Differential response of type I and type II corticosteroid receptors to changes in plasma steroid level and circadian rhythmicity

Corticosterone (CORT) binds to two receptor systems in rat brain: the type I CORT-preferring receptor (CR) and the type II glucocorticoid receptor (GR). Discrimination between the two receptor types can be achieved with the 'pure' synthetic glucocorticoid RU 28362. In this study, we show that the binding capacity of GR in the rat hippocampus exhibits a strikingly different response from CR to adrenalectomy (ADX), chronic steroid replacement, hypophysectomy (HYPOX) and during circadian variation. Under those experimental conditions neither receptor site showed changes in binding affinity. After ADX, CR number remained relatively constant for a period of 13 days, while GR capacity increased by 133%, a level which was reached 5 days post-surgery. CR capacity showed circadian variation, since CR number was 65% higher in the evening than in the morning. GR capacities at those two time points were not significantly different. Replacement with subcutaneous CORT implants (100-mg pellets) for 7 days following ADX rats did not affect CR number, but caused a 38% decrease in GR number compared to control animals (cholesterol-treated, 7-day-ADX rats). On the other hand, dexamethasone (DEX) implants (5-, 15-, 25-mg pellets) elicited a dose-dependent increase in CR capacity (up to 99%) and a dose-dependent decrease in GR capacity (40-44%). Finally, 2 weeks after HYPOX, CR and GR numbers were increased by 60 and 38%, respectively. We conclude that the type II GR capacity responds in an autoregulatory manner to changes in circulating plasma glucocorticoid levels, while type I CR does not.     

Basal and glucocorticoid induced changes of hepatic glucocorticoid receptor during aging: relation to activities of tyrosine aminotransferase and tryptophan oxygenase

The characteristics of glucocorticoid receptors, their sensitivity to glucocorticoid as well as the basal and glucocorticoid induced thyrosine aminotranferase (TAT) and tryptophan oxygenase (TO) activities were studied in rat liver during aging. The concentration (N) and dissociation constant (K_d) of glucocorticoid receptor (GR) significantly change during the aging both in untreated and dexamethasone treated animals. The level of receptors was lower in dexamethasone treated rats of all analyzed aged groups compared to untreated animals. In comparison to untreated groups, there was no correlation between the changes of N and K_d during the lifespan. According to immunochemical analysis, the decline of receptor protein content occurs during lifespan. Dexamethasone treatment reduced the level of receptor protein compare to respective age group of untreated rats. The glucocorticoid-receptor (G-R) complexes from both untreated and treated animals underwent thermal activation, although the extent of activation was more pronounced in the case of untreated groups compared to treated animals. The magnitude of heat activation of receptor complexes was more pronounced in the liver of the youngest untreated rats compared to elderly ones, while the receptor activation between treated groups of studied ages has shown less significant differences. Besides, basal as well as induced TAT and TO activities after dexamethasone injection also showed age-related alterations. The observed alterations in GR might play a role in the changes of the cell responses to glucocorticoid during the age. This presumption is supported by detected changes in basal and dexamethasone induced TAT and TO activities during aging.     

Effect of growth hormone on prolactin receptors and on estrogen receptors in the liver of prepubertal rats

The regulation of both the sex-differentiated prolactin (Prl) receptor and the non-sex-differentiated estrogen receptor in rat liver was studied. In prepubertal female rats both liver Prl and estrogen receptors were found at a lower concentration compared to the receptor level present in adult female rats. The effect of growth hormone (GH) treatment on Prl and estrogen receptor levels was investigated. In prepubertal female rats (17 and 22 days old), infusion of human GH using an infusion rate of 0.1 microgram/h per g body weight (BW) over a period of 6 days resulted in an increase in the number of Prl receptors to a level normally found in adult female rats. Human GH also induced Prl receptors in prepubertal male rats to an adult female level. In contrast, the same infusion rate of human GH given to female rats at 17 and 22 days of age did not increase the level of estrogen receptor. Addition of the glucocorticoid dexamethasone, 2.8 ng/h per g BW, to the human GH infusion did not result in estrogen receptor induction. These findings indicate a differential response to human GH administration of Prl receptor and estrogen receptor induction in prepubertal rats. Thus, the so-called 'feminizing effect' of GH (induction of Prl receptors) can be achieved in prepubertal rats whereas a non-sex-differentiated effect of GH (induction of estrogen receptors) could not be evoked in prepubertal rats using the same dose of human GH.     

