George Giannopoulos

Cortisol receptors in rabbit fetal lung

Abstract Rabbit fetal lung nuclei contain macromolecules which have the properties of physiological receptors for cortisol by the criteria of specificity of binding and saturation of binding sites at low concentrations of the hormone The number of nuclear receptor sites is relatively low at 20 days of gestation, reaches a maximum at about 28–30 days of gestation and drops slightly after birth. These results correlate well with previously reported changes in pulmonary epithelial cell maturation and surfactant concentrations in rabbit fetal lung extracts. Preliminary evidence for the presence of cytoplasmic cortisol-binding components in fetal lung cells is also presented.

Binding of testosterone to cytoplasmic components of the immature rat uterus.

Abstract The high speed supernatant of the immature rat uterus contains a macromolecule which binds 3 H-testosterone specifically and with high affinity (K d ca 2 × 10 −10 M at 4°). Mild heating (37°) or treatment with pronase results in release of 3 H-testosterone from the complex while nucleases have no effect. When examined in media of low ionic strength, the testosterone-protein complex aggregates with a predominant peak at ca 8S while in 0.4 M KCl buffered media a single component sedimenting at ca 4S is observed. Binding to this protein is specific for testosterone as 5α-dihydrotestosterone is a weak competitor for this binding site. The uterine cytosol contains at least one more type of binding site having a similar affinity for both testosterone and 5α-dihydrotestosterone. These binding sites are not saturable with hormone concentrations as high as 10 −6 M.

A comparative study of receptors for natural and synthetic glucocorticoids in fetal rabbit lung

Abstract The cytosol fraction of fetal rabbit lung contains three glucocorticoid-binding proteins. One of these binds only natural glucocorticoids and shares some of the properties of serum transcortin. The other two proteins, sedimenting at 4 S and 7 S, bind both cortisol and dexamethasone but they are stabilized to a different extent by each steroid. The 7 S component is detectable by sucrose density gradient centrifugation, charcoal adsorption or gel filtration when complexed with dexamethasone. However, 7 S cortisol-protein complexes could not be detected by any of these methods. The presence of the latter complexes is indicated by the ability of cytosol fractions sedimenting at 7 S to enhance specific nuclear uptake of cortisol in cell-free systems. In contrast to the 7 S receptor, the 4 S protein is more stable when complexed with cortisol and does not enhance significantly specific transfer of glucocorticoid into the nucleus. Binding of dexamethasone to the 4 S component can be demonstrated by its ability to displace pre-bound cortisol. The nuclear cortisol- and dexamethasone-receptor complexes formed after incubation of fetal lungs at 37°C are similar, both sedimenting at 4 S. The cortisol-receptor complexes are very labile and are partially stabilized in the presence of excess hormone.

Uptake and metabolism of cortisone and cortisol by the fetal rabbit lung

Abstract Lungs of rabbit fetuses at 28 days of gestation were incubated with tritium-labeled cortisone (17α,21-dihydroxy-4-pregnene-3,11,20-trione) or Cortisol (11β,17α,21-trihydroxy-4-pregnene-3,20-dione). The fetal lungs metabolized efficiently cortisone yielding cortisol as the major product (64–71% conversion). Cortisol was poorly metabolized, only 10–14% being converted to cortisone and 68–75% of the substrate being recovered unchanged. A small amount of cortisone (5–7% of tissue radioactivity) was also found in the lungs twenty minutes after injection of labeled cortisol to the fetus in utero . Incubation of fetal lungs with labeled cortisone at 37° resulted in specific uptake and binding of radioactivity (predominantly cortisol) to nuclear macromolecules. The amount of cortisol bound to nuclear macromolecules was similar whether the tissue was incubated with cortisol or cortisone. These results demonstrate that the lungs of the rabbit fetus have the capacity to convert the biologically inactive cortisone to the biologically active cortisol, the reverse reaction occurring only to a limited extent.

Early events in the action of glucocorticoids in developing tissues

Abstract The interaction of glucocorticoids with developing tissues has been examined. Saturable, high affinity, glucocorticoid-specific binding sites (receptors) have been detected in several fetal and mature tissues of the rabbit and other species. Extensive studies in fetal rabbit lung indicate that nuclear uptake and retention of glucocorticoids in the tissue involves a multi-step mechanism in which the steroid first associates with an extranuclear receptor protein. Subsequently, the steroid-receptor complex is activated to a form which can enter the nucleus where it interacts with acceptor sites in the chromatin. The nuclear acceptor sites appear to involve DNA and do not seem to be specific for target tissues. Fetal lungs of various species contain glucocorticoid receptors long before the normal appearance of surfactant in alveolar spaces. In some species the pulmonary receptor could be detected only during fetal life suggesting a maturation-dependent responsiveness of lung to glucocorticoids. In addition to its ability to bind and retain glucocorticoids, the fetal rabbit lung is very efficient in activating cortisone to cortisol. Inactivation of cortisol to cortisone is a minor reaction. The failure of glucocorticoids to induce tyrosine aminotransferase in fetal rat liver does not appear to be due to the absence of receptors in this tissue. Glucocorticoid receptors are present in fetal rat liver and their concentration increases after birth reaching mature levels by the 5th postnatal day. A complex pattern of glucocorticoid interaction with adult liver nuclei was observed. Both high affinity and low affinity binding sites were detected. The high affinity sites do not appear to be homogeneous since only a small fraction is extractable with 0·4 M KCl. In addition, a fraction of these sites is released by Triton X-100 suggesting an interaction of glucocorticoids with nuclear membranes. In contrast, fetal rat liver nuclei appear to contain only a single class of high affinity sites and no evidence for the presence of low affinity sites or for glucocorticoid interaction with nuclear membranes was obtained. Comparative studies suggest that the glucocorticoid receptors of fetal and adult rat liver may not be identical. Evidence for this includes differences in the relative affinity of cortisol and corticosterone for the binding sites of fetal and adult liver cytosol as well as differences in the dissociation constants and sedimentation coefficients of the steroid-receptor complexes.

