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

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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