The inquiry into the active chemical constituents of Cannabis turned out to be more time consuming than expected. Many other plant-derived compounds, such as morphine and atropine, had long been identified when the Cannabis plant finally yielded its active principle, the terpenoid derivative ∆9 -tetrahydrocannabinol. The psychoactive properties of THC were recognized immediately, but the drug’s unique chemical structure offered no hints as to its mechanism of action. To complicate matters further, the hydrophobic nature of THC delayed experimentation and indicated that the compound might act by influencing membrane fluidity, rather than by combining with a specific receptor. This impasse was resolved by the development of new classes of potent and selective THC analogue7 , which led eventually to the pharmacological identification of cannabinoid-sensitive sites in the brain . The CB1 cannabinoid receptor was molecularly cloned from rat brain in 1990 and its immune system counterpart, the CB2 receptor, was identified by sequence homology three years later. These discoveries not only established the mechanism of action of THC, thereby fuelling the development of sub-type-selective agonists and antagonists , but they also initiated a hunt for brain-derived cannabinoid ligands. Surprisingly, the first THC-like factor to be isolated was a lipid, rather than the peptide that had been expected on the basis of the precedent set by morphine and the enkephalins. It was identified as the amide of ARACHIDONIC ACIDwith ethanolamine, and named anandamide after the Sanskrit word for bliss, ananda. This small lipid molecule resembled no known neurotransmitter,drying room but it did share structural features with the EICOSANOIDS, mediators of inflammation and pain with various functions in neural communication.
Though initially controversial, the signalling roles of anandamide were confirmed by the elucidation of the compound’s unique metabolic pathways and the demonstration of its release in the live brain. As the search for THC-like compounds continued, other bioactive lipids were extracted from animal tissues. These include 2-arachidonoylglycerol, noladin ether, virodhamine and N-arachidonoyldopamine. In this article, I review the synthesis, release and deactivation of the endogenous cannabinoids . I then outline the properties and distribution of brain CB1 receptors. Last, I describe the function of the endocannabinoids as local modulators of synaptic activity and their contribution to memory, anxiety, movement and pain.The membranes of plant cells contain a family of unusual lipids that consist of a long chain FATTY ACID tethered to the head group of PHOSPHATIDYLETHANOLAMINE through an amide bond. When attacked by a PHOSPHOLIPASE D enzyme, these membrane constituents generate a set of FATTY ACID ETHANOLAMIDES, which are used by plants as intercellular signalling molecules. They are released from cells in response to stress or infection, and stimulate the expression of genes engaged in systemic plant immunity. This ancestral biochemical device is conserved in mammalian cells, which use the ethanolamide of arachidonic acid, anandamide, as a primary component of the endocannabinoid signalling system. Anandamide formation in neurons is a two-step process, which parallels fatty acid ethanolamide production in plants. The first step is the stimulus-dependent cleavage of the phospholipid precursor N-arachidonoyl-PE. This reaction is mediated by an uncharacterized PLD and produces anandamide and phosphatidic acid, a metabolic intermediate that is used by cells in the synthesis of other glycerol-derived phospholipids. Genes encoding two PLD isoforms have been cloned in mammals, but it is not known whether either of these enzymes is responsible for anandamide synthesis. The brain contains tiny quantities of N-arachidonoyl-PE — probably too little to sustain anandamide release for an extended time.