Heat stress and hydrocortisone are independent stimulators of triiodothyronine-induced growth hormone production in cultured rat somatotrophic tumour cells

We have reported that, in cultured GC cells, the stress of incubation at 41 degrees C enhances thyroid hormone stimulation of growth hormone (GH) in a manner similar to the effects observed in a model of nonthyroidal disease in rats. Since glucocorticoids are potentially involved in stress responses both in vivo and in cell culture, we studied the role of glucocorticoid in the enhancement of (which are rat somatotrophic tumor cells) triiodothyronine (T3)-induced GH synthesis due to heat stress. Hydrocortisone addition increased T3-induced GH synthesis and GH mRNA content in cultured GC cells at both 37 degrees C and 41 degrees C. Depletion of glucocorticoid endogenous to serum supplement of the tissue culture medium did not prevent the enhancement of T3-induced GH synthesis that occurred during incubation at 41 degrees C. The levels and affinity of glucocorticoid cytosolic receptors were not enhanced during incubation at 41 degrees C. Lastly, no change in the sedimentation coefficient of the cytosolic glucocorticoid receptor or in its translocation into the nucleus occurred during incubation at 41 degrees C. Thus, the enhancement of T3-induced GH production in GC cells by heat stress appeared independent of the effect of glucocorticoids and not mediated through glucocorticoid receptors.     

Lack of decrease in hypothalamic and hippocampal glucocorticoid receptor mRNA during starvation

We have shown in a previous study that high corticosterone levels during repeated immobilization stress result in a reduction of glucocorticoid receptor (GR) mRNA in the hypothalamic paraventricular nucleus (PVN) and the hippocampus. The reduction of GR presumably accounts for loss of or decrease in glucocorticoid-negative feedback, and thus hyperfunction of the hypothalamic-pituitary-adrenocortical (HPA) axis persists during chronic stress. Starvation is a stress state in which the counterregulatory responses against the loss of food occur in the central nervous system. We explored the impact of starvation on the HPA axis, GR and mineralocorticoid receptor (MR) mRNAs in the hippocampus, the PVN, and the anterior pituitary (AP) of rats. Rats were starved for 4 days and sacrificed in the morning. Starved rats showed high levels of plasma corticosterone, whereas neither plasma corticotropin (ACTH), AP proopiomelanocortin (POMC) mRNA nor AP type-1 corticotropin-releasing hormone (CRH) receptor mRNA was altered in the starved rats. In the presence of high corticosterone, starvation resulted in a decrease in both CRH mRNA and type-1 CRH receptor mRNA in the PVN. Consistently, the starved rats did not show any changes in GR mRNA in the hippocampus (CA1-2, CA3, and dentate gyrus), the PVN or the AP despite the elevation of plasma corticosterone. A significant decrease in MR mRNA was seen in the dentate gyrus and the AP, but not in CA1-2, CA3 or PVN. The lack of reduction of GR may be one of the organism's counterregulatory responses during starvation, which allows an intact glucocorticoid negative feedback, thereby resulting in decreased anorectic neuropeptide levels, namely CRH, in the PVN. The results also indicate that GR mRNA in the hippocampus and other brain regions is not solely regulated by circulating glucocorticoids. The mechanism underlying the regulation of GR mRNA in the central nervous system remains to be clarified.     

Stress and cortisol treatment changed affinity and number of glucocorticoid receptors in leukocytes and gill of coho salmon.

