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Previous Article | Next Article
Volume 17, Number 4, Issue of February 15, 1997 pp. 1226-1242
Copyright ©1997 Society for NeuroscienceOccurrence and Biosynthesis of Endogenous Cannabinoid Precursor, N-Arachidonoyl Phosphatidylethanolamine, in Rat Brain
Hugues Cadas, Emmanuelle di Tomaso, and Daniele Piomelli The Neurosciences Institute, San Diego, California 92121
ABSTRACT
It has been suggested that anandamide (N-arachidonoylethanolamine), an endogenous cannabinoid substance, may be produced through Ca2+-stimulated hydrolysis of the phosphatidylethanolamine (PE) derivative N-arachidonoyl PE. The presence of N-arachidonoyl PE in adult brain tissue and the enzyme pathways that underlie its biosynthesis are, however, still undetermined. We report here that rat brain tissue contains both anandamide (11 ± 7 pmol/gm wet tissue) and N-arachidonoyl PE (22 ± 16 pmol/gm), as assessed by gas chromatography/mass spectrometry. We describe a N-acyltransferase activity in brain that catalyzes the biosynthesis of N-arachidonoyl PE by transferring an arachidonate group from the sn-1 carbon of phospholipids to the amino group of PE. We also show that sn-1 arachidonoyl phospholipids are present in brain, where they constitute ~0.5% of total phospholipids. N-acyltransferase activity is Ca2+ dependent and is enriched in brain and testis. Within the brain, N-acyltransferase activity is highest in brainstem; intermediate in cortex, striatum, hippocampus, medulla, and cerebellum; and lowest in thalamus, hypothalamus, and olfactory bulb. Pharmacological inhibition of N-acyltransferase activity in primary cultures of cortical neurons prevents Ca2+-stimulated N-arachidonoyl PE biosynthesis. Our results demonstrate, therefore, that rat brain tissue contains the complement of enzymatic activity and lipid substrates necessary for the biosynthesis of the anandamide precursor N-arachidonoyl PE. They also suggest that biosynthesis of N-arachidonoyl PE and formation of anandamide are tightly coupled processes, which may concomitantly be stimulated by elevations in intracellular Ca2+ occurring during neural activity.
Key words: anandamide; N-acylethanolamines; phosphatidylethanolamine; N-acylphosphatidylethanolamine; N-acyltransferase; arachidonate; endogenous cannabinoids
INTRODUCTION
The psychoactive effects of
9-tetrahydrocannabinol, the major pharmacological ingredient of Cannabis sativa, are produced through the activation of selective G protein-coupled membrane receptors (see Howlett, 1995
, for review). The abundant expression of cannabinoid receptors in brain and the behavioral consequences of their activation, ranging in humans from euphoria to memory deficits, underscore the potential importance of the endogenous signaling system by which these receptors are thought to be engaged (Dewey, 1986
A primary candidate for the role of endogenous cannabinoid substance anandamide (N-arachidonoylethanolamine) has been identified recently (Devane et al., 1992
Two such mechanisms have been proposed. Anandamide may be synthesized through the energy-independent condensation of ethanolamine and arachidonate (Deutsch and Chin, 1993
An additional mechanism of anandamide formation, suggested by experiments carried out in cultures of rat brain neurons, is via the phosphodiesterase-mediated cleavage of a phospholipid precursor, N-arachidonoyl phosphatidylethanolamine (PE) (Di Marzo et al., 1994
In the present study, we have examined the occurrence and biosynthesis of N-arachidonoyl PE in adult rat brain tissue. Using highly sensitive and selective gas chromatography/mass spectrometry (GC/MS) techniques, we show that N-arachidonoyl PE and anandamide are constituents of brain lipids. Further, we identify and partially characterize an N-acyltransferase activity in brain that catalyzes the biosynthesis of N-arachidonoyl PE, using as substrates sn-1 arachidonoyl-phospholipids and PE. Even further, we describe a novel brain phospholipid that contains arachidonate at the sn-1 position, and may therefore serve as substrate for such enzyme activity. Finally, we show that stimulation of N-acyltransferase activity satisfactorily accounts for the Ca2+-evoked biosynthesis of N-arachidonoyl PE observed in intact neurons. During the preparation of this manuscript, results very similar to ours were reported in a study by Sugiura et al. (1996b)
MATERIALS AND METHODS
Materials. 1,2-Di[1-14C]arachidonoyl phosphatidylcholine (PC; 110 mCi/mmol; custom synthesized), 1-palmitoyl,2-[1-14C]arachidonoyl phosphatidylethanolamine (PE; 54 mCi/mmol), and [1-3H] ethanolamine hydrochloride (50 Ci/mmol) were from American Radiolabeled Chemicals (St. Louis, MO). 1,2-di[1-14C]palmitoyl PC (112 mCi/mmol), 1-stearoyl,2-[1-14C]palmitoyl PC (56 mCi/mmol), and 1-stearoyl,2-[1-14C]arachidonoyl PC (54 mCi/mmol) were from Amersham (Arlington Heights, IL). Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was from Supelco (Bellefonte, PA). Phospholipase D (PLD type IV, Streptomyces chromofuscus), phospholipase C (PLC type IV, Bacillus cereus), and phospholipase A2 (PLA2, Apis mellifera) were from Sigma (St. Louis, MO). (E)-6-(bromomethylene)-tetrahydro-3-(1-naphthalenyl)2H-pyran-2-one (BTNP) was from Biomol (Plymouth Meeting, PA). Phospholipids were from Avanti Polar Lipids (Alabaster, AL), monoacylglycerols and fatty acyl chlorides from Nu-Check Prep (Elysian, MN), and ionomycin from Calbiochem (San Diego, CA). All other chemicals were from Sigma (St. Louis, MO) or Fluka (Ronkonkoma, NY).
