Identification of transmembrane domain 6 & 7 residues that contribute to the binding pocket of the urotensin II receptor

Urotensin II (U-II), a cyclic undecapeptide, is the natural ligand of the urotensin II (UT) receptor, a G protein-coupled receptor. In the present study, we used the substituted-cysteine accessibility method to identify specific residues in transmembrane domains (TMDs) six and seven of the rat urotensin II receptor (rUT) that contribute to the formation of the binding pocket of the receptor. Each residue in the R256^(6.32)-Q283^(6.59) fragment of TMD6 and the A295^(7.31)-T321^(7.57) fragment of TMD7 was mutated, individually, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the positively charged methanethiosulfonate-ethylammonium (MTSEA) or the negatively charged methanethiosulfonate-ethylsulfonate (MTSES) sulfhydryl-specific alkylating agents. MTSEA treatment resulted in a significant reduction in the binding of TMD6 mutants F268C^(6.44) and W278C^(6.54) and TMD7 mutants L298C^(7.34), T302C^(7.38), and T303C^(7.39) to ¹²⁵I-U-II. MTSES treatment resulted in a significant reduction in the binding of two additional mutants, namely L282C^(6.58) in TMD6 and Y300C^(7.36) in TMD7. These results suggest that specific residues orient themselves within the water-accessible binding pocket of the rUT receptor. This approach, which allowed us to identify key determinants in TMD6 and TMD7 that contribute to the UT receptor binding pocket, enabled us to further refine our homology-based model of how U-II interacts with its cognate receptor.     

尾加压素Ⅱ与原发性高血压患者应用缬沙坦治疗疗效的相关性研究

目的探讨尾加压素Ⅱ（UU）与原发性高血压患者应用缬沙坦治疗疗效的相关性。方法选择确诊的原发性高血压患者60例,根据治疗方法不同随机分为观察组（缬沙坦）与对照组（卡托普利）各30例,比较两组治疗前后的血压变化情况。以及应用放免分析法检测两组患者尾加压素Ⅱ治疗前后的变化,同时采用Toshiba65A型彩色多普勒超声显像仪进行心功能检测,比较两组治疗前后左心室重量指数（LVMI）的变化。结果观察组的降压效果有效率达96．7％,对照组的有效率仅73．3％,观察组的降压疗效明显优于对照组,差异有统计学意义,P〈0．05。观察组的SBP治疗后降低程度较对照组更明显,差异有统计学意义,P〈0．05。观察组的DBP治疗后降低程度较对照组更明显,差异有统计学意义．P〈0．05。观察组高血压合并心肌肥厚患者的UⅡ均较治疗前及对照组明显降低,差异有统计学意义,P〈0．05。观察组高血压合并心肌肥厚患者的LVMI也较治疗前及对照组明显降低,差异有高度统计学意义,P〈0．01。结论缬沙坦通过扩张血管从而降低血压,进而抑制UⅡ的水平,从而改善心室重构及心室肥厚功能。     

