Urocortin 1 and Urocortin 2 induce macrophage apoptosis via CRFR2

Macrophages undergo apoptosis as a mechanism of regulating their activation and the inflammatory reaction. Macrophages express the Corticotropin-Releasing Factor Receptor-2 (CRFR2) the endogenous agonists of which, the urocortins, are also present at the site of inflammation. We have found that urocortins induced macrophage apoptosis in a dose- and time-dependent manner via CRFR2. In contrast to lipopolysaccharide (LPS)-induced apoptosis, the pro-apoptosis pathway activated by urocortins involved the pro-apoptotic Bax and Bad proteins and not nitric oxide, JNK and p38MAPK characteristic of LPS. In conclusion, our data suggest that endogenous CRFR2 ligands exert an anti-inflammatory effect via induction of macrophage apoptosis.     

Urocortin has cell-proliferative effects on cardiac non-myocytes

Urocortin (Ucn) is a member of the corticotropin-releasing hormone (CRH)-related peptides that has been reported to have cardiac inotropic and hypertrophic effects. In addition, Ucn mRNA was expressed in cardiac myocytes (MCs) and Ucn was suggested to have cardioprotective effects. Recently, it was reported that Ucn mRNA was expressed in cardiac non-myocytes (NMCs). Based on these facts, Ucn is assumed to affect not only MCs but also NMCs in an autocrine fashion. The present study was designed to elucidate a pathophysiological role of Ucn on NMCs. NMCs were prepared by the discontinuous Percoll gradient and adhesion method. Ucn increased [(3)H]-thymidine uptake into NMCs. Ucn also enhanced endothelin-1-induced increase of [(3)H]-thymidine uptake into NMCs. Effects of Ucn on [(3)H]-thymidine uptake into NMCs were significantly abolished by the protein kinase A inhibitor, H89 (10(-5) M), but not by a competitive antagonist of CRH receptors, astressin (10(-5) M). Ucn also increased intracellular cAMP accumulation more potently than CRH on a molar basis. Finally, both MCs and NMCs also secreted Ucn. Together with the recent findings, at least in NMCs, these data suggest that Ucn could exert its own actions via the cAMP signaling pathway, but not through known CRH type 2 receptors, in an autocrine fashion. In conclusion, the present study indicated that Ucn was secreted not only from MCs but also from NMCs and that the primary source of Ucn acting on heart was the heart itself. On the other hand, Ucn could proliferate NMCs as well as MCs, suggesting that Ucn could be involved in cardiac hypertrophy and fibrosis, i.e., cardiac remodeling, in spite of its putative cardioprotective actions.     

Urocortin in human placenta and maternal plasma.

Plasma immunoreactive (IR-) urocortin (Ucn) and corticotropin-releasing factor (CRF) levels in pregnant women were measured by their specific radioimmunoassays after extraction. Although plasma IR-CRF levels were increased in pregnant women as compared to men and non-pregnant women, there was no difference of plasma IR-Ucn levels among groups. Ucn mRNA was detected in cytotrophoblasts and syncytiotrophoblasts by in situ hybridization. A reverse-phase high-performance liquid chromatography (HPLC) showed the major peak of IR-Ucn in placenta and plasma that had similar chromatographic mobility to synthetic Ucn1-40. These data suggest that Ucn is produced and processed into the same form of synthetic Ucn in placenta, but not secreted into maternal blood.     

