Molecular Vision 2006; 12:1543-1551 <http://www.molvis.org/molvis/v12/a177/> Received 31 January 2006 | Accepted 2 November 2006 | Published 6 December 2006

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

Vikram Palamalai,¹ Ruth M. Darrow,² Daniel T. Organisciak,² Masaru Miyagi¹
 
 

Department of ¹Biochemistry and Molecular Biology, School of Medicine and Health Sciences, University of North Dakota, Grand Forks, ND; the ²Petticrew Research Laboratory, Department of Biochemistry and Molecular Biology, Wright State University, Dayton, OH

Correspondence to: Masaru Miyagi, Ph.D., Case Center for Proteomics, Departments of Pharmacology and Ophthalmology, School of Medicine, Case Western Reserve University, 10900 Euclid Avenue, BRB 928, Cleveland, OH, 44106; Phone: (216) 368-5917; FAX: (216) 368-6846; email: masaru.miyagi@case.edu

Abstract

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.

Introduction

Acute light-induced photoreceptor cell degeneration in experimental animals has been studied for 40 years as a model for visual cell loss arising from retinal degenerative diseases [1]. The rationale for these studies is based largely on morphological similarities between the end stages of human disease and experimental retinal degenerations [2]. Although the pathogenesis of many human ocular disorders is unknown, retinal degenerations arising from genetic inheritance or in experimental animal models, is characterized by apoptotic photoreceptor cell death [3].

Retinal light damage is initiated within ROS by rhodopsin bleaching, because the action spectrum of light damage is very similar to the rhodopsin absorption spectrum [1,4]. Rhodopsin knockout mice and animals with impaired visual cycle proteins such as cellular retinaldehyde-binding protein [5] and RPE65 [6] are protected from light damage. It also has been suggested that intense light induces oxidative stress in the retina, because natural antioxidants such as ascorbate [7] and synthetic antioxidants such as dimethylthiourea (DMTU) [8,9], WR-77913 [10], and phenyl-N-tert-butylnitrone [11] prevent retinal light damage and photoreceptor cell death. However, the molecular events that link oxidative stress with the initiation of the apoptotic cascade are poorly understood.

Oxidative stress can cause several modifications in the retina. Light is known to modulate retinal protein nitration [12]. Nitration has also been demonstrated in photoreceptor mitochondria in experimental uveitis [13]. Light exposure induces carboxyethylpyrrole and nitrotyrosine modifications in dark adapted mice [14]. Protein modifications by 4-hydroxynonenal and 4-hydroxyhexenal have also been found in light exposed rat retinas [15]. Increased retinal protein carbonyl immunoreactivity suggestive of oxidative damage has been reported to occur in a chronic pressure induced model of glaucoma [16]. Thus, nitration is one of several oxidative modifications that are observed in the retina under stressful conditions like exposure to intense light.

Nitric oxide (NO), a gaseous free radical, is synthesized from L-arginine by a family of nitric oxide synthase (NOS) isozymes; neuronal (nNOS), endothelial (eNOS), and inducible-NOS (iNOS) [17]. nNOS and eNOS are constitutive, Ca⁺⁺/calmodulin-dependent enzymes and are tightly controlled by mechanisms regulating intracellular Ca⁺⁺ levels [18]. iNOS is not present constitutively, but is induced in response to allergic or inflammatory challenges, leading to the production of large amounts of potentially cytotoxic nitric oxide [18]. A major physiological effect of NO production involves the activation of soluble guanylyl cyclase, which catalyzes the production of 3', 5'-cyclic guanosine monophosphate (cGMP). The major role of cGMP in ROS is to regulate cGMP-gated cation channels [19]. Light triggers a decrease in cytoplasmic cGMP by activating a ROS phosphodiesterase (PDE6), leading to closure of cGMP-gated cation channels and hyperpolarization of the visual cell membrane potential. Thus, NO may serve to restore cGMP levels during or after light stimulation of the phototransduction cascade in photoreceptors.

