Recovery of respiratory function in mdx mice co-treated with neutralizing interleukin-6 receptor antibodies and urocortin-2.

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-1ß 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-1ß, IL-6, KC/GRO and TNF-α were significantly increased in mdx diaphragm compared with WT. Drug co-treatment significantly decreased IL-1ß 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.

Combined XIL-6R and urocortin-2 treatment restores MDX diaphragm muscle force.

INTRODUCTION: Duchenne muscular dystrophy (DMD) is characterized by progressive muscle degeneration leading to immobility, respiratory failure, and premature death. As chronic inflammation and stress are implicated in DMD pathology, the efficacy of an anti-inflammatory and anti-stress intervention strategy in ameliorating diaphragm dysfunction was investigated. METHODS: Diaphragm muscle contractile function was compared in wild-type and dystrophin-deficient mdx mice treated with saline, anti-interleukin-6 receptor antibodies (xIL-6R), the corticotrophin-releasing factor receptor 2 (CRFR2) agonist, urocortin 2, or both xIL-6R and urocortin 2. RESULTS: Combined treatment with xIL-6R and urocortin 2 rescued impaired force in mdx diaphragms. Mechanical work production and muscle shortening was also improved by combined drug treatment. DISCUSSION: Treatment which neutralizes peripheral IL-6 signaling and stimulates CRFR2 recovers force-generating capacity and the ability to perform mechanical work in mdx diaphragm muscle. These findings may be important in the search for therapeutic targets in DMD. Muscle Nerve 56: E134-E140, 2017.

Stress hormone potentiates Zn(2+)-induced neurotoxicity via TRPM7 channel in dopaminergic neuron.

Zinc toxicity is one of the key factors responsible for the neuronal injuries associated with various neurological conditions. Zinc accumulation in some cells is accompanied by the increase of blood stress hormone levels, which might indicate a functional connection between stress and zinc toxicity. However, the cellular mechanism for the effect of stress on zinc toxicity is not known. Recently, it was reported that the zinc permeable transient receptor potential melastatin 7 (TRPM7) channel may represent a novel target for neurological disorders where zinc toxicity plays an important role. To investigate the effect of stress hormone on zinc-induced cell death, neuroblastoma SH-SY5Y cells were pretreated with urocortin, a corticotropin releasing factor (CRF)-related peptide. Urocortin potentiated zinc-induced cell death at µM range of extracellular zinc concentrations. It significantly increased TRPM7 channel expression, and zinc influx into cytosol. Moreover, application of TRPM7 channel blockers and RNA interference of TRPM7 channel expression attenuated the zinc-induced cell death in urocortin-pretreated cells, indicating that TRPM7 channel may serve as a zinc influx pathway. These results suggest that TRPM7 channel may play a critical role for zinc toxicity associated with stress.

Cardiovascular effects of urocortin 2 and urocortin 3 in patients with chronic heart failure.

AIMS: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Urocortin attenuates myocardial fibrosis in diabetic rats via the Akt/GSK-3ß signaling pathway.

OBJECTIVE: Urocortin, a novel identified corticotropin-releasing factor-related endocrinal peptide, has been shown to play an essential role in cardioprotection. Until recently, whether urocortin can protect the heart against diabetic cardiomyopathy (DCM) remained unclear. Herein, we evaluated the cardioprotective effect of urocortin on cardiac dysfunction, inflammation, and fibrosis and demonstrated the potential mechanism in a diabetic rat model. METHODS: Diabetic rats were randomly divided into 4 groups: diabetic control group, urocortin, urocortin + astressin (a selective CRF receptor 2 antagonist) and urocortin + triciribine (an Akt pathway blocker). Cardiac catheterization was performed to evaluate cardiac function. The levels of creatine phosphokinase isoenzyme (CK-MB), plasma brain natriuretic peptide (BNP), myocardial collagen volume fraction (CVF) and left ventricular mass index (LVWI) were measured. Inflammatory factors (transforming growth factor beta 1, TGF-ß1; connective tissue growth factor, CTGF) and activation of signaling proteins (Akt, GSK-3ß) were also detected using western blot. RESULTS: DCM was successfully induced by the injection of streptozotocin (STZ) as evidenced by abnormal heart mass and cardiac function as well as the imbalance of extracellular matrix homeostasis. Rats in the DCM group showed increased mRNA and protein levels of LVWI, BNP, CK-MB, CVF, TGF-ß1 and CTGF compared to the control group, which were accompanied with diminished phosphorylation of Akt and GSK-3ß. Interestingly, myocardial dysfunction, cardiac fibrosis, and inflammation were suppressed by urocortin in the heart of diabetic rats. Moreover, inhibition of phosphorylation of Akt and GSK-3ß was also reversed by urocortin. These effects of urocortin were suppressed by astressin. In addition, triciribine partially reduced the effects of urocortin on myocardial dysfunction, inflammation, and cardiac fibrosis. CONCLUSIONS: These results suggest that urocortin exhibits a therapeutic benefit in the treatment of DCM by attenuating fibrosis and inflammation. Furthermore, inhibition of the Akt/GSK-3ß signaling pathway may be partially responsible for these effects.

