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 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.
Department of Biochemistry, Kagawa University School of Medicine, 1750-1 Ikenobe, Miki, Kagawa 761-0793, Japan.
Yong-Xin
YX
Sun
Kazuhito
K
Tsuboi
Yasuo
Y
Okamoto
Takeharu
T
Tonai
Makoto
M
Murakami
Ichiro
I
Kudo
Natsuo
N
Ueda
D000577:Amides; D000818:Animals; D001095:Arachidonic Acids / Q000096:biosynthesis*; D001921:Brain / Q000201:enzymology; D002460:Cell Line; D063388:Endocannabinoids; D004983:Ethanolamines / Q000378:metabolism; D054500:Group IB Phospholipases A2; D006801:Humans; D006868:Hydrolysis; D007527:Isoenzymes / Q000378:metabolism; D007668:Kidney / Q000166:cytology / Q000196:embryology; D008297:Male; D009928:Organ Specificity; D010169:Palmitic Acids / Q000378:metabolism*; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D054466:Phospholipases A1; D054467:Phospholipases A2; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D053284:Polyunsaturated Alkamides; D051381:Rats; D017208:Rats, Wistar; D013270:Stomach / Q000201:enzymology; D013379:Substrate Specificity; D000099189:Lysophospholipase D
D000577:Amides; D001095:Arachidonic Acids; D063388:Endocannabinoids; D004983:Ethanolamines; D054500:Group IB Phospholipases A2; D007527:Isoenzymes; D010169:Palmitic Acids; D010714:Phosphatidylethanolamines; D010741:Phospholipases A; D054466:Phospholipases A1; D054467:Phospholipases A2; D010727:Phosphoric Diester Hydrolases; D053284:Polyunsaturated Alkamides; D000099189:Lysophospholipase D; C022203:N-acylethanolamines; C483858:phosphatidylethanolamine; C005958:palmidrol; C516202:PLA2G1B protein, human; C516204:Pla2g1b protein, rat; C078814:anandamide