Hormones of choice: the neuroendocrinology of partner preference in animals.

Partner preference behavior can be viewed as the outcome of a set of hierarchical choices made by an individual in anticipation of mating. The first choice involves approaching a conspecific verses an individual of another species. As a rule, a conspecific is picked as a mating partner, but early life experiences can alter that outcome. Within a species, an animal then has the choice between a member of the same sex or the opposite sex. The final choice is for a specific individual. This review will focus on the middle choice, the decision to mate with either a male or a female. Available data from rats, mice, and ferrets point to the importance of perinatal exposure to steroid hormones in the development of partner preferences, as well as the importance of activational effects in adulthood. However, the particular effects of this hormone exposure show species differences in both the specific steroid hormone responsible for the organization of behavior and the developmental period when it has its effect. Where these hormones have an effect in the brain is mostly unknown, but regions involved in olfaction and sexual behavior, as well as sexually dimorphic regions, seem to play a role. One limitation of the literature base is that many mate or 'partner preference studies' rely on preference for a specific stimulus (usually olfaction) but do not include an analysis of the relation, if any, that stimulus has to the choice of a particular sexual partner. A second limitation has been the almost total lack of attention to the type of behavior that is shown by the choosing animal once a 'partner' has been chosen, specifically, if the individual plays a mating role typical of its own sex or the opposite sex. Additional paradigms that address these questions are needed for better understanding of partner preferences in rodents.

Changes in interneuronal phenotypes regulated by estradiol in the adult rat hippocampus: a potential role for neuropeptide Y.

Ovarian hormones regulate pyramidal cell synapse formation and excitability and interneuronal GABAergic tone in the CA1 region of the adult female rat hippocampus. The role of 17beta-estradiol in these effects is complex and appears to involve a subset of hippocampal interneurons, which express different calcium-binding protein and neuropeptide phenotypes and nuclear estrogen receptor alpha. We found that, in the hippocampus, nuclear estrogen receptor alpha-immunoreactive interneurons co-express neuropeptide Y, calbindin-D28k and calretinin but do not parvalbumin or cholecystokinin. Moreover, a proportion of neuropeptide Y-immunoreactive interneurons co-expresses calbindin-D28k and calretinin. This pattern is similar in the presence or absence of 17beta-estradiol treatment in ovariectomized rats. We then used immunohistochemistry and in situ hybridization to determine whether 17beta-estradiol treatment regulates expression of CA1 interneuronal phenotypic markers via nuclear estrogen receptor alpha activation. We found that 17beta-estradiol treatment of ovariectomized rats increased neuropeptide Y mRNA levels (25%) and the neuropeptide Y mRNA-associated grain density per cell (11%), as well as the number of neuropeptide Y-immunoreactive cells (11%), predominantly in the pyramidal cell layer (stratum pyramidale). Treatment with CI628, a selective estrogen response modulator that acts as an antagonist for nuclear estrogen receptor, blocked 17beta-estradiol-induced increase of neuropeptide Y mRNA levels. 17beta-Estradiol treatment did not alter the number of parvalbumin, calretinin, and cholecystokinin immunoreactive cells, nor mRNA levels for parvalbumin and cholecystokinin. Therefore, the present study has identified neuropeptide Y expression as the main interneuronal phenotype that co-expresses nuclear estrogen receptor alpha and shown that neuropeptide Y is responsive to 17beta-estradiol in CA1 pyramidal cell layer. We suggest that 17beta-estradiol may regulate neuropeptide Y expression mediated by nuclear estrogen receptor alpha-dependent activation in a subset of hippocampal interneurons, and we speculate that subsequent neuropeptide Y release may indirectly contribute to regulate glutamate-dependent neuronal activity in the adult rat hippocampus.

Selective estrogen receptor modulators regulate phasic activation of hippocampal CA1 pyramidal cells by estrogen.

