Grape juice causes endothelium-dependent relaxation via a redox-sensitive Src- and Akt-dependent activation of eNOS.
ABSTRACT An enhanced endothelial formation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), is thought to contribute to the protective effect of moderate consumption of red wine on coronary diseases. The present study has characterized endothelium-dependent relaxations to Concord grape juice (CGJ), a non-alcoholic rich source of grape-derived polyphenols, in the coronary artery.
Porcine coronary artery rings were suspended in organ chambers for the measurement of changes in isometric tension in the presence of indomethacin. NO formation was assessed by electron spin resonance spectroscopy, and the phosphorylation of Src, Akt and endothelial NO synthase (eNOS) by Western blot analysis in cultured endothelial cells.
Endothelium-dependent relaxations to CGJ were slightly but significantly reduced by L-NA, not affected by charybdotoxin (CTX) plus apamin (APA, two inhibitors of EDHF-mediated responses) whereas the combination of L-NA, CTX plus APA reduced maximal relaxation to about 50%. In the presence of CTX plus APA, relaxations to CGJ were markedly reduced by the membrane permeant mimetic of superoxide dismutase (SOD), MnTMPyP, the membrane permeant analogue of catalase polyethyleneglycol-catalase (PEG-catalase), PP2, an inhibitor of Src kinase, and by wortmannin, an inhibitor of the PI3-kinase. CGJ stimulated the formation of reactive oxygen species and the N(omega)-nitro-L-arginine-, PP2- and wortmannin-sensitive formation of NO in endothelial cells. The formation of NO was associated with a redox-sensitive and time-dependent phosphorylation of Src, Akt and eNOS.
CGJ induces endothelium-dependent relaxations of coronary arteries, which involve a NO-mediated component and also, to a minor extent, an EDHF-mediated component. In addition, CGJ-induced NO formation is due to the redox-sensitive activation of Src kinase with the subsequent PI3-kinase/Akt-dependent phosphorylation of eNOS.
- SourceAvailable from: Pavel Kubát[Show abstract] [Hide abstract]
ABSTRACT: Considering the important roles of porphyrins in biological systems and their promising use in photodynamic therapy (PDT), the present work investigated the photophysical properties of palladium(II) 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrin (PdTSPP) and the effects of non-activated by light form of this porphyrin on contractile behaviour of isolated healthy endothelium-denuded human mesenteric arteries.Photodiagnosis and Photodynamic Therapy 06/2014; · 2.24 Impact Factor
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ABSTRACT: Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have vasoprotective effects, in part, by stimulating the endothelial formation of nitric oxide (NO). This study determined the role of the EPA:DHA ratio and amount, and characterized the mechanism leading to endothelial NO synthase (eNOS) activation.PLoS ONE 01/2014; 9(8):e105102. · 3.53 Impact Factor
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ABSTRACT: The effect of freeze-dried red wine (FDRW) on cardiac function and electrocardiogram (ECG) in Langendorff-isolated rat hearts was investigated. FDRW significantly decreased left ventricular pressure and coronary perfusion pressure, the latter being dependent on the activation of both phosphatidylinositol 3-kinase and eNOS. FDRW did not affect the QRS and QT interval in the ECG, although at 56 μg of gallic acid equivalents/mL, it prolonged PQ interval and induced a second-degree atrioventricular block in 3 out of 6 hearts. This is the first study demonstrating that at concentrations resembling a moderate consumption of red wine, FDRW exhibited negative inotropic and coronary vasodilating activity leaving unaltered ECG, whereas at very high concentrations, it induced arrhythmogenic effects.Canadian Journal of Physiology and Pharmacology 02/2014; 92(2):171-4. · 1.56 Impact Factor
Grape juice causes endothelium-dependent relaxation via a redox-sensitive
Src- and Akt-dependent activation of eNOS
Eric Anselm⁎, Marta Chataigneau, Mamadou Ndiaye1,
Thierry Chataigneau, Valérie B. Schini-Kerth
Département de Pharmacologie et Physico-Chimie, UMR 7175-LC1, Université Louis Pasteur de Strasbourg, France
Received 20 December 2005; received in revised form 21 July 2006; accepted 1 August 2006
Available online 8 August 2006
Time for primary review 30 days
Objectives: An enhanced endothelial formation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF), is thought to
contribute to the protective effect of moderate consumption of red wine on coronary diseases. The present study has characterized endothelium-
dependent relaxations to Concord grape juice (CGJ), a non-alcoholic rich source of grape-derived polyphenols, in the coronary artery.
