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。与过量的自由CF6孵育50％，将免疫反应性fodiboty降低至细胞表面ATP合酶的 - 亚基，但对放射性抗体的显着性不受影响。在3×10 ^<-7> m处，未标记的CF6 Bunit抗体抑制了 ^<125> i-cf6的结合，而α-亚基不受影响的α-亚基的结合。 10^-7> m。而在10 ^<-5> m处的fropeptin却被CF6 ^<-7> m抑制Syncti ons作为CF6的静态感染者，并且通过调节浓度外细胞氢的浓度在高血压的创世记中可能具有未引起的作用与对照组相比，微阵列（n = 3）倍数，p <0.05）和弛豫蛋白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因子受体（1.77±0.24，p = 0.06）和雌激素受体β（1.74±0.30，p = 0.08）都与血管炎症和庆祝有关，以及蛋白质甲基转移酶（PRMT-1； 1.73±0.20，p <0.05）。一氧化氮合酶的内源性抑制剂（NOS）与降解酶，Dimethylaminoh Ydrollose 2（DDAH-2）一起进一步检查了二甲基制度的内源性抑制剂（NOS）。 CF6在10^<-7> m后48小时在48小时内增加了反定差链链反应，而DDAH-2与GAPDH的比率降低了。 CF6将基因变成ynthesis，并抑制其降解