Previous studies demonstrated that estrogen induces two sequential waves of CA1 pyramidal cell activation, evidenced by induction of c-Fos at 2 and 24 h after a single estrogen treatment. The second wave of activation is paralleled by suppression of immunoreactivity for glutamic acid decarboxylase-65kD (GAD65) in CA1 and decreased synaptic inhibition of CA1 pyramidal cells. Here, we report that pretreatment with either of the selective estrogen receptor (ER) modulators, tamoxifen (T) or CI628, has no effect on the first wave of c-Fos expression at 2 h but completely blocks the second wave of c-Fos and the suppression of GAD65 at 24 h. Interestingly, T, given 4 h after estrogen, failed to block c-Fos expression or suppression of GAD65 at 24 h. Electrophysiological experiments showed that the T metabolite, 4OH-T, or CI628 can inhibit the so-called rapid estrogen effect, to potentiate excitatory postsynaptic currents (EPSCs) in CA1 pyramidal cells. Thus, estrogen seems to act within 4 h via classical ERs and/or a rapid estrogen effect, such as EPSC potentiation, to produce activation/disinhibition of pyramidal cells 24 h later. In contrast, the initial activation of pyramidal cells, at 2 h after estrogen, seems to involve neither classical ERs nor rapid potentiation of EPSCs.

Effects of estrogen antagonists and agonists on the ACTH response to restraint stress in female rats.

Previous studies have found that female rats are less sensitive than males to hypothalamic-pituitary-adrenal axis feedback inhibition by exogenous glucocorticoid administration. To determine whether estrogen contributes to this sex difference, we examined the effects of the estrogen antagonists tamoxifen and C1628 on the ACTH and corticosterone responses to restraint stress. CI628 increased both the ACTH and corticosterone response to restraint stress, and tamoxifen increased the ACTH response to restraint. Using overiectomized female rats, we also examined the effects of seven days of estradiol and/or progesterone replacement. Low dose estradiol decreased the ACTH but not the corticosterone response to restraint stress while progesterone had no effect on ACTH or corticosterone responses. The combination of estradiol and progesterone also decreased the ACTH response to stress, and the magnitude of the effect did not differ from that found with estradiol treatment alone. These data suggest that in the physiological range estradiol is an important inhibitory factor in the hypothalamic-pituitary-adrenal stress response of females.

Inhibition of dendritic spine induction on hippocampal CA1 pyramidal neurons by a nonsteroidal estrogen antagonist in female rats.

Estrogens regulate the formation of excitatory synaptic connections in the hippocampus of female rats. Because the adult hippocampus has a very low concentration of intracellular estrogen receptors, it is unclear whether a conventional genomic mechanism is involved. Nonsteroidal estrogen antagonists are useful tools to study estrogen action because they can provide pharmacological data in favor of a particular pathway of estrogen action and evidence against other pathways. To investigate the role of intracellular estrogen receptors in the estrogen induction of synapse formation, we took advantage of previous studies in which we had shown that an estrogen antagonist, CI-628, enters the brain and blocks estrogen induction of progestin receptors to study whether the same antagonist would either mimic or block effects of estradiol to induce excitatory spine synapses. Using silver impregnation of neurons by the single section Golgi technique and morphometric analysis, we found that CI-628 effectively prevented estrogen induction of spines on CA1 pyramidal neurons, without having any agonist effects of its own. This result is consistent with an action of estradiol via intracellular estrogen receptors that are known to be expressed by interneurons within the hippocampus.

Ovarian steroid regulation of angiotensin II-induced water intake in the rat.

Spontaneous water intake as well as thirst elicited by ANG II has been shown to be influenced by the stage of the estrous cycle in the female rat. In these experiments, the contribution of each of the ovarian steroid hormones to the regulation of water intake was examined. Ovariectomized female rats were given replacement doses of estrogen, progesterone, or both, and their responsiveness to an intracerebroventricular injection of ANG II was tested. Forty-eight-hour treatment with estradiol benzoate attenuated ANG II-induced thirst by as much as 70% compared with control animals. The effect of estrogen on drinking was dose dependent and could be completely blocked with concurrent administration of the antiestrogen CI-628. In contrast, progesterone, given alone or after estrogen, did not significantly affect ANG II-induced water intake when animals were tested at 4 or 24 h after steroid administration. A central interaction between the peptide hormone ANG II and estrogen, involving a genomic mechanism, may underlie the cyclicity in water intake behavior observed in the rat.

Sexual preference and feminine and masculine sexual behavior of male rats prenatally exposed to antiandrogen or antiestrogen.

