The FASEB Journal, Vol 11, 154-163, Copyright © 1997 by The Federation of American Societies for Experimental Biology

RESEARCH COMMUNICATIONS

Impairment of excitatory amino acid transporter activity by oxidative stress conditions in retinal cells: effect of antioxidants

P Agostinho, CB Duarte and CR Oliveira
Center for Neurosciences of Coimbra, University of Coimbra, Portugal.

In the present study we analyzed how oxidative stress conditions induced by ascorbate/ Fe2+ affect the excitatory amino acid (EAA) transport systems in cultured chick retina cells. The uptake of D- [3H]aspartate, which is transported by the same carrier as glutamate, was determined in control cells and in cells subjected to ascorbate/Fe2+. The uptake of this EAA was Na+ dependent and was inhibited by about 40% under oxidative stress conditions. To clarify the molecular mechanisms involved in the inhibition of D-[3H]aspartate uptake by ascorbate/Fe2+, we investigated the effect of vitamin E (Vit E), melatonin, reduced glutathione (GSH), and dithiothreitol (DTT) on the uptake of D-[3H]aspartate and on the extent of lipid peroxidation in control and in peroxidized cells. Preincubation with Vit E (100 microM) abolished lipid peroxidation, but had no significant effect on the inhibition of D-[3H]aspartate uptake evoked by ascorbate/Fe2+. Melatonin was more effective in reducing the formation of TBARS and conjugated dienes than in preventing the D-[3H]aspartate uptake inhibition evoked by the oxidant pair. Conversely, GSH (4 mM) and DTT (4 mM) completely prevented the inhibition of D-[3H]aspartate uptake in cells subjected to oxidative stress, but were without effect on the extent of peroxidation. Free fatty acids, such as arachidonic acid, seem not to be involved in reducing the activity of the D-[3H]aspartate uptake system, whereas the reduction of the Na+ electrochemical gradient that occurs under oxidative stress was in part involved in the reduction of D-[3H]aspartate uptake by the cells. The inhibition of D- [3H]aspartate uptake by ascorbate/Fe2+ persisted for at least 1 h, but could be partially reverted by disulfide reducing agents. It is concluded that oxidative stress causes long-lasting modifications of the glutamate/D-[3H]aspartate transport system (or systems), such as oxidation of protein sulfhydryl (SH) groups, which can be recovered by some antioxidants.

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