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.

Figure 1. Stable expression of recombinant NAPE-PLD in HEK-293 and CHO-K1 cells

Western blotting was performed with anti-NAPE-PLD antiserum as described in the Materials and methods section. Lane 1, HEK-293 cell homogenates (20 μg of protein) transfected with the insert-free vector; lane 2, HEK-293 cell homogenates (20 μg of protein) transfected with mouse NAPE-PLD cDNA; lane 3, CHO-K1 cell homogenates (20 μg of protein) transfected with the insert-free vector; lane 4, CHO-K1 cell homogenates (20 μg of protein) transfected with mouse NAPE-PLD cDNA; lane 5, COS-7 cell homogenates (5 μg of protein) transiently expressing mouse NAPE-PLD prepared as described previously [35].