A novel fast-response and highly selective AIEgen fluorescent probe for visualizing peroxynitrite in living cells, C. elegans and inflammatory mice.

Most of the ONOO- fluorescent probes have restricted applications because of their aggregation-caused quenching (ACQ) effect, long response time and low fluorescence enhancement. Herein, we developed a novel AIEgen fluorescent probe (PE-XY) based on a benzothiazole and quinolin scaffold with high sensitivity and selectivity for imaging of ONOO-. The results indicated that probe PE-XY exhibited fast response towards ONOO- with 2000-fold enhancement of fluorescence intensity ratio in vitro. Moreover, PE-XY exhibited a relatively high sensitivity (limit of detection: 8.58 nM), rapid response (<50 s), high fluorescence quantum yield (δ = 0.81) and excellent selectivity over other analytes towards ONOO-in vitro. Furthermore, PE-XY was successfully applied to detect endogenous ONOO- levels in living HeLa cells, C. elegans and inflammatory mice with low cytotoxicity. Overall, this work provided a novel fast-response and highly selective AIEgen fluorescent probe for real-time monitoring ONOO- fluctuations in living systems.

A novel p-dimethylaminophenylether-based fluorescent probe for the detection of native ONOO- in cells and zebrafish.

Peroxynitrite (ONOO-) is a highly reactive substance, and plays an essential part in maintaining cellular homeostasis. It is crucial to monitor the ONOO- level in cells in normal and abnormal states. We introduced a p-dimethylaminophenylether-based fluorescent probe PDPE-PN, which could be synthesized readily. The new probe had prominent sensitivity and specificity, and a fast response towards ONOO-. The spectral performance of probe PDPE-PN was outstanding and the limit of detection was 69 nM. Probe PDPE-PN with low toxicity was applied to detect endogenous/exogenous ONOO- in RAW 264.7 macrophages and zebrafish. Importantly, successful application of the new receptor opens up new ideas for the design of ONOO- probes.

Photoacoustic Cavitation-Ignited Reactive Oxygen Species to Amplify Peroxynitrite Burst by Photosensitization-Free Polymeric Nanocapsules.

Photoacoustic (PA) technology can transform light energy into acoustic wave, which can be used for either imaging or therapy that depends on the power density of pulsed laser. Here, we report photosensitizer-free polymeric nanocapsules loaded with nitric oxide (NO) donors, namely NO-NCPs, formulated from NIR light-absorbable amphiphilic polymers and a NO-releasing donor, DETA NONOate. Controlled NO release and nanocapsule dissociation are achieved in acidic lysosomes of cancer cells. More importantly, upon pulsed laser irradiation, the PA cavitation can excite water to generate significant reactive oxygen species (ROS) such as superoxide radical (O2.- ), which further spontaneously reacts with the in situ released NO to burst highly cytotoxic peroxynitrite (ONOO- ) in cancer cells. The resultant ONOO- generation greatly promotes mitochondrial damage and DNA fragmentation to initiate programmed cancer cell death. Apart from PA imaging, PA cavitation can intrinsically amplify reactive species via photosensitization-free materials for promising disease theranostics.

A two-photon excited near-infrared fluorescent probe for imaging peroxynitrite during drug-induced hepatotoxicity and its remediation.

Drug-induced liver injury (DILI) has been a hot issue of public health, owing to its unpredictability and serious harm to public health. Peroxynitrite (ONOO-) is an important biomarker for the assessment and diagnosis of DILI. In this article, based on a kind of rhodamine analogue with a near-infrared (NIR) emission (610 nm-800 nm) and a two-photon absorption cross section (54 GM), a two-photon excited NIR fluorescence probe (NIR-ONOO) for ONOO- was developed. With a high selectivity and a high sensitivity to ONOO-, NIR-ONOO has a linear range for detection of ONOO- from 5.0 × 10-8 to 1.0 × 10-5 M, a good detection limit (15 nM) and a large fluorescence enhancement (340-fold). In addition, NIR-ONOO has been used to monitor ONOO- in cells with satisfactory results. Because of its two-photon excied NIR emission, NIR-ONOO also showed excellent performances for imaging ONOO- including low autofluorescence, stable and persistent fluorescence, and a deep penetration (204 µm). Finally, NIR-ONOO was successfully employed to image ONOO- in inflammatory mouse, drug-induced hepatotoxicity in cells and its remediation. All the results indicated that NIR-ONOO is a powerful chemical tool to image ONOO- and assay drug-induced hepatotoxicity.

