Urocortin-positive nerve fibres and cells are present in the human choroid.

BACKGROUND: Choroidal vascular regulation is mediated by the autonomic nervous system in order to gain proper blood flow control. While the mechanisms behind this control are unknown, neuroregulatory peptides are involved in this process. To better understand choroidal function, we investigate the presence of urocortin-1 (UCN), a neuroregulatory peptide with vascular effects, in the human choroid and its possible intrinsic and extrinsic origin. METHODS: Human choroid and eye-related cranial ganglia (superior cervical ganglion- SCG, ciliary ganglion-CIL, pterygopalatine ganglion-PPG, trigeminal ganglion-TRI) were prepared for immunohistochemistry against UCN, protein-gene product 9.5 (PGP9.5), substance P (SP), tyrosine hydroxylase (TH) and vesicular acetylcholine transporter (VAChT). For documentation, confocal laser scanning microscopy was used. RESULTS: In choroidal stroma, UCN-immunoreactivity was present in nerve fibres, small cells and intrinsic choroidal neurons (ICN). Some UCN+ nerve fibres colocalised for VAChT, while others were VAChT. A similar situation was found with SP: some UCN+ nerve fibres showed colocalisation for SP, while others lacked SP. Colocalisation for UCN and TH was not observed. In eye-related cranial ganglia, only few cells in the SCG, PPG and TRI were UCN+, while many cells of the CIL displayed weak UCN immunoreactivity. CONCLUSION: UCN is part of the choroidal innervation. UCN+/VAChT+ fibres could derive from the few cells of the PPG or cells of the CIL, if these indeed supply the choroid. UCN+/SP+ fibres might originate from ICN, or the few UCN+ cells detected in the TRI. Further studies are necessary to establish UCN function in the choroid and its implication for choroidal autonomic control.

Urocortin 1 inhibits guinea pig gallbladder contractility in vitro via corticotropin-releasing factor receptor 2.

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

Cardiac release of urocortin precedes the occurrence of irreversible myocardial damage in the rat heart exposed to ischemia/reperfusion injury.

This study evaluates whether cardiac ischemia induces release of urocortin, before and independently from myocyte cell death. Urocortin levels rose after 5-min ischemia and peaked after 10-min ischemia, when cell death was not detected. However, myocyte apoptosis and/or necrosis occurred following 20- and 30-min ischemia, which paralleled a fall in urocortin levels, suggesting that urocortin expression and release are mainly sustained by metabolically challenged, though still viable myocytes. Hence, since cardiac release of urocortin, unlike that of conventional biomarkers, occurs before and apart from cell death, urocortin levels may be clinically useful in the diagnosis of sublethal myocardial ischemia.

A case of multiple endocrine neoplasia type II accompanied by thyroid medullary carcinoma and pheochromocytomas expressing corticotropin-releasing factor and urocortins.

A 38-year-old woman with RET gene mutation presented with tumors in her thyroid and bilateral adrenal glands. I-metaiodobenzylguanidine scintigraphy revealed accumulation of the radioisotope in both adrenal glands. Both plasma adrenaline and noradrenaline levels were elevated. The circadian rhythms for plasma adrenocorticotropic hormone (ACTH) and cortisol levels were disturbed. Plasma ACTH and cortisol levels failed to be suppressed by an overnight dexamethasone test, suggesting autonomic secretion of ACTH and cortisol, although the patient had no typical Cushingoid features, hypertension, or impaired glucose tolerance. Pathological examination showed that these tumors were pheochromocytoma and thyroid medullary carcinoma, respectively, both of which highly expressed corticotropin-releasing factor, urocortin1, and urocortin3. Together with the endocrinological and pathological observations, the patient was diagnosed as multiple endocrine neoplasia type II with corticotropin-releasing factor- and urocortin-producing tumors that stimulated ACTH and glucocorticoid secretion.

Urocortin 1 in colonic mucosa in patients with ulcerative colitis.

