IMPAIRMENT OF EXCITATORY AMINO-ACID TRANSPORTER ACTIVITY BY OXIDATIVESTRESS CONDITIONS IN RETINAL CELLS - EFFECT OF ANTIOXIDANTS

In the present study we analyzed how oxidative stress conditions induc ed by ascorbate/Fe2+ affect the excitatory amino acid (EAA) transport systems in cultured chick retina cells, The uptake of D-[H-3]aspartate , which is transported by the same carrier as glutamate, was determine d in control cells and in cells subjected to ascorbate/Fe2+. The uptak e of this EAA was Na+ dependent and was inhibited by about 40% under o xidative stress conditions. To clarify the molecular mechanisms involv ed in the inhibition of D-[H-3]aspartate uptake by ascorbate/Fe2+, we investigated the effect of vitamin E (Vit E), melatonin, reduced gluta thione (GSH), and dithiothreitol (DTT) on the uptake of D-[H-3]asparta te and on the extent of lipid peroxidation in control and in peroxidiz ed cells, Preincubation with Vit E (100 mu M) abolished lipid peroxida tion, but had no significant effect on the inhibition of D-[H-3]aspart ate uptake evoked by ascorbate/Fe2+ Melatonin was more effective in re ducing the formation of TEARS and conjugated dienes than in preventing the D-[H-3]aspartate uptake inhibition evoked by the oxidant pair, Co nversely, GSH (4 mM) and DTT (41 mM) completely prevented the inhibiti on of D-[H-3]aspartate uptake in cells subjected to oxidative stress, but were without effect on tile extent of peroxidation., Free fatty ac ids, such as arachidonic acid, seem not to be involved ill reducing th e activity of the D-[H-3]aspartate uptake system, whereas the reductio n of the Na+ electrochemical gradient that occurs under oxidative stre ss was ill part involved in the reduction of D-[H-3]aspartate uptake b y the cells, The inhibition of D-[H-3]aspartate uptake by ascorbate/Fe 2+ persisted for at least 1 h, but could be partially reverted by disu lfide reducing agents, It is concluded that oxidative stress causes lo ng-lasting modifications of the glutamate/D-[H-3]aspartate transport s ystem (or systems), such as oxidation of protein sulfhydryl (SR) group s, which cll he recovered by some antioxidants.