Methods: Porcine coronary artery rings were suspended in organ chambers for the measurement of changes in isometric tension in the
presence of indomethacin. NO formation was assessed by electron spin resonance spectroscopy, and the phosphorylation of Src, Akt and
endothelial NO synthase (eNOS) by Western blot analysis in cultured endothelial cells.
Results: Endothelium-dependent relaxations to CGJ were slightly but significantly reduced by L-NA, not affected by charybdotoxin (CTX)
plus apamin (APA, two inhibitors of EDHF-mediated responses) whereas the combination of L-NA, CTX plus APA reduced maximal
relaxation to about 50%. In the presence of CTX plus APA, relaxations to CGJ were markedly reduced by the membrane permeant mimetic
of superoxide dismutase (SOD), MnTMPyP, the membrane permeant analogue of catalase polyethyleneglycol-catalase (PEG-catalase), PP2,
an inhibitor of Src kinase, and by wortmannin, an inhibitor of the PI3-kinase. CGJ stimulated the formation of reactive oxygen species and
the Nω-nitro-L-arginine-, PP2- and wortmannin-sensitive formation of NO in endothelial cells. The formation of NO was associated with a
redox-sensitive and time-dependent phosphorylation of Src, Akt and eNOS.
Conclusions: CGJ induces endothelium-dependent relaxations of coronary arteries, which involve a NO-mediated component and also, to a
minor extent, an EDHF-mediated component. In addition, CGJ-induced NO formation is due to the redox-sensitive activation of Src kinase
with the subsequent PI3-kinase/Akt-dependent phosphorylation of eNOS.
© 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Endothelial function; Coronary disease; Endothelial factors; Nitric oxide
Several epidemiological studies have indicated that regular
intake of vegetables, fruit, and beverages such as red wine and
a reduced number of cancer and coronary diseases [1–3]. The
protective effect has been attributable, at least in part, to poly-
phenols [4,5]. Indeed, grape products such as red wine contain
high levels of polyphenols, which are predominantly found in
skins, seeds and stems. The protective effect of red wine
polyphenols on the vascular system is thought to include their
ability to prevent oxidation of low-density lipoproteins [6,7],
migration and proliferation [10,11]. Alternatively, vascular
protection might also be due tothe directaction of polyphenols
onendothelialcells resultinginanenhancedformationof nitric
oxide (NO) and endothelium-derived hyperpolarizing factor
(EDHF), two factors playing a major role in the control of
Cardiovascular Research 73 (2007) 404–413
⁎Corresponding author. UMR CNRS 7175-LC1,Université Louis Pasteur
de Strasbourg, Faculté de Pharmacie, 74, route du Rhin, B.P. 60024, F-
67401 Illkirch, France. Tel.: +33 3 90 24 41 27; fax: +33 3 90 24 43 13.
E-mail address: email@example.com (E. Anselm).
1Current address: Laboratoire de Pharmacologie, de Pharmacodynamie et
de Physiologie, Faculté de Médecine et de Pharmacie, Dakar, Sénégal.
