Biology and receptor interactions of estriol and estriol derivatives in vitro and in vivo

The biological effects of estriol (E3) have been studied in three estrogen targets, namely, the rat uterus in vivo and in vitro, in primary human endometrial cell cultures and in MCF-7 human breast cancer cells in culture. Studies on the temporal relationships between estrogen receptor binding and biological responses in the uterus using estriol and several more long-acting estriol derivatives, namely, 17α-ethynyl estriol, estriol-3-cyclopentyl ether, and 17α-ethynyl estriol-3-cyclopentyl ether, indicate that estriol is a short-acting compound with a brief duration of action. Estriol is a poor stimulator of uterine growth and plasminogen activator activity in vivo. Chemical modifications of the estriol molecule produce long-acting derivatives that result in a prolonged input of hormone receptor complexes into the nucleus and a prolonged and marked stimulation of uterine growth. In human endometrial cells in primary tissue culture, E₃ has 12% the affinity of estradiol (E₂) for cytosol estrogen receptor and it is quite effective yet slightly less potent than estradiol in stimulation of progesterone receptor synthesis. Low concentrations of E₃(10⁻¹⁰ M) stimulate growth of MCF-7 cells in vitro and dose-response curves show E₃ to be only slightly less effective than E₂. In these endometrial and breast cancer cell systems in vitro, there is no metabolism of E₃ while E₂ is metabolized to estrone.Hence, estriol is an effective estrogen in vitro. In vivo, it is short-acting, but it can be made a full estrogen agonist when given at a sufficiently high concentration or in a chemically modified form which prolongs its activity by enabling effective concentrations of the compound to be maintained in the blood and in target tissues.     

Metabolism of progesterone in rat uterus

The in vitro metabolism of progesterone was studied in uteri of untreated and estrogen stimulated immature rats. In intact uteri the rate of metabolism varied with the hormonal status of the animal in a concentration dependent manner. At a low (3 × 10^?9M) progesterone concentration the rate of ring A reduction was decreased in estrogen stimulated uteri. At a high progesterone concentration (3 × 10^?6M) the rate of ring A reduction was increased after estrogen treatment. The rate of reduction of the C20 ketone was increased after estrogen treatment at all concentrations of incubated progesterone. In dilute homogenates of uterus, estrogen stimulation always increased the rate of progesterone metabolism.Estrogen stimulation results in increased concentration of progesterone receptor in the uterus. It is proposed that increased activity of ring A reductases also occurs. The relative influence of these two factors on the metabolism of progesterone is dependent on the progesterone concentration in the incubation medium.     

Progestin binding sites in the rat hypothalamus pituitary and uterus

Specific, estrogen-inducible, nuclear radioactivity uptake has been demonstrated in the uterus, pituitary and hypothalamus of immature female rats after injection of a highly potent labelled progestin, R 5020 (17, 21-dimethyl-19-nor-pregna-4, 9-diene-3, 20-dione). A cytoplasmic progestin “receptor” has been characterized in these target tissues by an in vitro Dextran-coated charcoal adsorption method. R 5020 binds with the same intrinsic dissociation constant to the receptor present in these tissues ; it dissociates at the same rate from the uterine and pituitary receptor (k^?1 = 1×10^?2min^?1), but about 6 times slower than progesterone. This evidence, together with the similar hormone specificity in the uterus and pituitary, suggests that the progestin “receptor” is a similar entity in central and peripheral target tissues.     

