Nuclear Binding of Hydrocortisone-Receptors in the Embryonic Chick Retina and its Relationship to Glutamine Synthetase Induction

Binding of hydrocortisone (HC) to cytoplasmic receptors andof the resulting receptor-hydrocortisone complex (R-HC) to nuclearacceptor sites has been studied in neural retina cells of thechick embryo, in which this hormone induces glutamine synthetase(GS). These studies were done in a cell-free system, as wellas in intact retina tissue in culture. Optimal conditions, specificity, and the quantitative aspectsof R-HC binding to nuclei in the cell-free system were determined.Isolated nuclei retained their binding specificity for R-HCprepared from retina cytosol; at saturation, the total numberof nuclear acceptor sites for R-HC was estimated to be in therange of 1500 per nucleus. These sites were resistant to RNAsebut sensitive to DNAse. When retina tissue was cultured in the presence of progressivelyhigher doses (0–90 nM) of HC, increasing amounts of receptorswere translocated from the cytoplasm to the nuclei. Followingincubation of retina with 9–90 nM HC, the cytoplasm wasalmost completely depleted of receptors and the nuclei becamesaturated with R-HC. From the amount of R-HC bound to nucleiat saturation, the estimated total number of R-HC acceptor sitesper nucleus was comparable to that derived from the cell-freesystem. Increases in the level of GS induction in the retinacorrelated well with amounts of R-HC complex bound by nuclei.     

Binding of receptor-hydrocortisone complexes to isolated nuclei from embryonic neural retina cells

Hydrocortisone (HC) induces glutamine synthetase in the embryonic chick neural retina. The binding of cytoplasmic receptor-hydrocortisone (R-HC) complexes to isolated retina nuclei has been studied in a cell-free system. Optimal conditions, specificity and quantitative aspects of binding were determined. The isolated nuclei retained binding specificity for the R-HC complex prepared from retina cytosol. Free HC, estradiol-receptor complexes from retina cytosol and HC-receptor complexes from mouse brain cytosol or from chick serum did not bind to the nuclei. Assuming monovalency of the binding sites, the number of nuclear acceptor sites per retina cell for the R-HC complex was estimated to be in the range of 1500. These sites were resistant to RNAse but sensitive to DNAse.     

Effects of cytosine arabinoside on differential gene expression in embryonic neural retina. II. Immunochemical studies on the accumulation of glutamine synthetase

Cytosine arabinoside (Ara-C) elicits a significant increase in the level of the enzyme glutamine synthetase (GS) while it markedly reduces overall RNA and protein synthesis in cultures of embryonic chick neural retina. This increase was analyzed by radioimmunochemical procedures and compared with the induction of GS by hydrocortisone (HC). Accumulation of GS in Ara-C-treated retinas was found to be due to de novo synthesis of the enzyme; however, unlike the induction of GS by HC, Ara-C caused no measurable increase in the rate of GS synthesis. The results indicate that Ara-C facilitates GS accumulation largely by preventing degradation of the enzyme. Even though Ara-C inhibits the bulk of RNA synthesis in the retina, it does not stop the formation of GS-specific RNA templates. However, the progressive accumulation of these templates does not result in an increased rate of GS synthesis unless Ara-C is withdrawn from such cultures under suitable experimental conditions. Thus, it is suggested that the continuous presence of Ara-C imposes a reversible hindrance at the translational level which limits the rate of GS synthesis. The results demonstrate that the increase in retinal GS elicited by Ara-C is achieved through mechanisms which are quite different from those involved in the hydrocortisone-mediated induction of this enzyme.     

Cortisol receptors and inducibility of glutamine synthetase in embryonic retina

Glutamine synthetase (GS) is a marker enzyme for Müller glia cells in neural retina. In chick embryo retina GS begins to increase sharply on the 16th day of development, but can be precociously induced by premature supply of the inducer, cortisol, already on the 8th day. At this stage GS inducibility is low, but it increases progressively with embryonic age. We investigated whether there was a corresponding age-dependent increase of cortisol-binding molecules (cortisol receptors) and found that their level is highest in the early retina and decreases with development. In light of this inverse relationship, we examined whether functional characteristics of these receptors change with age, but detected no differences. In in vitro tests, receptors from older retina translocated cortisol into nuclei from young retina, and vice versa, with similar effectiveness. Also, cortisol receptors from liver cells (which differ from retina receptors) can translocate the hormone into retina nuclei, and vice versa. These findings indicate that translocation of cortisol receptors is neither tissue-specific or age-dependent, nor is it conditional on the total amount of receptors normally present in cells. Therefore, the age-dependent increase of GS inducibility in embryonic retina cannot be directly related to quantitative or functional differences of cortisol receptors and is evidently controlled primarily at the gene level. The very large amount of cortisol-binding molecules in early embryonic retina raises the possibility that they play some role in early differentiation of retina cells unrelated to hormone binding.     

