Anandamide, an endogenous cannabinomimetic eicosanoid: ‘Killing two birds with one stone’

PROSTAGLANDINSLEUKOTRIENES AND ESSENTIALFATTYACIDS Prostaglandins Leukotrienes and Essential Fatty Acids (1995) 53, 1-11 © Pearson Professional Ltd 1995

REVIEW Anandamide, an Endogenous Cannabinomimetic Eicosanoid: 'Killing Two Birds With One Stone' V. Di Marzo and A. Fontana

Istituto per la Chimica di Molecole di Interesse Biologico, C.N.R., Viale Toiano 6, 80072 Arco Felice, Naples, Italy (Reprint requests to VDM)

INTRODUCTION

The discovery of endogenous opiate receptors in mammalian neural tissue (1) and the finding that benzodiazepines and y-aminobutyric acid share the same receptor (2) aroused a great deal of interest in the 1970s and 1980s. In particular, the presence in mammalian tissues of receptors to opioids of plant origin led to the search and subsequent discovery of a new class of neuropeptides, the endorphins (3). Similarly, the finding of endogenous cannabinoid receptors in mammals initiated a concerted search for endogenously produced cannabinoids. Since cannabinoids possess a range of properties ranging from analgesic to anti-inflammatory activity, this search was of obvious importance. The existence and nature of endogenous cannabinoids (which, as with endogenous opiates, could become known as 'endo-cannabinoids') proved to be elusive. In 1992, however, the first endo-cannabinoid was discovered and found to be an eicosanoid in that it was an amide of arachidonic acid (AA) with ethanolamine (4). This novel eicosanoid was christened 'anandamide'. The present article, therefore, reviews the discovery, structure, biosynthesis, metabolism and cannabinomimetic properties of anandamide.

CANNABINOID RECEPTORS

The Indian hemp, Cannabis sativa, is probably one of the oldest medicinal plants known to humans (5). Although employed as an analgesic, anticonvulsant and antiemetic, and in the treatment of inflammation, glaucoma and helminthiasis (6), its most popular and widespread use is that of a recreational (i.e. mind altering) drug. Thus, the clinical and pharmaceutical applications of Cannabis preparations (i.e. hashish

and marijuana) have been limited by their psychotropic actions which are due to only a few cannabinoids, in particular to A9-tetrahydrocannabinol (THC). These limitations have led to increasing interest in the mechanism of action of psychoactive cannabinoids. Considerations similar to those that had previously promoted the discovery of opiate receptors and endorphins were applied to studies on cannabinoid receptors and agonists and led first to the development of new cannabinoid agonists, including synthetic derivatives of THC (7, 8), and, subsequently, to the pharmacological characterization of the THC receptor (9, 10). The cloning of the THC receptor, in 1990, provided conclusive evidence for the existence of specific cannabinoid binding sites and represented a true landmark in cannabinoid research (11). The neuronal THC receptor was found to be localized in specific areas of the brain, such as the cortex, the hippocampus, the striatum and the cerebellum (12, 13). In contrast, the brain THC receptor was absent in peripheral tissues, the only exception being the testes (14). A more recent development, however, has been the finding (and subsequent cloning) of a peripheral THC receptor localized preferentially in spleen macrophages (15, 16). The latter receptor has been proposed as a mediator of the anti-inflammatory and immunosuppressant actions of THC. Other peripheral THC binding sites have been also described and partially characterized in cells of the reproductive and immune systems (17, 18, 46). Transmembrane signalling studies showed that both central and peripheral THC receptors are coupled to the inhibition of adenylate cyclase and N-type calcium channel currents through pertussis toxin-sensitive GTPbinding (G) proteins (19-22). However, despite the finding of specific functional receptors, several other non-receptor mediated effects have been ascribed to cannabinoids, for example, the release of intracellular calcium and the activation of phospholipase A 2 (23, 24).

2 ProstaglandinsLeukotrienes and Essential Fatty Acids These effects may be caused, in part, by interactions with biological membranes which, in turn, may account for some of the pharmacological properties of cannabinoids.

THE 'DISCOVERIES' OF A N A N D A M I D E The finding of cannabinoid receptors in the CNS led to the search for endogenous cannabinoids (in much the same way as endorphins were searched for and discovered). Pseudophysiological conditions (such as calcium ionophore stimulation) were found to lead to the production of chemically unidentified THC-mimetic material from nervous tissue (25). However, the identity of the putative endogenous cannabinomimetic mediator eluded the efforts of many scientists until the end of 1992, when a substance capable of inhibiting both the specific binding of a radiolabelled cannabinoid probe to synaptosomal membranes and the electrically evoked twitch response of the mouse vas deferens (a response typical of THC) was isolated from porcine brain (4). The chemical structure of this metabolite, the amide of A A with ethanolamine (N-arachidonylethanolamine), was found to be simpler than that expected from the efforts needed to identify it. Its lipophilic and highly unsaturated nature, potentially sensitive to both oxidation and hydrolysis, explained the difficulties met during its isolation. Moreover, this first endogenous cannabinoid ligand, named anandamide (derived from the Sanskrit word ~ m a n d a for 'bliss') belongs to a class of widespread natural products, the N-acylethanolamines (NAEs), whose precise biological role has not yet been fully elucidated. (For a review see Schmid et al (26)). Notably, a few months after its 'first' discovery, N-arachidonylethanolamine/anandamide was again iso-

lated, this time from calf brain, by a bioassay aimed at searching for endogenous regulators of L-type calcium channels (27). In this latter study, the ethanolamide of A A was found to interact in a non-competitive fashion with 1,4-dihydropyridine binding sites in rat cardiac and cortex membranes. This finding, obtained independently from the previous discovery of anandamide, seems to strengthen the hypothesis that this novel eicosanoid is a physiological chemical modulator.

