Formation of N-acyl-phosphatidylethanolamines by cytosolic phospholipase A2ε in an ex vivo murine model of brain ischemia

N-Acyl-phosphatidylethanolamines (NAPEs), a minor class of membrane glycerophospholipids, accumulate along with their bioactive metabolites, N-acylethanolamines (NAEs) during ischemia. NAPEs can be formed through N-acylation of phosphatidylethanolamine by cytosolic phospholipase A₂ε (cPLA₂ε, also known as PLA2G4E) or members of the phospholipase A and acyltransferase (PLAAT) family. However, the enzyme responsible for the NAPE production in brain ischemia has not yet been clarified. Here, we investigated a possible role of cPLA₂ε using cPLA₂ε-deficient (Pla2g4e^?/?) mice. As analyzed with brain homogenates of wild-type mice, the age dependency of Ca²⁺-dependent NAPE-forming activity showed a bell-shape pattern being the highest at the first week of postnatal life, and the activity was completely abolished in Pla2g4e^?/? mice. However, liquid chromatography-tandem mass spectrometry revealed that the NAPE levels of normal brain were similar between wild-type and Pla2g4e^?/? mice. In contrast, post-mortal accumulations of NAPEs and most species of NAEs were only observed in decapitated brains of wild-type mice. These results suggested that cPLA₂ε is responsible for Ca²⁺-dependent formation of NAPEs in the brain as well as the accumulation of NAPEs and NAEs during ischemia, while other enzyme(s) appeared to be involved in the maintenance of basal NAPE levels.     

N-Acylated phospholipid metabolism and seedling growth: Insights from lipidomics studies in Arabidopsis

N-Acylphosphatidylethanolamines (NAPEs) are precursors of endogenous bioactive lipids, N-acylethanolamines (NAEs). NAPEs, which occur as a minor membrane lipid, are hydrolyzed in a single enzymatic step catalyzed by a type of phospholipase D (PLD) to generate fatty acid ethanolamides. Although, the occurrence of NAPE is widespread in the plant kingdom, the physiological roles remain under appreciated due to the lack of sensitive tools to quantify the pathway metabolites. In Kilaru et al. (2012, Planta, DOI 10.1007/s00425-012-1669-z), comprehensive mass spectrometry (MS)-based methods were developed to gain a clearer understanding of the complex network of metabolites that participate in NAE metabolic pathway. This targeted lipidomics approach allowed insights to be drawn into the implications of altered NAE levels on NAPE content and composition, and the overall regulation of PLD-mediated hydrolysis in Arabidopsis. Based on these results, we point out here the important need for the identification of the precise isoform(s) of PLD in plants that is (are) involved in the regulated hydrolysis of NAPE and formation of NAE lipid mediators in vivo.     

Intracellular Ca2+-dependent formation of N-acyl-phosphatidylethanolamines by human cytosolic phospholipase A2ε

N-Acyl-phosphatidylethanolamines (NAPEs) are known to be precursors of bioactive N-acylethanolamines (NAEs), including the endocannabinoid arachidonoylethanolamide (anandamide) and anti-inflammatory palmitoylethanolamide. In mammals, NAPEs are produced by N-acyltransferases, which transfer an acyl chain from the sn-1 position of glycerophospholipid to the amino group of phosphatidylethanolamine (PE). Recently, the ɛ isoform of cytosolic phospholipase A₂ (cPLA₂ɛ) was found to be Ca²⁺-dependent N-acyltransferase. However, it was poorly understood which types of phospholipids serve as substrates in living cells. In the present study, we established a human embryonic kidney 293 cell line, in which doxycycline potently induces human cPLA₂ɛ, and used these cells to analyze endogenous substrates and products of cPLA₂ɛ with liquid chromatography-tandem mass spectrometry. When treated with doxycycline and Ca²⁺ ionophore, the cells produced various species of diacyl- and alkenylacyl-types of NAPEs as well as NAEs in large quantities. Moreover, the levels of diacyl- and alkenylacyl-types of PEs and diacyl-phosphatidylcholines (PCs) decreased, while those of lysophosphatidylethanolamines and lysophosphatidylcholines increased. These results suggested that cPLA₂ɛ Ca²⁺-dependently produces NAPEs by utilizing endogenous diacyl- and alkenylacyl-types of PEs as acyl acceptors and diacyl-type PCs and diacyl-type PEs as acyl donors.     

