Progestin receptors in human uterine cytosol

Progestin(norethindrone and norethindrone acetate)-binding protein, exhibiting characteristics similar to uterine progesterone receptor, has been identified in human uterine cytosol. The progestin receptor was characterized by sedimentation coefficient 4.2 S; Stokes radius, 39 Å; frictional ratio 1.29; isoelectric pH 4.6; molecular radius 2.7 nm; and molecualr weight in the range 67 000–74 000. The ammonium-sulfate-precipitated progestin-receptor complex was eluted from a DEAE-cellulose column at 0.18 M KC1. The progestin binding was saturable and stereospecific. The sequential variation in receptor concentration (early proliferative, 3800–4300 sites/cell; late proliferative, 9500–11200 sites/cell; early secretory, 4900–6200 sites/cell; late secretory, 1800–2300 sites/cell) was in conformity for progesterone and the progestins, when concurrently measured. Oral administration of norethindrone significantly reduced the cytoplasmic and nuclear receptor concentration for estradiol and progesterone. A significant observation was that the progestins stabilized the progestin receptor by forming a slowly dissociating complex with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～ 110?130}\text{min}$ as compared with the progesteronereceptor complex dissociating with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～41}\text{min}$. Thus, the uterine progestin receptor recognizes progestins in general, although with a varying degree of affinity, and the altered rate constants could be of putative importance in determining the biological potency of the progestins.     

Cortisol metabolism in lymphocytes from cancer-bearing patients

A study was conducted to determine the pattern of cortisol metabolism by lymphocytes obtained from four groups of subjects: 27 male and female patients suffering from various types of malignancy other than malignancy of lymphatic tissues; and 26 healthy male and female controls. Known concentrations of cells were incubated with 1,2-³H-cortisol and the products were isolated by thin-layer and paper chromatography. Three metabolites were found to be produced by lymphocytes from both normal and cancer-bearing patients: 20α-hydroxycortisol, 20β-hydroxycortisol, and tetrahydrocortisol. Cells from the female control group were found to be more active than those from the male controls, while cells from cancer-bearing patients were markedly more active than the normal cells, regardless of sex. It is suggested that this finding of increased metabolism of cortisol by lymphocytes from patients with different types of malignancy other than lymphoma may provide the basis for a new diagnostic aid.     

Effect of 17 beta-estradiol on thyroliberin responsiveness in GH3/B6 rat prolactin cells

GH3/B6 rat prolactin cells were used to analyse at the cellular level the mechanisms by which 17 beta-estradiol (E2) regulates TRH responsiveness of prolactin cells. Before experiments, cells were grown for up to 7 days in 3 different media: normal medium (N) containing 15% horse serum and 2.5% fetal calf serum, CD medium prepared with charcoal-dextran extracted serum and CDE medium supplemented with 4 x 10(-8) M E2. The binding of 3H-TRH (30 min at 37 degrees C) and the TRH-induced percent increase of prolactin release as a function of TRH doses were compared in the 3 conditions. Preculture in E2 enriched medium increased by 50% the number of TRH high-affinity binding sites without modifying their affinity, increased by up to 3 times the percent of the TRH-induced stimulation of prolactin release and improved by one order of magnitude the ED50 of the TRH effect on prolactin release. The presence of HEPES (10 mM) during TRH challenge masked the effect of E2 on the increase in number of binding sites but respected its potentiating effect on prolactin release.     

Progestins inhibit fsh-stimulated steroidogenesis in cultured rat granulosa cells

It has been hypothesized that progesterone (P) exerts a direct inhibitory effect on ovarian follicular development, an effect which could be mediated by P receptors located in granulosa cells. We tested this hypothesis by examining the effect of several progestins on FSH-stimulated estrogen (E), P, and 20α-dihydroprogesterone (DHP) production by cultured rat granulosa cells, and correlated the results with the ability of the progestins to bind to the granulosa cell P receptor. Granulosa cells from immature hypophysectomized DES-treated rats produced 9 ng/ml E, 21 ng/ml P and 29 ng/ml DHP during a 2-day incubation in McCoy's 5a medium containing 10^?7 M androstenedione and 10 ng/ml oFSH. The FSH-induced increase in E production was inhibited by 50 and 95% following concomitant treatment with 3 × 10^?6 and 10^?5 M resp. of R5020, a potent synthetic progestin. Added R5020 at these concentrations also significantly inhibited P and DHP production. R5020 had no effect on granulosa cell viability or plating efficiency, and the inhibitory action of R5020 on E production was reversible. In studies of the specificity of the progestin inhibitory action, the relative abilities of various progestins to inhibit E production were: R5020 > P > DHP > 17α-hydroxyprogesterone (170HP). The relative abilities of these progestins to bind to the ovary P receptor were also: R5020 > P > DHP > 170HP. These results indicate that exogenous progestins directly inhibit the FSH-stimulation of granulosa cell steroidogenesis in vitro and suggest that the progestin effect may be mediated by the P receptor. Such results offer a possible mechanism whereby progesterone could exert a direct but reversible inhibitory action on ovarian follicular development.     

