Dexamethasone stimulates the expression of ghrelin and its receptor in rat hypothalamic 4B cells

Growth hormone (GH)-releasing peptides (GHRPs) are synthetic peptides that strongly induce GH release. GHRPs act via a specific receptor, the GHRP receptor (GHSR), of which ghrelin is a natural ligand. GHRPs also induce adrenocorticotropic hormone (ACTH) release in healthy subjects. GHRPs or ghrelin stimulate ACTH release via corticotropin-releasing factor (CRF) and arginin vasopressin in the hypothalamus. Stress-activated CRF neurons are suppressed by glucocorticoids in the hypothalamic paraventricular nucleus (PVN), while CRF gene is up-regulated by glucocorticoids in the PVN cells without the influence of input neurons. However, little is known about the regulation of ghrelin and GHSR type 1a (GHSR1a) genes by glucocorticoids in PVN cells. To elucidate the regulation of ghrelin and GHSR gene expression by glucocorticoids in PVN cells, here we used a homologous PVN neuronal cell line, hypothalamic 4B, because these cells show characteristics of the parvocellular neurons of the PVN. These cells also express ghrelin and GHSR1a mRNA. Dexamethasone increased ghrelin mRNA levels. A potent glucocorticoid receptor antagonist, RU-486, significantly blocked dexamethasone-induced increases in ghrelin mRNA levels. Dexamethasone also significantly stimulated GHSR1a mRNA and protein levels. Finally, ghrelin increased CRF mRNA levels, as did dexamethasone. Incubation with both dexamethasone and ghrelin had an additive effect on CRF and ghrelin mRNA levels. The ghrelin-GHSR1a system is activated by glucocorticoids in the hypothalamic cells.     

Regulation of hypoxia-induced release of corticotropin-releasing factor in the rat hypothalamus by norepinephrine

Corticotropin-releasing factor (CRF) peptide release was activated by hypoxia in the rat hypothalamus. The mechanisms, however, of the hypoxia-induced CRF release remains unclear. In this study, we demonstrated that the norepinephrine (NE) and its receptors in the paraventricular nucleus (PVN) mediated the CRF release in a simulated altitude hypoxia. When rats were exposed to 5 or 7 km altitude of hypoxia for a short or long term: (1) NE levels in the PVN and the CeA, using the HPLC analysis, were intensity and time course dependently increased, but the increase in the PVN were potential than in the CeA. Restraint-induced NE increase was much higher in both the PVN and the CeA, compared with hypoxia-induced response. (2) Hypoxia and restraint significantly enhanced CRF release in the ME and the PVN but not in the CeA, through RIA assay, which result in stimulating corticosterone secretion. (3) Hypoxia-induced CRF release was reversed by an injection of prazosin (i.c.v.), an alpha-1 adrenoceptor antagonist, while administration of yohimbine (i.c.v.), an alpha-2 receptor antagonist, facilitated further CRF release. These data suggested that hypoxia induced NE activation centrally, via alpha-1 and -2 receptors, leading to improving hypothalamic CRF release, which in turn stimulated pituitary and adrenal cortex. Restraint presented much potential action on NE activation than hypoxia.     

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF(1) receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF(2) receptor) with little or no binding affinity for the CRF(1) receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF(1) receptor and CRF(2) receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF(1) receptor, while Ucn2 increased the levels via the CRF(2) receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.     