Role of estrogen-induced uterine peroxidase in the metabolism of estradiol in vivo

The nature of the steroids present in the uteri of immature rats and in those of animals pretreated with estrogen which contained peroxidase was examined 10 min and 2 h after the in vivo administration of 0.25 μg of [³H]-estradiol. Only unchanged estradiol and some estrone were detected in the uteri of both groups of rats and most of the radioactivity was released into the medium after incubation with p-hydroxymercuribenzoate (PHMB), a sulfhydryl-blocking reagent. Similarly, an 80-fold excess of diethylstilbestrol (DES) injected together with [³H]-estradiol prevented the uterine uptake of the ³H-labelled compound and also displaced it from the uteri of both control and estrogen-pretreated rats. The concentration of estrogen-receptor in the cytosol of immature and estradiol-pretreated rats was determined by two different methods to show that the receptor had been replenished in the uteri of rats given estrogen 20 h before the experiment to induce peroxidase. These results indicate that even though uterine peroxidase can catalyze the metabolism and covalent binding of estradiol to protein in vitro, it is unlikely to limit the duration of estrogen action in the intact animal by this mechanism. Other possible roles for the induced enzyme are considered.     

Antiestrogenic and antifertility actions of anordrin (2α,17ga-diethynyl-A-nor-5α-androstane-2β,17β-diol 2,17-dipropionate)

Anordrin, administered in a single s.c. dose of 62.5 μg in sesame oil, stimulated sustained uterine growth (wet weight) when measured at 24 and 72 hr, but total soluble protein and total DNA per uterus was not increased. By comparison, 3 μg of estradiol-17β under the same conditions significantly increased all three parameters of uterine growth. Both of the above steroid treatments significantly increased nuclear estrogen receptor content of the uterus, but only the estradi-ol-17β treatment resulted in significantly elevated cytosol receptor content per uterus. Anordrin binds to the 8S estrogen receptor with an affinity of about 2 × 10⁵ M^-1 as determined by competition with [³H]estradiol-17β. The abortifacient activity of Anordrin when given orally (8 mg/kg b.w.) to mice on the 7th day of pregnancy was almost completely blocked by simultaneous oral administration of estradiol-17β (0.8 mg/kg b.w.). It is concluded that the actions of Anordrin on the uterus can be attributed to its antiestrogenic activities.     

Monoiodo-d-tyrosine,an artificial amino acid radiopharmaceutical for selective measurement of membrane amino acid transport in the pancreas

Using ¹¹C-labeled natural amino acids, the functional diagnosis of tissue metabolism has been actively studied. Our interest has been focused on developing a clinically available ¹²³I-labeled artificial amino acid with a single metabolic function. For this study, [¹²³I]3-iodo-d-tyrosine ([¹²³I]d-MIT) was selected. In vitro and in vivo studies using ¹²⁵I-labeled d-MIT indicated that it showed a high pancreatic accumulation, selective affinity for membrane active transport systems, and was stable against enzymatic deiodination. A canine scintigraphic study using ¹²³I-labeled d-MIT and kinetic analysis showed that it behaved as an “artificial amino acid” radiopharmaceutical with selective membrane amino acid transport affinity in the pancreas.     

Sex-specific alterations in estrogen receptor interactions following induced androgen imbalance in vivo

Androgens have been shown, under in vitro conditions, to be capable of impeding the rate of formation of estrogen-receptor complexes in target tissues of the rat. The present study was designed to investigate effects of abnormal androgen levels in vivo on various estrogen receptor systems. Serum levels of testosterone (T) and 5α-DHT were measured in adult neonatally-androgenized rats. The T/DHT ratio in the androgenized animal was 0.70, compared to 4.37 in the normal adult rat, and this was unaccompanied by any change in the sum of the 2 androgens. Estradiol levels were equivalent to those of normal rats in estrus. In addition to this animal model, castrate rats of both sexes which had been administered chronic high dosages of various androgens were examined. Equilibrium binding studies of cytosol from uterus, anterior putuitary and hypothalamus showed that estrogen receptors were not modified in either of these animal models, with specific reference to affinity of estradiol binding or concentration of binding sites. The association rate kinetics for estradiol-receptor complex formation in uterus and pituitary were unaffected by androgen administration to ovariectomized animals; however, in the corresponding male castrate model, interaction between estradiol and its pituitary cytosol receptor was accelerated by in vivo exposure to androgens, and the effect was dependent on the nature and level of the androgen used. Neonatally-androgenized rats also manifested an initial rate of estradiol-receptor interaction which was appreciably higher than control values. The reversibility of the androgen effect on the estrogen receptor was demonstrated in an in vitro protocol. The results indicate that the in vivo effects of androgens on estrogen receptor kinetics are sex-dependent and, where observed, any influence was of a stimulatory nature. Moreover, it appears that the nature of the androgen present in vivo is at least as important a determinant as the total androgen concentration, and that androgens do not engender permanent changes in the ability of the estrogen receptor to interact with estradiol.     

