eNOS S-glutathionylation Research Articles

Ambient ultrafine particles reduce endothelial nitric oxide production via S-glutathionylation of eNOS

Exposure to airborne particulate pollutants is intimately linked to vascular oxidative stress and inflammatory responses with clinical relevance to atherosclerosis. Particulate matter (PM) has been reported to induce endothelial dysfunction and atherosclerosis. Here, we tested whether ambient ultrafine particles (UFP, diameter <200nm) modulate eNOS activity in terms of nitric oxide (NO) production via protein S-glutathionylation. Treatment of human aortic endothelial cells (HAEC) with UFP significantly reduced NO production. UFP-mediated reduction in NO production was restored in the presence of JNK inhibitor (SP600125), NADPH oxidase inhibitor (Apocynin), anti-oxidant (N-acetyl cysteine), and superoxide dismutase mimetics (Tempol and MnTMPyP). UFP exposure increased the GSSG/GSH ratio and eNOS S-glutathionylation, whereas over-expression of Glutaredoxin-1 (to inhibit S-glutathionylation) restored UFP-mediated reduction in NO production by nearly 80%. Thus, our findings suggest that eNOS S-glutathionylation is a potential mechanism underlying ambient UFP-induced reduction of NO production.

Endothelial nitric oxide synthase S‐glutathionylation and Ser1177 phosphorylation modulate myocardial protection

Endothelial nitric oxide synthase (eNOS) plays an important role in cardiovascular function, myocardial injury and protection. The role of eNOS is well recognized in ischemic preconditioning (IPC); however, questions remain regarding how eNOS is regulated during ischemia‐reperfusion (I/R) and IPC. eNOS phosphorylation at Ser1177 enhances enzyme activation and nitric oxide (NO) production; however, S‐glutathionylation of eNOS uncouples the enzyme leading to loss of NO production and vascular dysfunction. We measure myocardial eNOS S‐glutathionylation and phosphorylation in the process of I/R injury and investigate the role of these modifications in cardioprotection. Isolated rat hearts were subjected to 30‐min of global ischemia and/or reperfusion with or without IPC stimuli. Ischemia induced a 4–5 fold increase in eNOS S‐glutathionylation that was completely reversed by â‐mercaptoethanol. Importantly, IPC reduced I/R‐induced eNOS S‐glutathionylation by 50%, decreased myocardial superoxide generation by &gt;;80% and attenuated myocardial infarction by 75%. Levels of eNOS phosphorylation at Ser1177 decreased with I/R but were increased by IPC. Thus, IPC stimuli inhibit I/R‐induced eNOS S‐glutathionylation but increase Ser1177 phosphorylation to maintain eNOS coupling, activation and NO production that protect myocardium against I/R injury. (Support: NIH grants HL63744, HL65608 and HL38324).

Integrated Redox Sensor and Effector Functions for Tetrahydrobiopterin- and Glutathionylation-dependent Endothelial Nitric-oxide Synthase Uncoupling

Endothelial nitric-oxide synthase (eNOS) is a critical regulator of vascular homeostasis by generation of NO that is dependent on the cofactor tetrahydrobiopterin (BH4). When BH4 availability is limiting, eNOS becomes "uncoupled," resulting in superoxide production in place of NO. Recent evidence suggests that eNOS uncoupling can also be induced by S-glutathionylation, although the functional relationships between BH4 and S-glutathionylation remain unknown. To address a possible role for BH4 in S-glutathionylation-induced eNOS uncoupling, we expressed either WT or mutant eNOS rendered resistant to S-glutathionylation in cells with Tet-regulated expression of human GTP cyclohydrolase I to regulate intracellular BH4 availability. We reveal that S-glutathionylation of eNOS, by exposure to either 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or glutathione reductase-specific siRNA, results in diminished NO production and elevated eNOS-derived superoxide production, along with a concomitant reduction in BH4 levels and BH4:7,8-dihydrobiopterin ratio. In eNOS uncoupling induced by BH4 deficiency, BCNU exposure further exacerbates superoxide production, BH4 oxidation, and eNOS activity. Following mutation of C908S, BCNU-induced eNOS uncoupling and BH4 oxidation are abolished, whereas uncoupling induced by BH4 deficiency was preserved. Furthermore, BH4 deficiency alone is alone sufficient to reduce intracellular GSH:GSSG ratio and cause eNOS S-glutathionylation. These data provide the first evidence that BH4 deficiency- and S-glutathionylation-induced mechanisms of eNOS uncoupling, although mechanistically distinct, are functionally related. We propose that uncoupling of eNOS by S-glutathionylation- or by BH4-dependent mechanisms exemplifies eNOS as an integrated redox "hub" linking upstream redox-sensitive effects of BH4 and glutathione with redox-dependent targets and pathways that lie downstream of eNOS.

