cytochrome-c-t and 3-nitrotyrosine

The present study aimed to investigate the ability of SS31, a novel mitochondria‑targeted peptide to protect against t‑BHP‑induced mitochondrial dysfunction and apoptosis in 661W cell lines. The 661W cells were treated with various concentrations of SS‑31 and an MTT assay was used to determine cell viability. The expression of nitrotyrosine and 8‑hydroxydeoxyguanosine (8‑OHdG) was detected using immunofluorescent staining. Apoptosis were assessed using Hoechst staining and an annexin V/propidium iodide flow cytometer. Reactive oxygen species (ROS) were detected using MitoSOXTM with confocal microscopy. Changes in mitochondrial membrane potential were analyzed using flow cytometry. In addition, the release of cytochrome c was analyzed using confocal microscopy. The viability of the cells improved following treatment with SS31 between 100 nM and 1 µM, compared with untreated control group. Compared with the t‑BHP treatment group (20.0±3.8%), the number of annexin V‑positive cells decreased dose‑dependently to 13.6±2.6, 9.8±0.5 and 7.4±2.0% in the SS‑31 treated group at concentrations of 10 nM, 100 nM and 1 µM, respectively. Treatment with SS‑31 significantly prevented the t‑BHP‑induced expression of nitrotyrosine and 8‑OHdG, decreased the quantity of mitochondrial ROS, increased mitochondrial potential, and prevented the release of cytochrome c from mitochondria into the cytoplasm. Therefore, the SS31 mitochondria‑targeted peptide protected the 661W cells from the sustained oxidative stress induced by t‑BHP.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Animals; Antioxidants; Apoptosis; Cell Line, Transformed; Cell Survival; Cytochromes c; Deoxyguanosine; Membrane Potential, Mitochondrial; Mice; Mitochondria; Oligopeptides; Oxidants; Oxidative Stress; Reactive Oxygen Species; Retinal Cone Photoreceptor Cells; tert-Butylhydroperoxide; Tyrosine

The protective effects of paeoniflorin isolated from Paeonia lactiflora against pharmacological inhibition of the respiratory chain were studied using osteoblastic MC3T3-E1 cells. Here we show that paeoniflorin decreases cell death induced by antimycin A, an inhibitor of mitochondrial complex III. Paeoniflorin restored antimycin A-induced inactivation of phosphoinositide 3-kinase (PI3K) and thioredoxin reductase, suggesting that PI3K and thioredoxin reductase may be involved in paeoniflorin-induced cytoprotective responses. We also examined the effect of paeoniflorin on mitochondrial dysfunction and oxidative stress induced by antimycin A. Paeoniflorin inhibited mitochondrial membrane potential dissipation, ATP loss, inactivation of complexes I and IV, cytochrome c release, and cardiolipin oxidation induced by antimycin A. In addition, paeoniflorin prevented antimycin A-induced ROS release and nitrotyrosine increase. These results imply that paeoniflorin protects osteoblasts from antimycin A-induced cell death via improved mitochondrial function.

Topics: 3T3 Cells; Animals; Antimycin A; Antioxidants; Benzoates; Bridged-Ring Compounds; Cardiolipins; Cell Survival; Cytochromes c; Glucosides; Membrane Potential, Mitochondrial; Mice; Mitochondria; Monoterpenes; Osteoblasts; Oxidative Stress; Paeonia; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Plant Extracts; Protective Agents; Reactive Oxygen Species; Thioredoxin-Disulfide Reductase; Tumor Necrosis Factor-alpha; Tyrosine

We have previously shown that homocysteine (Hcy) induces phosphatidylserine (PS) exposure, apoptosis and necrosis in human endothelial cells. Since it has been suggested that S-adenosylhomocysteine (SAH) is the main causative factor in Hcy-induced pathogenesis of cardiovascular disease, we evaluate here whether the cytotoxic Hcy effect in endothelial cells is also SAH dependent.. Human umbilical vein endothelial cells (HUVECs) were exposed to the following conditions: (1) non-treated control (resulting in 2.8 nM intracellular SAH and 3.1 μM extracellular l-Hcy); and incubation with (2) 50 μM adenosine-2,3-dialdehyde (ADA; resulting in 17.7 nM intracellular SAH and 3.1 μM extracellular l-Hcy), (3) 2.5 mM Hcy (resulting in 20.9 nM intracellular SAH and 1.8 mM extracellular l-Hcy), and (4) 1, 10 and 100 μM SAH. We then determined the effect of treatment on annexin V-positivity, caspase-3 activity, cytochrome c release (sub)cellular expression of NOX2, NOX4, p47(phox) and nitrotyrosine, and H(2)O(2). Both Hcy and ADA significantly increased PS exposure (n=5), caspase-3 activity (n=6) and cytochrome c release (n=3). Incubation with extracellular SAH alone did not affect cell viability. Both Hcy and ADA also induced similar increases in nuclear NOX2 and (peri)nuclear NOX4, coinciding with (peri)nuclear p47(phox) expression and local reactive oxygen species (ROS) (n=3). Inhibition of NOX-mediated ROS by the flavoenzyme inhibitor diphenylene iodonium (DPI) significantly decreased apoptosis induction (n=3) and ROS production (n=3).. SAH induces PS exposure and apoptosis in endothelial cells independently of Hcy. Our study therefore shows that Hcy-mediated endothelial dysfunction, as determined in the cell model used, is mainly due to SAH accumulation.

Topics: Adenosine; Apoptosis; Caspase 3; Cell Survival; Cells, Cultured; Cytochromes c; Endothelial Cells; Enzyme Inhibitors; Homocysteine; Humans; Hydrogen Peroxide; Membrane Glycoproteins; NADPH Oxidase 2; NADPH Oxidase 4; NADPH Oxidases; Onium Compounds; Phosphatidylserines; S-Adenosylhomocysteine; Tyrosine

Often, deregulation of protein activity and turnover by tyrosine nitration drives cells toward pathogenesis. Hence, understanding how the nitration of a protein affects both its function and stability is of outstanding interest. Nowadays, most of the in vitro analyses of nitrated proteins rely on chemical treatment of native proteins with an excess of a chemical reagent. One such reagent, peroxynitrite, stands out for its biological relevance. However, given the excess of the nitrating reagent, the resulting in vitro modification could differ from the physiological nitration. Here, we determine unequivocally the configuration of distinct nitrated-tyrosine rings in single-tyrosine mutants of cytochrome c. We aimed to confirm the nitration position by a non-destructive method. Thus, we have resorted to (1)H-(15)N heteronuclear single quantum coherence(HSQC) spectra to identify the (3)J(NH) correlation between a (15)N-tagged nitro group and the adjacent aromatic proton. Once the chemical shift of this proton was determined, we compared the (1)H-(13)C HSQC spectra of untreated and nitrated samples. All tyrosines were nitrated at ε positions, in agreement to previous analysis by indirect techniques. Notably, the various nitrotyrosine residues show a different dynamic behaviour that is consistent with molecular dynamics computations.

