Anesthesiology · 2017
Hypoxia Induces Internalization of κ-Opioid Receptor.
СтатьяПоддержка NIH: внешняя программаНазвание и аннотация: Перевод в очереди
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Xi Chunhua, Liang Xuan, Chen Chunhua, Babazada Hasan, Li Tianzuo, Liu Renyu
Аннотация
BACKGROUND: It has been demonstrated that κ-opioid receptor agonists can reduce hypoxia-ischemia brain injury in animal models. However, it is unclear how the κ-opioid receptor responds to hypoxia-ischemia. In the current study, the authors used an in vitro model of oxygen-glucose deprivation and reoxygenation to explore how κ-opioid receptors respond to hypoxia and reoxygenation.
METHODS: Mouse neuroblastoma Neuro2A cells were stably transfected with mouse κ-opioid receptor-tdTomato fusion protein or Flag-tagged mouse κ-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the κ-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen-glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of κ-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor.
RESULTS: Hypoxic conditions caused the κ-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific κ-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific κ-opioid receptor. κ-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular κ-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P < 0.01) after oxygen-glucose deprivation. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia-induced κ-opioid receptor internalization.
CONCLUSIONS: Hypoxia induces reversible κ-opioid receptor internalization, which was inhibited by selective κ-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between κ-opioid receptor and hypoxia via κ-opioid receptor activation and the dynamin-dependent mechanism.
METHODS: Mouse neuroblastoma Neuro2A cells were stably transfected with mouse κ-opioid receptor-tdTomato fusion protein or Flag-tagged mouse κ-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the κ-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen-glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of κ-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor.
RESULTS: Hypoxic conditions caused the κ-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific κ-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific κ-opioid receptor. κ-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular κ-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P < 0.01) after oxygen-glucose deprivation. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia-induced κ-opioid receptor internalization.
CONCLUSIONS: Hypoxia induces reversible κ-opioid receptor internalization, which was inhibited by selective κ-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between κ-opioid receptor and hypoxia via κ-opioid receptor activation and the dynamin-dependent mechanism.