Background/Aims: Drinking red wine is associated with a decreased mortality from coronary heart diseases. This study examined whether polyphenols contained in a grape skin extract (GSE) triggered the endothelial formation of nitric oxide (NO) and investigated the underlying mechanism. Methods: Vascular reactivity was assessed in organ chambers using porcine coronary artery rings in the presence of indomethacin (a cyclooxygenase inhibitor) and charybdotoxin plus apamin (inhibitors of endothelium-derived hyperpolarizing factor-mediated responses). The phosphorylation level of Src, Akt and endothelial NO synthase (eNOS) were assessed by Western blot analysis, and the formation of reactive oxygen species (ROS) was investigated using dihydroethidine and dichlorodihydrofluorescein. Results: GSE-induced endothelium-dependent relaxations were abolished by NG-nitro-L-arginine (an eNOS inhibitor) and ODQ (a soluble guanylyl cyclase inhibitor), and they were reduced by MnTMPyP, polyethyleneglycol catalase, PP2 (an inhibitor of Src kinase) and wortmannin (an inhibitor of phosphoinositide 3-kinase). GSE caused phosphorylation of Src, which was prevented by MnTMPyP. It also caused phosphorylation of Akt and eNOS, which were prevented by MnTMPyP, polyethyleneglycol catalase, PP2, wortmannin and LY294002. GSE elicited the formation of ROS in native and cultured endothelial cells, which was prevented by MnTMPyP. Conclusions: GSE causes endothelium-dependent NO-mediated relaxations of coronary arteries. This effect involves the intracellular formation of ROS in endothelial cells leading to the Src kinase/phosphoinositide 3-kinase/Akt-dependent phosphorylation of eNOS.

Keywords:
Grape skin extract, Phosphoinositide 3-kinase, Nitric oxide, Endothelium, Porcine coronary artery
1.
St. Leger AS, Cochrane AL, Moore F: Factors associated with cardiac mortality in developed countries with particular reference to the consumption of wine. Lancet 1979;1:1017–1020.
2.
Renaud SC, Gueguen R, Schenker J, D’houtaud A: Alcohol and mortality in middle-aged men from eastern France. Epidemiology 1998;9:184–188.
3.
Gronbaek M, Becker U, Johansen D, Gottschau A, Schnohr P, Hein HO, Jensen G, Sorensen TI: Type of alcohol consumed and mortality from all causes, coronary heart disease, and cancer. Ann Intern Med 2000;133:411–419.
4.
Stoclet JC, Chataigneau T, Ndiaye M, Oak MH, El Bedoui J, Chataigneau M, Schini-Kerth VB: Vascular protection by dietary polyphenols. Eur J Pharmacol 2004;500:299–313.
5.
Taubert D, Berkels R, Roesen R, Klaus W: Chocolate and blood pressure in elderly individuals with isolated systolic hypertension. JAMA 2003;290:1029–1030.
6.
Heiss C, Dejam A, Kleinbongard P, Schewe T, Sies H, Kelm M: Vascular effects of cocoa rich in flavan-3-ols. JAMA 2003;290:1030–1031.
7.
Fisher ND, Hughes M, Gerhard-Herman M, Hollenberg NK: Flavanol-rich cocoa induces nitric-oxide-dependent vasodilation in healthy humans. J Hypertens 2003;21:2281–2286.
8.
Hashimoto M, Kim S, Eto M, Iijima K, Ako J, Yoshizumi M, Akishita M, Kondo K, Itakura H, Hosoda K, Toba K, Ouchi Y: Effect of acute intake of red wine on flow-mediated vasodilatation of the brachial artery. Am J Cardiol 2001;88:1457–1460.
9.
Fitzpatrick DF, Hirschfield SL, Coffey RG: Endothelium-dependent vasorelaxing activity of wine and other grape products. Am J Physiol 1993;265:H774–H778.
10.
Flesch M, Schwarz A, Bohm M: Effects of red and white wine on endothelium-dependent vasorelaxation of rat aorta and human coronary arteries. Am J Physiol 1998;275:H1183–H1190.
11.
Schini-Kerth VB: Vascular biosynthesis of nitric oxide: effect on hemostasis and fibrinolysis. Transfus Clin Biol 1999;6:355–363.
12.
