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Section on Oxidative Stress Tissue Injury, Laboratory of Physiologic Studies, National Institutes of Health, National Institute of Alcohol Abuse and Alcoholism, Bethesda, Maryland; Linus Pauling Institute, Department of Biochemistry and Biophysics, 2011 Agricultural and Life Sciences, Oregon State University, Corvallis, Oregon; and Department of Intensive Care Medicine, University Hospital, Lausanne, Switzerland
ABSTRACT I. INTRODUCTION II. NITRIC OXIDE A. Discovery of Nitric Oxide as a Biological Molecule B. The Selective Reactivity of Nitric Oxide C. Diffusion and Signaling Properties of Nitric Oxide D. Cytotoxic Effects of Nitric Oxide E. Superoxide and the Hydroxyl Radical Theory of Radical Damage III. PEROXYNITRITE A. Historical Perspectives B. Biological Chemistry C. The Reverse Reaction of Peroxynitrite to Form Superoxide and Nitric Oxide IV. PEROXYNITRITE-INDUCED CYTOTOXICITY A. Mechanisms of Peroxynitrite-Mediated Oxidations B. Biological Targets of Peroxynitrite 1. Proteins A) REACTIONS OF PEROXYNITRITE WITH TRANSITION METAL CENTERS. B) REACTIONS OF PEROXYNITRITE WITH AMINO ACIDS. 2. Lipids 3. Nucleic acids C. Peroxynitrite, Mitochondria, and Cell Death 1. Peroxynitrite and apoptosis 2. Peroxynitrite and necrosis: the role of PARP V. PEROXYNITRITE AND CELL SIGNALING A. An Overview of Cell Signal Transduction B. Modulation of Cell Signaling by Peroxynitrite 1. Inhibition of phosphotyrosine-dependent signaling 2. Activation of phosphotyrosine-dependent signaling 3. Peroxynitrite and MAPK signaling A) ERK PATHWAY. B) JNK PATHWAY. C) P38 MAPK. 4. PI3K/protein kinase B (Akt) pathway 5. PKC pathway 6. NFkappaB VI. NITRIC OXIDE AND PEROXYNITRITE IN DISEASE A. Nitric Oxide and Peroxynitrite in Cardiac Diseases 1. Cardiovascular effects of peroxynitrite in vitro and in vivo, potential targets of peroxynitrite-induced toxicity in the cardiovascular system 2. Myocardial I/R injury 3. Preconditioning, postconditioning, and development of nitrate tolerance 4. Myocarditis, transplant coronary artery disease, and cardiac allograft rejection 5. Chronic heart failure B. Nitric Oxide and Peroxynitrite in Vascular Diseases 1. Atherosclerosis and restenosis 2. Aging 3. Hypertension C. Nitric Oxide and Peroxynitrite in Circulatory Shock 1. Mechanisms of tissue injury by peroxynitrite during circulatory shock 2. Role of peroxynitrite in the pathophysiological alterations of circulatory shock A) PERIPHERAL VASCULAR FAILURE. B) VASCULAR ENDOTHELIAL DYSFUNCTION. C) MYOCARDIAL DEPRESSION. D) SYSTEMIC INFLAMMATION. E) TISSUE LEUKOCYTE SEQUESTRATION. F) GUT MUCOSAL BARRIER FAILURE. D. Nitric Oxide and Peroxynitrite in Local Inflammation 1. Chronic arthritis 2. Inflammatory bowel diseases 3. Tissue inflammation from toxic origin E. Nitric Oxide and Peroxynitrite in Cancer F. Nitric Oxide and Peroxynitrite in Stroke and Other Forms of Reperfusion Injury 1. Stroke A) ROLE OF NO IN NORMAL BRAIN PHYSIOLOGY AND DURING ISCHEMIC STROKE. B) ROLE OF PEROXYNITRITE IN NO-DEPENDENT NEUROTOXICITY IN STROKE. 2. Other forms of reperfusion injury A) KIDNEY. B) GUT. C) LIVER. D) LUNG. G. Nitric Oxide and Peroxynitrite in Neurodegenerative Disorders 1. Multiple sclerosis (MS) 2. Parkinson's disease 3. Alzheimer's disease 4. Amyotrophic lateral sclerosis (ALS) 5. Huntington's disease 6. Traumatic brain injury H. Nitric Oxide and Peroxynitrite in Diabetes and Diabetic Complications 1. Primary diabetes 2. Diabetic cardiovascular dysfunction 3. Diabetic neuropathy, nephropathy, and retinopathy VII. CONCLUSIONS AND FUTURE PERSPECTIVES GRANTS ACKNOWLEDGMENTS REFERENCES
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I. INTRODUCTION |
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–) to form the much more powerful oxidant peroxynitrite (ONOO–) is a key element in resolving the contrasting roles of NO in physiology and pathology. Neither superoxide nor NO is particularly toxic in vivo because there are efficient means to minimize their accumulation (72, 74). Superoxide is rapidly removed by high concentrations of scavenging enzymes called superoxide dismutases (SOD) with distinct isoenzymes located in the mitochondria, cytoplasm, and extracellular compartments. NO is rapidly removed by its rapid diffusion through tissues into red blood cells (161, 639), where it is rapidly converted to nitrate by reaction with oxyhemoglobin (Fig. 1). This limits the biological half-life of NO in vivo to less than a second, whereas the concentrations of NO relevant for cellular signaling can persist in phosphate-buffered saline for an hour (79). However, when both superoxide and NO are synthesized within a few cell diameters of each other, they will combine spontaneously to form peroxynitrite by a diffusion-limited reaction (583). In essence, every time NO and superoxide collide, they form peroxynitrite. No enzyme is required to form peroxynitrite because no enzyme can possibly catalyze any reaction as fast. NO is the only known biological molecule that reacts faster with superoxide and is produced in high enough concentrations to outcompete endogenous levels of superoxide dismutase. Consequently, the kinetics and thermodynamics of the reaction of superoxide with NO make the formation of peroxynitrite inevitable in vivo.
