GLUTAMATE STIMULATES THE FORMATION OF N-ACYLPHOSPHATIDYLETHANOLAMINE AND N-ACYLETHANOLAMINE IN CORTICAL-NEURONS IN CULTURE

The formation of anandamide (N-arachidonoylethanolamine), N-acylethano lamine, and N-acylphosphatidylethanolamine was studied in primary cult ures of rat cortical neurons. The cells were incubated for 22 h with [ C-14]ethanolamine, [U-C-14]arachidonic acid, [H-3]arachidonic acid, [P -32]phosphate, [C-14]stearic acid, or [H-3]myristic acid. The lipids f rom the cells and media were separated by thin layer chromatography. [ C-14]Ethanolamine labelling revealed two compounds (I and II), which o n different thin layer chromatography systems migrated as N-acylethano lamine (0.05-0.55% of total radioactivity) and N-acylphosphatidylethan olamine (0.66-6.49% of total radioactivity), respectively. Compound II was also labelled with [P-32]phosphate, and radioactive fatty acids. Treatment of compound II with phospholipase D (Streptomyces chromofusc us) resulted in two compounds, one comigrating as phosphatidic acid an d the other as N-acylethanolamine, Compound I could be labelled with [ C-14]stearic acid and [H-3]myristic acid, but not with [H-3]- Or [C-14 ]arachidonic acid. Exogenous [H-3]anandamide was metabolised with a t( 1/2) of 2.6 h. The labelling of the two compounds identified as N-acyl ethanolamine and N-acylphosphatidylethanolamine were more pronounced t he older the culture. The neurotoxic amino acid, glutamate, stimulated within 2 h dose-dependently (ED(50)= 40 mu M) the formation of both c ompounds. It is suggested that N-acylethanolamine and N-acylphosphatid ylethanolamine are formed in relation to the cytotoxicity induced by g lutamate, and that these compounds may be markers of neurotoxicity. We could not detect any formation of anandamide using radioactive arachi donic acid.