0008-6363/$ - see front matter © 2006 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
by guest on June 7, 2013
vascular homeostasis [12,13]. Indeed, red wine polyphenols
cause pronounced endothelium-dependent relaxations of
isolated arteries, which are solely mediated by NO in the rat
aorta but involve both NO and EDHF in porcine coronary
arteries [14–16]. Besides red wines, grape juices, a non-
derived polyphenols. Studies have shown that ingestion of
purple grape juice improves flow-mediated vasodilatation,
platelet function and platelet-dependent inflammatory res-
ponses in patients with coronary artery disease [7,17,18], and
reduces blood pressure in moderately hypertensive patients
. In addition, consumption of purple grape juice also in-
creased serum antioxidant capacity and protected LDL against
oxidation in healthy subjects . In order to better understand
the protective effect of purple grape juice of the endothelial
function, experiments were performed to determine whether
purple grape juice induces endothelium-dependent relaxation
of coronary arteries and, if so, to characterize the relaxing
factors involved, and the signaling pathway leading to the
formation of NO.
Diethyldithiocarbamate sodium salt trihydrate (DETC),
apamin, charybdotoxin, superoxide dismutase (SOD), poly-
ethyleneglycol-SOD (PEG-SOD), catalase, PEG-catalase, in-
domethacin, bradykinin, hydroethidine, carboxyPTIO, and
Nω-nitro-L-arginine (L-NA) were from Sigma. 1H-[1,2,4]
oxadiazolo[4,3a]quinoxalin-1-one (ODQ) was from Tocris,
wortmannin, LY294002, and the superoxide dismutase (SOD)
(MnTMPyP) from Alexis Chemicals, U46619 (9,11-dideoxy-
9α-methanoepoxy prostaglandin F2α) from Cayman Chemi-
cal, and PP2 (4-amino-5-(4-chlorophényl)-7-(t-butyl)pyrazolo
[3,4-d]pyrimidine) from Calbiochem. Concord grape juice
containing 2.3 g/l polyphenols (total phenolics: 2307 mg/l
gallic acid equivalent; anthocyanins: 411 mg/l malvidin; pro-
provided by Welch Foods, Inc. (Concord, MA, USA).
2.2. Vascular reactivity studies
Left anterior descending coronary arteries (obtained from
of forceps. Rings were suspended in organ baths containing
oxygenated (95% O2and5% CO2) Krebs bicarbonatesolution
(composition in mM: NaCl 119, KCl 4.7, KH2PO41.18,
MgSO41.18, CaCl21.25, NaHCO325, and D-glucose 11, pH
7.4, 37 °C) and the cyclooxygenase inhibitor indomethacin
(10μM),forthe determinationof changes inisometric tension.
Following equilibration for 90 min under a resting tension of
5 g, rings were contracted with KCl (80 mM). After a 30-min
washout period, rings were contracted with the thromboxane
mimetic U46619 (1–60 nM) to about 80% of the maximal
presence of a functional endothelium. After washout and a 30-
min equilibration period, rings were again contracted with
U46619 before construction of a concentration–relaxation
curve to CGJ. In some experiments, rings were exposed to an
inhibitor for 30 min before the addition of U46619. In some
experiments, rings were exposed to CGJ for 5 min before
construction of a concentration–contraction curve to U46619.
Fig. 1. CharacterizationofConcordgrapejuice(CGJ)-inducedrelaxationsinporcinecoronaryarteryrings(A).Intactandendothelium-denudedringswerecontracted
APA for 30 min before addition of U46619. Effect of CGJ on contractile responses (B). As indicated intact rings were exposed to CGJ (44 mg/l) 5 min before the
addition of increasing concentrations of U46619. Rings with endothelium were also incubated with L-NA 30 min before the addition of CGJ. Experiments were
performed in the presence of indomethacin. n=3 to 8 different experiments. *Pb0.05 versus control.
405E. Anselm et al. / Cardiovascular Research 73 (2007) 404–413
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2.3. Cell culture
Porcine coronary artery segments were flushed with PBS
thelial cells were isolated by collagenase treatment (type I,
Worthington, 1 mg/ml for 12 min at 37 °C), and cultured in
culture dishes containing medium MCDB 131 (Invitrogen)
ml), streptomycin (100 U/ml), fungizone (250 μg/ml), and L-
All experiments were performed with confluent cultures of
cells used at first passage. Cells were exposed to serum-free
(QBiogene) for 6 h prior to treatment.
