Peroxynitrite-induced Apoptosis Research Articles

Peroxynitrite nitration of Tyr 56 in Hsp90 induces PC12 cell death through P2X7R-dependent PTEN activation

The diffusion-limited reaction of nitric oxide (NO) and superoxide (O2−) produces peroxynitrite (ONOO−), a biological oxidant that has been implicated in a number of pathological conditions, including neurodegenerative disorders. We previously reported that incubation of PC12 cells with peroxynitrite triggers apoptosis by simultaneously inhibiting the PI3K/Akt survival pathway, and activating the p38 and JNK MAP kinase pathways. We also reported that peroxynitrite-treated Heat Shock Protein 90 (Hsp90) stimulates PC12 cell death. Here, we show that nitrated Hsp90 mediates peroxynitrite-induced apoptosis by regulating specific signaling pathways triggered by activation of the purine receptor P2X7 (P2X7R) and downstream activation of PTEN. Intracellular delivery of peroxynitrite-treated Hsp90 was sufficient to stimulate PC12 cell death. In contrast, intracellular delivery of peroxynitrite-treated Hsp90 in which the five tyrosine (Tyr) residues susceptible to nitration were replaced by nitration-resistant phenylalanine had no effect on PC12 cell survival. Further, only nitration of Hsp90 at Tyr 56 was necessary and sufficient to stimulate PC12 cell apoptosis, and incubation of PC12 cells with peroxynitrite resulted in Hsp90 nitration at Tyr 56. Inhibition of P2X7R or downstream inhibition of PTEN prevented PC12 cell death stimulated by both incubation with peroxynitrite and nitrated Hsp90 (Hsp90NY). Peroxynitrite, Hsp90NY, and P2X7R activation all increased p38 and JNK MAP kinases activity, while inhibiting the Akt survival pathway. These results suggest that, in undifferentiated PC12 cells, peroxynitrite triggers apoptosis via nitration of Hsp90 at Tyr 56, which in turn activates P2X7R and PTEN. These results contrast with observations in motor neurons where the nitration of either Tyr 33 or Tyr 56 in Hsp90 stimulates apoptosis, suggesting that the targets of peroxynitrite may be different in different cell types. However, uncovering the pathways through which peroxynitrite triggers cell death in neurodegenerative conditions will provide new potential targets for therapeutic treatment.

Nitrated Hsp90 Mediates Peroxynitrite-Induced Apoptosis

To investigate the association between systemic nitrotyrosine (NT) levels and primary angle-closure glaucoma (PACG) and primary open-angle glaucoma (POAG) and the mechanism involved. A case control study was conducted in the Department of Ophthalmology & Visual Science, Eye & ENT Hospital, Fudan University from April 2017 to December 2017. A total of 400 participants were consecutively recruited into this study (100 PACG, 100 POAG and 200 controls). Multivariable logistic regression analysis was performed to identify the association between serum NT level and PACG or POAG. Clinical results were validated in cell and animal models. Among 200 glaucoma patients, 101 (50.5%) were women; the age was 57.07 ± 14.51 years. 106 (53%) control participants were women and age was 58.34 ± 14.04 years. Serum levels of NT in PACG and POAG patients are significantly higher than controls (1808.53 ± 417.76 nmol/L vs. 1270.62 ± 454.60 nmol/L, p < 0.001; 1718.63 ± 437.29 nmol/L vs. 1258.38 ± 460.72 nmol/L, p < 0.001). Further, elevated serum NT level increases the risk of developing PACG (OR = 1.003, 95% CI: 1.002 to 1.004, p < 0.001) and POAG (OR = 1.002, 95% CI: 1.002 to 1.003, p < 0.001). Consistent with the clinical data, serum and aqueous humour NT levels are significantly higher in caveolin 1 knockout (Cav1 KO) mice, an animal model of glaucoma. More importantly, peroxynitrite (PN) scavenger MnTMPyP and its transduction molecule PARP inhibitor significantly reduce intraocular pressure in Cav1 KO mice. Our data show for the first time that NT is a systemic risk factor and local treatment target of glaucoma.

