Tomoaki Urabe1, Hirotsugu Miyoshi1, Soshi Narasaki1, Yuhki Yanase2, Kazue Uchida3, Soma Noguchi4, Michihiro Hide3, Yasuo M Tsutsumi1, Norio Sakai4. 1. Department of Anesthesiology and Critical Care, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan. 2. Department of Pharmacotherapy, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. 3. Department of Dermatology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan. 4. Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.
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.
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.
Remimazolam is a medication for the induction and maintenance of general anesthesia like propofol. Remimazolam is from the same pharmacological class as the existing midazolam (category- benzodiazepines) and was approved for medical use in Japan in July 2020. 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. Remimazolam acts on GABAA receptors, but the mechanisms underlying its side effects are unclear [1, 2]. In general, anesthetic-induced elevation of intracellular calcium has been implicated in the development of a variety of side effects [3-5]. We have shown that propofol acts directly on intracellular organelles, such as the endoplasmic reticulum (ER) to elevate intracellular calcium [6]. This phenomenon may be involved in the development of excessive hypotension and propofol-induced vascular pain. 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]. In our recent study, we also found that high concentrations of remimazolam increase the intracellular calcium [7]. The aim of this study was to elucidate the mechanism underlying remimazolam-induced intracellular calcium elevation.
2. Materials and methods
2.1. Materials
Remimazolam was kindly provided from Mundipharma Japan and PAION Deutschland GmbH. Caffeine, dantrolene, and thapsigargin were obtained from FUJIFILM Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Acetylcholine was purchased from Sigma-Aldrich (St. Louis, Massachusetts). U-73122 and xestospongin C were purchased from Cayman Chemical Company (Ann Arbor, Michigan). ER-Tracker Red and Fluo-4 were purchased from Molecular Probes (Eugene, Oregon). Glass-bottom culture dishes were purchased from MatTek Corporation (Ashland, Oregon). All other chemicals used were of analytical grade.
2.2. Cell culture
SHSY-5Y cells and HEK293 cells were purchased from Riken Cell Bank (Tsukuba, Japan). Human umbilical vein endothelial cells (HUVECs) were obtained from American Type Culture Collection (ATCC, Manassas, Virginia). SHSY-5Y cells were maintained in Dulbecco’s modified Eagle’s medium/Ham’s F-12 medium (FUJIFILM Wako, Osaka, Japan). HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (FUJIFILM Wako, Osaka, Japan). The medium for SHSY-5Y and HEK293 cells were supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 μg/mL). HUVECs were cultured as previously described [8]. Cell cultures were maintained in a humidified atmosphere containing 5% CO2 at 37 °C in the dark. Cells were seeded in glass-bottomed culture dishes 1–2 days before imaging.
2.3. Loading of Fluo-4 and ER-Tracker Red
The culture medium of the cells was replaced with normal HEPES buffer composed of 165 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, and 10 mM glucose, pH 7.4. Then, the cells were incubated with calcium indicator Fluo-4 (125 μg/mL) and ER-Tracker Red (1 μM) under light shielding at 37 °C for 15–20 min prior to microscopic observations. In the experiments using HUVECs and COS-7 cells, Pluronic F-127 was also used to facilitate the loading of Fluo-4 and ER-Tracker Red.
2.4. Drug treatment
Remimazolam besylate was diluted in dimethyl sulfoxide (DMSO) to prepare a 100 mM stock solution. The same DMSO concentration was used as the negative control. Remimazolam or DMSO was diluted to the desired concentrations with normal HEPES buffer and was applied to the cells at the appropriate final concentrations. Sonication was performed immediately before the addition of remimazolam. Treatment concentrations and durations are described in each figure and caption. U-73122, xestospongin C, and dantrolene were applied 15 minutes before the treatment with remimazolam.
2.5. Observation of intracellular calcium elevation and morphological changes in intracellular organelles
Fluorescence images were taken with a BZ-9000 fluorescent microscope (KEYENCE, Osaka, Japan). We used GFP-B (KEYENCE, excitation wavelength 470/40 nm, emission wavelength 535/50 nm) as the excitation and emission filters and S Fluor X40/0.93 (NIKON) as the objective lens. To visualize remimazolam-triggered calcium elevation and morphological changes in the ER, time-lapse imaging at 15-second intervals was performed after treatment of the cells with these drugs.
2.6. Semiquantitative evaluation of remimazolam-induced intracellular calcium elevation
The time course of typical remimazolam-induced intracellular calcium elevation is shown in Fig 1, which was evaluated according to a previously described method [6].
(A) An example of a typical remimazolam-induced intracellular calcium elevation time series in HUVECs. Approximately one min after the administration of 300 μM remimazolam, an increase in the fluorescence intensity of Fluo-4 was observed, indicating that intracellular calcium was increased. Fluorescence intensity in the circled area was used as background. (B) The method of semi-quantifying the rate of calcium elevation. The red line indicates the average fluorescence change over the entire observed region. The blue line indicates the fluorescence change in the background region without cells. The differences between both lines before the remimazolam application (b) and at the peak of calcium elevation (a) were measured. The ratio a to b (a/b) was considered as an index of remimazolam-induced calcium elevation.
(A) An example of a typical remimazolam-induced intracellular calcium elevation time series in HUVECs. Approximately one min after the administration of 300 μM remimazolam, an increase in the fluorescence intensity of Fluo-4 was observed, indicating that intracellular calcium was increased. Fluorescence intensity in the circled area was used as background. (B) The method of semi-quantifying the rate of calcium elevation. The red line indicates the average fluorescence change over the entire observed region. The blue line indicates the fluorescence change in the background region without cells. The differences between both lines before the remimazolam application (b) and at the peak of calcium elevation (a) were measured. The ratio a to b (a/b) was considered as an index of remimazolam-induced calcium elevation.In Fig 1, fluorescence intensity in the circled area was used as background. The red and blue lines indicate the average fluorescence changes in cells in the total area and background, respectively. The differences between both lines before each drug application (b) and at the peak of calcium elevation (a) were measured. The a to b ratio (a/b) was considered as an index of remimazolam-induced calcium elevation.
2.7. Statistical analysis
Prism 4 software (GraphPad Software, San Diego, CA) was used to conduct statistical analyses. Statistical significance was determined by one-way ANOVA, followed by Dunnett’s post-test or Mann Whitney test. All data represent the mean ± SEM (standard error of the mean). Differences were considered significant when the p value was less than 0.05 (p < 0.05).
3. Results
3.1. Characterization of remimazolam-induced calcium elevation
We observed a dose-dependent remimazolam-induced elevation of intracellular calcium at concentrations between 0 and 500 μM in SHSY-5Y cells and HUVECs (Fig 2A and 2B).
Fig 2
Remimazolam-induced intracellular calcium elevation in SHSY-5Y cells and HUVECs.
