Abstract
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.
Highlights
Remimazolam is a medication for the induction and maintenance of general anesthesia like propofol
To elucidate whether the elevation of intracellular calcium was mediated by calcium influx from the extracellular buffer, we eliminated calcium from the extracellular buffer and observed a remimazolam-induced elevation of intracellular calcium at concentrations between 0 and 500 μM in SHSY-5Y cells and human umbilical vein endothelial cells (HUVECs)
Our present data indicate that high concentrations of remimazolam induced a dose-dependent elevation of intracellular calcium in SHSY-5Y cells and HUVECs
Summary
Remimazolam is a medication for the induction and maintenance of general anesthesia like propofol. Remimazolam is approved for procedural sedation in US, Europe and China and for general anesthesia in South Korea, and it is likely to be used more widely in the future. It has the same advantages as midazolam, such as less circulatory inhibitory effect, less injection site reaction at the time of administration, and is antagonized by flumazenil. Propofol-induced calcium elevation may induce vasodilation by activating the intracellular signaling pathway and promoting the phosphorylation of NO synthase, resulting in the synthesis of NO This may contribute to hypotension and vascular pain [6]. The aim of this study was to elucidate the mechanism underlying remimazolam-induced intracellular calcium elevation
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