Molecular and Cellular Biochemistry

, Volume 370, Issue 1–2, pp 141–150 | Cite as

(-)-Epicatechin-induced calcium independent eNOS activation: roles of HSP90 and AKT

  • Israel Ramirez-Sanchez
  • Hugo Aguilar
  • Guillermo Ceballos
  • Francisco Villarreal


Cardiovascular disease (CVD) is a leading determinant of mortality and morbidity in the world. Epidemiologic studies suggest that flavonoid intake plays a role in the prevention of CVD. Consumption of cocoa products rich in flavonoids lowers blood pressure and improves endothelial function in healthy subjects as well as in subjects with vascular dysfunction such as smokers and diabetics. The vascular actions of cocoa follow the stimulation of nitric oxide (NO). These actions can be reproduced by the administration of the cocoa flavanol (-)-epicatechin (EPI). Previously, using human endothelial cells cultured in calcium-free media, we documented EPI effects on eNOS independently of its translocation from the plasmalemma. To further define the mechanisms behind EPI-eNOS activation in Ca2+ -deprived endothelial cells, we evaluated the effects of EPI on the eNOS/AKT/HSP90 signaling pathway. Results document an EPI-induced phosphorylation/activation of eNOS, AKT, and HSP90. We also demonstrate that EPI induces a partial AKT/HSP90 migration from the cytoplasm to the caveolar membrane fraction. Immunoprecipitation assays of caveolar fractions demonstrate a physical association between HSP90, AKT, and eNOS. Thus, under Ca2+-free conditions, EPI stimulates NO synthesis via the formation of an active complex between eNOS, AKT, and HSP90.


eNOS Epicatechin Cocoa flavanols Endothelial cells 



This study was supported by NIH AT4277, HL43617, P60-MD000220 grants to Dr. Villarreal.


