Department of Anesthesia, Kyoto University Hospital, Kyoto City, Japan.
Graduate School of Medicine, Department of Anesthesia, Kyoto University, Kyoto City, Japan.
Pharmacol Res Perspect. 2022 Apr;10(2):e00933. doi: 10.1002/prp2.933.
Long-term opioid use develops tolerance and attenuates analgesic effects. Upon activation, µ-opioid receptors (MOPs) are internalized and directed to either recycling or degradation pathway. Ligand stimulation also promotes de novo MOP synthesis. These processes collaboratively regulate MOP expression and play critical roles in tolerance development. However, there is limited understanding of how the endogenous MOP expression changes after prolonged opioid administration because previous analyses have focused on individual processes using overexpression systems, which ignored physiological regulation. Another fundamental problem is the unavailability of commercial antibodies to detect the low expression of endogenous MOP in neuronal systems. Here, we established a neuronal cell line to detect endogenous MOP with sufficient sensitivity using CRISPR/Cas9 technology. We incorporated the hemagglutinin sequence into the MOP gene of the SH-SY5Y cell. The genome-editing did not significantly impair MOP functions such as MOP internalization or the downstream signaling. The clone was differentiated into a state similar to the primary culture undergoing treatment with all-trans retinoic acid, followed by brain-derived neurotrophic factor. Upon continuous stimulation with MOP ligands, endogenous MOP constantly decreased up to 48 h. The expression level was maintained at a certain level following this period, depending on the ligand properties. DAMGO reduced MOP from the cell surface by about 70%, while morphine did so by 40%. Our results indicate that even a few days of opioid administration could significantly reduce the MOP expression level. Our cell line could be a potential tool to investigate the molecular mechanisms underlying the problems caused by long-term opioid use.
长期使用阿片类药物会产生耐受性，并减弱其镇痛效果。µ-阿片受体（MOPs）在被激活后会被内化，并被定向到再循环或降解途径。配体刺激还会促进新的 MOP 合成。这些过程共同调节 MOP 的表达，并在耐受发展中发挥关键作用。然而，由于之前的分析主要集中在使用过表达系统的单个过程上，而忽略了生理调节，因此对于长期阿片类药物给药后内源性 MOP 表达的变化，我们的了解有限。另一个基本问题是缺乏商业抗体来检测神经元系统中内源性 MOP 的低表达。在这里，我们使用 CRISPR/Cas9 技术建立了一种能够检测内源性 MOP 的神经元细胞系，其具有足够的灵敏度。我们将血凝素序列插入到 SH-SY5Y 细胞的 MOP 基因中。基因组编辑并没有显著损害 MOP 的功能，如 MOP 的内化或下游信号转导。该克隆被分化为类似于原代培养物的状态，经历全反式视黄酸和脑源性神经营养因子的处理。在持续受到 MOP 配体的刺激下，内源性 MOP 在 48 小时内不断减少。在此期间之后，表达水平会根据配体的性质维持在一定水平。DAMGO 通过约 70%的细胞表面减少 MOP，而吗啡则减少 40%。我们的结果表明，即使是几天的阿片类药物给药也可能显著降低 MOP 的表达水平。我们的细胞系可能是研究长期阿片类药物使用引起的问题的分子机制的潜在工具。
