Reactive Nitrogen Species and Tyrosine Nitration in the Respiratory Tract
Epiphenomena or a Pathobiologic Mechanism of Disease?

ALBERT van der VLIET, JASON P. EISERICH, MARK K. SHIGENAGA, and CARROLL E. CROSS

Department of Internal Medicine, University of California, Davis; Division of Biochemistry and Molecular Biology, University of California, Berkeley, California; and Department of Anesthesiology, Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama

	INTRODUCTION

TOP INTRODUCTION REACTIVE NITROGEN SPECIES AND... FORMATION OF NITROTYROSINE: A... TYROSINE NITRATION IN... WHAT IS NEEDED? REFERENCES

Since its discovery as a biologic messenger molecule just over a decade ago, nitric oxide (NO^·) has become well recognized for its participation in diverse biologic processes in nearly all aspects of life, including vasodilation, bronchodilation, neurotransmission, inhibition of phagocyte and platelet aggregation, and antimicrobial activity (1). Excessive production of NO^· during inflammatory-immune processes of the respiratory tract is thought to provide a host defense mechanism, although this comes with a price, since high levels of NO^· can also cause respiratory tract injury and thus contribute to the pathobiology of respiratory tract disease. These detrimental effects of NO^· are generally assumed to be related to the formation of more reactive nitrogen intermediates via interactions of NO^· with partially reduced oxygen species, a common hallmark of inflammatory processes. Conversely, NO^· has in some cases been shown to also attenuate oxidant-induced lung injury, and NO^· inhalation has been proposed as a therapeutic strategy in the management of pulmonary hypertension and in some forms of adult respiratory distress syndrome (ARDS). This dual property of NO^· has been the subject of intense recent investigation, which has uncovered multifaceted biochemical pathways of NO^· that are highly dependent on dose and on local redox status. NO^·-derived reactive nitrogen intermediates can induce a number of covalent modifications in various biomolecules, such as nitroso- and nitro- adducts, that result in functional and/or structural changes. One such modification yields 3-nitrotyrosine, and detection of this adduct in proteins is now commonly used as a diagnostic tool to identify involvement of NO^·-derived oxidants in many disease states (4). Furthermore, a number of in vitro studies have established changes in enzyme activity upon nitration of critical tyrosine residues, which has raised suggestions that protein nitration in vivo may be causally linked to inflammation-related forms of lung injury. In this Pulmonary Perspective we will briefly summarize the involvement of NO^· in the pathophysiology of inflammatory diseases of the respiratory tract, and will address characteristic diagnostic modifications in proteins or in other biomolecules, with special emphasis on 3-nitrotyrosine and related modifications in other aromatic substrates. We will examine the scope of bioreactive mechanisms known to contribute to aromatic nitration during inflammatory-immune processes, and will critically evaluate analytical procedures that have been developed and used to detect such modifications. We will also discuss the potential pathophysiologic consequences of tyrosine nitration and related modifications.

	REACTIVE NITROGEN SPECIES AND THE LUNG

TOP INTRODUCTION REACTIVE NITROGEN SPECIES AND... FORMATION OF NITROTYROSINE: A... TYROSINE NITRATION IN... WHAT IS NEEDED? REFERENCES

In healthy human subjects, NO^· can be measured in exhaled breath, and this exhaled NO^· is known to originate from local synthesis by both constitutive and inducible forms of NO^· synthases (NOS) present in several cell types within the respiratory tract, including airway and alveolar epithelial cells, macrophages, neutrophils, mast cells, and vascular endothelial and smooth-muscle cells. Although most exhaled NO^· is now recognized to originate primarily from the nasal cavity and the sinuses, NO^· is known to be generated constitutively in all areas of the respiratory tract. Production of NO^· is generally increased during inflammatory diseases of the respiratory tract, such as asthma or bronchiectasis, or after exposure to irritant gases such as ozone, as demonstrated by induction of inducible NOS (iNOS), increased levels of the metabolic end products nitrite (NO₂) and/or nitrate (NO₃) in respiratory tract fluids, and/or increased levels of NO^· in exhaled breath (2). This enhanced production of NO^· is thought to provide increased host defense against invading pathogens (3), and some support for this notion has been obtained in studies with iNOS-deficient mice. Animals lacking iNOS were found to be more susceptible to infections by Mycobacterium tuberculosis or Leishmania major, although deficiency of iNOS did not significantly affect susceptibility to Pseudomonas aeruginosa or Legionella pneumophila (7). Moreover, iNOS-deficient mice were protected from death caused by influenza virus pneumonitis, and were found to be less susceptible than control mice to lung damage after injection of endotoxin, acute allograft rejection, or allergic airways eosinophilia (7). These latter findings imply that excessive production of NO^· during inflammatory diseases of the respiratory tract can also contribute to respiratory tract injury, which most likely involves the formation of more reactive nitrogen intermediates via interaction of NO^· with inflammatory oxidants (3). Despite the potential contribution of excessive NO^· production to respiratory tract injury, protective effects of NO^· against oxidant- induced cytotoxicity or lung injury have also been demonstrated in a number of investigations. These protective effects are most likely conferred by the ability of NO^· to inhibit leukocyte activation and adhesion, and to interfere with radical-mediated oxidative processes (8, 9). Administration of inhaled NO^· has therefore been advocated for patients with pulmonary hypertension, and has also been suggested as being useful in some forms and/or stages of ARDS (10). This dual aspect of NO^· biochemistry, with both salutary and deleterious functions within the respiratory tract, constitutes one of the major sources of debate in determining the significance of NO^· and/or NO^·-derived oxidants in inflammatory lung disease.