Synthesis and purification of N-acylethanolamines. N-acylethanolamines (NAEs) were synthesized as described previously (Devane et al., 1992
Preparation of rat tissue subcellular fractions. Male Wistar rats (Charles River, Wilmington, MA) were anesthetized with carbon dioxide and sacrificed by decapitation. Brains and other tissues were homogenized in 50 mM Tris buffer, pH 8, containing 0.32 M sucrose. Tissue extracts were centrifuged sequentially at 1000 × g (1 min), 22,000 × g (30 min), and 105,000 × g (60 min). The pellets obtained after the second and third centrifugations were solubilized for 1 hr at 0-4°C in Tris buffer containing 0.5% NP-40, and centrifuged again at 105,000 × g (60 min). In some experiments, individual brain regions were dissected and homogenized in the same buffer. The homogenates were centrifuged at 22,000 × g (30 min), and subcellular fractions were prepared as described above. All tissue samples were stored in liquid nitrogen until used.
Cell cultures. Primary cultures of cortical neurons were prepared from 18-d-old rat embryos, maintained in serum-supplemented culture medium (Huettner and Baughman, 1986
Extraction of N-acyl PEs and NAEs. Rats were sacrificed by decapitation, and the heads were immediately ( 2 sec) immersed in liquid nitrogen. After 10 sec, the heads were removed, and the whole brains were dissected while frozen and homogenized in 20 ml of an ice-cold sucrose-containing Tris buffer (50 mM, pH 8), or in a mixture of methanol/Tris buffer (1:1, 20 ml). Comparable results were obtained with either method. Lipids were extracted twice in a mixture containing chloroform, methanol, and buffer, adjusted to a volume ratio of 2:1:1. Before extraction, synthetic N-myristoylethanolamine (800 pmol) was added to the mixture as an internal standard. In initial experiments, we found that N-myristoylethanolamine was not detectable in rat brain tissue.
Phospholipid extraction. Phospholipids were extracted according to Folch et al. (1957)
Enzymatic digestions. Lipid fractions containing the N-acyl PEs were reconstituted in ethylether and incubated in 40 mM MOPS buffer, pH 5.7, containing 250 U/ml Streptomyces chromofuscus PLD and 15 mM CaCl2. Incubations were carried out for 2 hr at 37°C with shaking. The reaction products were extracted with chloroform/methanol (2:1) and fractionated by column chromatography. Purified brain phospholipids were subjected to sequential enzymatic digestions. They were first digested for 2 hr at 37°C with 50 U/ml Apis mellifera PLA2 in 100 mM Tris buffer, pH 9, containing 10 mM CaCl2. The reaction mixtures were acidified with Tris buffer, pH 4, and extracted with chloroform/methanol. The lysophospholipids produced during these reactions were further digested for 2 hr with 80 U/ml Bacillus cereus PLC in 200 mM phosphate buffer, pH 7, containing 0.4 mM) ZnCl2 and 1 mM) -mercaptoethanol. Sphingomyelin (0.5 mg/ml) was added to these incubations to enhance PLC activity (Wood and Snyder, 1969
Lipid purification and analysis. Brain lipid extracts were subjected to chromatography on silica gel G columns. NAEs and monoacylglycerols were eluted with chloroform/methanol (9:1), and N-acyl PEs with chloroform/methanol (6:4). In some experiments, the N-acyl PE fractions were analyzed by monodimensional TLC, using a solvent system of chloroform/methanol/ammonium hydroxide (80:20:1). Lipids were visualized by spraying the TLC plates with a solution of phosphomolybdic acid in ethanol (10%, w/v) and heating at 150°C for 5 min. Plastic-backed TLC plates were cut into 1 cm bands, and radioactivity in the bands was measured by liquid scintillation counting. In other experiments, N-acyl PEs were analyzed by bidimensional high-performance TLC (HPTLC, system 1: chloroform/methanol/ammonium hydroxide, 80:20:1; system 2: chloroform/methanol/acetic acid, 80:20:1), visualized by autoradiography using a Phosphor-Imager 445 SI apparatus (Molecular Dynamics, Sunnyvale, CA), and identified by comigration with an authentic standard. Yet in other experiments, N-acyl PEs were analyzed by reversed-phase HPLC with the use of a µBondapak C18 column (3.9 mm × 30 cm, Waters Associates) eluted with a gradient of water in methanol (from 30 to 0% over 10 min) at a flow rate of 1.5 ml/min. NAEs were purified after column chromatography by normal-phase HPLC with the use of a Resolve silica column (3.9 mm × 15 cm, 5 µm, Waters Associates) eluted with a gradient of 2-propanol in n-hexane (from 0 to 20% over 20 min) at a flow rate of 1.5 ml/min. NAEs were eluted from the HPLC at 12-13 min and collected in glass reaction vessels (Supelco, Bellefonte, PA) for chemical derivatization. NAEs and monoacylglycerols were derivatized by treatment with BSTFA (15-30 min at room temperature), dried under nitrogen, and resuspended in