Intestinal levels of anandamide and oleoylethanolamide in food-deprived rats are regulated through their precursors

The anorectic lipid oleoylethanolamide and the orexigenic lipid anandamide both belong to the group of N-acylethanolamines that are generated by the enzyme N-acylphosphatidylethanolamine-hydrolyzing phospholipase D. The levels of the two bioactive lipids were investigated in rat intestines after 24 h of starvation as well as after 1 and 4 h of re-feeding. Total levels of precursor phospholipids and N-acylethanolamines were decreased upon food-deprivation whereas the level of the anandamide precursor molecule was significantly increased. The level of 2-arachidonoyl-glycerol was unchanged as was the activity of N-acyltransferase, N-acylphosphatidylethanolamine-hydrolyzing phospholipase D, and fatty acid amide hydrolase upon starvation and re-feeding. It is concluded that remodeling of the amide-linked fatty acids of N-acylphosphatidylethanolamine is responsible for the opposite effects on levels of anandamide and oleoylethanolamide in intestines of food-deprived rats and not an alternative biochemical route for anandamide synthesis. Furthermore, linoleoylethanolamide, which accounted for more than 50 mol% of the endogenous pool of N-acylethanolamines, was found not to have the same inhibitory effect on food intake, as did oleoylethanolamide following oral administration.     

Expression and secretion of N-acylethanolamine-hydrolysing acid amidase in human prostate cancer cells

N-acylethanolamines (NAEs) are a class of bioactive lipid molecules in animal tissues, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. Enzymatic hydrolysis of NAEs is considered to be an important step to regulate their endogenous levels. Lysosomal NAE-hydrolysing acid amidase (NAAA) as well as fatty acid amide hydrolase (FAAH) is responsible for this reaction. Here, we report relatively high expression of NAAA in human prostate cancer cells (PC-3, DU-145 and LNCaP) and prostate epithelial cells (PrEC), with the highest mRNA level in LNCaP cells. FAAH and the NAE-forming enzyme N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) were also detected in these cells. NAAA activity in LNCaP cells could be distinguished from coexisting FAAH activity, based on their different pH dependency profiles and specific inhibition of FAAH activity by URB597. These results showed that both the enzymes were functionally active. We also found that NAAA was partly secreted from LNCaP cells, which underlined possible usefulness of this enzyme as a biomarker of prostate cancer.     

Modulation of the endocannabinoid system by focal brain ischemia in the rat is involved in neuroprotection afforded by 17beta-estradiol

Endogenous levels of the endocannabinoid anandamide, and the activities of the synthesizing and hydrolyzing enzymes, i.e. N-acylphosphatidylethanolamine-hydrolyzing phospholipase D and fatty acid amide hydrolase, respectively, were determined in the cortex and the striatum of rats subjected to transient middle cerebral artery occlusion. Anandamide content was markedly increased ( approximately 3-fold over controls; P < 0.01) in the ischemic striatum after 2 h of middle cerebral artery occlusion, but not in the cortex, and this elevation was paralleled by increased activity of N-acylphosphatidylethanolamine-hydrolyzing phospholipase D ( approximately 1.7-fold; P < 0.01), and reduced activity ( approximately 0.6-fold; P < 0.01) and expression ( approximately 0.7-fold; P < 0.05) of fatty acid amide hydrolase. These effects of middle cerebral artery occlusion were further potentiated by 1 h of reperfusion, whereas anandamide binding to type 1 cannabinoid and type 1 vanilloid receptors was not affected significantly by the ischemic insult. Additionally, the cannabinoid type 1 receptor antagonist SR141716, but not the receptor agonist R-(+)-WIN55,212-2, significantly reduced (33%; P < 0.05) cerebral infarct volume detected 22 h after the beginning of reperfusion. A neuroprotective intraperitoneal dose of 17beta-estradiol (0.20 mg x kg(-1)) that reduced infarct size by 43% also minimized the effect of brain ischemia on the endocannabinoid system, in an estrogen receptor-dependent manner. In conclusion, we show that the endocannabinoid system is implicated in the pathophysiology of transient middle cerebral artery occlusion-induced brain damage, and that neuroprotection afforded by estrogen is coincident with a re-establishment of anandamide levels in the ischemic striatum through a mechanism that needs to be investigated further.     

