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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. Since its discovery in 1992, N-arachidonoylethanolamide or anandamide in particular has attracted special interest ('ananda' means inner bliss and tranquility in Sanskrit). 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 receptors to produce similar biological results.

Formula of anandamide

Three years later, it was recognized that 2‑arachidonoylglycerol has very similar properties. Together anandamide and 2‑arachidonoylglycerol have been termed endogenous cannabinoids or 'endocannabinoids'. Some other simple amides, including oleamide and arachidonoyldopamine, are now known to interact with the same receptors, and they must also be classified as endocannabinoids. While some authors classify palmitoylethanolamide as an endocannabinoid, it does not interact with the same receptors and it is discussed on the web page dealing with other simple amides, some of which share the synthetic and degradative pathways of anandamide and thus may indirectly affect its function. Arguably, the definition of an endocannabinoid could be widened, but for reasons of practical convenience I have adhered to a strict definition here.

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.

1.   N-Arachidonoylethanolamide (Anandamide)

In the more reliable recent analyses, basal levels of anandamide in tissues are estimated to lie in the picomolar to low nanomolar range. It is synthesised upon demand from phospholipid precursors in cell membranes in almost all cells and tissues of the body 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 acyl donors; the reaction 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. 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.

Biosynthesis of the anandamide precursor N-acyl-phosphatidylethanolamine

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 a phosphodiesterase that is specific for this lipid. It has a similar function to phospholipase D, but it differs from all others of this type in its amino acid sequence. In addition to anandamide, phosphatidic acid is formed and this also has messenger functions.

Biosynthesis of anandamide

A second pathway has been described that does not use the specific phosphodiesterase. Rather it involves either single or double O-deacylation of N‑acyl- or N‑alkenyl-phosphatidylethanolamine catalysed by phospholipases prior to the action of specific phospholipase Ds 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 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. 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.

Scottish thistleFunction: Anandamide and other endocannabinoids are highly lipophilic and have a tendency to remain in membranes, where they can diffuse to encounter membrane-bound enzymes and receptors. However, they are also able to diffuse into the cytoplasm, where they are transported, probably by the fatty acid amide hydrolase (see below), to act on presynaptic cannabinoid receptors. It appears that cytoplasmic lipid droplets (‘adiposomes’) may act as a reservoir, although they are also an active site for metabolism. In plasma, anandamide binds reversibly to serum albumin and is presumably transported to other tissues in this form.

Anadamide exerts its effects at nanomolar to sub-micromolar concentrations mainly through binding to and activating specific cannabinoid receptors, designated ‘CB1’ and ‘CB2’, both of which are membrane-bound G-proteins (a large and diverse family of receptors with a characteristic membrane-spanning structure whose main function is to convert extracellular stimuli into intracellular signals). They 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 and small intestine, while the CB2 receptor is found in the 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. Thus, as with the bioactive constituents of marijuana, 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. It is believed to have beneficial effects in some neurological disorders, including Alzheimer's disease and Parkinson's disease, but not in all.

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 also 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. 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. Also, anandamide derived from macrophages has anti-inflammatory effects both in the peripheral and central nervous system. It can induce apoptosis in a number of cell types.

Some of these effects appear to be independent of CB1 and CB2, and anandamide is known to bind to a number of other receptors, including the peroxisome proliferator-activated receptors (PPARα, β and γ). For example, by binding to PPARβ/γ in brain it may regulate memory and learning. In addition, endocannabinoids are now known to also elicit activity at receptors such as the transient receptor potential vanilloid 1 (TRPV1), 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.

Formula of virodhamineO-Arachidonoylethanolamine, i.e. with an ester instead of an amide linkage to 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‑Dihomo-γ-linolenoylethanolamide, N‑eicosa-5,8,11-trienoylethanolamide, N‑eicosapentaenoylethanolamide and perhaps N‑docosahexaenoylethanolamide are other N‑acylethanolamides that may bind to the CB1 and CB2 receptors. However, a separate specific receptor for N-docosahexaenoylethanolamide has been identified in brain (see our web page on simple lipoamino acids).

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, it is rapidly degraded to free arachidonic acid and ethanolamine. In vivo, the concentrations of all 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 (primarily in the perinuclear membranes). It belongs to a large family of enzymes 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. 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.

Hydrolysis of anandamide

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-tumor 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 tumor cells is curtailed by inhibiting hydrolase activity and increasing the concentration of anandamide. Also, administration of inhibitors has beneficial effects against inflammatory pain with the potential to reduce the need for non-steroidal anti-inflammatory drugs with their side effects. It should not be forgotten that the arachidonic acid released has its own biological properties.

Oxygenated metabolites of anandamide (prostamides)

It has been demonstrated that anandamide can be converted by cellular systems in vitro to ethanolamides of the prostaglandins PGE2, PGD2, PGF, PGJ2 and PGI2 (but not thromboxanes) by the action of the enzyme cyclooxygenase-2 (COX-2), but not by COX-1. These metabolites have been termed 'prostamides'. The first significant product is PGG2-ethanolamide and this is acted upon by 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).

Oxygenated metabolites of anandamide

In addition, anandamide is a substrate for the action of 12- and 15-lipoxygenases 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 that might be expected together with 20-hydroxy-anandamide. Some representative examples are illustrated.

