Nitroso–Redox Interactions in the Cardiovascular System
2006; Lippincott Williams & Wilkins; Volume: 114; Issue: 14 Linguagem: Inglês
10.1161/circulationaha.105.605519
ISSN1524-4539
AutoresJeffrey Zimmet, Joshua M. Hare,
Tópico(s)Electron Spin Resonance Studies
ResumoHomeCirculationVol. 114, No. 14Nitroso–Redox Interactions in the Cardiovascular System Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessResearch ArticlePDF/EPUBNitroso–Redox Interactions in the Cardiovascular System Jeffrey M. Zimmet and Joshua M. Hare Jeffrey M. ZimmetJeffrey M. Zimmet From the Department of Medicine, Division of Cardiology, Institute for Cell Engineering, and Johns Hopkins University School of Medicine, Baltimore, Md. and Joshua M. HareJoshua M. Hare From the Department of Medicine, Division of Cardiology, Institute for Cell Engineering, and Johns Hopkins University School of Medicine, Baltimore, Md. Originally published3 Oct 2006https://doi.org/10.1161/CIRCULATIONAHA.105.605519Circulation. 2006;114:1531–1544Normal cardiovascular performance requires exquisite balancing of many complex biochemical processes. Perturbation of this balance may lead to myocardial dysfunction or may be a secondary result of structural heart disease such as myocardial infarction (MI) or cardiomyopathic processes. Altered signaling systems in turn contribute to the progression of myocardial dysfunction. The roles of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in normal and failing myocardium and vasculature have been the subject of intense investigation and continue to engender considerable debate. A disturbance in the oxidation–reduction state of the cell, in which ROS production exceeds antioxidant defenses, is called oxidative stress. By analogy, nitrosative stress is an impairment in nitric oxide (NO) signaling caused by increased amounts of RNS, which may be caused by or associated with a disturbance in the redox state. This review addresses the role of the redox state and nitroso–redox balance in determining cardiovascular function in health and disease.The Chemistry of Nitroso–Redox BalanceFree radicals are highly reactive molecules with unpaired electrons. Free radical chemistry is the underpinning of 2 broad classes of signaling molecules in biological systems: ROS, which are reactive intermediates of oxygen metabolism, and a closely related group of RNS. The forms of ROS that are relevant in biological systems include the superoxide radical (O2·−), hydrogen peroxide (H2O2), and hydroxyl radical (OH·).1 RNS of biological importance include NO, low- and high-molecular-weight S-nitrosothiols, and peroxynitrite (ONOO−)2 (Figure 1). Download figureDownload PowerPointFigure 1. Sources of ROS and RNS. Superoxide is produced by a variety of mechanisms, including the normal functioning of oxidase enzymes, hemoglobin, and mitochondria, as well as by uncoupled NOS. Superoxide dismutase catalyzes the dismutation of O2·− to hydrogen peroxide and water. H2O2 can be converted by catalase or peroxidases to water and molecular oxygen. It also may be converted to the hydroxyl radical through the Fenton reaction, which requires Fe2+ as a cofactor. NO is produced primarily by 1 of the 3 forms of NOS. NO may interact with O2−· to form the highly reactive peroxynitrite.Superoxide, Peroxide, and PeroxynitriteSuperoxide is formed by the 1-electron reduction of molecular oxygen, resulting in a free radical.3 This reductive process can be accomplished by a variety of oxidases, including xanthine oxidase (XO) and NADPH oxidases, or as a byproduct of oxidative phosphorylation. XO produces varying proportions of superoxide and hydrogen peroxide during reoxidation of the enzyme4 (Figure 2). Download figureDownload PowerPointFigure 2. A, XOR is reduced during the conversion of hypoxanthine to xanthine and of xanthine to uric acid. B, Reoxidation of fully reduced XOR involves electron transfer to oxygen, producing hydrogen peroxide and superoxide. Reproduced with permission from Berry and Hare.4 Copyright 2004, The Physiological Society.Peroxynitrite (ONOO−) is a product of the reaction of NO with superoxide. At physiological pH, ONOO− is protonated to form peroxynitrous acid, which is a highly reactive species with a very short half-life in vivo. ONOO− is able to react with DNA, proteins, and lipids, potentially leading to cellular damage and cytotoxicity. ONOO− reaction with CO2 is a physiologically relevant pathway, given the high rate constant for this reaction and the relatively high concentration of CO2 in vivo.5 This reaction forms intermediates that provide a route to nitration and oxidation. Tyrosine nitration by ONOO− has been demonstrated in vitro and has long been suspected as a mechanism of protein inactivation.6,7 However, the mechanisms of protein inactivation by ONOO− are multifactorial and nonspecific with multiple targets, including thiols, methionine, and amines.8 In the case of the sarcoplasmic reticulum (SR) calcium ATPase (SERCA), for example, oxidation at