Oxidative alterations of cyclooxygenase during atherogenesis

Nitric oxide (*NO) and eicosanoids are critical mediators of physiological and pathophysiological processes. They include inflammation and atherosclerosis. *NO production and eicosanoid synthesis become disrupted during atherosclerosis and thus, it is important to understand the mechanisms that may contribute to this outcome. We, and others, have shown that nitrogen oxide (NO(x)) species modulate cyclooxygenase (COX; also known as prostaglandin H(2) synthase) activity and alter eicosanoid production. We have determined that peroxynitrite (ONOO(-)) has multiple effects on COX activity. ONOO(-) can provide the peroxide tone necessary for COX activation, such that simultaneous exposure of COX to its arachidonic acid substrate and ONOO(-) results in increased eicosanoid production. Alternatively, in the absence of arachidonic acid, ONOO(-) can modify COX through nitration of an essential tyrosine residue (Tyr385) such that it is incapable of catalysis. In this regard, we have shown that COX nitration occurs in human atherosclerotic tissue and in aortic lesions from ApoE(-/-) mice kept on a high fat diet. Additionally, we have demonstrated that Tyr nitration in ApoE(-/-) mice is dependent on the inducible form of NO synthase (iNOS). Under conditions where ONOO(-) persists and arachidonic acid is not immediately available, the cell may try to correct the situation by responding to ONOO(-) and releasing arachidonic acid via a signaling pathway to favor COX activation. Other post-translational modifications of COX by NO(x) species include S-nitrosation of cysteine (Cys) residues (which may have an activating effect) and Cys oxidation. The central focus of this review will include a discussion of how NO(x) species alter COX activity at the molecular level and how these modifications may contribute to altered eicosanoid output during atherosclerosis and lesion development.     

Peroxynitrite and protein tyrosine nitration of prostacyclin synthase

When working on the regulation of prostacyclin synthase (PGIS), we found that PGIS was selectively inhibited by peroxynitrite (ONOO-), a potent oxidant formed by the combination of superoxide anion and nitric oxide (NO) at a rate of diffusion-controlled. None of the cellular antioxidants studied (i.e. GSH, Vitamins C and E, and others) prevented the inhibition of ONOO- on PGIS. This unexpected behavior was explained by a catalytic reaction of the iron-thiolate center of PGIS with ONOO- anion. In contrast, ONOO- activated both thromboxane A2-synthase and cyclooxygenases. In addition, we demonstrated that sub-micromolar levels of ONOO- inhibited PGI2-dependent vasorelaxation and triggered a PGH2-dependent vasospasm, indicating that ONOO- increased PGH2 formation as a consequence of PGIS nitration. We have subsequently demonstrated that endogenous ONOO- caused PGIS nitration and TxA2 activation in several diseased conditions such as atherosclerotic vessels, hypoxia-reperfusion injury, cytokines-treated cells, diabetes, as well as hypertension. Since NO is produced physiologically it seems that excessive formation of superoxide not only eliminates the vasodilatory, growth-inhibiting, anti-thrombotic and anti-adhesive effects of NO and PGI2 but also allows and promotes an action of the potent vasoconstrictor, prothrombotic agent, growth promoter, and leukocyte adherer, PGH2. We conclude that the nitration of PGIS nitration might be a new pathogenic mechanism for superoxide-induced endothelium dysfunction often observed in vascular diseases such as atherosclerosis, hypertension, ischemia, endotoxic shock, and diabetes.     

Involvement of the mitogen-activated protein kinase cascade in peroxynitrite-mediated arachidonic acid release in vascular smooth muscle cells

