Peroxynitrite increases iNOS through NF-kappa B and decreases prostacyclin synthase in endothelial cells

Peroxynitrite, a marker of oxidativestress, is elevated in conditions associated with vascular endothelialcell dysfunction, such as atherosclerosis, preeclampsia, and diabetes.However, the effects of peroxynitrite on endothelial cell function are not clear. The endothelium-derived enzymes nitric oxide synthase (NOS)and prostaglandin H synthase (PGHS) mediate vascular reactivity andcontain oxidant-sensitive isoforms (iNOS and PGHS-2) that can beinduced by nuclear factor (NF)-B activation. We investigated theeffect(s) of peroxynitrite on NOS and PGHS pathways in endothelial cells. We hypothesized that peroxynitrite will increase levels of iNOSand PGHS-2 through activation of NF-B. Western immunoblots ofendothelial cells show that 3-morpholinosydnonimine (SIN-1; 0.5 mM), aperoxynitrite donor, increased iNOS protein mass, which can beinhibited by pyrroline dithiocarbamate (an NF-B inhibitor) (167 ± 24.2 vs. 78 ± 19%, P < 0.05, n = 6). SIN-1 treatment also significantly increasedNF-B translocation into endothelial cell nuclei (135 ± 10%,P < 0.05). Endothelial NOS, PGHS-1, and PGHS-2 proteinlevels were not altered by SIN-1. However, prostacyclin synthaseprotein mass, but not mRNA, was significantly reduced in SIN-1-treatedendothelial cells (78 ± 8.9%, P < 0.05). Our results illustrate novel mechanisms through which peroxynitrite maymodulate vascular endothelial function.

     

Peroxynitrite and nitric oxide differ in their effects on pig coronary artery smooth muscle

Peroxynitrite generated in arteries fromsuperoxide and nitric oxide (NO) may damage their function. Here, wecompare the effects of peroxynitrite and peroxynitrite/NO-generatingagents SIN-1 (3-morpholinosydnonimine hydrochloride), SNAP(S-nitroso-N-acetyl-penicillamine), SNP (sodiumnitroprusside), and NONOate (spermine NONOate) on pig coronary artery.Deendothelialized artery rings were pretreated with these agents andthen washed before examining their contractility. Pretreatment with allagents (200 µM) results in a decrease in the force of contraction inresponse to the sarco(endo)plasmic Ca²⁺ (SERCA) pumpinhibitor cyclopiazonic acid (CPA): SNAP > NONOate  peroxynitrite  SIN-1 > SNP. Pretreatment with SNAP,NONOate, or SIN-1 also inhibits the force of contraction produced with 30 mM KCl, with SNAP being the most potent. Including catalase plussuperoxide dismutase (SOD) during the preincubation has no effect. Including an NO scavenger[2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide] or a guanylate cyclase inhibitor(1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) partially protects against SNAP. Pretreatment of cultured cells with peroxynitrite, but not with SNAP, inhibits the Ca²⁺transients produced in response to CPA. Pretreating isolated membranevesicles with peroxynitrite inhibits the Ca²⁺ uptake due tothe SERCA pump, with all the other agents being less effective. Thusperoxynitrite and NO both inhibit the CPA-induced contractions indeendothelialized artery rings, peroxynitrite by damage to the SERCApump and NO possibly by a step downstream from the increase incytosolic Ca²⁺.

     

Reactive nitrogen species control apoptosis and autophagy in K562 cells: implication of TAp73α induction in controlling autophagy

The biological outcome of nitric oxide (NO) and reactive nitrogen species (RNS) in regulating pro survival and pro death autophagic pathways still demand further investigation. In the present study, we investigated the effect of nitrosative stress in K562 cells using NO donor compound DETA-NONOate, peroxynitrite, and SIN-1. Exposure to NO, peroxynitrite, and SIN-1 caused decrease in K562 cell survival. NO induced autophagy but not apoptosis or necrosis in K562 cells. In contrast, peroxynitrite and SIN-1 treatment induced apoptosis in K562 cells. Surprisingly, inhibition of autophagic response using 3-methyladenine led to the induction of apoptosis in K562 cells. Increase in 5’adenosine monophosphate-activated protein kinase (AMPK) phosphorylation was only observed in the presence of NO donor indicated that AMPK was crucial to induce autophagy in K562 cells. We for the first time discovered a novel role of p73 in autophagy induction under nitrosative stress in K562 cells. TAp73α was only induced upon exposure to NO but not in the presence of peroxynitrite. Reduced glutathione (GSH)/oxidised glutathione (GSSG) ratio remained unaltered upon NO exposure. Our data suggest a complex network of interaction and cross regulations between NO and p73. These data open a new path for therapies based on the abilities of RNS to induce autophagy-mediated cell death.     

Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells

Peroxynitrite, formed by the reaction of nitric oxide (NO. ) with superoxide anions (O(2)(-).), may play a role in the pathophysiology of inflammation. The effects of 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, on the human bronchial epithelial cell line BEAS-2B, were examined. SIN-1 exposure resulted in cell death in a time- and dose-dependent manner. Depletion of intracellular glutathione increased the vulnerability of the cells. Pretreatment with Mn(III)tetrakis(N-methyl-4'-pyridyl)porphyrin (MnTMPyP) or hydroxocobalamin (HC), O(2)(-). and NO. scavengers, respectively, reduced significantly SIN-1-induced cell death (18.66 +/- 3.57 vs. 77.01 +/- 14.07 or 82.20 +/- 9.64, % cell viability SIN-1 vs. MnTMPyP or HC). Moreover, the mitogen-activated protein kinases (MAPK) p44/42 (ERK), p38, and p54/46 (JNK) were also activated in a time- and concentration-dependent manner. PD-98059 and SB-239063, specific inhibitors of ERK and p38 MAPK pathways, failed to protect cells against 1 mM SIN-1. However, PD-98059 partially inhibited (60% cell survival) SIN-1 effects at < or =0.25 mM, and this was increased with the inclusion of SB-239063. Therefore, MAPKs may mediate signal transduction pathways induced by peroxynitrite in lung epithelial cells leading to cell death.     

Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing alpha beta gamma -rENaC

We examined the effect of peroxynitrite(ONOO) on the cloned ratepithelial Na⁺ channel(-rENaC) expressed in Xenopusoocytes. 3-Morpholinosydnonimine (SIN-1) was used to concurrentlygenerate nitric oxide (· NO) and superoxide(O₂ ·), which react toform ONOO, a species knownto promote protein nitration and oxidation. Under control conditions,oocytes displayed an amiloride-sensitive whole cell conductance of 7.4 ± 2.8 (SE) µS. When incubated at 18°C with SIN-1 (1 mM) for 2 h (final ONOO concentration = 10 µM), the amiloride-sensitive conductance was reduced to0.8 ± 0.5 µS. To evaluate whether the observed inhibition was due to ONOO, as opposedto · NO, we also exposed oocytes to SIN-1 in the presence ofurate (500 µM), a scavenger ofONOO and superoxidedismutase, which scavengesO₂ ·, converting SIN-1from an ONOO to an· NO donor. Under these conditions, conductance values remained at control levels following SIN-1 treatment.Tetranitromethane, an agent that oxidizes sulfhydryl groups at pH6, also inhibited the amiloride-sensitive conductance. These datasuggest that oxidation of critical sulfhydryl groups within rENaC byONOO directly inhibitschannel activity.

     

Effects of nitric oxide on blood flow distribution and O2 extraction capabilities during endotoxic shock

Zhang, Haibo, Peter Rogiers, Nadia Smail, Ana Cabral,Jean-Charles Preiser, Marie-Odile Peny, and Jean-Louis Vincent.Effects of nitric oxide on blood flow distribution andO₂ extraction capabilities duringendotoxic shock. J. Appl. Physiol.83(4): 1164-1173, 1997.The effects of the nitric oxide (NO)synthase inhibitorN^G-monomethyl-L-arginine(L-NMMA) and the NO donor3-morpholinosydnonimine (SIN-1) were tested in 18 endotoxic dogs. L-NMMA infusion(10 mg · kg¹ · h¹)increased arterial and pulmonary artery pressures and systemic andpulmonary vascular resistances but decreased cardiac index, leftventricular stroke work index, and blood flow to the hepatic, portal,mesenteric, and renal beds. SIN-1 infusion (2 µg · kg¹ · min¹)increased cardiac index; left ventricular stroke work index; andhepatic, portal, and mesenteric blood flow. It did not significantly influence arterial and pulmonary artery pressures but decreased renalblood flow. The critical O₂delivery was similar in the L-NMMA group and in the controlgroup (13.3 ± 1.6 vs. 12.8 ± 3.3 ml · kg¹ · min¹)but lower in the SIN-1 group (9.1 ± 1.8 ml · kg¹ · min¹,both P < 0.05). The criticalO₂ extraction ratio was alsohigher in the SIN-1 group than in the other groups (58.7 ± 10.6 vs.42.2 ± 7.6% in controls, P < 0.05; 43.0 ± 15.5% inL-NMMA group,P = not significant). We conclude thatNO is not implicated in the alterations inO₂ extraction capabilitiesobserved early after endotoxin administration.

