Protein Nitration in Rat Retina Before and After Light Damage

Abstract

Abstract: : Purpose:Oxidative stress contributes to retinal light damage but the molecular mechanisms remain poorly understood. As part of ongoing efforts to better understand the role of protein oxidative modifications in retinal pathology, protein nitration in retina has been compared between rats exposed to damaging light or maintained in the dark. Methods:Albino rats maintained in a dark environment for 2-4 month were exposed to intense green light (1500 lux) for 3 hours and sacrificed immediately following light treatment. Proteomic and immunohistochemical analyses have been used to identify nitrated retinal proteins and their localization within the layers and cell types of the retina before and after light exposure. Results:Methodology for detecting nitrotyrosine containing proteins by Western analysis has been improved by incorporating chemical reduction of nitrotyrosine to aminotyrosine, allowing specific and nonspecific anti-nitrotyrosine antibody reactivity to be distinguished. Using 2D gel electrophoresis, Western and mass spectrometric analyses, several different nitrotyrosine immunoreactive proteins were identified in light exposed retina compared with those maintained in the dark. Immunocytochemical analyses of retina revealed that rats reared in darkness exhibited more nitrotyrosine immunoreactivity in the photoreceptor outer segments. After intense light exposure, immunoreactivity decreased in the outer segments and increased in the photoreceptor inner segments and retinal pigment epithelium. Conclusion:Light modulates retinal protein nitration in vivo. The present findings justify further consideration of nitration and nitric oxide as possible mediators in the light-induced biochemical sequela leading to photoreceptor cell death.

Keywords: 491 nitric oxide • 525 protein modifications-post translational • 561 retinal degenerations: cell biology