Heterogeneity and ontogenesis of progestin receptors in rabbit lung

In order to evaluate the possible role of progesterone in fetal lung development, the presence of specific pulmonary progestin receptors and their ontogenesis were investigated in the rabbit fetus. Scatchard analysis of binding in lung cytosol from 29-day fetuses over a wide range of [³H]-R5020 concentrations indicates the presence of at least two binding sites. One of these sites, type I, is of very high affinity (K_D = 0.12 nM) and low capacity (26fmol per mg protein). The second binding site, type II, is of lower affinity (K_D = 36 nM) and higher capacity (240 fmol per mg protein). These two binding sites can be distinguished by sucrose density gradient centrifugation, the type I component sedimenting at 7.1 S and the type II component sedimenting at 4.5 S. Similar type I and type II sites are present in adult lung cytosol except that the type II binding component in adult lung sediments at 2.8 S rather than 4.5 S. Progesterone and R5020 compete well with [³H]-R5020 for binding to both sites while dexamethasone and cortisol do not compete. Thus the type I and type II binding sites appear to represent specific progestin receptors distinct from transcortin or the glucocorticoid receptor. The concentration of the type I sites increases significantly between the 20th and 29th day of gestation, with a further increase being observed in adult animals. The type II site is not measurable until 26 days of gestation and attains adult levels by day 29. Among a large number of fetal tissues examined, the lung contained the highest concentration of type I progestin receptor sites.Although cortisol and dexamethasone, even at very high concentrations, do not compete with [³H]-R5020 for binding to lung cytosol, the binding of [³H]-dexamethasone is inhibited significantly by nonlabeled progesterone or R5020 and this inhibition appears to be due to dissociation of [³H]-dexamethasone-receptor complexes. These results indicate that, in addition to type I and type II progestin receptor sites, fetal lung cytosol contains a third binding site, type III, which appears to be different from the glucocorticoid receptor site. Occupation of the type III site by progestins interferes with the binding of glucocorticoids to glucocorticoid receptors perhaps by increasing the rate of dissociation of glucoeortieoid-receptor complexes.     

Levels and subcellular distribution of endogenous corticosterone in rat liver during development

The concentration of corticosterone in liver homogenates, liver cytosol and purified nuclear fractions, and in plasma of fetal, newborn, immature and adult rats has been measured by radioimmunoassay.Highest plasma corticosterone levels were found in fetal rats, decreasing close to the levels observed in the adrenalectomized rat by the 6th day of postnatal life followed by a rise in the adult rat. The concentration of corticosterone in liver during development paralleled the plasma levels, the liver to plasma corticosterone ratio ranging between 0.09 and 0.17 suggesting that the corticosterone retained by the tissue is related to the unbound fraction of the hormone in plasma.Both plasma and tissue corticosterone levels declined after adrenalectomy and they were elevated after ether stress.Fractionation of liver homogenates showed that the major fraction of liver corticosterone is localized in the cytosol. Purified liver nuclei contained between 9 and 16% of the total liver corticosterone. The amount of corticosterone in the nuclei seems to be related to the plasma and tissue hormone levels rather than the concentration of cytoplasmic glucocorticoid receptors. Since most of the nuclear corticosterone appears to be bound to receptors, it has been calculated that close to 60% of the cellular receptors in fetal liver are localized in the nucleus. In adult rat liver, only about 10% of the cellular receptors appear to be associated with nuclei. Changes in the concentration of glucocorticoid receptors in liver during development and after adrenalectomy are inversely related to changes in plasma corticosterone levels. It is suggested that corticosterone may regulate the levels of its own receptors in liver.     

Fetal and adult sheep adrenal cortical cells contain glucocorticoid receptors

Specific receptors for glucocorticoids were identified in the fetal and adult sheep adrenal cortex by a whole-cell binding assay using [3H]triamcinolone acetonide ([3H]TA) as the radiolabelled ligand. [3H]TA binding sites were saturable and of high affinity, with dissociation constant (Kd) of 2-3nM. Scatchard analysis revealed a single class of binding sites with a binding capacity (Bmax) of 207 and 5 fmol/10(6) cells for d100 fetuses and adults, respectively. By single point analyses at saturating [3H]TA concentration, we found that glucocorticoid receptors (GR) were present in the fetal adrenal cortex as early as d60. Highest concentrations were found at d100-110. GR decreased to d125, then increased to term (approx. d145) before declining again in newborn and adult animals. This demonstration of glucocorticoid receptors in ovine fetal adrenal cortical cells provides a mechanistic basis for the concept that glucocorticoids may act, perhaps in a paracrine or autocrine fashion, to influence adrenocorticotropin (ACTH)-induced activation of fetal adrenal function and the events leading to parturition.     

