[Clinical use of promegestone, a progestational agent with high specificity for receptors].
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Binding of progesterone with the oviduct cytosol fraction of estrogen-primed quail (Coturnix coturnix japonica).
Progesterone-specific binding components were detected in the cytosol fraction of enlarged oviducts from estrogen (diethylstilbestrol)-primed immature Japanese quail (Coturnix coturnix japonica) females by several techniques using [3H]promegestone. The oviduct as a target tissue of progesterone is the most efficient in [3H]promegestone binding, and muscle and intestine as nontarget tissues and plasma are less efficient as expected. By using [3H]promegestone for binding, the possibility of blood contamination of the oviduct may have been eliminated with the detection of a specific binding site. The participation of protein in the steroid-binding site was inferred from the destruction of the binding component by protease, but not by RNase or DNase. The interaction with [3H]promegestone in low salt conditions has a high affinity (Kd 0.69 nM) and low capacity (the number of binding sites per milligram of protein is about 1.3 pmol). Six unlabeled steroids were tested as competitors for binding to [3H]promegestone in vitro. Progesterone-like steroids competed specifically with [3H]promegestone: progesterone congruent to promegestone greater than deoxycorticosterone greater than testosterone much greater than estradiol-17 beta greater than cortisol. These chemical properties show that the progesterone binding protein present in the oviduct of estrogen-primed quail is essentially similar to that obtained from chick oviduct. In addition, heterogeneity of the [3H]promegestone binding components was shown. The binding component was eluted as an aggregate on gel (Bio-Gel A-0.5m) column chromatography in low salt conditions which reverted to two major peaks, tentatively named I (molecular weight, about 110,000) and II (about 41,000), in the presence of high salt (0.3 M KCl). The relative amounts of the two peaks differed. It was interesting that peak II of the small component was not found in the estrogen-primed chick and was a distinctive one in quail. On the other hand, both peaks were recovered with 0.3 M KCl elution on DEAE-cellulose column chromatography. These studies suggest that this binding component may function as a biological receptor for progesterone in the estrogen-enlarged oviduct, and the problems to be solved are under examination.
Animals↗
Cholesterol binding at the cholesterol recognition/ interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide.
We previously defined a cholesterol recognition/interaction amino acid consensus (CRAC; ATVLNYYVWRDNS) in the carboxyl terminus of the peripheral-type benzodiazepine receptor (PBR), an outer mitochondrial membrane protein involved in the regulation of cholesterol transport into the mitochondria, the rate-determining step in steroid biosynthesis. We examined (i) the PBR-cholesterol interaction by UV crosslinking of the C17 side-chain containing progestin, promegestone, and (ii) the role of the CRAC domain of PBR in Leydig cell steroidogenesis by using a transducible peptide composed of the TAT domain of HIV and the CRAC domain of PBR. [(3)H]Promegestone photoincorporated into recombinant PBR, and this labeling was displaced by cholesterol. [(3)H]Promegestone also photoincorporated into the TAT-CRAC peptide. [(3)H]Promegestone crosslinking to TAT-CRAC could be displaced by cholesterol and promegestone, with IC50 values of 1 and 200 microM, respectively. TAT-CRAC efficiently transduced into MA-10 Leydig cells and inhibited the hCG- and cAMP-stimulated steroid production in a dose-dependent manner. TAT-CRAC did not affect the hCG-induced cAMP synthesis and the 22R-hydroxycholesterol-supported steroidogenesis. Mutated TAT-CRAC lost its ability to bind [(3)H]promegestone and to inhibit the hCG-stimulated steroidogenesis. These results show that TAT-CRAC binds cholesterol and competes for cholesterol interaction with endogenous PBR, suggesting that the cytosolic carboxyl-terminal domain of PBR is responsible for taking up and bringing steroidogenic cholesterol into the mitochondria.
Amino Acid Sequence↗
Enzyme kinetic and inhibition analyses of cytochrome P450XVII, a protein with a bifunctional catalytic site. Quantification of effective substrate concentrations at the active site and their significance for intrinsic control of the hydroxylase/lyase reaction sequence.