PHARMACOLOGICAL AND BEHAVIOURAL PROPERTIES OF A N A N D A M I D E In order to establish whether anandamide was a true agonist of cannabinoid receptors, its pharmacological effects in vitro and in vivo had to be assessed and compared to the actions reported for THC (4, 5). This has been the rationale for most of the studies on anandamide so far published (see Table). Immediately after its discovery, anandamide was administered intraperitoneally to mice, where it produced (at doses ranging from 2 to 20 mg/kg body weight) hypothermia, antinociception and hypomotility in immobility and open field tests (28), thus paralleling the psychotropic effects caused by cannabinoids at slightly lower doses. Interestingly, the effects of anandamide, if compared to those of THC, occurred with a more rapid onset and with a rather shorter duration of time (30). Other research on the behavioural effects of anandamide was extended to rats and included other in vivo tests such as chow consumption in ad libitum fed animals, anxiolytic properties and delayed nonmatching to sample memory task, where the compound was found to be inactive (29). In vitro, conclusive evidence for the specific binding of anandamide to cannabinoid binding sites was sought

Table Summary of the pharmacologicalactions of anandamide in vivo and in vitro. The pharmacologicaland behavioural actions so far reported for anandamide are summarized. The type of assay (italics), the animal species/tissue and the range of concentrations (or Ki, IC50,etc.) used are shown Actions in vivo

Assay

Hypothermia Antinociception Catalepsy Inhibition of locomotor and rearing activity Enhancement of muscimol-inducedcatalepsy Effects on ACTH, corticosteroneand CRF-1 secretion Inhibition of sperm acrosomereaction

rectal A °C, mouse, rat, 2-20 mg/kg (28, 29) hotplate latency, mouse, 2-20 mg/kg (28, 30) ring immobility test, mouse, 2-20 mg/kg (28, 30) openfield test, mouse, rat, 2-20 mg/kg (28, 30)

rat globus pallidus, 30 gg/rat (35) rat, 50-150 gg/rat (36) sea urchin, 1 gM (47)

Actions in vitro

Assay

Displacement of specific THC agonists

binding assay, rat brain membranes, mouse brain P-2 membranes, L-cell

Inhibition of adenylate cyclase Inhibition of N-type Caa+channels Activation of phospholipase A2* Release of intracellular calcium* Dis-inhibition of L-type Ca2+channels* Inhibition of twitch response * Not mediated by the cannabinoid receptor.

membranes, transfected CHO cells, 37-543 nM (4, 30-33) rat brain membranes, transfected CHO cells, frog neuromuscularjunction, 0.16-1.9 gM (31-34) N18neuroblastoma cells, 10-100 nM (32, 38) arachidonate release, CHO cells, 1-100 gM (32) fura-2 assay, CHO cells, 10 p-M (32) displacement of 1,4-dihydropyridine, rat cardiac and cortex membranes, 15 gM (27) electrically stimulated mouse vas deferens, 52.7 nM (4, 37, 41)

Anandamide, an EndogenousCannabinomimeticEicosanoid 3 in binding assays performed in Chinese hamster ovary (CHO) cells transfected with the cDNA encoding the brain THC receptor (31, 32). In these cells, which express the cannabinoid receptor, the eicosanoid displaced specific cannabinoid agonists from their binding sites and inhibited the forskolin-induced activation of adenylate cyclase with a Ki and an ICs0 in the medium and high nanomolar range. The latter effect was also exerted in NG18 neuroblastoma cells (31), which possess constitutive THC binding sites and where anandamide was also found to exert an inhibition of N-type calcium currents (32), but not in non-transfected CHO cells, which do not express cannabinoid receptors. Furthermore, as for THC, anandamide inhibition of both cAMP formation and N-type calcium currents (the two typical cannabinoid receptor-coupled transmembrane signalling events) was blocked by pertussis toxin pretreatment (31, 32). Some of these data were subsequently corroborated by experiments conducted in rat brain membranes (33), where anandamide displayed an ICs0 of 90 nM versus the binding of the THC agonist WIN 55,212-2 to the cannabinoid receptor, provided that the general serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) was present during the incubations. This suggested the presence of enzymes, possibly of the amidohydrolase type, which catalyze the degradation of anandamide. In the above preparations, the ethanolamide of AA inhibited adenylate cyclase in a GTP-dependent manner with an ICs0 of 1.9 gM. The extent of this effect also depended on the brain region from which the membranes were prepared, and was found to be maximal in the cerebellum and striatum, two areas rich in THC receptors. In another study, anandamide was also reported to block adenylate cyclase at the frog neuromuscular junction, thus suggesting the presence of a cannabinoid receptor at the motor nerve terminal (34). Taken together, the above findings cast little doubt on the conclusion that anandamide is indeed capable of binding functionally to brain THC receptors, and activating all the second messenger pathways triggered thereafter. Anandamide was also found to exhibit some of THC's non-cannabinoid receptor-mediated actions, such as activation of phospholipase A2 and the release of intracellular calcium (32), albeit only in the mid-high micromolar range, thus supporting further the hypothesis of a pharmacological similarity between this novel eicosanoid and THC. This hypothesis has been tested even further by investigating whether anandamide could exert, in vivo, the more sophisticated pharmacological actions of THC and whether the two compounds would induce tolerance to each other's effects. Thus, both substances, at similar doses, were found to enhance GABAinduced catalepsy upon co-injection with the GABAA agonist, muscimol into the globus pallidus of rats (35). Moreover, again in rats, intracerebroventricular injection of either of the two 'cannabinoids' increased the serum levels of adrenocorticotropic hormone and cortisone dose-dependently while causing a depletion of cortico-

