title: Formation of OX-1R/CB1R heteromeric complexes in embryonic mouse hypothalamic cells: Effect on intracellular calcium, 2-arachidonoyl-glycerol biosynthesis and ERK phosphorylation.
abstract: Orexin 1 (OX-1R) and cannabinoid receptor (CB1R) belong to the superfamily of G-protein-coupled receptors (GPCRs) and are mostly coupled to Gq and Gi/o proteins, respectively. In vitro studies in host cells over-expressing OX-1R and CB1R revealed a functional interaction between these receptors, through either their ability to form heteromers or the property for OX-1R to trigger the biosynthesis of 2-arachidonoylglycerol (2-AG), an endogenous CB1R ligand. Since: i) OX-1R and CB1R co-espression has been described at postsynaptc sites in hypothalamic circuits involved the regulation of energy homeostasis, and ii) increased orexin-A (OX-A) and 2-AG levels occur in hypothalamic neurons during obesity, we sought here to investigate the OX-1R/CB1R interaction in embryonic mouse hypothalamic NPY/AgRP mHypoE-N41 neurons which express, constitutively, both receptors. Treatment of mHypoE-N41 cells with OX-A (0.1-0.3μM), but not with the selective CB1R agonist, arachidonyl-2-chloroethylamide (ACEA; 0.1-0.3μM), transiently elevated [Ca(2+)]i. Incubation with a subeffective dose of OX-A (0.1μM)+ACEA (0.1μM) led to stronger and longer lasting elevation of [Ca(2+)]i, antagonized by OX-1R or CB1R antagonism with SB-334867 or AM251, respectively. FRET and co-immunoprecipitation experiments showed the formation of OX-1R/CB1R heteromers after incubation with OX-A (0.2μM), or OX-A (0.1μM)+ACEA (0.1μM), but not after ACEA (0.2μM), in a manner antagonized by SB-334867 or AM251. OX-A (0.2μM) or OX-A (0.1μM)+ACEA (0.1μM) also led to 2-AG biosynthesis. Finally, a stronger activation of ERK1/2(Thr202/185) phosphorylation in comparison to basal or each agonist alone (0.1-0.2μM), was induced by incubation with OX-A (0.1μM)+ACEA (0.1μM), again in a manner prevented by OX-1R or CB1R antagonism. We suggest that OX-A, alone at effective concentrations on [Ca(2+)]i, or in combination with ACEA, at subeffective concentrations, triggers intracellular signaling events via the formation of OX-1R/CB1R heteromers and an autocrine loop mediated by 2-AG.
authors.affiliation: Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Imperatore
authors.affiliation: Department of Biomolecular Sciences, University of Urbino ⿿Carlo Bo⿿, Urbino, Italy.
authors.forename: Letizia
authors.initials: L
authors.lastname: Palomba
authors.affiliation: Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Morello
authors.affiliation: Institute of Experimental and Clinical Pharmacology and Toxicology, University of Luebeck, Luebeck, Germany.
authors.forename: Alessandro Di
authors.initials: AD
authors.lastname: Spiezio
authors.affiliation: Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy.
authors.forename: Fabiana
authors.initials: F
authors.lastname: Piscitelli
authors.affiliation: Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy.
authors.forename: Vincenzo Di
authors.initials: VD
authors.lastname: Marzo
authors.affiliation: Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy. Electronic address: luigia.cristino@icb.cnr.it.
authors.forename: Luigia
authors.initials: L
authors.lastname: Cristino
mesh_terms: D000818:Animals; D001095:Arachidonic Acids / Q000096:biosynthesis / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D063388:Endocannabinoids / Q000096:biosynthesis; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005989:Glycerides / Q000096:biosynthesis; D007031:Hypothalamus / Q000166:cytology*; D051379:Mice; D064446:Orexin Receptors / Q000378:metabolism*; D000068797:Orexins / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D043884:Receptor, Cannabinoid, CB1 / Q000378:metabolism*
chemical_list: D001095:Arachidonic Acids; D063388:Endocannabinoids; D005989:Glycerides; C497459:Hcrtr1 protein, mouse; D064446:Orexin Receptors; D000068797:Orexins; D043884:Receptor, Cannabinoid, CB1; C119325:arachidonyl-2-chloroethylamide; C094503:glyceryl 2-arachidonate; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium
keywords: 2-Arachidonoylglycerol; ACEA (PubChem CID: 5311006); AM251 (PubChem CID: 2125); Calcium imaging; Cannabinoid receptor type 1; Fluorescence resonance energy transfer; Orexin-A (PubChem CID: 56842143); Orexin-A/hypocretin 1; SB-334867 (PubChem CID: 6604926)