multiple cysteine residues leads to loss of function.9Nitric OxideNO mediates signaling events through 2 important mechanisms. First, NO binds and affects the activity of enzymes with transition metal centers, leading to activation of soluble guanylyl cyclase and inhibition of cytochrome c. NO activates guanylyl cyclase by binding to its heme moiety, leading to the production of cGMP. cGMP then activates protein kinase G, leading to a series of signaling events with diverse consequences, including vascular smooth muscle relaxation.10–12 Second, NO exerts widespread signaling through the aforementioned covalent nitrosylation of sulfhydryl groups on proteins and small molecules,13 which has been demonstrated in well over 100 proteins in multiple cells and tissues, including the heart.14–17 Various proteins involved in the regulation of myocardial contractility are modified by protein S-nitrosylation.18–20 Cysteines susceptible to nitrosylation tend to be located between an acidic and a basic amino acid, leading to the proposal of a protypic consensus acid-base motif for S-nitrosylation.21 This motif has been shown to be predictive in several examples in which the essential cysteines have been well characterized.15Biochemistry of Nitroso–RedoxROS and RNS as Second MessengersA key concept in understanding redox balance in the cardiovascular system is that the effects of ROS and RNS depend on the location, amount, and timing of their production.22 ROS are not intrinsically destructive; on the contrary, increasing evidence shows that they play necessary roles in normal signal transduction.1 In low concentrations, they are implicated as second messengers primarily through inhibition of phosphatases, acting downstream of effectors such as platelet-derived growth factor, epidermal growth factor, tumor necrosis factor-α, β-adrenergic agonists, and interleukin-1β.1,23 In higher concentrations, however, they take on pathophysiological roles. ROS affect the oxidative modification of diverse molecules, including DNA, proteins, lipids, and sugars, potentially leading to toxicity. This results not only in processes such as lipid peroxidation but also in the interruption of normal signaling pathways, which leads to organ malfunction,24 largely because of reversible and irreversible oxidative modification of proteins.In terms of signaling specificity, there has been a recent explosion of data on the role of S-nitrosylation in biological systems. As such, increasing evidence indicates that RNS subserve a signaling system with diverse and widespread effects mediated through the nitrosylation of specific cysteine thiol residues or metal centers, which modifies protein activity.15What Is the Nature of Oxidative and Nitrosative Modification?To understand the idea of nitroso–redox modulation of protein function, it is important first to grasp how proteins are affected by oxidative and nitrosative modification. As mentioned, NO exerts diverse biological effects via posttranslational nitrosylation of specific cysteine thiol residues.15 S-nitrosylation is a reversible covalent modification, similar in principle to phosphorylation, which affects proteins of all classes.14,15,25 This modification is responsible for a large part of the biological activity of NO and provides a mechanism for redox-based regulation of protein function. For example, in cardiac myocytes, S-nitrosylation modulates the function of ion channels that regulate excitation–contraction coupling and therefore normal systolic and diastolic function.18 The role of NO signaling in excitation–contraction coupling is discussed in more detail later.Oxidative mechanisms may "compete" directly with NO for interaction with the same cysteine thiols.26 In general, nitrosylation is a highly reversible modification that lends itself more easily to roles in protein regulation, whereas oxidation represents a spectrum of modification ranging from reversible to irreversible change that entails some loss of control (Figure 3A). What is the nature of these oxidative modifications? In contrast to alcohols, thiols are less polar and are more easily oxidized. When occurring in close proximity to another thiol, oxidation of thiols can lead to formation of disulfide bonds. This may involve other cysteine moieties or can entail interactions with small-molecule thiols such as glutathione to form mixed disulfides. These further oxidation reactions involve formation of sulfenic acids (S-OH), the less reversible sulfinic (S-O2H) acids, and the irreversible formation of sulfonic (SO3H) acids26,27 (Figure 3B). Download figureDownload PowerPointFigure 3. Potential oxidation fates of cysteine thiols. A, The progression from more to less reversible reactions entails a loss of modulatory control and a transition from signaling to toxicity. B, Thiols represent a ubiquitous target for modification by RNS and ROS. Protein thiols may undergo reversible reactions to form S-nitrosothiols or disulfides. S-nitrosothiol subserves a