title: S-glutathionylation uncouples eNOS and regulates its cellular and vascular function.
abstract: Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O(2)(•-)), which are key mediators of cellular signalling. In the presence of Ca(2+)/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 (BH(4)) and l-Arg. In the absence of BH(4), NO synthesis is abrogated and instead O(2)(•-) is generated. While NOS dysfunction occurs in diseases with redox stress, BH(4) 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 O(2)(•-) 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 O(2)(•-) 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.
authors.affiliation: Davis Heart and Lung Research Institute and Division of Cardiovascular Medicine, Department of Internal Medicine, College of Medicine, Ohio State University, Columbus, Ohio 43210, USA.
authors.forename: Chun-An
authors.initials: CA
authors.lastname: Chen
authors.forename: Tse-Yao
authors.initials: TY
authors.lastname: Wang
authors.forename: Saradhadevi
authors.initials: S
authors.lastname: Varadharaj
authors.forename: Levy A
authors.initials: LA
authors.lastname: Reyes
authors.forename: Craig
authors.initials: C
authors.lastname: Hemann
authors.forename: M A Hassan
authors.initials: MA
authors.lastname: Talukder
authors.forename: Yeong-Renn
authors.initials: YR
authors.lastname: Chen
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Druhan
authors.forename: Jay L
authors.initials: JL
authors.lastname: Zweier
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004229:Dithiothreitol / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D008297:Male; D008623:Mercaptoethanol / Q000494:pharmacology; D009154:Mutation; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D017207:Rats, Sprague-Dawley; D019163:Reducing Agents / Q000494:pharmacology; D015398:Signal Transduction; D014664:Vasodilation / Q000502:physiology
chemical_list: D019163:Reducing Agents; D008623:Mercaptoethanol; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D005978:Glutathione; D004229:Dithiothreitol