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Aim To determine the role of corticotropin releasing factor receptor (CRF2) in epithelial permeability and enterocyte cell differentiation. Methods For this purpose, we used rat Sprague Dawley and various colon carcinoma cell lines (SW620, HCT8R, HT-29 and Caco-2 cell lines). Expression of CRF2 protein was analyzed by fluorescent immunolabeling in normal rat colon and then by western blot in dissociated colonic epithelial cells and in the lysates of colon carcinoma cell lines or during the early differentiation of HT-29 cells (ten first days). To assess the impact of CRF2 signaling on colonic cell differentiation, HT-29 and Caco-2 cells were exposed to Urocortin 3 recombinant proteins (Ucn3, 100 nmol/L). In some experiments, cells were pre-exposed to the astressin 2b (A2b) a CRF2 antagonist in order to inhibit the action of Ucn3. Intestinal cell differentiation was first analyzed by functional assays: the trans-cellular permeability and the para-cellular permeability were determined by Dextran-FITC intake and measure of the transepithelial electrical resistance respectively. Morphological modifications associated to epithelial dysfunction were analyzed by confocal microscopy after fluorescent labeling of actin (phaloidin-TRITC) and intercellular adhesion proteins such as E-cadherin, p120ctn, occludin and ZO-1. The establishment of mature adherens junctions (AJ) was monitored by following the distribution of AJ proteins in lipid raft fractions, after separation of cell lysates on sucrose gradients. Finally, the mRNA and the protein expression levels of characteristic markers of intestinal epithelial cell (IEC) differentiation such as the transcriptional factor kruppel-like factor 4 (KLF4) or the dipeptidyl peptidase IV (DPPIV) were performed by RT-PCR and western blot respectively. The specific activities of DPPIV and alkaline phosphatase (AP) enzymes were determined by a colorimetric method. Results CRF2 protein is preferentially expressed in undifferentiated epithelial cells from the crypts of colon and in human colon carcinoma cell lines. Furthermore, CRF2 expression is down regulated according to the kinetic of HT-29 cell differentiation. By performing functional assays, we found that Ucn3-induced CRF2 signaling alters both para- and trans-cellular permeability of differentiated HT-29 and Caco-2 cells. These effects are partly mediated by Ucn3-induced morphological changes associated with the disruption of mature AJ in HT-29 cells and tight junctions (TJ) in Caco-2 cells. Ucn3-mediated activation of CRF2 decreases mRNA and protein expression levels of KLF4 a transcription factor involved in IEC differentiation. This signaling is correlated to a down-regulation of key IEC markers such as DPPIV and AP, at both transcriptional and post-transcriptional levels. Conclusion Our findings suggest that CRF2 signaling could modulate IEC differentiation. These mechanisms could be relevant to the stress induced epithelial alterations found in inflammatory bowel diseases.

Background Corticotropin-releasing factor (CRF) signaling at the CRF 1 receptor (CRF 1 R) in the ventral tegmental area (VTA) can modulate ethanol consumption in rodents. However, the effects of binge-like ethanol drinking on this system have not been thoroughly characterized, and little is known about the role of CRF 2 R or the CRF neurocircuitry involved. Methods The effects of binge-like ethanol consumption on the VTA CRF system were assessed following drinking-in-the-dark procedures. Intra-VTA infusions of selective CRF 1 R and/or CRF 2 R compounds were employed to assess the contributions of these receptors in modulating binge-like ethanol consumption ( n = 89). To determine the potential role of CRF projections from the bed nucleus of the stria terminalis (BNST) to the VTA, CRF neurons in this circuit were chemogenetically inhibited ( n = 32). Binge-induced changes in VTA CRF system protein and messenger RNA were also assessed ( n = 58). Results Intra-VTA antagonism of CRF 1 R and activation of CRF 2 R resulted in decreased ethanol intake, which was eliminated by simultaneous blockade of both receptors. Chemogenetic inhibition of local CRF neurons in the VTA did not alter binge-like ethanol drinking, but inhibition of VTA-projecting CRF neurons from the BNST significantly reduced intake. Conclusions We provide novel evidence that 1) blunted binge-like ethanol consumption stemming from CRF 1 R blockade requires intact CRF 2 R signaling, and CRF 2 R activation reduces binge-like drinking; 2) inhibiting VTA-projecting BNST CRF neurons attenuates binge-like drinking; and 3) binge-like ethanol drinking alters protein and messenger RNA associated with the VTA-CRF system. These data suggest that ethanol-induced activation of BNST-to-VTA CRF projections is critical in driving binge-like ethanol intake.

Prognosis of patients with chronic heart failure remains poor, emphasizing the need to identify additional pathophysiological factors. Tsuda et al. show that Crhr2 activation causes cardiac dysfunction and suggest Crhr2 blockade is a promising therapeutic strategy for chronic heart failure., Heart failure occurs when the heart is unable to effectively pump blood and maintain tissue perfusion. Despite numerous therapeutic advancements over previous decades, the prognosis of patients with chronic heart failure remains poor, emphasizing the need to identify additional pathophysiological factors. Here, we show that corticotropin releasing hormone receptor 2 (Crhr2) is a G protein–coupled receptor highly expressed in cardiomyocytes and continuous infusion of the Crhr2 agonist, urocortin 2 (Ucn2), reduced left ventricular ejection fraction in mice. Moreover, plasma Ucn2 levels were 7.5-fold higher in patients with heart failure compared to those in healthy controls. Additionally, cardiomyocyte-specific deletion of Crhr2 protected mice from pressure overload-induced cardiac dysfunction. Mice treated with a Crhr2 antagonist lost maladaptive 3′-5′-cyclic adenosine monophosphate (cAMP)–dependent signaling and did not develop heart failure in response to overload. Collectively, our results indicate that constitutive Crhr2 activation causes cardiac dysfunction and suggests that Crhr2 blockade is a promising therapeutic strategy for patients with chronic heart failure.

Background The cytoarchitecturally defined Edinger–Westphal nucleus (EW) is now referred to by many investigators as the centrally-projecting EW (EWcp) in humans. Although the mature structure is well-characterized, there have been few reports describing the precise morphology of this nucleus during the second half of gestation. Subjects/Design Eleven brains were examined from preterm infants, aged 20–39 postmenstrual weeks, who died of various causes. After fixation, the brains were embedded in celloidin and serial sections of 30-μm thickness were cut in the horizontal plane. Sections were stained using the Kluver–Barrera method. In addition to microscopic observations, computerized 3D reconstruction and morphometry were performed. Results From 21 weeks, the EWcp had a distinctive, complex 3D structure comprising two or three parts. The dorsal part was arcuate, half encircling the oculomotor somatic nuclei (OSN). The rostral part was the most voluminous, ventral to the rostral OSN, extending anteriorly. The caudal part was the smallest, and was composed of several neuronal groups near the ventral tip of the OSN. In three cases, the caudal part was absent. It could also be joined to the rostral part, forming a ventral part. The total volume of the EWcp increased exponentially with age, and the ventral part grew more rapidly than the dorsal part. The mean neuronal profile area increased linearly with age, and the rate of increase was almost equal between the dorsal and ventral parts. Conclusions This study suggests that a distinctive, complex, two- or three-part 3D structure of the EWcp is preserved after mid-gestation, and that the ventral part of the EWcp may expand in volume more rapidly than the dorsal part.