Topics: Cytochromes c; Nitrates; Nuclear Magnetic Resonance, Biomolecular; Peroxynitrous Acid; Proteins; Tyrosine

NADPH oxidase is a major complex that produces reactive oxygen species (ROSs) during the ischemic period and aggravates brain damage and cell death after ischemic injury. Although many approaches have been tested for preventing production of ROSs by NADPH oxidase in ischemic brain injury, the regulatory mechanisms of NADPH oxidase activity after cerebral ischemia are still unclear. The aim of this study is identifying apocynin as a critical modulator of NADPH oxidase and elucidating its role as a neuroprotectant in an experimental model of brain ischemia in rat. Treatment of apocynin 5min before of reperfusion attenuated cerebral ischemia in rats. Administration of apocynin showed marked reduction in infarct size compared with that of control rats. Medial carotid artery occlusion (MCAo)-induced cerebral ischemia was also associated with an increase in, nitrotyrosine formation, as well as IL-1β expression, IκB degradation and ICAM expression in ischemic regions. These expressions were markedly inhibited by the treatment of apocynin. We also demonstrated that apocynin reduces levels of apoptosis (TUNEL, Bax and Bcl-2 expression) resulting in a reduction in the infarct volume in ischemia-reperfusion brain injury. This new understanding of apocynin induced adaptation to ischemic stress and inflammation could suggest novel avenues for clinical intervention during ischemic and inflammatory diseases.

Topics: Acetophenones; Animals; Apoptosis; bcl-2-Associated X Protein; Brain; Cytochromes c; Disease Models, Animal; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; I-kappa B Proteins; In Situ Nick-End Labeling; Infarction, Middle Cerebral Artery; Intercellular Adhesion Molecule-1; Interleukin-1beta; Male; NADPH Oxidases; Neurologic Examination; Peptide Fragments; Proto-Oncogene Proteins c-bcl-2; Rats; Rats, Wistar; Reperfusion Injury; Tyrosine

Mitochondrial dysfunction, particularly respiratory chain disruption, is often responsible for aging-related bone diseases. In this study, the protective effects of glabridin, an isoflavan isolated from licorice root, against pharmacological inhibition of the respiratory chain were studied using osteoblastic MC3T3-E1 cells treated with antimycin A, which inhibits complex III of the electron transport system. Glabridin restored mitochondrial membrane potential dissipation, ATP loss, inactivation of complex IV, intracellular calcium elevation, and cytochrome c release that was induced by antimycin A treatment. This compound also prevented cell death. These results imply that glabridin protects osteoblasts from antimycin A-induced cell death via improved mitochondrial function. Glabridin scavenged ROS and mitochondrial superoxide anions generated by antimycin A. In addition, glabridin prevented antimycin A-induced nitrotyrosine increase and thioredoxin reductase inactivation, suggesting that glabridin may be useful for protecting mitochondria against a burst of oxidative stress. Since phosphoinositide 3-kinase (PI3K) and cAMP-response element-binding protein (CREB) signaling is known to be pro-survival, we determined whether PI3K and CREB activation is associated with the cytoprotective effects of glabridin in the MC3T3-E1 cells. Glabridin restored antimycin A-induced inactivation of PI3K and CREB, suggesting that PI3K and CREB-dependent pathways may be involved in glabridin-induced cytoprotective responses. Our study demonstrates that glabridin reduces mitochondrial dysfunction induced during aging, and could significantly prevent osteoblast damage in osteoporotic patients.

Topics: Animals; Antimycin A; Calcium; Cell Line; CREB-Binding Protein; Cytochromes c; Isoflavones; Mice; Osteoblasts; Oxidative Stress; Phenols; Phosphatidylinositol 3-Kinases; Phosphorylation; Reactive Oxygen Species; Thioredoxin-Disulfide Reductase; Tyrosine

Luteolin is a flavonoid found in many herbal extracts including celery, green pepper, parsley, perilla leaf and seeds, and chamomile. Antimycin A (AMA) is an inhibitor of the mitochondrial electron transport chain. In the present study, the protective effect of luteolin on AMA-induced cell damage was investigated in osteoblastic MC3T3-E1 cells. Luteolin significantly increased the viability of MC3T3-E1 cells in the presence of AMA and the effect of luteolin in increasing cell viability was completely prevented by the presence of LY294002, Akt inhibitor, or auranofin, suggesting that the effect of luteolin might be partly mediated from PI3K, Akt, and thioredoxin reductase. Pre-treatment with luteolin prior to AMA exposure significantly prevented mitochondrial membrane potential dissipation, ATP loss, inactivation of complex I and IV, ROS production, inactivation of thioredoxin reductase, intracellular calcium elevation, and cytochrome c release induced by AMA. Moreover, luteolin increased activities of PI3K (phosphoinositide 3-kinase) and Akt (protein kinase B), and CREB (cAMP-response element-binding protein) phosphorylation inhibited by AMA treatment. Collectively, these results suggest that luteolin protects MC3T3-E1 cells from AMA-induced cell damage through the improved mitochondrial function and activation of PI3K/Akt/CREB.

Topics: Adenosine Triphosphate; Animals; Antimycin A; Calcium; Cardiolipins; Cell Line; Cell Survival; Cyclic AMP Response Element-Binding Protein; Cytochromes c; Luteolin; Membrane Potential, Mitochondrial; Mice; Mitochondria; NADP; Osteoblasts; Phosphatidylinositol 3-Kinases; Phosphorylation; Protective Agents; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Thioredoxin-Disulfide Reductase; Tyrosine

Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.

