Calcium-dependent glutamate cytotoxicity in a neuronal cell line

Membranes from the neuroblastoma × embryonic retina cell hybrid cell line, N18-RE-105, bind l-[³H]glutamate with a pharmacologic profile consistent with a 'quisqualate-type' brain l-glutamate receptor. We describe here the cytotoxic effect of l-glutamate receptor agonists on intact N18-RE-105 cells. Cytotoxicity was quantitated by measurement of the release of the cytosolic enzyme, lactate dehydrogenase, into the culture medium after addition of l-glutamate and its analogs to the cell culture medium. l-Glutamate (10 mM) and its confirmationally restricted analogs, quisqualate (1 mM) and ibotenate (10 mM), caused cell lysis. In contrast, similar analogs which do not bind to N18-RE-105 cell membranes (kainic acid, N-methyl-d,l-aspartic acid and γ-aminobutyric acid) were not cytotoxic. l-Glutamate-induced cytotoxicity was eliminated when calcium-free medium was used. Addition of inorganic or organic calcium channel antagonists also reduced the cytotoxicity of l-glutamate, even when 1.8 mM calcium was present in the medium. Cadmium chloride (10 μM) completely blocked l-glutamate toxicity, whereas manganese chloride (150 μM) and lanthanum chloride (25 μM) reduced toxicity by greater than 50%. Dihydropyridine voltage-sensitive calcium channel agonists or antagonists, had little or no significant effect on l-glutamate-induced toxicity. In contrast, the verapamil derivatives, D600 and D888, and the diltiazem derivative, MDL 12,330A reduced l-glutamate toxicity by greater than 50%. These results suggest that a subtype of voltage-sensitive calcium channels is involved in the mechanism of l-glutamate receptor mediated cytotoxicity in this cell line.