European Journal of Clinical Pharmacology

, Volume 62, Supplement 1, pp 21–28 | Cite as

Antioxidants and endothelial nitric oxide synthesis

  • Regine HellerEmail author
  • Gabriele Werner-Felmayer
  • Ernst R. Werner
Review Article


Oxidative stress in the vasculature has been suggested to contribute to the development of endothelial dysfunction via different mechanisms including LDL oxidation, nitric oxide (NO) scavenging, or oxidation of tetrahydrobiopterin, a critical cofactor of endothelial NO synthase (eNOS). Antioxidants may interfere with these processes and protect NO formed in the endothelium. In particular, ascorbic acid at high concentrations seems to be a prerequisite for sufficient NO bioavailability. Moreover, there is accumulating evidence that ascorbic acid improves tetrahydrobiopterin availability in the vasculature most probably via recycling oxidized tetrahydrobiopterin back to the fully reduced pterin. In addition, ascorbic acid may reduce the α-tocopheroxyl radical and may be required for beneficial vascular effects of α-tocopherol. Recent data have shown that apart from indirect protection of NO from inactivation, α-tocopherol exerts a direct stimulatory effect on eNOS activation via serine 1177 phosphorylation. This effect was amplified by ascorbic acid suggesting that both compounds may act synergistically in optimizing endothelial NO synthesis. The data obtained in cell culture and animal studies are promising, but human long-term studies are needed to determine whether the described mechanisms are active in vivo and may provide a rationale for optimizing dietary or supplementary intake of antioxidant vitamins in certain subsets of patients.


Ascorbic acid α-Tocopherol Nitric oxide 



Experimental work was supported by the Interdisziplinäres Zentrum für Klinische Forschung, Klinikum der Friedrich-Schiller-Universität Jena (to R.H.), the Austrian research funds “Zur Förderung der wissenschaftlichen Forschung,” project P16059 (to G.W.-F.) and project 16 188 (to E.R.W.). We thank Gunda Guhr, Elke Teuscher and Petra Loitzl for their excellent technical assistance.


