arachidonoylserotonin and palmidrol

Regenerative activity in tissues of mesenchymal origin depends on the migratory potential of mesenchymal stem cells (MSCs). The present study focused on inhibitors of the enzyme fatty acid amide hydrolase (FAAH), which catalyzes the degradation of endocannabinoids (anandamide, 2-arachidonoylglycerol) and endocannabinoid-like substances (N-oleoylethanolamine, N-palmitoylethanolamine). Boyden chamber assays, the FAAH inhibitors, URB597 and arachidonoyl serotonin (AA-5HT), were found to increase the migration of human adipose-derived MSCs. LC-MS analyses revealed increased levels of all four aforementioned FAAH substrates in MSCs incubated with either FAAH inhibitor. Following addition to MSCs, all FAAH substrates mimicked the promigratory action of FAAH inhibitors. Promigratory effects of FAAH inhibitors and substrates were causally linked to activation of p42/44 MAPKs, as well as to cytosol-to-nucleus translocation of the transcription factor, PPARα. Whereas PPARα activation by FAAH inhibitors and substrates became reversed upon inhibition of p42/44 MAPK activation, a blockade of PPARα left p42/44 MAPK phosphorylation unaltered. Collectively, these data demonstrate FAAH inhibitors and substrates to cause p42/44 MAPK phosphorylation, which subsequently activates PPARα to confer increased migration of MSCs. This novel pathway may be involved in regenerative effects of endocannabinoids whose degradation could be a target of pharmacological intervention by FAAH inhibitors.

Topics: Adipose Tissue; Amides; Amidohydrolases; Arachidonic Acids; Benzamides; Carbamates; Cell Movement; Cells, Cultured; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Glycerides; Humans; Mesenchymal Stem Cells; Oleic Acids; Palmitic Acids; Polyunsaturated Alkamides; PPAR alpha; Receptor, Cannabinoid, CB1; Serotonin

The endocannabinoids anandamide and 2-arachidonoylglycerol, and the anandamide-congener, palmitoylethanolamide, are all substrates for the enzyme fatty acid amide hydrolase, and are endowed with anti-inflammatory actions exerted via cannabinoid receptors or, in the case of palmitoylethanolamide, also via other targets. We investigated the role of the endocannabinoid system during granuloma formation, a model of chronic inflammation sustained by neoangiogenesis, in rats. Granuloma was induced by subcutaneous lambda-carrageenin-soaked sponge implants on the back of male Wistar rats. After 96h, granulomas were detached and tissue formation was evaluated as wet weight; the endocannabinoid system was evaluated by the measurement of endocannabinoid levels, by LC-MS, and of cannabinoid receptor expression, by western blot analysis. Moreover, angiogenesis was evaluated by the measurement of both hemoglobin content and CD31 protein expression. Arachidonoylserotonin (AA-5-HT, 12.5-50mug/ml), an inhibitor of FAAH, and palmitoylethanolamide (PEA, 200-800mug/ml) were given locally only once at the time of implantation. Granuloma formation was accompanied by a significant decrease in endocannabinoid and palmitoylethanolamide levels paralleled by increased levels of the fatty acid amide hydrolase, responsible for their breakdown. Moreover, an increase of cannabinoid receptor expression was also observed. Pharmacological elevation of endocannabinoids and palmitoylethanolamide, obtained separately by arachidonoylserotonin and exogenous palmitoylethanolamide treatment, dose-dependently reduced inflammatory hallmarks including tumor necrosis factor-alpha as well as granuloma-dependent angiogenesis. The effect of arachidonoylserotonin was accompanied by near-normalization of 2-arachidonoylglycerol and palmitoylethanolamide levels in the tissue. These findings suggest that chronic inflammation might develop also because of endocannabinoid and palmitoylethanolamide tissue concentration impairment, the correction of which might be exploited to develop new anti-inflammatory drugs.