The distribution and properties of the glucocorticoid receptor from rat brain and pituitary

The distribution and properties of cytoplasmic binding sites for the synthetic glucocorticoid dexamethasone and the natural glucocorticoid corticosterone in the brain and the pituitary were studied in detail. Cortisol-17 beta acid, a derivative which does not bind to the glucocorticoid receptor but is a competitor of corticosterone binding to plasma, was used to overcome plasma interference. In vitro competition assays in the presence of excess cortisol acid reveal that dexamethasone is as effective a competitor for [3H]corticosterone binding as corticosterone itself. Scatchard analysis of equilibrium experiments with both steroids, using cytosol from various brain areas and from the pituitary yielded linear plots, suggesting one class of binding sites. The quantitative distribution of the sites follows the pattern: cortex greater than hippocampus greater than or equal to pituitary greater than hypothalamus greater than brain stem white matter. Furthermore, kinetic analysis of corticosterone dissociation showed a first order reaction, thus indicating the presence of one type of receptor in all brain areas examined. Rat brain cytosolic receptors for corticosterone and dexamethasone elute from DEAE-Sephadex A-50 anion exchange columns at 0.3 M NaCl in the presence of stabilizing sodium molybdate and at 0.15 M NaCl and/or in the buffer wash when heat-activated, thus exhibiting the characteristic activation pattern of rat liver cytosolic glucocorticoid receptor. The ratio of the buffer wash to the 0.15 M NaCl form is low for dexamethasone and very high for corticosterone. Receptor complexes from various brain parts showed the same activation pattern. In our experiments, brain corticosterone and dexamethasone receptors stabilized by sodium molybdate are indistinguishable by a number of techniques, thus indicating that it is unnecessary to evoke specific binding sites for each glucocorticoid.     

Insulin receptors in developing rat liver: Acquisition of down regulation in the immediate postnatal period

Alterations in the high and low affinity insulin receptor concentrations in developing rat liver were investigated. The number of high affinity receptors in partially purified plasma membranes from fetal rats increased from Days 19 through 22 of gestation, with no further increase in binding during the postnatal period. Fetuses of diabetic rats had approximately three times as many high affinity insulin receptors as age-matched fetuses of normal rats; however, by 1 day after birth the receptor number decreased to the normal level. Neither the number of low affinity receptors nor the affinity of insulin binding to high or low affinity receptors changed during development or between offspring of normal and diabetic rats. These changes in the number of high affinity hepatic insulin receptors from prenatal animals did not correlate with the concentration of plasma insulin. When suckling pups were rendered diabetic the changes in the number of high affinity insulin receptors correlated with alterations in plasma insulin concentrations. The number of high affinity sites/microgram DNA in hepatocytes from Day 18 fetal rats was not altered when cells were cultured for 48 h in medium containing 0, 250, or 5000 μU/ml of added insulin. When cultured hepatocytes derived from 1-day-old and adult rats were maintained in medium with added insulin concentrations of 250 or 5000 μU/ml the number of high affinity receptors/microgram DNA decreased as compared to the number of high affinity receptors in hepatocytes cultured in medium with no added insulin. This decrease in receptor number was accompanied by an increase in the affinity of insulin binding to its high affinity receptors. The data show that (i) only the high affinity insulin receptor number increases in rat liver during the prenatal period, (ii) fetuses of diabetic rats show a greater increase in high affinity receptors than do fetuses of normal animals, and (iii) the phenomenon of down regulation for high affinity insulin receptors is not observed in fetal rat liver, but is acquired in the immediate postnatal period.     

Binding of glucocorticoids to liver nuclei and chromatin of fetal,immature and adult rats

