Determination of the Phospholipid Precursor of Anandamide and Other N‐Acylethanolamine Phospholipids Before and After Sodium Azide‐Induced Toxicity in Cultured Neocortical Neurons

Abstract

Phospholipase d‐mediated hydrolysis of N‐acylethanolamine phospholipids (NAPEs) releases anandamide and other N‐acylethanolamines, resulting in different actions at cellular targets in the CNS. Recently, we have demonstrated that these N‐acyl lipids accumulate in cultured neocortical neurons subjected to sodium azide‐induced cell injury. We here extend the information on the NAPE response, reporting on the composition of N‐acylspecies of NAPE, employing a new methodological approach of HPLC‐coupled electrospray ionization mass spectrometry. Exposure to sodium azide (5 mM) increased the total amount of NAPE threefold over control levels ; however, no alteration of the relative composition of NAPE species was detected. The anandamide precursor (20 : 4‐NAPE) constituted only 0.1% of all NAPEs detected in the neurons. Total NAPE species in control cells amounted to 956‐1,060 pmol/107 cells. Moreover, we detected the presence of an unknown NAPE species with molecular weight identical to 20 : 4‐NAPE. This may suggest the presence of a putative stereoisomer of the anandamide precursor with at least one trans‐configured double bond in the N‐arachidonoyl moiety. These results show that with the present method, neuronal NAPE species can be identified and quantified with respect to N‐acyl composition, including a trans‐isomer of the anandamide precursor. The anandamide precursor is up‐regulated to the same extent as other NAPEs upon neuronal injury.