Stress and nicotine during adolescence disrupts adult hippocampal-dependent learning and alters stress reactivity.

Adolescence represents increased susceptibility to stress that increases risk for nicotine dependence. The present study examined the interactive effects of brief exposure to stress (shipping/transportation or experimentally induced) and chronic nicotine during adolescence on cognitive function and stress reactivity in adulthood. Adolescent (P31), but not young adult (P47), C57BL/6J mice had higher levels of corticosterone after shipping vs mice bred onsite. Shipped preadolescent (P23) and adolescent (P38) mice, but not those bred onsite, exposed to nicotine showed deficits in contextual fear learning when tested in adulthood. Adult learning deficits were replicated in adolescent mice bred onsite, exposed to experimentally induced stress, and administered chronic nicotine. Stress and nicotine during adolescence resulted in higher expression of hippocampal glucocorticoid receptors and corticotropin-releasing factor receptors and blunted restraint induced CORT release in adulthood. Importantly, studies examining adolescent behavior in mice should consider stress influences outcomes.

Chronic corticosterone increases ΔFOSB and CRFR1 immunoreactivity in brain regions that modulate aversive conditioning.

Previous studies showed that chronic treatment with corticosterone facilitates elevated T-maze (ETM) inhibitory avoidance and a step-down avoidance task, responses that have been used to investigate aversive conditioning and memory processes. On the other hand, chronic corticosterone does not alter ETM escape from the open arms. The purpose of the present study was to further investigate the effects of chronic corticosterone treatment (200 mg pellets, 21-day release) in an animal model of anxiety that does not involve aversive conditioning: the light/dark transition model. We also investigated the pattern of ΔFosB immunoreactivity (ΔFosB-ir) in different brain regions. To examine how treatment with chronic corticosterone interferes with CRFR1 expression we measured CRFR1 in the same brain structures that exhibited increased ΔFosB-ir. Results showed that chronic treatment with corticosterone did not alter behavioral measurements performed in the light/dark transition model. On the other hand, ΔFosB-ir was increased in several structures that modulate aversive conditioning: the cingulate cortex, the ventro and dorsolateral septum, the amygdala, the paraventricular, dorsomedial and ventromedial hypothalamus, the periaqueductal grey matter, the dorsal raphe, and the median raphe nucleus. Chronic treatment with corticosterone also increased CRFR1-immunoreactivity in the ventrolateral septum, central amygdala, dorsomedial hypothalamus, ventral region of the dorsal raphe and median raphe. These results contribute to a better understanding of the behavioral and neurobiological alterations induced by chronic exposure to glucocorticoids.

CRHR1 Mediates the Up-Regulation of Synapsin I Induced by Nesfatin-1 Through ERK 1/2 Signaling in SH-SY5Y Cells.

The anorexigenic molecule nesfatin-1 has recently been taken as a potential mood regulator, but the potential mechanisms remain unknown. Results of our previous study have demonstrated that nesfatin-1 could induce anxiety- and depression-like behaviors in rats, accompanied by the hyperactivity of the hypothalamic-pituitary-adrenal axis and the imbalanced mRNA expression of synaptic vesicle proteins. To explore the potential neurobiological mechanism underlying the effect of nesfatin-1 on the synaptic plasticity, the human neuroblastoma SH-SY5Y cells were cultured and treated with different concentrations of nesfatin-1 in the present study. The mRNA and protein expressions of corticotropin-releasing hormone (CRH) were measured via real-time fluorescent quantitative PCR and western blot, respectively. The protein expressions of extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated-ERK1/2 (p-ERK1/2), and synapsin I were detected via western blot. The results confirmed that nesfatin-1 (10-9~10-7 mol/L) could up-regulate the expression of CRH. Moreover, nesfatin-1 (10-9~10-7 mol/L) could also increase the protein expressions of p-ERK1/2 and synapsin I, and these effects could be blocked by CP376395, a selective antagonist of CRH type 1 receptor (CRHR1). Furthermore, the increased expression of synapsin I induced by nesfatin-1 could also be reversed by PD98059, a specific inhibitor of the p-ERK. These results indicated that CRHR1 might mediate the effect of nesfatin-1 on the expressions of synapsin I via ERK1/2 signaling pathway.