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. PGF-EA analogues are efficacious in the treatment of glaucoma, while PGF-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, while PGE2-EA induces apoptosis in rat decidual cells. The prostamides do not bind to either the cannabinoid or prostanoid receptors, but a distinct prostamide F 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 in some instances. They may even have their own as yet unidentified receptors. HETE-ethanolamides inhibit the fatty acid amide hydrolase involved in the catabolism of endocannabinoids.

2.  2-Arachidonoylglycerol

2-Arachidonoylglycerol, a distinctive monoacylglycerol, was the second endogenous ligand for cannabinoid receptors or endocannabinoid to be discovered. It was first detected in brain, where it occurs at levels of nmol/g tissue, but it is now known to be present in many other organs. 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. It is important as a biologically active lipid in its own right and as a precursor of other bioactive lipids.

Formula for 2-arachidonoylglycerol

As with anandamide, 2-arachidonoylglycerol is synthesised upon demand from phospholipid precursors in cell membranes, probably in raft microdomains, in response to a rise in intra-cellular calcium levels. The main biosynthetic route involves 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.

Biosynthesis of 2-arachidonoylglycerol

Alternatively, the diacylglycerol intermediate can be produced by the sequential action of phospholipase D and phosphatidic acid phosphatase on phosphatidylcholine. The latter can also 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.

The defining properties of the endogenous cannabinoids 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 cannabinoid 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 'postsynaptic depression' through the synapse, i.e. a sustained suppression of excitatory signalling.

In addition, 2-arachidonoylglycerol is involved as a mediator of inflammatory reactions and immune responses. 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 it suppresses the elevation of the expression of cyclooxygenase(COX)-2, a key enzyme in prostanoid biosynthesis, in response to pro-inflammatory stimuli and may ameliorate some neurodegenerative diseases.

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 via the CB2 receptor trigger a phenotypic switch from aggressive to tumour-tolerant cells, such as "tumour associated macrophages"; these promote tumor invasiveness and metastasis. In contrast, anandamide concentrations do not change in this way. 2-Arachidonoylglycerol regulates systemic energy metabolism, it may be relevant to cardiovascular disease, and is also important in human reproduction.

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.

Catabolism. When isomerized to the 1/3-isomer, 2-arachidonoylglycerol loses its biological potency, and this probably occurs fairly rapidly in vivo. In plasma and within cells, it has a short half-life, as do the prostanoid metabolites, and it is rapidly hydrolysed to arachidonic acid and glycerol mainly by the cytosolic monoacylglycerol lipase but also by two α/β hydrolases and the fatty acid amide hydrolase with some tissue differences. In brain and probably in other tissues, some of the arachidonic acid released in this way is re-cycled into phospholipids, but it is also a quantitatively important precursor for pro-inflammatory prostaglandins with implications for the regulation of many signalling pathways. Accordingly, inhibitors of the monoacylglycerol lipase especially appear to have pharmaceutical potential against a number of disorders including cancer, and neurodegenerative and inflammatory diseases.

Catabolism of 2-arachidonoylglycerol

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

Formula for 2-PGH2-glycerolAlthough 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 is formed first, and this is converted sequentially to esterified forms of PGD2, PGE2, PGF and PGI2 (but not thromboxanes) by additional enzymes. These constitute a new class of lipid mediator, although only PGD2- and PGE2-glyceryl esters have been confirmed as having biological activity to date. Similarly, oxygenated metabolites are produced by the action of two lipoxygenases (12-LOX and 15-LOX) and a cytochrome P450 enzyme. For example, 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.

Although they do not interact with the prostaglandin receptors, glycerol-linked prostanoids activate both cannabinoid receptors CB1 and CB2 with high affinity and elicit biological responses in cultured cells. It is also 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, it might be expected that it would be de-activated rapidly by acyl migration like 2-arachidonoylglycerol per se. On release from their link to glycerol, prostanoids may exert signalling functions directly.

3.   Oleamide

cis-9,10-Octadecenamide or 'oleamide' is a primary fatty acid amide. It was first isolated from the cerebrospinal fluid of sleep-deprived cats, and 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. 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. It also 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.

Biosynthesis of oleamide

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, modulation or activation of a number of receptors, and effects on the perception of pain. As with the N-acylethanolamines, 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.

Although other fatty acid primary amides in addition to cis-9,10-octadecenamide are present naturally in the cerebrospinal fluid of animals, only linoleamide is known to be biologically active, for example in increasing Ca2+ flux.

4.   N-Arachidonoyldopamine and Other Dopamine Conjugates

N-arachidonoyldopamine has been detected as an endogenous component of mammalian nervous tissue, especially the brain, and 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 influences 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. It has thus been termed a ‘vanilloid’ or ‘endovanilloid’. In addition, it induces COX-2 production and the biosynthesis of prostaglandin PGD2 while inhibiting synthesis of PGE2.

Formula of N-arachidonoyldopamine

Biosynthesis is believed to occur mainly 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-acyldopamines may be formed from CoA esters by the action of an arylalkylamine N-acyltransferase in Drosophila melanogaster.

The N-oleoyl analogue has also 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. 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 biological activities so far detected, docosahexaenoyl(DHA)-dopamine assists in the transport of dopamine to the brain and has antioxidant properties, while oxidized derivatives of arachidonic acid and DHA linked to dopamine may be involved in the pathogenesis of Parkinson’s disease. N-Hexanoyldopamine is highly cytotoxic, and N-octanoyldopamine inhibits T-cell proliferation.

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Lipid listings Credits/disclaimer Updated: June 22nd, 2017 Author: William W. Christie LipidWeb icon