n-oleoylethanolamine and virodhamine

In this issue of the British Journal of Pharmacology, Ryberg et al. present convincing in vitro evidence that the orphan GPCR, GPR55, is a cannabinoid receptor. GPR55 was activated by a range of plant, synthetic and endogenous cannabinoids and blocked by the non-psychoactive phytocannabinoid, cannabidiol. Their experiments have revealed several differences between the pharmacology of GPR55 and the established cannabinoid CB1 and CB2 receptors. For example, the CB1 receptor antagonist, AM251, activated GPR55 and the main psychoactive constituent of cannabis, Delta9-tetrahydrocannabinol, displayed greater efficacy at GPR55 than at CB1 or CB2 receptors. They also compared the distribution of GPR55 and CB1 mRNA in mouse and report that GPR55 couples to Galpha13, that it is activated by virodhamine, palmitoylethanolamide and oleoylethanolamide, and that virodhamine displays relatively high efficacy as a GPR55 agonist. Still to be identified are the main roles played by GPR55 in health and disease and any potential therapeutic benefits of activating or blocking this receptor.

Topics: Amides; Animals; Arachidonic Acids; Cannabinoids; Dronabinol; Endocannabinoids; Ethanolamines; Humans; Oleic Acids; Palmitic Acids; Piperidines; Pyrazoles; Receptors, Cannabinoid; Receptors, G-Protein-Coupled

Previous reports have shown improvements in mood and increases in endocannabinoids in healthy adults following a session of aerobic exercise, but it is unclear whether adults with posttraumatic stress disorder (PTSD) experience similar responses. The purpose of this study was to examine psychobiological responses (plasma endocannabinoids [eCBs], mood, and pain) to aerobic exercise in a sample of adults with a diagnosis of PTSD (n = 12) and healthy controls (n = 12). Participants engaged in an aerobic exercise session in which they ran on a treadmill for 30 min at a moderate intensity (70 to 75% maximum heart rate [MHR]). Results indicated improvements in mood states and reductions in pain for both groups following exercise, ds = 0.19 to 1.53. Circulating concentrations of N-arachidonylethanolamine (AEA), 2-arachidonoylglycerol (2-AG), and oleoylethanolamide (OEA) significantly increased (ps = .000 to .050) following the aerobic exercise session for both groups. There were no significant time, group, or interaction effects (ps = .062 to .846) for palmitoylethanolamide (PEA) and 2-oleoylglycerol (2-OG). Although eCBs increased significantly for both groups, within-group effect size calculations indicated the healthy controls experienced a greater magnitude of change for AEA when compared with adults with PTSD, d = 1.21 and d = 0.45, respectively; as well as for 2-AG, d = 0.43 and d = 0.21, respectively. The findings from this study indicated that adults with and without PTSD reported significant mood improvements following 30 min of moderate-intensity aerobic exercise. In addition, the endocannabinoid system was activated in adults with and without PTSD, although effect sizes suggest that adults with PTSD may have a blunted endocannabinoid response to exercise.

Topics: Adolescent; Adult; Affect; Arachidonic Acids; Cannabinoids; Case-Control Studies; Endocannabinoids; Exercise; Female; Glycerides; Humans; Male; Oleic Acids; Pain; Pilot Projects; Stress Disorders, Post-Traumatic; Young Adult

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 d(4)-AEA as the internal standard. The method is linear up to 100ng/ml with a limit of quantitation of 50pg/ml for AEA and 100pg/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.

Topics: Adolescent; Adult; Amides; Amidohydrolases; Arachidonic Acids; Calibration; Cannabinoids; Chromatography, High Pressure Liquid; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Molecular Structure; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; Reproducibility of Results; Semen; Solid Phase Microextraction; Tandem Mass Spectrometry; Young Adult

The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes.. Cells transfected with the human cDNA for GPR55 were tested for their ability to bind and to mediate GTPgammaS binding by cannabinoid ligands. Using an antibody and peptide blocking approach, the nature of the G-protein coupling was determined and further demonstrated by measuring activity of downstream signalling pathways.. We demonstrate that GPR55 binds to and is activated by the cannabinoid ligand CP55940. In addition endocannabinoids including anandamide and virodhamine activate GTPgammaS binding via GPR55 with nM potencies. Ligands such as cannabidiol and abnormal cannabidiol which exhibit no CB1 or CB2 activity and are believed to function at a novel cannabinoid receptor, also showed activity at GPR55. GPR55 couples to Galpha13 and can mediate activation of rhoA, cdc42 and rac1.. These data suggest that GPR55 is a novel cannabinoid receptor, and its ligand profile with respect to CB1 and CB2 described here will permit delineation of its physiological function(s).

