Hypoxia Induces Internalization of κ-Opioid Receptor

Abstract

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.

Herkinorin negatively regulates NLRP3 inflammasome to alleviate neuronal ischemic injury through activating Mu opioid receptor and inhibiting the NF‐κB pathway

“…17 KOR-related cerebral I/H injury studies showed 4 h OGD can damage KOR recirculation, reducing the total number of KOR and leading to receptor downregulation. 18 Our study found that both MOR inhibitor F I G U R E 2 Effects of Herkinorin and MOR inhibitor, β-funaltrexamine on NLRP3 inflammasome in rat primary neurons exposed to OGD/R. (A) The typical Western blot results showed the changes in the expression of pro-IL1β, IL-1β, pro-Caspase1, Caspase1 p20, and NLRP3 in rat primary neurons.…”

“…It was also found that the delayed brain protective effect of morphine was found in hippocampal slices, and was caused by the activation of MOR 17 . KOR‐related cerebral I/H injury studies showed 4 h OGD can damage KOR recirculation, reducing the total number of KOR and leading to receptor downregulation 18 . Our study found that both MOR inhibitor and MOR knockdown reversed the effects of Herkinorin downregulating NLRP3 to alleviate cerebral ischemic injury, suggesting that MOR plays an important role in Herkinorin neuroprotection.…”

Technical Feasibility and Physiological Relevance of Hypoxic Cell Culture Models

“…In vitro hypoxia may also be studied to uncover the molecular mechanisms ameliorating ischemia/reperfusion injury in neurons (118) or cardiomyocytes (119). A perfusion-based model of murine cardiomyocytes subjected to abrupt anoxia and reperfusion was discovered to be an optimal platform for demonstrating the opening of mitochondrial permeability transition pores (mPTP).…”

Opioids in cancer: The κ‑opioid receptor (Review)

“…Hypoxic tumor cells secrete VEGF and lactate, as well as a number of cytokines that modulate the tumor microenvironment, which increases their viability ( 160 , 161 ). The expression levels of KOR and hypoxia-inducible factor 1α (HIF-1α) are significantly increased in live human neurons following 24-h hypoxia ( 162 ). HIF-1α knockdown decreases KOR expression ( 162 ).…”

“…The expression levels of KOR and hypoxia-inducible factor 1α (HIF-1α) are significantly increased in live human neurons following 24-h hypoxia ( 162 ). HIF-1α knockdown decreases KOR expression ( 162 ). Thus, during hypoxia, KOR promotes cell viability and its expression may be regulated by HIF-1α ( 163 ).…”