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Cell Membranes and Free Radical Research Volume 5, Number 1, 2013 ISSN Numbers: 1308-4178 (On-line), 1308-416X Indexing: Google Scholar, Index Copernicus, Chemical Abstracts, Scopus (Elsevier), EBSCOhost Research Database EDITOR Editor in Chief Mustafa Nazıroğlu, Department of Biophysics, Medical Faculty, Suleyman Demirel University, Isparta, Turkey. Phone: +90 246 211 37 08. Fax:+90 246 237 11 65 E-mail: [email protected] Managing Editor A. Cihangir Uğuz, Department of Biophysics, Medical Faculty, Suleyman Demirel University, Isparta, Turkey. E-mail: [email protected] EDITORIAL BOARD Cell Membranes, Ion Channels and Calcium Signaling Alexei Tepikin, The Physiological Laboratory, University of Liverpool, Liverpool, UK Andreas Lückhoff, Institute of Physiology, Medical Faculty, RWTH-Aachen University, Germany Andreas Daiber, 2nd Medical Clinic, Molecular Cardiology, Medical Center of the Johannes Gutenberg University , Mainz, Germany Giorgio Aicardi, Department of Human and General Physiology, University of Bologna, Italy. Gemma A. Figtree, North Shore Heart Research Group Kolling Institute of Medical Research University of Sydney and Royal North Shore Hospital Sydney, AUSTRALIA. Jose Antonio Pariente, Department of Physiology, University of Extremadura, Badajoz, Spain. James W. Putney, Jr. Laboratory of Signal Transduction, NIEHS, NC, USA. Martyn Mahaut Smith, Department of Cell Physiology and Pharmacology, Universtiy of Leicester, Leicester, UK. Stephan M. Huber, Department of Radiation Oncology, Eberhard - Karls University Tubingen, Germany Enzymatic Antioxidants Michael Davies, Deputy Director, The Heart Research Institute, Sydney, Australia. Süleyman Kaplan, Department of Histology and Embryology, Medical Faculty, Samsun, Turkey Xingen G. Lei, Molecular Nutrition, Department of Animal Science, Cornell University, Ithaca, NY, USA Ozcan Erel, Department of Biochemistry, Medical Faculty, Yıldırım Beyazıt University. Nonenzymatic Antioxidants, Nutrition and Melatonin Ana B. Rodriguez Moratinos, Department of Physiology, University of Extremadura, Badajoz, Spain. Cem Ekmekcioglu, Department of Physiology, Faculty of Medical University of Vienna, Austria. Peter J. Butterworth, Nutritional Sciences Division, King’s College London, London, UK
Cell Membranes and Free Radical Research
AIM AND SCOPES Cell Membranes and Free Radical Research is a print and online journal that publishes original research articles, reviews and short reviews on the molecular basis of biophysical, physiological and pharmacological processes that regulate cellular function, and the control or alteration of these processes by the action of receptors, neurotransmitters, second messengers, cation, anions, drugs or disease. Areas of particular interest are four topics. They are; A- Ion Channels (Na+ - K+ Channels, Cl – channels, Ca2+ channels, ADP-Ribose and metabolism of NAD+, PatchClamp applications) B- Oxidative Stress (Antioxidant vitamins, antioxidant enzymes, metabolism of nitric oxide, oxidative stress, biophysics, biochemistry and physiology of free oxygen radicals C- Interaction Between Oxidative Stress and Ion Channels (Effects of the oxidative stress on the activation of the voltage sensitive cation channels, effect of ADP-Ribose and NAD+ on activation of the cation channels which are sensitive to voltage, effect of the oxidative stress on activation of the TRP channels) D- Gene and Oxidative Stress (Gene abnormalities. Interaction between gene and free radicals. Gene anomalies and iron. Role of radiation and cancer on gene polymorphism) READERSHIP Biophysics Biochemistry Biology Biomedical Engineering Pharmacology Physiology Genetics Cardiology Neurology Oncology Psychiatry Neuroscience Keywords Ion channels, cell biochemistry, biophysics, calcium signaling, cellular function, cellular physiology, metabolism, apoptosis, lipid peroxidation, nitric oxide synthase, ageing, antioxidants, neuropathy.