     

SIN-1-induced cytotoxicity in mixed cortical cell culture: peroxynitrite-dependent and -independent induction of excitotoxic cell death

3-Morpholinosyndnomine (SIN-1) has been reported to be a peroxynitrite (OONO(-)) donor because it produces both nitric oxide (NO) and superoxide (O(2)(-).) upon decomposition in aqueous solution. However, SIN-1 can decompose to primarily NO in the presence of electron acceptors, including those found in biological tissues, making it necessary to determine the release product(s) formed in any given biological system. In a mixed cortical cell culture system, SIN-1 caused a concentration-dependent increase in cortical cell injury with a parallel increase in the release of cellular proteins containing 3-nitrotyrosine into the culture medium. The increase in 3-nitrotyrosine immunoreactivity, a footprint of OONO(-) production, was specific for SIN-1 as exposure to neurotoxic concentrations of an NO donor (Z)-1-[2-aminoethyl)-N-(2-ammonioethyl) aminodiazen-1-ium-1,2-diolate (DETA/NO), or NMDA did not result in the nitration of protein tyrosine residues. Both SIN-1-induced injury and 3-nitrotyrosine staining were prevented by the addition of either 5,10,15,20-Tetrakis (4-sulfonatophenyl) prophyrinato iron (III) [FeTPPS], an OONO(-) decomposition catalyst, or uric acid, an OONO(-) scavenger. Removal of NO alone was sufficient to inhibit the formation of OONO(-) from SIN-1 as well as its cytotoxicity. Removal of O(2)(-). and the subsequently formed H(2)O(2) by superoxide dismutase (SOD) plus catalase likewise prevented the nitration of protein-bound tyrosine but actually enhanced the cytotoxicity of SIN-1, indicating that cortical cells can cope with the oxidative but not the nitrosative stress generated. Finally, neural injury induced by SIN-1 in unadulterated cortical cells was prevented by antagonism of AMPA/kainate receptors, while blockade of the NMDA receptor was without effect. In contrast, activation of both NMDA and non-NMDA receptors contributed to the SIN-1-mediated neurotoxicity when cultures were exposed in the presence of SOD plus catalase. Thus, whether SIN-1 initiates neural cell death in an OONO(-)-dependent or -independent manner is determined by the antioxidant status of the cells. Further, the mode of excitotoxicity by which injury progresses is determined by the NO-related species generated.     

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.     

15-Deoxy-Delta12,14-prostaglandin J(2) protects against nitrosative PC12 cell death through up-regulation of intracellular glutathione synthesis

Nitrosative stress with subsequent inflammatory cell death has been associated with many neurodegenerative disorders. Expression of inducible nitric-oxide synthase and production of nitric oxide (NO) have been frequently elevated in many inflammatory disorders. NO can rapidly react with superoxide anion, producing more reactive peroxynitrite. In the present study, exposure of rat pheochromocytoma (PC12) cells to the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1) induced apoptosis, which accompanied depletion of intracellular glutathione (GSH), c-Jun N-terminal kinase activation, mitochondrial membrane depolarization, the cleavage of poly(ADP-ribose)polymerase, and DNA fragmentation. During SIN-1-induced apoptotic cell death, expression of inducible cyclooxygenase (COX-2), and peroxisome proliferator-activated receptor-gamma (PPARgamma) was elevated. SIN-1 treatment resulted in elevated production of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), an endogenous PPARgamma activator. Preincubation with 15d-PGJ(2) rendered PC12 cells resistant to nitrosative stress induced by SIN-1. 15d-PGJ(2) fortified an intracellular GSH pool through up-regulation of glutamylcysteine ligase, thereby preventing cells from SIN-1-induced GSH depletion. The above findings suggest that 15d-PGJ(2) may act as a survival mediator capable of augmenting cellular thiol antioxidant capacity through up-regulation of the intracellular GSH synthesis in response to the nitrosative insult.     

