A primary candidate for the role of endogenous cannabinoid substance anandamide has been identified recently . Like D 9 – tetrahydrocannabinol, anandamide binds with high affinity to cannabinoid receptors, reduces contractions in mouse vas deferens, and modulates the activities of adenylyl cyclase and voltagedependent ion channels in neurons and other cells . Moreover, anandamide elicits, in vivo, a series of behavioral responses typical of cannabinoid drugs . Although the pharmacological properties of anandamide are beginning to be well understood, we still lack essential information on the biochemical mechanisms underlying the biogenesis of this signaling molecule. Two such mechanisms have been proposed. Anandamide may be synthesized through the energy-independent condensation of ethanolamine and arachidonate . This reaction, however, requires pH optima and substrate concentrations that are unlikely to be found in neurons. Also, various lines of evidence indicate that condensation of ethanolamine and arachidonate may result from the reverse reaction of “anandamide amidohydrolase,” an enzyme activity involved in anandamide breakdown . 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 . For this mechanism to be considered plausible,trim tray pollen two necessary conditions should be fulfilled. First, the occurrence of N-arachidonoyl PE in adult brain tissue should be demonstrated. Second, the enzyme pathway that is responsible for the biosynthesis of N-arachidonoyl PE should be identified.

Studies carried out in the laboratory of Schmid et al. before the discovery of anandamide have reported the occurrence in brain of an N-acyltransferase activity, which catalyzes the formation of other N-acyl PEs by transferring a saturated or monounsaturated fatty acyl group from the sn-1 ester bond of phospholipids to the primary amino group of PE . Is this activity implicated in the biosynthesisof N-arachidonoyl PE? Such a possibility has been raised. Arguing against it, though, is the currently accepted notion that tissue phospholipids contain no arachidonate at the sn-1 position, but rather saturated or monounsaturated fatty acids such as palmitate, stearate, or oleate . 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 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 Ca21 -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. .This circumstance suggests that these lipids may be generated exclusively during cerebral ischemia and, as such, may not participate in normal brain function . We examined, therefore, whether N-acyl PEs, and particularly N-arachidonoyl PE, were present in rat brain tissue in which metabolic changes associated with ischemic damage had been prevented by immersing the head of the animal in liquid nitrogen within 2 sec of decapitation . We extracted total brain lipids in methanol/chloroform, fractionated the extracts by column chromatography, and analyzed them by bidimensional HPTLC.

The results of these analyses, illustrated in Figure 1A, indicated that a lipid component with the chromatographic properties of N-acyl PEs is present in the extracts. 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 . Under appropriate conditions, this enzyme quantitatively hydrolyzes the distal phosphodiester bond of N-acyl PEs, releasing the corresponding NAEs . The NAEs produced in these digestions were then purifified by a combination of column chromatography and normal-phase HPLC, and analyzed by GC/MS as TMS derivatives. The electron-impact mass spectra of the TMS derivatives of synthetic anandamide and other NAEs are shown in Figure 2. From these mass spectra we chose characteristic ions for analysis by SIM, a technique that provides both high sensitivity and specificity of detection. We observed diagnostic fragments for anandamide , N-palmitoylethanolamine , N-stearoylethanolamine , and N-oleoylethanolamine , which were eluted from the GC at the retention times expected for these compounds . 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 6 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.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 Ca21 -containing incubations, we noted a lipid component that comigrated with N-acyl PEs on HPTLC . In contrast, this component was not detectable in Ca21 -free incubations or in incubations of heat-inactivated fractions . These results are in agreement with studies by Schmid et al. indicating the occurrence, in brain tissue, of a Ca21 -activated enzymatic activity that catalyzes the biosynthesis of N-acyl PEs. Was N-arachidonoyl PE also produced in these incubations? To address this question, we partially purifified 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 , we were able to collect complete mass spectra for each of them . Comparison of these mass spectra with those of synthetic NAEs unambiguously confirmed the identification of these compounds. Consequently, our results demonstrate that brain particulate fractions incubated in the presence of Ca21 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 were also generated in these incubations, as determined by GC/MS analysis .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 . To determine whether rat brain tissue contains such activity, we incubated detergent-solubilized particulate fractions in a reaction mixture containing CaCl2 and 1,2-di[14C]palmitoyl PC,cannabis grow set up and identified the radioactive products by HPTLC or reversed-phase HPLC. By either method, we found a major radioactive component that displayed the chromatographic properties of N-acyl PEs, indicating the formation of N-[14C]palmitoyl PE . In contrast, parallel incubations carried out in the absence of Ca21 or with heat-inactivated samples contained no detectable N-[14C]palmitoyl PE . Likewise, we found no N-[14C]palmitoyl PE when we used 1-stearoyl,2- [ 14C]palmitoyl PC as fatty acyl donor . Analysis of additional subcellular fractions of rat brain tissue showed that N-acyltransferase activity is present both in crude particulate fractions and in microsomes, and is enriched in the former . To characterize N-acyltransferase activity further, in three subsequent experiments we applied solubilized particulate fractions to a MonoQ FPLC column and measured the enzyme activity in the eluate. Using an assay buffer that contained 1,2-di[14C]palmitoyl PC, exogenous PE, and CaCl2, we detected one major peak of enzyme activity in association with a UV-absorbing component . Thus, our results confirm that rat brain tissue contains a N-acyltransferase activity that catalyzes the biosynthesis of saturated and monounsaturated N-acyl PEs. 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 . We incubated, therefore, various brain sub-cellular fractions in a reaction mixture containing one of three possible N-acyltransferase substrates, radioactively labeled on arachidonate: 1,2-di[14C]arachidonoyl PC, 1-stearoyl,2-[14C]arachidonoyl PC, and 1-stearoyl,2-[14C]arachidonoyl PE.

