Mitochondrial coupling factor 6 as a potent endogenous vasoconstrictor

Abstract

We demonstrated recently that coupling factor 6, an essential component of the energy-transducing stalk of mitochondrial ATP synthase, suppresses the synthesis of prostacyclin in vascular endothelial cells. Here, we tested the hypothesis that coupling factor 6 is present on the cell surface and is involved in the regulation of systemic circulation. This peptide is present on the surface of CRL-2222 vascular endothelial cells and is released by these cells into the medium. In vivo, the peptide circulates in the vascular system of the rat, and its gene expression and plasma concentration are higher in spontaneously hypertensive rats (SHRs) than in normotensive controls. Elevation of blood pressure with norepinephrine did not affect the plasma concentration of coupling factor 6. Intravenous injection of recombinant peptide increased blood pressure, apparently by suppressing prostacyclin synthesis, whereas a specific Ab to coupling factor 6 decreased systemic blood pressure concomitantly with an increase in plasma prostacyclin. Interestingly, the antibody's hypotensive effect could be abolished by treating with the cyclooxygenase inhibitor indomethacin. These findings indicate that mitochondrial coupling factor 6 functions as a potent endogenous vasoconstrictor in the fashion of a circulating hormone and may suggest a new mechanism for hypertension.

Figure 1

(a) Western blot analysis of coupling factor 6 (CF6) in the heart extract from SHRs. (b) Reverse-phase HPLC of plasma samples monitored by RIA for rat CF6. The filled bar indicates the elution position and concentration of rat CF6.

Figure 2

The gene expression of coupling factor 6 (CF6) in the aorta from WKYs and SHRs. (a) Representative bands of CF6 and GAPDH. (b) The ratio of CF6 to GAPDH mRNA in WKYs and SHRs (n = 3).

Figure 3

Immunofluorescence microscopy of coupling factor 6 (CF6) in ATP synthase on CRL-2222 cell surfaces. (a) CRL-2222 cells under epi-illumination showing immunofluorescent surface staining for the CF6 of ATP synthase. (b) The same field of CRL-2222 cells under visible light. (c) Incubation with preimmune serum alone. (d) Incubation with secondary Ab alone.

Figure 4

Kinetics of prostacyclin in the plasma after intravenous bolus injection of rat recombinant coupling factor 6 (CF6) at 1 μg/kg (n = 4) and anti–coupling factor 6 Ab (CF6 Ab) at 3 μg/kg (n = 4) in SHRs at the age of 16 weeks.

Figure 5

(a) Changes in the arterial blood pressure after intravenous injection of anti–coupling factor 6 Ab (CF6 Ab) in WKYs and SHRs at the age of 16 weeks. It is noted that pretreatment with indomethacin at 10 mg/kg for 30 minutes blocked the decrease in blood pressure after injection of CF6 Ab in SHRs. (b) Dose-response effects of anti–coupling factor 6 Ab on mean arterial blood pressure (BP) in WKYs (n = 7) and SHRs (n = 7) at the age of 16 weeks. Statistical analysis was performed by ANOVA for repeated measures and two-way ANOVA.

Figure 6

(a) Changes in the arterial blood pressure after intravenous injection of rat recombinant coupling factor 6 (CF6) at 1 μg/kg in WKYs and SHRs at the age of 16 weeks. It is noted that pretreatment with indomethacin at 10 mg/kg for 30 minutes blocked the increase in blood pressure after injection of CF6 in SHRs. (b) Dose-response effects of rat recombinant coupling factor 6 on mean blood pressure (BP) in WKYs (n = 7) and SHRs (n = 7) at the age of 16 weeks. Statistical analysis was performed by ANOVA for repeated measures and two-way ANOVA.

Figure 7

(a) Changes in the arterial blood pressure after intravenous injection of bradykinin (BK) with or without preadministration of anti–coupling factor 6 Ab (CF6 Ab) at 3 μg/kg in WKYs and SHRs at the age of 16 weeks. CF6 Ab(+) is the condition in which arterial blood pressure returned to the baseline after preadministration of anti–coupling factor 6 Ab at 3 μg/kg. (b) Effects of preadministration of anti–coupling factor 6 Ab (CF6 Ab) at 3 μg/kg on the dose-dependent blood pressure–lowering effect of bradykinin in WKYs (n = 7) and SHRs (n = 7) at the age of 16 weeks. Statistical analysis was performed by ANOVA for repeated measures and two-way ANOVA.