Anandamide, 2‑Arachidonoylglycerol and Related Lipids
Long-chain N-acylethanolamides are ubiquitous trace constituents of animal and human cells, tissues, and body fluids, with important biological and pharmacological properties. After its discovery in 1992, N‑arachidonoylethanolamine or anandamide attracted special interest when it was found to exert similar neurological effects to the cannabinoids, i.e., the pharmacologically active compounds in marijuana or cannabis (from Cannabis sativa), mainly Δ9-tetrahydrocannabinol, through binding to and activating the same G protein-coupled receptors to produce similar biological results ('ananda' means inner bliss and tranquility in Sanskrit).
Three years later, it was recognized that 2‑arachidonoylglycerol has very similar properties and functions to anandamide, and that it interacts with the same specific receptors, although it is produced by a very different biosynthetic route and is much more abundant in animal tissues. Together, anandamide and 2‑arachidonoylglycerol have been termed endogenous cannabinoids or 'endocannabinoids', as their receptors were first identified through their interactions with phytocannabinoids, shortly before the discovery of the endogenous ligands. These lipids are synthesised from membrane lipids on demand in response to increased intracellular Ca2+ concentration, membrane depolarization and/or receptor stimulation to exert their biological effects. The signalling process is rapidly attenuated by their catabolism, i.e., there is tight regulation of the endocannabinoid system.
Some other simple amides, including oleamide and arachidonoyldopamine, are now known to interact with these receptors so can be classified as endocannabinoids. On the other hand, while some authors classify palmitoylethanolamine as an endocannabinoid, there is no evidence for interaction with the same receptors, so it is discussed in the web page dealing with other bioactive simple amides, some of which share the synthetic and degradative pathways of anandamide and thus may indirectly affect its function ('entourage effects'). Arguably, the definition of an endocannabinoid could be widened, as a greater range of G-protein-coupled receptors may be involved in their functions than was initially thought, and for example, ethanolamides of some polyunsaturated fatty acids of the omega-3 family are borderline candidates for endocannabinoid status. However, for reasons of practical convenience, I have tried to adhere to a relatively strict definition here, i.e., those lipids that interact with the primary cannabinoid/endocannabinoid receptors (CB1 and CB2 - see below). N-Arachidonoyldopamine and oleamide fall into this category.
Endocannabinoids exist in dynamic equilibria with many other lipid-derived mediators, including eicosanoids and other oxylipins, as part of a versatile system involved in fine-tuning different physiological and metabolic processes, including inflammation, in a tissue- or cell-specific manner. Indeed, this general lipid signalling pathway appears to have aspects that are conserved evolutionarily in most eukaryotes.
1. N-Arachidonoylethanolamine (Anandamide)
Anandamide is synthesised in all animals, including primitive nematodes and parasitic helminths, and it has even been found in truffles. In the more reliable recent analyses, basal levels in tissues are estimated to lie in the picomolar to low nanomolar range. It is produced upon demand from phospholipid precursors in cell membranes in almost all cells and tissues of higher animals in response to a rise in intra-cellular calcium levels.
Biosynthesis: Although direct N-acylation of ethanolamine is possible, the main mechanism for the biosynthesis of anandamide and related amides requires as a first step the conversion of phosphatidylethanolamine (PE) to N-acyl-phosphatidylethanolamine, a lipid that is normally present in animal tissues at very low levels only other than during injury. Unusual transacylase reactions rather than hydrolysis and re-synthesis via CoA esters are involved, and only 1-O-acyl groups of phospholipids can serve as N-acyl donors by a reaction that is Ca2+-dependent and energy-independent. The main route involves a direct transfer of the fatty acids of position sn-1 of phosphatidylcholine (PC) to N-acylate phosphatidylethanolamine, with concomitant formation of lyso-phosphatidylcholine. The Ca2+-dependent-N-acyltransferase is now known to be the ε isoform of cytosolic phospholipase A2 (cPLA2ε), although other isoforms are known to be active including one specific for the plasmalogen form of phosphatidylethanolamine. A second mechanism may involve an intramolecular transfer of the fatty acid in position sn-1 of phosphatidylethanolamine to form a hypothetical intermediate, 2‑O-acyl-sn-glycero-3-phospho-(N-acyl)-ethanolamine (or N-acyl-lysoPE), which is then reacylated in position 1 by an analogous transfer reaction.
The high specificity of this reaction might be considered surprising in view of the fact that the arachidonic acid levels in position 1 of phospholipids are usually very low (typically <0.3%) other than in testis. In neurons, there is evidence that 1,2-diarachidonoyl-phosphatidylcholine produced by a specific acylation of 2‑arachidonoyl-lysophosphatidylcholine is the required intermediate. This is the key to the specificity of the process since subsequent reactions are independent of the N-acyl substituent.
The second step in the biosynthesis of anandamide and related amides is hydrolysis of the N‑acyl-phosphatidylethanolamine by an N‑acyl phosphatidylethanolamine-specific phospholipase D-like hydrolase (NAPE-PLD) that is specific for this lipid. While it has a similar function to phospholipase D, it differs from all others of this type in its amino acid sequence. It has a hydrophobic cavity that provides an entryway for N‑acyl-phosphatidylethanolamine into the active site, where uniquely bile acids are bound with high affinity to enhance dimer assembly and enable catalysis. In addition to anandamide, phosphatidic acid (PA) is formed as a by-product, and this also has messenger functions.