Regulation of Endothelial Nitric-oxide Synthase (NOS) S-Glutathionylation by Neuronal NOS: EVIDENCE OF A FUNCTIONAL INTERACTION BETWEEN MYOCARDIAL CONSTITUTIVE NOS ISOFORMS

Myocardial constitutive No production depends on the activity of both endothelial and neuronal NOS (eNOS and nNOS, respectively). Stimulation of myocardial β(3)-adrenergic receptor (β(3)-AR) produces a negative inotropic effect that is dependent on eNOS. We evaluated whether nNOS also plays a role in β(3)-AR signaling and found that the β(3)-AR-mediated reduction in cell shortening and [Ca(2+)](i) transient amplitude was abolished both in eNOS(-/-) and nNOS(-/-) left ventricular (LV) myocytes and in wild type LV myocytes after nNOS inhibition with S-methyl-L-thiocitrulline. LV superoxide (O(2)(·-)) production was increased in nNOS(-/-) mice and reduced by L-N(ω)-nitroarginine methyl ester (L-NAME), indicating uncoupling of eNOS activity. eNOS S-glutathionylation and Ser-1177 phosphorylation were significantly increased in nNOS(-/-) myocytes, whereas myocardial tetrahydrobiopterin, eNOS Thr-495 phosphorylation, and arginase activity did not differ between genotypes. Although inhibitors of xanthine oxidoreductase (XOR) or NOX2 NADPH oxidase caused a similar reduction in myocardial O(2)(·-), only XOR inhibition reduced eNOS S-glutathionylation and Ser-1177 phosphorylation and restored both eNOS coupled activity and the negative inotropic and [Ca(2+)](i) transient response to β(3)-AR stimulation in nNOS(-/-) mice. In summary, our data show that increased O(2)(·-) production by XOR selectively uncouples eNOS activity and abolishes the negative inotropic effect of β(3)-AR stimulation in nNOS(-/-) myocytes. These findings provide unequivocal evidence of a functional interaction between the myocardial constitutive NOS isoforms and indicate that aspects of the myocardial phenotype of nNOS(-/-) mice result from disruption of eNOS signaling.

Abstract 19796: Vascular eNOS S-Glutathionylation is Increased in Hypertension and Atherosclerosis with eNOS uncoupling: Data from Animal Models and Human Aortic Tissue of Patients Undergoing Surgical Revascularization

Background: In vascular diseases, including hypertension (HTN) and atherosclerosis, endothelial dysfunction (ED) occurs secondary to altered function of endothelial NO synthase (eNOS); however, questions remain regarding the mechanism by which this occurs and how it can be reversed. We recently identified a novel redox regulated pathway through which eNOS is uncoupled due to S-glutathionylation. In vessels from spontaneous hypertensive rats (SHR) and in aortic tissue from patients undergoing coronary bypass (CABG) surgery, we measure eNOS S-glutathionylation and its effect on eNOS coupling. Methods: Studies were performed in aortic, femoral and carotid vessels from adult SHR rats and in aortic tissue from patients undergoing CABG or other vascular surgery. S-glutathionylation of eNOS was studied using anti-glutathione antibody, anti-eNOS antibody and an antibody specific for eNOS C689 S-glutathionylation. NO and superoxide (•O2--) was measured using fluorescent probes CuFL and DHE, respectively, in the absence or presence of the NOS inhibitor, L-NAME, NO scavenger, PTIO, or the SOD mimetic, MnTBAP. Results &amp; Conclusion: Immunohistology and immunoblotting revealed increased eNOS S-glutathionylation in vessels of SHR rats compared to matched WKY controls. The reducing agent DTT reversed this glutathionylation and reversed the impairment in endothelial-dependent relaxation measured from aortic rings. Mass spectroscopy on immunoprecipitated eNOS demonstrated modification at C689 in aorta of SHR but not WKY rats. Immunohistology with a specific eNOS C689 S-glutathionylation antibody confirmed endothelial C689 S-glutathionylation. Decreased NO and increased •O2-- was seen in these vessels. In patients undergoing CABG, high levels of C689 S-glutathionylation was seen abolished by DTT. Thus, eNOS S-glutathionylation is increased in an animal model of HTN with resultant ED and present in patients with coronary disease undergoing bypass surgery.

