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

1.人脐静脉内皮细胞与过量游离CF6孵育后，细胞表面抗三磷酸腺苷合成酶β亚单位抗体的免疫反应性降低50%，但对α亚单位抗体的免疫反应性无明显影响。观察到放射性配基在3×10~(-9)~(-9)M处通过10~(-7)M未标记的CF6发生显著的位移，其Kd为7.6 nm。β亚基抗体和α亚基抗体对I-CF6的结合无明显影响。在10℃~(-7)M时，CF6可使ATP对ADP的水解度提高1.6倍，而在10^~(-5)~(-5)M时，三磷酸腺苷对ADP的水解力被阻断。CF6浓度为10~(-7)M时，BCECF负载的人脐静脉内皮细胞内pH降低。10^&lt；-4&gt；M的阿米洛利增强了CF6引起的pH下降，而10^&lt；-5&gt；M的埃夫拉普汀则阻断了这一作用。花生四烯酸的释放可被CF6抑制，并可被β亚单位抗体或β亚单位抗体逆转。β亚单位抗体可抑制偶联因子…比6更多的药物会导致血压升高。这些结果表明，膜结合的三磷酸腺苷合成酶作为CF6的受体发挥作用，并可能通过调节细胞内氢的浓度而在高血压的发生中发挥先前未知的作用。2.用基因芯片(n=3)评估CF6暴露24小时后，增加的基因包括neuRegin-1(与对照组相比，1.83±0.82倍，p&lt；0.05)和relasin-1(1.74±0.20，p&lt；M)。尿激酶型纤溶酶原激活物受体(1.77±0.24，p=0.06)和雌激素受体β(1.74±0.30，p=0.08)和蛋白精氨酸甲基转移酶(prt-1；1.73±0.20，p&lt；0.05)均与充血性心力衰竭有关。在这些基因中，与一氧化氮合酶(NOS)的内源抑制物不对称二甲基精氨酸(ADMA)合成相关的酶(PRMT-1)与降解酶DDAH-2(DDAH-2)一起被进一步检测。实时定量逆转录-聚合酶链式反应检测，CF6作用48h后，PRMT-1/GAPDHm RNA比值升高，而DDAH-2/GAPDHm RNA比值降低。CF6可降低DDAH-2蛋白和活性。CF6可促进ADMA释放，降低NOS活性。这些结果表明，CF6通过促进ADMA的合成和抑制其降解，改变了基因表达谱，使其成为一种新型的ADMA释放刺激剂。较少

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