Department of Anesthesiology and Critical Care, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.
Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
PLoS One. 2022 Feb 1;17(2):e0263395. doi: 10.1371/journal.pone.0263395. eCollection 2022.
Many anesthetics, including Propofol, have been reported to induce elevation of intracellular calcium, and we were interested to investigate the possible contribution of calcium elevation to the mechanism of the newly approved remimazolam actions. Remimazolam is an intravenous anesthetic first approved in Japan in July 2020, and is thought to exert its anesthetic actions via γ-aminobutyric acid A (GABAA) receptors; however, the precise mechanisms of how remimazolam elevates intracellular calcium levels remains unclear. We examined the remimazolam-induced elevation of intracellular calcium using SHSY-5Y neuroblastoma cells, COS-7 cells, HEK293 cells, HeLa cells, and human umbilical vein endothelial cells (HUVECs) loaded with fluorescent dyes for live imaging. We confirmed that high concentrations of remimazolam (greater than 300 μM) elevated intracellular calcium in a dose-dependent manner in these cells tested. This phenomenon was not influenced by elimination of extracellular calcium. The calcium elevation was abolished when intracellular or intraendoplasmic reticulum (ER) calcium was depleted by BAPTA-AM or thapsigargin, respectively, suggesting that calcium was mobilized from the ER. Inhibitors of G-protein coupled receptors (GPCRs)-mediated signals, including U-73122, a phospholipase C (PLC) inhibitor and xestospongin C, an inositol 1,4,5-triphosphate receptors (IP3R) antagonist, significantly suppressed remimazolam-induced calcium elevation, whereas dantrolene, a ryanodine receptor antagonist, did not influence remimazolam-induced calcium elevation. Meanwhile, live imaging of ER during remimazolam stimulation using ER-tracker showed no morphological changes. These results suggest that high doses of remimazolam increased intracellular calcium concentration in a dose-dependent manner in each cell tested, which was predicted to be caused by calcium mobilization from the ER. In addition, our studies using various inhibitors revealed that this calcium elevation might be mediated by the GPCRs-IP3 pathway. However, further studies are required to identify which type of GPCRs is involved.
许多麻醉剂，包括丙泊酚，已被报道能诱导细胞内钙离子升高，我们有兴趣研究钙离子升高对新批准的瑞马唑仑作用机制的可能贡献。瑞马唑仑是一种静脉麻醉剂，于 2020 年 7 月在日本首次批准使用，被认为通过γ-氨基丁酸 A（GABAA）受体发挥其麻醉作用；然而，瑞马唑仑如何升高细胞内钙离子水平的确切机制尚不清楚。我们使用加载荧光染料的 SHSY-5Y 神经母细胞瘤细胞、COS-7 细胞、HEK293 细胞、HeLa 细胞和人脐静脉内皮细胞（HUVEC）检查了瑞马唑仑诱导的细胞内钙离子升高情况，进行了实时成像。我们证实，高浓度瑞马唑仑（大于 300 μM）以剂量依赖的方式在这些细胞中升高细胞内钙离子。这种现象不受细胞外钙离子消除的影响。当通过 BAPTA-AM 或 thapsigargin 分别耗尽细胞内或内质网（ER）内的钙离子时，钙升高被消除，这表明钙是从 ER 中动员出来的。G 蛋白偶联受体（GPCR）介导信号的抑制剂，包括 U-73122（PLC 抑制剂）和 xestospongin C（三磷酸肌醇受体（IP3R）拮抗剂），显著抑制了瑞马唑仑诱导的钙升高，而肌醇 1,4,5-三磷酸受体（IP3R）拮抗剂丹曲林钠则不影响瑞马唑仑诱导的钙升高。同时，使用 ER-tracker 在瑞马唑仑刺激期间对 ER 进行实时成像显示没有形态变化。这些结果表明，高剂量的瑞马唑仑以剂量依赖的方式在每种测试的细胞中增加细胞内钙离子浓度，这预计是由 ER 中的钙动员引起的。此外，我们使用各种抑制剂的研究表明，这种钙升高可能是由 GPCRs-IP3 途径介导的。然而，需要进一步的研究来确定涉及哪种类型的 GPCRs。