(A) Remimazolam at a concentration greater than or equal to 300 μM significantly induced the elevation of intracellular calcium in a dose-dependent manner in SHSY-5Y cells (n = 3–7, * p < 0.05, compared to control, one-way ANOVA followed by Dunnett’s post-test). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) Remimazolam at a concentration greater than or equal to 300 μM significantly induced the elevation of intracellular calcium in a dose-dependent manner in HUVECs (n = 3–5, * p < 0.05, ** p < 0.01, compared to control, one-way ANOVA followed by Dunnett’s post-test). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.
Remimazolam-induced intracellular calcium elevation in SHSY-5Y cells and HUVECs.
(A) Remimazolam at a concentration greater than or equal to 300 μM significantly induced the elevation of intracellular calcium in a dose-dependent manner in SHSY-5Y cells (n = 3–7, * p < 0.05, compared to control, one-way ANOVA followed by Dunnett’s post-test). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) Remimazolam at a concentration greater than or equal to 300 μM significantly induced the elevation of intracellular calcium in a dose-dependent manner in HUVECs (n = 3–5, * p < 0.05, ** p < 0.01, compared to control, one-way ANOVA followed by Dunnett’s post-test). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.Remimazolam at a concentration greater than or equal to 300 μM significantly induced the elevation of intracellular calcium in a dose-dependent manner in both cell lines. In addition, remimazolam-induced intracellular calcium elevation was observed in HEK293 cells (S1 Fig).
3.2. Remimazolam-induced calcium elevation in the absence of extracellular calcium
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 HUVECs. Remimazolam at a concentration greater than or equal to 300 μM significantly induced a dose-dependent increase in intracellular calcium in SHSY-5Y cells (Fig 3A). Remimazolam at 500 μM also significantly induced a dose-dependent increase in intracellular calcium levels in HUVECs (Fig 3B).
Fig 3
Characterization of remimazolam-induced intracellular calcium elevation in the absence of extracellular calcium.
After eliminating calcium from the extracellular buffer, remimazolam-induced elevation of intracellular calcium was observed at concentrations between 0 and 500 μM in SHSY-5Y cells and HUVECs. (A) Remimazolam (300 μM)-induced elevation of intracellular calcium was not influenced by the elimination of extracellular calcium in SHSY-5Y cells (n = 3–7, * p < 0.05, compared to control, one-way ANOVA followed by Dunnett’s post-test). The same concentration of DMSO corresponding to 500 μM of remimazolam was administered as a control. The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) Remimazolam (300 μM)-induced elevation of intracellular calcium was not influenced by the elimination of extracellular calcium in HUVECs (n = 3–7, * p < 0.05, compared to control, one-way ANOVA followed by Dunnett’s post-test). The same concentration of DMSO corresponding to 500 μM of remimazolam was administered as a control. The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.
Characterization of remimazolam-induced intracellular calcium elevation in the absence of extracellular calcium.
After eliminating calcium from the extracellular buffer, remimazolam-induced elevation of intracellular calcium was observed at concentrations between 0 and 500 μM in SHSY-5Y cells and HUVECs. (A) Remimazolam (300 μM)-induced elevation of intracellular calcium was not influenced by the elimination of extracellular calcium in SHSY-5Y cells (n = 3–7, * p < 0.05, compared to control, one-way ANOVA followed by Dunnett’s post-test). The same concentration of DMSO corresponding to 500 μM of remimazolam was administered as a control. The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) Remimazolam (300 μM)-induced elevation of intracellular calcium was not influenced by the elimination of extracellular calcium in HUVECs (n = 3–7, * p < 0.05, compared to control, one-way ANOVA followed by Dunnett’s post-test). The same concentration of DMSO corresponding to 500 μM of remimazolam was administered as a control. The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.These results are very similar to those seen in Fig 2A and 2B, suggesting that the remimazolam-induced elevation of intracellular calcium levels may not be influenced by the elimination of extracellular calcium.
3.3. Remimazolam-induced calcium elevation in the absence of intracellular calcium
To elucidate the influence of intracellular calcium levels on remimazolam-induced calcium elevation, we treated the cells with BAPTA-AM (20 μM), a calcium chelator useful for manipulating intracellular free calcium levels. BAPTA-AM significantly reduced and almost abolished the remimazolam (300 μM)-induced calcium elevation in both SHSY-5Y cells and HUVECs (Fig 4A and 4B).
Fig 4
Characterization of remimazolam-induced calcium elevation in the absence of intracellular calcium.
(A) BAPTA-AM (20 μM), a calcium chelator, significantly reduced and almost abolished the 300 and 500 μM remimazolam-induced calcium elevation in SHSY-5Y cells (n = 3–10, * p = 0.028, compared to control, Mann Whitney test, and n = 4–7, * p = 0.0242, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) BAPTA-AM (20 μM), a calcium chelator, significantly reduced and almost abolished the remimazolam (300 μM)-induced calcium elevation in HUVECs (n = 4–5, * p = 0.0159, compared to control, Mann Whitney test). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.
Characterization of remimazolam-induced calcium elevation in the absence of intracellular calcium.
(A) BAPTA-AM (20 μM), a calcium chelator, significantly reduced and almost abolished the 300 and 500 μM remimazolam-induced calcium elevation in SHSY-5Y cells (n = 3–10, * p = 0.028, compared to control, Mann Whitney test, and n = 4–7, * p = 0.0242, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) BAPTA-AM (20 μM), a calcium chelator, significantly reduced and almost abolished the remimazolam (300 μM)-induced calcium elevation in HUVECs (n = 4–5, * p = 0.0159, compared to control, Mann Whitney test). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.Similar effects of BAPTA-AM were observed even with 500 μM remimazolam (Fig 4A and 4B).
3.4. Effects of thapsigargin on remimazolam-induced calcium elevation
To elucidate whether remimazolam-induced calcium elevation was mobilized from the ER, we investigated the effect of thapsigargin (TG), a Ca2+-ATPase inhibitor that eliminates calcium from the ER. As shown in Fig 5, after treatment with 5 μM TG (①), [Ca2+]i was elevated.
Fig 5
Effects of thapsigargin on remimazolam-induced calcium elevation.
(A) Following the administration of 5 μM thapsigargin (TG), a Ca2+-ATPase inhibitor (①), the [Ca2+]i was elevated in SHSY-5Y cells. Subsequently, remimazolam at 300 μM was administered 30 min after the TG administration (②). Remimazolam did not induce any calcium elevation, suggesting that remimazolam mobilized calcium from ER. (B) Following the administration of 5 μM thapsigargin (TG), a Ca2+-ATPase inhibitor (①), the [Ca2+]i was elevated in HUVECs. Subsequently, remimazolam at 300 μM was administered 30 minutes after the TG administration (②). Remimazolam did not induce any calcium elevation, suggesting that remimazolam mobilized calcium from ER. For both experiments, data from one representative experiment are shown.