Comparative effects of urocortins and stresscopin on cardiac myocyte contractility.

RATIONALE: There is a current need for the development of new therapies for patients with heart failure. OBJECTIVE: We test the effects of members of the corticotropin-releasing factor (CRF) family of peptides on myocyte contractility to validate them as potential heart failure therapeutics. METHODS AND RESULTS: Adult feline left ventricular myocytes (AFMs) were isolated and contractility was assessed in the presence and absence of CRF peptides Urocortin 2 (UCN2), Urocortin 3 (UCN3), Stresscopin (SCP), and the ß-adrenergic agonist isoproterenol (Iso). An increase in fractional shortening and peak Ca(2+) transient amplitude was seen in the presence of all CRF peptides. A decrease in Ca(2+) decay rate (Tau) was also observed at all concentrations tested. cAMP generation was measured by ELISA in isolated AFMs in response to the CRF peptides and Iso and significant production was seen at all concentrations and time points tested. CONCLUSIONS: The CRF family of peptides effectively increases cardiac contractility and should be evaluated as potential novel therapeutics for heart failure patients.

Intrathecal urocortin I in the spinal cord as a murine model of stress hormone-induced musculoskeletal and tactile hyperalgesia.

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, von Frey fibre sensitivity and grip force after injection of corticotropin-releasing factor (CRF), urocortin I and urocortin II were measured in mice. Urocortin I (a CRF1 and CRF2 receptor ligand) produced hyperalgesia in both assays when injected intrathecally (i.t.) but not intracerebroventricularly, 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 behaviour 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 co-administered i.t. with either NBI-35965 or astressin 2B, they were even more sensitive to inhibition by astressin, a non-selective 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.

Mechanisms of cardiovascular actions of urocortins in the hypothalamic arcuate nucleus of the rat.

The presence of urocortins (UCNs) and corticotropin-releasing factor (CRF) receptors has been reported in the hypothalamic arcuate nucleus (ARCN). We have previously reported that UCNs are involved in central cardiovascular regulation. Based on this information, we hypothesized that the ARCN may be one of the sites where UCNs exert their central cardiovascular actions. Experiments were done in artificially ventilated, adult male Wistar rats anesthetized with urethane. Unilateral microinjections (30 nl) of UCN1 (0.12-2 mM) elicited decreases in mean arterial pressure (MAP) and heart rate (HR). Maximum cardiovascular responses were elicited by a 1 mM concentration of UCN1. Microinjections of UCN2 and UCN3 (1 mM each) into the ARCN elicited similar decreases in MAP and HR. UCN1 was used as a prototype for the other experiments described below. HR responses elicited by UCN1 were significantly attenuated by bilateral vagotomy. Prior microinjections of NBI-27914 (CRF-1 receptor antagonist) and astressin (CRF-1 receptor and CRF-2 receptor antagonist) (1 mM each) into the ARCN significantly attenuated the cardiovascular responses elicited by UCN1 microinjections at the same site. Microinjections of UCN1 into the ARCN decreased efferent renal sympathetic nerve activity. It was concluded that microinjections of UCN1, UCN2, and UCN3 into the ARCN elicited decreases in MAP and HR. Decreases in MAP, HR, and renal sympathetic nerve activity elicited by UCN1 microinjections into the ARCN were mediated via CRF receptors. Bradycardic responses to UCN1 were mediated via the activation of vagus nerves, and decreases in MAP may be mediated via decreases in sympathetic nerve activity.