critical and widespread signaling mechanism and thus can be considered a posttranslational modification system akin to phosphorylation. Thiols are also susceptible to oxidation to sulfenic acids and to sulfinic and sulfonic acids, which are progressively less reversible reactions and therefore maladaptive to the extent that they block the more reversible and physiological regulation mediated by nitrosylation.26The targets of signaling by ROS are being increasingly appreciated, and there are multiple candidates. An important example is the tyrosine phosphatases, which, by virtue of a redox-sensitive cysteine residue in the region of the active site, represent a group of potential targets for ROS.1 In vitro experiments have shown that hydrogen peroxide inhibits protein phosphatase activity in a manner that is reversible by reducing agents.28 By similar modification of susceptible protein thiol groups, oxidants may regulate protein dimerization, protein-protein interactions, or modification by small molecules such as glutathione.1 In recent work, cardiac hypertrophy stimulated by α-adrenergic receptor stimulation has been shown to be associated with oxidative modification of redox-sensitive thiols on Ras.29Redox Balance and Oxidative and Nitrosative StressBoth ROS and RNS are produced in most cell and tissue types, including those of the cardiovascular system. Production of these agents is counterbalanced in each case by specific mechanisms that help maintain local concentrations within physiological limits. Superoxide, first proposed by Fridovich and Handler30 in the 1960s as a biologically important reducing agent, was ultimately identified as a normal product of XO activity that led to cytochrome c reduction.3 It was studied in earnest after the subsequent identification by McCord and Fridovich31 of superoxide dismutase, the enzyme that catalyzes the breakdown of superoxide to hydrogen peroxide and oxygen (Figure 1). Although superoxide spontaneously "dismutates" to these same breakdown products, superoxide dismutase has the fastest reaction rate with its substrate of any known enzyme. Catalase or peroxidases then promote the conversion of hydrogen peroxide to water and molecular oxygen (Figure 1). Nonenzymatic antioxidants also play a role in the regulation of ROS and include glutathione, ubiquinone, vitamins E and C, lipoic acid, beta carotene, and urate.32Similarly, NO signaling is regulated in part by enzymes that terminate its actions. The NO effector cGMP is metabolized by phosphodiesterase-5, which is found in close proximity to NO synthase (NOS). Levels of protein S-nitrosylation are kept in equilibrium with S-nitrosoglutathione (GSNO), which in turn is selectively metabolized by a GSNO reductase.33 This enzyme, by depleting the pool of GSNO, shifts the equilibrium and therefore limits levels of S-nitroso proteins.In addition, proteins may be denitrosylated by thioredoxin, a general protein oxidoreductase that interacts with a broad range of proteins via a redox mechanism. Thioredoxin reverses the NO-induced reduction of NOS activity, which is mediated by S-nitrosylation,34 and has a similar effect on NO-mediated inhibition of protein kinase C.35 Recent work has shown that thioredoxin is itself regulated by S-nitrosylation of multiple residues.15,36 Although S-nitrosylation of an essential thiol is necessary for the basal oxidoreductase and antiapoptotic functions of this protein, further nitrosylation of an active-site cysteine blocks these activities and is proapoptotic.36 This illustrates the complex and multifaceted nature of protein regulation through S-nitrosylation.Nitroso–Redox Balance and ImbalanceNitroso–redox balance may be operationally defined by the idea that RNS and ROS work together in biological systems to achieve optimal signaling.22,37 The concept of imbalance arises because this signaling can be disrupted by either increased ROS or decreased RNS. Moreover, there is cross-talk between the enzymes that produce ROS and RNS, so NO deficiency can in some cases result in increased ROS production.38 Thus, the interactions between ROS and RNS are multifaceted and strike a balance that can be disrupted at both the cell and organ levels in cardiovascular disease states.Sources of RNS and ROSNO SynthasesNO is formed primarily by a family of enzymes known as NOSs, which oxidize the terminal guanidino nitrogen of l-arginine to form NO and the amino acid l-citrulline.39 There are 3 NOS isoforms, each with specific localization and function. NOS1 (neuronal NOS) and NOS3 (endothelial NOS) are found in a variety of cell types and are regulated by binding to calcium and calmodulin. NOS2 (inducible NOS), on the other hand, has very high baseline affinity for calcium and calmodulin; therefore, its activity is effectively independent of calcium concentration.Sources of ROSThe activity of ROS depends on the amount and location of production and on temporal elements. For example, the neutrophil uses an NADPH oxidase to produce