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The formation of reactive nitrogen species is not an inescapable consequence of synthesizing NO. NO is efficiently removed by reacting with oxyhemoglobin to form nitrate, which prevents even the highest rates of NO synthesis from directly reacting with oxygen to form significant amounts of nitrogen dioxide. However, the simultaneous activation of superoxide synthesis along with NO will completely transform the biological actions of NO by forming peroxynitrite. Several enzyme complexes, such as NADPH oxidases (NADPHox) and xanthine oxidase, can be activated in many cellular systems to actively produce large amounts of superoxide. What happens when superoxide and NO are produced simultaneously in close proximity? Modestly increasing superoxide and NO each at a 10-fold greater rate will increase peroxynitrite formation by 100-fold. Under proinflammatory conditions, simultaneous production of superoxide and NO can be strongly activated to increase production 1,000-fold, which will increase the formation of peroxynitrite by a 1,000,000-fold (Fig. 2). Without superoxide, the formation of nitrogen dioxide by the reaction of NO with oxygen is miniscule by comparison. NO and superoxide do not even have to be produced within the same cell to form peroxynitrite, because NO can so readily move through membranes and between cells (Fig. 1).
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II. NITRIC OXIDE |
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The regulation and synthesis of NO by mammalian cells has been the focus of many excellent reviews (935), as have many of its physiological and pathological actions (595, 890, 893, 895, 1201). With around 80,000 references invoking NO listed in PubMed, it may be difficult to remember how controversial was the initial proposal that NO was a biological molecule. There were early indications that were ignored for many decades. Haldane and co-workers (56) reported in the 1920s of a man found dead of apparent carbon monoxide poisoning. However, analysis of the blood showed that the stable red color persisting in hemoglobin at autopsy was due to nitroso-hemoglobin rather than carboxy-hemoglobin (56). There was no evidence for the consumption of nitrite that might account for the nitroso-hemoglobin. Commoner and co-workers (1387) in the 1960s demonstrated the nitroso-heme signals could be detected by EPR spectroscopy. Tannenbaum's group (475), while investigating the role of nitrite and nitrosamines in food in the induction of carcinogenesis, characterized nitrite and nitrate metabolism in healthy student volunteers. Curiously, more nitrate was being secreted than could be accounted for by ingestion. When one of the students became ill with the flu, nitrate levels in urine increased enormously.
For a century, nitrovasodilators had been used clinically without understanding their mechanism of action. Alfred Nobel lamented the irony that he was taking nitroglycerin to treat angina after making his fortune developing dynamite. Increased levels of cGMP produced by guanylate cyclase within vascular smooth muscle were discovered to allow blood vessels to relax and thus increase blood flow (596). The endogenous agents responsible for activating guanylate cyclase remained mysterious, although nitrovasodilators were able to activate the enzyme. The enzyme contained a heme group that was essential for this activation. Murad's group (130) found that NO itself, as well as a variety of oxidants, was able to activate guanylate cyclase. However, the possibility that NO might be synthesized in mammals was considered to be too far fetched for another decade. The more likely candidates for activators of guanylate cyclase were considered to be nitroso thiols and possibility that NO, a major air pollutant, could be the endogenous regulator was generally dismissed.
Major progress came from the curiosity of Furchgott and Zawadzki (422) in understanding how a technician in his laboratory was able to isolate aortas that relaxed in vitro when exposed to acetylcholine. Acetylcholine was a well-known vasodilating agent when injected in vivo, but generally caused isolated blood vessels to constrict in vitro. Furchgott and Zawadzki (422) recognized that the difference was caused by mechanical damage to the vascular rings as they were being cut, which stripped the single layer of endothelial cells off the blood vessels. With the preparation of a spiral cut of blood vessels, the endothelium was preserved and responded by relaxing when acetylcholine was added. Furthermore, Furchgott and Zawadzki (422) established that acetylcholine-treated endothelium was releasing a diffusible factor that would relax endothelium-denuded blood vessels by activating guanylate cyclase. The diffusible factor, termed the endothelium-derived relaxing factor (EDRF), was quickly inactivated by oxyhemoglobin and was inherently unstable in the perfusion cascades used to study the vasorelaxation. EDRF had a half-life of only 6–8 s in Krebs-Henseleit buffer saturated with 100% oxygen and 30–60 s in the same buffer saturated with room air (21% oxygen). Addition of low concentrations of SOD to the perfusion cascade doubled the half-life of EDRF in Krebs-Henseleit buffer, while agents known to generate superoxide diminished its activity (484, 894, 1089). Consequently, the sensitivity to superoxide and oxyhemoglobin became standard criteria to verify the production of EDRF in isolated cells. Moreover, EDRF was proposed to be a protective factor by scavenging superoxide. In 1986, Furchgott proposed that NO was the elusive EDRF produced by endothelium, with Ignarro providing additional evidence supporting the identification (594, 596, 597). Moncada's group (892), adapting a gas-phase chemiluminescent detector used for monitoring NO pollution in the atmosphere, was able to directly measure NO produced in vivo.