A second pathway has been described that does not use NAPE-PLD. Rather it involves either single or double O-deacylation of N‑acyl- or N‑alkenyl-phosphatidylethanolamine catalysed by a phospholipase, sPLA2 or by ABHD4 - a member of the 'serine hydrolase' family, prior to the action of specific phospholipase Ds or a metal-dependent glycerophosphodiesterase (GDE1) on the resulting lysoglycerophospho- or glycerophospho-N-arachidonoylethanolamines. Indeed, it has been suggested that this may be the major route to anandamide from plasmalogens in brain, where the intermediate alkenyl-glycerophospho-N-acylethanolamines have been detected in a mouse model. In rodent brain, the endogenous precursor of anandamide is mainly the plasmalogen form of N‑arachidonoyl-phosphatidylethanolamine (N‑acylplasmenylethanolamine), and contains alkenyl groups (16:0, 18:0, 18:1) in position sn-1 and mono- (18:1) and polyunsaturated (20:4, 22:4, 22:6) acyl groups in position sn-2 of the glycerol backbone.
Yet another pathway is known in which N-acyl-phosphatidylethanolamine is again the main precursor but is acted upon by phospholipase C to release a phospho-anandamide, which is then de-phosphorylated to anandamide by a specific phosphatase (PTPN22). This may be the main route when anandamide is required rapidly in response to bacterial endotoxins. Further biosynthetic pathways to anandamide are known to exist, and the relative importance of these and the manner of their regulation pose complex questions.
Function: Anandamide and other endocannabinoids share many physical and functional properties. They are highly lipophilic and tend to remain in membranes, which they can cross by passive diffusion to encounter membrane-bound enzymes and receptors. However, they can diffuse into the cytoplasm, where they are transported, possibly by microvesicles or exosomes, or by fatty acid binding proteins, the fatty acid amide hydrolase (see below), or other lipid carriers to the cannabinoid receptors. It appears that cytoplasmic lipid droplets may act as a reservoir that is accessible upon demand, and they are an active site for metabolism. In plasma, anandamide binds reversibly to serum albumin and is presumably transported to other tissues in this form, although this has still to be established definitively, and transport by extracellular vesicles is possible. Biosynthesis, activation of receptors, and degradation of endocannabinoids are highly regulated and are initiated by an increase in intracellular Ca2+.
Although anandamide and other endocannabinoids have no structural relationships to the active ingredients of cannabis such as the phytocannabinoid Δ9‑tetrahydrocannabinol, they exert their biological effects at nanomolar to sub-micromolar concentrations mainly through binding to and activating the same cannabinoid receptors, designated ‘CB1’ (or CB1 or CB1R) and ‘CB2’ (or CB2 or CB2R). These are membrane-bound G‑proteins that contain seven transmembrane spanning domains and are generally coupled to the activation of heterotrimeric G-proteins of the Gi and Go classes. They are members a large and diverse family of receptors whose main function is to convert extracellular stimuli into intracellular signals and are present only in the chordate branch of the animal kingdom. CB1 is found in the central nervous system and in some other organs, including the heart, uterus, testis, skin and small intestine, while the CB2 receptor is found in the skin, spleen and other cells associated with immunochemical functions, and in activated glia cells in brain. Although their functions have obvious similarities, the CB2 receptor has only 44% homology with CB1 (but 68% in the membrane-spanning region). They are located mainly in the plasma membrane, although there are suggestions that some are present in other organelles.
Within a given tissue, expression of a receptor can vary between different locations, and isoforms of the two receptors in humans and rodents have been identified that differ in their tissue distributions. It has become evident that endocannabinoid receptors exert a wide variety of different cellular effects by specific interactions with many other G-proteins and that this is dependent on such factors as cell type, subcellular location and cellular functional state. In turn, variation in the location of CB1-interacting proteins may have a role in the cell-specific modulation of endocannabinoid signalling. Anandamide is a high-affinity, partial agonist of CB1 but is relatively inactive at CB2, while 2‑arachidonoylglycerol acts as a full agonist at both CB1 and CB2 with moderate-to-low affinity (see below). CB1 is the main receptor mediating the psycho-activities of cannabis.
In addition to CB1 and CB2, anandamide is known to bind to several other receptors, including the peroxisome proliferator-activated receptors (PPARα, β and γ) to function in metabolic, anti-inflammatory and neuroprotective responses. For example, by binding to PPARβ or PPARγ in brain, it may regulate memory and learning. Endocannabinoids are now known to elicit activity in such receptors as the transient receptor potential vanilloid 1 (TRPV1 - a non-selective cation channel ubiquitously expressed in all tissues), GPR55, GPR119, and possibly toll-like receptor 4 (TLR4). Some of its biological properties may be mediated through the production of nitric oxide, which functions as a versatile signalling intermediate and is ubiquitous in tissues.
As in many other membrane associated processes, lipid rafts and caveolae serve as important platforms for regulation of the endocannabinoid system, and especially in the modulation of binding and signalling of the CB1 receptor. Cholesterol in these membrane domains is believed to have a role in mediating the interaction between anandamide and the receptor.
