An N-ethyl-n-nitrosourea induced corticotropin-releasing hormone promoter mutation provides a mouse model for endogenous glucocorticoid excess

Cushing's syndrome, which is characterized by excessive circulating glucocorticoid concentrations, maybe due to ACTH-dependent or -independent causes that include anterior pituitary and adrenal cortical tumors, respectively. ACTH secretion is stimulated by CRH, and we report a mouse model for Cushing's syndrome due to an N-ethyl-N-nitrosourea (ENU) induced Crh mutation at -120 bp of the promoter region, which significantly increased luciferase reporter activity and was thus a gain-of-function mutation. Crh -120/+ mice, when compared with wild-type littermates, had obesity, muscle wasting, thin skin, hair loss, and elevated plasma and urinary concentrations of corticosterone. In addition, Crh-120/+ mice had hyperglycemia, hyperfructosaminemia, hyperinsulinemia, hypercholesterolemia, hypertriglyceridemia, and hyperleptinemia but normal adiponectin. Crh -120/+ mice also had low bone mineral density, hypercalcemia, hypercalciuria, and decreased concentrations of plasma PTH and osteocalcin. Bone histomorphometry revealed Crh-120/+ mice to have significant reductions in mineralizing surface area, mineral apposition, bone formation rates, osteoblast number, and the percentage of corticoendosteal bone covered by osteoblasts, which was accompanied by an increase in adipocytes in the bone marrow. Thus, a mouse model for Cushing's syndrome has been established, and this will help in further elucidating the pathophysiological effects of glucocorticoid excess and in evaluating treatments for corticosteroid-induced osteoporosis.

Effect of naloxone-precipitated morphine withdrawal on c-fos expression in rat corticotropin-releasing hormone neurons in the paraventricular hypothalamus and extended amygdala

Morphine withdrawal is characterized by physical symptoms and a negative affective state. The 41 amino acid polypeptide corticotropin-releasing hormone (CRH) is hypothesized to mediate, in part, both the negative affective state and the physical withdrawal syndrome. Here, by means of dual-immunohistochemical methodology, we examined the co-expression of the c-Fos protein and CRH following naloxone-precipitated morphine withdrawal. Rats were treated with slow-release morphine 50 mg/kg (subcutaneous, s.c.) or vehicle every 48 h for 5 days, then withdrawn with naloxone 5 mg/kg (s.c.) or saline 48 h after the final morphine injection. Two hours after withdrawal rats were perfused transcardially and their brains were removed and processed for immunohistochemistry. We found that naloxone-precipitated withdrawal of morphine-dependent rats increased c-Fos immunoreactivity (IR) in CRH positive neurons in the paraventricular hypothalamus. Withdrawal of morphine-dependent rats also increased c-Fos-IR in the central amygdala and bed nucleus of the stria terminalis, however these were in CRH negative neurons.

Transgenic mice as a tool for depression research: examples from the endocannabinoid and the corticotropin-releasing hormone system

Major depression belongs to the most serious and widespread psychiatric disorders in today’s society. There is a great need for the delineation of the underlying molecular mechanisms as well as for the identification of novel targets for its treatment. In this thesis, transgenic mice of the endocannabinoid and the corticotropin-releasing hormone (CRH) system were investigated to determine the putative role of these systems for depression-like phenotypes in mice. In the first part of the thesis, we found that the endocannabinoid system was prominently involved in a brain region-specific and temporally controlled manner in acute as well as in chronic stress processing. Genetic deletion in combination with pharmacological intervention revealed the importance of a fully functional endocannabinoid system for efficient neuroendocrine and behavioral stress coping. Accordingly, cannabinoid type 1 (CB1) receptor-deficient mice displayed several depression-like symptoms and molecular alterations, including “behavioral despair”, stress hormone hypersecretion and decreased glucocorticoid receptor and brain-derived neurotrophic factor expression in the hippocampus. However, the endocannabinoid system was dispensable for the efficacy of currently used antidepressant drugs. To facilitate future endocannabinoid research, a transgenic mouse was generated, which overexpressed the CB1 receptor protein fused to a fluorescent protein. In the second part of the thesis, conditional brain region-specific CRH overexpressing mice were evaluated as a model for pathological chronic CRH hyperactivation. Mutant mice showed aberrant neuroendocrine and behavioral stress coping and hyperarousal due to CRH-induced activation of the noradrenergic system in the brain. Mutant mice appeared to share similarities with naturally occurring endogenous CRH activation in wild-type mice and were sensitive to acute pharmacological blockade of CRH receptor type 1 (CRH-R1). Thus, CRH overexpressing mice serve as an ideal in vivo tool to evaluate the efficacy of novel CRH-R1 antagonists. Together, these findings highlight the potential of transgenic mice for the understanding of certain endo-phenotypes (isolated symptoms) of depression and their molecular correlates.