The overall biochemistry of NO^· and related nitrogen oxides in relation to respiratory biology and disease has recently been reviewed (2, 11), and is beyond the scope of this discussion. Instead, we will restrict this overview to a more specific aspect of NO^· biochemistry: the formation of characteristic covalent modifications in proteins and other biomolecules, with primary focus on 3-nitrotyrosine, a covalent adduct of NO^·- derived oxidants of the amino acid tyrosine or protein tyrosine residues. In many recent investigations, detection of 3-nitrotyrosine in various diseased tissues has been put forward as direct evidence for the formation of reactive nitrogen intermediates during inflammatory processes (6), although such findings do not provide direct proof for contribution of these nitrogen intermediates to the pathophysiology of inflammatory diseases of the lung. Moreover, despite many suggested implications of alterations in protein structure or function by nitration of tyrosine residues (5, 6), the precise targets for tyrosine nitration and the potential pathophysiologic significance of tyrosine nitration in vivo are still largely unknown.

	FORMATION OF NITROTYROSINE: A PATHWAY DEPENDENT ON OXIDATION OF NO·

TOP INTRODUCTION REACTIVE NITROGEN SPECIES AND... FORMATION OF NITROTYROSINE: A... TYROSINE NITRATION IN... WHAT IS NEEDED? REFERENCES

NO^· is a relatively long-lived free radical species, with high diffusibility and surprisingly selective reactivity, both of which are useful assets in its role as a signaling molecule. Most biologic actions of NO^· appear to be mediated by interactions with paramagnetic centers in effector proteins, such as heme- or iron-sulfur centers, but NO^· is also known to react rapidly, via radical termination reactions, with other targets that carry unpaired electrons. These reactions include reactions with reactive oxygen metabolites such as superoxide (·O₂), or with radical intermediates in proteins or lipids. Furthermore, NO^· reacts with O₂ to form higher oxides of nitrogen, in a relatively slow reaction that may have some significance in the respiratory tract because of the relatively high local oxygen tension compared with that in other anatomic regions. The eventual biologic fate of NO^· is oxidation to NO₂ and NO₃, end products of NO^· metabolism that are rapidly distributed throughout the body and excreted in the urine (12). Overall, this oxidative metabolism of NO^· involves the formation of a number of reactive nitrogen intermediates in which the oxidation state of nitrogen ranges from +1 to +5 (Table 1), and several of these intermediates are presumed to be actively involved in the various biologic actions of NO^·.

According to a multitude of chemical investigations, many of these reactive nitrogen species are reactive toward all classes of biomolecules, such as transition-metal ions in metalloproteins, amino acid residues (thiols, amines, tyrosine), unsaturated lipids, and DNA bases, and such interactions are commonly thought to contribute significantly to the many pathophysiologic processes associated with excessive NO^· production (5, 13, 14). Concerning the interactive role of NO^· with other (inflammatory) oxidant species, most recent research has focused on the reaction of NO^· with ·O₂ to yield the powerful oxidant peroxynitrite (ONOO), which is presumed to be largely responsible for most of the adverse effects of excessive generation of NO^· (4). Because this reaction occurs at a nearly diffusion-limited rate, it is assumed that NO^· can outcompete superoxide dismutases (SOD) for reaction with ·O₂, and that ONOO will be generated as a consequence of the simultaneous production of ·O₂ and NO^·, a condition almost invariably occurring at sites of active inflammatory-immune processes, generally characterized by the induction of NOS and activation of oxidant production by respiratory-burst oxidases. Among the many types of oxidative modifications induced by ONOO and other reactive nitrogen intermediates are characteristic addition or substitution products in which NO^· is essentially incorporated into the target molecule (i.e., nitrosation and nitration reactions). For instance, reactions with thiol residues to form S-nitrosothiols have been proposed as a mechanism of either enzyme regulation or NO^· transport, and may provide a unique signaling mechanism induced by nitrosative stress. S-Nitrosothiols in proteins (e.g., albumin) or in low-molecular-weight thiols, such as glutathione (GSH), have been detected in the circulation, as well as in respiratory tract lining fluids (2). The exact mechanism by which S-nitrosation occurs in vivo is still unclear, but it essentially involves the formation of NO^·-derived intermediates with the redox equivalence of NO⁺ (primary candidates are N₂O₃, ONOO, and (di)nitrosyl iron complexes (11). Nitrosation of amines by these reactive nitrogen intermediates has been implicated in the mutagenic properties of NO^·, presumably through nitrosative deamination of DNA bases (14). Other characteristic and more irreversible NO^·-induced modifications include nitration of aromatic amino acids, lipids, or DNA bases (5, 8, 14). The amino acid tyrosine appears to be a particularly susceptible target for nitration, and the formation of free or protein-associated 3-nitrotyrosine has received much recent interest as a potential biomarker for the generation of reactive nitrogen intermediates in vivo. Furthermore, there is considerable evidence in the protein chemistry literature that nitration of essential tyrosine residues can inactivate many enzymes or prevent phosphorylation of tyrosine kinase substrates, and these findings have supported the hypothesis that tyrosine nitration might result not only in the formation of inactive "footprints" of reactive nitrogen intermediates, but might also be functionally related to the pathobiology of inflammatory diseases.