In brain, some types of neuron contain very high levels of CB1 receptor protein, especially on synaptic terminals, whereas others have much lower levels with least in some regions of the hypothalamus and astroglial cells. The endocannabinoid system is an important factor in the regulation of neuron production during embryonal development, and the CB1 receptor participates in the modulation of neuronal and glial proliferation, while CB2 is involved in the proliferation of glial cells. As with the bioactive phytocannabinoids, the endocannabinoids produce neurobehavioral effects and may have important signalling roles in the central nervous system, especially in the perception of pain, anxiety and fear, in the regulation of body temperature, and in the control of appetite. The main mechanism is via retrograde signalling; in response to activation of postsynaptic neurons, endocannabinoids are synthesised and released to activate pre-synaptic CB1 receptors and suppress the release of neurotransmitters. They are reported to have beneficial effects in some neurological disorders, including Alzheimer's disease, Parkinson's disease, schizophrenia and some major depressive disorders, but not in all. Increased synthesis occurs during exercise, and this may contribute to healthy ageing. As there is evidence that glucocorticoids recruit endocannabinoid signalling, this property may mediate many of the neurobiological and physiological effects of glucocorticoids.
Anandamide has important anti-inflammatory and anti-cancer properties both in vivo and in vitro in animal models. It affects the cardiovascular system by inducing profound decreases in blood pressure and heart rate. In addition, it is an anabolic regulator of metabolism in that it increases the intake of food, promotes the storage of lipid, and decreases the expenditure of energy; it is involved in the regulation of body temperature, locomotion, feeding and anxiety. There are suggestions that modulation of anandamide levels in the gut has potential for treatment of inflammatory bowel disease and colon cancer. Anandamide is present in the reproductive fluids of both males and females and is believed to be important in reproduction in relation to both the production of spermatozoa and female fertility and pregnancy. Macrophages generate anandamide in response to the presence of bacterial endotoxin, and it is involved in the pathology of septic shock and cirrhosis of the liver. Anandamide derived from macrophages in this way has anti-inflammatory effects both in the peripheral and central nervous system. It can induce apoptosis in many cell types. Anandamide (and 2-arachidonoylglycerol and perhaps other long-chain ethanolamides) are able to modulate the activity of several antioxidant enzymes and control redox homeostasis through targeting the cannabinoid and other receptors.
O-Arachidonoylethanolamine, i.e., with an ester instead of an amide linkage between ethanolamine and arachidonic acid and termed virodhamine, has been isolated from brain tissues. It acts as a full agonist for the CB2 receptor and is a partial agonist for the CB1 receptor. While it has yet to be determined how it is synthesised, stored or degraded, inter-conversion with anandamide can occur. N-Acylethanolamides derived from other members of the n-6 family of polyunsaturated fatty acids (docosatetraenoic and docosapentaenoic acids in addition to arachidonic) activate both CB1 and CB2 receptors, as well as TRPV1 channels, so these should be considered true endocannabinoids (and ‘endovanilloids’). In mice genetically modified to lack the Δ6 desaturase, the ethanolamide of cis-5,cis-11,cis-14-eicosatrienoic acid and the 2-acylglycerol analogue are formed and function as ligands of CB1.
Catabolism: There are believed to be active transport systems for anandamide from the plasma membrane to other tissues, although the detailed mechanisms are poorly understood. Once it enters a cell, some may be converted to oxylipin metabolites (see below), but any excess is rapidly degraded to free arachidonic acid and ethanolamine. In vivo, the concentrations of these amides in many animal species are controlled by a single hydrolytic enzyme present in most tissues other than skeletal muscle and heart, i.e., a fatty acid amide hydrolase (FAAH-1), which is an integral membrane protein found primarily in the perinuclear membranes. It belongs to a large family of enzymes ('serine hydrolases') that share a highly conserved 130 amino acid motif termed the ‘amidase signature’ sequence and is well conserved in the primary structure. However, a second enzyme of this type (FAAH-2) has been found in humans and other primates that is absent in mice and rats, but is more active against N-oleoylethanolamine than anandamide.. The two enzymes are found in different tissues with the second present mainly in heart and ovary, where perhaps surprisingly it is located on the surface of cytoplasmic lipid droplets. There is also an N-acylethanolamine-hydrolysing acid amidase (NAAA), although N‑palmitoylethanolamine is the preferred substrate, and the lysosomal acid ceramidase is capable of hydrolysing bioactive amides.
There is currently great interest in the potential use of anandamide and other amides for therapeutic purposes, such as the alleviation of inflammation, asthma and some forms of chronic pain, and as anti-tumour drugs. Because of their role in terminating amide signalling, amide hydrolases are the subject of intensive study and are targets for potential drug therapies. For example, there is evidence that the growth of certain tumour cells is curtailed by inhibiting hydrolase activity and increasing the concentration of anandamide. Administration of such inhibitors has beneficial effects against inflammatory pain, depression and anxiety with the potential to reduce the need for non-steroidal anti-inflammatory drugs (or cannabis-derived drugs) with their well-known side effects. It should not be forgotten that any arachidonic acid released has its own biological properties.
