Nuclear progesterone-binding protein in the guinea pig adrenal cortex: distinction from the classical progesterone receptor

Nuclei purified from the guinea pig adrenal cortex contain a specific progesterone-binding activity which, based on enzyme degradation studies, appears to be proteinaceous. Saturation analysis revealed a Kd of about 15 nM and a binding capacity of about 33 pmol/mg DNA. The activity of the nuclear binding protein was specific essentially for progestational steroids; the two most potent progesterone competitors were 5 alpha-pregnane-3,20-dione and medroxyprogesterone (17 alpha-hydroxy-6 alpha-methylprogesterone), while 17 beta-estradiol, testosterone, cortisol, and other related steroids were poor competitors. The adrenocortical nuclear progesterone-binding protein was present to an equal extent in both male and female guinea pigs. The adrenocortical nuclear progesterone-binding protein differed from the classical progesterone receptor in that 1) the affinity of the adrenocortical binding protein for progesterone is an order of magnitude lower; 2) the potent synthetic progestin R5020 binds less tightly to the adrenocortical progesterone-binding protein; 3) the adrenocortical progesterone-binding protein is not modulated by estrogenic activity; 4) the adrenocortical progesterone-binding protein is more stable at 37 C; 5) the adrenocortical nuclear progesterone-binding protein is not salt extractable; and 6) Western blot analysis has revealed that an antiprogesterone receptor monoclonal antibody, which recognizes the guinea pig uterine classical nuclear progesterone receptor, does not recognize the adrenocortical nuclear progesterone-binding protein. Thus, the guinea pig adrenocortical nucleus contains a type of progesterone-binding protein that appears to be clearly different from the classical progesterone receptor.     

The nuclear conversion of pregnenolone to progesterone and subsequent binding to the nuclear progesterone-binding protein in the guinea pig adrenal cortex: a possible regulatory role for the pregnenolone-binding protein.

Progesterone, which is normally produced in the endoplasmic reticulum, was found to be rapidly degraded in the cytosolic fraction of the guinea pig adrenal cortex in vitro. Assuming this finding reflects what happens in vivo raises a question as to the source of progesterone for interacting with a nuclear progesterone-binding protein (P4-BP) that exists in this model system. It was subsequently found that pregnenolone, which in contrast to progesterone is relatively stable in the cytosol, was converted to progesterone by endogenous nuclear 3 beta-ol dehydrogenase. It was also determined that the nuclear-derived progesterone specifically bound to the nuclear P4-BP which is distinct from the classical progesterone receptor. The guinea pig adrenocortical cytosol contains a specific pregnenolone-binding protein (P5-BP) that could be virtue of its pregnenolone binding activity regulate the conversion of pregnenolone to progesterone in the nuclear compartment and thereby reduce the binding of progesterone to the nuclear P4-BP. A partially purified P5-BP preparation markedly inhibited the nuclear conversion of pregnenolone to progesterone and reduced the binding of progesterone to the nuclear P4-BP (P5-BP did not directly inhibit binding of progesterone to the nuclear P4-BP). The ability of P5-BP to inhibit the conversion of pregnenolone to progesterone was destroyed by heat and alkaline phosphatase treatment. The binding of pregnenolone to the P5-BP, as previously reported, is regulated by phosphorylation/dephosphorylation, and alkaline phosphatase-treated P5-BP loses the ability to bind pregnenolone; this process can be reversed by a cytosolic kinase. This provides a mechanism for controlled release of bound steroid. These results suggest that P5-BP regulates the nuclear conversion of pregnenolone to progesterone and thus the binding of progesterone to the nuclear P4-BP.     

Antibodies to rabbit progesterone receptor: crossreaction with human receptor.

