2. CARDIOVASCULAR AND CEREBROVASCULAR AGING-Nitric oxide (NO) and reactive oxygen species (ROS) play critical roles in hypoxia/reoxygenation in heart. Aged animals are more susceptible to oxidative assault during hypoxia/reoxygenation. Increased ROS, perturbed redox balance and apoptosis occur during heart hypoxia/reoxygenation. Mitochondria play a central role in oxidative damage and apoptosis. Mitochondria render one of the main cellular sources of ROS and they release key apoptogenic proteins such as cytochrome c. Recently we and others showed that mitochondria also produce NO and they possess mitochondrial NO synthase (mtNOS). NO generated by mtNOS can react with mitochondrial ROS to produce oxidizing species such as peroxynitrite. Mitochondrial peroxynitrite induces oxidative stress and releases the key mitochondrial pro-apoptosis protein, cytochrome c. The interplay between mitochondrial NO, ROS, peroxynitrite, and apoptosis components during hypoxia/reoxyqenation is not well understood. We have designed and established a novel in vitro model to study these factors with minimum cellular components that may confound the study. To mimic hypoxia we incubate isolated mitochondria in lowered oxygen concentrations, and to mimic hypoxia/reoxygenation we incubate isolated mitochondria in lowered oxygen concentrations followed by reoxygenation. Using mitochondria isolated from heart of young adult rats we found that hypoxia/reoxygenation, but not hypoxia pre se, causes the release of cytochrome c and induces oxidative stress in a reverse oxygen concentration-dependent manner. The release of cytochrome c Is prevented when mtNOS is inhibited or when mitochondria are provided exogenously with antioxidants: glutathione, vitamin C, or a vitamin E derivative peroxynitrite scavenger. We hypothesize that mtNOS is involved in hypoxia/reoxygenation-induced oxidative stress and cytochrome c release, and that mitochondria isolated from the heart of aged animals are more susceptible to hypoxia/reoxygenation-induced oxidative stress and cytochrome c release. We propose to address this hypothesis using our in vitro model. We will test heart mtNOS protein expression and activity during hypoxia/reoxygenation in young and awed rats. We will also test apoptosis, oxidative stress and peroxynitrite markers during hypoxia/reoxygenation in heart mitochondria of young and aged rats. The results of the proposed study will reveal novel insights into the molecular mechanisms underlying hypoxia/reoxygenation induced oxidative stress and apoptosis and aging.

National Institute of Health (NIH)
National Institute on Aging (NIA)
Small Research Grants (R03)
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National Institute on Aging Initial Review Group (NIA)
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Kohanski, Ronald A
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Marshall University
Schools of Medicine
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Parihar, Mordhwaj S; Parihar, Arti; Fujita, Masayo et al. (2009) Alpha-synuclein overexpression and aggregation exacerbates impairment of mitochondrial functions by augmenting oxidative stress in human neuroblastoma cells. Int J Biochem Cell Biol 41:2015-24
Parihar, Arti; Vaccaro, Patrick; Ghafourifar, Pedram (2008) Nitric oxide irreversibly inhibits cytochrome oxidase at low oxygen concentrations: evidence for inverse oxygen concentration-dependent peroxynitrite formation. IUBMB Life 60:64-7
Parihar, Arti; Parihar, Mordhwaj S; Chen, Zhonghai et al. (2008) mAtNOS1 induces apoptosis of human mammary adenocarcinoma cells. Life Sci 82:1077-82
Parihar, Mordhwaj S; Parihar, Arti; Chen, Zhonghai et al. (2008) mAtNOS1 regulates mitochondrial functions and apoptosis of human neuroblastoma cells. Biochim Biophys Acta 1780:921-6
Ghafourifar, Pedram; Mousavizadeh, Kazem; Parihar, Mordhwaj S et al. (2008) Mitochondria in multiple sclerosis. Front Biosci 13:3116-26
Parihar, M S; Parihar, A; Fujita, M et al. (2008) Mitochondrial association of alpha-synuclein causes oxidative stress. Cell Mol Life Sci 65:1272-84
Parihar, Mordhwaj S; Parihar, Arti; Villamena, Frederick A et al. (2008) Inactivation of mitochondrial respiratory chain complex I leads mitochondrial nitric oxide synthase to become pro-oxidative. Biochem Biophys Res Commun 367:761-7
Parihar, Mordhwaj S; Nazarewicz, Rafal R; Kincaid, Erick et al. (2008) Association of mitochondrial nitric oxide synthase activity with respiratory chain complex I. Biochem Biophys Res Commun 366:23-8
Zenebe, Woineshet J; Nazarewicz, Rafal R; Parihar, Mordhwaj S et al. (2007) Hypoxia/reoxygenation of isolated rat heart mitochondria causes cytochrome c release and oxidative stress;evidence for involvement of mitochondrial nitric oxide synthase. J Mol Cell Cardiol 43:411-9
Nazarewicz, Rafal R; Zenebe, Woineshet J; Parihar, Arti et al. (2007) 12(S)-hydroperoxyeicosatetraenoic acid (12-HETE) increases mitochondrial nitric oxide by increasing intramitochondrial calcium. Arch Biochem Biophys 468:114-20

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