Stimulation of soluble guanylate cyclase prevents cigarette smoke-induced pulmonary hypertension and emphysema.

Chronic obstructive pulmonary disease (COPD) is a major cause of death worldwide. No therapy stopping progress of the disease is available. To investigate the role of the soluble guanylate cyclase (sGC)-cGMP axis in development of lung emphysema and pulmonary hypertension (PH) and to test whether the sGC-cGMP axis is a treatment target for these conditions. Investigations were performed in human lung tissue from patients with COPD, healthy donors, mice, and guinea pigs. Mice were exposed to cigarette smoke (CS) for 6 hours per day, 5 days per week for up to 6 months and treated with BAY 63-2521. Guinea pigs were exposed to CS from six cigarettes per day for 3 months, 5 days per week and treated with BAY 41-2272. Both BAY compounds are sGC stimulators. Gene and protein expression analysis were performed by quantitative real-time polymerase chain reaction and Western blotting. Lung compliance, hemodynamics, right ventricular heart mass alterations, and alveolar and vascular morphometry were performed, as well as inflammatory cell infiltrate assessment. In vitro assays of cell adhesion, proliferation, and apoptosis have been done. The functionally essential sGC β1-subunit was down-regulated in patients with COPD and in CS-exposed mice. sGC stimulators prevented the development of PH and emphysema in the two different CS-exposed animal models. sGC stimulation prevented peroxynitrite-induced apoptosis of alveolar and endothelial cells, reduced CS-induced inflammatory cell infiltrate in lung parenchyma, and inhibited adhesion of CS-stimulated neutrophils. The sGC-cGMP axis is perturbed by chronic exposure to CS. Treatment of COPD animal models with sGC stimulators can prevent CS-induced PH and emphysema.

Alteration of discoidin domain receptor-2 expression: possible role in peroxynitrite-induced apoptosis in human cerebral vascular smooth muscle cells

Peroxynitrite(ONOO−), a product of nitric oxide and superoxide anion interaction, is a powerful and versatile oxidant. In this work, the human cerebral vascular smooth muscle cells (HCVSMCs) were cultured in vitro and subjected to different concentrations of ONOO−. Cell viability and morphological changes were measured by methylthiazoletetrazolium (MTT) assay and Ho33342/PI double staining respectively. The apoptotic rate was assessed by flow cytometry. The alterations in the expression of DDR2 at both mRNA and protein levels were examined by real-time PCR and Western-blot simultaneously. Direct exposure of HCVSMCs to ONOO− was able to inhibit the cell proliferation, which was further revealed via the apoptotic pathway. ONOO− exerted a dual-effect on DDR2 expression at both mRNA and protein levels that depended on the concentrations of ONOO−. Our results demonstrate for the first time that DDR2 gene may play an important role in ONOO−-induced apoptosis in HCVSMCs.

Intranuclear localization of apoptosis-inducing factor and endonuclease G involves in peroxynitrite-induced apoptosis of spiral ganglion neurons

Objectives: The present study was designed to determine whether or not the caspase-independent apoptotic pathway participated in the cellular death of spiral ganglion neurons (SGNs) after exposure to peroxynitrite (ONOO−), with particular attention given to the intranuclear translocation of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (Endo G) in this process.Methods: The rat SGNs were isolated and primary cultured in vitro and were exposed to ONOO− with pre-treatment of pan-caspase inhibitor. Morphological changes of SGNs were observed by acridine orange cytochemistry staining, and apoptosis was examined by flow cytometry. The translocation of mitochondrial AIF and Endo G was detected by immunocytochemistry and Western blot. The protein expressions of Bcl-2 family in SGNs exposed to ONOO− were determined by Western blot.Results: Treatment of SGNs with ONOO− resulted in the occurrence of caspase-independent apoptosis as evidenced by acridine orange staining and flow cytometry analysis. The immunocytochemical analysis showed that AIF and Endo G labeling were marked in neuronal nuclei, while the Western blot demonstrated the intranuclear localization of AIF and Endo G in SGNs treated with ONOO−. Western blot analysis demonstrated that ONOO− increased the Bax expression while reducing Bcl-2 expression, which was not prevented by pre-treatment with caspase inhibitor.Conclusion: These data indicate that ONOO− can trigger caspase-independent apoptosis in SGNs associated with mitochondrial AIF and Endo G intranuclear localization.