NO is the biological precursor molecule for protein nitrating agents such as peroxynitrite. Recent studies suggest that NO also mediates light-induced photoreceptor degeneration. Treating rats with NOS inhibitors partially protects rats against light-induced photoreceptor loss, as indicated by measurement of outer nuclear layer thickness in retina [20]. Inhibition of nNOS also prevents light-induced photoreceptor cell death in mice [21]. These results suggest that production of NO might be involved in light-induced photoreceptor cell death by an unknown mechanism.

It is well known that NO can cause chemical modifications in various biomolecules, including proteins, DNA, and lipids [22]. Nitration of tyrosine residues is one of the chemical modifications occurring in proteins when cells experience oxidative stress [23]. Our previous experiments suggested that a considerable amount of protein nitration occurs in the retina and ROS of dark-reared rats, and that the level of protein nitration is reduced by light treatment [12]. However, we had not examined whether light-induced changes in protein nitration also occur within the ROS of rats reared in a dim cyclic light environment, and whether the level of protein nitration changes in the dark following light exposure.

In this study, we determined ROS nitration levels in cyclic light-reared rats under normal physiological conditions and after intense light exposure. We found that ROS nitration changes after intense light exposure and following darkness, and that the antioxidant DMTU reduces these changes. We also identified nitrated target proteins within ROS, and determined changes in the levels of three isoforms of NOS during and after light exposure.

Methods

Animal maintenance and exposure to intense light

Weanling male Sprague-Dawley rats (Harlan Inc., Indianapolis, IN) were reared in a dim (20-40 lux) cyclic light environment for 12 h per day; lights were on at 8 AM and off at 8 PM. The rats were fed a standard rat chow (Teklad, Madison, WI) and given water ad libitum. 500 mg/kg of DMTU (Sigma-Aldrich, St. Louis, MO) was administered on day 59 (24 h before light exposure) by intraperitoneal injection (IP), and again 10 min before light exposure [8]. Untreated control animals received saline IP injections. Treated and untreated rats were dark-adapted for 16 h and then put into cylindrical green plexiglas (number 2092) chambers (Dayton Plastic, Dayton, OH), that transmit 490-580 nm light. They were then exposed to light (1,200-1,400 lux) starting at 1 AM. Eight h of light beginning at 1 AM resulted in approximately 50% visual cell loss as determined by rhodopsin and retinal DNA levels two weeks later [24]. After 8 h of light exposure, a subset of animals was sacrificed immediately in a chamber with a CO₂-saturated atmosphere under dim red illumination, while the remaining animals were placed in darkness for 24 h before being sacrificed. Six groups of four animals (0 h light with or lacking DMTU, 8 h light with or lacking DMTU, and 8 h light plus 24 h dark with or lacking DMTU) were used in these experiments. Three independent experiments were performed. To assess the expression levels of NOS, separate groups of P60 rats were exposed to green light, as already described, for different time periods (0, 0.5, 2, 4, and 8 h) starting at 1 AM. Three additional animal groups were maintained in darkness for different time periods (4, 8, or 24 h) following 8 h of light treatment. Four animals were used as replicates in each group. All procedures involving rats followed the protocols outlined in the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.

Preparation of rod outer segments

Both retinas from each animal were excised under dim red light within 2 min of death and rinsed in phosphate-buffered saline (PBS). ROS were prepared under dim red illumination from these retinas by sucrose density ultracentrifugation [25] and stored at -80 °C until use. All solvents used for ROS preparations contained protease inhibitors (1 mM EDTA, 0.2 mM PMSF, 0.7 μg/μl leupeptin, and 0.5 μg/μl pepstatin A) and 100 μM diethylenetriamine pentaacetic acid (DTPA) to inhibit protein degradation and oxidation, respectively. Only the band I fractions, which contain the purest ROS [25], were used in this study.

