John P. Crow

Oxidative chemistry of peroxynitrite.

Publisher Summary Nitric oxide (.NO) is an important and largely unrecognized mediator of oxygen radical injury because it contains an unpaired electron that readily combines with many free radicals. Endothelium and neurons produce nitric oxide as an intercellular messenger, which has important roles in vasoregulation and synaptic plasticity. Nitric oxide reacts rapidly with superoxide to form the strong oxidant, peroxynitrite anion (ONOO - ). Activated macrophages and neutrophils can produce nitric oxide and superoxide at similar rates. This chapter presents that essentially all of the nitric oxide produced by rat alveolar macrophages activated with phorbol ester is converted to peroxynitrite. Peroxynitrite is not a free radical because the unpaired electrons on nitric oxide and superoxide have combined to form a new N–O bond in peroxynitrite. Peroxynitrite anion can be stored for weeks in alkaline solution or even entrapped in solid forms. During its decomposition at physiological pH, peroxynitrite can produce some of the strongest oxidants known in a biological system, initiating reactions characteristic of hydroxyl radical, nitronium ion, and nitrogen dioxide. The unusual stability of peroxynitrite as an anion contributes to its toxicity by allowing it to diffuse far from its site of formation while being selectively reactive with cellular targets.

Decreased zinc affinity of amyotrophic lateral sclerosis-associated superoxide dismutase mutants leads to enhanced catalysis of tyrosine nitration by peroxynitrite

Abstract: Mutations to Cu/Zn superoxide dismutase (SOD) linked to familial amyotrophic lateral sclerosis (ALS) enhance an unknown toxic reaction that leads to the selective degeneration of motor neurons. However, the question of how >50 different missense mutations produce a common toxic phenotype remains perplexing. We found that the zinc affinity of four ALS‐associated SOD mutants was decreased up to 30‐fold compared to wild‐type SOD but that both mutants and wild‐type SOD retained copper with similar affinity. Neurofilament‐L (NF‐L), one of the most abundant proteins in motor neurons, bound multiple zinc atoms with sufficient affinity to potentially remove zinc from both wild‐type and mutant SOD while having a lower affinity for copper. The loss of zinc from wild‐type SOD approximately doubled its efficiency for catalyzing peroxynitrite‐mediated tyrosine nitration, suggesting that one gained function by SOD in ALS may be an indirect consequence of zinc loss. Nitration of protein‐bound tyrosines is a permanent modification that can adversely affect protein function. Thus, the toxicity of ALS‐associated SOD mutants may be related to enhanced catalysis of protein nitration subsequent to zinc loss. By acting as a high‐capacity zinc sink, NF‐L could foster the formation of zinc‐deficient SOD within motor neurons.

Reactions between Nitric Oxide, Superoxide, and Peroxynitrite: Footprints of Peroxynitrite in Vivo

Publisher Summary This chapter examines a number of reaction pathways for nitric oxide, with the emphasis on assessing their biological relevance. Till date, the fastest reaction for nitric oxide with clear toxicological significance is that with superoxide to produce ONOO − . Thus, the chemistry and reactivity of ONOO − are discussed at length. In addition, the interaction between ONOO − and nitric oxide is examined with respect to its effects on nitric oxide half-life as well as effects on peroxynitrite reactivity toward phenol. Reaction mechanisms are proposed to account for the nitrated, hydroxylated, and nitrosated phenolic products available. The primary reactions of nitric oxide are almost exclusively limited to other species possessing unpaired electrons, such as the iron in heme proteins, as well as nonheme iron and superoxide. Nitric oxide does react with molecular oxygen; however, this reaction occurs so slowly at physiological concentrations as to be toxicologically insignificant. Primary reactions of nitric oxide can result in a variety of secondary products ranging from innocuous nitrate (NO −3 ), nitrite (NO −2 ) and nitroxyl (NO − ) to reactive intermediates such as nitrosonium (NO + ), peroxynitrite (ONOO − ), and nitrogen dioxide (NO 2 ). The predominant end products of these reactive intermediates that are stable enough to be measured in biological systems include nitrite, nitrate, nitrotyrosine, and various nitrosothiols.