The identification of NO as EDRF was greatly facilitated by the independent work of John Hibbs, who was investigating how macrophages kill cancer cells. Hibbs found that different batches of fetal calf serum had widely varying effects on the tumoricidal activity of macrophages in vivo. He observed in the local slaughterhouse that calf serum at the time was prepared from blood collected in buckets and deduced that endotoxin from contaminating bacteria was responsible for activating the macrophages. He also noticed that activated macrophages rapidly depleted the media of some nutrient necessary for their tumoricidal activity (542, 543). By supplementing the depleted media with each component, he discovered that arginine was the major compound that could restore the tumoricidal activity of macrophages. He also showed that the macrophages were producing nitrite and nitrate by oxidizing arginine to citrulline, but did not make the connection that macrophages may be producing NO and peroxynitrite. The production of NO by macrophages was established by Stuehr and Nathan (1208) and peroxynitrite by Ischiropoulos et al. (611). He deduced that arginine derivatives might be useful inhibitors and discovered that methylarginine blocked the tumoricidal activity of macrophages. Thus he provided the background for Moncada's group to show that EDRF was synthesized from arginine and that methylarginine blocked its biological activity.
A major difficulty in the identification of EDRF is the fact that endothelium produces relatively small amounts and is not an abundant cell in vivo. But the identification of EDRF as NO made sense of many puzzling results in the brain, where cGMP was known to be involved in many intracellular functions. Deguchi and Yoshioka (305) painstakingly purified a low-molecular-weight compound from nervous tissue that was required to activate soluble guanylate cyclase and showed that it was unexpectedly arginine. For many years, it had been recognized that the brain had all of the enzymes necessary to convert citrulline to arginine, but curiously lacked arginase that is necessary for the complete urea cycle to be functional. Although the urea cycle was not functional in the brain, genetic deficiencies in these enzymes often resulted in phenotypes with neurological defects. Garthwaite and co-workers (433, 435) connected the dots and showed that under normal physiological conditions the brain produced 
20 times more NO than the entire vasculature. Since NO synthase produces citrulline from arginine, the presence of the remaining urea cycle enzymes was rationalized. Later, he identified ODQ, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one, as a potent inhibitor that prevented the activation of guanylate cyclase by NO (436), which has proven to be a crucial tool for unraveling the physiological action of NO in vitro.
The brain proved to be a rich source of NO synthesis and allowed the first NO synthase (NOS) to be cloned and purified (133–135). It is called nNOS (neuronal NOS) or NOS1 as it was the first synthase to be cloned. Eventually, it was realized that the histological stain for NADPH diaphorase in brain corresponded to the distribution of nNOS (292, 568). The NOS1 gene has the most complex genomic organization in humans with multiple splice variants being produced (468, 1164). The first knock-out mouse still retained substantial NOS1 activity in muscle and some neuronal populations because of alternative splicing that bypassed the second exon that had been targeted for deletion (574, 1394). Consequently, several studies finding no protection in the knockout of NOS1 do not necessarily prove the lack of involvement of NO (375).
The second NOS to be cloned was isolated from macrophages and known as NOS2 or iNOS (inducible NOS) because it is readily induced in many tissues by proinflammatory cytokines. Unlike the neuronal or endothelial NOS isoenzymes, NOS2 is not regulated by changes in intracellular calcium. However, careful examination of purified preparations of iNOS revealed that calmodulin was so tightly bound that it copurified with iNOS (215, 1395). Consequently, the enzyme appeared to be unresponsive to physiologically relevant changes in calcium concentrations. Because NOS2 can be strongly induced by proinflammatory stimuli, it is often called a high-output source of NO. However, the enzyme does not produce NO at a substantially greater rate measured for neuronal or endothelial NOS, just more of the protein can transiently be induced and normal calcium levels are sufficient to fully activate NOS2 (934, 935).
The first source of NO identified, endothelial NO synthase was the last to be cloned and is known as eNOS or NOS3. NOS3 binds to plasma membranes and is typically associated with caveolin (387). It is strongly activated by the entry of calcium through membrane-bound receptors and is also regulated by phosphorylation (1122). NOS3 is also found in neurons and other tissues in addition to endothelium.
B. The Selective Reactivity of Nitric Oxide
To understand why NO itself is not highly reactive, it is instructive to remember that NO is an intermediate between molecular oxygen (O2) and nitrogen (N2) (Fig. 3). All three have low solubility and readily diffuse through membranes as easily as through cytoplasm. While molecular oxygen is thermodynamically a strong oxidant, it has a limited kinetic reactivity as evidenced by life being possible in an atmosphere of 21% oxygen without spontaneous combustion. Molecular oxygen has two unpaired electrons in separate orbitals, which prevents this biradical from directly reacting with most biological molecules. However, the unpaired electrons on molecular oxygen allow it to bind strongly with metals such as the iron in hemoglobin and in cytochrome-c oxidase. Molecular oxygen will also react quickly with the unpaired electron on other free radicals, which leaves the second unpaired electron available for further reactions: R· + ·OO· 
R-OO· further reactions.