Depolarizing and calcium-mobilizing stimuli fail to enhance synthesis and release of endocannabinoids from rat brain cerebral cortex slices

The concentrations of the endocannabinoids 2-arachidonoylglycerol (2-AG) and N-arachidonylethanolamine (anandamide) were examined in rat brain cerebral cortex slices and surrounding medium. Basal concentrations of endocannabinoids were similar to those identified previously in rat brain, with anandamide content being much lower (19 pmol/g) than that of 2-AG (7300 pmol/g). In contrast, basal concentrations in the surrounding medium were proportionally much lower for 2-arachidonoylglycerol (16 pmol/mL) compared to anandamide (0.6 pmol/mL). Incubation of slices with glutamate receptor agonists, depolarizing concentrations of KCl, or ionomycin failed to alter tissue concentrations of endocannabinoids, while endocannabinoids in the medium were unaltered by elevated KCl. Cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester, an inhibitor of fatty acid amide hydrolase, significantly enhanced tissue concentrations of anandamide (and related N-acylethanolamines), without altering 2-AG, while evoking proportional elevations of anandamide in the medium. Removal of extracellular calcium ions failed to alter tissue concentrations of anandamide, but significantly reduced 2-AG in the tissue by 90% and levels in the medium to below the detection limit. Supplementation of the medium with 50 μM N-oleoylethanolamine only raised tissue concentrations of N-oleoylethanolamine in the presence of cyclohexyl carbamic acid 3'-carbamoyl-biphenyl-3-yl ester and failed to alter either tissue or medium anandamide or 2-AG concentrations. These results highlight the ongoing turnover of endocannabinoids, and the importance of calcium ions in maintaining 2-AG concentrations in this tissue.     

Cannabinoids and brain injury: therapeutic implications

Mounting in vitro and in vivo data suggest that the endocannabinoids anandamide and 2-arachidonoyl glycerol, as well as some plant and synthetic cannabinoids, have neuroprotective effects following brain injury. Cannabinoid receptor agonists inhibit glutamatergic synaptic transmission and reduce the production of tumour necrosis factor-alpha and reactive oxygen intermediates, which are factors in causing neuronal damage. The formation of the endocannabinoids anandamide and 2-arachidonoyl glycerol is strongly enhanced after brain injury, and there is evidence that these compounds reduce the secondary damage incurred. Some plant and synthetic cannabinoids, which do not bind to the cannabinoid receptors, have also been shown to be neuroprotective, possibly through their direct effect on the excitatory glutamate system and/or as antioxidants.     

Molecular characterization of a phospholipase D generating anandamide and its congeners

Anandamide (N-arachidonoylethanolamine) is known to be an endogenous ligand of cannabinoid and vanilloid receptors. Its congeners (collectively referred to as N-acylethanolamines) also show a variety of biological activities. These compounds are principally formed from their corresponding N-acyl-phosphatidylethanolamines by a phosphodiesterase of the phospholipase D-type in animal tissues. We purified the enzyme from rat heart, and by the use of the sequences of its internal peptides cloned its complementary DNAs from mouse, rat, and human. The deduced amino acid sequences were composed of 393-396 residues, and showed that the enzyme has no homology with the known phospholipase D enzymes but is classified as a member of the zinc metallohydrolase family of the beta-lactamase fold. As was overexpressed in COS-7 cells, the recombinant enzyme generated anandamide and other N-acylethanolamines from their corresponding N-acyl-phosphatidylethanolamines at comparable rates. In contrast, the enzyme was inactive with phosphatidylcholine and phosphatidylethanolamine. Assays of the enzyme activity and the messenger RNA and protein levels revealed its wide distribution in murine organs with higher contents in the brain, kidney, and testis. These results confirm that a specific phospholipase D is responsible for the generation of N-acylethanolamines including anandamide, strongly suggesting the physiological importance of lipid molecules of this class.     