We observed formation of N-[14C]arachidonoyl PE with only 1,2-di[14C]arachidonoyl PC . Such N-arachidonoyltransferase activity was ; 10-fold lower than the N-palmitoyltransferase activity we measured by using 1,2-di[14C]palmitoyl PC. In contrast, incubations with 1-stearoyl,2- [ 14C]arachidonoyl PC yielded no N-[14C]arachidonoyl PE , whereas incubations with 1-stearoyl,2-[14C]arachidonoyl PE yielded N-acyl PEs that were radioactively labeled only on the sn-2 acyl ester group, as the PE substrate from which they derived . From these observations, we conclude that N-acyltransferase activity in brain can catalyze the transfer to PE of either saturated or polyunsaturated fatty acyl groups, provided that these groups are esterified at the sn-1 position of phospholipids.It is generally thought that, in brain as well as other tissues, arachidonate is mainly esterified at the sn-2 position of phospholipids . Such selective distribution is in striking contrast with the substrate specificity of brain N-acyltransferase, which argues against the participation of this enzyme activity in the biosynthesis of N-arachidonoyl PE. Studies by Blank et al. and Chilton and Murphy have suggested, however, that small amounts of phospholipid species containing arachidonate at the sn-1 position may occur in non-neural tissues. Accordingly, we examined whether similar phospholipids may also be present in rat brain. 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 . We extracted the reaction products and incubated them with Bacillus cereus PLC, which hydrolyzes the vicinal phosphate ester bond of sn-1 lysophospholipids, yielding sn-1 monoacylglycerols. The latter, after conversion to bis-TMS derivatives, are readily resolved by GC, and can be unambiguously identified and distinguished from their sn-2 isomers by their typical retention times and mass spectral properties .We have previously reported that, in rat cortical mixed cultures, biosynthesis of N-acyl PEs is enhanced in a Ca21 -dependent manner by ionomycin, a Ca21 ionophore, or by membranedepolarizing agents . These results suggested that a Ca21 -activated N-acyltransferase activity may catalyze N-acyl PE biosynthesis in intact neurons. This possibility could not be stringently tested, however, because pharmacological inhibitors of this enzyme were not available. The finding that BTNP is an effective N-acyltransferase inhibitor in vitro prompted us to examine its effects on N-acyl PE biosynthesis in intact neurons. In a first series of experiments, we used cortical mixed cultures that had been labeled by incubation with [3 H]ethanolamine. We stimulated the cultures with 1 m M ionomycin and measured the radioactivity associated with N-acyl PEs after TLC purification. As reported, N-acyl[3 H]PE levels in ionomycin-treated cultures were approximately threefold greater than those of untreated controls . This effect was almost completely abolished when the neurons were exposed to 25 m M BTNP . To examine the effects of BTNP on N-arachidonoyl PE biosynthesis, in subsequent experiments we stimulated the cultures, purified the N-acyl PEs, and quantifified 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 m M 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.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 . In the present study, we have considered three aspects of this hypothetical mechanism that are essential to establish its physiological relevance. First, we have determined whether N-arachidonoyl PE is present in adult brain tissue. Second, we have examined the enzyme activity and lipid substrates that participate in the biosynthesis of N-arachidonoyl PE in vitro. Finally, we have assessed the contribution of this enzyme activity to N-arachidonoyl PE biosynthesis in intact neurons.