Superoxide Dismutase Catalyzes Nitration of Tyrosines by Peroxynitrite in the Rod and Head Domains of Neurofilament‐L

Abstract: Superoxide dismutase (SOD) catalyzes the nitration of specific tyrosine residues in proteins by peroxynitrite (ONOO−), which may be the damaging gain‐of‐function resulting from mutations to SOD associated with familial amyotrophic lateral sclerosis (ALS). We found that disassembled neurofilament‐L (light subunit) was more susceptible to tyrosine nitration catalyzed by SOD in vitro. Neurofilament‐L was selectively nitrated compared with the majority of other proteins present in brain homogenates. Assembled neurofilament‐L was more resistant to nitration, suggesting that the susceptible tyrosine residues were protected by intersubunit contacts in assembled neurofilaments. Electrospray mass spectrometry of trypsin‐digested neurofilament‐L showed that tyrosine 17 in the head region and tyrosines 138, 177, and 265 in α‐helical coil regions of the rod domain of neurofilament‐L were particularly susceptible to SOD‐catalyzed nitration. Nitrated neurofilament‐L inhibited the assembly of unmodified neurofilament subunits, suggesting that the affected tyrosines are located in regions important for intersubunit contacts. Neurofilaments are major structural proteins expressed in motor neurons and known to be important for their survival in vivo. We suggest that SOD‐catalyzed nitration of neurofilament‐L may have a significant role in the pathogenesis of ALS.

Chapter 31 Reactions of nitric oxide, superoxide and peroxynitrite with superoxide dismutase in neurodegeneration

Publisher Summary To be successful in any attempt to regenerate the nervous system, the processes responsible for the underlying neurodegeneration must be understood and controlled. This chapter discusses three points. First, nitric oxide is far less reactive and toxic at physiologically relevant concentrations than commonly thought. Many investigators have been mislead by using millimolar solutions of nitric oxide or various relatively uncharacterized nitric oxide donors to study chemical reactivity and then equating the results with the reactivity of nitric oxide per se. The chapter describes how low concentrations of nitric oxide do not react rapidly with oxygen to form nitrogen dioxide. Instead, nitric oxide is more likely to diffuse into red blood cells and be consumed by reaction with hemoglobin. Second, the reactivity and toxicity of nitric oxide is enormously increased by its near diffusion-limited reaction with superoxide to form peroxynitrite anion. From 1 to 5% of all oxygen consumed is partially reduced to produce the oxygen radical, superoxide. Third, paradoxically, peroxynitrite reacts with superoxide dismutase (SOD) to form a powerful nitrating agent with the reactivity of nitronium ion and modifies tyrosine residues on proteins to form nitrotyrosine. Nitrotyrosine cannot be phosphorylated by tyrosine kinases and thus SOD-mediated injury may affect critical signal transduction pathways. The chapter proposes that this mechanism may account for the pathological action of SOD mutations in amyotrophic lateral sclerosis.

[17] Detection and quantitation of nitrotyrosine residues in proteins: In vivo marker of peroxynitrite

Publisher Summary This chapter discusses the detection and quantitation methods of nitrotyrosine residues in proteins. Nitrotyrosine is detected in human diseases associated with oxidative stress and is visualized using immunological techniques in atherosclerotic plaques of human coronary vessels, in lungs of infants with acute lung injury and sepsis, and in adult respiratory distress syndrome (ARDS). High-performance liquid chromatography (HPLC) analysis is used to detect nitrotyrosine in synovial fluid from patients with rheumatoid arthritis. Peroxynitrite can be synthesized from sodium nitrite and acidified hydrogen peroxide. Selective tyrosine nitration can be accomplished by titrating protein with tetranitromethane (TNM) at neutral or alkaline conditions (pH 7–8). TNM is a potent carcinogen, which must be handled carefully. The residual TNM and trinitromethane must be removed prior to nitrotyrosine quantitation. Nitrotyrosine is essentially nonfluorescent whereas aminotyrosine is highly fluorescent and has a characteristic emission spectrum. Thus, fluorescent detection of aminotyrosine can be used as an alternative to direct detection of nitrotyrosine. Quantitation of nitrotyrosine using the solid-phase immunoradiochemical method has the advantage of high sensitivity and does not require sample manipulation.