n-hexane. The trimethylsilylether (TMS) derivatives were injected in the splitless mode into a Hewlett-Packard 5890 GC equipped with an HP-5MS capillary column (30 m; internal diameter, 0.25 mm), and interfaced with a Hewlett-Packard 5972 MS. One minute after the injection, the oven temperature was increased from 150°C to 280°C at a rate of 8°C/min. The injector temperature was kept at 250°C, and helium was used as carrier gas. To improve recovery of the NAEs, and to allow for their quantitative analysis, we included 800 pmol synthetic N-myristoylethanolamine in all samples as an internal standard. The TMS derivative of N-myristoylethanolamine was eluted from the GC at a retention time of ~13.5 min, and its electron impact mass spectrum displayed a series of informative ions which included: m/z 343 (M+, molecular ion); m/z 328 (M-15, loss of a methyl group); m/z 300 (M-43, loss of a propyl group); m/z 253 (M-90, loss of [HO-TMS]+); and m/z 175 (McLafferty rearrangement). For selected ion monitoring (SIM) analyses, we chose the fragment at m/z 328 (M-15). With this ion, the MS response was linear (r2 = 0.98) when amounts of N-myristoylethanolamine-TMS ranging between 0.2 and 400 pmol were injected into the GC.
N-acyltransferase assay. Standard N-acyltransferase assays were carried out for 1 hr at 37°C in 0.5 ml of Tris buffer (50 mM, pH 8), to which we added 0.3 mg protein, 3 mM CaCl2, and 1 × 106 dpm radioactive substrates. Parallel incubations carried out in the absence of tissue contained on average 100 dpm/sample; these blank values were subtracted in the calculations of enzyme activity. Incubations were stopped by adding chloroform/methanol (2:1), and the N-acyl PEs were fractionated by column chromatography. In some experiments, to verify that all the radioactive material eluting in the N-acyl PE fractions comigrated with authentic N-acyl PE, samples were analyzed by TLC or HPTLC.
Identification of N-acyltransferase by fast protein liquid chromatography (FPLC). Crude brain particulate fractions containing N-acyltransferase activity were solubilized and analyzed by FPLC (Pharmacia, Piscataway) using a MonoQ column (HR 10/10). The column was equilibrated at 2 ml/min with 20 mM Tris buffer, pH 8.1, containing 0.05% NP-40 and 3 mM CaCl2. Proteins were eluted with a gradient of NaCl (from 50 mM to 1 M). N-acyltransferase activity was measured in samples of the eluted fractions under standard conditions. The assay mixtures contained 1,2-di[14C]palmitoyl PC, 32 µg/ml PC, 42 µg/ml PE, and 26 µg/ml phosphatidylserine.
RESULTS
N-Arachidonoyl PE and other N-acyl PEs are normal constituents of rat brain lipids
The levels of N-acyl PEs in brain tissue increase dramatically as a result of hypoxic insults (Natarajan et al., 1981
Fig. 1. A, Identification of brain N-acyl PEs by bidimensional HPTLC. Phosphomolybdic acid staining revealed the presence of a lipid component, indicated by the arrow, with the chromatographic properties of N-acyl PEs. Results are from one experiment, repeated once with identical results. B, Analytical approach used to confirm the identification of brain N-acyl PEs and to determine their molecular composition. Partially purified N-acyl PEs were digested with S. chromofuscus PLD to release the corresponding NAEs. These were subsequently purified by HPLC and analyzed by GC/MS.
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To confirm this identification and to determine the molecular composition of brain N-acyl PEs, in six additional experiments we digested the lipid fractions containing N-acyl PEs with a bacterial PLD (Schmid et al., 1990 
Fig. 2. Electron-impact mass spectra of the TMS derivatives of synthetic anandamide (A), N-palmitoylethanolamine (B), N-stearoylethanolamine (C), and N-oleoylethanolamine (D).
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Fig. 3. Identification of N-arachidonoyl PE and other N-acyl PEs in brain by GC/MS. N-acyl PEs were digested with S. chromofuscus PLD, and the resulting NAEs were analyzed by GC/MS in the SIM mode as TMS derivatives. A, Anandamide, derived from the hydrolysis of N-arachidonoyl PE; B, NAEs derived from the hydrolysis of N-palmitoyl PE, N-stearoyl PE, and N-oleoyl PE. The arrows indicate the retention times of authentic standards. Results are from one experiment, representative of six.
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These results indicate that a family of N-acyl PEs, including N-arachidonoyl PE, are normal constituents of rat brain lipids. By comparison with an internal standard, we estimated that an average of 22 ± 16 pmol of N-arachidonoyl PE were recovered from 1 gm of wet brain tissue after lipid extraction and purification. The percent composition of brain N-acyl PEs determined in these experiments is illustrated in Table 1.