Effects of thapsigargin on remimazolam-induced calcium elevation.
(A) Following the administration of 5 μM thapsigargin (TG), a Ca2+-ATPase inhibitor (①), the [Ca2+]i was elevated in SHSY-5Y cells. Subsequently, remimazolam at 300 μM was administered 30 min after the TG administration (②). Remimazolam did not induce any calcium elevation, suggesting that remimazolam mobilized calcium from ER. (B) Following the administration of 5 μM thapsigargin (TG), a Ca2+-ATPase inhibitor (①), the [Ca2+]i was elevated in HUVECs. Subsequently, remimazolam at 300 μM was administered 30 minutes after the TG administration (②). Remimazolam did not induce any calcium elevation, suggesting that remimazolam mobilized calcium from ER. For both experiments, data from one representative experiment are shown.Remimazolam was added at a concentration of 300 μM 30 min after TG administration, when the [Ca2+]i had almost returned to the basal level (②). Remimazolam did not induce any calcium elevation, suggesting that remimazolam mobilized calcium from the ER.
3.5. Observation of remimazolam-induced morphological changes in the ER by ER-Tracker Red
We found that propofol alters the morphology of the ER, likely by fragmentation or aggregation, and causes calcium leakage into the cells. Therefore, we investigated the effects of remimazolam on ER morphology. ER-Tracker Red dye, is a cell-permeant marker capable of staining the ER in living cells with high selectivity. Cells were stained with this dye before remimazolam treatment, and ER status was observed during remimazolam stimulation. In this study, we did not see any morphological changes in the ER of both SHSY-5Y cells and HUVECs after the administration of remimazolam at any concentration (S2 Fig). These results showed that remimazolam-induced calcium elevation was not induced by morphological changes in the ER of both SHSY-5Y cells and HUVECs, unlike our previous research using propofol.
3.6. Mechanism underlying remimazolam-induced mobilization of calcium from the ER
3.6.1. Possible involvement of GPCRs in remimazolam-induced calcium elevation
Next, we investigated the mechanisms underlying remimazolam-induced intracellular calcium elevation. To elucidate the possible involvement of G-protein coupled receptors (GPCRs), we examined the effects of U-73122, a phospholipase C (PLC) inhibitor, on remimazolam-induced calcium elevation. Pretreatment with U-73122 at 5 μM significantly inhibited the 10 μM acetylcholine-induced calcium elevation through muscarinic receptors expressed in SHSY-5Y cells (Fig 6A) as well as the 10 μM histamine-induced calcium elevation through histamine (H1) receptors expressed in HUVECs (Fig 6B).
Fig 6
Characterization of remimazolam-induced calcium elevation; possible involvement of the G-protein coupled receptors.
(A) Fifteen minutes-pretreatment with U-73122, a phospholipase C (PLC) inhibitor, at 5 μM significantly inhibited the 10 μM acetylcholine (ACh)-induced calcium elevation in SHSY-5Y cells (n = 9–10, *** p = 0.0004, compared to control, Mann Whitney test). Similarly, 15 minutes-pretreatment with U-73122 significantly affect 300 and 500 μM remimazolam-induced calcium elevation in SHSY-5Y cells (n = 9–10, ** p = 0.0044, compared to control, Mann Whitney test, and n = 7–12, * p = 0.018, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) Fifteen minutes-pretreatment with U-73122 at 5 μM significantly inhibited the 10 μM histamine-induced calcium elevation in HUVECs (n = 6, ** p = 0.0022, compared to control, Mann Whitney test). Similarly, 15 minutes-pretreatment with U-73122 significantly affected 300 and 500 μM remimazolam-induced calcium elevation in HUVECs (n = 5, ** p = 0.0079, compared to control, Mann Whitney test, and n = 5–8, * p = 0.0295, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.
Characterization of remimazolam-induced calcium elevation; possible involvement of the G-protein coupled receptors.
(A) Fifteen minutes-pretreatment with U-73122, a phospholipase C (PLC) inhibitor, at 5 μM significantly inhibited the 10 μM acetylcholine (ACh)-induced calcium elevation in SHSY-5Y cells (n = 9–10, *** p = 0.0004, compared to control, Mann Whitney test). Similarly, 15 minutes-pretreatment with U-73122 significantly affect 300 and 500 μM remimazolam-induced calcium elevation in SHSY-5Y cells (n = 9–10, ** p = 0.0044, compared to control, Mann Whitney test, and n = 7–12, * p = 0.018, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) Fifteen minutes-pretreatment with U-73122 at 5 μM significantly inhibited the 10 μM histamine-induced calcium elevation in HUVECs (n = 6, ** p = 0.0022, compared to control, Mann Whitney test). Similarly, 15 minutes-pretreatment with U-73122 significantly affected 300 and 500 μM remimazolam-induced calcium elevation in HUVECs (n = 5, ** p = 0.0079, compared to control, Mann Whitney test, and n = 5–8, * p = 0.0295, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.Similarly, U-73122 significantly inhibited remimazolam-induced calcium elevation in both SHSY-5Y cells and HUVECs (Fig 6A and 6B). These results suggest that the elevation of intracellular calcium by remimazolam may be mediated by GPCRs coupled with PLC.
3.6.2. Possible involvement of IP3 and ryanodine receptors in remimazolam-induced calcium elevation
Calcium mobilization from the ER is mediated by inositol 1,4,5-triphosphate (IP3) or ryanodine receptors (RyRs). Therefore, we examined the effects of xestospongin C (Xc), an IP3 receptor (IP3R) antagonist, on remimazolam-induced calcium elevation. Pretreatment with Xc at 5 μM significantly inhibited the 10 μM acetylcholine-induced calcium elevation in SHSY-5Y cells (Fig 7A) and 10 μM histamine-induced calcium elevation in HUVECs (Fig 7B).
Fig 7
Characterization of remimazolam-induced calcium elevation; possible involvement of the IP3 receptors.
(A) Fifteen minutes-pretreatment with Xestospongin C (Xc), an inositol 1,4,5-triphosphate receptor (IP3R) antagonist, at 5 μM significantly inhibited the ACh 10 μM-induced calcium elevation in SHSY-5Y cells (n = 10–11, ** p = 0.0035, compared to control, Mann Whitney test). Similarly, 15 minutes-pretreatment with Xc at 5 μM affected 300 and 500 μM remimazolam-induced calcium elevation in SHSY-5Y cells (n = 4–7, * p = 0.0242, compared to control, Mann Whitney test, and n = 5–7, * p = 0.0177, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) Fifteen minutes-pretreatment with Xestospongin C (Xc) at 5 μM significantly inhibited the histamine 10 μM-induced calcium elevation in HUVECs (n = 4–6, ** p = 0.0095, compared to control, Mann Whitney test). Similarly, 15 minutes-pretreatment with Xc at 5 μM affected 300 and 500 μM remimazolam-induced calcium elevation in HUVECs (n = 5–6, * p = 0.0173, compared to control, Mann Whitney test, and n = 5–7, * p = 0.0303, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.