Ethanolamides of n-3 fatty acids: N-acylethanolamides derived from the n-3 family of polyunsaturated fatty acids (eicosapentaenoic (EPA), docosapentaenoic and docosahexaenoic (DHA) acids) may activate CB2 receptors, and of these, the C22 derivatives also activate TRPV1 channels but not the CB1 receptor. They are presumed to be synthesised and catabolized by the same mechanism as for anandamide. N‑Docosahexaenoylethanolamine or 'synaptamide' is present in brain tissue in amounts comparable to anandamide, but it binds only weakly to the CB2 receptor. However, it does bind strongly to the G-protein-coupled receptor GPR110 (ADGRF1) to trigger cAMP production and signalling with low nM potency, with the effect of inducing neurogenesis, neuritogenesis and synaptogenesis in developing neurons (hence the trivial name), a further mechanism by which DHA promotes brain development and function. Synaptamide may have neuroprotective effects after conversion to oxygenated metabolites such as the 17-hydroxy and 19,20-epoxy forms. There are several reports of anti-inflammatory effects that may be due to inhibition of eicosanoid production by COX‑2. It has been suggested that the preferential activation of CB2 receptors by N‑acylethanolamides of the n‑3 family of polyunsaturated fatty acids contributes in part to the broad anti-inflammatory profile of the latter. As synaptamide is present in human breast milk, it is possible that it has a role in the development of the new-born infant.
EPA-ethanolamine is barely detectable in animal tissues under normal conditions, although it has been found in human milk and has apparent beneficial properties in vitro at least, while tetracosahexaenoylethanolamine has been detected in rat brain and may have a neuroprotective function. Saturated and monoenoic N‑acylethanolamides have important biological activities but are not endocannabinoids by the strict definition, and they are discussed on a separate web page with other fatty amides and lipoamino acids.
Oxygenated metabolites of anandamide
It has been demonstrated that anandamide can be converted by cellular systems in vitro to ethanolamides of the prostaglandins PGE2, PGD2, PGF2α, PGJ2 and PGI2 (but not thromboxanes) initially by the action of the enzyme cyclooxygenase-2 (COX-2) but not COX‑1. These metabolites have been termed 'prostamides'. The first significant product is PGG2-ethanolamide and this is acted upon by the same prostaglandin synthases that operate to produce the unesterified prostanoids, though a quite distinct prostamide/PGF synthase has now been discovered that is especially abundant in brain and spinal cord. For cyclo-oxygenation to occur, there is an essential requirement for the hydroxyl group of anandamide. Similar reactions occur with the other endocannabinoid 2‑arachidonoylglycerol (see below).
In addition, anandamide is a substrate for the action of 12- and 15-lipoxygenases (but not 5-LOX) to produce ethanolamides of hydroxy- and epoxyeicosatrienoic acids (HETE and EET). The full range of HETE-ethanolamide products is formed. but in a tissue and species-specific manner. It is noteworthy that human 15-lipoxygenase-1 is able to convert anandamide to cysteine-containing metabolites ('eoxamides'), analogous to the eoxins, more rapidly than in the corresponding reaction with arachidonic acid per se. Cytochrome P450 enzymes produce the four epoxy metabolites (EET) and their dihydroxy derivatives that might be expected together with 20-hydroxy-anandamide. Mouse analogues of these enzymes exhibit a similar range of activities. Some representative examples are illustrated. In contrast to anandamide, prostamides are not efficiently hydrolysed by FAAH1/2 or NAAA, although they can be catabolized in a similar manner to the prostanoids per se.
Synaptamide can undergo a lipoxygenase reaction to form the 17-hydroperoxy-derivative, which can be reduced to 17S‑hydroxy-docosahexaenoylethanolamine, or it can be a precursor for further oxygenated metabolites. Similarly, cytochrome P450 (CYP450) products of synaptamide have been described, i.e., 19,20-epoxy-docosapentaenoylethanolamine and related n‑3 fatty acid derivatives, and these are also potent lipid mediators. COX‑2 generates 13- and 16‑hydroxy-docosahexaenoylethanolamines with immunomodulating properties in macrophages, while eicosapentaenoylethanolamine is converted into PGE3-ethanolamide and 11-, 14- and 18-hydroxy-eicosapentaenoylethanolamines.
The prostamides do not bind strongly to either the cannabinoid or prostanoid receptors, but a distinct prostamide F2α receptor has been identified. However, 12(S)‑hydroxy-eicosa-5Z,8Z,10E,14Z-tetraenoyl-N-(2‑hydroxyethyl)amine and the related cytochrome P450 metabolites bind to both CB receptors with an affinity that is similar to that of anandamide per se or even greater in some instances. For example, 5,6‑EET‑ethanolamide has a 300-fold selectivity for activation of the CB2 receptor over CB1, and a 1000 times greater affinity for CB2 than does anandamide. It is even possible that the oxygenated metabolites have their own as yet unidentified receptors. HETE-ethanolamides inhibit the fatty acid amide hydrolase involved in the catabolism of endocannabinoids.