EARLY EVENTS IN THE ACTION OF GLUCOCORTICOIDS IN DEVELOPING TISSUES

SUMMARY The interaction of glucocorticoids with developing tissues has been examined. Saturable, high affinity, glucocorticoid-specific binding sites (receptors) have been detected in several fetal and mature tissues of the rabbit and other species. Extensive studies in fetal rabbit lung indicate that nuclear uptake and retention of glucocorticoids in the tissue involves a multi-step mechanism in which the steroid first associates with an extranuclear receptor protein. Subsequently, the steroid-receptor complex is activated to a form which can enter the nucleus where it interacts with acceptor sites in the chromatin. The nuclear acceptor sites appear to involve DNA and do not seem to be specific for target tissues. Fetal lungs of various species contain glucocorticoid receptors long before the normal appearance of surfactant in alveolar spaces. In some species the pulmonary receptor could be detected only during fetal life suggesting a maturation-dependent responsiveness of lung to glucocorticoids. In addition to its ability to bind and retain glucocorticoids, the fetal rabbit lung is very efficient in activating cortisone to cortisol. Inactivation of cortisol to cortisone is a minor reaction. The failure of glucocorticoids to induce tyrosine aminotransferase in fetal rat liver does not appear to be due to the absence of receptors in this tissue. Glucocorticoid receptors are present in fetal rat liver and their concentration increases after birth reaching mature levels by the 5th postnatal day. A complex pattern of glucocorticoid interaction with adult liver nuclei was observed. Both high affinity and low affinity binding sites were detected. The high affinity sites do not appear to be homogeneous since only a small fraction is extractable with 0·4 M KCl. In addition, a fraction of these sites is released by Triton X-100 suggesting an interaction of glucocorticoids with nuclear membranes. In contrast, fetal rat liver nuclei appear to contain only a single class of high affinity sites and no evidence for the presence of low affinity sites or for glucocorticoid interaction with nuclear membranes was obtained. Comparative studies suggest that the glucocorticoid receptors of fetal and adult rat liver may not be identical. Evidence for this includes differences in the relative affinity of cortisol and corticosterone for the binding sites of fetal and adult liver cytosol as well as differences in the dissociation constants and sedimentation coefficients of the steroid-receptor complexes.

A specific glucocorticoid binding macromolecule of rabbit uterine cytosol.

Abstract A high affinity (Kd=2.7 × 10−10M at 0°) dexamethasone binding macro-molecule has been identified in the cytosol fraction of rabbit uteri. Competition studies show high specificity for glucocorticoids since binding of labeled dexamethasone is inhibited by cortisol and corticosterone but not by progesterone, testosterone, or estradiol 17β. The binding component has a sedimentation coefficient of 8S and its concentration in uterine cytosol is about 0.2 pmoles per mg protein. Uptake of labeled dexamethasone by isolated uterine nuclei requires the presence of cytosol and is temperature dependent. The KCl-extractable nuclear complex sediments at 4S. Thus the dexamethasone binding components of the rabbit uterus have properties similar to those described for steroid hormone receptors present in target tissues. Specific dexamethasone binding could not be demonstrated in rat uterine cytosol.

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 degrees. 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 KC1-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 KC1. This form of the receptor was not detected in fetal liver nuclei. In liver chromatin, bound dexamethasone exists in a KC1-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 KC1- and DNase-soluble, only 30 percent remaining in the residual pellet.

Effect of endogenous corticosterone on the determination of dexamethasone receptor levels in rat liver cytosol

The effect of endogenous corticosterone on the quantitative measurement of dexamethasone receptors in liver cytosols from developing rats has been studied. Liver cytosols from adrenalectomized rats were preincubated with increasing concentrations of nonlabeled corticosterone and the levels of detectable dexamethasone receptors were subsequently determined either directly or after removal of unbound corticosterone. Corticosterone concentrations of 50 nM or lower had no significant effect on the specific binding of labeled dexamethasone. Higher concentrations of corticosterone resulted in under-estimation of dexamethasone receptor levels. The mean levels of endogenous corticosterone in liver cytosols from 19.5- to 21.5- day fetuses, 22-day fetuses, 6-day-old immature rats and adult rats were 27.40, 11.91, 0.81 and 4.05 nM, respectively. It is concluded that variations in the levels of circulating corticosterone in the rat under normal physiological conditions have no significant effect on the quantitative measurement of total (occupied and unoccupied) receptor sites for dexamethasone in liver cytosol. This is supported by the finding that prior treatment of liver cytosols, from rats at different stages of development, with charcoal to remove unbound steroids has no effect on the amount of detectable dexamethasone receptors.

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

Abstract 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.