Male rats were prenatally (Day 10-19 of pregnancy) exposed to an antiestrogen, nitromifene citrate (CI628, 1 mg/rat), or an antiandrogen, cyproterone acetate (CA, 10 mg/rat), and in adulthood were examined for their exhibition of male-typical and female-typical behavior pattern. Treatment with CI628 abolished the capacity of the adult intact male to ejaculate, enhanced his potential to exhibit feminine sexual behavior, and decreased the intensity of the level of female-oriented behavior in a two-choice stimulus situation (estrous female vs active male). The administration of testosterone (T) did not alter these behaviors. Males exposed to CA showed low levels of lordosis behavior and normal levels of female-oriented preference. Further, they showed increased frequency of mounts and decreased number of intromissions, and only a few males ever ejaculated. Macroscopic inspection of the genital organs of the CI628-treated males revealed complete absence of the prostate. The dissections of the CA-treated males revealed a poorly developed penis and a blind-ending vagina. It was concluded that prenatal estrogen (E) is involved (1) in determining the development of mechanisms destined to mediate the display of male-typical behaviors in adulthood, (2) in suppressing the development of mechanisms of female-typical behaviors, and (3) seems to stimulate neural mechanisms influencing sexual preference behavior in the adult.

Sexual differentiation of copulatory behaviour in the male chick requires gonadal steroids.

1. Embryonic injections of 0.3 mg/egg of tamoxifen (TAM), 0.2 mg/egg CI-628 (both antioestrogens), 0.5 mg/egg (ATD (aromatisation inhibitor), or antibodies to oestradiol (E), all suppressed male copulatory activity (MCA) in young male chicks. 2. Embryonic injections with either flutamide (F, androgen antagonist) or high dose of antibodies to testosterone (T) only slightly suppressed MCA. 3. TAM had no effect on embryonic plasma LH levels, 24 and 48 h after injection. 4. It seems that at the embryonic stage oestradiol is required for the normal differentiation of MCA.

Aromatization mediates aggressive behavior in quail.

Although testosterone (T) stimulates aggressive and reproductive behaviors in males of many vertebrate species, it is now known that the full expression of T action in the brain requires aromatization to estradiol (E2) and subsequent interaction of locally formed E2 with nuclear estrogen receptors. In experiments reported here, we used a behavioral test which quantifies the response of an individual male Japanese quail (Coturnix coturnix japonica) to the visual stimulus of a conspecific. We have called this behavior aggression because it shares many features in common with traditional measures of aggression, e.g., predicting dominance and subordinance. Nevertheless, the behavior probably also combines a complex steroid-sensitive masculine behavior. The advantage of this test is that it allows the discrimination of individual differences in masculine behavior but avoids fighting and sexual encounters per se, thereby reducing effects of learning, a problem with previous tests of avian aggression. In addition, this test has been applied usefully to identify neuroendocrine correlates to male behavior. Using this test, the arousal of reproductively inactive males (hereafter referred to as aggression) is activated by administration of T or estradiol benzoate (EB), but not by 5 alpha-dihydrotestosterone (DHT). T-induced aggression was blocked by the aromatase inhibitor 4-hydroxyandrostenedione (OHA), an effect partially reversed by treatment with EB. In addition, OHA or the estrogen receptor blocker CI-628 reduced aggressiveness of reproductively active males whereas the androgen receptor blocker flutamide had no effect. Results with the 5 alpha-reductase inhibitor N,N-diethyl-4-methyl-3-oxo-4-aza-5 alpha-androstane-17 alpha-carboxyamide (4-MA) were equivocal. Additionally, treatment of reproductively inactive quail with T or E2 but not DHT increased aromatase activity in the hypothalamus-preoptic area (HPOA). We conclude, therefore, that T to E2 conversion is essential for the activation of aggressiveness in this species. Although locally formed estrogen exerts its effects on aggression in part by increasing activity of aromatase per se, analysis of the time course of behavioral induction or suppression by the various treatments suggests that the response has multiple components, including both short latency, receptor-independent and long latency, receptor-dependent events.

Differential effects of ovarian steroids and triphenylethylene compounds on macromolecular uptake and thymidine incorporation in the mouse uterus.