The role of endogenous and exogenous RasGAP-derived fragment N in protecting cardiomyocytes from peroxynitrite-induced apoptosis

Peroxynitrite (PN) is a potent nitrating and oxidizing agent generated during various pathological situations affecting the heart. The negative effects of PN result, at least in part, from its ability to activate caspases and apoptosis. RasGAP is a ubiquitously expressed protein that is cleaved sequentially by caspase-3. At low caspase-3 activity, RasGAP is cleaved into an N-terminal fragment, called fragment N, that protects cells by activating the Ras/PI3K/Akt pathway. At high caspase-3 activity, fragment N is further cleaved and this abrogates its capacity to stimulate the antiapoptotic Akt kinase. Fragment N formation is crucial for the survival of cells exposed to a variety of stresses. Here we investigate the pattern of RasGAP cleavage upon PN stimulation and the capacity of fragment N to protect cardiomyocytes. PN did not lead to sequential cleavage of RasGAP. Indeed, PN did not allow accumulation of fragment N because it induced its rapid cleavage into smaller fragments. No situations were found in cells treated with PN in which the presence of fragment N was associated with survival. However, expression of a caspase-resistant form of fragment N in cardiomyocytes protected them from PN-induced apoptosis. Our results indicate that the antiapoptotic pathway activated by fragment N is effective at inhibiting PN-induced apoptosis (as seen when cardiomyocytes express a capase-3-resistant form of fragment N) but because fragment N is too transiently generated in response to PN, no survival response is effectively produced. This may explain the marked deleterious consequences of PN generation in various organs, including the heart.

Involvement of MsrB1 in the regulation of redox balance and inhibition of peroxynitrite-induced apoptosis in human lens epithelial cells

Methionine sulfoxide reductases (Msrs) in lens cells are important for the maintenance of lens cell viability and resistance to oxidative stress damage. Peroxynitrite (ONOO−), as a strong oxidizing and nitrating agent, occurred in diabetic retinopathy patients and diabetic model animal. In an attempt to shed light on the roles of MsrB1, known as selenoprotein R, in protecting human lens epithelial (HLE) cells against peroxynitrite damage, and contribution of loss of its normal activity to cataract, the influences of MsrB1 gene silencing on peroxynitrite-induced apoptosis in HLE cells were studied. The results showed that both exogenous peroxynitrite and MsrB1 gene silencing by short interfering RNA (siRNA) independently resulted in oxidative stress, endoplasmic reticulum (ER) stress, activation of caspase-3 as well as an increase of apoptosis in HLE cells; moreover, when MsrB1-gene-silenced cells were exposed to 300 μM peroxynitrite, these indexes were further aggravated at the same conditions and DNA strand breaks occurred. The results demonstrate that in HLE cells MsrB1 may play important roles in regulating redox balance and mitigating ER stress as induced by oxidative stress under physiological conditions; MsrB1 may also protect HLE cells against peroxynitrite-induced apoptosis by inhibiting the activation of caspase-3 and oxidative damage of DNA under pathological conditions. Our results imply that loss of its normal activity is likely to contribute to cataract.

Cyclic GMP protects human macrophages against peroxynitrite-induced apoptosis.

