Mammalian cells stably overexpressing N-acylphosphatidylethanolamine-hydrolysing phospholipase D exhibit significantly decreased levels of N-acylphosphatidylethanolamines.
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.
Okamoto
Yasuo
Y
Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan.
Morishita
Jun
J
Wang
Jun
J
Schmid
Patricia C
PC
Krebsbach
Randy J
RJ
Schmid
Harald H O
HH
Ueda
Natsuo
N
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*
D010714:Phosphatidylethanolamines; D010739:Phospholipase D; C506062:Napepld protein, mouse