There is great interest in generating functionally mature beta cells from stem cells, as loss of functional beta cell mass contributes to the pathophysiology of diabetes. Identifying markers of beta cell maturity is therefore very helpful for distinguishing stem cells that have been successfully differentiated into fully mature beta cells from stem cells that did not. Urocortin 3 (UCN3) is a peptide hormone whose expression is associated with the acquisition of functional maturity in beta cells. The onset of its expression occurs after other beta cell maturity markers are already expressed and its loss marks the beginning of beta cell dedifferentiation. Its expression pattern is therefore tightly correlated with beta cell maturity. While this makes UCN3 an excellent marker of beta cell maturity, it is not established whether UCN3 is required for beta cell maturation. Here, we compared gene expression and function of beta cells from Ucn3-null mice relative to WT mice to determine whether beta cells are functionally mature in the absence of UCN3. Our results show that genetic deletion of Ucn3 does not cause a loss of beta cell maturity or an increase in beta cell dedifferentiation. Furthermore, virgin beta cells, first identified as insulin-expressing, UCN3-negative beta cells, can still be detected at the islet periphery in Ucn3-null mice. Beta cells from Ucn3-null mice also exhibit normal calcium response when exposed to high glucose. Collectively, these observations indicate that UCN3 is an excellent mature beta cell marker that is nevertheless not necessary for beta cell maturation.

Background Urocortin 1 (Ucn1), a stress-related peptide, is a member of the corticotropin-releasing factor (CRF) family and acts as a CRF1 receptor agonist. Ucn1 and CRF1 receptor immunoreactivity are present in the enteric nervous system (ENS), and Ucn1 elicits contraction of colonic muscle strips. Considering these findings, we have hypothesized that Ucn1 acts as an excitatory neurotransmitter in the ENS. The present study was conducted to determine whether exogenously applied Ucn1 causes contractions, whether it participates in neurally mediated contraction, and whether it is released from the ENS of the rat colon. Methods Isometric tension of the rat colonic muscle strips (middle to distal colon) in a longitudinal direction was measured. The effects of Ucn1 on phasic contractions were examined in the absence and presence of antalarmin (CRF1 receptor antagonist), tetrodotoxin (TTX), and atropine. The effects of antalarmin on electrical field stimulation (EFS)-induced contractions were examined in the absence and presence of atropine. Ucn1 peptide in the bath solution was measured after EFS using an EIA kit. Key results Ucn1 caused a significant and dose-dependent increase in phasic contractions. These effects were completely inhibited by antalarmin, TTX, and atropine. EFS-induced contractions were inhibited by antalarmin. Atropine markedly reduced EFS-induced contractions, and antalarmin did not decrease these contractions further. EFS elicited a significant increase in the concentration of Ucn1 in the bath solution, and this increase was completely inhibited by TTX. Conclusions and inferences These results suggest that Ucn1 acts as an excitatory neurotransmitter in the ENS enhancing the cholinergic neurotransmission.

No organ in the body is impervious to the effects of stress, and a coordinated response from all organs is essential to deal with stressors. A dysregulated stress response that fails to bring systems back to homeostasis leads to compromised function and ultimately a diseased state. The components of the corticotropin-releasing factor (CRF) family, an ancient and evolutionarily conserved stress hormone-receptor system, helps both initiate stress responses and bring systems back to homeostasis once the stressors are removed. The mammalian CRF family comprises of four known agonists, CRF and urocortins (UCN1–3), and two known G protein-coupled receptors (GPCRs), CRF1 and CRF2. Evolutionarily, precursors of CRF- and urocortin-like peptides and their receptors were involved in osmoregulation/diuretic functions, in addition to nutrient sensing. Both CRF and UCN1 peptide hormones as well as their receptors appeared after a duplication event nearly 400 million years ago. All four agonists and both CRF receptors show sex-specific changes in expression and/or function, and single nucleotide polymorphisms are associated with a plethora of human diseases. CRF receptors harbor N-terminal cleavable peptide sequences, conferring biased ligand properties. CRF receptors have the ability to heteromerize with each other as well as with other GPCRs. Taken together, CRF receptors and their agonists due to their versatile functional adaptability mediate nuanced responses and are uniquely positioned to orchestrate sex-specific signaling and function in several tissues.

Molecular therapy 28(1), 180-188 (2020). doi:10.1016/j.ymthe.2019.10.003, Published by Nature Publ. Group, New York, NY

Urocortin 3 (UCN3) is a peptide hormone playing a pivotal role in glucose and lipid metabolisms. However, its clinical implications remain unclear. Our aims were to investigate the altered levels of UCN3 in newly diagnosed type 2 diabetes mellitus (nT2DM) patients in comparison to subjects with normal glucose tolerance (NGT) and to determine the presence of any possible link between UCN3 levels and metabolic parameters.Eighty nT2DM and 80 age-, body mass index (BMI)-, and gender-matched NGT subjects were enrolled into this case-control study. The circulating UCN3 levels were measured using the enzyme-linked immunoabsorbent assay (ELISA). Metabolic parameters of enrolled subjects were also determined. A standard 75-g 2-hour oral glucose tolerance test was used for diagnosis of type 2 diabetes mellitus (T2DM).UCN3 levels were higher in subjects with nT2DM than in controls (115.64 ± 39.26 vs 86.16 ± 22.81 pg/mL, P .001). UCN3 levels were increased in subjects with metabolic syndrome compared to subjects without metabolic syndrome in both nT2DM and NGT groups. UCN3 levels showed a positive correlation with BMI in both groups. Moreover, UCN3 levels were positively and independently associated with insulin, fasting blood glucose, insulin resistance, 2-hour plasma glucose, glycosylated hemoglobin, and triglycerides, whereas UCN3 levels were negatively and independently associated with high-density lipoprotein cholesterol. According to logistic regression analysis, increased risk of T2DM and metabolic syndrome were parallel with the highest elevated levels of UCN3.Increased levels of UCN3 are associated with unfavorable metabolic profiles in T2DM, indicating a potential role of UCN3 in glucose and lipid metabolisms in T2DM.背景: 尿皮质激素3(urocortin 3, UCN3)是一种在糖脂代谢中起关键作用的肽类激素。然而, 其临床意义仍然不清楚。我们的目的是研究新诊断2型糖尿病(nT2DM)患者与正常糖耐量(NGT)患者UCN3水平的变化, 并确定UCN3水平与代谢参数之间是否存在任何可能的联系。 方法: 80例nT2DM患者和80例年龄、体重指数(BMI)、性别相匹配的NGT患者进入本病例对照研究。采用酶联免疫吸附试验(ELISA)测定循环UCN3水平。同时测定了受试者的代谢参数。使用标准的75g-2小时口服葡萄糖耐量试验用于2型糖尿病(T2DM)的诊断。 结果: nT2DM组患者血清UCN3水平明显高于对照组(115.64±39.26 vs 86.16±22.81pg/mL, P0.001)。在nT2DM和NGT组中, 有代谢综合征的受试者UCN3水平均高于无代谢综合征的受试者。两组UCN3水平均与BMI呈正相关。此外, UCN3水平与胰岛素、空腹血糖、胰岛素抵抗、2小时血糖、糖化血红蛋白和甘油三酯呈独立正相关, 与高密度脂蛋白胆固醇呈独立负相关。根据Logistic回归分析, T2DM和代谢综合征的风险增加与UCN3升高的最高水平是平行的。 结论: UCN3水平升高与T2DM的不良代谢相关, 提示UCN3在T2DM的糖脂代谢中具有潜在作用。.