Topics: Amino Acid Sequence; Animals; Cattle; Chromatography, High Pressure Liquid; Cytochromes c; Molecular Sequence Data; Nanotechnology; Nitrosation; Peptide Fragments; Serum Albumin, Bovine; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tyrosine

We studied the subcellular distribution of mitochondria and superoxide dismutase-1 (SOD1) in whole mounts of microdissected motor axons of rats expressing the ALS-linked SOD1-G93A mutation. The rationale was to determine whether physical interactions between the enzyme and mitochondria were linked to the axonopathy of motor fibers occurring in amyotrophic lateral sclerosis (ALS). Mitochondria and SOD1 displayed a homogeneous distribution along motor axons both in nontransgenic rats and in those overexpressing wild-type SOD1. In contrast, axons from SOD1-G93A rats (older than 35 days) showed accumulation of mitochondria in discrete clusters located at regular intervals. Most of SOD1 immunoreactivity was enriched in these clusters and colocalized with mitochondria, suggesting a recruitment of SOD1-G93A to the organelle. The SOD1/mitochondrial clusters were abundant in motor axons but scarcely seen in sensory axons. Clusters also were stained for neuronal nitric oxide synthase, nitrotyrosine, and cytochrome c. The later also was detected surrounding clusters. Ubiquitin colocalized with clusters only at late stages of the disease. The cytoskeleton was not overtly altered in clusters. These results suggest that mutant SOD1 and defective mitochondria create localized dysfunctional domains in motor axons, which may lead to progressive axonopathy in ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Axons; Cytochromes c; Disease Models, Animal; Humans; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Mutation; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Superoxide Dismutase; Tyrosine; Ubiquitin

To determine photoreceptor oxidative stress and damage in sympathetic ophthalmia (SO).. Immunohistologic study.. Eight formalin-fixed and paraffin-embedded human globes with typical histologic features of SO and five age-matched globes without intraocular inflammation (controls) were retrieved from the Doheny Eye Institute ophthalmic pathology files. Deparaffinized sections of the globes were processed to localize tumor necrosis factor-alpha (TNF-alpha), tumor necrosis factor receptor-1 (TNF-R1), acrolein, inducible nitric oxide synthase (iNOS), and nitrotyrosine by immunolocalization method. The latter two were localized to photoreceptor mitochondria using anti-cytochrome C antibody. Apoptotic cells were detected by Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) assay and were localized to the site of oxidative stress using antinitrotyrosine antibody.. Increased expression of TNF-alpha can be seen in the photoreceptor nuclear layer in all SO globes, whereas no such expression was observed in control globes. TNF-R1, iNOS, acrolein, and nitrotyrosine were immunolocalized to the inner segments of the photoreceptors in all SO globes, but only mild focal staining was observed in the control retinas. Both nitrotyrosine and iNOS immunolocalization revealed positive staining restricted primarily to mitochondria at the inner segments of the photoreceptors. Most of the TUNEL-positive cells were detected in the photoreceptors at the site of nitrotyrosine staining. In contrast, the age-matched control globes showed negative results.. In SO, photoreceptor mitochondrial oxidative stress occurs in the absence of leukocytic infiltration of the retina and may lead to photoreceptor apoptosis and subsequent vision loss. The oxidative stress seems to be mediated by iNOS and TNF-alpha. The current anti-inflammatory therapy combined with agents that could prevent oxidative stress may prevent photoreceptor damage in SO and may preserve vision.

Topics: Acrolein; Adult; Aged; Apoptosis; Biomarkers; Cytochromes c; Fluorescent Antibody Technique, Indirect; Humans; In Situ Nick-End Labeling; Middle Aged; Mitochondrial Diseases; Nitric Oxide Synthase Type II; Ophthalmia, Sympathetic; Oxidative Stress; Photoreceptor Cells, Vertebrate; Receptors, Tumor Necrosis Factor, Type I; Tumor Necrosis Factor-alpha; Tyrosine

Microsomal glutathione transferase (MGST1) is activated by oxidative stress. Although MGST1 is found in mitochondrial membranes (mtMGST1), there is no information about the oxidative activation of mtMGST1. In the present study, we aimed to determine whether mtMGST1 also undergoes activation and about its function. When rats were treated with galactosamine/lipopolysaccharide (GalN/LPS), mtMGST1 activity was significantly increased, and the increased activity was reduced by the disulfide reducing agent dithiothreitol. In mitochondria from GalN/LPS-treated rats, disulfide-linked mtMGST1 dimer and mixed protein glutathione disulfides (glutathionylation) were detected. In addition, cytochrome c release from mitochondria isolated from GalN/LPS-treated rats was observed, and the release was inhibited by anti-MGST1 antibodies. Incubation of mitochondria from control rats with diamide and diamide plus GSH in vitro resulted in dimer- and mixed disulfide bond-mediated activation of mtMGST1, respectively. The activation of mtMGST1 by diamide plus GSH caused cytochrome c release from the mitochondria, and the release was prevented by treatment with anti-MGST1 antibodies. In addition, diamide plus GSH treatment caused mitochondrial swelling accompanied by cytochrome c release, which was inhibited by cyclosporin A (CsA) and bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT) pore. Furthermore, mtMGST1 activity was also inhibited by CsA and BKA. These results indicate that mtMGST1 is activated through mixed disulfide bond formation that contributes to cytochrome c release from mitochondria through the MPT pore.