Dissociation between cortisol-induced pycnosis and inhibition of [3H]uridine incorporation in rat thymocytes.

Treatment of rat thymocytes with cortisol induced an inhibition of [3H]uridine incorporation after 30-90 min, an accumulation of pycnotic cells after 90 min, and a decrease in cell viability after several hours. No cortisol-resistant cells could be distinguished, and dose-response curves for a number of glucocorticoids showed a correlation to the saturation of the glucocorticoid receptors. The pycnotic effect of cortisol increased between pH 5.2--7.0 in parallel with a stimulation of the spontaneous development of pycnotic cells. The cortisol-induced accumulation of pycnotic cells and inhibition of [3H]uridine incorporation varied independently as a function of the cell density, and in a glucose-salt medium only the pycnotic effect of cortisol became inhibited. The inhibition of [3H] uridine incorporation is therefore not an integral part of the pycnotic change of the cells. The glucocorticoid sensitivity was found to increase with the age of the animals, before the onset of thymus involution.     

Glucocorticoid receptor in human embryo fibroblasts. I. Kinetic and physicochemical properties.

The kinetics of dexamethasone binding to L 809 E cell line cytosol have been investigated by means of the protamine sulfate precipitation assay. The KDeq for dexamethasone was 1.1--3.3 nM. Binding was specific for glucocorticoids. The mean association rate constant (k+1) was 8.5 x 10(5) M-1 x min-1 and the dissociation rate constant was 4.6 x 10(-5) min-1 at 0 degrees C. The concentration of binding sites was 0.3 pmol/mg of cytosol protein. Binding kinetics were compatible with a model of positive cooperativity. The receptor sedimented at 7.5--9 S in glycerol gradients. By a combination of calibrated ultracentrifugation and polyacrylamide gel electrophoresis, a Stokes radius of 8.5 nm, a molecular weight of 268 000 daltons and a frictional ratio of 1.8 were determined in low ionic strength conditions. When the cells were incubated with 10 nM [3H]dexamethasone for 1 h, a more than 90% depletion of cytosol receptor and an equivalent accumulation of nuclear dexamethasone--receptor complexes was observed.     

In vitro inhibition of aldosterone biosynthesis by 17-hydroxylated steroids in rat adrenal

The in vitro effect of the addition of non-17-hydroxylated and 17-hydroxylated steroids on the production of aldosterone has been studied in the rat. The biosynthesis of deoxycorticosterone, corticosterone, and aldosterone was increased when progesterone or pregnenolone was added to the incubation media, but neither cortisol nor 11-deoxycortisol was produced. The addition of 17α-hydroxyprogesterone or 17α-hydroxypregnenolone to the incubation media resulted in the biosynthesis of cortisol and 11-deoxycortisol by the rat adrenal; concomitantly, a decrease in aldosterone production occurred. However, no decrease in aldosterone production resulted when cortisol and 11-deoxycortisol were added directly to the incubation media. These studies show that neither cortisol nor 11-deoxycortisol inhibits the in vitro biosynthesis of aldosterone and suggest that other mechanisms are involved.     

Androgen metabolism in rat anterior pituitary cells in culture

The metabolism in vitro of 4 androgens, namely testosterone, androstenedione, 5α-dihydrotestosterone and 3α-androstanediol has been studied in male and female rat anterior pituitary cells in primary culture. When testosterone was used as precursor, androstenedione, 5a-dihydrotestosterone and 3α-androstanediol were the main metabolites whereas androstenedione was mainly converted into testosterone, 5α-androstanedione, 5α-dihydrotestosterone and androsterone. Studies on the metabolism of 5α-dihydrotestosterone and 3α-androstanediol showed that these compounds were easily interconverted and were also significantly metabolized to 5α-androstanedione and androsterone. No aromatized compounds could be detected suggesting that androgen action in the pituitary cell occurs directly via the androgen receptor rather than through prior conversion into estrogens.     

