Formation of OX-1R/CB1R heteromeric complexes in embryonic mouse hypothalamic cells: Effect on intracellular calcium, 2-arachidonoyl-glycerol biosynthesis and ERK phosphorylation

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 (0.1-0.3 mu M), but not with the selective CB1R agonist, arachidonyl-2-chloroethylamide (ACEA; 0.1-0.3 mu M), transiently elevated [Ca2+](i). Incubation with a subeffective dose of OX-A (0.1 mu M) + ACEA (0.1 mu M) led to stronger and longer lasting elevation of [Ca2+](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 (0.2 mu M), or OX-A (0.1 mu M) + ACEA (0.1 mu M), but not after ACEA (0.2 mu M), in a manner antagonized by SB-334867 or AM251. OX-A (0.2 mu M) or OX-A (0.1 mu M) + ACEA (0.1 mu M) also led to 2-AG biosynthesis. Finally, a stronger activation of ERK1/2(Thr202/185) phosphorylation in comparison to basal or each agonist alone (0.1-0.2 mu M), was induced by incubation with OX-A (0.1 mu M) + ACEA (0.1 mu M), again in a manner prevented by OX-1R or CB1R antagonism. We suggest that OX-A, alone at effective concentrations on [Ca2+](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. (C) 2016 Elsevier Ltd. All rights reserved.