Nitric oxide regulates oxygen uptake and hydrogen peroxide release by the isolated beating rat heart

Isolated rat heart perfused with 1.5-7.5µM NO solutions or bradykinin, which activates endothelial NOsynthase, showed a dose-dependent decrease in myocardial O₂uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 µM NO, n = 18,P < 0.05) and to 1.2 ± 0.1 µM O₂ · min¹ · gtissue¹ (10 µM bradykinin, n = 10,P < 0.05). Perfused NO concentrations correlated with aninduced release of hydrogen peroxide (H₂O₂) inthe effluent (r = 0.99, P < 0.01). NO markedlydecreased the O₂ uptake of isolated rat heart mitochondria(50% inhibition at 0.4 µM NO, r = 0.99,P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b andcytochrome c, which accounts for the effects in O₂uptake and H₂O₂ release. Most NO was bound tomyoglobin; this fact is consistent with NO steady-state concentrationsof 0.1-0.3 µM, which affect mitochondria. In the intact heart,finely adjusted NO concentrations regulate mitochondrial O₂uptake and superoxide anion production (reflected byH₂O₂), which in turn contributes to thephysiological clearance of NO through peroxynitrite formation.

     

Contrasting effects of NO and peroxynitrites on HSP70 expression and apoptosis in human monocytes

The free radicals nitric oxide(·NO) and superoxide (O₂·) react to formperoxynitrite (ONOO), a highly toxic oxidant species. Inthis study we investigated the respective effects of NO andONOO in monocytes from healthy human donors. Purifiedmonocytes were incubated for 6 or 16 h with a pure NO donor(S-nitroso-N-acetyl-DL-penicillamine, 0-2 mM), an ·NO/ONOO donor(3-morpholinosydnonimine chlorhydrate, 0-2 mM) with and withoutsuperoxide dismutase (200 IU/ml), or pure ONOO. Weprovide evidence that 3-morpholinosydnonimine chlorhydrate alonerepresents a strong stress to human monocytes leading to adose-dependent increase in heat shock protein-70 (HSP70) expression, mitochondrial membrane depolarization, and cell death by apoptosis andnecrosis. These phenomena were abolished by superoxide dismutase, suggesting that ONOO, but not ·NO, was responsible forthe observed effects. This observation was further strengthened by theabsence of a stress response in cells exposed toS-nitroso-N-acetyl-DL-penicillamine. Conversely, exposure of cells to ONOO alone also inducedmitochondrial membrane depolarization and cell death by apoptosis andnecrosis. Thus ONOO formation may well explain the toxiceffect generally attributed to ·NO.

     

Chemical evaluation of compounds as nitric oxide or peroxynitrite donors using the reactions with serotonin

The aim of this work was to assess the capacities of some ^·NO-donors to release ^·NO, and consequently NOx in aerobic medium, or to give peroxynitrite. The method was based on the differential reactivity of serotonin (5-HT) with either NO_x or peroxynitrite, leading in phosphate-buffered solutions to 4-nitroso- and 4-nitro-5-HT formation, respectively. Yields and formation rates of 5-HT derivatives with ^·NO-donor were compared to those obtained with authentic ^·NO or peroxynitrite in similar conditions. Aside from the capacity of diazenium diolates (SPER/NO and DEA/NO) to release ^·NO spontaneously, converting 5-HT exclusively to 4-nitroso-5-HT, all other ^·NO donors must undergo redox reactions to produce ^·NO. S-nitrosoglutathione (GSNO) and sodium nitroprus-side (SNP) modified 5-HT only in the presence of Cu²⁺, GSNO yielding 6 times more 4-nitroso-5-HT than SNP. Furthermore, in the presence of Cu⁺, the yield of ^·NO-release from GSNO was 45%. The molsidomine metabolite (SIN-1), which was presumed to release both ^·NO and O2/·- at pH 7.4, reacted with 5-HT differently, depending on the presence of reductant or oxidant. Under aerobic conditions, SIN-1 acted predominantly as a 5-HT oxidant and also as a poor ^·NO and peroxynitrite donor (15% yield of ^·NO-release and 14 % yield of peroxynitrite formation). The strong oxidant Cu²⁺, even in the presence of air oxygen, accelerated oxidation and increased ^·NO release from SIN-1 up to 86%. Only a small part of SIN-1 gave simultaneously ^·NO and O2/·- able to link together to give peroxynitrite, but other oxidants could enhance ^·NO release from SIN-1.     

