CHARACTERIZATION OF GLUTAMATE-INDUCED FORMATION OF N-ACYLPHOSPHATIDYLETHANOLAMINE AND N-ACYLETHANOLAMINE IN CULTURED NEOCORTICAL NEURONS

Glutamate-induced formation of N-acylethanolamine (NAE) and N-acylphos phatidylethanolamine (NAPE) was studied in primary cultures of mouse n eocortical neurons prelabeled with [C-14]ethanolamine. The formation o f these two lipids was dependent on the maturity of the cell culture; i.e., no glutamate-induced formation was seen in 2-day-old cultures, w hereas glutamate induced a pronounced formation in 6-day-old cultures. The calcium ionophore A23187 (2 mu M) stimulated, within 2 h, formati on of NAPE in 2-day-old cultures (fourfold) as well as in 6-day-old cu ltures (eightfold). Glutamate exerted its effect via NMDA receptors as seen by the inhibitory action of the NMDA-selective receptor antagoni sts D-(-)-2-amino-5-phosphonovalerate and N-(1-(2-thienyl)cyclohexyl)p iperidine and the lack of effect of the lpha-amino-3-hydroxy-5-methyli soxazole-4-propionic acid (AMPA)/kainate-receptor antagonist 6-cyano-7 -nitroquinoxaline-2,3-dione (CNQX), In 6-day-old cultures, exposure to NMDA (100 mu M for 24 h) induced a linear increase in the formation o f NAPE and NAE as well as a 40-50% neuronal death, as measured by a de crease in cellular formazan formation (4,5-dimethylthiazol-2-yl)-2,5-d iphenyltetrazolium bromide (MTT) assay]. The increase in NAPE and NAE could be detected earlier than the neuronal death, Neither cyclic AMP, cyclic GMP, nitric oxide, protein kinase C, nor peroxidation appears to be involved in the formation of NAPE and NAE, as assessed by the us e of different pharmacological agents. Exposure to 5 mM NaN3 for 8 h r esulted in a >80% decrease in the cellular MTT staining and a pronounc ed linear increase in the formation of NAE and NAPE (reaching 25-30% o f total labeling). [C-14]Anandamide was also formed in [C-14]arachidon ic acid-labeled neurons exposed to NaN3. No NAPE formation was detecte d in A23187-stimulated mouse astrocytes, rat Leydig cells and cardiomy ocytes, and several other cells, These results suggest that the glutam ate-induced formation of NAPE and NAE was mediated by the NMDA recepto r and the formation of these lipids may be associated with neuronal de ath.