Determination of anandamide and other fatty acyl ethanolamides in human serum by electrospray tandem mass spectrometry

We developed a new selective liquid chromatography-electrospray ionization-tandem mass spectrometry method for the identification and quantification of anandamide (AEA), an endogenous cannabinoid receptor ligand, and other bioactive fatty acid ethanolamides (FAEs) in biological samples. Detection limit (0.025 pmol for AEA and 0.1 pmol for palmitoylethanolamide (PEA) and oleoylethanolamide (OEA)) and quantification limit (0.2 pmol for AEA and 0.4 pmol for OEA and PEA) were in the high fmol to low pmol range for all analytes. Linear correlations (r(2)=0.99) were observed in the calibration curves for standard AEA over the range of 0.025-25 pmol and for standard PEA and OEA over the range of 0.1-500 pmol. This method provides a time-saving and sensitive alternative to existing methods for the analysis of FAEs in biological samples.     

Sex-dependent effects of neonatal maternal deprivation on endocannabinoid levels in the adipose tissue: influence of diet

Maternal deprivation (MD) during neonatal life has diverse long-term effects, including modification of metabolism. We have previously reported that MD modifies the metabolic response to high-fat diet (HFD) intake, with this response being different between males and females, while previous studies indicate that in mice with HFD-induced obesity, endocannabinoid (EC) levels are markedly altered in various brown and white adipose tissue depots. Here, we analyzed the effects of MD (24 h at postnatal day 9), alone or in combination with a HFD from weaning until the end of the experiment in Wistar rats of both sexes. Brown and white perirenal and subcutaneous adipose tissues were collected and the levels of anandamide (AEA), 2-arachidonoylglycerol (2-AG), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were determined. In males, MD increased the content of OEA in brown and 2-AG in subcutaneous adipose tissues, while in females the content of 2-AG was increased in perirenal fat. Moreover, in females, MD decreased AEA and OEA levels in perirenal and subcutaneous adipose tissues, respectively. HFD decreased the content of 2-AG in brown fat of both sexes and OEA in brown and subcutaneous adipose tissue of control females. In contrast, in subcutaneous fat, HFD increased AEA levels in MD males and OEA levels in control and MD males. The present results show for the first time that MD and HFD induce sex-dependent effects on the main ECs, AEA, and 2-AG, and of AEA-related mediators, OEA and PEA, in the rat brown and white (visceral and subcutaneous) adipose tissues.     

Validation and application of an LC–MS/MS method for quantitation of three fatty acid ethanolamides as biomarkers for fatty acid hydrolase inhibition in human plasma