In the rodent uterus, estrogen elicits a biphasic response i.e. an early phase (Phase I) and a late phase (Phase II). Estradiol-17 beta (E2) and estriol (E3), as well as triphenylethylene (TPE) compounds, CI-628 and clomiphene citrate (CC), were used to characterize Phase I and Phase II responses in uterine preparation for implantation in the mouse. While uterine macromolecular uptake (vascular permeability), a Phase I response, was studied in progesterone (P4)-primed animals, uterine [3H]thymidine incorporation (DNA synthesis), a Phase II response, was investigated with and without P4-priming. In the P4-primed uterus, all compounds, except CC, significantly increased uterine macromolecular uptake as determined by interstitial tissue accumulation of [125I]bovine serum albumin [( 125I]BSA). DNA synthesis as determined by cellular incorporation of [3H]thymidine was modulated by P4, estrogens and TPE compounds in a cell-type specific and temporal manner. As a single injection and in the absence of P4, E2 induced [3H]thymidine incorporation in the luminal and glandular epithelium at 18 and 24 h. E3 was inferior to E2 in this response. On the other hand, treatment with P4 for 1 day or 4 days induced [3H]thymidine incorporation primarily in stromal cells. However, stromal cell incorporation was potentiated when P4 treatment was combined with estrogens or TPE compounds. These results reveal the relative importance of Phase I and cell-type specific Phase II responses in uterine preparation for implantation.

Characterization of MCF 7 breast cancer cell growth inhibition by the antiestrogen nitromifene (CI 628) and selected metabolites.

Besides undergoing O-demethylation in vivo, the triarylethylene antiestrogen nitromifene [1-(4-(2-pyrrolidinylethoxy)phenyl)-1-(4-methoxy)-phenyl-2-phenyl- 2- nitroethene, 1] undergoes biotransformation via nitroreduction, ethene bond cleavage, and pyrrolidine ring oxidation affording ketone metabolites 2 and 3 and a lactam metabolite 4. Estrogen receptor (ER) affinities of 1, 2, and 4 were, in turn, 1.7, 0.1, and 3.8% that of estradiol in MCF 7 human breast cancer cells, and these compounds inhibited by 50% the proliferation of MCF 7 cells at respective concentrations of 1.1, 5.6, and 2.0 microM. The inhibitory effect of 4 was fully reversible by estradiol, but that of 2 was only partially reversible. Also 3, which did not interact with ER, inhibited proliferation by 44% at a concentration of 10 microM. These results suggested that in contrast to 4, the effects of 2 and 3 were due in part to interaction with sites distinct from ER. Antiestrogen binding sites and calmodulin have been suggested to mediate antiproliferative effects of drugs. Interaction of ligands with the former sites has been proposed to antagonize the growth promoting effect of histamine. Although 2 and 3 had high affinities for these sites, their inhibitory effects on MCF 7 cell growth were largely unaffected by the presence of histidine, the source of intracellular histamine. Thus, the relationship between antiestrogen binding site affinity and antiproliferative effects of 2 and 3 was not clarified. In contrast, MCF 7 cell growth suppression potencies paralleled calmodulin antagonist potencies of 1 and 2 suggesting that interaction of 1 and 2 with calmodulin may contribute to their anticancer effects.

Estrogenic effects of phenol red on rat pituitary cell responsiveness to gonadotropin-releasing hormone.

This study investigated whether phenolsulfonphthalein (PR), a common pH indicator in tissue culture media, affects luteinizing hormone (LH) secretion from rat pituitary cells or 17 beta-estradiol (E2) augmentation of pituitary responsiveness to gonadotropin-releasing hormone (GnRH). PR enhanced GnRH-stimulated LH secretion and shifted the GnRH dose-response curve leftward with a relative potency ratio of 0.24 +/- 0.09 (+/- SE; p less than 0.01). The effect of E2 on LH release was significantly diminished by PR, which elevated GnRH-stimulated LH secretion in the absence of E2. This phenomenon was elicited by PR from different sources and was inhibited by the antiestrogen Cl628. Thus, PR exerted estrogen-like effects on rat pituitary cells and caused an underestimation of the degree to which E2 enhanced GnRH-stimulated LH secretion.

