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.

  • Article
  • Published:

S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide

Nature Medicine volume 10, pages 1200–1207 (2004)Cite this article

Abstract

Nitric oxide (NO) physiologically stimulates the sarco/endoplasmic reticulum calcium (Ca2+) ATPase (SERCA) to decrease intracellular Ca2+ concentration and relax cardiac, skeletal and vascular smooth muscle. Here, we show that NO-derived peroxynitrite (ONOO−) directly increases SERCA activity by S-glutathiolation and that this modification of SERCA is blocked by irreversible oxidation of the relevant cysteine thiols during atherosclerosis. Purified SERCA was S-glutathiolated by ONOO− and the increase in Ca2+-uptake activity of SERCA reconstituted in phospholipid vesicles required the presence of glutathione. Mutation of the SERCA-reactive Cys674 to serine abolished these effects. Because superoxide scavengers decreased S-glutathiolation of SERCA and arterial relaxation by NO, ONOO− is implicated as the intracellular mediator. NO-dependent relaxation as well as S-glutathiolation and activation of SERCA were decreased by atherosclerosis and Cys674 was found to be oxidized to sulfonic acid. Thus, irreversible oxidation of key thiol(s) in disease impairs NO-induced relaxation by preventing reversible S-glutathiolation and activation of SERCA by NO/ONOO−.

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: ONOO− increases formation of GSS-SERCA and Ca2+-uptake activity of reconstituted purified SERCA.
Figure 2: The effects of ONOO− on GSS-SERCA formation and Ca2+-uptake activity in cell lysates of HEK293 cells overexpressing wild-type or C674S mutant SERCA.
Figure 3: Increased GSS-SERCA and sarcoplasmic reticulum Ca2+ uptake caused by either endothelium-derived or exogenous NO.
Figure 4: Role of intracellular ONOO− in GSS-SERCA formation and arterial relaxation.
Figure 5: Atherosclerosis highly oxidizes reactive thiols on SERCA and abolishes GSS-SERCA formation and SERCA activation by NO in rabbit abdominal aorta.
Figure 6: Summary of MALDI-TOF MS analysis of cysteine residues modified physiologically by NO and by atherosclerosis, and the proposed mechanism of cyclic GMP–independent vasodilatation to NO and the influence of disease.

Similar content being viewed by others

References

  1. Misquitta, C.M., Mack, D.P. & Grover, A.K. Sarco/endoplasmic reticulum Ca2+ (SERCA)-pumps: link to heart beats and calcium waves. Cell Calcium 25, 277–290 (1999).

    Article  CAS  Google Scholar 

  2. Cohen, R.A. et al. Mechanism of nitric oxide-induced vasodilatation. Refilling of intracellular stores by sarcoplasmic reticulum Ca2+ ATPase and inhibition of store-operated Ca2+ influx. Circ. Res. 84, 210–219 (1999).

    Article  CAS  Google Scholar 

  3. Adachi, T., Matsui, R., Weisbrod, R.M., Najibi, S. & Cohen, R.A. Reduced sarco/endoplasmic reticulum Ca2+ uptake activity can account for the reduced response to NO, but not sodium nitroprusside, in hypercholesterolemic rabbit aorta. Circulation 104, 1040–1045 (2001).

    Article  CAS  Google Scholar 

  4. Trepakova, E.S., Cohen, R.A. & Bolotina, V.M. Nitric oxide inhibits capacitative cation influx in human platelets by promoting sarcoplasmic/endoplasmic reticulum Ca2+-ATPase-dependent refilling of Ca2+ stores. Circ. Res. 84, 201–209 (1999).

    Article  CAS  Google Scholar 

  5. Paolocci, N. et al. cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation. Am. J. Physiol. Heart Circ. Physiol. 279, H1982–H1988 (2000).

    Article  CAS  Google Scholar 

  6. Barouch, L.A. et al. Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms. Nature 416, 337–339 (2002).

    Article  CAS  Google Scholar 

  7. Stamler, J.S., Lamas, S. & Fang, F.C. Nitrosylation:the prototypic redox-based signaling mechanism. Cell 106, 675–683 (2001).

    Article  CAS  Google Scholar 

  8. Jaffrey, S.R., Erdjument-Bromage, H., Ferris, C.D., Tempst, P. & Snyder, S.H. Protein S-nitrosylation: a physiological signal for neuronal nitric oxide. Nat. Cell Biol. 3, 193–197 (2001).

    Article  CAS  Google Scholar 

  9. Eu, J.P., Sun, J., Xu, L., Stamler, J.S. & Meissner, G. The skeletal muscle calcium release channel: coupled O2 sensor and NO signaling functions. Cell 102, 499–509 (2000).

