Laboratory of Cellular and Molecular Pharmacology, Section of Neurosciences, Department of Biomedical Sciences, University of Cagliari, Cittadella Universitaria di Monserrato, 09042, Monserrato, CA, Italy.
Neurochem Res. 2018 Feb;43(2):245-258. doi: 10.1007/s11064-017-2410-x. Epub 2017 Oct 9.
The 5'-adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of the cellular energy metabolism and may induce either cell survival or death. We previously reported that in SH-SY5Y human neuroblastoma cells stimulation of muscarinic acetylcholine receptors (mAChRs) activate AMPK by triggering store-operated Ca entry (SOCE). However, whether mAChRs may control AMPK activity by regulating additional mechanisms beyond SOCE remains to be investigated. In the present study we examined the effects of mAChRs on AMPK when SOCE was induced by the sarco-endoplasmic reticulum Ca-ATPase inhibitor thapsigargin. We found that in SH-SY5Y cells depleted of Ca by thapsigargin, the re-addition Ca to the medium stimulated AMPK phosphorylation at Thr172, which is required for full kinase activity. This response occurred through SOCE, as it was blocked by either the SOCE modulator 2-aminoethoxydiphephenyl borate, knockdown of the SOCE molecular component STIM1, or inhibition of Ca/calmodulin (CaM)-dependent protein kinase kinase β (CaMKKβ). In thapsigargin-pretreated cells, stimulation of pharmacologically defined M mAChRs potentiated SOCE-induced AMPK activation. This potentiation did not involve an increased Ca influx, but was associated with CaM mobilization from membrane to cytosol, increased CaM/CaMKKβ interaction, and enhanced CaMKK stimulation by thapsigargin-induced SOCE. In thapsigargin-pretreated cells Ca re-addition stimulated glucose uptake and increased the membrane expression of the glucose transporter GLUT1. Both responses were significantly potentiated by mAChRs. These data indicate that in human neuroblastoma cells mAChRs up-regulate AMPK and the downstream glucose uptake by triggering not only SOCE but also CaM translocation and enhanced formation of active CaM/CaMKKβ complexes.
5'- 腺苷一磷酸激活蛋白激酶（AMPK）是细胞能量代谢的关键调节因子，可诱导细胞存活或死亡。我们之前的研究报告表明，在 SH-SY5Y 人神经母细胞瘤细胞中，刺激毒蕈碱乙酰胆碱受体（mAChR）通过触发储存操纵的钙内流（SOCE）激活 AMPK。然而，mAChR 是否可以通过调节 SOCE 以外的其他机制来控制 AMPK 活性仍有待研究。在本研究中，我们研究了 mAChR 在毒蕈碱乙酰胆碱受体激动剂 thapsigargin 诱导的 SOCE 时对 AMPK 的影响。我们发现，在 thapsigargin 耗尽 Ca 的 SH-SY5Y 细胞中，向培养基中重新添加 Ca 可刺激 AMPK 在 Thr172 处磷酸化，这是充分激酶活性所必需的。这种反应是通过 SOCE 发生的，因为它被 SOCE 调节剂 2-氨基乙氧基二苯硼酸盐、SOCE 分子成分 STIM1 的敲低或钙/钙调蛋白（CaM）依赖性蛋白激酶激酶 β（CaMKKβ）的抑制所阻断。在 thapsigargin 预处理的细胞中，药理学定义的 M mAChR 的刺激增强了 SOCE 诱导的 AMPK 激活。这种增强不涉及增加 Ca 内流，而是与 CaM 从膜向细胞质易位、增加 CaM/CaMKKβ 相互作用以及 thapsigargin 诱导的 SOCE 增强 CaMKK 刺激有关。在 thapsigargin 预处理的细胞中，Ca 再添加刺激葡萄糖摄取并增加葡萄糖转运蛋白 GLUT1 的膜表达。这两种反应都被 mAChR 显著增强。这些数据表明，在人神经母细胞瘤细胞中，mAChR 通过触发不仅 SOCE，而且还通过 CaM 易位和增强形成活性 CaM/CaMKKβ 复合物来上调 AMPK 和下游葡萄糖摄取。
