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Psychosocial stress disrupts reproduction and interferes with pulsatile LH secretion. The posterodorsal medial amygdala (MePD) is an upstream modulator of the reproductive axis and stress. Corticotropin-releasing factor type 2 receptors (CRFR2s) are activated in the presence of psychosocial stress together with increased expression of the CRFR2 ligand Urocortin3 (Ucn3) in the MePD of rodents. We investigate whether Ucn3 signalling in the MePD is involved in mediating the suppressive effect of psychosocial stress on LH pulsatility. First, we administered Ucn3 into the MePD and monitored the effect on LH pulses in ovariectomized mice. Next, we delivered Astressin2B, a selective CRFR2 antagonist, intra-MePD in the presence of predator odor, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Subsequently, we virally infected Ucn3-cre-tdTomato mice with inhibitory designer receptor exclusively activated by designer drugs (DREADDs) targeting MePD Ucn3 neurons while exposing mice to TMT or restraint stress and examined the effect on LH pulsatility as well as corticosterone release. Administration of Ucn3 into the MePD dose-dependently inhibited LH pulses and administration of Astressin2B blocked the suppressive effect of TMT on LH pulsatility. Additionally, DREADDs inhibition of MePD Ucn3 neurons blocked TMT and restraint stress-induced inhibition of LH pulses and corticosterone release. These results demonstrate for the first time that Ucn3 neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator and corticosterone secretion. Ucn3 signalling in the MePD plays a role in modulating the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes, and this brain locus may represent a nodal center in the interaction between the reproductive and stress axes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society.)

There is little literature showing the effect of urocortin (UCN) on macrophage apoptosis. The underlying mechanism is also unclear. This work was to investigate the involvement of UCN in the regulation of LPS-induced macrophage apoptosis and hence in the prevention from the atherosclerotic lesion development through targeting PLA2. Flow cytometry analysis showed that cell apoptosis was increased by more than 50% after LPS treatment in human THP-1 macrophage. Lp-PLA2 and cPLA2 were found to mediate LPS-induced macrophage apoptosis and NF-κB differentially influenced the expression of Lp-PLA2 and cPLA2. However, the reverse regulation of the expression of Lp-PLA2 and cPLA2 by NF-κB suggested that NF-κB may not be a key target for regulating macrophage apoptosis. Interestingly, we found that the approximate three folds upregulation of cPLA2 was in line with the induction of S1P formation and cell apoptosis by LPS. Inversely, LPS obviously decreased UCN expression by about 50% and secretion by about 25%. Both the enzyme inhibitor and knockdown expression of cPLA2 could completely abolish LPS-induced cell apoptosis. In addition, suppression of S1P synthesis by Sphk1 inhibitor PF-543 reduced the expression of cPLA2 and cell apoptosis but at the same time restored the normal level of UCN in cell culture supernatant. Furthermore, addition of exogenous UCN also reversed LPS-induced expression of cPLA2 and apoptosis. Taken together, UCN may be the reverse regulator of LPS-S1P-cPLA2-apoptosis pathway, thereby contributing to the prevention from the formation of unstable plaques., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Intracerebral hemorrhage (ICH) causes an accumulation of blood in the brain parenchyma that disrupts the normal neurological function of the brain. Despite extensive clinical trials, no medical or surgical therapy has shown to be effective in managing ICH, resulting in a poor prognosis for the patients. Urocortin (UCN) is a 40-amino-acid endogenous neuropeptide that belongs to the corticotropin-releasing hormone (CRH) family. The effect of UCN is activated by binding to two G-protein coupled receptors, CRH-R1 and CRH-R2, which are expressed in brain neurons and glial cells in various brain regions. Current research has shown that UCN exerts neuroprotective effects in ICH models via anti-inflammatory effects, which generally reduced brain edema and reduced blood-brain barrier disruption. These effects gradually help in the improvement of the neurological outcome, and thus, UCN may be a potential therapeutic target in the treatment of ICH. This review summarizes the data published to date on the role of UCN in ICH and the possible protective mechanisms underlined.

Urocortin 2 (UCN2) is a peptide related to corticotropin-releasing factor, capable of activating CRF-R2. Among its multisystemic effects, it has actions in all 3 muscle subtypes. This study's aim was to determine its potential role in two of the intrinsic eye muscle kinetics. Strips of iris sphincter (rabbit) and ciliary (bovine) muscles were dissected and mounted in isometric force-transducer systems filled with aerated-solutions. Contraction was elicited using carbachol (10(-6) M for iris sphincter, 10(-5) M for ciliary muscle), prior adding to all testing substances. UCN2 induced relaxation in iris sphincter muscle, being the effect maximal at 10(-7) M concentrations (-12.2 % variation vs. control). This effect was abolished with incubation of indomethacin, antisauvagine-30, chelerytrine and SQ22536, but preserved with L-nitro-L-arginine. In carbachol pre-stimulated ciliary muscle, UCN2 (10(-5) M) enhanced contraction (maximal effect of 18.2 % increase vs. control). UCN2 is a new modulator of iris sphincter relaxation, dependent of CRF-R2 activation, synthesis of prostaglandins (COX pathway) and both adenylate cyclase and PKC signaling pathways, but independent of nitric oxide production. Regarding ciliary muscle, UCN2 enhances carbachol-induced contraction, in higher doses.

Alcohol use is pervasive in the United States. In the transition from nonhazardous drinking to hazardous drinking and alcohol use disorder, neuroadaptations occur within brain reward and brain stress systems. One brain signaling system that has received much attention in animal models of excessive alcohol drinking and alcohol dependence is corticotropin-releasing factor (CRF). The CRF system is composed of CRF, the urocortins, CRF-binding protein, and two receptors - CRF type 1 and CRF type 2. This review summarizes how acute, binge, and chronic alcohol dysregulates CRF signaling in hypothalamic and extra-hypothalamic brain regions and how this dysregulation may contribute to changes in alcohol reinforcement, excessive alcohol consumption, symptoms of negative affect during withdrawal, and alcohol relapse. In addition, it summarizes clinical work examining CRF type 1 receptor antagonists in humans and discusses why the brain CRF system is still relevant in alcohol research.

The aim of the present study was to determine the role of corticotropin-releasing factor (CRF), the urocortins (UCN 1, UCN 2 and UCN 3) and their receptors (CRF 1 and CRF 2 ) in the preference for social novelty of mice. Male CFLP mice were administered intracerebroventricularly (ICV) with CRF, UCN 1, UCN 2 or UCN 3 and/or antalarmin or astressin 2B, selective antagonists of CRF 1 receptor and CRF 2 receptor, respectively. The mice were investigated in a Crawley social interaction test arena consisting of three chambers: an unknown female was set in the first chamber and a known female, with which the male was familiarized previously for 24h, was set in the third chamber. First the tested male was habituated with the middle chamber for 5min and then allowed to explore the remaining chambers for 5min, during which the number of entries and the time of interaction were measured. CRF decreased significantly the number of entries and the time of interaction with the unknown female, but not the known female. UCN 1 decreased significantly the number of entries into the chamber of the unknown female, but not the known female, without changing the time of interaction. All decreasing effects were reversed by antalarmin, but not astressin 2B. UCN 2 and UCN 3 didn't influence significantly any of the parameters. The present study suggests that CRF and UCN 1 decrease the preference for social novelty by activating CRF 1 receptor, while UCN 2 and UCN 3, activating selectively CRF 2 receptor, do not participate to male-female interaction., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Dysregulation of the corticotropin-releasing factor (CRF)-urocortin (UCN) system has been implicated in stress-related psychopathologies such as depression and anxiety. It has been proposed that CRF-CRF receptor type 1 (CRFR1) signalling promotes the stress response and anxiety-like behaviour, whereas UCNs and CRFR2 activation mediate stress recovery and the restoration of homeostasis. Recent findings, however, provide clear evidence that this view is overly simplistic. Instead, a more complex picture has emerged that suggests that there are brain region- and cell type-specific effects of CRFR signalling that are influenced by the individual's prior experience and that shape molecular, cellular and ultimately behavioural responses to stressful challenges.