2.4. In situ detection of superoxide anions
The oxidative fluorescent dye hydroethidine was used to
evaluate in situ production of superoxide anions by use of a
method described by Miller et al. . Hydroethidine is freely
permeable to cells and in the presence of superoxide anions is
the DNA. Ethidium bromide is excited at 488 nm with an
emission spectrum of 610 nm. Endothelial cells were rinsed in
PBS and incubated in Hanks' balanced salt solution containing
hydroethidine (10 μM) in a light-protected humidified chamber
obtained with a Bio-Rad MRC-1024 laser scanning confocal
microscope equipped with krypton/argon laser. Fluorescence
was detected with a 605-nm long-pass filter.
2.5. Determination of NO formation
Determination of NO formation was assessed by electron
(DETC)2], a paramagnetic DETC iron complex with NO, in
cultured endothelial cells. The ESR methodology was used as
reported previously with minor modifications . Confluent
cultures of endothelial cells (first passage, ∼1 million cells per
well) were washed twice with Hanks balanced salt solution
(HBSS) buffered with 10 mM HEPES, and then they were
serum albumin (20.5 mg/ml), 1.5 mM CaCl2, 0.3 mM L-argi-
nine and antioxidants or inhibitors as indicated for 30 min at
37 °C. Spin trap chemicals FeSO4 (0.8 mM) and DETC
(1.6 mM) were rapidly mixed to obtain a colloid form [Fe(II)
(DETC)2], which was added to endothelial cells at a final
of NO was induced by addition of CGJ (44 mg/l) for 30 min.
Thereafter, dishes were placed on ice, and the incubation me-
dium was removed before addition of 0.2 ml of the HBSS–
HEPES buffer. Cells were then scraped, and the cell suspension
was collected in a calibrated tube. Tubes were rapidly frozen at
77 K for ESR measurements. ESR measurements were
Fig. 2. Characterization of the L-NA (100 μM) resistant relaxation to CGJ in intact coronary artery rings. Rings were incubated with (A) L-NA (300 μM), (B)
carboxyPTIO (300 μM, a NO scavenger), (C) ODQ (1 μM, an inhibitor of soluble guanylyl cyclase), and (D) ouabain (500 nM, an inhibitor of Na+, K+-ATPase) for
30 min before induction of contraction to U46619 and subsequent relaxation to CGJ. The effect of ouabain on relaxation to bradykinin is also shown (E). In A, B, C,
to 6 different experiments.⁎Pb0.05 versus control.
406 E. Anselm et al. / Cardiovascular Research 73 (2007) 404–413
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Fig. 3. Role of the redox-sensitive Src kinase and the PI3-kinase/Akt pathway in CGJ-induced endothelium-dependent relaxation. Coronary artery rings with
endothelium were incubated with (A) MnTMPyP (100 μM), a cell permeable superoxide dismutase (SOD) mimetic, or native SOD, (B) the membrane permeant
analogue of catalase polyethyleneglycol-catalase (PEG-catalase) (500 U/mL) or native catalase, (C) the PI3-kinase inhibitor, wortmannin (30 nM), and (D) the
Src kinase inhibitor, PP2 (10 μM) for 30 min before contraction to U46619 and subsequent relaxation to CGJ. Experiments were performed in the presence of
indomethacin, and charybdotoxin plus apamin. n=4 to 5 different experiments. *Pb0.05 versus control.
Fig. 4. CGJ-induced generation of ROS in cultured endothelial cells. Hydroethidine (HE)-loaded cells were stimulated with CGJ and, thereafter, the ethidium
bromide fluorescence was monitored over 10 min using a confocal microscope. (A) Original pictures showing ethidium bromide fluorescence in unstimulated
cells, and in cells stimulated with CGJ in the absence or presence of MnTMPyP (100 μM). (B) Corresponding cumulative data. n=3 to 4 different experiments.
*Pb0.05 versus control. #Pb0.05 versus CGJ.