Volume 5, Number 1, 2013
Oxidative Stress and Vascular Function Andreas Daiber, Michael Mader, Paul Stamm, Elena Zinßius, Swenja Kröller-Schön, Matthias Oelze, Thomas Münzel From the 2nd Medical Clinic, Department of Cardiology, Medical Center of the Johannes Gutenberg University, Mainz, Germany
List of abbreviations RONS reactive oxygen or nitrogen species mPTP mitochondrial permeability transition pore NOX1 NOX2 eNOS
Abstract Many drug-induced complications and diseases are known to be associated with or even based on a dysequilibrium between the formation of reactive oxygen or nitrogen species (RONS) and the expression/activity of antioxidant enzymes that catalyze the breakdown of these harmful reactive species. The “kindling radical” concept is based on the initial formation of RONS that in
Corresponding Address Prof. Dr. Andreas Daiber, Universitätsmedizin der Johannes Gutenberg-Universität Mainz, II. Medizinische Klinik – Labor für Molekulare Kadiologie, Gebäude 605, Langenbeckstr. 1, 55131 Mainz, Germany, Phone: +49 (0)6131 176280, Fax: +49 (0)6131 176293, E-mail: [email protected]
turn activate additional sources of RONS in certain pathological conditions. Recently, we and others have demonstrated such “cross-talk” between NADPH oxidases and mitochondria in the setting of nitroglycerin-induced nitrate tolerance, the aging process and angiotensin-II triggered arterial hypertension via redox pathways compromising the mitochondrial, ATP-sensitive potassium channel (mKATP), the mitochondrial permeability transition pore (mPTP), cSrc and protein kinases and the NADPH oxidase isoform Nox2 (and eventually Nox1). This review will focus on the uncoupling of endothelial nitric oxide synthase (eNOS) by initially formed “kindling radicals” (RONS) and on the different “redox switches” that are involved in the uncoupling process of eNOS. S-glutathionylation of the eNOS reductase domain, adverse phosphorylation of eNOS, and of course the oxidative depletion of tetrahydrobiopterin (BH4) will be highlighted as potential “redox switches” in eNOS. In addition, RONS-triggered increases in levels of asymmetric dimethylarginine (ADMA) and L-arginine depletion will be discussed as alternative reasons for dysfunctional eNOS. Finally, we present the clinical perspectives of eNOS uncoupling (and dysfunction) for the development and progression of cardiovascular disease and discuss the important prognostic value of the measurement of endothelial function (e.g. by flow-mediated dilation or forearm plethysmography) for patients with cardiovascular disease.
Introduction Oxidative stress has been demonstrated to be a hallmark of most cardiovascular and neurodegenerative diseases (Griendling and FitzGerald 2003; Ischiropoulos and Beckman 2003). The most common reactive oxygen and nitrogen species (RONS) include superoxide radicals, hydrogen peroxide, hydroxyl radicals, carbon-centered ,!.+4% !/ƫ* ƫ,!.+45(ƫ. %(/Čƫ*%0.%ƫ+4% !ƫ. %(/ƫĨđĩČƫ nitrogen dioxide radicals, peroxynitrite, and hypochlorite. In physiological concentrations, these oxidative species work as intracellular messengers. Among these, the !/0ƫ 1* !./0++ ƫ %/ƫ *%0.%ƫ +4% !ƫ ĨđĩČƫ 3$%$ƫ 0/ƫ /ƫ *ƫ important vasodilator and inhibitor of platelet activation (Furchgott and Zawadzki 1980). Oxidative stress defines the state of cellular redox imbalance in favor of free radicals, produced by sources like NADPH oxidase or mitochondrial respiratory chain, and reduced protecting antioxidant enzyms such as superoxide dismutases (mitochondrial MnSOD and cytosolic/extracellular Cu,ZnSOD) shown by Fridovich and coworkers in the 1960s (McCord et al. 1971).
