Intracellular Signaling for Vasoconstrictor Coupling Factor 6
Novel Function of β-Subunit of ATP Synthase as Receptor
Coupling factor 6 (CF6), a component of adenosine triphosphate (ATP) synthase, is circulating and functions as an endogenous vasoconstrictor by inhibiting cytosolic phospholipase A2. 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 125I-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.
Adenosine triphosphate (ATP) synthase is one of the most unique supermolecule enzymes in the mitochondria, and it is separated into 2 units.1 One is a water-soluble component, F1, containing 5 subunit types in the ratio α3β3γδε, and constructs the catalytic site of ATP synthase. The other is a detergent-soluble component, F0, and constructs an energy transduction part. The presence of ATP synthase at the cell surface of lymphocytes, hepatocytes, and human vascular endothelial cells has been recently verified2–4 and its novel function has been reported. In hepatocytes, β-subunit of ATP synthase has been identified as a receptor for apolipoprotein E-rich high-density lipoprotein (HDL) and their interaction triggers the endocytosis of HDL particles.3 In endothelial cells, the interaction of α- and β-subunits of ATP synthase with angiostatin was suggested to inhibit vascularization by suppression of endothelial-surface ATP metabolism.4 Coupling factor 6 is one component of ATP synthase that is normally associated with the detergent-soluble Fo segment. Coupling factor 6 has been reported to operate for restoration of inorganic phosphate (Pi)–ATP exchange and oligomycin-sensitive ATPase activity.5 While investigating the mechanism for inhibition of prostacyclin synthesis in spontaneously hypertensive rats (SHR),6 coupling factor 6 was unexpectedly identified as a novel inhibitor of prostacyclin synthesis.
We found that coupling factor 6 is present in the systemic circulation in SHR and exogenously administered coupling factor 6 consistently induces an increase in arterial blood pressure.7 We also showed that its vasoconstrictor effect is unaffected by a single pass through the pulmonary vascular bed and is operating in the fashion of a circulating hormone.7 Coupling factor 6 is released from the surface of vascular endothelial cells and its release is stimulated by tumor necrosis factor (TNF)-α and shear stress through activation of NF-kB signaling pathway.8–10 Despite a potent vasoconstrictor effect of coupling factor 6, the receptor and the post-receptor signaling pathway of the peptide have not been determined. Thereby, the present study focused on the identification and characterization of a receptor for vasoconstrictor coupling factor 6 and its post-receptor signaling pathway in vascular endothelial cells. We show the novel function of membrane-bound ATP synthase as a receptor for coupling factor 6.
Materials and Methods
Fetal bovine serum (FBS), trypsin (0.05%)–ethylenediaminetetraacetic acid (EDTA) (0.02%), and phosphate-buffered saline (PBS) were purchased from Gibco Grand. HuMedia was purchased from Kurabo Co, Ltd. [3H] arachidonic acid (AA) were purchased from New England Nuclear. An iodinated coupling factor 6 (12.9 MBq/mL, 5 μg/mL) was prepared by Daiichi Suntory Biomedical Research Co, LTD (Osaka, Japan). Fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG was from ICN Biomedicals, Inc. ATTO 550 was from ATTO-TEC GmbH. Angiostatin was from Hematologic Technologies, Inc. Antibodies for α- and β-subunit ATP synthase were from BD Biosciences. The 2′,7′-bis(carboxyethyl-56)-carboxyfluorescein (BCECF) was purchased from Molecular Probes. Protein A/G PLUS-Agarose beads were from Santa Cruz Biotechnology Inc. All other reagents were of the finest grade available from Sigma Chemical.
Primary human umbilical vein endothelial cells (HUVECs) (Kurabo Co, Ltd) were cultured in HuMedia supplemented with 2% FBS, 10 ng/mL recombinant epidermal growth factor, 1 mg/mL hydrocortisone, 5 ng/mL recombinant fibroblast growth factor, and 10 μg/mL heparin (complete medium) at 37°C under 5% CO2. HUVECs from the second to fourth passages were used for the study.
Synthesis of Recombinant Coupling Factor 6 and Its Antibody
Mature human coupling factor 6 was obtained from Escherichia coli using a cleavable fusion protein strategy.8 For the antibody, synthetic coupling factor 6 fragment (human Cys-10 to 27 amino acid) solution was emulsified with an equal volume of Freund’s complete adjuvant and used for immunizing New Zealand White rabbits as reported previously.8
Flow Cytometry Analysis
HUVECs were incubated with coupling factor 6 at 10−7 M for 30 minutes, and then trypsinized and reacted with saturating concentrations of α- and β-subunit ATP synthase antibodies (dilution of 1:1000) in PBS containing 1% bovine serum albumin (BSA) for 30 minutes on ice. After being washed 3 times with PBS, the cells were stained with FITC-conjugated goat anti-rabbit IgG in PBS for another 30 minutes, and then analyzed in a FACScan.