Detection of 3-Nitrotyrosine In Vivo: Technical Considerations

The recent recognition that nitration of tyrosine occurs in vivo and presents a footprint of reactive nitrogen oxides has spurred a large number of investigators to analyze free or protein-associated 3-nitrotyrosine in various tissue or fluid specimens as an indicator of oxidation reactions mediated by these NO^·-derived oxidants. Various procedures have been developed and utilized to this end, and in most previously published studies, protein-associated 3-nitrotyrosine has been analyzed either immunohistochemically or with enzyme-linked immunosorbent assays (ELISAs), using monoclonal or polyclonal antibodies that have been raised against nitrated proteins (15). Control experiments, using excess 3-nitrotyrosine and/or reduction of tissue 3-nitrotyrosine to the amino-derivative with sodium dithionite, are often but not always performed to assess the specificity of antibody binding in these assays. It is important to consider two potential sources of artifact when using immunohistochemistry to detect nitrotyrosine-containing proteins. First, when peroxidase-conjugated secondary antibodies are used to detect primary antibodies against nitrotyrosine, tissue sections are commonly rinsed with H₂O₂ to block endogenous peroxidase activity. The presence of NO₂ within these tissue sections may thereby give rise to artifactual protein nitration, via formation of reactive nitrogen intermediates through peroxidase-catalyzed oxidation of NO₂ (18, 19). Also, because residual peroxidase activity within these tissue sections may contribute to peroxidase-dependent tissue staining, it is critical to use appropriate controls to establish the specificity of the assay.

Although efforts have been made to quantitate the extent of tyrosine nitration with ELISA, based on comparison with chemically nitrated proteins, such approaches are at best semi-quantitative and do not allow the easy comparison of results of different studies using various immunoassay methods. The ability of various nitrated proteins to competitively inhibit the binding of antinitrotyrosine antibody to immobilized nitrated albumin was found to vary widely, with 50% inhibitory concentration (IC₅₀) values ranging over two orders of magnitude (17), illustrating the limited quantitative value of such ELISA procedures in determining the extent of protein tyrosine nitration. More quantitative analytical assay procedures have therefore been designed, using high-pressure liquid chromatography (HPLC)- or gas chromatography/mass spectroscopy (GC/ MS)-based methods. Although formation of 3-nitrotyrosine can easily be followed in experimental model systems using HPLC with ultraviolet (UV) detection to monitor the intrinsic absorptivity of aromatic amino acids (20), the sensitivity and selectivity of this technique is limited, and it does not allow the accurate determination of endogenous levels of 3-nitrotyrosine. More sensitive HPLC procedures, using coulometric electrochemical (EC) array detection, have been more successfully applied to the simultaneous detection of 3-nitrotyrosine and other tyrosine modifications in tissues (21). An alternative HPLC procedure involves prior derivatization of 3-nitrotyrosine with 4-fluoro-7-nitrobenzo-2-oxa-1,3-diazole and analysis through fluorescence detection (22). With this procedure, plasma levels of free 3-nitrotyrosine in healthy human subjects were found to be in the range of 30 nM. A recently developed HPLC procedure with EC detection is based on the derivatization of 3-nitrotyrosine to yield N-acetyl-3-aminotyrosine by acetylation, followed by solvent extraction, O-deacetylation, and dithionite reduction. A major advantage of this procedure is that the solvent extraction step results in substantial sample cleanup, thereby eliminating many sources of interference. Moreover, the resulting amino-derivative is EC active with a low oxidation potential, allowing its selective monitoring by EC detection (23). With this HPLC/EC-based approach, protein-associated nitrotyrosine levels in normal rat plasma were estimated to be approximately 0.3 µmol/mol tyrosine, or roughly 5 nM. Treatment of rats with a single intraperitoneal injection of the proinflammatory agent zymosan was found to result in a 30- to 40-fold increase in protein-associated 3-nitrotyrosine after 2 wk.

GC/MS-based approaches to assaying 3-nitrotyrosine have been more troublesome, since derivatization of 3-nitrotyrosine by most common derivatization procedures is relatively ineffective (24). Difficulties in derivatizing the phenolic hydroxyl group of 3-nitrotyrosine, caused by the strong electron-withdrawing properties of the nitro group, which results in incomplete derivatization and/or rapid hydrolysis, are often associated with poor chromatography and relatively low sensitivity. Nevertheless, the assay of n-propyl-heptafluorobutyryl derivatives with GC/MS has been successfully used to detect increased levels of 3-nitrotyrosine in low-density lipoproteins (LDLs) isolated from human atherosclerotic lesions (25). Quantitation by stable isotope dilution has led to the estimation that LDL-associated 3-nitrotyrosine is present at levels ranging from approximately 10 µmol/mol tyrosine to values about 100-fold higher than this in advanced atherosclerotic lesions, indicating the broad range of protein nitration that occurs under pathophysiologic conditions. A recent attempt to circumvent problems in analyzing 3-nitrotyrosine with GC/MS has involved dithionite reduction of 3-nitrotyrosine prior to derivatization, since the resulting n-propyl-tris-heptafluorobutyryl derivative can be analyzed with great sensitivity by using GC/MS with negative-ion chemical ionization (26). The recent development and improvement of analytical methodology for the measurement of in vivo (protein) tyrosine nitration is expected to soon yield more quantitative and comparative data with respect to this process.

Methods of protein extraction and acid hydrolysis commonly utilized to establish the 3-nitrotyrosine content in proteins present considerable sources of artifactual nitration when insufficient care is taken to eliminate contaminating NO₂ or NO₃ from the sample. At acidic pH, protonation of NO₂ and/or NO₃ causes significant nitration, which should be avoided in the processing of biologic samples. Rigorous removal of these contaminants has been shown to dramatically reduce measured levels of 3-nitrotyrosine and to better reflect the extent of nitration occurring in vivo (23). Since the efficiency of acid-catalyzed NO₂- and NO₃-driven nitration is high, failure to attend to their removal can result in detection of 3-nitrotyrosine at levels that reflect the levels of these generally unreactive end products of NO^· metabolism, rather than the reactive nitrogen oxides that are proposed to be responsible for endogenous nitration.