  1. 1.
    Alderton WK, Cooper CE, Knowles RG (2001) Nitric oxide synthases: structure, function and inhibition. Biochem J 357:593–615PubMedCrossRefGoogle Scholar
  2. 2.
    Alp NJ, Mussa S, Khoo J, Cai S, Guzik T, Jefferson A, Goh N, Rockett KA, Channon KM (2003) Tetrahydrobiopterin-dependent preservation of nitric oxide-mediated endothelial function in diabetes by targeted transgenic GTP-cyclohydrolase I overexpression. J Clin Invest 112:725–735PubMedGoogle Scholar
  3. 3.
    Alp NJ, McAteer MA, Khoo J, Choudhury RP, Channon KM (2004) Increased endothelial tetrahydrobiopterin synthesis by targeted transgenic GTP-cyclohydrolase I overexpression reduces endothelial dysfunction and atherosclerosis in apoE-knockout mice. Arterioscler Thromb Vasc Biol 24:445–450PubMedCrossRefGoogle Scholar
  4. 4.
    Baker TA, Milstien S, Katusic ZS (2001) Effect of vitamin C on the availability of tetrahydrobiopterin in human endothelial cells. J Cardiovasc Pharmacol 37:333–338PubMedCrossRefGoogle Scholar
  5. 5.
    Blair A, Shaul PW, Yuhanna IS, Conrad PA, Smart EJ (1999) Oxidized low density lipoprotein displaces endothelial nitric-oxide synthase (eNOS) from plasmalemmal caveolae and impairs eNOS activation. J Biol Chem 274:32512–32519PubMedCrossRefGoogle Scholar
  6. 6.
    Brigelius-Flohé R, Traber MG (1999) Vitamin E: function and metabolism. FASEB J 13:1145–1155PubMedGoogle Scholar
  7. 7.
    Brigelius-Flohé R, Kelly FJ, Salonen JT, Neuzil J, Zingg J-M, Azzi A (2002) The European perspective on vitamin E: current knowledge and future research. Am J Clin Nutr 76:703–716PubMedGoogle Scholar
  8. 8.
    Cai H, Harrison DG (2000) Endothelial dysfunction in cardiovascular disease: the role of oxidant stress. Circ Res 87:840–844PubMedGoogle Scholar
  9. 9.
    Carr AC, Zhu BZ, Frei B (2000) Potential antiatherogenic mechanisms of ascorbate (vitamin C) and alpha-tocopherol (vitamin E). Circ Res 87:349–354PubMedGoogle Scholar
  10. 10.
    d'Uscio LV, Milstien S, Richardson D, Smith L, Katusic ZS (2003) Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ Res 92:88–95PubMedCrossRefGoogle Scholar
  11. 11.
    Diaz MN, Frei B, Vita JA, Keaney JF Jr (1997) Antioxidants and atherosclerotic heart disease. N Engl J Med 337:408–416PubMedCrossRefGoogle Scholar
  12. 12.
    Fleming I, Busse R (2003) Molecular mechanisms involved in the regulation of the endothelial nitric oxide synthase. Am J Physiol 284:R1–R12Google Scholar
  13. 13.
    Freedman JE, Li L, Sauter R, Keaney JF Jr (2000) alpha-Tocopherol and protein kinase C inhibition enhance platelet-derived nitric oxide release. FASEB J 14:2377–2379PubMedGoogle Scholar
  14. 14.
    Guzik TJ, Mussa S, Gastaldi D, Sadowski J, Ratnatunga C, Pillai R, Channon KM (2002) Mechanisms of increased vascular superoxide production in human diabetes mellitus: role of NAD(P)H oxidase and endothelial nitric oxide synthase. Circulation 105:1656–1662PubMedCrossRefGoogle Scholar
  15. 15.
    Heitzer T, Brockhoff C, Mayer B, Warnholtz A, Mollnau H, Henne S, Meinertz T, Münzel T (2000) Tetrahydrobiopterin improves endothelium-dependent vasodilation in chronic smokers: evidence for a dysfunctional nitric oxide synthase. Circ Res 86:E36–E41PubMedGoogle Scholar
  16. 16.
    Heitzer T, Schlinzig T, Krohn K, Meinertz T, Münzel T (2001) Endothelial dysfunction, oxidative stress, and a risk of cardiovascular events in patients with coronary artery disease. Circulation 104:2673–2678PubMedCrossRefGoogle Scholar
  17. 17.
    Heller R, Münscher-Paulig F, Gräbner R, Till U (1999) L-Ascorbic acid potentiates nitric oxide synthesis in endothelial cells. J Biol Chem 274:8254–8260PubMedCrossRefGoogle Scholar
  18. 18.