Pronounced and sustained central hypernoradrenergic function in major depression with melancholic features: Relation to hypercortisolism and corticotropin-releasing hormone

Both stress-system activation and melancholic depression are characterized by fear, constricted affect, stereotyped thinking, and similar changes in autonomic and neuroendocrine function. Because norepinephrine (NE) and corticotropin-releasing hormone (CRH) can produce these physiological and behavioral changes, we measured the cerebrospinal fluid (CSF) levels each hour for 30 consecutive hours in controls and in patients with melancholic depression. Plasma adrenocorticotropic hormone (ACTH) and cortisol levels were obtained every 30 min. Depressed patients had significantly higher CSF NE and plasma cortisol levels that were increased around the clock. Diurnal variations in CSF NE and plasma cortisol levels were virtually superimposable and positively correlated with each other in both patients and controls. Despite their hypercortisolism, depressed patients had normal levels of plasma ACTH and CSF CRH. However, plasma ACTH and CSF CRH levels in depressed patients were inappropriately high, considering the degree of their hypercortisolism. In contrast to the significant negative correlation between plasma cortisol and CSF CRH levels seen in controls, patients with depression showed no statistical relationship between these parameters. These data indicate that persistent stress-system dysfunction in melancholic depression is independent of the conscious stress of the disorder. These data also suggest mutually reinforcing bidirectional links between a central hypernoradrenergic state and the hyperfunctioning of specific central CRH pathways that each are driven and sustained by hypercortisolism. We postulate that α-noradrenergic blockade, CRH antagonists, and treatment with antiglucocorticoids may act at different loci, alone or in combination, in the treatment of major depression with melancholic features.

Corticotropin-releasing hormone causes antidiuresis and antinatriuresis by stimulating vasopressin and inhibiting atrial natriuretic peptide release in male rats

In both normally hydrated and volume-expanded rats, there was a biphasic effect of corticotropin-releasing hormone (CRH) (1–10 μg, i.v.) on renal function. Within the first hour, CRH caused antidiuresis, antinatriuresis, and antikaliuresis together with reduction in urinary cGMP output that, in the fourth hour, were replaced by diuresis, natriuresis, and kaliuresis accompanied by increased cGMP output. Plasma arginine vasopressin (AVP) concentrations increased significantly within 5 min, reached a peak at 15 min, and declined by 30 min to still-elevated values maintained for 180 min. Changes in plasma atrial natriuretic peptide (ANP) were the mirror image of those of AVP. Plasma ANP levels were correlated with decreased ANP in the left ventricle at 30 min and increased ANP mRNA in the right atrium at 180 min. All urinary changes were reversed by a potent AVP type 2 receptor (V2R) antagonist. Control 0.9% NaCl injections evoked an immediate increase in blood pressure and heart rate measured by telemetry within 3–5 min. This elevation of blood pressure was markedly inhibited by CRH (5 μg). We hypothesize that the effects are mediated by rapid, direct vasodilation induced by CRH that decreases baroreceptor input to the brain stem, leading to a rapid release of AVP that induces the antidiuresis by direct action on the V2Rs in the kidney. Simultaneously, acting on V2Rs in the heart, AVP inhibits ANP release and synthesis, resulting in a decrease in renal cGMP output that is responsible for the antinatriuretic and antikaliuretic effects.

