Inhalational Anesthetics Induce Cell Damage by Disruption of Intracellular Calcium Homeostasis with Different Potencies

DT40 WT and its IP³R TKO cells were cultured in RPMI 1640 with 10% fetal calf serum, 1% chicken serum, 50 μm 2-mercaptoethanol, 4 mm l-glutamine, and antibiotics in a 95% air, 5% carbon dioxide humidified atmosphere at 38°C as we previously described.8,18 

DT40 cells (WT and IP³R TKO) were exposed to the equivalent of 2 minimum alveolar concentration (MAC) isoflurane (2.4%), sevoflurane (4%), and desflurane (12%) for 24 h in a gastight chamber inside the culture incubator (Bellco Glass, Inc., Vineland, NJ), with humidified 5% CO²–21% O²–balanced N²(AirGas East, Bellmawr, NJ) going through a calibrated agent-specific vaporizer as we have previously described.7–9Gas phase concentrations in the gas chamber were verified and maintained at the desired concentration throughout the experiments using an infrared Ohmeda 5330 agent monitor (Coast to Coast Medical, Fall River, MA). In a pilot study, the cell media was aspirated and extracted into hexane for high-performance liquid chromatography measurement (System Gold; Beckman Coulter, Fullerton, CA) to verify that the various anesthetic concentrations in the medium in millimolars are equivalent to the MACs in the gas phase inside the gas chamber using the concentration correlation previously described.19 

Translocation of membrane phospholipid phosphatidylserine from the inner to the outer leaflet of the plasma membrane is an early indication of cell damage. Annexin V, a phospholipid binding protein with a high affinity for phospholipid phosphatidylserine, can bind to phospholipid phosphatidylserine when it is exposed to the extracellular environment. Propidium iodide (PI) can bind to nucleic acid after penetrating a breached plasma membrane, as occurs in the later stages of cell damage. We treated DT40 cells, grown floating in the medium, with equivalent 2 MAC isoflurane (2.4%), sevoflurane (4%), and desflurane (12%) for 24 h. Our previous study demonstrated that the minimal exposure of 2 MAC for 24 h of isoflurane, but not its equivalent of sevoflurane, induced cell apoptosis in the rat primary cortical neurons.7However, it only required 1.2% isoflurane for 6 h to induce apoptosis in DT40 WT cells.8We hypothesized that sevoflurane or desflurane could still induce apoptosis but with much less potency than isoflurane in the vulnerable DT40 WT cells. As a result, we elected to use relatively high concentrations (2 MAC) for a prolonged time (24 h) of three inhalational anesthetics so that the possible toxic effects of sevoflurane or desflurane could be detected. Immediately after treatment, we determined annexin V– or PI–positive cells by the methods we have previously described.8Briefly, cells were dropped onto 25-mm coverslips and stained with annexin V or PI. Annexin V– and PI–positive cells and normal cells were counted by two persons blinded to the treatments. The percentage of annexin V– or PI–positive cells was calculated by dividing the positively stained cells by the total number of counted cells in at least four areas on each coverslip.

Increased caspase-3 activity is a hall marker for cell damage by apoptosis. We measured the caspase-3 activity immediately after treatment using a method we have described previously.8,18The assay is based on the ability of the active enzymes to cleave the fluorogenic substrates Ac-DEVD-AFC (caspase 3; Calbiochem, San Diego, CA). DT40 cells grown on six-well plates were treated with equipotent concentrations of isoflurane (2.4%), sevoflurane (4%), and desflurane (12%) for 24 h and then were harvested via  trypsinization and washed with phosphate-buffered saline. The cell pellet was gently resuspended in CelLytic M lysis buffer and protease inhibitor cocktail (Sigma, St. Louis, MO), lysed, and centrifuged; the supernatant was used for the assay. Caspase substrates were added to a final concentration of 50 μm, and the samples were incubated at 37°C for 45 min in caspase assay buffer. Incubated samples were measured at an excitation of 400 nm and an emission of 505 nm in a multiwavelength-excitation dual-wavelength-emission fluorometer (Delta RAM; Photon Technology International, Birmingham, NJ).