The Modulation of Cardiac Contractile Function by the Pharmacological and Toxicological Effects of Urocortin2.

Urocortin2 (Ucn2) has been revealed to enhance cardiac function in heart failure. However, the pharmacological and toxicological effects of Ucn2 on cardiomyocytes are incompletely understood. In this study, we investigated the possible mechanisms of Ucn2 on mediating the contractility of cardiomyocytes. Mechanical properties and intracellular Ca(2+) properties were measured in isolated cardiomyocytes from different treatment groups. The stress signaling was evaluated using Western blot. The results demonstrated that Ucn2 induced maximal velocity of shortening (+dL/dt), peak height, peak shortening (PS) amplitude, maximal velocity of relengthening (-dL/dt), accompanied by a significant rise in intracellular Ca(2+) level and a fall of the mean time constant of Ca(2+) transient decay (Tau) in WT cardiomyocytes. However, these effects were abolished by preincubation of type 2 CRF receptors (CRFR2) antagonist anti-sauvagine 30 (a-SVG-30). We also found that Ucn2 treatment activated the AMPK pathway in isolated cardiomyocytes via CRFR2. Furthermore, Ucn2 induced protein kinase A (PKA) and phospholamban (PLN) phosphorylation. Pretreatment of PKA inhibitor H89 reduced the inotropic and lusitropic effects of Ucn2 as well as decreased the intracellular Ca(2+) load and slowed down the Ca(2+) transient decay. We also showed that preincubation of Compound C, an inhibitor of AMPK, inhibited the phosphorylation of PKA and the intracellular Ca(2+) level in cardiomyocytes without affecting the contractile function and the Tau of cardiomyocytes. Taken together, it suggests that Ucn2 facilitate the contractility of cardiomyocytes via activating both AMPK and PKA.

Corticotropin-releasing factor receptor subtype 2 in human colonic mucosa: down-regulation in ulcerative colitis.

AIM: To assess corticotropin-releasing factor receptor 2 (CRF2) expression in the colon of healthy subjects and patients with ulcerative colitis (UC). METHODS: We examined CRF2 gene and protein expression in the distal/sigmoid colonic mucosal biopsies from healthy subjects and patients with UC (active or disease in remission), human immunodeficiency virus (HIV) and functional bowel disease (FBD) by reverse transcription-polymerase chain reaction and immunofluorescence. RESULTS: Gene expression of CRF2 was demonstrated in the normal human colonic biopsies, but not in the human colorectal adenocarcinoma cell line Caco2. Receptor protein localization showed immunoreactive CRF2 receptors in the lamina propria and in the epithelial cells of the distal/sigmoid biopsy samples. Interestingly, CRF2 immunoreactivity was no longer observed in epithelial cells of patients with mild-moderately active UC and disease in remission, while receptor protein expression did not change in the lamina propria. No differences in CRF2 expression profile were observed in distal/sigmoid intestinal biopsies from HIV infection and FBD patients, showing no signs of inflammation. CONCLUSION: The down-regulation of the CRF2 receptor in the distal/sigmoid biopsies of UC patients is indicative of change in CRF2 signalling associated with the process of inflammation.

Urocortin increased LPS-induced endothelial permeability by regulating the cadherin-catenin complex via corticotrophin-releasing hormone receptor 2.