Long-term, progressive hippocampal cell loss and dysfunction induced by early-life administration of corticotropin-releasing hormone reproduce the effects of early-life stress

Stress early in postnatal life may result in long-term memory deficits and selective loss of hippocampal neurons. The mechanisms involved are poorly understood, but they may involve molecules and processes in the immature limbic system that are activated by stressful challenges. We report that administration of corticotropin-releasing hormone (CRH), the key limbic stress modulator, to the brains of immature rats reproduced the consequences of early-life stress, reducing memory functions throughout life. These deficits were associated with progressive loss of hippocampal CA3 neurons and chronic up-regulation of hippocampal CRH expression. Importantly, they did not require the presence of stress levels of glucocorticoids. These findings indicate a critical role for CRH in the mechanisms underlying the long-term effects of early-life stress on hippocampal integrity and function.

Transcription of the human corticotropin-releasing hormone gene in NPLC cells is correlated with Z-DNA formation.

The intron of the corticotropin-releasing hormone (corticoliberin; CRH) gene contains a sequence of over 100 bp of alternating purine/pyrimidine residues. We have used binding of a Z-DNA-specific antibody in metabolically active, permeabilized nuclei to study the formation of Z-DNA in this sequence at various levels of transcription. In the NPLC human primary liver carcinoma cell line, activation of cAMP-dependent pathways increased the level of transcription, while adding glucocorticoids inhibited transcription of the CRH gene. These cells respond in a manner similar to hypothalamic cells. Z-DNA formation in this sequence was detected at the basal level of transcription, as well as after stimulation with forskolin. Inhibition of transcription by dexamethasone abolished Z-DNA formation. Z-DNA formation in the WC gene (c-myc) was affected differently in the same experiment. Thus, changes in Z-DNA formation in the CRH gene are gene specific and are linked to the transcription of the gene.

Effect of naloxone-precipitated morphine withdrawal on c-fos expression in rat corticotropin-releasing hormone neurons in the paraventricular hypothalamus and extended amygdala

Morphine withdrawal is characterized by physical symptoms and a negative affective state. The 41 amino acid polypeptide corticotropin-releasing, hormone (CRH) is hypothesized to mediate, in part, both the negative affective state and the physical withdrawal syndrome. Here, by means of dual-immunohistochemical methodology, we examined the co-expression of the c-Fos protein and CRH following naloxone-precipitated morphine withdrawal. Rats were treated with slow-release morphine 50 mg/kg (subcutaneous, s.c.) or vehicle every 48 It for 5 days, then withdrawn with naloxone 5 mg/kg (s.c.) or saline 48 h after the final morphine injection. Two hours after withdrawal rats were perfused transcardially and their brains were removed and processed for immunohistochemistry. We found that naloxone-precipitated withdrawal of morphine-dependent rats increased c-Fos immunoreactivity (IR) in CRH positive neurons in the paraventricular hypothalamus. Withdrawal of morphine-dependent rats also increased c-Fos-IR in the central amygdala and bed nucleus of the stria terminalis. however these were in CRH negative neurons. (C) 2004 Published by Elsevier Ireland Ltd.

Regulation of corticotropin-releasing factor concentration and overflow in the rat central nervous system

Examines alterations in corticotropin-releasing factor concentration and nerve release during maturation and aging in the rat brain. Release of neuropeptide Y was also measured. These studies may provide information leading to the effective treatment of age-related disorders.

The tale of a person and a peptide: Wylie W. Vale Jr. and the role of corticotropin- releasing factor in the stress response

The studies reviewed in this article certainly do not constitute the entire body of research conducted by Wylie Vale's group and his collaborators, they might constitute turning points in CRF research. In addition, the studies reviewed here show that, over the course of 31 years (from 1981 to 2012), Wylie tirelessly pursued the answers to fundamental questions regarding CRF. He was a man whose drive never seemed to falter

Enhanced nicotine-seeking behavior following pre-exposure to repeated cocaine is accompanied by changes in BDNF in the nucleus accumbens of rats