Cholecystokinin (CCK) had been the first gastrointestinal hormone known to exert anorexic effects. CCK had been inferred to contribute to the onset of functional dyspepsia (FD) symptoms. To understand the pathophysiology of FD, the roles of stress have to be clarified. In this study, we aimed to clarify the influence of stress on the action of cholecystokinin (CCK) on appetite and gastric emptying. Using rats, stress was simulated by giving restraint stress or intraperitoneal injection of the stress-related peptide hormone urocortin 1 (UCN1). The effects of CCK and restraint stress, alone or in combination, on food intake and gastric motility were examined, and c-Fos expression in the neurons of appetite control network in the central nervous system was assessed by immunohistochemical staining. CCK inhibited food intake and gastric emptying in a dose-dependent manner. Food intake for 1 h was significantly lower with UCN1 (2 nmol/kg) than with the saline control. Restraint stress amplified the suppressive effects of CCK on food intake for 1 h and on gastric emptying. With regard to brain function, the CCK induced c-Fos expression in the neurons of the nucleus tractus solitarius and paraventricular nucleus of the hypothalamus was markedly and significantly amplified by the addition of restraint stress with CCK. The results suggested that stress might amplify the anorexic effects of CCK through activation of the nuclei that comprise the brain neuronal network for satiation; this might play a role in the pathogenesis of the postprandial distress syndromes of functional dyspepsia.

Urocortins (Ucns), peptides belonging to the corticotropin-releasing hormone (CRH) family, are classified into Ucn1, Ucn2, and Ucn3. They are involved in regulating several body functions by binding to two G protein-coupled receptors: receptor type 1 (CRHR1) and type 2 (CRHR2). In this review, we provide a historical overview of research on Ucns and their receptors in the mammalian endocrine system. Although the literature on the topic is limited, we focused our attention particularly on the main role of Ucns and their receptors in regulating the hypothalamic–pituitary–adrenal and thyroid axes, reproductive organs, pancreas, gastrointestinal tract, and other tissues characterized by “diffuse” endocrine cells in mammals. The prominent function of these peptides in health conditions led us to also hypothesize an action of Ucn agonists/antagonists in stress and in various diseases with its critical consequences on behavior and physiology. The potential role of the urocortinergic system is an intriguing topic that deserves further in-depth investigations to develop novel strategies for preventing stress-related conditions and treating endocrine diseases.

CRF system is comprised of 4 homologous lineages, 2 main receptors (CRF-R1 and CRF-R2), and a binding protein CRF-BP. The homologous lineages are corticotropin-releasing factor (CRF), urotensin I (UI)/sauvagine (SVG)/urocortin 1 (UCN1), urocortin 2 (UCN2), and urocortin 3 (UCN3), and UI, SVG, UCN1 are orthologous genes. CRF system genes are widely distributed in the brain and gastrointestinal tract, which may relate to feeding regulation. According the research progress about CRF system on mammals and non-mammals, this paper summarized the discovery, structure, tissue distribution, appetite regulation and mechanism of CRF system in animals, which can provide the reference for further research and production of feeding regulation and growth in mammals and fish species.

Central administration of neuropeptide K (NPK), a 36-amino acid peptide, is associated with anorexigenic effects in rodents and chickens. The mechanisms underlying the potent anorexigenic effects of NPK are still poorly understood. Thus, the aim of the present study was to identify the hypothalamic nuclei and neuropeptides that mediate anorexic effects of NPK in 7 day-old Japanese quail (Coturnix japonica). After a 6 h fast, intracerebroventricular (ICV) injection of NPK decreased food and water intake for 180 min post-injection. Quail injected with NPK had more c-Fos immunoreactive cells in the arcuate nucleus (ARC), lateral hypothalamus, and paraventricular nucleus (PVN) compared to the birds that were injected with the vehicle. In the ARC of NPK-injected quail, there was decreased neuropeptide Y (NPY), NPY receptor sub-type 1, and agouti-related peptide mRNA, and increased CART, POMC, and neurokinin receptor 1 mRNA. NPK-injected quail expressed greater amounts of corticotropin-releasing factor (CRF), CRF receptor sub-type 2, melanocortin receptors 3 and 4, and urocortin 3 mRNA in the PVN. In conclusion, results provide insights into understanding NPK-induced changes in hypothalamic physiology and feeding behavior, and suggest that the anorexigenic effects of NPK involve the ARC and PVN, with increased CRF and melanocortin and reduced NPY signaling.

Helicobacter pylori (Hp) infection is related to the pathogenesis of chronic gastric disorders and extragastric diseases. Here, we examined the anorexigenic and anxiogenic effects of Hp vacuolating cytotoxin A (VacA) through activation of hypothalamic urocortin1 (Ucn1). VacA was detected in the hypothalamus after peripheral administration and increased Ucn1 mRNA expression and c-Fos-positive cells in the hypothalamus but not in the nucleus tractus solitarius. c-Fos and Ucn1-double positive cells were detected. CRF1 and CRF2 receptor antagonists suppressed VacA-induced anxiety and anorexia, respectively. VacA activated single paraventricular nucleus neurons and A7r5 cells; this activation was inhibited by phospholipase C (PLC) and protein kinase C (PKC) inhibitors. VacA causes anorexia and anxiety through the intracellular PLC-PKC pathway, migrates across the blood-brain barrier, and activates the Ucn1-CRF receptor axis.

Contains fulltext : 169819.pdf (Publisher’s version ) (Closed access) The interaction of early life stress (ELS) and the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) has been associated with increased risk to develop depression in later life. We have used the maternal separation paradigm as a model for ELS exposure in homozygous and heterozygous 5-HTT knockout rats and measured urocortin 1 (Ucn1) mRNA and/or protein levels, Ucn1 DNA methylation, as well as 5-HT innervation in the centrally projecting Edinger-Westphal (EWcp) and dorsal raphe (DR) nuclei, both implicated in the regulation of stress response. We found that ELS and 5-HTT genotype increased the number of 5-HT neurons in specific DR subdivisions, and that 5-HTT knockout rats showed decreased 5-HT innervation of EWcp-Ucn1 neurons. Furthermore, ELS was associated with increased DNA methylation of the promoter region of the Ucn1 gene and increased expression of 5-HT receptor 1A in the EWcp. In contrast, 5-HTT deficiency was associated with site-specific alterations in DNA methylation of the Ucn1 promoter, and heterozygous 5-HTT knockout rats showed decreased expression of CRF receptor 1 in the EWcp. Together, our findings extend the existing literature on the relationship between EWcp-Ucn1 and DR-5-HT neurons. These observations will further our understanding on their potential contribution to mediate affect as a function of ELS interacting with 5-HTTLPR.

