Corticotropin-releasing Hormone Peptide Research Articles

A corticotropin-releasing hormone antisense oligodeoxynucleotide reduces spontaneous waking in the rat

We have previously hypothesized that corticotropin-releasing hormone (CRH) is involved in the regulation of physiological waking. In this study, we tested the hypothesis that reduction of CRH peptide would reduce spontaneous wakefulness of rats. We administered intracerebroventricularly into rats at several circadian time points antisense or sense DNA oligodeoxynucleotides (ODNs) corresponding to the initiation codon of CRH mRNA and determined subsequent effects on wakefulness and sleep of the rat. Our results indicate that CRH antisense oligodeoxynucleotides reduce spontaneous wakefulness during the dark (active) period, but not during the light (rest) period of the light/dark cycle. The alterations in time spent awake are due to reduced wake bout numbers, rather than a change in wake bout duration. These reductions in wakefulness were mirrored by increases in slow-wave sleep, while rapid eye movement sleep was not affected. Corticosterone, used as an index of CRH in the hypothalamus, was reduced by CRH antisense oligodeoxynucleotides during the same time that spontaneous wakefulness was reduced, suggesting CRH peptide modulation as the mediator of this response. In contrast, CRH sense oligodeoxynucleotides did not alter any parameter of this study during either the dark or light period. These findings provide additional support for the hypothesis that CRH is involved in the regulation/modulation of wakefulness.

Reduced cerebral injury in CRH-R1 deficient mice after focal ischemia: a potential link to microglia and atrocytes that express CRH-R1.

Corticotropin releasing hormone (CRH) and its family of related peptides are involved in regulating physiologic responses to multiple stressors, including stroke. Although CRH has been implicated in the exacerbation of injury after stroke, the mechanism remains unclear. After ischemia, both excitotoxic damage and inflammation contribute to the pathology of stroke. CRH is known to potentiate excitotoxic damage in the brain and has been shown to modulate inflammatory responses in the periphery. Here the present authors examine the relative contribution of the two known CRH receptors, CRH-R1 and CRH-R2, to ischemic injury using CRH receptor knockout mice. These results implicate CRH-R1 as the primary mediator of ischemic injury in this mouse model of stroke. In addition, the authors examine a potential role for CRH in inflammatory injury after stroke by identifying functional CRH receptors on astrocytes and microglia, which are cells that are known to be involved in brain inflammation. By single cell PCR, the authors show that microglia and astrocytes express mRNA for both CRH-R1 and CRH-R2. However, CRH-R1 is the primary mediator of cAMP accumulation in response to CRH peptides in these cells. The authors suggest that astrocytes and microglia are cellular targets of CRH, which could serve as a link between CRH and inflammatory responses in ischemic injury via CRH-R1.

Stress-induced interleukin-6 release in mice is mast cell-dependent and more pronounced in Apolipoprotein E knockout mice.

Interleukin-6 (IL-6) is elevated in the serum of patients with coronary artery disease (CAD) and acute coronary syndromes (ACS). Intracoronary release of IL-6 was reported in ACS that can also be triggered by acute stress. In rats, acute restraint stress increases serum IL-6 and histamine, both of which may derive from mast cells. The current study was carried out in order to determine any possible difference in stress-induced IL-6 release in atherosclerotic mice and the contribution of mast cells, corticotropin-releasing hormone (CRH) and urocortin (Ucn). C57BL/6J, W/W(v) mast cell-deficient, Apolipoprotein E (ApoE) and CRH knockout (k/o) mice were stressed in plexiglass restraint chambers for 15 to 120 min. Serum corticosterone and IL-6 levels were measured. Some C57BL and ApoE k/o mice were pretreated with either the mast cell stabilizer disodium cromoglycate (cromolyn) or the peptide CRH receptor (CRH-R) antagonist Astressin. Cardiac mast cell activation was studied in both C57BL and ApoE k/o mice using light microscopy. Acute stress increased serum IL-6 in mice, an effect much greater in ApoE k/o atherosclerotic mice. Stress-induced IL-6 release was absent in W/W(v) mast cell-deficient mice and it was partially inhibited by cromolyn in C57BL and ApoE mice. Mast cells were found adjacent to atherosclerotic vessels in ApoE k/o mice and were activated after stress. CRH k/o mice released more IL-6 in response to stress than their wild types, but Astressin significantly inhibited IL-6 release. Stress-induced IL-6 release may, therefore, be at least partly due to peripheral Ucn and/or CRH, with Ucn possibly overcompensation for CRH deficiency. The present findings indicate that acute stress leads to mast cell-dependent serum IL-6 increase that may help explain stress-related coronary inflammation.