tropin releasing factor (CRF-41) in the median eminence, thus suggesting that anandamide, like THC, activates the hypothalamopituitary adrenal axis, possibly via the secretion of CRF-41 (36). Cross-tolerance between THC and anandamide was studied using two typical effects of cannabinoids: hypothermia and inhibition of electrically stimulated mouse vas deferens twitch response (37). Surprisingly, while inducing tolerance to anandamide in the latter assay, THC pre-treatment did not produce any reduction in the hypothermic effect caused by anandamide. This discrepancy may have not been given the appropriate consideration if a study on the cumulative effects of the brain cannabinoid receptor agonist WIN 55,212-2 and anandamide on N-type C a 2+ channels had not been reported previously (38). In this latter study it was observed that while WIN 55,212-2 always caused a further inhibition of Ca 2+ currents in cells exposed to maximally effective concentrations of anandamide, application of the latter always caused a partial recovery from the inhibition caused by maximally effective concentrations of WIN 55,212-2. This result suggested that anandamide acts only as a partial agonist at the brain cannabinoid receptor, thus raising the possibility that its mechanism of action in intact cells may be more complex than that which would be inferred by studies with THC, or, that other endogenous cannabinoid agonists may exist. These possibilities were supported by the recent finding (obtained in vivo in mice) that the k-opioid agonist nor-binaltorphimine blocks THC- but not anandamide-induced antinociception (30).

'ANANDAMIDES' AND OTHER 'ENDOCANNABINOIDS' The last three reports (30, 37, 38) described in the previous section, as well as the isolation and cloning of the peripheral cannabinoid receptor, which displayed a lower affinity for anandamide than the homologous brain receptor, seem to suggest the existence of other endo-cannabinoids (endogenous ligands of cannabinoid receptors). Indeed, a cannabinoid-like activity distinct from anandamide, released from nervous tissue by agents causing neuron depolarization and C a 2+ influx, and presumably of peptidic chemical nature, has been recently described (25, 39). A partially purified fraction of this unidentified metabolite was also found to inhibit adenylate cyclase (39), thus substantiating its physiological role as a ligand of the brain cannabinoid receptor. Nevertheless, the only other cannabinomimetic substances so far isolated and characterized are two chemical congeners of anandamide, the ethanolamides of di-homo-7-1inolenic (20:3, n-6) and docosatetraenoic (22:4, n-6) acids (40). Like anandamide, these two metabolites were purified from porcine brain and, apart from binding specifically to cannabinoid binding sites (32, 40), were found to inhibit both forskolin-stimulated adenylate cyclase (32) and mouse vas deferens twitch

4 ProstaglandinsLeukotrienesand EssentialFattyAcids response (41), albeit at concentrations slightly higher than those needed for the ethanolamide of AA. These findings led to the suggestion that a family of 'anandamides', e.g. of NAEs with cannabinomimetic and neuromodulatory properties, occurs in the CNS. As mentioned above, saturated and monounsaturated NAEs, such as N-palmitoyl- and N-stearoylethanolamine, are widespread fatty acid derivatives (26) had have also been found in mammalian brain in association with ischemic conditions (42). They are co-released with anandamide upon depolarization of neurons (see next section) and exert significant effects on calcium fluxes and fast sodium channels (43, 44). On the other hand, polyunsaturation of the fatty acyl moiety of NAEs seems to be an essential prerequisite for their binding to brain THC receptors (31, 32), a fact that would seem to restrict the field of 'anandamides' only to polyunsaturated NAEs. Both saturated and polyunsaturated NAEs, as well as chemically different compounds, may function as 'endo-cannabinoids' in the peripheral nervous system. N-Palmitoylethanolamine, for example, shares with cannabinoids some important non-psychotropic effects, such as anti-inflammatory, platelet anti-aggregatory and immunosuppressant activities (26, 45). Although unable to bind to the brain THC receptor, this compound, like any other saturated NAE, was not tested on the homologous receptor present in spleen macrophages (16), and, therefore, any speculation on its possible involvement in peripheral cannabinoid receptor-mediated physiological responses must await future investigations in this direction. Indeed, no evidence on the existence of anandamide itself in the periphery has been so far reported, although a clue on the possible presence of this or other related metabolites in sea urchin sperm, where specific THC receptors have been found (46), has been provided by a recent study where anandamide has been shown to inhibit the egg jelly-induced acrosome reaction (47), thus mimicking an effect previously reported for THC (48). It goes without saying that an effort similar to the one currently applied to the understanding of anandamide role in the CNS will have to be made also to isolate peripheral 'endo-cannabinoids' and to correlate them with non-psychotropic cannabinoid action.