Urocortin (Ucn1), a member of corticotrophin-releasing hormone (CRH) family, has been reported to be upregulated in inflammatory diseases and function as an autocrine or paracrine inflammatory mediator. Growing evidence shows that Ucn1 increases the endothelial permeability in inflammatory conditions; however, the detailed mechanisms are not clear. In the present study, we investigated the mechanisms of increased endothelial permeability by Ucn1 in human umbilical vein endothelial cells (HUVECs) exposed to lipopolysaccharide (LPS). Pretreatment of HUVECs with Ucn1 increased the endothelial cell permeability, which was augmented by LPS synergistically. Significant downregulation of VE-cadherin expression was also observed. Moreover, Ucn1 increased phosphorylation of protein kinase D (PKD) and heat shock protein 27 (HSP27) in a time- and CRHR(2) -dependent manner. Inhibition of PKD and HSP27 drastically attenuated Ucn1-induced downregulation of VE-cadherin expression. Further investigations demonstrated that Ucn1 phosphorylated ß-catenin at Ser552 to disrupt the cadherin-catenin complex and hence promote the disassociation of ß-catenin and VE-cadherin. Disassociation of ß-catenin and VE-cadherin resulted in decreased VE-cadherin expression while on the contrary ß-catenin was increased, which may due to the inactivation of GSK-3ß. Increased ß-catenin translocated into the nucleus and subsequently bound to TCF/LEF site, contributing to the elevated expression of vascular endothelial growth factor (VEGF). The above effects of Ucn1 were completely reversed by CRHR(2) receptor blocker, antisauvagine-30. Taken together, our data suggest that Ucn1 increase LPS-induced endothelial permeability by disrupting the VE-cadherin-ß-catenin complex via activation of CRHR(2) and PKD-HSP27 signaling pathway.

Contrasting effects of nitric oxide and corticotropin- releasing factor within the dorsal periaqueductal gray on defensive behavior and nociception in mice

The anxiogenic and antinociceptive effects produced by glutamate N-methyl-D-aspartate receptor activation within the dorsal periaqueductal gray (dPAG) matter have been related to nitric oxide (NO) production, since injection of NO synthase (NOS) inhibitors reverses these effects. dPAG corticotropin-releasing factor receptor (CRFr) activation also induces anxiety-like behavior and antinociception, which, in turn, are selectively blocked by local infusion of the CRF type 1 receptor (CRFr1) antagonist, NBI 27914 [5-chloro-4-(N-(cyclopropyl)methyl-N-propylamino)-2-methyl-6-(2,4,6-trichlorophenyl)aminopyridine]. Here, we determined whether i) the blockade of the dPAG by CRFr1 attenuates the anxiogenic/antinociceptive effects induced by local infusion of the NO donor, NOC-9 [6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine], and ii) the anxiogenic/antinociceptive effects induced by intra-dPAG CRF are prevented by local infusion of Nω-propyl-L-arginine (NPLA), a neuronal NOS inhibitor, in mice. Male Swiss mice (12 weeks old, 25-35 g, N = 8-14/group) were stereotaxically implanted with a 7-mm cannula aimed at the dPAG. Intra-dPAG NOC-9 (75 nmol) produced defensive-like behavior (jumping and running) and antinociception (assessed by the formalin test). Both effects were reversed by prior local infusion of NBI 27914 (2 nmol). Conversely, intra-dPAG NPLA (0.4 nmol) did not modify the anxiogenic/antinociceptive effects of CRF (150 pmol). These results suggest that CRFr1 plays an important role in the defensive behavior and antinociception produced by NO within the dPAG. In contrast, the anxiogenic and antinociceptive effects produced by intra-dPAG CRF are not related to NO synthesis in this limbic midbrain structure.

Members of CRF family and their receptors: from past to future.

Corticotropin releasing factor (CRF), originally isolated from the mammalian hypothalamus, is a 41 amino acid peptide that plays an important physiological role and is implicated in the pathophysiology of various diseases. In addition to CRF and its related peptides, a large number of small non-peptide CRF analogs have been recently synthesized, some currently in clinical trials having considerable therapeutic potential in the treatment of CRF-related illnesses. CRF and its related peptides exert their multiple actions by interacting with two types of plasma membrane G-protein coupled CRF receptors, the type 1 (CRF(1)) and type 2 (CRF(2)). These receptors, like all GPCRs consist of an amino-terminal extracellular region, a carboxyl-terminal intracellular tail and seven, membrane-spanning segments, connected by alternating intracellular and extracellular loops. This review describes the functional role of CRF receptors and their ligands emphasizing the structural elements that are important for their function and could potentially contribute in the development of future target-based approaches to design new CRF-related drugs which will enrich the pharmaceutical armoire against serious diseases.

