Brain corticosteroid receptor gene expression and neuroendocrine dynamics during aging

The present study examined the stress responsiveness of the hypothalamic-pituitary-adrenal axis in relation to the properties of corticosteroid receptors in the brain and pituitary in old (30 months) and young (3 months) male Brown Norway rats. The data demonstrate that circulating ACTH rather than the corticosteroid plasma level was elevated under basal conditions and following stress. Furthermore, a reduction of mineralocorticoid receptor (MR) number in the hippocampus and of glucocorticoid receptor (GR) number in the hypothalamus and the pituitary correspond to increased neuroendocrine responsiveness and negative feedback following stress. The changes in receptor binding do not parallel the changes in the amount of MR and GR mRNA measured with in situ hybridization. This suggests that the processing rather than the receptor gene expression is affected in senescence.     

Acute and chronic regulation of pituitary receptors for vasopressin and corticotropin releasing hormone

At least two hypothalamic peptides, corticotropin releasing hormone (CRH) and vasopressin (VP), are important in regulating adrenocorticotropin (ACTH) release from the anterior pituitary. Both are secreted in a pulsatile manner and stimulate ACTH secretion by interacting with G protein-coupled receptors (GPCRs), namely the type 1 CRH receptor and V1b receptor, respectively. Repeated or prolonged stimulation with either peptide can cause reduced ACTH responsiveness or desensitisation, both in vivo and in vitro. Desensitisation of perifused sheep anterior pituitary cells to VP was found to be rapid and occurred following treatment with 5 nM VP for 5 min. This is within the range of concentrations and durations of VP pulses seen in sheep portal blood during acute stress. In contrast, significant desensitisation of the ACTH response to CRH required pre-treatment for longer than 25 min with a CRH concentration of 1 nM, suggesting that endogenous pulses may not elicit desensitisation. Although rapid GPCR desensitisation involves uncoupling of receptors from their G proteins, commonly mediated by receptor phosphorylation, and internalisation of receptors, desensitisation of neither the CRH nor VP receptor was mediated by PKA or PKC, respectively. Desensitisation of the response to VP was found to be dependent upon receptor internalisation, and resensitisation could be delayed by treatment with a protein phosphatase 2B inhibitor. The rapid kinetics of desensitisation of the ACTH response to VP suggest that this process is important in regulating the response to acute rather than chronic stress. If, as has been suggested, CRH acts in a permissive way to set corticotrope gain, desensitisation to CRH could also be important in long term regulation of ACTH secretion.     

Vasopressinergic regulation of the hypothalamic-pituitary-adrenal axis: implications for stress adaptation

In addition to its role on water conservation, vasopressin (VP) regulates pituitary ACTH secretion by potentiating the stimulatory effects of corticotropin releasing hormone (CRH). The pituitary actions of VP are mediated by plasma membrane receptors of the V1b subtype, coupled to calcium-phospholipid signaling systems. VP is critical for adaptation of the hypothalamic-pituitary-adrenal (HPA) axis to stress as indicated by preferential expression of VP over CRH in parvocellular neurons of the hypothalamic paraventricular nucleus, and the upregulation of pituitary VP receptors during stress paradigms associated with corticotroph hyperresponsiveness. V1b receptor mRNA levels and coupling of the receptor to phospolipase C are stimulated by glucocorticoids, effects which may contribute to the refractoriness of VP-stimulated ACTH secretion to glucocorticoid feedback. The data suggest that vasopressinergic regulation of the HPA axis is critical for sustaining corticotroph responsiveness in the presence of high circulating glucocorticoid levels during chronic stress.     

Dimerization Between Vasopressin V1b and Corticotropin Releasing Hormone Type 1 Receptors