Urocortin 1 administered into the hypothalamic supraoptic nucleus inhibits food intake in freely fed and food-deprived rats.

Peptides of the corticotropin-releasing hormone/Urocortin (CRH/Ucn) family are known to suppress appetite primarily via CRH(2) receptors. In the rat hypothalamic supraoptic nucleus (SON), synthesis of both Ucn1 and CRH(2) receptors has been reported, yet little is known about the effects of Ucn1 in the SON on feeding behaviour. We first established the dose-related effects of Ucn1 injected into the SON on the feeding response in both freely fed and 24-h food-deprived rats. A conditioned taste avoidance paradigm was performed to investigate possible generalised effects of local Ucn1 treatment. Administration of Ucn1 into the SON at doses equal to or higher than 0.5 µg significantly decreased food intake in both freely fed and food-deprived rats. The Ucn1-mediated suppression of food intake was delayed in freely fed as compared to food-deprived animals. Conditioning for taste aversion to saccharine appeared at 0.5 and 1 µg of Ucn1. Both the early and the delayed onset of anorexia observed after intra-SON injection of Ucn1 under fasting and fed conditions, respectively, suggest the possible involvement of different CRH receptor subtypes in the two conditions, while the conditioned taste aversion seems to be responsible for the initial latency to eat the first meal in these animals.

Alteration of antral and proximal colonic motility induced by chronic psychological stress involves central urocortin 3 and vasopressin in rats.

Because of the difficulties in developing suitable animal models, the pathogenesis of stress-induced functional gastrointestinal disorders is not well known. Here we applied the communication box technique to induce psychological stress in rats and then examined their gastrointestinal motility. We measured upper and lower gastrointestinal motility induced by acute and chronic psychological stress and examined the mRNA expression of various neuropeptides in the hypothalamus. Chronic psychological stress disrupted the fasted motility in the antrum and accelerated motility in the proximal colon. mRNA expression of AVP, oxytocin, and urocortin 3 was increased by chronic psychological stress. Intracerebroventricular (ICV) injection of urocortin 3 disrupted the fasted motility in the antrum, while ICV injection of Ucn3 antiserum prevented alteration in antral motility induced by chronic psychological stress. ICV injection of AVP accelerated colonic motility, while ICV injection of SSR 149415, a selective AVP V1b receptor antagonist, prevented alteration in proximal colonic motility induced by chronic psychological stress. Oxytocin and its receptor antagonist L 371257 had no effect on colonic motility in either the normal or chronic psychological stress model. These results suggest that chronic psychological stress induced by the communication box technique might disrupt fasted motility in the antrum via urocortin 3 pathways and accelerates proximal colonic motility via the AVP V1b receptor in the brain.

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

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

Systemic administration of urocortin after intracerebral hemorrhage reduces neurological deficits and neuroinflammation in rats.