Endogenous ethanolamides (fatty acid amides), including arachidonyl ethanolamide (anandamide, AEA), oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA), are substrates of fatty acid amide hydrolase (FAAH). FAAH may play an important role for pain, anxiety/depression, and metabolic disorders. Ethanolamides are considered to be potential pharmacodynamic biomarkers to determine target engagement for FAAH inhibition by novel pharmaceutical agents. A highly selective, sensitive, and high-throughput liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous quantitation of AEA, OEA, and PEA in human plasma. The method employed D₄-AEA, D₄-OEA, and ¹³C₂-PEA as “surrogate analytes” to establish the concentration–mass response relationship, i.e. a regression equation. The concentrations of AEA, OEA, and PEA were calculated based on the regression equations derived from the surrogate analytes. This approach made it possible to prepare calibration standard and quality control (QC) samples in plasma devoid of interferences from the endogenous analytes. The analytical methodology required 150 μL of human plasma that was processed via liquid–liquid extraction (LLE) using a 96-well plate format. Chromatographic separation was achieved with a reversed-phase high performance liquid chromatography (HPLC) column using gradient elution, and the run time was 3 min. The method was fully validated and it demonstrated acceptable accuracy, precision, linearity, and specificity. The lower limit of quantitation (LLOQ) was 0.1/0.5/0.5 ng/mL for AEA/OEA/PEA, which was sensitive enough to capture the basal plasma levels in healthy subjects. Bench-top stability in plasma, freeze–thaw stability in plasma, frozen long-term stability in plasma, autosampler stability, and stock solution stability all met acceptance criteria (%Bias within ±12.0%). Characterization of stability in purchased/aged blood indicated that ethanolamides are subject to degradation mediated by intracellular membrane-bound FAAH, which has been shown to be inhibited by phenylmethylsulfonyl fluoride (PMSF). In the presence of PMSF, ethanolamide levels increased slightly over time, suggesting that blood cells release ethanolamides into plasma. Whole blood stability conducted in fresh blood immediately following collection revealed that there was significant elevation of ethanolamide concentrations (∼1.3–2.0-fold on ice and ∼1.5–3.0-fold at room temperature by 2 h), indicating that de novo synthesis and release from blood cells were the predominant factors affecting ethanolamide concentrations ex vivo. Accordingly, conditions that ensured rapid separation of plasma from blood cells and consistency in the blood harvesting procedures were established and implemented for clinical studies to minimize the ex vivo elevation of plasma ethanolamide concentrations. The variability (intra-subject and inter-subject) of plasma ethanolamide levels was evaluated in healthy subjects during a Phase 0 study (no drug administration) that simulated the design of single-ascending dose and multiple-ascending dose clinical trials in terms of sample collection time points, population, food, and activity. The data indicated there was relatively large inter- and intra-subject variation in plasma ethanolamide concentrations. In addition, apparent variations due to time of day and/or food effects were also revealed. Understanding the variability of ethanolamide levels in humans is very important for study design and data interpretation when changes in ethanolamide levels are used as target engagement biomarkers in clinical trials.     

Heteroarylureas with spirocyclic diamine cores as inhibitors of fatty acid amide hydrolase

A series of mechanism based heteroaryl urea fatty acid amide hydrolase (FAAH) inhibitors with spirocyclic diamine cores is described. A potent member of this class, (37), was found to inhibit FAAH centrally, elevate the brain levels of three fatty acid ethanolamides [FAAs: anandamide (AEA), oleoyl ethanolamide (OEA) and palmitoyl ethanolamide (PEA)], and was moderately efficacious in a rat model of neuropathic pain.     

Noladin ether, a putative novel endocannabinoid: inactivation mechanisms and a sensitive method for its quantification in rat tissues

The occurrence of the novel proposed endocannabinoid, noladin ether (2-arachidonyl glyceryl ether, 2-AGE) in various rat organs and brain regions, and its inactivation by intact C6 glioma cells, were studied. 2-AGE was measured by isotope dilution liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry, with a detection limit of 100 fmol. A compound with the same mass and chromatographic/chemical properties as 2-AGE was found in whole brain, with the highest amounts in the thalamus and hippocampus. Synthetic [(3)H]2-AGE was inactivated by intact rat C6 glioma cells by a time- and temperature-dependent process consisting of cellular uptake and partial incorporation into phospholipids. Further data suggested that 2-AGE is taken up by cells via the anandamide/2-arachidonoyl glycerol (2-AG) membrane transporter(s), and biosynthesized in a different way as compared to 2-AG.     

The endocannabinoid arachidonyl ethanolamide (anandamide) increases pulmonary arterial pressure via cyclooxygenase-2 products in isolated rabbit lungs