Colonic epithelium is the first line of defense against various pathological offenses in the gut. Previous studies have shown that the peptides of the corticotropin-releasing hormone (CRH) family modulate vascular endothelial growth factor (VEGF)-A production in other cells. Here we sought to investigate whether CRH and urocortin (Ucn) 3 regulate VEGF-A secretion in colonocytes through CRH receptors and to elucidate the underlying mechanism of action. CRH and Ucn 3 significantly increased the expression levels of VEGF-A mRNA and protein through CRH receptor 1 and 2, respectively, in human colonic epithelial cells and primary mouse intestinal epithelial cells. Underlying mechanisms involve activation of adenylyl cyclase with subsequent increase of intracellular cAMP level and increased DNA binding activity of transcription factor CREB on VEGF-A promoter region. Finally, genetic deficiency of CREB decreased intestinal inflammation and VEGF-A expression in a dextran sodium sulfate-induced colitis model. These results show that activation of CRH receptors by CRH ligands stimulates VEGF-A expression in intestinal epithelial cells through the cAMP/CREB pathway. Since VEGF-A boosts inflammatory responses through angiogenesis, these data suggest that CREB may be a key effector of CRH and Ucn 3-dependent inflammatory angiogenesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Our group has previously shown that corticotropin releasing factor (CRF) inhibits proliferation of human endocrine-related cancer cell lines via the activation of CRF type-1 receptors (CRF-R1). Tumors originating from the nervous system also express CRF receptors but their role on neoplastic cell proliferation was poorly investigated. Here we investigated the effect of CRF receptor stimulation on nervous system-derived cancer cells, using the SK-N-SH (N) human neuroblastoma cell line as an experimental model. We found that SK-N-SH (N) cells express functionally active CRF-R1, whose activation by CRF and the cognate peptide urocortin (UCN) is associated to reduced cell proliferation and motility, as well as neuronal-like differentiation. UCN did not interfere with cell viability and cell-cycle arrest. Those effects seem to be mediated by a mechanism involving the activation of cAMP/PKA/CREB pathway and the subsequent downstream increase in p27(Kip1) and underphosphorylated retinoblastoma protein levels, as well as reduced c-Myc mRNA accumulation., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Urocortin (UCN) is a newly identified vascular-active peptide that has been shown to reverse cardiovascular remodeling and improve left ventricular (LV) function. The effects and mechanism of urocortin 2 (UCN2) in vivo on the electrical remodeling of left ventricle and the hemodynamics of hypertensive objectives have not been investigated., Methods: UCN2 (1 μg/kg/d, 3.5 μg/kg/d or 7 μg/kg/d) was intravenously injected for 2 weeks and its effects on hemodynamics in spontaneously hypertensive rats (SHRs) observed. The whole-cell patch clamp technique was used to explore the effects of UCN2 on the electrical remodeling of left ventricular cardiomyocytes. The flow cytometry method was used to determine the content of fluorescence calcium in myocardium., Results: UCN2 improved the systolic and diastolic function of SHRs as demonstrated by decreased left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), increased +dp/dtmax and -dp/dtmax and decreased cAMP level. UCN2 inhibited the opening of L-type calcium channel and decreased the calcium channel current of cardiomyocytes. In addition, UCN2 also decreased the contents of fluorescence calcium in SHR myocardium. However, astressin2-B (AST-2B), the antagonist of corticotropin-releasing factor receptor 2 (CRFR2), could reverse the inhibitory effects of UCN2 on calcium channel., Conclusion: UCN2 can modulate electrical remodeling of the myocardium and hemodynamics in an experimental model of SHR via inhibition of L-type calcium channel and CRFR2 in cardiomyocytes., (Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Aim: Atherosclerosis is the complex lesion that consists of endothelial inflammation, macrophage foam cell formation, vascular smooth muscle cell (VSMC) migration and proliferation, and extracellular matrix production. Human urocortin 1 (Ucn1), a 40-amino acid peptide member of the corticotrophin-releasing factor/urotensin I family, has potent cardiovascular protective effects. This peptide induces potent and long-lasting hypotension and coronary vasodilation. However, the relationship of Ucn1 with atherosclerosis remains unclear. The present study was performed to clarify the effects of Ucn1 on atherosclerosis., Methods: We assessed the effects of Ucn1 on the inflammatory response and proliferation of human endothelial cells (ECs), human macrophage foam cell formation, migration and proliferation of human VSMCs, extracellular matrix expression in VSMCs, and the development of atherosclerosis in apolipoprotein E-deficient (Apoe-/-) mice., Results: Ucn1 significantly suppressed cell proliferation without inducing apoptosis, and lipopolysaccharide-induced up-regulation of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in human ECs. Ucn1 significantly reduced oxidized low-density lipoprotein-induced foam cell formation with a significant down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase 1 in human monocyte-derived macrophages. Ucn1 significantly suppressed the migration and proliferation of human VSMCs and increased the activities of matrix metalloproteinase-2 (MMP2) and MMP9 in human VSMCs. Intraperitoneal injection of Ucn1 into Apoe-/- mice for 4 weeks significantly retarded the development of aortic atherosclerotic lesions., Conclusions: This study provided the first evidence that Ucn1 prevents the development of atherosclerosis by suppressing EC inflammatory response and proliferation, macrophage foam cell formation, and VSMC migration and proliferation. Thus, Ucn1 could serve as a novel therapeutic target for atherosclerotic cardiovascular diseases.

Insulin resistance (IR) in skeletal muscle is an important component of both type 2 diabetes and the syndrome of sarcopaenic obesity, for which there are no effective therapies. Urocortins (UCNs) are not only well established as neuropeptides but also have their roles in metabolism in peripheral tissues. We have shown recently that global overexpression of UCN3 resulted in muscular hypertrophy and resistance to the adverse metabolic effects of a high-fat diet. Herein, we aimed to establish whether short-term local UCN3 expression could enhance glucose disposal and insulin signalling in skeletal muscle. UCN3 was found to be expressed in right tibialis cranialis and extensor digitorum longus muscles of rats by in vivo electrotransfer and the effects studied vs the contralateral muscles after 1 week. No increase in muscle mass was detected, but test muscles showed 19% larger muscle fibre diameter (P=0.030), associated with increased IGF1 and IGF1 receptor mRNA and increased SER256 phosphorylation of forkhead transcription factor. Glucose clearance into the test muscles after an intraperitoneal glucose load was increased by 23% (P=0.018) per unit mass, associated with increased GLUT1 (34% increase; P=0.026) and GLUT4 (48% increase; P=0.0009) proteins, and significantly increased phosphorylation of insulin receptor substrate-1, AKT, AKT substrate of 160 kDa, glycogen synthase kinase-3β, AMP-activated protein kinase and its substrate acetyl coA carboxylase. Thus, UCN3 expression enhances glucose disposal and signalling in muscle by an autocrine/paracrine mechanism that is separate from its pro-hypertrophic effects, implying that such a manipulation may have promised for the treatment of IR syndromes including sarcopaenic obesity., (© 2014 The authors.)

AAA (abdominal aortic aneurysm) is an important cause of sudden death in older adults, but there is no current effective drug therapy for this disease. The UCNs (urocortins1-3) and their receptors: CRFR (corticotrophin-releasing factor receptor)-1 and -2 have been implicated in various CVDs (cardiovascular diseases). We assessed the relative expression of UCN1-3 in AAA by qRT-PCR (quantitative reverse transcription-PCR) and ELISA, and examined in vitro how UCN2 affects human aortic VSMC (vascular smooth muscle cell) Akt phosphorylation, pro-inflammatory cytokine IL (interleukin)-6 secretion, proliferation, cell cycle and apoptosis. UCN2 and CRFR2 expression were significantly up-regulated in biopsies from the AAA body. AAA body biopsies released high amounts of UCN2 in vitro. Median plasma UCN2 concentrations were 2.20 ng/ml (interquartile range 1.14-4.55 ng/ml, n=67) in AAA patients and 1.11 ng/ml (interquartile range 0.76-2.55 ng/ml, n=67) in patients with non-aneurysmal PAD (peripheral artery disease) (P=0.001). Patients with UCN2 in the highest quartile had a 4.12-fold (95% confidence interval, 1.37-12.40) greater prevalence of AAA independent of other risk factors, P=0.012. In vitro, UCN2 significantly inhibited VSMC Akt phosphorylation and proliferation in a dose-dependent manner. UCN2 induced VSMC G1 cell-cycle arrest and increased IL-6 secretion over 24 h. The CRFR2 antagonist astressin-2B significantly abrogated the effects of UCN2 on VSMCs. In conclusion, UCN2 is significantly associated with AAA and inhibits VSMC proliferation by inducing a G1 cell cycle arrest suggesting a plausible regulatory role in AAA pathogenesis.

