Photoreceptor mitochondrial tyrosine nitration in experimental uveitis.
OBJECTIVE
In experimental autoimmune uveitis (EAU), phagocytes are thought to be the primary cells in the initiation and maintenance of pathologic tissue damage through the release of cytotoxic agents. Recently, the presence of nitric oxide synthase has been shown in mammalian mitochondria. In this study, the effect of mitochondrial peroxynitrite on the modification of cellular proteins was evaluated in the early phase of uveitis, before the infiltration of leukocytes.


METHODS
Tyrosine nitration in proteins was detected by UV/Vis (visible) absorption and Western blot analysis. The identity of the nitrated protein was obtained by liquid chromatography-tandem mass spectrometry. The release of cytochrome c was assessed in whole retinal extract and in isolated mitochondria. The protein nitration in the inflamed retina was also localized by immunohistochemistry.


RESULTS
Before the leukocyte infiltration in the early phase of EAU, the mitochondria-originated peroxynitrite initiated the inflammatory insult by specifically nitrating three mitochondrial proteins. In vitro nitration of the control retina by peroxynitrite donor resulted in nonspecific nitration of all major retinal proteins. After nitration, cytochrome c was displaced from its original binding site in the respiratory chain. Further, the nitration appeared to commence in the early phase of inflammation, on postimmunization day 5, long before the peak of inflammation on day 14. Immunohistochemically, tyrosine-nitrated proteins were localized exclusively in the photoreceptor inner segments, which are known to be densely populated with mitochondria.


CONCLUSIONS
These data indicate that mitochondrial proteins are the prime targets of inactivation by the mitochondrial peroxynitrite and that photoreceptor mitochondria initiate the subsequent irreversible retinal damage in experimental uveitis.
Doheny Eye Institute, University of South California, Keck School of Medicine, Los Angeles, CA 90033-1088, USA.
Guey-Shuang
GS
Wu
Terry D
TD
Lee
Roger E
RE
Moore
Narsing A
NA
Rao
D000595:Amino Acid Sequence; D000818:Animals; D001327:Autoimmune Diseases / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology; D015153:Blotting, Western; D045304:Cytochromes c / Q000378:metabolism; D004195:Disease Models, Animal; D007124:Immunoenzyme Techniques; D013058:Mass Spectrometry; D024101:Mitochondrial Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D008969:Molecular Sequence Data; D015538:Nitrosation; D030421:Peroxynitrous Acid / Q000378:metabolism; D010736:Phosphoglycerate Mutase / Q000378:metabolism; D020419:Photoreceptor Cells, Vertebrate / Q000378:metabolism*; D051381:Rats; D011917:Rats, Inbred Lew; D012160:Retina / Q000378:metabolism / Q000473:pathology; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000378:metabolism*; D014605:Uveitis / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology
D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D030421:Peroxynitrous Acid; C090759:mitochondrial import stimulation factor, rat; D014443:Tyrosine; D045304:Cytochromes c; D010736:Phosphoglycerate Mutase