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G. Wu, T.D. Lee, N.A. Rao; Mitochondrial Cytochrome C is Nitrated and Released in Rat Experimental Autoimmune Uveitis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):734.
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Purpose: Peroxynitrite (NOOO-)-mediated in vivo mitochondrial damage has been implicated in several pathogenic conditions in recent years. But, similar oxidative injury to retinal mitochondria in EAU has not been demonstrated. The mitochondrial density in photoreceptors is among the highest in mammalian cells. In this study, we investigated the inflammation-mediated injury to retinal mitochondria by detecting tyrosine (Tyr) nitration. Methods: EAU was induced in Lewis rats by S-antigen. Non-immunized naïve rats were used as controls. At days 5, 10, 12, 13, 14 and 15 postimmunization, the rats were sacrificed and six retinas were combined for one determination. The nitration was initially assessed by absorption at 370 nm and by an immunoblot probed with anti-nitrotyrosine polyclonal antibody. To identify cytochrome C (Cyt C), monoclonal anti-rat Cyt C antibody was used for immunoblot. Results: Without lysing mitochondria, Cyt C, a mitochondrial intermembrane protein, was detected abundantly in the cellular cytosolic fraction in EAU retinas, whereas only a trace quantity was detected in controls. The released Cyt C was also Tyr-nitrated as indicated by 370 nm absorption and by immunoblot. Moreover, nitration/release of Cyt C appears to precede any significant morphologic retinal alteration. In immunoblots, the intensity of nitrated/released Cyt C was highest at the early stage of inflammation, decreasing slowly thereafter as the disease progressed. Conclusions: The mitochondrial origin of iNOS has recently been described. Therefore, by combining this source of nitric oxide and superoxide, from the electron transport chain, mitochondria are also capable of producing ONOO- aside from phagocyte participation. The fact that the mitochondrial protein was targeted predominantly implies a local mitochondrial stress early in the disease progression, before phagocytic infiltration. The release of Cyt C leads especially to the activation of caspases, which are apoptosis effectors. These observations, therefore, implicate the protein nitration in pathogenesis of EAU and impart the putative role mitochondria play in initiating retinal destruction.
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