Progesterone receptor from rabbit uterine cytosol was purified to a specific activity of approximately 2 nmol of bound hormone per mg of protein. A goat was immunized with this preparation and, after two injections of 0.7-0.8 nmol, yielded antireceptor antibodies. The antiserum reacted with both cytosolic and nuclear rabbit progesterone receptor and also with progesterone receptor from other rabbit tissues (vagina and pituitary). A crossreaction was observed with progesterone receptors from other mammalian, especially human, tissues (cytosolic receptor from rat and guinea pig uterus, cytosolic receptor from human breast cancer, and nuclear receptor from human endometrium). On the contrary, there was no interaction with a nonmammalian receptor (chicken oviduct progesterone receptor). The antibodies did not crossreact with other rabbit steroid receptors (uterine estradiol receptor and liver glucocorticoid receptor) or with nonreceptor progesterone-binding proteins (transcortin from plasma and uteroglobin from uterine fluid).     

Differential activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase in zones of the adrenal cortex

Cholesterol metabolism and steroidogenesis in the outer (zona fasciculata/glomerulosa) and inner (zona reticularis) zones of the adrenal cortex were examined in the guinea pig. It is known from previous studies that the content of cholesterol in the inner zone is considerably lower than that in the outer zone, although basal low density lipoprotein (LDL) receptor activity is similar in the two zones. To further explore cholesterol metabolism in the guinea pig adrenal cortex, the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting step in cholesterol synthesis, has been examined for which this paper forms the initial report. It was found that the basal specific activity of HMG-CoA reductase was similar in the outer and inner adrenocortical zones (approximately 230 pmol mevalonate formed/min X mg microsomal protein). The administration of ACTH caused 4- and 5-fold increases in HMG-CoA reductase activity in the outer and inner zones, respectively. In fact, the increase in HMG-CoA reductase activity with ACTH treatment was always greater for the inner zone than for the outer zone. This is in contrast to LDL receptor activity, which does not increase in the inner zone as it does in the outer zone with ACTH treatment. When dexamethasone was administered, HMG-CoA reductase activity decreased in the outer zone by about 50%, while there was no change in reductase activity in the inner zone. The latter finding is similar to what happens with LDL receptor activity during dexamethasone administration. Why suppression of endogenous ACTH had no effect on HMG-CoA reductase activity in the inner zone while exogenous ACTH administration caused a marked increase in enzyme activity is not clear, but may be related to phosphorylation/dephosphorylation mechanisms. Based on the use of sodium fluoride in solutions to block HMG-CoA reductase phosphatase, evidence is presented which indicates that a pharmacological dose of ACTH alters the phosphorylation/dephosphorylation status of HMG-CoA reductase in the inner adrenocortical zone, but not in the outer cortical zone.     

Cytoplasmic and nuclear progesterone receptors in the guinea pig uterus.

Although progesterone receptors have been studied in the uterine cytoplasm of many species, relatively little was known about the nuclear content of these binding proteins. In the present study, a nuclear progesterone receptor was deteced in the guinea pig uterus. The binding of progesterone to the nuclear receptor was hormone and tissue specific. Furthermore, the nuclear localization of the progesterone receptor complex in the uterus was both time and temperature dependent. Since the nuclear receptor was extracted in high salt buffer, the effects of KCl on several physical properties of the cytoplasmic and nuclear binders were studied. In the presence of high salt, cytosol and nuclear receptors were virtually indistinguishable. These proteins were clearly distinguished upon removal of the KCl by rapid dialysis: the nuclear receptor had a slower sedimentation rate, a faster rate of dissociation and a higher binding affinity than did the cytosol receptor for progesterone. We conclude that the cytosol and nuclear receptors for progesterone in the guinea pig uterus are distinct macromolecules. These observations are consistent with the postulate that the cytoplasmic receptor is a precursor of that in the nucleus.     

Tightly bound nuclear progesterone receptor is not phosphorylated in primary chick oviduct cultures.