Structure and phase behavior of O-stearoylethanolamine: A combined calorimetric, spectroscopic and X-ray diffraction study

Recent studies show that O-acylethanolamines (OAEs), structural isomers of the putative stress-fighting lipids, namely N-acylethanolamines (NAEs), can be derived from NAEs and are present in biological membranes under physiological conditions. In view of this, we have synthesized O-stearoylethanolamine (OSEA) as a representative OAE and investigated its phase behavior and crystal structure. The thermotropic phase transitions of OSEA dispersed in water and in 150 mM NaCl were characterized using calorimetric, spectroscopic, turbidimetric and X-ray diffraction studies. These studies have revealed that when dispersed in water OSEA undergoes a cooperative phase transition centered at 53.8 °C from an ordered gel phase to a micellar structure whereas in presence of 150 mM NaCl the transition temperature increases to 55.8 °C and most likely the bilayer structure is retained above the phase transition. O-Stearoylethanolamine crystallized in the orthorhombic space group P2₁2₁2₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that OSEA molecules adopt a linear structure with all-trans conformation in the acyl chain region. The molecules are organized in a tail-to-tail fashion, similar to the arrangement in a bilayer membrane. These studies are relevant to understanding the role of salt on the phase properties of this new class of lipids.     

Discovery and Characterization of an Arabidopsis thaliana N-Acylphosphatidylethanolamine Synthase

N-Acylethanolamines (NAEs) are lipids involved in several physiological processes in animal and plant cells. In brain, NAEs are ligands of endocannabinoid receptors, which modulate various signaling pathways. In plant, NAEs regulate seed germination and root development, and they are involved in plant defense against pathogen attack. This signaling activity is started by an enzyme called N-acylphosphatidylethanolamine (NAPE) synthase. This catalyzes the N-acylation of phosphatidylethanolamine to form NAPE, which is most likely hydrolyzed by phospholipase D β/γ isoforms to generate NAE. This compound is further catabolized by fatty amide hydrolase. The genes encoding the enzymes involved in NAE metabolism are well characterized except for the NAPE synthase gene(s). By heterologous expression in Escherichia coli and overexpression in plants, we characterized an acyltransferase from Arabidopsis thaliana (At1g78690p) catalyzing the synthesis of lipids identified as NAPEs (two-dimensional TLC, phospholipase D hydrolysis assay, and electrospray ionization-tandem mass spectrometry analyses). The ability of free fatty acid and acyl-CoA to be used as acyl donor was compared in vitro with E. coli membranes and purified enzyme (obtained by immobilized metal ion affinity chromatography). In both cases, NAPE was synthesized only in the presence of acyl-CoA. β-Glucuronidase promoter experiments revealed a strong expression in roots and young tissues of plants. Using yellow fluorescent protein fusion, we showed that the NAPE synthase is located in the plasmalemma of plant cells.N-Acylethanolamines (NAEs)² are bioactive lipids composed of an ethanolamine headgroup amide-linked to an acyl chain varying in length and degree of saturation. In animals, NAEs are involved in different physiological processes, such as neuroprotective action (1), embryo development (2), cell proliferation (3), apoptosis (4), nociception, anxiety, inflammation, appetite/anorexia, learning, and memory (for review, see Ref. 5). Most studies carried out with animal cells/tissues have focused on N-arachidonoylethanolamine (anandamide, NAE20:4), which is synthesized in brain neurons but also, under certain conditions, in macrophage cells (6). NAE20:4 binds CB1 cannabinoid receptors located in brain neurons (7) and also acts as ligand of vanilloid receptors for pain modulation (8). In addition, it has been shown that NAE20:4 also promotes food intake, whereas NAE18:0 and NAE18:1 exert anorexic