Several cannabinoids elicit systemic vasodilation, mainly via CB1 cannabinoid and vanilloid receptors. However, effects in the pulmonary circulation are unknown. Using the isolated, ventilated, buffer-perfused rabbit lung, we have shown that the endocannabinoids arachidonyl ethanolamide (anandamide) and 2-arachidonyl glycerol (2-AG) dose-dependently increase pulmonary arterial pressure (+19.9 +/- 3.4 mmHg, 5 microM, and +39.5 +/- 10.8 mmHg, 0.4 microM, respectively). 2-AG induced lung edema. The CB1 receptor antagonist AM-251 (0.1 and 5 microM) and the VR1 vanilloid receptor antagonist capsazepine (10 microM) failed to reduce anandamide's effects. The metabolically stable anandamide and 2-AG analogs R-methanandamide and noladin ether, Delta9-tetrahydrocannabinol, and the synthetic cannabinoid HU-210, which is no arachidonic acid product, were without effect. The unspecific cyclooxygenase (COX) inhibitor aspirin (100 microM, P < 0.001) and the specific COX-2 inhibitor nimesulide (10 microM, P < 0.01) completely prevented pulmonary hypertension after 5 microM anandamide. COX-2 RNA was detected in rabbit lungs. The synthetic thromboxane receptor antagonist SQ 29,548 was without effect, but the specific EP1 prostanoid receptor antagonist SC-19220 (100 microM) inhibited the pressure increase after anandamide (P < 0.05). PCR analysis detected fatty acid amidohydrolase (FAAH), an enzyme that degrades endocannabinoids, in rabbit lung tissue. Furthermore, the specific FAAH inhibitor methyl arachidonyl fluorophosphonate (0.1 microM) blocked pressure effects of anandamide (P < 0.01). Finally, anandamide (99 +/- 55 pmol/g) and 2-AG (19.6 +/- 8.4 nmol/g) were found in native lungs. We conclude that anandamide increases pulmonary arterial pressure via COX-2 metabolites following enzymatic degradation by FAAH into arachidonic acid products.     

Ether-linked analogue of 2-arachidonoylglycerol (noladin ether) was not detected in the brains of various mammalian species

2-Eicosa-5',8',11',14'-tetraenylglycerol (2-AG ether, HU310, noladin ether) is a metabolically stable ether-linked analogue of 2-arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand. 2-AG ether has been used as a valuable experimental tool by a number of investigators. Recently, several groups reported that 2-AG ether is present in mammalian brains. We examined in detail whether 2-AG ether actually exists in the brains of various mammalian species. We found that 2-AG ether is not present, at least in an appreciable amount, in the rat brain by gas chromatography-mass spectrometry analysis and fluorometric high performance liquid chromatography analysis. The level of 2-AG ether in the rat brain was below 0.2 pmol/g brain, if at all present. Similar results were obtained for the mouse brain, hamster brain, guinea-pig brain and pig brain. The fact that 2-AG ether was not detected in the brains of various mammalian species is consistent with the fact that an ether bond is formed through enzymatic replacement of the fatty acyl moiety of 1-acyl dihydroxyacetone phosphate by a fatty alcohol, the resultant 1-O-alkyl dihydroxyacetone phosphate being a common intermediate of the biosynthesis of ether-linked lipids in mammalian tissues. It is rather questionable whether 2-AG ether is present in appreciable amounts in the brain and acts as an 'endogenous' cannabinoid receptor ligand.     

Hydrolysis of prostaglandin glycerol esters by the endocannabinoid-hydrolyzing enzymes, monoacylglycerol lipase and fatty acid amide hydrolase

Cyclooxygenase-2 (COX-2) can oxygenate the endocannabinoids, arachidonyl ethanolamide (AEA) and 2-arachidonylglycerol (2-AG), to prostaglandin-H2-ethanolamide (PGH2-EA) and -glycerol ester (PGH2-G), respectively. Further metabolism of PGH2-EA and PGH2-G by prostaglandin synthases produces a variety of prostaglandin-EA's and prostaglandin-G's nearly as diverse as those derived from arachidonic acid. Thus, COX-2 may regulate endocannabinoid levels in neurons during retrograde signaling or produce novel endocannabinoid metabolites for receptor activation. Endocannabinoid-metabolizing enzymes are important regulators of their action, so we tested whether PG-G levels may be regulated by monoacylglycerol lipase (MGL) and fatty acid amide hydrolase (FAAH). We found that PG-Gs are poor substrates for purified MGL and FAAH compared to 2-AG and/or AEA. Determination of substrate specificity demonstrates a 30-100- and 150-200-fold preference of MGL and FAAH for 2-AG over PG-Gs, respectively. The substrate specificity of AEA compared to those of PG-Gs was approximately 200-300 fold higher for FAAH. Thus, PG-Gs are poor substrates for the major endocannabinoid-degrading enzymes, MGL and FAAH.     