Ulcerative colitis (UC) is characterized by a long-standing chronic inflammation of the bowel with intermittent periods of exacerbation and remission. Its acute exacerbation appears to be related to various stresses. Urocortin 1 (Ucn1) may play important roles in integrated local responses to stress. We therefore examined local production of Ucn1 in patients with UC by immunohistochemistry and mRNA in situ hybridization. Ucn1 immunoreactivity was predominantly detected in lamina propria plasma cells and enterochromaffin cells. In UC patients without glucocorticoid treatment, Ucn1-positive cells and plasma cells increased in proportion to the severity of inflammation (P < 0.0001). Ucn1-positive cells significantly increased in UC patients with advanced inflammatory grades, compared with a control group (P < 0.0001) and nonspecific colitis group (P < 0.0001). In glucocorticoid-treated patients, Ucn1-positive cells were significantly lower in number, compared with the nonglucocorticoid-treated group. Ucn1 mRNA was expressed in lamina propria plasma cells, and both corticotropin-releasing factor(1) and corticotropin-releasing factor(2(a)) mRNAs were also partially coexpressed in these cells and macrophages. The present study showed that Ucn1-positive cells were correlated with the severity of inflammation in colonic mucosa with UC, and glucocorticoid treatment decreased these cells. Ucn1 therefore may act as a possible local immune-inflammatory mediator in UC.

A complete corticotropin releasing factor system localized in human fetal lung.

OBJECTIVE: The Corticotropin Releasing Factor (CRF) system (neuropeptides CRF, Ucn I, II, III and binding sites CRFR1, CRFR2, CRF-BP) is responsible for stress regulation and the homeostasis of an organism. Herein we study the CRF system in human normal and pathological fetal lungs. DESIGN: Lung tissues from 46 archival human fetuses were divided into Group A (normal), Group B (chromosomal abnormalities) and Group C (congenital disorders). Presence of elements of the CRF system was evaluated using immunohistochemistry and was correlated to pathology, lung developmental stage and clinicopathological characteristics. RESULTS: Immunoreactivity for all antigens was found in both epithelial and mesenchymal lung cells of the bronchi and alveoli. Ucn I and CRFR1 were more frequently present in Group A. Ucns were more frequently localized at the pseudoglandular stage. There was a positive correlation between the presence of the CRF neuropeptides and between CRFR1 and CRF. Two fetuses with lung malformations showed low or no detectable presence of the CRF system. CONCLUSIONS: We report the presence of a complete CRF system in human fetal lungs correlating its developmental stage and several pathologies. Our results are in agreement with findings in experimental animal models, implicating the CRF system in fetal lung development, its action being more significant in the early stages.

Evaluation of the effect of natural peptide 'Urocortin' on corticotrophin releasing factor (CRF) receptor expression in ND7/23 cells

CRF receptors are involved in the stress management of the cells and are believed to have a cytoprotective role in the body. CRF receptors have been reported to be potential drug targets for the treatment of neurodegenerative disorders. The cell line used in the study is ND7/23 (mouse neuroblastoma and rat dorsal root ganglion neuron hybridoma). The aim of the study was to confirm the expression of CRF receptors in ND7/23 cells and to determine if urocortin (Ucn) can enhance the expression of CRF receptors. ND7/23 cells were cultured in RPMI 1640 media and cells grown after the second passage were used for the experiments. RNA was extracted from the cells and amplified by RT-PCR to confirm the presence of CRF receptors. The cells were then subjected to oxidative stress by hydrogen peroxide (0.00375%) and divided into two groups i.e. control and Ucn (10-8 μM) treated. Later RNA was extracted from both group of cells and PCR was performed. Finally, densitometry analysis was conducted on the agarose gel to determine the quantity of PCR product formed. PCR experiment confirmed the expression of both CRF-R1 and CRF-R2 in the cell line, but CRF-R1 was found to be expressed more strongly. Densitometry analysis of the PCR product and calculation of the relative expression of CRF receptors indicated a higher level of expression of CRF receptors in samples treated with Ucn as compared to those that were kept untreated. The results indicate that Ucn may be useful for the management of neuro-degenerative disorders and further studies may be carried out to establish its use as a therapeutic agent.