Activation of corticotropin-releasing factor receptor 2 mediates the colonic motor coping response to acute stress in rodents.

BACKGROUND & AIMS: Corticotropin-releasing factor receptor-1 (CRF(1)) mediates the stress-induced colonic motor activity. Less is known about the role of CRF(2) in the colonic response to stress. METHODS: We studied colonic contractile activity in rats and CRF(2)-/-, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute partial-restraint stress (PRS), and/or intraperitoneal injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF(1) and CRF(2) using immunohistochemical and immunoblot analyses. We measured phosphorylation of extracellular signal-regulated kinase 1/2 by CRF ligands in primary cultures of LMMP neurons (PC-LMMPn) and cyclic adenosine monophosphate (cAMP) production in human embryonic kidney-293 cells transfected with CRF(1) and/or CRF(2). RESULTS: In rats, a selective agonist of CRF(2) (urocortin 2) reduced CRF-induced defecation (>50%), colonic contractile activity, and Fos expression in the colonic LMMP. A selective antagonist of CRF(2) (astressin(2)-B) increased these responses. Urocortin 2 reduced PRS-induced colonic contractile activity in wild-type and CRF-overexpressing mice, whereas disruption of CRF(2) increased PRS-induced colonic contractile activity and CRF-induced defecation. CRF(2) colocalized with CRF(1) and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of extracellular signal-regulated kinase in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF(1) (NBI35965) or astressin(2)-B, respectively. The half maximal effective concentration, EC(50), for the CRF-induced cAMP response was 8.6 nmol/L in human embryonic kidney-293 cells that express only CRF(1); this response was suppressed 10-fold in cells that express CRF(1) and CRF(2). CONCLUSIONS: In colon tissues of rodents, CRF(2) activation inhibits CRF(1) signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF(2) function impairs colonic coping responses to stress.

Effects of corticotropin-releasing factor receptor-1 antagonists on the brain stress system responses to morphine withdrawal.

The role of stress in drug addiction is well established. The negative affective states of withdrawal most probably involve recruitment of brain stress neurocircuitry [e.g., induction of hypothalamo-pituitary-adrenocortical (HPA) axis, noradrenergic activity, and corticotropin-releasing factor (CRF) activity]. The present study investigated t$he role of CRF receptor-1 subtype (CRF1R) on the response of brain stress system to morphine withdrawal. The effects of naloxone-precipitated morphine withdrawal on noradrenaline (NA) turnover in the paraventricular nucleus (PVN), HPA axis activity, signs of withdrawal, and c-Fos expression were measured in rats pretreated with vehicle, CP-154526 [N-butyl-N-ethyl-2,5-dimethyl-7-(2,4,6-trimethylphenyl)pyrrolo[3,2-e]pyrimidin-4-amine], or antalarmin (selective CRF1R antagonists). Tyrosine hydroxylase-positive neurons expressing CRF1R were seen at the level of the nucleus tractus solitarius-A(2) cell group in both control and morphine-withdrawn rats. CP-154526 and antalarmin attenuated the increases in body weight loss and irritability that were seen during naloxone-induced morphine withdrawal. Pretreatment with CRF1R antagonists resulted in no significant modification of the increased NA turnover at PVN, plasma corticosterone levels, or c-Fos expression that was seen during naloxone-induced morphine withdrawal. However, blockade of CRF1R significantly reduced morphine withdrawal-induced increases in plasma adrenocorticotropin levels. These results suggest that the CRF1R subtype may be involved in the behavioral and somatic signs and in adrenocorticotropin release (partially) during morphine withdrawal. However, CRF1R activation may not contribute to the functional interaction between NA and CRF systems in mediating morphine withdrawal-activation of brain stress neurocircuitry.