We currently face an alarming resurgence in infectious diseases characterized by antimicrobial resistance and therapeutic failure. This has generated the urgent need of developing new therapeutic approaches that include agents with nontraditional modes of action. A recent interest focused on approaches based on our natural immune defenses, especially on peptides that combine innate antimicrobial activity against diverse pathogens and immunoregulatory functions. In this study, to our knowledge, we describe for the first time the antimicrobial activity of the neuropeptide urocortin II (UCNII) against a panel of Gram-positive and Gram-negative bacteria and tropical parasites of the genus Leishmania. Importantly, this cytotoxicity was selective for pathogens, because UCNII did not affect mammalian cell viability. Structurally, UCNII has a cationic and amphipathic design that resembles antimicrobial peptides. Using mutants and UCNII fragments, we determined the structural requirements for the interaction between the peptide and the surface of pathogen. Following its binding to pathogen, UCNII caused cell death through different membrane-disrupting mechanisms that involve aggregation and membrane depolarization in bacteria and pore formation in Leishmania. Noteworthily, UCNII killed the infective form of Leishmania major even inside the infected macrophages. Consequently, UCNII prevented mortality caused by polymicrobial sepsis and ameliorated pathological signs of cutaneous leishmaniasis. Besides its presence in body physical and mucosal barriers, we found that innate immune cells produce UCNII in response to infections. Therefore, UCNII could be considered as an ancient highly-conserved host peptide involved in the natural antimicrobial defense and emerge as an attractive alternative to current treatments for microbial disorders with associated drug resistances.

The discovery of corticotropin-releasing factor (CRF) or CRH defining the upper regulatory arm of the hypothalamic-pituitary-adrenal (HPA) axis, along with the identification of the corresponding receptors (CRFRs 1 and 2), represents a milestone in our understanding of central mechanisms regulating body and local homeostasis. We focused on the CRF-led signaling systems in the skin and offer a model for regulation of peripheral homeostasis based on the interaction of CRF and the structurally related urocortins with corresponding receptors and the resulting direct or indirect phenotypic effects that include regulation of epidermal barrier function, skin immune, pigmentary, adnexal, and dermal functions necessary to maintain local and systemic homeostasis. The regulatory modes of action include the classical CRF-led cutaneous equivalent of the central HPA axis, the expression and function of CRF and related peptides, and the stimulation of pro-opiomelanocortin peptides or cytokines. The key regulatory role is assigned to the CRFR-1α receptor, with other isoforms having modulatory effects. CRF can be released from sensory nerves and immune cells in response to emotional and environmental stressors. The expression sequence of peptides includes urocortin/CRF→pro-opiomelanocortin→ACTH, MSH, and β-endorphin. Expression of these peptides and of CRFR-1α is environmentally regulated, and their dysfunction can lead to skin and systemic diseases. Environmentally stressed skin can activate both the central and local HPA axis through either sensory nerves or humoral factors to turn on homeostatic responses counteracting cutaneous and systemic environmental damage. CRF and CRFR-1 may constitute novel targets through the use of specific agonists or antagonists, especially for therapy of skin diseases that worsen with stress, such as atopic dermatitis and psoriasis.

The primary goal was to determine agonist-specific regulation of CRF2(a) receptor function. Exposure of human retinoblastoma Y79 cells to selective (UCN2, UCN3 or stresscopins) and non-selective (UCN1 or sauvagine) agonists prominently desensitized CRF2(a) receptors in a rapid, concentration-dependent manner. A considerably slower rate and smaller magnitude of desensitization developed in response to the weak agonist CRF. CRF1 receptor desensitization stimulated by CRF, cortagine or stressin1-A had no effect on CRF2(a) receptor cyclic AMP signaling. Conversely, desensitization of CRF2(a) receptors by UCN2 or UCN3 did not cross-desensitize Gs-coupled CRF1 receptor signaling. In transfected HEK293 cells, activation of CRF2(a) receptors by UCN2, UCN3 or CRF resulted in receptor phosphorylation and internalization proportional to agonist potency. Neither protein kinase A nor casein kinases mediated CRF2(a) receptor phosphorylation or desensitization. Exposure of HEK293 or U2OS cells to UCN2 or UCN3 (100nM) produced strong βarrestin2 translocation and colocalization with membrane CRF2(a) receptors while CRF (1μM) generated only weak βarrestin2 recruitment. βarrestin2 did not internalize with the receptor, however, indicating that transient CRF2(a) receptor-arrestin complexes dissociate at or near the cell membrane. Since deletion of the βarrestin2 gene upregulated Gs-coupled CRF2(a) receptor signaling in MEF cells, a βarrestin2 mechanism restrains Gs-coupled CRF2(a) receptor signaling activated by urocortins. We further conclude that the rate and extent of homologous CRF2(a) receptor desensitization are governed by agonist-specific mechanisms affecting GRK phosphorylation, βarrestin2 recruitment, and internalization thereby producing unique signal transduction profiles that differentially affect the stress response., (© 2013.)

Stresscopin-related peptide (SRP), which is a member of the corticotropin-releasing factor (CRF) family, is a high-affinity ligand for the type 2 corticotropin-releasing factor receptor (CRF-R2) and is involved in stress-coping responses. Central treatment with SRP suppresses food intake, delays gastric emptying and decreases heat-induced edema, but the effects of central administration of SRP on the cardiovascular system are unclear. Here we examined the effects of intracerebroventricular (i.c.v.) administration of SRP on cardiovascular function, and compared the cardiovascular effects of SRP and stresscopin (SCP). Our results showed that i.c.v. administration of SRP (0.5nmol) increased mean arterial blood pressure (MABP) and heart rate (HR), but failed to increase plasma norepinephrine and epinephrine levels. Compared with an equivalent dose of SCP, the area under the curve (AUC) values for the changes in MABP and HR were significantly smaller with SRP, indicating that the cardiovascular effects of SRP were weaker than those mediated by SCP. Pre-treatment with a selective CRF-R2 antagonist, antisauvagine-30 (4nmol, i.c.v.) abolished the SRP and SCP induced changes in MABP and HR. These results indicate that central administration of SRP induces a weaker enhancement of cardiovascular function through CRF-R2 than that induced by SCP and that these effects are mediated without increasing plasma norepinephrine and epinephrine levels., (© 2013.)

Urocortins (UCNs), peptides that belong to the corticotrophin-releasing hormone family, represent a novel group of inotropic agents that have a multifaceted effect on the body with significant effects on the cardiovascular, hemodynamic, neurohormonal, and renal systems. UCNs can potentially improve the overall picture of heart failure by targeting not only the cardiovascular and hemodynamic systems like many current inotropic agents but also other systemic tissues that contribute significantly to the mortality and morbidity of heart failure. The 3 types of UCNs (1, 2, and 3) have been shown in preclinical studies to be effective in improving cardiovascular, neurohormonal, and renal function. UCN 2 has been shown in clinical studies to induce significant cardiovascular benefit with limited systemic effects. UCNs, specifically UCNs 2 and 3, show great potential as additional treatment in the management of systolic heart failure.

Inhibition of the RAAS (renin-angiotensin-aldosterone system) plays a pivotal role in the prevention and treatment of diabetic nephropathy and a spectrum of other proteinuric kidney diseases. Despite documented beneficial effects of RAAS inhibitors in diabetic patients with nephropathy, reversal of the progressive course of this disorder or at least long-term stabilization of renal function are often difficult to achieve, and many patients still progress to end-stage renal disease. Incomplete inhibition of the RAAS has been postulated as one of reasons for unsatisfactory therapeutic responses to RAAS inhibition in some patients. Inhibition of renin, a rate-limiting step in the RAAS activation cascade, could overcome at least some of the abovementioned problems associated with the treatment with traditional RAAS inhibitors. The present review focuses on experimental and clinical studies evaluating the two principal approaches to renin inhibition, namely direct renin inhibition with aliskiren and inhibition of the (pro)renin receptor. Moreover, the possibilities of renin inhibition and nephroprotection by interventions primarily aiming at non-RAAS targets, such as vitamin D, urocortins or inhibition of the succinate receptor GPR91 and cyclo-oxygenase-2, are also discussed.