Eicosanoid production is reduced when the nitric oxide (NO·) pathway is inhibited or when the inducible NO synthase gene is deleted, indicating that the NO· and arachidonic acid pathways are linked. We hypothesized that peroxynitrite, formed by the reaction of NO· and superoxide anion, may cause signaling events leading to arachidonic acid release and subsequent eicosanoid generation. Western blot analysis of rat arterial smooth muscle cells demonstrated that peroxynitrite (100–500 µM) and 3-morpholinosydnonimine (SIN-1; 200 µM) stimulate phosphorylation of extracellular signal-regulated kinase (ERK), p38, and cytosolic phospholipase A₂ (cPLA₂). We found that peroxynitrite-induced arachidonic acid release was completely abrogated by the mitogen-activated protein/ERK kinase (MEK) inhibitor U0126 and by calcium chelators. With the p38 inhibitor SB-20219, we demonstrated that peroxynitrite-induced p38 phosphorylation led to minor arachidonic acid release, whereas U0126 completely blocked p38 phosphorylation. Addition of arachidonic acid caused p38 phosphorylation, suggesting that arachidonic acid or its metabolites are responsible for p38 activation. KN-93, a specific inhibitor of Ca²⁺/calmodulin-dependent kinase II (CaMKII), revealed no role for this kinase in peroxynitrite-induced arachidonic acid release in our cell system. Together, these results show that in response to peroxynitrite the cell initiates the MEK/ERK cascade leading to cPLA₂ activation and arachidonic acid release. Thus studies investigating the role of the NO· pathway on eicosanoid production must consider the contribution of signaling pathways initiated by reactive nitrogen species. These findings may provide evidence for a new role of peroxynitrite as an important reactive nitrogen species in vascular disease. reactive nitrogen species; prostaglandin H₂ synthase; extracellular signal-regulated kinase; p38; cytosolic phospholipase A₂     

S-nitrosylation of peroxiredoxin II E promotes peroxynitrite-mediated tyrosine nitration

Nitric oxide (NO) is a free radical product of cell metabolism that plays diverse and important roles in the regulation of cellular function. S-Nitrosylation is emerging as a specific and fundamental posttranslational protein modification for the transduction of NO bioactivity, but very little is known about its physiological functions in plants. We investigated the molecular mechanism for S-nitrosylation of peroxiredoxin II E (PrxII E) from Arabidopsis thaliana and found that this posttranslational modification inhibits the hydroperoxide-reducing peroxidase activity of PrxII E, thus revealing a novel regulatory mechanism for peroxiredoxins. Furthermore, we obtained biochemical and genetic evidence that PrxII E functions in detoxifying peroxynitrite (ONOO-), a potent oxidizing and nitrating species formed in a diffusion-limited reaction between NO and O2- that can interfere with Tyr kinase signaling through the nitration of Tyr residues. S-Nitrosylation also inhibits the ONOO- detoxification activity of PrxII E, causing a dramatic increase of ONOO--dependent nitrotyrosine residue formation. The same increase was observed in a prxII E mutant line after exposure to ONOO-, indicating that the PrxII E modulation of ONOO- bioactivity is biologically relevant. We conclude that NO regulates the effects of its own radicals through the S-nitrosylation of crucial components of the antioxidant defense system that function as common triggers for reactive oxygen species- and NO-mediated signaling events.     

Site-specific nitration differentially influences tau assembly in vitro

Previously, we reported that the microtubule-associated tau protein, the major constituent of neurofibrillary tangles (NFTs) in Alzheimer's brain, undergoes site-selective nitration by peroxynitrite (ONOO-) and that this event inhibits tau polymerization in vitro [Reynolds et al. (2005) Biochemistry 44, 1690-1700]. In the present study, we extend our analysis of tau nitration to include mutant tau proteins singly nitrated at each residue targeted by ONOO- in vitro (Tyr18, Tyr29, Tyr197, and Tyr394). Using our polymerization paradigm, we demonstrate that site-specific Tyr nitration differentially alters the rate and/or extent of tau assembly and generates robust changes in filament morphology. As determined by quantitative electron microscopy, select nitration of residues Tyr29 and Tyr197 increases the average length of synthetic tau filaments but does not alter the steady-state polymer mass. In contrast, site-specific nitration of residues Tyr18 and Tyr394 decreases the average length and/or number of synthetic filaments, resulting in a significant reduction in filamentous mass and an increase in tau critical concentration. Intriguingly, affinity measurements demonstrate that nitrative modifications do not preclude formation of the Alz-50 epitope, a pathological tau conformation detectable in authentic paired helical filaments (PHFtau). In fact, the Alz-50 antibody binds filaments assembled from nitrated mutant tau with higher avidity than wild-type filaments, even in instances where the overall filamentous mass is reduced. Taken together, our results suggest that site-specific nitration modulates the nucleation and/or elongation capacity of assembly-competent tau and that assumption of the Alz-50 conformation may be necessary, but not sufficient, to induce filament formation.     