The postmortal accumulation of brain N-arachidonylethanolamine (anandamide) is dependent upon fatty acid amide hydrolase activity

N-arachidonylethanolamine (AEA) accumulates during brain injury and postmortem. Because fatty acid amide hydrolase (FAAH) regulates brain AEA content, the purpose of this study was to determine its role in the postmortal accumulation of AEA using FAAH null mice. As expected, AEA content in immediately frozen brain tissue was significantly greater in FAAH-deficient (FAAH-/-) than in wild-type mice. However, AEA content was significantly lower in brains from FAAH-/- mice at 5 and 24 h postmortem. Similarly, wild-type mice treated in vivo with a FAAH inhibitor (URB532) had significantly lower brain AEA content 24 h postmortem compared with controls. These data indicate that FAAH contributes significantly to the postmortal accumulation of AEA. In contrast, the accumulations of two other N-acylethanolamines, N-oleoylethanolamine (OEA) and N-palmitoylethanolamine (PEA), were not reduced at 24 h postmortem in either the FAAH-/- mice or mice treated with URB532. FAAH-/- mice accumulated significantly less ethanolamine at 24 h postmortem compared with wild-type mice, suggesting that FAAH activity plays a role in the accumulation of ethanolamine postmortem. These data demonstrate that FAAH activity differentially affects AEA and OEA/PEA contents postmortem and suggest that AEA formation specifically occurs via an ethanolamine-dependent route postmortem.     

Effect of polyunsaturated fatty acids on endocannabinoid and N-acyl-ethanolamine levels in mouse adipocytes

The tissue concentrations of the endocannabinoids, 2-arachidonoylglycerol (2-AG) and N-arachidonoyl-ethanolamine (anandamide), are altered in the adipose tissue of mice fed a high fat diet. We have investigated here the effect on endocannabinoid levels of incubation of mouse 3T3-F442A adipocytes with several free polyunstaurated fatty acids (PUFAs), including linolenic acid (LA), alpha-linolenic acid (ALA), arachidonic acid (AA) and docosahexaenoic acid (DHA), as well as oleic acid (OA) and palmitic acid (PA). By using mass spectrometric methods, we quantified the levels of endocannabinoids, of two anandamide congeners, N-palmitoyl-ethanolamine (PEA) and N-oleoyl-ethanolamine (OEA), and of fatty acids esterified in triacylglycerols or phospholipids, which act as 2-AG and/or N-acyl-ethanolamine precursors. Incubation with AA strongly elevated 2-AG levels and the amounts of AA esterified in triacylglycerols and on glycerol carbon atom 2 (sn-2), but not 1 (sn-1), in phospholipids. Incubation with DHA decreased 2-AG and anandamide levels and the amounts of AA esterified on both the sn-2 and sn-1 position of phospholipids, but not on triacylglycerols. PEA levels augmented following incubation of adipocytes with OA and PA, with no corresponding changes in phospholipids and triacylglycerols. We suggest that dietary PUFAs might modulate the levels of adipocyte phospholipids that act as endocannabinoid precursors.     