Binding studies were performed with HUVECs on 24-well plate. Briefly, each well was incubated in the 200 μL serum-free Dulbecco’s modified Eagle’s medium and contained 125I-coupling factor 6 (0.25 to 0.5 nM) with unlabeled coupling factor 6 (10−10 M to 10−7 M) at 37°C under 5% CO2. After incubating for 30 minutes, bound and free radioligand were separated by rapidly diluting 1 mL PBS and immediately washing 4 times. The bound radioligand was collected after incubating with 20% sodium dodecyl sulfate PBS (200 μL) for 1 hour at 37°C and counted in a gamma counter. In some experiments, α- and β-subunits antibodies, ATP, adenosine diphosphate (ADP), angiostatin, and efrapeptin, an inhibitor of ATP synthase, were added in the standard mixture of 125I-coupling factor 6 without unlabeled coupling factor 6. Nonspecific binding was assessed by incubating the cells in the presence of 10−6 M unlabeled coupling factor 6 and found to be 20±5% of total binding in HUVECs. Specific binding was then determined by subtracting nonspecific from total binding.
Assays for ATP Synthase Activity
ATPase activity was assayed as described previously by a spectrophotometric procedure.11 Briefly, it was monitored spectrophotometrically at 340 nm by coupling the production of ADP to the oxidation of nicotinamide adenine dinucleotide (NADH) via the pyruvate kinase and lactate dehydrogenase reactions. The reaction mixture was contained in a final volume of 0.7 mL at 30°C and included the following: 100 mmol/L Tris, 4.0 mmol/L MgATP, 2 mmol/L MgCl2, 50 mmol/L KCl, 0.2 mmol/L EDTA, 0.23 mmol/L NADH, 1 mmol/L phosphoenol pyruvate, 1.4 U of pyruvate kinase, 1.4 U of lactate dehydrogenase, and the cells. The reaction was started by the addition of the cells.
Intracellular pH Measurement
HUVECs were harvested and resuspended in PBS containing BCECF-AM (1 mg/mL), and then incubated for 15 minutes at 37°C. Excess dye was removed by centrifugation and resuspension of the cells in nominally HCO3−-free Krebs-Henseleit buffered with 20 mmol/L Hepes containing 0.2% fatty acid-free BSA. The suspension was transferred to a cuvette that was placed in the temperature-controlled chamber of a Shimazu 5000 luminescence spectrophotometer, maintained at 37°C. Using a dual excitation fast filter accessory, the sample was excited at 495 nm and 440 nm successively, and the fluorescence emission was measured at 535 nm. The ratio of the fluorescence intensity measured using the 2 excitation wavelengths (495 nm/440 nm) provides a quantitative measure of pH. Calibration with the K+/H+ ionophore, nigericin at 5 μg/mL, was performed exactly as described.12
Measurement of AA Release
HUVECs on 24-well plates were labeled for 24 hours with [3H] AA (0.25μCi in 250 μL complete medium/well at 37°C). The labeling media was aspirated and the cells were washed 3 times at 5-minute intervals with 1 mL serum-free HuMedia and preincubated for 10 minutes in the same medium. This medium was removed, and the cells were incubated with various concentrations of coupling factor 6, or efrapeptine, a specific inhibitor of ATP synthase F1, or the α- and β-subunit antibodies, or ATP and ADP in 250 μL serum-free HuMedia at 37°C. After 30 minutes, the medium was collected and the [3H] level was counted with a liquid scintillation counter. In addition, the effect of coadministration of coupling factor 6 at 10−7 M and efrapeptin at 10−5 M, or the α- and β-subunit antibodies were also assessed by the same method.
Blood Pressure Measurement
Blood pressure was determined by direct arterial cannulation in anesthetized rats as previously reported.7 To examine whether coupling factor 6-induced hypertensive effect is mediated by β-subunit of ATP synthase, we evaluated the blood pressure-elevating effect of coupling factor 6 with or without pre-administration of anti-β-subunit antibody at 3.0 μg/kg body weight in 20-week-old Wistar Kyoto rats (Charles River, Kanagawa, Japan). This study protocol was approved by the Animal Committee of the Hirosaki University.
Human coupling factor 6 was labeled by ATTO 550, a new fluorescent dye for protein. HUVECs were incubated in PBS with ATTO 550-labeled human coupling factor 6, and the transition of the peptide into the cells was observed for 10 minutes.