Urocortin 2 induces tyrosine hydroxylase phosphorylation in PC12 cells

Urocotins (Ucns) are newly discovered members of the corticotropin-releasing factor (CRF) neuropeptide family. Ucn 2 is expressed in the adrenal medulla, and its receptor, CRF2 receptor, is also expressed in the adrenal gland. To predict the physiological significance of Ucn 2 expression in the adrenal medulla, we examined the effects of Ucn 2 on catecholamine secretion and intracellular signaling using PC12 cells, a rat pheochromocytoma cell line. PC12 cells were found to express CRF2 receptor, but not CRF1 receptor. Treatment with Ucn 2 increased noradrenaline secretion and induced phosphorylation of PKA and Erk1/2. Tyrosine hydroxylase (TH), a rate-limiting enzyme for catecholamine synthesis, was also phosphorylated by Ucn 2. Pretreatment with a PKA inhibitor blocked Ucn 2-induced NA secretion, and Erk1/2 and TH phosphorylation. Pretreatment with a MEK inhibitor did not block Ucn 2-induced noradrenaline secretion or PKA phosphorylation, although TH phosphorylation was blocked. Thus, Ucn 2 induces noradrenaline secretion and TH phosphorylation through the PKA pathway and the PKA-Erk1/2 pathway, respectively. These results suggest Ucn 2 in the adrenal gland may be involved in the regulation of catecholamine release and synthesis.     

Urocortin 3 elevates cytosolic calcium in nucleus ambiguus neurons

J. Neurochem. (2012) 122, 1129-1136. ABSTRACT: Urocortin 3 (also known as stresscopin) is an endogenous ligand for the corticotropin-releasing factor receptor 2 (CRF(2) ). Despite predominant G(s) coupling of CRF(2) , promiscuous coupling with other G proteins has been also associated with the activation of this receptor. As urocortin 3 has been involved in central cardiovascular regulation at hypothalamic and medullary sites, we examined its cellular effects on cardiac vagal neurons of nucleus ambiguus, a key area for the autonomic control of heart rate. Urocortin 3 (1?nM-1000?nM) induced a concentration-dependent increase in cytosolic Ca(2+) concentration that was blocked by the CRF(2) antagonist K41498. In the case of two consecutive treatments with urocortin 3, the second urocortin 3-induced Ca(2+) response was reduced, indicating receptor desensitization. The effect of urocortin 3 was abolished by pre-treatment with pertussis toxin and by inhibition of phospolipase C with U-73122. Urocortin 3 activated Ca(2+) influx via voltage-gated P/Q-type channels as well as Ca(2+) release from endoplasmic reticulum. Urocortin 3 promoted Ca(2+) release via inositol 1,4,5 trisphosphate receptors, but not ryanodine receptors. Our results indicate a novel Ca(2+) -mobilizing effect of urocortin 3 in vagal pre-ganglionic neurons of nucleus ambiguus, providing a cellular mechanism for a previously reported role for this peptide in parasympathetic cardiac regulation.     

Urocortin modulates inflammatory response and neurotoxicity induced by microglial activation

Microglia are the major inflammatory cells in the brain. Recent studies have highlighted the reciprocal roles of other brain cells in modulating the microglial inflammatory responses. Urocortin (UCN) is a member of the corticotropin-releasing hormone (CRH) family of neuropeptides that function to regulate stress responses. In the present study, we demonstrated that expression of UCN in rat substantia nigra was found to be localized principally to dopaminergic neurons. In cell culture models, the CRH receptors were expressed in microglia, and CRHR expression was up-regulated by treatment with LPS. Thus, it might be proposed that UCN regulates cellular communication between dopaminergic neurons and microglia. We show that femtomolar concentrations of UCN could inhibit LPS-induced TNF-alpha production in cultured microglia. Investigation of the underlying signaling pathway that mediated the anti-inflammatory effect of UCN the involved PI3K/Akt and glycogen synthase kinase-3beta pathway, but not cAMP pathway. Furthermore, UCN protected dopaminergic neurons against LPS-induced neurotoxicity by inhibiting microglial activation in LPS-treated mesencephalic neuron-glia cultures. These results suggest that endogenous UCN and its receptors might be involved in a complex network of paracrine interaction between dopaminergic neurons and glia.     