To determine whether glucocorticoid receptors were altered during observed changes in immune function after stress or cortisol treatment, we conducted a series of experiments in which juvenile coho salmon (Oncorhynchus kisutch) were acutely or chronically stressed or fed a single meal containing cortisol. We then determined glucocorticoid receptor (GR) binding in cell-free gill preparations and whole leukocytes from spleen and anterior kidney using the synthetic hormone triamcinolone acetonide as radioligand. The affinities of GR were consistently lower in all tissues from chronically stressed fish than in tissues from controls; however, numbers of GR increased in whole leukocytes and decreased in gill. Acute stress had no affect on GR in gill and did not affect the affinity of GR in anterior kidney leukocytes, but did increase the number of GR in those leukocytes. Acute stress reduced affinity and increased numbers of GR in splenic leukocytes, suggesting heterogeneity of response to stress. Feeding cortisol to fish resulted in changes in GR from gill similar to those caused by chronic stress. Incubating leukocytes in cortisol for 3 or 24 h prior to assaying GR resulted in increased number and decreased affinity of GR in anterior kidney leukocytes, but had no effect on cells from spleen.     

Enduring, handling-evoked enhancement of hippocampal memory function and glucocorticoid receptor expression involves activation of the corticotropin-releasing factor type 1 receptor

Early-life experience, including maternal care, influences hippocampus-dependent learning and memory throughout life. Handling of pups during postnatal d 2-9 (P2-9) stimulates maternal care and leads to improved memory function and stress-coping. The underlying molecular mechanisms may involve early (by P9) and enduring reduction of hypothalamic corticotropin-releasing factor (CRF) expression and subsequent (by P45) increase in hippocampal glucocorticoid receptor (GR) expression. However, whether hypothalamic CRF levels influence changes in hippocampal GR expression (and memory function), via reduced CRF receptor activation and consequent lower plasma glucocorticoid levels, is unclear. In this study we administered selective antagonist for the type 1 CRF receptor, NBI 30775, to nonhandled rats post hoc from P10-17 and examined hippocampus-dependent learning and memory later (on P50-70), using two independent paradigms, compared with naive and vehicle-treated nonhandled, and naive and antagonist-treated handled rats. Hippocampal GR and hypothalamic CRF mRNA levels and stress-induced plasma corticosterone levels were also examined. Transient, partial selective blockade of CRF1 in nonhandled rats improved memory functions on both the Morris watermaze and object recognition tests to levels significantly better than in naive and vehicle-treated controls and were indistinguishable from those in handled (naive, vehicle-treated, and antagonist-treated) rats. GR mRNA expression was increased in hippocampal CA1 and the dentate gyrus of CRF1-antagonist treated nonhandled rats to levels commensurate with those in handled cohorts. Thus, the extent of CRF1 activation, probably involving changes in hypothalamic CRF levels and release, contributes to the changes in hippocampal GR expression and learning and memory functions.     

Expression of GH receptor mRNA in regenerating skeletal muscle of normal and hypophysectomized rats. An in situ hybridization study.

Expression of growth hormone receptor mRNA was investigated by in situ hybridization in skeletal muscle from normal and hypophysectomized rats during the first seven days of regeneration after ischemic injury. A digoxigenin-labelled RNA probe directed against the extracellular part of the rat GH receptor was used. In both normal and hypophysectomized rats distinct expression of GH receptor mRNA could be demonstrated in the regenerating muscle cells at the myoblast/myotube stage. The GH receptor expression appeared to decline with increasing maturation of the regenerated muscle fibres. In hypophysectomized rats, the regeneration process and the expression of GH receptor mRNA was delayed compared with that in normal animals. It is concluded that growth hormone may affect also the early phase of muscle regeneration in normal animals. To what extent lack of growth hormone contributes to the delayed regeneration observed in the hypophysectomized rats remains to be elucidated.     

Sexually dimorphic effects of aging on rat somatotropes and lactotropes

During the aging process modified functions of hypothalamic factors may cause sexually dimorphic changes in pituitary somatotropes and lactotropes. To test this hypothesis, pituitary tissue from young adult (4 months) and old (20-22 months) male and female rats was labeled immunocytochemically for growth hormone (GH) and prolactin (PRL). The total amount of immunoreactive material as well as the total area and number of immunoreactive structures were evaluated. With increasing age the intracellular GH content was moderately increased in male and decreased in female rats. An age-dependent PRL increase, due both to increased cell number and intracellular hormone content, was present only in female rats. The amount of GH- and PRL-immunoreactive material, distributed into classes of increasing density, differed both between sex and age groups. Our results indicate that the aging process of the somatotrope and lactotrope cell populations in rats appears to be different in the two sexes.     

