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A.R. Whitetree, L. Carter-Dawson; Changes in Protein Nitration in Rats with Increased Extracellular Glutamate . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3147.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To investigate the hypothesis that increased extracellular glutamate increases protein nitration in retinal neurons. Methods: Right eyes of male Sprague-Dawley rats were injected intraocularly with 1.0 or 1.5 mM dihydrokainic acid (DHK; 2.0 µl total volume) to block GLT-1 glutamate transporters. Left eyes were injected with an equal volume of vehicle. Under deep anesthesia, eyes were enucleated at 7 or 10 days after injection, as immunohistochemistry suggested an increase in nitrotyrosine immunoreactivity at these times. Retinas were removed on ice and two to three retinas were pooled in lysis buffer and sonicated. After centrifugation, 500 µl of protein from experimental and control tissue were immuno-precipitated on nitrotyrosine-conjugated agarose beads (Upstate) and separated by SDS-PAGE. Equal aliquots of total protein were also loaded onto the gels. Gels were stained with Coomassie Blue to visualize all protein or transferred to nitrocelluose membranes and immunolabeled with antibodies to proteins of interest.. Results: Coomassie Blue staining of 7-day rats showed an increase in the amount of nitrated protein in the DHK-injected samples. Nitration of proteins with molecular weights similar to glutamate transporters were increased in retinas with increased extracellular glutamate, however, labeling for GLAST and GLT-1 showed no difference in amount of nitrated protein. In blots with total protein (not immunoprecipitated) from the same rats, there were differences in the amounts of GLAST and GLT-1 protein. Conclusions: Increasing extracellular glutamate by blocking the GLT-1 transporter increases protein nitration in rat retinas. GLAST and GLT-1 expression changed with increase in extracellular glutamate, but the amount of these proteins that were nitrated did not change.
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