    Article  CAS  Google Scholar 

  10. Sun, J., Xin, C., Eu, J.P., Stamler, J.S. & Meissner, G. Cysteine-3635 is responsible for skeletal muscle ryanodine receptor modulation by NO. Proc. Natl. Acad. Sci. USA 98, 11158–11162 (2001).

    Article  CAS  Google Scholar 

  11. Klatt, P. & Lamas, S. Regulation of protein function by S-glutathiolation in response to oxidative and nitrosative stress. Eur. J. Biochem. 267, 4928–4944 (2000).

    Article  CAS  Google Scholar 

  12. Viner, R.I., Williams, T.D. & Schöneich, C. Nitric oxide-dependent modification of the sarcoplasmic reticulum Ca-ATPase: localization of cysteine target sites. Free Radic. Biol. Med. 29, 489–496 (2000).

    Article  CAS  Google Scholar 

  13. Viner, R.I., Williams, T.D. & Schöneich, C. Peroxynitrite modification of protein thiols: oxidation, nitrosylation, and S-glutathiolation of functionally important cysteine residue(s) in the sarcoplasmic reticulum Ca-ATPase. Biochemistry 38, 12408–12415 (1999).

    Article  CAS  Google Scholar 

  14. Viner, R.I., Huhmer, A.F., Bigelow, D.J. & Schöneich, C. The oxidative inactivation of sarcoplasmic reticulum Ca(2+)-ATPase by peroxynitrite. Free Radic. Res. 24, 243–259 (1996).

    Article  CAS  Google Scholar 

  15. Pagano, P.J., Griswold, M.C., Najibi, S., Marklund, S.L. & Cohen, R.A. Resistance of endothelium-dependent relaxation elevation of O2− levels in rabbit carotid artery. Am. J. Physiol. Heart Circ. Physiol. 46, H2109–H2114 (1999).

    Google Scholar 

  16. Guzik, T.J., West, N.E., Pillai, R., Taggart, D.P. & Channon, K.M. Nitric oxide modulates superoxide release and peroxynitrite formation in human blood vessels. Hypertension 39, 1088–1094 (2002).

    Article  CAS  Google Scholar 

  17. Davidson, C.A., Kaminski, P.M. & Wolin, M.S. NO elicits prolonged relaxation of bovine pulmonary arteries via endogenous peroxynitrite generation. Am. J. Physiol. 273, L437–L444 (1997).

    CAS  Google Scholar 

  18. White, C.R. et al. Superoxide and peroxynitrite in atherosclerosis. Proc. Natl. Acad. Sci. USA 91, 1044–1048 (1994).

    Article  CAS  Google Scholar 

  19. Beckman, J.S. & Koppenol, W.H. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271, C1424–C1437 (1996).

    Article  CAS  Google Scholar 

  20. Adachi, T. et al. Antioxidant improves smooth muscle sarco/endoplasmic reticulum Ca(2+)-ATPase function and lowers tyrosine nitration in hypercholesterolemia and improves nitric oxide-induced relaxation. Circ. Res. 90, 1114–1121 (2002).

    Article  CAS  Google Scholar 

  21. Flesch, M. et al. Effect of beta-blockers on free radical-induced cardiac contractile dysfunction. Circulation 100, 346–353 (1999).

    Article  CAS  Google Scholar 

  22. Xu, K.Y., Zweier, J.L. & Becker, L.C. Hydroxyl radical inhibits sarcoplasmic reticulum Ca2+ -ATPase function by direct attack on the ATP binding site. Circ. Res. 80, 76–81 (1997).

    Article  CAS  Google Scholar 

  23. Viner, R.I., Ferrington, D.A., Williams, T.D., Bigelow, D.J. & Schöneich, C. Protein modification during biological aging: selective tyrosine nitration of the SERCA2a isoform of the sarcoplasmic reticulum Ca2+-ATPase in skeletal muscle. Biochem. J. 340 (Pt 3), 657–669 (1999).

    Article  Google Scholar 

  24. Yao, Q., Chen, L.T. & Bigelow, D.J. Affinity purification of the Ca-ATPase from cardiac sarcoplasmic reticulum membranes. Protein Expr. Purif. 13, 191–197 (1998).

    Article  CAS  Google Scholar 

  25. Kim, J.R., Yoon, H.W., Kwon, K.S., Lee, S.R. & Rhee, S.G. Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH. Anal. Biochem. 283, 214–221 (2000).

    Article  CAS  Google Scholar 

  26. Bishop, J.E., Squier, T.C., Bigelow, D.J. & Inesi, G. (Iodoacetamido)fluorescein labels a pair of proximal cysteines on the Ca2+-ATPase of sarcoplasmic reticulum. Biochemistry 27, 5233–5240 (1988).

    Article  CAS  Google Scholar 

  27. Adachi, T. & Cohen, R.A. Decreased aortic glutathione levels may contribute to impaired nitric oxide-induced relaxation in hypercholesterolaemia. Br. J. Pharmacol. 129, 1014–1020 (2000).