Figure 1. The chemical basis of vascular redox signaling. Superoxide formation from NADPH oxidases (Nox), the mitochondrial respiratory chain (Mito), xanthine oxidase (XO), an uncoupled NO synthase and P450 side reactions confers redox signaling mainly upon breakdown by self-dismutation or catalyzed by superoxide dismutases (SODs) to hydrogen peroxide. H2O2 modulates the thiol/disulfide equilibrium and thereby modifies enzymatic activities (e.g. in zinc-finger-motifs as found in transcription factors). Reaction with thiol groups is also a major route of detoxification for H2O2 via peroxiredoxins
Redox signaling versus oxidative stress Peroxynitrite (ONOO ), generated through a diffusion+*0.+((! ƫ .!0%+*ƫ !03!!*ƫ đƫ * ƫ /1,!.+4% !Čƫ %/ƫ ƫ )1$ƫ)+.!ƫ,+0!*0ƫ+4% *0ƫ0$*ƫđƫ* ƫ/1,!.+4% !ƫ* ƫ its contribution to cardiovascular and neurodegenerative disease has long been accepted (Ischiropoulos and Beckman 2003; Turko and Murad 2002). In the absence +"ƫ đČƫ /1,!.+4% !ƫ %/ƫ !%0$!.ƫ !#. ! ƫ 0$.+1#$ƫ ƫ spontaneous self-dismutation or catalytically scavenged by SODs. Due to its short half-life and limited reactivity, superoxide mainly reacts with metal centers such as ironsulfur-clusters (present for instance in aconitase (Flint et al. 1993) or calcineurin (Namgaladze et al. 2002)). Higher concentrations of superoxide anions cause the yield of hydroxyl radicals. This phenomenon is described as the Haber-Weiss-Cyle, with an ferric ion (Fe3+) as the essential katalysator. An increase of hydroxyl radicals enhances immediately the risk of DNA strand breaks and oxidation of any biomolecule. In the presence of đČƫ 0$!ƫ %/)100%+*ƫ +"ƫ /1,!.+4% !ƫ %/ƫ +10+),!0! ƫ 5ƫ 0$!ƫ"+.)0%+*ƫ+"ƫ,!.+45*%0.%0!ƫ/%*!ƫ0$!ƫ.!0%+*ƫ+"ƫđƫ 3%0$ƫ đ2- is 3- to 5-fold faster than the dismutation of đ2- catalyzed by SODs. ONOO- confers similar redox modifications (mainly oxidation of thiols, thioethers or metal-catalyzed nitration of tyrosines), leading to an outage of the protective proteins and oxidative stress. In the longterm the consequences is death of particluar cells (apotosis/necrosis) and the hole organism. These events are discussed in detail in previous reviews (Beckman and Koppenol 1996; Daiber and Ullrich 2002; Radi 2004) and in figure 1. -
Molecular proof of a damaging role of oxidative stress in cardiovascular diseases
of eNOS. Demonstrating the existence of a cross-talk between different cellular ROS sources, the mitochondrial
The first reports on the role of oxidative stress in
permeability transition pore (mPTP) blockade or genetic
the progression and pathophysiology of cardiovascular
deletion of subunits of the phagocytic NADPH oxidase
disease were published in the early 1990’s by Harrison and
both improved eNOS dysfunction in nitrate tolerance
Ohara in an experimental model of hypercholesterolemia
and angiotensin-II induced arterial hypertension (Wenzel
(Harrison and Ohara 1995; Ohara et al. 1993). Molecular
et al. 2008b)(Kroller-Schon et al. 2013). The hypothesis
proofs of the pathophysiological compound of oxidative
underlying this cross-talk is based on induction of
stress in cardiovascular diseases was provided by a
mitochondrial ROS formation, directly by mitochondrial
large number of preclinical studies using genetic tools
dysregulation in the aging process, under nitroglycerin
(e.g. knockout mice) which clarified the involvement
therapy, MnSOD deficiency, or by angiotensin-II driven
of ROS producing or degrading enzymes in the onset
activation of the NADPH oxidase (Nox2, Nox1) with
and progression of these diseases. Moreover, deletion
subsequent impairment of the mitochondrial respiration
of the NADPH oxidase subunits p47
and Nox1 has a
protective effect on blood pressure and endothelial
potassium channels (mKATP) (figure 2). Upon escape
of mtROS to the cytosol, they activate redox-sensitive
mice (Landmesser et al. 2002; Matsuno et al. 2005). In
cSrc kinase or protein kinase pathways that lead to the
contrast, overexpression of Nox1 in these transgenic mice
activation of Nox2 (or Nox1) and this secondary burst
causes a further increase in blood pressure (Dikalova et al.
