European Journal of Clinical Pharmacology

, Volume 62, Supplement 1, pp 5–12

Endothelial NO synthase as a source of NO and superoxide

Review Article

Abstract

Endothelial nitric oxide (NO) synthase (eNOS) is responsible for most of the vascular NO produced. A functional eNOS transfers electrons from nicotinamide adenine dinucleotide phosphate (NADPH) via flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) in the carboxy-terminal reductase domain to the heme in the amino-terminal oxygenase domain where the substrate L-arginine is oxidized to L-citrulline and NO. This normal flow of electrons requires dimerization of the enzyme, the presence of the substrate L-arginine, and presence of the cofactor (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), one of the most potent naturally occurring reducing agents. Cardiovascular risk factors, such as hypertension, hypercholesterolemia, diabetes mellitus, or chronic smoking, stimulate the production of reactive oxygen species (ROS) in the vascular wall. NADPH oxidases represent major sources of this ROS and have been found upregulated in animal models of hypertension, diabetes, and sedentary lifestyle. Superoxide avidly interacts with vascular NO to form peroxynitrite (ONOO). BH4 is highly sensitive to oxidation, e.g., by ONOO, and reduced levels of BH4 promote eNOS uncoupling. In fact, in many cases, supplementation with BH4 is capable of correcting eNOS dysfunction. Alternatively, an oxidation of the zinc-thiolate complex of eNOS by ONOO has been proposed as a mechanism for eNOS uncoupling. Under uncoupled conditions, superoxide is generated from the oxygenase domain of eNOS. eNOS uncoupling and its change from a protective enzyme to a contributor to oxidative stress has been observed in several in vitro models and in animals with cardiovascular pathophysiology such as spontaneously hypertensive rats (SHR), angiotensin-II-induced hypertension, or diabetes. Taken together, several mechanisms seem to underlie endothelial dysfunction, but an uncoupled eNOS markedly contributes to this phenomenon.

