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The endogenous cannabinoid signalling system: chemistry, biochemistry and physiology. Vincenzo Di Marzo 1 and Luciano De Petrocellis 2 1 Istituto per la Chimica di Molecole di Interesse Biologico and 2 Istituto di Cibernetica, C.N.R.,Via Toiano 6, 80072, Arco Felice, Napoli, Italy Received 28 March 1997 Accepted 3 Feb 1997 Published 6 Feb 1997 Copyright © 1997 Internet Journal of Science - Biological Chemistry Abstract The cloning of central and peripheral cannabinoid receptor subtypes and the finding, in animal tissues, of endogenous metabolites capable of selectively binding them, opened a new age in cannabinoid research. In this article, starting from the pharmacology and chemistry of Indian hemp-derived as well as synthetic cannabinoids, and ending with the most recent findings on the biochemistry of endocannabinoids, we review the current state of the art of cannabinoid research, and explore the possible physiological roles of a newly discovered chemical regulatory apparatus: the endogenous cannabinoid system. ## Contents Introduction Pharmacological properties of plant and synthetic cannabinoids Chemical signalling through cannabinoid receptors The discovery of the ‘endocannabinoids’ Metabolic aspects of the ‘endocannabinoids’: biosynthesis, uptake and degradation Possible physiological roles of the endogenous cannabinoid system Concluding remarks Acknowledgements References Introduction Several similarities exist in the centuries-old histories of scientific research on opium and cannabis, the two illicit drugs of plant origin most widely used in the world. In both cases, the isolation and chemical characterization of the plant major active principle allowed its chemical synthesis and, subsequently, the thorough investigation of its pharmacological properties. These studies opened the way to the discovery in mammals of membrane receptors (and of the trans-membrane signal transduction events therewith associated) that could explain these pharmacological actions at the cellular and molecular level. As the presence in animals of receptors for molecules of plant origin could be justified only by the existence of endogenous ligands capable of binding to them, the characterization (1) and subsequent cloning (2) of the first cannabinoid receptor in mammalian brain in 1990 allowed, two years later, the isolation and characterization of the first ‘endocannabinoid’, anandamide (3), much in the same way as the finding of opiate receptors (4) had led to the discovery of ‘endorphins’ in the 1970’s (5). The somehow slower evolution of cannabis research compared to opiate research cannot be explained by a lower interest of the scientific community in cannabinoid pharmacology, since in the early nineteenth century the potentially important therapeutic exploitation of Cannabis sativa preparations, promped by their wide use in Indian folklore medicine, had been already recognized also by several Western physicians (6). It was rather the peculiar chemical nature of cannabis most abundant active principle, the highly lipophilic (-)-D 9-tetrahydrocannabinol, that made difficult its isolation and characterization (which were completed only in 1964 [7]), as well as its chemical synthesis and pharmacological handling, thus delaying the discovery of cannabinoid binding sites until 1988 (3). Unlike the ‘endogenous opiate system’, the development of our knowledge on the recently discovered ‘endogenous cannabinoid system’ has greatly benefited from (and is still greatly relying upon) the effort of bio-organic and natural product chemists, whose experience with lipophilic molecules, integrated with the work of pharmacologists, biochemists and molecular biologists, turned out to be extremely useful. In this article, while laying particular emphasis on the role of chemistry in both basic and applied cannabinoid research, we review the milestones that have led to the discovery of the ‘endogenous cannabinoid system’, and discuss the current hypotheses on its possible physiological functions. Pharmacological properties of plant and synthetic cannabinoids Back to the contents list As it is often the case with recreational drugs, cannabis initial popularity was probably due not only to its pshychoactive properties, but also to its several medical applications, widely documented by Indian medicine, and ranging from treatment of cramps, migraine, convulsions and neuralgia to attenuation of nausea and vomiting, decreased intestinal motility during diarrhea, bronchodilation in asthma and appetite stimulation (6). It was certainly also with the hope of finding potentially useful drugs that the isolation and chemical characterization of cannabis major, and essentially only, psychotropic constituent were painstakingly attempted and finally achieved in 1964 (7) thanks to the efforts of two Israeli natural The hypothetic precursors for D 9-tetrahydrocannabinol biosynthesis in plants, i.e. geranylpyrophosphate and olivetol, are shown. product chemists, Y. Gaoni and R. Mechoulam, who brilliantly completed the pioneering work started in the 1940’s by R. Adams (8). This component, (-)-D 9-tetrahydrocannabinol (THC), is one of a family of about 60 bi- and tri-cyclic compunds, formally derived from geranyl-pyrophosphate and olivetol, and named cannabinoids. These natural products usually contain a 1,1’-di-methyl-pyrane ring (the B ring), a variedly derivatized aromatic ring (the C ring), and a variedly unsaturated ciclohexyl ring (the A ring), and include also the non-psychoactive cannabinol, cannabidiol and cannabinolic acid (Fig. 1). The latter compounds have been suggested to contibute to some of THC-mediated effects of cannabis on peripheral tissues, such as cell protection, immunosuppression and anti-inflammatory properties (9). The structure elucidation of THC opened the way to the design of synthetic strategies for this as well as other natural cannabis components and also for a series of analogs (10). By 1986 over 300 such compounds, reviewed by Razdan (11), were available, and an extensive study of the pharmacological properties of THC, previously prevented by the rapid deterioration of crude cannabis preparations, as well as of the structural pre-requisites necessary for this compound to exert its typical psychotropic properties, was finally possible. A long list of pharmacological properties, both in the CNS and in peripheral tissues, was soon compiled for THC (12, 13), including the analgesic, antiemetic, anti-inflammatory, bronchodilatory and anti-convulsant effects already known for cannabis preparations, but also the reduction of blood ocular pressure in glaucomic patients and the alleviation of neurological disorders such as multiple sclerosis, Huntington’s chorea, spinal cord injury-associated spasticity and seizures. Also the illicitly looked for psychotropic properties of marijuana and hashish could be more accurately studied by using synthetic THC, which could reproduce all the typical effects of cannabis in humans (tachycardia, impairment of memory, alteration of mood, motor coordination, posture, cognitive ability and sensory perception) as well as in animal models (dog static ataxia, mouse hypomotility, catalepsy, hypothermy and antinociception) (12, 13). It would be impossible to update the ever increasing inventory of the effects ascribed to THC, which include other beneficial as well as noxious properties such as abortive and anti-fertility actions, various metabolic effects and the modulation of the release and/or action of prostaglandins or of pituitary and steroid hormones (12-14). A thorough structure-activity relationship (SAR) investigation on cannabinoids was originally performed by Martin and coworkers (15) by using a multi-parameter mouse model that included the measurement of: a) antinociception, determined by tail-flick latency, b) catalepsy, by the ring stand test, c) rectal temperature, and d) spontaneous activity in the open-field test. This ‘tetrad’ of behavioural tests was based on four of the several psychotropic properties of THC, that, when evaluated individually, are not selective for any particular class of compounds, but, if assessed together, have ‘proven to be highly predictive of cannabinoids’ (15). Based on the work of Martin and colleagues as well as on previous data (see refs. [16-18] for recent reviews of SAR studies) it was possible to conclude that: 1) the psychoactive effects of THC are enantioselective for the (-)-trans isomers - in the Martin’s ‘tetrad’ of tests the ED 50 ranged between 4 and 21 m g/kg for the (-)-enantiomer, while the (+)-enantiomer was inactive at 30 mg/kg (19); 2) the length as well as the lipophilicity of the C 3 alkyl chain are extremely important for biological activity - methylation/elongation of the pentyl chain of THC to the 1,1’- or 1,2-di-methyl-heptyl-derivatives, and hydroxylation (the main route for THC metabolism in vivo) respectively increase and reduce its biological activity; 3) the phenolic hydroxy-group is another important pre-requisite for biological activity - its acetylation, glycosilation, sulfation, replacement with an amine, a carboxyl or a methyl group, all resulted in the partial or total loss of cannabimimetic activity (20); 4) the orientation and chemical nature of the C 9 