Glucocorticoid receptor inhibits microtubule assembly in vitro

The effect of glucocorticoid hormones, purified glucocorticoid receptor (GR) and purified heat shock protein M_r 90 000 (hsp90) on microtubule (MT) assembly in vitro was tested by a spectrophotometric MT assembly assay and electron microscopy. GR significantly prolonged the nucleation phase, slowed down the assembly rate and reduced the maximal amplitude of MT assembly compared with control. The effects were partially reversed by the addition of glucocorticoid hormone. GR associated with MTs. These results indicate that GR affects MT assembly in vitro, which may be a functional correlate to the structural association of GR with MTs. This implies that factors affecting GR may affect MT assembly in vivo.     

Thyroid hormone and glucocorticoid regulation of receptor and target gene mRNAs in pituitary GH3 cells

We have examined the effects of triiodothyronine (T₃), in dose-response and time-course studies, on T₃ receptor (T₃R) and β and glucocorticoid receptor (GR) mRNAs in rate pituitary GH₃ cells, in parallel with T₃ actions on expression of the growth hormone (GH) target gene. Modulatory influences of dexamethasone (dex) on T₃ action were studied by treatment with dex before and during T₃ treatment. T₃ treatment (1–100 nM) for 24 h reduced T₃R mRNA, while the presence of dex (1 μM) enhanced the T₃ effect on T₃R mRNA and induced T₃ inhibition of T₃R β mRNA. Stimulatory effects of T₃ treatment on GH mRNA and release were seen in the face of inhibition of T₃R mRNAs; these effects on GH were also enhanced by the presence of dex. T₃ treatment for 24 h increased GR mRNA; this effect was inhibited by the presence of dex. We next examined the influence of dex on GR and T₃R and β mRNAs, in parallel with effects of dex on the prolactin (PRL) target gene. Modulatory influences of T₃ on dex action were studied by treatment of cells with T₃ before and during dex treatment. Treatment with dex (0.1–10 μM) for 24 h reduced GR mRNA, an action enhanced by the presence of T₃ (100 nM). Dex treatment resulted in inhibition of PRL mRNA and release despite parallel inhibition of GR mRNA by dex; these effects were enhanced by the presence of T₃. In contrast to actions on GR, dex has no effect on T₃R mRNAs. These effects of T₃ and dex on receptor mRNAs suggest that glucocorticoid modulation of T₃ action is not related to direct actions on T₃R synthesis. In contrast, the mechanism of T₃ modulation of glucocorticoid action may be due in part to alteration of GR mRNA expression. Effects of T₃ and dex on target gene expression were observed in the presence of parallel reduction of their respective receptor mRNAs. This provides new evidence that interactions between these hormones are likely to be mediated by mechanisms other than regulation of receptor gene expression.     

Differential physiological and endocrine responses of rainbow trout, Oncorhynchus mykiss, transferred from fresh water to ion-poor or salt water

To understand the physiological and molecular endocrine changes that occur in response to a salinity challenge, we transferred rainbow trout from fresh water to an ion-poor or 24‰ saltwater treatment for 14 days. An increase in gill Na(+), K(+)-ATPase (NKA) activity in salt water was associated with higher mRNA expression for the NKA α1b subunit. In contrast, there was little change in gill NKA activity following transfer to ion-poor water, but the mRNA expression of NKA α1a was significantly elevated. Endocrine signals were assessed by measuring plasma cortisol concentrations and by quantifying changes in mRNA extracted from the gill for glucocorticoid receptors 1 and 2 (GR1 and GR2), mineralocorticoid receptor (MR), growth hormone receptor (GHR1), and prolactin receptor (PrlR). Cortisol increased after transfer to ion-poor and salt water, but both GR and MR mRNA in the gill showed little change. PrlR mRNA was significantly higher when fish were transferred to the ion-poor water and GHR1 mRNA was elevated during the saltwater challenge. This study demonstrated an increase in gill PrlR mRNA that parallels the changes in gill NKA α1a when rainbow trout were transferred to a lower salinity level. Furthermore, the increase in gill GHR1 mRNA supports the importance of GH for seawater acclimation as there is a corresponding increase in the expression of gill NKA α1b, the saltwater isoform. GH and Prl, therefore, may differentially determine the function of cortisol in both fresh- and saltwater ionoregulation.     