BACKGROUND: Intracerebral hemorrhage (ICH) remains a serious clinical problem lacking effective treatment. Urocortin (UCN), a novel anti-inflammatory neuropeptide, protects injured cardiomyocytes and dopaminergic neurons. Our preliminary studies indicate UCN alleviates ICH-induced brain injury when administered intracerebroventricularly (ICV). The present study examines the therapeutic effect of UCN on ICH-induced neurological deficits and neuroinflammation when administered by the more convenient intraperitoneal (i.p.) route. METHODS: ICH was induced in male Sprague-Dawley rats by intrastriatal infusion of bacterial collagenase VII-S or autologous blood. UCN (2.5 or 25 µg/kg) was administered i.p. at 60 minutes post-ICH. Penetration of i.p. administered fluorescently labeled UCN into the striatum was examined by fluorescence microscopy. Neurological deficits were evaluated by modified neurological severity score (mNSS). Brain edema was assessed using the dry/wet method. Blood-brain barrier (BBB) disruption was assessed using the Evans blue assay. Hemorrhagic volume and lesion volume were assessed by Drabkin's method and morphometric assay, respectively. Pro-inflammatory cytokine (TNF-α, IL-1ß, and IL-6) expression was evaluated by enzyme-linked immunosorbent assay (ELISA). Microglial activation and neuronal loss were evaluated by immunohistochemistry. RESULTS: Administration of UCN reduced neurological deficits from 1 to 7 days post-ICH. Surprisingly, although a higher dose (25 µg/kg, i.p.) also reduced the functional deficits associated with ICH, it is significantly less effective than the lower dose (2.5 µg/kg, i.p.). Beneficial results with the low dose of UCN included a reduction in neurological deficits from 1 to 7 days post-ICH, as well as a reduction in brain edema, BBB disruption, lesion volume, microglial activation and neuronal loss 3 days post-ICH, and suppression of TNF-α, IL-1ß, and IL-6 production 1, 3 and 7 days post-ICH. CONCLUSION: Systemic post-ICH treatment with UCN reduces striatal injury and neurological deficits, likely via suppression of microglial activation and inflammatory cytokine production. The low dose of UCN necessary and the clinically amenable peripheral route make UCN a potential candidate for development into a clinical treatment regimen.

Bradycardic effects of microinjections of urocortin 3 into the nucleus ambiguus of the rat.

The presence of urocortin 3 (UCN3) and CRF2 receptors (CRF2R) has been demonstrated in brain tissue. Nucleus ambiguus (nAmb) is the predominant brain area providing parasympathetic innervation to the heart. On the basis of these reports, it was hypothesized that activation of CRF2Rs in the nAmb may elicit cardiac effects. Experiments were carried out in urethane-anesthetized, artificially ventilated, and adult male Wistar rats. Microinjections of l-glutamate (l-GLU, 5 mM) were used to identify the nAmb. Different concentrations of UCN3 (0.031, 0.062, 0.125, 0.25, and 0.5 mM) microinjected into the nAmb elicited decreases in heart rate (HR) (5.3 ± 1, 22 ± 3.3, 38 ± 4.9, 45.7 ± 2.7, and 27.3 ± 2.3 bpm, respectively). The volume of all microinjections was 30 nl. Blood pressure changes concomitant with decreases in HR were not observed. Bradycardia elicited by microinjections of UCN3 (0.25 mM; maximally effective concentration) into the nAmb was significantly (P < 0.05) attenuated by microinjections of selective CRF2R antagonists (K41498, 0.5 mM, and astressin 2B, 0.25 mM) at the same site. Bilateral vagotomy abolished the bradycardic responses to UCN3. These results indicated that activation of CRF2Rs in the nAmb by UCN3 elicited bradycardia, which was vagally mediated. UCNs have been reported to exert cardioprotective effects in heart failure and ischemia/reperfusion injury. In this situation, centrally induced bradycardia by UCN3 would be beneficial. The results of the present investigation provide a platform for future studies on the role of CRF2Rs in the nAmb in pathological states such as heart failure.

Relevance of urocortins to cardiovascular disease.

Acquired cardiovascular diseases such as coronary heart disease, peripheral artery disease and related vascular problems contribute to more than one-third of worldwide morbidity and mortality. In many instances, particularly in the under developed world, cardiovascular diseases are diagnosed at a late stage limiting the scope for improving outcomes. A range of therapies already exist for established cardiovascular disease, although there is significant interest in further understanding disease pathogenesis in order to improve diagnosis and achieve primary and secondary therapeutic goals. The urocortins are a group of recently defined peptide members of the corticotrophin-releasing factor family. Previous pre-clinical work and human association studies suggest that urocortins have potential to exert some beneficial and other detrimental effects on the heart and major blood vessels. More current evidence however favours beneficial effects of urocortins, for example these peptides have been shown to inhibit production of reactive oxygen species and vascular cell apoptosis, and thus may have potential to antagonise the progression of cardiovascular disease. This review summarises published data on the potential role of urocortins in cardiovascular disease.