The biological importance of these novel lipids is now being actively explored. PGE2-ethanolamide is stable in human plasma and mobilizes calcium in cell preparations in vitro at picomolar concentrations. PGF2α-EA analogues are efficacious in the experimental treatment of glaucoma, while PGF2α-EA and PGE2-EA have variable effects in inflammatory conditions, although the former may be important for the perception of pain. PGD2-EA, 15-deoxy-Δ12,14-PGJ2-EA and Δ12‑PGJ2/PGJ2-EA are produced and induce apoptosis in various cancer cell types, especially those that over-express COX-2. In particular, PGD2-EA and its metabolite 15-deoxy-Δ12,14-PGJ2-EA are reported to function in this manner by inhibiting the antioxidant activities of glutathione and thioredoxin, thus causing oxidative stress and thence apoptosis. PGE2-EA induces apoptosis in rat decidual cells.
2-Arachidonoylglycerol, a distinctive species of monoacylglycerol, was the second endocannabinoid to be discovered. It was first detected in canine gut and was demonstrated to be as an endogenous ligand for the cannabinoid receptors in 1995. Subsequently, it was shown to be especially abundant in the central nervous system with the concentrations in brain ~170 to 1000 times higher than that of anandamide. As a neutral lipophilic molecule, it is possible that 2-arachidonoylglycerol can diffuse freely through membranes to reach the sites of activity, since no active transport system has been identified to date, although it does bind to the cytosolic carrier protein fatty acid binding protein 5 (FABP5). In addition to its own well-established biologically activities, 2‑arachidonoylglycerol acts as a precursor of other bioactive lipids.
As with anandamide, 2-arachidonoylglycerol is synthesised upon demand mainly from phospholipid precursors in cell membranes, probably in raft microdomains, in response to a rise in intra-cellular calcium levels. The main biosynthetic route is believed to involve generation of sn‑1,2‑diacylglycerols from phosphatidylinositol by the action of phospholipase Cβ; these are hydrolysed by sn-1 specific diacylglycerol lipases (DAGLs) to yield 2‑arachidonoylglycerol. DAGLα is expressed throughout the brain, while DAGLβ has a more limited occurrence in brain and in peripheral tissues. Phosphatidylinositol 4,5-bisphosphate is the primary precursor in neurons. Lysophosphatidylinositol is a direct precursor in a non-canonical pathway in humans via the action of glycerophosphodiesterase 3, a lysophospholipase C, which suppresses the normal receptor for lysophosphatidylinositol, i.e., GPR55, and so acts as a switch between GPR55 and CB2 signalling.
Alternatively, the diacylglycerol intermediate can be produced by the sequential action of phospholipase D and phosphatidic acid phosphatase on phosphatidylcholine. The latter can serve as precursor via the action of a specific phospholipase A1 to generate 2‑arachidonoyl-lysophosphatidylcholine, which is in turn acted upon by a lysophospholipase C to produce 2‑arachidonoylglycerol. In addition, triacylglycerols containing arachidonic acid in position sn‑2 can serve as precursors of the diacylglycerol intermediate after hydrolysis by hormone-sensitive lipase, carboxyl esterase or other lipases. The relative contributions of these various pathways are not yet known.
Function: The defining properties of the endogenous cannabinoids and their receptors are discussed above in relation to anandamide, but there is evidence to suggest that 2‑arachidonoylglycerol is the more important natural ligand for both the CB1 and the CB2 receptors. While anandamide may only act as a partial agonist at these cannabinoid receptors, 2-arachidonoylglycerol usually acts as a full agonist. The two molecules may interact to regulate some processes, but the latter has many activities that are distinct. For example, amongst innumerable metabolic functions that have been reported, 2‑arachidonoylglycerol is believed to be a messenger molecule that regulates the transmission of signals across synapses in the brain. At excitatory synapses, the 2‑arachidonoylglycerol 'signalosome' consists of a supra-molecular complex in a single functional unit containing three key players: phospholipase Cβ, an activator protein designated mGluR5 and DAGLα. Activation of this complex with synthesis of 2‑arachidonoylglycerol results in 'post-synaptic depression' through the synapse, i.e., a sustained suppression of excitatory signalling, for example by preventing the excessive activation of glutamate receptors. In damaged neurons, it enhances the clearance of myelin debris by microglia and the differentiation of oligodendrocyte progenitor cells to enable a complete re‑myelination.
Indeed, many of the important activities of 2-arachidonoylglycerol mediated by the CB1 receptor are expressed through brain metabolism, and evidence suggests that it is responsible for the regulation of food intake and energy metabolism; increased levels were found during fasting in limbic forebrain and hypothalamus. It is important in modulating anxiety and depressive behaviours and in addiction. By providing the main pool of arachidonic acid for the generation of inflammatory eicosanoids, it plays a key if indirect role in the regulation of numerous neuro-inflammatory processes in the brain. Anandamide may act similarly but at a much lower level.
2-Arachidonoylglycerol is involved as a mediator of inflammatory reactions and immune responses in many other tissues, and it can reduce the effects of pain. In immune cells such as platelets and macrophages, this lipid is produced in response to injury, and its production is thought to be a beneficial response aimed at decreasing pro-inflammatory mediators. For example, there is evidence that in response to pro-inflammatory stimuli it suppresses the elevation of the expression of COX-2, a key enzyme in prostanoid biosynthesis, as well as ameliorating some neurodegenerative diseases. It may be a factor in systemic lupus erythematosus. Via its regulation of systemic energy metabolism, 2-arachidonoylglycerol may be relevant to cardiovascular disease and to human reproduction.