1. Increasing evidence indicates that guanyl protein coupled receptors (GPCRs), including members of the vasopressin (VP) receptor family can act as homo- and heterodimers. Regulated expression and interaction of pituitary VP V1b receptor (V1bR) and corticotropin releasing hormone receptor type 1 (CRHR1) are critical for hypothalamic pituitary adrenal (HPA) axis adaptation, but it is unknown whether this involves physical interaction between these receptors. 2. Bioluminescence resonance energy transfer (BRET) experiments using V1bR and CRHR1 fused to either Renilla luciferase (Rluc) or yellow fluorescent protein (YFP) at the N-terminus, but not the carboxyl-terminus, revealed specific interaction (BRET₅₀ = 0.39 ± 0.08, V1bR) that was inhibited by untagged V1b or CRHR1 receptors, suggesting homo- and heterodimerization. The BRET data were confirmed by coimmunoprecipitation experiments using fully bioactive receptors tagged at the aminoterminus with c-myc and Flag epitopes, demonstrating specific homodimerization of the V1b receptor and heterodimerization of the V1b receptor with CRHR1 receptors. 3. Heterodimerization between V1bR and CRHR1 is not ligand dependent since stimulation with CRH and AVP had no effect on coimmunoprecipitation. In membranes obtained from cells cotransfected with CRHR1 and V1bR, incubation with the heterologous nonpeptide antagonist did not alter the binding affinity or capacity of the receptor. 4. The data demonstrate that V1bR and CRHR1 can form constitutive homo- and heterodimers and suggests that the heterodimerization does not influence the binding properties of these receptors.     

Corticotropin-releasing hormone and pituitary-adrenocortical responses in chronically stressed rats

Brain corticotropin-releasing hormone (CRH) concentration and pituitary adreno-cortical responses were examined in chronically stressed rats: body restraint stress (6 h/day) for 4 or 5 weeks. Stressed rats showed a reduction in weight gain. CRH concentration in the median eminence and the rest of the hypothalamus were not different between control and chronically immobilized rats. The anterior pituitary adenocorticotropic hormone (ACTH) concentration was elevated in chronically stressed rats, whereas plasma ACTH and corticosterone levels did not differ from the control values. The median eminence CRH concentration was reduced to the same extent at 5 min after onset of ether exposure (1 min) in chronically immobilized rats and controls. However, plasma ACTH and corticosterone showed greater responses to ether stress in chronically immobilized rats than in control rats. Plasma ACTH and corticosterone responses to exogenous CRH were not different between control and chronically immobilized rats, while the response to arginine vasopressin (AVP) was significantly greater in chronically immobilized rats. These results suggest that chronic stress caused an increase in the ACTH-secreting mechanism and that pituitary hypersensitivity to vasopressin might at least be partly responsible for this.     

Hyperglycemia does not increase basal hypothalamo-pituitary-adrenal activity in diabetes but it does impair the HPA response to insulin-induced hypoglycemia

Recently, we established that hypothalamo-pituitary-adrenal (HPA) and counterregulatory responses to insulin-induced hypoglycemia were impaired in uncontrolled streptozotocin (STZ)-diabetic (65 mg/kg) rats and insulin treatment restored most of these responses. In the current study, we used phloridzin to determine whether the restoration of blood glucose alone was sufficient to normalize HPA function in diabetes. Normal, diabetic, insulin-treated, and phloridzin-treated diabetic rats were either killed after 8 days or subjected to a hypoglycemic (40 mg/dl) glucose clamp. Basal: Elevated basal ACTH and corticosterone in STZ rats were normalized with insulin but not phloridzin. Increases in hypothalamic corticotrophin-releasing hormone (CRH) and inhibitory hippocampal mineralocorticoid receptor (MR) mRNA with STZ diabetes were not restored with either insulin or phloridzin treatments. Hypoglycemia: In response to hypoglycemia, rises in plasma ACTH and corticosterone were significantly lower in diabetic rats compared with controls. Insulin and phloridzin restored both ACTH and corticosterone responses in diabetic animals. Hypothalamic CRH mRNA and pituitary pro-opiomelanocortin mRNA expression increased following 2 h of hypoglycemia in normal, insulin-treated, and phloridzin-treated diabetic rats but not in untreated diabetic rats. Arginine vasopressin mRNA was unaltered by hypoglycemia in all groups. Interestingly, hypoglycemia decreased hippocampal MR mRNA in control, insulin-, and phloridzin-treated diabetic rats but not uncontrolled diabetic rats, whereas glucocorticoid receptor mRNA was not altered by hypoglycemia. In conclusion, despite elevated basal HPA activity, HPA responses to hypoglycemia were markedly reduced in uncontrolled diabetes. We speculate that defects in the CRH response may be related to a defective MR response. It is intriguing that phloridzin did not restore basal HPA activity but it restored the HPA response to hypoglycemia, suggesting that defects in basal HPA function in diabetes are due to insulin deficiency, but impaired responsiveness to hypoglycemia appears to stem from chronic hyperglycemia.     

