Effect Of ONOO Research Articles

The good, the bad and the ugly of nitric oxide, superoxide and peroxynitrite signaling during oxygen‐glucose deprivation in rat and arctic ground squirrel

Cerebral ischemia‐reperfusion (I/R) injury initiates a complex cascade of events, several of which involve the generation of nitic oxide (NO) and oxygen (O2•−) free radicals. NO and O2•− rapidly combine to form peroxynitrite (ONOO−) a potent oxidant and nitrating agent. Previous studies have revealed that the arctic ground squirrel (AGS; Urocitellus parryii) is a natural model of high tolerance to I/R injury, however, the mechanisms that contribute to the ability to tolerate this pathological scenario is still elusive. Here, we tested the hypothesis that tolerance to I/R injury modeled in an acute hippocampal slice preparation in AGS is modulated by reduced oxidative and nitrosative stress associated with oxygen glucose deprivation (OGD).Hippocampal slices (400 micron) from rat and AGS were subjected to oxygen glucose deprivation (OGD) to mimic I/R in vivo using a novel microperfusion technique. Injury consisted of exposing the brain slices to: 1) NO and O2•− donors with and without OGD; and 2) pretreatment of slices with inhibitors of NO, O2•− and ONOO− followed by OGD. In control slices, treatment was administered by switching to standard aCSF. Perfusates were collected every 15 min and analyzed for LDH release, an indicator of cell death; slices were analyzed for nitrosative and oxidative damage using the markers 3‐nitrotyrosine (3NT) and 4‐hydroxynonenal (4HNE).Results show the effect of NO, O2•− and ONOO− mediated injury in ischemic tolerant AGS and ischemic susceptible rat. 1) Effect of NO mediated Injury: An NO donor alone is not sufficient to cause cell death, but with OGD enhances cell death more in rat than in AGS; an inhibitor of nNOS attenuates OGD‐induced injury in rat but has no effect in AGS. 2) Effect of O2•− mediated injury: A O2•− donor alone causes slight injury in rat and even less injury in AGS. Combining the O2•− donor with OGD aggravates the injury in rat; a SOD mimetic attenuates OGD injury in rat but has no effect in AGS. 3) Effect of ONOO− mediated injury: A ONOO− inhibitor attenuates OGD injury in rat but has no effect in AGS. Rat also shows higher levels of 3NT and 4HNE after OGD insult than AGS. These data demonstrate that in rat both NO and O2•− are required for injury and point to ONOO− as the damaging agent. This same mechanism of OGD‐induced cell death is lacking in AGS supporting the hypothesis that resistance to oxidative and nitrosative stress contributes to tolerance to cerebral I/R injury in AGS.Support or Funding InformationThis work was supported by the US Army Medical research and Material Command, No 0517800; the National Institute of Neurological Disorder and Stroke, Nos. NS041069‐06 and R15NS070779; Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103395.

Relevance of peroxynitrite formation and 3-nitrotyrosine on spermatozoa physiology

Infertility is a clinical condition that affects around 15% of reproductive-aged couples worldwide. Around half of these cases are due to male factors, the most owing to idiopathic causes. The increase of reactive oxygen species (ROS), which leads to oxidative stress (OS), has been discussed in the last years as a possible cause of male idiopathic infertility. Superoxide anion (O2•−) and nitric oxide (NO) can react with each other contributing to the formation of peroxynitrite (ONOO−). This molecule can then act on spermatozoa proteins, leading to nitration of protein tyrosines – addition of a nitro (NO2) group – that is then manifested by the formation of 3-nitrotyrosine (3-NT). In turn, 3-NT may be responsible for the alteration or inactivation of the protein function.This review will focus on the description of spermatozoa ROS, namely O2•−, NO and ONOO− and in their contribution to protein tyrosine nitration, namely by 3-NT formation. Previous results about the effect of ONOO− and 3-NT in spermatozoa will be presented, as well as, the methods that can be performed to detect the protein oxidation by these species. The impact of measuring, at the clinical level, 3-NT, considered a marker of OS, in spermatozoa will be discussed.

Endothelium and the effect of activated neutrophils on arterial smooth muscle.