An enzyme kinetic analysis of the cytochrome P450XVII-dependent testicular steroid-17 alpha-hydroxylase/17,20-lyase system was performed which catalyzes simultaneously, at one active site, the formation of androstenedione from progesterone (productive pathway, coordinated performance of both hydroxylase and lyase activities) and the formation of 17 alpha-hydroxyprogesterone as the intermediate (abortive pathway, isolated performance of hydroxylase activity only). Progesterone was used as the substrate and promegestone (17 alpha, 21-dimethyl-4,9(10)-pregnadiene-3,20-dione) or estradiol as competitive inhibitors, and description of the data was based on the discrimination of those alternative routes of progesterone metabolism. The overall catalytic activity of P450XVII obeyed Michaelis-Menten kinetics (control incubations: V = 440 nM/min with 80 nM P450XVII, Km = 140 nM), and KI values amounted to 6.8 microM for promegestone and to 23.5 microM for estradiol. In contrast, analysis of the rates of both abortive and productive events yielded curvilinear Eadie-Hofstee plots. The former presented an apparent negative cooperative behavior with nh = 0.78 in the region of Kh, while the latter presented an apparent substrate inhibition behavior with the maximal rate occurring at that progesterone concentration where nh for the abortive path reached its minimum. Consequently, the ratio of productive versus abortive catalytic events decreased with increasing substrate supply, but did not reach zero. Effective substrate concentrations [S]eff with respect to either the productive or the abortive pathway were derived from Hill plots. Linearization of Eadie-Hofstee plots and parallel calculation methods using these [S]eff instead of [S] yielded V = 236 nM/min and [S]eff(0.5) = 131 nM for the abortive events and V = 217 nM/min and [S]eff(0.5) = 38 nM for the productive events. Both promegestone and estradiol were identified as competitive inhibitors of either reaction after consideration of effective progesterone concentrations. With the inhibitors, ratios of productive versus abortive events were constantly higher at a given substrate concentration than in control incubations, although neither promegestone nor estradiol affected progesterone or 17 alpha-hydroxyprogesterone accumulation in the endoplasmic reticulum membrane compartment. Rather, a linear correlation between ratios of productive versus abortive events and the overall catalytic rate as a measure of E.S complex concentration was obtained irrespective of the absence or presence of inhibitor. It was therefore concluded that promegestone and estradiol inhibit access or binding of progesterone to P450XVII, whereas the relative efficiency of androgen formation is solely dictated by the local substrate concentration being effective with respect to E.S formation. A model of P450XVII function is presented which proposes that excess substrate at the active site of the P450XVII enzyme protein hinders a certain fraction of a putative transient intermediate from being retained at the bifunctional catalytic site with the consequence that it cannot be further processed to androgen.
Animals↗
Hypothalamic sites of progestin action on aggression and sexual behavior in female Syrian hamsters.
The effects of intracranial implants of the synthetic progestin, promegestone (R5020), on aggression and sexual behavior were examined in female Syrian hamsters. Ovariectomized female hamsters showed high levels of aggression and no lordosis towards stimulus male and female hamsters, both prior to and after estradiol benzoate treatment. Forty-six hr after estradiol treatment (10 micrograms SC), 10% crystalline promegestone was applied bilaterally (27 ga cannulae) to the ventromedial hypothalamus. When tested 5-7 hr later, these animals had a significant reduction in the rate of attacks towards females, with no lordosis responding to a male. In contrast, promegestone in the caudal anterior hypothalamus activated low levels of lordosis, yet these females maintained high levels of aggression towards stimulus females. Females receiving promegestone in the rostral anterior hypothalamus, and control females receiving intracranial cholesterol, maintained high levels of aggression and no lordosis. Estradiol treatment alone enhanced pelvic movements in response to experimenter-applied perineal stimulation in a majority of females (18/22 compared with 4/22 prior to estradiol injection), with intracranial treatments having no further effect. These results indicate that progestins act on aggression and sexual behavior in female Syrian hamsters at different hypothalamic loci.
Aggression↗
Progestin receptor in the thymus of ovariectomized immature rats.
By using the synthetic progestin promegestone (R5020), the location and characteristics of progestin receptors in the thymic cytosols from immature ovariectomized oestrogen-treated rats were determined. Tritiated promegestone bound to the cytosol with high affinity (dissociation constant (Kd) = 2.0 +/- 0.3 nmol/l; promegestone greater than progesterone greater than oestradiol greater than corticosterone testosterone) and low capacity (number of binding sites (Bmax) = 143.0 +/- 13.5 fmol/mg protein). These values were appropriate for progestin receptors. However, an extremely high dose of dexamethasone (10 mumol/l; 1000-fold excess over [3H]promegestone) slightly inhibited the specific binding. Progestin receptors were predominantly located in the reticuloepithelial (RE)-cell fraction, with few in the thymocyte T-cell fraction. The receptor level was raised (24.9 +/- 11.3 (S.E.M.) to 143.0 +/- 13.5 fmol/mg protein) with increased doses of oestrogen (0-30 micrograms) administered in vivo. Using sucrose density gradient ultracentrifugation it was found that the thymic progestin receptor had a sedimentation coefficient of 9S under low-salt conditions. These results clearly suggest that the thymus of the immature female rat contains a specific progestin receptor which is mainly located in the RE cells.
