Cloning and sequence analysis of complementary DNA encoding a precursor for chicken natriuretic peptide

Chicken -natriuretic peptide (-chNP) has been identified in chicken heart, which showed higher homology to brain natriuretic peptide (BNP) than to atrial natriuretic peptide (ANP) [1]. Complementary DNA (cDNA) clone encoding a chNP precursor (pre-chNP) precursor (pre-chNP) was isolated from cardiac cDNA library and sequenced. Pre-chNP was 140-residue signal peptide at the N-terminus and -chNP at the C-terminus, and did not exhibit high homology to poreine BNP except for the C-terminal region. However, a characteristic AT-rich nucleotide sequence commonly found in mammalian BNPs was also present in the 3′-untranslated region. Thus, chNP is concluded to be classified into the BNP-type     

慢性心力衰竭患者N端脑钠肽前体、脑钠肽与患者心脏超声参数及炎症因子的关系

目的:探讨慢性心力衰竭(CHF)患者N端脑钠肽前体(NT-proBNP)、脑钠肽(BNP)与患者心脏超声参数及炎症因子的关系。方法:选取2015年3月～2018年2月期间我院收治的CHF患者135例为研究组。根据纽约心脏病学协会(NYHA)心功能分级将研究组分为Ⅰ级组26例,Ⅱ级组34例,Ⅲ级组42例,IV级组33例。另选取同期于我院体检的健康志愿者30例为对照组。检测并比较对照组与研究组、不同NYHA心功能分级的血清标志物、炎症因子、心功能超声参数,采用Pearson相关性分析NT-proBNP、BNP与炎症因子、心功能超声参数的相关性。结果:研究组血清BNP、NT-proBNP、白介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、超敏C反应蛋白(hs-CRP)水平及左心室舒张末期内径(LVEDD)、左心室收缩末期内径(LVESD)均显著高于对照组,左心室射血分数(LVEF)则低于对照组(P0.05)。BNP、NT-proBNP、IL-6、TNF-α、hs-CRP水平及LVEDD、LVESD随心功能分级的升高而升高,LVEF随心功能分级的升高而降低(P0.05)。经Pearson相关性分析显示,CHF患者BNP、NT-proBNP与IL-6、TNF-α、hs-CRP、LVEDD、LVESD呈正相关,而与LVEF呈负相关(P0.05)。结论:血清NT-proBNP、BNP与CHF患者的心脏超声参数及炎症因子密切相关,可考虑将其作为临床诊断CHF的生物学指标。     

Expression of C-type natriuretic peptide and of its receptor NPR-B in normal and failing heart

C-type natriuretic peptide (CNP) was recently found in the myocardium, but possible insights into differences between atrium and ventricle production are so far lacking. Our aim was to evaluate, in an experimental model of pacing-induced heart failure (HF), plasma and tissue levels of CNP and mRNA expression of the peptide and of its specific receptor, NPR-B. Cardiac tissue was collected from male adult minipigs without (control, n=5) and with pacing-induced HF (n=5). Blood samples were collected at baseline and after pacing (10 min, 1, 2, 3 weeks). CNP in plasma and in cardiac extracts was determined by a radioimmunoassay, while the expression of mRNA by real time PCR. Compared to control, plasma CNP was increased after 1 week of pacing stress (36.9+/-10.4 pg/ml vs.16.7+/-1.1, p=0.013, mean+/-S.E.M.). As to myocardial extract, at baseline, CNP was found in all cardiac chambers and its content was 10-fold higher in atria than in ventricles (RA: 13.7+/-1.9 pg/mg protein; LA: 8.7+/-3.8; RV: 1.07+/-0.33; LV: 0.93+/-0.17). At 3 weeks of pacing, myocardial levels of CNP in left ventricle were higher than in controls (15.8+/-9.9 pg/mg protein vs. 0.9+/-0.17, p=0.01). CNP gene expression was observed in controls and at 3 weeks of pacing. NPR-B gene expression was found in all cardiac regions analyzed, and a down-regulation was observed in ventricles after HF. The co-localization of the CNP system and NPR-B suggests a possible role of CNP in HF and may prompt novel therapeutical strategies.     