Urocortin 1 reduces food intake and ghrelin secretion via CRF(2) receptors.

Although it is known that urocortin 1 (UCN) acts on both corticotropin-releasing factor receptors (CRF(1) and CRF(2)), the mechanisms underlying UCN-induced anorexia remain unclear. In contrast, ghrelin, the endogenous ligand for the growth hormone secretagogue receptor, stimulates food intake. In the present study, we examined the effects of CRF(1) and CRF(2) receptor antagonists (CRF(1)a and CRF(2)a) on ghrelin secretion and synthesis, c-fos mRNA expression in the caudal brain stem, and food intake following intracerebroventricular administration of UCN. Eight-week-old, male Sprague-Dawley rats were used after 24-h food deprivation. Acylated and des-acylated ghrelin levels were measured by enzyme-linked immunosorbent assay. The mRNA expressions of preproghrelin and c-fos were measured by real-time RT-PCR. The present study provided the following important insights into the mechanisms underlying the anorectic effects of UCN: 1) UCN increased acylated and des-acylated ghrelin levels in the gastric body and decreased their levels in the plasma; 2) UCN decreased preproghrelin mRNA levels in the gastric body; 3) UCN-induced reduction of plasma ghrelin and food intake were restored by CRF(2)a but not CRF(1)a; 4) UCN-induced increase of c-fos mRNA levels in the caudal brain stem containing the nucleus of the solitary tract (NTS) was inhibited by CRF(2)a; and 5) UCN-induced reduction of food intake was restored by exogenous ghrelin and rikkunshito, an endogenous ghrelin secretion regulator. Thus, UCN increases neuronal activation in the caudal brain stem containing NTS via CRF(2) receptors, which may be related to UCN-induced inhibition of both ghrelin secretion and food intake.

Microinjections of urocortin1 into the nucleus ambiguus of the rat elicit bradycardia.

Urocortins are members of the hypothalamic corticotropin-releasing factor (CRF) peptide family. Urocortin1 (UCN1) mRNA has been reported to be expressed in the brainstem neurons. The present investigation was carried out to test the hypothesis that microinjections of UCN1 into the nucleus ambiguus (nAmb) may elicit cardiac effects. Urethane-anesthetized, artificially ventilated, adult male Wistar rats, weighing between 300-350 g, were used. nAmb was identified by microinjections of l-glutamate (5 mM, 30 nl). Microinjections (30 nl) of different concentrations (0.062, 0.125, 0.25, and 0.5 mM) of UCN1 into the nAmb elicited bradycardic responses (26.5 ± 1, 30.1 ± 1.7, 46.9 ± 1.7, and 40.3 ± 2.6 beats/min, respectively). These heart rate responses were not accompanied by significant changes in mean arterial pressure. The bradycardic responses to maximally effective concentration of UCN1 (0.25 mM) were significantly (P < 0.05) attenuated by prior microinjections of a selective antagonist (NBI 27914, 1.5 mM) for CRF type 1 receptor (CRF1R). Prior microinjections of ionotropic glutamate receptor (iGLUR) antagonists [d-(-)-2-amino-7-phosphono-heptanoic acid and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo-(f)quinoxaline-7-sulfonamide disodium] also attenuated the bradycardia elicited by UCN1 microinjections into the nAmb. Microinjections of NBI 27914 (1.5 mM) into the nAmb did not alter baroreflex responses. Bilateral vagotomy abolished the bradycardic responses to microinjections of UCN1 into the nAmb. These results indicated that 1) microinjections of UCN1 into the nAmb elicited bradycardia, 2) the bradycardia was vagally mediated, 3) activation of CRF1Rs in the nAmb was responsible for the actions of UCN1, and 4) activation of iGLURs in the nAmb also participated in the bradycardia elicited by UCN1.

Urocortin 3 inhibits cardiac sympathetic nerve activity in conscious sheep.