Hormonal induction of glutamine synthetase in cultures of embryonic retina cells: requirement for neuron-glia contact interactions

Cortisol induces glutamine synthetase (GS) in gliocytes of chick embryo neural retina. Using adherent cultures of retina cells we have demonstrated that responsiveness of the gliocytes to GS induction by the hormone requires contact with neurons. GS is not inducible in high-density cultures depleted of neurons and consisting only of gliocytes. In neuron-containing cultures, induced GS was detected immunohistochemically only in those gliocytes that were closely juxtaposed with clusters of neurons. Unlike the induction of GS, the expression of carbonic anhydrase-C (which does not require cortisol) persisted in these glia cells also in the absence of neurons. The nature and role of glia-neuron interactions in the hormonal induction of GS are briefly discussed.     

The induction of glutamine synthetase in cell aggregates of embryonic neural retina: correlations with differentation and multicellular organization

A rapid increase in the synthesis and accumulation of the enzyme glutamine synthetase (GS) in the neural retina of the chick embryo characterizes the functional differentiation and maturation of this tissue. A precocious increase of GS can be induced in the embryonic retina by hydrocortisone and related corticosteroids. This paper presents evidence that the responsiveness of neural retina cells to GS induction by the hormonal inducer is dependent on histotypic associations and organization. This was demonstrated, using retina from embryos of different ages, by comparing GS induction in cultures of intact retina tissue with that in aggregates of retina cells and in monolayer cultures of retina cells.     

Nuclear binding of progesterone in chick oviduct. Multiple binding sites in vivo and transcriptional response.

1. Varied doses of labelled or unlabelled progesterone were injected into immature chicks which had previously been stimulated with oestrogen. The concentrations of nuclear bound [3H]progesterone were correlated with the effects of the hormone on endogenous RNA polymerase I and II activities in isolated oviduct nuclei. 2. The extent of nuclear localization of [3H]progesterone in oviduct (a progesterone target tissue) was shown to be much greater than in lung (non-target tissue). The conccentration of bivalent cations in solvents used in the nuclei isolations has a marked effect on the amount of bound hormone in the nuclei. 3. Evidence for the existence of several classes of binding sites for progesterone in the oviduct nuclei is given. These classes represent about 1000) 10000 and 100000 molecules of the hormone per cell nucleus and are saturated by injecting approx. 10, 100 and 1000 mug of progesterone respectively. 4. When saturation of the first (highest affinity) class of nuclear sites occurs, a marked inhibition in RNA polymerase II (but not RNA polymerase I) activity was observed. When the second class of sites was saturated, alterations in both RNA polymerase I and II activities were observed. Binding to the third class of nuclear binding sites was not accompained by further changes in polymerase activity. It is suggested that the first two classes of nuclear binding sites may represent functional sites for progesterone action in the chick oviduct.     

Compared intracellular localization of the glucocorticosteroid and progesterone receptors: an immunocytochemical study

The intracellular distribution of the glucocorticosteroid and progesterone receptors (GR and PR, respectively) was studied immunohistochemically. In control adrenalectomized (Adx) rat liver, immunostaining of paraffin sections revealed GR in cell nuclei, with a wide range of intensity between individuals. Following dexamethasone (Dex) treatment, the nuclear staining was uniformly high in all animals; the cytoplasmic staining was always weak and remained unchanged after Dex treatment. In frozen sections, the GR immunoreactivity in cell nuclei was weak in the absence and very strong in the presence of Dex, while no GR-specific cytoplasmic staining was observed. In frozen sections fixed in vapor of formaldehyde to avoid any artifactual redistribution of the receptor, some GR immunostaining was observed in the cytoplasm and the nucleus. In contrast, in paraffin as well as in frozen sections of chick oviduct, fixed by immersion or in vapor, PR was exclusively nuclear, including in the absence of progesterone, and the intensity of immunostaining was not modified by progesterone treatment. In order to verify if loss of nuclear receptors during tissue preparation could explain the differences in nuclear immunostaining observed between hormone-free and hormone-occupied GR, and between GR and PR, frozen sections of Adx rat liver and chick oviduct were preincubated at 4 degrees C in buffer solutions before the fixation procedure. It was found that hormone-free GR diffused out of the nucleus faster than hormone-occupied GR nuclei, and that nuclear GR diffused faster than nuclear PR. Based on these results, we propose that, during the fixation procedure, the fraction of nuclear GR which diffuses out of the nucleus is much smaller in the presence than in the absence of Dex. This lesser loss of nuclear GR after Dex treatment results in an increase of immunostaining after hormonal administration, which might have been erroneously interpreted as a sign of translocation from cytoplasm to nucleus. That the nuclear PR detection is not modified by progesterone treatment may be explained by its reduced diffusibility as compared to nuclear GR. This hypothesis does not rule out the existence of some cytoplasmic GR, whose significance remains unclear, but it offers a unified mechanism of action for all steroid hormone receptors. In the case of glucocorticosteroids, as already proposed for estradiol and progesterone, no step of cytoplasm to nucleus translocation would be required for hormone action, and transformation-activation would occur in the nucleus, resulting in tighter binding of the hormone receptor complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ecdysteroid binding sites in gastrolith forming tissue and stomach during the molting cycle of crayfish Procambarus clarkii.