substituent is also an important feature of psychoactive cannabinoids - it must protrude into the same face as the phenolic hydroxy-group for the activity to be observed, while its hydroxylation increases biological potency (21, 22); this last pre-requisite, that keeps into account not only the chemical moieties necessary to the cannabinoid pharmacophore but also their three-dimensional conformation, may explain why the natural cannabis component cannabidiol is not active whereas the synthetic 9-nor-9 b-hydroxy-hexahydrocannabinol (HHC, Fig. 1) is ten-fold more active than THC. Chemical signalling through cannabinoid receptors Back to the contents list Despite the fact that a single mechanism of action is not likely to account for the multiple effects of THC, and that the existence of membrane receptors for this xenobiotic compound has been doubted for several years on the basis of its high lipophilicity, conclusive evidence exists today for the presence in the brain of high affinity and stereoselective cannabinoid binding sites. The stringent structural characteristics that cannabinoid compounds must possess in order to exert their psychological effects was the first strong evidence to a receptor-mediated mechanism of action. Only the existence of proteins with a three-dimensional binding site for cannabinoids could in fact explain, for example, the steric pre-requisites 1) and 4) outlined in the previous chapter. The development of cannabinoid analogs, designed on the basis of these structural requirements and using modern molecular modeling techniques, led eventually to the synthesis of CP-55,940 (Fig. 1), a compound which proved to be 4-25 times more potent than THC and could be radiolabelled and used as a probe for the first characterization of a selective, high affinity cannabinoid binding site (3). This receptor was identified in rat brain cortical membranes, where it exhibited K D and B max values respectively of 0.13-5 nM and of 0.9-3.3 pmol/mg protein; its binding with a selected series of cannabinoids perfectly correlated with their antinociceptive potencies (3, 23). The finding of the cannabinoid receptor explained why a completely different class of compounds, the aminoalkylindoles (such as WIN 55,212-2, Fig. 1, [24]), exhibit a range of pharmacological activities in the CNS superimposable to those of THC and other cannabinoids. With the help of computer-assisted molecular modelling it was in fact possible to obtain a three-dimensional model for the cannabinoid binding site whose steric and electrostatic properties kept into account the variations in the potencies of known cannabinoids and accomodated the chemically different aminoalkylindoles (25). The discovery of the ‘central’ cannabinoid receptor accounted for two widely described intra-cellular effects of THC, i.e. the inhibition of agonist-induced cyclic AMP (cAMP) formation and blockade of inward Ca 2+ currents mediated by N-type Ca 2+ channels (for reviews see [26, 27]). These effects, while suggesting that most of THC actions in the CNS may be due to the interference with neurotransmitters such as g-aminobutyric acid, acetylcholine and the cathecolamines (28), whose action is mediated by cAMP and Ca 2+, were shown to be blocked by pre-treatment of cells with pertussis toxin. The latter protein, in turn, catalyzes the ADP-ribosylation and subsequent inactivation of heterotrimeric GTP-binding proteins (the G-proteins) which are coupled to several receptors and deputed to translating an extra-cellular signal into a series of intracellular events. Therefore, it was reasonable to propose that THC, after binding to a receptor on the cell membrane, may activate one of such G-proteins (for example G i or G o) thereby inhibiting neurotransmitter-induced cAMP formation and Ca 2+ influx into neurons, and affecting, as a consequence, other neuronal biochemical events such as neurotransmitter release into and re-uptake from the synaptic interface (Fig. 2). Figure 2. Anandamide, 2-arachidonoyl-glycerol and psychoactive cannabinoids (e.g. THC) may act at pre-sinaptic CB1 receptors and inhibit, via a pertussis toxin sensitive G-protein, inward calcium currents and adenylate cyclase, thereby influencing, respectively, the release of other neurotransmitters (inhibition) and outward potassium currents (facilitation). The latter effect may lead to potentiation of GABA B receptor-mediated neurotransmission. Endocannabinoids may also act at post-synaptic CB1 receptors and inhibit the re-uptake of neurotransmitters or modulate the likelyhood of action potential generation and impulse propagation'. AC= adenylate cyclase; PKA= protein kinase A; Gi=inhibitory G-protein; GABA=g-aminobutyric acid; THC=D 9-tetrahydrocannabinol. After the first finding in 1988, cannabinoid binding sites were reported by several laboratories in specific brain regions as well as peripheral tissues, such as spleen cells (e.g. lymphocytes and monocytes/macrophages [29, 30]) and reproductive tissues (31), and their distribution was shown to well correlate with THC pharmacological actions. However, final and conclusive evidence for the existence of the central cannabinoid receptor was gained in 1990, when this protein was cloned and sequenced by Matsuda et al. (2) using the ‘homology screening’ approach. Based on the assumption that the protein possessed some sequence homology with other G-protein coupled receptors, an oligonucleotide probe based on the G-protein coupled receptor for substance K was synthesized and used to isolate, from a rat library, a clone encoding for an ‘orphan receptor’, i.e. a receptor with a unique sequence and no known ligand. The receptor was then expressed by transfection of its complementary DNA (cDNA) in host cells and several ligands were screened for their ability to selectively bind to membranes prepared from these cells. Only the cannabinoids were found to bind to these preparations and to inhibit adenylate cyclase-catalyzed cAMP formation. Almost concomitantly to this somehow fortuitous discovery, Gérard et al. reported the isolation and characterization of the human central cannabinoid receptor (32), which displayed a 98% amino acid homology with the rat receptor and was also expressed in the testes (31). The central cannabinoid receptor belongs to the ‘seven trans-membrane spanning receptor’ family, i.e. to those integral membrane proteins whose amino acid sequence assume a three-dimensional conformation with: 1) seven a-helices spanning from one side to the other of the cell membrane, 2) three extra-cellular and three intra-cellular loops, 3) a glycosylated extra-cellular N-terminal domain, and 4) an intra-cellular C-terminal domain involved (together with the intra-cellular loops) in the interaction with the G-protein responsible for the trans-membrane signal transduction of the receptor-mediated signal. In particular, the cannabinoid receptor had 32-39% homology and some structural similarities with the adrenocorticotropic hormone and melanocortin receptors, i.e. the lack of a disulfide bond between the first and second extra-cellular loops and of a proline residue in the fourth and/or fifth trans-membrane domain. The knowledge of the genomic and amino acid sequence of the central cannabinoid receptor had two important and immediate consequences. First, it was possible to synthesize oligonucleotide probes for the analysis and quantitation, assisted also by polymerase chain reaction technology, of the messenger RNA encoding for the receptor in several tissues (33). Direct visualization of cannabinoid receptor mRNA could be obtained also by in situ hybridization (34). These studies confirmed the overall CNS and peripheral distribution of the receptor obtained by previous binding experiments, even though discrepancies were observed, with some regions containing little amounts of mRNA and a high density of binding sites and vice versa. Secondly, three years after the cloning of the central receptor, a second human cannabinoid receptor could be identified in the marginal zone of the spleen, cloned and expressed (35). Following to this finding, the central and ‘peripheral’ cannabinoid receptors were named, respectively, CB1 and CB2. Also the CB2 receptor belongs to the ‘seven trans-membrane spanning receptor’ family, and exhibited an overall 44% identity with the CB1 receptor (with a 68% identity within the helical regions), was present in differentiated HL60 cells (35) and in RBL-2H3 basophils (36) but was not expressed in the brain nor (like the CB1 receptor) in the thymus, liver, lung and kidneys. Expression, by cDNA transfection, in CHO and COS cells of both CB1 and CB2 receptors, confirmed their coupling, through pertussis-toxin sensitive G-protein(s), to inhibition of N-type Ca2+ channel and/or adenylate cyclase (37, 38). Moreover, recent experiments with transfected cells have shown that CB1 receptors are also coupled to inhibition of Q-type Ca 2+ channels (39) and activation of inwardly rectifying K+ currents (40), whereas activation of CB2 receptors was found to induce sequentially the activation of mitogen-activated protein (MAP) kinase and the expression of the growth-related gene krox-24 (41). CB1 receptor-mediated activation of MAP kinase has been also recently described (42). The finding of the ‘peripheral’ cannabinoid