Urocortin protects coronary endothelial function during ischemia-reperfusion: a brief communication

Urocortin is a vasodilator peptide related to corticotrophin-releasing factor, which may protect myocardium during coronary ischemia-reperfusion. To study whether urocortin also protects coronary endothelial function during ischemia-reperfusion, hearts from Sprague-Dawley rats were perfused at constant flow and then exposed to 15 mins ischemia followed by 15 mins reperfusion. In one series of experiments, we found that the coronary relaxation to urocortin (10(-11) to 10(-8) M) was reduced by ischemia-reperfusion (51 +/- 4% vs. 79 +/- 4% of the active tone, for the 10(-10) Mdose). In other series of experiments, we observed that ischemia-reperfusion reduced the coronary relaxation to a test dose of acetylcholine (10(-6) M) (25 +/- 2% vs. 54 +/- 9% of active tone), without modifying the relaxation to sodium nitroprusside (10(-6) M). Treatment with a low threshold concentration of urocortin (10(-11) M), administered before ischemia and during reperfusion, partly improved the coronary relaxation to acetylcholine (36 +/- 3% of active tone). These results suggest that ischemia-reperfusion impairs the coronary vasodilation to urocortin and produces endothelial dysfunction and that this endothelial dysfunction may be improved by urocortin.     

Urocortin protects against ischemic and reperfusion injury via a MAPK-dependent pathway

Urocortin (UCN) is a peptide related to hypothalamic corticotrophin-releasing hormone and binds with high affinity to corticotrophin-releasing hormone receptor-2beta, which is expressed in the heart. In this study, we report that UCN prevented cell death when administered to primary cardiac myocyte cultures both prior to simulated hypoxia/ischemia and at the point of reoxygenation after simulated hypoxia/ischemia. UCN-mediated cell survival was measured by trypan blue exclusion, 3'-OH end labeling of DNA (TUNEL), annexin V, and fluorescence-activated cell sorting. To explore the mechanisms that could be responsible for this effect, we investigated the involvement of MAPK-dependent pathways. UCN caused rapid phosphorylation of ERK1/2-p42/44, and PD98059, which blocks the MEK1-ERK1/2-p42/44 cascade, also inhibited the survival-promoting effect of UCN. Most important, UCN reduced damage in isolated rat hearts ex vivo subjected to regional ischemia/reperfusion, with the protective effect being observed when UCN was given either prior to ischemia or at the time of reperfusion after ischemia. This suggests a novel function of UCN as a cardioprotective agent that could act when given after ischemia, at reperfusion.     

Urocortin and corticotropin-releasing factor receptor expression in the human colonic mucosa

Urocortin is a newly identified member of the CRF neuropeptide family. Urocortin has been found to bind with high affinity to CRF receptors. The present study investigated urocortin and CRF receptor expression in human colonic mucosa. Non-pathologic sections of adult colorectal tissues were obtained from patients with colorectal cancer at surgery. Urocortin expression was examined using immunohistochemistry and messenger (m) RNA in situ hybridization. Isolated lamina propria mononuclear cells (LPMC) and epithelial cells were also analyzed by flow cytometry for the characterization of urocortin-positive cells, and by RT-PCR for detection of urocortin, CRF, and CRF receptor mRNA. Urocortin peptide distribution at various stages of human development (n = 35, from 11 weeks of gestation to 6 years of age) was examined by immunohistochemistry using surgical and autopsy specimens. Immunoreactive urocortin and urocortin mRNA were predominantly detected in lamina propria macrophages. Urocortin peptide expression was detected from as early as three months of age, but not before birth or in neonates. Urocortin, CRF receptor type 1 and type 2 mRNA were detected in LPMC. CRF receptor type 2β mRNA, a minor isoform in human tissues, was also detected in LPMC, but at lower levels. Urocortin is locally synthesized in lamina propria macrophages and may act on lamina propria inflammatory cells as an autocrine/paracrine regulator of the mucosal immune system. The appearance of urocortin after birth indicates that the exposure to dietary intake and/or luminal bacteria after birth may contribute to the initiation of urocortin expression in human gastrointestinal tract mucosa.     

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 (CRF₂R) 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 μg) 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 μg 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 CRF₂R antagonist, astressin-2B (0.1 μg) normalized Ucn1 treatment-induced effects. These results suggest that Ucn1 released within the SON area interacts with CRF₂R to control the state of arousal.     