Heterooligomerization between vasotocin and corticotropin-releasing hormone (CRH) receptors augments CRH-stimulated 3',5'-cyclic adenosine monophosphate production

In birds, ACTH release from the anterior pituitary gland during stress is controlled by CRH and arginine vasotocin (AVT). Using 5-wk-old male chicks, simultaneous iv injections of CRH and AVT were found to result in a greater than additive increase in plasma corticosterone levels compared with that obtained with individual administration of either peptide hormone. In order to investigate molecular mechanisms underlying this observation, the chicken CRH receptor (CRHR) and vasotocin VT2 receptor (VT2R) were fused to cyan and yellow fluorescent proteins and expressed in HeLa cells. The resulting CRHR and VT2R fusion proteins were expressed appropriately in the plasma membrane and were found to couple to downstream signal transduction pathways. Quantitative fluorescence resonance energy transfer (FRET) analysis was used to determine whether the CRHR and VT2R formed heterodimers. In the absence of CRH and AVT, the FRET efficiency was 15-18%, and the distance between receptors was 5-6 nm. Treatment of the cells that expressed both cyan fluorescent protein-CRHR and yellow fluorescent protein-VT2R with CRH or AVT alone did not lead to a significant change in the FRET efficiency. However, simultaneous addition of these hormones increased the efficiency of the FRET signal and decreased the distance between the two receptors. In HeLa cells expressing both CRHR and VT2R, treatment with CRH and AVT resulted in a significant increase in cAMP production over that with CRH alone, indicating that heterodimer formation may enhance the ability of the CRHR to activate downstream signal transduction.     

Chronic administration of the non-peptide CRH type 1 receptor antagonist antalarmin does not blunt hypothalamic-pituitary-adrenal axis responses to acute immobilization stress.

Antalarmin is a pyrrolopyrimidine compound that antagonizes corticotropin-releasing hormone (CRH) type 1 receptors (CRHR1). In order to assess the effects of antalarmin treatment on hypothalamic-pituitary-adrenal (HPA) function we measured the plasma concentrations of adrenocorticotropic hormone (ACTH) and corticosterone in animals treated with either antalarmin or vehicle for 1 week or for 8 weeks. We found that antalarmin treatment for 1 week did not affect basal concentrations of ACTH or corticosterone. In contrast, treatment for 8 weeks significantly lowered basal ACTH and corticosterone concentrations and also significantly decreased the basal corticosterone to ACTH ratio, indicating decreased basal adrenocortical responsiveness to ACTH. However, immobilization stress resulted in ACTH and corticosterone concentrations that were the same in animals treated with vehicle or antalarmin for either 1 or 8 weeks. We conclude that even though 8-week antagonism of CRHR1 by the non-peptide antalarmin blunts basal concentrations of ACTH and corticosterone, and affects the adrenal responsiveness to ACTH, it does not blunt the HPA response to acute stress, and it does not appear to cause stress-induced adrenal insufficiency.     

Mouse models of altered CRH-binding protein expression

CRH is the key physiological mediator of the endocrine, autonomic, and behavioral responses to stress. The recent characterization of urocortin, a new mammalian CRH-like ligand, adds to the complexity of the CRH system. Both CRH and urocortin mediate their endocrine and/or synaptic effects via two classes of CRH receptors. Similarly, both CRH and urocortin bind to the CRH-binding protein (CRH-BP). This secreted binding protein is smaller than the CRH receptors, but binds CRH and urocortin with an affinity equal to or greater than that of the receptors, and blocks CRH-mediated ACTH release in vitro. Several regions of CRH-BP expression colocalize with sites of CRH synthesis or release, suggesting that this binding protein may have a profound impact on the biological activity of CRH (or urocortin). While in vitro and in vivo studies have characterized the biochemical properties and regulation of the CRH-BP, animal models of altered CRH-BP expression can provide additional information on the in vivo role of this important modulatory protein. This review focuses on three mouse models of CRH-BP overexpression or deficiency. These animal models show numerous physiological changes in the HPA axis and in energy balance, with additional alterations in anxiogenic behavior. These changes are consistent with the hypothesis that CRH-BP plays an important in vivo modulatory role by regulating levels of "free" CRH and other CRH-like peptides in the pituitary and central nervous system.     