Table 1. Molecular composition of rat brain N-acyl PEs
N-acyl PE Amount (% total ± SEM) N-palmitoyl 83.9 ± 1 N-stearoyl 10.6 ± 1 N-oleoyl 4.8 ± 0.3 N-arachidonoyl 0.6 ± 0.1 Analyses of rat brain N-acyl PEs were carried out as described in Materials and Methods. Results are expressed as percent of total N-acyl PEs present, and represent the mean ± SEM of six experiments. Anandamide and other NAEs are also present in brain tissue
We next examined whether rapidly frozen brain tissue contains anandamide and other NAEs. We purified the NAEs by column chromatography and normal-phase HPLC, and analyzed them by GC/MS. In six experiments, a component of the HPLC-purified brain extract was eluted at a retention time of 18.7 min, identical to that of synthetic anandamide (Fig. 4). Diagnostic ions were detected at m/z 419, m/z 404, and m/z 328 (Fig. 4A and data not shown). We also observed ions typical of three additional NAEs (Fig. 4B; compare with the mass spectra of synthetic NAEs shown in Fig. 2, B-D). In these experiments, we measured an average of 11 ± 7 pmol of anandamide/gm wet tissue, accounting for ~8% of the total NAEs detected (Table 2).
Fig. 4. Identification of anandamide and other NAEs in brain by GC/MS. The NAEs were purified chromatographically and analyzed by GC/MS in the SIM mode as TMS derivatives. A, Anandamide; B, additional NAEs. The arrows indicate the retention times of authentic standards. Results are from one experiment, representative of six.
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Table 2. Molecular composition of rat brain N-acylethanolamines
NAE Amount (% total ± SEM) N-palmitoyl 65.5 ± 6 N-stearoyl 19.7 ± 4 N-oleoyl 7.6 ± 5 Anandamide 8.1 ± 5 Analyses of rat brain NAEs were carried out as described in Materials and Methods. Results are expressed as percent of total NAEs present, and represent the mean ± SEM of six experiments. Enzymatic biosynthesis of N-arachidonoyl PE
We have shown that small amounts of the putative anandamide precursor, N-arachidonoyl PE, are present in brain tissue. To explore the physiological roles of this lipid, it was first essential to characterize the biochemical mechanisms responsible for its biosynthesis. We examined, therefore, whether N-arachidonoyl PE may be produced de novo in brain subcellular fractions. We solubilized crude particulate fractions and incubated them for 60 min with or without 3 mM CaCl2. When we analyzed the lipid extracts of Ca2+-containing incubations, we noted a lipid component that comigrated with N-acyl PEs on HPTLC (Fig. 5A). In contrast, this component was not detectable in Ca2+-free incubations (Fig. 5B) or in incubations of heat-inactivated fractions (data not shown). These results are in agreement with studies by Schmid et al. (1990)
Fig. 5. Brain particulate fractions contain an enzymatic activity that catalyzes the biosynthesis of N-acyl PEs. Particulate fractions were solubilized with the nonionic detergent, NP-40, and incubated either with 3 mM Ca2+ (A) or without Ca2+ (B). The lipids were analyzed by HPTLC and visualized with phosphomolybdic acid. A lipid component with the chromatographic properties of synthetic N-acyl PEs, indicated by the arrow, was present only in the Ca2+-containing incubations. Results are from one experiment, representative of four.
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Was N-arachidonoyl PE also produced in these incubations? To address this question, we partially purified the N-acyl PEs; digested them with bacterial PLD; and analyzed, by GC/MS, the NAEs produced. In seven experiments, we found components that eluted from the GC at the retention times of anandamide, N-palmitoylethanolamine, N-stearoylethanolamine, and N-oleoylethanolamine. Because the NAEs were present in relatively high amounts (>100 pmol/sample), we were able to collect complete mass spectra for each of them (Fig. 6). Comparison of these mass spectra with those of synthetic NAEs (shown in Fig. 1) unambiguously confirmed the identification of these compounds. 
Fig. 6. Identification of N-arachidonoyl PE and other N-acyl PEs produced by brain detergent-solubilized particulate fractions. The fractions (4.5 mg of protein) were incubated for 1 hr at 37°C, and the lipids were extracted. N-acyl PEs were partially purified and digested with PLD, and the NAEs produced in the digestions were analyzed by GC/MS as TMS derivatives. Complete mass spectra were obtained for each product. Results are from one experiment, repeated once with identical results.
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Consequently, our results demonstrate that brain particulate fractions incubated in the presence of Ca2+ produce a family of N-acyl PEs, which include N-arachidonoyl PE. A quantitative account of these findings is presented in Table 3. In addition to N-acyl PEs, the four NAEs identified in brain tissue (anandamide, N-palmitoylethanolamine, N-stearoylethanolamine, and N-oleoylethanolamine) were also generated in these incubations, as determined by GC/MS analysis (data not shown).
