Intracellular Signaling for Vasoconstrictor
Coupling Factor 6
Novel Function of ␤-Subunit of ATP Synthase as Receptor
Tomohiro Osanai, Koji Magota, Makoto Tanaka, Michiko Shimada, Reiichi Murakami, Satoko Sasaki,
Hirofumi Tomita, Naotaka Maeda, Ken Okumura
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Abstract—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 ␤-subunit of ATP synthase at the cell surface, but
unaffected that for the ␣-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 ␤-subunit antibody
suppressed the binding of 125I-CF6 by 81.3⫾9.7% and 32.0⫾2.0%, respectively, whereas the ␣-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 ␤-subunit antibody or ADP at 10⫺7 M. The ␤-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.
(Hypertension. 2005;46:1140-1146.)
Key Words: hypertension 䡲 prostacyclin
A
denosine 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 ␣3␤3␥␦⑀, 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, ␤-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 ␣- and ␤-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 oligomycinsensitive 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)-␣ 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 postreceptor signaling pathway of the peptide have not been
determined. Thereby, the present study focused on the identification and characterization of a receptor for vasoconstric-
Received April 18, 2005; first decision May 9, 2005; revision accepted September 1, 2005.
From the Second Department of Internal Medicine (T.O., M.T., M.S., R.M., S.S., H.T., N.M, K.O.), Hirosaki University School of Medicine, Hirosaki,
Japan; Daiichi Suntory Biomedical Research Co, Ltd (K.M.), Osaka, Japan.
Correspondence to Tomohiro Osanai, MD, the Second Department of Internal Medicine, Hirosaki University School of Medicine, 5 Zaifu-cho,
Hirosaki, 036-8562 Japan. E-mail [email protected]
© 2005 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000186483.86750.85
1140
Osanai et al
tor 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
Materials
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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 ␮g/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 ␣- and ␤-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.
Cell Culture
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 ␮g/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 ␣- and ␤-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 Assay
Binding studies were performed with HUVECs on 24-well plate.
Briefly, each well was incubated in the 200 ␮L 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 ␮L) for 1 hour at 37°C and counted in a gamma counter. In
some experiments, ␣- and ␤-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.
Intracellular Signaling of Coupling Factor 6
1141
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 temperaturecontrolled 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 ␮g/mL,
was performed exactly as described.12
Measurement of AA Release
HUVECs on 24-well plates were labeled for 24 hours with [3H] AA
(0.25␮Ci in 250 ␮L 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 ␣and ␤-subunit antibodies, or ATP and ADP in 250 ␮L 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 ␣- and ␤-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
␤-subunit of ATP synthase, we evaluated the blood pressureelevating effect of coupling factor 6 with or without preadministration of anti-␤-subunit antibody at 3.0 ␮g/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.
Immunofluorescence Microscopy
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 ␤-subunit antibody by amplified alkaline
phosphatase immunoblot kits.
1142
Hypertension
November 2005
Figure 1. Immunofluorescence and
immunoprecipataion analyses for ATP
synthase subunits and coupling factor 6.
Flow cytometry: The cell surfaceassociated coupling factor 6 (A),
␤-subunit of ATP synthase (B), and
␣-subunit of ATP synthase (C) before
(solid line) and after (dotted line) preincubating with coupling factor 6 at 10⫺7 M
for 30 minutes. D, Immunoprecipitation.
Western blot for coupling factor 6 (lane
1) and ␤-subunit of ATP synthase (lane
2). E, Immunofluorescence microscopy
for ATTO-labeled coupling factor 6.
Upper panel, Permeabilized cells. Bottom panel, nonpermeabilized cells.
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Statistical Analysis
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.
Results
Immunofluorescence and
Immunoprecipitaion Analyses
Fluorescence-assisted flow cytometry experiments in intact
cells confirmed the cell-surface localization of the ␣- and
␤-subunits in HUVECs. The selectivity of the response to the
␣- and ␤-subunit antibodies was clearly shown by substantially low signal obtained with isotypic IgG. After preincubating 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 ␤-subunit antibody by
⬇50% (Figure 1B) but did not affect that for the ␣-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 ␤-subunit of ATP synthase. Immunoprecipitation
analysis directly showed the binding of coupling factor 6 and
␤-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.
Binding Assay
125
I-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 ␤-subunit of ATP synthase
was 7.6⫾0.3 nM. ADP at 10⫺7 M suppressed the timedependent binding of 125I-coupling factor 6 (P⬍0.05 by
2-way ANOVA) (Figure 2B). As shown in Figure 2C, the
␤-subunit antibody suppressed by 32.0⫾2.0% the binding of
125
I-labeled coupling factor 6 (n⫽5, P⬍0.05). In contrast, the
␣-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
125
I-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 sodiumhydrogen 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
Osanai et al
Intracellular Signaling of Coupling Factor 6
1143
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Figure 2. The binding assay of 125Icoupling factor 6. A, Competitive displacement analysis of 125I-coupling factor 6
(n⫽4). B, Time-dependent effect of ADP at
10⫺7 M on the binding of 125I-coupling factor 6 (n⫽4). C, Effect of various compounds on the binding of 125I-coupling factor 6 (n⫽3 to 5). C indicates absence of
compounds; ␣ Ab, antibody for ␣-subunit
of ATP synthase (dilution of 1:1000); ␤ Ab,
antibody for ␤-subunit of ATP synthase
(dilution of 1:1000); ATP (10⫺7 M); ADP
(10⫺7 M); EF, efrapeptin (10⫺5 M)
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 ␤-subunit antibody
also dampened the suppressant effect of coupling factor 6,
whereas the ␣-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 ␤-Subunit of ATP Synthase
Figure 3. ATP synthase activity for hydrolysis of ATP to ADP at
the cell surface (n⫽4). CF6 indicates coupling factor 6 (10⫺7 M);
EF, efrapeptin (10⫺5 M).