Chemical modification of pro-inflammatory proteins by peroxynitrite increases activation of TLR4 and NF-κB: Implications for the health effects of air pollution and oxidative stress.

Environmental pollutants like fine particulate matter can cause adverse health effects through oxidative stress and inflammation. Reactive oxygen and nitrogen species (ROS/RNS) such as peroxynitrite can chemically modify proteins, but the effects of such modifications on the immune system and human health are not well understood. In the course of inflammatory processes, the Toll-like receptor 4 (TLR4) can sense damage-associated molecular patterns (DAMPs). Here, we investigate how the TLR4 response and pro-inflammatory potential of the proteinous DAMPs α-Synuclein (α-Syn), heat shock protein 60 (HSP60), and high-mobility-group box 1 protein (HMGB1), which are relevant in neurodegenerative and cardiovascular diseases, changes upon chemical modification with peroxynitrite. For the peroxynitrite-modified proteins, we found a strongly enhanced activation of TLR4 and the pro-inflammatory transcription factor NF-κB in stable reporter cell lines as well as increased mRNA expression and secretion of the pro-inflammatory cytokines TNF-α, IL-1ß, and IL-8 in human monocytes (THP-1). This enhanced activation of innate immunity via TLR4 is mediated by covalent chemical modifications of the studied DAMPs. Our results show that proteinous DAMPs modified by peroxynitrite more potently amplify inflammation via TLR4 activation than the native DAMPs, and provide first evidence that such modifications can directly enhance innate immune responses via a defined receptor. These findings suggest that environmental pollutants and related ROS/RNS may play a role in promoting acute and chronic inflammatory disorders by structurally modifying the body's own DAMPs. This may have important consequences for chronic neurodegenerative, cardiovascular or gastrointestinal diseases that are prevalent in modern societies, and calls for action, to improve air quality and climate in the Anthropocene.

Peroxiredoxin 1 plays a primary role in protecting pancreatic ß-cells from hydrogen peroxide and peroxynitrite.

Both reactive nitrogen and oxygen species (RNS and ROS), such as nitric oxide, peroxynitrite, and hydrogen peroxide, have been implicated as mediators of pancreatic ß-cell damage during the pathogenesis of autoimmune diabetes. While ß-cells are thought to be vulnerable to oxidative damage due to reportedly low levels of antioxidant enzymes, such as catalase and glutathione peroxidase, we have shown that they use thioredoxin reductase to detoxify hydrogen peroxide. Thioredoxin reductase is an enzyme that participates in the peroxiredoxin antioxidant cycle. Peroxiredoxins are expressed in ß-cells and, when overexpressed, protect against oxidative stress, but the endogenous roles of peroxiredoxins in the protection of ß-cells from oxidative damage are unclear. Here, using either glucose oxidase or menadione to continuously deliver hydrogen peroxide, or the combination of dipropylenetriamine NONOate and menadione to continuously deliver peroxynitrite, we tested the hypothesis that ß-cells use peroxiredoxins to detoxify both of these reactive species. Either pharmacological peroxiredoxin inhibition with conoidin A or specific depletion of cytoplasmic peroxiredoxin 1 (Prdx1) using siRNAs sensitizes INS 832/13 cells and rat islets to DNA damage and death induced by hydrogen peroxide or peroxynitrite. Interestingly, depletion of peroxiredoxin 2 (Prdx2) had no effect. Together, these results suggest that ß-cells use cytoplasmic Prdx1 as a primary defense mechanism against both ROS and RNS.

Metal ions modify DNA-protecting and mutagen-scavenging capacities of the AV-153 1,4-dihydropyridine.