The cellular stores of this precursor are replenished by the enzyme N-acyltransferase , which catalyses the intermolecular passage of an arachidonic acid group from the SN-1 position of PHOSPHATIDYLCHOLINE to the head group of PE . In cultures of rat cortical neurons, two intracellular second messengers control NAT activity: Ca2+ and cyclic AMP. Ca2+ is required to engage NAT, which is inactive in its absence, whereas cAMP works through protein kinase A-dependent phosphorylation to enhance NAT activity. Although catalysed by separate enzymes, the syntheses of anandamide and its parent lipid are thought to proceed in parallel because Ca2+-stimulated anandamide production is generally accompanied by de novo formation of N-arachidonoyl-PE. As expected of a Ca2+-activated process, anandamide formation can be elicited by Ca2+ ionophores, which carry Ca2+ ions across cell membranes. For example, in cultures of rat striatal neurons labelled by incubation with [3 H]ethanolamine, the Ca2+ ionophore ionomycin stimulates accumulation of [3 H]anandamide.A similar stimulation is produced by kainate , 4-aminopyridine or membrane-depolarizing concentrations of K+, and can be prevented by chelating extracellular Ca2+ . The Ca2+ dependence of anandamide synthesis was also demonstrated using MICRODIALYSIS. Administration of a high-K+ pulse in the rat striatum caused a reversible increase in interstitial anandamide concentrations, which was prevented by removal of Ca2+ from the perfusing solution. Although neural activity induces anandamide release in a Ca2+-dependent manner, Ca2+ entry into neurons is not the only determinant of anandamide generation: there is evidence that G-protein-coupled receptors canalso trigger this process. For example, application of the dopamine D2 -receptor agonist quinpirole causes an eight fold increase in anandamide outflow in the rat striatum, which is prevented by the D2 -receptor antagonist raclopride. This response is accompanied by an elevation in tissue anandamide content, indicating that it might be due to a net increase in anandamide formation rather than to extracellular release of preformed anandamide. Muscarinic acetylcholine receptors and metabotropic glutamate receptors can also cause endocannabinoid release in hippocampal slices in a Ca2+-independent manner, but the substance involved have not been identified.
How does occupation of D2 receptors initiate anandamide synthesis? Inhibition of cAMP formation, a hallmark of D2 -receptor signalling, is unlikely to be responsible for this effect because cAMP positively regulates NAT activity. Alternatively, D2 receptors could interact with the Rho family of small G proteins to stimulate PLD activity, or they might engage β−γ subunits of G proteins to activate phospholipase Cβ. PLCβ catalyses the cleavage of phosphatidylinositol-4,5- bisphosphate to produce inositol-1,4,5-trisphosphate, which might then recruit the NAT/PLD pathway by mobilizing Ca2+ from internal stores.Like other MONOACYLGLYCEROLS, 2-AG is at the crossroads of multiple routes of lipid metabolism, where it can serve interchangeably as an end-product for one pathway and precursor for another. These diverse metabolic roles can explain its high concentration in brain tissue, and imply that a significant fraction of brain 2-AG is engaged in housekeeping functions rather than in signalling. The place occupied by 2-AG at central intersections of lipid metabolism also complicates efforts to define the biochemical pathway responsible for its physiological synthesis. There is, however, enough information to indicate two possible routes . The first begins with the phospholipase-mediated formation of 1,2-diacylglycerol . This product regulates protein kinase C activity — an important second messenger function — and is a substrate for two enzymes: DAG kinase,vertical farming units which attenuates DAG signalling by catalysing its phosphorylation to phosphatidic acid; and DAG lipase , which hydrolyses DAG to monoacylglycerol. The fact that drug inhibitors of PLC and DGL block Ca2+-dependent 2-AG accumulation in rat cortical neurons indicates primary involvement of this pathway in 2-AG formation. An alternative pathway of 2-AG synthesis begins with the production, mediated by phospholipase A1, of a 2-arachidonoyl-LYSOPHOSPHOLIPID, which might be hydrolysed to 2-AG by lyso-PLC activity . Although there is no direct evidence for this mechanism in 2-AG formation, the high level of PLA1 expression in brain tissue makes it an intriguing target for future investigation. In addition to the phospholipase-operated pathways outlined above, monoacylglycerols can be produced by hormonesensitive lipase acting on triacylglycerols or by lipid phosphatases acting on lysophosphatidic acid. In general, however, these enzymes preferentially target lipids that are enriched in saturated or monounsaturated fatty acids, rather than the polyunsaturated species that would give rise to 2-AG. Irrespective of its exact mechanism, neuronal 2-AG production can be initiated by an increase in the concentration of intracellular Ca2+. In cultures of rat cortical neurons, the Ca2+ ionophore ionomycin and the glutamate receptor agonist NMDA stimulate 2-AG synthesis in a Ca2+-dependent manner. Likewise, in freshly dissected hippocampal slices, high-frequency stimulation of the SCHAFFER COLLATERALS produces a Ca2+-dependent increase in tissue 2-AG content32. Importantly, this treatment has no effect on the concentrations of non-cannabinoid monoacylglycerols, such as 1-palmitoylglycerol, which indicatesthat 2-AG formation is not due to a generalized increase in the rate of lipid turnover. Furthermore, highfrequency stimulation does not alter hippocampal anandamide concentrations, indicating that the syntheses of 2-AG and anandamide can be independently regulated. In further support of this idea, activation of D2 receptors — a potent stimulus for anandamide formation in the rat striatum — has no effect on striatal 2-AG concentrations.Noladin ether is an ether-linked analogue of 2-AG that binds to and activates CB1 receptors. Its pathway of formation has not been characterized, and its occurrence in the normal brain has been questioned. Virodhamine, the ester of arachidonic acid and ethanolamine , might act as an endogenous CB1 antagonist. Its presence in brain tissue has been documented, but is intriguing because this chemically unstable molecule is rapidly converted to anandamide in aqueous environments.