4-[123I]Iodospiperone as a ligand for dopamine DA receptors: In vitro and in vivo experiments in a rat model

Radioiodinated spiperone is of interest for dopamine (DA) receptor studies in the living human brain by single photon emission computed tomography (SPECT). Stimulated by data obtained with [¹¹C]-N-methyl-spiperone we synthesized 4-[¹²³I]iodospiperone and investigated the in vitro binding characteristics of this ligand to the striatal membrane of the rat and the in vivo distribution over various rat brain regions. The in vitro binding experiments showed that this radioligand displays about 10 times less affinity for the DA receptor than spiperone and specific binding, as shown with [³H]spiperone, was not observed. Displacement by butaclamol was not observed. The in vivo studies demonstrated that both 4-[¹²³I]iodospiperone and [³H]spiperone concentrate in striatal tissue, respectively, 1.9 and 3.5 times as high as in cerebellar tissue.Haloperidol pretreatment largely prevented this accumulation. In view of the obtained target-to-non-target ratios we believe, however, that this accumulation in brain areas rich in DA-receptors does not offer prospects for clinical receptor imaging with SPECT.     

Identification of thiol group at the estrogen-binding site on the cytoplasmic receptor from rabbit uterus by affinity labeling

The interaction of estrogen with its receptor from rabbit uterus was studied using the affinity labeling technique. An affinity labeling reagent, 3-hydroxy-17β-(p-nitrophenyldithio)-1,3,5(10)-estratriene (Reagent A) was synthesized. The compound was designed to meet two requirements: (1) it binds specifically to the steroid-binding site of the receptor; (2) it has functional group capable of forming a covalent bond with an amino acid residue at or near the binding site. The first requirement was demonstrated by competitive binding assay and sedimentation analysis. If the binding affinity of 17β-estradiol is defined as 100, that of Reagent A is found to be 0.05. It was shown that the binding was specific for the estradiol receptor. The second requirement was examined by extraction of Reagent A with simple model compounds. 10 μM Reagent A gave rise to approx. 1.3 μM p-nitrothiophenol in the presence of a large excess of l-cysteine or reduced l-glutathione under the experimental conditions employed, indicating that a covalent bond was formed between Reagent A and the model compounds. Furthermore, I have presented evidence by using polyacrylamide gel electrophoresis that ³H-labeled Reagent A is linked covalently to the estrogen-binding site on the receptor of rabbit uterus. The experiment also fulfilled the first requirement. These results indicate that the thiol group present at the binding site is directly involved in the estradiol-receptor binding.     

Novel effects of diosgenin on skin aging

Extracts of Dioscorea coomposita or Dioscorea villosa are consumed as supplemental health foods at the time of climacteric. The extracts contain large amounts of the plant steroid, diosgenin. Here, we studied the safety and efficacy of diosgenin against skin aging at the time of climacteric. In vitro, diosgenin enhanced DNA synthesis in a human 3D skin equivalent model, and increased bromodeoxyuridine uptake and intracellular cAMP level in adult human keratinocytes. The increase of bromodeoxyuridine uptake by diosgenin was blocked by an adenylate cyclase inhibitor, but not by antisense oligonucleotides against estrogen receptor α, estrogen receptor β or an orphan G-protein-coupled receptor, GPR30, indicating the involvement of cAMP but not estrogen receptor α, estrogen receptor β or GPR30. In vivo, administration of diosgenin improved the epidermal thickness in the ovariectomized mice, a climacteric model, without altering the degree of fat accumulation. In order to examine the safety of diosgenin, diosgenin and 17β-estradiol were administered to breast cancer-burdened mice. The results revealed that while 17β-estradiol accelerated the tumor growth, diosgenin did not show this effect. Our finding, a restoration of keratinocyte proliferation in aged skin, suggests that diosgenin may have potential as a safe health food for climacteric.     