The distribution of ecdysteroid binding sites in the stomach and gastrolith disc tissue of crayfish (Procambarus clarkii) was examined in relation to the molting stage by thaw-mount autoradiography. The radiolabeled hormone analogue ponasterone A (25-deoxy-20-hydroxyecdysone) was used. Ecdysteroid binding sites were demonstrated only in certain molting stages, the small gastrolith period and the aftermolt stage. In gastrolith epithelium, ponasterone A binding sites first appeared in the cytoplasm, and then in the nuclei and cytoplasm. In the stomach epithelium, many nuclear binding sites were detectable during the period of gastrolith secretion. These periodical changes in specific ponasterone A binding when correlated with the molting stages clearly show that ecdysteroids may function as an initiator for gastrolith formation and reabsorption. The findings also suggest that ecdysteroids control calcium transport in the stomach epithelium. The time-related and functional differences of cytoplasmic and nuclear concentration of ecdysteroid receptors indicate the presence of cytoplasmic and nuclear receptors associated with specific actions.     

Modulation of cortisol receptors in embryonic retina cells by changes in cell-cell contacts: Correlations with induction of glutamine synthetase

Cortisol induces glutamine synthetase (GS) in neural retina tissue of chick embryos. GS induction represents a characteristic feature of embryonic retina differentiation. However, if the tissue is dissociated into single cells, the dispersed cells are not inducible for GS. We report that cell dispersion results in a rapid and marked reduction in the level of cortisol-binding cytoplasmic receptors. This reduction persists if the cells are maintained in a dispersed state. However, if the cells are reaggregated and they reconstruct tissue-like contacts and architecture, the level of cortisol receptors increases, and so does inducibility for GS. The results indicate that, in the embryonic neural retina histotypic cell contacts and interactions are involved in regulating the level of cortisol receptors. We propose that cell contact-dependent signals from the cell surface may modulate levels of cytoplasmic cortisol receptors necessary for GS induction.     

Nuclear localization and DNA binding of ecdysone receptor and ultraspiracle

The Ecdysone receptor (EcR) is distributed between cytoplasm and nucleus in CHO cells. Nuclear localization is increased by the ligand Muristerone A. The most important heterodimerization partner Ultraspiracle (Usp) is localized predominantly in the nucleus. We used the diethylentriamine nitric oxide adduct DETA/NO, which releases NO and destroys the zinc-finger structure of nuclear receptors, to investigate whether nuclear EcR and Usp interact with DNA. If expressed separately, Usp and EcR in the absence of hormone do not interact with DNA. The hormone-induced increase in nuclear EcR is due to enhanced DNA binding. In the presence of Usp, EcR is shifted nearly quantitatively into the nucleus. Only a fraction (approximately 30%) of the heterodimer is sensitive to DETA/NO. Interaction of the heterodimer with DNA is mediated mainly by the C-domain of EcR. Deletion of the DNA-binding domain of Usp only slightly reduces nuclear localization of EcR/Usp, although the nuclear localization signal of Usp is not present anymore. The results indicate that EcR and Usp can enter the nucleus independently, but cotransport of both receptors mediated by dimerization via the ligand binding domains is possible even in the absence of hormone.     