Peroxynitrite-mediated matrix metalloproteinase-2 activation in human hepatic stellate cells

To investigate whether hepatic stellate cells (HSCs) alter their expression of MMPs after exposure to nitrogen oxide intermediate (NOI), a human hepatic stellate cell line, LI90 cells, was stimulated with an NO donor, SNAP, or a peroxynitrite donor, SIN-1, and the culture supernatants were analyzed by gelatin zymography or anti-MMPs immunoblot. Although SIN-1 did not enhance the secretions of MMP-1 and 13, SIN-1 induced the NF-kappaB activation, MT1-MMP expression and the secretion of activated MMP-2 in LI90 cells. These results suggest that peroxynitrite may contribute to the remodeling of the extracellular matrix in liver by activating pro-MMP-2.     

In vivo and in situ visualization of early physiological events induced by heavy metals in pea root meristem

Heavy metals (HMs) are toxic pollutants, which can negatively affect the physiological processes of plants; moreover, HMs can be present in the food chain endangering people’s health. The aim of this study was to investigate the early physiological events during HM exposure in the root tips of the food plant Pisum sativum L. Ten-day-old pea plants were treated with 100 μM CdCl₂ or CuSO₄, in nutrient solution for 48 h. We studied the rapid formation of different reactive oxygen species (hydrogen peroxide H₂O₂ and superoxide radical O₂^·−) and reactive nitrogen species (nitric oxide NO· and peroxynitrite ONOO⁻) together with membrane damage and cell death in the meristem cells of pea roots using in vivo and in situ microscopic methods. In our experimental system, copper and cadmium induced the formation of H₂O₂ and NO. Two hours of heavy metal treatments resulted in an increased O₂^·− formation; however, later the level of this reactive molecule dramatically decreased. We found that high levels of NO were needed for ONOO⁻ production under HM exposure. A fast loss of membrane integrity and decreased cell viability were detected in root tips of copper-treated plants. The effects of cadmium seemed to be slower compared to copper, but this non-essential metal also caused cell death. We concluded that viability decreased when NO and H₂O₂ levels were simultaneously high in the same tissues. Using the NO scavenger it was also evidenced that NO generation is essential for cell death induction under copper or cadmium stress.     

Ethyl Pyruvate Inhibits Peroxynitrite-induced DNA Damage and Hydroxyl Radical Generation: Implications for Neuroprotection

Ethyl pyruvate (EP) has recently been reported to afford protection against neurodegenerative disorders. However, the mechanism underlying EP-mediated neuroprotection remains to be elucidated. Because peroxynitrite has been extensively implicated in the pathogenesis of various forms of neurodegenerative disorders via its cytotoxic effects, this study was undertaken to investigate whether the neuroprotective effect of EP is associated with inhibition of peroxynitrite-induced DNA strand breaks, a critical event leading to peroxynitrite elicited cytotoxicity. Incubation of φX-174 plasmid DNA with 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, led to the formation of both single- and double-stranded DNA breaks in a concentration- and time- dependent manner. The presence of EP (0.5–10 mM) was found to significantly inhibit SIN-1-induced DNA strand breaks in a concentration-dependent fashion. The consumption of oxygen induced by 250 μM SIN-1 was found to be decreased in the presence of EP (0.5–10 mM), indicating that EP might affect the auto-oxidation of SIN-1. It was observed that incubation of the plasmid DNA with authentic peroxynitrite caused significant DNA strand breaks, which could also be dramatically inhibited by EP (0.5–10 mM). EPR spectroscopy in combination with spin-trapping technique using 5,5-dimethylpyrroline-N- oxide (DMPO) as a spin trap demonstrated the formation of DMPO-hydroxyl radical adducts (DMPO-OH) from authentic peroxynitrite, and that EP at 0.5–10 mM inhibited the adduct signal in a concentration-dependent manner. Taken together, these results demonstrate for the first time that EP can inhibit peroxynitrite-mediated DNA damage and hydroxyl radical generation.     

神经元缺氧复氧损伤时氧自由基的毒性作用及其机制

在原代分离培养Ｗｉｓｔａｒ乳鼠大脑皮质神经元上研究了缺氧复氧损伤（Ｈ／Ｒ）对神经细胞乳酸脱氢酶（ＬＤＨ）,漏出率,死亡率和脂质过氧化物含量的影响,并选用一氧化氮（ＮＯ）合酶抑制剂Ｌ－ＮＧ－硝基－精氨酸（Ｌ－ＮＮＡ）巯基供体Ｎ－乙酰半胱氨酸（ＮＡＣ）和超氧化物歧化酶（Ｃｕ,Ｚｎ－ＳＯＤ）三种自由基清除剂进行预保护等方法来探讨机制。结果表明 Ｈ／Ｒ损伤引起ＬＤＨ漏出率,细胞死亡率和脂过氧化物含量极显著     