Table 3. Molecular composition of N-acyl PEs produced in rat brain particulate fraction
N-acyl PE Amount (% total ± SEM) N-palmitoyl 43.7 ± 1.5 N-stearoyl 46.1 ± 1.7 N-oleoyl 9.4 ± 0.6 N-arachidonoyl 0.1 ± 0.02 Analyses of the N-acyl PEs produced in particulate fractions were carried out as described in Materials and Methods. Results are expressed as percent of total N-acyl PEs produced, and represent the mean ± SEM of seven experiments. An N-acyltransferase activity catalyzes the biosynthesis of N-arachidonoyl PE and other N-acyl PEs
Previous studies with various preparations of neural tissue have suggested that N-acyl PE biosynthesis is mediated by an N-acyltransferase activity, which transfers a fatty acyl group from the sn-1 position of phospholipids to the amino group of PE (Natarajan et al., 1986
Fig. 7. A Ca2+-dependent N-acyltransferase activity is present in particulate fractions. The detergent-solubilized fractions were incubated with 3 mM Ca2+ (A, C) or without Ca2+ (B, D), in a medium containing 1,2-di[14-C]palmitoyl PC as substrate. Radioactive products were identified by HPTLC (A, B) or by reversed-phase HPLC (C, D).
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Table 4. Subcellular distribution of rat brain N-acyltransferase activity
Fraction Substrate di[14C] (16:0) di[14C] (20:4) 2-[14C] (16:0) 2-[14C] (20:4) Homogenate 10.0 ± 2.8 0.5 ± 0.2 ND ND Supernatant 1 0.8 ± 0.4 0.1 ± 0.1 ND ND Particulate 25.3 ± 4.7 1.8 ± 0.5 ND ND Soluble 73.8 ± 12.4 5.4 ± 2.2 0.7 ± 0.4 0.1 ± 0.1 Pellet 4.3 ± 0.9 0.5 ± 0.4 ND ND Supernatant 2 0.3 ± 0.2 0.05 ± 0.05 ND ND Microsomes 8.6 ± 0.3 0.4 ± 0.2 ND ND Soluble 23.5 ± 4.2 1 ± 0.5 ND ND Pellet 0.4 ± 0.5 0.2 ± 0.1 ND ND N-acyltransferase assays were carried out under standard conditions (see Materials and Methods). Radioactive N-acyl PEs were purified by monodimensional TLC and quantified by liquid scintillation counting. Results represent the mean ± SEM of 3-4 experiments. di[14C] (16:0), 1,2-di[14]palmitoyl PC; di[14C] (20:4), 1,2-di[14C]arachidonoyl PC; 2-[14C] (16:0), 1-stearoyl,2-[14C]palmitoyl PC; 2-[14C] (20:4), 1-stearoyl,2-[14C]arachidonoyl PC; ND, not determined. 
Fig. 8. Identification of brain N-acyltransferase by FPLC. Samples of the detergent-solubilized particulate fractions were injected into a MonoQ column, and the proteins were eluted with a gradient of NaCl from 50 mM to 1 M. The column eluate was collected in 1 min fractions, and N-acyltransferase activity measured as described in Materials and Methods. Results are from one experiment, representative of three.
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We considered that the biosynthesis of N-arachidonoyl PE may proceed through a similar mechanism. To address this possibility, it was first necessary to show that, in brain tissue, an arachidonoyl-containing phospholipid may serve as fatty acyl donor in the N-acyltransferase reaction, as recently reported in rat testis (Sugiura et al., 1996a
From these observations, we conclude that N-acyltransferase activity in brain can catalyze the transfer to PE of either saturated (e.g., palmitate) or polyunsaturated (e.g., arachidonate) fatty acyl groups, provided that these groups are esterified at the sn-1 position of phospholipids. A novel brain phospholipid serves as precursor for N-arachidonoyl PE
It is generally thought that, in brain as well as other tissues, arachidonate is mainly esterified at the sn-2 position of phospholipids (Shetty et al., 1996
The experimental approach we adopted to identify sn-1 arachidonoyl phospholipids in brain tissue is depicted in Figure 9A. We isolated brain phospholipids by column chromatography, and digested them with Apis mellifera PLA2, an enzyme that selectively hydrolyzes the sn-2 fatty acyl ester bond, yielding sn-1 lysophospholipids (Kuksis et al., 1983 
Fig. 9. Identification of brain sn-1 arachidonoyl phospholipids by GC/MS. A, Schematic illustration of the analytical approach used in these experiments. Phospholipids, purified by column chromatography, were digested with A. mellifera PLA2 to generate sn-1 acyl lysophospholipids. The latter were converted to the corresponding sn-1 monoacylglycerols, by treatment with B. cereus PLC. sn-1 monoacylglycerols were analyzed directly by GC/MS as bis(TMS) derivatives. B, Electron-impact mass spectrum of the bis(TMS) derivative of synthetic sn-1 arachidonoylglycerol. C, Mass spectrum of a brain-derived component with the GC retention time of bis(TMS) sn-1 arachidonoyl glycerol.