Expression of urocortin and its receptors in the rat epididymis

Urocortin (UCN; 40 aa) is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated by two types of G-protein-coupled receptors, CRH type 1 receptor (CRHR1) and CRH type 2 receptor (CRHR2). The biological effects of the peptides are mediated and modulated not only by CRH receptors but also by a highly conserved CRH-binding protein (CRHBP). The aim of the present study was to investigate the expression of UCN, CRHR1, CRHR2 and CRHBP by immunohistochemistry, Western blot, RT-PCR and real-time RT-PCR in the rat epididymis. Urocortin, CRHR1 and CRHR2, but not CRHBP, were expressed in all segments of the rat epididymis. Specifically, UCN- and CRHR2-immunoreactivities (IRs) were distributed in epididymal epithelial cells of the caput, corpus and cauda. CRHR1-IR was found in the fibromuscular cells surrounding the epididymal duct and in the smooth musculature of the blood vessels throughout the organ. UCN and CRHR2 mRNA expression levels were higher in the caput and corpus than in the cauda, while CRHR1 mRNA level was higher in the cauda than those in the caput and corpus. In summary, UCN, CRHR1 and CRHR2 are expressed in the rat epididymis. It is suggested that CRH-related peptides might play multiple roles in the maturation and storage of spermatozoa.     

Effects of adrenalectomy on CRH regulation of ACTH release: adenylate cyclase activity, cyclic AMP-dependent protein kinase activity and ACTH release

Corticotropin releasing hormone (CRH) stimulation of ACTH release and cyclic AMP-mediated events involved in the control of ACTH release were compared in sham-operated and adrenalectomized rats. CRH-stimulated adenylate cyclase activity was decreased in pituitary homogenates from adrenalectomized animals. CRH-stimulated cyclic AMP accumulation was essentially abolished and CRH-stimulated cyclic AMP-dependent protein kinase (A-kinase) activity was decreased in freshly prepared anterior pituitary cells from adrenalectomized animals. Basal and CRH-stimulated ACTH release was elevated in these cells. Since ACTH release is increased in adrenalectomized rats despite the down regulation of CRH-linked pituitary mechanisms, we speculate that the site of action of disinhibition by corticosterone of ACTH release (or synthesis) following adrenalectomy is distal to the generation of cyclic AMP and/or that non-CRH mediated mechanisms assume a greater role in ACTH regulation following adrenalectomy.     

The opposite effects of short- and long-term salt loading on pituitary adrenal axis activity in rats.

Osmotic stimulation has been shown to modify corticotropin responsiveness. We compared the effects of short- and long-term salt loading on pituitary-adrenal activity in control rats receiving tap water and rats submitted to salt loading for 1 day (S1) or 8 days (S8). Corticosterone (B) and adrenocorticotropic hormone (ACTH) plasma levels were determined at 8 a. m. under basal conditions or after immobilization stress for 15 min or corticotropin-releasing hormone (CRH) stimulation. S1 rats showed a similar ACTH response to immobilization, but an increased CRH response. In contrast, S8 rats showed blunted responses after immobilization or CRH stimulation. To evaluate the circadian variation of this inhibitory effect on the stress in the S8 group, immobilization was also performed at 8 p. m. Plasma ACTH and B levels under resting conditions were higher at 8 p. m. than 8 a. m. (p < 0.05) in control and S8 rats. The ACTH response to immobilization in the S8 group was lower than control at both 8 a. m. and 8 p. m. (p < 0.05); however, this reduction was more evident the morning, resulting in an inversion of the diurnal pattern with a higher ACTH response at 8 p. m. In conclusion, short osmotic stimulation results in an increased pituitary response to CRH, whereas prolonged stimulation decreases the pituitary response to CRH and immobilization, showing an interaction between osmoregulation and hypothalamus-pituitary-adrenocortical activity.     