Cannabimimetic fatty acid derivatives in cancer and inflammation

Evidence for the role of the cannabimimetic fatty acid derivatives (CFADs), i.e. anandamide (arachidonoylethanolamide, AEA), 2-arachidonoylglycerol (2-AG) and palmitoylethanolamide (PEA), in the control of inflammation and of the proliferation of tumor cells is reviewed here. The biosynthesis of AEA, PEA, or 2-AG can be induced by stimulation with either Ca(2+) ionophores, lipopolysaccharide, or platelet activating factor in macrophages, and by ionomycin or antigen challenge in rat basophilic leukemia (RBL-2H3) cells (a widely used model for mast cells). These cells also inactivate CFADs through re-uptake and/or hydrolysis and/or esterification processes. AEA and PEA modulate cytokine and/or arachidonate release from macrophages in vitro, regulate serotonin secretion from RBL-2H3 cells, and are analgesic in some animal models of inflammatory pain. However, the involvement of endogenous CFADs and cannabinoid CB(1) and CB(2) receptors in these effects is still controversial. In human breast and prostate cancer cells, AEA and 2-AG, but not PEA, potently inhibit prolactin and/or nerve growth factor (NGF)-induced cell proliferation. Vanillyl-derivatives of anandamide, such as olvanil and arvanil, exhibit even higher anti-proliferative activity. These effects are due to suppression of the levels of the 100 kDa prolactin receptor or of the high affinity NGF receptors (trk), are mediated by CB(1)-like cannabinoid receptors, and are enhanced by other CFADs. Inhibition of adenylyl cyclase and activation of mitogen-activated protein kinase underlie the anti-mitogenic actions of AEA. The possibility that CFADs act as local inhibitors of the proliferation of human breast cancer is discussed here.     

Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry

We developed a high-performance liquid chromatography/mass spectrometry (HPLC/MS) method for the identification and quantification of anandamide, an endogenous cannabinoid substance, and other fatty acid ethanolamides (AEs) in biological samples. Using a mobile-phase system of methanol/water and gradient elution, we achieved satisfactory resolution of all major AEs, including anandamide, palmitylethanolamide (PEA), and oleylethanolamide (OEA). Electrospray-generated quasi-molecular species were used as diagnostic ions and detected by selected ion monitoring (SIM). Synthetic deuterium-labeled AEs were used as internal standards, and quantification was carried out by isotope dilution. A linear correlation (r2 = 0.99) was observed in the calibration curves for standard AEs over the range 0-0.5 nmol. Detection limits between 0.1 and 0.3 pmol per sample and quantification limits between 0.5 and 1.2 pmol per sample were obtained. The method was applied to the quantification of anandamide, PEA, and OEA in plasma prepared from rat blood collected either by cardiac puncture or by decapitation. After cardiac puncture, AE levels were in the low-nanomolar range: anandamide, 3.1 +/- 0.6 pmol/ml; PEA, 9.4 +/- 1.6 pmol/ml; OEA, 9.2 +/- 1.8 pmol/ml (mean +/- SE, n = 9). By contrast, after decapitation AEs were dramatically elevated (anandamide, 144 +/- 13 pmol/ml; PEA, 255 +/- 55 pmol/ml; OEA, 175 +/- 48 pmol/ml). Thus, disruptive procedures of blood collection may result in gross overestimates in the concentrations of circulating AEs.     

Simultaneous quantitative analysis of N-acylethanolamides in clinical samples

A simple and rapid analytical method is described for the simultaneous quantitative analysis of three different N-acylethanolamides in human biological samples: anandamide (AEA), oleoylethanolamide (OEA), and palmitoylethanolamide (PEA). The method is based on a new hybrid solid phase extraction-precipitation technology followed by ultra-performance liquid chromatography/mass spectrometry (UPLC/MS) analysis using d₄-AEA as the internal standard. The method is linear up to 100 ng/ml with a limit of quantitation of 50 pg/ml for AEA and 100 pg/ml for OEA and PEA. Good reproducibility, accuracy, and precision were demonstrated during the method validation. Application of this new methodology to the analysis of clinical study samples is presented.     

