Light-induced changes in protein nitration in photoreceptor rod outer segments

Purpose: Light has been shown to modulate protein nitration in rat retinas. To better understand the role of protein nitration in photoreceptor cell death induced by intense light, we examined retinal protein nitration and identified target proteins in rod outer segments (ROS). Methods: Cyclic light-reared rats, treated or not with the antioxidant, dimethylthiourea (DMTU), were exposed to intense green light for 8 h. A subset of these rats was kept in the dark for 24 h after 8 h of light exposure. Western analysis of ROS proteins with an anti-nitrotyrosine antibody was performed to examine changes in protein nitration. 2D-immunoblots with anti-nitrotyrosine antibody followed by liquid chromatography tandem mass spectrometry was used to identify nitrated proteins in ROS. The expression levels of three nitric oxide synthase ( NOS) isoforms, inducible, neuronal-, and endothelial-NOS were semi-quantified by immunoblot analysis. Results: Western analysis revealed that the level of ROS protein nitration increased during the dark recovery period after 8 h of light treatment in both DMTU treated and untreated rats. However, DMTU effectively reduced protein nitration in ROS during light exposure and during the subsequent dark recovery period. Using 2D-immunoblotting followed by liquid chromatography tandem mass spectrometry analysis, we identified ten ROS proteins as nitration targets. Most of these proteins were glycolytic enzymes. The level of inducible-NOS in the retina was increased by light exposure. Conclusions: The effect of DMTU in reducing ROS protein nitration during and after light suggests the involvement of protein nitration during light-induced photoreceptor cell death. Nitration of glycolytic enzymes specifically may alter their activities. Increased levels of iNOS during and after intense light exposure suggest that this isoform is responsible for intense light induced protein nitration in ROS during the dark recovery period. The limited nitration seen in ROS during light exposure may reflect a quenching effect by endogenous antioxidants on the generation of reactive oxygen and nitrogen species.