RETRACTED: In vitro regulation of corticotropin-releasing hormone

Studies involving regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Many in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH regulation in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. Amygdalar CRH levels in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated signal transduction mechanisms and these studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Thus, much more investigative work in better understanding CRH regulation in vitro is needed.

Overexpression of corticotropin-releasing hormone in transgenic mice and chronic stress-like autonomic and physiological alterations.

To gain a greater insight into the relationship between hyperactivity of the corticotropin-releasing hormone (CRH) system and autonomic and physiological changes associated with chronic stress, we developed a transgenic mouse model of central CRH overproduction. The extent of central and peripheral CRH overexpression, and the amount of bioactive CRH in the hypothalamus were determined in two lines of CRH-overexpressing (CRH-OE) mice. Furthermore, 24 h patterns of body temperature, heart rate, and activity were assessed using radiotelemetry, as well as cumulative water and food consumption and body weight gain over a 7-day period. CRH-OE mice showed increased amounts of CRH peptide and mRNA only in the central nervous system. Despite the presence of the same CRH transgene in their genome, only in one of the two established lines of CRH-OE mice (line 2122, but not 2123) was overexpression of CRH associated with increased levels of bioactive CRH in the hypothalamus, increased body temperature and heart rate (predominantly during the light (inactive) phase of the diurnal cycle), decreased heart rate variability during the dark (active) phase, and increased food and water consumption, when compared with littermate wildtype mice. Because line 2122 of the CRH transgenic mice showed chronic stress-like neuroendocrine and autonomic changes, these mice appear to represent a valid animal model for chronic stress and might be valuable in the research on the consequences of CRH excess in situations of chronic stress.

The expression of corticotropin-releasing hormone in melanoma.

We previously demonstrated that advanced melanoma cells express high amounts of proopiomelanocortin (POMC) that correlate with tumor progression. We now investigated whether the high expression of POMC derives from increased expression of corticotropin-releasing hormone (CRH) and the possible role of CRH as a melanoma growth factor. Forty-five cases of melanoma [25 primary malignant melanoma; 20 metastatic melanoma (MetM)] were immunohistochemically analysed for coexpression of POMC and CRH peptides. The ability of CRH to induce POMC expression in cultured melanoma cells was examined using CRH and a CRH antagonist. In CRH positive melanomas, seven out of nine cases (78%) of primary melanoma, and 7 out of 12 cases (58%) of MetM showed colocalization of CRH and POMC peptides. CRH induced POMC mRNA expression, an effect that was inhibited by a CRH antagonist. These results provide evidence for the existence of the CRH/POMC axis in pigmented lesions.

Functional expression of corticotropin-releasing hormone (CRH) receptor 1 in cultured rat microglia.