Immunoprecipitation and Western Blotting
HUVECs were solubilized in ice-cold lysis buffer containing protease inhibitor and were coincubated with coupling factor 6. After incubation with anti-coupling factor 6 antibody for 1 hour, the proteins were immunoprecipitated by protein A/G PLUS-Agarose beads, subjected to SDS-PAGE, and stained with anti-coupling factor 6 antibody or β-subunit antibody by amplified alkaline phosphatase immunoblot kits.
All results are expressed as mean±SEM. An unpaired t test for comparison of 2 variables and 1-way or 2-way ANOVA for multiple comparisons followed by Fisher’s protected least significant difference test were used for statistical analysis. The level of significance was <0.05.
Immunofluorescence and Immunoprecipitaion Analyses
Fluorescence-assisted flow cytometry experiments in intact cells confirmed the cell-surface localization of the α- and β-subunits in HUVECs. The selectivity of the response to the α- and β-subunit antibodies was clearly shown by substantially low signal obtained with isotypic IgG. After pre-incubating the cells with coupling factor 6, the immunoreactivity for the anti-coupling factor 6 antibody was enhanced at the surface of HUVECs (Figure 1A), suggesting that the binding site of coupling factor 6 is present on the cell surface. Incubation of HUVECs with an excess of free coupling factor 6 reduced the immunoreactivity for the β-subunit antibody by &50% (Figure 1B) but did not affect that for the α-subunit antibody (Figure 1C). Identical results were observed in 4 separate experiments in different HUVEC strains. This confirmed the cell surface interaction of free coupling factor 6 with the β-subunit of ATP synthase. Immunoprecipitation analysis directly showed the binding of coupling factor 6 and β-subunit in vitro (Figure 1D). ATTO-labeled coupling factor 6 was undetected inside of the cells for 10 minutes (Figure 1E), suggesting that the effect of coupling factor 6 is receptor-dependent.
125I-coupling factor 6 bound to the surface of HUVECs and plateaued to the level of 2185±120 cpm/well at 30 minutes (n=4). As shown in Figure 2A, a significant displacement of radioligand was observed at 3×10−9 M unlabeled coupling factor 6, and maximum displacement was seen at 10−7 M (n=4). As estimated by the method of Cheng and Prusoff,13 the Kd of coupling factor 6 to the β-subunit of ATP synthase was 7.6±0.3 nM. ADP at 10−7 M suppressed the time-dependent binding of 125I-coupling factor 6 (P<0.05 by 2-way ANOVA) (Figure 2B). As shown in Figure 2C, the β-subunit antibody suppressed by 32.0±2.0% the binding of 125I-labeled coupling factor 6 (n=5, P<0.05). In contrast, the α-subunit antibody unaffected the binding of 125I-labeled coupling factor 6. ADP at 10−7 M suppressed by 81.3±9.7% the binding of coupling factor 6 (n=5, P<0.05). This suppression was unaffected by coadministration of ATP at 10−7 M and efrapeptin at 10−5 M (both n=4). Neither ATP at 10−7 M nor efrapeptin at 10−5 M influenced the binding of 125I-coupling factor 6 (both n=3). Angiostatin at 1 mmol/L did not affect the binding of 125I-coupling factor 6 (2210±85 versus 2015±65 cpm/well at 30 minutes, n=3).
ATP Synthase Activity
As shown in Figure 3, the hydrolysis activity of ATP to ADP on the surface of HUVEC was verified in the absence of coupling factor 6 by using a coupled enzymatic assay in which production of ADP is linked to oxidation of NADH via pyruvate kinase and lactate dehydrogenase. It was increased by 61.5±4.9% in the presence of coupling factor 6 at 10−7 M (n=4, P<0.05), whereas its increase in hydrolysis activity was abolished after pretreatment with efrapeptin at 10−5 M (n=4). Efrapeptin at 10−5 M alone did not affect the hydrolysis activity.
Effect of Coupling Factor 6 on Intracellular pH
Figure 4 illustrates representative tracings of changes in intracellular pH after administration of coupling factor 6 in HUVECs. Coupling factor 6 at 10−7 M decreased intracellular pH within seconds, reaching the maximal decrease within 2 minutes and returning to baseline after several minutes (Figure 4A). Amyloride at 10−3 M, an inhibitor of sodium-hydrogen exchanger, promptly and remarkably decreased intracellular pH, suggesting that sodium-hydrogen exchanger is intact after treatment with coupling factor 6 at 10−7 M (Figure 4A). Pretreatment with efrapeptin at 10−5 M abolished the reduction of intracellular pH in response to coupling factor 6 at 10−7 M (Figure 4B). Pretreatment with the lower concentration (10−4 M) of amyloride induced a transient decrease in intracellular pH and augmented the decrease in intracellular pH by subsequent administration of coupling factor 6 at 10−7 M (Figure 4C).