BackgroundNitric oxide (NO) can be both pro- and anti-apoptotic in various cell types, including macrophages. This apparent paradox may result from the actions of NO-related species generated in the microenvironment of the cell, for example the formation of peroxynitrite (ONOO-). In this study we have examined the ability of NO and ONOO- to evoke apoptosis in human monocyte-derived macrophages (MDMϕ), and investigated whether preconditioning by cyclic guanosine monophosphate (cGMP) is able to limit apoptosis in this cell type.MethodsCharacterisation of the NO-related species generated by (Z)-1- [2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO) and 1,2,3,4-oxatriazolium, 5-amino-3-(3,4-dichlorophenyl)-, chloride (GEA-3162) was performed by electrochemistry using an isolated NO electrode and electron paramagnetic resonance (EPR) spectrometry. Mononuclear cells were isolated from peripheral blood of healthy volunteers and cultured to allow differentiation into MDMϕ. Resultant MDMϕ were treated for 24 h with DETA/NO (100 – 1000 μM) or GEA-3162 (10 – 300 μM) in the presence or absence of BAY 41–2272 (1 μM), isobutylmethylxanthine (IBMX; 1 μM), 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ; 20 μM) or 8-bromo-cGMP (1 mM). Apoptosis in MDMϕ was assessed by flow cytometric analysis of annexin V binding in combination with propidium iodide staining.ResultsElectrochemistry and EPR revealed that DETA/NO liberated free NO radical, whilst GEA-3162 concomitantly released NO and O2-, and is therefore a ONOO- generator. NO (DETA/NO) had no effect on cell viability, but ONOO- (GEA-3162) caused a concentration-dependent induction of apoptosis in MDMϕ. Preconditioning of MDMϕ with NO in combination with the phosphodiesterase inhibitor, 3-Isobutyl-1-methylxanthine (IBMX), or the NO-independent stimulator of soluble guanylate cyclase, BAY 41–2272, significantly attenuated ONOO--induced apoptosis in a cGMP-dependent manner.ConclusionThese results demonstrate disparities between the ability of NO and ONOO- to induce apoptosis in human MDMϕ. Furthermore, this study provides evidence for a novel cGMP-dependent pre-conditioning mechanism to limit ONOO--induced apoptosis in human MDMϕ.

Exogenous Superoxide Dismutase Prevents Peroxynitrite-Induced Apoptosis in Non-Heart-Beating Donor Livers

Objective: To investigate the role of oxygen free radicals in the induction of apoptosis in non-heart-beating donor (NHBD) livers, and if superoxide dismutase (SOD) ameliorates these alterations. Methods: Rat livers were perfused via the portal vein with histidine/tryptophan/α-ketoglutarate solution from heart-beating donors (HBD) or 60-min warm ischemia from NHBD, with or without the addition of SOD. After 24 h, cold storage livers were evaluated by isolated reperfusion. Results: NHBD showed significantly higher enzyme leakage and elevated portal venous pressure (PVP) versus HBD. Bile and total adenine nucleotides (TAN) were significantly decreased. Apoptosis was prominent in sinusoidal lining cells, coupled with strong nitrotyrosine staining (NTR). The concentrations of nitric oxide and lipoperoxides were largely increased. SOD medication reduced hepatic enzyme release by 30% and lipoperoxides by nearly 50%. Apoptosis and NTR were significantly decreased, and PVP was strikingly reduced to normal values. A 3-fold enhancement in bile production and 1.5-fold increase in TAN of the liver tissue were also observed. Conclusion: NHBD livers are prone to severe reoxygenation injury promoted by oxygen free radicals, massive nitrite oxide production and peroxynitrite-induced apoptosis within the sinusoids. Antioxidant medication with SOD should be considered as a useful means of preserving NHBD livers.