Urocortins (Ucns) injected peripherally decrease food intake and gastric emptying through peripheral CRF(2) receptors in rodents. However, whether Ucns influence circulating levels of the orexigenic and prokinetic hormone, ghrelin has been little investigated. We examined plasma levels of ghrelin and blood glucose after intravenous (iv) injection of Ucn 1, the CRF receptor subtype involved and underlying mechanisms in ad libitum fed rats equipped with a chronic iv cannula. Ucn 1 (10 μg/kg, iv) induced a rapid onset and long lasting increase in ghrelin levels reaching 68% and 219% at 0.5 and 3h post injection respectively and a 5-h hyperglycemic response. The selective CRF(2) agonist, Ucn 2 (3 μg/kg, iv) increased fasting acyl (3h: 49%) and des-acyl ghrelin levels (3h: 30%) compared to vehicle while the preferential CRF(1) agonist, CRF (3 μg/kg, iv) had no effect. Ucn 1's stimulatory actions were blocked by the selective CRF(2) antagonist, astressin(2)-B (100 μg/kg, iv). Hexamethonium (10 mg/kg, sc) prevented Ucn 1-induced rise in total ghrelin levels while not altering the hyperglycemic response. These data indicate that systemic injection of Ucns induces a CRF(2)-mediated increase in circulating ghrelin levels likely via indirect actions on gastric ghrelin cells that involves a nicotinic pathway independently from the hyperglycemic response., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Urocortin 3 (also known as stresscopin) is an endogenous ligand for the corticotropin-releasing factor receptor 2 (CRF(2)). Despite predominant G(s) coupling of CRF(2), promiscuous coupling with other G proteins has been also associated with the activation of this receptor. As urocortin 3 has been involved in central cardiovascular regulation at hypothalamic and medullary sites, we examined its cellular effects on cardiac vagal neurons of nucleus ambiguus, a key area for the autonomic control of heart rate. Urocortin 3 (1 nM-1000 nM) induced a concentration-dependent increase in cytosolic Ca(2+) concentration that was blocked by the CRF(2) antagonist K41498. In the case of two consecutive treatments with urocortin 3, the second urocortin 3-induced Ca(2+) response was reduced, indicating receptor desensitization. The effect of urocortin 3 was abolished by pre-treatment with pertussis toxin and by inhibition of phospolipase C with U-73122. Urocortin 3 activated Ca(2+) influx via voltage-gated P/Q-type channels as well as Ca(2+) release from endoplasmic reticulum. Urocortin 3 promoted Ca(2+) release via inositol 1,4,5 trisphosphate receptors, but not ryanodine receptors. Our results indicate a novel Ca(2+) -mobilizing effect of urocortin 3 in vagal pre-ganglionic neurons of nucleus ambiguus, providing a cellular mechanism for a previously reported role for this peptide in parasympathetic cardiac regulation., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

CRF receptor 1 (CRF(1)), a key neuroendocrine mediator of the stress response, has two known agonists corticotropin-releasing factor (CRF) and urocortin 1 (Ucn1). Here we report that endothelin-converting enzyme-1 (ECE-1) differentially degrades CRF and Ucn1; ECE-1 cleaves Ucn1, but not CRF, at critical residue Arginine-34/35', which is essential for ligand-receptor binding. At near K(D) agonist concentration (30 nm), both Ucn1- and CRF-mediated Ca(2+) mobilization are ECE-1 dependent. Interestingly, at high agonist concentration (100 nm), Ucn1-mediated Ca(2+) mobilization remains ECE-1 dependent, whereas CRF-mediated mobilization becomes independent of ECE-1 activity. At high agonist concentration, ECE-1 inhibition disrupted Ucn1-, but not CRF-induced CRF(1) recycling and resensitization, but did not prolong the association of CRF(1) with β-arrestins. RNA interference-mediated knockdown of Rab suggests that both Ucn1- and CRF-induced CRF(1) resensitization is dependent on activity of Rab11, but not of Rab4. CRF(1) behaves like a class A G protein-coupled receptor with respect to transient β-arrestins interaction. We propose that differential degradation by ECE-1 is a novel mechanism by which CRF(1) receptor is protected from overactivation by physiologically relevant high concentrations of higher affinity ligand to mediate distinct resensitization and downstream signaling.

Urotensin II (UII) and urocortin (UCN) are potent contributors to the physiopathology of heart failure. Our study investigated the effects of UII and UCN on the expression of myostatin (Mstn) in primary culture of adult cardiomyocytes. Adult rat cardiomyocytes were stimulated for 48 h with UII and UCN. Cell size and protein content were determined. Mstn gene expression was determined by real time quantitative polymerase chain reaction. Treatment with UII and UCN stimulates hypertrophy of adult cardiomyocytes. This effect was associated with a twofold increase of Mstn gene expression. We have established for the first time that the two hypertrophic peptides UII and UCN stimulate the expression of Mstn., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Real-time measurements for gut motility in conscious rats or mice combined with intracerebroventricular or intravenous injection of peptide agonists or antagonists allow us to understand the regulatory mechanism of gastrointestinal motility. Neuropeptide Y (NPY) in the arcuate nucleus in the hypothalamus stimulates the fasted motility in the duodenum, while urocortin in the paraventricular nucleus inhibits fed and fasted motility in the antrum and duodenum. Acyl ghrelin exerts stimulatory effects on the motility of the antrum and duodenum in both the fed and fasted state of animals. NPY Y2 and Y4 receptors in the brain may mediate the action of acyl ghrelin, and vagal afferent pathways might be involved in this mechanism. Des-acyl ghrelin exerts inhibitory effects on the motility of the antrum but not on the motility of the duodenum in the fasted state of animals. CRF type 2 receptor in the brain may mediate the action of des-acyl ghrelin, and vagal afferent pathways might not be involved in this mechanism. Obestatin exerts inhibitory effects on the motility of the antrum and duodenum in the fed state but not in the fasted state of animals. CRF type 1 and type 2 receptors in the brain may mediate the action of obestatin, and vagal afferent pathways might be partially involved in this mechanism., (Copyright © 2012 S. Karger AG, Basel.)

Objectives: The aim of the present study was to evaluate the effect of Ucn2 and Ucn3 on cytokine expression and secretion from placental explants., Study Design: Placentas were collected from healthy pregnancies at term elective caesarean delivery and trophoblast explants were prepared and treated with Ucn2 or Ucn3 in presence/absence of the selective CRH-R2 antagonist, astressin 2b. The mRNA expression and secretion of IL-10 and TNF-α were evaluated by Real Time RT-PCR and ELISA, respectively., Main Outcome Measures: To evaluate the possible role of Ucn2 and Ucn3 in inflammatory pathways., Results: Ucn2 increased the mRNA expression and secretion of IL-10 and TNF-α, and Ucn3 increased the mRNA expression and secretion of IL-10, but did not modify the secretion of TNF-α. Ucn3 treatment reversed the LPS-induce increase of TNF-α expression and release, an effect blocked by astressin 2b. Ucn2 potentiated the LPS-induced increase of TNF-α expression and release, an effect reversed by astressin 2b., Conclusions: The present study showed that Ucn2 and Ucn3 differentially regulate the LPS-induced TNF-α and IL-10 expression and secretion in trophoblast explants acting through CRH-R2. A pro inflammatory effect of Ucn2 and an anti-inflammatory effect of Ucn3 in placental immunomodulatory mechanisms is suggested., (Published by Elsevier Ltd.)

Background and Purpose: Infusion of corticotropin-releasing factor (CRF)/urocortin (Ucn) family peptides suppresses feeding in mice. We examined whether rats show peripheral CRF/Ucn-induced anorexia and determined its behavioural and pharmacological bases., Experimental Approach: Male Wistar rats (n= 5-12 per group) were administered (i.p.) CRF receptor agonists with different subtype affinities. Food intake, formation of conditioned taste aversion and corticosterone levels were assessed. In addition, Ucn 1- and Ucn 2-induced anorexia was studied in fasted CRF(2) knockout (n= 11) and wild-type (n= 13) mice., Key Results: Ucn 1, non-selective CRF receptor agonist, reduced food intake most potently (~0.32 nmol·kg(-1) ) and efficaciously (up to 70% reduction) in fasted and fed rats. The peptides' rank-order of anorexic potency was Ucn 1 ≥ Ucn 2 > >stressin(1) -A > Ucn 3, and efficacy, Ucn 1 > stressin(1) -A > Ucn 2 = Ucn 3. Ucn 1 reduced meal frequency and size, facilitated feeding bout termination and slowed eating rate. Stressin(1) -A (CRF(1) agonist) reduced meal size; Ucn 2 (CRF(2) agonist) reduced meal frequency. Stressin(1) -A and Ucn 1, but not Ucn 2, produced a conditioned taste aversion, reduced feeding efficiency and weight regain and elicited diarrhoea. Ucn 1, but not Ucn 2, also increased corticosterone levels. Ucn 1 and Ucn 2 reduced feeding in wild-type, but not CRF(2) knockout, mice., Conclusions and Implications: CRF(1) agonists, Ucn 1 and stressin(1) -A, reduced feeding and induced interoceptive stress, whereas Ucn 2 potently suppressed feeding via a CRF(2) -dependent mechanism without eliciting malaise. Consistent with their pharmacological differences, peripheral urocortins have diverse effects on appetite., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)