Ndiaye M, Chataigneau T, Andriantsitohaina R, Stoclet JC, Schini-Kerth VB: Red wine polyphenols cause endothelium-dependent EDHF-mediated relaxations in porcine coronary arteries via a redox-sensitive mechanism. Biochem Biophys Res Commun 2003;310:371–377.
13.
Ndiaye M, Chataigneau T, Chataigneau M, Schini-Kerth VB: Red wine polyphenols induce EDHF-mediated relaxations in porcine coronary arteries through the redox-sensitive activation of the PI3-kinase/Akt pathway. Br J Pharmacol 2004;142:1131–1136.
14.
Ndiaye M, Chataigneau M, Lobysheva I, Chataigneau T, Schini-Kerth VB: Red wine polyphenol-induced, endothelium-dependent NO-mediated relaxation is due to the redox-sensitive PI3-kinase/Akt-dependent phosphorylation of endothelial NO-synthase in the isolated porcine coronary artery. FASEB J 2005;19:455–457.
15.
Feletou M, Vanhoutte PM: EDHF: new therapeutic targets? Pharmacol Res 2004;49:565–580.
16.
Soares De Moura R, Costa Vieira FS, Souza MAV, Kovary K, Guedes DC, Oliveira EPB, Rubenich LMS, Carvalho LCRM, Tano T, Gusmão Correia ML: Antihypertensive, vasodilator and antioxidant effects of vinifera grape-skin extract. J Pharm Pharmacol 2002;54:1515–1520.
17.
Soares De Moura R, Miranda DZ, Pinto AC, Sicca RF, Souza MA, Rubenich LM, Carvalho LC, Rangel BM, Tano T, Madeira SV, Resende AC: Mechanism of the endothelium-dependent vasodilation and the antihypertensive effect of Brazilian red wine. J Cardiovasc Pharmacol 2004;44:302–309.
18.
Madeira SV, De Castro Resende A, Ognibene DT, De Sousa MA, Soares De Moura R: Mechanism of the endothelium-dependent vasodilator effect of an alcohol-free extract obtained from a vinifera grape skin. Pharmacol Res 2005;52:321–327.
19.
Singleton VL, Rossi JA: Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic 1965;16:144–158.
20.
Miller FJ Jr, Gutterman DD, Rios CD, Heistad DD, Davidson BL: Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ Res 1998;82:1298–1305.
21.
Ullrich V, Bachschmid M: Superoxide as a messenger of endothelial function. Biochem Biophys Res Commun 2000;278:1–8.
22.
Corson MA, James NL, Latta SE, Nerem RM, Berk BC, Harrison DG: Phosphorylation of endothelial nitric oxide synthase in response to fluid shear stress. Circ Res 1996;79:984–991.
23.
Garcia-Cardena G, Fan R, Stern DF, Liu J, Sessa WC: Endothelial nitric oxide synthase is regulated by tyrosine phosphorylation and interacts with caveolin-1. J Biol Chem 1996;271:27237–27240.
24.
Michell BJ, Griffiths JE, Mitchelhill KI, Rodriguez-Crespo I, Tiganis T, Bozinovski S, De Montellano PR, Kemp BE, Pearson RB: The Akt kinase signals directly to endothelial nitric oxide synthase. Curr Biol 1999;9:845–848.
25.
Harris MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ, Chen ZP, Kemp BE, Venema RC: Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation. J Biol Chem 2001;276:16587–16591.
26.
Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, Jo H: Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A. J Biol Chem 2002;277:3388–3396.
27.
Mineo C, Yuhanna IS, Quon MJ, Shaul PW: High density lipoprotein-induced endothelial nitric-oxide synthase activation is mediated by Akt and MAP kinases. J Biol Chem 2003;278:9142–9149.
28.
Andriambeloson E, Kleschyov AL, Muller B, Beretz A, Stoclet JC, Andriantsitohaina R: Nitric oxide production and endothelium-dependent vasorelaxation induced by wine polyphenols in rat aorta. Br J Pharmacol 1997;120:1053–1058.
29.
Anselm E, Chataigneau M, Ndiaye M, Chataigneau T, Schini-Kerth VB: Grape juice causes endothelium-dependent relaxation via a redox-sensitive Src- and Akt-dependent activation of eNOS. Cardiovasc Res 2007;73:404–413.
30.