Mechanisms of replenishment of nuclear and rogen receptor in rat ventral prostate

1. The concentration of androgen receptor in the nucleus of the prostatic cell is rapidly elevated by the administration in vivo of 2μg of [³H]testosterone to 1-day-castrated rats. From a concentration of 2300 receptors/nucleus at 5min after intravenous injection of hormone, there is an increase to 21000 receptors/nucleus at 60min. At the same time, the amount of binding of androgen in the cytoplasm remains constant at a relatively low value. 2. An identical dose of [³H]testosterone administered to 7-day-castrated rats produces a much smaller change in the concentration of nuclear receptor, from 700 receptors/nucleus at 5min to only 4300 receptors/nucleus at 60min. Thus the reservoir from which nuclear receptor is replenished is considerably smaller in regressed prostatic cells. Again, the amount of binding of androgen in the cytoplasm remains unchanged at a low value over the experimental time course of 60min. 3. In contrast with the scant labelling of cytoplasmic receptor achieved by injecting animals with [³H]testosterone, labelling in vitro, by incubation of tissue slices with radioisotope, indicates that prostate of 1-day-castrated animals actually contains 21400 receptors/cell in the cytoplasmic compartment, and prostate of 7-day-castrated animals 3000 receptors/cell. 4. Owing to the similarity between the concentration of nuclear receptor measured in vivo and the concentration of cytoplasmic receptor measured in vitro, the labelling techniques in vivo and in vitro were used in sequence to demonstrate the movement of most of the cytoplasmic receptor into the nucleus. In the 5–60min interval after the administration of [³H]testosterone to 1-day-castrated rats, a decrease of 17400 receptor molecules in the cytoplasm is exactly mirrored by an increase of 17200 receptor molecules in the nucleus. 5. These results imply that, in prostate of 1-day-castrated rats, nuclear receptor is replenished exclusively by translocation of cytoplasmic receptor. However, in the regressed prostate of 7-day-castrated rats, only about 25% of the nuclear receptor is replenished through translocation of existing cytoplasmic receptor. The remainder is ultimately synthesized during new rounds of cell division induced by hormone.     

Progesterone-binding components of chick oviduct. IX. The kinetics of nuclear binding.

A cell-free system was used to study the kinetics of progesterone-receptor interaction with purified nuclei prepared from estrogen-primed chick oviducts. The binding process was a saturable phenomenon in both target and nontarget tissues. More nuclear acceptor sites were available to target tissue (similar to 9000 sites per oviduct nucleus) than in nontarget tissues (similar to 1000 to 3000 sites per nucleus), but the binding constant was essentially the same (Kd similar to 10-8 M). A second much smaller class of higher affinity sites (Kd similar to 10-11M) may exist. Its presence was detected by Scatchard plot nonlinearity at very low concentrations of added receptor-hormone complex (similar to 10-10 to 10-12M). The current study focused on the prevalent class of acceptor sites which was more readily detectable. Receptor binding to these sites was highly sensitive to salt. More sites were exposed at 25 degrees than at 0 degrees. Binding to these sites was inhibited in a nonselective fashion by the addition of protein. Although receptors may be activated by temperature or conditions of high ionic strength, these conditions could not capacitate more than 30 to 40% of the progesterone-receptor proteins for binding. Rate studies suggested that temperature plays a minimal role in nuclear uptake of activated receptors. Such a finding is consistent with a diffusion-limited uptake process.     

Decrease in triiodothyronine binding sites in chick embryo erythrocytes during early development

Specific thyroid hormone (TH) binding sites have been detected in nuclei of erythrocytes obtained from developing chick embryos. The binding characteristics and relative affinities for TH analogs were those expected of TH receptors. Nuclear triiodothyronine (T3) saturation analysis was carried out in vitro by incubating intact erythrocytes in M199 medium with 3-200 pM [125I]T3 for 1 hr at 37 degrees C or 20-24 hr at 21 degrees C. Nuclei were obtained by centrifugation after lysing the erythrocytes in a stabilizing buffer containing 0.3% saponin, followed by addition of Triton X-100 (final concentration 0.2%) to minimize the nonspecific binding. Scatchard analysis of equilibrium binding data suggested that the nuclei possess a single class of binding sites. The binding is reversible and the rate of dissociation is temperature dependent. T3 and T4 appear to bind to the same sites, but the affinity of T3 was 16 times greater. Among TH analogs tested, Triac had the highest affinity followed by L-T3, D-T3, Tetrac, L-T4, D-T4, T2, and rT3. Serial studies performed on different days of chick embryogenesis demonstrated a rapid and significant decrease of the erythrocyte nuclear T3 receptor. On Day 5, the number of T3 binding sites was maximal at 1600 +/- 100 per nucleus. The number declined steadily until, by Day 20, it had reached about 60 +/- 10 sites/nucleus. RBC from adult and baby chickens had less than 1% as many binding sites as those from Day 5 embryos. There was no significant change in the affinity of the sites (Kd approximately equal to 20 pM at 37 degrees C). The reason for the loss of T3 binding sites during embryogenesis is not known. Since the plasma level of the TH increases during embryogenesis, this may reflect down regulation. Another possibility is that the change in erythrocyte population which occurs during this period involves production of erythrocytes which contain fewer T3 binding sites.     

