Functional characterization of α7 nicotinic acetylcholine and NMDA receptor signaling in SH-SY5Y neuroblastoma cells in an ERK phosphorylation assay
Functional characterization of α7 nicotinic acetylcholine and NMDA receptor signaling in SH-SY5Y neuroblastoma cells in an ERK phosphorylation assay
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DOI：
10.1016/j.ejphar.2018.02.047
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发表时间：
2018-05-05
影响因子：
5
通讯作者：
Jensen, Anders A.
中科院分区：
文献类型：
--
作者：
Elnagar, Mohamed R.;Walls, Anne Byriel;Jensen, Anders A.
在本研究中，α 7烟碱乙酰胆碱受体（α 7 nAChR）和N-甲基-D-天冬氨酸受体（NMDAR）内源性表达在SH-SY 5 Y人神经母细胞瘤细胞的功能特性的特点是在细胞外信号调节激酶（ERK）磷酸化试验。胆碱和N-甲基-D-天冬氨酸（NMDA）均介导SH-SY 5 Y细胞中ERK磷酸化的浓度依赖性增强，其EC 50值分别与重组α 7 nAChR和NMDAR激动剂的EC 50值一致。重要的是，胆碱（10 mM）和NMDA（50 μ M）引起的反应显着抑制α 7-选择性拮抗剂α-银环蛇毒素（100 nM）和NMDAR-选择性拮抗剂MK-801（50 μ M），分别。与两种激动剂单独产生的ERK磷酸化水平相比，在胆碱（1、3、10 mM）和NMDA（50 μ M）共同应用时观察到的ERK磷酸化水平增加与相加效应完全一致，并且没有揭示α 7 nAChR和NMDAR信号传导之间的实质性协同作用。然而，有趣的是，由“胆碱（10 mM）- NMDA（50 μ M）”组合引起的反应几乎完全被α-银环蛇毒素（100 nM）以及MK-801（50 μ M）抑制，这表明α 7 nAChR-和NMDAR-介导的ERK磷酸化之间存在某种联系。最后，寡聚淀粉样蛋白-β（1-42）肽（1000 nM）介导对胆碱（10 mM）、NMDA（50 μ M）和“胆碱（10 mM）- NMDA（50 μ M）”组合诱导的ERK磷酸化的强力抑制。总之，SH-SY 5 Y细胞中ERK磷酸化测量为研究α 7 nAChR和NMDAR介导的信号传导和受体之间假定的功能相互作用提供了可靠的测定。
In the present study, the functional properties of alpha 7 nicotinic acetylcholine receptors (alpha 7 nAChRs) and N-methyl-D-aspartate receptors (NMDARs) endogenously expressed in SH-SY5Y human neuroblastoma cells were characterized in an extracellular-signal regulated kinase (ERK) phosphorylation assay. Both choline and N-methyl-D-aspartate (NMDA) mediated robust concentration-dependent increases in ERK phosphorylation in the SH-SY5Y cells, exhibiting EC50 values in good agreement with those reported for the agonists at recombinant alpha 7 nAChRs and NMDARs, respectively. Importantly, the responses evoked by choline (10 mM) and by NMDA (50 mu M) were significantly inhibited by the alpha 7-selective antagonist alpha-bungarotoxin (100 nM) and by the NMDAR-selective antagonist MK-801 (50 mu M), respectively. The increased ERK phosphorylation levels observed upon co-application of choline (1, 3, 10 mM) and NMDA (50 mu M) compared to those produced by the two agonists on their own were fully reconcilable with additive effects and did not reveal substantial synergy between alpha 7 nAChR and NMDAR signaling. Interestingly, however, the responses evoked by the "choline (10 mM) - NMDA (50 mu M)" combination were almost completely inhibited by a-bungarotoxin (100 nM) as well as by MK-801 (50 mu M), suggesting some sort of a link between alpha 7 nAChR- and NMDAR-mediated ERK phosphorylation. Finally, oligomeric amyloid-beta(1-42) peptide (1000 nM) mediated robust inhibition of the ERK phosphorylation induced by choline (10 mM), NMDA (50 mu M) and the "choline (10 mM) - NMDA (50 mu M)" combination. In conclusion, ERK phosphorylation measurements in SH-SY5Y cells provides a robust assay for studies of alpha 7 nAChR-and NMDAR-mediating signaling and putative functional interactions between the receptors.