Corticotropin-releasing hormone (CRH), known as a key regulator of the hypothalamic-pituitary-adrenal axis response to stress, elicits its biological effects by binding to two membrane receptors (CRH-R1 and CRH-R2). The present studies examined the presence of functional expression of CRH receptors in cultured microglia of rat. CRH-R1 mRNA and protein were detected by reverse transcriptase polymerase chain reaction (RT-PCR), western blotting and receptor chemical cross-linking assay in cultured microglia. CRH-R2 mRNA was undetectable by RT-PCR. The radioligand binding analysis using [125I]Tyr-rat/human CRH revealed a high affinity binding site (Kd of 1.2 nm and Bmax of 84 fmol/mg of protein). Competition studies using CRH and related peptides indicated kinetic and pharmacological characteristics consistent with the CRH-R1 receptor subtype. Receptor chemical cross-linking assay demonstrated a single band of CRH receptor with a molecular weight of -77 kDa, which was inhibited in the presence of excess unlabeled rat/human CRH in a dose-dependent manner and inhibited by a CRH receptor antagonist astressin. Functional coupled cAMP production in cultured microglia was stimulated by exogenous addition of CRH and related peptides in a dose-dependent manner and blocked by astressin. Our findings suggest the functional expression of CRH-R1 receptor in rat microglia, indicating an important mechanism of interaction between immune and neuroendocrine systems in brain physiological and pathological conditions.

Neuropeptides: Modulators of immune responses in health and disease

Publisher Summary Glucocorticoids remain the most powerful of all anti-inflammatory agents, but their clinical application in chronic inflammation and autoimmune diseases is limited by systemic side effects. The challenge that needs to be addressed is to develop selective anti-inflammatory drugs with efficacy equal to or better than glucocorticoids. Immunoneuropeptides have exciting potential for development as a new generation of nonglucocorticoid anti-inflammatory drugs. One area of promise is the direct injection into inflamed tissue of small amounts of a long-acting compound or local implants of slow-release polymer beads impregnated with an anti-inflammatory agent. This would be a useful strategy for the application of low dose/high potency opioid or corticotropin-releasing hormone (CRH) analogs to exert anti-inflammatory effects without systemic release and consequent nonspecific effects. Expression of some immunoneuropeptides is responsive to glucocorticoids and catecholamines, and these neuropeptides can modulate the effects of stress on immune functions. Stress can influence the production of CRH and pro-opiomelanocortin (POMC) peptides from leukocytes and also determine T-cell differentiation into distinctive Th1/Th2 cytokine secretory profiles. Therefore, neuropeptides may be involved in mediating the effects of stress on production of specific cytokines with pro- or anti-inflammatory activity.

Expression and regulation of corticotropin-releasing hormone binding protein (CRH-BP) in rat adrenals.

Corticotropin-releasing hormone (CRH) is present in the adrenal gland acting as a paracrine factor via stimulation of the locally expressed CRH receptors. In this study, we examined if the adrenal CRH system also contains a key component of the neuronal CRH-containing system, the CRH-binding protein (CRH-BP). Our data show that: (i) the CRH-BP transcript is detectable using RT-PCR in total RNA isolated from rat adrenals, and (ii) its protein product is also found by western blot analysis in cell lysates. (iii) Immunohistochemical staining showed that adrenomedullary chromaffin cells produce the bulk of adrenal CRH-BP, an ability retained by the PC12 rat pheochromocytoma cell line. (iv) Regulation of adrenal CRH-BP expression by major modulators of the CRH system was also examined. Protein expression appears to be under the positive control of CRH itself, protein kinase A effector cAMP, glucocorticoids and interleukin (IL)-6. It is thus evident that CRH-BP may play a role in mediating their effects in the adrenal. (v) Differentiation of PC12 into neuron-like cells resulted in a significant increase in CRH-BP, parallel to the induction of the CRH peptide itself. In conclusion, CRH-BP mRNA and protein are present in normal rat adrenomedullary chromaffin cells and in the PC12 rat pheochromocytoma cell line, making the adrenal CRH system directly comparable with those described in the CNS.

The effects of the selective serotonin reuptake-inhibitor fluvoxamine on body weight in Zucker rats are mediated by corticotropin-releasing hormone.