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In contrast, nitrogen gas (N2) is one of the most inert molecules known. Because NO is essentially a hybrid between molecular nitrogen and oxygen, NO is generally less reactive than molecular oxygen. In particular, NO has only one unpaired electron, which allows it to bind strongly to the iron in heme groups, which is crucial to its biological activity of activating guanylate cyclase and slowing mitochondrial respiration by binding to cytochrome-c oxidase. Like molecular oxygen, NO also reacts with free radicals quickly, but NO is chain terminating. For example, NO can convert thiyl radicals into nitrosothiols by acting as a chain-terminating agent: RS· + ·NO RS-NO.
The chain-terminating reactions account for many of the antioxidant properties attributed to NO, but this is an oversimplification. NO does not directly repair radical damage as does ascorbate, tocopherol, or glutathione, but rather forms transient intermediate products that have distinct biological activities. These intermediate products can often be repaired by antioxidants to regenerate the original compound. Depending on one's point of view, nitrosative stress can also be viewed as an antioxidant activity.
C. Diffusion and Signaling Properties of Nitric Oxide
NO is often considered to be just another signaling molecule. But it is important to consider how NO communicates information to understand why NO has so many physiological roles in vivo. The production of cGMP by guanylate cyclase is the major signal transduction mechanism of NO. Soluble guanylate cyclase contains the same heme protoporphyrin IX as hemoglobin with iron in the ferrous form that binds NO with great affinity. Deoxyhemoglobin binds NO with a 10,000-fold greater affinity than molecular oxygen (1151, 1286). Only 5–10 nM NO is necessary to activate guanylate cyclase. NO can diffuse from where it is synthesized into surrounding cells where it will activate soluble guanylate cyclase in the target tissue to produce cGMP. In turn, cGMP activates cGMP-dependent kinases in the target tissue that modulates intracellular calcium levels to modulate many diverse activities in the target tissues.
However, the unique properties of NO confer an important distinction that allows NO to be used for local signaling in virtually every organ system. For many signaling molecules, receptors in target tissues can distinguish two closely related molecules like norepinephrine or epinephrine because the appropriate receptors can recognize subtle variations in shape. With only two atoms, NO cannot be readily distinguished by its shape. Thus information is reflected by changes in its local concentration. The longer NO is present, the greater the amount of cGMP that will be formed. The system depends on NO being constantly removed or else guanylate cyclase will remain fully activated.
The rapid infusion of NO has major implications for how information is communicated. NO is a small hydrophobic molecule that crosses cell membranes without channels or receptors as readily as molecular oxygen and carbon dioxide. The diffusion coefficient of NO in water at 37°C is slightly faster than oxygen and carbon dioxide (1377), which is ideal for quickly transmitting information over short distances. Because NO is freely permeable to membranes, NO will repeatedly diffuse in as well as out of a cell over the time span of a second. The average molecular velocity of a molecule with the mass of NO is 400 m/s at room temperature. The trajectory of NO in solution is repeatedly changed by making 10 billion collisions each second. Consequently, the path of a NO molecule will follow long but highly convoluted trails that can repeatedly cross cell membranes during its half-life of 1 s. A single molecule of NO can readily move between cells many times within this time span.
The hydrophobicity of NO will allow slightly faster diffusion in a lipid membrane than in water so that membranes provide effectively no barrier to NO. Hence, diffusion away from a single cell producing NO contributes far more substantially to the loss of NO than do the reactions of NO within the cell (728). When a group of cells simultaneously produces NO, the concentration of NO within a single cell will be much greater than if that cell was producing NO alone. Most of the NO one breathes when stuck in a traffic jam results from the surrounding cars rather than the car itself. An important consequence is that NO can integrate and average the activity of the local group of cells (728).
Red blood cells provide a drain for NO that creates a sharp diffusion gradient leading to the vasculature (161, 1294, 1385). The addition of a red blood cell outside of a cell will capture much of the NO produced inside of this cell, because the hemoglobin will greatly reduce the reentry of NO into the cell (728). For example, endothelium can make 10- to 40-fold more NO than needed to activate guanylate cyclase as measured by microelectrodes placed against individual isolated cells. However, red blood cells will be major sinks in vivo, so the majority of the NO will be quickly lost to the vascular compartment (196, 1293). In fact, the packaging of hemoglobin into red blood cells is important to limit the rate of scavenging of NO. Lysis of red blood cells causes vasoconstriction by more efficient scavenging of NO and is a major limitation of giving hemoglobin-based blood substitutes. Even with hemoglobin packaged into red blood cells, the production of 100 nM NO by endothelium would be necessary to achieve 5–10 nM concentrations in smooth muscle containing guanylate cyclase, because such a large fraction is consumed by red blood cells. NO that has diffused into the smooth muscle can also diffuse back down the diffusion gradient to the red blood cell (Fig. 1). In the perfusion cascades used to originally assay EDRF, the excess production of NO in the absence of hemoglobin allowed the dilation of endothelium-denuded artery rings far removed down the perfusion cascade.