The interaction of dexamethasone with nuclei and chromatin was investigated following incubation of liver slices from fetal, immature (6-day-old) and adult rats with the labeled steroid at 37°. The number of specific binding sites for dexamethasone in purified liver nuclei increases with the age of the animal in a manner similar to that previously reported for the cytoplasmic receptor. The high affinity nuclear binding approaches saturation at 40 and 500 nM dexamethasone in fetal and adult liver, respectively. In comparison with dexamethasone, the relative efficiency of corticosterone to accumulate in the nucleus is 9 percent in fetal liver and only 1 percent in adult liver.Specifically bound dexamethasone in adult nuclei exists in at least three forms; a Tris-soluble, a KCl-soluble, and a residual (non-extractable with KC1 or DNase) form. Part of the Tris-soluble steroid is associated with macromolecules sedimenting at about 4 S both in the presence and absence of 0.4 M KCl. This form of the receptor was not detected in fetal liver nuclei. In liver chromatin, bound dexamethasone exists in a KCl-soluble and a residual form, the latter comprising the major fraction of steroid associated with chromatin from both fetal and adult tissue (60 and 75 percent, respectively). Treatment with Triton X-100 releases about 20 percent of the radioactivity in adult liver nuclei, but has no effect on fetal liver nuclei.In contrast with the above observations in the intact tissue, the major fraction of steroid bound to chromatin in cell-free systems is KCl- and DNase-soluble, only 30 percent remaining in the residual pellet.     

Glucocorticoid receptors in WI-38 fibroblasts: characterization and changes with population doubling in culture.

A high-affinity dexamethasone binding macromolecule was identified in WI-38 human fetal lung fibroblasts. High specificity of binding for glucocorticoids was shown by competition studies in which binding of dexamethasone was inhibited by cortisol and corticosterone but not by testosterone or 17 beta-estradiol. WI-38 cells exposed to [3H]dexamethasone at 30 degrees C were able to transfer the 3H-labeled steroid-receptor complex to the nuclear materal. A reduction of 30--50% was observed in the number of [3H]dexamethasone-receptor binding sites per cell as well as in the nuclear fraction of the cells as a function of age (passage levels 27 and 54). However, in the same cells no significant changes in affinity of receptor for [3H]dexamethasone as a function of the two passage levels were detected.     

Androgen receptors in fetal rabbit lung and the effect of fetal sex on the levels of circulating hormones and pulmonary hormone receptors

High affinity (KD = 0.2 nM), low capacity (3.6-5.0 fmol/mg protein), androgen-specific binding proteins with characteristics typical of androgen receptors were identified in the lungs of rabbit fetuses between the 26 and 29th day of gestation and in the lungs of adult rabbits. While androgen receptor concentrations increased significantly from late gestation to adulthood (P less than 0.01), no sex-related differences were observed in either the binding affinities or concentrations of the receptors at any age tested. Similarly, no sex-related differences were found in the levels of progesterone, cortisol and cortisone in the fetal circulation, or in the levels of progesterone receptors, glucocorticoid receptors and beta-adrenergic receptors in the fetal lung at 26 days of gestation. It is concluded that the fetal lung interacts directly with circulating androgens via specific androgen receptors and that the suggested male disadvantage with respect to lung maturation in the perinatal period does not appear to be associated with sex-related differences in the levels of pulmonary androgen, glucocorticoid, progesterone or beta-adrenergic receptors.     

Corticosteroid-binding globulin (CBG) in fetal development

In fetal sheep the prepartum increase in plasma cortisol concentration is associated with an increase in high affinity corticosteroid binding activity in plasma. This appears to reflect an increase in corticosteroid-binding globulin (CBG) biosynthesis from the fetal liver, and evidence is presented that hepatic CBG gene expression is increased by exposure to glucocorticoids in the fetus. Immunoreactive CBG is found in other fetal tissues, and CBG mRNA is present in fetal pituitary. CBG reduces the ability of cortisol to exert negative feedback on basal or CRH-stimulated ACTH output by fetal sheep pituitary cells in culture. We suggest that CBG interacts with cortisol in a manner that maintains a low negative feedback on the pituitary, and perhaps hypothalamus. This constitutes a component of the cascade of events that is associated with hypothalamic-pituitary-adrenal activation in the late gestation fetus, and with the onset of parturition.     

Androgen receptors from the epididymis of the rhesus monkey

The demonstration and partial characterization of androgen "receptors" in the epididymis of the rhesus monkey are reported. The affinity of specific binding sites for dihydrotestosterone was higher than that for testosterone while cortisol and corticosterone did not compete. Density gradient analysis of the cytosol revealed that macromolecules responsible for specific binding have a sedimentation coefficient of 8 S. The isoelectric point of the complex was 5.5 and thermoability was evidenced by the release of hormone during incubation at 27 degrees C (20% and 44% after 30 and 60 minutes, respectively.) A concomitant decrease in radioactivity bound to 8 S cytoplasmic components suggests the translocation into the nuclei of a modified 8 S-receptor complex. These data indicate the presence of similar cytoplasmic and nuclear androgen receptors in the monkey seminal vesicles and prostate gland.     