Satiety induced by central stresscopin is mediated by corticotrophin-releasing factor receptors and hypothalamic changes in chicks.

The central mechanism that mediates stresscopin (SCP)-induced satiety is poorly understood, and its effect on avian appetite is not documented. Thus, this study was conducted to elucidate some of the central and behavioral mechanisms that are associated with SCP-induced satiety using broiler- and layer-type chicks (Gallus gallus) as model organisms. In Experiment 1, broiler-type chicks responded with decreased food and water intake but had increased plasma corticosterone concentration after intracerebroventricular (ICV) SCP injection. However, the effect on water intake was secondary to food intake, since food-restricted SCP-treated broiler-type chicks did not reduce water intake in Experiment 2. In Experiment 3, layer-type chicks responded with decreased food intake at much lower doses than broiler-type chicks. In Experiment 4, astressin (a non-selective corticotrophin-releasing factor [CRF] receptor antagonist) prevented SCP-induced anorexia in broiler-type chicks. In Experiment 5, SCP-treated broiler-type chicks had an increased number of c-Fos immunoreactive cells in the ventromedial hypothalamus, parvicelluar and magnocellular divisions of the paraventricular nucleus and the periventricular nucleus. In Experiment 6, SCP-treated broiler-type chicks had decreased feeding pecks and increased jumping, distance moved and more escape attempts. Thus, we conclude that central SCP causes anorexigenic and other behavioral effects in chicks, and the hypothalamus and CRF receptors are involved.

Anorexigenic effects of central neuropeptide K are associated with hypothalamic changes in juvenile Gallus gallus.

The central mechanisms that mediate neuropeptide K (NPK) associated anorexia are poorly understood in any species, and information in this area of avian biology is totally lacking. Thus, the effects of intracerebroventricular NPK treatment were studied in Cobb-500 chicks (Gallus gallus). In Experiment 1, NPK caused decreased feed intake, but did not affect water intake or whole blood glucose concentration. In Experiment 2, NPK-treated chicks had increased c-Fos immunoreactivity in the parvicellular division of the paraventricular nucleus and arcuate nucleus. The lateral hypothalamus, ventromedial hypothalamus, dorsomedial hypothalamus, periventricular nucleus, magnocellular division of the paraventricular nucleus, and the superchiasmatic nucleus were not affected by NPK treatment. In Experiment 3, the number of feed pecks, exploratory pecks, jumps, escape attempts, and distance moved were decreased, while time spent standing was increased. None of the NPK-treated chicks sat or entered deep rest. In Experiment 4, blockage of corticotrophin releasing factor receptors did not affect NPK-induced anorexia. Thus, we conclude that NPK is a regulator of chick appetite and the effects may be mediated directly in the arcuate nucleus and parvicellular division of the paraventricular nucleus.

Corticotropin-releasing hormone stimulates SGK-1 kinase expression in cultured hippocampal neurons via CRH-R1.

Corticotropin-releasing hormone (CRH) has been shown to exhibit various functions in hippocampus. In the present study, we examined the effect of CRH on the expression of serum/glucocorticoid-inducible protein kinase-1 (SGK-1), a novel protein kinase, in primary cultured hippocampal neurons. A dose-dependent increase in mRNA and protein levels of SGK-1 as well as frequency of SGK-1-positive neurons occurred upon exposure to CRH (1 pmol/l to 10 nmol/l). These effects can be reversed by the specific CRH-R1 antagonist antalarmin but not by the CRH-R2 antagonist astressin 2B. Blocking adenylate cyclase (AC) activity with SQ22536 and PKA with H89 completely prevented CRH-induced mRNA and protein expression of SGK-1. Blockage of PLC or PKC did not block CRH-induced SGK-1 expression. Our results suggest that CRH act on CRH-R1 to stimulate SGK-1 mRNA and protein expression in cultured hippocampal neurons via a mechanism that is involved in AC/PKA signaling pathways.