407E. Anselm et al. / Cardiovascular Research 73 (2007) 404–413
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performed on an MS100 spectrometer (Magnettech Ltd.) under
the following conditions: temperature 77 K, microwave fre-
quency 9.34 GHz, microwave power 20 mW, modulation fre-
quency 100 kHz, modulation amplitude 1 mT. The third
component of the ESR signal was used for relative comparison
of the concentration of NO trapped in each sample.
2.6. Western blot analysis
After treatment,cells were washed twicewithPBS andthen
7.5; QBiogene), NaCl 150, Na3VO41, sodium pyrophosphate
10, NaF 20, okadaic acid 0.01 (Sigma), a tablet of protease
inhibitor (Roche) and 1% Triton X-100 (QBiogen)). Total
proteins (20 μg) were separated on 8% SDS-polyacrylamide
(Sigma) gels at 70 V for 2.5 h. Separated proteins were trans-
ferred electrophoretically onto polyvinylidine difluoride mem-
branes (Amersham) at 100 V for 120 min. Membranes were
blocked with blocking buffer containing 3% bovine serum
albumin for p-Akt and p-eNOS, and I-block for p-Src, Tris-
buffered saline solution (Biorad) and 0.1% Tween 20 (Sigma)
(TBS-T) for 1 h. For detection of phosphorylated proteins,
membranes were incubated with the respective primary anti-
body (p-Src Tyr418, Biosource; p-Akt Ser473 and p-eNOS
Ser1177, Cell Signaling Technology; dilution of 1:1000)
overnight at 4 °C. After washing, membranes were incubated
with the secondary antibody (peroxidase-labeled anti–rabbit
IgG, dilution of 1:5000; Cell Signaling Technology) at room
temperature for 60 min. Prestained markers (Invitrogen) were
used for molecular mass determinations. Immunoreactive
bands were detected by enhanced chemiluminescence (Amer-
sham). Ponceau staining was performed to verify the quality of
the transfer and equal amounts of proteins in each lane.
2.7. Statistical analysis
Values are expressed as means±SEM. Statistical evalu-
ation was performed with Student's t test for paired data or
ANOVA followed by Fischer's protected least significant
difference test where appropriate. Values of Pb0.05 were
considered statistically significant.
Fig. 5. CGJ stimulates the formation of NO in cultured endothelial cells as
assessed by electron spin resonance spectroscopy. Cells were exposed to either
solvent, L-NA, PP2 or wortmannin for 30 min before the addition of CGJ for
Fig. 6. CGJ caused a time-dependent phosphorylation of Src at Tyr418, Akt at Ser473 and eNOS at Ser1177 in endothelial cells. Cells were exposed to CGJ for
the indicated times at 37 °C. Thereafter, the level of p-Src, p-Akt and p-eNOS was determined by Western blot analysis. (A) Representative immunoblots, and
(B), (C), and (D) corresponding cumulative data. n=3 to 4 different experiments. *Pb0.05 versus control.
408E. Anselm et al. / Cardiovascular Research 73 (2007) 404–413
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3.1. Concord grape juice induces endothelium-dependent
relaxation and inhibition of contractile responses in
dependent relaxations in coronary artery rings with endothe-
lium but only minor ones in those without endothelium (Fig.
by L-NA (100 μM, a competitive inhibitor of NO synthase)
without affecting the maximal relaxation, not affected by
charybdotoxin plus apamin whereas thecombination ofL-NA,
charybdotoxin plus apamin reduced maximal relaxation to
about 50%. These findings indicate that CGJ induces en-
dothelium-dependent relaxations of coronary arteries, which
include a NO-mediated component, a modest EDHF-mediated
component and also L-NA-, charybdotoxin plus apamin-resis-
tant component. Next, experiments were performed to cha-
racterize the residual relaxation to CGJ. Increasing the
concentration of L-NA from 100 to 300 μM further reduced
relaxations to CGJ (Fig. 2A). They were also significantly
reduced by a NO scavenger, carboxyPTIO and by an inhibitor
of soluble guanylyl cyclase, ODQ (Fig.2B and C). In addition,
the Na+, K+-ATPase inhibitor ouabain markedly blunted
relaxations to CGJ whereas those to bradykinin were not
affected (Fig. 2D and E).
