Determination of the phospholipid precursor of anandamide and other N-acylethanolamine phospholipids before and after sodium azide-induced toxicity in cultured neocortical neurons.
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/10(7) 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.
Hansen
H H
HH
Department of Pharmacology, Royal Danish School of Pharmacy, Copenhagen, Denmark. hsh@mail.dfh.dk
Hansen
S H
SH
Schousboe
A
A
Hansen
H S
HS
D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D004622:Embryo, Mammalian; D063388:Endocannabinoids; D051379:Mice; D008815:Mice, Inbred Strains; D019579:Neocortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010739:Phospholipase D / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D010955:Plasmalogens / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D019810:Sodium Azide / Q000633:toxicity*; D018629:Spectrometry, Mass, Secondary Ion
D001095:Arachidonic Acids; D063388:Endocannabinoids; D010714:Phosphatidylethanolamines; D010743:Phospholipids; D010955:Plasmalogens; D053284:Polyunsaturated Alkamides; D019810:Sodium Azide; D010739:Phospholipase D; C078814:anandamide