1,4-Dihydropyridines (1,4-DHP) possess important biochemical and pharmacological properties, including antioxidant and antimutagenic activities. AV-153-Na, an antimutagenic and DNA-repair enhancing compound was shown to interact with DNA by intercalation. Here we studied DNA binding of several AV-153 salts to evaluate the impact of AV-153 modifications on its DNA binding capacity, the ability to scavenge the peroxynitrite, to protect HeLa and B-cells cells against DNA damage. Affinity of the AV-153 salts to DNA measured by a fluorescence assay was dependent on the metal ion forming a salt in position 4 of the 1,4-DHP, and it decreased as follows: Mg > Na > Ca > Li > Rb > K. AV-153-K and AV-153-Rb could not react chemically with peroxynitrite as opposed to AV-153-Mg and AV-153-Ca, the latter increased the decomposition rate of peroxynitrite. AV-153-Na and AV-153-Ca effectively reduced DNA damage induced by peroxynitrite in HeLa cells, while AV-153-K and AV-153-Rb were less effective, AV-153-Li did not protect the DNA, and AV-153-Mg even caused DNA damage itself. The Na, K, Ca and Mg AV-153 salts were also shown to reduce the level of DNA damage in human B-cells from healthy donors. Thus, metal ions modify both DNA-binding and DNA-protecting effects of the AV-153 salts.

H2 O2 -induced microvessel barrier dysfunction: the interplay between reactive oxygen species, nitric oxide, and peroxynitrite.

Elevated H2 O2 is implicated in many cardiovascular diseases. We previously demonstrated that H2 O2 -induced endothelial nitric oxide synthase (eNOS) activation and excessive NO production contribute to vascular cell injury and increases in microvessel permeability. However, the mechanisms of excessive NO-mediated vascular injury and hyperpermeability remain unknown. This study aims to examine the functional role of NO-derived peroxynitrite (ONOO- ) in H2 O2 -induced vascular barrier dysfunction by elucidating the interrelationships between H2 O2 -induced NO, superoxide, ONOO- , and changes in endothelial [Ca2+ ]i and microvessel permeability. Experiments were conducted on intact rat mesenteric venules. Microvessel permeability was determined by measuring hydraulic conductivity (Lp). Endothelial [Ca2+ ]i , NO, and O2- were assessed with fluorescence imaging. Perfusion of vessels with H2 O2 (10 µmol/L) induced marked productions of NO and O2- , resulting in extensive protein tyrosine nitration, a biomarker of ONOO- . The formation of ONOO- was abolished by inhibition of NOS with NG -Methyl-L-arginine. Blocking NO production or scavenging ONOO- by uric acid prevented H2 O2 -induced increases in endothelial [Ca2+ ]i and Lp. Additionally, the application of exogenous ONOO- to microvessels induced delayed and progressive increases in endothelial [Ca2+ ]i and microvessel Lp, a pattern similar to that observed in H2 O2 -perfused vessels. Importantly, ONOO- caused further activation of eNOS with amplified NO production. We conclude that the augmentation of NO-derived ONOO- is essential for H2 O2 -induced endothelial Ca2+ overload and progressively increased microvessel permeability, which is achieved by self-promoted amplifications of NO-dependent signaling cascades. This novel mechanism provides new insight into the reactive oxygen and/or reactive nitrogen species-mediated vascular dysfunction in cardiovascular diseases.

Unique Regulation of Enterocyte Brush Border Membrane Na-Glutamine and Na-Alanine Co-Transport by Peroxynitrite during Chronic Intestinal Inflammation.

Na-amino acid co-transporters (NaAAcT) are uniquely affected in rabbit intestinal villus cell brush border membrane (BBM) during chronic intestinal inflammation. Specifically, Na-alanine co-transport (ASCT1) is inhibited secondary to a reduction in the affinity of the co-transporter for alanine, whereas Na-glutamine co-transport (B0AT1) is inhibited secondary to a reduction in BBM co-transporter numbers. During chronic intestinal inflammation, there is abundant production of the potent oxidant peroxynitrite (OONO). However, whether OONO mediates the unique alteration in NaAAcT in intestinal epithelial cells during chronic intestinal inflammation is unknown. In this study, ASCT1 and B0AT1 were inhibited by OONO in vitro. The mechanism of inhibition of ASCT1 by OONO was secondary to a reduction in the affinity of the co-transporter for alanine, and secondary to a reduction in the number of co-transporters for B0AT1, which were further confirmed by Western blot analyses. In conclusion, peroxynitrite inhibited both BBM ASCT1 and B0AT1 in intestinal epithelial cells but by different mechanisms. These alterations in the villus cells are similar to those seen in the rabbit model of chronic enteritis. Therefore, this study indicates that peroxynitrite may mediate the inhibition of ASCT1 and B0AT1 during inflammation, when OONO levels are known to be elevated in the mucosa.

