LAUSR

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.