As shown in Figure 6, an intravenous bolus injection of
coupling factor 6 at 1.0 ␮g/kg body weight increased mean
arterial blood pressures by 6⫾1 mm Hg, but pretreatment
with anti-␤-subunit antibody for 2 minutes suppressed by
1144
Hypertension
November 2005
Figure 4. Effect of coupling factor 6 on
intracellular pH. A, Intracellular pH after
administration of coupling factor 6 (CF6) at
10⫺7 M and followed by amyloride (AM) at
10⫺3 M. B, Effect of pretreatment with
efrapeptin (EF) at 10⫺5 M on intracellular
pH in response to CF6 at 10⫺7 M. C, Effect
of pretreatment with AM at 10⫺4 M on
intracellular pH in response to CF6 at
10⫺7 M.
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47⫾6% compared with that produced by coupling factor 6
alone (n⫽4, P⬍0.01).
Discussion
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 ␤ subunit of ATP synthase, which is
distinct from the original position of the peptide, suggesting that coupling factor 6 binds to the ␤ subunit of ATP
synthase at the cell surface. The association between
coupling factor 6 and the ␤-subunit was further verified by
a type of competition experiment showing the suppression
of 125I-coupling factor 6 binding by anti-␤-subunit antibody
and immunoprecipitation analysis. Displacement analysis
revealed that coupling factor 6 binds to the ␤-subunit of
ATP synthase at the surface of HUVECs with Kd for 7.6
nM. This Kd for the ␤-subunit of ATP synthase was
similar to that for another agonist binding to the ␣ and
␤-subunits of its enzyme, angiostatin.4
Of the 3 catalytic ␤-subunits in the F1–ATP synthase
complex (␣3␤3␥␦⑀), one ␤ subunit was reported to contain an
ATP analogue, adenosine 5⬘-[␤,␥-imido] triphosphate (AMPPNP), and the other ADP; these 2 subunits have been referred
to as the ␤TP and ␤DP subunits, respectively. The third site is
empty or half-closed in the structure, thereby called the ␤E or
␤HC 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 ␤TP subunit is
identified as the “tight” binding site for nucleotide, whereas
the ␤DP 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 ␤DP
subunit) rather than the “tight” binding site for nucleotide (the
␤TP 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 ␤E 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 ␤DT subunit rather than the ␤TP and ␤E
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 ␤-subunit of ATP
synthase triggers the endocytosis of HDL particle and ADP
suppresses it.3
Figure 5. Effect of coupling factor 6 on
arachidonic acid (AA) release. A, Dosedependent effect of coupling factor 6
(CF6) on AA release (n⫽4). B, Effect of
various compounds on AA release (n⫽5).
C, Absence of compounds. CF6 indicates coupling factor 6 (10⫺7 M); EF,
efrapeptin (10⫺5 M); ␣ Ab, antibody for
␣-subunit of ATP synthase (dilution of
1:1000); ␤ Ab, antibody for ␤-subunit of
ATP synthase (dilution of 1:1000); ATP
(10⫺7 M); ADP (10⫺7 M); PPAD, pyridoxal
phosphate-6-azophenyl-2⬘,4⬘-disulfonic
acid (a blocker for ATP receptors) (10⫺5
M). C, Effect of CF6 at 10⫺7M and EF at
10⫺5 M on bradykinin (BK) at 10⫺5
M-induced increase in AA release (n⫽4).
Osanai et al
Intracellular Signaling of Coupling Factor 6
1145
0.4 inhibit cPLA2 activity directly or through the modulators
of cPLA2 activity such as annexins.22 Further study is needed.
The ␤-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.
Figure 6. Effect of anti-␤-subunit of ATP synthase antibody on
coupling factor 6-induced increase in blood pressure. A, Representative tracings. B, The changes in mean blood pressure.
Post-Receptor Signaling Pathway
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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 ␤-subunit. Because ADP suppressed
the binding of coupling factor 6 to the ␤-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
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.
Perspectives
The present study identified the ␤-subunit of membranebound 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.
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Intracellular Signaling for Vasoconstrictor Coupling Factor 6: Novel Function of β
-Subunit of ATP Synthase as Receptor
Tomohiro Osanai, Koji Magota, Makoto Tanaka, Michiko Shimada, Reiichi Murakami, Satoko
Sasaki, Hirofumi Tomita, Naotaka Maeda and Ken Okumura
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Hypertension. 2005;46:1140-1146; originally published online October 17, 2005;
doi: 10.1161/01.HYP.0000186483.86750.85
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
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