Vasoprotective Effects of Urocortin 1 against Atherosclerosis In Vitro and In Vivo

Aim

Atherosclerosis is the complex lesion that consists of endothelial inflammation, macrophage foam cell formation, vascular smooth muscle cell (VSMC) migration and proliferation, and extracellular matrix production. Human urocortin 1 (Ucn1), a 40-amino acid peptide member of the corticotrophin-releasing factor/urotensin I family, has potent cardiovascular protective effects. This peptide induces potent and long-lasting hypotension and coronary vasodilation. However, the relationship of Ucn1 with atherosclerosis remains unclear. The present study was performed to clarify the effects of Ucn1 on atherosclerosis.

Methods

We assessed the effects of Ucn1 on the inflammatory response and proliferation of human endothelial cells (ECs), human macrophage foam cell formation, migration and proliferation of human VSMCs, extracellular matrix expression in VSMCs, and the development of atherosclerosis in apolipoprotein E-deficient (Apoe ^−/−) mice.

Results

Ucn1 significantly suppressed cell proliferation without inducing apoptosis, and lipopolysaccharide-induced up-regulation of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in human ECs. Ucn1 significantly reduced oxidized low-density lipoprotein-induced foam cell formation with a significant down-regulation of CD36 and acyl-CoA:cholesterol acyltransferase 1 in human monocyte-derived macrophages. Ucn1 significantly suppressed the migration and proliferation of human VSMCs and increased the activities of matrix metalloproteinase-2 (MMP2) and MMP9 in human VSMCs. Intraperitoneal injection of Ucn1 into Apoe ^−/− mice for 4 weeks significantly retarded the development of aortic atherosclerotic lesions.

Conclusions

This study provided the first evidence that Ucn1 prevents the development of atherosclerosis by suppressing EC inflammatory response and proliferation, macrophage foam cell formation, and VSMC migration and proliferation. Thus, Ucn1 could serve as a novel therapeutic target for atherosclerotic cardiovascular diseases.     

Urocortin 2 expression in the rat gastrointestinal tract under basal conditions and in chemical colitis

It is becoming increasingly evident that the urocortins (Ucns) and their receptors are involved in the initiation and development of inflammation in the gastrointestinal (GI) tract. There has not been a systematic study of the basal expression of Ucns or their receptors in the GI tract. Here, we examined basal expression of Ucn 2 and its high-affinity receptor, CRF-R2 in the rat GI tract. Ucn 2 mRNA was expressed throughout the small and large intestine. Surprisingly, CRF-R2 mRNA expression was detected in only a subset of GI regions that expressed Ucn 2. Immunohistochemical study showed that both Ucn 2 immuno-reactivity (Ucn 2-IR) and CRF-R2-IR were consistently seen in the neurons of the myenteric plexus and the nerve fibers innervating the circular muscle. By and large, Ucn 2-IR was detected in all layers, including the mucosal and the submucosal layers throughout the GI regions. In contrast, CRF-R2-IR was very low or undetectable in the mucosal layers of all regions examined. The role of Ucn 2 and CRF-R2 was then examined in a rat model of chemically-induced colitis. In the early phase of colitis, Ucn 2 mRNA levels peaked, whereas, in striking contrast, CRF-R2 mRNA expression decreased approximately 2.5-fold below control levels. At the peptide level, Ucn 2-IR was specifically induced in a large population of immune cells that infiltrated the lamina propria and submucosa of the distal colon, whereas CRFR2-IR was detected in only a small fraction of infiltrated immune cells. CRF-R2-IR was dramatically reduced in the neurons of the myenteric plexus. Thus, we show, for the first time, that in the acute phase of inflammation, Ucn 2 levels are increased whereas expression levels of its only identified receptor, CRF-R2, are decreased. This suggests that Ucn 2 exerts its effects only in part via CRF-R2.     

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.     