血浆尾加压素Ⅱ在老年高血压合并心肌肥厚患者中的作用机制的临床研究

目的探讨血浆尾加压素Ⅱ（urotensinll,UⅡ）在老年高血压病以及合并心肌肥厚患者中的作用机制。方法选取2009年1月-2010年1月来我院就诊的老年高血压患者67例作为观察组（年龄均〉60岁）,其中单纯性高血压患者32例,高血压合并心肌肥厚患者35例。同时选取我院34例进行健康体检者作为对照组,应用放射免疫分析法测定各组血浆UⅡ含量,并作相关性分析。结果①67例老年高血压组的UⅡ含量平均为（8.02±2.59）pg/mL,34例正常对照组的UⅡ含量平均为（6.75±1.25）pg/mL,两组比较,差异有统计学意义（P〈0.05）。②不同分级的高血压患者血浆UⅡ的含量明显高于正常对照组（P〈0.05）,且随着高血压分级的升高,高血压患者血浆UⅡ的含量也明显增加,3级高血压患者血浆UⅡ的含量明显高于1、2级高血压组血浆UⅡ的含量（P〈0.05）。③单纯高血压组、高血压合并心肌肥厚组的血浆UⅡ含量均明显高于正常对照组（P〈0.05）。且高血压合并心肌肥厚组的UⅡ水平明显高于单纯高血压组（P〈0.05）。单纯高血压组、高血压合并心肌肥厚组的左心室重量指数（LVMI）均明显高于正常对照组（P〈0.01）。且高血压合并心肌肥厚组的LVMI水平明显高于单纯高血压组（P〈0.01）。④老年高血压组心脏室间隔厚度（IVST）为（12.45±0.79）mm,左室后壁厚度（LPWD）为（12.67±0.29）mm,二者分别与UⅡ呈正相关（r分别为0.762,0.891,P〈0.05）。结论 UII可能在老年高血压病心肌肥厚的发病机制中具有重要作用,但其作用机制及其意义有待深入研究。     

低氧性肾间质纤维化和游泳运动对大鼠尾加压素Ⅱ及其受体表达的影响

目的: 探讨慢性低氧性肾纤维化大鼠尾加压素Ⅱ(UⅡ)及其受体(GPR14)的变化,以及游泳运动在其中的作用。方法: 45只雄性SD大鼠随机分成3组,即对照组、低氧组(低氧7周)和游泳组(低氧+游泳锻炼7周组,即低氧3周后,每天1 h无负重游泳4周)。测定血清肌酐(Scr)、血尿素氮(BUN)和血桨UⅡ含量,以及肾组织羟脯氨酸(Hyp)含量、UⅡ及GPR14 mRNA表达和UⅡ蛋白表达。结果: (1)Scr和BUN含量:低氧组较对照组分别低18.5%和14.1%(均P< 0.05),而游泳组与低氧组间无显著差别;(2)肾组织Hyp含量:低氧组较对照组高42.9%(P<0.01),而游泳组较低氧组低26.1%(均P<0.05);(3)血浆UⅡ含量:低氧组较对照组高380.8%(P<0.01),而游泳组较低氧组低42.6%(P<0.01);(4)肾组织UⅡ mRNA表达:低氧组较对照组上调104.5%,游泳组较低氧组下调33.2%(均P<0.01);GPR14 mRNA表达:低氧组较对照组上调35.4%(P<0.01),而游泳组与低氧组间无显著差异;(5)肾组织UII蛋白的表达:低氧组明显高于对照组(P<0.01),而游泳组低于低氧组(P<0.01);(6)van Gieson染色显示低氧组肾血管胶原纤维增生明显,而游泳组明显改善。结论: 慢性低氧性肾纤维化大鼠尾加压素Ⅱ及其受体的表达上调;适度游泳运动有减轻慢性低氧肾间质纤维化的作用,并下调尾加压素Ⅱ基因与蛋白的表达。     

nCPAP治疗重度OSAS合并高血压患者血浆尾加压素Ⅱ的研究

目的探讨经鼻持续气道正压通气（nCPAP）治疗重度阻塞性睡眠呼吸暂停综合征（OSAS）合并高血压病患者尾加压素（U—II）在其过程中病理生理的作用及临床意义。方法采用放射免疫法分析30例患者经nCPAP治疗前后U—II的水平;采用夜间多导睡眠（PSG）监测患者治疗前后SaO2及夜间7h血压水平。结果治疗前、后患者血浆U—II的水平及Sa02分别为（9．01±1．0）pg／ml、（7．2±0．8）pg/ml,（51．2±3．64）％、（85．3±3．6）％,均有统计学意义（P〈0．005）;治疗前、后夜间7h平均收缩压及舒张压分别为（156．67±9．56）mmHg、（137．57±15．309）mmHg、（105．50±4．89）mmHg、（83．45±14．9）mmHg,均有统计学意义（P〈0．001）。结论U—II的增高可能是重度阻塞性睡眠呼吸暂停综合征合并高血压病患者疾病发生、发展的重要因素,通过nCPAP治疗后可使患者血浆中血浆U—II的水平明显降低,氧饱和度改善,具有明显的临床疗效。     