Distribution of angiotensinogen in diseased human hearts

Extrahepatic synthesis and localization of angiotensinogen (ATN) have been described in animals, thus establishing the tissue renin-angiotensin (RA) system. However, there had been no reports of tissue RA systems in human organs, including the heart. In earlier, we have reported the possibility of ATN synthesis in the human heart using ribonuclease protection assay system. ATN mRNA was detected not only in the liver, but also in both the atrial and ventricular heart tissues, suggesting that ATN is synthesized in the human heart. In this report, we looked for the distribution of ATN in diseased human heart.Northern blot hybridization of cDNA with total RNA extracted from human liver, brain, kidney, atrial and ventricular tissues revealed that ATN mRNA exists in cardiac ventricule.Immunohistochemical studies using a specific antibody to ATN revealed a stronger reaction in the endocardial layer of the human left ventricle, than in the epicardial layer, and intense immunoreactivity in the conduction system and right atrium. This distribution pattern was similar to that of human atrial natriuretic peptide (hANP), which functions a smooth muscle relaxant. Double immunostaining of ATN and hANP demonstrated that all myocytes in the right atrium had immunopositive reactions to ATN, hANP or both of ATN and hANP. Double immunoelectron staining enabled us to show more detailed localization of ATN and hANP; hANP only existed in the specific granules and ATN existed in the myofibril, but not in the granule. Furthermore, our experiments provide evidence of ATN in healthy human hearts and also reveal a widespread immunopositive reaction for ATN in the left ventricle of diseased hearts.     

静息心率与慢性心力衰竭患者血清NT-proBNP的相关性分析

目的:探讨入院时静息心率与慢性心力衰竭患者血清N末端B型钠尿肽原(NT-pro BNP)相关性。方法:选取我院收治的242例慢性心力衰竭患者为研究对象,按静息心率将患者分为Ⅰ组(心率70次/min),Ⅱ组(心率:70%~90次/min),Ⅲ组(心率90次/min);测量患者入院时血清NT-pro BNP水平,按照其中位数分为两组:NT-pro BNP2087.63 pg/m L组和NT-pro BNP≥2087.63 pg/m L,比较各组相关指标的差异。用多元线性回归分析静息心率与血清NT-pro BNP的关系。结果:三组在收缩压、心功能分级、左室射血分数(LVEF)、左室舒张末内径(LVEDD)、空腹血糖(FPG)比较具有统计学差异(P0.05),与Ⅰ组及Ⅱ组比较,Ⅲ组患者收缩压、LVEDD、FPG,NYHA心功能分级Ⅲ~Ⅳ比例较高、LVEF偏低;三组在血清NT-pro BNP比较亦存在显著的统计学差异(P0.05),Ⅲ组高于Ⅰ组及Ⅱ组、Ⅱ组高于Ⅰ组;与NT-pro BNP2087.63 pg/m L组比较,NT-pro BNP≥2087.63 pg/m L组静息心率偏快,且患者中90(次/min)的比例较高(P0.05)。静息心率与与NT-pro BNP呈正相关(r=0.281,P=0.035);静息心率是影响NT-pro BNP水平的独立危险因素。结论:静息心率水平与慢性心力衰竭患者血清NT-pro BNP水平密切相关。     

The in vitro chronotropic and inotropic effects of vasoactive intestinal peptide (VIP) on the atria and ventricular papillary muscle from Cynomolgus monkey heart

The in vitro chronotropic and inotropic effects of vasoactive intestinal peptide (VIP) and of isoproterenol, two agents known to stimulate cardiac adenylate cyclase were compared on the heart from Cynomolgus monkey using the spontaneously beating right atrium, the electrically stimulated left atrium, and the electrically-stimulated ventricular papillary muscle. VIP increased concentration-dependently the rate of beating of the right atrium as well as the contractility of both atria but its efficiency was lower than that of D,L-isoproterenol. VIP also stimulated concentration-dependently, and this time as efficiently as D,L-isoproterenol, the contractility of papillary muscle. These VIP effects were unaltered by the neuronal blocker tetrodotoxin. In addition, the moderate inhibition exerted by the beta-adrenergic blocker D,L-propranolol on VIP effects argued against the implication of beta-adrenergic receptors in VIP effects. These results indicate that VIP exerts a direct stimulatory influence on the rate and contractility of Cynomolgus monkey heart.     