Although the biological half-life of NO is only a scant few seconds in vivo, a second is long compared with a simple neural reflex or to the time needed to contract a muscle. A sprinter can run 10 m within the reported half-life of NO (far shorter than the total distance covered of a molecule of NO as it bounces around inside and between cells in the same time). The relatively short overall distance that NO can diffuse limits its action to only a few cells near the source of production. Thus NO produced in the gut, for example, will not influence its actions in the central nervous system (CNS). On the other hand, the intermediate lifetime of NO coupled with its rapid diffusion through most tissues allows NO to integrate and modulate complex physiological processes (1293).
In effect, NO is the equivalent of a shock absorber that dampens the oscillations of a car driving over a bumpy road. By affecting levels of calcium in target tissues through the actions of cGMP, NO can modulate the extent that target tissues respond to stress. When a distal vascular bed dilates to supply more blood flow to an actively working muscle, the upstream blood vessels must also dilate to support the increased blood flow. Exceeding the Reynolds number results in turbulent flow, increasing the local stress on blood vessels (479). The stress of turbulent flow is counterpoised by myogenic contraction that would cause a further increase in shear stress in the absence of endothelial production of NO. Left unopposed, further muscle contraction amplifies turbulent flow that in the extreme will cause a catastrophic collapse of blood flow to distal vascular beds. However, turbulence induces endothelium to synthesize NO via shear-induced stress. The resulting local relaxation of the underlying vascular smooth muscle increases the diameter of the blood vessel to restore laminar flow and thereby ensures a laminar distribution of blood between vessels (479). With prolonged turbulence, additional mechanisms are activated that can lead to the formation of superoxide and the pathogenic conversion of NO to peroxynitrite as described below.
NO can convey information by more subtle means than just simply its local concentration. The life span of NO is relatively long compared with the firing of nerve or even the activation of a neural network, and thus can help to integrate neuronal activity in small volumes of the brain (1385). Gally et al. (426) proposed that NO provides an important signal-averaging mechanism to control synaptic plasticity in the brain. The spatial organization of the nervous system is determined by the temporally correlated activation of neurons. A general principle controlling the organization of the developing brain is that groups of neurons activated in synchrony tend to project to similar regions, whereas neurons with uncorrelated activity will project into different regions (897). NO appears to be one determinant of this neuronal localization because it is ideally suited to carry information about neuronal activity in a retrograde manner, opposite to the normal mode of an activated neuron passing information down its axon and releasing a neurotransmitter across the synaptic junction. NO produced by dendrites will diffuse radially to surrounding synapses and will not distinguish between synapses in direct contact with that particular dendrite versus those that are localized in the same region (434, 1385, 1391).
The nNOS (NOS1) is particularly well suited to produce NO in a manner that facilitates synaptic plasticity (336, 1188). The amino terminus of NOS1 contains an additional sequence lacking on NOS2 and NOS3 that anchors the enzyme to the cytoskeleton in postsynaptic boutons beneath the N-methyl-D-aspartate (NMDA) receptor (547). The NMDA receptor has been implicated in learning and development as well as in many forms of excitotoxic neurodegeneration (149) (see sect. VI, F and G). The NMDA receptor only activates when a neuron has been partially depolarized, as occurs when a neuron has been firing frequently. In addition, the receptor must bind glutamate and glycine to the extracellular surface for the channel to open. A local group of neurons that are firing repeatedly for a few milliseconds is sufficient to cause local increase in NO. The synthesis of NO is initiated by extracellular calcium entering the neuron through the NMDA receptor. NO plays an important role in long-term potentiation, the most widely studied neuronal equivalent of learning in vitro (126, 1137).
Local neuronal activity can be temporally integrated because NO will only be synthesized by localized groups of neurons that had been depolarized through repeated activation that is necessary to open NMDA receptors (426). NO can help enhance the synaptic efficiency of surrounding axonal arbors of neurons that have been active, whereas the axonal arbors from neurons that have not been activated will be weakened. The ability to modulate local groups of neurons on a moderate time scale is one in the reasons why the CNS is a major source of NOS and why it undergoes radical changes in expression throughout development (154, 462, 647, 1188).
D. Cytotoxic Effects of Nitric Oxide
Although NO is reported to have many potentially toxic effects, many of them are more likely mediated by its oxidation products rather than NO itself. Thus NO does not directly attack DNA, as was initially believed, but this effect instead depends on its conversion into higher nitrogen oxides (1375). It also does not directly cause the ribosylation of glyceraldehyde-3-phosphate dehydrogenase (323), but rather reacts with a sulfhydryl anion in the active site of the enzyme (888). Furthermore, the early consideration that NO produced by activated macrophages would inactivate the iron/sulfur centers in tumor cell mitochondria (329, 542) has been reevaluated by a series of experiments indicating that the NO-dependent inactivation of iron/sulfur centers was in fact mediated by peroxynitrite (182, 523). Indeed, activated macrophages produce both NO and superoxide, so the inactivation of mitochondria in tumor cells could well have been mediated by peroxynitrite (1062).