Cytochrome c: a catalyst and target of nitrite-hydrogen peroxide-dependent protein nitration

Nitration of protein tyrosine residues to 3-nitrotyrosine (NO2Tyr) serves as both a marker and mediator of pathogenic reactions of nitric oxide (*NO), with peroxynitrite (ONOO-) and leukocyte peroxidase-derived nitrogen dioxide (*NO2) being proximal mediators of nitration reactions in vivo. Cytochrome c is a respiratory and apoptotic signaling heme protein localized exofacially on the inner mitochondrial membrane. We report herein a novel function for cytochrome c as a catalyst for nitrite (NO2-) and hydrogen peroxide (H2O2)-mediated nitration reactions. Cytochrome c catalyzes both self- and adjacent-molecule (hydroxyphenylacetic acid, Mn-superoxide dismutase) nitration via heme-dependent mechanisms involving tyrosyl radical and *NO2 production, as for phagocyte peroxidases. Although low molecular weight phenolic nitration yields were similar for cytochrome c and the proteolytic fragment of cytochrome c microperoxidase-11 (MPx-11), greater extents of protein nitration occurred when MPx-11 served as catalyst. Partial proteolysis of cytochrome c increased both the peroxidase and nitrating activities of cytochrome c. Extensive tyrosine nitration of Mn-superoxide dismutase occurred when exposed to either cytochrome c or MPx-11 in the presence of H2O2 and NO2-, with no apparent decrease in catalytic activity. These results reveal a post-translational tyrosine modification mechanism that is mediated by an abundant hemoprotein present in both mitochondrial and cytosolic compartments. The data also infer that the distribution of specific proteins capable of serving as potent catalysts of nitration can lend both spatial and molecular specificity to biomolecule nitration reactions.     

Nitric oxide-dependent generation of reactive species in sickle cell disease. Actin tyrosine induces defective cytoskeletal polymerization

The intermittent vascular occlusion occurring in sickle cell disease (SCD) leads to ischemia-reperfusion injury and activation of inflammatory processes including enhanced production of reactive oxygen species and increased expression of inducible nitric-oxide synthase (NOS2). Appreciating that impaired nitric oxide-dependent vascular function and the concomitant formation of oxidizing and nitrating species occur in concert with increased rates of tissue reactive oxygen species production, liver and kidney NOS2 expression, tissue 3-nitrotyrosine (NO(2)Tyr) formation and apoptosis were evaluated in human SCD tissues and a murine model of SCD. Liver and kidney NOS2 expression and NO(2)Tyr immunoreactivity were significantly increased in SCD mice and humans, but not in nondiseased tissues. TdT-mediated nick end-label (TUNEL) staining showed apoptotic cells in regions expressing elevated levels of NOS2 and NO(2)Tyr in all SCD tissues. Gas chromatography mass spectrometry analysis revealed increased plasma protein NO(2)Tyr content and increased levels of hepatic and renal protein NO(2)Tyr derivatives in SCD (21.4 +/- 2.6 and 37.5 +/- 7.8 ng/mg) versus wild type mice (8.2 +/- 2.2 and 10 +/- 1.2 ng/mg), respectively. Western blot analysis and immunoprecipitation of SCD mouse liver and kidney proteins revealed one principal NO(2)Tyr-containing protein of 42 kDa, compared with controls. Enzymatic in-gel digestion and MALDI-TOF mass spectrometry identified this nitrated protein as actin. Electrospray ionization and fragment analysis by tandem mass spectrometry revealed that 3 of 15 actin tyrosine residues are nitrated (Tyr(91), Tyr(198), and Tyr(240)) at positions that significantly modify actin assembly. Confocal microscopy of SCD human and mouse tissues revealed that nitration led to morphologically distinct disorganization of filamentous actin. In aggregate, we have observed that the hemoglobin point mutation of sickle cell disease that mediates hemoglobin polymerization defects is translated, via inflammatory oxidant reactions, into defective cytoskeletal polymerization.     