We examined the influence of adrenalectomy on NSAID-induced small intestinal damage in rats and investigated the possible involvement of adrenal glucocorticoids in the protective effects of urocortin I, a corticotropin-releasing factor (CRF) agonist. Male SD rats without fasting were administered indomethacin s.c. and killed 24 h later in order to examine the hemorrhagic lesions that developed in the small intestine. Urocortin I (20 μg/kg) was given i.v. 10 min before the administration of indomethacin. Bilateral adrenalectomy was performed a week before the experiment. Indomethacin (10 mg/kg) caused multiple hemorrhagic lesions in the small intestine, which were accompanied by a decrease in mucus secretion and increases in intestinal motility, enterobacterial invasion, and iNOS expression. Adrenalectomy markedly increased the ulcerogenic and motility responses caused by indomethacin, with further enhancements in bacterial invasion and iNOS expression; severe lesions occurred at 3 mg/kg, a dose that did not induce any damage in sham-operated rats. This worsening effect was also observed by the pretreatment with mifepristone (a glucocorticoid receptor antagonist). Urocortin I prevented indomethacin-induced enteropathy, and this effect was completely abrogated by the pretreatment with astressin 2B, a CRF2 receptor antagonist, but was not significantly affected by either adrenalectomy or the mifepristone pretreatment. These results suggested that adrenalectomy aggravated the intestinal ulcerogenic response to indomethacin, the intestinal hypermotility response may be a key element in the mechanism for this aggravation, and endogenous glucocorticoids played a role in intestinal mucosal defense against indomethacin-induced enteropathy, but did not account for the protective effects of urocortin I, which were mediated by the activation of peripheral CRF2 receptors.

Intrahepatic cholestasis of pregnancy(ICP) is complicated by acute placental-fetal hypoxia. Corticotropin-releasing hormone(CRH) and urocortin(UCN) are vasodilatory regulators of blood flow in the placenta. An ethinylestradiol(EE)-induced cholestasis rat model was reproduced and serum/placental CRH/UCN were detected during 14–21 days of gestation(DG). Maternal serum or placental CRH/UCN levels in the control rats were relatively consistent during 14–21DG. Serum CRH was reduced in the EE-treated rats compared with the control rats at 21DG. Regarding serum UCN, we observed a decrease at 17DG as well as an increase at 21DG in the EE-treated rats compared with the controls. Moreover, we observed a noticeable reduction of placental CRH/UCN expression at 17 or 19DG in the EE-treated rats compared with the control rats. The serum bile acids levels exhibited an inverse correlation with placental CRH/UCN expression. EE-induced cholestasis rats might serve as a good model to further investigate the pathological mechanism underlying CRH/UCN dysregulation in ICP.

Ovarian endometrioma (OMA) and deep infiltrating endometriosis (DIE) are the most severe forms of endometriosis, but different pathogenetic mechanisms and clinical symptoms distinguish these two forms. Corticotrophin-releasing hormone (CRH) and urocortin (Ucn) are endometrial neuropeptides involved in tissue differentiation and inflammation. The expression of CRH, Ucn, Ucn2, CRH-receptors (type-1 and type-2) and inflammatory enzymes phospholipase-A2 group IIA (PLA2G2A) and cycloxygenase-2 (COX2) were evaluated in OMA (n = 22) and DIE (n = 26). The effect of CRH or Ucn on COX2 mRNA expression was evaluated in cultured human endometrial stromal cells. In DIE lesions, CRH, Ucn and CRH-R2 mRNA levels were significantly higher than in OMA (P < 0.01, P < 0.001 and P < 0.05, respectively); DIE lesions showed a higher expression of COX2 (P < 0.01) and PLA2G2A (P < 0.05) mRNA than OMA, which was positively correlated with CRH-R2 mRNA expression (P < 0.05). Intense immunostaining for CRH and Ucn was shown in DIE. Treatment of cultured endometrial stromal cells with Ucn significantly increased COX2 mRNA expression (P < 0.01); this effect was reversed by the CRH-R2 antagonist astressin-2B. In DIE, DIE lesions highly express neuropeptide and enzyme mRNAs, supporting a strong activation of inflammatory pathways.

Chun Yeh,1 Ching-Heng Ting,2 Ming-Luen Doong,3 Chin-Wen Chi,4,5 Shou-Dong Lee,1 Chih-Yen Chen6–8 1Division of Gastroenterology, Department of Internal Medicine, Cheng-Hsin General Hospital, 2Department of Pathology, Mackay Memorial Hospital, 3Institute of Physiology, 4Institute of Pharmacology, National Yang-Ming University School of Medicine, 5Department of Medical Research, Taipei Veterans General Hospital, 6Division of Gastroenterology and Hepatology, Department of Medicine, Taipei Veterans General Hospital, 7Faculty of Medicine, National Yang-Ming University School of Medicine, Taipei, 8Taiwan Association for the Study of Small Intestinal Diseases, Guishan, Taiwan Purpose: Urocortin 3 is a key neuromodulator in the regulation of stress, anxiety, food intake, gut motility, and energy homeostasis, while ghrelin elicits feeding behavior and enhances gastric emptying, adiposity, and positive energy balance. However, the interplays between urocortin 3 and ghrelin on food intake and gastric emptying remain uninvestigated.Methods: We examined the differential effects of central O-n-octanoylated ghrelin, des-Gln14-ghrelin, and urocortin 3 on food intake, as well as on charcoal nonnutrient semiliquid gastric emptying in conscious rats that were chronically implanted with intracerebroventricular (ICV) catheters. The functional importance of corticotropin-releasing factor (CRF) receptor 2 in urocortin 3-induced responses was examined by ICV injection of the selective CRF receptor 2 antagonist, astressin2-B.Results: ICV infusion of urocortin 3 opposed central acyl ghrelin-elicited hyperphagia via CRF receptor 2 in satiated rats. ICV injection of O-n-octanoylated ghrelin and des-Gln14-ghrelin were equally potent in accelerating gastric emptying in fasted rats, whereas ICV administration of urocortin 3 delayed gastric emptying. In addition, ICV infusion of urocortin 3 counteracted central acyl ghrelin-induced gastroprokinetic effects via CRF receptor 2 pathway.Conclusion: ICV-infused urocortin 3 counteracts central acyl ghrelin-induced hyperphagic and gastroprokinetic effects via CRF receptor 2 in rats. Our results clearly showed that enhancing ghrelin and blocking CRF receptor 2 signaling in the brain accelerated gastric emptying, which provided important clues for a new therapeutic avenue in ameliorating anorexia and gastric ileus found in various chronic wasting disorders. Keywords: corticotropin-releasing factor receptor, des-Gln14-ghrelin, food intake, gastric emptying, intracerebroventricular, urocortin 3&nbsp

It is well established, that epidermal keratinocytes express functional equivalent of hypothalamus-pituitary-adrenal axis (HPA) in order to respond to changing environment and maintain internal homeostasis. We are presenting data indicating that differentiation of primary neonatal human keratinocytes (HPEKp), induced by prolonged incubation or calcium is accompanied by significant changes in the expression of the elements of skin analog of HPA (sHPA). Expression of CRF, UCN1-3, POMC, ACTH, CRFR1, CRFR2, MC1R, MC2R, and GR (coded by NR3C1 gene) were observed on gene/protein levels along differentiation of keratinocytes in culture with similar pattern seen by immunohistochemistry on full thickness skin biopsies. Expression of CRF was more pronounced in less differentiated keratinocytes, which corresponded to the detection of CRF immunoreactivity preferentially in the stratum basale. POMC expression was enhanced in more differentiated keratinocytes, which corresponded to detection of ACTH immunoreactivity, predominantly in the stratum spinosum and stratum granulosum. Expression of urocortins was also affected by induction of HPEKp differentiation. Immunohistochemical studies showed high prevalence of CRFR1 in well differentiated keratinocytes, while smaller keratinocytes showed predominantly CRFR2 immunoreactivity. MC2R mRNA levels were elevated from days 4 to 8 of in vitro incubation, while MC2R immunoreactivity was the highest in the upper layers of epidermis. Similar changes in mRNA/protein levels of sHPA elements were observed in HPEKp keratinocytes treated with calcium. Summarizing, preferential expression of CRF and POMC (ACTH) by populations of keratinocytes on different stage of differentiation resembles organization of central HPA axis suggesting their distinct role in physiology and pathology of the epidermis. J. Cell. Physiol. 232: 154-166, 2017. © 2016 Wiley Periodicals, Inc.

