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X. Zhan, Y. Du, J. S. Crabb, T. S. Kern, J. W. Crabb; Identification of Nitrated Proteins in Diabetic Rat Retina. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4962.
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Diabetic retinopathy is inhibited and retinal protein nitration is reduced in animals treated with aminoguanidine. We sought the identity of retinal proteins that are nitrated in diabetes as an approach to better understanding the pathogenic mechanisms of this retinopathy.
In vivo studies utilized retinas collected from STZ-diabetic rats (2 month duration), and in vitro studies used a transformed rat Muller cell line (rMC-1) incubated in normal (5mM) and high (25 mM) glucose. Western analysis was used to compare levels of nitrotyrosine. For nitrated protein identification, retinas or cells were homogenized and nitrated proteins immunoprecipitated using anti-nitrotyrosine antibody. Immunoprecipitation products were fractionated by SDS-PAGE, gel bands excised, digested in situ with trypsin and analyzed by QTOF LC MS/MS. Protein identification utilized the ProteinLynx Global Server and Mascot search engines and the Swiss-Protein and NCBI protein databases. Tyrosine-nitration sites were determined by manual examination of MS/MS spectra.
Western analysis showed that nitrotyrosine immunoreactivity was greater in retinas from diabetic rats than from nondiabetic rats, and greater in rMC-1 cells incubated in 25 mM glucose than in rMC-1 cells incubated in 5 mM glucose. To date, tyrosine nitration sites in diabetic retina have been identified in insulin-responsive glucose transporter type 4, inositol 1,4,5-trisphosphate receptor type 3, BTB/POZ domain-containing protein 5, and serine-protein kinase ATM. In Müller cells incubated in 25 mM glucose, nitrotyrosine has been identified in sensory sodium channel 2, alpha. Additional analyses are in progress.
Hyperglycemia appears to increase tyrosine nitration in retina and Müller cells. Currently identified nitrotyrosine-containing proteins from diabetic rat retina function in glucose metabolism and signal transduction pathways. The present technology offers promise for insights into the mechanisms of diabetic retinopathy.
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