Cytosolic calcium concentration was measured using fura-2/AM fluorescence (Molecular Probes, Eugene, OR) with a photometer coupled to an Olympus 1Χ70 inverted microscope (Olympus America Inc., Center Valley, PA) and IPLab version 3.7 imaging processing and analysis software (Biovision Technologies, Exton, PA). The protocol to determine [Ca²⁺]^cwas same as that which we previously described.8,9Briefly, cells attached to Cell-Tak (BD Biosciences, Bedford, MA)–coated glass coverslips were washed three times with Krebs-Ringer’s buffer without addition of calcium and then loaded with 2.5 μm fura-2/AM (Molecular Probes) for 30 min at room temperature. The cells were then placed in a sealed chamber (Warner Instrument Inc., Hamden, CT) connected with multiple inflow infusion tubes and one outflow tube, which provided constant flow to the chamber. All bubbles in the chamber were flushed out at the beginning so that there was no gas phase in the sealed chamber during measurement of calcium concentration in the buffer. The cells were first washed with Krebs-Ringer’s buffer through one inflow tube for the baseline measurement of [Ca²⁺]^cand then were exposed to the approximate equivalent of 2 MAC isoflurane (0.64 mm), sevoflurane (0.96 mm), and desflurane (1.2 mm)19,via  a separate inflow infusion tube driven by a syringe pump (Braintree Scientific Inc., Braintree, MA). The anesthetic concentrations in the buffer were determined with high-performance liquid chromatography (System Gold; Beckman Coulter) as we described previously.9,20The fluorescence signals were measured with excitation at 340 and 380 alternatively and emission at 510 nm for a period up to 15 min for each treatment. The ratios of F³⁴⁰/F³⁸⁰reflected the cytosolic calcium concentrations and were used for comparison among three inhalational anesthetics. The final result of F³⁴⁰/F³⁸⁰was averaged from the cells of at least three separate experiments. A pilot study confirmed that the cells were still viable at the end of experiments for calcium measurement.

We measured the calcium concentrations inside the ER ([Ca²⁺]^e) using the dye mag-fura-2/AM (Molecular Probes) and a previously described protocol.21Briefly, DT40 cells were incubated with 5 μm mag-fura-2/AM in modified Hanks balanced salt solution buffered with HEPES (HBSS-H: 137 mm NaCl, 5.4 mm KCl, 1.3 mm CaCl², 0.41 mm MgSO⁴, 0.49 mm MgCl², 0.34 mm Na2HPO⁴, 0.44 mm NaH2PO⁴, 5.5 mm glucose, and 20 mm HEPES/NaOH [pH 7.4]) containing 1% bovine serum albumin for 45 min at 37°C in a dark room. The mag-fura-2/AM–loaded cells were then washed with bovine serum albumin–free HBSS-H and attached to Cell-Tak–coated glass coverslips placed at the bottom of the recording chambers. The cells were then exposed to intracellular-like medium containing 125 mm KCl, 19 mm NaCl, 10 mm HEPES (pH7.3 with KOH), 330 μm CaCl², and 100μg/ml (wt/vol) saponin (Sigma) for 1–2 min to permeabilize the cells. Thereafter, the permeabilized cells were washed with intracellular-like medium and then incubated in intracellular-like medium containing 3 mm ATP and 1.4 mm MgCl²for at least 5 min. The coverslips were then placed in a sealed chamber (Warner Instrument Inc., Hamden, CT) connected with multiple inflow infusion tubes and one outflow tube, which provided constant flow to the chamber. The cells were washed with running buffer through one inflow tube for the baseline measurement of [Ca²⁺]^eat first and then were exposed to equivalent concentrations of isoflurane (0.64 mm), sevoflurane (0.96 mm), or desflurane (1.2 mm) via  a separate inflow infusion tube driven by a syringe pump (Braintree Scientific Inc.). The fluorescence signals were measured with excitation at 340 and 380 alternatively and emission at 510 nm for a period up to 6 min for each treatment. The ratios of F³⁴⁰/F³⁸⁰reflected the ER calcium concentrations relatively and were used for comparison among three inhalational anesthetics. The final results of F³⁴⁰/F³⁸⁰were averaged from the cells of at least three separate experiments and compared among three inhalational anesthetics. All these experiments were performed at room temperature.