    Heller R, Unbehaun A, Schellenberg B, Mayer B, Werner-Felmayer G, Werner ER (2001) L-Ascorbic acid potentiates endothelial nitric oxide synthesis via a chemical stabilization of tetrahydrobiopterin. J Biol Chem 276:40–47PubMedCrossRefGoogle Scholar
  19. 19.
    Heller R, Werner ER (2002) Ascorbic acid and endothelial NO synthesis. In: Packer L, Traber MG, Kraemer K, Frei B (eds) The antioxidant vitamins C and E. AOCS Press, Champaign, Illinois, pp 66–88Google Scholar
  20. 20.
    Heller R, Hecker M, Stahmann N, Thiele J, Werner-Felmayer G, Werner ER (2004) α-Tocopherol amplifies phosphorylation of endothelial nitric oxide synthase at serine 1177 and its short-chain derivative trolox stabilizes tetrahydrobiopterin. Free Radic Biol Med 37:620–631PubMedCrossRefGoogle Scholar
  21. 21.
    Huang A, Vita JA, Venema RC, Keaney JF Jr (2000) Ascorbic acid enhances endothelial nitric-oxide synthase activity by increasing intracellular tetrahydrobiopterin. J Biol Chem 275:17399–17406PubMedCrossRefGoogle Scholar
  22. 22.
    Jackson TS, Xu A, Vita JA, Keaney JF Jr (1998) Ascorbate prevents the interaction of superoxide and nitric oxide only at very high physiological concentrations. Circ Res 83:916–922PubMedGoogle Scholar
  23. 23.
    Katusic ZS (2001) Vascular endothelial dysfunction: does tetrahydrobiopterin play a role? Am J Physiol 281:H981–H986Google Scholar
  24. 24.
    Keaney JF Jr, Guo Y, Cunningham D, Shwaery GT, Xu A, Vita JA (1996) Vascular incorporation of alpha-tocopherol prevents endothelial dysfunction due to oxidized LDL by inhibiting protein kinase C stimulation. J Clin Invest 98:386–394PubMedCrossRefGoogle Scholar
  25. 25.
    Kuzkaya N, Weissmann N, Harrison DG, Dikalov S (2003) Interactions of peroxynitrite, tetrahydrobiopterin, ascorbic acid, and thiols: implications for uncoupling endothelial nitric oxide synthase. J Biol Chem 278:22546–22554PubMedCrossRefGoogle Scholar
  26. 26.
    Landmesser U, Dikalov S, Price SR, McCann L, Fukai T, Holland SM, Mitch WE, Harrison DG (2003) Oxidation of tetrahydrobiopterin leads to uncoupling of endothelial cell nitric oxide synthase in hypertension. J Clin Invest 111:1201–1209PubMedGoogle Scholar
  27. 27.
    Laursen JB, Somers M, Kurz S, McCann L, Warnholtz A, Freeman BA, Tarpey M, Fukai T, Harrison DG (2001) Endothelial regulation of vasomotion in ApoE-deficient mice. Implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation 103:1282–1288PubMedGoogle Scholar
  28. 28.
    Li D, Saldeen T, Romeo F, Mehta JL (2001) Different isoforms of tocopherols enhance nitric oxide synthase phosphorylation and inhibit human platelet aggregation and lipid peroxidation: implications in therapy with vitamin E. J Cardiovasc Pharmacol Therapeut 6:155–161CrossRefGoogle Scholar
  29. 29.
    Liao JK, Clark SL (1995) Regulation of G protein alpha i2 subunit expression by oxidized low-density lipoprotein. J Clin Invest 95:1457–1463PubMedCrossRefGoogle Scholar
  30. 30.
    Liao JK, Shin WS, Lee WY, Clark SL (1995) Oxidized low-density lipoproteins decrease the expression of endothelial nitric oxide synthase. J Biol Chem 270:319–324PubMedCrossRefGoogle Scholar
  31. 31.
    May JM, Qu Z-C, Mendiretta S (1998) Protection and recycling of alpha-tocopherol in human erythrocytes by intracellular ascorbic acid. Arch Biochem Biophys 349:281–289PubMedCrossRefGoogle Scholar
  32. 32.
    May JM (2000) How does ascorbic acid prevent endothelial dysfunction? Free Radic Biol Med 28:1421–1429PubMedCrossRefGoogle Scholar
  33. 33.
    Milstien S, Katusic Z (1999) Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem Biophys Res Commun 263:681–684PubMedCrossRefGoogle Scholar
  34. 34.