Characterization of remimazolam-induced calcium elevation; possible involvement of the IP3 receptors.
(A) Fifteen minutes-pretreatment with Xestospongin C (Xc), an inositol 1,4,5-triphosphate receptor (IP3R) antagonist, at 5 μM significantly inhibited the ACh 10 μM-induced calcium elevation in SHSY-5Y cells (n = 10–11, ** p = 0.0035, compared to control, Mann Whitney test). Similarly, 15 minutes-pretreatment with Xc at 5 μM affected 300 and 500 μM remimazolam-induced calcium elevation in SHSY-5Y cells (n = 4–7, * p = 0.0242, compared to control, Mann Whitney test, and n = 5–7, * p = 0.0177, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) Fifteen minutes-pretreatment with Xestospongin C (Xc) at 5 μM significantly inhibited the histamine 10 μM-induced calcium elevation in HUVECs (n = 4–6, ** p = 0.0095, compared to control, Mann Whitney test). Similarly, 15 minutes-pretreatment with Xc at 5 μM affected 300 and 500 μM remimazolam-induced calcium elevation in HUVECs (n = 5–6, * p = 0.0173, compared to control, Mann Whitney test, and n = 5–7, * p = 0.0303, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.Similarly, 5 μM Xc significantly reduced the remimazolam-induced calcium elevation in both SHSY-5Y cells and HUVECs (Fig 7A and 7B). These results suggest that the elevation of intracellular calcium by remimazolam may be mediated by IP3 receptors.In addition, we investigated the effects of dantrolene, a selective RyR antagonist, on remimazolam-induced calcium elevation. First, we examined dantrolene effects on calcium mobilization induced by caffeine, a RyR agonist. The results showed that pretreatment with 50 μM dantrolene significantly inhibited 10 mM caffeine-induced calcium mobilization in both SHSY-5Y cells and HUVECs (Fig 8A and 8B).
Fig 8
Characterization of remimazolam-induced calcium elevation; possible involvement of ryanodine receptors.
(A) Caffeine, a ryanodine receptor (RYR) agonist, at 10 mM induced the intracellular calcium in SHSY-5Y cells. Fifteen minutes-pretreatment with dantrolene, a RYR antagonist, at 50 μM significantly inhibited the caffeine-induced calcium mobilization (n = 9, * p = 0.024, compared to control, Mann Whitney test), however, 15 minutes-pretreatment with dantrolene at 50 μM did not significantly influence the 300 and 500 μM remimazolam-induced calcium elevation (n = 5–7, p = 0.149, compared to control, Mann Whitney test, and n = 6–7, p = 0.5338, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) Caffeine at 10 mM induced the intracellular calcium in HUVECs. Fifteen minutes-pretreatment with dantrolene at 50 μM significantly inhibited the caffeine-induced calcium mobilization (n = 5–8, * p = 0.0186, compared to control, Mann Whitney test), however, 15 minutes-pretreatment with dantrolene at 50 μM did not significantly influence the 300 and 500 μM remimazolam-induced calcium elevation (n = 5–7, p = 0.5303, compared to control, Mann Whitney test, and n = 5–7, p = 0.2468, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.
Characterization of remimazolam-induced calcium elevation; possible involvement of ryanodine receptors.
(A) Caffeine, a ryanodine receptor (RYR) agonist, at 10 mM induced the intracellular calcium in SHSY-5Y cells. Fifteen minutes-pretreatment with dantrolene, a RYR antagonist, at 50 μM significantly inhibited the caffeine-induced calcium mobilization (n = 9, * p = 0.024, compared to control, Mann Whitney test), however, 15 minutes-pretreatment with dantrolene at 50 μM did not significantly influence the 300 and 500 μM remimazolam-induced calcium elevation (n = 5–7, p = 0.149, compared to control, Mann Whitney test, and n = 6–7, p = 0.5338, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR. (B) Caffeine at 10 mM induced the intracellular calcium in HUVECs. Fifteen minutes-pretreatment with dantrolene at 50 μM significantly inhibited the caffeine-induced calcium mobilization (n = 5–8, * p = 0.0186, compared to control, Mann Whitney test), however, 15 minutes-pretreatment with dantrolene at 50 μM did not significantly influence the 300 and 500 μM remimazolam-induced calcium elevation (n = 5–7, p = 0.5303, compared to control, Mann Whitney test, and n = 5–7, p = 0.2468, compared to control, Mann Whitney test, respectively). The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR.However, dantrolene did not significantly influence remimazolam-induced calcium elevation in either cell lines (Fig 8A and 8B), suggesting that remimazolam-induced calcium elevation may not be mediated via RyRs. We also examined the effects of tetracaine, another type of RyR antagonist, on remimazolam-induced calcium elevation in SHSY-5Y cells. Tetracaine (500 μM) did not significantly affect the calcium elevation, although it significantly inhibited caffeine (10 mM)-induced calcium elevation (S3 Fig).
