Biology of PN

The biology of PN is rather characterized by its participation in pathophysiologi-cal processes. These actions can be summarized by modifications of biomolecules as shown in 1.3.3, especially nitration of Tyr-residues and oxidation of Cys- and Met-residues in proteins [148, 149, 150, 151]. All these protein modifications are not specific for PN, thiols are also oxidized by hydrogen peroxide, superoxide, hydroxyl radicals and hypochlorite [137] and tyrosines are nitrated by ·NO₂ or by the MPO-catalyzed reaction of nitrite with hydrogen peroxide [15, 152]. 3-NT is often taken as a marker for PN presence in vivo [5, 153], but is also critizized for its unspecificity [155, 154, 156] and some reports even question the relevance of PN-mediated nitration under physiological conditions [22]. 3-NT can be also formed by nitrogen dioxide, the autoxidation product of nitric oxide [15], by the myeloperoxidase- or HRP-catalyzed reaction of hydrogen peroxide with nitrite [152], by the CPO-catalyzed reaction of hypochlorite with nitrite [53] and by the addition of nitric oxide to a once formed tyrosyl radical with subsequent oxidation [34, 157, 158]. Protein-bound 3-NT is popular as a footprint of PN

Biomolecule k_sec [M⁻¹s⁻¹] [Biomol.][M] k_scav [s⁻¹]

Self-decompos. - - 0.4-0.6

Carbon dioxide 3-4.6x10⁴ / - 10⁻³ 3046 /

-Ascorbate 50-90 / 230 10⁻² 0.5-0.9 / 2.3

Glutathione 580 / 1.5x10⁶ 10⁻² 5.8 / 15000

Myeloperoxidase 2.5x10⁵ / 6.2x10⁶ 2x10⁻⁷-5x10⁻⁴ 0.05-125 / 0.12-3100 oxyHemoglobin - / 2.5x10⁴ 2.3-5x10⁻³ - / 58-125

GPx 2x10⁴-8x10⁶ / - 1.5-2x10⁻⁶ 0.0316 /

-Albumin 3-6x10³ 6x10⁻⁴ 1.8-3.6

Table 2: Velocities for PN decay in presence of physiological concentrations of biomolecules (effective rate constant). Second column: k-value for ONOO⁻ / k-value for ONOOH. Fourth column: k_scav for ONOO⁻ / k_scav for ONOOH. Values are taken from reviews [144, 162] and corrected with values fromTab.1.

in vivo, because antibodies are available for its sensitive detection and the spectroscopic and electronic properties of 3-NT for UV/Vis and electrochemical HPLC detection are suitable for a sensitive monitoring [154, 159]. During the last year another issue was brought up, speaking against 3-NT as a biomarker for PN:

Muradand coworkers have observed that 3-NT is not stable under physiological conditions and that there may be enzymatic and non-enzymatic pathways for reduction of PN [160, 161]. These findings also revitalized the discussion, that PN could be an unspecific messenger, on the basis that PN-mediated nitrations are reversible.

Depletion of antioxidants or modification of low molecular weight biomolecules requires extremely high concentrations of PN, to be harmful to cells or the whole organism. The halflife of PN in vivois short regarding the cellular defense, which is able to intercept PN. Tab.2 shows possible pathways for PN consumption in vivo [144, 162]. Important for the PN scavenging ability are the effective velocities, that means the second order rate constant of PN with an antioxidative system multiplied with its physiological concentration. Most important is the decay velocity (kscav) for the PN-anion. The reactivity of ONOOH is almost completely suppressed by glutathione (k_scav=15000 s⁻¹). The difference of the values is due to the different second order rate constants and physiological concentration that were used for calculation of the effective velocities.