Abstract 19795: S-glutathionylation of eNOS occurs in Clinical Obstructive Sleep Apnea (OSA)

Obstructive sleep Apnea (OSA) is a risk factor for cardiovascular disease and causes vascular endothelial dysfunction (VED); however, the basis of this VED is unknown. Increased oxidant production is observed in the endothelium of OSA patients. We have recently demonstrated that under oxidant stress eNOS uncoupling is triggered by S-glutathionylation of specific cysteines including C689 of the reductase domain. Since OSA is associated with oxidant stress, we hypothesized that S-glutathionylation of eNOS may occur in OSA and trigger eNOS uncoupling. Methods: 13 patients with recently diagnosed OSA and 10 healthy individuals with low cardiovascular risk were studied. OSA patients underwent subcutaneous forearm biopsy and Doppler measurements of endothelial function by flow mediated dilation (FMD) prior to, and 12 weeks after initiation of treatment. Non-OSA control patients also provided FMD measurements and biopsies. eNOS S-glutathionylation was studied using specific anti-eNOS C689 S-glutathionylation antibody. Superoxide ·( O 2 _ ) levels in the microcirculation were determined using fluorescent probe DHE in the absence or presence of the SOD mimetic, MnTBAP. Results: OSA patients had impaired FMD, compared to non-OSA controls, which decreased after 12 weeks of treatment with CPAP. Immunohistochemical analysis of frozen sections from OSA patients using anti-eNOS C689 glutathionylation antibody revealed that prominent eNOS S-glutathionylation was present in the endothelium of OSA patients that was abolished by treatment with the reducing agent DTT, and was attenuated following CPAP treatment and absent in healthy control subjects. In association with these findings, · O 2 _ production from the endothelium was also decreased with CPAP treatment in OSA patients. Conclusions: These findings demonstrate that eNOS S-glutathionylation occurs in OSA and is associated with endothelial dysfunction with increased superoxide and decreased NO availability.

Tetrahydrobiopterin- and glutathionylation-dependent regulation of eNOS coupling: Relationships revealed by eNOS glutathionylation mutants in cells with tet-regulated GTP cyclohydrolase I expression

Endothelial nitric oxide synthase (eNOS) is critical in the regulation of vascular homeostasis and generates nitric oxide (NO) by a process that is dependent on the binding of the cofactor tetrahydrobiopterin (BH4). When BH4 availability is limiting, electron transfer from NOS flavins becomes ‘uncoupled’ from l -arginine oxidation and superoxide is produced in place of NO. This process of eNOS ‘uncoupling’ is associated with many pathophysiologic conditions including atherosclerosis and hypercholesterolemia, although BH4 repletion only partially restores NOS activity and NOS-dependent vasodilation. Recent evidence suggests that eNOS uncoupling can also be induced by S-glutathionylation, however the exact mechanisms remain unknown. To address a possible role for BH4 in S-glutathionylation-induced eNOS uncoupling, we developed novel cell lines with tet-regulated expression of human GTP cyclohydrolase I (GTPCH; the rate-limiting enzyme in BH4 synthesis) and either WT or mutant eNOS rendered resistant to S-glutathionylation by mutation of two critical cysteine residues (C689S and C908S). We reveal that S-glutathionylation of eNOS by exposure of endothelial cells to 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) or glutathione reductase (GR)-specific siRNA results in diminished NO production and elevated eNOS-derived superoxide production, along with a concomitant reduction in BH4 levels and BH4:BH2 ratio. In our model of BH4-dependent eNOS uncoupling, BCNU exposure further exacerbated superoxide production, BH4 oxidation and eNOS activity, effects that were totally abolished following mutation of either C689S or C908S. These data provide the first evidence that BH4 deficiency- and S-glutathionylation-induced mechanisms of eNOS uncoupling occur independently and their effects are additive.