Recent research has demonstrated that it has a role in the regulation of the proliferation and invasion of certain types of cancer cells with concentrations both in the primary tumour and in plasma increasing appreciably during cancer development and metastasis. It has been suggested that this increase is due to the activity of activated immune cells, which trigger a phenotypic switch from aggressive to tumour-tolerant cells via the CB2 receptor. The result is that 2‑arachidonoylglycerol exerts anticancer activities by inhibiting the proliferation, migration and/or invasion of cancer cells, and tumour angiogenesis. In contrast, anandamide concentrations do not change in this way in cancers. The expression of the CB1 and CB2 receptors increases markedly in some types of cancer in relation to healthy tissue but not in others. Therefore, increasing the concentrations of endocannabinoids by direct administration or by inhibiting their catabolism is seen as a potential therapeutic strategy. Although they may present ethical problems, the phytocannabinoids such as Δ9‑tetrahydrocannabinol isolated from the plant Cannabis sativa interfere with the endocannabinoid pathways and are being tested to alleviate pain in cancer.
The 2-glycerol ether analogue (termed ‘noladin ether’) has similar biological activity in vitro, and it has been reported to occur naturally in pig brain but at very low levels. It is metabolically more stable than 2‑arachidonoylglycerol, and it is an agonist of both CB1 and CB2 receptors and a partial agonist of TRPV1 and PPARα; it serves a neuroprotective role among other functions. 2-Glycerol esters of EPA and DHA have been detected in embryonic stem cells of mice and have been shown to have anti-inflammatory effects in vitro.
An endocannabinoid system utilizing 2-arachidonoylglycerol and anandamide has been identified in the primitive organism Hydra vulgaris (Cnidaria) and in molluscs, annelids and some crustaceans. However, the fruit fly Drosophila melanogaster, widely used as a model species of insect, is a vegetarian and derives much of its unsaturated fatty acids from its diet; these do not include arachidonic, which can be lethal in some developmental stages. It has now been determined that this species utilizes 2-linoleoyl-linoleate as a signalling molecule in an endocannabinoid-like way, suggesting that this may a signalling system from which endocannabinoids developed during evolution.
Catabolism. When isomerized to the more thermodynamically stable 1/3-sn-glycerol isomers, arachidonoylglycerol interacts much less efficiently with the CB1 receptor and loses much of its biological potency; this probably occurs rapidly and spontaneously in vivo. In plasma and within cells, 2-arachidonoylglycerol has a short half-life as do the prostanoid metabolites (see below), and it is rapidly hydrolysed to arachidonic acid and glycerol mainly by the cytosolic monoacylglycerol lipase present in most cell types but also by two α/β hydrolases, especially the ubiquitously expressed serine hydrolase α/β-hydrolase domain 6 (ABHD6), and the fatty acid amide hydrolase, with some tissue differences. ABHD6 may be of most importance in regulating the signalling effects of 2-arachidonoylglycerol. It is worthy of note that most monoacylglycerol lipases target the 1/3‑isomers preferentially and are less active against 2-isomers.
In brain and probably in other tissues, some of the arachidonic acid released in this way is re-cycled into phospholipids and some is a quantitatively important precursor for pro-inflammatory prostaglandins and other eicosanoids with implications for the regulation of many signalling pathways. Accordingly, inhibitors of the monoacylglycerol lipase especially appear to have pharmaceutical potential against several disorders including cancer, and neurodegenerative and inflammatory diseases, with fewer side-effects than many alternatives. They can promote hypothermia, hypomotility, and analgesia. It is hoped that inhibitors of ABHD6 may prove efficacious against diseases such as epilepsy.
In addition, phosphorylation of 2-arachidonoylglycerol by an acylglycerol kinase may in effect remove this lipid while generating in its place lysophosphatidic acid, another important signalling molecule.
Oxygenated metabolites of 2-arachidonoylglycerol
Although the reaction is not as efficient as with arachidonic acid per se, 2‑arachidonoylglycerol can function as a precursor of 2-glycerol-linked prostanoids through a specific interaction with the enzyme COX-2, but not COX-1, followed by further downstream processing as with anandamide. Thus, prostaglandin H2‑glycerol ester (2PGH2-glycerol) is formed first, and this is converted sequentially by other prostaglandin synthases to esterified forms of PGD2, PGE2, PGF2α and PGI2 (thromboxane analogues are only just detectable) by additional enzymes. This may be a mechanism for terminating signalling by 2-arachidonoylglycerol in the central nervous system. These metabolites constitute a new class of lipid mediator, although only PGD2- and PGE2-glycerol esters have been confirmed as having biological activity to date. For example, PGE2-glycerol is a multifunctional signalling molecule that has a function in pain, immunomodulation and synaptic plasticity. Similarly, oxygenated metabolites are produced by the action of two lipoxygenases (12‑LOX and 15‑LOX) and a cytochrome P450 enzyme. The last enzyme produces two metabolites of 2‑arachidonoylglycerol, i.e., 2‑(11,12-epoxyeicosatrienoyl)-glycerol and 2‑(14,15-epoxyeicosatrienoyl)-glycerol, in various animal tissues.
