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

Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular function, and can generate both nitric oxide (NO) and superoxide (O₂^-2), which are key mediators of cellular signalling. In the presence of Ca²⁺/calmodulin, eNOS produces NO, endothelialderived 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^1-3. In the absence of BH₄, NO synthesis is abrogated and instead O₂^-2 is generated^4-7. While NOS dysfunction occurs in diseases with redox stress,BH₄ repletion only partly restoresNOS 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 adaptation^8,9. Under oxidative stress, S-glutathionylation occurs through thiol-disulphide exchange with oxidized glutathione or reaction of oxidant-induced protein thiyl radicals with reduced glutathione^10,11. Cysteine residues are critical for the maintenance of eNOS function^12,13; 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^-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 Sglutathionylation in endothelial cells, with loss of NO and gain of O₂^-2generation, is associated withimpaired 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.