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Under our conditions, synthetic sn-1 arachidonoylglycerol was eluted from the GC at 18.3 min and yielded the mass spectrum depicted in Figure 9B. Informative ions in this mass spectrum included: m/z 522 (M+), 507 (M-15, loss of a methyl group), 451 (M-71, loss of a pentyl group), 432 (M-90, loss of [HO-TMS]+), 419 (M-103, loss of [CH2=O-TMS]+, fragmentation typical of sn-1 MAGs), and 329 (M-193, loss of [CH3 CH
CH
Material derived from the enzymatic hydrolysis of brain phospholipids also contained a component that was eluted at 18.3 min, the mass spectrum of which was very similar to that of synthetic sn-1 arachidonoylglycerol, except that it lacked the molecular ion at m/z 522 (Fig. 9C). Analysis by SIM revealed the presence of the molecular ion, however, and confirmed the identification of this component as sn-1 arachidonoylglycerol (Fig. 10). 
Fig. 10. Identification of brain sn-1 arachidonoyl phospholipids by SIM. Three diagnostic ions were chosen from the mass spectrum of the bis(TMS)-derivative of sn-1 arachidonoylglycerol: m/z 522 (M+), m/z 507 (M-15), and m/z 419 (M-103, typical of sn-1 arachidonoylglycerol). The arrow indicates the retention time of standard bis(TMS) sn-1 arachidonoyl glycerol. Results are from one determination, representative of three.
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We conclude from these results that brain tissue contains molecular species of phospholipids in which arachidonate is esterified at the sn-1 position of glycerol. Although further experiments are necessary to identify and quantify precisely brain sn-1 arachidonoyl phospholipids, initial analyses show that they represent ~0.5% of the total brain phospholipid pool (Table 5).
Table 5. Fatty acids at the sn-1 position of brain phospholipids
Fatty acid Amount (% total) Palmitate 19.4 Stearate 56.7 Oleate 23.3 Arachidonate 0.5 Purified brain phospholipids were sequentially digested with A. mellifera PLA2 and B. cereus PLC. The resulting sn-1 monoacylglycerols were identified by GC/MS. Results, expressed in percentage of total sn-1 monoacylglycerols, are from one determination, typical of three. Properties and distribution of N-acyltransferase activity
We investigated some properties of brain N-acyltransferase activity in solubilized particulate fractions, using either 1,2-di[14C]palmitoyl PC or 1,2-di[14C]arachidonoyl PC as fatty acyl donors. With either substrate, the enzyme activity was dependent on protein concentration and incubation time, and was optimal at pH 6 and 8 (data not shown). Ca2+ was necessary for the activity and it could be replaced with Sr2+ and Ba2+, but not with Mg2+ (Table 6). Moreover, several divalent and monovalent cations, including Be2+, Zn2+, and Ag+, were potent in inhibiting the activity (Table 6).
Table 6. Effects of various divalent and monovalent cations on rat brain N-acyltransferase activity
Ion N-acyltransferase activity (%) di-[14C] (16:0) PC di-[14C] (20:4) PC Ca2+ 100 ± 1.8 100 Ba2+ 63 ± 1.6 ND Sr2+ 94 ± 4.6 ND Mg2+ 9 ± 0.6 ND BE2+ 4 ± 0.7 5 Cd2+ 0.2 ± 0.1 6.5 Zn2+ 0.7 ± 0.7 0.8 Ag+ 0.1 ± 1.1 1 Ca2+ plus Ba2+ 95 ± 4.3 ND Ca2+ plus Sr2+ 100 ± 3.6 ND Ca2+ plus Mg2+ 95 ± 4.1 ND Ca2+ plus Be2+ 5 ± 0.8 7 Ca2+ plus Cd2+ 0.1 ± 0.2 5.5 Ca2+ plus Zn2+ 0.2 ± 0.2 2.3 Ca2+ plus Ag+ 0.8 ± 0.3 2.5 EDTA 16 ± 1.6 10 ± 4 EGTA 12 ± 0.9 17 ± 0.8 N-acyltransferase assays were carried out under standard conditions, as described in Materials and Methods. CaCl2 was used at 3 mM. All other cations, EGTA and EDTA, at 10 mM. Results obtained with 1,2-di[14-C]palmitoyl PC are from two experiments carried out in triplicate, in which control N-acyltransferase activity was 179 ± 8 pmol/min/mg protein. Results obtained with 1,2-di[14-C]arachidonoyl PC are from two experiments, in which control N-acyltransferase activity was 22 ± 2 pmol/min/mg protein.