Interleukin-6 stimulates gonadotropin-releasing hormone secretion from rat hypothalamic cells

There are reports that both interleukin-1 beta and interleukin-6 (IL-6) stimulate the release of adrenocorticotropin through stimulation of hypothalamic corticotropin-releasing factor. We established a primary culture system for hypothalamic neurons producing gonadotropin-releasing hormone (GnRH) and examined whether IL-6 stimulated their GnRH secretion. We demonstrated immunohistochemically that some of these neurons contained GnRH-like immunoreactivity. In primary cultures of these GnRH neurons, we found that the calcium ionophore A23187 stimulated GnRH secretion in a dose- and time-dependent manner. These hypothalamic cells secreted IL-6 spontaneously, producing about 10 ng/l in 24 h, and their IL-6 secretion was significantly stimulated by E2 at 10(-9)-10(-8) mol/l. This stimulatory effect was observed within 3 h. IL-6 also stimulated the release of GnRH in a dose- and time-dependent manner, and these effects of IL-6 were significantly blocked by anti-IL-6 antiserum. These results suggest that the central action of IL-6 on the GnRH neurons may be an important physiological event in the hypothalamus.     

Impaired adaptation of gastrointestinal motility following chronic stress in maternally separated rats

Exposure to early life stress causes increased stress responsiveness and permanent changes in the central nervous system. We recently showed that delayed gastric emptying (GE) and accelerated colonic transit (CT) in response to acute restraint stress (ARS) were completely restored following chronic homotypic stress (CHS) in rats via upregulation of hypothalamic oxytocin (OXT) expression. However, it is unknown whether early life stress affects hypothalamic OXT circuits and gastrointestinal motor function. Neonatal rats were subjected to maternal separation (MS) for 180 min/day for 2 wk. Anxiety-like behaviors were evaluated by the elevated-plus-maze test. GE and CT were measured under nonstressed (NS), ARS, and CHS conditions. Expression of corticotropin-releasing factor (CRF) and OXT in the paraventricular nucleus (PVN) of the hypothalamus was evaluated by real time RT-PCR and immunohistochemistry. MS increased anxiety-like behaviors. ARS delayed GE and accelerated CT in control and MS rats. After CHS, delayed GE and accelerated CT were restored in control, but not MS, rats. CRF mRNA expression was significantly increased in response to ARS in control and MS rats. Increased CRF mRNA expression was still observed following CHS in MS, but not control, rats. In response to CHS, OXT mRNA expression was significantly increased in control, but not MS, rats. The number of OXT-immunoreactive cells was increased following CHS in the magnocellular part of the PVN in control, but not MS, rats. MS impairs the adaptation response of gastrointestinal motility following CHS. The mechanism of the impaired adaptation involves downregulation of OXT and upregulation of CRF in the hypothalamus in MS rats.     

Urocortins and corticotropin releasing factor type 2 receptors in the hypothalamus and the cardiovascular system

In addition to urocortin (Ucn I), Ucn II and Ucn III were identified as endogenous ligands for corticotropin-releasing factor type 2 receptor (CRF2 receptor). CRF2 receptor is abundantly located in central hypothalamic ventromedial nucleus (VMH) and in peripheral cardiovascular system. In this mini-review, we focused on the roles of these urocortins and CRF2 receptor in the hypothalamus and the cardiovascular system. Ucn II mRNA was increased in the parvocellular part or the magnocellular part of the hypothalamic paraventricular nucleus (PVN) following immobilization stress or 3 days of water deprivation, respectively. Therefore, it is thought that Ucn II may modulate CRF and vasopressin synthesis in the PVN in a paracrine or autocrine fashion through PVN CRF2 receptor. The early and later phases of Ucn I-mediated feeding suppression may be CRF1 and CRF2 receptor-mediated events, respectively. Ucn II decreases food intake at a later phase, beyond 4 h post injection. A large dose of corticosterone increased plasma leptin and insulin levels as well as the levels of CRF2 receptor mRNA. Adrenalectomy, starvation, and immobilization each lowered plasma leptin and insulin levels and were associated with decrements in CRF2 receptor mRNA levels in the VMH. Peripheral injection of leptin increased VMH CRF2 receptor mRNA, as can induce reductions of food intake and body weight, indicating that circulating leptin is involved in the regulation of VMH CRF2 receptor mRNA expression. Therefore, it is also plausible that VMH CRF2 receptor transduces the anorexogenic effects of leptin as well as those of urocortins. The systemic administration of Ucn II decreases mean arterial pressure (arterial vascular tone) and causes tachycardia via vascular CRF2 receptor in rats, similar to the effects of Ucn I. Thus, CRF2 receptor seems to mediate cardioprotective effects of urocortins.     