the respiratory burst that has a killing effect on target cells.40 The NADPH oxidase present in vascular cells is similar in structure to the neutrophil enzyme but produces superoxide in lesser amounts over longer periods of time.41 Thus, similar systems for production of ROS are used under normal circumstances to accomplish very different objectives. Another key oxidase is XO, which is involved in the final steps of purine degradation. XO is physiologically present in the heart, and increased XO activity and the resultant imbalance in redox activity have an important role in cardiac disease, including ischemia–reperfusion injury and heart failure.4 Superoxide and other ROS are produced in the mitochondria during oxidative phosphorylation as a normal byproduct of aerobic respiration, and agents such as ceramide are able to increase mitochondrial ROS production by inhibiting complex III respiration, resulting in increased peroxide formation.42 Hemoglobin can likewise serve as a significant producer of superoxide under hypoxic conditions,43 as can occur during myocardial ischemia. And the NOSs, when deprived of the substrate l-arginine and the cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4), become uncoupled from NO production and produce superoxide.44 Other important oxidase pathways include lipoxygenase, auto-oxidation of catecholamines, and the cytochrome P450 class of enzymes, at least one of which is present in coronary arteries and is known to produce superoxide after stimulation with bradykinin (see the Table).45Potential Sources of ROS in the Cardiovascular SystemMitochondrial respirationXanthine oxidoreductaseNADPH oxidasesHeme oxidasesCytochrome P450 oxygenasesCyclooxygenasesLipoxygenasesPeroxidasesMyeloperoxidaseNADPH OxidasesThe NADPH oxidases comprise a family of enzymes that are present in a wide variety of tissues. The best-known member is that which is responsible for generating the reactive species required for the killing oxidative burst in phagocytic cells. These enzymes are each composed of 2 basic parts: a membrane-bound catalytic core known as cytochrome b558 and cytosolic regulatory subunits that affect catalytic activity by translocating to and binding the catalytic core (Figure 4). Cytochrome b558 is comprised of one each of the proteins known as gp91phox and p22phox. The regulatory subunits include the Rac1 GTPase and the proteins p47phox, p67phox, and p40phox. Of these proteins, the catalytic subunit gp91phox is the central component of the enzyme complex and has been studied the most. Over the past several years, multiple isoforms of gp91phox have been identified, so it is now recognized to be a family of homologues that are called NADPH oxidases (Nox). Download figureDownload PowerPointFigure 4. The multisubunit NADPH oxidases are composed of the membrane-bound catalytic subunits NOX (gp91phox in the prototypical phagocyte oxidase) and p22phox, as well as the regulatory subunits p67phox, p47phox, p40phox, and Rac. Enzymatic activity produces superoxide as a byproduct. Several gp91 isoforms have been isolated and characterized as the NOX family of proteins.The best-described Nox is gp91phox, now known as Nox2, which was originally described in neutrophils as the enzyme responsible for the respiratory burst. In the cardiovascular system, Nox2 is found in endothelial cells, fibroblasts, and cardiomyocytes, whereas Nox1 has been identified in vascular smooth muscle and colon carcinoma cells. Unlike the neutrophil oxidase, the enzyme in cardiac cells produces lower levels of ROS with slower kinetics. Production of ROS is modulated in response to a host of stimuli that includes mechanical factors such as stretch or shear forces, as well as signaling molecules such as angiotensin II, endothelin 1, and tumor necrosis factor-α.46 Cardiomyocytes and endothelial cells coexpress Nox2 and Nox4. It has been suggested that the effects of ROS produced by the NADPH oxidase depend on the specific isoform involved. Recent work has suggested that specificity of NADPH oxidases containing different Nox subunits may be achieved, at least in part, by precise subcellular localization of the Nox proteins. In this regard, the Nox1- and Nox4-containing NADPH oxidases in vascular smooth muscle have been localized to caveolae and focal adhesions, respectively.47MitochondriaO2·− production occurs at a measurable rate during even normal oxidative phosphorylation,48 making mitochondria the greatest cellular source of ROS. The majority of O2·− produced in the mitochondria has a relatively short half-life; it is acted on by manganese superoxide dismutase in the mitochondrial matrix or by copper/zinc superoxide dismutase in the intermembrane space.49 Nonetheless, mitochondrial ROS production increases in stress states, including heart failure.50,51 NOS1 localization to the mitochondria provides an additional mechanism for cross-talk between ROS and RNS.52 See elsewhere53,54 for reviews of mitochondrial ROS.Xanthine OxidoreductaseXanthine