Roques SC, Landrault N, Teissedre PL, Laurent C, Besancon P, Rouane JM, Caporiccio B: Hydrogen peroxide generation in caco-2 cell culture medium by addition of phenolic compounds: effect of ascorbic acid. Free Radic Res 2002;36:593–599.
31.
Long LH, Clement MV, Halliwell B: Artifacts in cell culture: rapid generation of hydrogen peroxide on addition of (–)-epigallocatechin, (–)-epigallocatechin gallate, (+)-catechin, and quercetin to commonly used cell culture media. Biochem Biophys Res Commun 2000;273:50–53.
32.
Yeh LH, Park YJ, Hansalia RJ, Ahmed IS, Deshpande SS, Goldschmidt-Clermont PJ, Irani K, Alevriadou BR: Shear-induced tyrosine phosphorylation in endothelial cells requires Rac1-dependent production of ROS. Am J Physiol 1999;276:C838–C847.
33.
Colavitti R, Pani G, Bedogni B, Anzevino R, Borrello S, Waltenberger J, Galeotti T: Reactive oxygen species as downstream mediators of angiogenic signaling by vascular endothelial growth factor receptor-2/KDR. J Biol Chem 2002;277:3101–3108.
34.
Oldenburg O, Qin Q, Krieg T, Yang X, Philipp S, Critz SD, Cohen MV, Downey JM: Bradykinin induces mitochondrial ROS generation via NO, cGMP, PKG, and mitoKATP channel opening and leads to cardioprotection. Am J Physiol Heart Circ Physiol 2004;286:H468–H476.
35.
Thomas SR, Chen K, Keaney JF Jr: Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway. J Biol Chem 2002;277:6017–6024.
36.
Cai H, Li Z, Davis ME, Kanner W, Harrison DG, Dudley SC Jr: Akt dependent phosphorylation of serine 1179 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 cooperatively mediate activation of the endothelial nitric-oxide synthase by hydrogen peroxide. Mol Pharmacol 2003;63:325–331.
37.
Oak MH, Chataigneau M, Keravis T, Chataigneau T, Beretz A, Andriantsitohaina R, Stoclet JC, Chang SJ, Schini-Kerth VB: Red wine polyphenolic compounds inhibit vascular endothelial growth factor expression in vascular smooth muscle cells by preventing the activation of the p38 mitogen-activated protein kinase pathway. Arterioscler Thromb Vasc Biol 2003;23:1001–1007.
38.
Vitseva O, Varghese S, Chakrabarti S, Folts JD, Freedman JE: Grape seed and skin extracts inhibit platelet function and release of reactive oxygen intermediates. J Cardiovasc Pharmacol 2005;46:445–451.
39.
Hu JP, Calomme M, Lasure A, De Bruyne T, Pieters L, Vlietinck A, Vanden Berghe DA: Structure-activity relationship of flavonoids with superoxide scavenging activity. Biol Trace Elem Res 1995;47:327–331.
40.
Sato M, Maulik G, Ray PS, Bagchi D, Das DK: Cardioprotective effects of grape seed proanthocyanidin against ischemic reperfusion injury. J Mol Cell Cardiol 1999;31:1289–1297.
41.
Aldini G, Carini M, Piccoli A, Rossoni G, Facino RM: Procyanidins from grape seeds protect endothelial cells from peroxynitrite damage and enhance endothelium-dependent relaxation in human artery: new evidences for cardio-protection. Life Sci 2003;73:2883–2898.
42.
Ying CJ, Xu JW, Ikeda K, Takahashi K, Nara Y, Yamori Y: Tea polyphenols regulate nicotinamide adenine dinucleotide phosphate oxidase subunit expression and ameliorate angiotensin II-induced hyperpermeability in endothelial cells. Hypertens Res 2003;26:823–828.
43.
Lin JK, Chen PC, Ho CT, Lin-Shiau SY: Inhibition of xanthine oxidase and suppression of intracellular reactive oxygen species in HL-60 cells by theaflavin-3,3′-digallate, (–)-epigallocatechin-3-gallate, and propyl gallate. J Agric Food Chem 2000;48:2736–2743.
44.
Sarr M, Chataigneau M, Martins S, Schott C, El Bedoui J, Oak MH, Muller B, Chataigneau T, Schini-Kerth VB: Red wine polyphenols prevent angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase. Cardiovasc Res 2006;71:794–802.
© 2009 S. Karger AG, Basel
2009
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.