A N A N D A M I D E BIOSYNTHESIS: T W O INDEPENDENT PATHWAYS?

For an endogenous metabolite to be defined a neurotransmitter or a neuromodulator it is necessary to demonstrate the presence in neurons not only of receptors and of receptor-coupled signalling events mediating its response, but also of the enzymatic machinery necessary for its biosynthesis, release and rapid inactivation. Therefore, a few studies have also focused on the molecular mechanisms underlying anandamide formation and clearance in the CNS. Owing to its chemical nature as a NAE, it was reasonable to predict for this

endo-cannabinoid two alternative biosynthetic models analogous to those suggested in the past for saturated and monounsaturated NAEs (schematically depicted in Fig. 1). The first, suggested by studies conducted in the 1960s, consisted of the energy-independent, enzymecatalyzed condensation of fatty acids with ethanolamine (49). The enzyme involved in this condensation was found in microsomal preparations of brain, kidney and, particularly, liver from both rat and guinea pig (49) and displayed the highest specificity for unsaturated fatty acids, low affinity for ethanolamine (a 30 mM concentration was needed) and an alkaline pH optimum (50). This pathway, if also responsible for anandamide formation, would require the presence in the cell of high concentrations of both ethanolamine and AA. This situation does not occur in resting neurons and can be achieved only by the simultaneous activation of both phospholipase A2 (PLA2) and phospholipase D (PLD). Investigations conducted in the 1980s led to the discovery of another, and possibly more likely, route for NAE biosynthesis. This consisted of the enzymatic hydrolysis of the phosphoester bond of a preformed membrane precursor belonging to a widespread phospholipid class, the N-acylphosphatidylethanolamines (NAPEs), with direct formation of NAEs and phosphatidic acid (51, 52 and, for a review, ref. 26). This mechanism was reported to occur in many mammalian tissues, including the brain. Here, the phosphodiesterase responsible for NAPE hydrolysis was partially characterized as a microsomal enzyme of the phospholipase D type, probably distinct from phosphatidyl-ethanolamine- and -choline-dependent PLD, and stimulated or inhibited, respectively, by low or high concentrations of Ca 2÷ (53). Thus, when investigations on anandamide started, two possible routes were available from the literature that could provide a mechanism for its formation: one, leading to the de novo synthesis of the eicosanoid, would occur through PLD and PLA 2 activation, AA and ethanolamine liberation, followed by their enzymatic condensation; the other route would be made possible by a new type of PLD, specific for a putative anandamide precursor, (N-arachidonyl)phosphatidylethanolamine, which is obtained in turn by N-acylation (a sort of re-modelling) of membrane phosphatidylethanolamine (Fig. 1). Initial efforts were directed towards the finding, in brain homogenates, of the putative 'anandamide synthase' activity catalyzing the condensation between arachidonate and ethanolamine. The first preliminary study on such an activity was published in 1993 (54), and was conducted with rat brain homogenates using the same conditions reported initially to lead to enzymatic formation of Npalmitoyl ethanolamine (i.e. pH = 9.0, ethanolamine concentration = 1.6 raM, [49]). Under these conditions only very little anandamide-like material, characterized by using a single thin layer chromatographic step, was obtained unless a high concentration (1.5 raM) of the alkylating agent PMSF was added to the incubation mixture. Subsequent experiments, however, have shown

Anandamide, an Endogenous CannabinomJmetic Eicosanoid UEMBR*NE

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Fig. 1 Two alternative pathways for anandamide biosynthesis. Like for saturated and monounsaturated N-acylethanolamines (26), two anabolic routes have been suggested for anandamide production by cells (54, 56, 57, 59). The first has been shown to occur in synaptic and microsomal membranes of both neurons and glial cells through the enzymatic, energy-independent condensation of the two basic constituents of anandamide. AA and ethanolamine. These two molecules being present only in very low intracellular levels, the activation of the 'condensing enzyme', named anandamide synthase (Knl= 27-130 mM for ethanolamine) (56, 57) can occur only through the previous stimulation of both PLA2 and PLD, which catalyze membrane phospholipid breakdown yielding increased concentrations of the enzyme's two substrates (57). As evidence for this pathway has been obtained so far only in cellfree systems treated with exogenous AA and ethanolamine, no proof for the actual participation of 'anandamide synthase' activity or of simultaneous PLAjPLD activation in the 'physiological' biosynthesis of anandamide exists at the present date. An alternative anandamide biosynthetic pathway (59) occurs selectively in intact neurons upon depolarization and subsequent calcium influx, and is effected through the single-step, hydrolytic breakdown of a preformed membrane phospholipid precursor, (Narachidonyl)phosphatidylethanolamine (NAPE). The enzyme responsible for this hydrolysis would be a partially CaZ+-dependent phospodiesterase of the D type (NAPE-PLD), absent in astrocytes, and capable of recognizing also other (Nacylphosphatidylethanolamines, but not non-acylated phosphogtycerides (53). The biosynthesis of NAPE is activated by the same stimuli leading to anandamide formation (59), and, as shown previously for other N-acylethanolamine phospholipids (26), may well occur via N-trans-acylation of membrane phosphatidylethanolamine by other arachidonate-containing membrane phospholipids. Whatever the mechanism of its biosynthesis, no evidence exists for the storage into presynaptic vescicles of anandamide (Piomelli and Di Marzo, unpublished observations) which, like other eicosanoids, is likely to be directly released by the neurons once synthesized de novo.