triggers eNOS uncoupling with endothelial dysfunction
2005). Just as well, partial deletion of the mitochondrial
(via the “redox switches” in eNOS that are described
below) or further increase mitochondrial dysfunction
(MnSOD ) +/-
dependent mitochondrial oxidative stress and endothelial
initiating a vicious circle (Kroller-Schon et al. 2013).
dysfunction (Wenzel et al. 2008c). These data (along with
Other origins of oxidative stress have similar redox
several other ones in the literature (Chen et al. 2012; Daiber
switches: Consequently the uncoupling of eNOS (but
2010; Schulz et al. 2012)) provide molecular proof of the
also other isoforms) is based on increased formation of
crucial role of oxidative stress in causing cardiovascular
oxidants and various reports exist that demonstrate that
eNOS function is improved and uncoupling is reversed when the sources of oxidative stress are either inhibited
The “kindling radical” hypothesis
by pharmacological manipulations (e.g. by PKC inhibitors,
Under the terms of the concept of “kindling radicals”
NADPH oxidase inhibitors or AT1-receptor-blockers)
(or also “bonfire” hypothesis), initial formation of ROS
(Guzik et al. 2002; Knorr et al. 2011; Li et al. 2006; Loomis
(e.g. from NADPH oxidases) triggers further damage such
et al. 2005; Mollnau et al. 2002; Wenzel et al. 2008d) or
as eNOS uncoupling by different mechanisms (see “redox
there is a genetic deletion of the p47phox or gp91phox subunit
switches” below). The ROS-induced ROS production
resulting in dysfunctional phagocytic NADPH oxidase
concept can be extended to almost any kind of source
(Landmesser et al. 2003; Xu et al. 2006; Zhang et al.
of RONS as almost all of these sources contain “redox
2011). Similar observations were made when antioxidants
switches”. Beyond cytoplasmic enzymes, there is clear
were added acutely and at high concentrations to the
evidence of a cross-talk between mitochondrial ROS
system (e.g. infusion of vitamin C) (Heitzer et al. 1996a;
formation and NADPH oxidases (Daiber 2010)(Schulz et
Heitzer et al. 2001b). The interconnection between
different sources of oxidative stress also explains the
Examples for this cross-talk between mitochondrial
observation that inhibition of only one source of RONS
and NADPH oxidase-derived ROS are based on the
is able to completely normalize a cardiovascular disease
state. This was shown for the inhibition of NADPH oxidase
increases mitochondrial oxidative stress, a phenomenon
by apocynin in diabetes, hypertension and ischemia/
which has been attributed a mechanistic importance
reperfusion (Dodd and Pearse 2000; Hayashi et al. 2005;
in nitrate tolerance (loss of nitroglycerin bioactivation
Li et al. 2003).
by mitochondrial aldehyde dehydrogenase [ALDH2]), but also with NADPH oxidase-induced dysfunction
Cell Membranes and Free Radical Research
Volume 5, Number 1, 2013
Oxidative stress and vascular function
and Munzel 2006; Munzel et al. 2005). It should be noted that not only eNOS may be uncoupled and produce superoxide but also neuronal NOS (type 1) (Miller et al. 1997; Pou et al. 1999; Vasquez-Vivar et al. 1999a; VasquezVivar et al. 1999b) and inducible NOS (type 2) (Miller et al. 2000; Ungvari et al. 2003; Xia et al. 1998a; Xia and Zweier 1997b) are subject to the uncoupling phenomenon and produce superoxide in the uncoupled state.