effects by increasing satiety (9–11). NAE16:0 is accumulated during inflammation and has several anti-inflammatory effects (for a review, see Ref. 12).In plants, NAEs are thought to be involved in various physiological functions. For example, because NAE levels observed in various dry seeds decline rapidly after imbibition, a possible role of these compounds in the regulation of seed germination has been proposed (13). It was further observed that the addition of 25 μm NAE12:0 to growth medium of Arabidopsis thaliana leads to a decrease in the size of the main and lateral roots and in root hair formation. This reduction in growth was associated with a modification of cytoskeletal organization (14). NAE12:0 is also able to delay cut Dianthus caryophyllus (carnation) senescence by decreasing oxidative damage and enhancing antioxidant defense (15), whereas NAE14:0 inhibits the elicitor-induced medium alkalinization and activates phenylalanine ammonia lyase gene expression involved in plant defense against pathogen attack (16).Both in plant and animal cells (for a review, see Ref. 17), NAEs are formed by the hydrolysis (by PLDs) of N-acylphosphatidylethanolamine (NAPE). NAPE is an unusual derivative of phosphatidylethanolamine (PE) with a third fatty acid linked to the amine position of the ethanolamine headgroup. In animals, the formation of NAEs is catalyzed by a PLD with a high specificity toward NAPE (NAPE-PLD). In plants, PLDβ and PLDγ isoforms, but not PLDα, hydrolyzed NAPE into NAE in vitro, and this is thought to operate in response to several biotic and abiotic stresses. Both in animals and in plants, NAEs signaling is terminated by the action of fatty acid amide hydrolases, which hydrolyze NAEs to free fatty acid and ethanolamine. FAAH has been identified and characterized in mammals and plants (for a review, see Ref. 17). In Arabidopsis, FAAH has been shown to modulate NAE content. Moreover, lines overexpressing FAAH displayed enhanced seedling growth as well as increased cell size (18) and were also more susceptible to bacterial pathogens (19).Although the role of NAEs and their catabolism have been extensively investigated, little is known about their precursors, the NAPEs. NAPEs represent a minor phospholipid class but are present in all tissues of plants and animals. The principal function of NAPEs is to serve as a precursor for the production of lipid mediator NAEs, but it has also been suggested that NAPEs could serve as a membrane stabilizer to maintain cellular compartmentalization during tissue damage (20). More recently, N-palmitoyl-PE was proposed to act as an inhibitor of macrophage phagocytosis through inhibition of the activation of Rac1 and Cdc42 (21).In the animal and plant kingdoms, therefore, the signaling events mediated by NAEs appear to be involved in many physiological processes that have been extensively studied. The genes encoding the enzymes involved in the synthesis (from NAPEs) and the degradation of NAEs have been cloned and characterized. By contrast, little is known about the physiological roles of NAPEs or about the first step of this lipid signaling pathway, namely the N-acylation of PE to form NAPEs. In animals, the synthesis of NAPEs is catalyzed by an N-acyltransferase, where the O-linked acyl unit from a phospholipid donor is transferred to the ethanolamine headgroup of PE (22). Recently, a rat LRAT-like protein 1 or RLP1 was shown to display such an activity, but according to the authors, RLP-1 can function as a PE N-acyltransferase, catalytically distinguishable from the known Ca²⁺-dependent N-acyltransferase (23). However, a different situation is observed in plants. NAPE synthase activity was shown to directly acylate PE with free fatty acids (24, 25), but a gene encoding a NAPE synthase activity remained unidentified until now. The present work shows that the A. thaliana acyltransferase At1g78690p catalyzes the synthesis of NAPEs from PE and acyl-CoAs in vitro as well as in vivo when this enzyme is expressed in E. coli and overexpressed in plants.     