Aims:\ud \ud Urocortin 2 and urocortin 3 may play a role in the pathophysiology of heart failure and are emerging therapeutic targets. We aimed to examine the local and systemic cardiovascular effects of urocortin 2 and urocortin 3 in healthy subjects and patients with heart failure.\ud Methods:\ud \ud Patients with heart failure (n = 8) and age and gender-matched healthy subjects (n = 8) underwent bilateral forearm arterial blood flow measurement using forearm venous occlusion plethysmography during intra-arterial infusions of urocortin 2 (3.6–36 pmol min−1), urocortin 3 (360–3600 pmol min−1) and substance P (2–8 pmol min−1). Heart failure patients (n = 9) and healthy subjects (n = 7) underwent non-invasive impedance cardiography during incremental intravenous infusions of sodium nitroprusside (573–5730 pmol kg−1 min−1 ), urocortin 2 (36–360 pmol min−1 ), urocortin 3 (1.2–12 nmol min−1) and saline placebo.\ud Results:\ud \ud Urocortin 2, urocortin 3 and substance P induced dose-dependent forearm arterial vasodilatation in both groups (P < 0.05 for both) with no difference in magnitude of vasodilatation between patients and healthy subjects. During systemic intravenous infusions, urocortin 3 increased heart rate and cardiac index and reduced mean arterial pressure and peripheral vascular resistance index in both groups (P < 0.01 for all). Urocortin 2 produced similar responses to urocortin 3, although increases in cardiac index and heart rate were only significant in heart failure (P < 0.05) and healthy subjects (P < 0.001), respectively.\ud Conclusion:\ud \ud Urocortins 2 and 3 cause vasodilatation, reduce peripheral vascular resistance and increase cardiac output in both health and disease. These data provide further evidence to suggest that urocortins 2 and 3 continue to hold promise for the treatment of heart failure.

We investigated whether corticotropin-releasing factor receptor 2 (CRF2) and its high-affinity agonist urocortin 1 (Ucn1) mediate sex-specific signaling and immune responses. Intrarectal trinitrobenzene sulfonic acid was used to induce experimental colitis in wild-type, CRF2knockout (CRF2KO), and heterozygous (CRF2Ht) mice of both sexes. Changes in plasma extravasation, organ weight, survival, immune cell numbers, inflammatory cytokines, and the MAPK signaling pathway were assessed. Stored intestinal biopsies from patients with Crohn's disease (CD) and age- and sex-matched individuals without inflammatory bowel disease (IBD) were examined by immunofluorescence and confocal microscopy to characterize Ucn1 and CRF receptor expression. CRF2Ht mice of both sexes showed decreased survival during colitis compared with other genotypes. Ucn1 improved survival in male mice alone. Ucn1 restored colon length and spleen and adrenal weight and decreased colonic TNF-α, IL-6, and IL-1β levels in male CRF2Ht mice alone. CRF2Ht mice of both sexes showed decreased phosphorylation of MAPK p38 and heat shock protein 27 (Hsp27) levels. Ucn1 restored p-Hsp27 levels in male CRF2Ht mice alone. Expression of the chaperone protein Hsp90 decreased during colitis, except in male CRF2Ht mice. Taken together, our data indicate that sex shows significant interaction with genotype and Ucn1 during colitis. Human duodenal and colonic biopsies revealed that sex-specific differences exist in levels of CRF receptors and Ucn1 expression in patients with CD compared with the matched non-IBD subjects. To conclude, Ucn1 mediates sex-specific immune and cellular signaling responses via CRF2, emphasizing the need for inclusion of females in preclinical studies.

Only few reports exist regarding the coexistence of multiple sclerosis (MS) and autoimmune myopathies. We describe the case of a man with a long history of undiagnosed left lower limb motor impairment who was hospitalized for subacute onset of a myopathic syndrome. In addition, neurological examination revealed sensory impairment and pyramidal signs in the left limbs. Muscle biopsy revealed a necrotizing myopathy and serum anti-signal recognition particle (SRP) antibodies were found. Brain and spinal cord MRI displayed several non-enhancing demyelinating lesions, and CSF-restricted oligoclonal bands were detected. Multimodal evoked potentials showed increased latency of central conduction. Total body PET-CT did not reveal malignancies. A final diagnosis of anti-SRP necrotizing autoimmune myopathy (NAM) and MS was made, and subsequent therapy with azathioprine resulted in a complete stability for both diseases during the follow up. To the best of our knowledge this is the first reported case of concomitant NAM and MS.

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.

Urocortin 2 (Ucn2) - corticotropin-releasing hormone receptor 2 signalling has favourable effects in the cardiovascular system, including vasodilation, lowering of blood pressure and systemic peripheral resistance, increase in cardiac output and cardiac contractility, as well as cardioprotection against ischemia-reperfusion injury. Vasodilation and lowering of blood pressure seem to be very interesting and important effects, but their mechanism and interaction with the antihypertensive drugs have not been evaluated. The aim of the present study was to assess the relationship between Ucn2 concentration and antihypertensive therapy in patients with primary hypertension. We examined a group of 65 patients with primary hypertension receiving at least 3 antihypertensive drugs. In all of them plasma level of Ucn2, anthropometric measurements, biochemical tests, ambulatory blood pressure monitoring (ABPM), and echocardiography were performed. There were no differences in Ucn2 level related to beta-blockers, calcium channel blockers or diuretics, but we observed that in patients treated with angiotensin converting enzyme inhibitors (ACEI) (n = 52) serum Ucn2 levels were significantly higher than in patients treated with angiotensin-receptor blockers (ARBs) (n = 13) (10.93 versus 5.56 ng/mL; P0.05). Moreover, we did not observe any differences in terms of blood pressure on ABPM, biochemical measurements, left ventricular mass index, or presence of diabetes. In addition, in a small subgroup receiving alpha-blockers we also found a lower level of Ucn2, with coexisting higher systolic blood pressure at night, higher left ventricle mass index (LVMI) and more frequent occurrences of diabetes compared to non-alpha-blockers. Our findings suggest that the hypotensive action of renin-angiotensin-aldosterone system blockade may be related to the urocortin system. Ucn2 may be an important element in the mosaic of blood pressure-lowering factors in patients treated for essential hypertension.

