Light–Induced Protein Nitration in the Photoreceptor Rod Outer Segments

Abstract

Abstract: : Purpose: Light–induced retinal degeneration in rats is characterized by apoptosis of photoreceptor cells. However, the molecular events that initiate the apoptotic cascade are poorly understood. Our recent immunohistochemical study on rat retina suggested that protein nitration increases in the rod outer segments of rats kept in the dark after intense green light exposure. This study was conducted to identify proteins that are nitrated in the rod outer segments after the light treatment and to define molecular effects of this modification in the functions of the nitrated proteins. Methods: Cyclic light reared rats (2 month old) were exposed to intense green light (1,200–1,400 lux; 490–580 nm) for various time periods up to 8 hours. Some animals were kept in the dark for various time periods up to 24 hours after the 8 hours of exposure to the intense light. The amount of nitrated proteins in the photoreceptor rod outer segments was semi–quantitatively determined by 1D–Western analysis using anti–nitrotyrosine antibody. The identities of the nitrated proteins in the photoreceptor rod outer segments were determined by 2D–Western analysis followed by liquid chromatography tandem mass spectrometry analyses for the immunoreactive protein spots. Results: Western analysis clearly showed that protein nitration increases in the photoreceptor rod outer segments in rats kept in the dark after intense light exposure. The nitrated proteins identified in the photoreceptor rod outer segments were glyceraldehyde–3–phosphate dehydrogenase, fructose bisphosphate aldolase C, glutamine synthetase 1, α–enolase, ATP synthase, triose phosphate isomerase 1, phosphoglycerate mutase, carbonic anhydrase 2, fructose bisphosphate aldolase A, and peptidylprolyl isomerase D. Conclusions:Glycolytic enzymes appear to be major nitration targets in the rod outer segment. Intense light results in increased rod outer segments protein nitration during the dark recovery period after exposure. Further studies to define the molecular effects of this modification in the functions of the proteins are in progress.

Keywords: radiation damage: light/UV • nitric oxide • retina: distal (photoreceptors, horizontal cells, bipolar cells)