Ser123 Is Essential for the Water Channel Activity of McPIP2;1 from Mesembryanthemum crystallinum

The increased expression of McPIP2;1 (MipC), a root-specific aquaporin (AQP) from Mesembryanthemum crystallinum, under salt stress has suggested a role for this AQP in the salt tolerance of the plant. However, whether McPIP2;1 transports water or another solute and how its activity is regulated are so far unknown. Therefore, wild type (wt) or mutated McPIP2;1 protein was expressed in Xenopus laevis oocytes. Then, the osmotic water permeability (P_f) of the oocytes membrane was assessed by hypotonic challenges. Selectivity of McPIP2;1 to water was determined by radiolabeled glycerol or urea uptake assays. Moreover, swelling and in vitro phosphorylation assays revealed that both water permeation and phosphorylation status of McPIP2;1 were significantly increased by the phosphorylation agonists okadaic acid (OA), phorbol myristate acetate (PMA), and 8-Br-cAMP, and markedly decreased by the inhibitory peptides PKI 14-22 and PKC 20-28, inhibitors of protein kinases A (PKA) and C (PKC), respectively. Substitution of Ser¹²³ or both, Ser¹²³ and Ser²⁸², abolished the water channel activity of McPIP2;1 while substitution of Ser²⁸² only partially inhibited it (51.9% inhibition). Despite lacking Ser¹²³ and/or Ser²⁸², the McPIP2;1 mutant forms were still phosphorylated in vitro, which suggests that phosphorylation may have a dual role on this AQP. Our results indicate that McPIP2;1 water permeability depends completely on Ser¹²³ and is positively regulated by PKA- and PKC-mediated phosphorylation. Regulation of the phosphorylation status of McPIP2;1 may contribute to control water transport through root cells when the plant is subjected to high salinity conditions.     

Effects of experimental ventilation and ambient Po2 on O2 uptake of the isolated cutaneous tissue in the carp,Cyprinus carpio

Effects of experimental ventilation and ambient Po₂ on cutaneous O₂ uptake in vitro were studied in the carp, Cyprinus carpio. Oxygen uptake rate of the isolated cutaneous tissue was determined by ventilating the epidermis side of the skin with normoxic water in flow-through respirometers. Oxygen uptake rate of the skin increased with ventilation rate across the skin between 2.5 and 40 ml/min and became 3.2 nmol/cm²/min at a flow rate of 40 ml/min, which corresponds to an apparent water velocity of 1.1 cm/sec. At a ventilation rate of 10 ml/min, oxygen uptake rate of the skin increased with the ambient Po₂ between 115 and 230 Torr and became constant (3.8 nmol/cm²/min) between 230 and 295 Torr. When both sides of the skin were ventilated with normoxic water, oxygen uptake rate of the skin increased and became 3.7 nmol/cm²/min at a flow rate of 20–40 ml/min. These results suggest that the oxygen requirement of the skin is 3.7–3.8 nmol/cm²/min at 21.3°C and that cutaneous O₂ uptake in vitro depends on experimental ventilation and ambient Po₂, consistent with values measured in vitro in the carp (ref).     

