title: Mammalian cells stably overexpressing N-acylphosphatidylethanolamine-hydrolysing phospholipase D exhibit significantly decreased levels of N-acylphosphatidylethanolamines.
abstract: In animal tissues, NAEs (N-acylethanolamines), including N-arachidonoylethanolamine (anandamide), are primarily formed from their corresponding NAPEs (N-acylphosphatidylethanolamines) by a phosphodiesterase of the PLD (phospholipase D) type (NAPE-PLD). Recently, we cloned cDNAs of NAPE-PLD from mouse, rat and human [Okamoto, Morishita, Tsuboi, Tonai and Ueda (2004) J. Biol. Chem. 279, 5298-5305]. However, it remained unclear whether NAPE-PLD acts on endogenous NAPEs contained in the membrane of living cells. To address this question, we stably transfected two mammalian cell lines (HEK-293 and CHO-K1) with mouse NAPE-PLD cDNA, and investigated the endogenous levels and compositions of NAPEs and NAEs in these cells, compared with mock-transfected cells, with the aid of GC-MS. The overexpression of NAPE-PLD caused a decrease in the total amount of NAPEs by 50-90% with a 1.5-fold increase in the total amount of NAEs, suggesting that the recombinant NAPE-PLD utilizes endogenous NAPE as a substrate in the cell. Since the compositions of NAEs and NAPEs of NAPE-PLD-overexpressing cells and mock-transfected cells were very similar, the enzyme did not appear to discriminate among the N-acyl groups of endogenous NAPEs. These results confirm that overexpressed NAPE-PLD is capable of forming NAEs, including anandamide, in living cells.
authors.affiliation: Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Okamoto
authors.forename: Jun
authors.initials: J
authors.lastname: Morishita
authors.forename: Jun
authors.initials: J
authors.lastname: Wang
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Schmid
authors.forename: Randy J
authors.initials: RJ
authors.lastname: Krebsbach
authors.forename: Harald H O
authors.initials: HH
authors.lastname: Schmid
authors.forename: Natsuo
authors.initials: N
authors.lastname: Ueda
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D006224:Cricetinae; D015870:Gene Expression / Q000235:genetics; D006801:Humans; D051379:Mice; D010714:Phosphatidylethanolamines / Q000378:metabolism*; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism*
chemical_list: D010714:Phosphatidylethanolamines; D010739:Phospholipase D; C506062:Napepld protein, mouse