Although HPLC or GC/MS procedures may provide more quantitative information about the extent of tyrosine nitration or other protein modifications, they do not establish which proteins present preferential targets for nitration or which amino acid residues are targeted, nor do they provide information about the regional anatomic sites at which tyrosine nitration occurs. Immunofluorescence techniques or Western blot analysis with antinitrotyrosine antibodies will address these issues more directly, and should therefore be viewed as complementary tools for disclosing the extent and localization of tyrosine nitration in vivo. Other complementary techniques, such as HPLC-coupled electrospray ionization/tandem mass spectrometry (27, 28), will be useful in the determination of sites of nitration within proteins. However, it should again be emphasized that in each case extreme care should be taken to avoid artifactual nitration during sample processing.

Protein Tyrosine Nitration in the Respiratory Tract: What Are the Culprits?

A summary of several recent observations of 3-nitrotyrosine in animal models of respiratory tract diseases or in diseased human lung tissues is presented in Table 2, which indicates that tyrosine nitration is a general feature observed in a wide range of inflammatory-immune processes, conditions known to be associated with increased oxidative stress. Because of the nearly diffusion-limited rate of reaction of NO^· with ·O₂, and the ability of the reaction product ONOO to readily induce tyrosine nitration in vitro, it is often assumed that detection of 3-nitrotyrosine in vivo indicates local formation of ONOO. However, based on reaction mechanisms involved in aromatic nitration, and the multiple metabolic fates of NO^·, especially during inflammatory processes, it is premature to infer that ONOO is the exclusive source of tyrosine nitration in vivo, and alternative reaction mechanisms need to be considered.

Tyrosine nitration via radical mechanisms. Chemical studies of tyrosine nitration by ONOO have indicated that such nitration is promoted in the presence of ferric heme proteins, SOD, or Fe(III)ethylenediamine tetraacetic acid (Fe[III]EDTA) (5), in a process presumed to involve heterolytic cleavage of the peroxide bond in ONOO to produce a nitryl cation (NO₂⁺)- like intermediate. Nitryl cation (NO₂⁺) is classically known to nitrate aromatic rings by electrophilic aromatic substitution (41), although nitration of activated aromatic rings (such as phenolic substrates) instead proceeds via an electron-transfer mechanism followed by combination of the resulting radical pair (42). Because NO₂⁺ has an extremely short lifetime in aqueous solution, owing to its rapid hydrolysis to NO₃ (t_1/2  1.4 ns; [43]), formation of free NO₂⁺ is unlikely to contribute significantly to nitration reactions in biologic systems. Direct reaction of ONOO (or its conjugate acid ONOOH) with phenolic substrates such as tyrosine also occurs via an electron-transfer mechanism, initially generating a phenoxyl radical (which is in resonance with the more stable, carbon-centered tyrosyl radical), and H₂O and NO₂^· as products (Figure 1). Radical combination reactions will then yield 3-nitrotyrosine and 3,3'-dityrosine, with formation of the latter being favored at high relative concentrations of tyrosine because of additional formation of tyrosyl radicals by reaction of NO₂^· with tyrosine (Figure 1, Reaction 4) (43, 44). Cross-linking of tyrosine residues in proteins by such a mechanism will be less favorable because of kinetic and steric constraints. Evidence for the formation of (protein) tyrosyl radical intermediates by ONOO was recently demonstrated in intact biologic systems such as human blood plasma, through the use of electron paramagnetic resonance (45). The facile reaction of ONOO with ferric heme centers, such as in various peroxidases, results in formation of a Fe(IV)=O intermediate plus NO₂^· (46, 47), both of which are potent oxidants capable of oxidizing and/or nitrating tyrosine via one-electron oxidation mechanisms, in accord with observed increased nitration yields by ONOO in the presence of Fe(III)EDTA or heme peroxidases (5). Hence, irrespective of the initial nitrating species (ONOO, NO₂⁺, NO₂Cl, NO₂^·), the mechanism of phenolic nitration by these species proceeds via electron transfer, with the intermediate formation of tyrosyl radicals and NO₂^· (except when NO₂^· itself is the initial oxidant, in which case NO₂ is formed as a coproduct [44]).

View larger version (14K): [in this window] [in a new window]   Figure 1.   Mechanism of reaction of ONOO with tyrosine. Peroxynitrite (ONOO) reacts with tyrosine in a first-order reaction via an electron transfer mechanism to form tyrosyl radicals and nitrogen dioxide (NO2·) as products (Reaction 1). Subsequent radical combination reactions then yield 3-nitrotyrosine and 3,3'-dityrosine as products (Reactions 2 and 3). Reaction of NO2· with tyrosine also results in formation of tyrosyl radicals, thus favoring the formation of dityrosine over nitrotyrosine when tyrosine is present in excess.