Characterization of cortisol binding sites in chicken liver plasma membrane

1. The presence of sites specifically binding [3H]cortisol in plasma membrane isolated from chicken liver has been determined. The kinetic parameters of this binding are: Kd = 4.5 nM and Bmax = 2225 fmol/mg protein in presence of 10(-6) M progesterone. 2. The affinities of several natural and synthetic steroids for the membrane binding site respect to the binding of 4 nM [3H]cortisol without competitor increased in the following order: Testosterone less than pregnenone less than dexamethasone less than progesterone less than prednisolone less than corticosterone less than deoxycorticosterone. 3. Other steroids such as estradiol, ouabain and triamcinolone acetonide does not bind to the plasma membrane. 4. Metal ions such as Ca2+ and Mg2+ did not modify the binding of [3H]cortisol. 5. Neither propranolol nor phentolamine, beta- and alpha-adrenergic antagonists affected [3H]cortisol binding to the plasma membranes. 6. The result suggest that the binding site detected is more specific for glucocorticoids and it is different of nuclear glucocorticoid receptor and progesterone receptor.     

Identification and ontogeny of beta-adrenergic receptors in fetal rabbit lung

High affinity (Kd=0.2 nM), low capacity (48 fmoles per mg protein), stereospecific binding sites, with properties characteristic of the β₁-subtype of β-adrenergic receptors, have been detected in fetal rabbit lung membranes as early as the 22nd day of gestation. The concentration of the receptor did not change significantly between the 22nd and 26th day of gestation, but increased 3-fold between the 26th and 29th day, reaching a level of 198 fmoles per mg protein. A further increase (from 198 to 315 fmoles per mg protein) in receptor concentration was observed in adult female rabbits. The increase in the levels of pulmonary β-adrenergic receptors between the 26th and 29th day of gestation is temporally related to the increase in surfactant release into the alveolar spaces of the fetal lung. Thus β-adrenergic agonists may act directly on the fetal lung to regulate surfactant secretion.     

Characterization of specific binding sites for corticosterone in mouse liver plasma membrane

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.     

Pregnancy-related changes of galactose recognition system on sinusoidal rat liver cells

We have studied the galactose specific receptor activities of sinusoidal liver cells of pregnant rat from the 13th day of gestation to the 10th day after delivery. Both liver macrophages and endothelial cells modulate the expression of hepatic binding protein during rat pregnancy and lactation. Except for endothelial cells of rats at the 22nd day of gestation, we have measured an overall decrease of binding sites. In addition, in liver macrophages the clustered distribution of galactose specific receptors, which is typical for adult animals, is reduced or completely absent. These results indicate that the galactose specific receptors on non-parenchymal liver cells are modulated in relation with the physiological state of the organ like the asialoglycoprotein receptor of hepatocytes.     

Proliferin secreted by cultured cells binds to mannose 6-phosphate receptors

Proliferin is a prolactin-related glycoprotein secreted by proliferating mouse cell lines and by mouse placenta. In an attempt to identify target sites for proliferin action, we looked for proliferin receptors in murine fetal and maternal tissues during pregnancy using proliferin purified from the conditioned medium of a constructed Chinese hamster ovary cell line carrying amplified copies of proliferin cDNA. Purified proliferin bound to membrane preparations from fetal or maternal liver and from placenta with a Kd of 1 to 2 nM. The amount of proliferin bound per microgram of membrane protein varied markedly during pregnancy; maximal binding to day 16 fetal liver membranes was approximately 25 times that to liver membranes from adult animals. Binding to fetal and maternal receptors was specifically and completely inhibited by mannose 6-phosphate, with half-maximal inhibition at 10 microM. Furthermore, non-glycosylated proliferin did not inhibit the binding of the glycosylated protein. A approximately 300 Kd proliferin receptor was purified from the liver of pregnant mice using a proliferin affinity column and elution with mannose 6-phosphate. This receptor reacted with antibodies directed against the rat cation-independent mannose 6-phosphate receptor. We conclude that 1) proliferin secreted by cultured cell binds to cation-independent mannose 6-phosphate receptors and therefore may be a lysosomal protein or targeted to lysosomes, and 2) the concentration or activity of mannose 6-phosphate receptors in murine fetal and maternal liver and in placenta is regulated during pregnancy.     