Brain serotonin plays a crucial role in the regulation of food intake and body weight homeostasis. Previous data suggest an interaction with corticotropin-releasing hormone (CRH). To further examine the interaction between these neurotransmitters, the selective serotonin reuptake-inhibitor (SSRI) fluvoxamine was given intraperitoneally in fa/fa Zucker rats with and without the CRH-receptor-antagonist alpha-helical CRH administered intracerebroventricularly (i.c.v.). The saline treated animals gained weight. Fluvoxamine led to a significant weight loss while not affecting food intake. Furthermore, insulin levels in this animal model were reduced following fluvoxamine administration. These effects were antagonized by alpha-helical CRH and are thus most likely mediated via CRH or CRH-like peptides.

In situ expression of corticotropin-releasing hormone (CRH) and proopiomelanocortin (POMC) genes in human skin.

Systemic stresses induce corticotropin-releasing hormone (CRH) expression in hypothalamus. CRH is released to the pituitary gland, where it stimulates proopiomelanocortin (POMC) production acting via the CRH receptor (CRH-R). CRH and POMC peptides are also detected in sites outside of the central nervous system (CNS), such as the skin. However, it has not been elucidated whether these peptides detected in the skin are derived from CNS or are produced locally. Using immunohistochemical and in situ reverse-transcription (RT)-PCR techniques, we demonstrated coexpression of CRH and POMC mRNAs in the epidermis and pilosebaceous units of the human skin. This coexpression was confirmed by the combination of laser-capture microdissection (LCM) with RT-PCR, analyzing mRNA expressions in captured sebaceous cells. Immunoreactivities and expressions of CRH and POMC mRNAs were strong in inflammatory lesions, melanocytic nevus, seborrheic keratosis, and also in the periphery of the benign tumor. These findings suggest that CRH and POMC peptides are produced locally in the skin and are regulated by inflammatory cells as well as by autocrine mechanisms. The skin may have "a local stress response system," whose activity is mediated by CRH and POMC peptides, in an equivalent to hypothalamus-pituitary adrenal axis.

Cutaneous expression of corticotropin-releasing hormone (CRH), urocortin, and CRH receptors.

Studies in mammalian skin have shown expression of the genes for corticotropin-releasing hormone (CRH) and the related urocortin peptide, with subsequent production of the respective peptides. Recent molecular and biochemical analyses have further revealed the presence of CRH receptors (CRH-Rs). These CRH-Rs are functional, responding to CRH and urocortin peptides (exogenous or produced locally) through activation of receptor(s)-mediated pathways to modify skin cell phenotype. Thus, when taken together with the previous findings of cutaneous expression of POMC and its receptors, these observations extend the range of regulatory elements of the hypothalamic-pituitary-adrenal axis expressed in mammalian skin. Overall, the cutaneous CRH/POMC expression is highly reactive to common stressors such as immune cytokines, ultraviolet radiation, cutaneous pathology, or even the physiological changes associated with the hair cycle phase. Therefore, similar to its central analog, the local expression and action of CRH/POMC elements appear to be highly organized and entrained, representing general mechanism of cutaneous response to stressful stimuli. In such a CRH/POMC system, the CRH-Rs may be a central element.

Regulation of corticotropin-releasing hormone in vitro

Studies examining regulation of corticotropin-releasing hormone (CRH) in vitro have been used to validate findings obtained in vivo and more importantly have been used as model systems to better understand signalling mechanisms responsible for the expression of the CRH gene and peptide. Most in vitro studies examining CRH have utilized hypothalamic tissue while a few have focused on the amygdala. Furthermore, clonal cell lines have also been utilized as models of central nervous system CRH neurons. Stimuli that have been implicated in regulating hypothalamic CRH in vitro include protein kinase A (PKA) and protein kinase C (PKC) activators, glucocorticoids, biogenic amines, cytokines and the gaseous neurotransmitters. CRH levels in the amygdala in vitro are affected by some of the same stimuli that regulate hypothalamic CRH; however there is evidence supporting differential regulation of CRH in these two brain regions by some of the same stimuli. Only a few studies in aggregate have investigated the signal transduction mechanisms responsible for CRH expression. These mechanistic studies have focused on PKA- and glucocorticoid-mediated changes in CRH expression. Clearly much more investigative work in better understanding CRH regulation in vitro is needed.