We investigated the behavioral and molecular interactions between cocaine and nicotine, through evaluating locomotor activity, nicotine intravenous self-administration and gene expression. Locomotor sensitization was induced in male Wistar rats by repeated cocaine (20 mg/kg; i.p.) or saline injections once a day over 7 days. Three days after the last injection, rats were challenged with either saline or cocaine (15 mg/kg; i.p.) and the locomotor activity was measured. The very next day animals received either saline or nicotine (0.4 mg/kg; s.c.) and the locomotor cross-sensitization was tested. Animals were then prepared with intrajugular catheters for nicotine self-administration. Nicotine self-administration patterns were evaluated using fixed or progressive ratio schedules of reinforcement and a 24-h unlimited access binge. Immediately after the binge sessions animals were decapitated, the brains were removed and the nucleus accumbens was dissected. The dynorphin (DYN), μ-opioid receptor (mu opioid), neuropeptide Y (NPY), brain-derived neurotrophic factor (BDNF), tropomyosin-related tyrosine kinase B receptor (TrkB) and corticotropin- releasing factor receptor type 1 (CRF-R1) gene expression were measured by the reverse transcription-polymerase chain reaction (RT-PCR). Pretreatment with cocaine caused sensitization of cocaine motor response and locomotor cross-sensitization with nicotine. In the self-administration experiments repeated cocaine administration caused an increase in the nicotine break point and nicotine intake during a 24 h binge session. © 2013 Elsevier Inc.

Five gonadotrophin-releasing hormone receptors in a teleost fish: isolation, tissue distribution and phylogenetic relationships

Gonadotrophin-releasing hormone (GnRH) is the main neurohormone controlling gonadotrophin release in all vertebrates, and in teleost fish also of growth hormone and possibly of other adenohypophyseal hormones. Over 20 GnRHs have been identified in vertebrates and protochoordates and shown to bind cognate G-protein couple receptors (GnRHR). We have searched the puffer fish, Fugu rubripes, genome sequencing database, identified five GnRHR genes and proceeded to isolate the corresponding complementary DNAs in European sea bass, Dicentrachus labrax. Phylogenetic analysis clusters the European sea bass, puffer fish and all other vertebrate receptors into two main lineages corresponding to the mammalian type I and II receptors. The fish receptors could be subdivided in two GnRHR1 (A and B) and three GnRHR2 (A, B and C) subtypes. Amino acid sequence identity within receptor subtypes varies between 70 and 90% but only 50–55% among the two main lineages in fish. All European sea bass receptor mRNAs are expressed in the anterior and mid brain, and all but one are expressed in the pituitary gland. There is differential expression of the receptors in peripheral tissues related to reproduction (gonads), chemical senses (eye and olfactory epithelium) and osmoregulation (kidney and gill). This is the first report showing five GnRH receptors in a vertebrate species and the gene expression patterns support the concept that GnRH and GnRHRs play highly diverse functional roles in the regulation of cellular functions, besides the ‘‘classical’’ role of pituitary function regulation.

Affinity purification of 61- and 65-kDa rat brain corticotropin-releasing factor receptors and receptor-associated G proteins.

Corticotropin-releasing factor (CRF) has been shown to have a central role in physiological adaptation to stress. It is recognized for stimulating the release of adrenocorticotropin from the anterior pituitary gland, and has more recently been implicated as a regulator of autonomic and immunological responses to stress. Much confusion has surrounded the characterization of CRF receptors, with proteins of varying molecular weights having been identified but never purified and characterized. Recently, two CRF receptors have been cloned from brain and pituitary gland, but evidence from in-situ hybridization studies suggests that further CRF receptor types exist. We therefore developed two techniques which enable the isolation of CRF receptors from whole rat brain. The use of a solid-phase CRF analogue affinity column and elution using a competing ligand resulted in the purification of a single protein of 61 kDa. A second technique was devised which allowed the co-isolation of associated signalling proteins and the identification of CRF bound species following purification. CRF was covalently cross-linked to receptors and the complex purified using antibodies specific for the ligand. This enabled the purification of a CRF receptor of approximately 65 kDa and associated alpha and beta gamma G protein subunits. This study demonstrates the successful isolation of CRF receptors which are of different molecular weights to those previously observed from affinity cross-linking studies or predicted from cloned genes. In addition, we confirm the involvement of G proteins in CRF stimulated cell signalling by demonstrating their association with purified CRF receptor.