Determination of endocannabinoids in nematodes and human brain tissue by liquid chromatography electrospray ionization tandem mass spectrometry

A simple and highly sensitive liquid chromatography/tandem mass spectrometric (LC/MS/MS) method was developed to compare endogenous cannabinoid levels in nematodes and in brains of rats and humans, with and without prior exposure to ethanol. After liquid-liquid extraction of the lipid fraction from homogenized samples, a reversed-phase sub 2 μm column was used for separating analytes with an isocratic mobile phase. Deuterated internal standards were used in the analysis, and detection was made by triple quadrupole mass spectrometer with multiple reaction monitoring (MRM). Ionization was performed with positive electrospray ionization (ESI). The nematode Caenorhabditis elegans fat-3 mutant, that lacks the necessary enzyme to produce arachidonic acid, the biologic precursor to 2-arachidonoyl glycerol and anandamide, was used as an analyte-free surrogate material for selectivity and calibration studies. The matrix effect was further investigated by in-source multiple reaction monitoring (IS-MRM) and standard addition studies. Selectivity studies demonstrated that the method was free from matrix effects. Good accuracy and precision were obtained for concentrations within the calibration range of 0.4-70 nM and 40-11,000 nM for monitored N-acylethanolamides (NAEs) and acyl glycerols, respectively.     

Biosynthesis and degradation of anandamide and 2-arachidonoylglycerol and their possible physiological significance

N -arachidonoylethanolamine (anandamide) was the first endogenous cannabinoid receptor ligand to be discovered. Dual synthetic pathways for anandamide have been proposed. One is the formation from free arachidonic acid and ethanolamine, and the other is the formation from N -arachidonoyl phosphatidylethanolamine (PE) through the action of a phosphodiesterase. These pathways, however, do not appear to be able to generate a large amount of anandamide, at least under physiological conditions. The generation of anandamide from free arachidonic acid and ethanolamine is catalyzed by a degrading enzyme anandamide amidohydrolase/fatty acid amide hydrolase operating in reverse and requires large amounts of substrates. As for the second pathway, arachidonic acids esterified at the 1-position of glycerophospholipids, which are mostly esterified at the 2-position, are utilized for the formation of N -arachidonoyl PE, a stored precursor form of anandamide. In fact, the actual levels of anandamide in various tissues are generally low except in a few cases. 2-Arachidonoylglycerol (2-AG) was the second endogenous cannabinoid receptor ligand to be discovered. 2-AG is a degradation product of arachidonic acid-containing glycerophospholipids such as inositol phospholipids. Several investigators have demonstrated that 2-AG is produced in a variety of tissues and cells upon stimulation. 2-AG acts as a full agonist at the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating and indicates that 2-AG is the most efficacious endogenous natural ligand for the cannabinoid receptors.In this review, we summarize the tissue levels, biosynthesis, degradation and possible physiological significance of two endogenous cannabimimetic molecules, anandamide and 2-AG.     

Endothelium-derived 2-arachidonylglycerol: an intermediate in vasodilatory eicosanoid release in bovine coronary arteries

Acetylcholine stimulates the release of endothelium-derived arachidonic acid (AA) metabolites including prostacyclin and epoxyeicosatrienoic acids (EETs), which relax coronary arteries. However, mechanisms of endothelial cell (EC) AA activation remain undefined. We propose that 2-arachidonylglycerol (2-AG) plays an important role in this pathway. An AA metabolite isolated from bovine coronary ECs was identified as 2-AG by mass spectrometry. In ECs pretreated with the fatty acid amidohydrolase inhibitor diazomethylarachidonyl ketone (DAK; 20 micromol/l), methacholine (10 micromol/l)-stimulated 2-AG release was blocked by the phospholipase C inhibitor U-73122 (10 micromol/l) or the diacylglycerol lipase inhibitor RHC-80267 (40 micromol/l). In U-46619-preconstricted bovine coronary arterial rings, 2-AG relaxations averaging 100% at 10 micromol/l were inhibited by endothelium removal, by DAK, by the hydrolase inhibitor methyl arachidonylfluorophosphate (10 micromol/l), by the cyclooxygenase inhibitor indomethacin (10 micromol/l), but not by the CB1 cannabinoid receptor antagonist SR-141716 (1 micromol/l). The cytochrome P-450 inhibitor SKF-525a (10 micromol/l) and the 14,15-epoxyeicosa-5Z-enoic acid EET antagonist (14,15-EEZE; 10 micromol/l) further attenuated the indomethacin-resistant relaxations. The nonhydrolyzable 2-AG analogs noladin ether, 2-AG amide, and 14,15-EET glycerol amide did not induce relaxation. N-nitro-L-arginine-resistant relaxations to methacholine were also inhibited by U-73122, RHC-80267, and DAK. 14,15-EET glycerol ester increased opening of large-conductance K(+) channels 12-fold in cell-attached patches of isolated smooth muscle cells and induced relaxations averaging 95%. These results suggest that methacholine stimulates EC 2-AG production through phospholipase C and diacylglycerol lipase activation. 2-AG is further hydrolyzed to AA, which is metabolized to vasoactive eicosanoids. These studies reveal a role for 2-AG in EC AA release and the regulation of coronary tone.     

N-acylphosphatidylethanolamine-hydrolysing phospholipase D lacks the ability to transphosphatidylate.

The N-acylphosphatidylethanolamine-hydrolysing phospholipase D (NAPE-PLD) generates N-acylethanolamines, including N-arachidonoyl-ethanolamine (anandamide), that may be neuroprotective and analgesic. The properties of NAPE-PLD from rat heart and brain microsomes are investigated and compared to those of other PLDs. NAPE-PLD is inhibited by the fatty acid aminohydrolase inhibitor MAFP in high concentrations (> or = 100 microM) while PMSF in high concentrations (10 mM) tends to stabilise NAPE-PLD activity. Oleate inhibits NAPE-PLD but the enzyme is not affected by PIP2, alpha-synuclein or mastoparan. Furthermore, it is for the first time reported that NAPE-PLD is not capable of catalysing a transphosphatidylation reaction like most other known PLDs.     

Occurrence and possible biological role of the endocannabinoid system in the sea squirt Ciona intestinalis

A cannabinoid receptor orthologue (CiCBR) has been described in the sea squirt Ciona intestinalis. Here we report that CiCBR mRNA expression is highest in cerebral ganglion, branchial pharynx, heart and testis of C. intestinalis, and that this organism also contains cannabinoid receptor ligands and some of the enzymes for ligand biosynthesis and inactivation. Using liquid chromatography-mass spectrometry, the endocannabinoid anandamide was found in all tissues analysed (0.063-5.423 pmol/mg of lipid extract), with the highest concentrations being found in brain and heart. The endocannabinoid 2-arachidonoylglycerol (2-AG) was fivefold more abundant than anandamide, and was most abundant in stomach and intestine and least abundant in heart and ovaries (2.677-50.607 pmol/mg of lipid extract). Using phylogenomic analysis, we identified orthologues of several endocannabinoid synthesizing and degrading enzymes. In particular, we identified and partly sequenced a fatty acid amide hydrolase (FAAH) orthologue, showing 44% identity with human FAAH and containing nearly all the amino acids necessary for a functional FAAH enzyme. Ciona intestinalis also contained specific binding sites for cannabinoid receptor ligands, and an amidase enzyme with pH-dependency and subcellular/tissue distribution similar to mammalian FAAHs. Finally, a typical C. intestinalis behavioural response, siphon reopening after closure induced by mechanical stimulation, was inhibited by the cannabinoid receptor agonist HU-210, and this effect was significantly attenuated by mammalian cannabinoid receptor antagonists.     

The endogenous cannabinoid system and its role in nociceptive behavior

The analgesic properties of exogenous cannabinoids have been recognized for many years and suggest a regulatory role for the endogenous cannabinoid ("endocannabinoid") system in mammalian nociceptive pathways. The endocannabinoid system includes: (1) at least two families of lipid signaling molecules, the N-acyl ethanolamines (e.g., anandamide) and the monoacylglycerols (e.g., 2-arachidonoyl glycerol); (2) multiple enzymes involved in the biosynthesis and degradation of these lipids, including the integral membrane enzyme fatty acid amide hydrolase; and (3) two G-protein coupled receptors, CB1 and CB2, which are primarily localized to the nervous system and immune system, respectively. Here, we review recent genetic, behavioral, and pharmacological studies that have tested the function of the endocannabinoid system in pain sensation. Collectively, these investigations support a role for endocannabinoids in modulating behavioral responses to acute, inflammatory, and neuropathic pain stimuli.     