Alterations in insulin binding induced by changes in vivo in the levels of glucocorticoids and growth hormone.

Previous studies have shown that the sensitivity of tissues to insulin is diminished in states of glucocorticoid and GH excess and is increased when these hormones are deficient. To evaluate the role of the insulin receptor in these states, we have studied [125I]insulin binding to purified liver plasma membranes obtained from rats with a variety of perturbations of both glucocorticoids and GH. Glucocorticoid excess was produced in rats by administration of ACTH (40 U/day for 4 days) or dexamethasone (1 mg/day for 4 days). This resulted in an insulin-resistant state. Associated with this insulin resistance, there was a 50-60% decrease in insulin binding to its specific receptors in liver. Conversely, adrenalectomy, which produces an increase in insulin sensitivity, was associated with an increase in insulin binding to liver. Computer-assisted Scatchard analysis using a negative cooperative model for the inulin-receptor interaction indicated that, in contrast to our findings with obesity, the changes in insulin binding in these states were most likely due entirely to changes in receptor affinity, with no change in receptor concentration. GH administration also produced mild insulin resistance and a decrease in receptor concentration. This was associated with a reciprocal increase in receptor affinity and thus, no major alteration in insulin binding occurred at low physiological insulin concentrations. Hypophysectomized rats, on the other hand, showed an increase in receptor concentration and a decrease in affinity, and GH treatment only partially corrected these changes. Rats implanted with the MtT tumor (which secretes ACTH, GH, and PRL) have the combined effects of excess glucocorticoids and GH and are very insulin resistant. Liver membranes prepared from these rats showed a decrease in insulin binding and receptor affinity similar to that observed in other states of glucocorticoid excess. Further, adrenalectomy of the tumor-bearing rats resulted in an increase in insulin binding despite the persistence of the elevated levels of GH, ACTH, and PRL. These findings suggest that alterations in insulin receptor affinity and number may play a major role in the states of altered insulin sensitivity which accompany glucocorticoid excess and deficiency, and follow hypophysectomy. In contrast, the insulin resistance associated with GH excess is mediated at either a site on the receptor distal to the insulin-binding site (i.e. transduction) or at one or more of the intracellular reactions important in insulin action.     

Dose-dependent effects of a glucocorticoid on prolactin production

Glucocorticoids are known to stimulate growth hormone (GH) production but to suppress prolactin (PRL) production. However, previous data were obtained with rather high doses of corticosterone. In this study we examined the effects of various doses (10 (-12) -10 (-7) M) of corticosterone on GH and PRL production in a rat pituitary somatomammotropic cell line, MtT/SM cells, and found that GH mRNA expression was facilitated by high doses (10 (-7) and 10 (-8) M). In contrast, a biphasic effect of corticosterone on PRL mRNA expression and secretion was observed, i.e., high doses (10 (-7) and 10 (-8) M) suppressed and low doses (10 (-12) -10 (-10) M) facilitated them. In an immunofluorescent staining study, the number of PRL immunopositive cells increased with low doses of corticosterone while it decreased with high doses of it, which corresponded to PRL mRNA expression and hormone secretion, respectively. These effects of corticosterone on PRL production were abolished by a glucocorticoid receptor (GR) antagonist, mifepristone. In addition, co-treatment with low doses of corticosterone (10 (-12) -10 (-10) M) and 17beta-estradiol (E(2), 10 nM) additively increased the number of PRL immunopositive cells. Moreover, a 24 h BrdU incorporation experiment suggested that the increase in the number of PRL immunopositive cells treated with low dose corticosterone was caused by novel synthesis of PRL while, on the other hand, that of those treated with E(2) resulted from PRL cell proliferation. Thus, we concluded that corticosterone biphasically regulates PRL production and the sensitivity of E(2) to different degrees.