Letter | Published:

S-glutathionylation uncouples eNOS and regulates its cellular and vascular function

Nature volume 468, pages 11151118 (23 December 2010) | Download Citation


Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O2), which are key mediators of cellular signalling. In the presence of Ca2+/calmodulin, eNOS produces NO, endothelial-derived relaxing factor, from l-arginine (l-Arg) by means of electron transfer from NADPH through a flavin containing reductase domain to oxygen bound at the haem of an oxygenase domain, which also contains binding sites for tetrahydrobiopterin (BH4) and l-Arg1,2,3. In the absence of BH4, NO synthesis is abrogated and instead O2 is generated4,5,6,7. While NOS dysfunction occurs in diseases with redox stress, BH4 repletion only partly restores NOS activity and NOS-dependent vasodilation7. This suggests that there is an as yet unidentified redox-regulated mechanism controlling NOS function. Protein thiols can undergo S-glutathionylation, a reversible protein modification involved in cellular signalling and adaptation8,9. Under oxidative stress, S-glutathionylation occurs through thiol–disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione10,11. Cysteine residues are critical for the maintenance of eNOS function12,13; we therefore speculated that oxidative stress could alter eNOS activity through S-glutathionylation. Here we show that S-glutathionylation of eNOS reversibly decreases NOS activity with an increase in O2 generation primarily from the reductase, in which two highly conserved cysteine residues are identified as sites of S-glutathionylation and found to be critical for redox-regulation of eNOS function. We show that eNOS S-glutathionylation in endothelial cells, with loss of NO and gain of O2 generation, is associated with impaired endothelium-dependent vasodilation. In hypertensive vessels, eNOS S-glutathionylation is increased with impaired endothelium-dependent vasodilation that is restored by thiol-specific reducing agents, which reverse this S-glutathionylation. Thus, S-glutathionylation of eNOS is a pivotal switch providing redox regulation of cellular signalling, endothelial function and vascular tone.

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  1. 1.

    et al. Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase. Nature 351, 714–718 (1991)

  2. 2.

    , & Vascular endothelial cells synthesize nitric oxide from l-arginine. Nature 333, 664–666 (1988)

  3. 3.

    , & Endothelium-dependent relaxation in rat aorta may be mediated through cyclic GMP-dependent protein phosphorylation. Nature 306, 174–176 (1983)

  4. 4.

    , , & Superoxide generation from endothelial nitric-oxide synthase. A Ca2+/calmodulin-dependent and tetrahydrobiopterin regulatory process. J. Biol. Chem. 273, 25804–25808 (1998)

  5. 5.

    et al. Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc. Natl Acad. Sci. USA 95, 9220–9225 (1998)

  6. 6.

    , , , & Update on mechanism and catalytic regulation in the NO synthases. J. Biol. Chem. 279, 36167–36170 (2004)

  7. 7.

    et al. Myocardial ischemia results in tetrahydrobiopterin (BH4) oxidation with impaired endothelial function ameliorated by BH4. Proc. Natl Acad. Sci. USA 104, 15081–15086 (2007)

  8. 8.

    , , , & S-glutathionylation: from redox regulation of protein functions to human diseases. J. Cell. Mol. Med. 8, 201–212 (2004)

  9. 9.

    , & Redox modifications of protein-thiols: emerging roles in cell signaling. Biochem. Pharmacol. 71, 551–564 (2006)

  10. 10.

    , , , & Thiol oxidation in signaling and response to stress: detection and quantification of physiological and pathophysiological thiol modifications. Free Radic. Biol. Med. 43, 1099–1108 (2007)

  11. 11.

    & Role of glutathiolation in preservation, restoration and regulation of protein function. IUBMB Life 59, 21–26 (2007)

  12. 12.

    & Thiol dependence of nitric oxide synthase. Biochemistry 34, 13443–13452 (1995)

  13. 13.

    et al. Glutathione regulates nitric oxide synthase in cultured hepatocytes. Ann. Surg. 225, 76–87 (1997)

  14. 14.

    et al. Regulation of endothelium-derived nitric oxide production by the protein kinase Akt. Nature 399, 597–601 (1999)

  15. 15.

    , , , & Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme. J. Biol. Chem. 283, 27038–27047 (2008)

  16. 16.

    et al. Structural basis for isozyme-specific regulation of electron transfer in nitric-oxide synthase. J. Biol. Chem. 279, 37918–37927 (2004)

  17. 17.

    , , & Biphasic lindane-induced oxidation of glutathione and inhibition of gap junctions in myometrial cells. Toxicol. Sci. 86, 417–426 (2005)

  18. 18.

    , , , & Molecular mechanism of glutathione-mediated protection from oxidized low-density lipoprotein-induced cell injury in human macrophages: role of glutathione reductase and glutaredoxin. Free Radic. Biol. Med. 41, 775–785 (2006)

  19. 19.

    , & Glutaredoxin mediates Akt and eNOS activation by flow in a glutathione reductase-dependent manner. Arterioscler. Thromb. Vasc. Biol. 27, 1283–1288 (2007)

  20. 20.

    & Thiol-metabolizing proteins and endothelial redox state: differential modulation of eNOS and biopterin pathways. Am. J. Physiol. Heart Circ. Physiol. 298, H194–H201 (2010)

  21. 21.

    et al. On the selectivity of superoxide dismutase mimetics and its importance in pharmacological studies. Br. J. Pharmacol. 140, 445–460 (2003)

  22. 22.