Protective potential of thymoquinone against peroxynitrite induced modifications in histone H2A: In vitro studies.

Peroxynitrite (ONOO-) is a reactive oxidant involved in numerous pathological conditions. Thymoquinone (TQ) is an active constituent of Nigella sativa and is reported to have anti-disease activities, but its role on ONOO- has never been investigated. This study was undertaken to investigate the role of TQ on ONOO--induced damage of histone-H2A. Our novel data showed TQ significantly inhibited ONOO--induced oxidative damage in histone-H2A. ONOO- induces UV-hypochromicity of histone-H2A, whereas TQ reversed this effect to hyperchromicity. Tyrosine fluorescence was significantly reduced by ONOO- and was significantly increased upon TQ treatment. TQ reduces ONOO--induced hydrophobicity in histone-H2A and also reduces thermal stability of ONOO--histone H2A complex. SDS-PAGE of native histone-H2A showed a single band, which disappeared when treated with ONOO- alone. This changed was retained when protein samples were treated with TQ. Similar protective effects of TQ were found when protein carbonyl contents were estimated. In conclusion, this is the first study that shows the potential of TQ against ONOO--induced damaged of histone-H2A. TQ inhibits oxidative modification of tyrosine, lysine, arginine, proline and threonine in histone-H2A. These results have importance for the development of novel therapeutic strategies for the treatment of disorders, where ONOO- plays a role.

Phenelzine reduces the oxidative damage induced by peroxynitrite in plasma lipids and proteins.

Peroxynitrite is a reactive nitrogen species produced in the intravascular compartment from superoxide anion and nitric oxide. Peroxynitrite destroys blood plasma proteins and membranes of red blood cells and of platelets. This explains why excessive production of peroxynitrite contributes to diseases and to ageing. Therapeutics that antagonize peroxynitrite may delay ageing and the progression of disease. We developed an in vitro assay that allows the investigation of the oxidative damage caused by peroxynitrite in the intravascular compartment. This assay correlates the damage with the rate of formation of protein carbonyl groups, 3-nitrotyrosine (3-NT) and thiobarbituric acid reactive substances. Using this assay, we evaluated the ability of phenelzine, a scavenger of reactive aldehydes, to antagonize the effects of peroxynitrite. Herein, we showed that phenelzine significantly decreased the lipid peroxidative damage caused by peroxynitirite in blood plasma and platelets. Moreover, it inhibited carbonyl group and 3-NT formation in blood plasma and platelet proteins.

Arctic ground squirrel resist peroxynitrite-mediated cell death in response to oxygen glucose deprivation.

Cerebral ischemia-reperfusion (I/R) injury initiates a cascade of events, generating nitric oxide (NO) and superoxide(O2•-) to form peroxynitrite (ONOO-), a potent oxidant. Arctic ground squirrels (AGS; Urocitellus parryii) show high tolerance to I/R injury. However, the underlying mechanism remains elusive. We hypothesize that tolerance to I/R modeled in an acute hippocampal slice preparation in AGS is modulated by reduced oxidative and nitrative stress. Hippocampal slices (400µm) from rat and AGS were subjected to oxygen glucose deprivation (OGD) using a novel microperfusion technique. Slices were exposed to NO, O2.- donors with and without OGD; pretreatment with inhibitors of NO, O2.- and ONOO- followed by OGD. Perfusates collected every 15min were analyzed for LDH release, a marker of cell death. 3-nitrotyrosine (3NT) and 4-hydroxynonenal (4HNE) were measured to assess oxidative and nitrative stress. Results show that NO/O2.- alone is not sufficient to cause ischemic-like cell death, but with OGD enhances cell death more in rat than in AGS. A NOS inhibitor, SOD mimetic and ONOO- inhibitor attenuates OGD injury in rat but has no effect in AGS. Rats also show a higher level of 3NT and 4HNE with OGD than AGS suggesting the greater level of injury in rat is via formation of ONOO-.

Yunnaneic Acid B, a Component of Pulmonaria officinalis Extract, Prevents Peroxynitrite-Induced Oxidative Stress in Vitro.