Solubilization of a tamoxifen-binding protein. Assessment of its molecular mass.

Recent findings point to a role of Antioestrogen-Binding Site (ABS) in some of the growth-modulatory effects of antioestrogens. In the present study, a method for the solubilization of ABS from rat uterus microsomal fractions by using 3-(3-cholamidopropyl)dimethylammonio-1-propanesulphonate (CHAPS; 20 mM) and KCl (0.4 M) is described. Decreasing the CHAPS concentration below the critical micelle concentration led to long-term stabilization of the protein. All of the membrane-bound ABS was recovered in the extract, and only one class of binding site, with a high affinity for [3H]tamoxifen (KA = 5 x 10(8) M-1) was detectable. This binding was time-dependent and reversible: at 4 degrees C, the association rate constant was ka = 7.2 x 10(4) M-1.s-1, and the reverse rate constant was kd = 1.0 x 10(-4) s-1. Solubilized ABS exhibited an affinity and specificity similar to those of the membrane-bound sites. Under disaggregating conditions, solubilized ABS had an apparent sedimentation coefficient, s20,w, of 5.2 S and a Stokes radius of 6.4 nm. From these two values, molecular masses of 160,000 Da for the detergent-ABS complex, and 110,000 for the protein moiety, were estimated. Assessment of the size of the membrane-bound ABS by a radiation inactivation technique is also described. The 'radiation inactivation size', corresponding to the mass of 1 mol of protein structure(s) whose associated tamoxifen-binding activity is abolished after a single hit by ionizing radiation, was estimated to be 80,000 Da.

Pharmacodynamic and biological effects of anti-estrogens in different models.

The biological response to anti-estrogens is very variable and depends on the animal species considered, the target organ, the parameter studied, and the experimental conditions. Anti-estrogens can bind specifically, (1) to the estrogen receptor, (2) to the typical anti-estrogen specific binding site, and (3) to low density lipoproteins in the plasma. Using a monoclonal antibody against the estrogen receptor, different immunological characteristics of the anti-estrogen-receptor complex can be observed. This difference could explain some of the different biological effects. Studies using different human mammary cancer cell lines (hormone-dependent) show that anti-estrogens are active in decreasing cell proliferation. Also, anti-estrogens can block proteins specifically produced by these cells. Some of these proteins could act as growth or inhibitory factors. Estrogen sulfates are the main precursors of estradiol in breast tissues and this conversion is significantly decreased by anti-estrogens. It is accepted that the main pathway of action of anti-estrogens is through the estrogen receptor, but recent information suggests the possibility that this is not the only step in the mechanism of action of anti-estrogens.

Differential inhibition of estrogen and antiestrogen binding to the estrogen receptor by diethylpyrocarbonate.

Diethylpyrocarbonate differentially inhibited the specific binding, in lamb uterine cytosol, of estradiol (inhibition approximately 90% with 4 mM reagent) and 4-hydroxytamoxifen (inhibition approximately less than 50% with 4-16 mM reagent), a potent triphenylethylene antiestrogen. Saturation analysis experiments indicated that the effects of diethylpyrocarbonate were due to progressive but differing decreases in the concentration of binding sites for the two ligands, with no apparent change in the affinity constants. However, competitive binding and dissociation experiments evidenced that steroidal and nonsteroidal estrogens still bound, but with very low affinities, to diethylpyrocarbonate-modified receptor (greater than 1000-fold decrease in affinity) whereas the affinities of triphenylethylene antiestrogens were much less affected (less than 10-fold decrease). Both ligands prevented the inactivation of the estrogen receptor by diethylpyrocarbonate, estradiol being more efficient than 4-hydroxytamoxifen. These data indicate that the action of diethylpyrocarbonate results in the formation of two populations of estrogen receptor that are quantitatively nearly equivalent: the first does not bind estrogens or antiestrogens; the second does not bind estrogens significantly but still interacts with antiestrogens at a high affinity. The simplest interpretation is that these two populations arise from mutually exclusive modifications by diethylpyrocarbonate of at least two aminoacid residues located at or close to the ligand binding site; modification of one residue totally prevents the binding of estrogens and antiestrogens; the modification of the second impairs only the binding of estrogens. Considering that (i) hydroxylamine, which specifically reverses the diethylpyrocarbonate-induced modification of histidine and tyrosine residues, restored a large part (greater than 80%) of the estradiol- and 4-hydroxytamoxifen-binding capacity of diethylpyrocarbonate-inactivated cytosol, and that (ii) similar differential inhibition of estrogen and antiestrogen binding was observed following the action of tetranitromethane, it is likely that these residues are histidine(s) and/or tyrosine(s). These results evince a marked difference in the interaction of estrogens and triphenylethylene antiestrogens with the estrogen receptor, which could account for the altered activation of the receptor by triphenylethylene antiestrogens. Consequently, the screening of ligands with modified steroid receptors could be a useful method for distinguishing between potential hormone agonists and antagonists.

