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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−.

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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.

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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.).

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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

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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)

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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

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