High-affinity binding of the antiestrogen [3H]tamoxifen to the 8S estradiol receptor

The interaction of tamoxifen (ICI 46,474), a synthetic antiestrogen, with uterine cytosol proteins of immature calf and rat has been studied directly using the tritiated compound labeled with a high specific activity. The binding complexes were measured by the dextrancoated charcoal, protamine sulfate and hydroxyapatite assays. Scatchard plots revealed a single class of high-affinity (KD congruent to 1.7 nM) binding sites, with a binding capacity similar to that of estradiol. Competitive experiments showed the same binding specificity for estrogens and antiestrogens. Sucrose gradient analysis revealed an 8S binding protein which could be partially proteolysed by trypsin into a 4S binding protein. Kinetic studies showed that the association rate of tamoxifen was 5 times lower than that of estradiol and reacted according to a second order kinetics. The first-order kinetics of dissociation was considerably higher than that of estradiol, giving a half-dissociation time of 20--40 min at 0--2 degrees C. In some cases tamoxifen displayed two slopes of dissociation, but the proportion of the slow-dissociating complex was always inferior to that found with estradiol. In contrast to estradiol, the kinetic constants ratio (k-/k+) gave a calculated dissociation constant, similar to that determined in equilibrium conditions (KD), agreeing with a simple reactional scheme. We conclude that the antiestrogen tamoxifen binds directly to the 8S cytosol receptor for estrogens and not to another receptor for the antagonists. In contrast to estradiol, the antagonist is rapidly dissociated from the receptor sites and is unable to protect them against thermal inactivation. The affinity of tamoxifen for its receptor sites as determined directly is surprisingly high when compared to its affinity evaluated indirectly by competitive experiments. It is then suggested that the two ligands either bind on two different sites of the same protein or induce a different conformational change of the same binding site.     

Competitive binding studies with glucocorticoid receptors from rat-thymus cells: differential temperature-dependence of steroid binding.

Competitive steroid-binding studies were performed with intact rat thymus cells and with cytosol preparations at different temperatures using [1,2-3H]dexamethasone as the labelled ligand. Steroids lacking a 17 alpha-hydroxyl group, such as corticosterone, were better able to compete with [1,2-3H]dexamethasone for binding to glucocorticoid receptors at 0 degrees C than compounds containing a 17 alpha-hydroxyl substituent, such as cortisol. At 37 degrees C the reverse was true. This temperature-dependent change in relative affinities appeared to be unrelated to steroid metabolism or receptor activation, and to depend only on the thermodynamic parameters of the steroid--receptor interaction. Relative biological activities for different steroids agree more closely with the relative affinities determined at 37 degrees C than with those determined at lower temperatures.     

Effect of aminoglutethimide on extraglandular metabolism of exogenous testosterone

Aminoglutethimide (AG), an inhibitor of steroid biosynthesis, seems to have an extraglandular site of action on steroid catabolism. To study this effect, five males with peripheral hypogonadism were first given testosterone propionate and then the same dose was repeated combined with AG and urinary testosterone, and its metabolites were measured. AG was shown to have a very evident effect on the peripheral degradation of exogenous testosterone. This may be responsible for a few signs of virilization and fetal masculinization in women taking AG.     

Glucocorticoid-induced cell-size changes and nuclear fragility in rat thymocytes

The events preceding glucocorticoid-induced lymphocytolysis have been studied in isolated rat thymocytes. Incubation of thymocytes at 37°C in the presence of 1 μM dexamethasone resulted in the progressive appearance of pyknotic cells of modal diameter 4.6 μm, distinct from normal cells of diameter 5.2 μm. The rate of appearance of the pyknotic cells was determined by selective electronic cell counting, and was shown to be accompanied by increased nuclear fragility. The production of pyknotic cells was glucocorticoid-specific, dose-dependent, blocked by cycloheximide, and preceded the loss of cell viability as determined by dye exclusion. The pyknotic cells were separated from the non-pyknotic cells by density-gradient centrifugation and shown to be solely responsible for the observed nuclear fragility.     