Ecdysteroid binding sites in gastrolith forming tissue and stomach during the molting cycle of crayfishProcambarus clarkii

Summary The distribution of ecdysteroid binding sites in the stomach and gastrolith disc tissue of cryafish (Procambarus clarkii) was examined in relation to the molting stage by thaw-mount autoradiography. The radiolabeled hormone analogue ponasterone A (25-deoxy-20-hydroxyecdysone) was used. Ecdysteroid binding sites were demonstrated only in certain molting stages, the small gastrolith period and the aftermolt stage. In gastrolith epithelium, ponasterone A binding sites first appeared in the cytoplasm, and then in the nuclei and cytoplasm. In the stomach epithelium, many nuclear binding sites were detectable during the period of gastrolith secretion. These periodical changes in specific ponasterone A binding when correlated with the molting stages clearly show that ecdysteroids may function as an initiator for gastrolith formation and reabsorption. The findings also suggest that ecdysteroids control calcium transport in the stomach epithelium. The time-related and functional differences of cytoplasmic and nuclear concentration of ecdysteroid receptors indicate the presence of cytoplasmic and nuclear receptors associated with specific actions.     

Comparison of the Binding of [3H]Spiperone and [3H]Domperidone in Homogenates of Mammalian Retina and Caudate Nucleus

Abstract— The specific binding of [³H]spiperone and [³H]domperidone, as defined by 1 μ m -(+)butaclamol, was compared in homogenates of bovine retina and caudate nucleus. Scatchard analyses of saturation data for [³H]spiperone binding yielded dissociation constants ( K _d) of 0.35 n m in the retina and 0.64 n m in the caudate nucleus. Comparison of the maximum number of binding sites (B_max) present in each tissue indicated that the density of sites in bovine caudate nucleus (270 fmol/mg protein) was approximately three times higher than in bovine retina (92 fmol/mg protein). This difference was even more marked in guinea pig tissues, with a ratio of 7:1 between corpus striatum and retina. The pharmacological analysis of [³H]spiperone binding in both the bovine retina and caudate nucleus indicated an interaction with dopaminergic rather than serotonergic sites. However, inhibition curves obtained to dopaminergic agonists in the bovine retina were significantly steeper than those observed in the bovine caudate nucleus, as reflected in the greater Hill coefficients obtained for these agents in the retina. Furthermore, only a small amount of specific [³H]domperidone binding was observed in either the bovine caudate nucleus or the guinea pig striatum, whilst no specific [³H]domperidone binding was detectable in homogenates of either bovine or guinea pig retina. These data suggest that the retina possesses only a small population of dopaminergic D2 sites and that these binding sites may differ from those present in the caudate nucleus.     

Dynamic Aspects of Glucocorticoid Receptors in the Spinal Cord of the Rat

In spite of biochemical and autoradiographic evidence for glucocorticoid binding sites in the spinal cord (SC), events occurring after the preliminary step of hormone binding were not studied. In this investigation, we have examined the transformation (activation) of the cytosolic receptor coupled to [3H]dexamethasone (DEX) and the in vivo interaction of adrenal hormone [corticosterone (CORT)] with purified nuclei from the SC, in addition to the CORT content of the SC before and after stress. Binding of [3H]DEX in the SC was 40% lower than in the hippocampus (HC), although the KD values were comparable. Transformation of [3H]DEX-receptor complexes in the cytosol was demonstrated by diethylaminoethane-cellulose chromatography, by DNA-cellulose binding, and by a combined minicolumn procedure including hydroxyapatite in addition to the last two techniques for separation of transformed, nontransformed, and meroreceptor complexes. In all these situations, SC glucocorticoid binding sites behaved similarly to those in the HC. Nuclear uptake of a tracer dose of [3H]CORT was much lower in the SC than in the HC; nuclear retention of CORT was more easily detected by radioimmunoassay after injection of 1 mg of CORT into adrenalectomized rats. Substantial amounts of CORT, which increased in level after stress, were measured in five regions in the SC, with higher concentrations in the cervical regions. These studies suggest that although SC and HC receptors show similar properties in vitro, differences emerged at the level of nuclear uptake in vivo, in that glucocorticoid action in the SC was similar to that in the optic nerve, where receptors seem to be localized mostly in glial cells.