that the substance p r o d u c e d under these conditions is not a n a n d a m i d e but rather an artefactual adduct b e t w e e n P M S F and e t h a n o l a m i n e (55), and that the condensating e n z y m e is not insensitive to P M S F but is in fact strongly inhibited by it (56). M o r e recent studies, c o n d u c t e d in b o v i n e (56) and rabbit (57) brain, h a v e s h o w n that m u c h h i g h e r e t h a n o l a m i n e concentrations (apparent Km ranging f r o m 27 to 130 m M ) are n e e d e d to o b s e r v e the e n z y m a t i c synthesis o f 'true' anandamide. T h e e n z y m e i n v o l v e d in this reaction had the highest affinity for A A as the fatty acyl constituent and was specific for e t h a n o l a m i n e as the polar m o i e t y ; it was A T P - and C o A i n d e p e n d e n t and was inhibited by P M S F in b o v i n e brain, or by dithiobisnitrobenzoic acid in rabbits, thus suggesting

a reaction m e c h a n i s m that utilizes a critical sulfhydryl residue e n g a g e d in a thioester b o n d with A A (56, 57). T h e highest levels o f this e n z y m a t i c activity (which had an alkaline p H o p t i m u m [56]) w e r e found in synaptic vescicles, myelin, and m i c r o s o m a l and s y n a p t o s o m a l m e m b r a n e s o f specific b o v i n e brain regions such as the h i p p o c a m p u s , the thalamus, the cortex and the striatum (56), as w e l l as in m i c r o s o m a l fractions o f rabbit brain, lung, kidney and liver (57). Interestingly, m a n y o f the characteristics reported for these 'anandamide synthases' are identical to those o f a ' N A E synthase' activity which was described, in a detailed paper dating b a c k to 1985 (58), simply as a N A E a m i d o h y d r o l a s e w o r k i n g 'in r e v e r s e ' . Saturated and m o n o u n s a t u r a t e d N A E s are, in

6 ProstaglandinsLeukotrienesand EssentialFatty Acids fact, degraded to fatty acids and ethanolamine by a well known enzyme, NAE amidohydrolase (see next section and, for a review, ref. 26), having the same tissue distribution as 'NAE synthase'. This enzyme, which is also inhibited by PMSF and has, again, an alkaline pH optimum, was shown, indeed, to work 'backwards' (58), thus leading to NAE formation, with a Km for ethanolamine (20 raM) almost identical to that reported for both NAE (50) and anandamide (56) 'synthase'. The following question then arises: is 'anandamide synthase' an anandamide amidohydrolase working 'backwards'? Obviously, only the purification and structural characterization of the two enzymes can provide a final answer to this question. In any event, bearing in mind the high K m for ethanolamine reported for the synthetic enzyme, it is reasonable to ask: is the 'condensation route', be it effected through one or the other of the above mentioned enzymes, actually responsible for the physiological formation of anandamide in stimulated neurons? The quest for an answer to the latter question aroused renewed interest in the hypothesis of the occurrence of a phospholipid precursor for anandamide. Initially, the fact that no report existed in the literature on the presence in mammalian tissues of (N-arachidonyl)phosphatidylethanolamine (26) had somehow discouraged the testing of this hypothesis. However, the knowledge that such a phospholipid, if present in very small amounts, due to the high unsaturation of the N-fatty acyl chain, might have easily escaped detection in previous studies because of its likely oxidation during the purification and characterization procedures, stimulated investigations also in this direction. It was thought that a more sensitive approach, using radiolabelled precursors, had to be used in order to track down this phospholipid. More importantly, it was reasoned that any physiologically relevant study on anandamide biosynthesis had to be conducted in intact central neurons stimulated by pseudophysiological conditions such as those leading to membrane depolarization and already used in the past to produce cannabinomimetic metabolites (25, 39). Therefore, initial efforts were directed towards the finding of the appropriate stimulatory conditions in primary cultures of rat cortical and striatal neurons. In these systems, treatment with substances inducing membrane depolarization and C a 2+ influx, such as ionomycin, kainate, high potassium and 4-aminopyridine, was found to lead to a substantial production of anandamide (59). If the treatment was conducted in the presence of bovine serum albumin (BSA), almost 50% of newly produced anandamide was found to be released outside the cells. It was estimated that the amount of anandamide formed (about 2 pmol/dish, when using kainate as the stimulus, determined by using gas chromatographic-mass spectrometric [GC-MS] analyses) was, indeed, sufficient to produce (in a brain volume containing the same number of neurons present in one Petri dish) concentrations near to those necessary to activate the brain cannabinoid