扩张型心肌病慢性心力衰竭患者血浆脑利钠肽水平的临床意义

目的:探究扩张型心肌病慢性心力衰竭患者血浆脑利钠肽水平的临床意义。方法:收集2012年3月至2016年3月我院收治的90例扩张型心肌病慢性心力衰竭患者,将患者按照NYHA心功能分级分为A组(II级)20例、B组(III级)38例、C组(IV级)32例。比较各组患者的血浆脑力钠肽(BNP)以及超声心动图相关指标,包括左心室射血分数(LVEF)、左心房内径(LA)、左室舒张末期内径(LVEDD)以及左室收缩末期内径(LVESD),分析血浆BNP与NYHA分级和超声心动图相关指标的相关性,以及比较血浆BNP和LVEF在慢性心力衰竭病情程度中的能力。结果:C组患者的血浆BNP浓度显著高于A组和B组(P0.05),而B组患者的血浆BNP浓度显著高于A组,比较差异具有统计学意义(P0.05)。心脏超声检测发现,C组患者的LA显著高于A组(P0.05),而LVEF、LVEDD及LVESD比较差异无统计学意义(P0.05)。血浆BNP与NYHA分级呈正相关关系,但与LVEDD、LVESD、LVEF、LA无明显相关关系(P0.05)。血浆BNP对评价心力衰竭患者病情程度呈现出较强的能力(受试者工作特征曲线下面积=0.902,P0.001)。血浆BNP=523.5 pg/mL为中重度心力衰竭患者的诊断最佳值。LVEF对评价心力衰竭患者病情程度无明显能力(受试者工作特征曲线下面积=0.392,P=0.276)。结论:血浆BNP浓度对扩张型心肌病慢性心力衰竭患者的诊断、筛查以及心功能分级具有重要的临床意义。     

Natriuretic peptides in cardiovascular diseases

The natriuretic peptide family comprises atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), dendroaspis natriuretic peptide (DNP), and urodilatin. The activities of natriuretic peptides and endothelins are strictly associated with each other. ANP and BNP inhibit endothelin-1 (ET-1) production. ET-1 stimulates natriuretic peptide synthesis. All natriuretic peptides are synthesized from polypeptide precursors. Changes in natriuretic peptides and endothelin release were observed in many cardiovascular diseases: e.g. chronic heart failure, left ventricular dysfunction and coronary artery disease.     

Identification and gene expression analyses of natriuretic peptide system in the ovary of goat (Capra hircus)

Natriuretic peptides (NPs) are involved in maintaining cardiovascular and fluid homeostasis, regulating reproductive processes and bone growth, and other numerous functions. To better understand the role of NPs in goat (Capra hircus), in the present study, full-length cDNAs of goat Nppa (natriuretic peptide precursor A), Nppb (natriuretic peptide precursor B) and Nppc (natriuretic peptide precursor C), respectively encoding ANP, BNP and CNP, were cloned from adult goat heart and ovary. The putative prepropeptide ANP (prepro-ANP) and prepro-CNP share a high amino acid sequence identity with other species. Real-time PCR showed that Nppa, Nppb and Nppc were widely expressed in adult goat tissues. The mRNA expression of Nppa and Nppb in the heart was extremely higher compared with other tissues. Nppc mRNA expression in the lung and uterus was also higher than in other tissues. The expression of Nppa, Nppb and Nppc genes was examined at different ovarian follicle stages using RT-PCR. The mRNAs of Nppa and Nppb were detected in secondary follicles as well as in COCs (cumulus–oocyte-complexes) and granulosa cells of antral follicles. However, the mRNA expression of Nppc was observed throughout ovarian follicle development, and it was especially higher in granulosa cells of antral follicles. In vitro, stimulating goat granulosa cells with FSH led to an increase in the expression of Nppc by dose- and time-dependent manners and a rapid decline was induced by LH stimulation, but the expression of Nppa and Nppb did not change after FSH or LH treatment. These results suggest that Nppc is a gonadotropin-induced gene in granulosa cells of goat ovary and CNP may be involved in the regulation of ovarian follicle development and oocyte maturation.     