This tyrosine nitration mechanism predicts that 3-nitrotyrosine can theoretically be formed under any condition in which tyrosyl radicals and/or NO₂^· are generated simultaneously. There is increasing evidence for the presence of stable or transient protein radicals (including tyrosyl radicals) in a large variety of enzymes, such as ribonucleotide reductase, prostaglandin H synthase, cytochrome c peroxidase, photosystem II, and galactose oxidase (48), and these radicals may present susceptible sites for nitration by NO₂^·. Furthermore, a wide variety of heme peroxidases utilize free tyrosine or protein tyrosine residues in the synthesis of protein cross-links (e.g., by horseradish peroxidase or ovoperoxidase), or in the synthesis of thyroid hormone by thyroid peroxidase (51). Chemical studies have also indicated that aromatic substances such as tyrosine can act as substrates for the mammalian peroxidases myeloperoxidase (MPO), eosinophil peroxidase, and lactoperoxidase (LPO), although the physiologic significance of this is still unknown. The presence of large amounts of MPO or eosinophil peroxidase in various leukocytes that participate in inflammatory-immune processes incites local oxidation of tyrosine residues at sites of leukocyte activation, as has been demonstrated indirectly via detection of 3,3'-dityrosine in inflamed tissues (52). Although NO₂^· is capable of directly inducing nitration of tyrosine residues (44), a role for NO₂^· in tyrosine nitration in vivo is often ignored, on the basis of the argument that nitration requires two NO₂^· molecules per tyrosine residue (Figure 1, Reactions 2 and 4), which renders such a mechanism relatively inefficient. However, because protein tyrosyl radicals can be generated by various distinct mechanisms not necessarily involving NO₂^·, and may be relatively long-lived, with half-lives of several seconds, tyrosine nitration by NO₂^· via diffusion-limited radical combination with tyrosyl radicals (43, 44) may be a highly feasible event under inflammatory conditions. Additionally, radical combination reactions of NO^· with protein tyrosine radicals has been proposed to evenually result in tyrosine nitration, as has been shown in prostaglandin H synthase (48).

Formation of nitrating intermediates from NO₂. Various biochemical pathways may cause the formation of NO₂^· in vivo, including autoxidation of NO^· by reaction with O₂ (although this is presumed to be relatively insignificant at physiologic levels of NO^·), one-electron reduction of ONOO by various biomolecular targets (e.g., antioxidants, thiols, aromatic amino acids, unsaturated lipids) (13), and one-electron oxidation of the NO^· metabolite NO₂. Furthermore, NO₂ can be protonated under acidic conditions to nitrous acid (HNO₂; pK_a = 3.4), which can subsequently decompose to NO^· and NO₂^· and thereby induce both nitrosation and nitration of endogenous proteins (24). This latter mechanism may be particularly significant in acidic environments such as in gastric compartments or phagosomes, or under conditions of acidosis as a result of tissue anoxia.

Nitrite does not accumulate in vivo but is further metabolized to NO₃ by a mechanism involving various heme proteins, and NO₂^· may be generated as an intermediate (18). Recent investigations have established that activation of various heme peroxidases (MPO or LPO) by H₂O₂ can promote oxidation of NO₂ to intermediates that are capable of nitrating aromatic substrates and proteins (18). The biologic significance of such NO₂ oxidation mechanisms depends on whether NO₂ effectively competes with other known physiologic substrates (e.g., chloride [Cl], bromide [Br], or thiocyanate [SCN]) for oxidation by these heme peroxidases. Studies with purified enzymes have indeed shown that pathophysiologic levels of NO₂ are metabolized by MPO or LPO, as indicated by detectable aromatic nitration in the presence of either Cl or SCN. Moreover, reduction of MPO compound II by NO₂ promotes enzyme turnover and enhances MPO-mediated oxidation of Cl (18), as illustrated in Figure 2. Other oxidants that can oxidize NO₂ to nitrating intermediates include hydroxyl radical (·OH) and hypochlorous acid (HOCl), the product of MPO-catalyzed Cl oxidation (18, 53). Oxidation by HOCl of NO₂ to NO₃ was recently determined to proceed via formation of reactive chlorinated intermediates (nitryl chloride [ClNO₂] or chlorine nitrite [ClONO]) that are capable of inducing both aromatic nitration and chlorination (53).

View larger version (17K): [in this window] [in a new window]   Figure 2.   Schematic representation of NO2 oxidation by myeloperoxidase. The ferric (FeIII) heme center of myeloperoxidase is oxidized by hydrogen peroxide to an FeIV=O/porphyrin radical cation (Compound I), which can oxidize various substrates by either one- or two-electron mechanisms, yielding the FeIV=O species (Compound II) or the native ferric enzyme, respectively. MPO Compound II can also be reduced by NO2 to the native ferric (FeIII) enzyme, thereby enhancing MPO-catalyzed oxidation of other substrates such as Cl.

The significance of these various oxidative processes depends on local levels of NO₂, which is normally present in the micromolar range within various extracellular fluids (0.5 to 3.6 µM in plasma, 0.4 to 60 µM in gastric juice, 30 to 210 µM in saliva, and 10 to 15 µM in alveolar epithelial lining fluids [18, 19], and 10 to 40 µM in nasal epithelial lining fluids [A. van der Vliet, unpublished results]), but extracellular NO₂ levels are markedly increased during active inflammation because of enhanced NO^· synthesis (18, 19), making such NO₂ oxidation pathways more feasible. Studies with isolated polymorphonuclear neutrophils (PMN) have indicated that activation of PMN in the presence of NO^·-donor molecules results in phenolic nitration that involves MPO-dependent NO₂ oxidation rather than ONOO, even though the latter may have been formed initially via radical combination between NO^· and ·O₂ (19). Unequivocal evidence for NO₂ oxidation under these conditions was found through similar experiments performed in the presence of ¹⁵NO₂, which showed enhanced incorporation of ¹⁵N into aromatic substrates (19). Aromatic nitration of substrate molecules or within proteins from cultured respiratory tract epithelial cells by MPO/H₂O₂/NO₂ (using oxidant-generating systems) was found to be relatively more efficient than aromatic nitration by comparable fluxes of ONOO, generated from the donor compound 3-morpholinosydnonimine (SIN-1) (18, 44, and unpublished results). Furthermore, although tyrosine nitration by ONOO has been well established in chemical experiments, continuous generation of ONOO through the use of NO^· and ·O₂-generating systems under physiologic conditions failed to induce significant aromatic nitration (54).