NO may reversibly inhibit enzymes with transition metals or with free radical intermediates in their catalytic cycle. NO in micromolar concentrations will reversibly inhibit catalase and cytochrome P-450 (153, 1376). It also can inhibit ribonucleotide reductase, the enzyme responsible for DNA synthesis that contains a tyrosine radical. Subsequent inhibition of DNA may inhibit viral replication. However, the inhibition of ribonucleotide reductase is rapidly reversible and lost when NO is less than a few micromolar in concentration. Large continuous fluxes of NO are necessary to keep ribonucleotide reductase inhibited, which would occur only under major inflammatory conditions or in the neighborhood of an activated macrophage. Enormous amounts of oxygen are required to maintain synthesis of NO in micromolar concentrations. Since it takes two oxygens per NO produced, and if the half-life of NO in vivo was as long as 7 s, then 120 nmol O2 would be needed per gram tissue per minute to maintain NO at a steady-state concentration of 1 µM! NO in the submicromolar range can also reversibly inhibit cytochrome-c oxidase (141, 220, 1161), which may transiently increase the leakage of superoxide from the electron transport chain. The superoxide so formed could then react with NO to generate peroxynitrite, which would cause irreversible injury to the mitochondria.
E. Superoxide and the Hydroxyl Radical Theory of Radical Damage
The recognition that superoxide could be a biologically significant molecule was at one time viewed as unlikely as NO. Many thought superoxide was chemically far too reactive to be produced in vivo. Inorganic chemists produce superoxide by burning molten potassium metal with pure molecular oxygen. As a solid, potassium superoxide (KO2·) is a powerful oxidant that reacts vigorously when added to water. Originally, chemists wrote the structure as K2O4. In 1934, Pauling and Neuman (1013) deduced from quantum mechanics that 2KO2· would be more stable than a molecule with four oxygen atoms and proposed the name superoxide for this one-electron reduced state of molecular oxygen.
In the mid 1960s, Fridovich (410) investigated the oxygen-dependent reduction of cytochrome c by the flavin-containing xanthine oxidase. Oxygen would be expected to oxidize cytochrome c rather than to facilitate reduction. A protein was found in blood cells that would inhibit the reduction of cytochrome c, which was purified by McCord and Fridovich in 1969 (859). This protein had previously been purified as a copper binding protein, but McCord and Fridovich (688) showed this protein was an efficient scavenger of superoxide. Fridovich's group soon isolated a manganese-containing version from bacteria and also present in mitochondria (1026). The ability of SOD to scavenge superoxide provided strong evidence that superoxide was a biologically relevant molecule. This provided strong impetus to the theory that the univalent reduction of molecular oxygen to produce the free radicals superoxide, hydrogen peroxide, and hydroxyl radical were responsible for the toxicity of oxygen in biological systems. Oxygen has been known since the 1950s to greatly amplify the toxicity resulting from radiation by amplifying radical damage.
While the name superoxide implies that superoxide should be a powerful oxidant, superoxide more generally behaves as a mild reductant under physiological conditions rather than a "super"-oxidizing agent. This is because superoxide exists naturally as a small anion (O2–), which is more likely to surrender its electron than to accept a second electron from another biological molecule. Hence, the reduction potential for superoxide is about –0.1 V at physiological oxygen concentrations. Superoxide is a strong oxidant when it is protein mated, but its pKa is 4.3 so that it will directly oxidize positively charged chemical moieties such as iron/sulfur centers. The destruction of iron/sulfur centers in mitochondria has been well described in SOD knockout mice (761, 910). Aconitase is another iron/sulfur protein that is particularly susceptible to inactivation by superoxide (523).
The limited chemical reactivity of superoxide created considerable controversy about the role of superoxide in cellular toxicity (1125). To explain how a more potent oxidant might be generated by superoxide, the iron-catalyzed formation of hydroxyl radical from hydrogen peroxide was proposed. Because hydroxyl radical was well known to be a potent oxidant and important in radiation-induced damage to biological molecules, it became widely accepted as the major toxin produced in vivo. While widely described in many pathology textbooks, there are many limitations to the hydroxyl radical theory. In particular, the formation of hydroxyl radical requires the presence of an appropriately chelated iron atom reacting first with superoxide to become reduced and then with hydrogen peroxide to form hydroxyl radical. Scavengers of either superoxide or hydrogen peroxide such as SOD can effectively inhibit this reaction. The reaction of iron with hydrogen peroxide tends to have rather slow rate constants. Finally, hydroxyl radical is so reactive that it will react with virtually every biological molecule within a very short diffusion distance. Hydroxyl radical will diffuse on average less than the diameter of the typical protein. Thus the biological relevance of hydroxyl radical is limited because it is formed by a slow reaction while being so highly reactive that its toxicity becomes limited by reacting with too many irrelevant biological targets.
Still administration of SOD to experimental animals subjected to ischemia and other inflammatory stresses could provide substantial benefits. At the same time that McCord and Fridovich had purified SOD, another group purified an anti-inflammatory compound from cow blood that turned out to be identical to SOD (578). By the mid 1980s, 6,000,000 doses of SOD had been administered as an anti-inflammatory agent to patients in Europe. These studies clearly implicated superoxide as having a major role in promoting tissue pathology. However, the biological targets of superoxide remained obscure. The protection of EDRF by SOD suggested that it might be a major target of superoxide in vivo. When EDRF was identified as NO, it raised the possibility that its product, peroxynitrite, could be a significant and unrecognized oxidant in vivo.
