Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy.
Department of Biomolecular Sciences, University of Urbino ⿿Carlo Bo⿿, Urbino, Italy.
Pharmacol Res. 2016 Sep;111:600-609. doi: 10.1016/j.phrs.2016.07.009. Epub 2016 Jul 18.
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.
食欲素 1（OX-1R）和大麻素受体（CB1R）属于 G 蛋白偶联受体（GPCR）超家族，分别主要与 Gq 和 Gi/o 蛋白偶联。在过表达 OX-1R 和 CB1R 的宿主细胞中的体外研究表明，这些受体之间存在功能相互作用，通过形成异源二聚体或通过 OX-1R 触发内源性 CB1R 配体 2-花生四烯酸甘油（2-AG）的生物合成。由于：i）在参与能量稳态调节的下丘脑回路的突触后部位已描述了 OX-1R 和 CB1R 的共表达，并且 ii）在肥胖期间下丘脑神经元中出现增加的食欲素-A（OX-A）和 2-AG 水平，我们在这里研究了在胚胎小鼠下丘脑 NPY/AgRP mHypoE-N41 神经元中 OX-1R/CB1R 相互作用，该神经元组成型表达这两种受体。用 OX-A（0.1-0.3μM）处理 mHypoE-N41 细胞，但不用选择性 CB1R 激动剂花生四烯酸 2-氯乙基酰胺（ACEA；0.1-0.3μM），可瞬时升高 [Ca(2+)]i。用亚效剂量的 OX-A（0.1μM）+ACEA（0.1μM）孵育可导致 [Ca(2+)]i 更强和更长时间的升高，分别用 OX-1R 或 CB1R 拮抗剂 SB-334867 或 AM251 拮抗。FRET 和共免疫沉淀实验表明，在用 OX-A（0.2μM）或 OX-A（0.1μM）+ACEA（0.1μM）孵育后形成 OX-1R/CB1R 异源二聚体，但在用 ACEA（0.2μM）孵育后未形成，这可被 SB-334867 或 AM251 拮抗。OX-A（0.2μM）或 OX-A（0.1μM）+ACEA（0.1μM）也导致 2-AG 生物合成。最后，与基础水平或每种激动剂（0.1-0.2μM）单独作用相比，用 OX-A（0.1μM）+ACEA（0.1μM）孵育诱导更强的 ERK1/2（Thr202/185）磷酸化激活，再次通过 OX-1R 或 CB1R 拮抗剂预防。我们认为，OX-A 单独在有效浓度下作用于 [Ca(2+)]i，或在亚效浓度下与 ACEA 联合作用，通过形成 OX-1R/CB1R 异源二聚体和由 2-AG 介导的自分泌环触发细胞内信号事件。