Evidence for involvement of MPO in tyrosine nitration in vivo. Table 2 indicates that 3-nitrotyrosine is commonly detected in inflammatory/infectious diseases characterized by the infiltration and activation of leukocytes. Indeed, positive immunostaining with antinitrotyrosine antibodies is often observed in and around neutrophils and monocytes, as well as macrophages and epithelial cells (15, 34, 39). Furthermore, a striking correlation between the extent of tyrosine nitration and the presence of inflammatory cells was observed in patients with idiopathic pulmonary fibrosis (IPF), with both the nitration and cell levels being relatively high in early to intermediate stages of IPF but markedly reduced in end-stage IPF (35). Hence, these various observations strongly implicate a role for inflammatory cells (neutrophils, monocytes, macrophages) in the generation of nitrating intermediates. Although the observed tyrosine nitration was generally attributed to ONOO in these cases, oxidation of NO₂ by neutrophil- derived MPO, or by other, related peroxidases could easily be invoked in any of these conditions. Similary, support for MPO-dependent oxidation and nitration mechanisms has also been found in other inflammatory conditions, including atherosclerosis (52) and neurodegenerative diseases such as multiple sclerosis (55), both of which are conditions associated with the presence of substantial amounts of active MPO (52, 56). Involvement of MPO-dependent oxidative processes in vivo has furthermore been established by the detection of 3-chlorotyrosine, a characteristic product of HOCl-mediated oxidation, and 3,3'-dityrosine in atherosclerotic lesions (52). With the recent development and refinement of sensitive analytical techniques, it can be expected that other protein modifications characteristic of MPO activity will be detected in many tissues in which increased concentrations of 3-nitrotyrosine have been found.

In summary, formation during inflammatory-immune processes of NO^·-derived reactive nitrogen species that are capable of inducing aromatic nitration involves multiple distinct reaction mechanisms. Although evidence for the formation of ONOO in vivo is compelling (4), any contribution of ONOO to aromatic nitration in vivo would most likely occur indirectly, via kinetically favored reactions of ONOO with CO₂ or with metalloproteins such as MPO (43, 46), both of which are known to promote ONOO-induced nitration of aromatic substrates (4, 5). Alternatively, many reactions of ONOO (e.g., with nucleophilic agents such as thiols and selenium-containing compounds) generate NO₂, which in turn becomes a substrate for oxidation reactions by MPO or related peroxidases (18, 19). Hence, it appears that MPO or other heme peroxidases may play a central role in the formation of nitrating intermediates at sites of active inflammatory- immune processes, as schematically illustrated in Figure 3. Future studies with MPO-deficient animals or selective peroxidase inhibitors will be needed to unequivocally establish the importance of MPO or related peroxidases in aromatic nitration reactions in vivo.

View larger version (28K): [in this window] [in a new window]   Figure 3.   Formation of NO·-derived oxidants by neutrophils. Stimulation of neutrophils results in activation of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to form ·O2 and secretion of MPO into the phagosome or the extracellular space. Simultaneous production of NO· either by neutrophils or adjacent cells causes formation of ONOO or NO2, which can either activate or act as a substrate for MPO.

	TYROSINE NITRATION IN RESPIRATORY TRACT DISEASE: MARKER OR MEDIATOR?

TOP INTRODUCTION REACTIVE NITROGEN SPECIES AND... FORMATION OF NITROTYROSINE: A... TYROSINE NITRATION IN... WHAT IS NEEDED? REFERENCES

Although there is ample evidence for the formation of 3-nitrotyrosine in a number of inflammatory diseases, as well as for other modifications of tyrosine, such as the formation of 3-chlorotyrosine or 3,3'-dityrosine, it is not yet clear whether such modifications contribute significantly to the pathobiology of respiratory tract disease. Despite suggested relationships between tyrosine nitration and cytotoxicity and/or apoptosis (4, 57, 58), tyrosine nitration does not always parallel cell toxicity. For instance, the bactericidal activity of ONOO is dramatically reduced in the presence of CO₂, in coincidence with decreases in most oxidative modifications brought about by ONOO, even though radical-induced modifications such as aromatic nitration are enhanced (43). Additionally, toxicity of MPO/H₂O₂ to A431 epidermoid carcinoma cells was found to be inhibited in the presence of 0.1 to 1 mM NO₂, despite increased nitration of tyrosine in cellular proteins (A. van der Vliet, unpublished results). Although tyrosine nitration may not be directly involved in acute cytotoxicity or apoptosis (59), such irreversible modifications might have a long-lasting impact on a number of cellular pathways that affect processes involved in cell proliferation or differentiation. Nitration of protein tyrosine residues, or related modifications (e.g., nitration of guanine and the formation of abasic sites, which may result in DNA mutations [60, 61]), might be involved in disturbances of cell signaling pathways and/or processes linked to mutagenesis and carcinogenesis.