Urocortin's inhibition of tumor growth and angiogenesis in hepatocellular carcinoma via corticotrophin-releasing factor receptor 2.

Urocortin (UCN) functions via corticotrophin-releasing factor receptors (CRFRs), CRFR1 & 2. CRFR2 is reported to be a tonic suppressor of vascularization, implying its role in tumor angiogenesis. Here, it was found that UCN inhibited the growth of hepatocellular carcinoma (HCC) and reduced tumor microvessel density in nude mice. Hepatoma cells didn't express CRFRs whereas vessels expressed CRFRs, mainly CRFR2. In vitro three-dimensional culture assay showed UCN inhibited angiogenesis, this effect was abolished by CRFR2 antagonist, anti-sauvagine-30, demonstrating involvement of CRFR2. Furthermore, UCN inhibited the proliferation and promoted the apoptosis of endothelial cells and down-regulated VEGF expression in vivo via CRFR2.

Feeding microstructure in diet-induced obesity susceptible versus resistant rats: central effects of urocortin 2.

With one billion people overweight worldwide, the need to identify risk factors and treatments for obesity is urgent. The present study determined whether rats genetically prone to diet-induced obesity (DIO) show preexisting differences in meal microstructure and are sensitive to central anorectic effects of corticotropin-releasing factor type 2 (CRF(2)) receptor stimulation. Male, selectively bred DIO rats and their diet resistant (DR) counterparts (n = 9/genotype) were weaned onto low-fat chow and compared as young adults for spontaneous or intracerebroventricular urocortin 2 administration-induced (0, 0.3, 1, 3 microg) differences in ingestion. DIO rats were hyperphagic selectively at the dark cycle onset, showing shorter latencies to initiate feeding, faster returns to eating following meal completion, and a lower satiety ratio than DR rats. At other times, DIO rats had briefer postmeal intervals, but ate smaller and briefer meals, resulting in normal intake. DIO rats also ate faster than DR rats. Urocortin 2 was less potent in DIO rats, ineffective at the 0.3 microg dose, but produced CRF(2) antagonist-reversible anorexia at higher doses. Though heavier, chow-maintained DIO rats were proportionately as or more lean than DR rats. Thus, DIO rats showed signs of a preexisting, heritable deficit in the maintenance of postmeal satiety and a reduced sensitivity to anorectic CRF(2) agonist stimulation. The meal patterns of DIO rats temporally resemble human 'snacking' behaviour, which predicts adult obesity. Because central CRF(2) stimulation retains full anorectic efficacy at higher doses in the DIO model, manipulating this neuropeptidergic system might yield new therapeutic approaches for diet-induced obesity.

Corticotropin releasing factor receptor 1 (CRF1) and CRF2 agonists exert an anti-inflammatory effect during the early phase of inflammation suppressing LPS-induced TNF-alpha release from macrophages via induction of COX-2 and PGE2.

Corticotropin-releasing factor (CRF), the principal regulator of the hypothalamus-pituitary-adrenal (HPA) axis, also modulates the inflammatory response directly, via its effect on mast cells and macrophages. On macrophages, it augments production of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines. CRF and its related peptides may also act as anti-inflammatory agents. Aim of the present work was to examine the role of macrophages on the anti-inflammatory effects of CRF-peptides and the mechanism involved. Thus, we examined if CRF receptor 1 (CRF1) and CRF2 agonists exert any anti-inflammatory effect on primary mouse macrophages. We have found that: (a) CRF, Urocortin (UCN)1 and UCN2 transiently suppressed the release of Tumor Necrosis Factor-alpha (TNF-alpha) in LPS-activated macrophages, an effect peaking at 4 h. This effect did not involve changes on TNF-alpha transcription. (b) CRF peptide-induced suppression of TNF-alpha release depended on induction of COX-2 and PGE2 synthesis. (c) Use of specific CRF1 and CRF2 antagonists suggested that this effect involved both CRF receptor types. (d) The effect of CRF-peptides on COX-2 was mediated via PI3K and p38MAPK. (e) Longer exposure of macrophages to CRF-peptides resulted in induction of TNF-alpha production via enhancement of its transcription. In conclusion, this is the first report suggesting that CRF1 and CRF2 agonists exert a biphasic effect on macrophages. During the early stages of the inflammatory response, they suppress TNF-alpha release via induction of COX-2/PGE2 while later on they induce TNF-alpha transcription. Hence, the reported anti-inflammatory effect of CRF-peptides appears to involve macrophages and is confined at the early stage of inflammation.