receptor (about 40 nM). Importantly, no anandamide formation was observed in cortical and striatal astrocytes incubated under the same experimental conditions. Therefore, this was the first report of the stimulusinduced, neuron-specific production/release of anandamide. Remarkably, other five NAEs were produced together with the eicosanoid, and in higher amounts, i.e. the 16:0, 18:0, 18:1, 18:2, and 18:3 anandamide congeners (59), substantiating the hypothesis that other components of this class of compounds may have a physiological role in the CNS, although not necessarily as 'endo-cannabinoids' (see previous section). Having found the conditions suitable to produce anandamide in intact cells, the mechanism underlying this production was investigated. If the 'condensation pathway' was involved it was reasonable to expect that inhibitors of PLA 2 would block the ionomycin-induced production of anandamide (Fig. 1) (57). Two inhibitors of this enzyme, however, did not exert any effect on the biosynthesis of the eicosanoid, even though they were very effective in blocking the ionomycin-induced formation of AA. Moreover, when intact neurons were incubated with exogenous PLA2 and ethanolamine, no anandamide formation was observed, although a considerable increase of arachidonate levels was measured. Finally, incubation of intact neurons with exogenous PLD resulted in the formation of ethanolamine and, more importantly, of anandamide, without altering the endogenous levels of arachidonate, which remained under the detection limit. Moreover, PLD-induced anandamide formation was not enhanced by addition of exogenous AA, as one would have expected if a condensation with ethanolamine took place. These results suggested that, in intact central neurons, little, if any, anandamide is produced by a 'condensation mechanism' ([59] and Cadas H., Gaillet S., Di Marzo, V., Piomelli D., in preparation). Accordingly, when neuronal homogenates were incubated with ethanolamine and AA (or PLAz), under conditions identical to those used to identify NAE synthase for the first time (pH = 7.5, [ethanolamine] = 1 mM, [arachidonate] = 0.1 mM, [49]) but different from those reported (58) to activate the 'reversal' of NAE amidohydrolase (an enzyme abundant in central neurons, as explained in the next section), no anandamide formation was observed. Conversely, since the experiments conducted with exogenous PLD suggested that a preformed anandamide precursor existed in membranes, it was possible to isolate and characterize this precursor from central neurons labelled with [3H]arachidonate or [3H]ethanolamine. (N-Arachidonyl)phosphatidylethanolamine was thus described for the first time as a minor component of neuronal membrane phosphoglycerides (59 and Cadas H., Gaillet S., Di Marzo, V., Piomelli D., in preparation). Neuronal membranes were found to contain also the NAPEs corresponding to the NAEs co-released with anandamide upon membrane depolarization, thus suggesting that in

Anandamide, an EndogenousCannabinomimeticEicosanoid 7 neurons all NAEs are produced by phosphodiesterase (very probably PLD)-catalyzed hydrolysis of membrane NAPEs, as shown previously in other tissues only for saturated and monounsaturated NAEs (26). This suggestion was supported further by the finding that neuronal (but not astroglial) homogenates contain an enzymatic activity capable of converting both natural and synthetic (N-arachidonyl)phosphatidylethanolamine into anandamide, and that the same stimulus leading to anandamide formation also leads to (N-arachidonyl)phosphatidylethanolamine biosynthesis in neurons (59), thus conclusively establishing for the two ethanolamine lipids a precursor/product relationship. The same mechanism, in different neurons, may also underlie the formation of the two other 'anandamides' so far found in the brain, that is 20:3 and 22:4 ethanolamides (40). At the present date, too little is still known of the types of stimuli leading to anandamide formation, of its brain distribution and of its functions to draw any conclusion as to which of the two mechanisms described here is the one actually operative in vivo or whether they are mutually exclusive. It is possible that the 'condensation pathway' (whether it occurs through a 'true' synthase enzyme or a 'reversed' anandamide amidohydrolase), by displaying a higher selectivity towards the formation of N-arachidonylethanolamine (56), may occur under circumstances where the synthesis of other NAEs (whose precise physiological role in the CNS needs now to be assessed) is not required. Nevertheless, it is important to obtain evidence for its occurrence also in intact neurons, following the onset of physiological stimulation, and to find an explanation for the extremely low affinity for ethanolamine displayed by the 'condensating enzymes' so far identified. On the other hand, the 'N-acylphospholipid pathway', where anandamide is produced by a phosphodiesterase-mediated mechanism (59), is a biosynthetic route typical of phospholipid-derived second messengers. Owing to the non-cannabinoid receptor-mediated effects exhibited by anandamide at the intracellular level (32), it is possible that this metabolite (as with AA and other eicosanoids) may function as both an intra- and extra-cellular messenger, and that the above phospholipase-mediated pathway is activated when this particular multifunctional role is required. With regard to the possible coexistence of the two pathways, it is worthwhile drawing attention to the striking resemblance that this putative 'biosynthetic plasticity' would have with that of another lipid mediator, platelet-activating factor (PAF). For the production of this metabolite in vivo, two routes have been shown to occur: (i) de novo synthesis, including the participation of three separate enzymes, and (ii) a remodelling pathway, again mediated by a phospholipase (in this case PLA2) and starting from a preformed phospholipid precursor. Interestingly, while the latter route is activated by various stimuli (e.g. by inflammatory agents), the de novo pathway seems to be unaffected by this type

of stimulation and is thought to be the primary source for basal levels of PAF (for a review see ref. 60); this observation, applied to anandamide biosynthesis, may again provide a matter for speculation.

