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 μM), but not with the selective CB1R agonist, arachidonyl-2-chloroethylamide (ACEA; 0.1-0.3 μM), transiently elevated [Ca2+]i. Incubation with a subeffective dose of OX-A (0.1 μM) + ACEA (0.1 μM) 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 μM), or OX-A (0.1 μM) + ACEA (0.1 μM), but not after ACEA (0.2 μM), in a manner antagonized by SB-334867 or AM251. OX-A (0.2 μM) or OX-A (0.1 μM) + ACEA (0.1 μM) also led to 2-AG biosynthesis. Finally, a stronger activation of ERK1/2Thr202/185 phosphorylation in comparison to basal or each agonist alone (0.1-0.2 μM), was induced by incubation with OX-A (0.1 μM) + ACEA (0.1 μM), 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.

Graphical abstractmHypoE-N41 cells express both CB1R and OX-1R coupled to Gi/o and Gq, respectively. OX-A + ACEA at concentrations otherwise ineffective per se (0.1 mM) induce a prolonged strong increase of [Ca2+]i possibly because of CB1R/OX-1R heteromerization and Gq-mediated activation of: 1) non-selective Ca2+ operated channels (i.e. voltage-gated Ca2+ channels, a reverse-acting Na+/Ca2+ exchanger, store-operated Ca2+ channels or non-selective cation channels); 2) PLCÿ, with consequent production of IP3 and DAG. IP3 enhances the [Ca2+]i following store-operated Ca2+ release. DAG is the DAGLα substrate and the precursor of 2-AG. Finally, upon stimulation with OX-A (0.1 μM) + ACEA (0.1 μM) ERK1/2 stimulation goes up to ⿼3 or 5 fold more than baseline levels or when only OX-1R is activated, respectively.In conclusion, OX-A in combination with ACEA, each at subeffective concentrations on [Ca2+]i, triggers stronger intracellular signaling events via the formation of OX-1R/CB1R heteromers and an autocrine loop mediated by 2-AG.Download high-res image (122KB)Download full-size image