胍丁胺对离体大鼠主动脉张力的影响及其受体机制

采用离体血管环灌流方法,观察了胍丁胺（agmatine,Agm)对大鼠胸主动脉张力的影响,并探讨其受体机制,实验结果如下：（1）在苯肾上腺素PE,10^-6mol/L)引起血管预收缩的 基础上,Agm(10^-7-10^-2mol/L)剂量依赖性地舒张大鼠胸主动脉。（2）上述舒张反应在去除内皮和应用NOS抑制剂N^-G-mnitro-L-arginine methyl ester(L-NAME,0.5mmol/L)后依然存在,提示Agm的舒血管作用为非内皮依赖性,并无NO的参与。（3）在高Ca^2 (3mmol/L)引起血管预收缩的基础上,Agm也可剂量依赖性地舒张大鼠主动脉。（4）预先应用α2－肾上腺素能受体（α2－adrenergic receptor,α2－AR）和咪唑啉受体（IR）阻断剂idazoxan(10^-4mol/L)则可完全阻断Agm的上述作用。（5）应用α2－AR拮抗剂yohimbine(10^-4mol/L)可部分阻断Agm对大鼠主动脉的舒张反应,以上结果表明,Agm对大鼠主动脉血管的舒张作用是由α2－AR和IR共同介导。     

双环醇减轻超氧阴离子诱导的大鼠胸主动脉舒张功能损伤

目的：探讨双环醇（bicyclol）对超氧阴离子（O2）诱导的血管舒张功能损伤的影响。方法：采用离体器官灌流技术,观察bicyclol对离体大鼠胸主动脉环张力的影响。采用焦酚（O2的供体）建立O2损伤模型,观察bicyclol预孵育对氧化应激损伤后血管内皮依赖性舒张功能的改善作用。结果：bicyclol（10-8~10-5mol/L）对由苯肾上腺素预收缩的内皮完整主动脉环产生舒张作用,该作用可被NO合酶抑制剂L-NAME和环氧化酶抑制剂吲哚美辛阻断。500μmol/L焦酚可引起乙酰胆碱诱导的主动脉环内皮依赖性舒张反应减弱,bicyclol（10-5mol/L）预孵育45 min可减轻焦酚的损伤作用。对于吲哚美辛处理的主动脉环,bicyclol（10-5mol/L）可抑制焦酚所致的血管舒张反应降低,但这一效应未见于L-NAME处理的主动脉环。结论：bicyclol具有内皮依赖性舒血管作用,并能对抗O2引起的血管舒张功能损伤,该作用通过NO途径介导。     

血管钠肽、 C型钠尿肽和心房钠尿肽舒血管作用的对比

本实验采用离体血管灌流方法,观察和比较血管钠肽（ＶＮＰ）,Ｃ型钠尿肽（ＣＮＰ）和心房钠尿肽（ＡＮＰ）对大鼠肺动脉,腹主动脉和腹腔静脉的舒张作用。．结果表明,ＶＮＰ,ＣＮＰ和ＡＮＰ对离体大鼠的保留内皮与去内皮的肺动脉,腹主动脉和腹腔静脉均有浓度依赖性舒张作用。     

三羟异黄酮对离体家兔股动脉张力的影响及其机制

植物雌激素三羟异黄酮（genistein,GST)使离体的预先收缩的动脉舒张,其舒张的机制仍然不完全清楚。本研究旨在观察植物雌激素三羟异黄酮对离体家兔股动脉的作用及其机制,结果如下：（1）在苯肾上腺素（PE,1umol/L)引起血管收缩的基础上,GST（10－40umol/L)剂量依赖性地舒张离体家兔股动脉;（2）去除血管内皮显著地抑制GST引起的舒张;（3）在内皮完整情况下,预先应用NOS抑制剂L－NAME（100umol/L)也可显著地抑制GST引起的舒张,提示GST的舒血管作用是内皮依赖的,并与一氧化氮有关;（4）在内皮完整的扣去除内皮的股动脉环,预先应用L－型钙通道激动剂Bay M8644(0.5umol/L)也显著抑制由GST引起的血管舒张,以上结果表明,GST引起的兔股动脉的舒张是部分内皮依赖的,且与拮抗钙有关。     

