Elucidation of physiological finction of anandamide, an endogenous marijuana-like substance, in mammals

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N-Acylethanolamines (NAEs) constitute a large and diverse class of signaling lipids that includes the endogenous cannabinoid anandamide. It is widely accepted that in mammalian tissues all NAEs are principally released from their corresponding N-acyl phosphatidylethanolamines (NAPEs) by the catalysis by a membrane-associated phospholipase D generally abbreviated as NAPE-PLD. Recently, NAPE-PLD was identified as a candidate enzyme involved in the biosynthesis of NAEs by us. To understand the physiological and pathophysiological significance of anandamide and other bioactive NAEs in mammals, we generated and characterized mice with a targeted disruption in the NAPE-PLD gene (NAPE-PLD-1). (1) lb generate mice lacking NAPE-PLD, exon 3 of the NAPE-PLD gene was removed by Cre/loxP system. (2) NAPE-PLD-1- were born at the expected Mendelian frequency, were viable and healthy, and showed no overt differences in their cage behavior compared to wild type littermates. (3) RT-PCR and Western blotting with anti-NAPE-PLD antibodies confirmed the absence of NAPE-PLD mRNA and protein in tissues from NAPE-PLO-h. (4) Tissues from NAPE FED" showed significantly lower NAPE-PLD activity with an N-palmitoyl-PE as substrate compared to wild type tissues. (5) Significant reductions in the levels of saturated NAEs were observed in NAPE-PLO-I brains. On the other hand, complementary profiles of NAPEs were found in NAPE-PLD+/+ and NAPE-PLD-1- brains, with the latter samples possessing 15-20-fold higher levels of saturated N-acyl NAPEs, These data suggest that NAPE-PLD is a principal enzyme responsible for the conversion of NAPEs to NAEs in mammals.