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Review
. 2023 May;38(13-15):1001-1021.
doi: 10.1089/ars.2023.0006. Epub 2023 Apr 6.

Vascular Redox Signaling, Endothelial Nitric Oxide Synthase Uncoupling, and Endothelial Dysfunction in the Setting of Transportation Noise Exposure or Chronic Treatment with Organic Nitrates

Thomas Münzel  1   2 , Andreas Daiber  1   2
Affiliations
Review

Vascular Redox Signaling, Endothelial Nitric Oxide Synthase Uncoupling, and Endothelial Dysfunction in the Setting of Transportation Noise Exposure or Chronic Treatment with Organic Nitrates

Thomas Münzel et al. Antioxid Redox Signal. 2023 May.

Abstract

Significance: Cardiovascular disease and drug-induced health side effects are frequently associated with-or even caused by-an imbalance between the concentrations of reactive oxygen and nitrogen species (RONS) and antioxidants, respectively, determining the metabolism of these harmful oxidants. Recent Advances: According to the "kindling radical" hypothesis, the initial formation of RONS may further trigger the additional activation of RONS formation under certain pathological conditions. The present review specifically focuses on a dysfunctional, uncoupled endothelial nitric oxide synthase (eNOS) caused by RONS in the setting of transportation noise exposure or chronic treatment with organic nitrates, especially nitroglycerin (GTN). We further describe the various "redox switches" that are proposed to be involved in the uncoupling process of eNOS. Critical Issues: In particular, the oxidative depletion of tetrahydrobiopterin and S-glutathionylation of the eNOS reductase domain are highlighted as major pathways for eNOS uncoupling upon noise exposure or GTN treatment. In addition, oxidative disruption of the eNOS dimer, inhibitory phosphorylation of eNOS at the threonine or tyrosine residues, redox-triggered accumulation of asymmetric dimethylarginine, and l-arginine deficiency are discussed as alternative mechanisms of eNOS uncoupling. Future Directions: The clinical consequences of eNOS dysfunction due to uncoupling on cardiovascular disease are summarized also, providing a template for future clinical studies on endothelial dysfunction caused by pharmacological or environmental risk factors.

Keywords: NADPH oxidase; nitrate tolerance; nitric oxide synthase uncoupling; oxidative stress; peroxynitrite; superoxide; transportation noise exposure.

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Conflict of interest statement

No competing financial interests exist.