Keywords

eNOS uncoupling (6R)-5,6,7,8-tetrahydro-L-biopterin Zinc thiolate cluster 

References

  1. 1.
    Förstermann U, Mülsch A, Böhme E, Busse R (1986) Stimulation of soluble guanylate cyclase by an acetylcholine-induced endothelium-derived factor from rabbit and canine arteries. Circ Res 58:531–538PubMedGoogle Scholar
  2. 2.
    Ignarro LJ, Harbison RG, Wood KS, Kadowitz PJ (1986) Activation of purified soluble guanylate cyclase by endothelium-derived relaxing factor from intrapulmonary artery and vein: stimulation by acetylcholine, bradykinin and arachidonic acid. J Pharmacol Exp Ther 237:893–900PubMedGoogle Scholar
  3. 3.
    Rapoport RM, Draznin MB, Murad F (1983) Endothelium-dependent relaxation in rat aorta may be mediated through cyclic GMP-dependent protein phosphorylation. Nature 306:174–176PubMedCrossRefGoogle Scholar
  4. 4.
    Alheid U, Frölich JC, Förstermann U (1987) Endothelium-derived relaxing factor from cultured human endothelial cells inhibits aggregation of human platelets. Thromb Res 47:561–571PubMedCrossRefGoogle Scholar
  5. 5.
    Busse R, Luckhoff A, Bassenge E (1987) Endothelium-derived relaxant factor inhibits platelet activation. Naunyn-Schmiedeberg's Arch Pharmacol 336:566–571CrossRefGoogle Scholar
  6. 6.
    Radomski MW, Palmer RM, Moncada S (1987) The anti-aggregating properties of vascular endothelium: interactions between prostacyclin and nitric oxide. Br J Pharmacol 92:639–646PubMedGoogle Scholar
  7. 7.
    Arndt H, Smith CW, Granger DN (1993) Leukocyte-endothelial cell adhesion in spontaneously hypertensive and normotensive rats. Hypertension 21:667–673PubMedGoogle Scholar
  8. 8.
    Kubes P, Suzuki M, Granger DN (1991) Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci U S A 88:4651–4655PubMedCrossRefGoogle Scholar
  9. 9.
    Garg UC, Hassid A (1989) Nitric oxide-generating vasodilators and 8-bromo-cyclic guanosine monophosphate inhibit mitogenesis and proliferation of cultured rat vascular smooth muscle cells. J Clin Invest 83:1774–1777PubMedCrossRefGoogle Scholar
  10. 10.
    Nakaki T, Nakayama M, Kato R (1990) Inhibition by nitric oxide and nitric oxide-producing vasodilators of DNA synthesis in vascular smooth muscle cells. Eur J Pharmacol 189:347–353PubMedCrossRefGoogle Scholar
  11. 11.
    Nunokawa Y, Tanaka S (1992) Interferon-gamma inhibits proliferation of rat vascular smooth muscle cells by nitric oxide generation. Biochem Biophys Res Commun 188:409–415PubMedCrossRefGoogle Scholar
  12. 12.
    Hogan M, Cerami A, Bucala R (1992) Advanced glycosylation endproducts block the antiproliferative effect of nitric oxide. Role in the vascular and renal complications of diabetes mellitus. J Clin Invest 90:1110–1115PubMedCrossRefGoogle Scholar
  13. 13.
    Southgate K, Newby AC (1990) Serum-induced proliferation of rabbit aortic smooth muscle cells from the contractile state is inhibited by 8-Br-cAMP but not 8-Br-cGMP. Atherosclerosis 82:113–123PubMedCrossRefGoogle Scholar
  14. 14.
    Crane BR, Arvai AS, Ghosh DK, Wu C, Getzoff ED, Stuehr DJ, Tainer JA (1998) Structure of nitric oxide synthase oxygenase dimer with pterin and substrate. Science 279:2121–2126PubMedCrossRefGoogle Scholar
  15. 15.
    Alderton WK, Cooper CE, Knowles RG (2001) Nitric oxide synthases: structure, function and inhibition. Biochem J 357:593–615PubMedCrossRefGoogle Scholar
  16. 16.
    Nishimura JS, Martasek P, McMillan K, Salerno J, Liu Q, Gross SS, Masters BS (1995) Modular structure of neuronal nitric oxide synthase: localization of the arginine binding site and modulation by pterin. Biochem Biophys Res Commun 210:288–294PubMedCrossRefGoogle Scholar
  17. 17.
    Hemmens B, Goessler W, Schmidt K, Mayer B (2000) Role of bound zinc in dimer stabilization but not enzyme activity of neuronal nitric-oxide synthase. J Biol Chem 275:35786–35791PubMedCrossRefGoogle Scholar
  18. 18.
    Raman CS, Li H, Martasek P, Kral V, Masters BS, Poulos TL (1998) Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell 95:939–950PubMedCrossRefGoogle Scholar
  19. 19.