Topics: Amides; Amidohydrolases; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Carrageenan; Chronic Disease; Disease Models, Animal; Dose-Response Relationship, Drug; Endocannabinoids; Ethanolamines; Granuloma; Hemoglobins; Inflammation; Male; Neovascularization, Pathologic; Palmitic Acids; Phospholipase D; Platelet Endothelial Cell Adhesion Molecule-1; Rats; Rats, Wistar; Receptors, Cannabinoid; Serotonin; TRPV Cation Channels

We evaluated the effects of intra-periaqueductal grey (PAG) N-arachidonoyl-serotonin (AA-5-HT), a compound with a "dual" ability to inhibit the fatty acid amide hydrolase (FAAH) and to antagonize transient receptor vanilloid type 1 (TRPV1) receptors, on endocannabinoid levels, rostral ventromedial medulla (RVM) ON and OFF cell activities, thermal nociception (tail flick in anaesthetized rats) and formalin-induced nocifensive responses in awake rats. AA-5-HT increased endocannabinoid levels in the PAG and induced analgesia. Paradoxically, it also depressed the RVM OFF cell, as well as the ON cell activities. The effect of AA-5-HT was mimicked by co-injecting the selective FAAH inhibitor URB597 and the selective TRPV1 antagonist I-RTX into the PAG, which also induced analgesia and inhibition of ON and OFF cell ongoing activities. The recruitment of "alternative" pathways, such as PAG-locus coeruleus (LC)-spinal cord might be responsible for AA-5-HT effect since we found evidence that (i) intra-PAG AA-5-HT increased LC neuron firing activities, and (ii) intrathecal phentolamine or ketanserin prevented the analgesic effect of AA-5-HT. Moreover, intra-PAG AA-5-HT prevented the changes in the ON and OFF cells firing activity induced by intra-paw formalin, and it inverted the formalin-induced increase in LC adrenergic cell activity. All AA-5-HT effects were antagonized by cannabinoid CB1 and TRPV1 receptor antagonists thus suggesting that co-localization of these receptors in the PAG can be an appropriate neural substrate for AA-5-HT-induced analgesia.

Topics: Adrenergic Antagonists; Amides; Amidohydrolases; Analgesics; Animals; Arachidonic Acids; Electrophysiology; Endocannabinoids; Ethanolamines; Extracellular Space; Formaldehyde; Locus Coeruleus; Male; Medulla Oblongata; Microinjections; Oleic Acids; Pain Measurement; Palmitic Acids; Rats; Reaction Time; Serotonin; Serotonin Antagonists; TRPV Cation Channels

N-arachidonoyl-serotonin (AA-5-HT) is an inhibitor of fatty acid amide hydrolase (FAAH)-catalysed hydrolysis of the endocannabinoid/ endovanilloid compound, anandamide (AEA). We investigated if AA-5-HT antagonizes the transient receptor potential vanilloid-1 (TRPV1) channel and, as FAAH and TRPV1 are targets for analgesic compounds, if it exerts analgesia in rodent models of hyperalgesia.. AA-5-HT was tested in vitro, on HEK-293 cells overexpressing the human or the rat recombinant TRPV1 receptor, and in vivo, in rats and mice treated with formalin and in rats with chronic constriction injury of the sciatic nerve. The levels of the endocannabinoids, AEA and 2-arachidonoylglycerol, in supraspinal (periaqueductal grey, rostral ventromedial medulla), spinal or peripheral (skin) tissues were measured.. AA-5-HT behaved as an antagonist at both rat and human TRPV1 receptors (IC(50)=37-40 nM against 100 nM capsaicin). It exerted strong analgesic activity in all pain models used here. This activity was partly due to FAAH inhibition, elevation of AEA tissue levels and indirect activation of cannabinoid CB(1) receptors, as it was reversed by AM251, a CB(1) antagonist. AA-5-HT also appeared to act either via activation/desensitization of TRPV1, following elevation of AEA, or as a direct TRPV1 antagonist, as suggested by the fact that its effects were either reversed by capsazepine and 5'-iodo-resiniferatoxin, two TRPV1 antagonists, or mimicked by these compounds administered alone.. Possibly due to its dual activity as a FAAH inhibitor and TRPV1 antagonist, AA-5-HT was highly effective against both acute and chronic peripheral pain.

Topics: Amides; Amidohydrolases; Analgesics, Non-Narcotic; Animals; Arachidonic Acids; Cannabinoid Receptor Modulators; Cell Line; Endocannabinoids; Enzyme Inhibitors; Ethanolamines; Injections, Subcutaneous; Male; Mice; Pain Measurement; Palmitic Acids; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Recombinant Proteins; Serotonin; TRPV Cation Channels