In this study, we tested the effect of urocortin 1 (Ucn1) on the contractility of gallbladder smooth muscle (GBSM) strips from guinea pigs and studied the involvement of corticotropin-releasing factor (CRF) receptors in this effect. The effect of Ucn1 on the isometric contractions of non-contracted and acetylcholine (Ach)-contracted GBSM, and the effects of CRF-R antagonists antalarmin and astressin 2B on the effect of Ucn1 were studied. In addition, the expression of receptors for CRF-R1 and CRF-R2 in guinea pig gallbladder were investigated using reverse transcription - polymerase chain reaction (RT-PCR). Ucn1 dose-dependently inhibited the contractility of GBSM. Moreover, Ucn1 decreased the resting tension, the mean contractile amplitude, and the contractile frequency in both non-contracted and Ach-contracted strips of GBSM. Furthermore, Ucn1 induced rightward shift of the Ach concentration-response curve of Ach in Ach-contracted strips. This inhibitory effect of Ucn1 on both non-contracted and Ach-contracted strips was inhibited by astressin 2B, but not by antalarmin. RT-PCR demonstrated that the CRF-R2, but not CRF-R1 receptor subtype is expressed in the muscularis muscle of guinea pig gallbladder. In conclusion, Ucn1 has an inhibitory effect on the contractility of GBSM of guinea pig, mediated through stimulating CRF-R2 receptors in GBSM. More studies are needed to clarify the intracellular signaling events involved in this effect.

Objectives: The challenge in management of acute decompensated heart failure (ADHF) is relieving congestion and improving symptoms while preserving renal and systemic perfusion at a level that can tolerate introduction of drugs such as beta-blockers which may have adverse effects acutely yet have proven benefit in the longer term. Urocortin 2 (Ucn2) is a novel peptide with therapeutic potential in heart failure. The present study investigated the effects of combined Ucn2 and beta-blockade in heart failure., Methods: Ucn2 and metoprolol were administered for 3 h, separately and together, in eight sheep with pacing-induced congestive heart failure., Results: Compared with time-matched controls, metoprolol significantly reduced heart rate (HR), left ventricular contractility, cardiac output (CO) and mean arterial pressure (MAP), together with increases in peripheral resistance and left atrial pressure (LAP). In contrast, Ucn2 elevated HR, contractility, CO and MAP, and decreased peripheral resistance and LAP. Combined Ucn2 + metoprolol produced intermediate haemodynamic effects closer to those observed with Ucn2 than with beta-blockade. All three active treatment regimes decreased plasma renin activity, whereas only Ucn2 and Ucn2 + metoprolol significantly reduced plasma aldosterone. Compared with metoprolol alone (which tended to reduce urine output and creatinine excretion/clearance), Ucn2 + metoprolol increased urine volume, sodium and creatinine excretion and clearance., Conclusion: Ucn2 coupled with beta-blockade in experimental heart failure improves CO and MAP, sustains HR, reduces peripheral resistance, LAP and aldosterone and augments renal function. These results suggest a role for Ucn2 as a short-term parenteral therapy in the initial stages of managing ADHF that improves tolerance to introduction of beta-blockers.

Urocortin (UCN) is a member of the corticotropin-releasing hormone (CRH) family of neuropeptides that regulates stress responses. Although UCN is principally expressed in dopaminergic neurons in rat substantia nigra (SN), the function of UCN in modulating dopaminergic neuronal survival remains unclear. Using primary mesencephalic cultures, we demonstrated that dopaminergic neurons underwent spontaneous cell death when their age increased in culture. Treatment of mesencephalic cultures with UCN markedly prolonged the survival of dopaminergic neurons, whereas neutralization of UCN with anti-UCN antibody accelerated dopaminergic neurons degeneration. UCN increased intracellular cAMP levels followed by phosphorylating glycogen synthase kinase-3β (GSK-3β) on Ser9. Moreover, UCN directly inhibited the histone deacetylase (HDAC) activity and induced a robust increase in histone H3 acetylation levels. Using pharmacological approaches, we further demonstrated that inhibition of GSK-3β and HDAC contributes to UCN-mediated neuroprotection. These results suggest that dopaminergic neuron-derived UCN might be involved in an autocrine protective signaling mechanism., (Copyright © 2009 Elsevier Inc. All rights reserved.)

This review summarizes some of the milestones of the research on the biological functions(s) of midbrain urocortin 1 (Ucn1) since its discovery 15 years ago. Detailed characterization of Ucn1 in the midbrain revealed its overall significance in food intake and regulation of homeostatic equilibrium and mood under stress. In addition, we have recently found a conspicuous alteration in midbrain Ucn1 levels in brains of depressed suicide victims. Furthermore, from the results from the genetically modified animals, a picture is emerging where corticotrophin-releasing factor promotes the initial reactions to stress, whereas Ucn1 seems to be crucial for management of the later adaptive phase. In the case of imbalance in action of these principle stress mediators, vulnerability to stress-related brain diseases is enhanced.

Urocortins (UCNs) and their receptors are potent immunoregulators in the gastrointestinal (GI) tract, where they can exert both pro- and anti-inflammatory effects. We examined the contribution of Ucn1 and its receptors to the pathogenesis, progression, and resolution of colitis. Trinitrobenzene sulfonic acid was used to induce colitis in rats. Ucn1 mRNA and immunoreactivity (IR) were ubiquitously expressed throughout the GI tract under basal conditions. During colitis, Ucn1 mRNA levels fell below basal levels on day 1 then increased again by day 6, in association with an increase in the number of Ucn1-IR inflammatory cells. Ucn1-IR cells were also numerous in proliferating granulation tissue. In contrast to Ucn1 expression, average phosphorylated ERK1/2 (pERK1/2) expression rose above controls levels on day 1 and was very low on day 6 of colitis. Knockdown of corticotropin-releasing factor 2 (CRF(2)) but not CRF(1) by RNA interference during colitis significantly decreased the macroscopic lateral spread of ulceration compared with uninjected controls or animals with CRF(1) knockdown. After knockdown of CRF(2), but not of CRF(1) during colitis, edema resolution assessed microscopically was slowed, and myeloperoxidase activity remained elevated even at day 6. Ucn1 and TNF-α mRNA peaked earlier, whereas pERK1/2 activation was attenuated after CRF(2) knockdown. Thus we conclude that local CRF(2) and pERK1/2 activation is pivotal for macroscopic spread of colitis and resolution of edema. Elimination of CRF(2), but not CRF(1), results in uncoordinated immune and pERK1/2 signaling responses.

The corticotropin releasing hormone-related ligand, urocortin-I (UcnI), suppresses food intake when injected into multiple hypothalamic and extrahypothalamic areas. UcnI also alters energy substrate utilization, specifically via enhanced fat oxidation as reflected in reductions in respiratory quotient (RQ). In the present study we compared the feeding and metabolic effects of ghrelin and NPY following pretreatment with UcnI. Direct PVN injections of NPY (50 pmol) and ghrelin (50 pmol) were orexigenic while UcnI (10-40 pmol) reliably suppressed food intake. Both ghrelin and NPY increased RQ, indicating enhanced utilization of carbohydrates and the preservation of fat stores. UcnI alone suppressed RQ responses. PVN UcnI attenuated the effects of both ghrelin and NPY on food intake and energy substrate utilization. While ghrelin (5 pmol) potentiated the effect of NPY (25 pmol) on RQ and food intake, these responses were inhibited by pretreatment with UcnI (10 pmol). In conclusion, PVN NPY and ghrelin stimulate eating and promote carbohydrate oxidation while inhibiting fat utilization. These effects are blocked by UcnI which alone suppresses appetite and promotes fat oxidation. Overall these findings are consistent with a possible interactive role of PVN NPY, ghrelin and urocortin in the modulation of appetite and energy metabolism., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Context: Women with endometriosis have altered endometrial function. CRH and urocortin (Ucn) are neuropeptides produced by human endometrium and modulate endometrial decidualization., Objective: To evaluate endometrial mRNA expression of CRH and Ucn, their role in in vitro decidualization of cultured human endometrial stromal cells (HESCs) in patients with endometriosis, and the role of CRH receptors (CHR-Rs)., Design: Obstetrics and Gynecology, University of Siena., Patients: Endometrial specimens were obtained from patients with and without endometriosis., Interventions: Endometrial biopsy obtained at both phases of menstrual cycle. In vitro decidualization of HESCs collected from endometriosis or control was done in the presence of CRH, Ucn, or CRH receptor type 1 (CRH-R1, antalarmin) or type 2 (CRH-R2, astressin 2b) antagonists., Outcome Measures: Endometrial mRNA expression of CRH and Ucn during endometrial cycle; prolactin, CRH-R1, and CRH-R2 mRNA expression during in vitro decidualization., Results: In healthy women CRH and Ucn expression were significantly higher (P < 0.05) in secretory than in proliferative phase; no differences were observed in endometriotic women. During in vitro decidualization, prolactin mRNA expression and release in endometriosis was lower than in control (P < 0.001). CRH and Ucn were able to significantly increase (P < 0.01) prolactin release only in control group; moreover, in this group antalarmin reduced prolactin release (P < 0.01). CRH-R1 mRNA expression increased during in vitro decidualization of HESCs in control (P < 0.01) but not in endometriosis., Conclusions: Women with endometriosis show an impaired endometrial expression of CRH and Ucn mRNA, and these neuropeptides are no more active in modulating the in vitro decidualization of HESCs, associated with a reduced expression of CRH-R1 mRNA.