There is an increasing body of evidence suggesting a role for the urocortin (Ucn) peptides in blood pressure regulation and in the pathophysiology of cardiovascular disease. Both Ucn1 and Ucn2 have recently been shown to inhibit cardiac sympathetic nerve activity (CSNA) in sheep. However, there are few studies reporting the effects of Ucn3 on the sympathetic nervous system. Hence, this study performed in normal conscious sheep examines the effects of Ucn3 on CSNA. Intravenous Ucn3 (administered at doses of 25 and 100 µg) caused transient falls in arterial pressure (P = 0.025), more prolonged rises in heart rate (P < 0.001), and falls in stroke volume (P < 0.001) and peripheral resistance (P = 0.018). CSNA, measured as burst area and burst incidence, fell in a dose-dependent manner after Ucn3 bolus (all P < 0.001). CSNA burst frequency tended to be reduced after high-dose Ucn3. Ucn3 had no significant effect on plasma catecholamine levels. In conclusion, this study reports for the first time that Ucn3 induces potent inhibition of sympathetic traffic directed toward the heart. This provides further support for a role for Ucn3 in pressure and volume homeostasis and suggests that further investigation of potential therapeutic applications for Ucn3 in cardiovascular disease is warranted.

Injection of Urocortin 3 into the ventromedial hypothalamus modulates feeding, blood glucose levels, and hypothalamic POMC gene expression but not the HPA axis.

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.

Urocortin 1 administered into the hypothalamic supraoptic nucleus affects open-field behaviour in rats.

The presence of both Urocortin 1 (Ucn1) and corticotropin-releasing factor 2 receptors (CRF(2)R) in the hypothalamic supraoptic nucleus (SON) suggests that endogenous Ucn1 released within this brain area acts as a local signal that might be involved in the regulation of not only endocrine but also behavioural stress responses. To test this hypothesis, we monitored the effects induced by the administration of a range of doses of synthetic Ucn1 (0.001-1.0 microg) bilaterally into the SON of rats in the open field test (OFT). Ucn1 administration produced an inverted U-shaped dose-response curve on OFT behaviour, in particular the dose of 0.01 microg of Ucn1 significantly increased the number of rearing and grooming episodes without affecting locomotion. In addition, this dosage augmented also the latency to visit the centre of the open field. Pre-treatment with the CRF(2)R antagonist, astressin-2B (0.1 microg) normalized Ucn1 treatment-induced effects. These results suggest that Ucn1 released within the SON area interacts with CRF(2)R to control the state of arousal.

Urocortin prevents indomethacin-induced small intestinal lesions in rats through activation of CRF2 receptors.

PURPOSE: The role of corticotropin-releasing factor (CRF) in the pathogenesis of indomethacin-induced small intestinal lesions was examined in rats. METHODS: Animals were given indomethacin (10 mg/kg) subcutaneously and killed 24 h later. Urocortin I [a nonselective CRF receptor (CRFR) agonist], astressin (a nonselective CRFR antagonist), NBI-27914 (a CRFR1 antagonist), or astressin-2B (a CRFR2 antagonist) was given intravenously 10 min before the administration of indomethacin. RESULTS: Indomethacin caused hemorrhagic lesions in the small intestine, accompanied by intestinal hypermotility, mucosal invasion of enterobacteria, up-regulation of inducible nitric oxide synthase (iNOS) expression, and an increase of mucosal myeloperoxidase (MPO) activity. Pretreatment of the animals with astressin, a non-selective CRFR antagonist, aggravated the lesions in a dose-dependent manner. Likewise, astressin-2B also exacerbated the intestinal ulcerogenic response induced by indomethacin, while NBI-27914 did not. Urocortin I prevented indomethacin-induced intestinal lesions, together with the suppression of bacterial invasion and an increase in mucosal MPO activity and iNOS expression; these effects were significantly reversed by co-administration of astressin-2B but not NBI-27914. Urocortin I suppressed the hypermotility response to indomethacin, and this effect was also abrogated by astressin-2B but not NBI-27914. CONCLUSIONS: These results suggest that urocortin 1 prevents indomethacin-induced small intestinal lesions, and that this action is mediated by the activation of CRFR2 and is functionally associated with the suppression of the intestinal hypermotility response caused by indomethacin. It is assumed that endogenous CRF contributes to the maintenance of the mucosal defensive ability of the small intestine against indomethacin through the activation of CRFR2.