Next, the possibility that CGJ, besides inducing endothe-
lium-dependent relaxations, also affects contractile respon-
ses was assessed. Exposure of coronary artery rings to CGJ
5 min prior to the addition of increasing concentrations of
U46619 significantly blunted contractions in rings with
endothelium but not in those without endothelium (Fig. 1B
and data not shown). The inhibitory effect of CGJ was
prevented by L-NA indicating that this effect is due to an
enhanced endothelial formation of NO.
3.2. Role of reactive oxygen species (ROS), Src kinase and
the PI3-kinase/Akt pathway in CGJ-induced relaxation
Previous studies have shown that red wine polyphenols-
induced NO-mediated relaxations in coronary arteries are
critically dependent on the redox-sensitive activation of the
PI3-kinase/Akt pathway leading to the phosphorylation of
eNOS .Therefore, experimentswere plannedto determine
whether this signaling pathway is also involved in CGJ-
induced relaxations. In the presence of charybdotoxin plus
apamin, NO mediated relaxations to CGJ were markedly
reduced by the membrane permeant SOD mimetic MnTMPyP
and by the cell permeable PEG-catalase whereas native SOD
Fig. 7. Role of ROS in CGJ-induced phosphorylation of Src, Akt and eNOS in endothelial cells. Cells were incubated with either solvent, PEG-catalase (500 U/
mL), PEG-SOD (500 U/mL) or MnTMPyP (100 μM) for 30 min before the addition of CGJ. The level of p-Src, p-Akt and p-eNOS was determined by Western
blot analysis. (A) Representative immunoblots, and (B), (C), and (D) corresponding cumulative data. n=3 to 4 different experiments. *Pb0.05 versus control.
#Pb0.05 versus CGJ.
409E. Anselm et al. / Cardiovascular Research 73 (2007) 404–413
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PI3-kinase inhibitor, wortmannin also significantly reduced
relaxations to CGJ (Fig. 3C). Since the Src family of kinases
are redox-sensitive kinases that have been shown to act as
upstream activators of the PI3-kinase/Akt pathway ,
experiments were performed to determine the role of Src
kinase in relaxations to CGJ.Inhibition of Src kinase with PP2
significantly reduced relaxations to CGJ (Fig. 3D). In contrast,
to bradykinin 1 μM were 91.4±3.0 and 87.7±5.0% in the
absence and presence of PP2, respectively; n=6).
3.3. CGJ causes the formation of ROS in endothelial cells
To provide further evidence that CGJ is able to cause an
intracellular pro-oxidant response, the in situ formation of
ROS was assessed in coronary artery endothelial cells using
theredox-sensitive fluorescentprobe hydroethidine.Exposure
of cells to CGJ markedly increased the fluorescent signal, and
MnTMPyP abolished this effect (Fig. 4).
3.4. CGJ stimulates the formation of NO in endothelial cells
Next, experiments were performed to provide direct
evidence that CGJ induces NO formation in porcine coronary
artery endothelial cells using electron spin resonance spec-
troscopy. Exposure of cells to CGJ induced about a 1.5-fold
was abolished by L-NA, PP2 and wortmannin (Fig. 5).
3.5. CGJ causes the redox-sensitive activation of Src with
subsequent PI3-kinase/Akt-dependent phosphorylation of
To better characterize the signaling pathway involved in
p-eNOS wereassessed in endothelialcells byimmunoblotting.
of p-Src, p-Akt and p-eNOS (Fig. 6). CGJ increased within
signals evolved differentially. Indeed, phosphorylation of Src
was an early and transient event, which reached a peak value
within 1 to 3 min and then returned to baseline at 10 min. In
contrast, the level of p-Akt and p-eNOS was detected after
1 min and then increased steadily until 30 min (Fig. 6). CGJ-
induced phophorylation of Src was abolished by PEG-SOD
and MnTMPyP but not by PEG-catalase (Fig. 7A, B). In
contrast, PEG-SOD, MnTMPyP and PEG-catalase prevented
addition, the PI3-kinase inhibitors wortmannin and LY294002
Fig. 8. Role of the PI3-kinase/Akt pathway and the Src kinase in CGJ-induced phosphorylation of Akt and eNOS in endothelial cells. Cells were incubated either with
analysis. Top, representative immunoblots, and bottom, corresponding cumulative data. n=3 to 4 different experiments. *Pb0.05 versus control.#Pb0.05 versus CGJ.