Chronic cold exposure potentiates CRH-evoked increases in electrophysiologic activity of locus coeruleus neurons

Background: Chronic stress exposure can produce sensitization of norepinephrine release in the forebrain in response to subsequent stressors. Furthermore, the increase in norepinephrine release in response to the stress-related peptide corticotropin-releasing hormone (CRH) is potentiated by prior chronic stress exposure. To explore possible mechanisms underlying these alterations in norepinephrine release, we examined the effect of chronic stress on the electrophysiologic activity of locus coeruleus (LC) neurons in response to centrally applied CRH. Methods: Single-unit recordings of LC neurons in halothane-anesthetized rats were used to compare the effect of intraventricular administration of CRH (0.3–3.0 μg) in control and previously cold-exposed (2 weeks at 5°C) rats. Results: The CRH-evoked increase in LC neuron activity was enhanced following chronic cold exposure, without alteration in basal activity of LC neurons. The enhanced CRH-evoked activation was apparent at higher doses of CRH but not at lower ones, resulting in an increased slope of the dose-response curve for CRH in previously cold-exposed rats. Conclusions: These data, in combination with previous data, suggest that the sensitivity of LC neurons to excitatory inputs is increased following chronic cold exposure. The altered functional capacity of LC neurons in rats after continuous cold exposure may represent an experimental model to examine the role of central noradrenergic neurons in anxiety and mood disorders.

Characterising the Corticotropin-releasing Hormone (CRH) Receptors Mediating CRH and Urocortin Actions During Human Pregnancy and Labour

The mechanism of human labour remains unresolved. One of the most important regulatory signals, however, appears to be corticotropin-releasing hormone (CRH), a hypothalamic peptide that controls the body's response to stress, which is also produced by the placenta and intrauterine tissues during pregnancy. CRH belongs to a family of peptides that includes urocortin, which shares sequence homology with CRH and is also expressed by the placenta and intrauterine tissues. During human pregnancy circulating CRH appears to have five main target tissues; the myometrium, the placenta, the fetal membranes, the fetal adrenal cortex and the vasculature. In these tissues CRH plays a role in the control of myometrial contractility, placenta vasodilation, peptide and prostaglandin production and adrenal steroidogenesis and probably many more, yet unidentified processes. The actions of CRH in these tissues are mediated via specific G-protein coupled membrane-bound receptors. These receptors have different functional characteristics, depending on where they are expressed and on the stage of pregnancy. In addition, their function depends upon other intracellular signals via communication between signalling cascades. These findings led us to propose a hypothesis for a dual role of CRH and other CRH-like peptides during pregnancy and labour.

Urocortin, but not corticotropin-releasing hormone (CRH), activates the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium: an effect mediated via R1alpha and R2beta CRH receptor subtypes and stimulation of Gq-proteins.

CRH and CRH-related peptides such as urocortin mediate their actions in the human myometrium via activation of two distinct classes of CRH receptors, R1 and R2. These heptahelical receptors are able to stimulate a number of different intracellular signals; one key mediator of G protein-activated intracellular signaling is the cascade of p42/p44, mitogen-activated protein kinase (MAPK). We therefore hypothesized that activation of MAPK might mediate CRH and or/urocortin actions in the myometrium. In cultured human pregnant myometrial cells, urocortin but not CRH was able to induce MAPK phosphorylation and activation, suggesting that in the human myometrium these two peptides have distinct actions and biological roles. To identify the particular receptor subtypes mediating this phenomenon, all known CRH receptors present in the human myometrial cells were stably expressed individually in HEK293 and CHO cells, and their ability to activate MAPK was tested. The R1alpha and R2beta, but not the R1beta, R1c, or R1d, receptor subtypes were able to mediate urocortin-induced MAPK activation. The signaling components were further investigated; activation of Gs, Go, or Gi proteins did not appear to be involved, but activation of Gq with subsequent production of inositol triphosphates (IP3) and protein kinase C (PKC) activation correlated with MAPK phosphorylation. Studies on Gq protein activation using [alpha-32P]-GTP-gamma-azidoanilide and IP3 production in cells expressing the R1alpha or R2beta CRH receptors demonstrated that urocortin was 10 times more potent than CRH. Moreover, urocortin (UCN) generated peak responses that were 50-70% greater than CRH in activating the Gq protein and stimulating IP3 production. In conclusion, UCN acting thought multiple receptor subtypes can stimulate myometrial MAPK via induction of the Gq/phospholipase C/IP3/PKC pathway, whereas CRH-induced activation of this pathway appears to be insufficient to achieve MAPK activation.