Progestins inhibit FSH-stimulated granulosa estrogen production at a post-cAMP site

The purpose of this investigation is to examine the mechanism by which progestins inhibit FSH-induced estrogen (E) production by cultured rat ovary granulosa cells. We have demonstrated that the highly potent synthetic progestin, R5020, is able to inhibit the induction of granulosa cell aromatase activity by cholera toxin, prostaglandin E2, dibutyryl cAMP or oFSH. Since the induction of E synthesis by these compounds is mediated through activation of adenylate cyclase and increased cellular cAMP production, these observations indicate that the progestin inhibitory effect is a post-cAMP event. In addition, we have demonstrated that R5020 does not inhibit FSH-stimulated granulosa cell cAMP production. The involvement of the granulosa cell progesterone (P) receptor as a mediator of this post-cAMP progestin effect is suggested by the relative abilities of various progestins to both bind the P receptor and to block the induction of granulosa cell aromatase activity by dibutyryl cAMP. While the precise mechanism of progestin action remains unclear, the kinetic analysis of aromatase enzyme activity demonstrates that progestins are not acting as competitive inhibitors of granulosa cell aromatase. Since E is necessary for follicular development, our in vitro data are consistent with the hypothesis that P is a factor which can inhibit FSH-induced follicular growth and development in the rat ovary.     

Activation of the progesterone receptor of rabbit uterus

The influence of several parameters on the kinetics of activation of the progesterone receptor in the cytosol of rabbit uterus is described. The estimation of the proportion of activated receptor is based on the differential affinity of the activated and non-activated forms of the receptor for phosphocellulose. Under appropriate conditions binding to phosphocellulose can be used as a test of activation and gives results similar to those obtained with DNA--cellulose, or isolated cell nuclei. The kinetics of receptor activation is temperature-dependent and compatible with a first-order reaction at all temperatures tested. The thermodynamic activation energy of this reaction is 67.8 kcal mol-1. The progesterone receptor can be activated to various extents by increased ionic strength or by dilution of the cytosol with buffers of low ionic strength, and in all cases the activation follows apparent first order kinetics. At a concentration of 0.4 M NaCl, 70--80% of the receptor can be converted into the activated form. The activated and non-activated forms of the receptor appear to be in equilibrium. Salt-activated and heat-activated receptor can be transformed to a non-activated form by decreasing either the salt concentration, or the temperature of incubation. The rate of dissociation of the steroid from the activated form of the receptor is indistinguishable from that observed with the non-activated form, but the activated receptor is more thermolabile. Upon centrifugation on sucrose gradients there are no major differences in the sedimentation behaviour of the two forms of the receptor.     

The impact of radioimmunoassay on our understanding of human adrenal physiology

Radioimmunoassay has only been used for a relatively short time to study the human hypothalamic-pituitary-adrenal system, but the findings have already contributed immeasurably toward a better understanding of the physiology of this system. In some instances classical concepts have been confirmed and extended. Other findings have led to revisions of old concepts and the formulation of new ones. These physiologic studies have produced new and better laboratory tests for the pituitary-adrenal function that are simple and reliable enough to be used not only in clinical research but in the routine practice of medicine. Even greater contributions from radioimmunoassay can be expected in the future.     

Evidence suggesting 11 beta-hydroxylase inhibition during aminoglutethimide administration

Steroid hormone excretion during amino-glutethimide administration was studied in a patient with Cushing's syndrome due to bilateral adrenocortical hyperplasia. Plasma and urinary 17-OHCS showed a persistent decrease, as did urinary total 17-KS. Chromatographic fractionation of urinary 17-KS demonstrated a dramatic reduction of 11-oxy-17-KS, while 11-deoxy-17-KS, after a transient fall, tended to recover. Urinary THS showed an absolute increase, while pregnanetriolone diminished. Δ⁵-pregnenetriol and pregnanetriol, after initial decreases, showed a gradual trend upward. The sum of these data, and particularly the increase in THS excretion, suggests that, in this case, aminoglutethimide exerted an inhibitory effect not only on the early steps of adrenocortical steroidogenesis but also on 11 β-hydroxylation.     

Interaction between the hepatic growth hormone receptor and concanavalin A.