A paradoxical effect of an antiestrogen, CI-628, on implantation in the mouse.

Some triphenylethylene compounds (TPE), commonly known as antiestrogens, have been reported to interfere with pregnancy. This effect of these TPE compounds was attributed to their antagonistic effects to estrogen in the uterus and/or the embryo. Paradoxically, recent evidence suggests that several TPE compounds act as estrogen agonists in inducing embryo implantation in the uterus of ovariectomized, progesterone-treated, delayed-implanting mice. The present study was undertaken to determine the effect of CI-628, a TPE compound, on normal pregnancy and its site of action. Mice were given a single injection of CI-628 (1.5, 5, or 15 micrograms/mouse, ip) on Day 3, Day 4, or both days and killed on Day 8 for examination of implantation sites or unimplanted embryos. CI-628 at 5 and 15 micrograms reduced the implantation rate when injected on Day 3 or Days 3 and 4, but not when administered only on Day 4. The lower dose of the compound (1.5 micrograms) was ineffective in altering implantation. The implantation failure was overcome to various degrees by simultaneous injection of progesterone (2 mg/mouse, sc), but not by estradiol-17 beta (20 ng/mouse, sc). These results suggest that Day 3 is critical for CI-628 to exert its inhibitory effect and that this compound appears to affect ovarian progesterone synthesis and/or secretion. This suggestion is consistent with our findings that animals that showed implantation following injection of 5 or 15 micrograms of CI-628 had higher serum progesterone levels than those without implantation. Serum progesterone levels of animals bearing implantation sites following 5 micrograms of CI-628 on Day 3 or Day 4 were comparable to those of vehicle-treated controls, whereas the level of this steroid in animals treated with 15 micrograms of CI-628 and having implantation sites was relatively lower. However, a small number of animals that had implantation following treatment with 5 micrograms of CI-628 on Days 3 and 4 showed lower progesterone levels compared to those of the controls, but not different from those of some of the treated animals without implantation. Treatment with estrogen could not restore progesterone levels or implantation. In summary, the results suggest that CI-628 acts as an estrogen antagonist in interfering with implantation at the ovarian level in intact, pregnant mice.

Role of early and late oestrogenic effects on implantation in the mouse.

Oestrogen action in the uterus is expressed in an early phase (Phase I) and a late phase (Phase II). The role of this biphasic oestrogen action in implantation is not clear. To determine the relative importance of Phase I and II responses, triphenylethylene compounds (CI-628, LY-117018, nafoxidine, clomiphene citrate and tamoxifen) and oestrogens (oestriol and oestradiol-17 beta) were used in a physiologically relevant experimental system for studying implantation. All compounds elicited uterine water imbibition to various degrees in ovariectomized-progesterone-treated mice at 6 h (Phase I response) and their effectiveness in inducing implantation in delayed implanting mice correlated with their respective potency to increase uterine wet weight. This suggests that Phase I might be an essential component of oestrogen action in implantation and that the efficiency of a compound to elicit a Phase I response might serve as a predictive indicator of its potential action on implantation.

Studies on the ligand specificity and potential identity of microsomal antiestrogen-binding sites.

