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.     

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.     

Mammalian enzymes responsible for the biosynthesis of N-acylethanolamines

Bioactive N-acylethanolamines (NAEs) are ethanolamides of long-chain fatty acids, including palmitoylethanolamide, oleoylethanolamide and anandamide. In animal tissues, NAEs are biosynthesized from membrane phospholipids. The classical “transacylation-phosphodiesterase” pathway proceeds via N-acyl-phosphatidylethanolamine (NAPE), which involves the actions of two enzymes, NAPE-generating Ca²⁺-dependent N-acyltransferase (Ca-NAT) and NAPE-hydrolyzing phospholipase D (NAPE-PLD). Recent identification of Ca-NAT as Ɛ isoform of cytosolic phospholipase A₂ enabled the further molecular biological approaches toward this enzyme. In addition, Ca²⁺-independent NAPE formation was shown to occur by N-acyltransferase activity of a group of proteins named phospholipase A/acyltransferases (PLAAT)-1–5. The analysis of NAPE-PLD-deficient mice confirmed that NAEs can be produced through multi-step pathways bypassing NAPE-PLD. The NAPE-PLD-independent pathways involved three members of the glycerophosphodiesterase (GDE) family (GDE1, GDE4 and GDE7) as well as α/β-hydrolase domain-containing protein (ABHD)4. In this review article, we will focus on recent progress made and latest insights in the enzymes involved in NAE synthesis and their further characterization.     

Inactivation of N-acyl phosphatidylethanolamine phospholipase D reveals multiple mechanisms for the biosynthesis of endocannabinoids

N-Acyl ethanolamines (NAEs) constitute a large and diverse class of signaling lipids that includes the endogenous cannabinoid anandamide. Like other lipid transmitters, NAEs are thought to be biosynthesized and degraded on-demand rather than being stored in vesicles prior to signaling. The identification of enzymes involved in NAE metabolism is therefore imperative to achieve a complete understanding of this lipid signaling system and control it for potential therapeutic gain. Recently, an N-acyl phosphatidylethanolamine phospholipase D (NAPE-PLD) was identified as a candidate enzyme involved in the biosynthesis of NAEs. Here, we describe the generation and characterization of mice with a targeted disruption in the NAPE-PLD gene [NAPE-PLD(-/-) mice]. Brain tissue from NAPE-PLD(-/-) mice showed more than a 5-fold reduction in the calcium-dependent conversion of NAPEs to NAEs bearing both saturated and polyunsaturated N-acyl chains. However, only the former group of NAEs was decreased in level in NAPE-PLD(-/-) brains, and these reductions were most dramatic for NAEs bearing very long acyl chains (>or=C20). Further studies identified a calcium-independent PLD activity in brains from NAPE-PLD(-/-) mice that accepted multiple NAPEs as substrates, including the anandamide precursor C20:4 NAPE. The illumination of distinct enzymatic pathways for the biosynthesis of long chain saturated and polyunsaturated NAEs suggests a strategy to control the activity of specific subsets of these lipids without globally affecting the function of the NAE family as a whole.     

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.     

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.     

Age dependent accumulation of N-acyl-ethanolamine phospholipids in ischemic rat brain. A (31)P NMR and enzyme activity study

N-acyl-ethanolamine phospholipids (NAPE) can be formed as a stress response during neuronal injury, and they are precursors for N-acyl-ethanolamines (NAE), some of which are endocannabinoids. The levels of NAPE accumulated during post-decapitative ischemia (6 h at 37 degrees C) were studied in rat brains of various age (1, 6, 12, 19, 30, and approximately 70 days) by the use of (31)P NMR spectroscopy of lipid extracts. This ability to accumulate NAPE was compared with the activity of N-acyltransferase and of NAPE-hydrolyzing phospholipase D (NAPE-PLD) in brain microsomes. These two enzymes are involved in the formation and degradation of NAPE, respectively.The results showed that 1) the ability to accumulate NAPE during post-decapitative ischemia is especially high in the youngest rats and is markedly reduced in older brains [in 1-day-old rat brains NAPE accumulated to 1.5% of total phospholipids, while in 30-day-old rat brains NAPE accumulation could not be detected (detection limit 0.09%)] and 2) this age pattern of accumulation can be explained by a combination of the decreased activity of N-acyltransferase and the increased activity of NAPE-PLD during development. These results point out that it would be advantageous to investigate a potential cytoprotective role of NAPE in the brains of very young rats.     

Biology of endocannabinoid synthesis system

Endocannabinoids (endogenous ligands of cannabinoid receptors) exert diverse physiological and pathophysiological functions in animal tissues. N-Arachidonoylethanolamine (anandamide) and 2-arachidonoylglycerol (2-AG) are two representative endocannabinoids. Both the compounds are arachidonic acid-containing lipid molecules generated from membrane glycerophospholipids, but their biosynthetic pathways are totally different. Anandamide is principally formed together with other N-acylethanolamines (NAEs) in a two-step pathway, which is composed of Ca²⁺-dependent N-acyltransferase and N-acylphosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD). cDNA cloning of NAPE-PLD and subsequent analysis of its gene-disrupted mice led to the discovery of alternative pathways comprising multiple enzymes. As for the 2-AG biosynthesis, recent results, including cDNA cloning of diacylglycerol lipase and analyses of phospholipase Cβ-deficient mice, demonstrated that these two enzymes are responsible for the in vivo formation of 2-AG functioning as a retrograde messenger in synapses. In this review article, we will focus on recent progress in the studies on the enzymes responsible for the endocannabinoid biosyntheses.     

Enzymological studies on the biosynthesis of N-acylethanolamines

Ethanolamides of different long-chain fatty acids constitute a class of endogenous lipid molecules generally called N-acylethanolamines (NAEs). They contain N-arachidonoylethanolamine (anandamide), N-palmitoylethanolamine, and N-oleoylethanolamine, which receive considerable attention because of their actions as an endogenous cannabinoid receptor ligand (endocannabinoid), an anti-inflammatory substance, and an appetite-suppressing substance, respectively. Identification of their biosynthetic routes in animal tissues and molecular characterization of the enzymes involved are essential for better understanding of physiological importance of NAEs as well as development of enzyme inhibitors as possible therapeutic drugs. In the classical “transacylation–phosphodiesterase pathway”, NAEs are formed from glycerophospholipids via N-acylphosphatidylethanolamine (NAPE), an unusual derivative of phosphatidylethanolamine with a third acyl chain attached to the amino group, by sequential catalyses by Ca²⁺-dependent N-acyltransferase and NAPE-hydrolyzing phospholipase D. However, recent studies reveal that NAE-generating pathways are more complex than presumed before. In this review article, we will focus on recent findings regarding mammalian enzymes that are involved or might be involved in the biosynthesis of NAEs.     

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.     

Discovery and characterization of a Ca2+-independent phosphatidylethanolamine N-acyltransferase generating the anandamide precursor and its congeners

N-Acylphosphatidylethanolamines (NAPEs) are precursors of bioactive N-acylethanolamines, including the endocannabinoid anandamide. In animal tissues, NAPE is formed by transfer of a fatty acyl chain at the sn-1 position of glycerophospholipids to the amino group of phosphatidylethanolamine (PE), and this reaction is believed to be the principal rate-limiting step in N-acylethanolamine synthesis. However, the Ca2+-dependent, membrane-associated N-acyltransferase (NAT) responsible for this reaction has not yet been cloned. In this study, on the basis of the functional similarity of NAT to lecithin-retinol acyltransferase (LRAT), we examined a possible PE N-acylation activity in two rat LRAT homologous proteins. Upon overexpression in COS-7 cells, one protein, named rat LRAT-like protein (RLP)-1, catalyzed transfer of a radioactive acyl group from phosphatidylcholine (PC) to PE, resulting in the formation of radioactive NAPE. However, the RLP-1 activity was detected mainly in the cytosolic rather than membrane fraction and was little stimulated by Ca2+. Moreover, RLP-1 did not show selectivity with respect to the sn-1 and sn-2 positions of PC as an acyl donor and therefore could generate N-arachidonoyl-PE (anandamide precursor) from 2-arachidonoyl-PC and PE. In contrast, under the same assay conditions, partially purified NAT from rat brain was highly Ca2+-dependent, membrane-associated, and specific for the sn-1-acyl group of PC. RLP-1 mRNA was expressed predominantly in testis among various rat tissues, and the testis cytosol exhibited an RLP-1-like activity. These results reveal that RLP-1 can function as a PE N-acyltransferase, catalytically distinguishable from the known Ca2+-dependent NAT.     

Accumulation of the anandamide precursor and other N-acylethanolamine phospholipids in infant rat models of in vivo necrotic and apoptotic neuronal death

It has been demonstrated that the endogenous cannabinoid receptor ligand, anandamide, and other N-acylethanolamines (NAEs), accumulate during neuronal injury in vitro, a process that may be linked to the neuroprotective effects of NAEs. The crucial step for generation of NAEs is the synthesis of the corresponding precursors, N-acylethanolamine phospholipids (NAPEs). However, it is unknown whether this key event for NAE formation is regulated differently in the context of insults causing necrotic or apoptotic neuronal death. To address this question, we monitored a range of cortical NAPE species in three infant rat models of in vivo neurodegeneration: (i) necrosis caused by intrastriatal injection of NMDA (25 nmol); (ii) apoptosis induced by systemic administration of the NMDA-receptor antagonist (+)MK-801 (3 x 0.5 mg/kg, i.p.); and (iii) apoptosis following focal necrosis triggered by concussive head trauma. A marked increase of all NAPE species was observed in both hemispheres 4 and 24 h after NMDA-induced injury, with a relatively larger increase in N-stearoyl-containing NAPE species. Thus, the percentage of the anandamide precursor fell from 1.1 to 0.5 mol %. In contrast, administration of (+)MK-801 did not alter cortical NAPE levels. Concussion head trauma resulted in a similar but less pronounced upregulation of NAPE levels at both 4 and 24 h as compared to NMDA injections. Increased levels of NAPE 24 h post-trauma possibly reflect that necrosis is still ongoing at this time point. Consequently, our data suggest that excitotoxic necrotic mechanisms of neurodegeneration, as opposed to apoptotic neurodegeneration, have a profound effect on in vivo NAE precursor homeostasis.     

Expression and secretion of N-acylethanolamine-hydrolysing acid amidase in human prostate cancer cells

N-acylethanolamines (NAEs) are a class of bioactive lipid molecules in animal tissues, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. Enzymatic hydrolysis of NAEs is considered to be an important step to regulate their endogenous levels. Lysosomal NAE-hydrolysing acid amidase (NAAA) as well as fatty acid amide hydrolase (FAAH) is responsible for this reaction. Here, we report relatively high expression of NAAA in human prostate cancer cells (PC-3, DU-145 and LNCaP) and prostate epithelial cells (PrEC), with the highest mRNA level in LNCaP cells. FAAH and the NAE-forming enzyme N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) were also detected in these cells. NAAA activity in LNCaP cells could be distinguished from coexisting FAAH activity, based on their different pH dependency profiles and specific inhibition of FAAH activity by URB597. These results showed that both the enzymes were functionally active. We also found that NAAA was partly secreted from LNCaP cells, which underlined possible usefulness of this enzyme as a biomarker of prostate cancer.     

Anandamide and its congeners inhibit human plasma butyrylcholinesterase. Possible new roles for these endocannabinoids?

Butyrylcholinesterase (BChE), a serine hydrolase biochemically related to the cholinergic enzyme Acetylcholinesterase (AChE), is found in many mammalian tissues, such as serum and central nervous system, but its physiological role is still unclear. BChE is an important human plasma esterase, where it has detoxifying roles. Furthermore, recent studies suggest that brain BChE can have a role in Alzheimer’s disease (AD). The endocannabinoid arachidonoylethanolamide (anandamide) and other acylethanolamides (NAEs) are almost ubiquitary molecules and are physiologically present in many tissues, including blood and brain, where they show neuroprotective and anti-inflammatory properties. This paper demonstrates that they are uncompetitive (oleoylethanolamide and palmitoylethanolamide) or non competitive (anandamide) inhibitors of BChE (Ki in the range 1.32-7.48 nM). On the contrary, NAEs are ineffective on AChE kinetic features. On the basis of the X-ray crystallographic structure of human BChE, and by using flexible docking procedures, an hypothesis on the NAE-BChE interaction is formulated by molecular modeling studies. Our results suggest that anandamide and the other acylethanolamides studied could have a role in the modulation of the physiological actions of BChE.     

Regional distribution and age-dependent expression of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D in rat brain

The endocannabinoid anandamide (N-arachidonoylethanolamine) and other bioactive long-chain N-acylethanolamines are thought to be formed from their corresponding N-acylphosphatidylethanolamines by a specific phospholipase D (NAPE-PLD) in the brain as well as other tissues. However, regional distribution of NAPE-PLD in the brain has not been examined. In the present study, we investigated the expression levels of NAPE-PLD in nine different regions of rat brain by enzyme assay, western blotting and real-time PCR. The NAPE-PLD activity was detected in all the tested brain regions with the highest activity in thalamus. Similar distribution patterns of NAPE-PLD were observed at protein and mRNA levels. We also found a remarkable increase in the expression levels of protein and mRNA of the brain NAPE-PLD with development, which was in good agreement with the increase in the activity. The age-dependent increase was also seen with several brain regions and other NAPE-PLD-enriched organs (heart and testis). p-Chloromercuribenzoic acid and cetyltrimethylammonium chloride, which inhibited recombinant NAPE-PLD dose-dependently, strongly inhibited the enzyme of all the brain regions. These results demonstrated wide distribution of NAPE-PLD in various brain regions and its age-dependent expression, suggesting the central role of this enzyme in the formation of anandamide and other N-acylethanolamines in the brain.     

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.     

N-Acylethanolamines: Formation and Molecular Composition of a New Class of Plant Lipids

Recently, the biosynthesis of an unusual membrane phospholipid, N-acylphosphatidylethanolamine (NAPE), was found to increase in elicitor-treated tobacco (Nicotiana tabacum L.) cells (K.D. Chapman, A. Conyers-Hackson, R.A. Moreau, S. Tripathy [1995] Physiol Plant 95: 120–126). Here we report that before induction of NAPE biosynthesis, N-acylethanolamine (NAE) is released from NAPE in cultured tobacco cells 10 min after treatment with the fungal elicitor xylanase. In radiolabeling experiments [¹⁴C]NAE (labeled on the ethanolamine carbons) increased approximately 6-fold in the culture medium, whereas [¹⁴C]NAPE associated with cells decreased approximately 5-fold. Two predominant NAE molecular species, N-lauroylethanolamine and N-myristoylethanolamine, were specifically identified by gas chromatography-mass spectrometry in lipids extracted from culture medium, and both increased in concentration after elicitor treatment. NAEs were found to accumulate extracellularly only. A microsomal phospholipase D activity was discovered that formed NAE from NAPE; its activity in vitro was stimulated about 20-fold by mastoparan, suggesting that NAPE hydrolysis is highly regulated, perhaps by G-proteins. Furthermore, an NAE amidohydrolase activity that catalyzed the hydrolysis of NAE in vitro was detected in homogenates of tobacco cells. Collectively, these results characterize structurally a new class of plant lipids and identify the enzymatic machinery involved in its formation and inactivation in elicitor-treated tobacco cells. Recent evidence indicating a signaling role for NAPE metabolism in mammalian cells (H.H.O. Schmid, P.C. Schmid, V. Natarajan [1996] Chem Phys Lipids 80: 133–142) raises the possibility that a similar mechanism may operate in plant cells.NAPE is a widespread, albeit minor, membrane phospholipid in animal and plant tissues (Schmid et al., 1990; Chapman and Moore, 1993). Its unusual structural features (a third fatty acid moiety linked to the amino head group of PE) impart stabilizing properties to membrane bilayers (Domingo et al., 1994; LaFrance et al., 1997). NAPE and its hydrolysis products, NAEs, are known to accumulate in vertebrate tissues under pathological conditions (for review, see Schmid et al., 1990). Recently, there has been renewed interest in NAEs because of the contention that anandamide (N-arachidonylethanolamine) is an endogenous ligand for the cannabinoid receptor in mammalian brain (Devane et al., 1992; Fontana et al., 1995; Schmid et al., 1996). The likely route for NAE formation in neural and nonneural tissues, although the matter of some debate, is via the signal-mediated hydrolysis of NAPE (DiMarzo et al., 1994; Schmid et al., 1996; Sugiura, et al., 1996).In plants little is known regarding the catabolism of NAPE. In cottonseed microsomes NAPE was metabolized to NAE or NAlysoPE by PLD- or PLA-type activities, respectively (Chapman et al., 1995b). However, the metabolic fate of NAPE in vivo and the factors that regulate NAPE hydrolysis remain largely unknown. We previously noted that the biosynthesis of NAPE was increased in elicitor-treated cell suspensions of tobacco (Nicotiana tabacum L.). Here we extend our investigations with this model system to examine NAPE catabolism by plant cells in vivo. NAE was released from NAPE, and it accumulated extracellularly. We identified by GC-MS these tobacco NAEs as N-lauroylethanolamine and N-myristoylethanolamine. These NAEs were increased in elicitor-treated cell suspensions. Furthermore, we detected the enzymatic machinery capable of the release and the degradation of NAEs in tobacco cells. To our knowledge this represents the first identification of the NAE molecular species in plant cells. It is tempting to speculate that NAPE hydrolysis in elicitor-treated plant cells may be involved in a signaling pathway analogous to that found in mammalian cells.     

Alteration of endocannabinoid system in human gliomas

Endocannabinoids are neuromodulatory lipids that mediate the central and peripheral neural functions. Endocannabinoids have demonstrated their anti-proliferative, anti-angiogenic and pro-apoptotic properties in a series of studies. In the present study, we investigated the levels of two major endocannabinoids, anandamide and 2-arachidonylglycerol (2-AG), and their receptors, CB1 and CB2, in human low grade glioma (WHO grade I-II) tissues, high grade glioma (WHO grade III-IV) tissues, and non-tumor brain tissue controls. We also measured the expressions and activities of the enzymes responsible for anandamide and 2-AG biosynthesis and degradation, that is, N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD), fatty acid amide hydrolase (FAAH), monoacylglycerol lipase (MGL), and diacylglycerol lipase-alpha (DGL), in the same samples. Liquid chromatography-mass spectometry analysis showed that the levels of anandamide decreased, whereas the levels of 2-AG increased in glioma tissues, comparing to the non-tumor controls. The expression levels and activities of NAPE-PLD, FAAH and MGL also decreased in glioma tissues. Furthermore, quantitative-PCR analysis and western-blot analysis revealed that the expression levels of cananbinoid receptors, CB1 and CB2, were elevated in human glioma tissues. The changes of anandamide and 2-AG contents in different stages of gliomas may qualify them as the potential endogenous biomarkers for glial tumor malignancy.     

Emerging physiological roles for N-acylphosphatidylethanolamine metabolism in plants: signal transduction and membrane protection

The activation of N-acylphosphatidylethanolamine (NAPE) metabolism in plants appears to be associated mostly with cellular stresses. In response to pathogen elicitors, NAPE is hydrolzyed by phospholipase-D (PLD), and corresponding medium-chain, saturated N-acylethanolamines (NAEs) are released by plant cells where they act as lipid mediators to modulate ion flux and activate defense gene expression. In desiccated seeds of higher plants, long-chain, saturated and unsaturated NAEs are prevalent, but are rapidly metabolized during the first few hours of imbibition, a period of substantial osmotic stress. NAPE synthesis is increased in seeds during this same period of rapid rehydration. A membrane-bound enzyme designated NAPE synthase has been purified from imbibed cottonseeds and its unusual biochemical properties suggest that it may scavenge free fatty acids in vivo. This feature of NAPE metabolism may be unique to higher plants a may be a mechanism for the rapid recycling of fatty acids back into membrane-associated NAPE. Altogether, increasing evidence indicates that NAPE metabolism in plants shares functional similarities with NAPE metabolism in animal systems, including signal transduction and cellular protection. In particular, the emerging role of released NAEs as lipid mediators in plant defense signaling represents an intriguing parallel to 'endocannabinoid signaling' in several mammalian cell types.     

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.