In vitro and in vivo D2-dopamine receptor binding with [123I]S(−)iodobenzamide ([123I]IBZM) in rat and human brain

As a promising dopamine D2-receptor imaging agent for single photon emission computerized tomography (SPECT), [¹²³I](S)-(−)-2-hydroxy-3-iodo-6-methoxy-N [(1-ethyl-2-pyrrolidinyl)methyl]benzamide ([¹²³I]IBZM) has recently been synthesized in a modified way along with its precursor, S(−)BZM, and the stereoisomer R(+)BZM. The present study applied this new product to investigate in vitro and in vivo D2-receptor binding in rat brain and in postmortem human brain. In vitro saturation binding curves with [¹²³I]IBZM for rat crude striatal membrane preparations yielded an affinity constant (K_d) of 0.28 nM confirming data in the literature. Displacement curves revealed an order of increasing potency as follows: R(+)BZM < S(−)sulpiride = < S(−)BZM < S(−)IBZM. A similar order was obtained when [³H]spiperone was used as ligand. For human putamen and caudate nucleus membranes slightly higher K_d values (0.49 nM) were obtained. Rank order of displacing potency for the various drugs was similar to that found in the rat preparations. In vivo uptake of [¹²³I]IBZM in rat brain following injection of 50 μCi (12–16pmol) in the tail vein revealed an increase in the striatum-to-cerebellum ratio from 1.5 at 5 min to 6.9 at 2 h. The olfactory tubercle-to-cerebellum ratio was also raised from 1.6 to 3.3. Other brain regions tested failed to show statistically significant enhancements. Coinjection of 40 nmol S(-)IBZM, 4μ mol S(−)BZM or 200 nmol haloperidol displaced [¹²³I]IBMZ when tested at 90 min. The use of 4μ mol R(+)BZM resulted in minor displacement only, demonstrating that stereospecificity of the displacement was present in vivo and in vitro. Displacements were also observed in substantia nigra and pons-medulla oblongata, but not in hippocampus or frontal and occipital cortex. The data provide the required background needed in order to initiate in vivo binding studies for D2-receptors in basal ganglia of human patients using [¹²³I]IBZM in SPECT analyses.     

Estrogen-induced metabolism of estradiol-17β in the rat uterus: A possible mechanism for the termination of estrogen action

Treatment of immature female rats with estrogen resulted in the induction of a peroxidase in the uterus which is able to convert estradiol-4-¹⁴C to water-soluble products in vitro. It is suggested that this reaction may be part of an adaptive response to limit the duration of action of estradiol-17β upon its target tissue.     

Hormonal regulation of macrophage collagenase activity.

Whereas peritoneal macrophages from nonpregnant guinea pigs were stimulated $\text{in vitro}$ by endotoxin to produce collagenase on the second day of culture, those from pregnant guinea pigs were incapable of this response. However, if the cells from pregnant animals were preincubated for one day prior to endotoxin stimulation, collagenase activity could be detected. Injection of either estrogen or progesterone into guinea pigs at doses comparable to those found during pregnancy prior to removal of the peritoneal cells also inhibited the $\text{in vitro}$ stimulation of collagenase production. The addition of these hormones $\text{in vitro}$ revealed that at 5 × 10^?6 M estrogen and progesterone inhibited 53% and 100% respectively of the collagenase activity. Addition of both hormones at a final concentration of 5 × 10^?7 M of each inhibited 87% of the activity indicating a synergistic effect since this concentration of either hormone alone was ineffective.     

Estriol and estradiol interactions with the estrogen receptor in vivo and in vitro