Our work reveals that the aerial parts of Pulmonaria officinalis L. are a new source of yunnaneic acid B. We studied antioxidant activity and cytotoxicity of this compound (1-50 µg/mL) and its contents in various plant extracts. This is the first study confirming the presence of yunnaneic acid B in P. officinalis L. and Pulmonaria obscura Dumort and hence in the Boraginaceae family. Determination of 1,1-diphenyl-2-picrylhydrazyl radical reduction and peroxynitrite-scavenging efficacy in inorganic experimental systems provided EC50 values of 7.14 and 50.45 µg/mL, respectively. Then we examined the antioxidant action of yunnaneic acid B in blood plasma under peroxynitrite-induced oxidative stress in vitro. Yunnaneic acid B effectively diminished oxidative damage to blood plasma proteins and lipids. Furthermore, it was able to prevent the peroxynitrite-induced decrease in nonenzymatic antioxidant capacity of blood plasma. Additionally, cytotoxicity of yunnaneic acid B (at concentrations ≤50 µg/mL) toward peripheral blood mononuclear cells was excluded.

Nitrosative stress by peroxynitrite impairs ATP production in human spermatozoa.

The most toxic species in live systems include reactive nitrogen species such as peroxynitrite, which at high levels induces nitrosative stress. In human spermatozoa, the negative effect of peroxynitrite on motility and mitochondrial membrane potential was recently demonstrated, and the hypothesis of this work is that impairment of ATP production could be one cause of the effect on motility. Therefore, the aim here was to evaluate ATP production by both glycolysis and oxidative phosphorylation (OXPHOS) in spermatozoa exposed to peroxynitrite in vitro. Human spermatozoa were incubated with SIN-1, a molecule which generates peroxynitrite, and the ATP level was evaluated. Then, to inactivate glycolysis or OXPHOS, spermatozoa were incubated with pharmacological inhibitors of these pathways. Spermatozoa treated for inactivating one or the other pathway were exposed to SIN-1, and the ATP level was compared to the control without SIN-1 in each condition. The ATP level fell after peroxynitrite exposure. The ATP in spermatozoa treated for inactivating one or the other metabolic pathway and subsequently exposed to peroxynitrite was reduced compared with the control. These results show for the first time that an important mechanism by which peroxynitrite reduces sperm function is the inhibition of ATP production, affecting both glycolysis and OXPHOS.

RhoA/rock signaling mediates peroxynitrite-induced functional impairment of Rat coronary vessels.

BACKGROUND: Diabetes-induced vascular dysfunction may arise from reduced nitric oxide (NO) availability, following interaction with superoxide to form peroxynitrite. Peroxynitrite can induce formation of 3-nitrotyrosine-modified proteins. RhoA/ROCK signaling is also involved in diabetes-induced vascular dysfunction. The study aimed to investigate possible links between Rho/ROCK signaling, hyperglycemia, and peroxynitrite in small coronary arteries. METHODS: Rat small coronary arteries were exposed to normal (NG; 5.5 mM) or high (HG; 23 mM) D-glucose. Vascular ring constriction to 3 mM 4-aminopyridine and dilation to 1 µM forskolin were measured. Protein expression (immunohistochemistry and western blot), mRNA expression (real-time PCR), and protein activity (luminescence-based G-LISA and kinase activity spectroscopy assays) of RhoA, ROCK1, and ROCK2 were determined. RESULTS: Vascular ring constriction and dilation were smaller in the HG group than in the NG group (P < 0.05); inhibition of RhoA or ROCK partially reversed the effects of HG. Peroxynitrite impaired vascular ring constriction/dilation; this was partially reversed by inhibition of RhoA or ROCK. Protein and mRNA expressions of RhoA, ROCK1, and ROCK2 were higher under HG than NG (P < 0.05). This HG-induced upregulation was attenuated by inhibition of RhoA or ROCK (P < 0.05). HG increased RhoA, ROCK1, and ROCK2 activity (P < 0.05). Peroxynitrite also enhanced RhoA, ROCK1, and ROCK2 activity; these actions were partially inhibited by 100 µM urate (peroxynitrite scavenger). Exogenous peroxynitrite had no effect on the expression of the voltage-dependent K+ channels 1.2 and 1.5. CONCLUSIONS: Peroxynitrite-induced coronary vascular dysfunction may be mediated, at least in part, through increased expressions and activities of RhoA, ROCK1, and ROCK2.

Caveolin-1 protects against hepatic ischemia/reperfusion injury through ameliorating peroxynitrite-mediated cell death.

