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. 2018 Jan 3;13(1):e0190213.
doi: 10.1371/journal.pone.0190213. eCollection 2018.

General anesthetics cause mitochondrial dysfunction and reduction of intracellular ATP levels

Jun-Ichi Kishikawa  1 , Yuki Inoue  1 , Makoto Fujikawa  2 , Kenji Nishimura  1 , Atsuko Nakanishi  1 , Tsutomu Tanabe  2 , Hiromi Imamura  3 , Ken Yokoyama  1
Affiliations

General anesthetics cause mitochondrial dysfunction and reduction of intracellular ATP levels

Jun-Ichi Kishikawa et al. PLoS One. .

Abstract

General anesthetics are indispensable for effective clinical care. Although, the mechanism of action of general anesthetics remains controversial, lipid bilayers and proteins have been discussed as their targets. In this study, we focused on the relationship between cellular ATP levels and general anesthetics. The ATP levels of nematodes and cultured mammalian cells were decreased by exposure to three general anesthetics: isoflurane, pentobarbital, and 1-phenoxy-2-propanol. Furthermore, these general anesthetics abolished mitochondrial membrane potential, resulting in the inhibition of mitochondrial ATP synthesis. These results suggest that the observed decrease of cellular ATP level is a common phenomenon of general anesthetics.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Anesthetic treatment reduced ATP levels in nematodes.
The level of ATP in each animal was measured after treatment with anesthetics for the indicated times (n = 24 for each measurement). Nematodes were exposed to IF under saturated vapor pressure in a sealed box. For the other anesthetics, nematodes were soaked in each anesthetic solution at concentrations of 0.2% PBNa, or 0.3% 1PP. The numbers under the graphs indicate the treatment time. Error bars indicate S.D. *P < 0.05, ANOVA with Bonferroni test vs control.
Fig 2
Fig 2. In vivo ATP imaging of pharyngeal muscle cells in nematodes.
In vivo ATP imaging after treatment with each anesthetic. A, The panel shows typical YFP/CFP ratio images of nematodes after anesthetic treatment. The YFP/CFP ratio on the pharyngeal cells is pseudo-colored. B, The panel shows the averages of YFP/CFP ratios of ATeam after anesthetic treatment. Nematodes were exposed under the same conditions as used in for assessment of ATP levels. Error bars indicate S.D. *P < 0.05, ANOVA with Bonferroni test vs control.
Fig 3
Fig 3. ATP level changes in Neuro2A cells by general anesthetics.
ATP levels in Neuro2A cells were measured after each anesthetic treatment (n = 3). The values were normalized by protein concentration. The cells were treated with IF under saturated vapor pressure conditions. C, control treated without anesthetics for 30 min. Other anesthetics were added into the culture medium (0.3% 1PP, or 0.2% PBNa). The numbers under the graphs indicate the treatment time. Error bars indicate S.D. *P < 0.05, ANOVA with Bonferroni test vs control.
Fig 4
Fig 4. ATP imaging of Neuro2a cells during anesthetics treatments.
(A) Live cell imaging after treatment with the indicated anesthetics. The YFP/CFP ratios in the cells are pseudo-colored. The number in each panel indicates the treatment time (min). The cells were treated in sealed boxes in the cases of IF (left top and bottom panels). In other cases, anesthetics added into the culture medium. (B) Time courses of the average YFP/CFP ratios of the Neuro2a cells expressing ATeam. The black and red lines indicate control (n = 31) and IF (saturated vapor pressure, n = 24) conditions, respectively. IF treatment was initiated at time 0 (min). (C) Black, blue, and green lines show control (n = 26), 1PP (0.2%, n = 9), and PBNa (0.2%, n = 32) treatment, respectively. Anesthetics were added at time 5 (min). Error bars indicate S.D.
Fig 5
Fig 5. Anesthetic effect on mitochondrial ATP synthesis.
Time course of ATP synthesis by mitochondria with or without anesthetics (n = 3). Mitochondrial ATP synthesis was measured using the MASC assay [21]. Colors are the same as in Fig 4; control (black), 0.3% IF (red), 0.1% 1PP (blue), and 0.05% PBNa (green). We used 10 μg/ml oligomycin as a control inhibitor (purple). Error bars indicate S.D. The results are also summarized in Table 1.
Fig 6
Fig 6. Anesthetic effects on mitochondrial membrane potential.
Mitochondrial membrane potentials were measured using TMRE (see Materials and methods). The cells were treated by TMRE for 30 min prior to anesthetic treatment. Values represent the fluorescence intensity of TMRE. (A) IF was applied to the cells under saturated vapor pressure conditions. The intensity of IF treated cells is indicated by a red line (n = 18). The intensity of the control cells is indicated as black line (n = 12). (B) Black, blue, and green lines show the time courses of TMRE intensity of control (n = 18), 0.3% 1PP (n = 16), and 0.2% PBNa (n = 17), respectively. These anesthetics were added into each cell culture medium after the incubation with TMRE for 30 min; then, the TMRE intensity was measured at the indicated time. (C) The fraction of TMRE intensity at 30 min from results of (A) and (B). For 1PP, the value at 20 min is indicated. The colors are same as in (A) and (B). *P < 0.05, ANOVA with Bonferroni test vs control.
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Grants and funding

This work was supported by Grants-in-Aid from the Ministry of Education, Science, Sports and Culture of Japan No. 16K21472 to JK and No. 17H03648 to KY. The funder had no role in study design, data collection and analysis, decision of publish, or preparation of the manuscript.

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