Oviduct cells from estradiol-treated chicks were grown in primary culture. After 3-5 days of culture in medium containing estradiol, 90% of the cellular progesterone binding sites were detected in the cytosol. After exposure to [3H]progesterone at 37 degrees C, 80% of the progesterone binding sites were found in nuclear fractions. Progesterone receptor phosphorylation was assessed after incubating the cells with [32P]orthophosphate. Receptor components were immunoprecipitated with a specific polyclonal antibody (IgG-G3) and analyzed by NaDodSO4/PAGE and autoradiography. In the cytosol, constant amounts of 32P-labeled 110-kDa subunit (the B subunit, one of the progesterone-binding components of the receptor) and of the non-steroid-binding heat shock protein hsp90 were found, whether cells had been exposed to progesterone or not. No 32P-labeled 79-kDa subunit (the A subunit, another progesterone-binding subunit) was detected. Various procedures were used to solubilize nuclear progesterone receptor (0.5 M KCl, micrococcal nuclease, NaDodSO4), and in no case was 32P-labeled B subunit detected in the extracts. However, nonradioactive B subunit was detected by immunoblot in a nuclear KCl extract of progesterone-treated cells. These results suggest that the fraction of the B subunit that becomes strongly attached to nuclear structures is not phosphorylated upon exposure of cells to progesterone.     

Sequential expression of high and low density lipoprotein receptors in differentiating fetal rat adrenocortical cells in primary culture

Binding of fluorescent human high density lipoprotein-3 (DIL-HDL3) and fluorescent human low density lipoprotein (DIL-LDL) to differentiating fetal rat adrenocortical cells was studied in primary culture, a system in which addition of ACTH induces differentiation of zona glomerulosa cortical cells into zona fasciculata-type cells. Undifferentiated adrenocortical cells did not bind DIL-HDL3 or DIL-LDL. Differentiation induced by ACTH was accompanied by intense binding of DIL-HDL3 and DIL-LDL to plasma membranes of adrenocortical cells. Binding of DIL-HDL3 and DIL-LDL was saturable and specific, and resulted in enhanced secretion of corticosterone and 18-hydroxydeoxycorticosterone, indicating that the binding sites for these lipoproteins represented functional receptors. Binding of DIL-HDL3 to the surface of adrenocortical cells was detected 6 h after the start of ACTH stimulation, whereas binding of DIL-LDL could not be detected until 12 h after ACTH stimulation was started. Thus, in the ACTH-induced differentiation of adrenocortical cells from zona glomerulosa-type into zona-fasciculata type, exogenous lipoprotein cholesterol is needed for enhanced growth and steroidogenesis, and an initial event in the differentiation of these cells is the sequential expression of specific receptors for HDL3 and LDL.     

Expression of activin/inhibin receptor and binding protein genes and regulation of activin/inhibin peptide secretion in human adrenocortical cells

Activins and inhibins are glycoprotein hormones produced mainly in gonads but also in other organs. They are believed to be important para/autocrine regulators of various cell functions. We investigated activin/inhibin receptor and binding protein gene expression and the regulation of activin/inhibin secretion in human adrenal cells. RT-PCR revealed inhibin/activin alpha-, betaA/B-subunit, follistatin, activin type I/II receptor, and inhibin receptor (betaglycan and inhibin-binding protein) mRNA expression in fetal and adult adrenals and cultured adrenocortical cells. Cultured cells secreted activin A and inhibin A/B as determined by specific ELISAs. ACTH stimulated inhibin A/B secretion in fetal (1.8- and 1.8-fold of control, respectively) and in adult cells (3.4- and 1.7-fold of control, respectively) without significant effect on activin A. 8-bromoadenosine cAMP (protein kinase A activator) increased activin A and inhibin A/B secretion in the human adrenocortical NCI-H295R cell line (32-, 17-, and 3-fold of control, respectively). 12-O-tetradecanoyl phorbol-13-acetate (protein kinase C activator) stimulated both activin A and inhibin A secretion (764- and 32-fold of control, respectively), and activin treatment increased inhibin B secretion in these cells (25-fold of control). In conclusion, human adrenocortical cells produce dimeric activins and inhibins. ACTH stimulates inhibin secretion and decreases activin/inhibin secretion ratio, probably via the protein kinase A signal transduction pathway. This, together with the adrenocortical activin/ inhibin receptor and binding protein expression, suggests a physiological role for activins and inhibins in the human adrenal gland.     