Protein chemists have extensively used the nitrating agent tetranitromethane to investigate the location and essentiality of tyrosine residues in a large number of proteins and enzymes (5), and such studies have indicated that nitration of key tyrosine residues is often associated with a loss of either enzyme or protein function. Similarly, studies with isolated tyrosine kinase systems, in which kinase substrates were chemically nitrated or tyrosine residues were replaced with 3-nitrotyrosine, have shown that tyrosine nitration results in inhibition of tyrosine phosphorylation (62, 63). However, the significance of such modifications in vivo remains unknown, since efforts to demonstrate a direct causal relationship between tyrosine nitration and phosphorylation in intact cellular systems have produced inconclusive results (63). Recently, effects of ONOO on tyrosine phosphorylation in erythrocytes were found to be biphasic, with low concentrations of ONOO causing stimulation of tyrosine phosphorylation of the cytoplasmic domain of band 3, presumably via inhibition of tyrosine phosphatase activity, and higher concentrations of ONOO causing inhibition of tyrosine phosphorylation, which was accompanied by nitration of tyrosine residues (65). Exposure of A431 epidermoid carcinoma cells to nontoxic levels of ONOO was found to cause nitration within the epidermal growth factor receptor (EGFR), but this was not accompanied by significant decreases in EGF-stimulated receptor autophosphorylation on tyrosine residues. Moreover, although EGF-stimulated tyrosine phosphorylation of the target protein phospholipase C-1 was temporally inhibited after exposure to ONOO, this was not accompanied by significant nitration of the enzyme (66). Because the overall extent of tyrosine nitration in vivo may only amount to one nitrotyrosine per 10³ tyrosine residues (23, 25), it appears unlikely that tyrosine nitration in vivo significantly impairs signaling pathways involving tyrosine phosphorylation, unless specific tyrosine kinase substrates are preferential targets for nitration. Conversely, tyrosine nitration might in fact mimic tyrosine phosphorylation or adenylation (67) and thereby represent a more irreversible signaling mechanism, although experimental evidence for this notion is marginal.

In several studies with purified enzymes, ONOO was found to be a potent and selective inhibitor of prostacyclin (prostaglandin I₂ [PGI₂]) synthase (49), and nitration of a critical tyrosine residue in the active site of this enzyme via initial interaction of ONOO with the heme-thiolate center was presumed to be responsible for this inhibition. Similarly, ONOO has been found to be capable of activating prostaglandin H₂ (PGH₂) synthase by serving as a hydroperoxide substrate for the peroxidase activity of this enzyme (68). However, the protein radical formed on tyrosine 385 during PGH₂ synthase activation is a susceptible target for NO^·-derived oxidants, resulting in nitration of the tyrosine residue and inactivation of cyclooxygenase activity (48). Collectively, these findings suggest that ·O₂ may serve as a biochemical link between NO^· and prostaglandin synthesis, and that ONOO-mediated tyrosine nitration may represent an important mechanism in the regulation of arachidonate metabolism. However, no direct evidence has so far been presented for tyrosine nitration within these enzymes in vivo.

Cytoskeletal proteins such as actin or neurofilaments have been suggested to represent important targets for tyrosine nitration reactions, since they are abundant proteins and contain several tyrosine residues that are involved in their structural assembly. Indeed, stimulation of rat pulmonary microvascular smooth-muscle cells with interleukin-1 was recently found to result in nitration of primarily cytoskeletal proteins (69). Chemical nitration of actin or neurofilaments has been shown to disrupt assembly of these proteins, and modification of only a few protein subunits appears necessary to cause disruption of a structure involving thousands of subunits (5). Also, extensive tyrosine nitration has been shown to occur in the myocardium during inflammatory forms of myocarditis. This nitration was suggested to occur primarily in actin, although it is still unclear whether the nitration plays a pathophysiologic role in mediating myocardial dysfunction (70). Interestingly, free 3-nitrotyrosine was recently found to be a competing substrate for tubulin tyrosine ligase, an enzyme that post-translationally incorporates L-tyrosine into -tubulin. Nitrotyrosination of -tubulin was found to induce changes in microtubule organization and cell morphology, and to affect the intracellular distribution of motor proteins (71).

More directly relevant to the respiratory tract is the finding that ONOO and related reactive nitrogen species can nitrate tyrosine residues in surfactant protein A (SP-A), and that such modifications are causatively linked to the decreased ability of SP-A to aggregate lipids or bind to mannose receptors present on immune effector cells (72, 73). Given the proposed role of SP-A in host defense, such irreversible modification may compromise its function to facilitate phagocytic uptake and killing of bacteria. However, the significance of tyrosine nitration in SP-A during inflammatory diseases of the respiratory tract is unclear, since evidence of nitration of SP-A in vivo is still lacking. In fact, despite the detection of increased levels of 3-nitrotyrosine in various respiratory tract diseases (Table 2), the precise protein targets undergoing tyrosine nitration are largely unknown. Subfractionation of rat liver tissue has shown more extensive protein nitration in mitochondrial fractions than in cytosolic fractions or in whole-tissue homogenates, suggesting increased nitration at sites of increased superoxide formation. Future efforts to identify specific targets for protein nitration or to colocalize protein nitration with NOS or enzymatic systems that are believed to catalyze nitration (such as heme peroxidases) can be expected to reveal functional consequences of protein nitration in vivo.

Related tyrosine modifications, such as chlorination or dimerization, may similarly affect cellular pathways that rely on critical tyrosine residues. Recently, covalent homodimerization of the EGF receptor was observed in epithelial cells exposed to ONOO or to H₂O₂ in the presence of heme peroxidases, via putative formation of dityrosine cross-links (66). Such irreversible cross-linking might result in aberrant receptor activation, and might thus affect regulation of cell growth and/or differentiation. With the increasing availability of sensitive and reliable techniques to measure and characterize these various modifications (23, 26), our understanding of their potential role in tissue pathobiology will continue to increase.

	WHAT IS NEEDED?