N-Acylethanolamines and precursor phospholipids - relation to cell injury

The present review focuses on the relationship between formation of N-acylethanolamine phospholipids (NAPEs) and N-acyletransferase (NAEs) catalyzed by N-acyltranferase and NAPE-hydrolyzing phospholipase D, respectively, and cell injury in tissues like brain, heart, and testis. A number of mechanisms are proposed by which these two groups of lipids may have cytoprotective properties. The mechanisms may involve activation of cannabinoid receptors, as well as non-receptor-mediated effects such as stabilization of membrane bilayers, antioxidant mechanisms, inhibition of calcium leakage from mitochondria, and direct inhibition of ceramidase. Anandamide (20:4-NAE) is formed as a minor component along with other NAEs during cell injury. Whether 20:4-NAE has a separate physiological role is at present not known, but some data suggest that 20:4-NAE may be formed, e.g. in the uterus, by a more selective mechanism without being accompanied by a vast majority of saturated and monounsaturated NAEs.     

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.     

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.     

N-acyl phosphatidylethanolamines affect the lateral distribution of cholesterol in membranes

N-Acyl phosphatidylethanolamines are negatively charged phospholipids, which are naturally occurring albeit at low abundance. In this study, we have examined how the amide-linked acyl chain affected the membrane behavior of the N-acyl-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-POPE) or N-acyl-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-DPPE), and how the molecules interacted with cholesterol. The gel → liquid crystalline transition temperature of sonicated N-acyl phosphatidylethanolamine vesicles in water correlated positively with the number of palmitic acyl chains in the molecules. Based on diphenylhexatriene steady state anisotropy measurements, the presence of 33 mol% cholesterol in the membranes removed the phase transition from N-oleoyl-POPE bilayers, but failed to completely remove it from N-palmitoyl-DPPE and N-palmitoyl-POPE bilayers, suggesting rather weak interaction of cholesterol with the N-saturated NAPEs. The rate of cholesterol desorption from mixed monolayers containing N-palmitoyl-DPPE and cholesterol (1:1 molar ratio) was much higher compared to cholesterol/DPPE binary monolayers, suggesting a weak cholesterol interaction with N-palmitoyl-DPPE also in monolayers. In bilayer membranes, both N-palmitoyl-POPE and N-palmitoyl-DPPE failed to form sterol-rich domains, and in fact appeared to displace sterol from sterol/N-palmitoyl-sphingomyelin domains. The present data provide new information about the effects of saturated NAPEs on the lateral distribution of cholesterol in NAPE-containing membranes. These findings may be of relevance to neural cells which accumulate NAPEs during stress and cell injury.     

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.     

Pitfalls in the sample preparation and analysis of N-acylethanolamines

N-acylethanolamines (NAEs) are a group of lipid mediators synthesized in response to a number of physiological and pathological stimuli. Because of the low tissue concentrations of NAEs, analyses often include liquid extraction followed by solid-phase extraction and subsequent quantitation by LC/MS or GC/MS. Reported levels of NAEs vary considerably, however, and often no explanation is given for these discrepancies. Brought on by difficulties encountered during method development, the effects of using four different brands of silica-containing solid phase extraction (SPE) columns and five different brands of chloroform for sample preparation were investigated. Considerable variation in the retention and recoveries of seven NAEs and 2-arachidonoylglycerol existed between the SPE columns. Furthermore, it was found that some chloroforms contained quantifiable amounts of N-palmitoylethanolamine and N-stearoylethanolamine. Finally, it was found that use of one of the chloroforms resulted in a loss of N-oleoylethanolamine from solution due to addition of chlorine to the ω-9 bond. The identity of this reaction product was confirmed by LC-MS/MS and NMR. It is recommended that these aspects of sample preparation and analysis should be thoroughly validated during method development and the relevant information on specific brands used be reported in future communications in order to better estimate the validity of reported quantitative data.     

Synthesis, calorimetric studies, and crystal structures of N, O-diacylethanolamines with matched chains

Recent studies show that N-, O-diacylethanolamines (DAEs) can be derived by the O-acylation of N-acylethanolamines (NAEs) under physiological conditions. Because the content of NAEs in a variety of organisms increases in response to stress, it is likely that DAEs may also be present in biomembranes. In view of this, a homologous series of DAEs with matched acyl chains (n = 10–20) have been synthesized and characterized. Transition enthalpies and entropies obtained from differential scanning calorimetry show that dry DAEs with even and odd acyl chains independently exhibit linear dependence on the chainlength. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-, O-Didecanoylethanolamine (DDEA), N-, O-dilauroylethanolamine (DLEA), and N-, O-dimyristoylethanolamine (DMEA) crystallized in the orthorhombic space group Pbc₂₁ with four symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that DDEA, DLEA, and DMEA are isostructural and adopt an L-shaped structure with the N-acyl chain and the central ethanolamine moiety being essentially identical to the structure of N-acylethanolamines, whereas the O-acyl chain is linear with all-trans conformation. In all three DAEs, the lipid molecules are organized in a bilayer fashion wherein the N-acyl and O-acyl chains from adjacent layers oppose each other.     

Relationships between the Transition of the Physical Phase of Membrane Lipids and Photosynthetic Parameters in Anacystis nidulans and Lettuce and Spinach Chloroplasts

The transition of the physical phase of lipids in membrane fragments of a blue-green alga Anacystis nidulans was studied by a spin labeling technique. The maximum hyperfine splitting of the electron spin resonance spectrum of the N-oxyl-4′, 4′-dimethyloxazolidine derivative of 5-ketostearic acid plotted against the reciprocal of the absolute temperature gave a discontinuity point that was characteristic of a transition of the physical phase of the hydrocarbon region of membrane lipids. The phase transition appeared at approximately 13 or 24 C in the organisms grown at 28 or 38 C, respectively.     

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.     

