Intracellular Signaling for Vasoconstrictor Coupling Factor 6

Abstract

Coupling factor 6 (CF6), a component of adenosine triphosphate (ATP) synthase, is circulating and functions as an endogenous vasoconstrictor by inhibiting cytosolic phospholipase A 2 . We showed a high plasma level of CF6 in human hypertension. The present study focused on the identification and characterization of a receptor for CF6 and its post-receptor signaling pathway. Incubation of human umbilical vein endothelial cells (HUVECs) 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 7.6 nM. Adenosine diphosphate (ADP) at 10 −7 M and β-subunit antibody suppressed the binding of 125 I-CF6 by 81.3±9.7% and 32.0±2.0%, respectively, 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 2′,7′-bis(carboxyethyl-5 (6))-carboxyfluorescein-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 factor 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.