Animals↗
[Growth inhibition by progestins in human endometrial cancer cells with progesterone receptors].
The presence of estrogen-independent progesterone receptors (PR) was demonstrated in a subline of a human endometrial cancer cell line, Ishikawa cells. Scatchard plot analysis of cytoplasmic binding data in a subline (IK-90) revealed a high affinity binding site for promegestone (Kd, 1.0 nM) with maximum binding sites of 158 fmol/mg protein. Competition experiments showed a binding specificity similar to that of typical PR. By low-salt sucrose gradient centrifugation, radioactive 8S and 4S peaks were found. The addition of 1 microM progesterone in culture medium resulted in a rapid nuclear translocation of cytoplasmic PR. Accumulation of glycogen in cytoplasm of the cells in response to 0.1 microM promegestone was observed by periodic acid-Schiff staining. Addition of various concentrations (1 nM-1 microM) of promegestone reduced the cell growth in a dose-dependent manner. The growth inhibition by promegestone was totally prevented by the concomitant addition of equimolar RU 486, a progestin antagonist. The other steroids including tamoxifen, cortisol, and testosterone had no significant effects on the growth of the cells. The growth-inhibitory effect of progestin on cultured cancer cells from human endometrial adenocarcinomas obtained by hysterectomy was also investigated. Addition of medroxyprogesterone acetate (1 nM-1 microM) caused a marked decrease in [3H]thymidine incorporation in cultured cancer cells from tumors with PR. From the viewpoint obtained in the present studies using both IK-90 cells and endometrial cancer cells in primary culture, it can be concluded that progestins produce regression of endometrial cancer directly via the PR system.
Cell Division↗
Nuclear localization of a glucocorticoid antagonist in cultured hepatoma cells.
The binding characteristics of promegestone, a typical antagonist of glucocorticoid action, has been investigated. We localized [3H] promegestone in the nuclei purified from steroid-treated HTC cells but the radioactivity specifically bound to the nucleus was much lower for the antagonist than for an inducer steroid, for instance dexamethasone. We attempted to define the nature of the "nuclear" activity and found that promegestone does not bind to the perinuclear membrane but is associated with the chromatin fraction.
Animals↗
Progestin effects on growth in the human breast cancer cell line T-47D--possible therapeutic implications.
In order to determine growth effects of the progestin R5020, (promegestone), we have utilized the progesterone-receptor rich human breast cancer cell line T-47D, growing the cells in the absence of the pH indicator phenol red, which has recently been found to be estrogenic. In contrast to reports on cells grown in the presence of phenol red, we find that promegestone alone, at physiological progestin concentration, significantly stimulates growth. Estradiol alone, at physiological concentration, stimulates growth much more. Promegestone in combination with estradiol is antiestrogenic for growth; that is, it significantly decreases the growth stimulatory effect of estradiol. These results raise the possibility that estrogen receptor and progesterone receptor-rich breast cancer patients might benefit more from a combination of anti-progestin and anti-estrogen therapy than from anti-estrogens alone.
Breast Neoplasms↗
Active immunization of female rats against 17 beta-estradiol. Preliminary studies on 17 beta-estradiol binding in uterine and pituitary cytosols.
Female rats were immunized with 17 beta-estradiol-6-carboxymethyloxime-bovine serum albumin. They developed antibodies to estradiol and, to a very low extent, antibodies to BSA. Anti-estradiol antibodies possessed tight specificity to estradiol-17 beta, without cross-reactivities with other estrogens. It was demonstrated that the specific estradiol binding in uterine and pituitary cytosols gradually decreased when antiserum titres increased. In uterine cytosols, the presence of progesterone receptor was studied using promegestone (R50 20) as ligand. No significant variations in promegestone binding were observed. Competition experiments however, questioned the permanence in immunized rats of the actual progesterone receptor or of a promegestone binding protein.
Aging↗
Antagonism of glucocorticoid action in cultured hepatoma cells.