Oreochromis mossambicus (tilapia) corticotropin-releasing hormone: cDNA sequence and bioactivity.

Although hypothalamic corticotropin-releasing hormone (CRH) is involved in the stress response in all vertebrate groups, only a limited number of studies on this neuroendocrine peptide deals with non-mammalian neuroendocrine systems. We determined the cDNA sequence of the CRH precursor of the teleost Oreochromis mossambicus (tilapia) and studied the biological potency of the CRH peptide in a homologous teleost bioassay. Polymerase chain reaction (PCR) with degenerate and specific primers yielded fragments of tilapia CRH cDNA. Full-length CRH cDNA (988 nucleotides) was obtained by screening a tilapia hypothalamus cDNA library with the tilapia CRH PCR products. The precursor sequence (167 amino acids) contains a signal peptide, the CRH peptide and a motif conserved among all vertebrate CRH precursors. Tilapia CRH (41 aa) displays between 63% and 80% amino acid sequence identity to CRH from other vertebrates, whereas the degree of identity to members of the urotensin I/urocortin lineage is considerably lower. In a phylogenetic tree, based on alignment of all full CRH peptide precursors presently known, the three teleost CRH precursors (tilapia; sockeye salmon, Oncorhynchus nerka; white sucker, Catostomus commersoni) form a monophyletic group distinct from amphibian and mammalian precursors. Despite the differences between the primary structures of tilapia and rat CRH, maximally effective concentrations of tilapia and rat CRH were equally potent in stimulating adrenocorticotropic hormone (ACTH) and alpha-MSH release by tilapia pituitaries in vitro. The tilapia and salmon CRH sequences show that more variation exists between orthologous vertebrate CRH structures, and teleost CRHs in particular than previously recognized. Whether the structural differences reflect different mechanisms of action of this peptide in the stress response remains to be investigated.

Central corticotropin-releasing hormone receptors modulate hypothalamic–pituitary–adrenocortical and sympathoadrenal activity during stress