Piceatannol attenuates hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells by blocking down-regulation of Bcl-X L and activation of JNK

There is mounting evidence implicating the accumulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. Recently, considerable attention has been focused on identifying naturally occurring antioxidants that are able to reduce excess ROS and RNS, thereby protecting against oxidative stress and neuron death. The present study investigated the possible protective effects of piceatannol ( trans-3,4,3′,5′-tetrahydroxystilbene), which is present in grapes and other foods, on hydrogen-peroxide- and peroxynitrite-induced oxidative cell death. PC12 rat pheochromocytoma (PC12) cells treated with hydrogen peroxide or SIN-1 (a peroxynitrite-generating compound) exhibited apoptotic death, as determined by nucleus condensation and cleavage of poly(ADP-ribose)polymerase (PARP). Piceatannol treatment attenuated hydrogen-peroxide- and peroxynitrite-induced cytotoxicity, apoptotic features, PARP cleavage and intracellular ROS and RNS accumulation. Treatment of PC12 cells with hydrogen peroxide or SIN-1 led to down-regulation of Bcl-X L and activation of caspase-3 and -8, which were also inhibited by piceatannol treatment. Hydrogen peroxide or SIN-1 treatment induced phosphorylation of the c-Jun-N-terminal kinase (JNK), which was inhibited by piceatannol treatment. Moreover, SP600125 (a JNK inhibitor) significantly inhibited hydrogen-peroxide- and peroxynitrite-induced PC12 cell death, revealing inactivation of the JNK pathway as a possible molecular mechanism for the protective effects of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells. Collectively, these findings suggest that the protective effect of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells is associated with blocking the activation of JNK and the down-regulation of Bcl-X L.

Prevention of Peroxynitrite-induced Apoptosis of Motor Neurons and PC12 Cells by Tyrosine-containing Peptides

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radical-mediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.

Carbon monoxide protects PC12 cells from peroxynitrite-induced apoptotic death by preventing the depolarization of mitochondrial transmembrane potential

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in catalyzing heme degradation into biliverdin, free iron, and carbon monoxide (CO), serves as a protective enzyme against oxidative and nitrosative stresses. In the present study, we investigated the cytoprotective effects of HO-1 upregulation and its product CO against the peroxynitrite-induced PC12 cell death. PC12 cells treated with 3-morphoinosydonimine (SIN-1), a generator of peroxynitrite (ONOO −), underwent apoptotic cell death as evidenced by dissipation of mitochondrial transmembrane potential (ΔΨm), release of mitochondrial cytochrome c into cytoplasm, cleavage of poly(ADP-ribose)polymerase and fragmentation of internucleosomal DNA. Pretreatment of PC12 cells with a low non-toxic concentration of SIN-1 (0.5 mM) induced HO-1 expression and abrogated the cell death caused by subsequent challenge with high dose SIN-1 (2.5 mM). Furthermore, pretreatment of PC12 cells with SnCl 2, a potent inducer of HO-1 expression, increased endogenous production of CO (HO activity) and rescued the PC12 cells from peroxynitrite-induced apoptosis. The cytoprotective effect of SnCl 2 was abolished when the HO activity was inhibited by zinc protoporphyrin IX (ZnPP IX). PC12 cells treated directly with the CO-releasing molecule, tricarbonyldichlororuthenium (II) dimer ([Ru(CO) 3Cl 2] 2) became tolerant to the depolarization of ΔΨm and apoptosis induced by high dose peroxynitrite. Taken together, these data demonstrate that the adaptive protection against peroxynitrite-induced apoptotic death in PC12 cells is mediated by CO formed as a consequence of HO-1 induction.

Two distinct signaling pathways regulate peroxynitrite-induced apoptosis in PC12 cells

The mechanisms of peroxynitrite-induced apoptosis are not fully understood. We report here that peroxynitrite-induced apoptosis of PC12 cells requires the simultaneous activation of p38 and JNK MAP kinase, which in turn activates the intrinsic apoptotic pathway, as evidenced by Bax translocation to the mitochondria, cytochrome c release to the cytoplasm and activation of caspases, leading to cell death. Peroxynitrite induces inactivation of the Akt pathway. Furthermore, overexpression of constitutively active Akt inhibits both peroxynitrite-induced Bax translocation and cell death. Peroxynitrite-induced death was prevented by overexpression of Bcl-2 and by cyclosporin A, implicating the involvement of the intrinsic apoptotic pathway. Selective inhibition of mixed lineage kinase (MLK), p38 or JNK does not attenuate the decrease in Akt phosphorylation showing that inactivation of the Akt pathway occurs independently of the MLK/MAPK pathway. Together, these results reveal that peroxynitrite-induced activation of the intrinsic apoptotic pathway involves interactions with the MLK/MAPK and Akt signaling pathways.

