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-Arg1–3. In the absence of BH4, NO synthesis is abrogated and instead O2•− is generated4–7. 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.

Mitochondrial ROS have emerged as an important mechanism of disease and redox signaling in the cardiovascular system. Under basal or pathological conditions, electron leakage for ROS production is primarily mediated by the electron transport chain and proton motive force consisting of a membrane potential (ΔΨ) and a proton gradient (ΔpH). Several factors controlling ROS production in mitochondria include FMN and the FMN-binding domain of complex I, ubisemiquinone and quinone-binding domain of complex I, FAD binding moiety and quinone-binding pocket (Qp) of complex II, and unstable semiquinone •Qo− mediated by the Q cycle of complex III. In mitochondrial complex I, specific cysteinyl redox domains modulate ROS production from the FMN moiety and iron sulfur clusters. In the cardiovascular system, mitochondrial ROS have been linked to mediating physiological effects of metabolic dilation and preconditioning-like mKATP channel activation. Furthermore, oxidative post-translational modification by glutathione in complex I and complex II has been shown to affect enzymatic catalysis, protein-protein interactions, and enzyme-mediated ROS production. Conditions associated with oxidative or nitrosative stress, such as myocardial ischemia and reperfusion, increase mitochondrial ROS production via oxidative injury of complexes I and II, and •O2−-induced hydroxyl radical production by aconitase. Further insight into cellular mechanisms by which specific redox post-translational modifications regulate ROS production in mitochondria will enrich our understanding of redox signal transduction and identify new therapeutic targets for cardiovascular diseases in which oxidative stress perturbs normal redox signaling.

Reactive oxygen species and reactive nitrogen species are biological molecules that play important roles in cardiovascular physiology and contribute to disease initiation, progression, and severity. Because of their ephemeral nature and rapid reactivity, these species are difficult to measure directly with high accuracy and precision. In this statement, we review current methods for measuring these species and the secondary products they generate and suggest approaches for measuring redox status, oxidative stress, and the production of individual reactive oxygen and nitrogen species. We discuss the strengths and limitations of different methods and the relative specificity and suitability of these methods for measuring the concentrations of reactive oxygen and reactive nitrogen species in cells, tissues, and biological fluids. We provide specific guidelines, through expert opinion, for choosing reliable and reproducible assays for different experimental and clinical situations. These guidelines are intended to help investigators and clinical researchers avoid experimental error and ensure high-quality measurements of these important biological species.

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. With oxidative stress, the critical cofactor BH 4 is depleted, and NADPH oxidation is uncoupled from NO generation, leading to production of (O 2 . . generation from the enzyme at low Ca 2؉ concentrations, and PKC␣-mediated phosphorylation alters the sensitivity of the enzyme to other negative regulatory signals. Nitric-oxide synthase (NOS)2 is a critical enzyme that converts L-arginine (L-Arg) to L-citrulline and nitric oxide (NO) with the consumption of NADPH. NO is a signaling molecule that promotes vascular smooth muscle relaxation and functions as an endogenous mediator of a wide range of effects in different tissues (1, 2). After oxidant stress, as occurs in postischemic tissues, production of O 2. and its derived oxidants, including peroxynitrite (ONOO Ϫ ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radical (⅐OH), induce NOS dysfunction with uncoupling of the enzyme leading to the production of NOSderived O 2. instead of NO (3, 4). It has been reported that an imbalance between NO and O 2 . can contribute to the onset of a variety of cardiovascular diseases, including hypertension, atherosclerosis, and heart failure (5). Therefore, tight coupling of the enzyme is important for normal cardiovascular function and prevention of disease. The catalytic domains of NOS include a flavin-containing NADPH binding reductase and a heme-binding oxygenase that also contains the binding sites for the redox labile cofactor tetrahydrobiopterin (BH 4 ) and the substrate L-Arg. In the presence of Ca 2ϩ and calmodulin (CaM), electrons flow from NADPH through the reductase domain to the oxygenase domain resulting in the activation of oxygen at the heme center followed by substrate monooxygenation. This process requires the presence of the fully reduced BH 4 . Our laboratory and several others have demonstrated that besides synthesizing NO, all three isoforms of NOS can also generate O 2 . , depending on substrate and cofactor availability (3, 6 -9). One of the primary mechanisms implicated in the oxidant-induced switch of NOS from the production of NO to the generation of O 2 . is the oxidation of the enzyme bound BH 4 (10, 11). Various extracellular signals, including shear stress and additional stimuli such as vascular endothelial growth factor (VEGF), estrogen, sphingosine 1-phosphate, bradykinin, and aldosterone, modulate eNOS NO generation through several signal transduction pathways (12)(13)(14)(15)(16). Cellular studies have demonstrated that phosphorylation of eNOS at specific amino acids regulates enzyme-mediated NO production (17). The majority of previous work has focused on two residues, serine 1177 and threonine 495. It has been shown that Akt specifically induces phosphorylation of Ser-1177 (18, 19) and that PKC specifically phosphorylates . Although phosphorylation of Ser-1177 has been shown to increase NO production * This work was supported, in whole or in part, by National Institu...