Receptores de CRF estão envolvidos na gestão do estresse das células e são acreditados para ter um papel de cito-proteção no organismo. Os receptores do CRF têm sido relatados como alvos potenciais de fármacos para o tratamento de doenças neurodegenerativas. A linhagem celular utilizada no estudo é ND7/23 (neuroblastoma de camundongo e hibridoma de raíz dorsal do neurônio ganglionar de rato). O objetivo do estudo foi confirmar o que a expressão de receptores de CRF em células ND7/23 determinar se urocortina (Ucn) pode aumentar a expressão de receptores de CRF. Cultivaram-se células ND7/23 em meio RPMI 1640 e as células que cresceram após a segunda passagem foram usadas para os experimentos. O RNA foi extraído células e amplificado por RT-PCR para confirmar a presença de receptores de CRF. As células foram, então, submetidas a estresse oxidativo por peróxido de hidrogênio (0.00375 %) e divididas em dois grupos, ou seja, controle e tratadas com UCN (10-8 µM). Em seguida, o RNA foi extraído de ambos os grupo de células e realizou-se o PCR. Finalmente, realizou-se análise densitométrica em gel de agarose para determinar a quantidade de produto formado por PCR. O PCR confirmou a expressão de CRF-R1 e CRF-R2 na linhagem celular, mas o CRF-R1 expresso mais fortemente. A análise densitométrica do produto de PCR e o cálculo da expressão relativa de receptores de CRF indicaram um nível mais elevado de expressão de receptores de CRF em amostras tratadas com Ucn, em comparação com aqueles sem tratamento. Os resultados indicam que a Ucn pode ser útil no tratamento de doenças neurodegenerativas e mais estudos podem ser realizados para estabelecer seu uso como agente terapêutico.

Acute effects of urocortin 2 on cardiac function and propensity for arrhythmias in an animal model of hypertension-induced left ventricular hypertrophy and heart failure.

AIMS: To test acute effects of the corticotropin-releasing factor-related peptide urocortin 2 (Ucn2) on left ventricular (LV) function and the propensity for ventricular arrhythmias in the isolated heart of an animal model of hypertension-induced heart failure. METHODS AND RESULTS: Hearts from Dahl salt-sensitive rats with severe LV dysfunction were perfused according to Langendorff. Left ventricular developed pressure and the positive and negative derivatives of LV pressure were analysed before and after perfusion with Ucn2 (n = 15) or normal perfusion solution (control, n = 9). Intracellular calcium cycling parameters were assessed by surface fluorometry. Furthermore, monophasic action potential duration (MAPD) and ventricular fibrillation threshold (VFT) were determined, the latter by a train-of-pulses method at increasing voltage to scan the vulnerable period of repolarization. Urocortin 2 significantly affected intracellular calcium cycling and improved LV contractile function and relaxation. Compared with baseline values, Ucn2 significantly decreased MAPD at 30, 50, and 90% repolarization and significantly increased VFT compared with baseline values. No changes were observed in control experiments. CONCLUSION: Administration of Ucn2 rapidly improves LV function and increases VF threshold in failing, isolated rat hearts with increased propensity for ventricular arrhythmias. These observations suggest a potential use of Ucn2 as a safe and novel agent for the treatment of acute heart failure.

Placental urocortins and CRF in late gestation.

Placental corticotropin-releasing factor (CRF) are thought to induce labor via activation of CRF receptor type 1 (CRF-R1) leading to several feed forward mechanisms in the placental, fetal and maternal compartments. Recently, receptor type 2 (CRF-R2) selective ligands called urocortin 2 and 3 (Ucn 2, Ucn 3) were characterized as neuropeptides in the brain. We studied the expression of Ucn 1, 2 and 3 in feto-placental tissues qualitatively (by immunohistochemistry) and quantitatively (by radioimmunoassay) and compared these with expression of placental CRF. The presented placental Ucn 2 and 3 peptide quantification, characterization and ex-vivo release results are novel. Reversed-phase HPLC fractionation of placental extracts revealed several peaks containing immune-reactive (ir)-like Ucn 2 or 3, of which the main peaks had the same retention time as the synthetic Ucn 2 and 3 peptides. Placental tissues contained between 6 and 10 times more ir-CRF than ir-Ucn 1, 2 or 3. The placental Ucn 1, 2 and 3 peptide contents correlated with each other. Our immunohistochemical results showed that all urocortins were mainly localized in the syncytiotrophoblasts of the placental villi. Placental urocortins were actively released during ex-vivo perfusion of cotyledons. From these results it can be concluded that Ucn 2 and 3 peptides are present in placental and fetal membrane tissues, and released by ex-vivo perfused cotyledons. Therefore, placental urocortins may function as paracrine or endocrine messengers during pregnancy and parturition.

Superiority of a new gradient system utilizing a critical threshold for the adsorption capacity of polypeptides to column packing when compared with a standard gradient system for the simultaneous and quantitative analysis of polypeptides.