MECHANISMS FOR THE INACTIVATION OF ANANDAMIDE: UPTAKE AND CATABOLISM The finding that central neurons are capable of biosynthesizing and releasing anandamide is strongly suggestive of a role as neurotransmitter or neuromodulator for this 'endo-cannabinoid'. It is implicit, however, that such mediator be rapidly metabolized or inactivated in order to terminate its signal quickly. A preliminary investigation (54) suggested the occurrence, in neuroblastoma and glioma cells, of systems which both uptake and degrade anandamide (by a PMSF-sensitive amidohydrolase activity). However, the experimental procedures used in this latter study failed to evince a mechanism rapid enough to be of true physiological significance (maximal effects were observed only after 2.5-h incubations!). Subsequently, both uptake and amidohydrolase-mediated degradation of anandamide were studied in greater detail in primary cultures of striatal and cortical neurons, which contain high concentrations of the cannabinoid receptor and were shown to synthesize the eicosanoid upon membrane depolarization (59). It was shown that exogenous radiolabelled anandamide was rapidly sequestered by cells from the incubation medium (Fig. 2). This process reaches 50% of its maximum (tin) after a 2.5-3-min incubation, and fulfills several criteria of a carrier-mediated uptake by being: (a) temperature dependent; (b) saturable at 37°C (apparent Km= 30 ~tM) and consequently dose dependently inhibited by addition of unlabelled anandamide; and (c) selective (since radiolabelled Npalmitoylethanolamine was neither cleared from medium nor incorporated into cells, and other NAEs did not compete for [3H]anandamide uptake). Interestingly, astrocytes were found to contribute to this uptake mechanism, which was always inhibited by 0.5% BSA (59). Once taken up by cells, anandamide is immediately (tm = 6 min, in intact neurons) hydrolyzed by an amidohydrolase (amidase) activity to ethanolamine and AA (59). Experiments conducted with the anandamide radiolabelled either on the ethanolamine moiety or on the fatty acyl chain were used to find out the destiny of the two products of hydrolysis. Ethanolamine was found outside the cell (as the free amine) as well as inside the cell (either in free or in esterified form as phosphatidylethanolamine), while AA was immediately and quantitatively acylated into membrane phosphoglycerids (Fig. 2). Neuron-free cultures of astrocytes were also shown to hydrolyze anandamide (59). A recent investigation has described the preliminary characterization of

8

Prostaglandins Leukotrienes and Essential Fatty Acids

0

__~~OH

H2N~ O H

~

H2N~ O H

~

ANANDAMIDE AMIDOHYDROLASE

II

NH~ O H

N

H

~OH

ENZYMES OFTHE'AACASCADE'

./ t?

HETE-ETHANOLAMIDEs

L T-ETHANOLAMIDEs

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Fig. 2 Two possible routes for anandamide metabolism. Anandamide inactivation occurs through uptake and degradative mechanisms. A rapid, specific, saturable and temperature sensitive carrier is responsible for the recapture of the eicosanoid by both neurons and astrocytes (59). Once inside the cell, anandamide is readily hydrolyzed by a specific anandamide amidohydrolase into ethanolamine and AA. The latter product is immediately re-acylated into phosphatidyl-serine, -inositol, -ethanolamine and -choline (PS, PI, PE, PC) and, presumably, into other membrane phospholipids. Anandamide-derived ethanolamine is also re-incorporated into PE, although at a slower rate, since it is possible to find it both inside and outside cells immediately after incubation with radiolabelled anandamide. Bearing in mind that anandamide can be derived from membrane phospholipids (see Fig. 1), the reincorporation of the products of its hydrolysis into the latter metabolites represents the closing of what can be indicated as the anandamide cycle. Anandamide is recognized and subsequently oxidized in vitro by some enzymes of the 'AA cascade' (63-65), and this raises the possibility that the ethanolamide of AA, prior to hydrolysis of the amide bond, can be subjected in vivo to an oxidative metabolism also. This, in turn, might generate ethanolamine eicosanoids (or eicosanoid ethanolamides, such as prostaglandin (PG), leukotriene (LT) or hydroxyeicosatetraenoic (HETE) ethanolamides) active either at the cannabinoid receptor or at other targets.