Brain regional distribution of endocannabinoids: implications for their biosynthesis and biological function

The amounts, in nine different rat brain regions, of the two endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoylglycerol (2-AG), and of the putative AEA precursor N-arachidonoyl-phosphatidylethanolamine (NArPE), were determined by isotope-dilution gas chromatography-mass spectrometry and compared to the number of cannabinoid binding sites in each region. The distribution of NArPE, reported here for the first time, exhibited a good correlation with that of AEA, the former metabolite being 3-13 times more abundant than the endocannabinoid in all regions. The highest amounts of both metabolites (up to 358.5 and 87 pmol/g wet weight tissue, respectively) were found in the brainstem and striatum, and the lowest in the diencephalon, cortex, and cerebellum. These data support the hypothesis that, in the brain, AEA is a metabolic product of NArPE and may reach levels compatible with its proposed neuromodulatory function. The brain distribution of 2-AG, also described in this study for the first time, was found to correlate with that of AEA with levels ranging from 2.0 to 14.0 nmol/g (in the diencephalon and brainstem, respectively). The distribution of the endocannabinoids did not match exactly with that of cannabinoid binding sites, suggesting either that these compounds are not necessarily produced near their molecular targets, or that they play functional roles additional to the activation of cannabinoid receptors. Regional differences in the ligand/receptor ratios may also lead to predict corresponding differences in the efficiency of receptor activation, as shown by previous studies.     

Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide,into prostaglandin,thromboxane, and prostacyclin glycerol esters and ethanolamides

Cyclooxygenase-2 (COX-2) action on the endocannabinoids, 2-arachidonylglycerol (2-AG) and anandamide (AEA), generates prostaglandin glycerol esters (PG-G) and ethanolamides (PG-EA), respectively. The diversity of PG-Gs and PG-EAs that can be formed enzymatically following COX-2 oxygenation of endocannabinoids was examined in cellular and subcellular systems. In cellular systems, glycerol esters and ethanolamides of PGE(2), PGD(2), and PGF(2alpha) were major products of the endocannabinoid-derived COX-2 products, PGH(2)-G and PGH(2)-EA. The sequential action of purified COX-2 and thromboxane synthase on AEA and 2-AG provided thromboxane A(2) ethanolamide and glycerol ester, respectively. Similarly, bovine prostacyclin synthase catalyzed the isomerization of the intermediate endoperoxides, PGH(2)-G and PGH(2)-EA, to the corresponding prostacyclin derivatives. Quantification of the efficiency of prostaglandin and thromboxane synthase-directed endoperoxide isomerization demonstrated that PGE, PGD, and PGI synthases catalyze the isomerization of PGH(2)-G at rates approaching those observed with PGH(2). In contrast, thromboxane synthase was far more efficient at catalyzing PGH(2) isomerization than at catalyzing the isomerization of PGH(2)-G. These results define the in vitro diversity of endocannabinoid-derived prostanoids and will permit focused investigations into their production and potential biological actions in vivo.     

Full Inhibition of Spinal FAAH Leads to TRPV1-Mediated Analgesic Effects in Neuropathic Rats and Possible Lipoxygenase-Mediated Remodeling of Anandamide Metabolism

Neuropathic pain elevates spinal anandamide (AEA) levels in a way further increased when URB597, an inhibitor of AEA hydrolysis by fatty acid amide hydrolase (FAAH), is injected intrathecally. Spinal AEA reduces neuropathic pain by acting at both cannabinoid CB1 receptors and transient receptor potential vanilloid-1 (TRPV1) channels. Yet, intrathecal URB597 is only partially effective at counteracting neuropathic pain. We investigated the effect of high doses of intrathecal URB597 on allodynia and hyperalgesia in rats with chronic constriction injury (CCI) of the sciatic nerve. Among those tested, the 200 µg/rat dose of URB597 was the only one that elevated the levels of the FAAH non-endocannabinoid and anti-inflammatory substrates, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), and of the endocannabinoid FAAH substrate, 2-arachidonoylglycerol, and fully inhibited thermal and tactile nociception, although in a manner blocked almost uniquely by TRPV1 antagonism. Surprisingly, this dose of URB597 decreased spinal AEA levels. RT-qPCR and western blot analyses demonstrated altered spinal expression of lipoxygenases (LOX), and baicalein, an inhibitor of 12/15-LOX, significantly reduced URB597 analgesic effects, suggesting the occurrence of alternative pathways of AEA metabolism. Using immunofluorescence techniques, FAAH, 15-LOX and TRPV1 were found to co-localize in dorsal spinal horn neurons of CCI rats. Finally, 15-hydroxy-AEA, a 15-LOX derivative of AEA, potently and efficaciously activated the rat recombinant TRPV1 channel. We suggest that intrathecally injected URB597 at full analgesic efficacy unmasks a secondary route of AEA metabolism via 15-LOX with possible formation of 15-hydroxy-AEA, which, together with OEA and PEA, may contribute at producing TRPV1-mediated analgesia in CCI rats.     