We report here our recent data on the effects of antiglucocorticoids in two established cell lines (HTC, FAZA). These steroid hormone target tissues were designed to consider the problem of differential antagonism and lack of correlation between antiglucocorticoid activity and competition for the glucocorticoid receptor. In the systems chosen, several responses were considered which may be differentially antagonized: induction of tyrosine aminotransferase (TAT), alanine aminotransferase (AAT) and tryptophan oxygenase (TPO). By testing the anti-inducing capacities of a number of steroids, we found a few synthetic compounds like promegestone and R 25055 which exert a stronger antagonism against TAT and AAT induction than natural steroids like progesterone. The availability of highly radioactive antagonists (promegestone, progesterone) has greatly facilitated our "whole cell" study and allowed us to detect the antagonists in isolated nuclei whose purity and morphological integrity were controlled by specific criteria; our results suggest that the binding of the antagonists to the nucleus proceeds via the glucocorticoid receptor. The appearance of promegestone and progesterone in the nucleus suggests that the nucleus may be involved in the mechanism of action of anti-glucocorticoids.
Animals↗
Pre-eclampsia-like conditions produced by nitric oxide inhibition: effects of L-arginine, D-arginine and steroid hormones.
The aim of this study was to establish that inhibiting nitric oxide (NO) production with NG-nitro-L-arginine methyl ester (L-NAME) results in high blood pressure conditions in chronically treated pregnant rats. To validate the model, the effects of L-arginine (the substrate for NO) and D-arginine (the stereoisomer of L-arginine which is not a substrate for NO synthesis) were studied on blood pressure and fetal weights. The effects of a progesterone agonist, promegestone (R5020) and 17 beta-oestradiol were also explored. The NO synthase inhibitor L-NAME was chronically infused s.c. into pregnant rats from day 17 of gestation, either alone or with the simultaneous infusion of L-arginine and injections of sex steroid hormones (promegestone and oestradiol), compounds that may act in the pathogenic pathways of pre-eclampsia. Systolic blood pressure was measured daily. Weight and mortality of pups were recorded immediately after delivery. Blood pressure was elevated significantly in rats treated with L-NAME for only 1 day following infusion; there was a consistent decline during the next 3 days of pregnancy followed by a dramatic and significant rise just prior to delivery and post-partum. Fetal weights were reduced significantly in the L-NAME-treated rats. Co-treatment of L-NAME-infused rats with L-arginine reversed both the increase in blood pressure and the decrease in fetal weights observed with L-NAME alone. R5020, but not oestradiol, also reduced blood pressure and increased fetal weights in the L-NAME-treated animals. NO appears to play essential roles in the regulation of blood pressure during pregnancy, as well as in fetal perfusion and fetal weights at delivery. This study also indicates that progesterone, and not oestrogen, may regulate the vascular adaptations during normal pregnancy. L-Arginine and progesterone agonists like promegestone may have beneficial effects on the high blood pressure levels and reduced fetal weights associated with pre-eclampsia.
Animals↗
Progestin binding in benign hyperplastic prostatic tissue.
The identification of saturable binding sites for promegestone (17 alpha, 21-dimethyl-19-norpregna-4,9-diene-3,20-dione) in human prostatic cytosol as progesterone receptors is complicated by some differences in binding behaviour between progesterone and promegestone. These differences seem to result from the presence in cytosol of a second class of progestin-binding sites, which has a higher affinity for progesterone than for promegestone.
Binding, Competitive↗
Estrogen and progestin receptors in meningiomas: clinicopathological correlations.
Estradiol and progesterone receptors were studied in 44 patients with meningiomas and correlated to the clinicopathological features and amount of preoperative corticosteroid therapy. Thirty-four (77%) of the meningiomas contained high titers of specific high-affinity cytosol [3H]promegestone (R 5020) binding sites (mean 2,902 fmol/g tumor; range 0-9,598 fmol/g tumor) whereas only miniscule amounts of a nonspecific cytoplasmic [3H]estradiol binding component (mean 48 fmol/g tumor; range 0-201 fmol/g tumor) were detectable. No nuclear binding activity for [3H]estradiol was demonstrable. There was no convincing correlation between high PR activity and the age, sex, or menopausal status of the patients. The correlation study between the amount of preoperative corticosteroid therapy with the amount of [3H]promegestone binding revealed no dose relationship. Correlating [3H]promegestone content with the histologic type, we found 96% of meningothelial, 71% of transitional, and 40% of fibroplastic meningiomas to contain progesterone receptors. The necessity of in vitro studies is stressed to assess the biosynthesis and biological activity of the progesterone receptor in meningiomas, which is apparently not estrogen regulated, as is the case in other estrogen target tissues.