Urocortin 1 administration from onset of rapid left ventricular pacing represses progression to overt heart failure

Urocortin 1 (Ucn1) may be involved in the pathophysiology of heart failure (HF), but the impact of Ucn1 administration on progression of the disease is unknown. The aim of this study was to investigate the effects of Ucn1 in sheep from the onset of cardiac overload and during the subsequent development of HF. Eight sheep underwent two 4-day periods of HF induction by rapid left ventricular pacing (225 beats/min) in conjunction with continuous infusions of Ucn1 (0.1 microg.kg(-1).h(-1) iv) and a vehicle control (0.9% saline). Compared with control, Ucn1 attenuated the pacing-induced decline in cardiac output (2.43 +/- 0.46 vs. 3.70 +/- 0.89 l/min on day 4, P < 0.01) and increases in left atrial pressure (24.9 +/- 1.0 vs. 11.9 +/- 1.1 mmHg, P < 0.001) and peripheral resistance (38.7 +/- 9.4 vs. 25.2 +/- 6.1 mmHg.l(-1).min, P < 0.001). Ucn1 wholly prevented increases in plasma renin activity (4.02 +/- 1.17 vs. 0.87 +/- 0.1 nmol.l(-1).h(-1), P < 0.001), aldosterone (1,313 +/- 324 vs. 413 +/- 174 pmol/l, P < 0.001), endothelin-1 (3.8 +/- 0.5 vs. 2.0 +/- 0.1 pmol/l, P < 0.001), and vasopressin (10.8 +/- 4.1 vs. 1.8 +/- 0.2 pmol/l, P < 0.05) during pacing alone and blunted the progressive increases in plasma epinephrine (2,132 +/- 697 vs. 1,250 +/- 264 pmol/l, P < 0.05), norepinephrine (3.61 +/- 0.73 vs. 2.07 +/- 0.52 nmol/l, P < 0.05), and atrial (P < 0.05) and brain (P < 0.01) natriuretic peptide levels. Ucn1 administration also maintained urine sodium excretion (0.75 +/- 0.34 vs. 1.59 +/- 0.50 mmol/h on day 4, P < 0.05) and suppressed pacing-induced declines in creatinine clearance (P < 0.05). These findings indicate that Ucn1 treatment from the onset of cardiac overload has the ability to repress the ensuing hemodynamic and renal deterioration and concomitant adverse neurohumoral activation, thereby delaying the development of overt HF. These data strongly support a use for Ucn1 as a therapeutic option early in the course of the disease.     

Urocortin in the lateral septal area modulates feeding induced by orexin A in the lateral hypothalamus

The intermediate portion of the lateral septum (LSi) contains high levels of urocortin (UCN) peptide and type 2 corticotropin-releasing hormone (CRH) receptor (CRHR2) and has anatomic and functional connections with the lateral hypothalamus (LH). We tested the effect of UCN in the LSi on feeding. Injection of 10 or 30 pmol UCN into LSi significantly decreased feeding in food-deprived rats for 24 h without producing conditioned taste aversion (CTA). Pretreatment with a CRH receptor antagonist, alpha-helical CRH (alpha-hCRH), blocked the inhibitory effect of UCN on deprivation-induced feeding at 1 and 2 h postinjection. Furthermore, UCN in the LSi significantly decreased feeding induced by LH-injected orexin A at 2 and 4 h postinjection, and addition of alpha-hCRH blocked the inhibitory effect of UCN on orexin A-induced feeding. In conclusion, UCN significantly inhibits feeding induced by deprivation and LH-injected orexin A without producing a CTA, an effect that is mediated by CRHR2. These data define the LSi as an important site for UCN-induced anorexia and indicate that LSi UCN may influence orexin A feeding signals in the LH.     

Urocortin 1 modulates immunosignaling in a rat model of colitis via corticotropin-releasing factor receptor 2

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

Urocortin, a member of the corticotropin-releasing factor family, in normal and diseased heart

In the present study we investigated the form of expression, action, second messenger, and the cellular location of urocortin, a member of the corticotropin-releasing factor (CRF) family, in the heart. Urocortin mRNA, as shown by quantitative RT-PCR analysis, is expressed in the cultured rat cardiac nonmyocytes (NMC) as well as myocytes (MC) in the heart, whereas CRF receptor type 2beta (CRF-R2beta), presumed urocortin receptor mRNA, is predominantly expressed in MC compared with NMC. Urocortin mRNA expression is higher in left ventricular (LV) hypertrophy than in normal LV, whereas CRF-R2beta mRNA expression is markedly depressed in LV hypertrophy compared with normal LV. Urocortin more potently increased the cAMP levels in both MC and NMC than did CRF, and its effect was more potent in MC than in NMC. Urocortin significantly increased protein synthesis by [(14)C]Phe incorporations and atrial natriuretic peptide secretion in MC and collagen and increased DNA synthesis by [(3)H]prolin and [(3)H]Thy incorporations in NMC. An immunohistochemical study revealed that urocortin immunoreactivity was observed in MC in the normal human heart and that it was more intense in the MC of the human failing heart than in MC of the normal heart. These results, together with the recent evidence of urocortin for positive inotropic action, suggest that increased urocortin in the diseased heart may modulate the pathophysiology of cardiac hypertrophy or failing heart, at least in part, via cAMP signaling pathway.     