4. Discussion
Remimazolam has already been used in many clinical settings since it was approved in Japan in July 2020, prior to the rest of the world. Its structure is comparable to that of the existing benzodiazepines. Its sedative effect is on GABAA receptors, and it has relatively little effect on circulation. One of the benefits of remimazolam is that its actions can be antagonized by flumazenil [1, 2]. At this point, remimazolam is only approved for Induction and maintenance of general anesthesia, but it is likely that it will play a leading role in intravenous anesthetics in the future, similar to propofol.Remimazolam and propofol are considered to exert their anesthetic effects by acting on GABAA receptors. It has been suggested that elevated intracellular calcium is involved in the development of adverse effects of propofol. We have previously reported that propofol mobilizes calcium into cells by penetrating them and causing morphological changes in intracellular organelles [6]. In our recent report, we also showed that high concentrations of remimazolam increase intracellular calcium concentration in a dose-dependent manner [7]. In this study, we examined the effects of remimazolam on the dynamics of intracellular calcium in each cell, including the morphology of intracellular organelles.In the present work, we used SHSY-5Y cells and HUVECs derived from neuronal and endothelial cells, respectively, which are considered important sites of action for intravenous anesthetics. Although the therapeutic plasma concentrations are considerably lower than those used in our experiments, the results showed that remimazolam increased intracellular calcium in a dose-dependent manner in both cell lines. These results were similar to those of our previous experiments in which propofol was used [6]. In addition to SHSY-5Y cells and HUVECs, remimazolam-induced intracellular calcium elevation was observed in HEK293, suggesting that this effect is universal, regardless of the cell type.Next, we examined how remimazolam-induced intracellular calcium elevation could occur. When remimazolam was administered using extracellular calcium-free buffer, we observed an increase in intracellular calcium levels in both SHSY-5Y cells and HUVECs. Therefore, it is unlikely that this increase was caused by a calcium influx from extracellular sources.We further confirmed that the remimazolam-induced elevation of intracellular calcium could be largely eliminated by BAPTA-AM in both SHSY-5Y cells and HUVECs. Moreover, the remimazolam-induced increase in intracellular calcium was almost eliminated by removing calcium from the ER by thapsigargin. Based on these findings, we hypothesized that the remimazolam-induced increase in intracellular calcium levels might be due to calcium mobilization from the ER. We subsequently attempted to elucidate the mechanism of calcium mobilization from the ER. Since GPCR-IP3 and ryanodine receptors may be involved in the mechanism of calcium mobilization from the ER, we investigated the effects of a PLC inhibitor (U-73122), an IP3 receptor inhibitor (Xc), and a ryanodine receptor inhibitor (dantrolene). U-73122 and Xc significantly inhibited remimazolam-induced intracellular calcium elevation in both cell lines, whereas dantrolene had no effect, suggesting that this elevation is involved in the GPCR-IP3 pathway. While remimazolam does not seems to affect RyR1 directly, the IP3-dependent calcium elevation mediated by remimazolam could play a role in sensitizing RyR1 in an individual with Malignant Hyperthermia (MH) susceptibility. A recent study reported that there were no changes in the sensitivity to intracellular calcium elevation of remimazolam in cells transfected with the MH mutation [7]. This is very different from propofol-induced intracellular calcium elevation, the mechanism of which is involved in the direct destruction of intracellular organelles, such as the ER. In this meaning, the effects of remimazolam might be reversible since it elevates calcium by pharmacological mechanisms rather than by non-reversible morphological changes.In this study, we could not identify the type of GPCRs affected. Remimazolam-induced elevation of intracellular calcium was observed in all the investigated cells, including SHSY5Y, HUVECs, and HEK293 (S2 Fig). Based on these results, it is expected that GPCRs, which are related to remimazolam action, are universally expressed in a variety of cells. As an example, we considered the involvement of purinergic receptors, particularly P2 purinergic receptor [9]. In the future, it is expected that studies will be performed to identify GPCRs involved in remimazolam-induced calcium elevation by focusing on GPCRs universally expressed by these cells.Remimazolam concentrations used in this study were considerably higher than those used clinically (appropriately 5 μM for maintaining anesthesia) [10]. However, concentrations like the ones used in this study or higher could occur locally near the injection site. Currently, the indication for remimazolam is limited to general anesthesia in adults, but it is expected that this indication will be expanded to general anesthesia for pediatric patients in the future. Since the early 2000s, many basic studies on neurological disorders caused by general anesthetics have been conducted, and it has been reported that many general anesthetics have neurotoxic effects on juvenile central neurons and are associated with occurrence of developmental disorders in the long-term [11-14]. In such cases, prolonged exposure can lead to the accumulation of high concentrations of anesthetics in the cells, causing side effects. It is well-known that GABAA receptors, NMDA receptors, and IP3 receptors are associated with the receptors to which general anesthetics bind [15, 16]. Evidence has shown that overexposure or prolonged exposure of general anesthetics to these receptors induces an excessive increase in intracellular calcium and consequently, apoptosis [17-20]. Therefore, it is worthwhile to investigate the effects of high concentrations of remimazolam on cellular functions. Our findings that high concentrations of remimazolam universally elevated intracellular calcium levels will benefit future research.
5. Conclusion
Our present data indicate that high concentrations of remimazolam induced a dose-dependent elevation of intracellular calcium in SHSY-5Y cells and HUVECs. We also found that remimazolam acted on the GPCR-IP3 pathway to increase intracellular calcium levels. As the clinical uses of remimazolam are expected to expand worldwide in the future, elucidating its mechanism of action is very important to understand its advantages and disadvantages.
Remimazolam-induced intracellular calcium elevation in HEK293 cells.
(A) Remimazolam at a concentration greater than or equal to 300 μM significantly induced the elevation of intracellular calcium in a dose-dependent manner in HEK293 cells. (n = 3–5, * p < 0.05, compared to control, one-way ANOVA followed by Dunnett’s post-test).(TIF)Click here for additional data file.
Fluorescent microscopic imaging.
ER-tracker was capable of staining ER in living HUVECs and SHSY-5Y cells, remimazolam did not elicit the morphological changes of ER. (above: HUVECs, below: SHSY-5Y cells).(TIF)Click here for additional data file.Caffeine, a ryanodine receptor (RYR) agonist, at 10 mM induced the intracellular calcium in SHSY-5Y cells. Fifteen minutes-pretreatment with tetracaine, a RYR antagonist, at 500 μM significantly inhibited the caffeine-induced calcium mobilization (n = 5, * p = 0.0159, compared to control, Mann Whitney test), however, 15 minutes-pretreatment with tetracaine at 500 μM did not significantly influence the 300 μM remimazolam-induced calcium elevation. Data represent the mean ± SEM (n = 4, p = 0.3429, compared to control, Mann Whitney test).(TIF)Click here for additional data file.14 Sep 2021
PONE-D-21-15802
Characterization of intracellular calcium mobilization induced by remimazolam, a newly approved intravenous anesthetic
PLOS ONE