anandamide amidohydrolase as well as its distribution in the CNS (61). The enzyme was inhibited by the serine protease inhibitors (PMSF, p-bromophenacylbromide, N-ethylmaleimide) and was found to have the highest affinity for AA, to have a pH optimum of 9.0-10.0 and to have a tissue distribution similar to that of the previously reported 'anandamide synthase' (56). Taken together, these data may substantiate the hypothesis that the two catalytic activities of anandamide synthase and amidohydrolase are exerted by the same enzyme working in the two opposite directions. A second possible metabolic fate of anandamide can be predicted from the knowledge that many of the enzymes which catalyze AA oxidation to one of the largest class of bioactive lipids (the eicosanoids) appear to have a broad substrate specificity (62, and references cited therein) and may also recognize the ethanolamide of this polyunsaturated fatty acid. This possibility may generate an alternative branch of anandamide metabolism that may be utilized by cells not only to inactivate the mediator, but also to produce metabolites possibly active either on the cannabinoid receptor or on other extra- and intra-cellular targets. Therefore, this second process may potentially lead to a novel class of lipids,

the ethanolamide eicosanoids (Fig. 2), which, like the eicosanoids, may function as both primary and second messengers. In support of this second metabolic route, three studies showing that enzymes of the 'AA cascade' may indeed recognize anandamide and catalyze its oxidation have been conducted so far. The 12- and 15lipoxygenases purified from porcine leukocytes and rabbit reticulocytes were shown to catalyze, in vitro, the formation of 12-hydroxy and 15-hydroxy derivatives of anandamide, respectively, of which only the latter was found to preserve the cannabinoid-like activity in the mouse vas deferens assay (63). A hepatic, microsomal cytochrome P450-dependent hydroxylase was found to catalyze the oxidation of anandarnide to 10 different metabolites whose structure and biological activity was not fully elucidated (64). Finally, an unusual enzyme identified in the red alga Ptilota filicina was shown to mediate the conversion of both AA and anandamide into their conjugated (5Z, 7E, 9E)-triene derivatives (65). The physiological relevance of these findings, that have only demonstrated how some enzymes which metabolize arachidonate can also recognize its ethanolamide, remains to be assessed by attempting to find out whether similar anandamide derivatives occur in vivo and what

Anandamide, an Endogenous Cannabinomimetic Eicosanoid

type of biological function, if any, they exert. Preliminary experiments have shown that no oxidative metabolism of exogenous anandamide occurs in cortical and striatal neurons (Piomelli and Di Marzo, unpublished observations). In these cells, however, the occurrence of rapid hydrolysis of the compound by the amidase activity described above may have prevented the formation of its oxidation products. Therefore, future studies aimed at exploring the possibility of an oxidative metabolism of anandamide will have to be conducted in the presence of 'anandamide amidase' inhibitors. In any event, chemical modification of anandamide by the use of the large variety of arachidonate metabolizing enzymes available from natural sources may provide, along with the chemical synthesis of anandamide derivatives, material for structure/activity studies on this eicosanoid and for the development of new cannabinoid agonists and antagonists.

CONCLUDING REMARKS The dualistic nature of anandamide, since its 'double' discovery first as an 'endo-cannabinoid' (4) and then as an endogenous regulator of L-type calcium currents (27), can be summarized by the English proverb: 'to kill two birds with one stone'. Firstly, anandamide's 'double identity' as an eicosanoid and an N-acylethanolamine, while adding a new and atypical members to the already large family of AA derivatives, has aroused new interest in NAEs and in their likely precursors, the (N-acyl)phosphatidylethanolamines, whose precise biological role had been previously poorly understood (26). Subsequently, metabolic studies on anandamide have proceeded under the banner of dualism by providing two possible biosynthetic pathways (56, 59), as well as two routes for its degradation (59, 61, 63-65). Finally, the finding of a membrane phospholipid precursor and of a physiologically stimulated, phosphodiesterase-mediated biosynthetic mechanism (59), together with the knowledge that anandamide may have as targets calcium ion channels, extracellular G-protein-coupled cannabinoid receptors and intracellnlar signal transducing proteins (27, 31, 32), has suggested the possibility that this new eicosanoid may play a dual role as primary and second messenger. While providing answers to several questions, the findings reviewed here have opened a number of issues about anandamide: how can a molecule so different from THC recognize the THC receptor? Is there a real 'anandamidergic' network of neurons and how is it implicated in the control of behavioural and sensorial responses? Do saturated and monounsaturated NAEs play a role in the CNS? Since N-acylserines have been also reported (26), is there a bioactive N-arachidonylserine? Or, is there an anandamide-like metabolite or any other NAE in the peripheral nervous system? Do these metabolites participate in an endogenous cannabinomimefic control of immunological and repro-

9

ductive functions? Is 'anandamide synthase' simply an amidohydrolase working 'backwards'? Are the enzymes previously described to catalyze the biosynthesis of saturated and monounsaturated NAPEs or their metabolism to the corresponding NAEs (26, 53) also responsible for the formation and hydrolysis of the NAPE precursors of polyunsaturated, cannabinomimetic NAEs? Are the latter compounds metabolized in vivo by the same enzymes that catalyze the oxidation of polyunsaturated fatty acids? Can the stimulation of neurons evoke the formation, along with PLAz-derived eicosanoids, of PLD-derived ethanolarnide eicosanoids, such as, for example, ethanolamide prostaglandins or ethanolamide leukotrienes? These and many other questions will have to be addressed in the immediate future, thus requiring the joint efforts of neurobiologists, biochemists and chemists, and possibly opening up a new era both in neurochemistry and in eicosanoid research.

Acknowledgements The authors are grateful to Dr. D. Piomelli for letting them know about data on anandamide amidohydrolase, and to Mr. R. Turco, for artwork. This research was funded by the C.N.R. Strategic Project 'Tecnologie chimiche innovative'.

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