Converging lines of evidence point to the involvement of neurons of the centrally projecting Edinger-Westphal nucleus (EWcp) containing the neuropeptide Urocortin-1 (Ucn1) in excessive ethanol (EtOH) intake and EtOH sensitivity. Here, we expanded these previous findings by using a continuous-access, two-bottle choice drinking paradigm (3%, 6%, and 10% EtOH vs. tap water) to compare EtOH intake and EtOH preference in Ucn1 genetic knockout (KO) and wild-type (WT) mice. Based on previous studies demonstrating that electrolytic lesion of the EWcp attenuated EtOH intake and preference in high-drinking C57BL/6J mice, we also set out to determine whether EWcp lesion would differentially alter EtOH consumption in Ucn1 KO and WT mice. Finally, we implemented well-established place conditioning procedures in KO and WT mice to determine whether Ucn1 and the corticotropin-releasing factor type-2 receptor (CRF-R2) were involved in the rewarding and aversive effects of EtOH (2 g/kg, i.p.). Results from these studies revealed that (1) genetic deletion of Ucn1 dampened EtOH preference only in mice with an intact EWcp, but not in mice that received lesion of the EWcp, (2) lesion of the EWcp dampened EtOH intake in Ucn1 KO and WT mice, but dampened EtOH preference only in WT mice expressing Ucn1, and (3) genetic deletion of Ucn1 or CRF-R2 abolished the conditioned rewarding effects of EtOH, but deletion of Ucn1 had no effect on the conditioned aversive effects of EtOH. The current findings provide strong support for the hypothesis that EWcp-Ucn1 neurons play an important role in EtOH intake, preference, and reward.

We have previously shown that urocortin 2 (Ucn 2), a member of the corticotropin-releasing factor (CRF) peptide family that binds to CRF type 2 receptor, is expressed in proopiomelanocortin (POMC) cells of rat pituitary and that its secretion and expression are increased by CRF in both the anterior and intermediate lobes and suppressed by glucocorticoids in the anterior lobe. We have also shown that Ucn 2 secreted by POMC cells acts on gonadotrophs expressing CRF type 2 receptors and inhibits the expression and secretion of gonadotropins. In the present study, we examined whether pituitary Ucn 2 is involved in stress-induced inhibition of gonadotropin secretion. A 90-min period of immobilization stress increased POMC mRNA expression without influencing Ucn 2 mRNA expression and suppressed luteinizing hormone (LH) β-subunit mRNA expression in the anterior lobe and plasma LH levels, while it increased both POMC and Ucn 2 mRNA expression in the intermediate lobe of the pituitary. Pretreatment with anti-CRF IgG blocked immobilization-induced increases in plasma ACTH and corticosterone and in POMC mRNA expression in both pituitary lobes and Ucn 2 mRNA expression in the intermediate pituitary. It also blocked immobilization-induced suppression of plasma LH and LH β-subunit mRNA expression. Pretreatment with anti-Ucn 2 IgG blocked immobilization-induced suppression of plasma LH and LH β-subunit expression without affecting immobilization-induced ACTH and corticosterone release and POMC or Ucn 2 mRNA expression. These results suggest that CRF suppresses the secretion and expression of LH probably through pituitary Ucn 2 in stress.

Background: The current therapies for alcohol abuse disorders are not effective in all patients, and continued development of pharmacotherapies is needed. One approach that has generated recent interest is the antagonism of ghrelin receptors. Ghrelin is a gut-derived peptide important in energy homeostasis and regulation of hunger. Recent studies have implicated ghrelin in alcoholism, showing altered plasma ghrelin levels in alcoholic patients as well as reduced intakes of alcohol in ghrelin receptor knockout mice and in mice treated with ghrelin receptor antagonists. The aim of this study was to determine the neuroanatomical locus/loci of the effect of ghrelin receptor antagonism on alcohol consumption using the ghrelin receptor antagonist, D-Lys3-GHRP-6., Methods: In Experiment 1, male C57BL/6J mice were injected with saline 3 hours into the dark cycle and allowed access to 15% (v/v) ethanol or water for 2 hours in a 2-bottle choice experiment. On test day, the mice were injected with either saline or 400 nmol of the ghrelin receptor antagonist, D-Lys3-GHRP-6, and allowed to drink 15% ethanol or water for 4 hours. The preference for alcohol and alcohol intake were determined. In Experiment 2, the same procedure was followed as in Experiment 1 but mice were only allowed access to a single bottle of 20% ethanol (v/v), and alcohol intake was determined. Blood ethanol levels were analyzed, and immunohistochemistry for c-Fos was carried out to investigate changes in neural activity. To further elucidate the mechanism by which D-Lys3-GHRP-6 affects alcohol intake, in Experiment 3, the effect of D-Lys3-GHRP-6 on the neural activation induced by intraperitoneal ethanol was investigated. For the c-Fos studies, brain regions containing ghrelin receptors were analyzed, i.e. the perioculomotor urocortin population of neurons (pIIIu), the ventral tegmental area (VTA), and the arcuate nucleus (Arc). In Experiment 4, to test if blood ethanol concentrations were affected by D-Lys3-GHRP-6, blood samples were taken at 2 time-points after D-Lys3-GHRP-6 pretreatment and systemic ethanol administration., Results: In Experiment 1, D-Lys3-GHRP-6 reduced preference to alcohol and in a follow-up experiment (Experiment 2) also dramatically reduced alcohol intake when compared to saline-treated mice. The resulting blood ethanol concentrations were lower in mice treated with the ghrelin receptor antagonist. Immunohistochemistry for c-Fos showed fewer immunopositive cells in the pIIIu of the antagonist-treated mice but no difference was seen in the VTA or Arc. In Experiment 3, D-Lys3-GHRP-6 reduced the induction of c-Fos by intraperitoneal ethanol in the pIIIu but had no effect in the VTA. In the Arc, there was a significant increase in the number of c-Fos immunopositive cells after D-Lys3-GHRP-6 administration, but the antagonist had no effect on ethanol-induced expression of c-Fos. D-Lys3-GHRP-6-pretreatment also did not affect the blood ethanol concentrations observed after a systemic injection of ethanol when compared to saline-pretreated mice (Experiment 4)., Conclusions: These findings indicate that the action of ghrelin on the regulation of alcohol consumption may occur via the pIIIu.

Urocortin is a 40 amino acid peptide of the corticotrophin-releasing factor (CRF) family that is synthesized and released by cardiac myocytes. Endogenous urocortin expression is increased during ischemia/reperfusion (I/R) and addition of exogenous urocortin reduces cell death caused by I/R injury. Studies have also showed that the protective action of urocortin is mediated by the activation of ERK1/2. We discovered that a non-receptor tyrosine kinase, Src, is involved in the urocortin-induced activation of ERK1/2 in mouse atrial HL-1 myocytes. The selective Src family kinase inhibitor, PP2, reduced the urocortin-induced phosphorylation of ERK1/2, and so did the expression of a dominant-negative mutant of Src in transfected HL-1 cells. Inhibition of Src by PP2 also reduced urocortin's protective effects in HL-1 cells after hypoxia/reoxygenation (H/R), as assessed by flow cytometry and caspase-3 activation assay. Titration studies indicated that as little as 10(-8)M urocortin was sufficient to induce Src activation. Maximal phosphorylation/activation of Src and ERK1/2 were both detected after 5 min incubation with urocortin. These effects of urocortin were largely mediated by CRF receptor-1, although a minor contribution of CRF receptor-2 cannot be excluded. Here we report for the first time that short-term treatment with urocortin causes rapid phosphorylation of Src, and that the urocortin-activated Src kinase serves as an upstream modulator of ERK1/2 activation, playing an essential role in urocortin-mediated cardioprotection., (Copyright (c) 2010 Elsevier Ireland Ltd. All rights reserved.)