Evidence that corticotropin-releasing factor receptor type 1 couples to Gs- and Gi-proteins through different conformations of its J-domain.

BACKGROUND AND PURPOSE: According to the two-domain model for the corticotropin-releasing factor receptor type 1 (CRF(1)), peptide antagonists bind to the N-terminal domain (N-domain), non-peptide antagonists to the transmembrane region (J-domain), whereas peptide agonists attach to both the N- and J-domain of the receptor to express activity. The aim of this study was to search for possible differences in the antagonism of the Gs- and Gi-protein coupling of CRF(1) by a peptide (alpha-helical CRF(9-41)) and non-peptide antagonist (antalarmin), to determine whether the conformational requirements of the activated CRF(1) states for Gs and Gi coupling are similar or different. EXPERIMENTAL APPROACH: We studied the inhibitory effect of alpha-helical CRF(9-41) and antalarmin on the coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using the [(35)S]-GTPgammaS binding stimulation assay. KEY RESULTS: The non-peptide antagonized the receptor coupling to Gs competitively but that to Gi noncompetitively, and its antagonistic potency was different for urocortin- and sauvagine-evoked G-protein activation. In contrast, the peptide antagonist exhibited uniformly competitive antagonism. CONCLUSIONS AND IMPLICATIONS: The results allow us to extend the two-domain model of CRF(1) activation by assuming that CRF(1) agonists activate the receptor by binding to at least two ensembles of J-domain configurations which couple to Gs or Gi, that are in turn antagonized by a non-peptide antagonist competitively and allosterically, respectively. It is further concluded that the allosteric mechanism of non-peptide antagonism is not valid for the Gs-mediated physiological activities of CRF(1).

Injections of urocortin 1 into the basolateral amygdala induce anxiety-like behavior and c-Fos expression in brainstem serotonergic neurons.

The amygdala plays a key role in emotional processing and anxiety-related physiological and behavioral responses. Previous studies have shown that injections of the anxiety-related neuropeptide corticotropin-releasing factor or the related neuropeptide urocortin 1 into the region of the basolateral amygdaloid nucleus induce anxiety-like behavior in several behavioral paradigms. Brainstem serotonergic systems in the dorsal raphe nucleus and median raphe nucleus may be part of a distributed neural system that, together with the basolateral amygdala, regulates acute and chronic anxiety states. We therefore investigated the effect of an acute bilateral injection of urocortin 1 into the basolateral amygdala on behavior in the social interaction test and on c-Fos expression within serotonergic neurons in the dorsal raphe nucleus and median raphe nucleus. Male rats were implanted with bilateral cannulae directed at the region of the basolateral amygdala; 72 h after surgery, rats were injected with urocortin 1 (50 fmol/100 nl) or vehicle (100 nl of 1% bovine serum albumin in distilled water). Thirty minutes after injection, a subgroup of rats from each experimental group was exposed to the social interaction test; remaining animals were left in the home cage. Two hours after injection rats were perfused with paraformaldehyde and brains were removed and processed for immunohistochemistry. Acute injection of urocortin 1 had anxiogenic effects in the social interaction test, reducing total interaction time without affecting locomotor activity or exploratory behavior. These behavioral effects were associated with increases in c-Fos expression within brainstem serotonergic neurons. In home cage rats and rats exposed to the social interaction test, urocortin 1 treatment increased the number of c-Fos-immunoreactive serotonergic neurons within subdivisions of both the dorsal raphe nucleus and median raphe nucleus. These results are consistent with the hypothesis that the basolateral amygdala and serotonergic neurons within the midbrain raphe complex are part of an integrated neural system modulating anxiety state.