  1. 1.
    Shimbo D, Graham-Clarke C, Miyake Y, Rodriguez C, Sciacca R, Di Tullio M, Boden-Albala B, Sacco R, Shunichi Homma (2007) The association between endothelial dysfunction and cardiovascular outcomes in a population-based multi-ethnic cohort. Atherosclerosis 192:197–203PubMedCrossRefGoogle Scholar
  2. 2.
    Buitrago-Lopez A, Sanderson J, Johnson L, Warnakula S, Wood A, Di Angelantonio E, Franco OH (2011) Chocolate consumption and cardiometabolic disorders: systematic review and meta-analysis. BMJ 343:1–8CrossRefGoogle Scholar
  3. 3.
    Corti R, Flammer AJ, Hollenberg NK, Lüscher TF (2009) Cocoa and cardiovascular health. Circulation 119:1433–1441PubMedCrossRefGoogle Scholar
  4. 4.
    Schroeter H, Heiss C, Balzer J, Kleinbongard P, Keen CL, Hollenberg NK, Sies H, Kwik-Uribe C, Schmitz HH, Kelm M (2006) (-)-Epicatechin mediates beneficial effects of flavanol-rich cocoa on vascular function in humans. Proc Natl Acad Sci USA 103:1024–1029PubMedCrossRefGoogle Scholar
  5. 5.
    Ramirez-Sanchez I, Maya L, Ceballos G, Villarreal F (2011) (-)-Epicatechin induces calcium and translocation independent eNOS activation in arterial endothelial cells. Am L Physiol Cell Physiol 300:C880–C887CrossRefGoogle Scholar
  6. 6.
    Ramirez-Sanchez I, Maya L, Ceballos G, Villarreal F (2010) Epicatechin activation of endothelial cell eNOS, NO and related signaling pathways. Hypertension 55:1398–1405PubMedCrossRefGoogle Scholar
  7. 7.
    Ramirez-Sanchez I, Ceballos-Reyes G, Rosas-Vargas H, Cerecedo-Mercado D, Zentella-Dehesa A, Salamanca F, Coral-Vazquez RM (2007) Expression and function of utrophin associated protein complex in stretched endothelial cells: dissociation and activation of eNOS. Front Biosci 12:1956–1962PubMedCrossRefGoogle Scholar
  8. 8.
    Melkonian KA, Ostermeyer AG, Chen JZ, Roth MG, Brown DA (1999) Role of lipid modifications in targeting proteins to detergent-resistant membrane rafts. Many raft proteins are acylated, while few are prenylated. J Biol Chem 274:3910–3917PubMedCrossRefGoogle Scholar
  9. 9.
    Ichiro Shiojima, Kenneth Walsh (2006) Regulation of cardiac growth and coronary angiogenesis by the Akt/PKB signalin pathway. Genes Dev 20:3347–3365CrossRefGoogle Scholar
  10. 10.
    Lenasi H, Kohlstedt K, Fichtlscherer B, Mülsch A, Busse R, Fleming I (2003) Amlodipine activates the endothelial nitric oxide synthase by altering phosphorylation on Ser 1177 and Thr495. Cardiovasc Res 59:844–853PubMedCrossRefGoogle Scholar
  11. 11.
    Gratton J-P, Fontana J, O’Connor DS, Garcıa-Cardeña G, McCabe TJ, Sessa WC (2000) Reconstitution of an endothelial nitric-oxide synthase (eNOS), hsp90, and caveolin-1 complex in vitro. J Biol Chem. 275:22268–22272Google Scholar
  12. 12.
    Ju H, Zou R, Venema VJ, Venema RC (1997) Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem 272:18522–18525PubMedCrossRefGoogle Scholar
  13. 13.
    Michel JB, Feron O, Sacks D, Michel T (1997) Reciprocal regulation of endothelial nitric-oxide synthase by Ca2+-calmodulin and caveolin. J Biol Chem 272:15583–15586PubMedCrossRefGoogle Scholar
  14. 14.
    Boo YC, Sorescu G, Boyd N, Shiojima I, Walsh K, Du J, Jo H (2002) Shear stress stimulates phosphorylation of endothelial nitric-oxide synthase at Ser1179 by Akt-independent mechanisms: role of protein kinase A. J Biol Chem 277:3388–3396PubMedCrossRefGoogle Scholar
  15. 15.
    Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM (1999) Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399:601–605PubMedCrossRefGoogle Scholar
  16. 16.
    Fulton D, Gratton JP, McCabe TJ, Fontana J, Fujio Y, Walsh K, Franke TF, Papapetropoulos A, Sessa WC (1999) Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399:597–601PubMedCrossRefGoogle Scholar
  17. 17.
    McCabe TJ, Fulton D, Roman LJ, Sessa WC (2000) Enhanced electron flux and reduced calmodulin dissociation may explain “calcium-independent” eNOS activation by phosphorylation. J Biol Chem 275:6123–6128PubMedCrossRefGoogle Scholar
  18. 18.
