1型大麻素受体(CB1R)介导中枢神经体系神经递质释放和突触可塑性。内源性,植物衍生的,合成大麻素与CB1R结合,启动抑制性G蛋白(Gi)和β-抑制蛋白信号通路。在Gi信号通路中,CB1R激活G蛋白门控,向内整流钾(GIRK)通道。β-抑制蛋白途径通过受体内化减少细胞表面的CB1R表达。由于它们与镇痛和药物耐受性有关,GIRK通道和受体内化对药物的开发具有重要意义。这项研究使用了具有pH敏感性的永生化小鼠垂体细胞,荧光标记的人CB1R(AtT20-SEPCB1)以测量GIRK通道活性和CB1R内化。通过使用荧光膜电位敏感染料测量大麻素诱导的GIRK通道活性。我们开发了一种动力学成像测定法,可可视化和测量CB1R内化。所有大麻素刺激GIRK通道反应的排序效能为WIN55,212-2>(±)CP55,940>Δ9-THC>AEA。功效相对于(±)CP55,940表示,其排序功效为(±)CP55,940>WIN55,212-2>AEA>Δ9-THC。所有大麻素均以(±)CP55,940>WIN55,212-2>AEA>Δ9-THC的等级顺序刺激CB1R内化。内化功效归一化为(±)CP55,940,排序功效为WIN55,212-2>AEA>(±)CP55,940>Δ9-THC。(±)CP55,940在刺激GIRK通道反应方面比AEA和Δ9-THC显着更有效和有效;CB1R内化在效力和功效之间没有观察到显着差异。比较大麻素的GIRK通道和CB1R内化反应时,没有发现显着差异。总之,AtT20-SEPCB1细胞可用于评估大麻素诱导的CB1R内化。虽然大麻素显示差异Gi信号时,彼此比较,这并没有扩展到CB1R内化.
The type 1 cannabinoid receptor (CB1R) mediates neurotransmitter release and synaptic plasticity in the central nervous system. Endogenous, plant-derived, synthetic cannabinoids bind to CB1R, initiating the inhibitory G-protein (Gi) and the β-arrestin signaling pathways. Within the Gi signaling pathway, CB1R activates G protein-gated, inwardly-rectifying potassium (GIRK) channels. The β-arrestin pathway reduces CB1R expression on the cell surface through receptor internalization. Because of their association with analgesia and drug tolerance, GIRK channels and receptor internalization are of interest to the development of pharmaceuticals. This research used immortalized mouse pituitary gland cells transduced with a pH-sensitive, fluorescently-tagged human CB1R (AtT20-SEPCB1) to measure GIRK channel activity and CB1R internalization. Cannabinoid-induced GIRK channel activity is measured by using a fluorescent membrane-potential sensitive dye. We developed a kinetic imaging assay that visualizes and measures CB1R internalization. All cannabinoids stimulated a GIRK channel response with a rank order potency of WIN55,212-2 > (±)CP55,940 > Δ9-THC > AEA. Efficacy was expressed relative to (±)CP55,940 with a rank order efficacy of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. All cannabinoids stimulated CB1R internalization with a rank order potency of (±)CP55,940 > WIN55, 212-2 > AEA > Δ9-THC. Internalization efficacy was normalized to (±)CP55,940 with a rank order efficacy of WIN55,212-2 > AEA > (±)CP55,940 > Δ9-THC. (±)CP55,940 was significantly more potent and efficacious than AEA and Δ9-THC at stimulating a GIRK channel response; no significant differences between potency and efficacy were observed with CB1R internalization. No significant differences were found when comparing a cannabinoid\'s GIRK channel and CB1R internalization response. In conclusion, AtT20-SEPCB1 cells can be used to assess cannabinoid-induced CB1R internalization. While cannabinoids display differential Gi signaling when compared to each other, this did not extend to CB1R internalization.