    Article  CAS  Google Scholar 

  28. Grover, A.K., Kwan, C.Y. & Samson, S.E. Effects of peroxynitrite on sarco/endoplasmic reticulum Ca2+ pump isoforms SERCA2b and SERCA3a. Am. J. Physiol. Cell Physiol. 285, C1537C1543 (2003).

  29. Sharov, V.S. et al. Two-dimensional separation of the membrane protein sarcoplasmic reticulum Ca-ATPase for high-performance liquid chromatography-tandem mass spectrometry analysis of posttranslational protein modifications. Anal. Biochem. 308, 328–335 (2002).

    Article  CAS  Google Scholar 

  30. Cohen, R.A. et al. Nitric oxide is the mediator of both endothelium-dependent relaxation and hyperpolarization of the rabbit carotid artery. Proc. Natl. Acad. Sci. USA 94, 4193–4198 (1997).

    Article  CAS  Google Scholar 

  31. Plane, F., Wiley, K.E., Jeremy, J.Y., Cohen, R.A. & Garland, C.J. Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery. Br. J. Pharmacol. 123, 1351–1358 (1998).

    Article  CAS  Google Scholar 

  32. Radi, R., Beckman, J.S., Bush, K.M. & Freeman, B.A. Peroxynitrite oxidation of sulfhydryls. J. Biol. Chem. 266, 4244–4250 (1991).

    CAS  Google Scholar 

  33. Woo, H.A. et al. Reversible oxidation of the active site cysteine of peroxiredoxins to cysteine sulfinic acid. Immunoblot detection with antibodies specific for the hyperoxidized cysteine-containing sequence. J. Biol. Chem. 278, 47361–47364 (2003).

    Article  CAS  Google Scholar 

  34. Adachi, T. et al. S-glutathiolation of Ras mediates redox-sensitive signaling by angiotensin II in vascular smooth muscle cells. J. Biol. Chem. 279, 29857–29862 (2004).

    Article  CAS  Google Scholar 

  35. Aracena, P., Sanchez, G., Donoso, P., Hamilton, S.L. & Hidalgo, C. S-glutathionylation decreases Mg2+ inhibition and S-nitrosylation enhances Ca2+ activation of RyR1 channels. J. Biol. Chem. 278, 42927–42935 (2003).

    Article  CAS  Google Scholar 

  36. Morikawa, K. et al. Pivotal role of Cu,Zn-superoxide dismutase in endothelium-dependent hyperpolarization. J. Clin. Invest. 112, 1871–1879 (2003).

    Article  CAS  Google Scholar 

  37. d'Uscio, L.V., Milstien, S., Richardson, D., Smith, L. & Katusic, Z.S. Long-term vitamin C treatment increases vascular tetrahydrobiopterin levels and nitric oxide synthase activity. Circ. Res. 92, 88–95 (2003).

    Article  CAS  Google Scholar 

  38. Go, Y.M. et al. Evidence for peroxynitrite as a signaling molecule in flow-dependent activation of c-jun NH2-terminal kinase. Am. J. Physiol. 277, H1647–H1653 (1999).

    CAS  Google Scholar 

  39. Ido, Y., Chang, K., Woolsey, T.A. & Williamson, J.R. NADH: sensor of blood flow need in brain, muscle, and other tissues. FASEB J. 15, 1419–1421 (2001).

    Article  CAS  Google Scholar 

  40. Barry-Lane, P.A. et al. p47phox is required for atherosclerotic lesion progression in ApoE−/− mice. J. Clin. Invest. 108, 1513–1522 (2001).

    Article  CAS  Google Scholar 

  41. Laursen, J.B. et al. Endothelial regulation of vasomotion in apoE-deficient mice: implications for interactions between peroxynitrite and tetrahydrobiopterin. Circulation 103, 1282–1288 (2001).

    Article  CAS  Google Scholar 

  42. White, C.R. et al. Circulating plasma xanthine oxidase contributes to vascular dysfunction in hypercholesterolemic rabbits. Proc. Natl. Acad. Sci. USA 93, 8745–8749 (1996).

    Article  CAS  Google Scholar 

  43. Fukai, T. et al. Vascular expression of extracellular superoxide dismutase in atherosclerosis. J. Clin. Invest. 101, 2101–2111 (1998).

    Article  CAS  Google Scholar 

  44. Abu-Soud, H.M. & Hazen, S.L. Nitric oxide is a physiological substrate for mammalian peroxidases. J. Biol. Chem. 275, 37524–37532 (2000).

    Article  CAS  Google Scholar 

  45. Weisbrod, R.M., Griswold, M.C., Du, Y., Bolotina, V.M. & Cohen, R.A. Reduced responsiveness of hypercholesterolemic rabbit aortic smooth muscle cells to nitric oxide. Arterioscler. Thromb. Vasc. Biol. 17, 394–402 (1997).

