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N-Acetylaspartate (NAA) is a largely neuron specific dianionic amino acid present in high concentration in vertebrate brain. Many fundamental questions concerning N-acetylaspartate in brain remain unanswered. One such issue is the predominantly neuronal synthesis and largely glial catabolism which implies the existence of a regulated efflux from neurons. Here we show that transient (5 min) NMDA-receptor activation (60 microM) induces a long lasting Ca2+ -dependent efflux of N-acetylaspartate from organotypic slices of rat hippocampus. The NMDA-receptor stimulated efflux was unaffected by hyper-osmotic conditions (120 mM sucrose) and no efflux of N-acetylaspartate was evoked by high K+ -depolarization (50 mM) or kainate (300 microM). These results indicate that the efflux induced by NMDA is not related directly to either cell swelling or depolarization but is coupled to Ca2+ -influx via the NMDA-receptor. The efflux of N-acetylaspartate persisted at least 20 min after the omission of NMDA, similar to the efflux of the organic anions glutathione and phosphoethanolamine. The efflux of taurine and hypotaurine was also stimulated by NMDA but returned more quickly to basal levels. The NMDA-receptor stimulated efflux of N-acetylaspartate, glutathione, phosphoethanolamine, taurine and hypotaurine correlated with delayed nerve cell death measured 24 h after the transient NMDA-receptor stimulation. However, exogenous administration of high concentrations of N-acetylaspartate to the culture medium was non-toxic. The results suggest that Ca2+ -influx via the NMDA-receptor regulates the efflux of N-acetylaspartate from neurons which may have both physiological and pathological importance.

The concentrations of gamma-glutamylglutamate, gamma-glutamylglutamine, gamma-glutamylcysteine, glutamate, aspartate, glutamine, cyst(e)ine and glutathione (including disulfides) were determined by HPLC analysis of both the tissue and the surrounding medium of incubated rat hippocampal slices. High potassium concentrations (50 mM; 2 x 4 min) increased the medium concentration of gamma-glutamylglutamate (maximal net efflux 0.07 +/- 0.06 pmol/mg protein/min; n = 8 +/- SD) with a relative time delay compared to the increase in glutamate (maximal net efflux 264 +/- 88 pmol/mg protein/min). Release of gamma-glutamylcysteine, the glutathione precursor, demonstrated an immediate response and gradually approached prestimulus levels (maximal net efflux 0.36 +/- 0.13 pmol/mg protein/min). Addition of acivicin (0.2 mM), a gamma-glutamyl transpeptidase (EC 2.3.2.2.) blocker, during preincubation for 45 min reduced the tissue concentrations (pmol/mg protein) of gamma-glutamylglutamate (19.4 +/- 8.2 (control) vs. 5.8 +/- 3.6 (+ acivicin)), gamma-glutamylglutamine (40.3 +/- 6.7 vs. 25.7 +/- 4.2 pmol/mg protein), glutamine (9.9 +/- 2.0 vs. 4.6 +/- 1.2 nmol/mg protein) and cysteine (1.0 +/- 0.2 vs. 0.56 +/- 0.18 nmol/mg protein). Incubation with acivicin (0.2 mM) reduced the net efflux of gamma-glutamylglutamine (0.79 +/- 0.19 vs. 0.21 +/- 0.07 pmol/mg protein/min) whereas that of the glutathione was increased (4.7 +/- 1.0 vs. 20 +/- 3 pmol/mg protein/min). The medium concentrations of glutamate in both low and high potassium were unaffected by acivicin, while the high potassium induced increase in gamma-glutamylglutamate was blocked. The results demonstrate differential efflux patterns of gamma-glutamyl dipeptides from brain slices and show that in vitro the activity of gamma-glutamyl transpeptidase regulates extracellular concentrations of glutathione, gamma-glutamylglutamine and gamma-glutamylglutamate.

The effects of chronic administration of 2-guanidinoethane sulfonic acid on the levels of intra- and extracellular amino acids in the rat hippocampus were studied. The tissue content of taurine was selectively reduced by almost one third after 9 days of peroral administration of 1% 2-guanidinoethane sulfonate. Extracellular levels of amino acids were monitored with the brain microdialysis method. The taurine concentration in the extracellular fluid was depressed in relation to the decrease in intracellular taurine. Unexpectedly, extracellular (but not intracellular) glutamate was doubled in 2-guanidinoethane sulfonate treated animals. The kainic acid evoked release of taurine was suppressed in the 2-guanidinoethane sulfonate group, whereas the kainate stimulated efflux of glutamate was elevated after 2-guanidinoethane sulfonate administration. The acute metabolic effects of kainate were studied by measuring the efflux of the adenosine triphosphate breakdown products hypoxanthine, xanthine, inosine and adenosine. No differences were found between control and 2-guanidinoethane sulfonate treated rats with respect to basal or kainic acid evoked release of purine catabolites. Also, the neuronal loss caused by kainate injection into the hippocampus was not modified by 2-guanidinoethane sulfonate treatment, suggesting that endogenous taurine does not affect these responses. We conclude that chronic administration of 2-guanidinoethane sulfonate does not sensitize central neurons to the metabolic and toxic actions of kainate.

The in vivo release of six radiolabeled amino acids or amines in the hippocampus was investigated in genetically seizure-resistant and seizure-susceptible rats. No differences were found in the spontaneous or high K(+)-evoked efflux of ?-aminobutyric acid, norepinephrine or its metabolites, taurine or ?-aminoisobutyrate. However, seizure-susceptible rats released significantly more d-aspartic acid on K(+)-depolarization. The initial rate of release (flux) was also higher. In view of the ubiquity of synapses in the mammalian brain utilizing excitatory amino acids as their transmitter, we propose that an exaggerated release of these excitants might be a determinant of the innate seizure susceptibility in these animals.