NO在CCK—8减轻肿瘤坏死因子诱导兔肺动脉反应性变化中的作用

为探讨八肽胆囊收缩素（CCK－8）缓解内毒素休克时肺动脉高压的作用机制,应用离体血管环张力测定技术及一氧化氮合酶（NOS）检测方法,观察了一氧化氮（NO）在CCK－8减轻肿瘤坏死因子－α（tumor necrosis factor-al-pha,TNF-α）的抑制肺动脉内皮依赖性舒张反应中的作用。结果显示：TNF－α（4000U／ml）孵育2h时,肺动脉对10^-6mol/L苯肾上腺素(phenylephrine,PE）和10^-6mol/L乙酰胆碱（ACh）的收缩反应及内皮依赖性舒张反应均无明显变化。TNF－α孵育7或14h时,肺动脉对10^-6mol/L ACh介导的内皮依赖性舒张反应降低,CCK－8（0.5μg/ml）可逆转TNF－α的上述作用,CCK－8本身对正常肺动脉反应性无明显影响。TNF－α、CCK－8对PE引起的收缩反应无显著影响。L－精氨酸（L－Arg）可使TNF－α7h内皮依赖性舒张作用恢复。氨基胍（AG）不影响各组肺动脉对10^-6mol/L ACh的内皮依赖性舒张反应,而使TNF－α组肺动脉环对10^-6mol/L PE的收缩反应显著增加。L－硝基精氨酸（L－NNA）使各组肺动脉环对10^-6mol/L ACh反应由舒张变为收缩,对10^-6mol/L PE的收缩反应显著增强。检测7h各组NOS活性,TNF－α组、TNF－α＋CCK－8组均较对照组显著增加,CCK－8组与对照组比较无显著差异。上述结果提示,CCK－8可逆转TNF－α对内皮依赖性舒张反应的抑制作用,此作用可能与NO有关。     

白细胞介素-2引起离体大鼠主动脉环舒张及其作用机制

本文旨在研究白细胞介素－2（interleukin-2,IL-2）以离体大鼠胸主动脉环收缩张力的作用及其可能机制。采用累积加药法,检测IL－2对去氧肾上腺素（PE）和KCl预收缩的胸主动脉环收缩张力的影响。结果表明,IL－2（1、10、100、1000U／ml）对PE（10μmol/L）预收缩的内皮完整血管环产生浓度依赖性的舒张作用,而对KCl (120mmol/L）预收缩的血管无作用,去除内皮后,IL－2的舒张作用被取消。用一氧化氮合酶抑制剂L－NAME（0.1mmol/L）和鸟苷酸环化酶抑制剂亚甲蓝（10μmol/L）预处理,均可阻断IL－2的舒张血管作用。用环氧合酶抑制剂吲哚美辛（Indo,10μmol/L）预处理可阻断IL－2的血管舒张作用。从上述观察结果推论,IL－2通过NO－鸟苷酸环化酶和环氧合酶途径产生内皮依赖的血管舒张作用。     

利用微血管环技术检测肠系膜动脉三级分支张力的方法

目的：以肠系膜动脉三级分支为样本,观察利用微血管技术检测血管张力功能的整个过程,以便研究各种微血管相关疾病中的微血管功能状态。方法：利用DMT张力测定仪和PowerLab数据采集系统检测微血管收缩、舒张功能,将肠系膜三级动脉血管游离,固定,标准化和激活后,通过加入血管收缩药物及舒张药物,完成对微血管张力功能的检测。结果：本文制备的肠系膜动脉三级分支血管环对血管活性药物出现良好的收缩、舒张反应,加入10^-5mol／L的去甲肾上腺素（NE）后收缩张力达19mN,之后依次加入10^-9～10^-5mol／L的乙酰胆碱（ACh）或硝普钠（SNP）,血管张力呈梯度降低,ACh和SNP引起的最大舒张率分别为80％和95％。结论：利用该微血管环技术成功检测出肠系膜动脉三级分支的收缩舒张功能。     

不同pH条件下离体大鼠胸主动脉对多巴胺反应性的变化

目的：观察不同pH值条件下大鼠胸主动脉对多巴胺反应性的变化。方法：采用离体血管灌流方法,观察①胸主动脉对不同浓度多巴胺的反应。②检测不同pH值备件下血管对多巴胺反应性的变化。结果：①不同浓度多巴胺（10^-6mol·L-1,10^-5mol·L^-1,10^-4mol·L^-1）均能增加正常大鼠胸主动脉收缩力。②pH值依次降低（7．4,7．3,7．2,7．1,7．0,6．9,618,6．7）,大鼠离体胸主动脉对多巴胺10^-5mol／L反应性下降,血管在pH6．6时完全失去对多巴胺的反应性。结论：大鼠胸主动脉对多巴胺刺激的反应性随环境pH值的变化而变化,表现为随着环境pH值的下降,离体动脉对多巴胺刺激的反应性也随之下降。     