Topics: Animals; Apoptosis; Blotting, Western; Bongkrekic Acid; Cyclosporine; Cytochromes c; Diamide; Dimerization; Disulfides; Dithiothreitol; Enzyme Activation; Galactosamine; Glutathione; Glutathione Transferase; Lipopolysaccharides; Male; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Mitochondrial Swelling; Oxidation-Reduction; Oxidative Stress; Rats; Rats, Sprague-Dawley; Reducing Agents; Sulfhydryl Reagents; Time Factors; Tyrosine

Intracerebral hemorrhage (ICH) remains a major medical problem and currently has no effective treatment. Hemorrhaged blood is highly toxic to the brain, and catabolism of the pro-oxidant heme, mainly released from hemoglobin, is critical for the resolution of hematoma after ICH. The degradation of the pro-oxidant heme is controlled by heme oxygenase (HO). We have previously reported a neuroprotective role for HO2 in early brain injury after ICH; however, in vivo data that specifically address the role of HO2 in brain edema and neuroinflammation after ICH are absent. Here, we tested the hypothesis that HO2 deletion would exacerbate ICH-induced brain edema, neuroinflammation, and oxidative damage. We subjected wild-type (WT) and HO2 knockout ((-/-)) mice to the collagenase-induced ICH model. Interestingly, HO2(-/-) mice had enhanced brain swelling and neuronal death, although HO2 deletion did not increase collagenase-induced bleeding; the exacerbation of brain injury in HO2(-/-) mice was also associated with increases in neutrophil infiltration, microglial/macrophage and astrocyte activation, DNA damage, peroxynitrite production, and cytochrome c immunoreactivity. In addition, we found that hemispheric enlargement was more sensitive than brain water content in the detection of subtle changes in brain edema formation in this model. Combined, these novel findings extend our previous observations and demonstrate that HO2 deficiency increases brain swelling, neuroinflammation, and oxidative damage. The results provide additional evidence that HO2 plays a critical protective role against ICH-induced early brain injury.

Topics: Analysis of Variance; Animals; Brain Edema; Calcium-Binding Proteins; Cerebral Hemorrhage; Cytochromes c; Disease Models, Animal; Encephalitis; Fluoresceins; Functional Laterality; Glial Fibrillary Acidic Protein; Granulocyte Colony-Stimulating Factor; Heme Oxygenase (Decyclizing); Interleukin-3; Mice; Mice, Inbred C57BL; Mice, Knockout; Microfilament Proteins; Nerve Degeneration; Organic Chemicals; Recombinant Fusion Proteins; Recombinant Proteins; Spectrophotometry; Time Factors; Tyrosine

Human immunodeficiency virus dementia (HIVD) is the most common form of dementia occurring among young adults. In HIVD, neuronal cell loss occurs in the absence of neuronal infection. With the advent of highly active anti-retroviral therapy (HAART), the incidence of HIVD has drastically reduced, though prevalence of milder forms of HIVD continues to rise. Though these agents have been used successfully in suppressing viral production, they have also been associated with a number of side effects. Here we examine the possible role of NRTIs, in particular 2',3'-dideoxycytidine (ddC), in the neuropathology of HIVD. Synaptosomes and isolated mitochondria treated and incubated for 6 h with CSF-achievable concentrations of ddC, i.e., 6-11 ng/ml, were found to show a significant increase in oxidative stress with 40 nM ddC as measured by protein carbonyls and 3-nitrotyrosine (3NT), effects that were not observed in the more tolerable NRTI, 3TC. Protection against protein oxidation induced by ddC was observed when brain mitochondria were isolated from gerbils 1 h after injection i.p. with the brain accessible antioxidant and glutathione mimetic, tricyclodecan-9-yl-xanthogenate (D609). In addition, there is a significant reduction in the levels of anti-apoptotic protein Bcl-2 and a significant increase in cytochrome c release and also a significant increase in the expression of pro-apoptotic protein caspase-3 after mitochondria were treated with 40 nM ddC. The results reported here show that ddC at 40 nM can induce oxidative stress, cause the release of cytochrome c, and in addition, reduce the levels of anti-apoptotic proteins, increase the levels of pro-apoptotic proteins, thereby increasing the possibility for induction of apoptosis. These findings are consistent with the notion of a possible role of the NRTIs, and in particular, ddC, in the mechanisms involved in HIVD.

Topics: AIDS Dementia Complex; Animals; Antioxidants; Apoptosis; bcl-2-Associated X Protein; Bridged-Ring Compounds; Caspase 3; Cerebral Cortex; Cytochromes c; Disease Progression; Gerbillinae; Humans; Lamivudine; Male; Mitochondria; Nerve Tissue Proteins; Neuroprotective Agents; Norbornanes; Oxidation-Reduction; Oxidative Stress; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Inhibitors; Synaptosomes; Thiocarbamates; Thiones; Tyrosine; Zalcitabine

Peroxynitrite, a potent oxidative stress inducer, inhibits the mitochondrial electron transfer, induces cell death, and is considered to be involved in the pathology of various diseases. However, the intracellular mechanisms involved in the cell death process are not fully understood. Here we demonstrate that the enhanced nitration of specific tyrosine residues of cytochrome c, which are induced by continuous peroxynitrite exposure, attenuates cytochrome c-induced caspase-9 activation in vitro. Interestingly, cytochrome c nitrated with a single high dose of peroxynitrite preserved its potency, while this did not occur when cytochrome c was treated with continuous peroxynitrite exposure. Although both of these experiments resulted in cytochrome c nitration at the tyrosine residues, it was found that nitration at specific residues was enhanced only when cytochrome c was exposed to continuous peroxynitrite. This is the first report to demonstrate that cytochrome c nitration affects the apoptotic pathway by means of enhancement of nitration at specific tyrosine residues. This result implies that the nitration pattern of cytochrome c may affect the efficacy of the mitochondrial pathway in apoptotic cell death.

Topics: 5-Methoxytryptamine; Animals; Apoptosis; Caspase 3; Caspase 9; Caspases; Cell Line, Tumor; Chromatography, High Pressure Liquid; Cytochromes c; Enzyme Activation; Free Radical Scavengers; Nitrosation; Peptide Mapping; Peroxynitrous Acid; Protein Processing, Post-Translational; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Time Factors; Trypsin; Tyrosine