Chronic therapy with isosorbide-5-mononitrate causes endothelial dysfunction, oxidative stress, and a marked increase in vascular endothelin-1 expression

Isosorbide-5-mononitrate (ISMN) is one of the most frequently used compounds in the treatment of coronary artery disease predominantly in the USA. However, ISMN was reported to induce endothelial dysfunction, which was corrected by vitamin C pointing to a crucial role of reactive oxygen species (ROS) in causing this phenomenon. We sought to elucidate the mechanism how ISMN causes endothelial dysfunction and oxidative stress in vascular tissue. Male Wistar rats (n= 69 in total) were treated with ISMN (75 mg/kg/day) or placebo for 7 days. Endothelin (ET) expression was determined by immunohistochemistry in aortic sections. Isosorbide-5-mononitrate infusion caused significant endothelial dysfunction but no tolerance to ISMN itself, whereas ROS formation and nicotinamide adenine dinucleotidephosphate (NADPH) oxidase activity in the aorta, heart, and whole blood were increased. Isosorbide-5-mononitrate up-regulated the expression of NADPH subunits and caused uncoupling of the endothelial nitric oxide synthase (eNOS) likely due to a down-regulation of the tetrahydrobiopterin-synthesizing enzyme GTP-cyclohydrolase-1 and to S-glutathionylation of eNOS. The adverse effects of ISMN were improved in gp91phox knockout mice and normalized by bosentan in vivo/ex vivo treatment and suppressed by apocynin. In addition, a strong increase in the expression of ET within the endothelial cell layer and the adventitia was observed. Chronic treatment with ISMN causes endothelial dysfunction and oxidative stress, predominantly by an ET-dependent activation of the vascular and phagocytic NADPH oxidase activity and NOS uncoupling. These findings may explain at least in part results from a retrospective analysis indicating increased mortality in post-infarct patients in response to long-term treatment with mononitrates.

Ambient ultrafine particles (UFP) inhibit eNOS activity via S‐glutathionylation

Atherosclerosis is a systemic disease, and its development is dependent upon a complex interplay among numerous genetic, epigenetic, and environmental risk factors. It is increasingly recognized that ambient UFP exposure promotes vascular oxidative stress and inflammatory responses. In this study, we tested the hypothesis that UFP attenuates eNOS activity via S‐glutathionylaiton. Treatment of human aortic endothelial cells (HAEC) with ambient UFP (Dp&lt;100nm) significantly inhibited nitric oxide (NO) production as measured by Griess assay (Control: 3.645, UFP: 1.662, P&lt;.01, n=4). To assess whether oxidative stress induced eNOS uncoupling via S‐glutathionylation, we demonstrated that UFP treatment increased eNOS S‐glutathionylation in HAEC by a modified ELISA procedure. To inhibit S‐glutahionylation, we over‐expressed glutaredoxin‐1 (Grx‐1), and showed a significant attenuation of UFP‐mediated decrease in NO production (LacZ+UFP: .338, Grx1+UFP: .630, P&lt;.01, n=6). Thus, our findings suggest that UFP inhibited eNOS activity via an increase in S‐ glutathionylation.

Chronic Therapy With Isosorbide-5-Mononitrate Causes Endothelial Dysfunction, Oxidative Stress and a Marked Increase in Vascular Endothelin-1 Expression

Aims Isosorbide-5-mononitrate (ISMN) is one of the most frequently used compounds in the treatment of coronary artery disease predominantly in the USA. However, ISMN was reported to induce endothelial dysfunction, which was corrected by vitamin C pointing to a crucial role of reactive oxygen species (ROS) in causing this phenomenon. We sought to elucidate the mechanism how ISMN causes endothelial dysfunction and oxidative stress in vascular tissue. Methods and results Male Wistar rats ( n = 69 in total) were treated with ISMN (75 mg/kg/day) or placebo for 7 days. Endothelin (ET) expression was determined by immunohistochemistry in aortic sections. Isosorbide-5-mononitrate infusion caused significant endothelial dysfunction but no tolerance to ISMN itself, whereas ROS formation and nicotinamide adenine dinucleotidephosphate (NADPH) oxidase activity in the aorta, heart, and whole blood were increased. Isosorbide-5-mononitrate up-regulated the expression of NADPH subunits and caused uncoupling of the endothelial nitric oxide synthase (eNOS) likely due to a down-regulation of the tetrahydrobiopterin-synthesizing enzyme GTP-cyclohydrolase-1 and to S-glutathionylation of eNOS. The adverse effects of ISMN were improved in gp91phox knockout mice and normalized by bosentan in vivo / ex vivo treatment and suppressed by apocynin. In addition, a strong increase in the expression of ET within the endothelial cell layer and the adventitia was observed. Conclusion Chronic treatment with ISMN causes endothelial dysfunction and oxidative stress, predominantly by an ET-dependent activation of the vascular and phagocytic NADPH oxidase activity and NOS uncoupling. These findings may explain at least in part results from a retrospective analysis indicating increased mortality in post-infarct patients in response to long-term treatment with mononitrates.