Urocortin-3 (UCN3) as a brain-gut peptide inhibits food intake of animal, but the underlying mechanism is not clear. To explore the appetite mechanism about the action of UCN3 in fish, intraperitoneal injection of UCN3 with CCK8, Lorglumide (CCK1R antagonist) or LY225910 (CCK2R antagonist) were conducted. Siberian sturgeon administrated with UCN3 and CCK8 showed a drastic reduction in food intake. The anorectic effect of UCN3 was significantly blocked by LY225910, but not affected by Lorglumide. Furthermore, LY225910 could effectively reverse appetite factor mRNA expressions, including cck, pyy, cart, npy, ucn3, apelin and nucb2 in the whole brain, stomach and intestinum valvula, but Lorglumide could only partially reverse these effects, suggesting the anorectic effect of UCN3 may be primarily mediated CCK2R in Siberian sturgeon. This study indicates for the first time in fish that UCN3 may inhibit food intake in coordination with CCK and CCK2R.

Because little is known about the role of corticotropin-releasing factor (CRF) agonists in regulating responses in pancreatitis, we evaluated the effects of urocortin 2 (UCN2) and stressin1 in caerulein-induced acute pancreatitis (AP) model in rats. Male rats were pretreated with UCN2 or stressin1 for 30 min followed by induction of AP with supraphysiologic doses of caerulein. Serum amylase and lipase activity, pancreatic tissue necrosis, immune cell infiltrate, nuclear factor (NF)-κB activity, trypsin levels, and intracellular Ca2+ ([Ca2+]i) were ascertained. UCN2, but not stressin1 attenuated the severity of AP in rats. UCN2, but not stressin1, reduced serum amylase and lipase activity, cell necrosis and inflammatory cell infiltration in AP. NF-κB activity in pancreatic nuclear extracts increased in AP and UCN2 treatment reduced caerulein-induced increases in NF-κB activity by 42%. UCN2 treatment prevented caerulein-induced degradation of IκB-α in the cytosolic fraction as well as increased levels of p65 subunit of NF-κB in the cytosolic fraction. Pancreatic UCN2 levels decreased in AP compared with saline. UCN2 evoked [Ca2+]i responses in primary acinar cells and abolished caerulein-evoked [Ca2+]i responses at 0.1nM, and decreased by ~50% at 1.0nM caerulein. UCN2 stimulation resulted in redistribution of a portion of F-actin from the apical to the basolateral pole. UCN2 prevented the massive redistribution of F-actin observed with supraphysiologic doses of caerulein. UCN2, but not stressin1 attenuated severity of an experimental pancreatitis model. The protective effects of UCN2, including anti-inflammatory and anti-necrotic effects involve activation of the CRF2 receptor, [Ca2+]i signaling, and inhibition of NF-κB activity.

IntroductionUrocortin 2 (Ucn2) is a 38-amino acid peptide of the corticotropin-releasing factor family. Intravenous (IV) delivery of an adeno-associated virus vector serotype 8 encoding Ucn2 (AAV8.Ucn2) increases insulin sensitivity and glucose disposal in mice with insulin resistance.ObjectiveTo determine the effects of Ucn2 on liver metabolome.MethodsSix-week-old C57BL6 mice were divided into normal chow (CHOW)-fed and high fat diet (HFD)-fed groups. The animals received saline, AAV8 encoding no gene (AAV8.Empt) or AAV8.Ucn2 (2x1013 genome copy/kg, IV injection). Livers were isolated from CHOW-fed and HFD-fed mice and analyzed by untargeted metabolomics. Group differences were statistically analyzed.ResultsIn CHOW-fed mice, AAV8.Ucn2 gene transfer (vs. saline) altered the metabolites in glycolysis, pentose phosphate, glycogen synthesis, glycogenolysis, and choline-folate-methionine signaling pathways. In addition, AAV8.Ucn2 gene transfer increased amino acids and peptides, which were associated with reduced protein synthesis. In insulin resistant (HFD-induced) mice, HFD (vs CHOW) altered 448 (112 increased and 336 decreased) metabolites and AAV8.Ucn2 altered 239 metabolites (124 increased and 115 reduced) in multiple pathways. There are 61 metabolites in 5 super pathways showed interactions between diet and AAV8.Ucn2 treatment. Among them, AAV8.Ucn2 gene transfer reversed HFD effects on 13 metabolites. Finally, plasma Ucn2 effects were determined using a 3-group comparison of HFD-fed mice that received AAV8.Ucn2, AAV.Empt or saline, where 18 metabolites that altered by HFD (15 increased and 3 decreased), but restored levels to that seen in CHOW-fed mice by increased plasma Ucn2.ConclusionsMetabolomics study revealed that AAV8.Ucn2 gene transfer, through increased plasma Ucn2, provided counter-HFD effects in restoring hepatic metabolites to normal levels, which could be the underlying mechanisms for Ucn2 effects on increasing glucose disposal and reducing insulin assistance.

Peptide infusions of peptides the corticotropin releasing factor family, including urocortin 2, stresscopin, and urocortin 3 (UCn3), have favorable acute effects in clinical heart failure (HF), but their short half-lives make them unsuitable for chronic therapy. This study asked whether UCn3 gene transfer, which provides sustained elevation of plasma UCn3 levels, increases the function of the failing heart. HF was induced by transmural left ventricular (LV) cryoinjury in mice. LV function was assessed 3 weeks later by echocardiography. Those with ejection fractions (EF)

Corticotropin-releasing factor (CRF) and the urocortins (Ucn1, Ucn2, and Ucn3) belong to the mammalian CRF family of peptides, having similar amino acidic structures but different pharmacological profiles. Their actions are mediated by two distinct receptors, CRF1 and CRF2. CRF and Ucn1 bind to both CRF receptors, whereas Ucn2 and Ucn3 bind selectively to CRF2. Several studies reported that the administration of selective CRF1 antagonists prevents the dysphoria and the reward deficit observed during nicotine withdrawal. Another study referred that the administration of nonselective CRF2 agonists, such as CRF and Ucn1, prevents the hyperphagia and the weight gain assessed during nicotine withdrawal. However, our study was the first to demonstrate that intracerebroventricular administration of Ucn2 and Ucn3 ameliorates the anxiety- and depression-like state developed during chronic nicotine treatment and consequent acute withdrawal, suggesting that the selective CRF2 agonists can be potential candidates in the therapy of nicotine withdrawal.

PURPOSE OF REVIEW To highlight the advances in the diagnosis and management of acute heart failure (AHF) from recent clinical trials and observational studies. RECENT FINDINGS We highlight date from recent studies examining the use of novel biomarkers (micro RNAs, osteopontin, insulin-like growth-factor binding protein-7) for the diagnosis of AHF. In addition, we summarize the evidence for novel pharmacological therapies including: serelaxin, ularitide, clevidipine, urocortins, BMS-986231, TRB027, vericiguat, omecamtiv, and torsemide. Finally, we explore the evidence behind the use of novel management algorithms. SUMMARY Despite many advances in understanding the pathophysiology of AHF, there is currently insufficient evidence to suggest changes to standards of care.

