Effect of oxidative stress on the release of [ 3H]GABA in cultured chick retina cells

Abstract

The effect of ascorbate (1.5 mM)/Fe 2+ (7.5 μM)-induced oxidative stress on the release of pre-accumulated [ 3H]γ-aminobutyric acid ([ 3H]GABA) from cultured chick retina cells was studied. Depolarization of control cells with 50 mM K + increased the release of [ 3H]GABA by 1.01 ± 0.16% and 2.5 ± 0.3% of the total, in the absence and in the presence of Ca 2+, respectively. Lipid peroxidation increased the release of [ 3H]GABA to 2.07 ± 0.31% and 3.6 ± 0.39% of the total, in Ca 2+-free or in Ca 2+-containing media, respectively. The inhibitor of the GABA carrier, 1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride (NNC-711) blocked almost completely the release of [ 3H]GABA due to K +-depolarization in the absence of Ca 2+, but only 65% of the release occurring in the presence of Ca 2+ in control and peroxidized cells. Under oxidative stress retina cells release more [ 3H]GABA than control cells, being the Ca 2+-independent mechanism, mediated by the reversal of the Na +/GABA carrier, the most affected. MK-801 (1 μM), a non-competitive antagonist of the NMDA receptor-channel complex, blocked by 80% the release of [ 3H]GABA in peroxidized cells, whereas in control cells the inhibitory effect was of 40%. The non-selective blocker of the non-NMDA glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), inhibited the release of [ 3H]GABA by 30% and 70% in control and peroxidized cells, respectively. Glycine (5 μM) stimulated [ 3H]GABA release evoked by 50 mM K +-depolarization in control but not in peroxidized cells. The release of d-[ 3H]aspartate (a non-metabolized analog of l-glutamate) evoked by 50 mM K +, in the absence of Ca 2+, was significantly higher in peroxidized cells (6.76 ± 0.64% of the total) than in control cells (3.79 ± 0.27% of the total). The results suggest that oxidative stress induced by ascorbate/Fe 2+ causes an excessive release of endogenous excitatory amino acids upon K +-depolarization. The glutamate released may activate NMDA and non-NMDA receptors, raising the intracellular Na + concentration and consequently stimulating the release of [ 3H]GABA by reversal of the Na +/GABA carrier.