Ischemic preconditioning (IPC) strongly protects against ischemia-reperfusion injury; however, its effect on subsequent myocardial oxygenation is unknown. Therefore, we determine in an in vivo mouse model of regional ischemia and reperfusion (I/R) if IPC attenuates postischemic myocardial hyperoxygenation and decreases formation of reactive oxygen/nitrogen species (ROS/RNS), with preservation of mitochondrial function. The following five groups of mice were studied: sham, control (I/R), ischemic preconditioning (IPC + I/R, 3 cycles of 5 min coronary occlusion/5 min reperfusion) and IPC + I/R N(G)-nitro-L-arginine methyl ester treated, and IPC + I/R eNOS knockout mice. I/R and IPC + I/R mice were subjected to 30 min regional ischemia followed by 60 min reperfusion. Myocardial Po(2) and redox state were monitored by electron paramagnetic resonance spectroscopy. In the IPC + I/R, but not the I/R group, regional blood flow was increased after reperfusion. Po(2) upon reperfusion increased significantly above preischemic values in I/R but not in IPC + I/R mice. Tissue redox state was measured from the reduction rate of a spin probe, and this rate was 60% higher in IPC than in non-IPC hearts. Activities of NADH dehydrogenase (NADH-DH) and cytochrome c oxidase (CcO) were reduced in I/R mice after 60 min reperfusion but conserved in IPC + I/R mice compared with sham. There were no differences in NADH-DH and CcO expression in I/R and IPC + I/R groups compared with sham. After 60 min reperfusion, strong nitrotyrosine formation was observed in I/R mice, but only weak staining was observed in IPC + I/R mice. Thus IPC markedly attenuates postischemic myocardial hyperoxygenation with less ROS/RNS generation and preservation of mitochondrial O(2) metabolism because of conserved NADH-DH and CcO activities.

Topics: Animals; Coronary Vessels; Cytochromes c; Ischemic Preconditioning, Myocardial; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mitochondria, Heart; Myocardial Infarction; Myocardium; NADH Dehydrogenase; Nitric Oxide Synthase Type III; Oxidation-Reduction; Oxidative Stress; Oxygen Consumption; Random Allocation; Reactive Oxygen Species; Regional Blood Flow; Tyrosine

We demonstrate that X chromosome-linked inhibitor of apoptosis protein (XIAP) counteracts oxidative stress in two essentially different disease-related models of brain injury, hypoxia-ischemia and irradiation, as judged by lower expression of nitrotyrosine (5-fold) and 4-hydroxy-2-nonenal (10-fold) in XIAP-overexpressing compared with wild-type mice. XIAP overexpression induced up-regulation of at least three antioxidants residing in mitochondria, superoxide dismutase 2, thioredoxin 2 and lysine oxoglutarate reductase. Cytochrome c release from mitochondria was reduced in XIAP-overexpressing mice. Hence, in addition to blocking caspases, XIAP can regulate reactive oxygen species in the brain, at least partly through up-regulation of mitochondrial antioxidants. XIAP-induced prevention of oxidative stress was not secondary to tissue protection because although XIAP overexpression provides tissue protection after hypoxia-ischemia, it does not prevent tissue loss after irradiation. This is a previously unknown role of XIAP and may provide the basis for development of novel protective strategies for both acute and chronic neurodegenerative diseases, where oxidative stress is an integral component of the injury mechanisms involved.

Topics: Aldehydes; Animals; Antioxidants; Brain; Brain Ischemia; Cytochromes c; Female; Gene Expression; Hypoxia, Brain; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitochondria; Oxidative Stress; Tyrosine; Up-Regulation; X-Linked Inhibitor of Apoptosis Protein

Nitration of tyrosine residues is taken as evidence for intracellular formation of peroxynitrite. Cytochrome c (cyt c) can be nitrated by peroxynitrite and nitrated cyt c has been observed in cells and tissues under stress conditions. Here we studied the biochemical properties of nitrated cyt c in order to understand its potential roles in nitrative stress. Nitration of cyt c resulted in disruption of the heme-methionine bond and rapid binding to cyanide. Equilibrium unfolding by guanidine hydrochloride showed that cyt c was slightly destabilized upon nitration but the unfolding transition of nitrated cyt c was highly cooperative indicating that the overall folding was largely preserved. Nitrated cyt c could not be reduced by superoxide and did not support electron transfer between ascorbate and cyt c oxidase. Nitration of cyt c resulted in a tremendous increase in peroxidase activity so that nitrated cyt c rapidly oxidized dihydrodichlorofluorescein even in the presence of a high concentration of glutathione. Enhanced peroxidase activity of nitrated cyt c was responsible for H2O2-induced oxidation of phospholipid membranes and H2O2/NO2--mediated nitration of other proteins. These results suggest that nitration of cyt c by peroxynitrite may exacerbate oxidative damage to mitochondrial proteins and membranes.

Topics: Animals; Cyanides; Cytochromes c; Enzyme Stability; Fluoresceins; Oxidation-Reduction; Peroxidases; Peroxynitrous Acid; Protein Denaturation; Protein Folding; Superoxides; Tyrosine

Senescence is accompanied by oxidative stress and cardiac dysfunction, although the link between the two remains unclear. This study examined the role of antioxidant metallothionein on cardiomyocyte function, superoxide generation, the oxidative stress biomarker aconitase activity, cytochrome c release, and expression of oxidative stress-related proteins, such as the GTPase RhoA and NADPH oxidase protein p47phox in young (5-6 mo) and aged (26-28 mo) FVB wild-type (WT) and cardiac-specific metallothionein transgenic mice. Metallothionein mice showed a longer life span (by approximately 4 mo) than FVB mice evaluated by the Kaplan-Meier survival curve. Compared with young cardiomyocytes, aged myocytes displayed prolonged TR(90), reduced tolerance to high stimulus frequency, and slowed intracellular Ca2+ decay, all of which were nullified by metallothionein. Aging increased superoxide generation, active RhoA abundance, cytochrome c release, and p47phox expression and suppressed aconitase activity without affecting protein nitrotyrosine formation in the hearts. These aging-induced changes in oxidative stress and related protein biomarkers were attenuated by metallothionein. Aged metallothionein mouse myocytes were more resistant to the superoxide donor pyrogallol-induced superoxide generation and apoptosis. In addition, aging-associated prolongation in TR90 was blunted by the Rho kinase inhibitor Y-27632. Collectively, our data demonstrated that metallothionein may alleviate aging-induced cardiac contractile defects and oxidative stress, which may contribute to prolonged life span in metallothionein transgenic mice.

