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.
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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
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ABSTRACT: To evaluate antioxidant, anti-inflammatory, hepatoprotective and vasorelaxant activities of Populus nigra flower buds ethanolic extract. Antioxidant and anti-inflammatory activities of the extract were assessed using respectively the ABTS test and the animal model of carrageenan-induced paw edema. Protection from hepatic toxicity caused by aluminum was examined by histopathologic analysis of liver sections. Vasorelaxant effect was estimated in endothelium-intact and -rubbed rings of porcine coronary arteries precontracted with high concentration of U46619. The results showed a moderate antioxidant activity (40%), but potent anti-inflammatory activity (49.9%) on carrageenan-induced mice paw edema, and also as revealed by histopathologic examination, complete protection against AlCl3-induced hepatic toxicity. Relaxant effects of the same extract on vascular preparation from porcine aorta precontracted with high concentration of U46619 were considerable at 10(-1) g/L, and comparable (P>0.05) between endothelium-intact (67.74%, IC50=0.04 mg/mL) and -rubbed (72.72%, IC50=0.075 mg/mL) aortic rings. The extract exerted significant anti-inflammatory, hepatoprotective and vasorelaxant activities, the latter being endothelium-independent believed to be mediated mainly by the ability of components present in the extract to exert antioxidant properties, probably related to an inhibition of Ca(2+) influx.Asian Pacific Journal of Tropical Biomedicine 09/2013; 3(9):697-704.
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ABSTRACT: This study examined the ability of Aronia melanocarpa (chokeberry) juice, a rich source of polyphenols, to cause NO-mediated endothelium-dependent relaxations of isolated coronary arteries and, if so, to determine the underlying mechanism and the active polyphenols. Aronia melanocarpa juice caused potent endothelium-dependent relaxations in porcine coronary artery rings. Relaxations to Aronia melanocarpa juice were minimally affected by inhibition of the formation of vasoactive prostanoids and endothelium-derived hyperpolarizing factor-mediated responses, and markedly reduced by N(ω)-nitro-L-arginine (endothelial NO synthase (eNOS) inhibitor), membrane permeant analogues of superoxide dismutase and catalase, PP2 (Src kinase inhibitor), and wortmannin (PI3-kinase inhibitor). In cultured endothelial cells,Aronia melanocarpa juice increased the formation of NO as assessed by electron paramagnetic resonance spectroscopy using the spin trap iron(II)diethyldithiocarbamate, and reactive oxygen species using dihydroethidine. These responses were associated with the redox-sensitive phosphorylation of Src, Akt and eNOS. Aronia melanocarpa juice-derived fractions containing conjugated cyanidins and chlorogenic acids induced the phosphorylation of Akt and eNOS. The present findings indicate that Aronia melanocarpa juice is a potent stimulator of the endothelial formation of NO in coronary arteries; this effect involves the phosphorylation of eNOS via the redox-sensitive activation of the Src/PI3-kinase/Akt pathway mostly by conjugated cyanidins and chlorogenic acids.Nitric Oxide 08/2013; · 3.27 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: firstname.lastname@example.org (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.
405 E. 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.
407 E. 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.
408 E. Anselm et al. / Cardiovascular Research 73 (2007) 404–413
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