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

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 PLA₂α 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.