Cannabinoid CB1 receptor restrains accentuated activity of hypothalamic corticotropin-releasing factor and brainstem tyrosine hydroxylase neurons in endotoxemia-induced hypophagia in rats

It is well known that endocannabinoids play an important role in the regulation of food intake and body weight. Endocannabinoids and cannabinoid receptors are found in the hypothalamus and brainstem, which are central areas involved in the control of food intake and energy expenditure. Activation of these areas is related to hypophagia observed during inflammatory stimulus. This study investigated the effects of cannabinoid (CB1) receptor blockade on lipopolysaccharide (LPS)-induced hypophagia. Male Wistar rats were pretreated with rimonabant (10 mg/kg, by gavage) or vehicle; 30 min later they received an injection of either LPS (100 mu g/kg, intraperitoneal) or saline. Food intake, body weight, corticosterone response, CRF and CART mRNA expression, Fos-CRF and Fos-alpha-MSH immunoreactivity in the hypothalamus and Fos-tyrosine hydroxylase (TH) immunoreactivity in the brainstem were evaluated. LPS administration decreased food intake and body weight gain and increased plasma corticosterone levels and CRF mRNA expression in the PVN. We also observed an increase in Fos-CRF and Fos-TH double-labeled neurons after LPS injection in vehicle-pretreated rats, with no changes in CART mRNA or Fos-alpha-MSH immunoreactive neurons in the ARC. In saline-treated animals, rimonabant pretreatment decreased food intake and body weight gain but did not modify hormone response or Fos expression in the hypothalamus and brainstem compared with vehicle-pretreated rats. Rimonabant pretreatment potentiated LPS-induced hypophagia, body weight loss and Fos-CRF and Fos-TH expressing neurons. Rimonabant did not modify corticosterone, CRF mRNA or Fos-alpha-MSH responses in rats treated with LPS. These data suggest that the endocannabinoid system, mediated by CB1 receptors, modulates hypothalamic and brainstem circuitry underlying the hypophagic effect during endotoxemia to prevent an exaggerated food intake decrease. This article is part of a Special Issue entitled 'Central Control of Food Intake'. (C) 2011 Elsevier Ltd. All rights reserved.

Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers

The proliferation of various tumors is inhibited by the antagonists of growth hormone-releasing hormone (GHRH) in vitro and in vivo, but the receptors mediating the effects of GHRH antagonists have not been identified so far. Using an approach based on PCR, we detected two major splice variants (SVs) of mRNA for human GHRH receptor (GHRH-R) in human cancer cell lines, including LNCaP prostatic, MiaPaCa-2 pancreatic, MDA-MB-468 breast, OV-1063 ovarian, and H-69 small-cell lung carcinomas. In addition, high-affinity, low-capacity binding sites for GHRH antagonists were found on the membranes of cancer cell lines such as MiaPaCa-2 that are negative for the vasoactive intestinal peptide/pituitary adenylate cyclase-activating polypeptide receptor (VPAC-R) or lines such as LNCaP that are positive for VPAC-R. Sequence analysis of cDNAs revealed that the first three exons in SV1 and SV2 are replaced by a fragment of retained intron 3 having a new putative in-frame start codon. The rest of the coding region of SV1 is identical to that of human pituitary GHRH-R, whereas in SV2 exon 7 is spliced out, resulting in a 1-nt upstream frameshift, which leads to a premature stop codon in exon 8. The intronic sequence may encode a distinct 25-aa fragment of the N-terminal extracellular domain, which could serve as a proposed signal peptide. The continuation of the deduced protein sequence coded by exons 4–13 in SV1 is identical to that of pituitary GHRH-R. SV2 may encode a GHRH-R isoform truncated after the second transmembrane domain. Thus SVs of GHRH-Rs have now been identified in human extrapituitary cells. The findings support the view that distinct receptors are expressed on human cancer cells, which may mediate the antiproliferative effect of GHRH antagonists.