Gastrin-releasing peptide mediated regulation of 5-HT neuronal activity in the hypothalamic paraventricular nucleus under basal and restraint stress conditions

The purpose of this study was to examine the gastrin-releasing peptide (GRP) mediated regulation of 5-HT neuronal activity in the paraventricular nucleus of the hypothalamus under basal and restraint stress conditions. Intracerebroventricular (icv) administration of GRP (1, 10, 100 ng/rat) increased 5-HIAA concentrations in the paraventricular nucleus (PVN) of the hypothalamus, but was without effect in the accumbens, suprachiasmatic and arcuate nuclei. Administration of (Leu(13)-psi-CH(2)NH-Leu(14)) Bombesin (10, 100 and 1000 ng/rat; icv), a GRP antagonist, had no effect by itself on PVN serotonergic activity; however, a dose of 1 microg/rat of this compound, completely blocked the increase of 5-HIAA concentrations induced by GRP (10 ng). Restraint stress increased serotonergic activity -as shown by an elevation of 5-HIAA in the PVN- as well as plasma ACTH and corticosterone. This stress-induced activation of both the serotonergic neurons and the hypothalamus-pituitary-adrenal axis was blocked by CRF and GRP antagonists. Interestingly, when the activation of hypothalamic 5-HT neurons was induced by GRP administration, alpha-helical (9-41) CRF was ineffective.These data suggest that GRP, by acting on GRP receptors but not via CRF receptors, increases 5-HT neuronal activity in the PVN. In turn, it appears that endogenous GRP and CRF receptor ligands are both simultaneously involved in the regulation of the increase in 5-HT neuronal activity, ACTH and corticosterone secretion, under stress conditions.     

Estrogen receptor alpha/beta isoforms, but not betacx, modulate unique patterns of gene expression and cell proliferation in Hs578T cells

The actions of 17beta-estradiol (E2) and selective estrogen receptor modulators (SERMs) have been extensively investigated regarding their ability to act through estrogen receptor-alpha (ERalpha) to perturb estrogen receptor positive (ER+) breast cancer (BC) growth. However, many BCs also express ERbeta, along with multiple estrogen receptor (ER) splice variants such as ERbetacx, an ERbeta splice variant incapable of binding ligand. To gain a more comprehensive understanding of ER action in BC cells, we stably expressed ERalpha, ERbeta, or ERbetacx under doxycycline (Dox) control in Hs578T cells. Microarrays performed on E2 or 4OH-tamoxifen (4HT) treated Hs578T ERalpha and ERbeta cells revealed distinct ligand and receptor-dependent patterns of gene regulation, while the induction of ERbetacx did not alter gene expression patterns. E2 stimulation of Hs578T ERbeta cells resulted in a 27% decrease in cellular proliferation, however, no significant change in proliferation was observed following the exposure of Hs578T ERalpha or ERbeta cells to 4HT. Expression of ERbetacx in Hs578T cells did not effect cellular proliferation. Flow cytometry assays revealed a 50% decrease in E2-stimulated Hs578T ERbeta cells entering S-phase, along with a 17% increase in G0/G1 cell-cycle arrest. We demonstrate here that ERalpha and ERbeta regulate unique gene expression patterns in Hs578T cells, and such regulation likely is responsible for the observed isoform-specific changes in cell proliferation. Hs578T ER expressing cell-lines provide a unique BC model system, permitting the comparison of ERalpha, ERbeta, and ERbetacx actions in the same cell-line.     