The short-term consumption of a moderately high-fat diet alters nitric oxide bioavailability in lean female Zucker rats

To determine whether short-term consumption of a moderately high-fat diet (MHFD) affects nitric oxide (NO) production, the concentration of stable NO metabolites (NOx) in urine and plasma of rats fed a MHFD (15.6?%g fat) or control diet (4.5?%g fat) was measured weekly for 4?weeks. Plasma and urine NOx levels were significantly depressed in the MHFD group by week 1 and remained so for the duration of the study. Decreased NO bioavailability may result from a decrease in NO production or the scavenging of NO by reactive oxygen species (ROS). Because endothelial NOS (eNOS) is the major contributor to NO production and circulating levels of NOx, eNOS expression was measured in several tissues. At week 1, there was a MHFD-associated decrease in eNOS expression in the liver. Subsequently, eNOS expression declined in the heart and kidney medulla of MHFD-fed rats at weeks 3 and 4, respectively. The expression of eNOS in the kidney cortex and adipose tissue did not change. These results suggest that a MHFD alters eNOS expression in a time-dependent and tissue-specific manner. In the liver, NOS activity and tissue levels of NOx and nitrotyrosine were measured. Nitrotyrosine levels were used as an indirect measure of the NO scavenged by ROS. There was a decrease in NOS activity, suggesting that the low levels of hepatic NOx were due, in part, to a decrease in NO production. In addition, there was a dramatic increase in nitrotyrosine formation, suggesting that the decline in hepatic NOx was also due to an increased interaction of NO with ROS. Tyrosine nitration commonly has detrimental effects on proteins. The decrease in NO and increase in protein nitration could potentially have adverse effects on tissue function.     

Site-specific nitration and oxidative dityrosine bridging of the tau protein by peroxynitrite: implications for Alzheimer's disease

Alzheimer's disease (AD) is a progressive amnestic disorder typified by the pathological misfolding and deposition of the microtubule-associated tau protein into neurofibrillary tangles (NFTs). While numerous post-translational modifications influence NFT formation, the molecular mechanisms responsible for tau aggregation remain enigmatic. Since nitrative and oxidative injury have previously been shown to play a mechanistic role in neurodegeneration, we examined whether these events influence tau aggregation. In this report, we characterize the effects of peroxynitrite (ONOO-)-mediated nitration and oxidation on tau polymerization in vitro. Treatment of tau with ONOO- results in 3-nitrotyrosine (3-NT) immunoreactivity and the formation of heat-stable, SDS-insoluble oligomers. Using ESI-MS and HPLC with fluorescent detection, we show that these higher-order aggregates contain 3,3'-dityrosine (3,3'-DT). Tyrosine (Tyr) residues are critical for ONOO(-)-mediated oligomerization, as tau proteins lacking all Tyr residues fail to generate oligomers upon ONOO- treatment. Further, tau nitration targets residues Y18, Y29, and to a lesser degree Y197 and Y394, and nitration at these sites inhibits in vitro polymerization. The inhibitory effect of nitration on tau polymerization is specific for the 3-NT modification, as pseudophosphorylation at these same Tyr residues does not inhibit tau assembly. Our results suggest that the nitrative and oxidative roles of ONOO- differentially affect tau polymerization and that ONOO(-)-mediated cross-linking could facilitate tau aggregation in AD.     

Ajulemic acid, a synthetic cannabinoid acid, induces an antiinflammatory profile of eicosanoids in human synovial cells

AIMS: To better understand mechanisms whereby Ajulemic acid (AjA), a synthetic antiinflammatory cannabinoid, promotes resolution of acute and chronic inflammation in animal models, we investigated its influence on cyclooxygenase 2 (COX2) expression and eicosanoid production in human fibroblast-like synovial cells (FLS). MAIN METHODS: FLS isolated from tissue obtained at joint replacement surgery or cultured from synovial fluid were treated for 60 min with AjA (10-30 microM), then stimulated with tumor necrosis factor alpha (TNFalpha). COX2 mRNA was measured by hybridization/colorimetric assay of whole cell lysates collected 4 h after stimulation. To determine effects on arachidonic acid release, FLS were incubated with (14)C-arachidonic acid for 20 h then treated with AjA (8-32 microM). Arachidonic acid release was measured by scintillation counting. Prostaglandins (PG) were measured by enzyme linked immunosorbent assay (ELISA) in cell supernatants collected 4 and 24 h after stimulation. KEY FINDINGS: AjA increased the steady state levels of COX2 mRNA in and arachidonic acid release from FLS. Treatment of FLS with AjA increased 15-deoxy-delta(12,14)-PGJ(2) (15d-PGJ(2)) production in a concentration dependent manner, but did not affect PGE(2) production significantly. SIGNIFICANCE: The capacity of AjA to increase selectively and markedly 15d-PGJ(2), an eicosanoid which facilitates resolution of inflammation, suggests that AjA may have value as a therapeutic agent for the treatment of rheumatoid arthritis (RA) and other diseases characterized by acute and chronic inflammation.     