410E. Anselm et al. / Cardiovascular Research 73 (2007) 404–413
by guest on June 7, 2013
prevented phosphorylation of Akt and eNOS (Fig. 8A). These
data indicate that ROS, especially superoxide anions, act as
upstream mediators of Src kinase whereas both superoxide
anions and hydrogen peroxide are involved in the activation of
the PI3-kinase/Akt pathway leading to eNOS phosphorylation
Akt and eNOS was abolished by the Src kinase inhibitor PP2,
indicating that the redox-sensitive Src kinase is the major
activator of the PI3-kinase/Akt pathway leading to eNOS
activation (Fig. 8B).
The present findings indicate that Concord grape juice is a
powerful endothelium-dependent vasodilator of coronary
relaxationstoCGJ were slightlybut significantlyreducedbyL-
NA without affecting the maximal relaxation indicating a role
for NO. Moreover, the present findings provide also direct
evidence that CGJ activates eNOS as indicated by the L-NA-
eNOS at Ser1177 in endothelial cells. Interestingly, CGJ
induced a 1.5-fold increase in NO formation, which is similar
to that induced by the physiological agonist bradykinin .
Although inhibition of EDHF-mediated responses with char-
ybdotoxin plus apamin alone did not affect relaxations to CGJ,
their combination with L-NA reduced maximal relaxation to
about 50%, indicating that EDHF plays also a role, although
only to some extent. Surprisingly, a substantial relaxation
persisted in the presence of L-NA, charybdotoxin and apamin.
In contrast to CGJ, the combination of L-NA, charybdotoxin
and apamin abolished relaxations to a red wine extract in the
coronary artery . The persistent relaxation to CGJ was not
due to the formation of relaxing prostanoids since all
experiments were performed in the presence of indomethacin.
In addition, it was not affected by the combination of a beta
blocker (propranolol) and an alpha2blocker (yohimbine), and
byan inhibitor of adenylyl cyclase (MDL12330),ruling out the
involvement of endogenous catecholamines and the cyclic
AMP relaxing pathway (data not shown). However, it was
inhibited by increasing the concentration of L-NA, by a NO
cyclase, ODQ. Thus, the ability of CGJ to stimulate the
endothelial formationof NO persists inthe presence of 100μM
of L-NA whereas no such effect is observed with a red wine
extract [present findings and 16]. The possibility that CGJ is
determined. In addition, relaxations to CGJ were also markedly
reduced by ouabain whereas those to bradykinin were not
affected. These findings suggest a role for the Na+, K+-ATPase
in CGJ-induced but not in bradykinin-induced endothelial
formation of NO. In addition, previous studies have indicated
solely mediated by NO [14,15,25,26]. Altogether, the present
findings indicate that CGJ causes potent endothelium-depen-
dent relaxations involving predominantly NO and also to some
extent EDHF in coronary arteries.