Nitrative stress is considered as an important pathological process of hepatic ischemia and reperfusion injury but its regulating mechanisms are largely unknown. In this study, we tested the hypothesis that caveolin-1 (Cav-1), a plasma membrane scaffolding protein, could be an important cellular signaling against hepatic I/R injury through inhibiting peroxynitrite (ONOO(-))-induced cellular damage. Male wild-type mice and Cav-1 knockout (Cav-1(-/-)) were subjected to 1h hepatic ischemia following 1, 6 and 12h of reperfusion by clipping and releasing portal vessels respectively. Immortalized human hepatocyte cell line (L02) was subjected to 1h hypoxia and 6h reoxygenation and treated with Cav-1 scaffolding domain peptide. The major discoveries included: (1) the expression of Cav-1 in serum and liver tissues of wild-type mice was time-dependently elevated during hepatic ischemia-reperfusion injury. (2) Cav-1 scaffolding domain peptide treatment inhibited cleaved caspase-3 expression in the hypoxia-reoxygenated L02 cells; (3) Cav-1 knockout (Cav-1(-/-)) mice had significantly higher levels of serum transaminases (ALT&AST) and TNF-α, and higher rates of apoptotic cell death in liver tissues than wild-type mice after subjected to 1h hepatic ischemia and 6hour reperfusion; (4) Cav-1(-/-) mice revealed higher expression levels of iNOS, ONOO(-) and 3-nitrotyrosine (3-NT) in the liver than wild-type mice, and Fe-TMPyP, a representative peroxynitrite decomposition catalyst (PDC), remarkably reduced level of ONOO(-) and 3-NT and ameliorated the serum ALT, AST and TNF-α levels in both wild-type and Cav-1(-/-) mice. Taken together, we conclude that Cav-1 could play a critical role in preventing nitrative stress-induced liver damage during hepatic ischemia-reperfusion injury.

Adverse Outcomes Associated with Cigarette Smoke Radicals Related to Damage to Protein-disulfide Isomerase.

Identification of factors contributing to the development of chronic obstructive pulmonary disease (COPD) is crucial for developing new treatments. An increase in the levels of protein-disulfide isomerase (PDI), a multifaceted endoplasmic reticulum resident chaperone, has been demonstrated in human smokers, presumably as a protective adaptation to cigarette smoke (CS) exposure. We found a similar increase in the levels of PDI in the murine model of COPD. We also found abnormally high levels (4-6 times) of oxidized and sulfenilated forms of PDI in the lungs of murine smokers compared with non-smokers. PDI oxidation progressively increases with age. We begin to delineate the possible role of an increased ratio of oxidized PDI in the age-related onset of COPD by investigating the impact of exposure to CS radicals, such as acrolein (AC), hydroxyquinones (HQ), peroxynitrites (PN), and hydrogen peroxide, on their ability to induce unfolded protein response (UPR) and their effects on the structure and function of PDIs. Exposure to AC, HQ, PN, and CS resulted in cysteine and tyrosine nitrosylation leading to an altered three-dimensional structure of the PDI due to a decrease in helical content and formation of a more random coil structure, resulting in protein unfolding, inhibition of PDI reductase and isomerase activity in vitro and in vivo, and subsequent induction of endoplasmic reticulum stress response. Addition of glutathione prevented the induction of UPR, and AC and HQ induced structural changes in PDI. Exposure to PN and glutathione resulted in conjugation of PDI possibly at active site tyrosine residues. The findings presented here propose a new role of PDI in the pathogenesis of COPD and its age-dependent onset.

Modification of HSP proteins and Ca2+ are responsible for the NO-derived peroxynitrite mediated neurological damage in PC12 cell.

Peroxynitrite as one crucial metabolite of NO-derived agents has been well multi-investigated to inspect its potential role and sought to define its concrete mechanism underlying the memory loss and impaired cognition involved in pathological processes. In this investigation, the cell viability was assessed by the MTT assay. The neurotoxicity of peroxynitrite was analyzed by using immunohistochemical measurements in cultured PC12 cells to explore the underlying mechanisms. The generation of ROS was evaluated by a fluorometry assay by a fluorometry assay. Apoptosis was assayed by annexin V-FITC and PI staining with flow cytometry. [Ca2+]i was examined by using the microspectrofluorometer. Hsp70 was detected by western blot assay. The results revealed that PC12 cells were inhibited by peroxynitrite both in a dose-dependent and time-dependent manner. The level of ROS in PC12 cells exposed to SIN-1 was increased in a dose-dependent manner. The result indicated that the SIN-1 induced apoptosis of PC12 cells in a dose-dependent manner. Quercetin inhibited the viability of PC12 cells in a concentration-dependent manner. [Ca2+]i was increased gradually when cells treated with quercetin alone and also increased with treatment of dantrolene-containing. Hsp70 was significantly decreased in SIN-1-treated group compared with that of control group (P<0.01). In conclusion, Ca2+ homeostasis and chaperone Hsp70 were critically involved in peroxynitrite induced nitrosative stress as protective. Peroxynitrite acts as the pathological agent in learning and memory defects in CNS disorders associated with challenge.