The aim of the study was to analyze the involvement of the endothelium in the effects of neutrophils (PMNL) on phenylephrine-precontracted isolated rings of the rat thoracic aorta and to compare their effects with those of peroxynitrite (ONOO−) and hypochlorous acid (HOCl). Activated PMNL-induced contraction of the precontracted aorta was prevented by the blockade of NO-synthase and by endothelium removal. In the endothelium-free preparations, the effect of PMNL reappeared in the presence of sodium nitroprusside. The effect of ONOO– and HOCl significantly differed from that of activated PMNL both in the presence and absence of the endothelium. It is therefore likely that neither ONOO– nor HOCl generated by transformation of superoxide anion radical (O2•–) produced by PMNL is involved in their action. Reduction of the relaxant effect of nitric oxide derived from the endothelium by O2•– seems to be the keystone mechanism in generation of PMNL-induced contraction.

Trifolium pallidum and Trifolium scabrum extracts in the protection of human plasma components

Clovers (genus: Trifolium) have been used in traditional medicine by many cultures, but the biological activity of the most of these plants still remains unknown. The aim of our in vitro study was to assess the antioxidative action of phenolic extracts from aerial parts of Trifolium scabrum and Trifolium pallidum in human blood plasma, exposed to oxidative stress. In the present study we also demonstrate, for the first time the effects of the tested extracts on coagulative properties and fibrinolytic activity of blood plasma. The protective properties of the examined extracts (0.5-50 μg/ml) against peroxynitrite-induced oxidative stress were estimated by the measurements of 3-nitrotyrosine, thiol groups and the thiobarbituric acid-reactive substances levels. The extracts considerably prevented the oxidative and nitrative damage to plasma proteins. Even the lowest doses of the Trifolium extracts (0.5 μg/ml) were able to markedly reduce 3-nitrotyrosine formation (by about 50%) and to increase the level of -SH groups (by about 30%), in comparison to the plasma exposed to ONOO(-) in the absence of the extracts. The protective action of all the used concentrations of the Trifolium extracts in the prevention of lipid peroxidation was also found. The tested extracts influenced neither the coagulative properties nor fibrinolytic activity of plasma. Moreover, the extracts were able to significantly reduce the inhibitory effect of ONOO(-) on fibrinolytic activity of plasma (assessed with the use of a chromogenic substrate for plasmin).

Enhancement of cellular Na+K+ATPase activity in the mouse renal tissue in‐vitro with low concentration of peroxynitrite

Endogenous production of peroxynitrite (ONOO°−) in normal condition has been suggested to play a renoprotective role. However, the exact mechanism associated with this protection is not yet clearly defined. ONOO°− at higher concentrations (μM to mM; in pathology) can inhibit cellular Na+K+ATPase (NKA) activity. However, the effect of ONOO°− on NKA activity at low conc. (nM; which is physiologically more relevant) remains unknown. To examine the hypothesis that ONOO°− at low conc. enhances NKA activity, preparations of cellular membrane fraction from mouse renal tissue (n=5 in each case) were incubated for 10 min each with different conc. of ONOO°− (10 nM to 200 μM). NKA activity in these samples was measured via a colorimetric assay capable of detecting inorganic phosphate. At high conc. (1–200 μM), ONOO°− caused dose‐dependent inhibition of NKA activity (−3.0±0.6 % to – 36.4±1.4 %) as reported previously. However, NKA activity remained unchanged at 100 and 500 nM ONOO°− conc., but interestingly, at lower conc.(10 and 50 nM), ONOO°− caused a small but significant increases in the NKA activity (3.3±1.1 % & 3.1±0.6 %). These responses were prevented at preparations co‐incubated with ONOO°− scavenger, mercapto ethyl guanadine (200 μM). These data indicate that NKA activity remains unaffected or rather increased at the physiological conc. of ONOO°− that facilitates the reno‐protective role for this oxidant molecule.

Reactive Nitrogen Species-Dependent Effects on Soybean Chloroplasts

Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was studied by electron paramagnetic resonance (EPR) spin-trapping technique1. Both nitrite and L-arginine (arg) are the required substrates for enzymatic activities considered as possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 ± 0.2 nmol min-1 mg-1 protein in the presence of 1 mM NaNO2. Chloroplasts incubated with 1 mM arg showed a NO production of 0.76 ± 0.04 nmol min-1 mg-1 protein. This production was inhibited when chloroplasts were incubated in presence of NOS-inhibitors L-NAME and L-NNA. In vitro exposure of chloroplasts to a NO-donor (GSNO) decreased both ascorbyl radical content and the activity of ascorbate peroxidase, without modification of the total ascorbate content. Exposure of the isolated chloroplasts to a NO-donor decreased lipid radical content in membranes, however, incubation in the presence of 25 μM peroxynitrite (ONOO-) led to an increase in lipid-derived radicals (34%). The effect of ONOO- on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to control. Taken as a whole, NO seems to be an endogenous metabolite in soybean chloroplasts and reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplasts, since both a decreased lipid radical content in membranes and a decrease in the activity of ascorbate peroxidase were observed after exposure to a NO donor.