Human mast cells take up and hydrolyze anandamide under the control of 5-lipoxygenase and do not express cannabinoid receptors

Human mast cells (HMC-1) take up anandamide (arachidonoyl-ethanolamide, AEA) with a saturable process (K(m)=200+/-20 nM, V(max)=25+/-3 pmol min(-1) mg protein(-1)), enhanced two-fold over control by nitric oxide-donors. Internalized AEA was hydrolyzed by a fatty acid amide hydrolase (FAAH), whose activity became measurable only in the presence of 5-lipoxygenase, but not cyclooxygenase, inhibitors. FAAH (K(m)=5.0+/-0.5 microM, V(max)=160+/-15 pmol min(-1) mg protein(-1)) was competitively inhibited by palmitoylethanolamide. HMC-1 cells did not display a functional cannabinoid receptor on their surface and neither AEA nor palmitoylethanolamide affected tryptase release from these cells.     

Human platelets bind and degrade 2-arachidonoylglycerol, which activates these cells through a cannabinoid receptor.

The endocannabinoid 2-arachidonoylglycerol (2-Delta(4)Ach-Gro) activates human platelets in platelet-rich plasma at physiological concentrations. The activation was inhibited by selective antagonists of CB(1) and CB(2) cannabinoid receptors, but not by acetylsalicylic acid. Human platelets can metabolize 2-Delta(4)Ach-Gro by internalization through a high affinity transporter (K(m) = 300 +/- 30 nM, V(max) = 10 +/- 1 pmol.min(-1).mg protein(-1)), followed by hydrolysis by a fatty acid amide hydrolase (K(m) = 8 +/- 1 microM, V(max) = 400 +/- 50 pmol.min(-1).mg protein(-1)). The anandamide transport inhibitor AM404, and anandamide itself, were ineffective on 2-Delta(4)Ach-Gro uptake by platelets, whereas anandamide competitively inhibited 2-Delta(4)Ach-Gro hydrolysis (inhibition constant = 10 +/- 1 microM). Platelet activation by 2-Delta(4)Ach-Gro was paralleled by an increase of intracellular calcium and inositol-1,4,5-trisphosphate, and by a decrease of cyclic AMP. Moreover, treatment of preloaded platelet-rich plasma with 2-Delta(4)Ach-Gro induced an approximately threefold increase in [(3)H]2-Delta(4)Ach-Gro release, according to a CB receptor-dependent mechanism. On the other hand, ADP and collagen counteracted the activation of platelets by 2-Delta(4)Ach-Gro, whereas 5-hydroxytryptamine (serotonin) enhanced and extended its effects. Remarkably, ADP and collagen also reduced [(3)H]2-Delta(4)Ach-Gro release from 2-Delta(4)Ach-Gro-activated platelets, whereas 5-hydroxytryptamine further increased it. These findings suggest a so far unnoticed interplay between the peripheral endocannabinoid system and physiological platelet agonists.     

Modulation of anxiety through blockade of anandamide hydrolysis

The psychoactive constituent of cannabis, Delta(9)-tetrahydrocannabinol, produces in humans subjective responses mediated by CB1 cannabinoid receptors, indicating that endogenous cannabinoids may contribute to the control of emotion. But the variable effects of Delta(9)-tetrahydrocannabinol obscure the interpretation of these results and limit the therapeutic potential of direct cannabinoid agonists. An alternative approach may be to develop drugs that amplify the effects of endogenous cannabinoids by preventing their inactivation. Here we describe a class of potent, selective and systemically active inhibitors of fatty acid amide hydrolase, the enzyme responsible for the degradation of the endogenous cannabinoid anandamide. Like clinically used anti-anxiety drugs, in rats the inhibitors exhibit benzodiazepine-like properties in the elevated zero-maze test and suppress isolation-induced vocalizations. These effects are accompanied by augmented brain levels of anandamide and are prevented by CB1 receptor blockade. Our results indicate that anandamide participates in the modulation of emotional states and point to fatty acid amide hydrolase inhibition as an innovative approach to anti-anxiety therapy.     