    Article  CAS  Google Scholar 

  46. Miller, F.J., Gutterman, D.D., Rios, C.D., Heistad, D.D. & Davidson, B.L. Superoxide production in vascular smooth muscle contributes to oxidative stress and impaired relaxation in atherosclerosis. Circ. Res. 82, 1298–1305 (1998).

    Article  CAS  Google Scholar 

  47. Tribble, D.L. et al. Fatty streak formation in fat-fed mice expressing human copper-zinc superoxide dismutase. Arterioscler. Thromb. Vasc. Biol. 17, 1734–1740 (1997).

    Article  CAS  Google Scholar 

  48. Okuda, M. et al. Expression of glutaredoxin in human coronary arteries: its potential role in antioxidant protection against atherosclerosis. Arterioscler. Thromb. Vasc. Biol. 21, 1483–1487 (2001).

    Article  CAS  Google Scholar 

  49. Takagi, Y. et al. Expression of thioredoxin is enhanced in atherosclerotic plaques and during neointima formation in rat arteries. Lab. Invest. 78, 957–966 (1998).

    CAS  Google Scholar 

  50. Sullivan, D.M., Wehr, N.B., Fergusson, M.M., Levine, R.L. & Finkel, T. Identification of oxidant-sensitive proteins: TNF-α induces protein glutathiolation. Biochemistry 39, 11121–11128 (2000).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We appreciate discussions with D. Bigelow and Y. Ido and acknowledge support for mass spectrometry from T. Heibeck and the Boston University Mass Spectrometry Resource Center and from the Mass Spectrometry Laboratory at the University of Kansas. The studies were supported by R01 HL31607-21 (T.A., R.W. and R.A.C.), SCOR HL55993-06 (T.A., R.W. and R.A.C.), the US National Institutes of Health Boston University Cardiovascular Proteomics Center, N01-HV-28178 (T.A. and R.A.C.), AG P01 12993 (V.S. and C.S.), a Grant-in-Aid (T.A.) and a Fellow-to-Faculty grant (D.R.P.) from the American Heart Association, the 21st Century Center-of-Excellence Program for Systems Biology, and the Leading Project for Biosimulation of the Ministry of Education, Sciences and Technology in Japan (T.A.).

Author information

Authors and Affiliations

  1. Vascular and Myocardial Biology Units, Whitaker Cardiovascular Institute, Boston University Medical Center, X707, 650 Albany Street, Boston, 02118-2393, Massachusetts, USA

    Takeshi Adachi, Robert M Weisbrod, David R Pimentel, Jia Ying & Richard A Cohen

  2. Department of Pharmaceutical Chemistry, University of Kansas School of Pharmacy, 2095 Constant Avenue, Lawrence, 66047, Kansas, USA

    Victor S Sharov & Christian Schöneich

Authors
  1. Takeshi Adachi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Robert M Weisbrod
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David R Pimentel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jia Ying
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Victor S Sharov
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christian Schöneich
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Richard A Cohen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Takeshi Adachi or Richard A Cohen.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Peroxynitrite, but not NO modifies thiols on SERCA. (PDF 82 kb)

Supplementary Fig. 2

Effects of peroxynitrite on SERCA activity in rabbit aortic homogenates. (PDF 58 kb)

Supplementary Fig. 3

S-glutathiolation of SERCA by bradykinin in pig carotid arteries. (PDF 59 kb)

Supplementary Fig. 4

S-glutathiolation and activation of SERCA by NO in cultured smooth muscle cells from rabbit aorta. (PDF 60 kb)

Supplementary Table 1

MALDI-TOF mass spectrometry analysis of Cys-modifications on SERCA purified from rabbit aortic homogenates treated with GSH (0.5 mM) and ONOO− (100 μm) (PDF 29 kb)

Supplementary Table 2

MALDI-TOF mass spectrometry analysis of Cys-modifications (GSS-Cys and SNO-Cys) on SERCA purified from pig carotid arteries treated with bradykinin (BK) (PDF 28 kb)

Supplementary Table 3

MALDI-TOF and ESI/LC mass spectrometry analysis of Cys-modifications (GSS-Cys and SNO-Cys) on SERCA purified from endothelium-denuded rabbit aorta treated with NO gas solution (PDF 34 kb)

Supplementary Table 4

MALDI-TOF mass spectrometry analysis of Cys-sulfonylation (RSO3H) on SERCA purified from atherosclerotic rabbit aorta (PDF 28 kb)

Supplementary Methods (PDF 289 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Adachi, T., Weisbrod, R., Pimentel, D. et al. S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide. Nat Med 10, 1200–1207 (2004). https://doi.org/10.1038/nm1119

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm1119

This article is cited by

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