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III. PEROXYNITRITE |
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At the time when NO was discovered, the controversy about the reactivity of superoxide and its significance to pathology was at its nadir. A few publications concerning peroxynitrite were scattered in the older chemical literature or found in studies of atmospheric pollution. The diffusion-limited reaction of NO with HO2· was recognized to be the major source of nitrogen dioxide and hydroxyl radical in the atmosphere and strongly implicated in the formation of smog.
The historical perspective of the discovery of peroxynitrite has been nicely reviewed by Koppenol (708). As early as 1901, the unusual oxidizing power of acidified nitrite mixed with hydrogen peroxide was noted. As an oxidant, peroxynitrite attracted little attention because it produced a bewildering array of products even with fairly simple starting substrates such as phenol. In 1970, several investigators more thoroughly characterized the chemistry of peroxynitrite showing that it decomposed to form hydroxyl radical and nitrogen dioxide (85, 401, 580, 581, 832). Hughes and Nicklin (580) established the most commonly used extinction coefficient (1.67 mM–1 · cm–1) through indirect measurements. Later, Bohle et al. (108) prepared pure peroxynitrite and determined the extinction coefficient as 1.70 mM–1 · cm–1.
Peroxynitrite can also be formed through the ultraviolet radiation of solid crystals of nitrate, turning clear crystals yellow (678). This is potentially more than a laboratory curiosity. The attempts by the Viking missions to Mars in 1976 to detect life may have been confronted by ultraviolet-induced peroxynitrite formation in nitrate found in the Martian soil (1031). Peroxynitrite may also prove useful to solubilize chromium III by oxidizing it to chromium VI from the radioactive sludge found in the Hanford nuclear storage facilities in Washington state (808). Solid chromium III causes glass to weaken more quickly and requires more glass to be used for vitrification of radioactive waste. Treatment with peroxynitrite can reduce the amount of glass needed to entrapp trans-uranium elements by a factor of two, which has huge economic benefits to reduce the cost of permanent storage of radioactive waste.
In studies of the fate of NO in the ocean, Zafirou and co-workers (104, 1428) showed that superoxide reacts with NO to form peroxynitrite. The most commonly cited rate constant for this reaction was measured as 6.7 x 109 M–1 · s–1 (583). Koppenol et al. (937) found using three different flash photolysis methods that the rate was slightly faster (16–20 x 109 M–1 · s–1), while others report slightly slower rate constants as determined by pulse radiolysis (3–4 x 109 M–1 · s–1). The rate of superoxide reacting with SOD is 2 x 109 M–1 · s–1. Hence, NO is the one molecule produced in high enough concentrations and reacts fast enough to outcompete endogenous SOD for superoxide. As described below, the interactions of superoxide, NO, and peroxynitrite with SOD are more complex than this simple analysis indicates.
In 1990, the first papers suggesting that peroxynitrite could be a biological oxidant were published (71, 75). At the time, much of the literature suggested that NO was a scavenger of superoxide and thus acting as an antioxidant (379, 855). However, Beckman et al. (75) showed peroxynitrite was a far more effective means of producing hydroxyl radical than the widely accepted reaction of reduced iron with hydrogen peroxide (known as the Fenton reaction or the iron-catalyzed Haber-Weiss reaction). These results were confirmed by Hogg et al. using systems to cogenerate superoxide and NO (287, 559). In addition, peroxynitrite produced nitrogen dioxide, which could lead to novel oxidation products that were previously only suspected to occur after exposure to cigarette smoke or to air pollution.
Peroxynitrite itself is also a strong oxidant and can react directly with electron-rich groups, such as sulfhydryls (1056), iron-sulfur centers (182), zinc-thiolates (245), and the active site sulfhydryl in tyrosine phosphatases (1254). Curiously, the reaction rate constants are relatively slow for these second-order reactions, ranging from 103 to 106 M–1 · s–1. Peroxynitrite is surprisingly stable in solution, considering its strong oxidizing potential and that it is 36 kcal/mol higher in energy than its isomer nitrate. The unusual stability of peroxynitrite results in part because it folds into a stable cis-conformation where the negative charge is localized over the entire molecule (1291). The molecules are further stabilized by forming strong hydrogen bonds with two or three waters (1290). The limited reactivity of peroxynitrite with most molecules makes it unusually selective as an oxidant, which increases its influence over biological processes.
Although peroxynitrite is a strong oxidant, the anion (ONOO–) also reacts directly with nucleophiles, molecules with a partial positive charge. One example of major importance is carbon dioxide. The carbon is surrounded by two oxygens, which effectively pull electron density away. Hence, carbon dioxide reacts with hydroxyl anion to form bicarbonate. In the same way, carbon dioxide reacts with peroxynitrite to form a transient intermediate nitrosoperoxycarbonate that rapidly decomposes homolytically to nitrogen dioxide and carbonate radical. Because carbon dioxide is nearly 1 mM in cells (10,000 times greater than hydrogen ions), the formation of carbonate radicals is more likely to occur in vivo than the formation of hydroxyl radical per se from HOONO (peroxynitrous acid, the conjugated acid of peroxynitrite). Carbonate radical is more selective than hydroxyl radical but will initiate many of the damaging reactions commonly attributed to hydroxyl radical in the biological literature and is perhaps more significant as a biological oxidant (873).