血浆肾上腺髓质素和尾加压素Ⅱ动态变化在毛细支气管炎中的临床意义

目的探讨毛细支气管炎患儿急性期和恢复期血浆肾上腺髓质素（ADM）及尾加压素Ⅱ（U-Ⅱ）变化的临床意义。方法选择153例毛细支气管炎患儿和36名健康对照儿童,测定患儿急性期（病程〈7d）及恢复期（病程〉14d）血浆ADM和U—Ⅱ水平,分析与疾病症状评分的相关性。结果患儿急性期血浆ADM和U—Ⅱ水平均高于恢复期和健康对照儿童（血浆ADM：t=20．57和26．26,P〈O．01;血浆U—Ⅱ：t=14．27和7．61,P〈0．01）,且疾病不同严重程度患儿间也存在明显差异（F=245．94和304．79,P值均〈0．01）。恢复期患儿血浆U-Ⅱ水平低于健康对照儿童（t=6．99,P〈0．01）,但ADM水平仍高于对照组（t=8．98,P〈0．01）,疾病不同严重程度患儿间血浆ADM水平相近（F=2．25,P〉0．05）,而U-Ⅱ水平比较则差异具有统计学意义（F=25．69,P〈0．01）。毛细支气管炎患儿急性期症状评分与血浆ADM和U-Ⅱ水平呈正相关（r值分别为0．884和0．943,P值均为0．000）;恢复期症状评分与血浆ADM和U-Ⅱ水平虽在统计学上P值小于0．05,但相关系数较小,因此实际意义不大。结论毛细支气管炎患儿急性期血浆ADM和u-Ⅱ均显著升高,水平与患者病情呈正相关,提示ADM和U-Ⅱ参与了毛细支气管炎的发病,可作为毛细支气管炎严重程度的参考指标之一。     

支气管哮喘患者血浆中尾加压素Ⅱ的变化及其意义

目的探讨尾加压素Ⅱ（UⅡ）在支气管哮喘发病中的意义。方法采用放射免疫法测定哮喘患者在急性发作期和临床缓解期血浆中UⅡ含量,并常规进行肺功能检测。结果28例哮喘患者在急性发作期和临床缓解期血浆UⅡ含量分别为（1．65±0．31）μg/L及（1．27±0．38）μg/L,二者差异显著（t=4．692,P〈0．01）;急性发作期血浆UⅡ含量随病情恶化而升高,但差异无统计学意义（P〉0．05）;男性与女性之间、吸烟与不吸烟之间血浆uⅡ含量差异无统计学意义（P〉0．05）;哮喘急性发作期血浆UⅡ含量与第1s用力呼气容积（FEV1）占预计值百分比呈负相关（r=-0．325,P〈0．05）。结论哮喘发作时血浆UⅡ含量增加,UⅡ可能参与哮喘的发病。     

Irisin levels are associated with urotensin II levels in diabetic patients

Aims/Introduction

Irisin is a newly identified myokine that can promote energy expenditure. Previous studies showed that circulating urotensin II (UII) levels were increased in diabetes, and UII could inhibit the glucose transport in skeletal muscle in diabetic mice and aggravated insulin resistance. We presumed that irisin levels are associated with UII in diabetic patients.

Materials and Methods

A total of 71 patients with type 2 diabetes and 40 healthy subjects were recruited. Blood and urinary irisin concentrations were measured by using enzyme-linked immunosorbent assay, and UII concentrations were measured by bioelectrical impedance analysis. Every participant''s body composition was analyzed by bioelectrical impedance.

