Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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

Nature volume 468, pages 1115–1118 (2010)Cite this article

Subjects

Abstract

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: S-glutathionylation of heNOS occurs and inhibits NOS activity.
Figure 2: Cysteine mutants (C689S, C908S and C689S/C908S) of heNOS resist S-glutathionylation and secondary uncoupling.
Figure 3: Effect of redox stress on eNOS S-glutathionylation and function in endothelial cells.
Figure 4: Effect of redox stress on eNOS S-glutathionylation and function in vessels.

Similar content being viewed by others

References

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

    Article  ADS  CAS  Google Scholar 

  2. Palmer, R. M., Ashton, D. S. & Moncada, S. Vascular endothelial cells synthesize nitric oxide from l-arginine. Nature 333, 664–666 (1988)

    Article  ADS  CAS  Google Scholar 

  3. Rapoport, R. M., Draznin, M. B. & Murad, F. Endothelium-dependent relaxation in rat aorta may be mediated through cyclic GMP-dependent protein phosphorylation. Nature 306, 174–176 (1983)

    Article  ADS  CAS  Google Scholar 

  4. Xia, Y., Tsai, A. L., Berka, V. & Zweier, J. L. Superoxide generation from endothelial nitric-oxide synthase. A Ca2+/calmodulin-dependent and tetrahydrobiopterin regulatory process. J. Biol. Chem. 273, 25804–25808 (1998)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  6. Stuehr, D. J., Santolini, J., Wang, Z. Q., Wei, C. C. & Adak, S. Update on mechanism and catalytic regulation in the NO synthases. J. Biol. Chem. 279, 36167–36170 (2004)

    Article  CAS  Google Scholar 

  7. Dumitrescu, C. 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)

    Article  ADS  CAS  Google Scholar 

  8. Giustarini, D., Rossi, R., Milzani, A., Colombo, R. & Dalle-Donne, I. S-glutathionylation: from redox regulation of protein functions to human diseases. J. Cell. Mol. Med. 8, 201–212 (2004)

    Article  CAS  Google Scholar 

  9. Biswas, S., Chida, A. S. & Rahman, I. Redox modifications of protein-thiols: emerging roles in cell signaling. Biochem. Pharmacol. 71, 551–564 (2006)

    Article  CAS  Google Scholar 

  10. Ying, J., Clavreul, N., Sethuraman, M., Adachi, T. & Cohen, R. A. 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)

    Article  CAS  Google Scholar 

  11. Hill, B. G. & Bhatnagar, A. Role of glutathiolation in preservation, restoration and regulation of protein function. IUBMB Life 59, 21–26 (2007)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  15. Chen, C. A., Druhan, L. J., Varadharaj, S., Chen, Y. R. & Zweier, J. L. Phosphorylation of endothelial nitric-oxide synthase regulates superoxide generation from the enzyme. J. Biol. Chem. 283, 27038–27047 (2008)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Caruso, R. L., Upham, B. L., Harris, C. & Trosko, J. E. Biphasic lindane-induced oxidation of glutathione and inhibition of gap junctions in myometrial cells. Toxicol. Sci. 86, 417–426 (2005)

    Article  CAS  Google Scholar 

  18. Wang, Y., Qiao, M., Mieyal, J. J., Asmis, L. M. & Asmis, R. 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)

    Article  CAS  Google Scholar 

  19. Wang, J., Pan, S. & Berk, B. C. Glutaredoxin mediates Akt and eNOS activation by flow in a glutathione reductase-dependent manner. Arterioscler. Thromb. Vasc. Biol. 27, 1283–1288 (2007)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Huang, A., Xiao, H., Samii, J. M., Vita, J. A. & Keaney, J. F., Jr Contrasting effects of thiol-modulating agents on endothelial NO bioactivity. Am. J. Physiol. Cell Physiol. 281, C719–C725 (2001)

    Article  CAS  Google Scholar 

  23. Sayed, N., Baskaran, P., Ma, X., van den Akker, F. & Beuve, A. Desensitization of soluble guanylyl cyclase, the NO receptor, by S-nitrosylation. Proc. Natl Acad. Sci. USA 104, 12312–12317 (2007)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Xia, Y., Dawson, V. L., Dawson, T. M., Snyder, S. H. & Zweier, J. L. 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)

    Article  ADS  CAS  Google Scholar 

  27. Cardounel, A. J., Xia, Y. & Zweier, J. L. 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)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  31. Rodriguez-Crespo, I., Gerber, N. C. & Ortiz de Montellano, P. R. Endothelial nitric-oxide synthase. Expression in Escherichia coli, spectroscopic characterization, and role of tetrahydrobiopterin in dimer formation. J. Biol. Chem. 271, 11462–11467 (1996)

    Article  CAS  Google Scholar 

  32. Berka, V., Palmer, G., Chen, P. F. & Tsai, A. L. Effects of various imidazole ligands on heme conformation in endothelial nitric oxide synthase. Biochemistry 37, 6136–6144 (1998)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  34. Giraldez, R. R., Panda, A., Xia, Y., Sanders, S. P. & Zweier, J. L. Decreased nitric-oxide synthase activity causes impaired endothelium-dependent relaxation in the postischemic heart. J. Biol. Chem. 272, 21420–21426 (1997)

    Article  CAS  Google Scholar 

  35. Xia, Y., Cardounel, A. J., Vanin, A. F. & Zweier, J. L. 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)

    Article  CAS  Google Scholar 

  36. Vanin, A. F., Liu, X., Samouilov, A., Stukan, R. A. & Zweier, J. L. 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)

    Article  CAS  Google Scholar 

  37. Roubaud, V., Sankarapandi, S., Kuppusamy, P., Tordo, P. & Zweier, J. L. Quantitative measurement of superoxide generation using the spin trap 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide. Anal. Biochem. 247, 404–411 (1997)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  41. Alzawahra, W. F., Talukder, M. A., Liu, X., Samouilov, A. & Zweier, J. L. Heme proteins mediate the conversion of nitrite to nitric oxide in the vascular wall. Am. J. Physiol. Heart Circ. Physiol. 295, H499–H508 (2008)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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.

Author information

Author notes

  1. Yeong-Renn Chen & Lawrence J. Druhan

    Present address: 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.).,

Authors and Affiliations

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

Authors
  1. Chun-An Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tse-Yao Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Saradhadevi Varadharaj
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Levy A. Reyes
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Craig Hemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. M. A. Hassan Talukder
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yeong-Renn Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Lawrence J. Druhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jay L. Zweier
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Jay L. Zweier.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

The file contains Supplementary Figures 1-10 with legends. (PDF 878 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, CA., Wang, TY., Varadharaj, S. et al. S-glutathionylation uncouples eNOS and regulates its cellular and vascular function. Nature 468, 1115–1118 (2010). https://doi.org/10.1038/nature09599

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature09599

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing