Endocannabinoid biosynthesis proceeding through glycerophospho-N-acyl ethanolamine and a role for alpha/beta-hydrolase 4 in this pathway

N-Acyl ethanolamines (NAEs) are a large class of signaling lipids implicated in diverse physiological processes, including nociception, cognition, anxiety, appetite, and inflammation. It has been proposed that NAEs are biosynthesized from their corresponding N-acyl phosphatidylethanolamines (NAPEs) in a single enzymatic step catalyzed by a phospholipase D (NAPE-PLD). The recent generation of NAPE-PLD(-/-) mice has revealed that these animals possess lower brain levels of saturated NAEs but essentially unchanged concentrations of polyunsaturated NAEs, including the endogenous cannabinoid anandamide. These findings suggest the existence of additional enzymatic routes for the production of NAEs in vivo. Here, we report evidence for an alternative pathway for NAE biosynthesis that proceeds through the serine hydrolase-catalyzed double-deacylation of NAPE to generate glycerophospho-NAE, followed by the phosphodiesterase-mediated cleavage of this intermediate to liberate NAE. Furthermore, we describe the functional proteomic isolation and identification of a heretofore uncharacterized enzyme alpha/beta-hydrolase 4 (Abh4) as a lysophospholipase/phospholipase B that selectively hydrolyzes NAPEs and lysoNAPEs. Abh4 accepts lysoNAPEs bearing both saturated and polyunsaturated N-acyl chains as substrates and displays a distribution that closely mirrors lysoNAPE-lipase activity in mouse tissues. These results support the existence of an NAPE-PLD-independent route for NAE biosynthesis and suggest that Abh4 plays a role in this metabolic pathway by acting as a (lyso)NAPE-selective lipase.     

Biosynthesis and turnover of anandamide and other N-acylethanolamines in peritoneal macrophages.

Polyunsaturated N-acylethanolamines (NAEs), including anandamide (20:4n-6 NAE), elicit a variety of biological effects through cannabinoid receptors, whereas saturated and monounsaturated NAEs are inactive. Arachidonic acid mobilization induced by treatment of intact mouse peritoneal macrophages with Ca2+ ionophore A23187 had no effect on the production of NAE or its precursor N-acylphosphatidylethanolamine (N-acyl PE). Addition of exogenous ethanolamine resulted in enhanced NAE synthesis by its N-acylation with endogenous fatty acids, but this pathway was not selective for arachidonic acid. Incorporation of (18)O from H2 (18)O-containing media into the amide carbonyls of both NAE and N-acyl PE demonstrated a rapid, constitutive turnover of both lipids.     

Substantial species differences in relation to formation and degradation of N-acyl-ethanolamine phospholipids in heart tissue: an enzyme activity study

The formation of N-acyl-ethanolamines (NAEs), including the cannabinoid receptor ligand anandamide, and their precursors N-acyl-ethanolamine phospholipids (NAPEs) are catalyzed by NAPE-hydrolyzing phospholipase D (NAPE-PLD) and N-acyl-transferase, respectively. NAPE and NAE are suggested to have beneficial effects on the heart, but in the literature there are indications of species differences in the activity of these enzymes. We have examined heart microsomes from rats, mice, guinea pigs, rabbits, frogs, cows, dogs, cats, mini pigs and human beings for activities of these two enzymes. N-Acyl-transferase activity was very high in dogs and cats (>13 pmol/min/mg protein) whereas it was very low to barely detectable in the other species (<3 pmol/min/mg protein). NAPE-PLD activity was very high in rats and guinea pigs (>45 pmol/min/mg protein) whereas it was 9 pmol/min/mg protein in frogs and below that in the other species. The ratio of activity between the two enzymes varied from 0.002 to 15 in the investigated species. The activity of the two enzymes in rat hearts as opposed to rat brain did not change during development. These results indicate that there may be substantial species differences in the generation of anandamide and other NAEs as well as NAPEs in heart tissues.     

N-acylphosphatidylethanolamine-hydrolyzing phospholipase D: a novel enzyme of the beta-lactamase fold family releasing anandamide and other N-acylethanolamines

N-acylethanolamines (NAEs) are a lipid class present in brain and other animal tissues and contains anandamide (an endocannabinoid) and other bioactive substances. NAEs are formed from N-acylphosphatidylethanolamines (NAPEs) by a phospholipase D (PLD)-type enzyme abbreviated to NAPE-PLD. Although this enzyme has been recognized for more than 20 years, its molecular cloning has only recently been achieved by us. We highly purified NAPE-PLD from the particulate fraction of rat heart, and on the basis of peptide sequences with the purified enzyme cloned its cDNA from mouse, rat and human. The deduced primary structures revealed no homology with any PLDs so far reported, but was suggested to belong to the beta-lactamase fold family. When overexpressed in COS-7 cells, the NAPE-PLD activity increased about 1000-fold in comparison with the endogenous activity. The recombinant enzyme generated various long-chain NAEs including anandamide from their corresponding NAPEs at similar rates. However, the enzyme was inactive with phosphatidylethanolamine and phosphatidylcholine and did not catalyze transphosphatidylation, a reaction characteristic of PLD. The enzyme was widely expressed in murine organs with higher levels in brain, testis and kidney. The existence of NAPE-PLD specifically hydrolyzing NAPEs to NAEs emphasizes physiological significance of NAEs including anandamide in brain and other tissues.     

An anatomical and temporal portrait of physiological substrates for fatty acid amide hydrolase

Fatty acid amide hydrolase (FAAH) regulates amidated lipid transmitters, including the endocannabinoid anandamide and its N-acyl ethanolamine (NAE) congeners and transient receptor potential channel agonists N-acyl taurines (NATs). Using both the FAAH inhibitor PF-3845 and FAAH(-/-) mice, we present a global analysis of changes in NAE and NAT metabolism caused by FAAH disruption in central and peripheral tissues. Elevations in anandamide (and other NAEs) were tissue dependent, with the most dramatic changes occurring in brain, testis, and liver of PF-3845-treated or FAAH(-/-) mice. Polyunsaturated NATs accumulated to very high amounts in the liver, kidney, and plasma of these animals. The NAT profile in brain tissue was markedly different and punctuated by significant increases in long-chain NATs found exclusively in FAAH(-/-), but not in PF-3845-treated animals. Suspecting that this difference might reflect a slow pathway for NAT biosynthesis, we treated mice chronically with PF-3845 for 6 days and observed robust elevations in brain NATs. These studies, taken together, define the anatomical and temporal features of FAAH-mediated NAE and NAT metabolism, which are complemented and probably influenced by kinetically distinguishable biosynthetic pathways that produce these lipids in vivo.     

Anandamide biosynthesis catalyzed by the phosphodiesterase GDE1 and detection of glycerophospho-N-acyl ethanolamine precursors in mouse brain

Anandamide (AEA) is an endogenous ligand of cannabinoid receptors and a well characterized mediator of many physiological processes including inflammation, pain, and appetite. The biosynthetic pathway(s) for anandamide and its N-acyl ethanolamine (NAE) congeners remain enigmatic. Previously, we proposed an enzymatic route for producing NAEs that involves the double-O-deacylation of N-acyl phosphatidylethanolamines (NAPEs) by alpha/beta-hydrolase 4 (ABDH4 or Abh4) to form glycerophospho (GP)-NAEs, followed by conversion of these intermediates to NAEs by an unidentified phosphodiesterase. Here, we report the detection and measurement of GP-NAEs, including the anandamide precursor glycerophospho-N-arachidonoylethanolamine (GP-NArE), as endogenous constituents of mouse brain tissue. Inhibition of the phosphodiesterase-mediated degradation of GP-NAEs ex vivo resulted in a striking accumulation of these lipids in brain extracts, suggesting a rapid endogenous flux through this pathway. Furthermore, we identify the glycerophosphodiesterase GDE1, also known as MIR16, as a broadly expressed membrane enzyme with robust GP-NAE phosphodiesterase activity. Together, these data provide evidence for a multistep pathway for the production of anandamide in the nervous system by the sequential actions of Abh4 and GDE1.     

Lipidomics profile of a NAPE-PLD KO mouse provides evidence of a broader role of this enzyme in lipid metabolism in the brain

A leading hypothesis of N-acyl ethanolamine (NAE) biosynthesis, including the endogenous cannabinoid anandamide (AEA), is that it depends on hydrolysis of N-acyl-phosphatidylethanolamines (NAPE) by a NAPE-specific phospholipase D (NAPE-PLD). Thus, deletion of NAPE-PLD should attenuate NAE levels. Previous analyses of two different NAPE-PLD knockout (KO) strains produced contradictory data on the importance of NAPE-PLD to AEA biosynthesis. Here, we examine this hypothesis with a strain of NAPE-PLD KO mice whose lipidome is uncharacterized. Using HPLC/MS/MS, over 70 lipids, including the AEA metabolite, N-arachidonoyl glycine (NAGly), the endocannabinoid 2-arachidonyl glycerol (2-AG) and prostaglandins (PGE₂ and PGF_2α), and over 60 lipoamines were analyzed in 8 brain regions of KO and wild-type (WT) mice.Lipidomics analysis of this third NAPE-PLD KO strain shows a broad range of lipids that were differentially affected by lipid species and brain region. Importantly, all 6 NAEs measured were significantly reduced, though the magnitude of the effect varied by fatty acid saturation length and brain region. 2-AG levels were only impacted in the brainstem, where levels were significantly increased in KO mice. Correspondingly, levels of arachidonic acid were significantly decreased exclusively in brainstem. NAGly levels were significantly increased in 4 brain regions and levels of PGE₂ increased in 6 of 8 brain regions in KO mice. These data indicate that deletion of NAPE-PLD has far broader effects on the lipidome than previously recognized. Therefore, behavioral characteristics of suppressing NAPE-PLD activity may be due to a myriad of effects on lipids and not simply due to reduced AEA biosynthesis.     

Pathways and mechanisms of N-acylethanolamine biosynthesis: can anandamide be generated selectively?

Long-chain N-acylethanolamines (NAEs) and their precursors, N-acylethanolamine phospholipids, are ubiquitous trace constituents of animal and human cells, tissues and body fluids. Their cellular levels appear to be tightly regulated and they accumulate as the result of injury. Saturated and monounsaturated congeners which represent the vast majority of cellular NAEs can have cytoprotective effects while polyunsaturated NAEs, especially 20:4n-6 NAE (anandamide), elicit physiological effects by binding to and activating cannabinoid receptors. It is the purpose of this article to review published data on the pathways and mechanisms of NAE biosynthesis in mammals and to evaluate this information for its physiological significance. The generation and turnover of NAE via N-acyl PE through the transacylation-phosphodiesterase pathway may represent a novel cannabinoid receptor-independent signalling system, analogous to and possibly related to ceramide-mediated cell signalling.     

Biosynthetic pathways of the endocannabinoid anandamide

Anandamide (=N-arachidonoylethanolamine) is the first discovered endocannabinoid, and belongs to the class of bioactive, long-chain N-acylethanolamines (NAEs). In animal tissues, anandamide is principally formed together with other NAEs from glycerophospholipid by two successive enzymatic reactions: 1) N-acylation of phosphatidylethanolamine to generate N-acylphosphatidylethanolamine (NAPE) by Ca2+-dependent N-acyltransferase; 2) release of NAE from NAPE by a phosphodiesterase of the phospholipase D type (NAPE-PLD). Although these anandamide-synthesizing enzymes were poorly understood until recently, our cDNA cloning of NAPE-PLD in 2004 enabled molecular-biological approaches to the enzymes. NAPE-PLD is a member of the metallo-beta-lactamase family, which specifically hydrolyzes NAPE among glycerophospholipids, and appears to be constitutively active. Mutagenesis studies suggested that the enzyme functions through a mechanism similar to those of other members of the family. NAPE-PLD is widely expressed in animal tissues, including various regions in rat brain. Its expression level in the brain is very low at birth, and remarkably increases with development. Analysis of NAPE-PLD-deficient mice and other recent studies revealed the presence of NAPE-PLD-independent pathways for the anandamide formation. Furthermore, calcium-independent N-acyltransferase was discovered and characterized. In this article, we will review recent progress in the studies on these enzymes responsible for the biosynthesis of anandamide and other NAEs.     

Structure-based design of a FAAH variant that discriminates between the N-acyl ethanolamine and taurine families of signaling lipids

Fatty acid amide hydrolase (FAAH) inactivates a large and diverse class of endogenous signaling lipids termed fatty acid amides. Representative fatty acid amides include the N-acyl ethanolamines (NAEs) anandamide, which serves as an endogenous ligand for cannabinoid receptors, and N-oleoyl and N-palmitoyl ethanolamine, which produce satiety and anti-inflammatory effects, respectively. Global metabolite profiling studies of FAAH (-/-) mice have recently identified a second class of endogenous FAAH substrates: the N-acyl taurines (NATs). To determine the metabolic and signaling functions performed by NAEs and NATs in vivo, a FAAH variant that discriminates between these two substrate classes would be of value. Here, we report the structure-guided design of a point mutant in the active site of FAAH that selectively disrupts interactions with NATs. This glycine-to-aspartate (G268D) mutant was found to exhibit wild-type kinetic parameters with NAEs, but more than a 100-fold reduction in activity with NATs attributable to combined effects on Km and kcat values. These in vitro properties were also observed in living cells, where WT-FAAH and the G268D mutant displayed equivalent hydrolytic activity with NAEs, but the latter enzyme was severely impaired in its ability to catabolize NATs. The G268D FAAH mutant may thus serve as a valuable research tool to illuminate the unique roles played by the NAE and NAT classes of signaling lipids in vivo.     

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

Bioactive N-acylethanolamines (NAEs), including N-palmitoylethanolamine, N-oleoylethanolamine, and N-arachidonoylethanolamine (anandamide), are formed from membrane glycerophospholipids in animal tissues. The pathway is initiated by N-acylation of phosphatidylethanolamine to form N-acylphosphatidylethanolamine (NAPE). Despite the physiological importance of this reaction, the enzyme responsible, N-acyltransferase, remains molecularly uncharacterized. We recently demonstrated that all five members of the HRAS-like suppressor tumor family are phospholipid-metabolizing enzymes with N-acyltransferase activity and are renamed HRASLS1-5 as phospholipase A/acyltransferase (PLA/AT)-1-5. However, it was poorly understood whether these proteins were involved in the formation of NAPE in living cells. In the present studies, we first show that COS-7 cells transiently expressing recombinant PLA/AT-1, -2, -4, or -5, and HEK293 cells stably expressing PLA/AT-2 generated significant amounts of [(14)C]NAPE and [(14)C]NAE when cells were metabolically labeled with [(14)C]ethanolamine. Second, as analyzed by liquid chromatography-tandem mass spectrometry, the stable expression of PLA/AT-2 in cells remarkably increased endogenous levels of NAPEs and NAEs with various N-acyl species. Third, when NAPE-hydrolyzing phospholipase D was additionally expressed in PLA/AT-2-expressing cells, accumulating NAPE was efficiently converted to NAE. We also found that PLA/AT-2 was partly responsible for NAPE formation in HeLa cells that endogenously express PLA/AT-2. These results suggest that PLA/AT family proteins may produce NAPEs serving as precursors of bioactive NAEs in vivo.     

Dietary fatty acids augment tissue levels of n-acylethanolamines in n-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) knockout mice

N-acylethanolamines (NAEs) are lipid signaling mediators, which can be synthesized from dietary fatty acids via n-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD) and in turn influence physiological outcomes; however, the roles of NAPE-PLD upon dietary fatty acid modulation are not fully understood. Presently, we examine if NAPE-PLD is necessary to increase NAEs in response to dietary fatty acid manipulation. Post-weaning male wild-type (C57Bl/6), NAPE-PLD (−/+) and NAPE-PLD (−/−) mice received isocaloric fat diets containing either beef tallow, corn oil, canola oil or fish oil (10% wt/wt from fat) for 9 weeks. Brain docosahexaenoic acid (DHA) levels were higher (P<.01) in NAPE-PLD (−/+) (10.01±0.31 μmol/g) and NAPE-PLD (−/−) (10.89±0.61 μmol/g) than wild-type (7.72±0.61 μmol/g) consuming fish oil. In NAPE-PLD (−/−) mice, brain docosahexaenoylethanolamide (DHEA) levels were higher (P<.01) after fish oil feeding suggesting that NAPE-PLD was not necessary for DHEA synthesis. Liver and jejunum arachidonoylethanolamide, 1,2-arachidonoylglycerol and DHEA levels reflected their corresponding fatty acid precursors suggesting that alternate pathways are involved in NAE synthesis. NAPE-PLD (−/−) mice had lower oleoylethanolamide levels in the jejunum and a leaner phenotype compared to wild-type mice. Overall, these results demonstrate that dietary fatty acid can augment tissue NAEs in the absence of NAPE-PLD.     

Lipidomic analysis of N-acylphosphatidylethanolamine molecular species in Arabidopsis suggests feedback regulation by N-acylethanolamines

N-Acylphosphatidylethanolamine (NAPE) and its hydrolysis product, N-acylethanolamine (NAE), are minor but ubiquitous lipids in multicellular eukaryotes. Various physiological processes are severely affected by altering the expression of fatty acid amide hydrolase (FAAH), an NAE-hydrolyzing enzyme. To determine the effect of altered FAAH activity on NAPE molecular species composition, NAE metabolism, and general membrane lipid metabolism, quantitative profiles of NAPEs, NAEs, galactolipids, and major and minor phospholipids for FAAH mutants of Arabidopsis were determined. The NAPE molecular species content was dramatically affected by reduced FAAH activity and elevated NAE content in faah knockouts, increasing by as much as 36-fold, far more than the NAE content, suggesting negative feedback regulation of phospholipase D-mediated NAPE hydrolysis by NAE. The N-acyl composition of NAPE remained similar to that of NAE, suggesting that the NAPE precursor pool largely determines NAE composition. Exogenous NAE 12:0 treatment elevated endogenous polyunsaturated NAE and NAPE levels in seedlings; NAE levels were increased more in faah knockouts than in wild-type or FAAH overexpressors. Treated seedlings with elevated NAE and NAPE levels showed impaired growth and reduced galactolipid synthesis by the "prokaryotic" (i.e., plastidic), but not the "eukaryotic" (i.e., extraplastidic), pathway. Overall, our data provide new insights into the regulation of NAPE-NAE metabolism and coordination of membrane lipid metabolism and seedling development.     

The N-acylation-phosphodiesterase pathway and cell signalling

Long-chain N-acylethanolamines (NAEs) elicit a variety of biological and pharmacological effects, Anandamide (20:4n-6 NAE) and other polyunsaturated NAEs bind to the cannabinoid receptor and may thus serve as highly specific lipid mediators of cell signalling. NAEs can be formed by phospholipase D-catalyzed hydrolysis of N-acylethanolamine phospholipids or by direct condensation of ethanolamine and fatty acid, So far, most of the latter biosynthetic activity has been shown to be the reverse reaction of the NAE amidohydrolase that catalyzes NAE degradation. Thus, increasing evidence supports the hypothesis that the N-acylation-phosphodiesterase pathway yields not only saturated-monounsaturated NAEs, but polyunsaturated ones, including anandamide, as well.     

Endogenous unsaturated C18 N-acylethanolamines are vanilloid receptor (TRPV1) agonists

The endogenous C18 N-acylethanolamines (NAEs) N-linolenoylethanolamine (18:3 NAE), N-linoleoylethanolamine (18:2 NAE), N-oleoylethanolamine (18:1 NAE), and N-stearoylethanolamine (18:0 NAE) are structurally related to the endocannabinoid anandamide (20:4 NAE), but these lipids are poor ligands at cannabinoid CB(1) receptors. Anandamide is also an activator of the transient receptor potential (TRP) vanilloid 1 (TRPV(1)) on primary sensory neurons. Here we show that C18 NAEs are present in rat sensory ganglia and vascular tissue. With the exception of 18:3 NAE in rat sensory ganglia, the levels of C18 NAEs are equal to or substantially exceed those of anandamide. At submicromolar concentrations, 18:3 NAE, 18:2 NAE, and 18:1 NAE, but not 18:0 NAE and oleic acid, activate native rTRPV(1) on perivascular sensory nerves. 18:1 NAE does not activate these nerves in TRPV(1) gene knock-out mice. Only the unsaturated C18 NAEs elicit whole cell currents and fluorometric calcium responses in HEK293 cells expressing hTRPV(1). Molecular modeling revealed a low energy cluster of U-shaped unsaturated NAE conformers, sharing several pharmacophoric elements with capsaicin. Furthermore, one of the two major low energy conformational families of anandamide also overlaps with the cannabinoid CB(1) receptor ligand HU210, which is in line with anandamide being a dual activator of TRPV(1) and the cannabinoid CB(1) receptor. This study shows that several endogenous non-cannabinoid NAEs, many of which are more abundant than anandamide in rat tissues, activate TRPV(1) and thus may play a role as endogenous TRPV(1) modulators.     