Gel electrophoresis and protein transfer to polyvinyl difluoride membrane

ROS pellets were prepared by mixing with an equal volume of PBS containing 20% sucrose followed by centrifugation at 10,000x g for 5 min using a refrigerated tabletop centrifuge (Kendro Laboratory Products, Newtown, CT) at 4 °C. For one-dimensional gel electrophoresis, proteins were extracted from the ROS pellet with a pH 6.8 buffer consisting of 62.5 mM Tris-HCl, 2% (w/v) sodium dodecyl sulfate (SDS) and 2% (v/v) protease inhibitor cocktail (Sigma-Aldrich, catalog number P2714). All procedures for the extraction of ROS proteins were performed under dim red illumination. Protein in the extract was estimated using Bio-Rad DC Protein assay kit (Bio-Rad, Hercules, CA) as per the manufacturer's instructions. The protein extract (20 μg/sample) was then subjected to electrophoresis using a 4% stacking and 10% resolving SDS-polyacrylamide gel [26]. After electrophoresis the gel was equilibrated in transfer buffer consisting of 25 mM Tris, 192 mM glycine, and 20% methanol for 15 min. Proteins were partially transferred to 0.45 μm polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA) using a constant current of 1.28 mA/cm² gel for 25 min in a Bio-Rad Trans-Blot SD semidry electrophoretic transfer cell [27]. The resulting membranes were used for western analyses. The remaining SDS-gels were stained with EZ Blue staining reagent (Sigma-Aldrich).

For two-dimensional gel electrophoresis, ROS proteins were extracted with an isoelectric focusing (IEF) solution consisting of 7 M Urea, 2 M thiourea, 4% CHAPS, 1% (v/v) ampholytes (pH 3-10), 0.5% (v/v) Triton-X 100, 2 mM tributylphosphine, 0.05 mg/ml DNase, 0.05 mg/ml RNase, 0.25 mM MgCl₂, and 2% (v/v) protease inhibitor cocktail (Sigma-Aldrich, catalog number P2714). Proteins in the extracts were estimated by a modified Bradford method [28]. ROS proteins (70 μg) were mixed with 10 ng of nitrated bovine serum albumin (BSA; internal standard) and rehydrated in a 11-cm nonlinear immobilized pH gradient (pH 3-10) strip (Bio-Rad) overnight at 50 V, followed by isoelectric focusing using a programmed voltage gradient. After the first dimension isoelectric focusing, the strips were subjected to reduction with 2 mM DTT in pH 8.0 equilibration buffer consisting of 6 M urea, 50 mM Tris-HCl, 2% (w/v) SDS and 30% (v/v) glycerol for 15 min. This was followed by S-alkylation with 5 mM iodoacetamide in the same buffer for 15 min. The strips were placed on discontinuous 4% stacking and 10% resolving SDS-polyacrylamide gels and proteins subjected to electrophoresis in a Bio-Rad Criterion Dodeca Cell. Following electrophoresis, the proteins were electroblotted onto PVDF membranes as described above, and used for western analyses. Proteins remaining in the gels were stained with SyproRuby (Bio-Rad) as per the manufacturer's instructions. The images of the stained gels were taken by Bio-Rad Versa Doc Model 3000 imaging system. All images were analyzed with Bio-Rad PD Quest software, version 7.1.1.

Retinal proteins-retinal tissues were homogenized on ice using a pestle and mortar (Kontes Glass Company, Vineland, NJ) in a pH 6.8 buffer consisting of 62.5 mM Tris-HCl, 2% (w/v) SDS, 25% glycerol (v/v), and 2% (v/v) protease inhibitor cocktail (Sigma-Aldrich, catalog number P2714). The extracted proteins were subjected to one-dimensional SDS-PAGE electrophoresis, after which the gels were equilibrated in transfer buffer consisting of 25 mM Tris, 192 mM glycine, 20% methanol and 0.1% SDS (w/v) for 15 min. The proteins were electrotransferred to PVDF membranes at a constant voltage of 70 V for 30 min using a Bio-Rad Mini Trans-Blot electrophoretic transfer cell, and used for subsequent western analyses.