The effects of various protein-modifying reagents on brain N-acyltransferase activity are summarized in Table 7. Chemicals that alkylate serine (e.g., PMSF), cysteine [dithionitrobenzoic acid (DTNB)], and histidine [p-bromophenacylbromide (pBPB)] inhibited the activity, whereas a cystine-reducing agent (DTT) enhanced it significantly. In the study by Sugiura et al. (1996b)
Table 7. Effects of various enzyme inhibitors on rat brain N-acyltransferase activity
Inhibitor Concentration (mM) N-acyltransferase activity (%) di-[14C] (16:0) PC di-[14C] (20:4) PC Control 100 100 DNPP 1 76 ± 0.5 ND DIFP 1 61 ± 1 ND DTNB 1 0.6 ± 0.2 6.5 DTT 1 139 ± 1.2 150 pBPB 0.025 74 ± 0.8 ND 0.1 1 ± 0.3 14.5 BTNP 0.001 91 ± 1 62 ± 1 0.010 13 ± 2 8 ± 3 0.025 13 ± 2 10 ± 1 PMSF 0.1 69 ± 0.3 ND 1 9 ± 1.0 13.5 PBA 1 44 ± 5.2 45 O-Phe 0.2 106 ± 1.0 ND N-acyltransferase assays were carried out under standard conditions, as described in Materials and Methods. Control N-acyltransferase activities and number of experimental determinations are as in Table 6. DNPP, diethyl p-nitrophenylphosphate (Ser-His reagent); DIFP, diisopropylfluorophosphate (Ser-His reagent); DTNB, dithionitrobenzoic acid (Cys reagent); O-Phe, o-phenanthroline (Zn2+ chelator). Drugs were diluted in standard assay buffer from DMSO or methanol stock solutions, yielding maximal solvent concentrations of 0.1%, which had no effect on N-acyltransferase activity.
The distribution of N-acyltransferase activity in particulate fractions from various rat tissues, assayed with either 1,2-di[14C]palmitoyl PC or 1,2-di[14C]arachidonoyl PC, is illustrated in Figure 11A. We found highest levels of activity in brain and testis. Lower but significant levels were observed also in skeletal muscle, whereas little or no activity was detected in other tissues. Within the CNS, differences were less marked: highest levels of N-acyltransferase activity were measured in the brainstem; intermediate levels were measured in the cortex, striatum, cerebellum, hippocampus, and medulla; and lowest levels were measured in olfactory tubercle, thalamus, hypothalamus, and olfactory bulb (Fig. 11B). 
Fig. 11. Distribution of N-acyltransferase activity in detergent-solubilized particulate fractions from various tissues (A) and from various brain regions (B) of the rat. N-acyltransferase assays were carried out under standard conditions (see Materials and Methods), using either 1,2-di[14C]arachidonoyl PC (A1, B1) or 1,2-di[14C]palmitoyl PC (A2, B2) as fatty acyl donor. Radioactive N-acyl PEs were purified by monodimensional TLC (A1, A2) or column chromatography (B1, B2) (see Materials and Methods). TLC purification was used in the experiments on tissue distribution because a PLA2 activity was present in some tissues, which produced large quantities of [14C]fatty acids, interfering with the assay by column chromatography (data not shown). Results are expressed as the mean ± SEM of at least three experiments.
[View Larger Version of this Image (32K GIF file)]
N-Acyltransferase activity mediates N-arachidonoyl PE formation in intact neurons
We have previously reported that, in rat cortical mixed cultures, biosynthesis of N-acyl PEs is enhanced in a Ca2+-dependent manner by ionomycin, a Ca2+ ionophore, or by membrane-depolarizing agents (Cadas et al., 1996a
In a first series of experiments, we used cortical mixed cultures that had been labeled by incubation with [3H]ethanolamine. We stimulated the cultures with 1 µM ionomycin and measured the radioactivity associated with N-acyl PEs after TLC purification. As reported, N-acyl[3H]PE levels in ionomycin-treated cultures were approximately threefold greater than those of untreated controls (Table 8). This effect was almost completely abolished when the neurons were exposed to 25 µM BTNP (Table 8).
Table 8. Effect of N-acyltransferase inhibition on N-acyl[3H]PE biosynthesis in cultures of rat cortical neurons
Condition Concentration (mM) N-acyl[3H]PE (dpm/dish) (%) Control 2825 ± 364 100 Ionomycin 1 9038 ± 1114 320 Ionomycin/BTNP 1/25 4347 ± 562 154 Cultures of rat cortical neurons, prelabeled with [3H]ethanolamine, were incubated for 10 min in DMEM with or without BTNP (25 µM), and then incubated for additional 10 min with ionomycin (1 µM) and BTNP, as indicated. N-[3H]acyl PE were purified by monodimensional TLC and quantified by liquid scintillation counting. Results are from one experiment performed in four separate cultures, repeated once with identical results.
To examine the effects of BTNP on N-arachidonoyl PE biosynthesis, in subsequent experiments we stimulated the cultures, purified the N-acyl PEs, and quantified them by GC/MS after PLD digestion. We found that the cultures contained 0.82 pmol/dish of N-arachidonoyl PE when unstimulated, 2.54 pmol/dish when stimulated with ionomycin, and 0.95 pmol/dish when stimulated with ionomycin in the presence of 25 µM BTNP. Together, these results support an essential role for N-acyltransferase activity in the biosynthesis of N-arachidonoyl PE and other N-acyl PEs in situ.