Glucocorticoids regulate peptidyl-glycine alpha-amidating monooxygenase gene expression in the rat hypothalamic paraventricular nucleus

Peptidyl-glycine alpha-amidating monooxygenase (PAM) is a posttranslational processing enzyme which catalyzes the formation of biologically active alpha-amidated peptides. The two major neuropeptides involved in the regulation of ACTH secretion [CRF and arginine vasopressin (AVP)], synthesized in the parvocellular part of the hypothalamic paraventricular nucleus (PVN), are amidated, and their synthesis and/or release is negatively regulated by glucocorticoids. In this study, using in situ hybridization, we have shown that PAM mRNA is abundantly expressed in the hypothalamic paraventricular and supraoptic nucleus. Surgical adrenalectomy (ADX) induced increases in PAM, CRF, and AVP mRNA in the parvocellular part of the PVN, while corticosterone treatment normalized these values. PAM and AVP gene expression were not changed in the magnocellular part of the PVN or in the supraoptic nucleus. These observations suggest that in addition to stimulation of CRF and AVP synthesis, ADX induces an increase in PAM synthesis in the PVN and, thus, support the hypothesis of increased secretion of both CRF and AVP after ADX.     

Different signaling molecules responsible for IL-1beta-induced oxytocinergic and vasopressinergic neuron activation in the hypothalamic paraventricular nucleus of the rat

It has been well known that oxytocin (OT)-ergic and arginine vasopressin (AVP)-ergic neurons located in the hypothalamic paraventricular nucleus (PVN) and super optic nucleus (SON) are two kinds of neuroendocrine cells with diverse functions. It has also been demonstrated that immune stimuli can activate these neurons to secret OT and AVP. However, the intracellular signal transduction molecules responsible for the activation of these OT-ergic and AVP-ergic neurons in PVN by immune stimuli are still unclear. In this experiment, the roles of Fos, a protein product of immediate early gene c-fos, and extracellular signal-regulated protein kinase (ERK) 1/2, a signal transduction molecule of mitogen-activated protein kinase (MAPK) family, in these processes were studied in the PVN of the rat following IL-1beta stimulation. The Sprague-Dawley rats were received either 750 ng/kg IL-1beta or equal volume normal saline (NS) injection intravenously (i.v.), and perfused transcardially by 4% paraformaldehyde 3h later. Fos and phosphorylated ERK1/2 (pERK1/2)-immunoreactivity (-ir) was observed in PVN by ABC immunohistochemical staining. Meanwhile, the double staining for OT/Fos, AVP/Fos, OT/pERK1/2 and AVP/pERK1/2 were also processed. The ABC immunohistochemical staining results showed that after an i.v. injection of IL-1beta, the expressions of Fos and pERK1/2 increased evidently in the PVN. Double-staining results showed that a large number of OT-ir cells contained strong Fos-ir products in their nuclei, while only a few of OT cells were double labeled with pERK1/2. As to AVP neurons, great quantities of AVP cells were strongly double labeled with pERK1/2 while there were nearly no Fos-ir nuclei in AVP-ir cells. We conclude from these results that the intracellular IL-1beta-induced events in OT and AVP neurons in PVN are quite different. The OT neurons are mainly activated via Fos without involvement of ERK1/2 pathway, while the latter, but not Fos, involves the intracellular event in AVP neurons activated by IL-1beta.     

Centrally administered orexin-A activates corticotropin-releasing factor-containing neurons in the hypothalamic paraventricular nucleus and central amygdaloid nucleus of rats: possible involvement of central orexins on stress-activated central CRF neurons

We examined the effects of centrally administered orexin-A on corticotropin-releasing factor (CRF)-containing neurons in the hypothalamic paraventricular nucleus (PVN) and the central amygdaloid nucleus (CeA) of rats, using dual immunostaining for CRF and Fos. Ninety minutes after intracerebroventricular administration of orexin-A, approximately 96% and 45% of CRF-containing neurons expressed Fos-like immunoreactivity (LI) in the PVN and the CeA, respectively. We also examined the effects of immobilized stress and cold exposure on orexin-A-containing neurons in the rat hypothalamus using dual immunostaining for orexin-A and Fos. After immobilized stress for 20 min and cold exposure at 4 degrees C for 30 min, approximately 24% and 15% of orexin-A-containing neurons expressed Fos-LI, respectively. These results suggest that orexins in the central nervous system may be involved in the activation of central CRF neurons induced by stress.     

