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

• Published in: Nature (London) Vol. 468; no. 7327; pp. 1115 - 1118
• Main Authors: Zweier, Jay L, Chen, Chun-An, Wang, Tse-Yao, Varadharaj, Saradhadevi, Reyes, Levy A, Hemann, Craig, Talukder, M. A. Hassan, Chen, Yeong-Renn, Druhan, Lawrence J
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 23.12.2010; Nature Publishing Group
• Subjects: 631/443/1338/1872; 631/80/86; 692/699/75/593; Animals; Biological and medical sciences; Blood vessels and receptors; Cattle; Cell physiology; Cells, Cultured; Cellular biology; Dithiothreitol - pharmacology; Endothelial Cells - metabolism; Endothelium, Vascular - metabolism; Enzymes; Fundamental and applied biological sciences. Psychology; Glutathione - metabolism; Humanities and Social Sciences; Humans; letter; Male; Mercaptoethanol - pharmacology; Miscellaneous; Molecular and cellular biology; multidisciplinary; Mutation; Nitric oxide; Nitric Oxide Synthase Type III - genetics; Nitric Oxide Synthase Type III - metabolism; Oxidation-Reduction; Oxidative stress; Oxidizing agents; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Reducing Agents - pharmacology; Science; Science (multidisciplinary); Signal Transduction; Vasodilation - physiology; Vertebrates: cardiovascular system; Oxidative stress; Regulation(control); Cell function; Oxidoreductases; Enzyme; Nitric-oxide synthase
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature09599

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-Arg. In the absence of BH4, NO synthesis is abrogated and instead O2*− is generated. While NOS dysfunction occurs in diseases with redox stress, BH4 repletion only partly restores NOS activity and NOS-dependent vasodilation. 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 adaptation. Under oxidative stress, S-glutathionylation occurs through thiol-disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione. Cysteine residues are critical for the maintenance of eNOS function; 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.