Western analysis

Nitrotyrosine in ROS-PVDF membranes containing ROS proteins were extensively washed with deionized water and incubated for 1 h in blocking buffer (pH 7.5) composed of 20 mM Tris, 150 mM NaCl, 0.2% (v/v) Tween 20, and 1% (w/v) bovine serum albumin (BSA). Blots were probed for 2 h at 4 °C with a monoclonal antibody against nitrotyrosine (1:1,000 dilution, Upstate Cell Signaling, Lake Placid, NY) in blocking buffer. The membranes were then washed three times in buffer (pH 7.5) consisting of 20 mM Tris, 150 mM NaCl, and 0.2% (v/v) Tween 20 before being incubated with a goat anti-mouse IgG, horseradish peroxidase-conjugated secondary antibody (1:10,000 dilution; Pierce Biotechnology, Rockford, IL) in blocking buffer for 1 h at room temperature. Membranes were washed three times as before, and developed using a chemiluminescence detection kit (Pierce Biotechnology, Rockford, IL, number 34076). Immunoreactive proteins were visualized by exposing blots to autoradiography film (CL-XPosure film, Pierce Biotechnology, Rockford, IL, number 34091) and protein bands quantified by densitometry. The optical density of an entire lane was obtained using Quantity One software (Bio-Rad). If obvious speckles were present in the lane, the intensities of these speckles were separately obtained and subtracted from the total intensity. Background was subtracted from a reference region on the individual blots. Three blots each from three independent experiments were used for quantification. The mean optical density in the DMTU untreated control lane was considered to be 100%.

Reduction of nitrotyrosine to aminotyrosine on PVDF membranes was achieved by treating with 10 mM sodium dithionite in 50 mM pyridine-acetate buffer, pH 5.0 for 1 h at room temperature following previously reported guidelines [12]. After washing with deionized water, we subjected the dithionite-treated membranes to western analysis using the previously described protocol.

NOS in retina-PVDF membranes electroblotted with retinal proteins were probed with anti-nNOS, anti-iNOS, or anti-eNOS (Upstate Cell Signaling) antibody at 1:1,000 dilution, and then subjected to western analyses, except this time chemiluminesence was detected by Lumiimager (Boehringer Mannheim GmbH, Mannheim, Germany). The immunoreactive protein bands were quantified by densitometry with the help of Molecular Analyst software, version 1.4 (Bio-Rad). The mean optical density in each NOS band was determined, and the fold change for each band was normalized to the band of unexposed NOS optical density.

Identification of nitrated proteins

Two-dimensional western blots were compared with the SyproRuby stained parental gels to identify immunoreactive protein spots. Immunoreactive proteins in the parental gels were excised and subjected to in-gel tryptic digestion. Briefly, the excised spots were washed with 25 mM ammonium bicarbonate for 1 h, twice with 25 mM ammonium bicarbonate containing 50% acetonitrile for 1 h, and twice with acetonitrile for 10 min. An adequate amount (just enough to cover the spots) of trypsin in 25 mM ammonium bicarbonate (0.1 μg/15 μl) was added to the gel spots, and proteins digested overnight at 37 °C. After trypsin treatment, the digest was collected and the gel washed twice with a solution containing 0.1% trifluoroacetic acid (TFA)/60% acetonitrile (v/v) for 30 min, and once with acetonitrile for 10 min. The digest and wash solutions were combined and solvent evaporated in a speed-vac concentrator. The dried digests were then redissolved in 0.1% TFA/5% acetonitrile (v/v) and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) using an UltiMate nano high performance liquid chromatography (HPLC) system (Dionex, San Francisco, CA) and a QStar quadrupole/time-of-flight mass spectrometer (Applied Biosystem-MDS Sciex, Foster City, CA).

Protein digests were injected onto a reverse-phase C4 trapping column (300 μm inner diameter, 1 mm length; Dionex, Sunnyvale, CA) equilibrated with 0.1% formic acid/2% acetonitrile (v/v) and washed for 5 min with the equilibration solvent at a flow rate of 10 μl/min. After washing, the trapping column was switched in-line with the reverse-phase analytical column and underwent chromatography on a 0.075x50 mm column (New Objective Inc., Woburn, MA) packed in-house with Jupiter C18 media (10 μm, 300 Å, Phenomenex, Torrance, CA). A linear gradient of acetonitrile from 2% to 42% in water in the presence of 0.1% formic acid over a period of 30 min was used, at a flow rate of 200 nl/min. The column effluent was passed directly into the nanoelectrospray ion source. The total ion current was obtained in the mass range of m/z 300-2000 at 2,050 V and 65 V of electrospray and orifice voltage, respectively, in the positive ion mode. AnalystQS software (version 1.0, Applied Biosystem-MDS Sciex) was used for instrument control, data acquisition, and processing. The mass spectrometer was operated in the data-dependent MS to MS/MS switching mode, with the three most intense ions in each MS scan subjected to MS/MS analysis.