Topics: Amino Acid Sequence; Animals; Arachidonic Acids; Binding Sites; Binding, Competitive; Cannabidiol; Cannabinoids; Cell Line; Cloning, Molecular; Cyclohexanols; Down-Regulation; Endocannabinoids; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Ligands; Mice; Molecular Sequence Data; Organ Specificity; Polymerase Chain Reaction; Polyunsaturated Alkamides; Rats; Receptors, Cannabinoid; Receptors, G-Protein-Coupled; RNA, Messenger; Signal Transduction; Structure-Activity Relationship

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method is described for the simultaneous identification and quantification of eight endocannabinoid (EC) or related "entourage" compounds in rat brain tissue. Analytes were extracted and purified from rat brain tissue using an ethyl acetate/hexane solvent extraction, followed by a solid phase extraction (SPE) protocol. Chromatographic separation was achieved using a gradient elution, with a mobile phase of acetonitrile, formic acid, and ammonium acetate, at pH 3.6. A Thermo Hypersil C8 HyPurity Advance column (100x2.1 mm i.d., 3 microm) was used with a flow rate of 0.3 ml/min). Anandamide (AEA), 2-arachidonyl glycerol (2-AG), 2-arachidonylglyceryl ether (noladin ether), O-arachidonyl ethanolamide (virodhamine), 2-linoleoyl glycerol (2-LG), arachidonyl glycine, oleoyl ethanolamide (OEA), and palmitoyl ethanolamide (PEA) were quantified by positive ion tandem electrospray ionization mass spectrometry. Internal standards were deuterated AEA, deuterated 2-AG, and heptadecanoyl ethanolamide (HEA). Linearity was proven over the range of 25 fmol to 250 pmol, with a limit of detection of 25 fmol on column for all analytes except 2-AG, noladin ether, and 2-LG (250 fmol). This corresponded to a limit of quantification in biological tissue of 10 pmol/g for all analytes except 2-AG (100 pmol/g). Intra- and interday precision in biological tissue was routinely approximately 20% or lower, and accuracy was between 65% and 155%. This method was used to quantitatively profile regional differences in nine discrete rat brain regions for AEA, 2-AG, 2-LG, OEA, PEA, noladin ether, virodhamine, and arachidonyl glycine.

Topics: Animals; Arachidonic Acids; Brain Chemistry; Cannabinoid Receptor Modulators; Cannabinoids; Chromatography, Liquid; Endocannabinoids; Glycerides; Glycine; Male; Oleic Acids; Polyunsaturated Alkamides; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

Although CB(1) receptor activation evokes neuroprotection in response to cannabinoids, some cannabinoids have been reported to be peroxisome proliferator activated receptor (PPAR) ligands, offering an alternative protective mechanism. We have, therefore, investigated the ability of a range of cannabinoids to activate PPAR alpha and for N-oleoylethanolamine (OEA), an endogenous cannabinoid-like compound (ECL), to evoke neuroprotection.. Assays of PPAR alpha occupancy and gene transactivation potential were conducted in cell-free and transfected HeLa cell preparations, respectively. In vivo estimates of PPAR alpha activation through fat mobilization and gene transcription were conducted in mice. Neuroprotection in vivo was investigated in wild-type and PPAR alpha gene-disrupted mice.. The ECLs OEA, anandamide, noladin ether and virodhamine were found to bind to the purified PPAR alpha ligand binding domain and to increase PPAR alpha-driven transcriptional activity. The high affinity synthetic CB(1/2) cannabinoid agonist WIN 55212-2 bound to PPAR alpha equipotently with the PPARalpha agonist fenofibrate, and stimulated PPARalpha-mediated gene transcription. The phytocannabinoid delta 9 tetrahydrocannabinol was without effect. OEA and WIN 55212-2 induced lipolysis in vivo, while OEA pre-treatment reduced infarct volume from middle cerebral artery occlusion in wild-type, but not in PPAR alpha-null mice. OEA treatment also led to increased expression of the NFkappa B-inhibitory protein, Ikappa B, in mouse cerebral cortex, while expression of the NFkappa B-regulated protein COX-2 was inhibited.. These data demonstrate the potential for a range of cannabinoid compounds, of diverse structures, to activate PPAR alpha and suggest that at least some of the neuroprotective properties of these agents could be mediated by nuclear receptor activation.

Topics: Animals; Arachidonic Acids; Benzoxazines; Cannabinoids; Cerebral Cortex; Cyclooxygenase 2; Dronabinol; Endocannabinoids; Fatty Acids, Unsaturated; Fenofibrate; HeLa Cells; Humans; I-kappa B Kinase; Ligands; Luciferases; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Morpholines; Naphthalenes; Neuroprotective Agents; Oleic Acids; Polyunsaturated Alkamides; PPAR alpha; Protein Binding; Recombinant Fusion Proteins; Transfection