Chloroplasts as a nitric oxide cellular source. Effect of reactive nitrogen species on chloroplastic lipids and proteins.

Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and l-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 +/- 0.2 nmol min(-1) mg(-1) protein in the presence of 1 mm NaNO(2). Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 +/- 0.04 nmol min(-1) mg(-1) protein) of NO generation. Chloroplasts incubated with 1 mm Arg showed NO production of 0.76 +/- 0.04 nmol min(-1) mg(-1) protein that was not affected either by omission of Ca(2+) or by supplementation with Ca(2+) and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors N(omega)-nitro-l-Arg methyl ester hydrochloride and N(omega)-nitro-l-Arg. In vitro exposure of chloroplasts to an NO donor (250 mum S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 mum peroxynitrite (ONOO(-)) led to an increase in lipid-derived radicals (34%). The effect of ONOO(-) on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO(-) treatment significantly affected both O(2) evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.

Peroxynitrite induces arteriolar smooth muscle cells membrane hyperpolarization with arteriolar hyporeactivity in rats

Peroxynitrite (ONOO −) has been recently known to act as a potent cytotoxin during pathogenesis of various diseases. This study aimed to investigate the possible effect of ONOO − on the cremaster muscle arteriolar reactivity in response to noradrenaline and subsequently determined whether membrane hyperpolarization and potassium channel activation were involved in ONOO −-induced alteration of arteriolar reactivity. The results demonstrated that 1) ONOO − could decrease arteriolar reactivity in a time- and concentration-dependent manner with no significant alteration of arteriolar diameter; 2) Superfusion with 20 μM ONOO − over 40 minutes showed slight but not significant influence on the resting potential (Em) of arteriolar smooth muscle cells (ASMCs). However, ASMCs subjected to 50 or 100 μM ONOO − administration were significantly hyperpolarized. As control, treatment with 50 μM decomposed ONOO − or Kreb's solution had little effect on the Em of ASMCs; 3) ONOO −-induced arteriolar hyporeactivity could be greatly reversed by co-administration of KCl and partially by TEA. The above results indicated that membrane hyperpolarization and potassium channel activation were preferentially responsible for the reduction of cremaster muscle arteriolar reactivity after exposure to ONOO −.

Peroxynitrite inactivates the human dopamine transporter by modification of cysteine 342: potential mechanism of neurotoxicity in dopamine neurons.

Peroxynitrite (ONOO(-)) has been implicated as a causative factor in dopamine neuronal damage resulting from exposure to methamphetamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and it may be involved in the etiology of Parkinson's Disease. ONOO(-) causes a concentration-dependent and irreversible reduction in dopamine uptake by EM4 cells stably expressing the human dopamine transporter (hDAT). The effect of ONOO(-) is manifested as a reduction in V(max). Cysteine, dithiothreitol, glutathione, and N-acetyl-cysteine, reagents that interact directly with ONOO(-), prevent this inhibition, whereas a scavenger of hydroxyl radical (dimethylsulfoxide), hydrogen peroxide (catalase), and superoxide (superoxide dismutase) did not. Dopamine in the extracellular medium protects the hDAT from ONOO(-), whereas intracellular dopamine does not. Parachloromercuribenzoic acid and 2-aminoethyl methanethiosulfonate (MTSEA), which share with ONOO(-) the ability to modify cysteine sulfhydryls, also inhibit hDAT function. ONOO(-) treatment lowers cysteine-specific labeling of the hDAT by MTSEA-biotin, suggesting that ONOO(-) reacts with one or more cysteines in hDAT. A mutant of hDAT (X7C) in which all intracellular and extracellular loop cysteines were mutated was resistant to inhibition by ONOO(-). Sensitivity to ONOO(-) was restored in mutants of hDAT in which reduced cysteines were present only in the first (C135) and third (C342) intracellular loops (CD-DAT), or in which C342 alone had been reintroduced into X7C (X7C-M342C). These results indicate that the hDAT is inhibited by ONOO(-) through oxidation of cysteine 342. Our studies also substantiate the possibility that drugs known to decrease DAT function in vivo (e.g., methamphetamine and MPTP) may exert their effects through ONOO(-)-mediated oxidative stress.