    Nuszkowski A, Gräbner R, Marsche G, Unbehaun A, Malle E, Heller R (2001) Hypochlorite-modified low density lipoprotein inhibits nitric oxide synthesis in endothelial cells via an intracellular dislocalization of endothelial nitric-oxide synthase. J Biol Chem 276:14212–14221PubMedGoogle Scholar
  35. 35.
    Ozaki M, Kawashima S, Yamashita T, Hirase T, Namiki M, Inoue N, Hirata K, Yasui H, Sakurai H, Yoshida Y, Masada M, Yokoyama M (2002) Overexpression of endothelial nitric oxide synthase accelerates atherosclerotic lesion formation in apoE-deficient mice. J Clin Invest 110:331–340PubMedGoogle Scholar
  36. 36.
    Patel KB, Stratford MR, Wardman P, Everett SA (2002) Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate. Free Radic Biol Med 32:203–211PubMedCrossRefGoogle Scholar
  37. 37.
    Ricciarelli R, Zingg J-M, Azzi A (2001) Vitamin E: protective role of a Janus molecule. FASEB J 15:2314–2325PubMedCrossRefGoogle Scholar
  38. 38.
    Spiekermann S, Landmesser U, Dikalov S, Bredt M, Gamez G, Tatge H, Reepschlager N, Hornig B, Drexler H, Harrison DG (2003) Electron spin resonance characterization of vascular xanthine and NAD(P)H oxidase activity in patients with coronary artery disease: relation to endothelium-dependent vasodilation. Circulation 107:1383–1389PubMedCrossRefGoogle Scholar
  39. 39.
    Tiefenbacher CP (2001) Tetrahydrobiopterin: a critical cofactor for eNOS and a strategy in the treatment of endothelial dysfunction. Am J Physiol 280:H2484–H2488Google Scholar
  40. 40.
    Tomasian D, Keaney JF Jr, Vita JA (2000) Antioxidants and the bioactivity of endothelium-derived nitric oxide. Cardiovasc Res 47:426–435PubMedCrossRefGoogle Scholar
  41. 41.
    Toth M, Kukor Z, Valent S (2002) Chemical stabilization of tetrahydrobiopterin by L-ascorbic acid: contribution to placental endothelial nitric oxide synthase activity. Mol Hum Reprod 8:271–280PubMedCrossRefGoogle Scholar
  42. 42.
    Upston JM, Terentis AC, Stocker R (1999) Tocopherol-mediated peroxidation of lipoproteins: implications for vitamin E as a potential antiatherogenic supplement. FASEB J 13:977–994PubMedGoogle Scholar
  43. 43.
    Vasquez-Vivar J, Kalyanaraman B, Martasek P, Hogg N, Masters BS, Karoui H, Tordo P, Pritchard KA (1998) Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A 95:9220–9225PubMedCrossRefGoogle Scholar
  44. 44.
    Vasquez-Vivar J, Whitsett J, Martasek P, Hogg N, Kalyanaraman B (2001) Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical. Free Radic Biol Med 31:975–985PubMedCrossRefGoogle Scholar
  45. 45.
    Vergnani L, Hatrik S, Ricci F, Passaro A, Manzoli N, Zulianin G, Brovkovych V, Fellin R, Malinski T (2000) Effect of native and oxidized low-density lipoproteins on endothelial nitric oxide and superoxide production. Key role of L-arginine availability. Circulation 101:1261–1266PubMedGoogle Scholar
  46. 46.
    Werner ER, Gorren ACF, Heller R, Werner-Felmayer G, Mayer B (2003) Tetrahydrobiopterin and nitric oxide: mechanistic and pharmacological aspects. Exp Biol Med 228:1291–1302Google Scholar
  47. 47.
    Xia Y, Tsai A-L, Berka V, Zweier JL (1998) Superoxide generation from endothelial nitric-oxide synthase. A Ca2+/calmodulin-dependent and tetrahydrobiopterin regulatory process. J Biol Chem 273:25804–25808PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Regine Heller
    • 1
    Email author
  • Gabriele Werner-Felmayer
    • 2
  • Ernst R. Werner
    • 2
  1. 1.Institute of Molecular Cell BiologyFriedrich-Schiller-University of JenaJenaGermany
  2. 2.Biocentre, Division of Biological ChemistryInnsbruck Medical UniversityInnsbruckAustria

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