Progesterone „Receptors” in the Cytoplasm and Nucleus of Chick Oviduct Target Tissue

This report demonstrates that the chick oviduct, a specific target organ for progesterone, contains both cytoplasmic and nuclear macromolecules which bind progestins. These binding molecules can be clearly distinguished from transcortin by centrifugation through sucrose gradients of low ionic strength and by agarose gel filtration. The cytoplasmic progesterone-binding molecules also bind 5-α-pregnane-3,20-dione, but have significantly lower affinity for cortisol, estrone, or aldosterone. They are absent from blood and nontarget organs such as lung and spleen. The tissue-specific binding components appear to be heat-labile proteins with an average dissociation constant for progesterone of about 8 × 10^-10 M at 2°C. These results are consistent with the identification of the progesterone-binding molecules as the functional hormone receptors. In further support of this concept is the finding that treatment of the chicks with estrogen coordinately induces a 20-fold increase in the number of progesterone-binding molecules and enhances the capacity of progesterone to induce avidin synthesis.     

Progesterone receptor in the chick bursa of fabricius: characterization and immunohistochemical localization

A high affinity progesterone-binding site was studied in the chick bursa of Fabricius. The dissociation constant for progesterone was 1.4 nM, and the concentration of progesterone-binding sites increased with estradiol treatment. In estradiol-treated bursas, the receptor concentration was about 240 fmol/mg protein. The binding site was specific for progestins, with the following order of affinities: ORG 2058 greater than progesterone greater than promegestone. Androgens, dexamethasone, and estradiol were weak competitors for progesterone binding in the bursa cytosols from estradiol-treated chicks. Immunoglobulin G fraction of antiserum (immunoglobulin G-RB) raised in rabbit against the B-subunit of chick oviduct progesterone receptor (PR) was used for an immunohistochemical study. The PR was found only in the interfollicular cells, which were most probably nonlymphoid cells. Staining was localized exclusively in the elongated nuclei of these cells. No staining was seen in the bursal epithelium or inside the lymphoid follicles. The results indicate that the interfollicular cells of the bursa contain specific PRs which are under estrogen regulation as in the oviduct. Thus, these cells might be under direct progesterone regulation.     

Low density lipoprotein receptor activity in the guinea pig adrenal cortex. I. Zonal characterization and response to adrenocorticotropin

The binding of [125I]iodo-low density lipoprotein ([125I]iodo-LDL) to isolated membranes was determined for the outer (glomerulosa/fasciculata) and inner (reticularis) zones of the guinea pig adrenal cortex. Binding of [125I]iodo-LDL to membranes from both zones was found to be highly specific and dependent on the presence of divalent cations, such as calcium. Saturation analysis revealed that the maximum high affinity binding capacity was similar for both zones under control conditions (approximately 400 ng/mg protein). When ACTH was administered, however, the maximum high affinity binding capacity increased in the outer zone by at least 3-fold, while there was no significant change for the inner zone. It was also noted that dexamethasone administration decreased [125I]iodo-LDL high affinity binding activity to less than half the control level in the outer zone without altering the binding activity in the inner zone. The cholesterol content in the outer zone was 2-3 times greater than that in the inner zone. When ACTH was administered, the concentration of cholesterol decreased significantly in both zones. Thus, ACTH appeared to influence cholesterol metabolism in the outer and inner adrenocortical zones, but was able to modulate LDL receptor activity only in the outer zone.     