Synthetic nonsteroidal antiestrogens are bound intracellularly by two high affinity saturable bindings sites, the estrogen receptor and the microsomal antiestrogen-binding site (AEBS). In order to further define the structural requirements for ligand binding to AEBS from rat liver and the MCF 7 human breast cancer cell line, the relative binding affinities of an extensive series of structurally related ligands were investigated using competitive binding assay techniques. The groups of compounds studied were: analogues of the triphenylethylene antiestrogens, Cl 628 and tamoxifen; analogues of cyclofenil; bibenzyl and stilbene derivatives; analogues of the cytochrome P-450 inhibitor SKF-525A; phenothiazine derivatives; and a series of structurally related compounds with a variety of pharmacological activities. High affinity binding to AEBS required the presence of both a hydrophilic basic aminoether side chain and a hydrophobic aromatic ring structure (di- or tricyclic for maximal affinity). Structural modifications to either influenced binding affinity. Aromatic substitution either raised (CF3) or lowered (OH, OCH3) affinity, apparently by electronic effects transmitted through the benzene nucleus. Side chain structure was the major determinant of binding affinity, but its influence was complex and dependent upon terminal amino group structure, side chain branching and substitution, and tissue source of AEBS. Optimal binding affinity was shown by side chains bearing basic heterocyclic amino terminal groups. Other cellular sites that are known to bind antiestrogens with relatively high affinity include calmodulin, cytochrome P-450, and histamine, dopamine, and muscarinic receptors. Binding studies using a variety of pharmacologically active and radiolabeled ligands selective for these sites, including those for dopamine D1 and D2 receptors ([3H]fluphenazine, [3H]flupenthixol, [3H]spiperone, and [3H]SCH 23390) and histamine H1 receptors ([3H]pyrilamine), demonstrated that several of these compounds interact with AEBS with high affinity. However, the ligand specificity and other binding properties of the AEBS as determined by competitive binding studies and Scatchard analysis show this site to be a molecular entity truly distinct from these other cellular binding sites.

Inhibition of testosterone metabolism in the brain and cloacal gland of the quail by specific inhibitors and antihormones.

The effects of antioestrogens, antiandrogens and of various inhibitors of testosterone metabolism on testosterone metabolism in the quail hypothalamus and cloacal gland were studied by an in-vitro radioenzymatic assay. It was found that antioestrogens and antiandrogens generally had little or no effect on aromatase and 5 alpha- and 5 beta-reductases of testosterone, except when used at very high doses. The 5 alpha-reductase inhibitor, 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one, inhibited both 5 alpha- and 5 beta-dihydrotestosterone production without markedly affecting aromatase activity. Surprisingly, the aromatase inhibitor, 1,4,6-androstatriene-3,17-dione, inhibited not only the production of oestradiol but also that of 5 beta-dihydrotestosterone and, to a lesser extent, 5 alpha-dihydrotestosterone. These unexpected properties should be taken into account when interpreting the results of in-vivo experiments using these compounds.

An evaluation of the interactions of antiestrogens with pituitary and striatal dopamine receptors.

We have examined the ability of various antiestrogens (AE's) to compete with 3H-spiroperidol for binding to membrane preparations from striatal tissue and anterior pituitary glands of immature female rats in order to determine the affinity of binding of AE's to D-2 dopamine receptors. Scatchard analyses revealed the presence of a single class of high affinity receptor sites in both the striatum and pituitary with a dissociation constant (Kd) of 0.33 nM and 0.40 nM, respectively, for the dopamine antagonist spiroperidol. The AE's tamoxifen, 4-hydroxy-tamoxifen (TAM-OH), CI-628, LY 117018, and a structurally related compound t-butyl-phenoxyethyl diethylamine (BPEA) were all able to compete with spiroperidol for binding to D-2 receptors and demonstrated relative binding affinities of 0.4-0.06%, with spiroperidol set at 100%. Dopamine displayed a lower affinity, 0.01%. Estradiol failed to compete with spiroperidol for D-2 receptor binding while the non-steroidal estrogen diethylstilbestrol (DES) showed very weak competition. For the lipophilic AE's, alteration of the level of their non-specific binding greatly affected their relative affinities in these competitive binding assays. The amine side chain on an aromatic ring appears to be a critical structural requirement in allowing the AE's to bind to the dopamine receptor. The relatively low affinity of AE's for the dopamine receptor and the high degree of interaction of AE's with other proteins suggest that only limited occupancy of D-2 receptors by AE's is likely in vivo.