receptor stimulated new chemical studies aimed at the development of cannabinoid antagonists and agonists selective for either of the two receptors. It was felt that agonists capable of selectively activating the CB2 receptor, being devoid of any psychotropic action, might be used as therapeutically useful compounds. Moreover, the finding of cannabinoid analogs capable of binding selectively only to one of the two cannabinoid receptors, without triggering the activation of the G-protein therewith coupled, might provide pharmacologically and therapeutically useful antagonists. Two landmarks in these new series of experiments were: 1) the synthesis of SR 141716A (Fig. 1), a potent and highly specific CB1 antagonist (43), and 2) the finding that THC binds to the CB2 receptor without inducing a typical cannabinoid receptor-coupled intra-cellular response, i.e. adenylate cyclase inhibition, and behaves as a weak functional antagonist at this receptor (44, 106). More potent, and rather selective, indole ligands for the CB2 receptors have also been described (45, 46), but their functional action on CB2-mediated intracellular events has not been yet assessed. Interestingly, cannabinol, which is not very active at CB1 receptors, was find to bind to CB2 receptors with a relatively low Ki (45). These findings open now the way to studies on the structural features of cannabinoid receptors involved in their interactions, on the one hand, with cannabimimetic ligands, and on the other, with G-proteins (47). A first example of such studies was the identification of lys-192 in the third transmembrane domain of the CB1 receptor as one of the aminoacid residues important for the interaction with HU-210 and CP-55490 but not with WIN 55212-2 (166). Several pieces of evidence seem to suggest the existence of other cannabinoid receptor types both in the CNS and the periphery. Some compounds have been shown to exert typical cannabimimetic actions, such as activation of phospholipase A 2, down-regulation of mast cells or inhibition of Ca 2+ currents through gap junctions, which cannot be specifically reproduced in cells transfected with either CB1 or CB2 receptors, even though they are blocked by pertussis toxin and/or SR 141716A (37, 54, 104, 114, 119, 153). However, no progress has been made toward the finding of ‘CBn’ receptors, even though an amino-truncated and functionally active isoform of the CB1 receptor has been recently characterized and christened CB1A (48). The discovery of the ‘endocannabinoids’ Back to the contents list The presence of cannabinoid receptors in mammalian cells could be explained only by assuming the existence of endogenous ligands for these receptors. Studies attempting to test this hypothesis had started already in the late 1980’s but proved to be inconclusive by leading to the isolation of various water soluble brain components whose chemical structure and pharmacological activity could not be fully assessed (49). The idea that an endogenous cannabimimetic metabolite might be, like THC, a lipophilic molecule and not necessarily a peptide, provided a successful lead for the isolation and complete characterization of the first ‘endocannabinoid’. The attention of chemists and biochemists was turned towards the extraction and purification of water insoluble fractions, and it was from such fractions that Devane et al. (3) isolated in 1992 a pig brain constituent capable of binding selectively and with high affinity to CB1 receptor-containing membrane preparations and to inhibit the electrically-stimulated mouse vas deferens twitch response, a typical cannabinoid pharmacological action (50). The chemical structure of this component was elucidated by means of proton NMR and GC/MS analyses. It was immediately clear that the first putative ‘endocannabinoid’ was a derivative of arachidonic acid, a polyunsaturated fatty acid that serves as precursor for a plethora of bioactive metabolites including prostaglandins, thromboxanes, leukotrienes etc. NMR data indicated the presence of amidated ethanolamine too, and MS data confirmed the structure of the metabolite as that of cis-5,8,11,14-eicosatetraenoyl-N-(2-hydroxy-ethyl)-amine (Fig. 3). Due to its possible cannabimimetic psychotropic properties, the compound was named anandamide after the Sanskrit word for ‘bliss’, ananda. During the four years following to its discovery, anandamide was shown to share with THC and other cannabinoids most of their pharmacological properties in both the CNS and peripheral systems, ranging from the basic actions in the ‘tetrad’ of behavioural tests in rodents (51-53), and on the intra-cellular second messengers cAMP and Ca 2+ (54-57), to the more peculiar: a) inhibitory effects on memory, motor activity and turning behaviour (58-62), and on ocular blood pressure and heart rate (63, 64), and b) regulatory actions on the levels of hormones of the hypothalamus-pituitary-adrenal axis (65-67), on dopamine-, acetylcholine-, noradrenaline-, endorphin-, glutamate- and GABA-mediated neurotransmission (68-71), and on immune (72, 73) and reproductive (74-76) functions. Cross-tolerance to THC for some of these effects, i.e. inhibition of mouse vas deferens twitch response (77), decrease of motor activity in an open field, catalepsy on a ring, hypothermia, analgesia on a hot plate (78, 79), was also observed with high doses of anandamide, thus further substantiating that this endogenous metabolite acts, at least in part, through the same mechanism as THC. Finally, anandamide, like THC, was found to increase both the affinity and number of rat cerebellum and hippocampus cannabinoid receptors after chronic and acute exposure (80). However, some differences between anandamide and THC actions have been also pointed out. First, the endogenous compound, unlike THC, was suggested to act as a partial agonist for the inhibition of N-type Ca 2+ channels in N18 neuroblastoma cells (56). Subsequently, biphasic effects for anandamide were reported by more than a laboratory, with opposite actions with respect to those of THC being observed, both in vivo and in vitro, with very low concentrations of the endocannabinoid (81). The biochemical bases for these effects were not investigated, and it is possible that they be mediated by a high affinity CBn receptor for anandamide coupled to adenylate cyclase through the a subunit of stimulatory (G s) G-proteins. Another possibility, depicted recently for other G i-coupled receptors such as the a 2-adrenoceptor and the D 2-dopamine receptor (82), is that low concentrations of anandamide may still induce the dissociation of pertussis-sensitive inhibitory G i-proteins with formation of a i subunits in concentrations too low to inhibit type I adenylaye cyclase, and bg subunits capable of activating type II and IV adenylate cyclase in the presence of GTP-bound a s subunits. Following to the finding of anandamide in pig brain, novel analytical methods for its purification and quantitative measurement were developed based mainly on HPLC, GC and GC-MS analyses of various derivatives (83-86). Preparation of the 4-(N-chloroformyl-methyl-N-methyl)amino-7-N,N-dimeyhyl-amino-sulphonyl-2,1,3-benzoxadiazole derivatives followed by HPLC and fluorometric detection, provided the first highly sensitive quantitative method (83). Another HPLC method exploited the preparation of the 1-anthroyl-derivatives of anandamide (84). Acetylation of the 2’-hydroxyl group or its derivatization with either trimethyl-silyl-, t-butyl-dimethyl-silyl-, bis-pentafluro-benzoyl- or bis-pentafluoro-propionyl-groups were used for GC and GC/MS methods (85-87). The use of deuterated d 8-anandamide (in turn prepared from d 8-arachidonic acid) allowed the development of a stable isotope dilution MS technique for anandamide quantitation (87). Anandamide was also directly quantitated without derivatization by using a sensitive LC/MS/MS method (88). By using these methods, anandamide was also identified and quantitated in human, bovine and rat brain (84-89), rat testis (84), rat skin and human and rat spleen (88). Some discrepancies exist in the data, reported by three laboratories (87-89), on the amounts of anandamide in rat brain. SAR studies have been also performed on anandamide in an attempt: a) to explain how a molecule apparently so chemically different from THC could bind to cannabinoid receptors and exert the same actions, b) to design novel drugs potentially useful in therapeutical applications and resistant to enzymatic degradation. It was, in fact, soon clear that anandamide was quickly degraded during both in vivo and in vitro pharmacological tests, as shown by the more rapid onset and termination of its action with respect to THC (51-53), and by the fact that non-specific inhibitors of esterases and proteases such as phenyl-methyl-sulphonyl-fluoride (PMSF) could both prolong and potentiate anandamide effects (90). The affinity for the CB1 receptor was shown to depend on both the length and degree of unsaturation of the fatty acid chain - aliphatic chains with less than three double bonds and with more or less than 20 carbon atoms were less potent or inactive (54). Ethanolamides of w 6 polyunsaturated fatty acids were more active than the ones of the corresponding w 3 polyunsaturated fatty acids. Hydroxylation of one of the four 1,4-diene