Effects of experimental hypothyroidism on the development of the hypothalamo-pituitary-adrenal axis in the rat

Hypothyroid pups were obtained by adding methimazole in the mother's drinking water from day 15 of gestation and sacrificed at 4, 8 or 15 days. Circulating corticosterone decreased at all ages, while CBG concentrations diminished at day 4, increased at day 8 and did not change at day 15 in hypothyroid rats. As opposed to controls, plasma ACTH concentrations decreased steadily with age while there was an accumulation of ACTH in the anterior pituitary of hypothyroid 15-day-old rats. Anterior pituitary POMC contents were unaffected by the treatment. In the hypothalamic PVN, CRF mRNA levels in the total population of CRF-synthesizing cells and in the CRF+/AVP+ subpopulation were below those of controls whatever the age considered while AVP mRNA in the CRF+/AVP+ subpopulation did not change at day 4 and decreased at day 8 and 15 in hypothyroid animals. Both the number of cell bodies expressing detectable levels of CRF mRNA and the percentage of CRF and AVP colocalization decreased at day 4 and were unchanged thereafter. CRF and AVP immunoreactivity in the zona externa of the median eminence increased with age but was not affected by methimazole treatment. The concentration of AVP mRNA in the magnocellular cell bodies of the PVN and the SON as well as AVP immunoreactivity in the zona interna of the median eminence were not changed by the treatment at days 4 and 8. In hypothyroid 15-day-old rats, SON AVP mRNA increased, AVP immunoreactivity decreased while plasma osmolality was enhanced. In conclusion, our data demonstrate that experimental hypothyroidism impairs specifically the maturation of hypothalamic parvocellular CRF and AVP gene expression during the stress hyporesponsive period. These observations suggest that the physiological peak in plasma thyroxine concentrations that occur between day 8-12 may participate in the maturation of hypothalamic CRF- and AVP-synthesizing cells.     

Interleukin-6 and nitric oxide synthase expression in the vasopressin and corticotrophin-releasing factor systems of the rat hypothalamus.

Nitric oxide synthase (NOS) and interleukin-6 (IL-6) are constitutively expressed in hypothalamic cells. However, phenotypic and functional aspects of these cells remain unknown. We have studied the expression pattern of these two molecules in hypothalamic cells expressing corticotropin-releasing factor (CRF) and arginin-vasopressin (AVP), two major regulatory peptides in the hypothalamus-pituitary system, using immunofluorescence, intracerebroventricular injection of colchicine, and the study in parallel of the labeling pattern of axons in the median eminence. Within AVP cells, we distinguished two different populations: large, intensely stained AVP cells coexpressing IL-6; and large, intensely stained AVP cells coexpressing IL-6 and NOS. Within the CRF cells, we distinguished three different populations: large, intensely stained CRF cells immunonegative for AVP, NOS, and IL-6; large cells weakly stained for CRF and AVP, immunopositive for NOS and immunonegative for IL-6; and small cells intensely stained for CRF and AVP and immunonegative for IL-6 and NOS. In addition, we also found AVP cells containing IL-6 in the suprachiasmatic nucleus. These results suggest that neuronal NOS and IL-6 may be involved in different modulatory processes in hypophysiotropic and non-hypophysiotropic cells.     