Proteins were identified by comparing all of the experimental product ion spectra of the peptides to the NCBI database using the Mascot database search software (version 2.0.0, Matrix Science, London, UK). Only mammalian proteins (282,498 sequences) were searched. S-carbamidomethylation of cysteine was set as a fixed modification. Oxidation of methionine (methionine sulfoxide) was set as variable modification in the database search. Mass tolerances for protein identification on precursor and product ions were both set to 0.2 Da. Strict trypsin specificity was applied, while allowing for one missed cleavage. Only peptides with a minimum score of 23 were considered to be significant. The score for a product ion spectrum match is based on the absolute probability (P) that the observed match between the experimental data and the database sequence is a random event. The reported score is -10Log(P).

Statistical analysis

Statistical analyses were performed using a one-way repeated measure analysis of variance (RM ANOVA) test for nitration samples, or a one way analysis of variance (ANOVA) for NOS samples, with pair-wise comparisons using the Holm-Sidak test on SigmaStat 3.0 (SPSS Inc, Chicago,IL) software. A p value of less than 0.05 was considered statistically significant (p<0.05).

Results

Western analyses on rod outer segment proteins for nitrotyrosine

Retinal protein extracts from animals unexposed to light, immediately after, or animals kept in the dark 24 h after light exposure were analyzed by western analyses to quantify the levels of ROS protein nitration. Figure 1A, shows that the relative amount of protein loaded on the gels was equal for all samples. A representative western blot is shown in Figure 1B. As can be seen in the figure, several nitrotyrosine immunoreactive protein bands from DMTU treated (Figure 1B, lane 1) and untreated rats (Figure 1B, lane 2) were present before light exposure. The intensities of the immunoreactive bands in DMTU-treated rats were reduced immediately after light exposure (Figure 1B, lane 3); however, the intensities of the immunoreactive bands in the untreated rats appeared to be unchanged (Figure 1B, lane 4). The intensities of the immunoreactive bands in both DMTU treated (Figure 1B-lane 5) and untreated rats (Figure 1B, lane 6) were significantly increased 24 h after light exposure. Based on the migrations of the immunoreactive bands, major nitration target proteins appear to be same in all ROS samples, with differing degrees of nitration for individual proteins.

Figure 1C shows the results of density analyses performed on nine western blots obtained from nine different gels from three independent experiments (triplicate analyses for each experiment). The relative optical density for each lane is plotted. As can be seen, the total optical density of the immunoreactive bands in DMTU-treated ROS decreased from 77% to 53% immediately after light exposure, and increased significantly from 53% to 143% (p<0.05) 24 h after light exposure. In the untreated rats, total optical density after light exposure was not significantly altered, but it increased significantly from 122% to 203% (p<0.05) 24 h after light exposure. In comparison to the untreated samples, DMTU reduced the levels of nitration by 23% prior to light exposure, by 57% immediately after light exposure, and by 30% 24 h after light exposure. In a separate experiment, three blots were treated with dithionite to reduce nitrotyrosine in proteins to aminotyrosine and then subjected to western analysis. The dithionite-treated blots did not show immunoreactivities seen in Figure 1B (data not shown), suggesting that there were no detectable false positive bands on the blot [12].

Identification of nitrated proteins in rod outer segments

ROS proteins from rats 24 h after light exposure were separated by 2D gel electrophoresis, and nitrotyrosine immunoreactive protein spots determined by western analysis. A representative SyproRuby-stained 2D gel and corresponding western blot of ROS proteins from unexposed rats (Figure 2A,C) and rats 24 h after light exposure (Figure 2B,D) are shown. A total of 11 immunoreactive spots were detected on the blot of ROS proteins from rats 24 h after light exposure (Figure 2D), while only three spots were detected on the blot from rats unexposed to light (Figure 2C), confirming the results obtained by 1D-western analysis on ROS proteins.

