Excessive stimulation of excitatory amino acid (EAA) receptors and abnormal production of oxygen-derived free radicals have repeatedly been implicated in the series of events linking brain hypoxia or ischemia to neuronal death. We report here that in rat hippocampal slices the KCl-stimulated output of labeled D-3H aspartate or of endogenous aspartate and glutamate significantly increased under in vitro simulated hypoxic, hypoglycemic, or ischemic conditions. In particular, when the slices were incubated for 10 min at 32 degrees C under "ischemic" conditions (namely, lack of oxygen and glucose), endogenous aspartate and glutamate in the supernatant increased by 10 and 20 times, respectively. Since radical scavengers (D-mannitol), drugs reducing free radical formation (indomethacin, corticosteroid), or enzymes able to metabolize them (catalase and superoxide dismutase) significantly reduced this output, it was supposed that free radicals caused EAA release. A direct demonstration of this concept was obtained by showing a significant release of EAA after incubation of hippocampal slices with enzymes and substrates known to cause the formation of free radicals, such as xanthine plus xanthine oxidase or arachidonic acid plus prostaglandin synthase. Neither ischemia nor the enzymatic reactions leading to free radical production increased the activity of the cytoplasmic enzyme lactate dehydrogenase in the incubation medium, thus ruling out a nonspecific cellular lysis. It appears therefore that during ischemic states, brain production of reactive molecules (free radicals) causes an increased output of EAA. This may trigger a series of events which could help to explain the delayed loss of neurons after a transient ischemic period.

The release of D-[3H]aspartate, [3H]noradrenaline, and of endogenous glutamate and aspartate from rat hippocampal slices was significantly increased when the slices were incubated with xanthine oxidase plus xanthine to produce superoxide and hydroxyl free radicals locally. Allopurinol, a specific xanthine oxidase inhibitor, the hydroxyl-radical scavenger D-mannitol, or the superoxide-radical scavenger system formed by superoxide dismutase plus catalase prevented this release. These results suggest that endogenous excitatory amino acids are released consequent to the formation of free radicals. The excess of glutamate and aspartate released by this mechanism could be one of the factors contributing to the death of neurons after anoxic or ischemic injuries.

1 The effects of several agonists of the metabotropic glutamate receptor (mGluR) were studied in adult rat striatal slices by measuring (i) KCl (30 mM while Quis appears to act preferentially on the mGluR receptor linked to the metabolism of PIs.

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