Intracellular pH at baseline was 7.20±0.03 in HUVECs (n=5). The maximum decrease in intracellular pH in response to coupling factor 6 at 10−7 M was 0.26±0.02 (n=5, P<0.05). Pretreatment with amyloride at 10−4 M enhanced the maximum decrease in intracellular pH to 0.42±0.03 (n=5, P<0.05).
Effect of Coupling Factor 6 on AA Release
Incorporation into cellular lipid was >85% of added [3H] AA, and by 30 minutes 0.3% to 0.5% of incorporated [3H] AA was released. Baseline [3H] AA release count was 5541±154 cpm/well per 30 minutes in HUVECs, and coupling factor 6 suppressed AA release from HUVECs in a dose-dependent manner (all n=4, P<0.05 by 1-way ANOVA) (Figure 5A). The suppressant effect of coupling factor 6 at 10−7 M (51.1±2.9%) was reversed by pretreatment with efrapeptin at 10−5 M (n=5, P<0.05) (Figure 5B). The β-subunit antibody also dampened the suppressant effect of coupling factor 6, whereas the α-subunit antibody unaffected it. ADP at 10−7 M reversed the suppressant effect of coupling factor 6 (n=5, P<0.05). ATP at 10−7 M suppressed AA release by 21.7±4.4% (n=5, P<0.05). Efrapeptin at 10−7 M reversed the decrease in AA release in response to ATP at 10−7 M, whereas pyridoxal phosphate-6-azophenyl- 2′,4′-disulfonic acid (PPAD) at 10−5 M, a blocker for ATP receptors, unaffected it. Coadministration of ATP at 10−7 M and coupling factor 6 at 10−7 M had no additive effect on AA release. As shown in Figure 5C (all n=4), bradykinin at 10−5 M enhanced by 27.7±8.6% the release of AA. Its increase was suppressed by coupling factor 6 and reversed by pretreatment with efrapeptin at 10−5 M.
In Vivo Role of β-Subunit of ATP Synthase
As shown in Figure 6, an intravenous bolus injection of coupling factor 6 at 1.0 μg/kg body weight increased mean arterial blood pressures by 6±1 mm Hg, but pretreatment with anti-β-subunit antibody for 2 minutes suppressed by 47±6% compared with that produced by coupling factor 6 alone (n=4, P<0.01).
Identification and Characterization of Coupling Factor 6 Receptor
The present study focused on the identification and characterization of a receptor for vasoconstrictor coupling factor 6 and its post-receptor signaling pathway in vascular endothelial cells, and showed a novel function of membrane-bound ATP synthase as a receptor for coupling factor 6. Although coupling factor 6 is found mostly at the inner side of mitochondrial membrane,1 we showed the colocalization of ATP synthase and coupling factor 6 at the surface of vascular endothelial cells. Coupling factor 6 was cross-reacted with the β subunit of ATP synthase, which is distinct from the original position of the peptide, suggesting that coupling factor 6 binds to the β subunit of ATP synthase at the cell surface. The association between coupling factor 6 and the β-subunit was further verified by a type of competition experiment showing the suppression of 125I-coupling factor 6 binding by anti-β-subunit antibody and immunoprecipitation analysis. Displacement analysis revealed that coupling factor 6 binds to the β-subunit of ATP synthase at the surface of HUVECs with Kd for 7.6 nM. This Kd for the β-subunit of ATP synthase was similar to that for another agonist binding to the α and β-subunits of its enzyme, angiostatin.4
Of the 3 catalytic β-subunits in the F1–ATP synthase complex (α3β3γδε), one β subunit was reported to contain an ATP analogue, adenosine 5′-[β,γ-imido] triphosphate (AMP-PNP), and the other ADP; these 2 subunits have been referred to as the βTP and βDP subunits, respectively. The third site is empty or half-closed in the structure, thereby called the βE or βHC subunit.14–16 To date, there are a tight binding site for nucleotide (Kd=10−12 M), a loose site (Kd=5×10−7 M), and a weak binding site (Kd=1.5×10−5 M), and the βTP subunit is identified as the “tight” binding site for nucleotide, whereas the βDP subunit is the “loose” site.17,18
The Kd of the binding of coupling factor 6 ranged near to that of the “loose” binding site for nucleotide (the βDP subunit) rather than the “tight” binding site for nucleotide (the βTP subunit). The binding of 125I-coupling factor 6 to the cells was modulated by ADP but not by ATP or efrapeptin. Efrapeptin blocks the conversion of subunit βE to a nucleotide binding conformation and inhibits the activity of ATP synthase.18 Taken together, the binding site of coupling factor 6 is likely to be the βDT subunit rather than the βTP and βE subunits. It is of interest that the binding fashion of coupling factor 6 is similar to that of HDL particle in hepatocytes: the binding of apolipoprotein E-rich HDL to β-subunit of ATP synthase triggers the endocytosis of HDL particle and ADP suppresses it.3