Different suicide inactivation processes for the peroxidase and cyclooxygenase activities of prostaglandin endoperoxide H synthase-1.

Prostaglandin endoperoxide H synthases (PGHSs)-1 and -2 have a cyclooxygenase (COX) activity involved in forming prostaglandin G2 (PGG2) from arachidonic acid and an associated peroxidase (POX) activity that reduces PGG2 to PGH2. Suicide inactivation processes are observed for both POX and COX reactions. Here we report COX reaction conditions for PGHS-1 under which complete COX inactivation occurs but with > or = 60% retention of POX activity. The rates of POX inactivation were compared for native oPGHS-1 versus Y385F oPGHS-1, a mutant that cannot form the Tyr385 radical of COX Intermediate II; the rates were the same for both native and Y385F oPGHS-1. Our data indicate that a COX Intermediate II/acyl or product complex is the precursor in COX inactivation. However, another species, probably an Intermediate II-like species but with a radical centered on a tyrosine other than Tyr385, is the immediate precursor for POX inactivation.     

Ancestral genetic complexity of arachidonic acid metabolism in Metazoa

Eicosanoids play an important role in inducing complex and crucial physiological processes in animals. Eicosanoid biosynthesis in animals is widely reported; however, eicosanoid production in invertebrate tissue is remarkably different to vertebrates and in certain respects remains elusive. We, for the first time, compared the orthologs involved in arachidonic acid (AA) metabolism in 14 species of invertebrates and 3 species of vertebrates. Based on parsimony, a complex AA-metabolic system may have existed in the common ancestor of the Metazoa, and then expanded and diversified through invertebrate lineages. A primary vertebrate-like AA-metabolic system via cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) pathways was further identified in the basal chordate, amphioxus. The expression profiling of AA-metabolic enzymes and lipidomic analysis of eicosanoid production in the tissues of amphioxus supported our supposition. Thus, we proposed that the ancestral complexity of AA-metabolic network diversified with the different lineages of invertebrates, adapting with the diversity of body plans and ecological opportunity, and arriving at the vertebrate-like pattern in the basal chordate, amphioxus.     

6]-Gingerol inhibits nitric oxide synthesis in activated J774.1 mouse macrophages and prevents peroxynitrite-induced oxidation and nitration reactions

Reactive nitrogen species (RNS), such as nitric oxide (NO) and its derivatives, e.g. peroxynitrite (ONOO-), have been proposed as being able to influence signal transduction and cause DNA damage, contributing to carcinogenic processes. In this study, the effect of [6]-gingerol, a pungent phenolic compound present in ginger (Zingiber officinale Roscoe), on NO synthesis in lipopolysaccharide (LPS)-activated J774.1 macrophages was tested, and the protective ability of this compound against peroxynitrite-mediated oxidation and nitration reactions were evaluated. [6]-Gingerol exhibited dose-dependent inhibition of NO production and significant reduction of inducible NO synthase (iNOS) in LPS-stimulated J774.1 cells. Moreover, [6]-gingerol effectively suppressed peroxynitrite-induced oxidation of dichlorodihydrofluorescein, oxidative single strand breaks in supercoiled pTZ 18U plasmid DNA, and formation of 3-nitrotyrosine in bovine serum albumin (BSA) and J774.1 cells. Our results indicate that [6]-gingerol is a potent inhibitor of NO synthesis and also an effective protector against peroxynitrite-mediated damage.     