The method used was the same as we described previously.8,18DT40 cells (WT and IP³R TKO) were loaded with 2 μm rhod-2/AM in cell medium containing 2.0% bovine serum albumin in the presence of 0.003% pluronic acid at 37°C for 50 min, then attached to Cell-Tak–coated glass coverslips. The cells were washed and placed on a stage and exposed to equivalent concentrations of isoflurane (0.64 mm), sevoflurane (0.96 mm), or desflurane (1.2 mm) dissolved in the perfusion buffer. The images were recorded using the Radiance 200 imaging system (Bio-Rad Laboratories, Hercules, CA) with excitation at 568 nm. The relative changes of fluorescence signals were determined and compared among three inhalational anesthetics.

We used GraphPad Prism 4 software (GraphPad Software, Inc., San Diego, CA) for all statistical analysis. Annexin V and propidium iodide staining were expressed as percentage of total cells examined. Changes of calcium concentrations in ER ([Ca²⁺]^e), cytosol ([Ca²⁺]^c), and mitochondria ([Ca²⁺]^m) were expressed as a percentage of their own baseline. All data were expressed as mean ± SD. We analyzed all the data with one-way analysis of variance followed by Tukey multiple comparison tests; P < 0.05 was considered statistically significant.

We have previously demonstrated that isoflurane induced apoptosis only in DT40 WT and not in IP³R TKO in a dose- and time-dependent manner.8Because sevoflurane and desflurane, at equipotent exposure to isoflurane, seemed not to induce cell damage in neuronal cultures in our previous studies,7,9we investigated whether the three inhalational anesthetics also have different potencies to induce apoptosis. All three inhalational anesthetics induced significant cell damage determined by both annexin V and PI staining only in DT40 WT and not in IP³R TKO (fig. 1A). Isoflurane significantly increased the percentage of annexin V– and PI–positive cells from 9.1 ± 2.4% and 8.7 ± 3.9% in controls to 28.4 ± 5.6% and 25.4 ± 6.7%, respectively, after treatments only in DT40 WT cells (P < 0.001, n = 29 for all controls, n = 28 for all treatments; figs. 1B and C). Compared with control, sevoflurane and desflurane also significantly increased the percentage of annexin V–positive cells from 9.4 ± 2.2% and 9.2 ± 2.3% in controls to 21.4 ± 2.9% and 20.2 ± 3.1%, respectively, after treatment only in DT40 WT cells (P < 0.001 for all, n = 29 for all controls, n = 28 for all treatments; fig. 1B). Similarly, sevoflurane and desflurane significantly increased the percentage of PI–positive cells from 8.1 ± 2.6% and 8.6 ± 2.4% in controls to 17 ± 2.7% and 16.9 ± 2.1%, respectively, after treatments (P < 0.001 for all, n = 29 for all controls, n = 28 for all treatments; fig. 1C) only in WT DT40 cells. Consistent with our previous finding,7,9isoflurane, at equipotent exposure to sevoflurane and desflurane, induced significantly greater cell damage than either sevoflurane or desflurane in WT DT40 cells (P < 0.001 for both annexin V and PI; figs. 1B and C). We also compared the potency of three commonly used inhalational anesthetics on induction of apoptosis in chicken B lymphocytes by measuring caspase-3 activity, which is one of the hallmarks of cell death by apoptosis. Isoflurane dramatically increased caspase-3 activity as a percentage of control to 358 ± 60% (P < 0.001, n = 8 for control, n = 9 for isoflurane treatment; fig. 2) only in WT DT40 cells, whereas sevoflurane and desflurane did not increase caspase-3 activity significantly (102 ± 2.6% and 87 ± 14%, respectively, as a percentage of control, P > 0.05, n = 4 for both controls and treatments; fig. 2). Isoflurane at equipotent concentrations caused a markedly greater caspase-3 activity in comparison with sevoflurane and desflurane (P < 0.001; fig. 2) only in DT40 WT cells. All three inhalational anesthetics did not induce significant increases of caspase-3 activity in DT40 IP³R TKO cells.

