Cystine/glutamate antiporter expression in retinal mu¨ller glial cells: Implications for dl-alpha-aminoadipate toxicity

Abstract

A cytotoxicity of glutamate or related amino acids (10 mM) mediated by a cystine/glutamate antiporter (system Xc¯) has recently been demonstrated in N18 neuroblastoma-rat retina hybrid (N18RE105) cells and C6 glioma cells. The antiporter usually transports glutamate outside and cystine inside, thereby maintaining cellular concentrations of glutathione. High concentrations of glutamate inhibit cystine uptake and lead to depletion of cellular levels of glutathione. Among related amino acids, dl-alpha-aminoadipic acid ( dl-α-AAA), which is well known as a selective gliotoxin in the retina, is also toxic to these cells. However, this does not explain why dl-α-AAA acts gliospecifically on the retina. To answer this question we first examined the effects of dl-α-AAA on the [ 35S]cystine uptake with parental N18 neuroblastoma cells and rat retina of the hybrid cells. dl-α-AAA showed a competitive inhibition of [ 35S]cystine uptake in the rat retina but not in the N18 cells. Such a competitive inhibition of cystine uptake by dl-α-AAA could also be seen in the carp retina. The cystine uptake with carp retina was mainly Na +-independent and Cl −-dependent as already described as a characteristic ion dependency of the Xc¯antiporter. We next examined the effects of exogenous cystine on the glutamate release from the retina. Cystine (1 mM) actually induced a glutamate release approximately twice that of the control. Furthermore, the glutamate release induced by cystine was also Na +-independent and Cl −-dependent, and was blocked by dl-α-AAA. An autoradiogram of [ 35S]cystine uptake in the carp retina showed typical radial glial Mu¨ller cells. A large incorporation of [ 35S]cystine into retinal glutathione fraction was detected by a high pressure liquid chromatography method during a 1–4-h incubation. A significant or large decrease of retinal levels of glutathione was observed one day after an intravitreal injection of 8 μmol dl-α-AAA or l-α-AAA, respectively. Buthionine sulfoximine (2.5 μmol), a specific inhibitor of glutathione synthesis, induced a large decrease of retinal levels of glutathione and a loss of electroretinographic b-wave 20–30 h after treatment. Taken together, our present data with rat and carp retinas strongly indicate that the expression of cystine/glutamate antiporter is enriched in the retina, particularly in the glial Mu¨ller cells which have a rapid turnover pool for glutathione. The gliotoxin dl-α-AAA inhibits cystine uptake through this antiporter on the glial cells and elicits reduction of cellular levels of glutathione. The site-specific action of gliotoxin dl-α-AAA in the retina will be discussed on the basis of the hypothesis that the expression of cystine/glutamate antiporter is enriched in the glial Mu¨ller cells.