Pregnancy-blocking progesterone antibody targets specifically the uterus through its progesterone-binding sites

Passive immunization with a mouse monoclonal antibody against progesterone, designated DB3, blocks pregnancy in several species. We have previously reported that DB3 localizes in the mouse uterine epithelium shortly before normal implantation. This phenomenon is pregnancy dependent and specific for the progesterone antibody. In this study we demonstrate that DB3 is present in the lumen of the uterus 36 h after an i.p. injection; this correlates with the time of maximum antibody reaction on the uterine epithelium. Incubation of DB3 with free progesterone, progesterone-hemisuccinate or progesterone-bovine serum albumin before administration prevented its localization on the epithelium, indicating that the localization requires free progesterone-binding sites and thus probably depends upon progesterone binding. In addition, studies in vitro show that DB3 can effectively bind to progesterone carried by high-affinity progesterone-binding protein purified from coypu plasma. We suggest that specific targeting of DB3 may be through progesterone associated with a progesterone-binding molecule on the membrane of the uterine epithelia. This may be an important part of the mechanism of antibody action against implantation.     

Monoclonal antibodies to rabbit progesterone receptor: crossreaction with other mammalian progesterone receptors.

A mouse was immunized with purified rabbit uterine cytosolic progesterone receptor (specific activity: 3 nmol of steroid bound per mg of protein). After fusion of its spleen cells with Sp2-OAg myeloma cells, supernatants of 11 hybrid cultures were found to react in both an immunoenzymatic test and a double-immunoprecipitation test with the progesterone receptor. Clones were obtained from the five hybrid cells that gave the strongest response in both tests. Antibodies from cell culture supernatants and ascitic fluids were characterized. Three are of the IgG1 and two of the IgG2a isotype. Their apparent affinity for the progesterone receptor was measured by immunoprecipitation in physiological salt conditions. The equilibrium dissociation constants were between 0.1 and 4 nM. All five monoclonal antibodies crossreacted with the rabbit nuclear receptor, the human cytosolic receptor, and other mammalian (rat, guinea pig) but not avian (chicken) cytosolic progesterone receptors. There was no interaction with the glucocorticoid receptor and corticosteroid binding globulin.     

Search for a progesterone-binding protein in secretory granules of the ovine corpus luteum

The hypothesis that electron-dense granules present in the corpus luteum contain progesterone in a protein-bound form was examined using differential and sucrose gradient centrifugation. Ovine luteal tissue was fractionated by differential centrifugation at 1,000 X g (P1 pellet), 10,000 X g (P2), and 82,000 X g (P3; supernatant S3). Samples of P2, P3, and S3 were further fractionated o 20-40% (P2 and P3) or 5-25% (S3) sucrose gradients and examined for progesterone-binding activity by measuring the progesterone content and/or the specific binding of [3H]progesterone of sucrose gradient samples. In addition, saturation binding assays were performed with steroid-free samples of P2 and S3. Saturable binding of progesterone was not found in P2, the fraction containing electron-dense granules. In S3, two progesterone-binding proteins with sedimentation rates of 3.2S and 8.6S and an affinity of 7.1 X 10(5) M-1 for progesterone were detected. The sedimentation behavior of these proteins was distinct from that of ovine plasma transcortin, a 4S protein. The view that a binding protein is released into the interstitial fluid during the exocytosis of granules was examined by measuring the progesterone-binding activity of protein released by slices of corpus luteum in vitro. No binding activity was found. The results of this investigation do not support the hypothesis that putative progesterone-secreting granules observed in luteal tissue contain a binding protein     

Oviduct progesterone receptor: Physical and chemical studies

Chicken oviduct progesterone receptor has been purified to homogeneity. The protein consists of two dissimilar hormone-binding subunits, A and B, present in equal amounts in the complex. They have molecular weights of 79,000 and 108,000, respectively, as shown by both SDS-gel electrophoresis of the purified proteins and photoaffinity labeling of both with a labeled synthetic progestin. The two subunits show considerable homology (or identity) of structure at the hormone-binding domain, located at the N-terminus of the proteins. Considerable divergence of sequence must exist elsewhere in A and B, as shown by tryptic peptide mapping and by the fact that subunit A has a strong DNA-binding site lacking in B. Both are phosphorylated in vitro by cAMP-dependent protein kinase; this phosphorylation appears to be responsible for creation of a second, weaker progesterone-binding site on each subunit.     