Post-Receptor Signaling Pathway
The F1-Fo ATP synthase holoenzyme efficiently catalyzes both the forward ATP synthase reaction and reverse ATP hydrolysis reaction. Moser et al showed that the HUVEC surface-associated ATP synthase is active in ATP synthesis by dual-label thin-layer chromatography and bioluminescence assays and that both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin.4 The present nucleotide analysis clearly showed that coupling factor 6 stimulated ATPase activity at the cell surface of HUVECs, indicating that at the plasma membrane of vascular endothelial cells, there is a complete F1–ATPase that functions as an ATP hydrolase and is activated after binding of coupling factor 6 to the β-subunit. Because ADP suppressed the binding of coupling factor 6 to the β-subunit of membrane-bound ATP synthase, the biological effect of coupling factor 6 is regulated by a negative feedback mechanism. This might explain the discontinuity of the vasoconstrictor effect of coupling factor 6 in vitro:7 injected coupling factor 6 increased arterial blood pressure within seconds, reaching the maximal increase and returning to baseline after several minutes in SHR.
Because the activation of ATP synthase is accompanied by a transient flux of hydrogen ion through Fo, we measured intracellular pH using hydrogen-sensitive dye BCECF and characterized the post-receptor signaling pathway. As expected, intracellular pH was decreased within seconds after administration of coupling factor 6 and lasted for several minutes. It is of interest that this time profile is similar to that of vasoconstrictor effect of coupling factor 6. In addition, efrapeptin dampened not only the decrease in intracellular pH by coupling factor 6 but also the decrease in AA release by the peptide, suggesting that ATPase activity is associated with the biological action of coupling factor 6. It was reported that the optimal pH for the action of cytosolic phospholipase A2 (cPLA2) ranges between 7.5 and 8.5 in the proximal tubular epithelium and 9.0 in the lung.19,20 Furthermore, the sulfite-induced activation of cPLA2 activity is pH-dependent and it is abolished by the decease in intracellular pH at 1.0,21 suggesting that coupling factor 6-induced intracellular acidification might inhibit cPLA2 activity. It still remains unclear whether the small changes in intracellular pH around 0.2 to 0.4 inhibit cPLA2 activity directly or through the modulators of cPLA2 activity such as annexins.22 Further study is needed.
The β-subunit antibody partially antagonized the biological action of coupling factor 6. ATP stimulated ATPase activity without affecting the binding of coupling factor 6, and this effect was suppressed by efrapeptin but unaffected by PPAD, a blocker of ATP receptors for P2X (ion channel) and P2Y (G-protein-coupled receptors).23 Furthermore, there was no additive effect in ATP and coupling factor 6, suggesting that the action of these 2 compounds may be yielded through the common signaling pathway.
Implications of Coupling Factor 6
In clinical settings, we showed that circulating coupling factor 6 is elevated in patients with essential hypertension and modulated by salt intake presumably via reactive oxygen species.24 We further showed that the plasma level of coupling factor 6 is related to the development of ischemic heart disease complicated with end-stage renal disease.25 A number of vasoconstrictors and growth factors induce intracellular alkalinization by activating sodium–hydrogen exchanger. However, coupling factor 6 is a unique vasoconstrictor that leads to the reverse response of pH, acidification, thereby inhibiting prostacyclin synthesis. In light of the current findings, it appears that a new signaling pathway is involved in the development of hypertension.
The present study identified the β-subunit of membrane-bound ATP synthase as a receptor for coupling factor 6 and showed that its post-receptor signaling pathway is mediated through the elevation of intracellular hydrogen concentration. These would provide more understanding for the regulation mechanism of arterial blood pressure and a novel therapeutic strategy for human hypertension. Because intracellular acidification might influence the gene expression profile, the present evidence further stimulates interest in identification of the novel effects of coupling factor 6 other than the genesis of hypertension.
- Received April 18, 2005.
- Revision received May 9, 2005.
- Accepted September 1, 2005.
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