Anti-proliferative effect of a putative endocannabinoid, 2-arachidonylglyceryl ether in prostate carcinoma cells

Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.     

Endocannabinoids control spasticity in a multiple sclerosis model.

Spasticity is a complicating sign in multiple sclerosis that also develops in a model of chronic relapsing experimental autoimmune encephalomyelitis (CREAE) in mice. In areas associated with nerve damage, increased levels of the endocannabinoids, anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG), and of the AEA congener, palmitoylethanolamide (PEA), were detected here, whereas comparable levels of these compounds were found in normal and non-spastic CREAE mice. While exogenously administered endocannabinoids and PEA ameliorate spasticity, selective inhibitors of endocannabinoid re-uptake and hydrolysis-probably through the enhancement of endogenous levels of AEA, and, possibly, 2-arachidonoyl glycerol-significantly ameliorated spasticity to an extent comparable with that observed previously with potent cannabinoid receptor agonists. These studies provide definitive evidence for the tonic control of spasticity by the endocannabinoid system and open new horizons to therapy of multiple sclerosis, and other neuromuscular diseases, based on agents modulating endocannabinoid levels and action, which exhibit little psychotropic activity.     

N-Acylethanolamines in human reproductive fluids

N-Acylethanolamines (NAEs) are an important family of lipid-signaling molecules. Arachidonylethanolamide (anandamide) (AEA), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) are co-produced from similar phospholipid precursors when neurons are stimulated. AEA is an endogenous agonist (endocannabinoid) for cannabinoid receptors. It binds with higher affinity to type CB1 than to type CB2 cannabinoid receptors. PEA does not bind to CB1, while the hypothesis that it reacts with putative CB2-like receptors has been questioned. OEA does not activate currently known cannabinoid receptors, but it mimics the effects of AEA and cannabinoids in reducing the fertilizing capacity of sea urchin sperm. OEA and PEA also act as entourage compounds by inhibiting the hydrolysis of AEA by fatty acid amide hydrolase. Cannabinoid receptors and/or AEA are present in mammalian reproductive organs including the testis, epididymis, prostate, ovary, uterus, sperm, preimplantation embryo and placenta, as well as prostatic and mammary carcinomas. We now report that analysis by high-performance liquid chromatography/mass spectrometry (HPLC/MS) shows the presence of AEA, PEA, and OEA in human seminal plasma, mid-cycle oviductal fluid, follicular fluid, amniotic fluid, milk, and fluids from malignant ovarian cysts. Previous studies showed that AEA-signaling via cannabinoid receptors regulates capacitation and fertilizing potential of human sperm, early embryonic development and blastocyst implantation into the uterine mucosa of rodents, as well as proliferation of human mammary and prostatic carcinomas. Current results imply that NAEs also may modulate follicular maturation and ovulation, normal and pathological ovarian function, placental and fetal physiology, lactation, infant physiology, and behavior. Collectively, these findings suggest that NAEs in human reproductive fluids may help regulate multiple physiological and pathological processes in the reproductive system, and imply that exogenous cannabinoids delivered by marijuana smoke might impact these processes. This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adolescents and adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.     

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.