groups, obtained by either autoxidation or enzymatic oxidation of anandamide (see also next chapter) and disregarding the stereochemistry of the alcoholic carbon atoms, variedly affected the CB1 binding activity of anandamide, with 12- and 15-hydroxy-anandamides being almost equipotent as anandamide and 8-, 9-, 11- and 5-hydroxy-anandamides being less active or inactive (91-93). Finally, ethanolamides of prostaglandins and leukotrienes were all completely inactive. This suggested that the steric restrictions induced by the formation of prostaglandin-like hairpins or the major mobility and hydrophilicity caused by the introduction of hydroxyl groups disrupt the three-dimensional conformation necessary for anandamide to mimick the THC pharmacophore and correctly orientate its pentyl chain. Modification of the N-(2-hydroxy-ethyl)- moiety of anandamide also proved to cause dramatic effects on the binding to CB1 receptors - its substitution with GABA and L-serine (97), with amides, ethers or esters of various length (94, 95) significantly decreased or abolished the activity, whereas its elongation by one carbon atom increased the potency of one order of magnitude. N-2-(4-hydroxy-phenyl)-ethyl- and N-2-(4-hydroxy-phenyl)-anandamide were less active than anandamide but more resistent to amidase-catalyzed inactivation (96). Finally, substitution of the hydroxyl group with a fluorine atom also increased the activity (95), while the introduction of a methyl group, as in N-(2-hydroxy-isopropyl)-arachidonoyl-ethanolamide (methanandamide), produced a compound less sensitive to enzymativ hydrolysis (98). The ability of this derivative to bind to CB1 receptors depends on the absolute configuration of the new chiral center which has to be R in order to observe an increased activity compared to anandamide (98). These data prompted the formulation of a model for the conformational relationships between anandamide and the cannabinoid pharmacophore (99) which was subsequently ameliorated by computer-assisted molecular dynamics studies (100). The latter showed that a looped, low-energy conformation of anandamide can overlap to the three-dimensional conformation of classical cannabinoids through the superimposition of: 1) the hydroxyl group of the ethanolamide and the phenolic group of THC; 2) the oxygen of the carboxyamide and the pyran oxygen in THC; 3) the w 6 aliphatic region of anandamide and the C 3 pentyl chain of THC; 4) the polyunsaturated loop and the cannabinoid tricyclic structure. This model can accomodate also other polyunsaturated N-acyl-ethanolamines such as the ethanolamides of di-homo-g-linolenic (20:3, w 6) and docosatetraenoic (22:4, w 6) acids, which were found in pig brain and, at concentrations slightly higher than those required for anandamide, shown: a) to bind to CB1 receptors (101, 54) and inhibit both forskolin-stimulated adenylate cyclase (54) and mouse vas deferens twitch response (102); and b) to be active in the ‘tetrad’ of mice behavioural tests (103). Recently another study, conducted with over 35 anandamide derivatives, provided further insights into the SAR of the endocannabinoid (104). Surprisingly, the hydroxyl group of the alkanolamine moiety was not found to be essential for CB1 binding activity, as arachidonoyl-n-propylamide and -isopropylamide were both found to be more active than anandamide. On the other hand, a secondary amide group in the molecule was shown to be a necessary pre-requisite for activity. Relevantly to the possible phospholipid origin of anandamide (see next chapter), its phosphorylation abolished binding activity. Finally introduction of one or two methyl groups on the carbon atom a to the carbonyl group in anandamide did not cause any change in the binding activity whereas analogous derivatizations of arachidonoyl-n-propylamide and -isopropyl-amides resulted in the two most active anandamide derivatives so far described (104). Another recent SAR study was carried out with head group analogs of anandamide and (R)-methanandamide, and confirmed the above model (105). More importantly, in this study some of the most active compounds at CB1 receptors, i.e. anandamide, 2’-fluoro-anandamide and (R)-methanandamide, were tested for their binding to CB2 receptors using a mouse spleen preparation and were found to bind very weakly, in agreement with the original finding by Munro et al. (35), and with the suggestion that anandamide, like THC, behaves as a weak agonist at CB2 receptors (106). These findings raise the question of whether there might be other ‘endocannabinoids’ selective for the CB2 cannabinoid receptor and produced in peripheral tissues. Two series of investigations attempted to find an answer to this question and led, respectively, to isolate another cannabimimetic arachidonic acid derivative, 2-arachidonoyl-glycerol, from canine gut (107), and to suggest for a saturated congener of anandamide, palmitoyl-ethanolamide, a role as endogenous CB2 receptor ligand with anti-inflammatory actions on mast cells/basophils (36). 2-Arachidonoyl-glycerol was shown to bind to both CB2 and CB1 receptors, transiently expressed in COS-7 cells, with Ki values higher than those previously reported for anandamide (107). This novel putative endocannabinoid produced in mice the typical ‘tetrad’ of cannabinoid behavioural actions, inhibited forskolin-induced cAMP accumulation (107), and shared with THC a down-regulatory action on mouse splenocyte proliferation (108), while it was much less active than THC and anandamide in inhibiting the mouse vas deferens twitch response (107). Later, 2-arachidonoyl-glycerol was shown to be present also in rat brain, in levels higher than those of anandamide (109), and in dog spleen and pancreas (110). Recently, the cannabimimetic monoglyceride was found to induce a rapid, transient elevation of intracellular Ca 2+ concentration in neuroblastoma X glioma hybrid cells, an effect that was also exerted by WIN 55,212-2 and blocked by the CB1 antagonist SR 141716A (167). Although a thorough analysis of its pharmacological properties in both the CNS and peripheral tissues has not been carried out yet, it is possible to hypothesize for 2-arachidonoyl-glycerol a role as either ‘primary’ or ‘auxiliary’ endocannabinoid on those target cells that selectively express either CB2 or CB1 receptors, where anandamide, respectively, has either no significant effect or is the ‘primary’ cannabinoid ligand. The picture is much less clear for palmitoyl-ethanolamide, an N-acyl-ethanolamide which is inactive at the CB1 receptor (54, 55) and is co-synthesized with anandamide in all tissues and cells examined so far (see next chapter). This lipid was shown, together with classical cannabinoids, to potently inhibit serotonin release from rat basophilic leukaemia (RBL-2H3) cells, which express CB2 but not CB1 receptors, and to displace the binding of [3 H]WIN 55,212-2 from membrane preparations from the same cells (36). Anandamide was found to antagonize the down-modulatory effects of palmitoyl-ethanolamide and cannabinoids and did not exert any effect on its own. These findings suggested that palmitoyl-ethanolamide and cannabinoid anti-inflammatory effect in RBL-2H3 cells were mediated by CB2 receptors, which cannot be efficiently activated by anandamide (106). However, when palmitoyl-ethanolamide was tested for its binding to membrane preparations from cells selectively transfected with CB2 receptor cDNA, it was found to be inactive (104). This discrepancy can be explained by hypothesizing the presence in RBL-2H3 cell membranes, apart of CB2 receptors, also of a ‘CBn’ receptor different from CB1 and responsible for the immunosuppressive actions of cannabinoids and palmitoyl-ethanolamide, or by assuming for the latter metabolite a non-cannabinoid receptor-mediated mechanism of action. Whatever its mode of action, the mast cell/basophil-mediated anti-inflammatory effects of palmitoyl-ethanolamide, in many aspects similar to those ascribed to synthetic cannabinoids (9), are substantiated by in vivo pharmacological studies conducted both in the 1960-70’s (111) and very recently (112), and by its de novo synthesis by stimulated basophils and macrophages (113). Moreover, a neuroprotective action against glutamate-induced excitotoxicity has been recently described for palmitoyl-ethanolamide in mouse cerebellar granule neurons (114). This effect was again suggested to be mediated by the CB2 receptor (whose mRNA was shown to be expressed in these cells), since it was not exerted, but, on the contrary, was antagonized, by anandamide. Also in this case the effect of palmitoyl-ethanolamide may be due to a non-CB1 non-CB2 receptor, and it is Figure 3. The proposed target of each metabolite is shown in parentheses. CB1=‘central’ cannabinoid receptor; CB2 ‘peripheral’ cannabinoid receptor; CBn=non-CB1-non-CB2 cannabinoid receptor(s); FAAH=fatty acid amide hydrolase. noteworthy that neuroprotective actions have been reported for non-psychoactive synthetic cannabinoids such as dexanabinol ((+)-HU-211, Fig. 1) that, like palmitoyl-ethanolamide, do not bind the CB1 receptor, but, unlike palmitoyl-ethanolamide (114), may act as non-competitive antagonists at the N-methyl-D-aspartate receptor for glutamate (115). Apart from 2-arachidonoyl-glycerol and N-acyl-ethanolamines, another potentially cannabimimetic long chain fatty acid derivative, cis-9-octadecenoamide (oleamide), has been recently isolated from the cerebrospinal fluid of cats and humans deprived of sleep and shown to induce sleep in mammals (116). Although not yet demonstrated for anandamide, a sleep-inducing action has been often suggested for the endocannabinoid and THC because of their sedative and motor inhibitory properties, and both slow-wave and REM sleep have been found to be inhibited by the CB1 antagonist SR 141716A (117), suggesting that " .... an endogenous cannabimimetic system may regulate the organization of the sleep-waking cycle". Accordingly, oleamide, like anandamide and/or 2-arachidonoyl-glycerol, was recently shown: a) to be active in the ‘tetrad’ of mice behavioural tests (118) which is highly predictive of cannabinoids, b) to inhibit gap junction-mediated Ca 2+ fluxes in astrocytes (119, 120), and c) to inhibit lymphocyte proliferation (121). However, the cannabimimetic actions of this compound cannot be due to the activation of any of the known cannabinoid receptors, since it binds to either CB1 or CB2 receptors in transfected cells only at very high concentrations (> 10 m M, [104, 117, 122]). An alternative, and indirect, way of exerting cannabimimetic actions has been suggested for oleamide (118) based on its ability to inhibit the enzymatic hydrolysis of anandamide (123) and, therefore, to raise the endogenous levels of the latter, by competing for the same inactivating enzyme, i.e. the recently characterized ‘fatty acid amide hydrolase’ (120) (see next chapter). A similar action may be expected also by other long chain fatty acid ethanolamides, which are co-synthesized with anandamide in neurons (124) but do not bind the CB1 receptor (54), and whose biological role is still unclear. Metabolic aspects of the ‘endocannabinoids’: biosynthesis, uptake and degradation Back to the contents list A necessary pre-requisite for an endogenous metabolite to play a role as a physiological (neuro)modulator in a certain organism is that molecular mechanisms leading to its de novo biosynthesis and inactivation must exist in that organism. Since 1992, based on previous knowledge and, again, on the work of bio-organic chemists, two biosynthetic pathways have been proposed for both anandamide and 2-arachidonoyl-glycerol. The two routes suggested in the 1960’s and 80’s (for a review, see [111]) for the biosynthesis of saturated, mono- and di-unsaturated fatty acid ethanolamides, i.e. the direct and energy-free enzymatic condensation between the fatty acid and ethanolamine, and the phospholipase D-catalyzed hydrolysis of N-acyl-phosphatidyl-ethanolamines, have been shown to apply also to anandamide (Fig. 4). The first pathway was found to occur in mammalian brain particulate fractions independently from ATP and coenzyme A and in the presence of m M concentrations of arachidonic acid and high mM concentrations of ethanolamine (126, 127). The enzyme responsible for this reaction was named ‘anandamide synthase’, was active at alkaline pH (8.5-10) and selective for arachidonic acid and ethanolamine versus other fatty acids or amines. As the cellular concentrations of arachidonic acid and ethanolamine are much lower than those necessary to the enzyme, it was suggested that ‘anandamide synthase’-mediated anandamide biosynthesis would require the prior activation of the two phospholipases mostly responsible for arachidonic acid and ethanolamine release from membrane phospholipids, i.e. phospholipase A 2 and D. Later, ‘anandamide synthase’ was partially purified from porcine brain (128) and shown to co-chromatograph with, and to exhibit the same pH/temperature dependency and inhibition profile as, the enzyme catalyzing anandamide hydrolysis to arachidonic acid and ethanolamine, ‘anandamide amidohydrolase’ (see below). This finding, together with the high substrate concentrations required for ‘anandamide synthase’ to work, allowed to suggest that this enzyme was an ‘anandamide amidohydrolase’ working ‘in reverse’ (128), and that another pathway, occurring with more physiologically relevant substrate concentrations, may be responsible for anandamide biosynthesis in intact cells. This second pathway was shown to underly the phospholipase A 2-independent production of anandamide and other acyl-ethanolamides in intact rat neurons stimulated with membrane-depolarizing agents (e.g. ionomycin, kainate and high potassium), and to occur through the phosphodiesterase-catalyzed hydrolysis of a pre-formed membrane phospholipid precursor, N-arachidonoyl-phosphatidyl-ethanolamine (N-ArPE) (124). Both N-ArPE and other N-acyl-phosphatidyl-ethanolamines, with the same N-fatty acid chains as those of the acyl-ethanolamides co-released with anandamide from stimulated neurons, were characterized in rat neuronal lipid fractions and their turnover was shown to be induced by ionomycin (124). Moreover, a phospholipase-D like enzyme catalyzing the conversion of synthetic radioactive N-ArPE to anandamide was found in rat neuronal homogenates (124), wheras a similar enzyme for the hydrolysis of other N-acyl-phosphatidyl-ethanolamines to acyl-ethanolamides had been described and partially characterized from dog brain as early as 1983 (125). Subsequently, these data were confirmed by another study (89) where acyl-ethanolamides present as lipid components of rat brain lipid extracts or released by the hydrolysis of the corresponding N-acyl-phosphatidyl-ethanolamines, purified from the same extracts, were identified and quantitated as 1-anthroyl-derivatives by HPLC. The presence of a Ca 2+-dependent trans-acylase activity catalyzing the transfer of arachidonic acid from the sn-1 position of 1,2-sn-di-arachidonoyl-phosphatidyl-choline to phosphatidyl-ethanolamine, with the subsequent formation of N-ArPE (Fig. 4), was also reported in this study. The enzyme displayed optimal activity at pH=7.0 and K m values of about 20 and 120 m M, respectively for 1,2-sn-di-arachidonoyl-phosphatidyl-choline and phosphatidylethanolamine. Following the development of a new HPLC methodology for the quantitation of N-acyl-phosphatidyl-ethanolamines, the trans-acylase activity was suggested to be up-regulated, in rat central neurons, by the same stimuli leading to anandamide and acyl-ethanolamide formation, e.g. augmentation of intracellular Ca 2+ and cAMP concentrations and activation of cAMP-dependent protein kinase (129). Trans-acylase-catalyzed N-acyl-phosphatidyl-ethanolamine formation was already known to be induced also by pathological stimuli such as ischemia and, more generally, tissue injury (111, 130), and anandamide and acyl-ethanolamide levels in bovine and rat CNS were accordingly shown to be increased post-mortem or following noxious stimulation (86-88). The phospholipase D-mediated pathway was also implicated in anandamide and palmitoyl-ethanolamide formation in N18 neuroblastoma cells (131) and in two immortalized immune cell types, stimulated with either ionomycin or immunological challenge (113), as well as in sea urchin ovaries (132) and rat testes (84). Therefore, several pieces of evidence support the involvement in physiopathological situations of this second pathway of anandamide biosynthesis, which may explain also the formation of other potential cannabimimetic acyl-ethanolamides (101) in both the CNS and peripheral tissues. However, the recent finding in the mouse uterus (133) of two distinct enzymatic activities for the synthesis and degradation of anandamide, respectively from and to arachidonic acid and ethanolamine, has re-opened the question as to which of the two proposed pathways is Figure 4. Possible biosynthetic and catabolic pathways for anandamide and related acyl ethanolamides. Anandamide (and palmitoyl-ethanolamide) produced by either a ‘synthase’ enzyme or N-acyl-phosphatidyl-ethanolamine-specific phospholipase D (NAPE-PLD) following membrane depolarization is released outside the cell and acts at neighboring cells. In order to be catalyzed by the ’synthase’ enzyme the formation of anandamide must be preceeded by phospholipase A 2 (PLA 2) activation. Once released by cells, anandamide or palmitoyl-ethanolamide can be uptaken by selective carrier mechanisms and degraded by anandamide amidohydrolase (fatty acid amide hydrolase=FAAH), thereby producing ethanolamine and fatty acids. The latter are readily incorporated into membrane phospholipids (PC, PS, PE, PC). Alternative catabolic pathways for anandamide may utilize the enzymes of the arachidonic acid (AA) cascade; however the formation of the ethanolamides of hydroxy-eicosatetraenoic acids (HETE), prostaglandins (PG) or leukotrienes (LT) has never been in intact cells. B= phospholipid bases. actually responsible for the biosynthesis of the endocannabinoid in vivo (for a recent review see also [134]). As with anandamide, also the biosynthesis of 2-arachidonoyl-glycerol was shown to be stimulated by ionomycin in neuronal cells (135). In mouse neuroblastoma cells, this effect was Ca 2+-dependent, accompanied by 1-acyl-2-arachidonoyl-glycerol formation, potentiated by incubation of cells with exogenous phospholipase A 2 , and not sensitive to an inhibitor of one of the enzymes responsible for 1-acyl-2-arachidonoyl-glycerol formation in cells, i.e. phospholipase C. An abundant enzymatic activity catalyzing 1-acyl-2-arachidonoyl-glycerol hydrolysis to 2-arachidonoyl-glycerol was also found in neuroblastoma cell homogenates (135) together with phospholipase C-like enzymes capable of slowly but significantly catalyzing the conversion of sn-2-arachidonoyl-containing (lyso)phosphatidylcholine species into 1-acyl-2-arachidonoyl-glycerol (136). It was suggested that 2-arachidonoyl-glycerol biosynthesis may follow either phospholipase A 2- or phospholipase C-mediated pathways depending on whether the neuron is stimulated with agents that are coupled only to Ca 2+ influx or also to phospholipase C Fig. 5 Fig. 5. Potential biosynthetic connections between anandamide and 2-arachidonyl glycerol in mouse neuroblastoma cells. a= Phospholipase A 2-dependent pathway; b= pathway in competition with anandamide biosynthesis; c= pathways occurring concomitantly to anandamide biosynthesis. MAG-AcCOA=monoacylglycerol-3-phosphate:acyl-coenzymeA; 2-AG= 2-arachidonoylglycerol; NAPE-PLD= Phospholipase D selective for N-acyl-phosphatidyl-ethanolamines. activation. The participation of phosphatidic acid, produced either from de novo biosynthesis or by phospholipase D-activation, as precursor for 1-acyl-2-arachidonoyl-glycerol may be also hypothesized, but still needs to be investigated. It is also possible that, under certain circumstances, the de novo formation of 2-arachidonoyl-glycerol may occur concomitantly to that of acyl-ethanolamides, including anandamide, starting from sn-2-arachidonate-containing 1-lyso-phosphatidylcholine (136) or phosphatidic acid, two by-products of acyl-ethanolamide biosynthesis (Fig. 5). A preliminary study has recently cast some light also on the biosynthesis of oleamide in rat brain. An enzymatic activity catalyzing the formation of the cannabimimetic sleep-inducing factor from oleic acid and ammonia was in fact identified in rat brain microsomes (168), and suggested to be the same as anandamide synthase-amidohydrolase. Metabolic pathways, leading to partial or complete inactivation of anandamide and 2-arachidonoyl-glycerol, have also been described. For the former compound, enzymatic hydrolysis of the amide bond is the major degradative pathway (111, 124, 125). This reaction is catalyzed by an enzyme originally named ‘anandamide amidohydrolase’, characterized from rat, mouse and porcine brain particulate fractions, and shown to be optimally active at alkaline pH (8.5-10), quite selective for anandamide versus other acyl-ethanolamides, and inhibited by typical serine- and cysteine-protease inhibitors such as PMSF, p-hydroxy-mercuri-benzoate, p-bromo-phenacyl-bromide and N-ethyl-maleimide (123, 128, 137-140), as well as by the phospholipase A 2 inhibitor arachidonoyl-trifluoro-methyl-ketone (141). Interestingly, this enzyme displayed a tissue distribution and a pH dependency profile almost identical to those of an amidohydrolase previously reported to catalyze the hydrolysis of oleoyl-ethanolamide in dog brain (125). Initial efforts toward the full purification of this integral membrane protein proved to be inconclusive. It was only at the end of 1996 that, once again thanks to the contribution of organic chemists, it was possible to fully purify the enzyme. Cravatt et al. were working on the enzyme ‘oleamide hydrolase’ that catalyzes the hydrolysis of the amide bond of oleamide (116, 142). This enzyme had been previously partially characterized first from mouse neuroblastoma N18 cells and subsequently from rat liver, and in both cases suggested to be the same protein as ‘anandamide amidohydrolase’ (123, 143). A chromatographic resin was derivatized using oleoyl-trifluoro-methyl-ketone, a compound previously shown (143) to inhibit ‘oleamide hydrolase’ by forming a reversible, albeit relatively stable, hemiketal adduct with the catalytically active sulphidryl or hydroxyl group (Fig. 6). The derivatized resin was then used for the purification of the enzyme, solubilized from rat liver membranes, by affinity chromatography, which led to the protein pure to homogeneity in basically one single step (120). Once purified the enzyme, it was then not difficult to obtain some amino acid sequence necessary to synthesize oligonucleotide probes or to raise antibodies for the screening of rat DNA libraries and the isolation of the clone containing the gene encoding for the enzyme. The gene was then sequenced and yielded the complete amino acid sequence of the protein, whose cDNA was expressed in COS-7 cells. It was thus possible to confirm the molecular weight of ‘oleamide hydrolase’ (60-65 kDa) and the fact that the enzyme recognizes anandamide as the preferential substrate. This prompted the authors the use of the name ‘fatty acid amide hydrolase’ (FAAH) for this enzyme (120), as previously suggested also for mouse neuroblastoma anandamide amidohydrolase (123). The sequence analysis of FAAH showed a) a hydrophobic region predicted to assume a trans-membrane a-helical conformation, b) a significant homology with other amide hydrolase enzymes, and c) a consensus class II-SH3-domain binding sequence, which suggested that other proteins may interact with FAAH, thereby regulating its activity and/or subcellular localization. Southern and Northern blots of DNA and mRNA from various rat tissues, probed with an internal 800 base pair fragment of FAAH cDNA, confirmed previous data (138) on the tissue distribution of this protein, whose levels are highest in the liver, brain and kidneys, measurable in testes and lung, low in the spleen and undetectable in the skeletal muscle and heart (120). FAAH-like enzymes have been recently described also in rat cortical astrocytes (124) as well as in other peripheral tissues such as sea urchin ovaries (132), mouse uterus (133), ocular tissues (144) and RBL-2H3 basophils (113). In the latter cell type, FAAH, unlike the enzyme from brain and neuronal cells, displayed a high affinity also toward palmitoyl-ethanolamide, in support of the putative role suggested for this compound as endocannabinoid down-regulator of basophils/mast cells. By using several long chain acyl-ethanolamides as substrates, it was shown that the basophilic enzyme had the higher catalytic efficiency the shorter and the more unsaturated the substrate fatty acid chain (113), with the highest efficiency being observed with anandamide, linoleoyl-ethanolamide and palmitoyl-ethanolamide. The possibility of RBL-2H3 cells expressing an isoform of FAAH structurally different from that characterized in brain and rat liver particulate fractions is currently under investigation. Chemical studies have been performed with FAAH. Recently, inhibitors with different mechanisms of action have been obtained from the derivatization of arachidonic acid and/or oleic acid with functional groups potentially reactive towards catalytically active serine and cysteine residues (Fig. 6). Diazo-methyl- and chloro-methyl-derivatives (143, 145) were found Upper panel: reversible inhibitors forming tetrahedral hemiketal intermediates with catalytically active serine or cysteine groups (a shows the reversal reaction). Lower panel: irreversible, covalent inhibitors forming stable adducts with catalytically active serine or cysteine groups. X= sulphur(II) or oxygen atom; E= strongly electrophilic group; Y=phosphorus or sulphur (VI) atom; L= leaving group. to potently inhibit FAAH from different sources (for the arachidonoyl-analogs, IC 50 values ranged between 2 and 23 m M, [145]), possibly by forming reversible tetrahedral adducts with the active site -SH or -OH groups. The methyl-arachidonoyl-fluoro-phosphonate derivative was the most potent FAAH inhibitor so far described, with IC 50 values between 1 and 3 nM, and formed a covalent, enzymatically inactive adduct with the enzyme (145). Palmityl-sulphonyl-fluoride, although less active than arachidonoyl-methyl-fluoro-phosphonate, is another very potent inhibitor of anandamide enzymatic hydrolysis (IC 50 =50 nM, [146]) and is also likely to behave as irreversible inhibitor of FAAH, since its analog PMSF has been shown to covalently inhibit the enzyme (145). Data on the mechanism of action of FAAH inhibitors may provide important informations on the sterical and chemical requirements of the enzyme active site as well as on the possible use of these substances for the affinity-labelling of FAAH enzymes. Apart from amide bond hydrolysis, anandamide has been shown to undergo, in tissue homogenates, also to another series of metabolic reactions consisting of the oxidation of its double bonds with subsequent formation of hydroperoxy- and hydroxy-anandamide derivatives. These reactions may be catalyzed by cytochrome p450 hydroxylases (92), even though anandamide has been shown to be recognized as a substrate also by lipoxygenase enzymes (91, 93). Indeed, it is quite possible that anandamide may be recognized also by other enzymes involved in arachidonic acid and polyunsaturated fatty acid metabolism. For example, the endocannabinoid has been shown to be a good substrate for an unusual algal enzyme, an ‘arachidonic acid conjugated-triene isomerase’ (147), with formation of the conjugated 5Z, 7E, 9E-triene derivative, a compound which was found to retain significant CB1 receptor binding activity (148). This finding opens the possibility that anandamide