Red wine polyphenols-induced NO- and EDHF-mediated
relaxations in coronary arteries are both redox-sensitive events
fore, the role of ROS in the NO-mediated CGJ-induced endo-
thelium-dependent relaxation was assessed. In the presence of
indomethacin and charybdotoxin plus apamin,both membrane
permeant analogs of either SOD or catalase strongly reduced
relaxations to CGJ whereas native SOD and native catalase
were inactive indicating a key role of both intracellular super-
oxide anions and H2O2. In addition, PEG-SOD and PEG-
catalase also prevented the CGJ-induced phosphorylation of
cause a modest MnTMPyP-sensitive pro-oxidant response in
endothelial cells was obtained with the redox-sensitive fluor-
escent probe hydroethidine. An increased endothelial forma-
red wine polyphenols . Although the specific endothelial
source of ROS remains to be determined, the redox-sensitive
NO- and EDHF-mediated relaxations to red wine polyphenols
were not affected by pharmacological inhibitors of mitochon-
drial function, xanthine oxidase, and cytochrome P450 ruling
out these potential sources [16,27]. Moreover, the observation
that NO-mediated relaxation to red wine polyphenols was
similar in aortas from gp91phox knockout and wild-type mice
is not consistent with a role of NADPH oxidase . Potential
polyphenolic compound itself [29–31].
Numerous lines of evidence indicate that ROS act as key
intracellular mediators activating redox-sensitive protein
kinases to induce biological responses such as cell growth,
survival, and apoptosis . Moreover, the redox-sensitive
latory effect of red wine polyphenols on both the endothelial
formation ofNO and EDHF [16,28]. Therefore, the role ofthe
redox-sensitive PI3-kinase/Akt pathway on the endothelial
formation of NO to CGJ was studied. The present findings
indicate that CGJ caused activation of the PI3-kinase/Akt
pathway in a redox-sensitive manner as indicated by the time-
dependent phosphorylation of Akt at Tyr418, and its pre-
PI3-kinase in endothelial cells. Moreover, the PI3-kinase/Akt
pathway mediates activation of eNOS since this response is
abolished by wortmannin. The present findings also indicate
thatSrckinase,a redox-sensitive proteinkinase,actsupstream
of the PI3-kinase/Akt pathway since the Src kinase inhibitor
PP2 prevented CGJ-induced phosphorylation of Akt in
endothelial cells, and relaxations to CGJ. It is also consistent
with the fact that CGJ caused an early and transient phos-
phorylation of Src that is followed by a delayed and more
induced phosphorylation of Src was prevented selectively by
intracellular scavengers of superoxide anions but not of
catalase, whereas both types of scavengers prevented phos-
phorylation of Akt and eNOS. Altogether, these findings
411E. Anselm et al. / Cardiovascular Research 73 (2007) 404–413
by guest on June 7, 2013
suggest thatthe intracellular formationof superoxideanions is
an early event, which triggers the cascade of events leading to
an enhanced endothelial formation of NO by increasing the
Additionally activation of the PI3-kinase/Akt pathway might
also result from the partial conversion of superoxide anions to
H2O2, a potent activator of the PI3-kinase/Akt pathway .
Although polyphenols caused a pro-oxidant response in
endothelial cells to activate signal transduction pathways such
in other types of vascular cells. Indeed, red wine polyphenols
prevented growth factor-induced NADPH oxidase-dependent
formation of ROS in vascular smooth muscle, and grape seed
and skin extracts the stimulation-induced superoxide release in
be explained by their ability to scavenge superoxide anions,
peroxyl radicals, hydroxyl radicals, and peroxynitrite [36–38].
Alternatively, the protective effect might also be due to their
ability to inhibit the expression and the activity of pro-oxidant
increase that of antioxidant enzymes such as catalase [39,40].
Altogether these findings suggest that the vasoprotective effect
of polyphenols is due to their dual action on vascular cells: a
moderate pro-oxidant action in endothelial cells to enhance
vasoprotective mechanisms involving NO and EDHF, and an
to keep them in a quiescent state.
In conclusion, the present findings indicate that Concord
grape juice is a powerful endothelium-dependent vasodilator
of coronary arteries by stimulating the endothelial formation
of NO and EDHF. They further indicate that the intracellular
formation of ROS in particular superoxide anions, is a
crucial and early event leading to activation of eNOS via the
Src/PI3-kinase/Akt pathway. Thus, the beneficial effect of
CGJ consumption on endothelial and platelet function in
patients with coronary heart disease [7,18] might be, in part,
due to an enhancement of the formation of vasoprotective
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