The role of brain corticotropin-releasing hormone receptors in modulating hypothalamic–pituitary–adrenal and sympathoadrenal responses to acute immobilization stress was studied in conscious rats under central corticotropin-releasing hormone receptor blockade by intracerebroventicular injection of a peptide corticotropin-releasing hormone receptor antagonist. Blood for catecholamines, adrenocorticotropic hormone and corticosterone levels was collected through vascular catheters, and brains were removed at 3 h for in situ hybridization for tyrosine hydroxylase messenger RNA in the locus coeruleus, and corticotropin-releasing hormone and corticotropin-releasing hormone receptor messenger RNA in the hypothalamic paraventricular nucleus. Central corticotropin-releasing hormone receptor blockade reduced the early increases in plasma epinephrine and dopamine, but not norepinephrine, during stress. Immobilization stress increased tyrosine hydroxylase messenger RNA levels in the locus coeruleus by 36% in controls, but not in corticotropin-releasing hormone antagonist-injected rats. In control rats, corticotropin-releasing hormone messenger RNA and type 1 corticotropin-releasing hormone receptor messenger RNA in the paraventricular nucleus increased after stress ( P<0.01), and these responses were attenuated by central corticotropin-releasing hormone receptor blockade. In contrast, central corticotropin-releasing hormone antagonist potentiated plasma adrenocorticotropic hormone responses, but slightly attenuated plasma corticosterone responses to stress. The inhibition of plasma catecholamine and locus coeruleus tyrosine hydroxylase messenger RNA responses to stress by central corticotropin-releasing hormone receptor blockade supports the notion that central corticotropin-releasing hormone regulates sympathoadrenal responses during stress. The attenuation of stress-induced corticotropin-releasing hormone and corticotropin-releasing hormone receptor messenger RNA responses by central corticotropin-releasing hormone receptor blockade suggests direct or indirect positive feedback effects of corticotropin-releasing hormone receptor ligands on corticotropin-releasing hormone expression, whereas additional mechanisms potentiate adrenocorticotropic hormone responses at the pituitary level. In addition, changes in neural activity by central corticotropin-releasing hormone are likely to modulate adrenocortical responsiveness during stress.

Altered anxiety and weight gain in corticotropin-releasing hormone-binding protein-deficient mice.

Corticotropin-releasing hormone (CRH) is widely recognized as the primary mediator of the neuroendocrine and behavioral responses to stress, including stress-induced anxiety. The biological activity of CRH and other mammalian CRH-like peptides, such as urocortin, may be modulated by CRH-binding protein (CRH-BP). To assess directly the CRH-BP function, we created a mouse model of CRH-BP deficiency by gene targeting. Basal adrenocorticotropic hormone and corticosterone levels are unchanged in the CRH-BP-deficient mice, and the animals demonstrate a normal increase in adrenocorticotropic hormone and corticosterone after restraint stress. In contrast, adult male CRH-BP-deficient mice show significantly reduced body weight when compared with wild-type controls. CRH-BP-deficient mice also exhibit a significant increase in anxiogenic-like behavior as assessed by the elevated plus maze and defensive withdrawal tests. The increased anorectic and anxiogenic-like behavior most likely is caused by increased "free" CRH and/or urocortin levels in the brain of CRH-BP-deficient animals, suggesting an important role for CRH-BP in maintaining appropriate levels of these peptides in the central nervous system.

Effects of Blocking GABA Degradation on Corticotropin‐Releasing Hormone Gene Expression in Selected Brain Regions

The gamma-aminobutyric acid (GABA) degradation blocker gamma-vinyl-GABA (VGB) is used clinically to treat seizures in both adult and immature individuals. The mechanism by which VGB controls developmental seizures is not fully understood. Specifically, whether the anticonvulsant properties of VGB arise only from its elevation of brain GABA levels and the resulting activation of GABA receptors, or also from associated mechanisms, remains unresolved. Corticotropin-releasing hormone (CRH), a neuropeptide present in many brain regions involved in developmental seizures, is a known convulsant in the immature brain and has been implicated in some developmental seizures. In certain brain regions, it has been suggested that CRH synthesis and release may be regulated by GABA. Therefore we tested the hypothesis that VGB decreases CRH gene expression in the immature rat brain, consistent with the notion that VGB may decrease seizures also by reducing the levels of the convulsant molecule, CRH. VGB was administered to immature, 9-day-old rats in clinically relevant doses, whereas littermate controls received vehicle. In situ hybridization histochemistry demonstrated a downregulation of CRH mRNA levels in the hypothalamic paraventricular nucleus but not in other limbic regions of VGB-treated pups compared with controls. In addition, VGB-treated pups had increased CRH peptide levels in the anterior hypothalamus, as shown by radioimmunoassay. These findings are consistent with a reduction of both CRH gene expression and secretion in the hypothalamus, but do not support an indirect anticonvulsant mechanism of VGB via downregulation of CRH levels in limbic structures. However, the data support a region-specific regulation of CRH gene expression by GABA.