The mechanism of its synthesis is unknown, and its deactivation might share anandamide’s pathways of uptake and intracellular hydrolysis. Finally, the endogenous vanilloid agonist, N-arachidonoyldopamine, also exhibits affinity for cannabinoid receptors in vitro.How are endocannabinoids released from cells and how do they reach their targets? Classical transmitters and neuropeptides can diffuse through the water-filled space that surrounds neurons, but hydrophobic compounds such as anandamide and 2-AG tend to remain associated with lipid membranes. One possibility is that endocannabinoids might not leave the cell where they are produced; rather, they could move sideways within the plasmalemma until they collide with membrane embedded CB1 receptors. This hypothesis is supported by the role of an intramembranous amino-acid residue in the binding of anandamide to CB1 , as well as by the finding that certain cannabinoid agonists can approach the receptor by lateral membrane diffusion. Nevertheless, it does not account for two pieces of evidence. First, anandamide is found in incubation media of cells and in brain interstitial fluid, implying that it can overcome its tendency to partition in membrane. Perhaps more importantly, physiological experiments have shown that an endocannabinoid substance does leave postsynaptic cells to activate CB1 receptors on adjacent axon terminals. This unidentified compound might travel as far as 20 µm from its cell of origin before being eliminate. If endocannabinoids are released from neurons, what is the mechanism of their release? The fact that plasma membranes contain precursor molecules for both anandamide and 2-AG indicates that they could leave the cell as soon as they are formed. Extracellular lipid-binding proteins such as the lipocalins, which are expressed at high levels in the brain, might facilitate this step and help to deliver endocannabinoids to their cellular targets. Although this scenario awaits confirmation, it does mirror what happens in the bloodstream, where anandamide’s movements are made possible by its reversible binding to serum albumin.Anandamide and 2-AG can diffuse passively through lipid membranes, but this process is accelerated by a rapid and selective carrier system that is present in both neurons and glial cells.Although it is superficially similar to other transmitter systems, endocannabinoid transport is not driven by transmembrane Na+ gradients, indicating that it might be mediated by a FACILITATED DIFFUSION mechanism. In this respect, neural cells seem to internalize anandamide and 2-AG in a manner similar to fatty acids, eicosanoids and other biologically relevant lipids, by using energy-independent carriers. Several lipid-carrier proteins have been molecularly cloned, inspiring optimism that, despite current controversy , endocannabinoid transporter will eventually be characterized. Meanwhile, to gain insight into the role of transport in endocannabinoid inactivation, we can rely on an expanding series of pharmacological transport inhibitors. The prototype is AM404, which slows the elimination of both anandamide and 2-AG, magnifying their biological effects. This inhibitor has helped to unmask important roles of the endocannabinoid system in the regulation of neurotransmission and synaptic plasticity, but suffers from various limitations, including an affinity for VANILLOID RECEPTORS and susceptibility to enzymatic attack by fatty acid amide hydrolase . FAAH is an intracellular membrane-bound serine hydrolase that breaks down anandamide into arachidonic acid and ethanolamine. It has been molecularly cloned and its catalytic mechanism, which allows it to recognize a broad spectrum of amide and ester substrates, has been elucidated in detail.