Glucocorticoid receptors in developing rat brain and liver

Dexamethasone receptors were measured by conventional equilibrium steroid binding studies in rat liver and brain cytosol, during late prenatal and postnatal development, Receptor binding could be detected in both cytosol preparations as early as the 17th day of gestation. Receptor levels in the cytosol from intact animals reached adult values by the 1st day after birth in both tissues. Using adrenalectomized animals an increase which reached adult values was observed during the first postnatal week for liver and the second postnatal week for brain. At physiological concentrations of endogenous glucocorticoids depletion of receptor from the cytosol of intact animals was minimal at postnatal day 1 and reached adult levels by day 7. chromatographie analysis in DEAE-Sephadex A50 minicolumns of unactivated and activated receptor constituents revealed the same pattern as that of adult animals. Glucocorticoid receptor complex from developing liver and brain was shown to be capable of binding to isolated adult liver nuclei after in vitro activation. However full capacity, for nuclear binding was observed in vivo, after injection of inducing doses of [³H]dexamethasone: By the end of the first week after birth adult nuclear binding capacity was observed in experiments in vivo while values peaked during the second week, in both tissues studied.     

The presence in bovine trachealis muscle of cytosol receptors with high affinity for glucocorticoids

Whereas receptors with high affinity for glucocorticoids have been reported present in both fetal and adult lungs, there has been no attempt to identify such receptors in airways smooth muscle. We have examined the binding characteristics of glucocorticoids for a cytosol fraction prepared from bovine trachealis muscle. The cytosol fraction, which was prepared in 10 mM Tris, 1.5 mM Na2 EDTA and 2 mM dithiothreitol, contained macromolecular high affinity receptors for 3H-dexamethasone (Kd = 5.6 nM) at a concentration of 0.20 pmol/mg protein. The comparative affinities of other steroids in descending order were: beclomethasone-17, 21-dipropionate, dexamethasone, beta-methasone, triamcinolone and triamcinolone acetonide. Progesterone and deoxycorticosterone only bound to a limited extent, whereas the binding with 5 alpha-dihydrotesterone and pregnenolone was negligible. We conclude that airways smooth muscle is a target tissue for glucocorticoids and postulate that the binding of glucocorticoids to these receptors may receptors may represent the initial step in the anti-asthmatic action of these drugs.     

Steroid hormone receptors in the thymus: a site of immunomodulatory action of melatonin.

1. Melatonin and glucocorticoids are known to affect the immune response in an opposite mode. The probability for an interaction between these hormones in the thymus gland has been investigated in rats following chronic administration of exogenous melatonin and long-term exposure to variable levels of circulating glucocorticoids. 2. Daily melatonin administration was shown to affect the properties of corticosterone and progestin receptors in the thymus in the presence of normal and increased systemic corticosterone concentrations, but not in adrenalectomized animals. 3. In intact rats melatonin caused a marked increase in the affinity and a decrease in the density of thymic receptors for adrenal steroids. Following corticosterone overdosage, simultaneously with melatonin treatment, a decrease in receptor affinity and a relative increase in the number of binding sites was observed. 4. The results suggest that steroid hormone receptors in the thymus might be considered as a target site for the interaction between melatonin and adrenal steroids in the modulation of the immune response.     

Glucocorticoids upregulate the high affinity receptors for vasoactive intestinal peptide (VIP) on human mononuclear leucocytes in vitro.

Glucocorticoids were shown to induce a time- and dose-dependent increment of specific [125I]VIP-binding on human mononuclear leucocytes in culture. Cortisol (0.5 microM) increased specific [125I]VIP-binding to 132% of control after 48 h preincubation, to 162% after 96 h, and to 175% after 144 h. Dexamethasone (0.5 microM) increased specific [125I]VIP-binding to 140%, 194% and 210% after the same time periods. Analysis of the binding data revealed an increase in Bmax to 119% by cortisol (0.5 microM, 48 h) and to 194% by dexamethasone (0.5 microM, 48 h), and no change in Kd for the high affinity receptor after preincubation. The number of low affinity binding sites for VIP was also increased by glucocorticoids. However, in contrast to the high affinity receptor, low affinity binding sites were initially downregulated in culture, and glucocorticoids induced a restitution to number and affinity close to those obtained for freshly isolated leucocytes. This increase in low affinity binding sites was blocked by actinomycin D, in contrast to the high affinity receptor upregulation which was independent of de novo protein synthesis. Furthermore, corresponding to the glucocorticoid induced high affinity receptor upregulation, an increase in VIP stimulated cyclic AMP production was observed. The results of this study suggest that leucocyte responsiveness to VIP can be influenced by glucocorticoids.