oxidoreductase (XOR) is a member of the molybdoenzyme family that includes enzymes such as aldehyde oxidase and sulfite oxidase.4,55 XOR is encoded by a single gene, but the protein exists as a homodimer in 2 potentially interconvertible forms, xanthine dehydrogenase (XDH) and XO. XOR catalyzes the final 2 steps of the purine degradation pathway, converting hypothanthine to xanthine and xanthine to uric acid. XO is best known to most clinicians for this activity and as the target for the antigout drug allopurinol, which efficiently blocks XO activity. XDH can be converted to XO either by a reversible process involving thiol oxidation or by an irreversible process through proteolytic cleavage.XOR is reduced during the reaction of xanthine to form uric acid. During the reoxidation process, 2 electrons are transferred to oxygen in each of the first 2 steps, thus generating hydrogen peroxide. In the final steps, the remaining 2 electrons are transferred to oxygen in separate steps to yield superoxide (Figure 2). Thus, each cycle of XOR activity produces 2 molecules of hydrogen peroxide and 2 molecules of superoxide.4 Between the 2 forms of XOR, XO appears to be responsible for most of the ROS production. Although XO readily transfers electrons to molecular oxygen, XDH prefers NAD+ as an electron acceptor. It is important to note, however, that once NAD+ has been reduced to NADH, XDH is able to act as an NADH oxidase. This process ultimately leads to generation of superoxide and is not inhibited by allopurinol.4 XOR thus produces both superoxide and hydrogen peroxide in the course of its normal activity.The location of XOR expression has been the subject of some controversy, specifically whether it is expressed in the heart. The highest levels of XOR in mammals occur in liver, small intestine, and mammary gland. Early studies showed that XOR also is present in significant quantities in endothelial cells and serum,56 but identification in the heart has been confirmed by most57,58 but not all investigations.59 Current evidence supports XOR expression as a low-abundance protein57,58 in the SR38 of cardiac myocytes. In addition to its well-known activities, XO also has the capacity to act as a GSNO reductase.22,60 This has given rise to speculation that XO is present in the SR to regulate the S-nitrosylation of the ryanodine receptor (RyR) and other SR proteins.Interactions Between ROS and RNSAs previously mentioned, there is linkage between the systems regulating ROS production and NO signalling. RNS–ROS cross-talk is evident at multiple levels. Interactions of RNS with ROS can take several forms, depending on the respective concentrations and rates of formation. NO can quench superoxide or combine with it to form peroxynitrite. Superoxide production by XOR is regulated by NO, and NO and ROS compete for the same thiols on target proteins.19,37,38In the failing heart, the beneficial effects of XO inhibition are dependent on NOS function. In an animal model of heart failure, treatment with NOS inhibitors abolishes the effects of allopurinol on contractility and myocardial efficiency without affecting basal function.61 XOR and NOS1 interact in the cardiac SR, and NOS regulates XO activity so that a deficiency in NOS1 translates to increased XOR activity38(Figure 5). Furthermore, XOR not only is present in the SR but coimmunoprecipitates with NOS1.38 Within the SR, then, the calcium channel proteins essential to excitation–contraction coupling (RyR2 and SERCA2a) localize with enzymes that affect their function (Figure 6). NOS1 may lead to NO-based modifications, as discussed above. XOR may act not only through production of ROS but also potentially through its activity as a GSNO reductase, thus regulating the nitrosylation state of these important target proteins. Download figureDownload PowerPointFigure 5. XO-derived superoxide production in cardiac tissue from NOS knockout mice. Basal lucigenin-enhanced chemiluminescence recordings were elevated in NOS1−/− but not NOS3−/−. Incubation with xanthine (solid bars) produced dramatic increases in lucigenin-detected superoxide. Importantly, this increase was 4-fold greater in NOS1−/− compared with wild-type and NOS3−/− hearts (†P<0.05; n=4 mice of each strain), indicating that NOS1 deficiency leads to augmented cardiac XOR superoxide production. Inhibition by allopurinol (striped bars) demonstrates that this increase is caused by XO production of superoxide. These experiments demonstrate ROS/RNS cross-talk within the cardiac myocyte, as well as NOS isoform specificity based on subcellular localization of NOS1 and NOS3. Reproduced with permission from Khan et al.38 Copyright 2004, The National Academy of Sciences of the United States of America.Download figureDownload PowerPointFigure 6. Nitroso-redox regulation of cardiac excitation-contraction coupling. Depicted are potential mechanisms for S-nitrosylation and oxidation regulation of the cardiac RyR channel. The tetrameric RyR is inserted in