C型钠尿肽的扩血管作用及机制

目的和方法：用常规离体血管灌流方法,观察钠尿肽家族新成员C型钠尿钛（CNP）对家兔腹主静脉及腹主动脉的作用及作用机制。结果：CNP在10^-1-10^-6mol/L浓度范围内对家兔静脉及动脉均呈剂量依赖性的舒张效应。其对静脉的作用与硝酸甘油（NTG）相似,且扩血管作用无ANP强,以腹主动脉为主对象分别施加阿托品（10^-7mol/L）,酚妥拉明（20μg）或消炎痛（20μg）等均不影响CNP的舒血管作用,优降糖和心得安可明显降低CNP对腹主动脉的舒张作用。CNP在基础状态下提前加入不抑制NE的缩血管反应。结论：CNP可能是一种静脉系统的扩张剂,同时亦是调节动脉张力的选择性调节肽,CNP舒血管作用机制至少有两途径：一是与K^ -ATP通道的开放有关,二是与激活β受体有关。     

血BNP 监测对慢性心力衰竭患者治疗的指导价值及其对预后的影响研究

目的:探讨血浆脑钠肽(BNP)监测对慢性心力衰竭(CHF)患者治疗的指导价值及其对预后的影响。方法:根据出院后是否进行BNP监测,将2009年5月至2010年12月我院心内科出院的CHF患者100例随机分为监测组和对照组,每组各50例。平均随访时间为1年,观察两组的治疗效果和预后包括心血管性死亡、因心血管事件再次住院例数、NYHA心功能分级、明尼苏达心衰生活质量问卷(MLHFQ)评分。结果:随访1年期间,监测组心血管性死亡率、因心血管事件再次住院率分别为2%、18.0%,对照组分别为16.0%、54.0%,差异均有统计学意义(P<0.05)。出院时与第1次随访,两组患者明尼苏达心衰生活质量问卷评分结果比较差异无统计学意义(P>0.05);而第2、3、4次随访时,监测组试验结果均显著高于对照组,差异有统计学意义(P<0.05)。随访1年时,监测组患者的NYHA心功能分级、抗心衰药物的靶剂量率显著优于对照组,差异有统计学意义(P<0.05)。结论:通过血浆BNP水平监测,及时调整治疗方案,可延缓心衰的进展,改善患者预后。     

脑钠肽在慢性心功能不全诊治的临床观察

目的：评价脑钠肽在慢性心功能不全过程中的诊治效果,探讨其临床适用性。方法：选择从2012年3月至2013年3月于我院就诊的82例心功能不全患者作为实验组,另选择同期的80例健康体检者作为对照组,分析实验组和对照组人员的脑钠肽的浓度,以及实验组患者经过治疗后的脑钠肽浓度变化情况,所有人员同时给予超声心动并观察实验组血浆脑钠肽浓度与左室射血分数及心功能分级之间的关系。结果：实验组患者的血浆脑钠肽浓度明显高于对照组人员,差异有统计学意义（P〈0．05）;实验组患者治疗前后血浆脑钠肽浓度有明显改变,差异也有统计学意义（P〈0．05）;心功能和左室射血分数不同分级之间的血浆脑钠肽浓度也不同,各级差异有明显统计学意义（P〈0．05）;脑钠肽浓度的不同参考值测定的灵敏度和特异性也不同。结论：脑钠肽在慢性心功能不全的诊治过程中有重要价值,为临床心功能的判断可以提供较为敏感的指示,适合临床长期推广应用。     