Despite modern advances in the treatment of the causes and consequences of cardiovascular illness, heart disease and heart failure remain a leading cause of death in the western world. Many novel peptides are emerging as biomarkers and potential therapeutic tools for this debilitating condition. Urocortins represent one such group of peptides whose role in normal cardiovascular physiology and disease states is now increasingly being recognized. The cardiovascular effects of the urocortins are mediated via corticotrophin-releasing hormone (CRH) receptors through a variety of intra-cellular signaling pathways. Studies to date have demonstrated a favourable effect of urocortins on hemodynamic and neurohumoral regulation. They cause relaxation of the vasculature as well as having positive inotropic, chronotropic and lusitropic effects on the heart. This makes the urocortins a potentially attractive target in the treatment of heart failure. Indeed, a number of studies have demonstrated increased urocortin activity in experimental and clinical heart failure, with apparent augmented responses in these states. This article provides a review of the role of urocortins in normal cardiovascular physiology and in the pathophysiology of heart failure., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Three urocortins (Ucns) are known as members of the corticotropin-releasing factor (CRF) family of peptides and serve as natural ligands for CRF receptors. Ucn1 and Ucn3 exhibit potent effects on the adrenal system via the CRF receptors. This study aimed to explore the regulation and roles of Ucns in the adrenal system using human adrenal carcinoma H295R cells, which express Ucn1, Ucn2, Ucn3, CRF receptor type 1 (CRF(1) receptor), and CRF receptor type 2a (CRF(2a) receptor) mRNA. Forskolin, which stimulates adenylate cyclase and then increases intracellular cAMP production, was shown to transiently decrease Ucn1 and Ucn2 mRNA levels, but increase Ucns 1-3 mRNA levels in H295R cells. Steroidogenic acute regulatory protein, Cyp11beta1, and Cyp11beta2 mRNA levels, and both cortisol and aldosterone secretions were elevated by Ucn1. Cell viability was reduced by both Ucn1 and Ucn3 via the CRF(2) receptor in H295R cells. Ucn1 and Ucn3 increased the expression of the cAMP-response element binding protein and extracellular signal-related kinase (ERK) phosphorylations. The ERK and protein kinase A pathways were involved in Ucn3-decreased cell viability., (Copyright 2010 Elsevier B.V. All rights reserved.)

Despite considerable therapeutic advances over recent years, chronic heart failure remains associated with significant morbidity and mortality. Further improvements in the treatment of this syndrome are therefore needed and this will require advances in the understanding of its underlying pathophysiology. This article reviews the literature regarding recently identified neurohormonal pathways that are declaring themselves as potential therapeutic targets in chronic heart failure.

Our previous acute study on urocortin (Ucn) demonstrated that Ucn altered serum and tissue angiotensin-converting enzyme (ACE) activity in rats. Therefore, the present investigation was designed to explore the effect of long-term treatment with Ucn on somatic ACE (sACE) and other components of the renin-angiotensin system (RAS). After 8 weeks of intravenous administration of Ucn in spontaneously hypertensive rats (SHR), serum and tissue sACE, angiotensin II (Ang II), nitric oxide (NO), Ang-(1-7), and tissue chymase activities were evaluated. RT-PCR analysis was performed to determine the quantity of tissue sACE mRNA. Serum sACE activity was reduced by Ucn, although tissue sACE activity and tissue sACE mRNA were elevated. Chymase activity was observed to be enhanced by Ucn, whereas the ACE inhibitor enalapril failed to influence chymase. Serum and tissue Ang II activity was reduced, but NO and Ang-(1-7) production was increased in a concentration-dependent manner after Ucn treatment. Meanwhile, a significant decrease of the systolic blood pressure (SBP) was observed after the long-term Ucn administration, and there was a significant positive correlation (r2 = 0.6993) between serum ACE activity and SBP. Pretreatment with the corticotropin-releasing factor (CRF) blocker astressin and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway blocker PD98059 abolished these effects of Ucn. Our findings further support the hypothesis that the changes of sACE activity and the production of other RAS components may play roles in the vasodilatory property of Ucn via the activation of the ERK1/2 pathway.

Emerging evidence suggests that the urocortin (UCN) peptides contribute to pressure and volume regulation with possible involvement in the pathophysiology of cardiovascular disease. We have recently reported that i.v. UCN1 potently inhibits cardiac sympathetic nerve activity (CSNA) in normal sheep. However, little is known about possible interactions between UCN2 and the sympathetic nervous system. Accordingly, we have examined the effects of i.v. UCN2 on CSNA, hemodynamics, and plasma catecholamines in normal conscious sheep. Bolus i.v. administration of UCN2 (25 and 100 mug) resulted in the expected hemodynamic actions including transient falls in arterial pressure (P=0.016) and more sustained rises in heart rate (P<0.001) and cardiac output (P<0.001) and falls in peripheral resistance (P<0.001). CSNA burst frequency showed a biphasic response (P<0.001) with an acute rise followed by a more prolonged fall. CSNA burst area and incidence showed prolonged, dose-dependent falls in response to UCN2 administration (all P<0.001). UCN2 also induced a short-lived rise in plasma norepinephrine levels (P=0.006). The marked rise in heart rate in response to UCN2 is preserved in sheep undergoing pharmacological ganglionic blockade with hexamethonium. In conclusion, this is the first study to report the effects of UCN2 on SNA and indicates potent inhibition of sympathetic traffic to the heart despite a generalized baroreceptor-induced activation of sympathetic activity. These findings suggest an important role for UCN2 in cardiovascular homeostasis and warrant further investigation for potential therapeutic applications in acute myocardial injury and heart disease.

Urocortin 3 (Ucn 3) is a corticotropin-releasing factor (CRF)-related peptide with high affinity for the type 2 CRF receptor (CRFR2). Central administration of Ucn 3 stimulates the hypothalamic-pituitary-adrenal axis, suppresses feeding, and elevates blood glucose levels, suggesting that activation of brain CRFR2 promotes stress-like responses. Several CRFR2-expressing brain areas, including the ventromedial hypothalamus (VMH) and the posterior amygdala (PA), may be potential sites mediating the effects of Ucn 3. In the present study, Ucn 3 or vehicle was bilaterally injected into the VMH or PA, and food intake and plasma levels of ACTH, corticosterone, glucose, and insulin were determined. Food intake was greatly reduced in rats following Ucn 3 injection into the VMH. Ucn 3 injection into the VMH rapidly elevated plasma levels of glucose and insulin but did not affect ACTH and corticosterone secretion. Injection of Ucn 3 into the PA did not alter any of the parameters measured. We determined that the majority of CRFR2-positive neurons in the VMH were excitatory glutamatergic, and a subset of these neurons project to the arcuate nucleus of the hypothalamus (ARH). Importantly, stimulation of CRFR2 in the VMH increased proopiomelanocortin mRNA expression in the ARH. In conclusion, the present study demonstrates that CRFR2 in the VMH mediates some of the central effects of Ucn 3, and the ARH melanocortin system may be a downstream target of VMH CRFR2 neurons.

The small peptide urocortin (Ucn) has the ability to protect the heart by reducing cardiac cell loss during myocardial ischemia/reperfusion, and improving post-ischemic cardiac performance. Although its mechanism of action is not clearly defined, investigations have revealed that Ucn acts through several kinase pathways, and modulates a group of genes which synergistically minimize mitochondrial damage. Besides cardioprotection, most recent findings suggest a role for Ucn as a cardiac biomarker. Serum Ucn levels may be clinically useful to diagnose cases of mild sub-lethal ischemia, lacking elevation of cardiac enzymes and electrocardiogram changes. Infusion of Ucn may also help reduce the extent of the iatrogenic ischemic/reperfusion injury, associated with cardioplegic arrest.