Peroxynitrite Inactivates the Human Dopamine Transporter by Modification of Cysteine 342: Potential Mechanism of Neurotoxicity in Dopamine Neurons

Peroxynitrite (ONOO⁻) has been implicated as a causative factor in dopamine neuronal damage resulting from exposure to methamphetamine and1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and it may be involved in the etiology of Parkinson9s Disease. ONOO⁻ causes a concentration-dependent and irreversible reduction in dopamine uptake by EM4 cells stably expressing the human dopamine transporter (hDAT). The effect of ONOO⁻ is manifested as a reduction in<i>V</i>_max. Cysteine, dithiothreitol, glutathione, and <i>N</i>-acetyl-cysteine, reagents that interact directly with ONOO⁻, prevent this inhibition, whereas a scavenger of hydroxyl radical (dimethylsulfoxide), hydrogen peroxide (catalase), and superoxide (superoxide dismutase) did not. Dopamine in the extracellular medium protects the hDAT from ONOO⁻, whereas intracellular dopamine does not. Parachloromercuribenzoic acid and 2-aminoethyl methanethiosulfonate (MTSEA), which share with ONOO⁻ the ability to modify cysteine sulfhydryls, also inhibit hDAT function. ONOO⁻ treatment lowers cysteine-specific labeling of the hDAT by MTSEA-biotin, suggesting that ONOO⁻ reacts with one or more cysteines in hDAT. A mutant of hDAT (X7C) in which all intracellular and extracellular loop cysteines were mutated was resistant to inhibition by ONOO⁻. Sensitivity to ONOO⁻ was restored in mutants of hDAT in which reduced cysteines were present only in the first (C135) and third (C342) intracellular loops (CD-DAT), or in which C342 alone had been reintroduced into X7C (X7C-M342C). These results indicate that the hDAT is inhibited by ONOO⁻ through oxidation of cysteine 342. Our studies also substantiate the possibility that drugs known to decrease DAT function <i>in vivo</i> (e.g., methamphetamine and MPTP) may exert their effects through ONOO⁻-mediated oxidative stress.

Effect of peroxynitrite on glutaredoxin.

Glutaredoxin is an important enzyme in thiol homeostasis. As a thioltransferase, it reduces oxidized thiols. It also has dehydroascorbate reductase (DHAR) activity to reduce dehydroascorbate (DHA) to ascorbic acid. Peroxynitrite (ONOO-) is one of the most active elements of oxidative stress that can be formed wherever nitric oxide and superoxide are produced simultaneously. ONOO- is known to react with free thiols easily. To observe the effect of ONOO on glutaredoxin, rat liver cytosolic fractions were incubated with 0-250 microM ONOO-. Thioltransferase activity was found to be decreased as ONOO concentration increased. The inhibition was not reversible with dithiothreitol (DTT). In cytosol besides glutaredoxin, another enzyme with DHAR activity is also present. In our study, the cytosolic DHAR activity which consisted both enzymes, was also inhibited by ONOO-, but DTT was able to return the activity almost completely.

Peroxynitrite Targets the Epidermal Growth Factor Receptor, Raf-1, and MEK Independently to Activate MAPK

Activation of ERK-1 and -2 by H(2)O(2) in a variety of cell types requires epidermal growth factor receptor (EGFR) phosphorylation. In this study, we investigated the activation of ERK by ONOO(-) in cultured rat lung myofibroblasts. Western blot analysis using anti-phospho-ERK antibodies along with an ERK kinase assay using the phosphorylated heat- and acid-stable protein (PHAS-1) substrate demonstrated that ERK activation peaked within 15 min after ONOO(-) treatment and was maximally activated with 100 micrometer ONOO(-). Activation of ERK by ONOO(-) and H(2)O(2) was blocked by the antioxidant N-acetyl-l-cysteine. Catalase blocked ERK activation by H(2)O(2), but not by ONOO(-), demonstrating that the effect of ONOO(-) was not due to the generation of H(2)O(2). Both H(2)O(2) and ONOO(-) induced phosphorylation of EGFR in Western blot experiments using an anti-phospho-EGFR antibody. However, the EGFR tyrosine kinase inhibitor AG1478 abolished ERK activation by H(2)O(2), but not by ONOO(-). Both H(2)O(2) and ONOO(-) activated Raf-1. However, the Raf inhibitor forskolin blocked ERK activation by H(2)O(2), but not by ONOO(-). The MEK inhibitor PD98059 inhibited ERK activation by both H(2)O(2) and ONOO(-). Moreover, ONOO(-) or H(2)O(2) caused a cytotoxic response of myofibroblasts that was prevented by preincubation with PD98059. In a cell-free kinase assay, ONOO(-) (but not H(2)O(2)) induced autophosphorylation and nitration of a glutathione S-transferase-MEK-1 fusion protein. Collectively, these data indicate that ONOO(-) activates EGFR and Raf-1, but these signaling intermediates are not required for ONOO(-)-induced ERK activation. However, MEK-1 activation is required for ONOO(-)-induced ERK activation in myofibroblasts. In contrast, H(2)O(2)-induced ERK activation is dependent on EGFR activation, which then leads to downstream Raf-1 and MEK-1 activation.