Background-Nitric oxide (NO) production is increased in postischemic myocardium, and NO can control mitochondrial oxygen consumption in vitro. Therefore, we investigated the role of endothelial NO synthase (eNOS)-derived NO on in vivo regulation of oxygen consumption in the postischemic heart. Methods and Results-Mice were subjected to 30 minutes of coronary ligation followed by 60 minutes of reperfusion.Myocardial oxygen tension (PO 2 ) was monitored by electron paramagnetic resonance oximetry. In wild-type, N-nitro-L-arginine methyl ester (L-NAME)-treated (with 1 mg/mL in drinking water), and eNOS knockout (eNOS Ϫ/Ϫ ) mice, no difference was observed among baseline myocardial PO 2 values (8.6Ϯ0.7, 10.0Ϯ1.2, and 10.1Ϯ1.2 mm Hg, respectively) or those measured at 30 minutes of ischemia (1.4Ϯ0.6, 2.3Ϯ0.9, and 3.1Ϯ1.4 mm Hg, respectively). After reperfusion, myocardial PO 2 increased markedly (PϽ0.001 versus baseline in each group) but was much lower in L-NAME-treated and eNOS Ϫ/Ϫ mice (17.4Ϯ1.6 and 20.4Ϯ1.9 mm Hg) than in wild-type mice (46.5Ϯ1.7 mm Hg; PϽ0.001). A transient peak of myocardial PO 2 was observed at early reperfusion in wild-type mice. No reactive hyperemia was observed during early reperfusion. Endothelial NO decreased the rate-pressure product (PϽ0.05), upregulated cytochrome c oxidase (CcO) mRNA expression (PϽ0.01) with no change in CcO activity, and inhibited NADH dehydrogenase (NADH-DH) activity (PϽ0.01) without alteration of NADH-DH mRNA expression. Peroxynitrite-mediated tyrosine nitration was higher in hearts from wild-type mice than in eNOS Ϫ/Ϫ or L-NAME-treated hearts. Conclusions-eNOS-derived

Mitochondria are the major cellular source of oxygen free radicals (1, 2). The generation of reactive oxygen species (ROS) 2 and free radical(s) in mitochondria is particularly relevant under the physiological conditions of low oxygen tension such as state 4 respiration or certain pathological conditions such as inflammation and ischemia-reperfusion injury (3-5). To understand disease processes associated with oxidative stress, it is necessary to understand the fundamental mechanisms of mitochondrial-derived oxygen-free radical generation. A decrease in the rate of mitochondrial oxidative phosphorylation can increase the production of superoxide anion radical (O 2 . ) in the early stages of the electron transport chain (ETC). Two segments of the ETC have been widely hypothesized to be responsible for O 2 . generation in mitochondria (6). One site is located on complex III. At this site, O 2 .production is mediated through the Q-cycle mechanism, in which electron leakage results from the auto-oxidation of ubisemiquinone and reduced cytochrome b 566 (7-10). The other site is located on complex I (11). Purified bovine heart complex I (or NADH ubiquinone reductase) contains up to 46 different subunits with a total molecular mass of 980 kDa (12). With the use of chaotropic anions such as perchlorate, complex I can be resolved into three fractions: a flavoprotein fraction (Fp), an iron-sulfur (Fe-S) protein fraction (Ip), and a hydrophobic protein fraction (Hp). The redox centers of complex I that are involved in mediation of two-electron transfer from NADH to ubiquinone include a non-covalent binding FMN, and ubiquinone (14,15).In addition to the function of electron transfer required for energy transduction in mitochondria, complex I is also responsible for O 2 . generation, which is frequently linked to disease pathophysiology (16). The oxidative damage of complex I has been identified in a number of diseased conditions such as ischemia-reperfusion injury (17)(18)(19) and Parkinson's disease (20,21). Two redox components of complex I are logically hypothesized to be involved in the electron leakage for O 2. generation: one located on the FMN-containing Fp fraction, and one on the Q-binding site that mediates ubiquinone reduction. Based on this hypothesis, reduced FMN semiquinone radical (FMNH ⅐ ) (22) and ubisemiquinone radical (Q ៛ ) can provide the sources of O 2 . generation.The Fp fraction of complex I contains the enzymatic activity of NADH dehydrogenase (NDH), and can be isolated as a three subunit subcomplex from submitochondrial particles (SMP) (13). Therefore, the Fp-containing three subunit subcomplex is normally termed as NDH. Of the three subunits, the 51-kDa subunit is an NADH-binding polypeptide hosting one FMN and one [4Fe-4S] iron-sulfur center (N3 center) with an E m,7 ϳ Ϫ250 mV. The 24-kDa subunit hosts one [2Fe-2S] iron-sulfur center (N1a) with an E m,7 ϳ Ϫ370 mV (14, 23). The 9-kDa subunit does not contain any cofactor. NDH retains the same kinetic parameters for NADH dehydrogenation, NADH bin...

. . Thus, the decreasing S-glutathionylation and ETA in mitochondrial complex II are marked during myocardial ischemia/reperfusion. This redox-triggered impairment of complex II occurs in the post-ischemic heart and should be useful to identify disease pathogenesis related to reactive oxygen species-induced mitochondrial dysfunction.

An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.