To support our hypothesis that inhalational anesthetics can induce cell damage by causing calcium release from the ER via  activation of IP³R, we measured the change of ER calcium after exposure of DT40 cells to these anesthetics at equipotent concentrations. All three inhalational anesthetics significantly decreased ER calcium selectively only in DT40 WT and not in IP³R TKO cells (fig. 3A), indicating that calcium is released from the ER via  activation of IP³R. Isoflurane, sevoflurane, and desflurane significantly reduced ER calcium concentration as percentage decrease below their initial baseline maximally by 17.6 ± 6.6% (n = 21), 11.6 ± 3.8% (n = 18), and 11 ± 4.2% (n = 22), respectively (P < 0.001 for all; figs. 3B and C), in DT40 WT cells. However, isoflurane, sevoflurane, and desflurane only reduced ER calcium level as percentage decrease below their baseline level by 1.4 ± 0.9% (n = 18), 1.9 ± 0.8% (n = 21), and 1.2 ± 0.3% (n = 35), respectively, in DT40 IP³R TKO cells, significantly less than in their corresponding WT DT40 cells (P < 0.001 for all; fig. 3C). These results were consistent with the greater potency of isoflurane than sevoflurane or desflurane to induce cell damage by apoptosis (figs. 1 and 2).

To confirm that the calcium released from the ER by inhalational anesthetic–mediated activation of IP³R was transferred into mitochondria via  the cytosolic space, we measured the changes of calcium concentrations in both the cytosolic space and the mitochondria. Isoflurane, sevoflurane, and desflurane significantly increased peak cytosolic calcium (F³⁴⁰/F³⁸⁰ratio) as percentages above their own baseline controls to 162.7 ± 64% (n = 39), 79.4 ± 19% (n = 15), and 67.5 ± 29% (n = 21) (P < 0.001 for all; figs. 4A and B) only in DT40 WT cells, which were also significantly higher than their effects in DT40 IP³R TKO cells (6.3 ± 3.5%, n = 23, 3 ± 1.4%, n = 38, and 3 ± 1.4%, n = 39, respectively, P < 0.001 for all; figs. 4A and B). Similarly, all three inhalational anesthetics significantly increased mitochondrial calcium concentrations only in DT40 WT cells (fig. 5). Isoflurane, sevoflurane, and desflurane significantly increased mitochondrial calcium concentrations as percentages of increase above their own baseline control to 121.1 ± 80% (n = 26), 51.5 ± 22.5% (n = 25), and 64.8 ± 34% (n = 27) (P < 0.001 for all; figs. 5A and B) only in DT40 WT cells, which were also significantly higher than their effects in DT40 IP³R TKO cells (2.7 ± 2.7%, n = 27, 1.4 ± 2.8%, n = 28, and 1.3 ± 2.3%, n = 28, respectively, P < 0.001 for all; figs. 5A and B). These results were consistent with their effects on decreasing ER calcium (fig. 3), increasing cytosolic calcium (fig. 4), and inducing cell damage (fig. 1 and 2).