Ghost protein damage by peroxynitrite and its protection by melatonin.

We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 microM ONOO-) disappearance at pH 7. 4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ss-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 microM ONOO-). However, a lack of effect of ss-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that (ONOO-)-induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 microM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.

Endothelium-dependent relaxation of rabbit atherosclerotic aorta was not restored by control of hyperlipidemia: the possible role of peroxynitrite (ONOO −)

We determined the role of ONOO − in nitric oxide (NO) mediated vascular response in atherosclerosis and regression following removal of dietary cholesterol. The effect of ONOO − on NO-mediated vascular responses was examined in vitro. Basal and stimulated NO release was estimated by an NO-selective electrode as well as vascular response and the plasma NO metabolites. An immunohistochemical study was also carried out. Responses were compared in normal controls, atherosclerotic rabbits fed 1% cholesterol diet for 6 or 9 weeks (atherosclerotic group) and animals fed a normal diet for 6–36 weeks after the high cholesterol diet for 6 or 9 weeks (regression group). ONOO − impaired the basal and acetylcholine-stimulated NO release, but did not affect endothelium-independent relaxation. After 15 weeks on a normal diet, the acetylcholine-stimulated and basal NO-mediated relaxation, which was diminished in the aorta induced by 6 weeks high cholesterol diet, became restored. However, the vascular response in the 9 weeks high cholesterol diet group did not return to normal after 36 weeks on a normal diet. iNOS was observed in atherosclerotic plaques in atherosclerotic and regression groups along with ONOO − in the 9 weeks high cholesterol diet group, but not in the 6 weeks group. Conclusively, ONOO − can play a role in impairment of NO-mediated vascular response during the regression of dietary cholesterol-induced atherosclerosis, not in the initiation of atherosclerosis.

Peroxynitrite anion stimulates arginine release from cultured rat astrocytes.

The biosynthesis of the physiological messenger nitric oxide (*NO) in neuronal cells is thought to depend on a glial-derived supply of the *NO synthase substrate arginine. To expand our knowledge of the mechanism responsible for this glial-neuronal interaction, we studied the possible roles of peroxynitrite anion (ONOO-), superoxide anion (O2*-), *NO, and H2O2 in L-[3H]arginine release in cultured rat astrocytes. After 5 min of incubation at 37 degrees C, initial concentrations of 0.05-2 mM ONOO- stimulated the release of arginine from astrocytes in a concentration-dependent way; this effect was maximum from 1 mM ONOO- and proved to be approximately 400% as compared with control cells. ONOO(-)-mediated arginine release was prevented by arginine transport inhibitors, such as L-lysine and N(G)-monomethyl-L-arginine, suggesting an involvement of the arginine transporter in the effect of ONOO-. In situ xanthine/xanthine oxidase-generated O2*- (20 nmol/min) stimulated arginine release to a similar extent to that found with 0.1 mM ONOO-, but this effect was not prevented by arginine transport inhibitors. *NO donors, such as sodium nitroprusside, S-nitroso-N-acetylpenicillamine, or 1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium+ ++-1,2-diolate, and H2O2 did not significantly modify arginine release. As limited arginine availability for neuronal *NO synthase activity may be neurotoxic due to ONOO- formation, our results suggest that ONOO(-)-mediated arginine release from astrocytes may contribute to replenishing neuronal arginine, hence avoiding further generation of ONOO- within these cells.