Vascular Dysfunction in Experimental Diabetes Is Improved by Pentaerithrityl Tetranitrate but Not Isosorbide-5-Mononitrate Therapy

OBJECTIVEDiabetes is associated with vascular oxidative stress, activation of NADPH oxidase, and uncoupling of nitric oxide (NO) synthase (endothelial NO synthase [eNOS]). Pentaerithrityl tetranitrate (PETN) is an organic nitrate with potent antioxidant properties via induction of heme oxygenase-1 (HO-1). We tested whether treatment with PETN improves vascular dysfunction in the setting of experimental diabetes.RESEARCH DESIGN AND METHODSAfter induction of hyperglycemia by streptozotocin (STZ) injection (60 mg/kg i.v.), PETN (15 mg/kg/day p.o.) or isosorbide-5-mononitrate (ISMN; 75 mg/kg/day p.o.) was fed to Wistar rats for 7 weeks. Oxidative stress was assessed by optical methods and oxidative protein modifications, vascular function was determined by isometric tension recordings, protein expression was measured by Western blotting, RNA expression was assessed by quantitative RT-PCR, and HO-1 promoter activity in stable transfected cells was determined by luciferase assays.RESULTSPETN, but not ISMN, improved endothelial dysfunction. NADPH oxidase and serum xanthine oxidase activities were significantly reduced by PETN but not by ISMN. Both organic nitrates had minor effects on the expression of NADPH oxidase subunits, eNOS and dihydrofolate reductase (Western blotting). PETN, but not ISMN, normalized the expression of GTP cyclohydrolase-1, extracellular superoxide dismutase, and S-glutathionylation of eNOS, thereby preventing eNOS uncoupling. The expression of the antioxidant enzyme, HO-1, was increased by STZ treatment and further upregulated by PETN, but not ISMN, via activation of the transcription factor NRF2.CONCLUSIONSIn contrast to ISMN, the organic nitrate, PETN, improves endothelial dysfunction in diabetes by preventing eNOS uncoupling and NADPH oxidase activation, thereby reducing oxidative stress. Thus, PETN therapy may be suited to treat patients with cardiovascular complications of diabetes.

A molecular switch of “Yin and Yang”: S-glutathionylation of eNOS turns off NO synthesis and turns on superoxide generation

A molecular switch of “Yin and Yang”: S -glutathionylation of eNOS turns off NO synthesis and turns on superoxide generation

Targeting Dimethylarginine Dimethylaminohydrolases in Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is a devastating and life-threatening clinical syndrome characterized by elevated pulmonary artery pressures leading to progressive symptoms, including shortness of breath, fatigue, and a decline in functional ability. The hemodynamic definition of PAH is a mean pulmonary artery pressure of >25 mm Hg at rest or >30 mm Hg during exercise, with a normal pulmonary arterial wedge pressure ≤15 mm Hg.1 Recent data from a French registry suggests that the prevalence of PAH is about 15 cases per 1 million,1 and the Registry to Evaluate Early and Long-Term Pulmonary Arterial Hypertension Disease Management (REVEAL) demonstrates that the mean age at diagnosis is 48 years and that mostly women (80%) are affected.2 The pathophysiology of PAH includes endothelial dysfunction, vascular remodeling with progressive obstruction and obliteration of pulmonary arteries, and ultimately, high right atrial pressure, right ventricular hypertrophy, right ventricular failure, and death.1 Endothelial dysfunction is believed to be an early event in PAH, and is characterized by overproduction of vasoconstrictor/mitogenic compounds, such as endothelin and thromboxane A2, and by insufficient production of vasodilators, such as prostacyclin and nitric oxide (NO). These observations led to the development of 3 different classes of therapies that are currently in clinics, either alone or in combination: prostacyclin analogs, endothelin receptor antagonists, and phosphodiesterase (PDE) inhibitors. Although current therapies can improve symptoms and reduce severity of the hemodynamic disorders, gradual deterioration of pulmonary and cardiac functions often necessitates lung transplantation. The prognosis of PAH is reportedly poor, with ≈15% mortality within 1 year of modern therapy.1 Therefore, a renewed interest has been focused on the mechanisms of PAH pathogenesis to identify a novel therapeutic target. Article see p 1194 It is now well established that disruption or dysfunction of the endothelium promotes vascular lesion formation. This …

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 (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.