Hypoxia Induces Internalization of κ-Opioid Receptor.
Hypoxia Induces Internalization of κ-Opioid Receptor.
复制标题
缺氧诱导γ-阿片受体的内化。
DOI：
10.1097/aln.0000000000001571
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发表时间：
2017
期刊：
影响因子：
8.8
通讯作者：
Liu,Renyu
中科院分区：
文献类型：
--
作者：
Xi,Chunhua;Liang,Xuan;Chen,Chunhua;Babazada,Hasan;Li,Tianzuo;Liu,Renyu
研究背景kappa阿片受体（KOR）激动剂可以减轻动物模型缺氧缺血（HI）脑损伤。然而，目前尚不清楚KOR对HI的反应。在本研究中，我们采用体外氧葡萄糖剥夺（OGD）和再氧合模型来探讨KORs对缺氧和再氧合的反应。方法小鼠神经母细胞瘤Neuro2A细胞稳定转染mKOR- tdtomato融合蛋白（N2A-mKOR-tdT）或flag标记的mKOR (N2A-mKOR-FLAG)，并分为若干组（n=6 ~ 12），观察其KOR运动情况。在正常氧条件下，OGD后30分钟至1小时，以及再氧合后1小时，使用高分辨率成像技术，包括免疫电镜，在存在和不存在KOR拮抗剂、动力蛋白抑制剂、钾通道阻滞剂和多巴胺受体抑制剂的情况下进行观察。结果缺氧可使KOR内化到细胞内。dygo -4a对dynamin的抑制阻止了受体的内化。有趣的是，一种特异性的KOR拮抗剂norbinaltorphimine阻断了内化，表明特异性的KOR激活参与。再氧化可逆转KOR内化。免疫电镜显示，OGD后细胞内与kor相关的金颗粒的数量从37%增加到85% （P < 0.01）。钾通道阻滞剂和多巴胺受体抑制剂未能阻断缺氧诱导的KOR内化。结论低氧诱导的KOR内化是可逆的，可被选择性KOR拮抗剂或动力蛋白抑制剂抑制，可通过神经母细胞瘤细胞的再氧化逆转，表明低氧对KOR的调节作用是通过KOR激活和动力蛋白依赖机制实现的。
Background It has been demonstrated that kappa opioid receptor (KOR) agonists can reduce hypoxia–ischemia (HI) brain injury in animal models. However, it is unclear how KOR responds to HI. In the current study, we used in vitro model of oxygen-glucose deprivation (OGD) and re-oxygenation to explore how KORs respond to hypoxia and re-oxygenation. Methods Mouse neuroblastoma Neuro2A cells stably transfected with mKOR–tdTomato fusion protein (N2A-mKOR-tdT) or Flag-tagged mKOR (N2A-mKOR-FLAG) and divided into several groups (n=6 to 12), and were used to investigate the KOR movement. Observations were performed under normal oxygen, at 30 min to 1 h after OGD, and at 1 h after re-oxygenation using high resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of KOR antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor. Results Hypoxic conditions caused KOR to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific KOR antagonist norbinaltorphimine blocked internalization, suggesting a specific KOR activation involvement. KOR internalization appears to be reversed by re-oxygenation. Quantities of intracellular KOR-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 % to 85% (P < 0.01) after OGD. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia induced KOR internalization. Conclusion Hypoxia induces reversible KOR internalization, which was inhibited by selective KOR antagonists or dynamin inhibitor, and can be reversed by re-oxygenation in neuroblastoma cells, indicating the modulating effects between KOR and hypoxia via KOR activation and dynamin dependent mechanism.