Peroxynitrite-mediated tau modifications stabilize preformed filaments and destabilize microtubules through distinct mechanisms

Alzheimer's disease (AD) is a progressive amnestic dementia typified by abnormal modifications of the microtubule (MT)-associated tau protein that promote its pathological self-assembly and displacement from the MT lattice. Previously, we showed that peroxynitrite (ONOO-) induces the oxidative 3,3'-dityrosine (3,3'-DT) cross-linking and site-selective nitration of tau monomers [Reynolds et al. (2005) Biochemistry 44, 1690-1700]. In the present study, we examined the effects of ONOO(-)-mediated modifications on two key elements of tau pathobiology: (1) the stability of preformed tau filaments and (2) the ability of monomeric tau to promote tubulin assembly. Here, we report that treatment of synthetic tau filaments with ONOO- generates heat-stable, SDS-insoluble aggregates with a significantly reduced mobility by SDS-PAGE compared to that of nontreated filaments. Ultrastructurally, these aggregates appear to be cross-linked via interfilament bridges. Using LC-MS/MS and HPLC with fluorescent detection, we demonstrate that covalent 3,3'-DT linkages are present within these higher-order aggregates. Similar to monomeric tau, filamentous tau exhibits a hierarchical pattern of nitration following ONOO- treatment with site selectivity toward the amino-terminal residues Tyr18 and Tyr29. Further, select nitration of residues Tyr18, Tyr29, Tyr197, and Tyr394, events known to stabilize the pathological Alz-50 conformation [Reynolds et al. (2005) Biochemistry 44, 13997-14009], inhibits the ability of monomeric tau to promote tubulin assembly. This effect is specific for the 3-NT modification, as mutant tau proteins pseudophosphorylated at each Tyr residue are fully competent to stabilize MTs. Collectively, our results suggest that ONOO(-)-mediated modifications stabilize tau filaments via 3,3'-DT bonding and destabilize MTs by site-selective nitration of tau monomers. Moreover, assumption of the Alz-50 conformation may be the mechanism through which tau nitration modulates MT stability.     

Relationship between protein nitration and oxidation and development of hyperoxic seizures.

Recent studies have implicated nitric oxide (NO*) as a mediator of CNS hyperbaric O2 (HBO2) toxicity. One mechanism by which NO* may contribute to HBO2-induced brain toxicity involves a neurotoxic, pro-oxidative action of NO* via the formation of the potent oxidant peroxynitrite (ONOO-). The present study compares: (a) the formation of protein nitrotyrosine as a marker of ONOO- accumulation and (b) protein oxidation as an indicator of reactive oxygen species production during HBO2 exposure. Rats were exposed to 5 atm 100% O2 to pre-convulsive exposure or until the occurrence of electroencephalographic (EEG) seizures. After exposures, brains were analyzed for protein nitrotyrosine (NT) and protein carbonyl measurement by Western blot and for superoxide dismutase (SOD) activity by NBT assay. The results show a significant increase in protein NT, exceeding control level by several fold. There was only a slow and non-significant increase in the quantity of oxidized proteins during the pre-convulsive phase of HBO2 exposure. Levels of both protein NT and protein carbonyls were significantly (p<0.05) elevated after seizures. Total SOD activity was not changed during preconvulsive exposures, but was significantly (p<0.05) elevated post-seizures. The specific neuronal nitric oxide synthase (NOS) inhibitor, 7-nitroindazole (7-NI), significantly reduced the increases in seizure-induced protein NT and protein carbonyl and at the same time very effectively (p<0.05) delayed onset of HBO2 seizures. Pre-seizure increases in protein NT might indicate its role in the mechanism of HBO2-induced brain toxicity. This is supported by the observed capacity of 7-NI to inhibit tyrosine nitration and increase time to seizure.     

Endothelial NOS-derived nitric oxide prevents injury resulting from reoxygenation in the hypoxic lung

Abstract

To date, the role that NO derived from endothelial NO synthase (eNOS) plays in the development of the injuries occurring under hypoxia/reoxygenation (H/R) in the lung remains unknown and thus constitutes the subject of the present work. A follow-up study was conducted in Wistar rats submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 48 h and 5 days), with or without prior treatment using the eNOS inhibitor L-NIO (20 mg/kg). Lipid peroxidation, apoptosis, protein nitration and NO production (NOx) were analysed. The results showed that L-NIO administration lowered NOx levels in all the experimental groups. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose, implying that eNOS-derived NO may have a protective effect against the injuries occurring during H/R in the lung. These findings could open the possibility of future studies to design new therapies for this type of hypoxia based on NO-pharmacology.     