Corticotropin-releasing hormone (CRH) and urocortin act through type 1 CRH receptors to stimulate dehydroepiandrosterone sulfate production in human fetal adrenal cells.

CONTEXT: Near term, the human fetal adrenal increases the production of cortisol and dehydroepiandrosterone sulfate (DHEAS). DHEAS, which acts as substrate for placental estrogen production, induces key changes involved in parturition. OBJECTIVE: The objective of this study was to determine quantitatively the effect of CRH on mRNA levels of enzymes needed for DHEAS production (steroidogenic acute regulatory protein, CYP11A, CYP17, and SULT2A1), to determine the CRH receptor (CRH-R) subtype(s) responsible for CRH action, and to determine the effect of CRH on CRH-R mRNA expression in human adrenal fetal zone (FZ) cells. DESIGN: Human adrenal FZ cells were treated with CRH, ACTH, urocortin (Unc), and CRH antagonists, and RNA was analyzed by microarray and real-time RT-PCR. SETTING: This study was performed at an academic research laboratory. MAIN OUTCOME MEASURE: The main outcome measure was the expression of steroidogenic enzymes and CRH-R. RESULTS: Microarray analysis of human FZ cells treated for 24 h with CRH or ACTH showed increased mRNA expression levels of the genes needed for DHEAS production. Real-time RT-PCR analysis confirmed these data. Induction was lost in the presence of CRH-R1 antagonists, but not CRH-R2 antagonists. Stimulation was reproduced by Unc. The CRH-R1alpha mRNA splice variant was the only type 1 receptor isoform expressed in the fetal adrenal, and treatment with CRH up-regulates its mRNA levels. CONCLUSIONS: CRH, Unc, and ACTH stimulate all elements of the DHEAS synthetic pathway and activate CRH-R1 as well. The resulting increased DHEAS levels can be used for placental estrogen synthesis and contribute to the process leading to parturition in humans.

Discovery of corticotropin releasing factor 2 receptor selective sauvagine analogues for treatment of skeletal muscle atrophy.

The corticotropin release factor 2 receptor (CRF2R) has many biological activities including modulation of the stress response. Recently, we have demonstrated that CRF2R activation functions to prevent skeletal muscle wasting resulting from a variety of physiological stimuli. Thus we are interested in identifying CRF2R selective agonists with optimal pharmacological properties for use in treating muscle wasting diseases. Several CRF2R agonists are known including the frog peptide sauvagine (Svg), which display superior pharmacological properties compared to other CRF2R agonists. Unfortunately sauvagine is a nonselective CRFR agonist, thus making it of less utility due to side effects resulting from corticotropin release factor 1 receptor (CRF1R) activation. Because our initial modifications of Svg at position 11 improved CRF2R selectivity, we investigated the role of amino acids at positions 12 and 13 in Svg. We observed that phenylalanine, leucine, isoleucine, threonine, glutamine, histidine, and tyrosine at the 12th position were the strongest promoters of CRF2R selectivity whereas phenylalanine, glutamine, trytophane, tyrosine, valine, isoleucine, leucine, and 2-naphthylalanine were the preferred residues at the 13th position. Selective sauvagine peptides demonstrated improved antiatrophy effects in a mouse-casting model when compared to sauvagine itself. Thus, we demonstrate that the CRF2R selectivity can be improved by optimizing amino acids at positions 12 and 13 (all with proline at position 11) and that the selective sauvagine analogues demonstrate better in vivo efficacy than sauvagine itself.