The monoacylglycerol lipase, a lysophospholipase A2 (LYPLA2) and carboxylesterases are the main enzymes responsible for hydrolysis of these oxygenated metabolites, and on release from their link to glycerol, prostanoids may exert signalling functions directly.
Although they do not interact with the prostaglandin receptors, glycerol-linked prostanoids may interact weakly with CB1 and CB2 and elicit biological responses in cultured cells, but this has been difficult to establish definitively because they are hydrolysed so readily. It is considered possible that these compounds have as yet unknown receptors of their own. Together with analogous lipoxygenase metabolites, they are new members of the endocannabinoid family. Although they are present in tissues at much lower levels than the free acid forms, some of the glycerol-linked prostanoids have greater biological activity with some being pro-inflammatory and others anti-inflammatory. While 2‑(15‑HETE)-glycerol is a specific agonist for the PPARα receptor with moderate affinity for the CB2 but not for CB1 receptor, it might be expected that it would be de-activated rapidly by acyl migration like 2-arachidonoylglycerol per se. It has yet to be established whether it is formed in vivo.
3. N-Arachidonoyldopamine and Other Dopamine Conjugates
N-Arachidonoyldopamine has been detected as an endogenous component of mammalian nervous tissue, especially the brain, with high concentrations in the hippocampus, cerebellum and striatum, and it has distinctive biological effects. It binds to the CB1 receptor for endocannabinoids and shows cannabimimetic properties in that it affects the sensation of pain and relieves inflammation, while influencing renal and cardiovascular functions. In addition, it interacts with the same receptor (vanilloid type 1) as capsaicin, the active ingredient of chili peppers, with which it has some structural similarity, so has thus been termed a ‘vanilloid’ or ‘endovanilloid’. However, it does not interact with dopamine receptors per se. N-Arachidonoyldopamine induces COX-2 production and the biosynthesis of prostaglandin PGD2, while inhibiting synthesis of PGE2.
Biosynthesis probably occurs by conjugation of dopamine with arachidonic acid, catalysed by a fatty acid amide hydrolase (not via the CoA ester), although there are suggestions that some might be derived from arachidonoyltyrosine. However, N‑acyldopamides may be formed from CoA esters by the action of an arylalkylamine N-acyltransferase in Drosophila melanogaster.
The N-oleoyl analogue has been found in brain and has characteristic biological properties of its own, although it interacts with the same receptors as N‑arachidonoyldopamine; it induces COX-2 production, but inhibits 5-lipoxygenase, for example. In addition, it induces interleukin 10 (IL-10) by acting as an agonist for TRPV1 in the central nervous system with beneficial effects upon inflammation and sepsis. While the N-palmitoyl and N-stearoyl derivatives of dopamine do not interact with these receptors to a significant extent, they appear to act together with N‑arachidonoyldopamine and anandamide to enhance calcium mobilization. N‑acetyldopamine is also present in many animal tissues. Among the other biological activities so far detected, docosahexaenoyl(DHA)-dopamine assists in the transport of dopamine to the brain and has antioxidant and anti-inflammatory properties. Similarly, eicosapentaenoyl(EPA)-dopamine is anti-inflammatory. DHA‑dopamine is of interest because of potential physiological properties that are linked to brain synapses and retinal neuroprotection. Oxygenated metabolites are produced from N‑arachidonoyldopamine and DHA by the action of cytochrome P450 enzymes, and they may be involved in the pathogenesis of Parkinson’s disease. N-Hexanoyldopamine is highly cytotoxic, and N‑octanoyldopamine inhibits T-cell proliferation.
cis-9-Octadecenamide or 'oleamide' is a primary fatty acid amide. It was first isolated from the cerebrospinal fluid of sleep-deprived cats, and it has been characterized and identified as the signalling molecule responsible for causing sleep. For example, it induced physiological sleep when injected directly into the brain of rats (linoleamide is reported to have the same property). Although it bears very little structural relationship to other endocannabinoids, it is an agonist for the CB1 receptor, which is believed to be a mediator for its biological activity, and it activates peroxisome proliferator-activated receptor gamma (PPARγ) in vitro.
Two routes have been proposed for the biosynthesis of oleamide of which that generally favoured involves first synthesis of N‑oleoylglycine followed by oxidation by peptidylglycine α-amidating monooxygenase.
The second rather unusual mechanism suggested involves the enzyme cytochrome c and oleoyl-CoA and ammonium ions as the substrates, with hydrogen peroxide as an essential cofactor (this reaction is illustrated in the web page dealing with the biosynthesis of simple lipoamino acids, which may follow the same route).
In addition to its sleep-inducing properties, oleamide has other neurological activities, which include regulation of memory processes, decreasing body temperature and locomotive activity, stimulating Ca2+ release, blocking potassium channels, modulation or activation of a number of receptors, effects on the perception of pain, and anti-cancer activity. As with the N‑acylethanolamides, the concentration of oleamide is controlled by the specific fatty acid amide hydrolase in vivo, but it is not known how these simple molecules avoid hydrolysis by the innumerable proteases, lipases and amidases present in brain.
