in brain cell nuclei, results strongly suggested that the principal nuclear androgen receptor ligand in HYP, POA and PIT was unchanged [3H]T rather than [3H]DHT as occurs in the genital tract. In the amygdala the situation was quite different. Here, nuclear concentrations of [3H]T were reduced by 67% following TP pretreatment but were not changed following DHTP pretreatment, indicating a different uptake mechanism in this region that could have particular relevance for testosterone's central actions on behavior.     

Somatostatin enhances binding of [3H]estradiol to a cytosolic protein in rat pancreas. Possible role of oligopeptide coligands in secretion

The cytosol fraction of rat pancrease can bind [3H] estradiol specifically and extensively. In contrast to the rat uterus, the binding protein in pancreas requires an accessory factor as a coligand in the steroid-binding reaction. Removal of this accessory factor by passage of the cytosol through CM Affi-Gel blue columns renders eluate fractions virtually incompetent with respect to binding of [3H]estradiol (10 nM). Certain synthetic oligopeptides such as N-benzoyl-L-argininyl-p-nitroanilide, as well as an endogenous accessory factor, can reactivate binding of [3H]estradiol. Thus, localization of the protein that binds [3H]estradiol following chromatography with CM Affi-Gel blue columns can be determined readily by assaying eluate fractions in the absence and presence of either accessory factor or N-benzoyl-L-argininyl-p-nitroanilide. Addition of somatostatin (tetradecapeptide referred to as SRIF14; somatotropin release inhibiting factor) to the activatable, but incompetent, eluate fractions, also enhanced binding of [3H]estradiol. The effect of SRIF14 was biphasic. The threshold concentration required for activation of [3H]estradiol binding was about 1 microM, and maximal stimulation occurred at 25 microM. At higher concentrations of SRIF14, binding declined and reached basal levels at about 75 microM. The concentrations of somatostatin required for activation of binding of [3H]estradiol in vivo may be lower than those indicated above since 1) preparations containing [3H]estradiol-binding protein also contained an SRIF14 peptidase. Following incubation of [125I-Tyr1]SRIF14 with these preparations there was loss of binding of radiolabeled peptide with SRIF14 antiserum. 2) The biphasic nature of SRIF14 activation may reflect feedback inhibition of [3H]estradiol binding by a degradation product of SRIF14. Since SRIF14 has been identified in the delta- (or D-) islet cells of the pancreas, and in concentrations that may be in the microM range, the possibility is raised that these cells serve a paracrine function with respect to acinar cell secretion.     

Comparison of the In Vitro Receptor Binding Properties of N-[3H]Methylspiperone and [3H]Raclopride to Rat and Human Brain Membranes

The aim of the present investigation was to study and compare the in vitro binding properties of the two radioligands N-[3H]methylspiperone ([3H]NMSP) and [3H]raclopride. These compounds, labeled with 11C, have been extensively used in positron emission tomography studies on central dopamine D2 receptors in schizophrenic patients, although with diverging results. One study (using [11C]NMSP) showed an increased dopamine receptor density in drug-naive schizophrenic patients, whereas in another study (using [11C]raclopride) the density in schizophrenic patients was no different from that in healthy controls. In the present study, using in vitro binding techniques, the density of the binding sites was found to be similar irrespective of which of the two radioligands was used (20 fmol/mg wet weight in rat striatum and 10 fmol/mg in human putamen; the 5-hydroxytryptamine 2 receptors were blocked with 40 nM ketanserin). [3H]NMSP had a 10-fold higher affinity (KD, 0.3 nM in rat striatum and 0.2 nM in human putamen) than [3H]raclopride (KD, 2.1 nM in rat striatum and 3.9 nM in human putamen), which was consistent with the longer dissociation half-life of [3H]NMSP compared with [3H]raclopride (14.8 and 1.19 min, respectively). There was an approximate overall similarity between the inhibition constants for five dopamine antagonists, chlorpromazine, haloperidol, raclopride, remoxipride, and NMSP, when using either radioligand. The Ki values were, however, two- to four-fold higher when using [3H]NMSP as the radioligand, irrespective of inhibiting compound, except for chlorpromazine (and haloperidol in human putamen). NMSP was found to inhibit the binding of [3H]raclopride competitively, whereas raclopride inhibited the binding of [3H]NMSP both competitively and noncompetitively. This difference suggests that part of the binding site is exclusively used by NMSP and can only be allosterically interfered with by raclopride. It is proposed that [3H]NMSP binds to an additional set of accessory binding sites, presumably located more distantly from the agonist binding active site than the sites to which [3H]raclopride binds.     