Regulation and role of p21-activated kinase 3 by corticotropin-releasing factor in mouse pituitary

Corticotropin-releasing factor (CRF) is a major regulatory peptide in the hypothalamic-pituitary-adrenal (HPA) axis under stress conditions. In response to stress, CRF, produced in the hypothalamic paraventricular nucleus, releases adrenocorticotropic hormone (ACTH) from the anterior pituitary (AP). ACTH in turn stimulates the release of glucocorticoid from the adrenal glands. Glucocorticoid then inhibits hypothalamic production of CRF and pituitary production of ACTH. Mice lacking a functional gene for CRF (CRF KO) showed severe impairment of the HPA axis, indicating that CRF is required for its regulation. We applied oligonucleotide microarray analysis to the AP of CRF KO to identify gene expression induced by CRF. Twenty-four genes showed less than 60% expression in CRF KO compared with normal mice. Real-time PCR analysis revealed that p21-activated kinase 3 (Pak3), prohormone convertase type 1 (PC1), and CRF-binding protein (BP) mRNA expression levels were increased by CRF in AP cells. Both Pak3 and PC1 were also increased by dexamethasone in AP cells, while CRF-BP mRNA levels were reduced. Therefore, both Pak3 and PC1 mRNA levels would be regulated by both CRF and glucocorticoids. Pak3 knockdown inhibited CRF-induced cell viability in AtT-20 cells, suggesting the important role of Pak3 in the proliferation of corticotrophs.     

ERbeta protein expression in female cynomolgus monkey and CF-1 mouse brain: Western analysis

In humans and rodents, multiple ERbeta variants with sizes ranging from 477-549 amino acids (aa) have been described. The identification of these variants in target tissues has important implications for estrogen signaling and cellular responsiveness. Western blot analysis using two anti-ERbeta antibodies specific for mammalian ERbeta sequences (PA1-310B and PA1-311) was employed to examine ERbeta protein expression in neural tissues from ovariectomized (OVX) cynomolgus macaques and CF-1 mice as well as to assess potential regulatory effects of acute and extended estradiol (E(2)) treatment. In hypothalamic extracts from both species, a single ERbeta immunoreactive (ERbeta-ir) band was detected at approximately 54 kDa, corresponding to the expected molecular weight for ERbeta477 and/or 485. In cynomolgus females, oral E(2) administration for 16 weeks had no apparent effect on hypothalamic ERbeta protein expression. In mouse, a single injection of E(2) did not change hypothalamic ERbeta protein levels 1.5, 4, 8, 16, or 24 h after injection. Extending the hormonal treatment to 4 or 21 days in OVX female mice also had no effect on the level of hypothalamic ERbeta protein. Additional regional analyses in female mouse brain with PA1-310B antibody showed that a second, 59 kDa ERbeta-ir band was present in cortex, striatum, hippocampus, and amygdala that could represent one or both of the larger ERbeta variants (530 and 549aa). The expression level of the second ERbeta isoform exhibited regional variation, with the strongest immunoreactivity detected in cortex and amygdala. Elucidating the functions of these ERbeta isoforms in the CNS will facilitate our understanding of the tissue- and promoter-specific actions of estrogen.     

Adrenergic innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat. A combined light and electron microscopic immunocytochemical study

Corticotropin releasing factor (CRF), a neuropeptide synthesized in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN), takes part in the regulation of different stress evoked responses of the organism. In order to elucidate the role of the central adrenergic system in the regulation of these CRF-synthesizing neurons, a novel ultrastructural immunocytochemical dual localization technique was utilized. Phenylethanolamine-N-methyltransferase (PNMT), a specific enzyme marker for the central adrenaline system, and CRF-immunoreactive elements were simultaneously visualized in hypothalamic sections. PNMT-immunoreactive axon terminals established synaptic connections with somata, dendrites and spinous structures of CRF-producing neurons. This morphological finding indicates that the central adrenergic system directly influences CRF-synthesizing neurons in the PVN and provides basis for a more definitive pharmacological manipulation of this system.