Ontogeny and effect of cortisol on vasopressin-1b receptor expression in anterior pituitaries of fetal sheep

Corticotroph responsiveness to arginine vasopressin (AVP) increases during late gestation in fetal sheep. The mechanism of this increase in AVP responsiveness is currently unknown but could be related to an increase in vasopressin type 1b (V1b) receptor expression in the pituitary during development. To determine if there are ontogenic changes in V1b receptor expression that may help explain the changes in ACTH responses to AVP, we studied pituitaries from three groups of fetal sheep [100, 120, or 140 days gestational age (dGA)]. V1b receptor mRNA and protein significantly decreased by 140 dGA. Peak V1b mRNA levels were detected at 100 dGA, while peak V1b protein levels were detected at 120 dGA. The reduction in V1b receptor expression in late gestation may be due to the naturally occurring peripartum increase in fetal plasma cortisol because cortisol infusion at 122-130 dGA decreased V1b receptor mRNA. Thus there is a marked decrease in the expression of the V1b receptor in the pituitary during fetal development, leaving the role of the V1b receptor in increasing AVP responsiveness uncertain.     

Differential Expression of CRH,UCN, CRHR1 and CRHR2 in Eutopic and Ectopic Endometrium of Women with Endometriosis

Endometriosis is considered as a benign aseptic inflammatory disease, characterised by the presence of ectopic endometrium-like tissue. Its symptoms (mostly pain and infertility) are reported as constant stressors. Corticotropin releasing hormone (CRH) and urocortin (UCN) are neuropeptides, strongly related to stress and inflammation. The effects of CRH and UCN are mediated through CRHR1 and CRHR2 receptors which are implicated in several reproductive functions acting as inflammatory components. However, the involvement of these molecules to endometriosis remains unknown. The aim of this study was to examine the expression of CRHR1 and CRHR2 in endometriotic sites and to compare the expression of CRHR1 and CRHR2 in eutopic endometrium of endometriotic women to that of healthy women. We further compared the expression of CRH, UCN, CRHR1 and CRHR2 in ectopic endometrium to that in eutopic endometrium of women with endometriosis. Endometrial biopsy specimens were taken from healthy women (10 patients) and endometrial and endometriotic biopsy specimens were taken from women with endometriosis (16 patients). Τhe expression of CRH, UCN, CRHR1, and CRHR2 was tested via RT-PCR, immunohistochemistry and Western blotting. This study shows for the first time that CRH and UCN receptor subtypes CRHR1β and CRHR2α are expressed in endometriotic sites and that they are more strongly expressed (p<0.01) in eutopic endometrium of women with endometriosis compared to healthy women endometrium at the mRNA and protein level. CRH, UCN, CRHR1 and CRHR2 mRNA were also more highly expressed in ectopic rather than eutopic endometrium (CRH, UCN, CRHR2α: p<0.01, CRHR1β: p<0.05) and protein (CRH and UCN: p<0.05, CRHR1 and CRHR2: p<0.01) in women with endometriosis. These data indicate that CRH and UCN might play an immunoregulatory role in endometriotic sites by affecting reproductive functions such as decidualization and implantation of women with endometriosis.     

Vasopressin pressor receptor-mediated activation of HPA axis by acute ethanol stress in rats

The plasma arginine vasopressin (AVP), ACTH, and corticosterone levels and the hypothalamic corticotropin-releasing hormone (CRH) content were measured after oral administration of 1 ml of 75% ethanol to rats, a model known to induce acute gastric erosions and stress. Elevated plasma AVP, ACTH, and corticosterone levels were detected 1 h after ethanol administration. Treatment with the vasopressin pressor (V(1)) receptor antagonist [d(CH(2))(5)Tyr(Me)-AVP] before ethanol administration significantly reduced the ACTH and corticosterone level increases. A higher hypothalamic CRH content was measured at 30 or 60 min after ethanol administration. V(1) receptor antagonist injection, 5 min before ethanol administration, inhibited the rise in hypothalamic CRH content. The protein synthesis blocker cycloheximide prevented the hypothalamic CRH content elevation after stress. The AVP-, CRH-, and AVP + CRH-induced in vitro ACTH release in normal anterior pituitary tissue cultures was also prevented by pretreatment with the V(1) receptor antagonist. The results support the hypothesis that stress-induced AVP may not only act directly on the ACTH producing anterior pituitary cells but also indirectly at the hypothalamic level via the synthesis and release of CRH.