The interaction between the plant lectin concanavalin A (Con A) and hepatic receptors for human growth hormone (GH) has been studied in particulate and soluble microsomal membrane preparations from rabbit and rat liver. Con A shows a dose-dependent, partial (30%) inhibition of 125I-human GH binding which is reversed by the Con A competitor, alpha-methyl mannoside. The Con A effect is dependent on the receptor concentration. The inhibition by Con A in rabbit liver is a reflection of a decreased number of available binding sites--there is no effect on binding affinity. It would appear that Con A binds directly to the GH-binding protein and not to an adjacent membrane glycoprotein. The GH receptor may consist of more than one molecular species, differing only in the carbohydrate type or content.     

Some properties of androgen-binding activity in rat testis.

High-affinity (Ka approximately equal to 5 X 10(8) M-1 for testosterone) androgen-binding activity in rat testis was shown to have a rapid dissociation rate constant (t1/2 = 3 min, 0 degrees C, 30% glycerol buffer) using dextran-coated charcoal to separate bound from free hormone. Because of this fact, exchange of endogenous and labeled hormone was complete in the assay incubation time (16 h, 0 degrees C) and Scatchard plots of the high-affinity binding data were shown to measure total as contrasted to available sites. The binding was highly specific for androgens. Polyacrylamide gel electrophoresis separated high-affinity androgen-binding protein (Rf 0.54) from albumin (Rf 0.62). Binding site estimates under saturating conditions or by Scatchard analysis of electrophoresis data utilizing [3H]dihydrotestosterone agreed reasonably well with estimates made by the charcoal technique using [3H]testosterone.     

Androgen-binding proteins in rat epididymis: properties of a cytoplasmic receptor for androgen similar to the androgen receptor in ventral prostate and different from androgen-binding protein (ABP)

The cytoplasmic receptor (CR) in rat epididymal 105,000 $\text{g}$ supernatant was separated from the androgen-binding protein (ABP) by gel electrophoresis following labeling with [1,2,6,7-³H]-testosterone in vivo. ABP disappeared from epididymal supernatants after castration 01 hypophysectomy, while CR remained unchanged. CR was evenly distributed between caput and cauda, while much more ABP was present in caput. Properties of CR in epididymis and prostate were similar and distinctly different from ABP. Binding to CR was destroyed by charcoal treatment (1 mg/mg protein) of supernatant at 0 °C for 6 h, heating at 50 °C for 30 min, or exposure to the sulfhydryl blocking reagent, $\text{p}$-chloromercuriphenylsulfonate (1 mM) at 25 °C for 30 min, while binding to ABP was unaffected. The isoelectric pH of CR (5.8) was higher than that of ABP (4.6). Dissociation of radioactive 5α-dihydrotestosterone (DHT) from CR and nuclear receptors was extremely slow (half-time at 0 °C >2 days), while dissociation from ABP was rapid (half-time at 0 °C ~ 6 min). Cyproterone acetate (250 mg/100g body weight) inhibited binding to CR both in epididymis and ventral prostate but did not affect binding to ABP. Nuclear uptake was inhibited by cyproterone to the same extent as binding to CR, indicating that nuclear uptake and binding are dependent on CR and independent of ABP. The time-course of uptake and binding in epididymal supernatant and nuclear fractions was essentially the same 1 day after bilateral castration when both CR and ABP were present or 8 days after castration when CR alone was present. It is concluded that the cytoplasmic receptor for androgen in rat epididymis has properties very similar to the androgen receptor in ventral prostate but different from ABP.     

Relation of membrane property of microsomes to androgen biosynthesis

Spin-labelled fatty acids I(12,3) and I(1,14) were incorporated into microsomal membrane of cryptorchid mouse testis and Leydig cell tumor as well as liver. The freedom of motion of spin of I (12,3) was more restricted in testis microsome than in liver. At the lower temperatures, the freedom of motion of spin in the tumor microsomes was similar to that in the testis, but at higher temperature (20–50°C) was much greater. Plotting of the empirical parameter, h₀/h-_?1, calculated by the spectra of I(1,14), against the reciprocal of the absolute temperature clearly showed two inflection points in both liver and testis microsomes, one at 19°C and the other at 30°C. On the other hand, tumor microsomes lacked these break points and permitted spin to move more freely.These results suggest that tumor microsomes contain the increased fluidity. The importance of membrane fluidity in relation to steroid biosynthesis was also discussed.