TPEN prevents rapid pacing-induced calcium overload and nitration stress in HL-1 myocytes.

INTRODUCTION: Atrial fibrillation (AF) is the most common cardiac arrhythmia. However, the current drug interference of antiarrhythmia has limited efficacy and off-target effects. Accumulating evidence has implicated a potential role of nitration stress in the pathogenesis of AF. The aim of the study was to determine whether TPEN provided antinitration effects on atrial myocytes during AF, especially under circumstances of nitration stress. METHODS: We utilized a rapid paced HL-1 cells model for AF. The changes of electrophysiological characteristics and structure of paced HL-1 cells were determined by a patch clamp and a TEM method. The effects of TPEN on pacing and ONOO(-) pretreated HL-1 cells were examined using MTT assay, TUNEL technique, confocal microscope experiment, and Western blot analysis. RESULTS: The results revealed that ONOO(-) reduced the viability of HL-1 cells in a dose-dependent manner, and 1 µmol/L TPEN significantly ameliorated the damage caused by 50 µmol/L ONOO(-) (P < 0.05). Pacing and/or ONOO(-) -induced marked shortening of APD, myolysis, and nuclear condensation. TPEN inhibited the Ca(2+) overload induced by rapid pacing (P < 0.05) and ONOO(-) stimulation (P < 0.05). The application of TPEN significantly prevented the protein nitration caused by pacing or pacing plus ONOO(-) (P < 0.05). Additionally, pacing in combination with ONOO(-) treatment led to increase in apoptosis in HL-1 cells (P < 0.01), which could be reduced by pretreatment with TPEN (P < 0.05). CONCLUSIONS: TPEN prevents Ca(2+) overload and nitration stress in HL-1 atrial myocytes during rapid pacing and circumstances of nitration stress.

Protective effect of molecular hydrogen against oxidative stress caused by peroxynitrite derived from nitric oxide in rat retina.

BACKGROUND: Oxidative and nitrative processes have an important role in the pathogenesis of glaucomatous neurodegeneration. Oxidative stress occurs when cellular production of reactive oxygen species outweighs the protective capacity of antioxidant defences. Reactive oxygen species are generated as by-products of cellular metabolism, primarily in the mitochondria. Herein, we present a novel investigation of the effects of molecular hydrogen (H2 ) on retinal cells exposed to oxidative stress. METHODS: We cultured adult rat retinal tissues in an organotypic culture system with a nitric oxide donor, S-nitroso-N-acetylpenicillamine, in the presence or absence of H2 . Loss of mitochondrial membrane potential and apoptosis of retinal cells were analysed using a MitoTMRE detection kit and TdT-mediated dUTP nick end labeling (TUNEL) assay, respectively. Tyrosine nitration levels and oxidative stress damage in the retina were evaluated using immunohistochemical staining. Retinal damage was quantified by measuring the numbers of cells in the ganglion cell and inner nuclear layers and the thickness of the retina. RESULTS: H2 suppressed loss of mitochondrial membrane potential and apoptosis in retinal cells. Moreover, H2 decreased the tyrosine nitration level and suppressed oxidative stress damage in retinal cells. S-nitroso-N-acetylpenicillamine treatment decreased the cell numbers in the ganglion cell layer and inner nuclear layer, but the presence of H2 inhibited this reduction. These findings suggest that H2 has a neuroprotective effect against retinal cell oxidative damage, presumably by scavenging peroxynitrite. CONCLUSIONS: H2 reduces cellular peroxynitrite, a highly toxic reactive nitrogen species. Thus, H2 may be an effective and novel clinical tool for treating glaucoma and other oxidative stress-related diseases.