Synthesis of Phenoxyacyl-Ethanolamides and Their Effects on Fatty Acid Amide Hydrolase Activity

N-Acylethanolamines (NAEs) are involved in numerous biological activities in plant and animal systems. The metabolism of these lipids by fatty acid amide hydrolase (FAAH) is a key regulatory point in NAE signaling activity. Several active site-directed inhibitors of FAAH have been identified, but few compounds have been described that enhance FAAH activity. Here we synthesized two sets of phenoxyacyl-ethanolamides from natural products, 3-n-pentadecylphenolethanolamide and cardanolethanolamide, with structural similarity to NAEs and characterized their effects on the hydrolytic activity of FAAH. Both compounds increased the apparent V_max of recombinant FAAH proteins from both plant (Arabidopsis) and mammalian (Rattus) sources. These NAE-like compounds appeared to act by reducing the negative feedback regulation of FAAH activity by free ethanolamine. Both compounds added to seedlings relieved, in part, the negative growth effects of exogenous NAE12:0. Cardanolethanolamide reduced neuronal viability and exacerbated oxidative stress-mediated cell death in primary cultured neurons at nanomolar concentrations. This was reversed by FAAH inhibitors or exogenous NAE substrate. Collectively, our data suggest that these phenoxyacyl-ethanolamides act to enhance the activity of FAAH and may stimulate the turnover of NAEs in vivo. Hence, these compounds might be useful pharmacological tools for manipulating FAAH-mediated regulation of NAE signaling in plants or animals.     

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.     

N-acylethanolamine-hydrolyzing acid amidase and fatty acid amide hydrolase inhibition differentially affect N-acylethanolamine levels and macrophage activation

N-acylethanolamines (NAEs) such as N-palmitoylethanolamine and anandamide are endogenous bioactive lipids having numerous functions, including the control of inflammation. Their levels and therefore actions can be controlled by modulating the activity of two hydrolytic enzymes, N-acylethanolamine-hydrolyzing acid amidase (NAAA) and fatty acid amide hydrolase (FAAH). As macrophages are key to inflammatory processes, we used lipopolysaccharide-activated J774 macrophages, as well as primary mouse alveolar macrophages, to study the effect of FAAH and NAAA inhibition, using PF-3845 and AM9053 respectively, on macrophage activation and NAE levels measured by HPLC-MS. Markers of macrophage activation were measured by qRT-PCR and ELISA. Activation of macrophages decreased NAAA expression and NAE hydrolytic activity. FAAH and NAAA inhibition increased the levels of the different NAEs, although with different magnitudes, whether in control condition or following LPS-induced macrophage activation. Both inhibitors reduced several markers of macrophage activation, such as mRNA expression of inflammatory mediators, as well as cytokine and prostaglandin production, with however some differences between FAAH and NAAA inhibition. Most of the NAEs tested – including N-docosatetraenoylethanolamine and N-docosahexaenoylethanolamine – also reduced LPS-induced mRNA expression of inflammatory mediators, again with differences depending on the marker and the NAE, thus offering a potential explanation for the differential effect of the inhibitors on macrophage activation markers. In conclusion, we show different and complementary effects of NAE on lipopolysaccharide-induced macrophage activation. Our results support an important role for inhibition of NAE hydrolysis and NAAA inhibition in particular in controlling macrophage activation, and thus inflammation.     

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.     

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.     

Involvement of acid ceramidase in the degradation of bioactive N-acylethanolamines

Bioactive N-acylethanolamines (NAEs) include palmitoylethanolamide, oleoylethanolamide, and anandamide, which exert anti-inflammatory, anorexic, and cannabimimetic actions, respectively. The degradation of NAEs has been attributed to two hydrolases, fatty acid amide hydrolase and NAE acid amidase (NAAA). Acid ceramidase (AC) is a lysosomal enzyme that hydrolyzes ceramide (N-acylsphingosine), which resembles NAAA in structure and function. In the present study, we examined the role of AC in the degradation of NAEs. First, we demonstrated that purified recombinant human AC can hydrolyze various NAEs with lauroylethanolamide (C12:0-NAE) as the most reactive NAE substrate. We then used HEK293 cells metabolically labeled with [¹⁴C]ethanolamine, and revealed that overexpressed AC lowered the levels of ¹⁴C-labeled NAE. As analyzed with liquid chromatography-tandem mass spectrometry, AC overexpression decreased the amounts of different NAE species. Furthermore, suppression of endogenous AC in LNCaP prostate cells by siRNA increased the levels of various NAEs. Lastly, tissue homogenates from mice genetically lacking saposin D, a presumable activator protein of AC, showed much lower hydrolyzing activity for NAE as well as ceramide than the homogenates from wild-type mice. These results demonstrate the ability of AC to hydrolyze NAEs and suggest its physiological role as a third NAE hydrolase.