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You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This manuscript aimed to examine the source of increased Ca2+ in response to exposure to the drug remimazolam. The authors have done a lot of work and have endeavoured to pharmacologically block multiple cellular pathways that could account for the Ca2+ release observed. Ultimately, the authors suggest that the increased Ca2+ is likely mediated via a GPCR-IP3 pathway, although more work needs to be done to establish the specific pathway.Major Points:• What is the rational for the choices of cell lines used? One of the cell lines reported, but no data was shown, is from a monkey kidney line.• The introduction is quite brief with minimal references provided throughout the submission.• A high proportion of supportive data is not shown as figures. For example, it is reported as data not shown but how did the authors designate a “normal morphology” of the ER in the SHSY-5Y and HUVEC cells? Images would help substantiate this statement.• What is the equivalent clinical dose for remimazolam? The authors state that the doses they use are higher than those used in clinical situations but seem to only get cellular effects at with high doses. How do these experimental doses correspond to the maximum dosage a patient would receive?• A non-ratiometric, AM loading, Ca2+ indicating dye has been used. What is the justification for the ratio that was created for the Ca2+ trace analysis? Convention is to subtract the background intensity and then convert to F/F0 to take into account the differences in dye loading between individual cells and different cell lines. The summarised percentage change in fluorescence may not reflect a large change in Ca2+ concentration due to the use of a non-ratiometric dye. It is also not appropriate to apply statistics to percentage data.• 30 mins after the TG treatment an agonist should be applied to show that there is no residual Ca2+ in the stores to further support the assumption that remimazolam is not causing a release of ER Ca2+.• The authors state that Ca2+ release due to remimazolam is likely due to IP3 receptor activation. It would be beneficial to apply a cocktail of the inhibitors used in this study (minus the IP3 inhibitor) and see if the same increased Ca2+ response is seen with treatment with remimazolam.• In Figure2 and 3, can the authors comment on why the same trends of Ca2+ release were not observed in the HUVEC cells but are conserved in the SHSY-5Y?• Figure 8 A&B the data suggests that dantrolene was not providing a complete block of the RyR. Tetracaine would be a more efficient inhibitor.Minor Points:• Histogram formats should be changed to include individual data points.• Figure 1, states “the rate” of calcium, is this a typo that should state ratio as the authors have not measured the rate of Ca2+ release/increase?• Figure 4, it appears the data panel of HUVECS with 500uM remimazolam is absent, was the data unable to be gathered under these conditions?Reviewer #2: Urabe et al. provide important information about the effect of remimazolam, a newly approved intravenous anesthetic. This work presents an empirical characterization of intracellular calcium alterations initiated by this new drug. The authors present results on calcium signaling using different mammalian cell lines such as neuronal-, fibroblast-like cells and human umbilical endothelial cells. Using calcium imaging, ionic substitution, calcium buffering, and pharmacological tools, the authors identified that supratherapeutic doses of remimazolam initiated intracellular calcium elevations from the ER. These Ca signals were GPCR and IP3 dependent when assessed in several cell types. The work is well-designed and presents empirical evidence of an interesting biological event that has a therapeutic interest.For this reviewer, the work is novel and original, necessary for the general understanding of the additional (side) effects of remimazolam. The experiments are, in general, well-conducted and the paper is well written and easy to follow; however, as far as this reviewer concerns, some issues require attention.*Specific CommentsAbstract:Briefly mention that remimazolam causes an elevation in intracellular calcium and why this may be of interest.Introduction:Line 75. Propose a mechanism by which propofol (or remimazolam) could cause hypotension or vascular pain. This mechanism could be used to justify the study of calcium mobilization and the use of neuronal-like cells and endothelial cells.Methods:Lines 126-130. For reproducibility purposes, please provide more details about the apparatus and software used to measure calcium signals. Was a photodiode, PMT, or camera used (include manufacturer and model)? Also, add details about excitation light source, excitation, and emission filters, and objective used. What was the sampling rate?Statistical: Please clarify whether the normality test was assessed and how. When reporting results (significant or non-significant), please give the exact p-value, adjusted significance level (when appropriate), and the test used.Results:Lines 194-204. “Observation of remimazolam…” Delete this section or show the results, a negative results still a result. Data could be show as supplementary information.To further characterize the involvement of GPCR, the authors could use GDPbetaS, to prevent non-specific activation of GPCR. If remimazolam effect depend on GPCR activation GDPbetaS should prevent its effects.Discussion:Lines 262. “ a newly marketed” delete this, it has been already mention few lines above.The authors wrote: “Remimazolam concentrations used in this study were considerably higher than those used clinically.”You could add something like: However, concentrations like the ones used in this study or higher could occur locally near the injection site.It will be worth mentioning somewhere in the discussion that while remimazolam does not seems to affect RyR1 directly, the IP3-dependent elevation in Ca2+ mediated by remimazloam could play a role in sensitizing RyR1 in an individual with HM-susceptibility.Figure 1:Traces in panel B appear identical to those previously published by the authors (see Ref #6 Urabe T et al. 2020, Fig. 1B). This issue is very problematic and needs to be fixed or request permission to reuse already published data. Also, in the same panel, how comes that the background signal is not flat. Would you please indicate the region of interest used to measure both the cell and background on the image?Figures 2, 3, 4, 6-8.Use box plot with data overlap instead of plunger bars (show the data).Figures 2 and 3 add the number of observations to boxes as in figures 4,6-8.Minor Comments:Figure 2-8. Adding labels to the figure panels (i.e., Control solution for Fig 2, Zero Ca2+ for figure 3) may help the reader quickly identify the results.Figure 3A, remove data for DMSO 0.05-0.3 as in panel B.Figure 4. Increase Font size for BAPTA-AM and symbolsFigures 6-8. Increase the font size for x- and y-axis labels********** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.1 Dec 2021GENERAL COMMENTS TO THE EDITORCOMMENTS TO THE EDITOR:1. COMMENT: Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.RESPONSE: I have checked and corrected. Fig legend has been added after each Fig.2. COMMENT: Please provide additional information about the source of the HUVEC cells. If the cells are a named cell line, please provide the name. If they were primary cells derived from clinical samples, please state whether the cells were isolated for the purposes of research and whether any identifying information was provided with them.RESPONSE: We have added information on the availability of HUVECs. （page 7, line 107 - 108）3. COMMENT: We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section.RESPONSE: We have added our Funding Information including the grant numbers in our cover letter.4. COMMENT: In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available.RESPONSE: We have attached the data as Supporting information.5. COMMENT: PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager.RESPONSE: Corresponding author obtained the ORCID ID and confirmed that it is enabled in Editorial Manager.6. COMMENT: We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data.RESPONSE: We have presented the data as Supporting information. The description of the paper has been corrected accordingly. (page 12, line 199; page 16, line 289; page 20, line 391; page 24, line 453)7. COMMENT: We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed:https://www.sciencedirect.com/science/article/abs/pii/S0014299920303952?via%3DihubRESPONSE: We have identified that many of the duplications overlap with papers we have published in the past. We have made every effort to minimize duplication where possible.GENERAL COMMENTS TO THE REVIEWER #1RESPONSES TO THE REVIEWER #1’s