The immunoreactive proteins 24 h after light exposure were excised from the SyproRuby stained gel (Figure 2B) and digested by trypsin. The resulting peptides were analyzed by LC-MS/MS and the data subjected to protein database analyses for protein identification. As can be seen in Table 1, 10 out of 11 immunoreactive spots were glycolytic enzymes. These were glyceraldehyde-3-phosphate dehydrogenase, fructose bisphosphate aldolase A, fructose bisphosphate aldolase C, α-enolase, and triose phosphate isomerase, suggesting that these glycolytic enzymes are major nitration target proteins in ROS. Spot number 9 did not exhibit a significant match to any protein in the database.

Western analyses of retina for nitric oxide synthase

NOS produces NO, which is the precursor molecule of nitrating agents (e.g., peroxynitrite) that cause protein nitration. Therefore, we examined light-dependent changes in the expression of the three NOS isoforms in retina to identify the isoform(s) that might lead to ROS protein nitration. Retinal proteins from rats unexposed to light, exposed to light for 0.5, 2, 4, or 8 h, or exposed to light for 8 h and kept in the dark for 4, 8, or 24 h were subjected to western analysis. Representative western blots and the corresponding relative density of nNOS, iNOS, and eNOS are presented in Figure 3. The quantitative analysis of nNOS showed that the amount of immunoreactivity for anti-nNOS antibody increased 2.0 fold after 8 h of light exposure (p<0.05) and then decreased to only a 1.3 fold elevation 24 h later (p<0.05). The amount of iNOS increased significantly (2.4 fold) following 8 h of light exposure (p<0.05) and remained elevated (2.6 fold) for another 24 h. eNOS showed only a marginal increase (1.15 fold) after 8 h of light exposure, which remained at that level for another 24 h.

Discussion

We previously found that a considerable amount of protein nitration occurs in the retina and ROS in dark-reared rats before light exposure [12]. In this study we observed the same phenomenon in the dark adapted ROS of cyclic light reared rats (Figure 1). This suggests that protein nitration occurs under normal physiological conditions in rats reared in a normal 12 h light:dark environment. Such basal protein nitration occurring under normal physiological conditions has been detected in numerous other tissues [29]. These data are consistent with the emerging perspective that low levels of tyrosine nitration may be a physiological regulator of a signaling pathway [30,31]. However, the physiological relevance of basal ROS nitration remains to be elucidated.

Our immunoblotting results showed that the level of protein nitration in ROS is maintained at a basal level in rats not treated with the antioxidant, DMTU, but is decreased in DMTU-treated rats during 8 h of light exposure. However, protein nitration increased significantly during the dark recovery period after light exposure in both DMTU-treated and untreated rats (Figure 1). Thus, it appears that ROS nitration is reduced during light and is increased during darkness. These results imply that there is a cellular mechanism to reduce the level of protein nitration, which is modulated by light. This could be a selective degradation of nitrated proteins in the proteasome [32], or enzymatic denitration of nitrated proteins [33]. Such dynamic changes in protein nitration within a relatively short period of time have been observed in isolated mitochondria that were subjected to hypoxia-anoxia and reoxygenation [34,35]. Further study will be needed to elucidate the fate of nitrated ROS proteins during light exposure. Increasing ROS protein nitration during the dark recovery period has also been observed in the retina in several animal models, including experimental autoimmune uveitis [13], ischemic proliferative retinopathy [36], and experimental diabetes [37], all of which are characterized by apoptotic photoreceptor cell death. These findings suggest that protein nitration may contribute to the biochemical sequelae leading to photoreceptor cell death.