Urocortin 3 Marks Mature Human Primary and Embryonic Stem Cell-Derived Pancreatic Alpha and Beta Cells

The peptide hormone Urocortin 3 (Ucn 3) is abundantly and exclusively expressed in mouse pancreatic beta cells where it regulates insulin secretion. Here we demonstrate that Ucn 3 first appears at embryonic day (E) 17.5 and, from approximately postnatal day (p) 7 and onwards throughout adult life, becomes a unifying and exclusive feature of mouse beta cells. These observations identify Ucn 3 as a potential beta cell maturation marker. To determine whether Ucn 3 is similarly restricted to beta cells in humans, we conducted comprehensive immunohistochemistry and gene expression experiments on macaque and human pancreas and sorted primary human islet cells. This revealed that Ucn 3 is not restricted to the beta cell lineage in primates, but is also expressed in alpha cells. To substantiate these findings, we analyzed human embryonic stem cell (hESC)-derived pancreatic endoderm that differentiates into mature endocrine cells upon engraftment in mice. Ucn 3 expression in hESC-derived grafts increased robustly upon differentiation into mature endocrine cells and localized to both alpha and beta cells. Collectively, these observations confirm that Ucn 3 is expressed in adult beta cells in both mouse and human and appears late in beta cell differentiation. Expression of Pdx1, Nkx6.1 and PC1/3 in hESC-derived Ucn 3⁺ beta cells supports this. However, the expression of Ucn 3 in primary and hESC-derived alpha cells demonstrates that human Ucn 3 is not exclusive to the beta cell lineage but is a general marker for both the alpha and beta cell lineages. Ucn 3⁺ hESC-derived alpha cells do not express Nkx6.1, Pdx1 or PC1/3 in agreement with the presence of a separate population of Ucn 3⁺ alpha cells. Our study highlights important species differences in Ucn 3 expression, which have implications for its utility as a marker to identify mature beta cells in (re)programming strategies.     

Urocortin reduces the viability of adult rat vascular smooth muscle cells via inhibiting L-type calcium channels

The newly isolated peptide, urocortin (UCN), is a member of the corticotropin-releasing factor (CRF)-related peptides that has been found to have potent cardiovascular protective effects. In order to investigate the effect of UCN on the viability of adult rat vascular smooth muscle cells (VSMC) and the relevant mechanisms, we exposed the VSMC to UCN to observe the change in cell viability using MTT assay and intracellular calcium concentration using confocal laser scanning microscope methods. Our results showed that UCN (10(-7)M) inhibited the viability of VSMC by about 26% (P<0.05, compared to control). The effect was concentration-dependent, but it was not dependent on the affecting time. Glybenclamide (Gly, 10(-5)M), the ATP-sensitive potassium channel (K(ATP) channel) blocker, and astressin (10(-6)M), a competitive antagonist of CRF receptors, had no influence on this inhibition. Bay K8644 (10(-6)M), a special L-type calcium channel activator, increased the viability of VSMC. Pre-treatment of the cells with UCN diminished the effect of Bay K8644 (n=6, P<0.05). UCN was also observed to reduce the intracellular Ca2+ increase induced by KCl and Bay K8644. There was no significant difference in nitrite accumulation between UCN groups and the control. In conclusion, UCN reduced the viability of VSMC through L-type calcium channels. These interesting results might suggest that UCN may be a new vasoactive agent involved in hindering vascular remodeling in combination with previous reports about UCN's hypotensive effects.