On the pH-dependent yield of hydroxyl radical products from peroxynitrite

Nitric oxide reacts rapidly with superoxide to give the strongly oxidizing peroxynitrite anion (ONOO-), which undergoes spontaneous first-order decomposition when protonated. The oxidative chemistry of peroxynitrite (ONOO-) is highly pH-dependent. At acidic pH, peroxynitrous acid (ONOOH) oxidizes dimethylsulfoxide to formaldehyde and 2,2-azino-bis-(3-ethyl-1,2- dihydrobenzothiazoline 6-sulfonate) (ABTS) to the greenish-colored ABTS+ radical cation. The product yield from dimethylsulfoxide and ABTS decreased at more alkaline pH with apparent pK(a)s of 7.9 and 8.2, respectively. Decreasing yield with increasing pH could not be explained by the oxidation of either formaldehyde or ABTS+ by peroxynitrite. In the presence of 50 mM dimethylsulfoxide, nitrogen dioxide was formed in approximately equimolar amounts to the other reaction product, formaldehyde. The yield of nitrogen dioxide also decreased with an apparent pK(a) of 8.0. We propose that the complex oxidative chemistry of peroxynitrite is controlled by the pH-dependent isomerization of the relatively stable cis-configuration (predominant at high pH) to the trans-configuration. Trans-peroxynitrous acid can form a vibrationally excited intermediate capable of reacting like hydroxyl radical. The vibrationally excited intermediate can also directly rearrange to nitric acid, reducing the apparent hydroxyl radical yield to less than 30%. The loss of hydroxyl radical-like reactivity can be explained on the basis of ionization of trans-peroxynitrous acid to the trans-anion, which in turn undergoes internal rearrangement to nitrate without forming a strong oxidant.(ABSTRACT TRUNCATED AT 250 WORDS)

Superoxide dismutase and the death of motoneurons in ALS

Amyotrophic lateral sclerosis (ALS) is a lethal disease that is characterized by the relentless death of motoneurons. Mutations to Cu-Zn superoxide dismutase (SOD), though occurring in just 2-3% of individuals with ALS, remain the only proven cause of the disease. These mutations structurally weaken SOD, which indirectly decreases its affinity for Zn. Zn-deficient SOD induces apoptosis in motoneurons through a mechanism involving peroxynitrite. Importantly, Zn-deficient wild-type SOD is just as toxic as Zn-deficient ALS mutant SOD, suggesting that the loss of Zn from wild-type SOD could be involved in the other 98% of cases of ALS. Zn-deficient SOD could therefore be an important therapeutic target in all forms of ALS.

Manganese porphyrin given at symptom onset markedly extends survival of ALS mice

Mice that overexpress the human Cu,Zn superoxide dismutase‐1 mutant G93A develop a delayed and progressive motor neuron disease similar to human amyotrophic lateral sclerosis (ALS). Most current studies of therapeutics in these mice to date have involved administration of agents long before onset of symptoms, which cannot currently be accomplished in human ALS patients. We examined the effects of the manganese porphyrin AEOL 10150 (manganese [III] tetrakis[N‐N′‐diethylimidazolium‐2‐yl]porphyrin) given at symptom onset and found, in three separate studies, that it extended the survival after onset up to 3.0‐fold. Immunohistochemical analysis of spinal cord for SMI‐32, an abundant protein in motor neurons, indicated better preservation of motor neuron architecture, less astrogliosis (glial fibrillary acidic protein), and markedly less nitrotyrosine and malondialdehyde in porphyrin‐treated spinal cords relative to vehicle‐treated mice. These results show that the catalytic antioxidant AEOL 10150 provides a pronounced therapeutic benefit with onset administration and is, therefore, a promising agent for the treatment of ALS. Ann Neurol 2005;58:258–265

The CB2 cannabinoid agonist AM-1241 prolongs survival in a transgenic mouse model of amyotrophic lateral sclerosis when initiated at symptom onset

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron loss, paralysis and death within 2–5u2003years of diagnosis. Currently, no effective pharmacological agents exist for the treatment of this devastating disease. Neuroinflammation may accelerate the progression of ALS. Cannabinoids produce anti‐inflammatory actions via cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2), and delay the progression of neuroinflammatory diseases. Additionally, CB2 receptors, which normally exist primarily in the periphery, are dramatically up‐regulated in inflamed neural tissues associated with CNS disorders. In G93A‐SOD1 mutant mice, the most well‐characterized animal model of ALS, endogenous cannabinoids are elevated in spinal cords of symptomatic mice. Furthermore, treatment with non‐selective cannabinoid partial agonists prior to, or upon, symptom appearance minimally delays disease onset and prolongs survival through undefined mechanisms. We demonstrate that mRNA, receptor binding and function of CB2, but not CB1, receptors are dramatically and selectively up‐regulated in spinal cords of G93A‐SOD1 mice in a temporal pattern paralleling disease progression. More importantly, daily injections of the selective CB2 agonist AM‐1241, initiated at symptom onset, increase the survival interval after disease onset by 56%. Therefore, CB2 agonists may slow motor neuron degeneration and preserve motor function, and represent a novel therapeutic modality for treatment of ALS.