Massive accumulation of N‐acylethanolamines after stroke. Cell signalling in acute cerebral ischemia?

We investigated levels and compositions of N-acylethanolamines (NAEs) and their precursors, N-acyl phosphatidylethanolamines (N-acyl PEs), in a rat stroke model applying striatal microdialysis for glutamate assay. Rats (n = 18) were treated with either intravenous saline (control), NMDA receptor antagonist MK801 (1 mg/kg), or CB1 receptor antagonist SR141716A (1 mg/kg) 30 min after permanent middle cerebral artery occlusion (MCAO). MK801 significantly attenuated the release of glutamate in the infarcted striatum (79 +/- 22 micromol/L) as compared with controls (322 +/- 104 micromol/L). The administration of CB1 antagonist SR141716A had no statistically significant effect on glutamate release (340 +/- 89 micromol/L), but reduced infarct volume at 5 h after MCAO significantly by approximately 40%, whereas MK801 treatment resulted in a non-significant (18%) reduction of infarct volume. In controls, striatal and cortical NAE concentrations were about 30-fold higher in the infarcted than in the non-infarcted hemisphere, whereas ipsilateral N-acyl phosphatidylethanolamine (N-acyl PE) levels exceeded contralateral levels by only a factor of two to three. Treatment with MK801 or SR141716A, or glutamate release in the infarcted tissue, had no significant effect on these levels. NAE accumulation during acute stroke may be due to increased synthesis as well as decreased degradation, possibly by inhibition of fatty acid amide hydrolase (FAAH).     

Enhancement of endocannabinoid signaling by fatty acid amide hydrolase inhibition: A neuroprotective therapeutic modality

AimsThis review posits that fatty acid amide hydrolase (FAAH) inhibition has therapeutic potential against neuropathological states including traumatic brain injury; Alzheimer's, Huntington's, and Parkinson's diseases; and stroke.Main methodsThis proposition is supported by data from numerous in vitro and in vivo experiments establishing metabolic and pharmacological contexts for the neuroprotective role of the endogenous cannabinoid (“endocannabinoid”) system and selective FAAH inhibitors.Key findingsThe systems biology of endocannabinoid signaling involves two main cannabinoid receptors, the principal endocannabinoid lipid mediators N-arachidonoylethanolamine (“anandamide”) (AEA) and 2-arachidonoyl glycerol (2-AG), related metabolites, and the proteins involved in endocannabinoid biosynthesis, biotransformation, and transit. The endocannabinoid system is capable of activating distinct signaling pathways on-demand in response to pathogenic events or stimuli, thereby enhancing cell survival and promoting tissue repair. Accumulating data suggest that endocannabinoid system modulation at discrete targets is a promising pharmacotherapeutic strategy for treating various medical conditions. In particular, neuronal injury activates cannabinoid signaling in the central nervous system as an intrinsic neuroprotective response. Indirect potentiation of this salutary response through pharmacological inhibition of FAAH, an endocannabinoid-deactivating enzyme, and consequent activation of signaling pathways downstream from cannabinoid receptors have been shown to promote neuronal maintenance and function.SignificanceThis therapeutic modality has the potential to offer site- and event-specific neuroprotection under conditions where endocannabinoids are being produced as part of a physiological protective mechanism. In contrast, direct application of cannabinoid receptor agonists to the central nervous system may activate CB receptors indiscriminately and invite unwanted psychotrophic effects.     

Discovery of endocannabinoids and some random thoughts on their possible roles in neuroprotection and aggression

A short history of the discovery of the main plant cannabinoid, Delta(9)-tetrahydrocannabinol and of the endogenous cannabinoids anandamide, 2-arachidonoyl glycerol and 2-arachidonyl glyceryl ether (noladin ether) is presented. The role of the cannabinoids in neuroprotection, with emphasis on the endocannabinoids, is described. The unexpected production of aggression by Cannabis and cannabinoids under stressful conditions, published mainly in the past, is summarized.