the SR membrane and is closely associated with the plasma membrane L-type calcium channel (LTCC), facilitating calcium-mediated calcium release from the SR. The top figure (A) illustrates the close association of these channel proteins in the dyad. Although multiple cysteine thiols are found on the RyR (≈50) that are candidates for modification by S-nitrosylation or oxidation, cys 3635 is implicated as a specific target for signaling in RYR1. Physiological and specific nitrosylation and denitrosylation (B and C) occur on a millisecond time scale and therefore may be able to participate in regulation of contraction in systole and relaxation in diastole. Excess S-nitrosylation or oxidation of multiple cysteine thiols on RyR leads to irreversible channel activation32 with maladaptive loss of regulatory control (D). Oxidation may occur at thiols that are nitrosylated or at other sites, which could change permissiveness to S-NO via allosteric effects. The LTCC and SERCA are similarly regulated by redox mechanisms at cysteine thiols. ROS leading to cysteine oxidation are derived from multiple sources, including XO. NOS1 is localized to the SR under normal circumstances but translocates to the plasma membrane under conditions of stress, including MI and heart failure.Some authors have suggested that XO also may contribute to the generation of NO.62,63 For example, in hypoxic states, NOSs are unable to produce NO, and XOR reduction of nitrates has been suggested as a mechanism of NO production; however, the ability of XOR to metabolize nitrates at physiological levels has not been demonstrated. In hypoxic rat heart, NO production continues even in the presence of NOS inhibitors, but the addition of allopurinol stops NO generation, presumably through inhibition of XOR.63 Interestingly, XOR is able to catalyze the reduction of nitrites to NO under experimental conditions, which has led to the suggestion that XOR may be involved in the vasodilator activity of nitrate drugs, including isosorbide dinitrate and nitroglycerin.64 However, this has not been demonstrated under physiological conditions. Further cross-talk between these 2 systems is suggested by the observation that NO may regulate XOR activity.38NO-Redox in Excitation–Contraction CouplingDepolarization of the cardiac myocyte plasma membrane triggers a cascade of events leading to a rapid increase in cytosolic calcium and resulting muscle contraction. The initial entry of calcium, via the plasmalemmal L-type calcium channel, leads to a larger release of calcium from the SR through the coupled RyR channel, a process known as calcium-induced calcium release18 (Figure 6). Relaxation of the myocytes requires diastolic calcium removal from the cytoplasm, which is mediated by the SR reuptake via the calcium ATPase (SERCA2a) and the sarcolemmal extrusion via the sodium–calcium exchanger (NCX). NO and the cellular redox state affect excitation–contraction coupling through interactions with calcium-handling proteins (primarily the L-type Ca2+ channel and the RyR), the contractile apparatus, and respiratory complexes.18 Both cGMP-dependent and -independent mechanisms are involved in NO effects on myocardial contractility.65Taking the interactions with plasma membrane and SR calcium channels as a prototype for this type of interaction, we know that NO may affect L-type Ca2+ channel opening either by cGMP formation or by nitrosylation of the channel protein.66 In the case of the L-type channel, cGMP inhibits channel activity, whereas S-nitrosylation and oxidation of the protein have a biphasic effect that is stimulatory at low concentration and inhibitory at high concentration.20,66Similarly, the SR Ca2+-release channel (RyR) is regulated by target cysteine nitrosylation or oxidation19,67,68 (Figure 6). The cardiac RyR2 has low-level basal S-NO. S-nitrosylation of additional cysteines leads to further activation of the channel.19 This is a highly reversible modification that can occur on a time scale commensurate with excitation–contraction coupling. In contrast, oxidation of multiple cysteine residues on RyR ultimately leads to irreversible activation of the channel (Figure 3A), a situation that favors SR leak and thus SR calcium depletion19 (Figure 6D). In the case of the skeletal muscle RyR1, the calcium-sensitizing effects of NO are mediated by specific nitrosylation of a particular cysteine (cysteine 3635) located within the calmodulin-binding domain of each subunit in the tetrameric receptor complex.67 By analogy, it is likely that a similar situation based on a single target cysteine regulates the cardiac-specific RyR, RyR2 (Figure 6).Some investigators have proposed other variations of the nitroso–redox posttranslational modifications concept. As noted, the range of oxidative modifications also includes the formation of mixed disulfides with small molecules such as glutathione.26,69 The resulting modification, S-glutathionylation, ha
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