The reaction of nitric oxide and superoxide yields PN in the anionic form and although protonation is in a fast equilibrium, it is mainly present as ONOO⁻ at

1 INTRODUCTION 22 physiological pH due to its pK_a of 6.8 [56], therefore scavengers for the PN-anion are more important than for ONOOH. Most of ONOOH is trapped by GSH, MPO or oxyHb (see Tab.2). Uric acid also reacts very fast with ONOOH and is present in cytosol and plasma at concentrations of 100-300 µM [4]. Regarding the fact that heme- and heme-thiolate proteins increase the nitration potential of PN [163, 164], these compounds cannot be considered as real scavengers for PN. On the other hand it had been shown, that such proteins coupled with antioxidants represent possibly the most effective scavenging systems for PN [50, 165]. Biomolecules that react with ONOO⁻ are more likely to be oxidized by PN, since only GPx and GSH have a certain protective potential. Since many metal-containing proteins and also carbon dioxide react with PN-anion and increase its oxidative properties, these two reactions could be the major activation pathways for PN in vivoand contribute the major part to PN-toxicity [163, 116, 146].

Tab.3 shows a list of human diseases, in which 3-NT could be detected and an involvement of PN was suggested or proved [166]. Tab.4 shows the same for animal models of disease [166] and Tab.5 for cellular models of disease [166].

Atherosclerotic plaques of coronary vessels Chronic renal failure in septic patients LDL isolated from atherosclerotic lesions Inflammatory bowel disease

Lungs with sepsis and/or respiratory disease Heliobacterium pylori gastritis Idiopathic pulmonary fibrosis Necrotizing enterocolitis Lung transplants with obliterative bronchiolitis Colliac disease

Plasma of infants with BPD Fluid of patients with arthritis Amyotrophic lateral sclerosis Early prosthesis failure Multiple sclerosis plaques Inclusion body myositis

Alzheimer’s lesions Placenta of preeclamptic pregnancies Rejected renal allografts Skin lesion with anaphylactoid purpura

Table 3: Detection of 3-NT in human disease [166].

These tables show that PN may have a major impact on the pathophysiology of disease and how important the development of scavengers for PN is. The impact of PN on the vascular system could be established in our group by Zou et al [167, 168, 170] and this finding is supported by other groups [171, 172].

PN not only eliminates the vasorelaxating PGI₂, but also consumes·NO, another potent vasorelaxating factor (EDRF), for its formation. Therefore PN could contribute seriously to vasodysfunction. Nitration and inactivation of PGIS by PN was found to be involved in hypoxia-reoxygenation triggered vasospasm [169],

Aorta of septic rats Brain vasculature of CO-poisoned rats Cardiac allocraft rejection Chronic cerebral vasospasm

Ischemia-reperfusion-injured rat heart Allergic encephalomyelitis

Cholesterol-induced atherosclerosis Central nervous system inflammation Autoimmune myocarditis Liver transplantation

Myocardial inflammation Dimercaptosuccinic acid nephrosclerosis Rabbit lungs following exposure to hyperoxia Guinea pig ileitis

Lungs of endotoxin-treated rats Placenta of LPS-treated rats Ischemia-reperfusion-injured rat lungs Carrageenan paw edema Influenza-induced pneumonitis Murine leishmaniasis Pulmonary granulomatous inflammation Zymosan peritonitis

HSV-1-induced pneumonia Autoimmune diabetes in NOD mice Malonate- and MPTP-induced neurotoxicity Autoimmune uveitis

Excitoxicity model of neuronal injury Dermal tumor promotion Transgenic model for ALS Mice bearing ResX tumor

ApoE-deficient mice Aged rat skeletal muscle SERc2a isoform

Table 4: Detection of 3-NT in animal models of disease [166].

Ingested bacteria in human PMN H₂O₂-exposed kidney epithelium IL-1β-stimulated VSMC Cytokine-stimulated fetal glial cultures Arginine-depleted neuronal cells TNF-α-treated endothelium

Native LDL-treated endothelium CO-exposed endothelium Table 5: Detection of 3-NT in cellular models of disease [166].

in atherosclerosis [173] and in endotoxic shock [manuscripts submitted] and was found to be stimulated by IL-1β in rat mesangial cells [12].