Transacylase‐Mediated and Phosphodiesterase‐Mediated Synthesis of N‐Arachidonoylethanolamine, an Endogenous Cannabinoid‐Receptor Ligand, in Rat Brain Microsomes

Abstract

The levels of N-arachidonoylethanolamine (anandamide), an endogenous cannabinoid-receptor ligand, and a relevant molecule, N-arachidonoylphosphatidylethanolamine (N-arachidonoylPtdEtn), in rat brain were investigated using a newly developed sensitive analytical method. We found that rat brain contains small but significant amounts of these two types of N-arachidonoyl lipids (4.3 pmol/g tissue and 50.2 pmol/g tissue, respectively). Then, we investigated how N-arachidonoylethanolamine (anandamide) is produced in the brain. We found that anandamide can be formed enzymatically via two separate synthetic pathways in the brain : enzymatic condensation of free arachidonic acid and ethanolamine; and formation of N-arachidonoylPtdEtn from PtdEtn and arachidonic acid esterified at the I-position of phosphatidyl-choline (PtdCho), and subsequent release of anandamide from N-arachidonoylPtdEtn through the action of a phosphodiesterase. We confirmed that rat brain contains both the enzyme activities and lipid substrates involved in these reactions. Several lines of evidence strongly suggest that the second pathway, rather than the first one, meets the requirements and conditions for the synthesis of various species of N-acylethanolamine including anandamide in the brain.

Changes in anandamide levels in mouse uterus are associated with uterine receptivity for embryo implantation

“…36 and 37). Very recent evidence strongly suggests that anandamide is synthesized in rat brain by the transacylation-phosphodiesterase pathway rather than by Nacylation of ethanolamine (38). However, our present results show that arachidonic acid is only a minor component of the N-acyl groups of NAPE in the mouse uterus during early pregnancy (Table 3), whereas the corresponding anandamide represents up to 95% of total NAEs (Table 1).…”

“…However, our present results show that arachidonic acid is only a minor component of the N-acyl groups of NAPE in the mouse uterus during early pregnancy (Table 3), whereas the corresponding anandamide represents up to 95% of total NAEs (Table 1). Thus, biosynthesis of anandamide in the uterus by the N-acylationphosphodiesterase pathway (36)(37)(38) would require either the action of a phosphodiesterase exhibiting extraordinary selec-FIG. 1.…”

The endocannabinoid system: a general view and latest additions

“…Neither the PLD nor the trans-acylase appeared to exhibit any selectivity for a particular fatty acid moiety. Indeed, this route was shown to underlie also AEA biosynthesis in central neurons after depolarization (Di Marzo et al, 1994), and subsequent studies (Di Marzo et al, 1996;Sugiura et al, 1996;Cadas et al, 1997) confirmed the occurrence of N-arachidonoyl-phosphatidylethanolamine (NArPE), the NAPE precursor of AEA, in murine brain, testes and leukocytes, and showed that cell-free preparations could convert NArPE into AEA and biosynthesize NArPE from phospholipid precursors (Figure 2). The enzymes catalyzing these reactions were only partially characterized, and, in particular, the NAPE-PLD was shown later to lack the transphosphatidylating activity typical of other PLD enzymes (Petersen & Hansen, 1999), to be dependent on Ca 2 þ for optimal activity (Ueda et al, 2001) and to be stimulated by polyamines (Liu et al, 2002).…”

Biosynthesis and turnover of anandamide and other N‐acylethanolamines in peritoneal macrophages

“…Unlabeled ethanolamine and calcium ionophore A23187 were from Sigma (St. Louis, MO, USA). [1,[2][3][4][5][6][7][8][9][10][11][12][13][14] C]Ethanolamine (speci¢c activity, 2.5 mCi/ mmol) was from NEN Life Science Products (USA). L-Glutamine and all other chemicals used in the incubation experiments were analytical grade from Wako Pure Chemical Industries (Osaka, Japan).…”