Our results suggest that inhalational anesthetics induce apoptosis in lymphocytes via  excessive activation of IP³R on the ER membrane. The apoptosis induced by inhalational anesthetics was associated with a decrease of calcium concentrations in the ER, and the sequential increase of calcium concentrations in the cytosolic space and then the mitochondria. This most likely represented transfer of ER calcium into mitochondria via  the cytosolic space, which is much more efficient than the calcium transfer originating from the calcium influx from the extracellular space.22It is generally believed that calcium ions in cytosolic space are transferred into mitochondria by a calcium ion uniporter.22The excessive cytosolic calcium originating from the ER can cause overloading of mitochondria with calcium, collapse of mitochondrial membrane potential, and subsequent release of cytochrome c  from mitochondria, triggering cell death by apoptosis.23The current study provides a novel finding that all three inhalational anesthetics actually decrease ER calcium by directly measuring ER calcium concentrations. The significant decrease of ER calcium concentrations after exposing DT40 to isoflurane in WT but not IP³R TKO cells further supports the finding from our previous reports8,9that isoflurane induced calcium release from the ER via  activation of IP³R. We did not test the effect of the IP³R antagonist xestospongin C on cell damage induced by inhalational anesthetics in the current study because xestospongin C does not behave consistently as a selective IP³R antagonist24and we have previously demonstrated the inhibitory effects of xestospongin C on isoflurane-mediated cell damage in other types of cells.8The major advantage of using the DT40 IP³R TKO cell line is to selectively study the role of this receptor on excessive calcium release from the ER and the subsequent apoptosis mediated by inhalational anesthetics, which is a more powerful tool than the relatively less selective IP³R antagonist xestospongin C. More studies are needed to investigate whether inhalational anesthetics can directly activate IP³R on the ER membrane or simply increase the production of IP³or augment the opening probability of IP³R upon activation by their agonists.

Isoflurane seems to behave consistently throughout our studies in having greater potency to induce apoptosis and calcium release from the ER than sevoflurane or desflurane.7–9This is especially obvious on comparison of activation of caspase 3 induced by three inhalational anesthetics, with isoflurane but not sevoflurane or desflurane inducing remarkable elevation of caspase-3 activity, a hallmark of cell death by apoptosis in WT DT40 cells. It is still not clear why sevoflurane and desflurane significantly increased annexin V and PI but not caspase 3–positive cells compared with their corresponding controls. Annexin V, a phospholipid-binding protein with a high affinity for phospholipid phosphatidylserine, can bind to phospholipid phosphatidylserine when it is exposed to the extracellular environment during early stage of cell damage. Propidium iodide (PI) can bind to nucleic acid after penetrating a breached plasma membrane, as occurs in the later stages of cell damage. Caspase-3 activation is the hallmark of apoptosis. We speculated that annexin V might also detect some damaged cells without apoptosis process, whereas caspase-3 activity assay detected only apoptotic cells. It is possible that sevoflurane or desflurane did not activate caspase 3 (fig. 2). DT40 IP³R TKO chicken lymphocytes were resistant to the transfer of ER calcium into mitochondria via  cytosolic space and apoptosis induced by all three inhalational anesthetics. Isoflurane has greater potency to induce calcium release from the ER, most likely derived from its greater activation of IP³Rs. It seems that neurons are more resistant to inhalational anesthetic–induced apoptosis than lymphocytes because the minimum exposure of isoflurane required to induce neuronal damage was 2 MAC for 24 h.7,8,25It should be noted that sevoflurane or desflurane could also induce cell damage determined by annexin V and PI staining in the vulnerable lymphocytes, but with significantly less potency than isoflurane. Our previous studies suggested that isoflurane induced apoptosis in DT40 WT cells and primary cortical neurons in a concentration- and time-dependent manner.7,8The minimal exposure for isoflurane to induce apoptosis in DT40 cells was approximately 1.2% for 6 h.8In normal human peripheral lymphocytes, only 0.85% isoflurane was needed to induce apoptosis.4We have chosen a treatment regiment with relatively high concentrations (equivalent to 2 MAC) for a long duration (24 h) to test the hypothesis that sevoflurane and desflurane could still induce apoptosis as isoflurane, but with much lower potencies. An intervention with low concentration and duration would be less likely to cause any detectible sevoflurane or desflurane cytotoxicity, as we have demonstrated previously in other types of cells.7,25Our results endorse the hypothesis that sevoflurane and desflurane at equipotent exposure of 2 MAC for 24 h induces cell damage determined with annexin V and PI staining in DT40 lymphocytes, but with significantly less potency than isoflurane. In peripheral human lymphocytes, both isoflurane and sevoflurane induced apoptosis,4,5with isoflurane having greater potency than sevoflurane at the same concentrations.4Loop et al.  5also demonstrated that isoflurane and sevoflurane, but not desflurane, induced apoptosis dose dependently in human T lymphocytes. Further animal and clinical studies are needed to determine whether desflurane may be a better choice than other inhalational anesthetics in patients with peripheral lymphocytopenia.