    Li H, Raman CS, Glaser CB, Blasko E, Young TA, Parkinson JF, Whitlow M, Poulos TL (1999) Crystal structures of zinc-free and -bound heme domain of human inducible nitric-oxide synthase. Implications for dimer stability and comparison with endothelial nitric-oxide synthase. J Biol Chem 274:21276–21284PubMedCrossRefGoogle Scholar
  20. 20.
    Miller RT, Martasek P, Raman CS, Masters BS (1999) Zinc content of Escherichia coli-expressed constitutive isoforms of nitric-oxide synthase. Enzymatic activity and effect of pterin. J Biol Chem 274:14537–14540PubMedCrossRefGoogle Scholar
  21. 21.
    Hemmens B, Mayer B (1998) Enzymology of nitric oxide synthases. Methods Mol Biol 100:1–32PubMedGoogle Scholar
  22. 22.
    Noble MA, Munro AW, Rivers SL, Robledo L, Daff SN, Yellowlees LJ, Shimizu T, Sagami I, Guillemette JG, Chapman SK (1999) Potentiometric analysis of the flavin cofactors of neuronal nitric oxide synthase. Biochemistry 38:16413-16418PubMedCrossRefGoogle Scholar
  23. 23.
    Stuehr D, Pou S, Rosen GM (2001) Oxygen reduction by nitric-oxide synthases. J Biol Chem 276:14533–14536PubMedCrossRefGoogle Scholar
  24. 24.
    Martasek P, Miller RT, Liu Q, Roman LJ, Salerno JC, Migita CT, Raman CS, Gross SS, Ikeda-Saito M, Masters BS (1998) The C331A mutant of neuronal nitric-oxide synthase is defective in arginine binding. J Biol Chem 273:34799–34805PubMedCrossRefGoogle Scholar
  25. 25.
    Masters BS, McMillan K, Sheta EA, Nishimura JS, Roman LJ, Martasek P (1996) Neuronal nitric oxide synthase, a modular enzyme formed by convergent evolution: structure studies of a cysteine thiolate-liganded heme protein that hydroxylates L-arginine to produce NO. as a cellular signal. FASEB J 10:552–558PubMedGoogle Scholar
  26. 26.
    Pou S, Pou WS, Bredt DS, Snyder SH, Rosen GM (1992) Generation of superoxide by purified brain nitric oxide synthase. J Biol Chem 267:24173–24176PubMedGoogle Scholar
  27. 27.
    Heinzel B, John M, Klatt P, Böhme E, Mayer B (1992) Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase. Biochem J 281 (Pt 3):627–630PubMedGoogle Scholar
  28. 28.
    Xia Y, Tsai AL, 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
  29. 29.
    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
  30. 30.
    Vaziri ND, Ni Z, Oveisi F (1998) Upregulation of renal and vascular nitric oxide synthase in young spontaneously hypertensive rats. Hypertension 31:1248–1254PubMedGoogle Scholar
  31. 31.
    Hink U, Li H, Mollnau H, Oelze M, Matheis E, Hartmann M, Skatchkov M, Thaiss F, Stahl RA, Warnholtz A, Meinertz T, Griendling K, Harrison DG, Förstermann U, Münzel T (2001) Mechanisms underlying endothelial dysfunction in diabetes mellitus. Circ Res 88:E14–E22PubMedGoogle Scholar
  32. 32.
    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
  33. 33.
    Mollnau H, Wendt M, Szocs K, Lassegue B, Schulz E, Oelze M, Li H, Bodenschatz M, August M, Kleschyov AL, Tsilimingas N, Walter U, Förstermann U, Meinertz T, Griendling K, Münzel T (2002) Effects of angiotensin II infusion on the expression and function of NAD(P)H oxidase and components of nitric oxide/cGMP signaling. Circ Res 90:E58–E65PubMedCrossRefGoogle Scholar
  34. 34.
    Honing ML, Morrison PJ, Banga JD, Stroes ES, Rabelink TJ (1998) Nitric oxide availability in diabetes mellitus. Diabetes Metab Rev 14:241–249PubMedCrossRefGoogle Scholar
  35. 35.
    Li H, Oehrlein SA, Wallerath T, Ihrig-Biedert I, Wohlfart P, Ulshöfer T, Jessen T, Herget T, Förstermann U, Kleinert H (1998) Activation of protein kinase C alpha and/or epsilon enhances transcription of the human endothelial nitric oxide synthase gene. Mol Pharmacol 53:630–637PubMedGoogle Scholar
  36. 36.
    Drummond GR, Cai H, Davis ME, Ramasamy S, Harrison DG (2000) Transcriptional and posttranscriptional regulation of endothelial nitric oxide synthase expression by hydrogen peroxide. Circ Res 86:347–354PubMedGoogle Scholar
  37. 37.
    Griendling KK, Sorescu D, Ushio-Fukai M (2000) NAD(P)H oxidase: role in cardiovascular biology and disease. Circ Res 86:494–501PubMedGoogle Scholar
  38. 38.
    Fukui T, Ishizaka N, Rajagopalan S, Laursen JB, Capers Q IV, Taylor WR, Harrison DG, de Leon H, Wilcox JN, Griendling KK (1997) p22phox mRNA expression and NADPH oxidase activity are increased in aortas from hypertensive rats. Circ Res 80:45–51PubMedGoogle Scholar