COMMENTS:Major points1. COMMENT: What is the rational for the choices of cell lines used? One of the cell lines reported, but no data was shown, is from a monkey kidney line.RESPONSE: SHSY-5Y cells are neuronal cell lines; HUVECs are vascular endothelial cells. Remimazolam is expected to have anesthetic effects and side effects on neuronal and vascular endothelial cells. Hence, we used these cells in our study. The other cells were used to determine if the effects of remimazolam are specific to neurons and endothelial cells or if it is a generalized effect. There was not enough data accumulated for COS-7 and Hela cells to provide examples, so the descriptions of these cells were removed in the revised paper.2. The introduction is quite brief with minimal references provided throughout the submission.RESPONSE: In accordance with the reviewer's comment, we added a new description of the side effects of anesthetics in the introduction.3. COMMENT: A high proportion of supportive data is not shown as figures. For example, it is reported as data not shown but how did the authors designate a “normal morphology” of the ER in the SHSY-5Y and HUVEC cells? Images would help substantiate this statement.RESPONSE: We have presented data showing that administration of remimazolam did not cause morphological changes in the ER as Supplemental figure 1. (page 15 - 16, line 286 - 289)4. COMMENT: What is the equivalent clinical dose for remimazolam? The authors state that the doses they use are higher than those used in clinical situations but seem to only get cellular effects at with high doses. How do these experimental doses correspond to the maximum dosage a patient would receive?RESPONSE: Although the concentration of remimazolam used in this study is certainly higher than that of clinical concentration, there is a high possibility that remimazolam will be expanded to pediatric general anesthesia in the future. In pediatric patients, various neurological disorders are likely to occur during long-term exposure to anesthetics. Given the possibility that remimazolam will be accumulated due to long-term exposure, remimazolam may exert its effects at higher than clinical concentrations. Therefore, the findings of our experiments may have clinical significance. These ideas have already been mentioned in Discussion.5. COMMENT: A non-ratiometric, AM loading, Ca2+ indicating dye has been used. What is the justification for the ratio that was created for the Ca2+ trace analysis? Convention is to subtract the background intensity and then convert to F/F0 to take into account the differences in dye loading between individual cells and different cell lines. The summarised percentage change in fluorescence may not reflect a large change in Ca2+ concentration due to the use of a non-ratiometric dye. It is also not appropriate to apply statistics to percentage data.RESPONSE: We do not intend to measure the absolute intracellular calcium concentration. We are using this method only as a semi-quantification method that can be used for the purpose of analyzing the effects of drugs on intracellular calcium elevation. As reported in our previous research paper using propofol (Urabe T, et al. Eur J Pharmacol. 2020;884:173303.), time-lapse imaging was carried out using a fluorescence microscope after the application of propofol in order to observe propofol-triggered calcium elevation.In this previous study, to examine the validity of this method, we have confirmed that the number of cells in the observation area does not influence the degree of drug-induced intracellular calcium elevation. Therefore, we believe that this semi-quantification method can be used to determine the effects of various drugs on the calcium elevation of remimazolam. Since we averaged the calcium elevation of all cells in an area, we believe this method would be less biased and more accurate, compared to averaging the calcium elevations of intentionally picked individual cells, in measuring the degree of calcium elevation.In this study, we showed the percentage increase in the fluorescence intensity of the entire area by remimazolam before administration of remimazolam. We have no intention to measure absolute values of fluorescence intensity. We performed a number of experiments with different experimental conditions, calculated the percentage increase in presentation for each experiment, and compared them across experiments. This type of analysis is general, not limited to calcium elevation experiments, and we do not believe that the use of statistical methods is inappropriate. We have shown the area where the background was measured in Fig. 1 in order to give an accurate understanding of the method used in this experiment.6. COMMENT: 30 mins after the TG treatment an agonist should be applied to show that there is no residual Ca2+ in the stores to further support the assumption that remimazolam is not causing a release of ER Ca2+.RESPONSE: In our previous study, we found that propofol mobilized intracellular calcium from ER by disrupting the membrane of the endoplasmic reticulum (Urabe T, et al. Eur J Pharmacol. 2020;884:17330). In this previous study, we investigated the effect of propofol on the mobilization of calcium from ER treated with TG. We proved that propofol did not increase intracellular calcium 30 minutes after the TG administration, indicating that 30-min treatment with TG sufficiently depleted calcium in ER. Therefore, we believe that calcium in the endoplasmic reticulum is thoroughly depleted at 30 minutes after TG administration.7. COMMENT: The authors state that Ca2+ release due to remimazolam is likely due to IP3 receptor activation. It would be beneficial to apply a cocktail of the inhibitors used in this study (minus the IP3 inhibitor) and see if the same increased Ca2+ response is seen with treatment with remimazolam.RESPONSE: The reviewer has proposed to use a cocktail containing an inhibitor other than the IP3 inhibitor used in this study, and to test whether intracellular calcium is elevated in the presence of this inhibitor cocktail. However, since the cocktail contains PLC inhibitors, the results would probably show that no calcium increase will be observed. It is unlikely that the results of this experiment will provide beneficial information.8. COMMENT: In Figure2 and 3, can the authors comment on why the same trends of Ca2+ release were not observed in the HUVEC cells but are conserved in the SHSY-5Y?RESPONSE: With regard to the results of Figs 2. and 3., we consider that remimazolam caused a dose-dependent elevation of intracellular calcium in both SHSY-5Y cells and HUVECs. We consider that the tendencies of calcium elevation seen in SHSY-5Y cells and HUVECs were same both in the presence or absence of extracellular calcium. In order to avoid confusion, we have revised the descriptions concerning this point. (page 12, line 209-210)9. COMMENT: Figure 8 A&B the data suggests that dantrolene was not providing a complete block of the RyR. Tetracaine would be a more efficient inhibitor.RESPONSE: In accordance with the reviewer’s comment, we investigated the effects of tetracaine on remimazolam-induced intracellular calcium elevation. As shown in a Supplemental Figure.3, tetracaine also did not significantly affect the calcium elevation, although it significantly inhibited caffeine (10 mM)-induced calcium elevation. We have shown these data as Supplemental Figure.3 in revised manuscript.Minor Points10. COMMENT: Histogram formats should be changed to include individual data points.RESPONSE: We were suggested the similar question by reviewer #2, so we modified the figures using the box plot.11. COMMENT: Figure 1, states “the rate” of calcium, is this a typo that should state ratio as the authors have not measured the rate of Ca2+ release/increase?RESPONSE: As the reviewer pointed out, we have changed the title of the figure to "a/b indicates the ratio of calcium elevation".12. COMMENT: Figure 4, it appears the data panel of HUVECS with 500uM remimazolam is absent, was the data unable to be gathered under these conditions?RESPONSE: As the reviewer mentioned, we could not collect data under these conditions.GENERAL COMMENTS TO THE REVIEWER #2RESPONSES TO THE REVIEWER #2’s COMMENTS:I appreciate your very thoughtful comments as a reviewer.