All inhalational anesthetics evidently affect intracellular calcium homeostasis. A transient and moderate elevation of cytosolic calcium by isoflurane may provide cytoprotection through up-regulation of host preconditioning responses,26,27but prolonged exposure to higher concentrations of isoflurane, such as this study, may maintain the IP³R in an open state, in turn excessively elevating [Ca²⁺]^cand [Ca²⁺]^m, depleting ER calcium, and ultimately leading to cell damage.28,29It seemed that the potency for inhalational anesthetics to induce cell apoptosis correlated with their potency to decrease ER calcium concentrations and subsequently their capacity to increase cytosolic and mitochondrial calcium. The greater likelihood of isoflurane to induce calcium release from ER than sevoflurane may also contribute to its greater ability to precondition neurons and provide neuroprotection against neurotoxicity induced by the prolonged use of isoflurane, as demonstrated in our previous study.25Future studies are needed to quantify the amount of ER calcium decrease required to convert the short-exposure neuroprotective effects into neurotoxic effects by a prolonged isoflurane exposure.

This study has several limitations that should influence in vivo  interpretations: (1) DT40 chicken lymphocytes are from immortal transformed cell lines. Although clearly different from normal cells, in general, such cells are more resistant to stressors, making the results of our study somewhat more relevant to the in vivo  situation. Although the mechanisms for this vulnerability variance among different types of cells are elusive, it seems that neurons in cell cultures are relatively less vulnerable to isoflurane-mediated neurotoxicity.7–9,25(2) Because the DT40 cells are chicken lymphocytes, not human lymphocytes, it is difficult to refer the finding in this study to clinical importance. Previous studies have demonstrated that inhalational anesthetics induced apoptosis in human lymphocytes with different potencies,4,5whereas the current study provided mechanisms of how the inhalational anesthetics induce apoptosis in lymphocytes. (3) We used inhalational anesthetics at relatively high concentrations for a prolonged time, which is less ideal to mimic the clinical scenario in patients. Further studies are needed to compare the potency of three commonly used inhalational anesthetics on intracellular calcium homeostasis and apoptosis in animals and patients in the future.

Taken together, our findings suggest that the commonly used inhalational anesthetic isoflurane may induce apoptosis in lymphocytes by significantly decreasing ER calcium and subsequently increasing cytosolic and mitochondrial calcium, which was associated with an excessive activation of IP³R. Sevoflurane and desflurane have significantly less potency to induce calcium release from the ER and apoptosis in these lymphocytes.

The authors thank Tomohiro Kurosaki, M.D. Ph.D. (Professor, Institute of Physical and Chemical Research, Research Center for Allergy and Immunology, Turumi-ku, Yokohama, Kanagawa, Japan), for providing total IP³R knockout chicken lymphocytes; Qingcheng Meng, Ph.D. (Senior Research Scientist, Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, Pennsylvania), for technical support; and Roderic Eckenhoff, M.D. (Professor of Anesthesia), Maryellen Eckenhoff, Ph.D. (Research Associate), and Randall Pittman, Ph.D. (Professor of Pharmacology, University of Pennsylvania), for discussions.