  39. 39.
    Rajagopalan S, Kurz S, Munzel T, Tarpey M, Freeman BA, Griendling KK, Harrison DG (1996) Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. J Clin Invest 97:1916–1923PubMedCrossRefGoogle Scholar
  40. 40.
    Morawietz H, Weber M, Rueckschloss U, Lauer N, Hacker A, Kojda G (2001) Upregulation of vascular NAD(P)H oxidase subunit gp91phox and impairment of the nitric oxide signal transduction pathway in hypertension. Biochem Biophys Res Commun 285:1130–1135PubMedCrossRefGoogle Scholar
  41. 41.
    Warnholtz A, Nickenig G, Schulz E, Macharzina R, Brasen JH, Skatchkov M, Heitzer T, Stasch JP, Griendling KK, Harrison DG, Bohm M, Meinertz T, Münzel T (1999) Increased NADH-oxidase-mediated superoxide production in the early stages of atherosclerosis: evidence for involvement of the renin-angiotensin system. Circulation 99:2027–2033PubMedGoogle Scholar
  42. 42.
    Sorescu D, Weiss D, Lassegue B, Clempus RE, Szocs K, Sorescu GP, Valppu L, Quinn MT, Lambeth JD, Vega JD, Taylor WR, Griendling KK (2002) Superoxide production and expression of nox family proteins in human atherosclerosis. Circulation 105:1429–1435PubMedCrossRefGoogle Scholar
  43. 43.
    Ohishi M, Ueda M, Rakugi H, Naruko T, Kojima A, Okamura A, Higaki J, Ogihara T (1997) Enhanced expression of angiotensin-converting enzyme is associated with progression of coronary atherosclerosis in humans. J Hypertens 15:1295–1302PubMedCrossRefGoogle Scholar
  44. 44.
    Diet F, Pratt RE, Berry GJ, Momose N, Gibbons GH, Dzau VJ (1996) Increase accumulation of tissue ACE in human atherosclerotic coronary artery disease. Circulation 94:2756–2767PubMedGoogle Scholar
  45. 45.
    Vergnani L, Hatrik S, Ricci F, Passaro A, Manzoli N, Zuliani G, Brovkovych V, Fellin R, Malinski T (2000) Effect of native and oxidized low-density lipoprotein on endothelial nitric oxide and superoxide production: key role of L-arginine availability. Circulation 101:1261–1266PubMedGoogle Scholar
  46. 46.
    Nickenig G, Baumer AT, Temur Y, Kebben D, Jockenhovel F, Bohm M (1999) Statin-sensitive dysregulated AT1 receptor function and density in hypercholesterolemic men. Circulation 100:2131–2134PubMedGoogle Scholar
  47. 47.
    Ohara Y, Peterson TE, Harrison DG (1993) Hypercholesterolemia increases endothelial superoxide anion production. J Clin Invest 91:2546–2551PubMedCrossRefGoogle Scholar
  48. 48.
    White CR, Darley-Usmar V, Berrington WR, McAdams M, Gore JZ, Thompson JA, Parks DA, Tarpey MM, Freeman BA (1996) Circulating plasma xanthine oxidase contributes to vascular dysfunction in hypercholesterolemic rabbits. Proc Natl Acad Sci U S A 93:8745–8749PubMedCrossRefGoogle Scholar
  49. 49.
    Cardillo C, Kilcoyne CM, Cannon R III, Quyyumi AA, Panza JA (1997) Xanthine oxidase inhibition with oxypurinol improves endothelial vasodilator function in hypercholesterolemic but not in hypertensive patients. Hypertension 30:57–63PubMedGoogle Scholar
  50. 50.
    Butler R, Morris AD, Belch JJ, Hill A, Struthers AD (2000) Allopurinol normalizes endothelial dysfunction in type 2 diabetics with mild hypertension. Hypertension 35:746–751PubMedGoogle Scholar
  51. 51.
    O'Driscoll JG, Green DJ, Rankin JM, Taylor RR (1999) Nitric oxide-dependent endothelial function is unaffected by allopurinol in hypercholesterolaemic subjects. Clin Exp Pharmacol Physiol 26:779–783PubMedCrossRefGoogle Scholar
  52. 52.
    Pritchard KA Jr, Groszek L, Smalley DM, Sessa WC, Wu M, Villaon P, Wolin MS, Stemerman MB (1995) Native low-density lipoprotein increases endothelial cell nitric oxide synthase generation of superoxide anion. Circ Res 77:510–518PubMedGoogle Scholar
  53. 53.
    Cosentino F, Luscher TF (1998) Tetrahydrobiopterin and endothelial function. Eur Heart J 19(Suppl G):G3–G8PubMedGoogle Scholar
  54. 54.
    Kerr S, Brosnan MJ, McIntyre M, Reid JL, Dominiczak AF, Hamilton CA (1999) Superoxide anion production is increased in a model of genetic hypertension: role of the endothelium. Hypertension 33:1353–1358PubMedGoogle Scholar
  55. 55.
    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
  56. 56.