    , , , & Contrasting effects of thiol-modulating agents on endothelial NO bioactivity. Am. J. Physiol. Cell Physiol. 281, C719–C725 (2001)

  23. 23.

    , , , & Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation. Proc. Natl Acad. Sci. USA 104, 12312–12317 (2007)

  24. 24.

    et al. L-2-Oxothiazolidine-4-carboxylic acid reverses endothelial dysfunction in patients with coronary artery disease. J. Clin. Invest. 101, 1408–1414 (1998)

  25. 25.

    et al. Intracoronary infusion of reduced glutathione improves endothelial vasomotor response to acetylcholine in human coronary circulation. Circulation 97, 2299–2301 (1998)

  26. 26.

    , , , & Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury. Proc. Natl Acad. Sci. USA 93, 6770–6774 (1996)

  27. 27.

    , & Endogenous methylarginines modulate superoxide as well as nitric oxide generation from neuronal nitric-oxide synthase: differences in the effects of monomethyl- and dimethylarginines in the presence and absence of tetrahydrobiopterin. J. Biol. Chem. 280, 7540–7549 (2005)

  28. 28.

    et al. Regulation of eNOS-derived superoxide by endogenous methylarginines. Biochemistry 47, 7256–7263 (2008)

  29. 29.

    et al. Free radical biology and medicine: it's a gas, man!. Am. J. Physiol. Regul. Integr. Comp. Physiol. 291, R491–R511 (2006)

  30. 30.

    Hydrogen sulphide and its therapeutic potential. Nature Rev. Drug Discov. 6, 917–935 (2007)

  31. 31.

    , & Endothelial nitric-oxide synthase. Expression in Escherichia coli, spectroscopic characterization, and role of tetrahydrobiopterin in dimer formation. J. Biol. Chem. 271, 11462–11467 (1996)

  32. 32.

    , , & Effects of various imidazole ligands on heme conformation in endothelial nitric oxide synthase. Biochemistry 37, 6136–6144 (1998)

  33. 33.

    et al. Site-specific S-glutathiolation of mitochondrial NADH ubiquinone reductase. Biochemistry 46, 5754–5765 (2007)

  34. 34.

    , , , & Decreased nitric-oxide synthase activity causes impaired endothelium-dependent relaxation in the postischemic heart. J. Biol. Chem. 272, 21420–21426 (1997)

  35. 35.

    , , & Electron paramagnetic resonance spectroscopy with N-methyl-d-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: applications in identifying nitrogen monoxide products from nitric oxide synthase. Free Radic. Biol. Med. 29, 793–797 (2000)

  36. 36.

    , , , & Redox properties of iron-dithiocarbamates and their nitrosyl derivatives: implications for their use as traps of nitric oxide in biological systems. Biochim. Biophys. Acta 1474, 365–377 (2000)

  37. 37.

    , , , & Quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide. Anal. Biochem. 247, 404–411 (1997)

  38. 38.

    & Characterization of Saccharomyces cerevisiae Atm1p: functional studies of an ABC7 type transporter. Biochim. Biophys. Acta 1760, 1857–1865 (2006)

  39. 39.

    et al. Acylation targets emdothelial nitric-oxide synthase to plasmalemmal caveolae. J. Biol. Chem. 271, 6518–6522 (1996)

  40. 40.

    & Ultrathin cryosections: an important tool for immunofluorescence and correlative microscopy. J. Histochem. Cytochem. 51, 707–714 (2003)

  41. 41.

    , , , & Heme proteins mediate the conversion of nitrite to nitric oxide in the vascular wall. Am. J. Physiol. Heart Circ. Physiol. 295, H499–H508 (2008)

  42. 42.

    et al. Nitric oxide promotes endothelial cell survival signaling through S-nitrosylation and activation of dynamin-2. J. Cell Sci. 120, 492–501 (2007)

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We thank L. Zhang and K. Green-Church for support with mass spectrometric analysis. This work was supported by R01 grants HL63744, HL65608, HL38324 (J.L.Z.), HL83237 (Y.-R.C.) and HL103846 (C.-A.C.) from the National Institutes of Health.

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Author notes

    • Yeong-Renn Chen
    •  & Lawrence J. Druhan

    Present addresses: Northeastern Ohio Universities College of Medicine Department of Integrative Medical Sciences, Rootstown, Ohio 44272, USA (Y.-R.C); Department of Anesthesiology, College of Medicine, Ohio State University, Columbus, Ohio 43210, USA (L.J.D.).


  1. Davis Heart and Lung Research Institute and Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, Ohio State University, Columbus, Ohio 43210, USA

    • Chun-An Chen
    • , Tse-Yao Wang
    • , Saradhadevi Varadharaj
    • , Levy A. Reyes
    • , Craig Hemann
    • , M. A. Hassan Talukder
    • , Yeong-Renn Chen
    • , Lawrence J. Druhan
    •  & Jay L. Zweier


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C.-A.C., the primary author, performed most of the experiments and data analysis with assistance from T.-Y.W., S.V. and L.J.D. L.R. and T.-Y.W. performed the vessel studies. S.V. performed the confocal microscopy and immunohistology work. C.H. performed molecular modelling and protein expression and purification. Y.-R.C. provided mass spectrometry expertise and guidance. M.A.H.T. coordinated physiology experiments and data analysis. J.L.Z. envisioned, directed, guided and fully supported all of the work and prepared the final manuscript with input from all the authors. All authors discussed the results and commented on the manuscript.

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Jay L. Zweier.

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