Cytosol↗
Effects of progestins and antiprogestins on mitochondria in uterine glandular cells in the rat. A quantitative investigation.
The administration of progesterone to ovariectomized rats induces an increase in the volume density (Vv) of the mitochondria and the appearance of giant mitochondria in the uterine glandular cells. This experimental model, including a stereological analysis, allowed us to investigate and quantify a direct effect of progesterone on a well-defined cellular structure without the intervention of estrogen in a priming phase. Synthetic compounds, promegestone, gestrinone and RU 38486, were tested in this model either in place of progesterone or simultaneously with progesterone. The potent progestomimetic activity of promegestone was confirmed by the proliferation of giant mitochondria and a high Vv value for the mitochondria, the two other compounds being inactive even at higher doses. At lower doses, gestrinone and RU 38486 partially inhibit the action of progesterone and at higher doses they both show a complete antagonist effect by preventing the development of the mitochondria.
Animals↗
Reversibility of antigestagenic action of antiprogestin onapristone by exogenous progestagens during early pregnancy in guinea pig.
Ability of progesterone, gestodene, promegestone and cyproterone acetate (CPA) to reverse antigestagenic action of onapristone in adult female guinea pigs was investigated. Onapristone (10 mg/kg, s.c.) administered on post-conception days 8-11 caused resorption of implantations and vaginal bleeding in all animals. Simultaneous administration of progesterone, gestodene or promegestone on days 7-13 successfully reversed antigestagenic action of this antiprogestin, since most animals supplemented with these progestagens had viable implantations at autopsy on day 14. CPA was, however, ineffective and animals supplemented with it had only resorbed implantations and blood in uterus and vagina like that in onapristone per se treated animals. High plasma progesterone and low PGFM concentration were generally observed in all pregnant animals bearing viable implantations. PGFM (13, 14-dihydro-15-keto PGF2 alpha) was significantly elevated by day 14 in onapristone-treated (Group II) and CPA-supplemented (Group X) animals. No discernible effect on pregnancy or post-implantation embryonic development was observed in animals treated per se with these progestagens.
Animals↗
Progestagen-concentrating cells in the brain, uterus, vagina and mammary glands of the galago (Galago senegalensis).
Progestagen-concentrating cells were localized in the oestrogen-primed ovariectomized galago by radioautography after injection of [3H]promegestone (R5020). In the brain, radioactivity was concentrated in the nuclei of neurones in the preoptic region and in the mediobasal hypothalamus. Labelled cells were also observed in the anterior pituitary. In the uterus (uterine horns and cervix), the muscle and stromal cells showed greater labelling than did the glandular and luminal epithelia. Labelled cells were present in the different cell layers of the vagina. The majority of glandular epithelial cells of the mammary glands exhibited a high degree of labelling. Pretreatment with an excess of unlabelled promegestone but not with an excess of nonradioactive testosterone reduced the nuclear concentration of radioactivity in these target tissues. These results show that there are no major differences in the distribution of progestagen-concentrating cells in rodents and galago.
Animals↗
Estrogen and progestin receptors in acoustic and spinal neurilemmomas. Clinicopathologic correlations.
Estradiol and progestin receptors were studied in 20 patients with neuraxial Schwann cell tumors, and their presence was correlated to the clinicopathologic features and the amount of preoperative corticosteroid therapy. Based on an arbitrary cutoff value of 200 fmol per gram of tumor as indicative of a positive receptor value in breast cancer, 4 and 13 of the neurilemmoma tissue samples could be considered as positive for estrogen and progesterone receptors, respectively. Whereas there was no convincing correlation between the estrogen and progestin receptor activity and the age, sex, or menopausal status of the patients, overweight patients had significantly higher estrogen and progestin binding values. The correlation between the amount of preoperative prednisone therapy and the amount of [3H]estradiol and [3H]promegestone binding revealed no dose relationship. Correlating [3H]estradiol and [3H]promegestone content with the histologic type of the schwannomas (Antoni types A and B, respectively), we were not able to draw conclusions, because of the predominance of Antoni type A over Antoni type B tissues in our material. The necessity of nuclear receptor assays, ligand specificity testing, and in vitro studies is stressed as a prerequisite for answering the questions whether neurilemmomas contain genuine sexual steroid hormone receptors and whether these receptors are regulated via an estrogen-estrogen-receptor system as is the case in classical sexual steroid hormone target tissues.
Adult↗