Topics: Aconitate Hydratase; Aging; Animals; Apoptosis; Calcium; Cytochromes c; Diastole; Gene Expression Regulation; Heart Ventricles; Metallothionein; Mice; Mice, Transgenic; Myocardial Contraction; Myocytes, Cardiac; Oxidative Stress; Pyrogallol; rhoA GTP-Binding Protein; Superoxides; Tyrosine

In the present study, we investigated how cytochrome c catalyzed the nitration of tyrosine at various pHs. The cytochrome c-catalyzed nitration of tyrosine occurred in proportion to the concentration of hydrogen peroxide, nitrite or cytochrome c. The cytochromec-catalyzed nitration of tyrosine was inhibited by catalase, sodium azide, cystein, and uric acid. These results show that the cytochrome c-catalyzed nitrotyrosine formation was due to peroxidase activity. The rate constant between cytochrome c and hydrogen peroxide within the pH range of 3-8 was the largest at pH 6 (37 degrees C). The amount of nitrotyrosine formed was the greatest at pH 5. At pH 3, only cytochromec-independent nitration of tyrosine occurred in the presence of nitrite. At this pH, the UV as well as visible spectrum of cytochrome c was changed by nitrite, even in the presence of hydrogen peroxide, probably via the formation of a heme iron-nitric oxide complex. Due to this change, the peroxidase activity of cytochrome c was lost.

Topics: Animals; Biomarkers; Catalysis; Cattle; Cytochromes c; Hydrogen Peroxide; Hydrogen-Ion Concentration; Nitrates; Nitrites; Oxidants; Oxidation-Reduction; Reactive Nitrogen Species; Serum Albumin, Bovine; Tyrosine

We aimed to test whether attenuation of early-phase cardiac cell death can prevent diabetic cardiomyopathy.. Our previous study showed that cardiac apoptosis as a major early cellular response to diabetes is induced by hyperglycemia-derived oxidative stress that activates a mitochondrial cytochrome c-mediated caspase-3 activation pathway. Metallothionein (MT) as a potent antioxidant prevents the development of diabetic cardiomyopathy.. Diabetes was induced by a single dose of streptozotocin (STZ) (150 mg/kg) in cardiac-specific, metallothionein-overexpressing transgenic (MT-TG) mice and wild-type (WT) controls. On days 7, 14, and 21 after STZ treatment, cardiac apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and caspase-3 activation. Cardiomyopathy was evaluated by cardiac ultrastructure and fibrosis in the diabetic mice 6 months after STZ treatment.. A significant reduction in diabetes-induced increases in TUNEL-positive cells, caspase-3 activation, and cytochrome c release from mitochondria was observed in the MT-TG mice as compared to WT mice. Cardiac protein nitration (3-nitrotyrosine [3-NT]) and lipid peroxidation were significantly increased, and there was an increase in mitochondrial oxidized glutathione and a decrease in mitochondrial reduced glutathione in the WT, but not in the MT-TG, diabetic mice. Double staining for cardiomyocytes with alpha sarcomeric actin and caspase-3 or 3-NT confirmed the cardiomyocyte-specific effects. A significant prevention of diabetic cardiomyopathy and enhanced animal survival were observed in the MT-TG diabetic mice as compared to WT diabetic mice.. These results suggest that attenuation of early-phase cardiac cell death by MT results in a significant prevention of the development of diabetic cardiomyopathy. This process is mediated by MT suppression of mitochondrial oxidative stress.

Topics: Actins; Animals; Antioxidants; Apoptosis; Cardiomyopathies; Caspase 3; Cell Death; Cytochromes c; Diabetes Mellitus, Experimental; Enzyme Activation; Glutathione; Glutathione Disulfide; Lipid Peroxidation; Metallothionein; Mice; Mice, Transgenic; Mitochondria, Heart; Myocardium; Myocytes, Cardiac; Oxidative Stress; Sarcomeres; Survival Analysis; Tyrosine

Tricyclodecan-9-yl-xanthogenate (D609) has in vivo and in vitro antioxidant properties. D609 mimics glutathione (GSH) and has a free thiol group, which upon oxidation forms a disulfide. The resulting dixanthate is a substrate for glutathione reductase, regenerating D609. Recent studies have also shown that D609 protects brain in vivo and neuronal cultures in vitro against the potential Alzheimer's disease (AD) causative factor, Abeta(1-42)-induced oxidative stress and cytotoxicity. Mitochondria are important organelles with both pro- and antiapoptotic factor proteins. The present study was undertaken to test the hypothesis that intraperitoneal injection of D609 would provide neuroprotection against free radical-induced, mitochondria-mediated apoptosis in vitro. Brain mitochondria were isolated from gerbils 1 h post injection intraperitoneally (ip) with D609 and subsequently treated in vitro with the oxidants Fe(2+)/H(2)O(2) (hydroxyl free radicals), 2,2-azobis-(2-amidinopropane) dihydrochloride (AAPH, alkoxyl and peroxyl free radicals), and AD-relevant amyloid beta-peptide 1-42 [Abeta(1-42)]. Brain mitochondria isolated from the gerbils previously injected ip with D609 and subjected to these oxidative stress inducers, in vitro, showed significant reduction in levels of protein carbonyls, protein-bound hydroxynonenal [a lipid peroxidation product], 3-nitrotyrosine, and cytochrome c release compared to oxidant-treated brain mitochondria isolated from saline-injected gerbils. D609 treatment significantly maintains the GSH/GSSG ratio in oxidant-treated mitochondria. Increased activity of glutathione S-transferase, glutathione peroxidase, and glutathione reductase in brain isolated from D609-injected gerbils is consistent with the notion that D609 acts like GSH. These antiapoptotic findings are discussed with reference to the potential use of this brain-accessible glutathione mimetic in the treatment of oxidative stress-related neurodegenerative disorders, including AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Antioxidants; Apoptosis; Brain; Bridged-Ring Compounds; Cytochromes c; Gerbillinae; Glutathione; Glutathione Peroxidase; Glutathione Reductase; Glutathione Transferase; Injections, Intraperitoneal; Lipid Peroxidation; Male; Mitochondria; Neurodegenerative Diseases; Neuroprotective Agents; Norbornanes; Oxidation-Reduction; Oxidative Stress; Thiocarbamates; Thiones; Type C Phospholipases; Tyrosine

Unilateral hypoxia-ischemia (HI) was induced in C57/BL6 male mice on postnatal day (P) 5, 9, 21 and 60, corresponding developmentally to premature, term, juvenile and adult human brains, respectively. HI duration was adjusted to obtain a similar extent of brain injury at all ages. Apoptotic mechanisms (nuclear translocation of apoptosis-inducing factor, cytochrome c release and caspase-3 activation) were several-fold more pronounced in immature than in juvenile and adult brains. Necrosis-related calpain activation was similar at all ages. The CA1 subfield shifted from apoptosis-related neuronal death at P5 and P9 to necrosis-related calpain activation at P21 and P60. Oxidative stress (nitrotyrosine formation) was also similar at all ages. Autophagy, as judged by the autophagosome-related marker LC-3 II, was more pronounced in adult brains. To our knowledge, this is the first report demonstrating developmental regulation of AIF-mediated cell death as well as involvement of autophagy in a model of brain injury.