Figure 2. Postulated molecular mechanisms of the crosstalk between mitochondria and NADPH oxidase through reactive oxygen species based on studies in white blood cells and in genetic/pharmacological animal. The brown boxes represent fundamental processes involved in the process of the crosstalk between mitochondria and NADPH oxidase through reactive oxygen species and the genetic/pharmacological stress factors that trigger this crosstalk. The red boxes contain important enzymatic
Figure 3. X-ray structure of human eNOS with the iron-porphyrin
signaling axis and the red circle represents the important role of
(blue), the substrate L- arginine (green), the P450-forming axial
cytosolic calcium levels. The green boxes represent genetic and
iron-thiolate ligand from a cysteine residue (yellow), the cofactor
pharmacological inhibitors and activators of this crosstalk. The
BH4 (purple), the zinc-thiolate complex forming cysteines (red,
yellow boxes show the detection assays used for the involved ROS
two from each subunit) and the zinc ion (brown). The blue boxes
and RNS. The blue boxes represent previous findings providing
represent the “redox switches” in eNOS triggering regulatory
the basis for our understanding of the crosstalk concept (Kimura
pathways that depend on oxidants and reductants. Modified
et al. 2005, Doughan et al. 2008, Wenzel et al. 2008, Dikalova
from Daiber and Münzel, Steinkopff Verlag Darmstadt 2006
et al. 2010). Modified from Kröller-Schön et al., Antioxid. Redox
(Daiber and Münzel 2006). With permission of Steinkopff Verlag
Signal. 2013 (Kroller-Schon et al. 2013). With permission of Mary
Redox switches in endothelial nitric oxide synthase (eNOS) In addition to the classical regulation of enzymatic activity of eNOS (e.g. by calcium/calmodulin, caveolin, HSP90,
regulatory pathways such as phosphorylation and S-glutathionylation are forthrightly linked to the formation of redox-active species. These “redox switches“ in eNOS confer alterations in enzymatic eNOS activity and may contribute to uncoupling of eNOS as depicted in figure 3. Uncoupling of eNOS is a condition by which electrons leak from the transport in the reductase domain (from NADPH over FMN and FAD) and are transmitted to molecular oxygen to yield superoxide instead of NO. This reversion is even more disadventageous than inhibition of eNOS, because uncoupling will switch the enzyme from a nitric oxide to a superoxide source, changing from a protective/ beneficial to a harmful/toxic phenotype (Forstermann
Cell Membranes and Free Radical Research
Oxidative depletion of tetrahydrobiopterin Among the regulatory pathways (“redox switches”) of eNOS, the oxidative depletion of tetrahydrobiopterin (BH4) is the most prominent (figure 3). Several independent groups provided substantial evidence for a causative role of BH4 depletion in the process of NOS uncoupling characterized by superoxide formation in the presence of the electron source NADPH (Vasquez-Vivar et al. 1999a; Vasquez-Vivar et al. 1998; Xia et al. 1998b). Milstein and Katusic reported highly efficient oxidative degradation of BH4 to dihydrobiopterin (BH2) by peroxynitrite and thereby provided an explanation of how RONS (especially peroxynitrite) may contribute to oxidative uncoupling of eNOS (Milstien and Katusic 1999). The understanding of the importance of vitamin C for the recycling or rescue +"ƫ 0$!ƫ đ4+ radicals (once BH2 is formed only energyconsuming enzymatic reaction confers reduction to BH4) (Baker et al. 2001; d'Uscio et al. 2003; Kuzkaya et al. 2003; Whitsett et al. 2007), provided an attractive explanation for the highly beneficial effects of vitamin C infusion
Volume 5, Number 1, 2013
Daiber et al
on improvement of endothelial function (measured by
function in experimental hypertension as well as
plethysmography) in chronic smokers (Heitzer et al.
atherosclerosis (Laursen et al. 2001; Schuhmacher et al.
1996a) and diabetic patients (Heitzer et al. 2001a). The
extent of the effect of vitamin C on endothelial function
Furthermore pharmacological inhibition of eNOS in
even turned out to be a valuable prognostic marker for
vessels decreased superoxide formation (Munzel et al.
cardiovascular events (risk for cardiovascular disease)
2000b) and forearm blood flow in arteries of GTN-treated
(Heitzer et al. 2001b).
volunteers was improved by folic acid (Gori et al. 2001;
The first direct evidence of a role of BH4 depletion
Gori et al. 2003). Finally, BH4 levels in vessels from tolerant
rabbits were significantly decreased (Ikejima et al. 2008).