This article summarizes the proceedings of a symposium held at the conference on "Alcoholism and Stress: A Framework for Future Treatment Strategies" in Volterra, Italy, May 6-9, 2008. Chaired by Markus Heilig and Roberto Ciccocioppo, this symposium offered a forum for the presentation of recent data linking neuropetidergic neurotransmission to the regulation of different alcohol-related behaviors in animals and in humans. Dr. Donald Gehlert described the development of a new corticotrophin-releasing factor receptor 1 antagonist and showed its efficacy in reducing alcohol consumption and stress-induced relapse in different animal models of alcohol abuse. Dr. Andrey Ryabinin reviewed recent findings in his laboratory, indicating a role of the urocortin 1 receptor system in the regulation of alcohol intake. Dr. Annika Thorsell showed data supporting the significance of the neuropeptide Y receptor system in the modulation of behaviors associated with a history of ethanol intoxication. Dr. Roberto Ciccocioppo focused his presentation on the nociceptin/orphanin FQ (N/OFQ) receptors as treatment targets for alcoholism. Finally, Dr. Markus Heilig showed recent preclinical and clinical evidence suggesting that neurokinin 1 antagonism may represent a promising new treatment for alcoholism. Collectively, these investigators highlighted the significance of neuropeptidergic neurotransmission in the regulation of neurobiological mechanisms of alcohol addiction. Data also revealed the importance of these systems as treatment targets for the development of new medication for alcoholism.

Background and Purpose: Our previous study showed that urocortin (Ucn1) exacerbates the hypercoagulable state and vasculitis in a rat model of sodium laurate-induced thromboangiitis obliterans. Furthermore, the inflammatory molecules COX-2 and ICAM-1 may participate in this effect. In the present study, the effects of Ucn1 on COX-2 and ICAM-1 expression in lipopolysaccharide (LPS)-induced rat aortic endothelial cells (RAECs) were investigated and the mechanisms involved explored., Experimental Approach: RAECs were isolated from adult male Wistar rats, and identified at the first passage. Experiments were performed on cells, from primary culture, at passages 5-8. The expression of COX-2 and ICAM-1 at both mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blot analysis. Levels of PGE(2) and soluble ICAM-1 (sICAM-1) in culture medium were measured by enzyme-linked immunosorbent assay. Furthermore, the phosphorylation status of p38MAPK, ERK1/2, JNK, Akt and NF-kappaB was analysed by Western blot; nuclear translocation of NF-kappaB was observed by immunofluorescence., Key Results: Ucn1 augmented LPS-induced expression of COX-2 and ICAM-1 in RAECs in a time- and concentration-dependent manner. Ucn1 increased PGE(2) and sICAM-1 levels. These effects were abolished by the CRF(2) receptor antagonist, antisauvagine-30, but not by the CRF(1) receptor antagonist, NBI-27914. Moreover, Ucn2 activated p38MAPK and augmented NF-kappaB nuclear translocation and phosphorylation, whereas ERK1/2, JNK and Akt pathways were not involved in this process., Conclusions and Implications: These findings suggest that Ucn1 exerts pro-inflammatory effects by augmenting LPS-induced expression of COX-2 and ICAM-1 in RAECs via CRF(2) receptors and the activation of p38MAPK and NF-kappaB.

Aims: The aim of this study is to evaluate the positive inotropic effect of urocortin (Ucn) and to characterize its signalling pathways., Methods and Results: Contractility was measured in ex vivo Langendorff-perfused hearts isolated from Wistar rats. Isolated ventricular cardiomyocytes were used to analyse intracellular calcium ([Ca(2+)](i)) transients evoked by electrical stimulation and L-type Ca(2+) current by confocal microscopy and whole-cell patch-clamping, respectively. The application of Ucn to perfused hearts induced progressive, sustained, and potent inotropic and lusitropic effects that were dose-dependent with an EC(50) of approximately 8 nM. Ucn effects were independent of protein kinase A (PKA) activation but were significantly reduced by protein kinase C (PKC) and mitogen-activated protein kinase (MAPK) inhibitors and by brefeldin A, an antagonist of guanine nucleotide exchange factor, suggested to be an inhibitor of exchange protein activated by cAMP (Epac). These whole-organ effects were correlated with the inotropic effects observed in isolated cells: Ucn increased I(CaL) density, [Ca(2+)](i) transients, cell shortening and Ca(2+) content of sarcoplasmic reticulum., Conclusion: Our results show that Ucn evokes potent positive inotropic and lusitropic effects mediated, at least in part, by an increase in I(CaL) and [Ca(2+)](i) transient amplitude. These effects may involve the activation of Epac, PKC, and MAPK signalling pathways.

Background and Purpose: Urocortin is a locally expressed pro-inflammatory peptide. Here we have examined the effects of urocortin on sodium laurate-induced peripheral arterial vasculitis in rats, modelling the mechanisms of thromboangiitis obliterans (TAO)., Experimental Approach: Peripheral vasculitis in rats was induced by sodium laurate and graded by gross appearance on the 12th day after injection. Histological changes in rat femoral arteries were assessed by histopathology and transmission electron microscopy. Blood cell counts, blood rheology, blood coagulation and plasma urocortin, thromboxane B(2), prostaglandin E(2) and soluble intercellular adhesion molecule-1 levels were measured. Expression of urocortin, corticotrophin-releasing factor (CRF(1/2)) receptors, cyclooxygenase (COX)-2 and intercellular adhesion molecule-1 (ICAM-1) at both mRNA and protein levels were determined by RT-PCR and Western blot., Key Results: Rats showed grossly visible signs and symptoms of TAO on the 12th day after sodium laurate injection. In these rats, blood was in a hypercoagulable state; plasma urocortin, prostaglandin E(2) and soluble intercellular adhesion molecule-1 levels were elevated; and the expression of urocortin, CRF(1) and CRF(1alpha)-receptors, COX-2 and ICAM-1 in rat femoral arteries were markedly increased. Exogenous urocortin, given for 12 days after sodium laurate, exacerbated the hypercoagulable state and augmented expression of CRF(1alpha)-receptors, COX-2 and ICAM-1. These effects were abolished by a CRF(1)-receptor antagonist, NBI-27914, or a non-selective CRF-receptor antagonist, astressin, but not by the CRF(2)-receptor antagonist, antisauvagine-30, given with exogenous urocortin., Conclusion and Implications: Urocortin exacerbated the hypercoagulable state and vasculitis in a model of TAO induced by sodium laurate in rats, via CRF(1)-receptors. COX-2 and ICAM-1 might also have contributed to this exacerbation.

Non-alcoholic fatty liver disease (NAFLD) has become the most common liver disorder of our times. Simple steatosis, a seemingly innocent manifestation of NAFLD, may progress into steatohepatitis and cirrhosis, but this process is not well understood. Since NAFLD is associated with obesity and insulin resistance, mechanisms that link lipid metabolism to inflammation offer insights into the pathogenesis. An important parallel between obesity-related pathology of adipose tissue and liver pertains to the emerging role of macrophages and evidence is growing that Kupffer cells critically contribute to progression of NAFLD. Toll-like receptors, in particular TLR4, represent a major conduit for danger recognition linked to Kupffer cell activation and this process may be perturbed at multiple steps in NAFLD. Steatosis may interfere with sinusoid microcirculation and hepatocellular clearance of microbial and host-derived danger signals, enhancing responsiveness of Kupffer cells. Altered lipid homeostasis in NAFLD may unfavourably affect TLR4 receptor complex assembly and sorting, interfere with signalling flux redistribution, promote amplification loops, and impair negative regulation including alternative activation of Kupffer cells. These events are further promoted by altered adipokine secretion and reactive oxygen species production. Specific targeting of these interactions may provide more effective strategies in the treatment of NAFLD.

Stress and alcohol abuse are co-related. Acute alcohol is anxiolytic and stress is cited as a factor in relapse to alcohol use. A primary mediator of the stress response is the neuropeptide corticotropin-releasing factor (CRF). The CRF family of endogenous ligands includes urocortin 3 (Ucn 3), which binds selectively to the CRF type 2 receptor and has been implicated in ethanol consumption in dependent and withdrawing rats. The objective of this study was to examine the effect of Ucn 3, delivered centrally to nondependent mice, on limited-access ethanol consumption. Adult C57BL/6J mice were trained to self-administer 10% ethanol during daily, 2-h limited-access sessions, using lickometers to assess drinking patterns for both ethanol and water. Sterile saline or 0.3, 1, or 3 nmol of Ucn 3 was microinjected into the lateral ventricle immediately before the limited-access session in a within-subjects design. There was a significant decrease in ethanol (both ml and g/kg), but not water, intake following Ucn 3 treatment, explained by a change in size of the largest lick run. Food intake at both 2 h and 24 h after injection was statistically unaffected by Ucn 3 administration. These results establish a role for CRF type 2 receptors in a nondependent mouse model of ethanol self-administration.