Results

The serum irisin levels were significantly lower in diabetic patients than that of controls, whereas serum UII levels were significantly higher in diabetic patients than that in that of controls. Serum irisin levels were negatively associated with circulating UII, hemoglobin A1c and the natural logarithm transformation of urinary albumin excretion, whereas serum irisin was positively associated with estimated glomerular filtration rate, and low-density lipoprotein cholesterol and urinary irisin were positively associated with urinary UII. Furthermore, circulating irisin is positively associated with muscle mass, whereas circulating UII is negatively associated with muscle mass in diabetic patients. Hemoglobin A1c and circulating UII are independent determinants of circulating irisin by multiple regression analysis.

Conclusions

The present results provide the clinical evidence of an association between irisin and UII in diabetic patients. Hemoglobin A1c and circulating UII are independent determinants of circulating irisin. Our results hint that UII and high glucose might inhibit the release of irisin from skeletal muscle in diabetic patients.     

Decreased plasma urotensin Ⅱ levels inversely correlate with extent and severity of coronary artery disease

Objective To determine the plasma urolensin Ⅱ(UⅡ) levels in various types of coronary heart disease and to clarify how the plasma UⅡ levels correlate with the clinical presentation, extent and severity of coronary artery atherosclerosis (CAD). Methods: One hundred and three aged patients undergoing elective diagnostic coronary angiography for proven or clinical suspected coronary heart disease were enrolled in this study. The extent and severity of coronary artery disease were evaluated by vessel score and Gensini score, respectively. Plasma UⅡ levels were measured by radioimmunoassay. Results: The plasma UⅡ levels in the patients with modest to severe coronary stenosis (3.03±0.34 pg/ml, 1.83±0.67 pg/ml) were significantly lower than that in subjects with normal coronary artery (4.80±1.11 pg/ml, P＜0.001). The plasma UⅡ levels in patients with coronary heart disease were also significantly lower than that in patients with insignificant coronary stenosis (P ＜ 0.001). Compared to patients with stable angina pectoris, plasma UⅡ levels in patients with acute coronary syndrome were significantly decreased (1.89±0.51 pg/ml vs 2.42±0.77 pg/ml, P＜ 0.001). Plasma UⅡ levels were found to be negatively correlated with the severity of coronary artery stenosis (r = -0.488, P＜0.001), as well as the vessel score (r = -0.408, P＜0.05) in the patients with CAD. Conclusion: Significant inverse correlations exist between the plasma UⅡ levels, and the extent and severity of coronary artery stenosis. These findings suggest that plasma UⅡ contribute to the development and progression of coronary artery stenosis, and may be a novel marker to predict clinical types, as well as the extent and severity of coronary artery disease in the patients.     

尾加压素受体拮抗剂对急性肺损伤的干预效应

目的探讨尾加压素受体拮抗剂(urotensin receptor antagonist,URA)对损伤肺的影响。方法健康SPF级Sprague Dawley(SD)大鼠49只,随机分为A、B、C三组:抽取7只注射生理盐水作为正常对照(A组);B、C两组各21只,二组所有大鼠用油酸复制急性肺损伤(acute lung injury,ALI)模型,B组为模型对照组,C组在模型基础上加用URA为干预组。三组均于注射后3h各抽动脉血作血气分析测定血氧分压,A组抽动脉血后全部活杀取肺,B、C两组分别于3(抽动脉血后)及12、24h三个时间段各取7只活杀取肺,右肺称湿重后烘干,左肺立即以中性福尔马林固定作病理切片。结果A组大鼠注射生理盐水后无异常表现,B、C两组注射油酸后均出现呼吸急促、活动减少或不合群、紫绀等表现,与A组相比,B、C两组动脉血氧分压(PaO2)明显下降(P<0.05),肺湿/干(W/D)比值明显升高(P<0.05),但A、B两组间差异无统计学意义(P>0.05)。C组活动能力改善较B组更快,肺组织病理符合急性肺损伤变化,随时间延长,炎症细胞、红细胞、肺水肿进行性加重,但C组红细胞渗出较B组有所减少。结论尾加压素受体拮抗剂可能对急性肺损伤动物肺损害有保护作用。