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

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

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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.

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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.

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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.

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  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

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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.

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Correspondence to Jay L. Zweier.

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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

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