Peroxynitrite-induced apoptosis in photoreceptor cells

Purpose. To study the mechanisms of peroxynitrite-induced photoreceptor cell damage, using retinal cultures and a peroxynitrite donor, 3-morpholinosydnonimine (SIN-1). Methods. Retinal explants obtained from 20-day-old Lewis rat pups, were exposed to SIN-1 for varying lengths of time at varying concentrations. Apoptosis in the photoreceptor cells was detected using the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method and a DNA fragmentation assay. Selected retinal samples were processed for an ultra-structural analysis to confirm apoptosis. The retinas exposed to SIN-1 were tested for the expression of caspase-3 by immunohistochemistry and a Western blot analysis. The retinas were also evaluated for the prevention of apoptosis in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk). Results. The retinal explants exposed to SIN-1 showed a significant increase in the presence of TUNEL-positive photoreceptor cells. Similarly lineal increases in TUNELpositive cells were seen in the presence of increasing concentrations of SIN-1. DNA ladder formation was seen with the exposure of SIN-1. Ultrastructurally, SIN-1 exposed retinas revealed typical apoptotic changes in the photoreceptor cell nuclei. The retinas preincubated with urate for 6 hours and exposed to SIN-1 for 16 hours showed significantly fewer TUNEL-positive cells compared to the retinas exposed to SIN-1 alone (p < 0.05). Moreover, the retinas exposed to SIN-1 showed the expression of caspase- 3. This expression, as well as the number of apoptotic photoreceptors, significantly decreased in the presence of Z-VAD-fmk. Conclusions. These results show that peroxynitrite induces apoptosis in photoreceptor cells and that such retinal damage appears to be mediated by caspase-3. The apoptotic process can be minimized by peroxynitrite scavenger urate, as well as by the caspase inhibitor Z-VAD-fmk.

Reduced glucose transport sensitizes Iec-6 cells to peroxynitrite-induced apoptosis

Introduction: Formula-fed infants develop necrotizing enterocolitis at higher rates than breast-fed infants. We hypothesize that diminished enterocyte glucose transport sensitizes enterocytes to nitrosative stress and this results in mitochondrial-dependent apoptosis. Methods: IEC6 cells were treated with 3-morpholinosydnonimine (SIN-1) (500 uM-2 mM), a peroxynitrite generator, in either normal (NM) (4.5 g/ml) and low (LM) (1 g/ml) glucose media. IEC6 cell apoptosis was measured by 7-aminoactinomycin-D FACS analysis and histology. In order to test the role of glucose transport, we pretreated the cells with phloretin (100 uM), a specific GLUT-2 transport inhibitor. Cells were treated with SIN-1 in combination with phloretin in NM for 4 hours. Mitochondrial respiration was tested with tetrazolium salts (MTT) and transmembrane potential with 5,5′, 6,6′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), ratiometric dye. ANOVA or Student t-test was used where appropriate; p < 0.05 is considered significant. Results: IEC6 cells exposed to the combination of SIN-1 and LM experienced a 2-fold increase in apoptosis at 24 hours compared to NM and SIN-1 (22.2 ± 3.2, 10.7 ± 2.4, P < 0.05). Impaired GLUT-2 transport plus SIN-1 increased apoptosis compared to SIN-1 alone in NM. (Table)TABLE—ABSTRACT 74GroupsApoptosis (%)MTTNM5.4 ± 0.6100.5 ± 0.4NM + SIN-14.3 ± 2.678.0 ± 5.9NM + SIN-1 and Phloretin33.7 ± 9.6∗43.6 ± 4.4∧NM and phloretin3.2 ± 0.487.4 ± 10.8LM4.4 ± 1.594.9 ± 6.0LM and SIN-133.2 ± 13.7∗78.4 ± 7.2Apop. and MTT (∗p < 0.05 v NM + SIN-1;∧p < 0.01 v all). Mitochondrial respiration was impaired in NM plus SIN-1 and phloretin but not in NM and SIN-1 alone; transmembrane potential findings were consistent with these results (dns). Conclusions: These findings support the hypothesis that decreased glucose in the local enterocyte environment may render enterocytes susceptible to reactive nitrogen species.