dysfunction in vivo was obtained in hypertensive mice
in 2003 (Landmesser et al. 2003). Clinical evidence
infusion in mice caused a decrease in vascular BH4 levels,
for a role of oxidative BH4 is based on improvement of
vascular NO bioavailability, eNOS uncoupling, loss of eNOS
endothelial function1 in chronic smokers by BH4 but not
dimers and vascular dysfunction that were normalized
by tetrahydroneopterin (NH4), which shares the same
by BH4 supplementation (Dikalova et al. 2010b). Similar
antioxidant properties with BH4 but is not a cofactor
mechanisms were reported to be responsible for diastolic
for eNOS (Heitzer et al. 2000a; Heitzer et al. 2000b).
dysfunction (Silberman et al.) representing a potential
Likewise, supplementation with the BH4 analogue folic
therapeutic target in cardiovascular disease (Harrison et
acid improves endothelial function in human subjects
(Antoniades et al. 2006; Gori et al. 2001) or treatment
S-glutathionylation of the eNOS reductase domain
with the BH4 precursor sepiapterin restored endothelial
redox regulatory mechanism for many enzymes (e.g. mitochondrial aldehyde dehydrogenase (Wenzel et al. 2007), or SERCA (Adachi et al. 2004)) and was also reported for eNOS (figure 3). According to a recent report by Zweier and coworkers, eNOS is adversely regulated and uncoupled by S-glutathionylation at one or more cysteine residues of the reductase domain (Chen et al. 2010) mostly at 689 and 908. In the same year, Manevich et al. have detected S-glutathionylated eNOS in response to nitrosative stress but rather under artificial conditions (Manevich et al. 2010). In a subsequent study, Chen et al. have demonstrated superoxide-induced thiyl radical formation in eNOS with subsequent intracellular disulfide formation or S-glutathionylation, both leading to uncoupling of eNOS and superoxide formation by the enzyme (Chen et al. 2011). Based on recent observations by Knorr and Figure 4. Effects of telmisartan treatment on S-glutathionylation
of eNOS in nitroglycerin treated endothelial cells and nitrate
increased in nitroglycerin-treated endothelial cells and
tolerant rats. S-glutathionylation of eNOS was determined by
aortic tissue from nitroglycerin- infused rats (figure
eNOS immunoprecipitation from (A) and (B) samples, followed
4), probably contributing to eNOS uncoupling and
by anti- glutathione staining and normalization on eNOS.
endothelial dysfunction in the setting of nitrate tolerance,
Disappearance of the anti-glutathione staining in the presence
which was prevented by AT1-receptor blocker therapy
with telmisartan (Knorr et al. 2011). Therefore, eNOS S-glutathionylation may represent a new “redox master switch” controlling NO versus superoxide production by this enzyme.
Measured by acetylcholine-dependent increase in forearm blood flow by plethysmography. This technique is based on an increase in vessel diameter and blood flow by proximal infusion of acetylcholine in increasing doses by a catheter and simultaneous monitoring of the vessel diameter and/or blood flow using Doppler ultrasound. 1
Cell Membranes and Free Radical Research
Volume 5, Number 1, 2013
Oxidative stress and vascular function
Other Mechanisms Phosphorylation represents another redox-sensitive regulatory pathway of eNOS (figure 3). There are 3 different important phosphorylation modifications of eNOS: First, the activating phosphorylation at serine1177 (Dimmeler et al. 1999), second, the inactivating phosphorylation at
Identification of uncoupled endothelial nitric oxide synthase (eNOS) in vascular cells and tissue Direct detection of eNOS-derived superoxide formation
tyrosine657 (Loot et al. 2009) and third, the inactivating
According to previous reports on nitrate tolerance
phosphorylation at threonine495 (Fleming et al. 2001;
(Munzel et al. 2000, Munzel et al. 2000) and experimental
Lin et al. 2003). PKC, which is activated in endothelial
diabetes (Hink et al. 2001), atherosclerosis (Laursen et
cells in response to GTN treatment (Lin et al. 2003), can
al. 2001, Mollnau et al. 2003) as well as hypertension
actually cause eNOS uncoupling via phosphorylation of
(Mollnau et al. 2002, Landmesser et al. 2003) by Münzel
eNOS at Thr495. Therefore, both regulatory pathways,
and Harrison, superoxide formation from uncoupled
eNOS is best detected by direct measurement of
regarded as “redox switches” in eNOS.
superoxide formation in the presence and absence of
Another direct redox-regulatory pathway for eNOS
NOS inhibitors. Increase in superoxide signal in the
function is the oxidative disruption of the zinc-sulfur-
control tissue (excluding direct antioxidant effects of the
complex (ZnCys4) in the binding region of the eNOS
NOS inhibitors) and a decrease in superoxide signal in the
dimer resulting in a loss of SDS-resistant eNOS dimers,
diseased sample by NOS inhibitors, can be explained by
which has been first described by Zou and coworkers
for peroxynitrite-mediated oxidation of eNOS (Zou et al.
