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B.A. Mysona, Y. Dun, V. Ganapathy, S.B. Smith; GLAST and System Xc–: Transporter Response to Prolonged Hyperglycemia and Acute Oxidative Stress in Primary Mouse Müller Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2056.
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Oxidative stress is implicated in the pathogenesis of diabetic retinopathy; elevated glutamate (Glu) levels are also associated with this disease. The Na+–dependent Glu transporter, GLAST, clears neurotoxic Glu from the extracellular milieu. The Na+–independent, Cys–Glu exchanger, system xc–, transports cystine into cells for use, as cysteine, in glutathione synthesis. The present study asked whether long–term hyperglycemia alters the expression and/or function of these two transporters in Müller cells and whether short–term oxidative stress has similar effects.
Primary Müller cells were isolated from 7–10 day C57BL/6 mouse retinas. Cells were cultured 2, 4 or 8 days in media containing 5mM, 20mM or 35mM glucose. Transporter activities were determined by measuring the uptake of [3H]–Glu in the presence/absence of Na+. Protein levels of GLAST and system xc– (xCT and 4F2hc) were assayed by immunoblotting. In studies of oxidative stress, cells were treated 15 minutes with oxidizing agents DTNB (500µM) or xanthine/xanthine oxidase (500µM/50 mU/ml). Subsequently, cells were maintained in low serum media for 0 to 18 hours, after which the GLAST and system xc– activities were assessed.
GLAST and system xc– activities did not differ in Müller cells treated with 35mM glucose compared to cells grown in 5mM or 20mM glucose, however, the level of the xCT component of system xc– (detected by immunoblotting) was increased compared to ß–actin in cells exposed for 8 days to 35mM glucose. Exposure to oxidizing agents DTNB and X/XO caused an immediate significant decrease in GLAST and system xc– activities. Over several hours, system xc– activity increased significantly and by 18 h returned to control values.
Hyperglycemia alone does not affect GLAST or xc– activity in vitro, but does increase xCT protein levels. Oxidative stress has immediate effects on the activities of both transporters in vitro, eventually leading to increased activity by xc–. The alterations of glutamate transporters in diabetic retinopathy observed in in vivo models are likely due to numerous factors, such as oxidative stress, in addition to hyperglycemia.
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