DISCUSSION
Experiments with rat brain neurons in primary cultures have suggested that anandamide formation may result from the hydrolytic cleavage of a membrane phospholipid precursor, N-arachidonoyl PE (Fig. 12) (Di Marzo et al., 1994
Fig. 12. Hypothetical model of N-arachidonoyl PE biosynthesis in rat brain tissue. Rises in intracellular Ca2+ concentration produced by neuronal activity may stimulate a N-acyltransferase activity, which catalyzes the transfer of arachidonate from sn-1 arachidonoyl phospholipids (possibly PC) to PE. Ca2+ may also stimulate a D-type phosphodiesterase activity (phospholipase D), which cleaves the distal phosphodiester bond of N-arachidonoyl PE, forming anandamide. The mass of N-arachidonoyl PE found in brain is only approximately twofold greater than the mass of anandamide, suggesting that sustained anandamide formation in neurons may require the concomitant stimulation by Ca2+ of N-acyltransferase and phosphodiesterase activities.
[View Larger Version of this Image (15K GIF file)]
Occurrence of anandamide and N-arachidonoyl PE in brain tissue
Like other phospholipid metabolites, N-acyl PEs and NAEs accumulate rapidly in the CNS after the inception of an ischemic injury (Schmid et al., 1995
On average, we measured 11 ± 7 pmol of anandamide/gm of wet brain tissue, a value very similar to those obtained very recently by Schmid et al. (1995
Estimating the content of anandamide in the CNS may be useful to delineate its possible roles in neural signaling. The low amounts measured in our study accord better with those obtained with lipid mediators (e.g., the eicosanoids, produced in stimulated brain tissue at 10-100 pmol/gm) and neuropeptides (e.g., vasoactive intestinal peptide, present at 1-150 pmol/gm), than with those obtained with neurotransmitters (e.g., -aminobutyric acid,
1 µmol/gm; dopamine,
A comparison of the brain levels of anandamide with those of N-arachidonoyl PE may help clarify the metabolic relationship between these lipid products. The content of N-arachidonoyl PE (22 ± 16 pmol/gm), twice as large that of anandamide, supports its proposed role as anandamide precursor. Yet, the relatively small proportions of this precursor pool may indicate the existence of an active biochemical mechanism for the resynthesis of N-arachidonoyl PE depleted during neural activity. In support of this possibility, we have shown previously that biosynthesis of N-acyl PEs in cortical mixed cultures is stimulated by physiologically relevant rises in intracellular Ca2+, elicited by membrane-depolarizing agents or by Ca2+ ionophore (Cadas et al., 1996a Mechanism of Ca2+-dependent N-arachidonoyl PE biosynthesis
How may intracellular Ca2+ levels regulate N-arachidonoyl PE biosynthesis? Extensive investigations by the laboratory of Schmid and colleagues have shown that formation of other N-acyl PEs is mediated by a Ca2+-dependent N-acyltransferase activity, which transfers a saturated or monounsaturated fatty acyl group from the sn-1 position of phospholipids to the primary amino group of PE (Reddy et al., 1983
We have identified and partially characterized a Ca2+-dependent enzyme activity that catalyzes the biosynthesis of N-[14C]arachidonoyl PE, when a phospholipid containing [14C]arachidonate at the sn-1 position is provided as fatty acyl donor. We have also demonstrated that sn-1 arachidonoyl phospholipids are normal, albeit quantitatively minor, components of brain phospholipids. Thus, our results indicate that brain tissue contains the complement of enzymatic activity and lipid substrates necessary for the biosynthesis of N-arachidonoyl PE. While the present study was being drafted, Sugiura et al. (1996b)
The discrete tissue localization of N-acyltransferase activity, concentrated in brain and testis, and its relative enrichment in select areas of the CNS, highlight its potential importance in neural signaling. This distribution only partially parallels that of CB1 cannabinoid receptors. In fact, whereas these receptors are densely expressed in cortex, hippocampus, cerebellum, and striatum (regions in which N-acyltransferase activity is also relatively high), they are only sparsely present in the brainstem (the region of highest enzyme activity; Herkenham et al., 1991
Stimulation of N-acyltransferase activity may account for the Ca2+-dependent increase in N-arachidonoyl PE biosynthesis observed when cortical mixed cultures are exposed to membrane depolarizing agents or Ca2+ ionophore (Cadas et al., 1996a Physiological significance of the N-acyltransferase pathway
The preceding discussion has focused on the N-acyltransferase pathway as a possible biochemical mechanism for the formation of N-arachidonoyl PE and anandamide, but it is important to note that these lipids represent only a small fraction of the N-acyl PEs and NAEs found in brain. What, if any, are the physiological roles of saturated and monounsaturated N-acyl PEs and NAEs? Although it is not yet possible to provide an adequate answer to this question, several findings indicate that these lipids may be biologically active. For example, N-palmitoylethanolamine was shown to protect cerebellar granule neurons from excitotoxic death and to prevent antigen-induced mast cell activation, possibly by binding to CB2-like cannabinoid receptors (Facci et al., 1995
FOOTNOTES
Received Oct. 4, 1996; revised Nov. 18, 1996; accepted Nov. 25, 1996.
This work was supported by the Neurosciences Research Foundation, which receives major support from Sandoz Pharmaceutical Corporation. We thank Dr. M. Beltramo for discussion and Drs. N. Stella and J. Gally for critically reading this manuscript.
Correspondence should be addressed to Dr. Daniele Piomelli, The Neurosciences Institute, 10640 J. J. Hopkins Drive, San Diego, CA 92121.
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