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Functional analysis of novel vasoconstrictor coupling factor 6 and its role in pathophysiology

新型血管收缩偶联因子6的功能分析及其在病理生理学中的作用

基本信息

批准号：
17590698
负责人：
OSANAI Tomohiro
金额：
$ 2.18万
依托单位：
Hirosaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-17590698/
关键词：
coupling factor 6 microarray binding assay receptor intracellular acidosis nitric oxide prostacyclin mitochondria prostacyclin ATP synthase asymmetric dimethylarginine estrogen receptor β neuregulin relaxin

项目摘要

1.Incubation of human umbilical vein endothelial cells (HUVEC) with an excess of free CF6 reduced by 50% the immunoreactivity for the antibody to β-subunit of ATP synthase at the cell surface, but unaffected that for the α-subunit antibody. A significant displacement of radioligand was observed at 3×10^<-9> through 10^<-7>M unlabeled CF6, and the Kd was 7.6nM. ADP at 10^<-7>M and β-subunit antibody suppressed the binding of ^<125>I-CF6, whereas the α-subunit antibody unaffected it. The hydrolysis activity of ATP to ADP was increased by 1.6-fold by CF6 at 10^<-7>M, and efrapeptin at 10^<-5>M, an inhibitor of ATP synthase, blocked it. CF6 at 10^<-7>M decreased intracellular pH in BCECF-loaded HUVEC. Amyloride at 10^<-4>M augmented the pH decrease in response to CF6, whereas efrapeptin at 10^<-5>M blocked it. Arachidonic acid release was suppressed by CF6, and it was reversed by efrapeptin at 10^<-5>M or β-subunit antibody or ADP at 10^<-7>M. The β-subunit antibody suppressed coupling fac … More tor 6-induced increase in blood pressure. These indicate that membrane-bound ATP synthase functions as a receptor for CF6 and may have a previously unsuspected role in the genesis of hypertension by modulating the concentration of intracellular hydrogen.2.The increased genes after 24-hour exposure to CF6 at 10^<-7>M, assessed by cDNA microarray (n=3), included neuregulin-1 (l.83±0.82 fold compared with control, p<0.05) and relaxin-1 (1.74±0.20, p<0.05) both relating to congestive heart failure, urokinase type plasminogen activator receptor (1.77±0.24, p=0.06) and estrogen receptor β (1.74±0.30, p=0.08) both relating to vascular inflammation and cell infiltration, and protein arginine methyltransferase (PRMT-1;1.73±0.20, p<0.05). Out of these genes, the enzyme relating to the synthesis (PRMT-1) of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), was further examined concomitantly with the degradation enzyme, dimethylarginine dimethylaminohydrolase 2 (DDAH-2). The ratio of PRMT-1 to GAPDH mRNA, measured by real time quantitative reverse transcription-polymerase chain reaction, was increased at 48 hours after CF6 at 10^<-7>M, whereas the ratio of DDAH-2 to GAPDH was decreased. DDAH-2 protein and activity were decreased by CF6. ADMA release was enhanced and NOS activity was decreased by CF6. These indicate that CF6 changes the gene expression profile to be proatherogenic and functions as a novel stimulator for ADMA release via enhancing its synthesis and suppressing its degradation. Less

1.人脐静脉内皮细胞（HUVEC）与过量游离CF 6孵育后，细胞表面ATP合成酶β亚基抗体的免疫反应性降低50%，而α亚基抗体的免疫反应性不受影响。在3×10 ~（-1）mol/L浓度下，未标记的CF ~（6+）对放射性配体有明显的置换作用<-9><-7>，其Kd值为7.6nM。10 μ M的ADP<-7>和β-亚基抗体可抑制α-亚基抗体与△ I-CF 6的结合<125>，而α-亚基抗体则不影响△ I-CF 6的结合，10 μ M的CF 6可使ATP水解为ADP的活性增加1.6倍<-7>，<-5>ATP合成酶抑制剂efrapeptin可阻断△ I-CF 6水解ATP的活性，10 μ M的CF 6<-7>可降低BCECF负载的HUVEC细胞内pH。10 μ M的阿米洛利<-4>增加了CF 6引起的pH下降，而10 μ M的efrapeptin<-5>则阻断了这一作用。CF 6抑制花生四烯酸的释放，10 μ M的efrapeptin<-5>或10 μ M的β-亚单位抗体或ADP<-7>可逆转花生四烯酸的释放。β-亚单位抗体抑制偶联因子， ...更多信息 6引起的血压升高。这些结果表明，膜结合ATP合成酶作为CF 6的受体发挥功能，并且可能通过调节细胞内氢浓度而在高血压的发生中发挥先前未被怀疑的作用。2.通过cDNA微阵列（n=3）评估的10 μ M CF 6暴露24小时后增加的基因<-7>包括神经调节蛋白-1（neuregulin-1（与对照相比为1.83 ±0.82倍，p<0.05）和松弛素-1（1.74±0.20，P<0.05）均与充血性心力衰竭、尿激酶型纤溶酶原激活物受体有关（1.77±0.24，p=0.06）和雌激素受体β（1.74±0.30，p=0.08），两者均与血管炎症和细胞浸润有关;和蛋白质精氨酸甲基转移酶（PRMT-1;1.73±0.20，p<0.05）。在这些基因中，与不对称二甲基精氨酸（ADMA）（一氧化氮合酶（NOS）的内源性抑制剂）的合成相关的酶（PRMT-1）与降解酶二甲基精氨酸二甲氨基水解酶2（DDAH-2）一起被进一步检查。通过真实的时间定量逆转录-聚合酶链反应测量的PRMT-1与GAPDH mRNA的比率在10 μ M的CF 6后48小时增加<-7>，而DDAH-2与GAPDH的比率降低。CF 6降低DDAH-2蛋白和活性。CF 6可促进ADMA释放，降低NOS活性。这些表明，CF 6改变基因表达谱是促动脉粥样硬化和功能作为一种新的刺激剂ADMA释放，通过增强其合成和抑制其降解。少