Compared with a standard gradient system, the new gradient system which we developed has a major advantage because it permits a wide range of acetonitrile content, e.g. more than the critical threshold, in the polypeptide solution and allows the quantitative analysis of the polypeptide with satisfactory analytical precision. Additionally, this new gradient system allows the enhancement of the sensitivity of the polypeptide analysis proportionate to the increased volume of solution loaded with the same levels of precision. In contrast, when using a standard gradient system it is difficult to analyze a polypeptide quantitatively with good precision due to either adsorption to various materials or to irregular change in the ratio between a retained and a passed peak of the polypeptide. Additionally, the appearance of a passed peak results in a loss in the sensitivity of the polypeptide analysis, although no adsorption of a polypeptide to various materials occurs in a solution with acetonitrile content more than the critical threshold. Consequently, the new gradient system is effective for the simultaneous and quantitative analysis of different polypeptides with good precision and without any loss of sensitivity due to either adsorption to various materials or the appearance of a passed peak.

Elution mechanism of polypeptides in reversed-phase liquid chromatography based on the critical threshold of organic solvent to induce abrupt change in adsorption capacity to the column packing.

Adsorption capacity of polypeptides to the column packing in a solution containing multiple organic solvents was found to be expressed by means of an fn value, which is the sum of the ratios of the content of each organic solvent in the solution to the critical content of each organic solvent to cause abrupt change in the adsorption capacity, and to change abruptly at the point where the fn value becomes 1. Additionally, our results indicate that each polypeptide is eluted by the eluent containing a specific organic solvent content regardless of gradient elution rate in reversed-phase liquid chromatography, and that total organic solvent content in the eluent containing polypeptides is equal to the critical content. Considering the power law relationship between the retention times and the gradient elution rates, our results suggest that the elution of each polypeptide in reversed-phase liquid chromatography is mainly controlled by abrupt change in the adsorption capacity induced by change in the organic solvent content of the eluent during a gradient elution process, and that the abrupt change repeats across the critical threshold while a polypeptide moves through the column, and as a result, each polypeptide is concentrated in the eluent with the critical threshold.

Differences in the urocortin 1 system between long-sleep and short-sleep mice.

There is evidence that the peptide urocortin 1 (Ucn1) may be involved in mediating some of the effects of ethanol. The purpose of the present study was to characterize Ucn1 immunoreactivity in mice selectively bred for either high or low sensitivity to ethanol-induced sedation, with additional differences in their response to ethanol-induced hypothermia. The brains of naïve male mice of the inbred long sleep/short sleep (ILS/ISS) selected lines were analyzed by immunohistochemistry. Significant differences were found between lines in the number of Ucn1-containing cells in the non-preganglionic Edinger-Westphal nucleus (npEW, the main source of Ucn1 in the brain); with the ISS mice having more cells. However, significant differences in the optical density of Ucn1 immunoreactivity in individual npEW cells and differences in cell area were also found between lines, with ILS mice having a greater density of Ucn1 per cell and having larger cells in the npEW. Importantly, the ILS mice also had a significantly greater number of Ucn1-positive terminal fibers than ISS mice in the lateral septum and the dorsal raphe nucleus, projection areas of Ucn1-containing neurons. These results suggest that the greater sensitivity of ILS than ISS mice to the hypothermic effects of ethanol could be mediated by stronger innervation of the dorsal raphe by Ucn1-containing fibers. In addition, these results lend further support to previous findings implicating Ucn1-containing projections from npEW to the dorsal raphe in ethanol-induced hypothermia.

Highly sensitive and quantitative analysis of polyeptides using a new gradient system based on an abrupt change in adsorption of polypeptide to the reversed-phase column packing.

During a study of 100 microL aliquots of urocortin containing various acetonitrile contents, we hypothesized that a change in the acetonitrile content in the solution across a specific content of acetonitrile (critical threshold) causes an abrupt change in adsorption capacity to the column packing. Circular dichroism measurements suggest that the conformational change induced by acetonitrile in the solution causes the abrupt change in adsorption capacity, and this solvent-induced conformational change is reversible across the critical threshold. This hypothesis can apply to various polypeptides with molecular weights range from 1007 to 6789 and to other organic solvents. A new gradient system utilizing the instant recovery of the adsorption capacity across the critical threshold was designed, and applied to the analysis of a 100 microL aliquot of various polypeptide solutions. The results suggest that use of a solution containing organic solvents more than the critical threshold allows successful dilution of polypeptides up to picomolar concentration range without any loss due to its adsorption during handling procedures and loading onto the LC system, and that a new gradient system enables quantitative analysis of polypeptides at picomolar concentrations in such solutions.