The synthetic antioxidant, DMTU, has been demonstrated to protect photoreceptor cells from damaging light [8,9,38]. Therefore, if protein nitration in ROS is involved in light-induced photoreceptor cell death, ROS nitration should be reduced by DMTU. As expected, our western results showed that DMTU effectively reduced the levels of ROS nitration 23% prior to light exposure, 57% immediately after light exposure, and 30% 24 h later (Figure 1). This suggests that ROS nitration is involved in light-induced photoreceptor cell death, but the mechanism remains unclear. The protective effect of DMTU against the formation of ROS nitration also suggests that this antioxidant can effectively scavenge reactive oxygen and nitrogen species that can form nitrating agents such as peroxynitrite. The reduction of ROS nitration by DMTU prior to light exposure suggests that nitrating agents are constantly generated during the dark and that DMTU lowers the endogenous basal level of nitration. DMTU appears to be less effective after light exposure, suggesting that its effective concentration has been reduced by intense light. This may occur by light-induced breakdown of DMTU or by leakage from ROS into the extracellular space of the retina. Our results also showed that the levels of protein nitration were unaffected during light exposure even in the presence of increased expression levels of iNOS and nNOS. 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.

Our study is the first to show that major nitration target proteins in the ROS are glycolytic enzymes. It is possible that the actual nitration target proteins are low abundance proteins that comigrated with the abundant proteins identified in the 2D-PAGE gel. It will require determination of the sites of nitration in these proteins to exclude this possibility. However, such identification will require far greater amounts of sample than we can currently obtain. Although our study does not clarify specific effects of nitration on the functions of these proteins, two of the glycolytic enzymes identified, enolase [39], and aldolase A [40], have been shown to lose catalytic activity upon nitration in vitro. Glycolysis provides a significant amount of energy required by the ROS [41]. Therefore, if glycolysis is inhibited in ROS it could lead to ATP depletion. Since ATP is utilized for protein phosphorylation, such as the light-induced phosphorylation of rhodopsin by rhodopsin kinase and for the synthesis of GTP from GDP by nucleotide diphosphokinases [41], ATP depletion might affect phototransduction. In addition, the increased amount of protein nitration and iNOS expression during the dark recovery period indicates that a large quantity of NO is generated within photoreceptor cells. NO has been suggested to modulate mitochondrial energy production by inhibiting cytochrome oxidase [42]. Therefore, NO might also affect cellular respiration and cause energy depletion within the inner segment of photoreceptor cells. The extent of energy depletion caused by protein nitration on glycolytic enzymes in ROS or on cytochrome oxidase in photoreceptor inner segments remains to be determined.

nNOS has been shown to increase in bright light-exposed (6000 lux) mouse retinas [21] and rat retinas exposed to constant light [43]. In this study, the expression of nNOS was seen to increase upon light exposure, but this increased expression is reduced during the dark recovery period following light exposure. This suggests that nNOS plays a role mainly in the acute pathogenesis of light induced retinal damage. Inhibition of nNOS is known to prevent light-induced retinal degeneration [21].

iNOS expression is known to be enhanced during inflammatory processes [44]. The elevated iNOS levels found during the dark recovery period suggests that the pathological process of retinal light damage proceeds in the dark. This is consistent with the fact that light exposure results in apoptotic photoreceptor cell death, as evidenced by the increased number of single-stranded breaks and nucleosomal DNA laddering [45] in animals kept in darkness following light exposure. The elevated amount of iNOS during and after 8 h of light exposure (Figure 3) also suggests that iNOS might be the isoform responsible for increased ROS nitration during the dark recovery period. We are currently identifying the sites of nitration in the glycolytic enzymes identified in this study to determine the effects of this modification on these proteins and their possible involvement in light-induced photoreceptor cell death.

In summary, we have shown that exposure to intense light causes nitration of proteins, mainly glycolytic enzymes, in the ROS of cyclic light-reared rats. This nitration may be mediated by iNOS. These results suggest that nitration is one of the key mediators responsible for photoreceptor cell death upon exposure to intense light.

Acknowledgements

The authors thank Ryan T. Carruth for his technical assistance on the mass spectrometry analysis. This work was supported in part by the following NIH grants: EY014020 (M.M.), EY01959 (D.T.O.), NIH P20 RR016741, and NIH P20RR017699.

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