Figures

FIG. 1.
FIG. 1.
The chemical basis of vascular oxidative stress and redox signaling by superoxide and nitric oxide in animal models of nitrate tolerance and traffic noise exposure. Upper part: Superoxide (•O2−) formation from Nox, the mitochondrial respiratory chain (Mito), XO, a ucNOS, and P450 side reactions confer redox signaling mainly upon breakdown by self-dismutation or catalyzed by SODs to hydrogen peroxide. Reaction with thiol groups is a major route of detoxification for H2O2 via Prx and Trx or the selenol in GPx—these systems require energy-consuming recycling by NADPH-coupled reductases (GR, TR). Another route for H2O2 decomposition is catalyzed by catalase (Cat). When H2O2 accumulates, it may lead to the Fenton reaction (hydroxyl radical formation) upon reaction with ferrous iron (Fe2+), triggering severe oxidative damage at the protein, lipid, and DNA levels. Likewise, nitric oxide (•NO, previously termed EDRF) is formed from eNOS, nNOS, iNOS, or reduction of nitrite from nutrition conferring the diffusion-controlled reaction with superoxide to yield peroxynitrite anion (ONOO−). This rapid kinetics even outcompetes the extremely fast SOD-catalyzed breakdown of superoxide. Upon protonation, peroxynitrite undergoes either spontaneous isomerization to nitrate (representing a detoxifying route) or undergoes homolysis to form the hydroxyl radical (•OH) and the nitrogen dioxide (•NO2) radical leading to oxidative damage comparable with the Fenton reactivity of H2O2. Lower part: (Left) Hydroxyl radicals typically induce oxidative DNA damage in the form of 8-oxoGua as envisaged in GTN-treated, nitrate-tolerant rats (GTN with a dose of 50 mg/kg/day for 3.5 days) or aircraft noise-exposed mice [72 dB(A) around-the-clock for 4 days] by immunohistochemistry (brown staining). (Right) ONOOH and derived free radicals typically induce protein tyrosine nitration as envisaged in GTN-treated, nitrate-tolerant rats or noise-exposed mice by immunohistochemistry (brown staining), although MPO/H2O2/nitrite reaction will also lead to nitration. Scheme in the upper part summarized from Daiber and Ullrich (2002) and adapted from Daiber et al (2020c) and Daiber et al (2014) with permission. Images in the lower part were reproduced from Mikhed et al (2016) (for nitrate tolerance) and Kvandova et al (2020) (for noise exposure) with permission. 8-oxoGua, 8-oxoguanine; EDRF, endothelium-derived relaxing factor; eNOS, endothelial nitric oxide synthase; GPx, glutathione peroxidase; GR, glutathione reductase; GTN, nitroglycerin; iNOS, inducible nitric oxide synthase; MPO, myeloperoxidase; nNOS, neuronal nitric oxide synthase; Nox, NADPH oxidases; Prx, peroxiredoxins; SODs, superoxide dismutases; TR, thioredoxin reductase; Trx, thioredoxin; ucNOS, uncoupled nitric oxide synthase; XO, xanthine oxidase.
FIG. 2.
FIG. 2.
Proposed mechanisms underlying GTN-induced and traffic noise-mediated eNOS uncoupling and diminished •NO bioavailability. Upper part: GTN treatment causes a decrease in Ser1177 (1) and an increase in Thr495 (2) phosphorylation of the eNOS (Knorr et al, 2011), leading to a decreased activity and uncoupling, respectively. In addition, the key enzyme of the de novo synthetic pathway of the eNOS cofactor BH4 GCH-1 is downregulated by chronic GTN treatment (3), also leading to a dysfunctional, superoxide (•O2−)-producing nitric oxide synthase. S-Glutathionylation represents another regulatory modification of eNOS, which is increased in the setting of tolerance (Knorr et al, 2011) and upon noise exposure (Munzel et al, ; Steven et al, 2020) (4). Lower part: All of these adverse regulatory pathways that are activated by GTN treatment (50 or 100 mg/kg/day for 3.5 days) (Jabs et al, ; Knorr et al, 2011) or aircraft noise exposure [72 dB(A) around-the-clock for 1, 2, or 4 days] (Munzel et al, 2017a), including direct scavenging of •NO by •O2−, lead to diminished •NO bioavailability and enhanced endothelial •O2− formation that was blocked by the eNOS inhibitor L-NAME, supporting eNOS uncoupling. “?” nearby Cys689/908 means that discrimination between the S-glutathionylation sites is not possible since a pan-antibody for S-glutathionylation was used. “?” nearby BH4 means that so far rather indirect evidence exists for eNOS uncoupling via BH4 depletion by GTN tolerance and noise exposure (e.g., diminished GCH-1 and improvement of FMD by vitamin C or folic acid administration). Scheme in the upper part was reproduced from Knorr et al (2011) with permission. Images in the lower part were reproduced from Jabs et al (2015) and Knorr et al (2011) (for nitrate tolerance) and Munzel et al (2017a) (for noise exposure) with permission. Akt, protein kinase B; BH4, tetrahydrobiopterin; FAD, flavin adenine dinucleotide; FMD, flow-mediated dilation; FMN, flavin mononucleotide; GCH-1, GTP-cyclohydrolase-1; GSSG, glutathione disulfide; L-NAME, NG-nitro-l-arginine methyl ester; PKC, protein kinase C.