    Münzel T, Li H, Mollnau H, Hink U, Matheis E, Hartmann M, Oelze M, Skatchkov M, Warnholtz A, Duncker L, Meinertz T, Förstermann U (2000) Effects of long-term nitroglycerin treatment on endothelial nitric oxide synthase (NOS III) gene expression, NOS III-mediated superoxide production, and vascular NO bioavailability. Circ Res 86:E7–E12PubMedGoogle Scholar
  57. 57.
    Werner-Felmayer G, Werner ER, Fuchs D, Hausen A, Reibnegger G, Schmidt K, Weiss G, Wachter H (1993) Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide-mediated formation of cyclic GMP. J Biol Chem 268:1842–1846PubMedGoogle Scholar
  58. 58.
    Rosenkranz-Weiss P, Sessa WC, Milstien S, Kaufman S, Watson CA, Pober JS (1994) Regulation of nitric oxide synthesis by proinflammatory cytokines in human umbilical vein endothelial cells. Elevations in tetrahydrobiopterin levels enhance endothelial nitric oxide synthase specific activity. J Clin Invest 93:2236–2243PubMedCrossRefGoogle Scholar
  59. 59.
    Pieper GM (1997) Acute amelioration of diabetic endothelial dysfunction with a derivative of the nitric oxide synthase cofactor, tetrahydrobiopterin. J Cardiovasc Pharmacol 29:8–15PubMedCrossRefGoogle Scholar
  60. 60.
    Shinozaki K, Nishio Y, Okamura T, Yoshida Y, Maegawa H, Kojima H, Masada M, Toda N, Kikkawa R, Kashiwagi A (2000) Oral administration of tetrahydrobiopterin prevents endothelial dysfunction and vascular oxidative stress in the aortas of insulin-resistant rats. Circ Res 87:566–573PubMedGoogle Scholar
  61. 61.
    Stroes E, Kastelein J, Cosentino F, Erkelens W, Wever R, Koomans H, Luscher T, Rabelink T (1997) Tetrahydrobiopterin restores endothelial function in hypercholesterolemia. J Clin Invest 99:41–46PubMedCrossRefGoogle Scholar
  62. 62.
    Heitzer T, Brockhoff C, Mayer B, Warnholtz A, Mollnau H, Henne S, Meinertz T, Munzel T (2000) Tetrahydrobiopterin improves endothelium-dependent vasodilation in chronic smokers: evidence for a dysfunctional nitric oxide synthase. Circ Res 86:E36–E41PubMedGoogle Scholar
  63. 63.
    Milstien S, Katusic Z (1999) Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function. Biochem Biophys Res Commun 263:681–684PubMedCrossRefGoogle Scholar
  64. 64.
    White CR, Brock TA, Chang LY, Crapo J, Briscoe P, Ku D, Bradley WA, Gianturco SH, Gore J, Freeman BA, Tarpey MM (1994) Superoxide and peroxynitrite in atherosclerosis. Proc Natl Acad Sci U S A 91:1044–1048PubMedCrossRefGoogle Scholar
  65. 65.
    Shinozaki K, Kashiwagi A, Nishio Y, Okamura T, Yoshida Y, Masada M, Toda N, Kikkawa R (1999) Abnormal biopterin metabolism is a major cause of impaired endothelium-dependent relaxation through nitric oxide/O2- imbalance in insulin-resistant rat aorta. Diabetes 48:2437–2445PubMedCrossRefGoogle Scholar
  66. 66.
    Hong H-J, Hsiao G, Cheng T-H, Yen M-H (2001) Supplemention with tetrahydrobiopterin suppresses the development of hypertension in spontaneously hypertensive rats. Hypertension 38:1044–1048PubMedCrossRefGoogle Scholar
  67. 67.
    Zou MH, Shi C, Cohen RA (2002) Oxidation of the zinc-thiolate complex and uncoupling of endothelial nitric oxide synthase by peroxynitrite. J Clin Invest 109:817–826PubMedGoogle Scholar
  68. 68.
    Lin KY, Ito A, Asagami T, Tsao PS, Adimoolam S, Kimoto M, Tsuji H, Reaven GM, Cooke JP (2002) Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine and dimethylarginine dimethylaminohydrolase. Circulation 106:987–992PubMedCrossRefGoogle Scholar
  69. 69.
    Sydow K, Münzel T (2003) ADMA and oxidative stress. Atherosclerosis Suppl 4:41–51CrossRefGoogle Scholar
  70. 70.
    Böger RH, Sydow K, Borlak J, Thum T, Lenzen H, Schubert B, Tsikas D, Bode-Böger SM (2000) LDL cholesterol upregulates synthesis of asymmetrical dimethylarginine in human endothelial cells: involvement of S-adenosylmethionine-dependent methyltransferases. Circ Res 87:99–105PubMedGoogle Scholar
  71. 71.
    Schachinger V, Britten MB, Zeiher AM (2000) Prognostic impact of coronary vasodilator dysfunction on adverse long-term outcome of coronary heart disease. Circulation 101:1899–1906PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  1. 1.Department of PharmacologyJohannes Gutenberg UniversityMainzGermany

Personalised recommendations