Peroxynitrite induces apoptosis in canine cerebral vascular muscle cells: possible relation to neurodegenerative diseases and strokes

Considerable evidence is accumulating to suggest that in vivo formation of free radicals in the brain, such as peroxynitrite (ONOO −), and programmed cell death (i.e. apoptosis) play important roles in neurodegeneration and stroke. However, it is not known whether ONOO − can induce apoptosis in cerebral vascular smooth muscle cells (CVSMCs). The present study was designed to determine whether or not canine CVSMCs undergo apoptosis following treatment with ONOO −. Direct exposure of canine CVSMCs to ONOO − induced apoptosis in a concentration-dependent manner, as confirmed by means of fluorescence staining, TdT-mediated dUTP nick-end labeling and comet assays. Peroxynitrite treatment resulted in an elevation of [Ca 2+] i in the CVSMCs. Peroxynitrite-induced apoptosis may thus be brought about by activation of Ca 2+-dependent endonucleases. Although the precise mechanisms by which peroxynitrite induces apoptosis need to be further investigated, the present findings could be used to suggest that ONOO − formation in the brain may play important roles in neurodegenerative processes and strokes via detrimental actions on cerebral microvessels and blood flow.

Antioxidant activity of the cruciferous vegetable Maca (Lepidium meyenii)

Maca (Lepidium meyenii) is a plant from the Andes of Peru. Maca is used as a food for its nutritional value and ethnomedicinal properties linked to fertility and vitality. The purpose of this study was to evaluate the antioxidant activity of Maca. For all experiments an aqueous extract of Maca was used. The antioxidant activity of Maca was assessed by the inhibition of peroxynitrite, 1,1-diphenyl-2-picrylhydrazyl (DPPH), peroxyls and deoxyribose degradation. The cytoprotection capacity of Maca was assessed using macrophages (RAW 264.7) treated with peroxynitrite or hydrogen peroxide (H2O2). Catechins were quantified by reversed-phase HPLC. Addition of Maca extract (0.3–1 mg/ml) to peroxynitrite (300 μM) decreased peroxynitrite concentration by 15 and 41%, respectively (P<0.01). The IC50 for scavenging DPPH and peroxyls was 0.61 and 0.43 mg/ml, respectively. Deoxyribose protection by Maca (1–3 mg/ml) against hydroxyl radicals was in the order of 57 and 74%. Maca (1 mg/ml) protected RAW 264.7 cells against peroxynitrite-induced apoptosis (P<0.01), and increased ATP production in cells treated with H2O2 (1 mM). The concentration of catechins in Maca was lower than in green tea (2.5 mg/g vs 145 mg/g). Collectively, our results indicate that Maca has the capacity to scavenge free radicals and protect cells against oxidative stress.

Opposite roles of selenium-dependent glutathione peroxidase-1 in superoxide generator diquat- and peroxynitrite-induced apoptosis and signaling.