FIG. 3.
FIG. 3.
S-glutathionylation of eNOS in GTN-treated nitrate-tolerant rats and aircraft noise-exposed mice. Upper part: Oxidative stress causes S-glutathionylation of eNOS at cysteine 689 and 908, a surrogate marker for uncoupling of the protein. In the “coupled” eNOS homodimer, electrons are usually transferred from the NADPH and flavins to the heme iron. When cysteine residues 689 and/or 908 undergo S-glutathionylation, structural changes in eNOS are initiated that are followed by misdirection of the electrons to molecular oxygen with subsequent •O2− formation, termed “uncoupled” state of eNOS. Lower part: S-glutathionylation of eNOS was determined by eNOS immunoprecipitation from aortic tissue, followed by antiglutathione staining and normalization on eNOS. S-glutathionylation of eNOS was increased in nitrate-tolerant rats (GTN with a dose of 50 mg/kg/day for 3.5 days), which was normalized by telmisartan cotherapy (T8, 8 mg/kg/day for 10 days) (Knorr et al, 2011). *p < 0.05 versus Ctr, #p < 0.05 versus GTN group. S-glutathionylation of eNOS was also increased by aircraft noise exposure [72 dB(A) around-the-clock for 1, 2, or 4 days] (Munzel et al, 2017a). Disappearance of the antiglutathione staining in the presence of 2-mercaptoethanol served as a control. *p < 0.05 versus Ctr, #p < 0.05 versus noise (1 day) group. Representative blots are shown at the bottom of each densitometric quantification along with the respective loading control. Scheme in the upper part was adapted from Daiber et al (2020b) with permission. Data in the lower part were reproduced from Knorr et al (2011) (for nitrate tolerance) and Munzel et al (2017a) (for noise exposure) with permission.
FIG. 4.
FIG. 4.
Prevention of vascular ROS formation and endothelial dysfunction by GTN or traffic noise exposure by pharmacological or genetic NADPH oxidase inhibition. Left part: Oxidative stress assessed by DHE staining (red fluorescence indicates reactive oxygen species [ROS] formation, whereas green fluorescence represents the autofluorescence of the basal lamina). E=Endothelium, M = Media, A = Adventitia. GTN (50 mg/kg/day for 3.5 days) treatment leads to aortic oxidative stress in rats, which was prevented by the PKC inhibitor chelerythrine (chele) (Knorr et al, 2011). Aircraft noise exposure [72 dB(A) around-the-clock for 1, 2, or 4 days] leads to aortic oxidative stress in rats, which was prevented by genetic deficiency of gp91phox (Kroller-Schon et al, 2018). The phagocytic NADPH oxidase (NOX-2) plays a key role in the pathomechanisms underlying nitrate tolerance and noise exposure-mediated cardiovascular damage. NOX-2 is activated by ATII receptor activation, leading to DAG formation, a strong PKC activator. PKC activation can be envisaged by phosphorylation of its target protein MARCKS. Activated PKC will cause phosphorylation of p47phox at Ser328 leading to translocation of this cytosolic regulator of NOX-2 to the multienzyme membrane complex of gp91phox with subsequent activation of NOX-2 and superoxide formation. Right part: Endothelial dysfunction (impaired ACh-dependent relaxation) by GTN treatment was prevented in mice with genetic deficiency of p47phox or gp91phox (Wenzel et al, 2008). *p < 0.05 versus WT EtOH solvent control group. Endothelial dysfunction by noise exposure was prevented in mice with genetic deficiency of gp91phox (Kroller-Schon et al, 2018). *p < 0.05 versus WT control group and #p < 0.05 versus gp91phox−/− + noise group. Scheme in the left part was adapted from Frenis et al (2021c) with permission [Copyright © 2021 the authors. Open access (CC BY)]. Data in the left part were reproduced from Knorr et al (2011) (for nitrate tolerance) and Kroller-Schon et al (2018) (for noise exposure) with permission. Data in the right part were reproduced from Wenzel et al (2008) (for nitrate tolerance) and Kroller-Schon et al (2018) (for noise exposure) with permission. ACh, acetylcholine; ATII, angiotensin-II; DAG, diacylglycerol; DHE, dihydroethidine; ROS, reactive oxygen species.
