PT - JOURNAL ARTICLE AU - Pauli M. Turunen AU - Maria H. Jäntti AU - Jyrki P. Kukkonen TI - OX<sub>1</sub> Orexin/Hypocretin Receptor Signaling through Arachidonic Acid and Endocannabinoid Release AID - 10.1124/mol.112.078063 DP - 2012 Aug 01 TA - Molecular Pharmacology PG - 156--167 VI - 82 IP - 2 4099 - http://molpharm.aspetjournals.org/content/82/2/156.short 4100 - http://molpharm.aspetjournals.org/content/82/2/156.full SO - Mol Pharmacol2012 Aug 01; 82 AB - We showed previously that OX1 orexin receptor stimulation produced a strong 3H overflow response from [3H]arachidonic acid (AA)-labeled cells. Here we addressed this issue with a novel set of tools and methods, to distinguish the enzyme pathways responsible for this response. CHO-K1 cells heterologously expressing human OX1 receptors were used as a model system. By using selective pharmacological inhibitors, we showed that, in orexin-A-stimulated cells, the AA-derived radioactivity was released as two distinct components, i.e., free AA and the endocannabinoid 2-arachidonoyl glycerol (2-AG). Two orexin-activated enzymatic cascades are responsible for this response: cytosolic phospholipase A2 (cPLA2) and diacylglycerol lipase; the former cascade is responsible for part of the AA release, whereas the latter is responsible for all of the 2-AG release and part of the AA release. Essentially only diacylglycerol released by phospholipase C but not by phospholipase D was implicated as a substrate for 2-AG production, although both phospholipases were strongly activated. The 2-AG released acted as a potent paracrine messenger through cannabinoid CB1 receptors in an artificial cell-cell communication assay that was developed. The cPLA2 cascade, in contrast, was involved in the activation of orexin receptor-operated Ca2+ influx. 2-AG was also released upon OX1 receptor stimulation in recombinant HEK-293 and neuro-2a cells. The results directly show, for the first time, that orexin receptors are able to generate potent endocannabinoid signals in addition to arachidonic acid signals, which may explain the proposed orexin-cannabinoid interactions (e.g., in neurons).