    Gallis B, Corthals GL, Goodlett DR, Ueba H, Kim F, Presnell SR, Figeys D, Harrison DG, Berk BC, Aebersold R, Corson MA (1999) Identification of flow-dependent endothelial nitric-oxide synthase phosphorylation sites by mass spectrometry and regulation of phosphorylation and nitric oxide production by the phosphatidylinositol 3-kinase inhibitor LY294002. J Biol Chem 274:30101–30108PubMedCrossRefGoogle Scholar
  19. 19.
    Chen ZP, Mitchelhill KI, Michell BJ, Stapleton D, Rodriguez-Crespo I, Witters LA, Power DA, Ortiz de Montellano PR, Kemp BE (1999) AMP-activated protein kinase phosphorylation of endothelial NO synthase. FEBS Lett 443:285–289PubMedCrossRefGoogle Scholar
  20. 20.
    Harris MB, Ju H, Venema VJ, Liang H, Zou R, Michell BJ, Chen ZP, Kemp BE, Venema RC (2001) Reciprocal phosphorylation and regulation of endothelial nitric-oxide synthase in response to bradykinin stimulation. J Biol Chem 276:16587–16591PubMedCrossRefGoogle Scholar
  21. 21.
    Joy S, Siow RCM, Rowlands DJ, Becker M, Wyatt AW, Aaronson PI, Coen CW, Kallo I, Jacob R, Mann GE (2006) The isoflavone equol mediates rapid vascular relaxation. Ca2+-independent activation of endothelial nitric-oxide synthase/Hsp90 involving ERK1/2 and Akt phosphorylation in human endothelial cells. J Biol Chem 281:27335–27345Google Scholar
  22. 22.
    Omura M, Kobayashi S, Mizukami Y, Mogami K, Todoroki-Ikeda N, Miyake T, Matsuzaki M (2001) Eicosapentaenoic acid (EPA) induces Ca(2+)-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation. FEBS Lett 487:361–366PubMedCrossRefGoogle Scholar
  23. 23.
    Michel T, Feron O (1997) Nitric oxide synthases: which, where, how, and why? J Clin Invest 100:2146–2152PubMedCrossRefGoogle Scholar
  24. 24.
    Fleming I (2010) Molecular mechanisms underlying the activation of eNOS. Pflugers Arch 459:793–806PubMedCrossRefGoogle Scholar
  25. 25.
    Takahashi S, Mendelsohn ME (2003) Synergistic activation of endothelial nitric-oxide synthase (eNOS) by HSP90 and Akt: calcium-independent eNOS activation involves formation of an HSP90-Akt-CaM-bound eNOS complex. J Biol Chem 278:30821–30827PubMedCrossRefGoogle Scholar
  26. 26.
    Fleming I, Fisslthaler B, Dimmeler S, Kemp BE, Busse R (2001) Phosphorylation of Thr(495) regulates Ca(2+)/calmodulin-dependent endothelial nitric oxide synthase activity. Circ Res 88:E68–E75PubMedCrossRefGoogle Scholar
  27. 27.
    Kitakaze M, Asamuma H, Takashima S, Minamino T, Ueda Y, Sakata Y, Asakura M, Sanada S, Kuzuya T, Hori M (2000) Nifedipine-induced coronary vasodilation in ischemic hearts is attribuible to bradykinin- and NO- dependent mechanisms in dogs. Circulation 101:311–317PubMedCrossRefGoogle Scholar
  28. 28.
    Zhang XP, Hintze TH (1998) Amlodipine releases nitric oxide from canine coronary microvessels: an unexpected mechanism of action of a calcium channel-blocking agent. Circulation 97:580–876Google Scholar
  29. 29.
    Fleming I, Bauersachs J, Fisslthaler B, Busse R (1998) Ca2+-independent activation of the endothelial nitric oxide synthase in response to tyrosine phosphatase inhibitors and fluid shear stress. Circ Res 82:686–695PubMedCrossRefGoogle Scholar
  30. 30.
    Igarashi J, Thatte HS, Prabhakar P, Golan DE, Michel T (1999) Calcium-independent activation of endothelial nitric oxide synthase by ceramide. Proc Natl Acad Sci USA 96:12583–12588PubMedCrossRefGoogle Scholar
  31. 31.
    Goetz Regina M, Thatte Hemant S, Prabhakar Prakash, Cho Michael R, Michel Thomas, Golan David E (1999) Estradiol induces the calcium-dependent translocation of endothelial nitric oxide synthase. Proc Natl Acad Sci USA 96:2788–2793PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2012

Authors and Affiliations

  • Israel Ramirez-Sanchez
    • 1
    • 2
  • Hugo Aguilar
    • 1
  • Guillermo Ceballos
    • 1
    • 2
  • Francisco Villarreal
    • 1
    • 3
  1. 1.Department of MedicineUniversity of California, San DiegoSan DiegoUSA
  2. 2.Escuela Superior de Medicina, Instituto Politecnico NacionalMexicoMexico
  3. 3.UCSD CardiologyLa JollaUSA

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