Biosynthesis of anandamide and N-palmitoylethanolamine by sequential actions of phospholipase A2 and lysophospholipase D

Abstract

Anandamide (an endocannabinoid) and other bioactive long-chain NAEs (N-acylethanolamines) are formed by direct release from N-acyl-PE (N-acyl-phosphatidylethanolamine) by a PLD (phospholipase D). However, the possible presence of a two-step pathway from N-acyl-PE has also been suggested previously, which comprises (1) the hydrolysis of N-acyl-PE to N-acyl-lysoPE by PLA1/PLA2 enzyme(s) and (2) the release of NAEs from N-acyllysoPE by lysoPLD (lysophospholipase D) enzyme(s). In the present study we report for the first time the characterization of enzymes responsible for this pathway. The PLA1/PLA2 activity for N-palmitoyl-PE was found in various rat tissues, with the highest activity in the stomach. This stomach enzyme was identified as group IB sPLA2 (secretory PLA2), and its product was determined as N-acyl-1-acyl-lysoPE. Recombinant group IB, IIA and V of sPLA2s were also active with N-palmitoyl-PE, whereas group X sPLA2 and cytosolic PLA2a were inactive. In addition, we found wide distribution of lysoPLD activity generating N-palmitoylethanolamine from N-palmitoyl-lysoPE in rat tissues, with higher activities in the brain and testis. Based on several lines of enzymological evidence, the lysoPLD enzyme could be distinct from the known N-acyl-PE-hydrolysing PLD. sPLA2-IB dose dependently enhanced the production of N-palmitoylethanolamine from N-palmitoyl-PE in the brain homogenate showing the lysoPLD activity. N-Arachidonoyl-PE and N-arachidonoyl-lysoPE as anandamide precursors were also good substrates of sPLA2-IB and the lysoPLD respectively. These results suggest that the sequential actions of PLA2 and lysoPLD may constitute another biosynthetic pathway for NAEs, including anandamide.

PUFA-derived endocannabinoids: an overview

“…1). Since cells lacking NAPEphospholipase D are also able to synthesise anandamide, alternative pathways have been proposed (41)(42)(43)(44)(45)(46)(47) and they are summarised in Fig. 1.…”

Generation of N-Acylphosphatidylethanolamine by Members of the Phospholipase A/Acyltransferase (PLA/AT) Family

“…14 C]Palmitoyllyso-PE was prepared as described previously (18). sn-Glycero-3-phospho-N-heptadecanoylethanolamine was prepared by alkaline hydrolysis of 5 mol of N-heptadecanoyl-1,2-dipalmitoyl-PE with 0.1 N KOH in methanol at 60°C for 10 min.…”

Regulation of food intake by oleoylethanolamide

“…2) [11]. An additional mechanism of FAE biosynthesis has been proposed, which involves the hydrolysis of NAPE to N-acyl-lyso-PE (lyso-NAPE), catalyzed by a secretory phospholipase A 2 (sPLA 2 ), followed by the cleavage of lyso-NAPE to FAEs, catalyzed by a lysophospholipase D (lyso-PLD) [12]. The relative contribution of the NAT/PLD and sPLA 2 /lyso-PLD pathways to OEA formation is unknown at present.…”