The neuropeptide urocortin 2 (UCN2) and its receptor corticotropin-releasing hormone receptor 2 (CRHR2) are highly expressed in skeletal muscle and play a role in regulating energy balance and glucose metabolism. We investigated a modified UCN2 peptide as a potential therapeutic agent for the treatment of obesity and insulin resistance, with a specific focus on skeletal muscle. High-fat–fed mice (C57BL/6J) were injected daily with a PEGylated UCN2 peptide (compound A) at 0.3 mg/kg subcutaneously for 14 days. Compound A reduced body weight, food intake, whole-body fat mass, and intramuscular triglycerides compared with vehicle-treated controls. Furthermore, whole-body glucose tolerance was improved by compound A treatment, with increased insulin-stimulated Akt phosphorylation at Ser473 and Thr308 in skeletal muscle, concomitant with increased glucose transport into extensor digitorum longus and gastrocnemius muscle. Mechanistically, this is linked to a direct effect on skeletal muscle because ex vivo exposure of soleus muscle from chow-fed lean mice to compound A increased glucose transport and insulin signaling. Moreover, exposure of GLUT4-Myc–labeled L6 myoblasts to compound A increased GLUT4 trafficking. Our results demonstrate that modified UCN2 peptides may be efficacious in the treatment of type 2 diabetes by acting as an insulin sensitizer in skeletal muscle.

The most adverse perinatal outcome of intrahepatic cholestasis of pregnancy (ICP) is sudden fetal death related to acute fetoplacental hypoxia. Corticotrophin-releasing hormone (CRH), urocortin (UCN), and Wolfram syndrome 1 (WFS1) proteins may have a compensatory response to hypoxic stress.A total of 108 singleton pregnant women were divided into three groups: control, late-onset ICP, and early-onset ICP. Enzyme-linked immunosorbent assays were used to detected maternal serum CRH, UCN, and WFS1 levels. Western blotting and real-time polymerase chain reaction were conducted to quantify placental protein and mRNA levels of CRH, UCN, and WFS1. Pearson correlation scatterplots and Pearson correlation matrix were employed to testify the correlation.Placental WFS1 had a positive relation with placental UCN (r = 0.69, P 0.05) and serum UCN (r = 0.36, P 0.05). Placental CRH was positively correlated with maternal serum CRH (r = 0.53, P 0.05). Maternal serum and placental levels of CRH, UCN, and WFS1 significantly increased in the early-onset ICP group compared with the control group (P 0.05). Placental levels of UCN and WFS1 in the early-onset ICP group were significantly elevated and higher in comparison with the late-onset ICP group (P 0.05). However, the transcriptional levels of CRH, UCN, and WFS1 were impaired in the early-onset ICP group.Our study revealed that transcription and translation of WFS1, CRH, and UCN were altered during pregnancies complicated by early-onset ICP. This disrupted compensatory response mediated by WFS1 and CRH family peptides in early-onset ICP may play a significant role in the pathogenesis of sudden fetal death in acute fetal hypoxia.

Context: Prevalence of bronchial asthma massively increases worldwide, while the frequent therapies are still not sufficient. Polydatin, a naturally occurring glycoside, was known as to have anti-i...

KEY POINTS: Impaired ventilatory capacity and diaphragm muscle weakness are prominent features of Duchenne muscular dystrophy, with strong evidence of attendant systemic and muscle inflammation. We performed a 2‐week intervention in young wild‐type and mdx mice, consisting of either injection of saline or co‐administration of a neutralizing interleukin‐6 receptor antibody (xIL‐6R) and urocortin‐2 (Ucn2), a corticotrophin releasing factor receptor 2 agonist. We examined breathing and diaphragm muscle form and function. Breathing and diaphragm muscle functional deficits are improved following xIL‐6R and Ucn2 co‐treatment in mdx mice. The functional improvements were associated with a preservation of mdx diaphragm muscle myosin heavy chain IIx fibre complement. The concentration of the pro‐inflammatory cytokine interleukin‐1β was reduced and the concentration of the anti‐inflammatory cytokine interleukin‐10 was increased in mdx diaphragm following drug co‐treatment. Our novel findings may have implications for the development of pharmacotherapies for the dystrophinopathies with relevance for respiratory muscle performance and breathing. ABSTRACT: The mdx mouse model of Duchenne muscular dystrophy shows evidence of hypoventilation and pronounced diaphragm dysfunction. Six‐week‐old male mdx (n = 32) and wild‐type (WT; n = 32) mice received either saline (0.9% w/v) or a co‐administration of neutralizing interleukin‐6 receptor antibodies (xIL‐6R; 0.2 mg kg(−1)) and corticotrophin‐releasing factor receptor 2 agonist (urocortin‐2; 30 μg kg(−1)) subcutaneously over 2 weeks. Breathing and diaphragm muscle contractile function (ex vivo) were examined. Diaphragm structure was assessed using histology and immunofluorescence. Muscle cytokine concentration was determined using a multiplex assay. Minute ventilation and diaphragm muscle peak force at 100 Hz were significantly depressed in mdx compared with WT. Drug treatment completely restored ventilation in mdx mice during normoxia and significantly increased mdx diaphragm force‐ and power‐generating capacity. The number of centrally nucleated muscle fibres and the areal density of infiltrates and collagen content were significantly increased in mdx diaphragm; all indices were unaffected by drug co‐treatment. The abundance of myosin heavy chain (MyHC) type IIx fibres was significantly decreased in mdx diaphragm; drug co‐treatment preserved MyHC type IIx complement in mdx muscle. Drug co‐treatment increased the cross‐sectional area of MyHC type I and IIx fibres in mdx diaphragm. The cytokines IL‐1β, IL‐6, KC/GRO and TNF‐α were significantly increased in mdx diaphragm compared with WT. Drug co‐treatment significantly decreased IL‐1β and increased IL‐10 in mdx diaphragm. Drug co‐treatment had no significant effect on WT diaphragm muscle structure, cytokine concentrations or function. Recovery of breathing and diaphragm force in mdx mice was impressive in our studies, with implication for human dystrophinopathies.

Corticotropin-releasing factor (CRF) and the three homolog neuropeptides, urocortin (UCN) 1, 2 and 3, are the major neuroendocrine factors implicated in the response of the body to stress. Recent evidence suggests that UCNs have a significant role in the pathogenesis and management of cardiovascular disease, such as congestive heart failure, ischemic heart disease, and hypertension. These data led to the initiation of clinical trials testing a possible role of UCNs in the diagnosis and therapy of cardiovascular disease, with encouraging results. Here, we summarize the available literature concerning the role of UCNs in the cardiovascular system, focusing on the emerging data creating a potential for clinical applications.

Pulmonary arterial hypertension (PAH) is a devastating disease and treatment options are limited. Urocortin-2 (Ucn-2) has shown promising therapeutic effects in experimental and clinical left ventricular heart failure (HF). Our aim was to analyse the expression of Ucn-2 in human and experimental PAH, and to investigate the effects of human Ucn-2 (hUcn-2) administration in rats with monocrotaline (MCT)-induced pulmonary hypertension (PH).Tissue samples were collected from patients with and without PAH and from rats with MCT-induced PH. hUcn-2 (5 μg/kg, bi-daily, i.p., for 10 days) or vehicle was administered to male wistar rats subjected to MCT injection or to pulmonary artery banding (PAB) to induce right ventricular (RV) overload without PAH. Expression of Ucn-2 and its receptor was increased in the RV of patients and rats with PAH. hUcn-2 treatment reduced PAH in MCT rats, resulting in decreased morbidity, improved exercise capacity and attenuated pulmonary arterial and RV remodelling and dysfunction. Additionally, RV gene expression of hypertrophy and failure signalling pathways were attenuated. hUcn-2 treatment also attenuated PAB-induced RV hypertrophy.Ucn-2 levels are altered in human and experimental PAH. hUcn-2 treatment attenuates PAH and RV dysfunction in MCT-induced PH, has direct anti-remodelling effects on the pressure-overloaded RV, and improves pulmonary vascular function.

