A molecular switch of “yin and yang”: s-glutathionylation of enos turns off no synthesis and turns on superoxide generation

Nitric oxide (NO) is a short-lived free radical produced endogenously in biological tissues by nitric oxide synthases (NOSs)1, 2. Three NOS isoforms, namely NOS1 or neuronal NOS (nNOS), NOS2

or inducible NOS (iNOS), and NOS3 or endothelial NOS (eNOS) are present in most cell types, including cardiac myocytes and vascular endothelial cells. Vascular relaxation to mediators such

as acetylcholine or increased blood flow depends on NO produced by the eNOS. The discovery of NO as the endothelium-derived relaxing factor (EDRF) and its crucial function as a signaling

molecule in cardiovascular system was awarded the Nobel Prize in Physiology or Medicine in 19981. Under normal circumstances the main function of eNOS is to produce NO. It catalyzes the

conversion of _L_-arginine (_L_-Arg) to _L_-citrulline and NO via electron transfer from the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) through a flavin containing

reductase domain to oxygen bound at the heme of an oxygenase domain containing tetrahydrobiopterin (BH4) and _L_-Arg binding sites (Figure 1). The eNOS-derived NO activates the guanylate

cyclase/cGMP/protein kinase G (PKG) pathway and modulates protein properties and function through nitrosylation of tyrosine and thiol-groups of cysteine in proteins, which are usually

effective protection mechanisms against oxidative stress. Under pathophysiological conditions such as hypertension, diabetes, septic shock and atherosclerosis, oxidative stress alters many

functions of the endothelium and leads to endothelial dysfunction when the endothelium fails to serve its normal physiologic and protective mechanisms. A common feature of endothelial

dysfunction is the reduced bioavailability of NO and increased production of superoxide (·O2−) and other reactive oxygen species (ROS) in the vasculature3. Multiple mechanisms may underlie

the impaired NO availability4. These include a reduction in the expression level of eNOS mRNA or protein, changes in subcellular compartmentalization of eNOS activity, and compromised

availability of the substrates and/or enzymatic cofactors for eNOS5, 6. Depletion of the substrate _L_-arginine, accumulation of methylarginines, and oxidation of the cofactor BH4 of eNOS

can uncouple the electron transfer reactions and revert eNOS to function as an NADPH oxidase, thus producing ·O2− instead of NO (Figure 1). The rapid reaction of NO with ·O2− can form the

most potent oxidant peroxynitrite anion (OONO−) and causes cellular injury associated with many pathophysiologic conditions, such as hypertension, atherosclerosis, diabetes, myocardial

hypertrophy, heart failure, and ischemia/reperfusion injury7. The precise molecular mechanisms underlying the “switch” of the eNOS function from NO synthesis to ·O2− production under

oxidative stress conditions, however, are still not fully understood. Recently, Chen _et al_ reported that _S_-glutathionylation of eNOS may be a unique mechanism for the redox regulation of

eNOS8. It has been demonstrated previously that cysteine residues are critical for the maintenance of normal eNOS function9. Protein _S_-glutathionylation has been known as a specific

post-translational modification of protein cysteine residues by adding the tripeptide glutathione through reversible thiol-disulfide exchange with oxidized glutathione (GSSG) or reaction of

oxidant-induced protein thiyl radicals with reduced glutathione (GSH). Under oxidative stress, therefore, protein _S_-glutathionylation can serve to prevent irreversible oxidation of protein

thiols10. _S_-glutathionylation has now emerged as a potential mechanism for dynamic, post-translational regulation of a variety of regulatory, structural, and metabolic proteins.

Increasing lines of evidence point to the important role of S-glutathionylation in the regulation of signaling and metabolic pathways in intact cellular systems. Indeed, Chen _et al_ found

that GSSG induced dose-dependent _S_-glutathionylation of human eNOS that was reversed by reducing agents β-mercaptoethanol or dithiothreitol8. _S_-glutathionylation of eNOS reversibly

decreases NOS activity with a concomitant increase in ·O2− generation primarily from the reductase domain. Two highly conserved cysteine residuals are identified as sites of

_S_-glutathionylation and found to be critical for redox-regulation of eNOS function. They further demonstrated that _S_-glutathionylation of eNOS in endothelial cells turned off NO

synthesis and turned on ·O2− generation (Figure 1). This conversion of eNOS function by _S_-glutathionylation is closely associated with impaired endothelium-dependent vasodilatation. In

hypertensive vessels, _S_-glutathionylation of eNOS is increased with impaired endothelium-dependent vasodilatation. Thio-specific reducing agents that reverse the _S_-glutathionylation of

eNOS are able to restore the endothelium-dependent vasodilatation of the hypertensive vessels. Thus, _S_-glutathionylation of eNOS may represent a novel and pivotal molecular switch

providing redox regulation of cellular signaling and endothelial function. Control of this molecular switch will perhaps provide new therapeutic targets and specific strategy to correct

endothelial dysfunction under pathophysiological conditions. Importantly, it may also shed new mechanistic light on the action of some antihypertensive herbal extracts that elicit potent

antioxidant properties and relax blood vessels in an endothelium-dependent and NO-mediated manner. It can be speculated that herbs with varying degrees of redox potential may exert

different, or even opposite, vascular effects by flipping the switch to preferentially cause the formation of NO or ·O2 11, thus lending scientific support for the Yin-Yang temperaments

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Laboratory of Cardiovascular Phenomics, Center of Biomedical Research Excellence, University of Nevada School of Medicine, Reno, 89557, Nevada, USA Dayue Darrel Duan * Vascular Biology

Research Group, Research Institute of Basic Medical Sciences and Center for Faculty Development, China Medical University, Taichung, Taiwan, China Chiu-yin Kwan Authors * Dayue Darrel Duan

View author publications You can also search for this author inPubMed Google Scholar * Chiu-yin Kwan View author publications You can also search for this author inPubMed Google Scholar

CORRESPONDING AUTHOR Correspondence to Dayue Darrel Duan. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Duan, D., Kwan, Cy. A molecular switch of “Yin

and Yang”: _S_-glutathionylation of eNOS turns off NO synthesis and turns on superoxide generation. _Acta Pharmacol Sin_ 32, 415–416 (2011). https://doi.org/10.1038/aps.2011.21 Download

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