Multiple oxidative pathways can form both hydroxyl and carbonate radical independently of peroxynitrite or NO. However, peroxynitrite can also produce novel products such as nitrotyrosine, nitrotryptophan, and nitrated lipids that serve as important biological markers in vivo. Ohshima et al. (967) developed a mass spectrometric method to measure nitrotyrosine of smokers, but was surprised to find significant amounts in the urine of nonsmoking humans as well. At the same time, Ischiropoulos and co-workers found that peroxynitrite caused bovine SOD to turn yellow, which was eventually traced to the nitration of the sole tyrosine at position 108 (609, 612, 1174).
A major limitation to the acceptance of peroxynitrite or any reactive nitrogen species as a significant player in disease was whether enough could be produced to be damaging. Significant questions were raised as to whether human "macrophages" produced NO and whether its synthesis was mostly a rodent-specific phenomenon. However, in subsequent studies, it became clear that the regulation of inducible NOS was under tighter control in humans (936, 1196, 1353).
The development of antibodies that recognize nitrotyrosine also provided a major impetus to the study of peroxynitrite (80). Nitrotyrosine can easily be detected in Formalin-fixed tissue, and the antibodies are not species specific. This allowed the antibodies to be broadly applicable (1327, 1414). Nitrotyrosine was first shown to be localized in human atherosclerotic lesions and vascular muscle (80). Later, it was found in a huge number of most disease-affected tissues (see Tables 4 and 5 and sect. VI) and its presence confirmed by mass spectrometric analyses (244, 1098, 1397). One of the major values of nitrotyrosine is that it provided strong evidence that the production of NO was sufficient to produce observable products in virtually every human disease (606).
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Nitrotyrosine is extremely useful for measuring the formation of peroxynitrite, but this requires additional experimental validation. The first evidence for peroxynitrite formation came from SOD as a catalyst of tyrosine nitration to detect peroxynitrite being formed from freshly isolated rat alveolar macrophages (611). As isolated, these macrophages produced NO, but not superoxide. No nitration of a tyrosine analog could be observed under these basal conditions. When superoxide synthesis by NADPH oxidase was activated with a phorbol ester, nitration could be visually observed by the tyrosine analog turning a faint yellow color. The amount of nitration could be increased paradoxically by adding SOD (Fig. 4). SOD catalyzes tyrosine nitration by peroxynitrite, but would generally be expected to reduce peroxynitrite formation by scavenging superoxide. To parse these conflicting actions, SOD was chemically treated by a combination of two well-established methods to reduce its superoxide scavenging activity by >99%. The copper remained bound to the SOD after these treatments, and the ability to catalyze tyrosine nitration was unaffected. Unexpectedly, unmodified SOD was equally effective at catalyzing nitration as the inactivated SOD, suggesting that nitration was enhanced by metal-dependent catalysis (copper) at the active site of the enzyme. This also argued that NO was reacting so quickly with superoxide produced on the cell surface by NADPH oxidase that SOD added to the extracellular media could not effectively remove superoxide under these identical conditions. What was particularly important about this study was that oxygen consumption, NO production, superoxide formation, and nitration were all quantified as well as the effects of NOS inhibition on these quantities. When superoxide formation was stimulated to a threefold greater rate than NO synthesis, all of these data showed that NO was being quantitatively converted to peroxynitrite.
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C. The Reverse Reaction of Peroxynitrite to Form Superoxide and Nitric Oxide
Although the back reaction is 100,000,000,000 times slower than the formation of peroxynitrite, the reverse reaction of peroxynitrite forming superoxide and NO can be significant when working with bolus additions. When the production of superoxide and NO are both occurring at very low levels, the back reaction can be significant. However, the rapid reaction of peroxynitrite with carbon dioxide will pull the overall reaction to form carbonate radical and nitrogen dioxide. Thus carbon dioxide increases the amount of radical damage produced from NO plus superoxide cogeneration and ensures that peroxynitrite decomposes to form radicals even at very low fluxes of superoxide and NO.
The reverse reaction to form superoxide and NO also explains many of the puzzling aspects of peroxynitrite chemistry at alkaline pH (Fig. 6). The apparent radical generation from peroxynitrite decomposition decreases at alkaline pH (75). The details have been worked out by Goldstein, Meyreni, Lymar, Hurst, and co-workers (224, 452, 812, 868). In brief, the superoxide produced during the reverse reaction reacts with nitrogen dioxide to form peroxynitrate anion (O2NOO–), which decomposes to give nitrite and oxygen. The NO produced reacts with additional nitrogen dioxide to form dinitrogen trioxide, which is a well-known nitrosating agent. Peroxynitrite reacts quickly with dinitrogen trioxide to form two nitrogen dioxides plus nitrite. This catalytically increases the formation of nitrogen dioxide and accelerates the decomposition of peroxynitrite. This complex interplay of reactive intermediates illustrates how a slight excess of NO can interact with peroxynitrite to increase the formation of nitrosothiols and other unexpected products that greatly complicate the biological chemistry of peroxynitrite.
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IV. PEROXYNITRITE-INDUCED CYTOTOXICITY |
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A. Mechanisms of Peroxynitrite-Mediated Oxidations
Although not a free radical in nature, peroxynitrite is much more reactive than its parent molecules NO and O2– (71, 75, 78). The half-life of peroxynitrite is short (10–20 ms), but sufficient to cross biological membranes, diffuse one to two cell diameters (312), and allow significant interactions with most critical biomolecules (1041). Kinetic studies have indicated that peroxynit