Corticotropin-releasing factor (CRF) is a hypothalamic neurohormone and an extrahypothalamic neurotransmitter that regulates the hypothalamic-pituitary-adrenal (HPA) axis. The urocortins (UCN I, UCN II and UCN III) are CRF-related peptides, which may also regulate the HPA axis directly or indirectly, by modulation of extrahypothalamic neurotransmitters, such as amygdalar GABA and hippocampal glutamate. Our previous in vitro superfusion studies have already demonstrated that CRF and UCN I stimulate the amygdalar GABA release in rats. The aim of the present study was to investigate the effects of CRF, UCN I, UCN II and UCN III on the glutamate release elicited electrically from rat hippocampal slices in similar in vitro conditions. In order to investigate the participation of CRF receptors (CRFR1 and CRFR2) in this process, hippocampal slices were pretreated with antalarmin, a selective antagonist of CRFR1 or astressin 2B, a selective antagonist of CRFR2. CRF and UCN I at 100 nM decreased significantly the hippocampal glutamate release evoked by electrical stimulation. In contrast, 100 nM of UCN II and UCN III did not affect significantly the hippocampal glutamate release enhanced by electrical stimulation. The decreasing effects of CRF and UCN I were reversed by antalarmin, but not by astressin 2B, both being administered in equimolar doses. Our results demonstrate that CRF and UCN I inhibit the glutamate release in the hippocampus via CRFR1 and that CRFR2 does not participate to this process. Based on the previous and the present results we conclude that CRFR1 agonists can activate the HPA axis not only directly, but also indirectly by increasing the amygdalar GABA release and decreasing the hippocampal glutamate release.

Stress is antinociceptive in some models of pain but enhances musculoskeletal nociceptive responses in mice and muscle pain in patients with fibromyalgia syndrome. To test the hypothesis that urocortins are stress hormones that are sufficient to enhance tactile and musculoskeletal hyperalgesia, we measured von Frey fiber sensitivity and grip force after injection of corticotrophin releasing factor (CRF), urocortin I and urocortin II in mice. Urocortin I (a CRF1 and CRF2 receptor ligand) produced hyperalgesia in both assays when injected intrathecally (i.t.) but not intracerebroventricularly (i.c.v.), and only at a large dose when injected peripherally, suggesting a spinal action. Morphine inhibited urocortin I-induced changes in nociceptive responses in a dose-related fashion, confirming that changes in behavior reflect hyperalgesia rather than weakness. No tolerance developed to the effect of urocortin I (i.t.) when injected repeatedly, consistent with a potential to enhance pain chronically. Tactile hyperalgesia was inhibited by NBI-35965, a CRF1 receptor antagonist, but not astressin 2B, a CRF2 receptor antagonist. However, while urocortin I-induced decreases in grip force were not observed when coadministered i.t. with either NBI-35965 or astressin 2B, they were even more sensitive to inhibition by astressin, a nonselective CRF receptor antagonist. Together these data indicate that urocortin I acts at CRF receptors in the mouse spinal cord to elicit a reproducible and persistent tactile (von Frey) and musculoskeletal (grip force) hyperalgesia. Urocortin I-induced hyperalgesia may serve as a screen for drugs that alleviate painful conditions that are exacerbated by stress.

© 2015 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. 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 increasedDNAbinding 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.

Background Renal dysfunction is a frequent complication and crucial determinant of outcome in acute decompensated heart failure (ADHF). The aim of the study was to assess urocortin 2 (Ucn2) as a short-term therapy in ADHF with a focus on its renal effects. Comparison was made with dobutamine to distinguish effects beyond pure inotropism. Methods Sheep with ADHF received a 2-day infusion of a vehicle-control, Ucn2 or dobutamine (n=6/grp). Results Compared to controls, Ucn2 and dobutamine produced matched improvements in cardiac contractility and output and arterial pressure, whereas reductions in central venous and left atrial pressures were greater with Ucn2. Both agents comparably repressed ADHF-activated hormone systems with the exception of the natriuretic peptides, which fell significantly during dobutamine but not Ucn2. While Ucn2 and dobutamine increased creatinine clearance and urine volume similarly, only Ucn2 induced a significant natriuresis. Ucn2 also decreased collagen deposition in the heart and kidney and suppressed gene expression of collagen-1, transforming growth factor-β1, components of the angiotensin system (angiotensinogen, angiotensin-converting enzyme, type-1 receptor) and markers of hypertrophy (GATA binding protein-4, β-myosin heavy chain), apoptosis (caspase3) and inflammation/injury (interleukin-18, cystatin C, neutrophil gelatinase-associated lipocalin, kidney injury molecule-1) in these tissues, while increasing cardiac natriuretic peptide mRNA. In contrast, dobutamine produced reduced or opposite effects on expression of these factors. Conclusions Ucn2 administration in ADHF has favorable effects on hemodynamics, the natriuretic peptides and tissue mediators of inflammation, fibrosis, apoptosis and hypertrophy that stand in contrast to dobutamine. These data support Ucn2's potential as a renoprotective therapeutic in this setting.

Background: A highly efficient approach to select microRNA (miRNA) targets is a key to develop a miRNA-based therapeutic approach to cardiac ischemia–reperfusion (I/R). To reverse the change induced by disease, I/R in this case, is the traditional strategy to develop therapeutic drugs. However, examples show that it will not always serve the purpose. In this study, we demonstrate an additional approach of selecting miRNA targets with therapeutic potential following cues from cardioprotection-induced changes rather than by reversing disease-induced changes in cardiac I/R. Methods: Isolated perfused rat hearts subjected to I/R were treated with 50 μmol/L sodium hydrosulfide (NaHS) or 10 nmol/L urocortin 2 (UCN2). Cardiac miRNA regulations were determined by miRNA array. Functional screening of selected miRNA mimics, assessed by WST (2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) activity and lactate dehydrogenase (LDH) release, was performed in H9c2 and neonatal rat ventricular myocytes (NRVMs) with hypoxia/reoxygenation. RNA-induced silencing complex (RISC)-loaded miRNAs caused by mimic transfection were quantified following argonaute-2 immunoprecipitation. Gene regulations of 1 selected miRNA were determined by quantitative polymerase chain reaction and Western blot. Results: Treatment with NaHS and UCN2 significantly improved cardiac function and reduced LDH release. The miRNA array indicated a panel of commonly up- and downregulated miRNAs. Among them, 10 upregulated miRNAs with antiapoptotic and antiautophagy potentials were selected for further screening. Mimics of miRNA-221, -150, and -206 were protective in both H9c2 and NRVM. RISC-loaded miRNAs were up by ∼20-fold above. To further prove the feasibility of this approach, miRNA-221 was studied. It reduced I/R-induced caspase 3/7 activity and LC3-II (microtubule-associated protein 1 light chain 3). Measuring genes predicted to regulate apoptosis and autophagy, miRNA-221 mimic decreased Ddit4, TP53inp1, and p27 at both messenger RNA (mRNA) and protein levels, and reduced mRNA of Bak1 and Puma and proteins of Bim and Bmf. Conclusion: Mimicking miRNA changes caused by cardioprotective agents, combined with functional screening, enables investigators to efficiently identify novel miRNAs with therapeutic potential in cardiac I/R.

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.