Topics: Aging; Animals; Apoptosis; Apoptosis Inducing Factor; Autophagy; Brain Injuries; Calpain; Caspase 3; Caspases; Cell Death; Cytochromes c; Disease Models, Animal; Flavoproteins; Hypoxia-Ischemia, Brain; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins; Mitochondria; Necrosis; Neurons; Protein Transport; Tyrosine

The short- and long-term neuroprotective effects of 2-iminobiotin, a selective inhibitor of neuronal and inducible nitric oxide synthase, were studied in 12-day-old rats following hypoxia-ischemia. Hypoxia-ischemia was induced by occlusion of the right carotid artery followed by 90 minutes of hypoxia (FiO2 0.08). Immediately on reoxygenation, 12 and 24 hours later the rats were treated with vehicle or 2-iminobiotin at a dose of 5.5, 10, 30, or 60 mg/kg per day. Histologic analysis of brain damage was performed at 6 weeks after hypoxia-ischemia. To assess early changes of cerebral tissue, levels of HSP70, nitrotyrosine, and cytochrome c were determined 24 hours after reoxygenation. Significant neuroprotection was obtained using a dose of 30 mg/kg per day of 2-iminobiotin. Levels of HSP70 were increased in the ipsilateral hemisphere in both groups (P<0.05), but the increase was significantly (P<0.05) less in the rats receiving the optimal dose of 2-iminobiotin (30 mg/kg per day). Hypoxia-ischemia did not lead to increased levels of nitrotyrosine, nor did 2-iminobiotin influence levels of nitrotyrosine. In contrast, hypoxia-ischemia induced an increase in cytochrome c level that was prevented by 2-iminobiotin. In conclusion, 2-iminobiotin administered after hypoxia-ischemia provides long-term neuroprotection. This neuroprotection is obtained by mechanisms other than a reduction of nitrotyrosine formation in proteins.

Topics: Animals; Animals, Newborn; Biotin; Brain Chemistry; Cytochromes c; Female; HSP70 Heat-Shock Proteins; Hypoxia-Ischemia, Brain; Male; Neuroprotective Agents; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oxidation-Reduction; Rats; Rats, Wistar; Tyrosine

Iron deficiency is associated with multiple health problems, including the cardiovascular system. However, the mechanism of action of iron-deficiency-induced cardiovascular damage is unclear. The aim of the present study was to examine the effect of dietary iron deficiency on cardiac ultrastructure, mitochondrial cytochrome c release, NOS (nitric oxide synthase) and several stress-related protein molecules, including protein nitrotyrosine, the p47phox subunit of NADPH oxidase, caveolin-1 and RhoA. Male weanling rats were fed with either control or iron-deficient diets for 12 weeks. Cardiac ultrastructure was examined by transmission electron microscopy. Western blot analysis was used to evaluate cytochrome c, endothelial and inducible NOS, NADPH oxidase, caveolin-1 and RhoA. Protein nitrotyrosine formation was measured by ELISA. Rats fed an iron-deficient diet exhibited increased heart weight and size compared with the control group. Heart width, length and ventricular free wall thickness were similar between the two groups. However, the left ventricular dimension and chamber volume were significantly enhanced in the iron-deficient group compared with controls. Ultrastructural examination revealed mitochondrial swelling and abnormal sarcomere structure in iron-deficient ventricular tissues. Cytochrome c release was significantly enhanced in iron-deficient rats. Protein expression of eNOS (endothelial NOS) and iNOS (inducible NOS), and protein nitrotyrosine formation were significantly elevated in cardiac tissue or mitochondrial extraction from the iron-deficient group. Significantly up-regulated NADPH oxidase, caveolin-1 and RhoA expression were also detected in ventricular tissue of the iron-deficient group. Taken together, these results suggest that dietary iron deficiency may have induced cardiac hypertrophy characterized by aberrant mitochondrial and irregular sarcomere organization, which was accompanied by increased reactive nitrogen species and RhoA expression.

Topics: Animals; Body Weight; Cardiomegaly; Cytochromes c; Heart Ventricles; Heat-Shock Proteins; Iron Deficiencies; Male; Microscopy, Electron; Mitochondria, Heart; Myocardium; Myocytes, Cardiac; Nitric Oxide Synthase; Organ Size; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Tyrosine

The mitochondrial permeability transition pore (PTP) is a membrane protein complex assembled and opened in response to Ca(2+) and oxidants such as peroxynitrite (ONOO(-)). Opening the PTP is mechanistically linked to the release of cytochrome c, which participates in downstream apoptotic signaling. However, the molecular basis of the synergistic interactions between oxidants and Ca(2+) in promoting the PTP are poorly understood and are addressed in the present study. In isolated rat liver mitochondria, it was found that the timing of the exposure of the isolated rat liver mitochondria to Ca(2+) was a critical factor in determining the impact of ONOO(-) on PTP. Specifically, addition of Ca(2+) alone, or ONOO(-) and then Ca(2+), elicited similar low levels of PTP opening, whereas ONOO(-) alone was ineffective. In contrast, addition of Ca(2+) and then ONOO(-) induced extensive PTP opening and cytochrome c release. Interestingly, Cu/Zn-superoxide dismutase enhanced pore opening through a mechanism independent of its catalytic activity. These data are consistent with a model in which Ca(2+) reveals a molecular target that is now reactive with ONOO(-). As a test of this hypothesis, tyrosine nitration was determined in mitochondria exposed to ONOO(-) alone or to Ca(2+) and then ONOO(-), and mitochondrial membrane proteins were analyzed using proteomics. These studies suggest protein targets revealed by Ca(2+) include dehydrogenases and CoA - containing enzymes. These data are discussed in the context of the role of mitochondria, Ca(2+), and ONOO(-) in apoptotic signaling.

