Characterization of glutamate-induced formation of N-acylphosphatidylethanolamine and N-acylethanolamine in cultured neocortical neurons

Abstract

Glutamate-induced formation of N-acylethanolamine (NAE) and N-acylphosphatidylethanolamine (NAPE) was studied in primary cultures of mouse neocortical neurons prelabeled with [14C] ethanolamine. The formation of 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, whereas glutamate induced a pronounced formation in 6-day-old cultures. The calcium ionophore A23187 (2 microM) stimulated, within 2 h, formation of NAPE in 2-day-old cultures (fourfold) as well as in 6-day-old cultures (eightfold). Glutamate exerted its effect via NMDA receptors as seen by the inhibitory action of the NMDA-selective receptor antagonists D-(-)-2-amino-5-phosphonovalerate and N-(1-(2-thienyl)cyclohexyl)piperidine and the lack of effect of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate-receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In 6-day-old cultures, exposure to NMDA (100 microM for 24 h) induced a linear increase in the formation of NAPE and NAE as well as a 40-50% neuronal death, as measured by a decrease in cellular formazan formation [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 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 use of different pharmacological agents. Exposure to 5 mM NaN3 for 8 h resulted in a >80% decrease in the cellular MTT staining and a pronounced linear increase in the formation of NAE and NAPE (reaching 25-30% of total labeling). [14C]Anandamide was also formed in [14C]arachidonic acid-labeled neurons exposed to NaN3. No NAPE formation was detected in A23187-stimulated mouse astrocytes, rat Leydig cells and cardiomyocytes, and several other cells. These results suggest that the glutamate-induced formation of NAPE and NAE was mediated by the NMDA receptor and the formation of these lipids may be associated with neuronal death.