*Specific Comments1. COMMENT: Abstract:Briefly mention that remimazolam causes an elevation in intracellular calcium and why this may be of interest.RESPONSE: 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.We added the above sentence to the beginning of the Abstract.2. COMMENT: Introduction:Line 75. Propose a mechanism by which propofol (or remimazolam) could cause hypotension or vascular pain. This mechanism could be used to justify the study of calcium mobilization and the use of neuronal-like cells and endothelial cells.RESPONSE: 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.We added the above sentence to the middle of the Introduction. （page 5, line 87 – 90）3. COMMENT: Methods:Lines 126-130. For reproducibility purposes, please provide more details about the apparatus and software used to measure calcium signals. Was a photodiode, PMT, or camera used (include manufacturer and model)? Also, add details about excitation light source, excitation, and emission filters, and objective used. What was the sampling rate?RESPONSE: The observing images obtained by a BZ-9000 fluorescent microscope (KEYENCE) were used for the analysis. We did not use any software, photodiodes, or PMT. In this experiment, the excitation light source of a conventional fluorescence microscope was used. We used GFP-B (KEYENCE, excitation wavelength 470/40 nm, emission wavelength 535/50 nm) as the excitation and emission filters and S Fluor X40/0.93 (NIKON) as the objective lens. Sampling rate was every 15 seconds. These are described in the Material & Method (page 9, line 140-142).4. COMMENT: Statistical: Please clarify whether the normality test was assessed and how. When reporting results (significant or non-significant), please give the exact p-value, adjusted significance level (when appropriate), and the test used.RESPONSE: In the revised paper, the statistical analysis method was changed to a nonparametric test since some studies had a small number of cases. Specifically, we used the Mann-Whitney test and described the exact P value. The description of the text and figure legends were changed in accordance with this revision.5. COMMENT: Results:Lines 194-204. “Observation of remimazolam…” Delete this section or show the results, negative results still a result. Data could be show as supplementary information.RESPONSE: We followed the reviewers' comments and presented a new figure as supplemental figure.2.6. COMMENT: To further characterize the involvement of GPCR, the authors could use GDPbetaS, to prevent non-specific activation of GPCR. If remimazolam effect depend on GPCR activation GDPbetaS should prevent its effects.RESPONSE: GDPbetaS is difficult to use in our experimental system due to the fact that it is an inhibitor that does not penetrate cell membranes; GDPbetaS is commonly used in electrophysiological experiments by filling it into a pipette.7. COMMENT: Discussion:Lines 262. “ a newly marketed” delete this, it has been already mention few lines above. The authors wrote: “Remimazolam concentrations used in this study were considerably higher than those used clinically.”RESPONSE: As you noted, we have removed it from our text.8. COMMENT: You could add something like: However, concentrations like the ones used in this study or higher could occur locally near the injection site.RESPONSE: Thank you for pointing this out. We have added the description you suggested to the Discussion (page 25, line 462-463).9. COMMENT: It will be worth mentioning somewhere in the discussion that while remimazolam does not seems to affect RyR1 directly, the IP3-dependent elevation in Ca2+ mediated by remimazloam could play a role in sensitizing RyR1 in an individual with HM-susceptibility.RESPONSE: Thank you for pointing this out. We have added the description you suggested to the Discussion. In addition, since it has been reported that there was no change in the sensitivity of intracellular calcium elevation to remimazolam in cells transfected with the MH mutation. Subsequently, we added the following statement.“A recent study (Watanabe T et al. Biomed Res Int. 2021;2021:8845129) reported that there were no changes in the sensitivity to intracellular calcium elevation of remimazolam in cells transfected with the MH mutation.” (page 23-24,line 440-445).10. COMMENT: Figure 1:Traces in panel B appear identical to those previously published by the authors (see Ref #6 Urabe T et al. 2020, Fig. 1B). This issue is very problematic and needs to be fixed or request permission to reuse already published data. Also, in the same panel, how comes that the background signal is not flat. Would you please indicate the region of interest used to measure both the cell and background on the image?RESPONSE: We apologize for confused Figure.1B. Figure.1B was modified to fit the data of this study. The areas where the background was taken are indicated by circles. We have chosen areas that appear as flat as possible, but some variation is allowed. The region of interest is the full screen.11. COMMENT: Figures 2, 3, 4, 6-8.Use box plot with data overlap instead of plunger bars (show the data).Figures 2 and 3 add the number of observations to boxes as in figures 4,6-8.RESPONSE: As a reviewer pointed out, we redesigned the figure using the box plot. In addition, I have added the number of observations to the box in Figures 2 and 3.Minor Comments12. COMMENT: Figure 2-8. Adding labels to the figure panels (i.e., Control solution for Fig 2, Zero Ca2+ for figure 3) may help the reader quickly identify the results.RESPONSE: We have made the changes as the reviewer suggested as far as possible.13. COMMENT: Figure 3A, remove data for DMSO 0.05-0.3 as in panel B.RESPONSE: We have made the changes as the reviewer suggested. We also made the same changes in Figure 2 A. and B.14. COMMENT: Figure 4. Increase Font size for BAPTA-AM and symbolsRESPONSE: We have made the changes as the reviewer suggested.15. COMMENT: Figures 6-8. Increase the font size for x- and y-axis labelsRESPONSE: We have made the changes as the reviewer suggested.Submitted filename: Response to Reviewers.docxClick here for additional data file.27 Dec 2021
PONE-D-21-15802R1
Characterization of intracellular calcium mobilization induced by remimazolam, a newly approved intravenous anesthetic
PLOS ONE
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In line with the the comments of Reviewer#1, please redraw all figures where necessary using box plots.
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6 Jan 2022GENERAL COMMENTS TO THE REVIEWER #1RESPONSES TO THE REVIEWER #1’s COMMENTS:1. COMMENT: The authors have done much to address reviewer comments and improve the manuscript. However, Figures 1 and 2 are still displayed as summary histograms and have not been altered to show the spread of the individual data points. While the subsequent use of box plots is a positive adjustment, as the data is stated in the figure legends as mean and SEM this is still are not showing the spread of the data. The box plots could be altered to include the individual data points or to show mean with SD.RESPONSE: In accordance with the reviewer#1's suggestion, the histograms in figures 2 and 3 have been changed to box plots. We have also removed the description concerning mean and SEM in figure legends. And, we added the following statements: "The horizontal line in each box indicates the median, the box shows the interquartile range (IQR), and the whiskers represent 1.5 × IQR." The descriptions in Figure legend 4, 6, 7, 8 have been changed as well.Submitted filename: GENERAL COMMENTS TO THE REVIEWER #1.docxClick here for additional data file.19 Jan 2022Characterization of intracellular calcium mobilization induced by remimazolam, a newly approved intravenous anestheticPONE-D-21-15802R2Dear Dr. Miyoshi,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.Kind regards,Laszlo Csernoch, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments (optional):Reviewers' comments:21 Jan 2022PONE-D-21-15802R2Characterization of intracellular calcium mobilization induced by remimazolam, a newly approved intravenous anestheticDear Dr. Miyoshi:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.If we can help with anything else, please email us at plosone@plos.org.Thank you for submitting your work to PLOS ONE and supporting open access.Kind regards,PLOS ONE Editorial Office Staffon behalf ofDr. Laszlo CsernochAcademic EditorPLOS ONE
Authors: Alexandra M Wesolowski; Michael P Zaccagnino; Raymond J Malapero; Alan D Kaye; Richard D Urman Journal: Pharmacotherapy Date: 2016-09-01 Impact factor: 4.705
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