Other fatty acid primary amides in addition to cis-9-octadecenamide are present naturally in the cerebrospinal fluid of animals, and linoleamide is known to increase the Ca2+ flux while 13-docosenamide or erucamide may have biological activity. Receptors for these metabolites have not yet been identified.
5. Other Endocannabinoids
N-Arachidonoylglycine and N-arachidonoylserotonin have many important biological functions in animal tissues through activation of G‑protein-coupled receptors other than the CB1 and CB2 receptors. These are discussed on our web page dealing with lipoamino acids, although the different treatment in this web site from the endocannabinoids now looks somewhat arbitrary. There is a report that N-oleoylglycine promotes adipogenesis via activation of the CB1 receptor, implying that it is an endocannabinoid, with the potential to increase insulin sensitivity and suppress obesity and diabetes.
The main problems in the analysis of endocannabinoids relate to the low levels at which they occur naturally, and the ease of isomerization or hydrolysis on storage of samples or extracts. There is a concern that artefactually high results might be obtained because of the physiological effects of sampling methods. High-performance liquid chromatography allied to tandem mass spectrometry with electrospray ionization is now the preferred methodology.
- Balezina, O.P., Tarasova, E.O. and Gaydukov, A.E. Noncanonical activity of endocannabinoids and their receptors in central and peripheral synapses. Biochemistry (Moscow), 86, 818-832 (2021); DOI.
- Biringer, R.G. The rise and fall of anandamide: processes that control synthesis, degradation, and storage. Mol. Cell. Biochem., 476, 2753-2775 (2021); DOI
- Cao, J.K., Kaplan, J. and Stella, N. ABHD6: its place in endocannabinoid signaling and beyond. Trends Pharm. Sci., 40, 267-277 (2019); DOI.
- Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Woodhead Publishing and now Elsevier) (2010) - see Science Direct.
- de Bus, I., Witkamp, R., Zuilhof, H., Albada, B. and Balvers, M. The role of n-3 PUFA-derived fatty acid derivatives and their oxygenated metabolites in the modulation of inflammation. Prostaglandins Other Lipid Mediators, 144, 106351 (2019); DOI.
- Fraguas-Sánchez, A.I., Martín-Sabroso, C. and Torres-Suárez, A.I. Insights into the effects of the endocannabinoid system in cancer: a review. Brit. J. Pharm., 175, 2566-2580 (2018); DOI.
- Gil-Ordonez, A., Martin-Fontecha, M., Ortega-Gutierrez, S. and Lopez-Rodriguez, M.L. Monoacylglycerol lipase (MAGL) as a promising therapeutic target. Biochem. Pharm., 157, 18-32 (2018); DOI - and other articles in this journal issue.
- Grabiec, U. and Dehghan, F. N-Arachidonoyl dopamine: a novel endocannabinoid and endovanilloid with widespread physiological and pharmacological activities. Cannabis Cannabinoid Res., 2, 183-196 (2017); DOI.
- Howlett, A.C. and Abood, M.E. CB1 and CB2 receptor pharmacology. Adv Pharmacol., 80, 169-206 (2017); DOI.
- Hussain, Z., Uyama, T., Tsuboi, K. and Ueda, N. Mammalian enzymes responsible for the biosynthesis of N-acylethanolamines. Biochim. Biophys. Acta, Lipids, 1862, 1546-1561 (2017); DOI.
- Kilaru, A. and Chapman, K.D. The endocannabinoid system. Essays Biochem., 64, 485-499 (2020); DOI.
- Lu, H.C. and Mackie, K. Review of the endocannabinoid system. Biological Psychiatry-Cognitive Neuroscience Neuroimaging, 6, 607-615 (2021); DOI.
- Mechoulam, R. A delightful trip along the pathway of cannabinoid and endocannabinoid chemistry and pharmacology. Annu. Rev. Pharm. Toxic., 63, 1-13 (2023); DOI.
- Röhrig, W., Achenbach, S., Deutsch, B. and Pischetsrieder, M. Quantification of 24 circulating endocannabinoids, endocannabinoid-related compounds, and their phospholipid precursors in human plasma by UHPLC-MS/MS. J. Lipid Res., 60, 1475-1488 (2019); DOI.
- Ruiz-Contreras, H.A., Santamaría, A., Arellano-Mendoza, M.G., Sánchez-Chapul, L., Robles-Bañuelos, B. and Rangel-López, E. Modulatory activity of the endocannabinoid system in the development and proliferation of cells in the CNS. Neurotox. Res., 40, 1690–1706 (2022); DOI.
- Shin, M., Ware, T.B., Lee, H.C. and Hsu, K.L. Lipid-metabolizing serine hydrolases in the mammalian central nervous system: endocannabinoids and beyond. Biochim. Biophys. Acta, Lipids, 1864, 907-921 (2019); DOI.
- Simard, M., Archambault, A.S., Lavoie, J.P.C., Dumais, E., Di Marzo, V. and Flamand, N. Biosynthesis and metabolism of endocannabinoids and their congeners from the monoacylglycerol and N-acyl-ethanolamine families. Biochem. Pharm., 205, 115261 (2022); DOI.
- Watson, J.E., Kim, J.S. and Das, A. Emerging class of omega-3 fatty acid endocannabinoids and their derivatives. Prostaglandins Other Lipid Mediators, 143, 10633 (2019); DOI.
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