derivatives may be obtained in the future also by enzymatic methods, thus widening the range of compounds to be tested for cannabinoid activity and to be used for SAR studies. If anandamide, palmitoyl-ethanolamide and oleamide are mostly inactivated through the action of the same or highly homologous FAAH enzymes, the breakdown of 2-arachidonoyl-glycerol to arachidonic acid and glycerol was found to be catalyzed by a different type of enzyme(s), located in both particulate and cytosolic fractions of mouse neuroblastoma cells (135). In fact, the enzymatic hydrolysis of the mono-glyceride was not inhibited by either anandamide or arachidonoyl-trifluoro-methyl-ketone, thus ruling out the involvement of FAAH-like enzymes. It is possible that one or more of the several mono-, di- and tri-acylglycerol lipases present in animal cells contribute to 2-arachidonoyl-glycerol degradation, and, unlike anandamide, there is no evidence to date for the existence of a specific enzyme deputed to the physiological inactivation of the cannabimimetic glyceride. Preliminary results, however, have shown that, much in the same way as anandamide enzymatic degradation is inhibited by other acyl-ethanolamides, unsaturated mono-acyl-glycerols may counteract the degradation of 2-arachidonoyl-glycerol and enhance its cannabimimetic activity (149). Finally, uptake mechanisms for cannabimimetic acyl-ethanolamides have been also reported in cultured rat central neurons and cerebellar granule cells (124, 150) as well as RBL-2H3 cells and J774 mouse macrophages (113). These mechanisms are likely to be mediated by specific carrier proteins in asmuch they are: 1) saturable - with K m and V max values ranging between 25 and 41 m M and 0.4 and 0.6 nmols min-1, respectively, 2) temperature dependent - most of the uptake is abolished by lowering the temperature to 4°C, 3) selective for anandamide in the CNS and for either anandamide or palmitoyl-ethanolamide in leukocytes, and 4) inhibited by phloretin and alkylating agents such as PMSF or N-ethyl-maleimide. The purification and further characterization of these proteins have not yet been attempted. Possible physiological roles of the endogenous cannabinoid system Back to the contents list Despite the several studies carried out on the pharmacological activity of both plant and endogenous cannabinoids, the physiological role of the endogenous cannabinoid system has remained elusive. While the picture of cannabinoid receptor distribution is becoming more and more clear, still very little is known on the correlation of certain physiopathological responses with the production and inactivation of endocannabinoids and the expression/activity of the enzymes involved. The development of GC/MS techniques for the measurement of anandamide renders now feasible studies on the occurrence of altered levels of this endocannabinoid during certain disfunctions of both central and peripheral systems. The preparation of antibodies against anandamide, and the subsequent setting up of selective radioimmunoassays for this acyl-ethanolamide, is likely to provide, in the future, a more accessible technique for its quantitative determination, and, therefore, to stimulate further studies in this direction. The recent cloning and sequencing of FAAH, i.e. the enzyme most likely responsible for the inactivation of cannabimimetic fatty acid amides, will allow the assessment of its expression in several tissues under physiopathological conditions, which, like with other inactivating enzymes for (neuro)transmitters and (neuro)modulators, will help clarifying the physiological roles of these metabolites. Conversely, the enzymes responsible for acyl-ethanolamide biosynthesis have not been characterized yet, and little information is available on their regulation. From the limited amount of data acquired so far, the general impression is that the endogenous cannabimimetic system may be a widespread tuning system of numerous finely regulated tasks performed in multi-cellular organisms, and that its importance is not limited to central nervous functions but, on the contrary, may concern with several peripheral processes such as the modulation of neurotransmitter release/action at autonomic and sensory fibers, and the control of immunological, gastrointestinal, reproductive and cardiovascular performance. In the brain, this regulatory system, through the action at CB1-like receptors of anandamide, polyunsaturated acyl-ethanolamides and 2-arachidonoyl-glycerol, may interact with thermoregulatory centres as well as regulate perceptive (hearing, colour vision, taste), cognitive (sleep, long term potentiation, short-term memory) and motor (movement, coordination, posture, skeletal muscle tone) functions. Indeed, the presence of CB1 receptors and/or anandamide and/or FAAH in the thalamus, hippocampus and cortex or in the striatum, substantia nigra and cerebellum, respectively, supports a role of the endogenous cannabinoid system in cognitive and motor responses. The recently reported CB1-mediated activation by anandamide of neuronal focal adhesion kinase in hyppocampal neurons (151), has suggested for the endocannabinoid a role in synaptic plasticity. A regulatory action on astrocyte Ca 2+ homeostasis through inhibition of gap junctions has been also observed for anandamide and oleamide (119, 120). A role in brain development has also been suggested (169). The physiological relevance to these central functions of the recently reported effect by anandamide on protein kinase C in vitro (170) remains to be assessed. A neuroprotective role of acyl-ethanolamides in general and of palmitoyl-ethanolamide in particular have also been described also on the basis of their production at sites of neuronal damage or death (86-88, 111, 125). Finally, the presence of CB1 receptors in the hypothalamus may imply a role of the endogenous cannabinoid system in the fine tuning of the secretion of pituitary hormones (65-67). In particular, the activation of the release of the hypothalamic hormone causing the secretion of adrenocorticotropic hormone from the pituitary, i.e. corticotropin releasing factor-41, and the subsequent enhancement of corticosterone production from the adrenal gland, have been observed following intracerebroventricular injection of anandamide (65). Likewise, the release of other hormones, such as prolactin and the luteinizing, follicle stimulating and growth hormones may be inhibited by anandamide acting at the level of the hypothalamus (66, 67). The central role of the endogenous cannabinoid system may be effected through an action at both the presynaptic and post-synaptic level. In the first case, endocannabinoids, by acting at presynaptic cannabinoid receptors, and subsequently modifying the intracellular levels of cAMP, K+ and Ca 2+ and/or the activity of protein kinases, may modulate the action of other presynaptic neurotransmitters or the release of mediators acting at post-synaptic receptors. Recently a facilitatory action of THC on the release of dynorphins acting at either k- or d-opioid receptors has been described (152), and this mechanism may underly the well known analgesic actions of THC and anandamide. Post-synaptic cannabinoid receptors may mediate an endocannabinoid action on the uptake of neurotransmitters such as GABA, thereby potentiating their action (Fig. 2). Interactions with other neurotransmitters, such as dopamine, acetylcholine and glutamate, may be mediated by similar pre- and post-synaptic actions of endocannabinoids (68-71). In the periphery, the endogenous cannabinoid system may mediate the chemical communications between different types of immune cells or between sensory fibers and blood cells. In acute inflammatory reactions, basophils and mast cells, once activated by IgE receptor cross-linking and/or Ca 2+ influx, would release palmitoyl-ethanolamide and anandamide (113) together with histamine, serotonin and leukotrienes. While the latter mediators produce vascular permeability and chemotaxis, palmitoyl-ethanolamide would act as an autacoid signal on the same or on neighboring basophils and mast cells and inhibit their degranulation (36), thus keeping the inflammatory reaction under control. Anandamide might play a dual role by potentiating the activity of basophil/mast cell phospholipase A 2 (153), thus facilitating the formation of eicosanoids with often opposing actions on immune cell functionality, e.g. leukotrienes and prostaglandin E 2, which respectively induce the recruitment of neutrophils and macrophages and suppress the activity/proliferation of lymphocytes. Basophilic anandamide might enhance the production of prostaglandin E 2 from macrophages (where this eicosanoid is one of the major products of AA oxidation) or directly inhibit the proliferation of lymphocytes recruited in the late phase of the inflammatory reaction, while inducing their apoptosis (73). Palmitoyl-ethanolamide and anandamide produced by macrophages might: a) feed-back on macrophages (72), basophils and mast cells (36), again in order to prevent the excessive propagation of the inflammatory response and inhibit the subsequent delayed hypersensitivity (111, 112); b) co-adjuvate macrophage prostaglandin E 2 in the non-specific immunosuppression of T helper cells (73), thus participating in the down-regulation of autoimmune reactions. 