Glutathionylation Mediates Angiotensin II–Induced eNOS Uncoupling, Amplifying NADPH Oxidase‐Dependent Endothelial Dysfunction

Abstract

BackgroundGlutathionylation of endothelial nitric oxide synthase (eNOS) “uncouples” the enzyme, switching its function from nitric oxide (NO) to O2•− generation. We examined whether this reversible redox modification plays a role in angiotensin II (Ang II)‐induced endothelial dysfunction.Methods and ResultsAng II increased eNOS glutathionylation in cultured human umbilical vein endothelial cells (HUVECs), rabbit aorta, and human arteries in vitro. This was associated with decreased NO bioavailability and eNOS activity as well as increased O2•− generation. Ang II‐induced decrease in eNOS activity was mediated by glutathionylation, as shown by restoration of function by glutaredoxin‐1. Moreover, Ang II‐induced increase in O2•− and decrease in NO were abolished in HUVECs transiently transfected, with mutant eNOS rendered resistant to glutathionylation. Ang II effects were nicotinamide adenine dinucleotide phosphate (NADPH) oxidase dependent because preincubation with gp 91ds‐tat, an inhibitor of NADPH oxidase, abolished the increase in eNOS glutathionylation and loss of eNOS activity. Functional significance of glutathionylation in intact vessels was supported by Ang II‐induced impairment of endothelium‐dependent vasorelaxation that was abolished by the disulfide reducing agent, dithiothreitol. Furthermore, attenuation of Ang II signaling in vivo by administration of an angiotensin converting enzyme (ACE) inhibitor reduced eNOS glutathionylation, increased NO, diminished O2•−, improved endothelium‐dependent vasorelaxation and reduced blood pressure.ConclusionsUncoupling of eNOS by glutathionylation is a key mediator of Ang II‐induced endothelial dysfunction, and its reversal is a mechanism for cardiovascular protection by ACE inhibition. We suggest that Ang II‐induced O2•− generation in endothelial cells, although dependent on NADPH oxidase, is amplified by glutathionylation‐dependent eNOS uncoupling.

β3 Adrenergic Stimulation Restores Nitric Oxide/Redox Balance and Enhances Endothelial Function in Hyperglycemia

“…Although these effects are shown in relatively short‐term hyperglycemia, our recent report of protective effects of β 3 AR agonism in the heart in hyperglycemic state,25, 33 the close similarities of β 3 AR‐induced redox effects with angiotensin‐converting enzyme inhibitors,7 and presence of the β 3 AR‐mediated effects in vascular tissue of diabetic patients we show here point to potential therapeutic benefit of β 3 AR agonists for cardiovascular protection in diabetes.…”

“…Glutathionylation of eNOS has been reported in STZ‐induced diabetes 49. Our recent report of eNOS glutathionylation as a critical switch in AngII‐induced endothelial dysfunction7 suggested a mechanistic rationale for renin–angiotensin system–mediated diabetes‐induced complications 17. The current study shows the central role of this oxidative modification of eNOS in the pathophysiology of vascular dysfunction, its cellular regulatory mechanisms, a novel therapy that modulates it in experimental hyperglycemia, and provides supportive evidence for potential significance of this pathway in human diabetes.…”

“…In contrast to nonspecific antioxidants, disruption of the renin–angiotensin system in diabetes by angiotensin‐converting enzyme inhibitors or angiotensin receptor blockers protects against the diabetes‐induced vascular dysfunction 2. Angiotensin II (Ang II) receptors are co‐localized with NADPH oxidase and eNOS,5 and inhibition of Ang II receptor–coupled signaling re‐establishes NO/redox balance by decreasing NADPH oxidase–mediated ROS production2 and by restoring eNOS function 6, 7…”

Endothelial Monocyteactivating Factor Ii Cancels Oxidative Stress, Сonstitutive Nos Uncoupling and Induced Violations of Cardiac Hemodynamics in Hypertension (Part Ii)

“…Щодо оксидативного стресу, превалює дум-ка, що ключовим ініціатором реверсного (reversible) неспряження еNOS і утворення нею в цьому стані • О 2 -, який відіграє не лише токсичну, але і регуляторну роль у серцево-судинній системі, є активована ангіотен зи ном ІІ НАДФН-оксидаза. Генерований нею • О 2 -окис нює глутатіон, а вже останній здійснює ре верс не глутаніонування димерної молекули еNOS за залишками цистеїну [11,12]. Отже, фізіологічна роль механізму неспряження (uncoupling) cNOS (як еNOS, так і/чи nNOS) на наш погляд, полягає в тому, що цей процес -один із механізмів адаптації органів серцево-судинної системи, а саме механізм швидкого реагування на потребу підвищення генерації • О 2 -задля регуляції багатьох фі-зіо логічних процесів [13].…”

New Components of the Renin-Angiotensin-Aldosterone System and Oxidative Stress

“…RhoA/Rho-kinase activation by increased NAD(P)H oxidasedependent ROS are also reported, leading to vascular smooth muscle contraction [17]. Involvement of the glutathionylation-dependent uncoupling of endothelial nitric oxide synthase (eNOS) is also reported [18]. Pharmacological intervention to oxidtive stress or RAAS are also reported.…”