Effects of age and adrenocorticotropin on microsomal enzymes in guinea pig adrenal inner and outer cortices

Guinea pig adrenals possess a distinctive zona reticularis which increases in volume as the animal matures, coming to occupy over 50% of the gland in mature 800-g animals. The adrenocortical cells of this zone possess abundant smooth-surfaced endoplasmic reticulum. In this paper we demonstrate that microsomes obtained from guinea pig inner cortices of both young and mature animals, consisting predominantly of zona reticularis tissue, possess higher activities of 21-hydroxylase, an enzyme that hydroxylates steroids, and ethylmorphine demethylase, a drug-metabolizing enzyme, as well as higher contents of cytochromes b5 and P450, than microsomes derived from outer cortices, which contain predominantly zona fasciculata tissue. In addition, we show that while the inner cortex contains 90% of the total adrenal activity for ethylmorphine demethylase, ACTH suppresses this activity by 55% and decreases activity in the outer cortex to negligible levels. ACTH does not affect 21-hydroxylase activity or cytochrome b5 or P450 content in either cortical region. These observations suggest that differential regulation of microsomal functions for steroid hydroxylation and drug metabolism occurs largely in the zona reticularis. Furthermore, they suggest that other activities for xenobiotic metabolism, previously reported as a property of whole guinea pig adrenals, may reside in the inner cortex and be unique functions of the abundant smooth endoplasmic reticulum of the zona reticularis cells.     

Steroidogenesis activator polypeptide (SAP) in the guinea pig adrenal cortex

Steroidogenesis activator polypeptide (SAP), a cytosolic stimulator of cholesterol side-chain cleavage (cholesterol SCC) previously characterized in the rat, was isolated from guinea pig adrenal cortex. This factor exhibited behavior on reverse-phase high-performance liquid chromatography (HPLC) that was indistinguishable from authentic SAP and crossreacted fully in a SAP radioimmunoassay. In dexamethasone-suppressed guinea pigs neither the concentrations of immunoreactive adrenal SAP nor the levels of cholesterol SCC activity were significantly different between the outer zones (zonae glomerulosa and fasciculata) and the inner zone (zona reticularis). However, at 10 min after treatment of dexamethasone-suppressed animals with ACTH1-24, the outer zone content of SAP was increased 42-fold over unstimulated controls whereas inner zone SAP was elevated only 4-fold. At the same time, cholesterol SCC activity was increased 2-fold in the outer zones but unchanged in the inner zone. In addition to SAP itself, a crossreacting 82 kDa protein (p82)--similar to the putative SAP precursor identified in the rat--was detected on two-dimensional immunoblots of guinea pig whole adrenal homogenate. There were no significant differences in the protein concentrations of p82 or of cytochrome P-450scc between zones, either with or without ACTH treatment. We conclude that the widely reported contrast in corticosteroidogenic potential between the zona fasciculata and the zona reticularis of the guinea pig may reflect a differential capacity to generate SAP, and thus activate cholesterol SCC, in response to ACTH.     