Antioxidant enzyme inhibitors enhance peroxynitrite-induced cell death in U937 cells

Peroxynitrite, a potent physiological inorganic toxin, is known to play a critical role in cellular oxidative damage. The protective role of antioxidant enzymes against peroxynitrite-induced oxidative damage in U937 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole, and oxlalomalate, specific inhibitors of superoxide dismutase, catalase, and NADP⁺-dependent isocitrate dehydrogenase, respectively. Upon exposure to 1 mM 3-morpholinosydnomine N-ethylcarbamide (SIN-1), a generator of peroxynitrite through the reaction between nitric oxide and superoxide anion, to U937 cells, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage reflected by an increase in 8-hydroxy-2′-deoxyguanosine, were higher in the inhibitor-treated cells as compared to the control cells. We also observed the significant increase in the endogenous production of reactive oxygen species, as measured by the oxidation of 2′7′-dichlorodihydrofluorescin as well as the significant decrease in the intracellular GSH level in the inhibitor-treated U937 cells upon exposure to SIN-1. These results suggest that antioxidant enzymes play an important role in cellular defense against peroxynitrite-induced cell death.     

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     

Peroxynitrite causes endothelial cell monolayer barrier dysfunction

Nitric oxide (·NO) attenuates hydrogen peroxide(H₂O₂)-mediated barrier dysfunction in culturedporcine pulmonary artery endothelial cells (PAEC) (Gupta MP, Ober MD,Patterson C, Al-Hassani M, Natarajan V, and Hart, CM. Am JPhysiol Lung Cell Mol Physiol 280: L116-L126, 2001). However,·NO rapidly combines with superoxide (O) to formthe powerful oxidant peroxynitrite (ONOO), which wehypothesized would cause PAEC monolayer barrier dysfunction. To testthis hypothesis, we treated PAEC with ONOO (500 µM) or3-morpholinosydnonimine hydrochloride (SIN-1; 1-500 µM).SIN-1-mediated ONOO formation was confirmed by monitoringthe oxidation of dihydrorhodamine 123 to rhodamine. BothONOO and SIN-1 increased albumin clearance(P < 0.05) in the absence of cytotoxicity and alteredthe architecture of the cytoskeletal proteins actin and -catenin asdetected by immunofluorescent confocal imaging.ONOO-induced barrier dysfunction was partially reversibleand was attenuated by cysteine. Both ONOO and SIN-1nitrated tyrosine residues, including those on -catenin and actin,and oxidized proteins in PAEC. The introduction of actin treated withONOO into PAEC monolayers via liposomes alsoresulted in barrier dysfunction. These results indicate thatONOO directly alters endothelial cytoskeletal proteins,leading to barrier dysfunction.

     

Peroxynitrite induces HO-1 expression via PI3K/Akt-dependent activation of NF-E2-related factor 2 in PC12 cells

Peroxynitrite is a strong oxidant produced by rapid interaction between superoxide anion and nitric oxide radicals and induces oxidative stress and cell death. Treatment of PC12 cells with 3-morpholinosydnonimine (SIN-1), a generator of peroxynitrite, induced the expression of heme oxygenase-1 (HO-1), an antioxidant cytoprotective enzyme. Inhibition of the HO activity by zinc protoporphyrin IX or knockdown of HO-1 gene expression with siRNA exacerbated the SIN-1-induced apoptosis. After SIN-1 treatment, there was a time-related increase in nuclear localization and subsequent binding of NF-E2-related factor 2 (Nrf2) to the antioxidant-responsive element (ARE). Transfection of PC12 cells with dominant-negative Nrf2 abolished the SIN-1-induced increase in Nrf2-ARE binding and subsequent upregulation of HO-1 expression, leading to enhanced cell death. Upon exposure of PC12 cells to SIN-1, the phosphatidylinositol 3-kinase (PI3K) activity was increased in a time-dependent manner. Pretreatment of cells with LY294002, a pharmacologic inhibitor of PI3K or transfection with the kinase-dead mutant Akt abrogated the SIN-1-induced Nrf2 activation and HO-1 expression. Taken together, these results suggest that peroxynitrite activates Nrf2 via PI3K/Akt signaling and enhances Nrf2-ARE binding, which leads to upregulation of HO-1 expression. The SIN-1-induced HO-1 upregulation may confer the adaptive survival response against nitrosative stress.