Effects of reactive oxygen species on brain synaptic plasma membrane Ca 2+-ATPase

The regulation of free intracellular calcium [Ca 2+] i is altered in neurons from the aged brain, possibly due to reductions in the activity of Ca 2+transporters. The plasma membrane Ca 2+-ATPase (PMCA) plays a critical role in Ca 2+ homeostasis, and its kinetic properties change in aged rat brain. These changes could be due to oxidative modification of PMCA as a result of age-related chronic oxidative stresses. The present studies were undertaken to determine the sensitivity of the neuronal PMCA to in vitro exposure of synaptic plasma membranes (SPMs) to reactive oxygen species (ROS). We examined the effects of three oxidants including peroxyl radicals generated by azo-initiators, 2,2′-Azobis 2-amidinopropane dihydrochloride (AAPH) and 4,4′-Azobis 14-cyanovaleric acid (ACVA), hydrogen peroxide (H 2O 2), and peroxynitrite (ONOO −). Synaptic plasma membranes briefly exposed to these oxidants were analyzed for functional and structural alterations in PMCA. Although all three oxidants led to significant loss of PMCA activity, the effect of ONOO − was the most potent, followed by peroxyl radicals and H 2O 2. Kinetic analysis of PMCA activity after oxidant treatment showed decreases in V max without significant changes in K act. Immunoblots revealed oxidant-induced cross-linking of PMCA molecules that were partially reversed under reducing conditions and completely reversed with addition of urea. The PMCA appears to be very sensitive to inhibition by ROS and hence may be a target of oxidative stress in the aging brain. Reduction in its activity may contribute to age-related alterations in neuronal [Ca 2+] i regulation.

Peroxynitrite is a contractile agonist of cerebral artery smooth muscle cells.

On reperfusion of ischemic tissue, a prolonged phase of vasoconstriction occurs, the mechanism of which is poorly understood. However, it is known that peroxynitrite (ONOO-) is formed during reperfusion. In this study the contractile properties of ONOO- were investigated in Wistar rat middle cerebral arteries. The effects of ONOO- on vessel diameter were dose dependent. Low-dose ONOO- (10 microM) caused vessels to constrict by 15%. At an intermediate concentration of 25 microM, the effect of ONOO- was variable, whereas at the highest concentration (100 microM), vessels underwent persistent dilation and became insensitive to the endogenous vasoconstrictor 5-hydroxytryptamine. At the single cell level, ONOO- caused cerebral artery smooth muscle cells to contract. Reduced, but not oxidized, glutathione completely inhibited the contractile action of ONOO- on single cells. Vehicle and decomposed ONOO- each had minimal effect on cell length. These data show that ONOO- is a contractile agonist of middle cerebral arteries, at the single cell and whole vessel levels, suggesting that formation of ONOO- may contribute mechanistically to ischemic brain injury during stroke. Moreover, relatively high concentrations of ONOO- result in vascular paralysis.

Peroxynitrite induces both vasodilatation and impaired vascular relaxation in the isolated perfused rat heart.

The effects of the oxidant species peroxynitrite (ONOO-) on coronary perfusion pressure and vasodilatation elicited by acetylcholine, isoproterenol, and S-nitroso-N-acetyl-DL-penicillamine were investigated in the isolated perfused rat heart. ONOO- (0.3-1000 microM) caused a concentration-dependent vasodilatation of the coronary vasculature. This dilator response was inhibited by oxyhemoglobin, indicating that it was due to the generation of nitric oxide. Tachyphylaxis to ONOO- developed rapidly, so that the response disappeared after three or four applications of this compound. ONOO- not only induced tachyphylaxis but also inhibited the vasodilatation induced by the three vasodilators studied. This latter effect of ONOO- was critically dependent on its concentration, since it occurred at 3 microM, which was subthreshold as a dilator, and at 1000 microM, which was supramaximal, but not at 30 and 100 microM. These latter concentrations inhibited the responses to vasodilators only in the presence of oxyhemoglobin. Thus, a wide range of concentrations of ONOO- induce a vascular dysfunction, as evidenced by the tachyphylaxis to its own vasodilator actions and the long-lasting impairment of the responses to other vasodilators. However, at the same time ONOO- generates nitric oxide, which at certain concentrations of ONOO- is sufficient to counteract its deleterious action. Coinfusion of S-nitroso-N-acetyl-DL-penicillamine or prostacyclin at low concentrations that did not produce vasodilatation also protected against ONOO(-)-induced vascular dysfunction: these compounds may be protective through a common mechanism, as yet undefined.