The cytosolic estrogen receptor (calf uterus) bound to estradiol (E₂) at 0°C changes from a state with fast into a state with slow E₂ dissociation rates when placed at 28°C. This temperature accelerated transition in receptor affinity for its ligand takes place within 10 min at 28°C. Similarly, receptor bound to estriol (E₃) at 0°C changes, when heated, from a state with fast into a state with slow E₃ dissociation. The main difference between RE₂ and RE₃ was that E₃ dissociates from unheated 8S RE₃ and heat-transformed 5S RE₃ at a much faster rate than E₂ from RE₂;In the mature ovariectomized rat a slow dissociating 5S receptor estrogen complex is found in nuclei 1 h after injection of [³H]E₂ or [³H]E₃. In vitro dissociation of these 2 estrogens from this nuclear bound receptor formed in vivo takes place at rates similar to those from heat-transformed cytosolic RE₂ or RE₃ complexes.Addition of pyridoxal 5'-phosphate (PLP) to the slow-dissociating heat-transformed 5S estrogen receptor complexes causes rapid dissociation of E₂ or E₃; this effect is dose-dependent and is not due to disruption of 5S dimers, since after PLP addition RE₂; and RE₃ sediment unchanged as 5S dimers.The presence of a large excess of non-radioactive 4S RE₃ does not interfere with the temperature induced rapid transition of 4S R[³H]E₂ complexes from the state with fast into a state with slow E₂ dissociation kinetics.A model is presented to explain the temperature induced biphasic estrogen dissociation from the receptor. It is proposed that the low affinity 4S RE₂ monomer undergoes a temperature and estrogen dependent conformation change, such that the ligand is “locked” into the receptor's binding site. This conformational change results in the formation of a high affinity 4S monomer from which estrogen dissociates at a slower rate. This reaction is independent from subsequent 4S to 5S dimerization (transformation). The different rates of ligand dissociation from the low and high affinity 4S receptors reflect the different interactions (hydrophobic and hydrogen bonding) of E₂ and E₃ with the estrogen binding domain.     

Pharmacokinetics of the SPECT benzodiazepine receptor radioligand [123I]iomazenil in human and non-human primates

The pharmacokinetics of [¹²³I]iomazenil (Ro 16-0154) in 5 healthy human volunteers were compared to those in 2 hypothermic and 3 normothermic anesthetized monkeys. Following intravenous injection in humans and monkeys, [¹²³I]iomazenil rapidly diffused outside the vascular bed and was cleared from the arterial plasma triexponentially. The clearance half-times in hypothermic animals were protracted to values closer to those of the human. [¹²³I]lomazenil was metabolized mainly to a polar radiometabolite (not extracted by ethyl acetate) in the human whereas an additional lipophilic radiometabolite was detected in the monkey. In vitro and in vivo studies showed that [¹²³I]Iomazenil established equal concentrations in association with the cellular and plasma component of the blood, indicating that the plasma clearance of [¹²³I]iomazenil mirrors that of the blood. Analysis of organs from a monkey given [¹²³I]iomazenil showed that the parent compound was actively taken up by peripheral organs; the polar radiometabolite accumulated mainly in the bile and the kidneys whereas the non-polar radiometabolite accumulated in the urine and kidneys. Greater than 90% of the radioactivity in the different regions of the brain was unchanged parent [¹²³I]iomazenil.     

Estradiol stimulates Akt, AMP-activated protein kinase (AMPK) and TBC1D1/4, but not glucose uptake in rat soleus

Post-menopausal women exhibit decreases in circulating estrogen levels and whole body insulin sensitivity, suggesting that estrogen regulates skeletal muscle glucose disposal. Thus, we assessed whether estrogen stimulates glucose uptake or enhances insulin sensitivity in skeletal muscle. Ex vivo muscle stimulation with 17β-estradiol (10 nM) resulted in a rapid (?10 min) increase in the phosphorylation of Akt, AMP-activated protein kinase (AMPK), and TBC1D1/4, key signaling proteins that regulate glucose uptake in muscle. Treatment with the estrogen receptor antagonist, ICI 182,780, only partly inhibited signaling, suggesting both an estrogen receptor-dependent and independent mechanism of estradiol action. 17β-Estradiol did not stimulate ex vivo muscle [³H]-2-deoxyglucose uptake or enhance insulin-induced glucose uptake, demonstrating discordance between the estradiol-induced stimulation of signaling proteins and muscle glucose uptake. This study is the first to demonstrate that estradiol stimulates Akt, AMPK, and TBC1D1/4 in intact skeletal muscle, but surprisingly, estradiol does not stimulate muscle glucose uptake.