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

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• 作者：Roberta Imperatore^a ; Letizia Palomba^b ; Giovanna Morello^a ; Alessandro Di Spiezio^c ; Fabiana Piscitelli^a ; Vincenzo Di Marzo^a ; Luigia Cristino^a ; ^{luigia.cristino@icb.cnr.it" class="auth_mail" title="E-mail the corresponding author}
• 关键词：2-AG ; 2-arachidonoylglycerol ; ACEA ; arachidonyl-2-chloroethylamide ; AgRP ; agouti-related peptide ; CB1R ; cannabinoid receptor ; CHO ; chinese hamster ovary cells ; DAG ; diacylglycerol ; DAGL ; diacylglycerol lipase ; ERK ; extracellular-signal-regulated kinases ; FRET ; fluorescence resonance energy transfer ; GPCRs ; G protein coupled receptors ; IP3 ; inositol trisphosphate ; NPY ; neuropeptide Y ; OX-A ; orexin-A ; OX-1R ; orexin 1 receptor ; PLC ; phospholipase C
• 刊名：Pharmacological Research
• 出版年：2016
• 出版时间：September 2016
• 年：2016
• 卷：111
• 期：Complete
• 页码：600-609
• 全文大小：2899 K

文摘

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 ERK1/2^Thr202/185 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.