Effects of ACTH administration on zonation of the guinea pig adrenal cortex

Experiments were done to determine the actions of ACTH on the morphologic and functional characteristics of the zona fasciculata (ZF) and zona reticularis (ZR) in the guinea pig adrenal cortex. In control guinea pigs, a number of morphologic differences distinguished the ZF from the ZR, including the presence of far more lipid in the ZF than in the ZR. Treatment with ACTH decreased the lipid droplet content of the ZF cells, equalizing the amount of lipid in the two zones. Other morphologic differences between the ZF and ZR were also diminished by ACTH treatment. Immunohistochemical analyses indicated that CYP17 protein was found in both the ZF and ZR in control animals, but with greater immunostaining intensity in the ZF. The enzyme protein distribution corresponded with higher 17alpha-hydroxylase activity in the ZF than in the ZR. After ACTH treatment, the intensity of staining and enzyme activities in the two zones were similar, attributable largely to increases in the ZR. In situ hybridization-and immunohistochemistry showed that in control animals CYPD216 was highly expressed in the ZR but not in the ZF. ACTH treatment dramatically reduced the intensity of CYP2D16 mRNA and protein staining in the ZR. Bufuralol 1'-hydroxylase activity, a marker for CYP2D subfamily members, was also decreased significantly in the ZR by ACTH treatment. The data indicate that administration of ACTH to guinea pigs has opposite effects on the expression of CYP17 and CYP2D16 in the ZR, and diminishes or eliminates some of the structural and functional differences between the ZF and ZR. The results suggest a role for ACTH in establishing and maintaining adrenocortical zonation.     

Demonstration of glucocorticoid receptors in the adrenal cortex: evidence for a direct dexamethasone suppressive effect on the rat adrenal gland

The adrenal cortex was evaluated for the presence of glucocorticoid receptors and functions. Substantial binding of [3H]dexamethasone was observed in aminoglutethimide-treated, hypophysectomized, and intact rats. Further studies demonstrated binding in cultured bovine adrenocortical cells and in Y-1 cells, a cloned murine cell line of adrenal cortical origin. Scatchard analysis of specific binding data in cytosol from hypophysectomized rats revealed an apparent Kd of approximately 15 nM and a receptor content (Nmax) of 123 fmol/mg cytosol protein. Analysis of Y-1 cell cytosol showed a Kd of approximately 17 nM and Nmax of 190 fmol/mg protein. The binding site in hypophysectomized rats had the following steroid specificities: high affinity for dexamethasone, corticosterone, and progesterone; moderate affinity for 11 beta-cortisol, and low affinity for testosterone, estradiol, pregnenolone, and 11 alpha-cortisol. Sedimentation in sucrose density gradients revealed 8S binding peaks in cytosols prepared from intact rat adrenal glands, Y-1 cells, and cultured bovine adrenocortical cells. Time- and temperature-dependent nuclear uptake of [3H]dexamethasone in Y-1 cells was demonstrated. In vivo treatment of hypophysectomized rats with dexamethasone significantly enhanced the rate of adrenal atrophy. ACTH stimulation tests in hypophysectomized rats showed a decreased corticosterone response in dexamethasone-treated rats compared to that in control animals. However, in vitro, there was no evidence for an effect of dexamethasone on ACTH-stimulated corticosterone production. The data indicate that the adrenal cortex possesses a high affinity binding site that fulfills the criteria for a glucocorticoid receptor. Glucocorticoid administration enhances adrenal atrophy and impairs adrenal function. We speculate that this action contributes to the suppressive effect of glucocorticoids on the pituitary-adrenal axis.     

Uteroglobin mRNA activity and levels of nuclear progesterone receptor in endometrium

To investigate the mechanism of induction of uteroglobin by progesterone, the relationship between uteroglobin mRNA (mRNA_UG) activity and nuclear progesterone receptor levels has been examined in rabbit endometrium during early pregnancy. mRNA_UG activity was assessed by translation in vitro of poly A-rich endometrial RNA and immunoprecipitation of the synthesized peptides using anti-uteroglobin antibodies. Progesterone receptor was determined in purified endometrial nuclei by Scatchard analysis of the specific binding of ³H-R5020 under exchange conditions. mRNA_UG activity reached a peak on Day 4 of pregnancy and declined thereafter up to Day 8. Nuclear progesterone receptor levels were uniformly low through this period, except for a slight rise on Day 2. When mRNA_UG activity was high, progesterone receptor levels were low. A cause-and-effect relationship between nuclear progesterone receptors and specific mRNA or protein synthesis remains to be established.