FIG. 5.
FIG. 5.
Impairment of endothelial dysfunction by GTN therapy or traffic noise exposure and improvement by vitamin C coadministration. Left part: Endothelial function was determined by forearm plethysmography, ultrasound-dependent assessment of forearm blood flow in response to increasing doses of infused ACh. FBF was expressed as the ratio of infused to noninfused arm. Each column presents the percent change in FBF from baseline in response to each infused concentration of ACh (7.5, 15, and 30 μg/min) in the untreated control group, chronic GTN treatment for 7 days, and the chronic GTN+vitamin C administration group (Gori et al, 2010). *p < 0.05 versus lowest ACh dose (7.5 μg/min), #p < 0.05 versus GTN therapy without vitamin C. Right part: Endothelial function was determined by FMD, ultrasound-dependent assessment of vasodilation (widening) of a large vessel in the arm upon reactive hyperemia after vascular occlusion for 5 min. FMD was determined in subjects without aircraft noise exposure [Leq 35.4 dB(A)] and 30 or 60 aircraft noise events for 1 night [Leq 43.1 or 46.3 dB(A)] (Schmidt et al, 2013). Likewise, FMD was determined in subjects without train noise exposure [Leq 33 dB(A)] and 30 or 60 train noise events for 1 night [Leq 52 or 54 dB(A)] (Herzog et al, 2019). The effect of vitamin C oral administration on FMD was measured in both the noise exposure studies. *p < 0.05 versus same group without vitamin C, †p < 0.05 versus unexposed control group with vitamin C. The schemes in the upper parts were reused from Daiber et al (2017c) with permission. Data in the left part were adapted from Gori et al (2010) (for nitrate tolerance) with permission. Data in the right part were adapted from Herzog et al (2019) and Schmidt et al (2013) (for noise exposure) with permission. FBF, forearm blood flow.
FIG. 6.
FIG. 6.
Summarizing schemes on the similarities and differences of pathomechanisms underlying nitrate tolerance (upper part) and transportation noise exposure (lower part)-mediated cardiovascular damage. (1) Chronic GTN therapy leads to activation of the RAAS via nitrovasodilator-induced hypotension with subsequent angiotensin-II release, PKC and NOX-2 activation (also called pseudotolerance). In addition, GTN therapy causes substantial mtROS formation also causing inhibition of ALDH-2, the GTN-bioactivating enzyme (2), followed by redox-dependent NOX-2 activation (3) and peroxynitrite (ONOO−) formation with subsequent dysregulation of other critical regulators of vascular tone by nitration of prostacyclin synthase (PGI-S) (4), oxidation of sGC (5), oxidative induction of ET-1 (6), and uncoupling of eNOS by BH4 depletion and S-glutathionylation (7), all of which leads to endothelial dysfunction and true vascular tolerance (8). In contrast, traffic noise exposure causes neuronal stress responses via the hypothalamic–pituitary–adrenal axis (release of cortisol/corticosterone) and the sympathetic nervous system (release of NA) (I) with subsequent RAAS and NOX-2 activation (II) along with enhanced inflammation (III). The resulting oxidative stress leads to peroxynitrite formation, protein tyrosine nitration, and lipid peroxidation (IV), as well as oxidative induction of ET-1 (V) and eNOS uncoupling (VI), all of which lead to endothelial dysfunction and hypertension (VII), in part, mediated by direct vasoconstrictor effects (VIII). The major differences of the pathomechanisms of nitrate tolerance and noise exposure are the pathways that lead to activation of the RAAS, the substantial adverse effects of GTN on mitochondrial function and mtROS formation, and the strong inflammatory component underlying noise exposure-mediated damage. The major similarities are the central role of oxidative stress in both pathomechanisms of endothelial dysfunction, with NOX-2, RAAS, and eNOS uncoupling as key players. The schemes were reused from Munzel et al (2011) (upper part) and Munzel et al (2017a) (lower part) with permission. ALDH-2, mitochondrial aldehyde dehydrogenase; ET-1, endothelin-1; mtROS, mitochondrial reactive oxygen species; NA, noradrenalin; RAAS, renin–angiotensin–aldosterone system; sGC, soluble guanylyl cyclase.
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