Oxidative injuries including apoptosis can be induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) in aerobic metabolism. We determined impacts of a selenium-dependent glutathione peroxidase-1 (GPX1) on apoptosis induced by diquat (DQ), a ROS (superoxide) generator, and peroxynitrite (PN), a potent RNS. Hepatocytes were isolated from GPX1 knockout (GPX1-/-) or wild-type (WT) mice, and treated with 0.5 mm DQ or 0.1-0.8 mm PN for up to 12 h. Loss of cell viability, high levels of apoptotic cells, and severe DNA fragmentation were produced by DQ in only GPX1-/- cells and by PN in only WT cells. These two groups of cells shared similar cytochrome c release, caspase-3 activation, and p21(WAF1/CIP1) cleavage. Higher levels of protein nitration were induced by PN in WT than GPX1-/- cells. Much less and/or slower cellular GSH depletion was caused by DQ or PN in GPX1-/- than in WT cells, and corresponding GSSG accumulation occurred only in the latter. In conclusion, it is most striking that, although GPX1 protects against apoptosis induced by superoxide-generator DQ, the enzyme actually promotes apoptosis induced by PN in murine hepatocytes. Indeed, GSH is a physiological substrate for GPX1 in coping with ROS in these cells.

Protective effects of morphine in peroxynitrite-induced apoptosis of primary rat neonatal astrocytes: potential involvement of G protein and phosphatidylinositol 3-kinase (PI3 kinase)

Opiates, such as morphine, have been used extensively in the clinical management of pain due to their potent analgesic effect. Astrocytes, representing a major non-neuronal cell population in the CNS, contain opioid receptors that are actively involved in several brain functions. This study was designed to evaluate the effects by which morphine, a preferential mu-opioid receptor agonist, contributes to cytotoxicity of nitric oxide (NO) species, including NO and peroxynitrite (ONOO −), in primary rat neonatal astrocytes. Primary astrocytes isolated from the cerebral cortex of 1- to 2-day-old Sprague–Dawley rats were treated with morphine, naloxone, and 3-morpholinosydnonimine (SIN-1), a donor of peroxynitrite. Morphine significantly protected primary rat astrocytes from apoptosis mediated by sodium nitroprusside, an NO donor, and SIN-1 in a dose-dependent manner, whereas it did not in other types of cells including C6 glioma, RAW 264.7, and HL-60 cells. Moreover, naloxone antagonized the protective effects of morphine on SIN-1-induced apoptosis. Morphine also inhibited the nuclear condensation and fragmentation of SIN-1-treated cells that was antagonized by naloxone pretreatment. The protective role of morphine in SIN-1-induced apoptosis was dependent on an intracellular antioxidant system such as GSH. Furthermore, the effects of morphine on SIN-1-induced cytotoxicity were prohibited by pretreatment with the G i protein inhibitor, pertussis toxin, and the phosphatidylinositol 3-kinase (PI3 kinase) inhibitors, wortmannin and LY294002. Taken together, these results suggest that morphine may protect primary rat astrocytes from apoptosis by NO species via the signaling cascades that involve both G protein and PI3 kinase.

BCL-2 protects peroxynitrite-treated thymocytes from poly(ADP-ribose) synthase (PARS)-independent apoptotic but not from PARS-mediated necrotic cell death

In thymocytes, peroxynitrite induces poly(ADP-ribose) synthetase (PARS) activation, which results in necrotic cell death. In the absence of PARS, however, peroxynitrite-treated thymocytes die by apoptosis. Because Bcl-2 has been reported to inhibit not only apoptotic but also some forms of necrotic cell death, here we have investigated how Bcl-2 regulates the peroxynitrite-induced apoptotic and necrotic cell death. We have found that Bcl-2 did not provide protection against peroxynitrite-induced necrotic death, as characterized by propidium iodide uptake, mitochondrial membrane potential decrease, secondary superoxide production, and cardiolipin loss. In the presence of a PARS inhibitor, peroxynitrite-treated thymocytes from Bcl-2 transgenic mice showed no caspase activation or DNA fragmentation and displayed smaller mitochondrial membrane potential decrease. These data show that Bcl-2 protects thymocytes from peroxynitrite-induced apoptosis at a step proximal to mitochondrial alterations but fails to prevent PARS-mediated necrotic cell death. Activation of tissue transglutaminase (tTG) occurs in various forms of apoptosis. Peroxynitrite did not induce transglutaminase activity in thymocytes and did not have a direct inhibitory effect on the purified tTG. Basal tTG was not different in Bcl-2 transgenic and wild type cells.