A FAAH-regulated class of N-acyl taurines that activates TRP ion channels

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that catabolizes several bioactive lipids in vivo. Most of the physiological substrates of FAAH characterized to date belong to the N-acyl ethanolamine (NAE) class of fatty acid amides, including the endocannabinoid anandamide, the anti-inflammatory lipid N-palmitoyl ethanolamine, and the satiating factor N-oleoyl ethanolamine. We recently identified a second structural class of fatty acid amides regulated by FAAH in vivo: the N-acyl taurines (NATs). Global metabolite profiling revealed high concentrations of long chain (> or = C20) saturated NATs in the central nervous system (CNS) of FAAH(-/-) mice. Here, we use metabolite profiling to characterize the FAAH-NAT system in peripheral mouse tissues. Livers and kidneys of FAAH(-/-) mice possessed dramatic elevations in NATs, which, in contrast to those detected in the CNS, were enriched in polyunsaturated acyl chains (e.g., C20:4, C22:6). Peripheral NATs rose more than 10-fold within 1 h following pharmacological inactivation of FAAH and reached levels up to approximately 5000 pmol/g tissue (C22:6 in kidney), implicating a constitutive and highly active pathway for NAT metabolism in which FAAH plays an integral part. Interestingly, NATs were found to activate multiple members of the transient receptor potential (TRP) family of calcium channels, including TRPV1 and TRPV4, which are both expressed in kidney. The dramatic elevation in endogenous levels of NATs following acute or chronic inactivation of FAAH, in conjunction with the pharmacological effects of these lipids on TRP channels, suggests the existence of a second major lipid signaling system regulated by FAAH in vivo.     

Functional analysis of the purified anandamide-generating phospholipase D as a member of the metallo-beta-lactamase family

In animal tissues, bioactive N-acylethanolamines including the endocannabinoid anandamide are formed from their corresponding N-acylphosphatidylethanolamines (NAPEs) by the catalysis of a specific phospholipase D (NAPE-PLD) that belongs to the metallo-beta-lactamase family. Despite its potential physiological importance, NAPE-PLD has not yet been characterized with a purified enzyme preparation. In the present study we expressed a recombinant NAPE-PLD in Escherichia coli and highly purified it. The purified enzyme was remarkably activated in a dose-dependent manner by millimolar concentrations of Mg2+ as well as Ca2+ and, hence, appeared to be constitutively active. The enzyme showed extremely high specificity for NAPEs among various glycerophospholipids but did not reveal obvious selectivity for different long chain or medium chain N-acyl species of NAPEs. These results suggested the ability of NAPE-PLD to degrade different NAPEs without damaging other membrane phospholipids. Metal analysis revealed the presence of catalytically important zinc in NAPE-PLD. In addition, site-directed mutagenesis studies were addressed to several histidine and aspartic acid residues of NAPE-PLD that are highly conserved within the metallo-beta-lactamase family. Single mutations of Asp-147, His-185, His-187, Asp-189, His-190, His-253, Asp-284, and His-321 caused abolishment or remarkable reduction of the catalytic activity. Moreover, when six cysteine residues were individually mutated to serine, only C224S showed a considerably reduced activity. The activities of L207F and H380R found as single nucleotide polymorphisms were also low. Thus, NAPE-PLD appeared to function through a mechanism similar to those of the well characterized members of this family but play a unique role in the lipid metabolism of animal tissues.     

Enzymatic formation of N-acylethanolamines from N-acylethanolamine plasmalogen through N-acylphosphatidylethanolamine-hydrolyzing phospholipase D-dependent and -independent pathways