血管钠肽对低氧大鼠心脏钠尿肽C受体表达的影响

目的 :探讨低氧对大鼠心脏钠尿肽C受体 (NPR C)表达的调节作用 ,以及血管钠肽 (VNP)对这一过程的影响。方法 :将大鼠随机分为 3组 :对照组、低氧组 (3～ 2 8d)和VNP(2 5～ 75 μg/kgbw) 低氧组 ,采用放射免疫的方法测定大鼠血浆心房钠尿肽 (ANP)的浓度 ,并采用定量PCR的方法分析NPR C的mRNA水平。结果 :低氧 2 8d大鼠血浆ANP浓度显著高于正常大鼠 (P <0 .0 5 ) ,而且每天注射 75 μg/kgbw的VNP使ANP浓度进一步升高 (P <0 .0 1)。低氧 3d对大鼠心脏NPR C的mRNA的量没有显著影响 ;低氧 7d使大鼠心脏NPR C的mRNA的拷贝数显著升高 (P <0 .0 5 ) ;低氧 14d、2 8d使大鼠心脏NPR C的mRNA的拷贝数进一步升高 (P <0 .0 1)。每日注射 2 5μg/kgbw的VNP对低氧诱导的大鼠心脏NPR C表达没有显著影响 ;5 0 μg/kgbw的VNP显著降低低氧大鼠心脏NPR C的表达 (P <0 .0 5 ) ;75 μg/kgbw的VNP进一步降低低氧大鼠心脏NPR C的表达 (P <0 .0 1)。 结论 :VNP可以升高低氧大鼠的血浆ANP水平 ;低氧可以使大鼠心脏NPR C表达增加 ,而且具有时间依赖性 ,而VNP对这一过程有抑制作用 ,并且呈剂量依赖性     

Cloning and functional expression of the bovine natriuretic peptide receptor-B (natriuretic factor R1c subtype)

We describe the isolation of a 3,276 base pair cDNA for the bovine natriuretic peptide receptor-B (NPR-B). Expression of this clone in Cos-P cells demonstrates that it encodes an agonist-dependent guanylyl cyclase. Porcine CNP stimulates the activity of this receptor up to 200-fold with an ED₅₀ of 12±2 nM, whereas brain natriuretic peptide C-type natriuretic peptide (CNP) and atrial natriuretic factor (ANF) are less efficacious. In addition, ligand binding studies indicate that this receptor exhibits the pharmacology appropriate for the bovine NPR-B. CNP binds to Cos-P cell membranes expressing this clone with a K_d of 13±1 pM, and natriuretic peptides compete for [¹²⁵I]-CNP binding with a rank order of pCNP>pBNP>rANF. Thus, the expressed receptor-guanylyl cyclase exhibits the expected pharmacological profile for ligand binding and cyclase activation of the bovine NPR-B receptor.Abbreviations BSA bovine serum albumin - dNTP deoxynucleotide triphosphate - SDS sodium dodecyl sulfate - DEAE-dextran diethylaminoethyl-dextran - EDTA ethylenediamine tetraacetic acid - Tris Tris(hydroxymethyl)aminomethane - DMSO dimethyl sulfoxide - RP-HPLC reverse phase-high performance liquid chromatography - AMV avian myeloblastosis virus - Arg arginine - Lys lysine     

Atrial natriuretic peptide relation to plasma and heart zinc concentrations of wistar rats exposed to cold and hot ambients

Plasma atrial natriuretic peptide (ANP) and zinc levels, as well as heart tissue zinc concentrations were determined, in male Wistar rats after the exposure of 114 rats at low temperature (4°C) and 95 rats at high temperature (35–36°C) for 28 d. Plasma ANP was estimated by radioimmunoassay and Zn²⁺ concentrations by atomic absorption spectrometry. Values were compared to a control group exposed at 20–22°C (76 rats). Plasma ANP and Zn²⁺ levels, as well as heart tissue Zn²⁺ concentrations of control rats did not show statistically significant variations during the study, whereas rats exposed to cold and hot ambients showed significant variations of the parameters. A significant increase of plasma ANP and plasma zinc and heart tissue Zn²⁺ concentrations developed during cold exposure, whereas a gradual decrease of plasma ANP and Zn²⁺ levels was revealed during hot adaptation. Results also indicate that plasma ANP and zinc levels are proportionally related, whereas there is an inverse relationship between plasma ANP levels and heart Zn²⁺ concentrations, in both cold and hot exposed rats. In conclusion, our results show that ANP in relation to zinc probably play an important role in cold and hot acclimatization of rats.     