Topics: Animals; Calcium; Cytochromes c; Drug Administration Schedule; In Vitro Techniques; Ion Channels; Male; Mitochondria, Liver; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Mitochondrial Proteins; Peroxynitrous Acid; Proteomics; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Tyrosine

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

Topics: Animals; Catalysis; Cattle; Cytochromes c; Horses; Humans; Hydrogen Peroxide; Jurkat Cells; Mitochondria, Liver; Myocardium; Nitrates; Nitrites; Peroxidase; Phenylacetates; Rats; Recombinant Proteins; Serum Albumin, Bovine; Superoxide Dismutase; Tyrosine

Many models of Parkinson's disease (PD) have succeeded in replicating dopaminergic neuron loss or alpha-synuclein aggregation but not the formation of classical Lewy bodies, the pathological hallmark of PD. Our cybrid model of sporadic PD was created by introducing the mitochondrial genes from PD patients into neuroblastoma cells that lack mitochondrial DNA. Previous studies using cybrids have shown that information encoded by mitochondrial DNA in patients contributes to many pathogenic features of sporadic PD. In this paper, we report the generation of fibrillar and vesicular inclusions in a long-term cybrid cell culture model that replicates the essential antigenic and structural features of Lewy bodies in PD brain without the need for exogenous protein expression or inhibition of mitochondrial or proteasomal function. The inclusions generated by PD cybrid cells stained with eosin, thioflavin S, and antibodies to alpha-synuclein, ubiquitin, parkin, synphilin-1, neurofilament, beta-tubulin, the proteasome, nitrotyrosine, and cytochrome c. Future studies of these cybrids will enable us to better understand how Lewy bodies form and what role they play in the pathogenesis of PD.

Topics: Aged; alpha-Synuclein; Blotting, Western; Carrier Proteins; Case-Control Studies; Cell Line; Cysteine Endopeptidases; Cytochromes c; DNA, Mitochondrial; Electron Transport Complex I; Female; Humans; Immunohistochemistry; Lewy Bodies; Male; Microscopy, Confocal; Microscopy, Electron; Middle Aged; Multienzyme Complexes; Nerve Tissue Proteins; Neuroblastoma; Neurofilament Proteins; Neurons; Parkinson Disease; Precipitin Tests; Proteasome Endopeptidase Complex; Staining and Labeling; Synucleins; Transgenes; Tubulin; Tyrosine; Ubiquitin; Ubiquitin-Protein Ligases

Oxidative alteration of mitochondrial cytochrome c (cyt c) has been linked to disease pathophysiology and is one of the causative factors for pro-apoptotic events. Hydrogen peroxide induces a short-lived cyt c-derived tyrosyl radical as detected by the electron spin resonance (ESR) spin-trapping technique. This investigation was undertaken to characterize the fate and consequences of the cyt c-derived tyrosyl radical. The direct ESR spectrum from the reaction of cyt c with H(2)O(2) revealed a single-line signal with a line width of approximately 10 G. The detected ESR signal could be prevented by pretreatment of cyt c with iodination, implying that the tyrosine residue of cyt c was involved. The ESR signal can be enhanced and stabilized by a divalent metal ion such as Zn(2+), indicating the formation of the protein tyrosine ortho-semiquinone radical (ToQ.). The production of cyt c-derived ToQ. is inhibited by the spin trap, 2-methyl-2-nitrosopropane (MNP), suggesting the participation of tyrosyl radical in the formation of the ortho-semiquinone radical. The endothelium relaxant factor nitric oxide is well known to mediate mitochondrial respiration and apoptosis. The consumption of NO by cyt c was enhanced by addition of H(2)O(2) as verified by inhibition electrochemical detection using an NO electrode. The rate of NO consumption in the system containing cyt c/NO/H(2)O(2) was decreased by the spin traps 5,5-dimethyl pyrroline N-oxide and MNP, suggesting NO trapping of the cyt c-derived tyrosyl radical. The above result was further confirmed by NO quenching of the ESR signal of the MNP adduct of cyt c tyrosyl radical. Immunoblotting analysis of cyt c after exposure to NO in the presence of H(2)O(2) revealed the formation of 3-nitrotyrosine. The addition of superoxide dismutase did not change the cyt c nitration, indicating that it is peroxynitrite-independent. The results of this study may provide useful information in understanding the interconnection among cyt c, H(2)O(2), NO, and apoptosis.

Topics: Animals; Apoptosis; Blotting, Western; Cell Line; Chickens; Cytochromes c; Electron Spin Resonance Spectroscopy; Electrophoresis, Polyacrylamide Gel; Free Radicals; Horses; Hydrogen Peroxide; Immunoblotting; Magnetics; Models, Chemical; Muramidase; Nitric Oxide; Nitrites; Oxygen; Peroxynitrous Acid; Rats; Superoxide Dismutase; Time Factors; Tyrosine

Degeneration of the stria vascularis (SV) is amongst the major causes of cisplatin (CDDP)-induced hearing impairment. The pathways of apoptosis occurring in the SV due to CDDP were examined using a mouse experimental model. Temporal bones of adult C57BL/6 mice were collected on days 3, 7 and 14 after the local application of CDDP. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay and immunostaining for apoptosis-related proteins or reactive radical species were employed for analysis. Local application of CDDP caused apoptotic cell death of marginal cells 3 days after CDDP treatment. Immunohistochemical analyses demonstrated activation of caspase-3 and -9, but not -8, and redistribution of cytochrome c in affected marginal cells, indicating a caspase-dependent, mitochondrion-mediated apoptotic pathway in marginal cells. Temporary expression of hydroxynonenal, nitrotyrosine and inducible nitric oxide synthase in the SV was observed at the induction of apoptosis in marginal cells. CDDP toxicity generates reactive radical species in the SV, which causes mitochondrial membrane permeabilization leading to apoptosis of marginal cells.

Topics: Aldehydes; Animals; Antineoplastic Agents; Apoptosis; Caspases; Cisplatin; Cytochromes c; Enzyme Activation; Immunohistochemistry; In Situ Nick-End Labeling; Lipid Peroxidation; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Reactive Oxygen Species; Stria Vascularis; Tyrosine