0$!ƫ+*0.+(ƫ#.+1,/ƫ/%*!ƫ.!' +3*ƫ5ƫđƫ3/ƫ(+'! ƫ
2002). Assymetric dimethylarginine (ADMA) is probably the most potent endogenous inhibitor of eNOS (Boger 2003a) and it is still a matter of debate whether or not ADMA itself may lead to uncoupling of eNOS (Sydow and Munzel 2003). Thus, oxidative stress in the vasculature may to ADMA concentrations that significantly inhibit eNOS activity (Cooke 2000) or may even uncouple the enzyme and switch it to a superoxide synthase. Furthermore L-Arginine is the endogenous substrate of eNOS providing the nitrogen for NO synthesis. According to a number of publications, “L-arginine deficiency” may contribute to uncoupling of eNOS (Bode-
Figure 5. Detection of eNOS uncoupling by oxidative fluorescence
Boger et al. 2007). Many studies reported a positive
microtopography. (A) To determine eNOS-dependent ROS
effect of L-arginine supplementation in different settings
formation, vessels were preincubated with the NOS inhibitor
of endothelial dysfunction (e.g. hypercholesterolemia,
L-NAME (500 M, lower panel), embedded in Tissue Tek resin,
hypertension, diabetes mellitus, etc) as reviewed in
frozen, cryo- sectioned and stained with DHE (1
(Sydow and Munzel 2003), and controversy still exists on the protective effect of L-arginine (Bevers et al. 2006). To this date it is too early to decide on whether L-arginine therapy or deficiency significantly contributes to endothelial dysfunction and cardiovascular events, but the above mechanisms represent feasible hypotheses to justify further investigations on this subject.
Densitometric data are presented as bar graphs. Pictures and data shown are representative for
sepiapterin, BH4) (Gori et al. 2001, Laursen et al. 2001,
It has been shown that NO production in mice with
Alp et al. 2003, Antoniades et al. 2006, Wang et al.
2008, Dikalova et al. 2010). A prominent example for
hypertension approaches the normal level following
indirect detection of eNOS uncoupling is the impaired
genetic deletion of the NADPH oxidase subunit p47PHOX
NO synthesis in DOCA- salt induced hypertension
and simultaneous administration with the NOS cofactor
(indicated by the decreased EPR-NO-signal) and
tetrahydrobiopterin (Landmesser et al. 2003). These
improvement of NOS activity by supplementation with
results demonstrate that NADPH oxidase-derived ROS
BH4 or genetic deletion of the NADPH oxidase subunit
contribute to the pathogenesis of hypertension and
p47phox-/- (eliminating the Nox2-dependent superoxide
the associated endothelial dysfunction. Moreover, these
formation that could represent the “kindling radical” for
results show that uncoupled NOS is a major source of ROS
NOS uncoupling. These beneficial effects of BH4 and its
and that restoring eNOS function can cure hypertension
precursors in various disease models were reviewed in
in experimental systems.
detail by Schulz et al. (Schulz et al. 2008).
Several other conditions (or risk factors) associated with
endothelial dysfunction. Endothelial dysfunction has been
Clinical perspective on the role of endothelial dysfunction as a consequence of endothelial nitric oxide synthase (eNOS) uncoupling in cardiovascular diseases
shown in chronic smokers (Heitzer et al. 1996, Heitzer et al. 2000), patients with increased LDL levels (Drexler and Zeiher 1991, Heitzer et al. 1996), patients with type I and II diabetes mellitus or metabolic syndrome (Ting et al. 1996, Williams et al. 1998, Butler et al. 2000, Heitzer et
According to a number of reports in the literature
al. 2000, Heitzer et al. 2001, Mather et al. 2001) as well as
and as shown in figure 7, endothelial dysfunction,
hypertensive patients (Perticone et al. 2001, Tzemos et al.