Bioactive N-acylethanolamines include anandamide (an endocannabinoid), N-palmitoylethanolamine (an anti-inflammatory), and N-oleoylethanolamine (an anorexic). In the brain, these molecules are formed from N-acylphosphatidylethanolamines (NAPEs) by a specific phospholipase D, called NAPE-PLD, or through NAPE-PLD-independent multi-step pathways, as illustrated in the current study employing NAPE-PLD-deficient mice. Although N-acylethanolamine plasmalogen (1-alkenyl-2-acyl-glycero-3-phospho(N-acyl)ethanolamine, pNAPE) is presumably a major class of N-acylethanolamine phospholipids in the brain, its enzymatic conversion to N-acylethanolamines is poorly understood. In the present study, we focused on the formation of N-acylethanolamines from pNAPEs. While recombinant NAPE-PLD catalyzed direct release of N-palmitoylethanolamine from N-palmitoylethanolamine plasmalogen, the same reaction occurred in the brain homogenate of NAPE-PLD-deficient mice, suggesting that this reaction occurs through both the NAPE-PLD-dependent and -independent pathways. Liquid chromatography-mass spectrometry revealed a remarkable accumulation of 1-alkenyl-2-hydroxy-glycero-3-phospho(N-acyl)ethanolamines (lyso pNAPEs) in the brain of NAPE-PLD-deficient mice. We also found that brain homogenate formed N-palmitoylethanolamine, N-oleoylethanolamine, and anandamide from their corresponding lyso pNAPEs by a Mg(2+)-dependent "lysophospholipase D". Moreover, the brain levels of alkenyl-type lysophosphatidic acids, the other products from lyso pNAPEs by lysophospholipase D, also increased in NAPE-PLD-deficient mice. Glycerophosphodiesterase GDE1 can hydrolyze glycerophospho-N-acylethanolamines to N-acylethanolamines in the brain. In addition, we discovered that recombinant GDE1 has a weak activity to generate N-palmitoylethanolamine from its corresponding lyso pNAPE, suggesting that this enzyme is at least in part responsible for the lysophospholipase D activity. These results strongly suggest that brain tissue N-acylethanolamines, including anandamide, can be formed from N-acylated plasmalogen through an NAPE-PLD-independent pathway as well as by their direct release via NAPE-PLD.     

Endocannabinoid metabolism in the absence of fatty acid amide hydrolase (FAAH): discovery of phosphorylcholine derivatives of N-acyl ethanolamines

Lipid transmitters are tightly regulated by a balance of biosynthetic and degradative enzymes. Termination of the activity of the N-acyl ethanolamine (NAE) class of lipid-signaling molecules, including the endocannabinoid anandamide (AEA), is principally mediated by the integral membrane enzyme fatty acid amide hydrolase (FAAH) in vivo. FAAH(-/-) mice are highly sensitized to the pharmacological effects of AEA; however, these animals eventually recover from AEA treatment, implying the existence of alternative routes for NAE metabolism. Here, we have pursued the characterization of these pathways by profiling the metabolome of FAAH(-/-) mice treated with AEA. Multiple AEA-induced metabolites were observed in brains from FAAH(-/-) mice, including a major product with a mass shift of +165 Da (m/z 513). The structure of this product was determined to be O-phosphorylcholine (PC)-AEA. Analysis of untreated mice identified PC-NAEs as endogenous constituents of the central nervous system (CNS) that were highly elevated in FAAH(-/-) animals. PC-NAEs were very poor substrates for FAAH; however, a vanadate-sensitive enzymatic activity was detected in brain membranes that converted PC-NAEs back to their parent NAEs. The choline-specific phosphodiesterase NPP6 was identified as a candidate enzyme responsible for this activity. These data indicate the presence of a complete metabolic pathway for the production and degradation of PC-NAEs in the CNS that constitutes an alternative route for endocannabinoid metabolism.     

Determination of the phospholipid precursor of anandamide and other N-acylethanolamine phospholipids before and after sodium azide-induced toxicity in cultured neocortical neurons

Phospholipase D-mediated hydrolysis of N-acylethanolamine phospholipids (NAPEs) releases anandamide and other N-acylethanolamines, resulting in different actions at cellular targets in the CNS. Recently, we have demonstrated that these N-acyl lipids accumulate in cultured neocortical neurons subjected to sodium azide-induced cell injury. We here extend the information on the NAPE response, reporting on the composition of N-acylspecies of NAPE, employing a new methodological approach of HPLC-coupled electrospray ionization mass spectrometry. Exposure to sodium azide (5 mM) increased the total amount of NAPE threefold over control levels; however, no alteration of the relative composition of NAPE species was detected. The anandamide precursor (20 : 4-NAPE) constituted only 0.1% of all NAPEs detected in the neurons. Total NAPE species in control cells amounted to 956-1,060 pmol/10(7) cells. Moreover, we detected the presence of an unknown NAPE species with molecular weight identical to 20 : 4-NAPE. This may suggest the presence of a putative stereoisomer of the anandamide precursor with at least one trans-configured double bond in the N-arachidonoyl moiety. These results show that with the present method, neuronal NAPE species can be identified and quantified with respect to N-acyl composition, including a trans-isomer of the anandamide precursor. The anandamide precursor is up-regulated to the same extent as other NAPEs upon neuronal injury.