B型利钠肽在心衰应用的研究进展

利钠肽（natriuretic peptides,NPs）的发现已有30年历史,其中B型利钠肽（B-type natriuretic peptide,BNP）及氨基末端脑钠肽前体（ N-terminal pro-B type natriuretic peptide,NT-proBNP）的临床应用对心血管疾病诊治具有里程碑意义,特别是对心衰的综合价值最高。BNP具备强大的心血管保护效应,但心衰后BNP水平大幅升高,却没展现出相应的活性,被称为“利钠肽悖论”。近几年,随着质谱、核磁共振技术的运用,逐渐从代谢途径和检测技术上解开了“利钠肽悖论”这一谜题：外周循环中存在多种不同生物活性的BNP亚型且心衰后的BNP代谢与生理状态下不同。所以,纵然检测到心衰后BNP大幅升高,但本质上是由于传统检测技术的瓶颈,使各类BNP亚型与检测试剂交叉反应,活性成分被高估而造成假阳性。因此,要加强对BNP在病理生理等不同情况下的认识,还要借助生物化学手段建立具有敏感性和特异性的检测方法来识别BNP_1-32、BNP_1-30、BNP_3-32及B型利钠肽原（pro-B-type natriuretic peptide,proBNP）等特殊形式。从而有助于探索心衰更深层次的病理生理机制,还可协助临床对心衰的诊断及预后做出更准确的判断。     

应用NT-proBNP评价常规药物加心区交感神经阻滞对心衰治疗的有效性

目的:利用常规药物加心区交感神经阻滞（CSNB）技术治疗（以下简称治疗）慢性心力衰竭,观察治疗前后患者血浆N- 端脑利钠肽前体（NT-proBNP）水平变化情况及利用NT-proBNP评价常规药物加CSNB对心衰治疗的有效性。方法：对54 例慢性心衰患者给予常规药物加CSNB治疗,比较治疗前后血浆NT-proBNP 的水平、左室射血分数（LVEF）、左室舒张末内径（LVEDD）变化情况,随访6 个月内不良事件（以下简称事件）发生率（包括全因病死率和再入院率之和）。结果：治疗后患者3-8 分钟即感觉呼吸困难等临床症状体征明显减轻,住院期间患者尿量明显增多,无一例死亡;治疗14日及出院时血浆NT-proBNP 明显下降（出院时下降比例为61.4%）,与治疗前比较其差异有显著统计学意义（P＜0.01）;LVEF升高,与治疗前比较其差异有显著统计学意义（P＜0.01）;住院14日及出院时LVEDD较入院时缩小。随访6 个月,发生事件12 例（22.2%）。结论：NT-proBNP是评价心衰治疗有效性的可信指标,常规药物加CSNB治疗心衰的临床疗效明显优于单纯常规药物治疗。     

Arsenic contamination of groundwater and prevalence of arsenical dermatosis in the Hetao plain area,Inner Mongolia,China

The present study determined cardiac chamber-specific alterations of the expression of the atrial and brain natriuretic peptide (ANP and BNP) genes with a small increase in age beyond adulthood and with systemic hypertension of intermediate duration. The expression distributions of these genes was determined using in situ hybridization in the right and left atria (RA and LA), and the right and left ventricles (RV and LV) in Wistar Kyoto rats (WKY) and age-matched Spontaneously Hypertensive rats (SHR) at ages 6 months (adult) and 8 months (advanced-age beyond adulthood).In all rat groups, both genes were expressed (ANP > BNP) in the LA and LV, and were not expressed in the RA and RV. The genes were expressed in the LA in all rat groups; the ANP, but not the BNP, expression increased with advancing age and with superimposed hypertension. They were expressed in the LV of the advanced-age WKY, adult and advanced-age SHR, but not in the adult WKY. The ANP mRNA labeling in the LA was diffuse and interspersed with dense accumulations, whereas BNP labeling was diffuse. The labeling of both genes in the form of sparse clusters was seen in the LV of the advanced-age SHR. Our study showed that ANP and BNP expression in left heart chambers increased with a small increase in age, with hypertension of intermediate duration, and with modest left ventricular hypertrophy. The chamber-specific expression distribution could be due to special groups of cardiac cells, or to local chamber-specific factors.