雌二醇对家兔窦房结自律细胞的电生理学效应

本文旨在探讨雌二醇（17β-estradiol）对家兔窦房结自律细胞的电生理学效应及其作用机制。应用经典的细胞内玻璃微电极技术观察不同浓度雌二醇（1,10,100μmol／L）对家兔窦房结自律细胞动作电位的影响。结果显示：（1）雌二醇浓度依赖性地延长窦房结自律细胞动作电位复极化50％时间（APD50）和动作电位复极化90％时间（APD50）,降低窦房结自律细胞动作电位0期最大除极速率（Vmax）、动作电位幅值（amplitude of action potential,APA）,降低窦房结自律细胞放电频率（rate of pacemaker firing,RPF）、舒张期（4相）自动去极化速率[velocity of diastolic（phase4）depolarization,VDD]：而雌二醇对窦房结自律细胞的最大舒张电位（maximal diastolic potential,MDP）无明显影响。（2）雌激素受体阻断剂他莫昔芬（10μmol／L）不能阻断雌二醇（10μmol／L）对窦房结自律细胞动作电位的抑制效应。（3）一氧化氮合酶抑制剂L—NAME（100μmol／L）可完全阻断雌二醇（10μmol／L）对窦房结自律细胞动作电位的抑制效应。结果提示,雌二醇对家兔窦房结自律细胞的电生理活动具有明显的抑制作用,此作用可能是通过非基因组机制发挥,与一氧化氮作用有关。     

大、小动脉内皮细胞乙酰胆碱作用靶标药理学特性的比较

目的 :比较大、小动脉内皮细胞乙酰胆碱作用靶标药理学特性的差异。方法 :采用大鼠尾动脉螺旋状血管条和主动脉离体血管环两种组织 ,对比观察乙酰胆碱 (ACh)诱发大、小动脉内皮依赖性舒张反应特征的差异 ,从而进一步研究小动脉内皮细胞乙酰胆碱作用靶标的药理学特性。结果 :氯化钾 (6 0mmol/L)预致血管收缩的尾动脉和主动脉对不同浓度ACh (10 -8～ 10 -4mol/L)产生内皮依赖性舒张反应 ,且呈剂量依赖性。L Nω 硝基精氨酸甲酯 (L NAME :10 -4mol/L)或美蓝 (MB :10 -5mol/L)与吲哚美辛 (Indo :10 -4mol/L)联用仅可部分地阻断ACh诱发尾动脉内皮依赖性舒张反应 ;而L NAME或MB可完全阻断ACh诱发主动脉内皮依赖性舒张反应。结论 :ACh激活大、小动脉上内皮细胞乙酰胆碱作用靶标诱发内皮依赖性舒张反应的药理学性质不同 ,在小动脉上 ,除了NO和PGI2介导外 ,还有一种非NO和非PGI2 的舒血管因子参与 ;在大动脉上 ,内皮依赖性舒张反应主要由NO介导     

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.     

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 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     

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     

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.     

Binding of Brain and Atrial Natriuretic Peptides to Cultured Mouse Astrocytes and Effect on Cyclic GMP

125I-Porcine brain natriuretic peptide (125I-pBNP) bound to mouse astrocytes in primary culture in a time-dependent manner (t1/2 = 4.5 min), similar to 125I-human atrial natriuretic peptide (125I-hANP) (t1/2 = 5 min). Binding was saturable and reached equilibrium after 90 min at 22 degrees C for both radioligands. Scatchard analysis suggested a single class of binding sites for pBNP with a binding affinity and capacity (KD = 0.08 nM; Bmax = 78.3 fmol/mg of protein) similar to those of hANP1-28 (KD = 0.1 nM; Bmax = 90.3 fmol/mg of protein). In competition binding studies, pBNP or human/rat atrial natriuretic peptide (ANP) analogues [hANP1-28, rat ANP1-28 (rANP1-28), and rANP5-28] displaced 125I-hANP, 125I-pBNP, and 125I-rANP1-28 completely, all with IC50 values of less than nM (0.14-0.83 nM). All four peptides maximally stimulated cyclic GMP (cGMP) production by 10 min at 22 degrees C at concentrations of 1 microM with EC50 values ranging from 50 to 100 nM. However, maximal cGMP induction by brain natriuretic peptide (BNP) (25.9 +/- 2.1 pmol/mg of protein) was significantly greater than that by hANP1-28 (11.5 +/- 2.2 pmol/mg of protein), rANP1-28 (16.5 +/- 2.0 pmol/mg of protein), and rANP5-28 (15.8 +/- 2.2 pmol/mg of protein). These studies indicate that BNP and ANPs act on the same binding sites and with similar affinities in cultured mouse astrocytes. BNP, however, exerts a greater effect on cGMP production. The difference in both affinity and selectivity between binding and cGMP production may indicate the existence of receptor subtypes that respond differentially to natriuretic peptides despite similar binding characteristics.     