Tyrosine nitration in prostaglandin H(2) synthase

In this study, we investigated the effects of various nitrogen oxide (NO(x)) species on the extent of prostaglandin H(2) synthase-1 (PGHS-1) nitration in purified protein and in vascular smooth muscle cells. We also examined PGHS-1 activity under these conditions and found the degree of nitration to correlate inversely with enzyme activity. In addition, since NO(x) species are thought to invoke damage during the pathogenesis of atherosclerosis, we examined human atheromatous tissue for PGHS-1 nitration. Both peroxynitrite and tetranitromethane induced Tyr nitration of purified PGHS-1, whereas 1-hydroxy-2-oxo-3-(N-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7; a nitric oxide-releasing compound) did not. Smooth muscle cells treated with peroxynitrite showed PGHS-1 nitration. The extent of nitration by specific NO(x) species was determined by electrospray ionization mass spectrometry. Tetranitromethane was more effective than peroxynitrite, NOC-7, and nitrogen dioxide at nitrating a tyrosine-containing peptide (12%, 5%, 1%, and <1% nitration, respectively). Nitrogen dioxide and, to a lesser extent, peroxynitrite, induced dityrosine formation. Using UV/Vis spectroscopy, it was estimated that the reaction of PGHS-1 with excess peroxynitrite yielded two nitrated tyrosines/PGHS-1 subunit. Finally, atherosclerotic tissue obtained from endarterectomy patients was shown to contain nitrated PGHS-1. Thus, prolonged exposure to elevated levels of peroxynitrite may cause oxidative damage through tyrosine nitration.     

Early release of arachidonic acid prevents an otherwise immediate formation of toxic levels of peroxynitrite in astrocytes stimulated with lipopolysaccharide/interferon-gamma

Addition of bacterial lipopolysaccharides (LPS) and interferon-gamma (IFN-gamma) to rat astrocytes in primary culture promotes an early release of arachidonic acid (ARA) associated with an immediate inhibition of neuronal nitric oxide synthase (nNOS). Preventing the release of constitutive nitric oxide (NO) is indeed critical for activation of the nuclear factor kappa B, and for the expression of inducible nitric oxide synthase responsible for the formation of large amounts of NO. LPS/IFN-gamma also promotes an early release of superoxide, via activation of NADPH oxidase, but the generation of peroxynitrite (ONOO-) is prevented by the different timing of superoxide (minutes) and NO (hours) formation. Upstream inhibition of the ARA-dependent nNOS inhibitory signaling, however, caused the parallel release of superoxide and constitutive NO, thereby leading to formation of ONOO- levels triggering loss of ATP and mitochondrial membrane potential followed by the mitochondrial release of cytochrome c, activation of caspase 3 and morphological evidence of apoptosis. Nanomolar levels of exogenous ARA prevented all these events via inhibition of early ONOO- formation. Thus, the ARA-dependent nNOS inhibition observed in astrocytes exposed to pro-inflammatory stimuli, as LPS/IFN-gamma, is critical for both the expression of nuclear factor kappa B-dependent genes and for survival.     

Heme catalyzes tyrosine 385 nitration and inactivation of prostaglandin H2 synthase-1 by peroxynitrite

The mechanism by which the inflammatory enzyme prostaglandin H(2) synthase-1 (PGHS-1) deactivates remains undefined. This study aimed to determine the stabilizing parameters of PGHS-1 and identify factors leading to deactivation by nitric oxide species (NO(x)). Purified PGHS-1 was stabilized when solubilized in beta-octylglucoside (rather than Tween-20 or CHAPS) and when reconstituted with hemin chloride (rather than hematin). Peroxynitrite (ONOO(-)) activated the peroxidase site of PGHS-1 independently of the cyclooxygenase site. After ONOO(-) exposure, holoPGHS-1 could not metabolize arachidonic acid and was structurally compromised, whereas apoPGHS-1 retained full activity once reconstituted with heme. After incubation of holoPGHS-1 with ONOO(-), heme absorbance was diminished but to a lesser extent than the loss in enzymatic function, suggesting the contribution of more than one process to enzyme inactivation. Hydroperoxide scavengers improved enzyme activity, whereas hydroxyl radical scavengers provided no protection from the effects of ONOO(-). Mass spectral analyses revealed that tyrosine 385 (Tyr 385) is a target for nitration by ONOO(-) only when heme is present. Multimer formation was also observed and required heme but could be attenuated by arachidonic acid substrate. We conclude that the heme plays a role in catalyzing Tyr 385 nitration by ONOO(-) and the demise of PGHS-1.