Reconstitution of the type II [3H]estradiol binding site with recombinant histone H4

Previously, we identified the rat uterine nuclear type II [3H]estradiol binding site as histone H4 and an unknown 35 kDa protein with histone H4 immunoreactivity. Studies using calf thymus histones indicated that the 35 kDa protein was likely a dimer of histone H3 and H4. Further study of the type II site required methodology for producing sufficient quantities of recombinant histones, which retained ligand-binding properties. A variety of production methods produce sufficient quantities of histone for binding analyses were evaluated prior to finding a successful technique. The present studies describe techniques for the production of recombinant histones that retain the ligand binding properties of type II binding site. Binding studies with recombinant protein mirrored [3H]estradiol binding assays with rat uterine nuclear preparations. Histone H4 specifically binds [3H]estradiol with a low affinity (Kd approximately 20 nM) and in a cooperative fashion (curvilinear Scatchard plot; Hill coefficient approximately 4). Although histone H3 does not appear to bind ligand, regeneration of the histone H3/H4 pair produced a 35 kDa protein equivalent to the 35 kDa protein labeled with [3H]luteolin in rat uterine nuclear extracts and calf thymus histones. These data confirm the identification of histone H4 as a key component of the type II site. Future studies with recombinant proteins will lead to the identification of the "nucleosomal ligand-binding domain" for methyl-p-hydroxyphenyllactate (MeHPLA) and related ligands and delineation of their epigenetic control of gene expression and cell proliferation.     

Yeast alpha-factor and somatostatin enhance binding of [3H]estradiol to proteins in rat pancreas and Saccharomyces cerevisiae

Pancreatic tissue contains an [3H]estradiol-binding protein that requires a coligand in the steroid-binding reaction. The endogenous coligand appears to be the tetradecapeptide somatostatin. Yeast alpha-factor, a tridecapeptide pheromone that induces conjugation between haploid cells of opposite mating type, was found to be as effective as somatostatin in enhancing specific binding of [3H]estradiol to partially purified pancreatic protein. Supernatant fractions from yeast cells also contain an [3H]estradiol-binding protein. alpha-Factor can enhance specific binding of [3H]estradiol to such yeast fractions. Somatostatin, somatostatin analogues, and an analogue of alpha-factor enhanced binding of [3H]estradiol but did not inhibit cell growth or induce morphological changes in S. cerevisiae. Thus, it appears that coligand-requiring [3H]estradiol-binding activity and mating in yeast are not directly related.     

Estradiol binding by intact cells isolated from DMBA-induced mammary tumors of the rat

Study of hormone binding in intact cells enables one to examine binding under conditions which elicit a biological response. Cells from 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumors of the rat were enzymatically dispersed. More than 80% of these cells excluded trypan blue and were used to study binding of [3H] estradiol-17 beta. Specific binding was determined by subtracting the amount of [3H]estradiol bound in the absence and presence of 200-fold excess unlabeled estradiol. Specific binding at 37 degrees was maximal after 15 min. Steroid competition studies indicated that [3H]estradiol binding sites were relatively specific for estrogens, although there was a 9-18% inhibition of binding by androgens and progestins when present at 150-fold molar excess. Scatchard analyses of [3H]estradiol (0.15-5.0 nM) binding by whole cells suggest a single, high-affinity binding site (Kd = 7.5 x 10-10M) of low capacity (6.1 fmol/10(6) cells). More [3H]estradiol was translocated to the nucleus after 1 hr at 37 degrees than at 0 degrees. Preliminary studies indicated that incubations at 37 degrees result in appreciable metabolism of [3H]estradiol to other steroids and/or conjugates when examined by silica gel thin layer chromatography.     

[3H]Tryptamine Binding Sites Are Not Identical to Monoamine Oxidase in Rat Brain

Competition binding studies, subcellular distribution, and in vitro autoradiography were employed to compare the binding in rat brain of [3H]tryptamine with two radioligands for monoamine oxidase (MAO), [3H]pargyline, and [3H]1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine ([3H]MPTP). The MAO inhibitors pargyline, clorgyline, and deprenyl all yielded biphasic competition curves versus [3H]tryptamine. At low concentrations, these drugs stimulated binding by protecting the radioligand from MAO oxidation; at considerably higher concentrations, they inhibited binding by direct competition at the [3H]tryptamine binding site. In subcellular distribution studies, [3H]tryptamine was localized preferentially to the synaptosomal fraction, whereas [3H]pargyline showed greater binding to the mitochondrial fraction. Equilibrium binding studies revealed that the potencies of a series of seven compounds at inhibiting [3H]tryptamine binding were completely different from their potencies at inhibiting [3H]MPTP binding. Finally, the autoradiographic distribution of [3H]tryptamine binding in rat brain was different from that of [3H]MPTP and [3H]pargyline. We conclude that the [3H]tryptamine binding site in rat brain is not equivalent to MAO.