The receptor attributable to C-type natriuretic peptide-induced differentiation of osteoblasts is switched from type B- to type C-natriuretic peptide receptor with aging

C-type natriuretic peptide (CNP) stimulates the differentiation and inhibits the proliferation of osteoblastic lineage cells. In this study, we examined whether the effects of CNP on osteoblastic functions change with aging using calvarial osteoblast-like cells from 25-week-old (young) and 120-week-old (aged) rats. CNP inhibited DNA synthesis and stimulated collagen synthesis and mineralized bone nodule formation. These effects were less pronounced in aged rat cells, suggesting the age-related attenuation of CNP-induced signaling. They were also blocked by the treatment of young rat cells with KT5823, a protein kinase G (PKG) inhibitor, but not by the treatment of aged rat cells with KT5823. CNP stimulated cGMP production in young rat cells, but not in aged rat cells. Natriuretic peptide receptor (NPR)-B, which has a guanylyl cyclase activity domain, and NPR-C, which has no enzyme activity domain, were predominantly expressed in young and aged rat cells, respectively. C-ANF, an NPR-C agonist, mimicked the effects of CNP on the proliferation and differentiation of aged rat cells; these effects were inhibited by the treatment with pertussis toxin (PTX), a Gi protein inhibitor. CNP and C-ANF evoked intracellular levels of inositol-1,4,5-triphosphate and Ca(2+), which are markers for phospholiase C (PLC) activation, in aged rat cells, and the effects of these two peptides were also blocked by the treatment with PTX. From these results, we concluded that CNP acts as a positive regulator of bone formation by osteoblasts and that the signaling pathway for CNP is switched from NPR-B/cGMP/PKG to NPR-C/G(i) protein/PLC with aging.     

Glycosylation is critical for natriuretic peptide receptor-B function

Co-transfection of a truncated natriuretic peptide receptor-B (NPR-B) with the full length receptor results in a decrease of 60–80% in wild-type receptor activity. This reduction correlates with a loss of glycosylation of the full length NPR-B. This effect is dose-dependent, and occurs with no change in the glycosylation of the truncated receptor. Co-transfection of the full length NPR-B with other receptors yields similar results. These data suggest that glycosylation may be crucial for NPR-B function. Cross-linking studies further demonstrate that only fully glycosylated NPR-B receptors are able to bind ligand. Our data therefore argue that carbohydrate modification may be critical for NPR-B receptor ligand binding.Abbreviations as amino acids - ANF atrial natriuretic factor - ANOVA analysis of variance - BS³ bis(sulfosuccinimidyl) suberate - BSA bovine serum albumin - CNP C-type natriuretic peptide - DEAE dextran-diethylaminoethyl-dextran - DMEM Dulbecco's modified Eagle medium - DMSO dimethyl sulfoxide - dNTP deoxynucleotide triphosphate - EDTA ethylenediamine tetraacetic acid - IBMX 3-isobutyl-l-methyl-=xanthine - min minutes - N-linked asparagine-linked - NPR natriuretic peptide receptor - nt nucleotide - PCR polymerase chain reaction - RIA radioimmunoassay - RP-HPLC reverse phase-high performance liquid chromatography - RP-HPLC reverse phase-high performance liquid chromatography - SDS sodium dodecyl sulfate - UV ultraviolet Address for offprints:Department of Pharmacology, University of Montreal, 2900 Edouard Montpetit, Montreal, Quebec, H3C3J7, Canada     

Glycosylation of asparagine 24 of the natriuretic peptide receptor-B is crucial for the formation of a competent ligand binding domain

UV cross-linking studies of the natriuretic pepti de receptor- B (NPR-B )using radio labeled C-type natriuretic peptide (CNP) indicate that onlyfully glycosylated receptors are capable of binding ligand. We thereforeused site-directed mutagenesis to determine which potential glycosylationsites are occupied by carbohydrate, and the relevant mutants werecharacterized in order to understand the function of carbohydrate additionat those sites. Our results suggest that five of seven potential N-linkedglycosylation sites are modified. In addition, mutation of asparagine 24results in a loss of ~90% of receptor activity. This mutant isexpressed at levels comparable to the wild-type receptor, and its activityis not significantly different from that of wild-type NPR-B in terms of EC50for CNP. Ligand binding studies on this mutant further show that althoughthere is no change in affinity for ligand, ~90% of receptor bindingis lost. These data suggest that many of the mutant receptors are simply notproperly folded. Our results indicate that glycosylation of asparagine 24 ofNPR-B receptors may be critical for the formation of a competent ligandbinding domain.