2-arachidonoyl-glycerol might act as a natural substitute for anandamide on target cells which express CB2 but not CB1 receptors. Were biosynthetic mechanisms similar to those described previously for central neurons (124) to be found for acyl-ethanolamides and 2-arachidonoyl-glycerol also in sensory neurons of afferent C-fibers, these compounds might play a major role in the model of neurogenic inflammation proposed in the 1980’s (154), by acting, together with somatostatin and vasoactive intestinal polypeptide, as ultimate terminators of inflammatory responses generated by noxious stimuli through an ‘axon-reflex’. On the other hand, the finding of presynaptic cannabinoid receptors on sensory neurons, whereupon palmitoyl-ethanolamide and anandamide produced by stimulated leukocytes may act by modulating the release of inflammatory neuropeptides, would add further complexity to this scenario and widen the range of possibilities for feed-back signalling from immune cells to peripheral fibers. The recent report of AnNH-induced inhibition of capsaicin-evoked substance P and CGRP release from the dorsal horn of rat spinal cord (155) provides preliminary support to this hypothesis. Auxiliary leukocytes (basophils and mast cells) and macrophage-like cells, either recruited from local blood vessels upon request or as resident populations, are found in several tissues where they participate in tissue damage-induced immune defense. Therefore, the finding that some of these cells synthesize cannabimimetic acyl-ethyanolamides (113) opens the way to a novel mode for their chemical signalling with several tissues and cell types expressing cannabinoid receptors. For example, the cellular source of acyl-ethanolamides with saturated or monounsaturated N-acyl chains and of their phospholipid precursors in infarcted dog heart and ischemic brain (125) has never been investigated. It is possible that leukocytes recruited from blood following tissue damage may be responsible for the production of acyl-ethanolamides with vasodilatory and depressor activity (64) or neuroprotecting effects (114). If shown to share with macrophages and RBL-2H3 cells the capability of biosynthesizing and responding to acyl-ethanolamides, populations of macrophages (as microglia) and mast cells resident in the brain may establish a network of chemical communications also with central neurons, which contain cannabinoid receptors and synthesize acyl-ethanolamides during physiopathological responses. Finally, endocannabinoids may also act by modulating nitric oxide (NO) release from leukocytes. A strong enhancement of human monocyte and mussel microglia and immunocyte basal NO production, that could be blocked by SR 141716A, has been recently reported for anandamide and CP 55,940 (156); conversely, THC, by decreasing cAMP levels, was found to inhibit inducible NO synthase expression and NO production by a macrophage cell line that does not express CB1 receptors (157). Therefore, different responses seem to be observed depending on whether constitutive or induced NO formation is measured. It is also possible that, as for other immunomodulatory effects of cannabinoids, the action on NO release from immune cells may depend on which cannabinoid receptor is expressed in these cells. To date, it is not yet clear which of the immune actions of endocannabinoids are mediated by CB1, CB2 or CBn cannabinoid receptors, and a thorough examination of the types of cannabinoid receptors expressed by the different sub-populations of each immune cell type must be carried out in order to obtain a complete picture of the immunomodulatory role of the endogenous cannabinoid system. Mechanisms analogous to the ones described above have been proposed to occur during a hypothetic modulation by the endogenous cannabimimetic system of cardiovascular, respiratory, digestive and escretory functions, particularly for what concerns with the control of smooth muscle tone. Endocannabinoids and THC were shown to inhibit guinea-pig small intestine and mouse urinary bladder acetylcholine-induced contractions by a pre-synaptic, CB1 receptor-mediated inhibitory action on the release of acetylcholine from autonomic fibres (158, 159). The hypothensive effects of anandamide and THC were also shown to be due in part to a presynaptic action on CB1 receptors and to the subsequent inhibition of noradrenaline release from peripheral sympathetic nerve terminals innervating the heart and vasculature (160, 161). Very recently, Randall and colleagues reported intriguing observations suggesting that the endothelium-derived hyperpolarizing factor (EDHF), whose structure has remained elusive since its existence was suggested some years ago, may be anandamide. EDHF-mediated relaxations in the rat mesenteric arterial bed were blocked by SR 141716A, and were accompanied by the formation of an arachidonate metabolite co-eluting with anandamide on TLC. Moreover, synthetic anandamide was found to relax the mesentery through an hyperpolarizing mechanism (171). The physiological function of the endogenous cannabinoid system is not necessarily mediated by either central or peripheral neurons. Apart from immune cells, also tissues of the reproductive system have been shown to both contain cannabinoid receptors and synthesize as well as degrade putative endocannabinoids such as the cannabimimetic acyl-ethanolamides, which may act as local autacoid mediators. Anandamide and/or CB1 receptors have been found in rat testes and mouse vas deferens (32, 84, 162), thus allowing to hypothesize a role of the ‘anandamidergic’ system in the control of spermatogenesis and male fertility. Cannabinoid receptors have been found also on sea urchin sperm cell membrane and suggested to mediate THC inhibition of the acrosome reaction (163, 164). Anandamide has been shown to inhibit the acrosome reaction (74) and sea urchin ovaries have been found to synthesize and degrade anandamide and palmitoyl-ethanolamide (132). It was proposed that sea urchin eggs may synthesize anandamide during the acrosome reaction with the purpose of inhibiting polyspermic fertilization. It remains to be assessed whether an analogous mechanism also occurs in mammals. Both CB1 and CB2 receptors have been found in mouse uterus, which contain also two distinct ‘anandamide synthase’ and FAAH-like enzymatic activities, whose levels appear to vary within the embryo implantation and inter-implantation sites (75, 76, 133). Apart from a possible metabolic action on uterin lactoferrin production (75), the endogenous cannabinoid system was suggested to mediate the chemical communication between the uterus and the embryo, as indicated by the presence of CB1 and CB2 receptors also in embryos starting from very early stages of their development (from the one-cell to the blastocyst stages [76]). Due to its inhibitory effect on embryonic cell division (76), anandamide may act as a negative signal for embryo development and implantation, as shown also by the higher and lower levels, respectively, of anandamide synthase and amide hydrolase detected when the uterus is least receptive to implantation (133). Therefore, the synthesis of anandamide may be used by the uterus in order to direct the timing and placing of embryo implantation. After birth, behavioural responses to anandamide develop gradually until adulthood, as shown by recent ontogenetic studies carried out in young mice. This suggests that, although being present since the early stages of development, CB1 cannabinoid receptors do not reach immediately their full development in the brain (165). Concluding remarks Back to the contents list As suggested by a recent article, the discoveries of the first endocannabinoid, anandamide, in 1992, and of peripheral cannabinoid receptors in 1993, have represented ‘a new dawn for cannabinoid research’ (178), by substantiating, respectively, the existence of a new endogenous regulatory system and the expectancy of new drugs, devoid of psychotropic actions, from cannabimimetic compounds. However, the general feeling among those working on this subject that the present knowledge of the endogenous cannabinoid system may be only the ‘tip of the iceberg’ allows to foresee that the potential of these discoveries may be even higher than that initially predicted. A renewed and unprecedented multi-disciplinary research effort from chemists, biochemists, molecular and cell biologists, physiologists, pharmacologists and psychologists is now needed in order to provide grounds to this feeling, and, possibly, to improve our understanding of some basic aspects of mammalian physiology and to exploit this knowledge for the development of novel therapeutic strategies for the treatment and cure of some CNS and peripheral dysfunctions. Acknowledgements Back to the contents list The authors wish to thank Prof. Raphael Mechoulam, The Hebrew University of Jerusalem, Israel, for fruitul discussions and for sharing results from work in progress. This article was supported by funds from the Human Frontiers in Science Program Organization (RG 26/95 to VDM) and from the P.O. CNR-MURST ‘Fondi Strutturali 1994-1999’. References Back to the contents list 1) Devane WA, Dysarz FA, Johnson MR, Melvin LS and Howlett AC. 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