atherosclerosis and the late cardiovascular complications
2001). Several mechanisms could account for the reduction
of these adverse phenomena are associated with a
in endothelium-dependent vascular relaxation observed
chronic activation of the local and/or circulating renin-
in these settings, including changes in the activity and/
angiotensin-aldosterone system (RAAS). Suggesting a
or expression of eNOS, decreased sensitivity of vascular
role of an inappropriate RONS production in this setting,
smooth muscle cells to NO, or increased degradation
diabetic patients show particularly beneficial responses
of NO via its interaction with ROS such as superoxide.
to mineralocorticoid receptor blockade (Pitt et al. 2003),
Suggesting an interaction between an inappropriately
ACE inhibitor (Mehler et al. 2003), AT1-receptor blockade
high bioavailability of ROS and endothelial dysfunction,
(Kintscher et al. 2007) and renin inhibitor (Parving et al.
vascular responses in these settings are improved by the
2009) therapy. Likewise, nitrate tolerance is associated
antioxidant vitamin C (Heitzer et al. 1996, Duffy et al.
with an activation of the RAAS (Kurz et al. 1999) and
2001) (also reviewed in (Schulz et al. 2008)). Importantly,
experimental nitrate tolerant animals and tolerant
the extent of the improvement in endothelial function
patients take profit from AT1- receptor blockade (Hirai
observed in response to vitamin C (i.e., a surrogate
et al. 2003, Knorr et al. 2011) and ACE inhibitor (Heitzer
evidence of oxidative stress) has been shown to predict
et al. 1998, Watanabe et al. 1998, Berkenboom et al.
patients’ prognosis (Heitzer et al. 1996, Heitzer et al. 2001,
1999) therapy. The similarities between the mechanisms
Warnholtz et al. 2007). In line with this evidence, there
underlying endothelial dysfunction in diabetes mellitus
are a number of reports supporting the importance of
and nitrate tolerance were recently discussed in detail
endothelial function in predicting patients prognosis: for
(Oelze et al. 2010): they include the activation of a
instance, the study by Gokce et al. in patients undergoing
superoxide source such as the NADPH oxidase to produce
peripheral or coronary bypass surgery (Gokce et al.
the “kindling radicals” leading to uncoupling of eNOS
2002). as well as a study by Perticone et al in patients
via the above described “redox switches” and may also
with essential hypertension (Perticone et al. 2001).
extend to direct uncoupling of eNOS by PKC-dependent
Taken together, there is no doubt that measurement
phosphorylation of eNOS at Thr495. A number of
of endothelial function, and particularly the impact
experimental studies using transgenic or knockout mouse
of oxidative stress on endothelial function, provides
models have provided molecular evidence that oxidative
stress leads to vascular (endothelial) dysfunction and
cardiovascular events in secondary prevention, whereas
associated cardiovascular diseases (see chapter 1.2).
its role in primary prevention remains to be established.
Cell Membranes and Free Radical Research
Volume 5, Number 1, 2013
Daiber et al
Whether this can be clinically exploited remains a complicated question. As of now, there is sound
Duffy SJ, Gokce N, Holbrook M, Hunter LM, Biegelsen ES, Huang A, Keaney JF,
evidence from clinical and experimental data for a role of
Jr. and Vita JA. 2001. Effect of ascorbic acid treatment on conduit vessel
uncoupled NOS in the development and progression of
endothelial dysfunction in patients with hypertension. Am J Physiol Heart
cardiovascular disease (Munzel et al. 2005, Forstermann
Circ Physiol 280: H528-534.
and Munzel 2006, Schulz et al. 2008). More detailed information on this topic can be found in our upcoming
Forstermann U and Munzel T. 2006. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation 113: 1708-1714.
book chapter in Systems Biology of Free Radicals and
Furchgott RF and Zawadzki JV. 1980. The obligatory role of endothelial cells
Anti-Oxidants (edited by I. Laher) and published by
in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:
Springer-Verlag Berlin Heidelberg in 2014.
373-376. Gokce N, Keaney JF, Jr., Hunter LM, Watkins MT, Menzoian JO and Vita JA. 2002.
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