TOP INTRODUCTION REACTIVE NITROGEN SPECIES AND... FORMATION OF NITROTYROSINE: A... TYROSINE NITRATION IN... WHAT IS NEEDED? REFERENCES

Involvement of induced NO^· synthesis in respiratory tract disease is often indicated by analysis of its metabolic end products NO₂ and NO₃. However, suggestions that NO₂ primarily represents autoxidation of NO^·, whereas NO₃^- is formed via intermediate formation of ONOO^- and isomerization, are simplistic and should be viewed with caution. For instance, oxidation of NO₂ via oxyhemoglobin or other (pseudo)peroxidase-catalyzed reactions, either intracellularly or extracellularly, yields NO₃, whereas many reactions of ONOO with cellular constituents (such as thiols or selenium-containing compounds and/or enzymes) generate NO₂ instead of NO₃. Furthermore, the lack of knowledge about the localization of NO^· production or oxidation, or about cellular transport mechanims for either NO₂ or NO₃, further precludes any predictions about cellular NO^· metabolism based on extracellularly measured levels of NO₂ and NO₃. Attempts to estimate NO^· production within the respiratory tract by quantitation of NO^· in exhaled air or by indirect measurement of either NO₂ and NO₃ levels in breath condensates, lavage fluids, or induced sputa are cumbersome because of incomplete understanding of the biochemical fates of NO^· within the respiratory tract. For instance, production of reactive oxygen intermediates during inflammatory-immune processes may accelerate NO^· metabolism and thereby reduce levels of exhaled NO^·, despite increased NO^· production. A more complete understanding of the biochemical fates of NO^· within the respiratory tract, especially during local inflammatory-immune processes, will be needed to better evaluate such measurements.

Studies with iNOS-deficient mice have not offered an unambigious picture with regard to whether induced synthesis of NO^· during inflammatory processes confers resistance against invading pathogens or aggravates host tissue injury. Careful monitoring of characteristic products of NO^·-mediated reactions, such as nitroso- or nitro- adducts in various biomolecules (e.g., 3-nitrotyrosine), will contribute to a better understanding of the metabolic fates of NO^· produced in the respiratory tract and to its biochemical effects. Moreover, identification of reaction mechanisms resulting in protein nitration in vivo will allow the development of better intervention strategies (i.e., should we be designing suicide inhibitors for MPO or specific "scavengers" of ONOO?). Approaches with transgenic animals deficient in MPO and/or iNOS are likely to contribute to resolving this important issue. Conversely, because these various oxidant mechanisms may be essential in host defense, intercepting these pathways might result in increased susceptibility to infectious diseases. Indeed, the potential significance of interactions between NO^·-derived metabolites and MPO in host defense has been underscored by findings that cytokine-stimulated human PMNs contain increased levels of iNOS colocalized with MPO in primary granules and nitrated tyrosine residues around ingested bacteria, observations consistent with MPO-dependent NO₂ oxidation (74). However, a role for NO₂ in host defense is still equivocal, since NO₂ inhibits the bactericidal activity of the MPO-derived oxidant HOCl (75) and studies with phagocytosed aromatic probes failed to detect aromatic nitration within the phagolysosome in the presence of NO₂ (76). Oxidation of NO₂ to more bactericidal intermediates by lactoperoxidases in saliva or in airway secretions (77) may also contribute to antimicrobial mechanisms in the oral cavity or in the upper respiratory tract.

In order to demonstrate a role for protein nitration in respiratory tract disease, identification of specific protein targets for nitration is of crucial importance. For instance, tyrosine nitration might occur in extracellular matrix proteins or within respiratory tract mucus, as demonstrated in cystic fibrosis (37), and this may protect more crucial cellular constituents against similar modifications. Hence, it remains to be established whether 3-nitrotyrosine and related modifications are merely biomarkers of inflammatory oxidants or whether they actively contribute to cellular dysfunction and the development of tissue injury associated with acute and chronic inflammation. In this respect, the recent discovery of apparent enzymatic "nitrotyrosine dinitrase" activity in rat spleen and lung homogenates (78) may further hint at the potential significance of nitration (and dinitration) as a signaling mechanism. Increased efforts to colocalize 3-nitrotyrosine with iNOS and/or with enzymatic systems that are thought to catalyze aromatic nitration (or dinitration) will be required to obtain further support for this notion. Future investigations, aimed at the identification of tyrosine residues most susceptible to nitration (e.g., surface, active site, protein-protein interaction sites) in relation to effects on protein and cellular function, are also needed. Preliminary data suggest that the spatial proximity of tyrosine to acidic amino acid residues may promote tyrosine nitration (6), but more research on this issue is warranted. Correspondingly, further investigations of the pathophysiologic relevance of similar modifications in lipids or DNA bases, that might result in the formation of bioactive products or in DNA mutations, are expected to reveal additional characteristic signaling mechanisms associated with nitrosative stress. In analogy to the proposed physiological role of other NO^·-dependent modifications such as S-nitrosation (e.g., 2, 4, 11), aromatic nitration of specific cellular targets may represent a previously unrecognized unique signaling mechanism activated during conditions of nitrosative stress accompanying inflammatory processes.

	Footnotes

Correspondence and requests for reprints should be addressed to Dr. A. van der Vliet, Center for Comparative Respiratory Biology and Medicine, University of California, Davis, 1121 Surge I Annex, Davis, CA 95616. E-mail: avandervliet{at}ucdavis.edu

(Received in original form July 9, 1998 and in revised form December 14, 1998).

Acknowledgments: Supported by the National Institutes of Health (Grants HL 57452 and HL 60812), the American Lung Association of California, and the Cystic Fibrosis Foundation.

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