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Anesth Analg 2008; 106:492-500
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e3181605b71
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ANESTHETIC PHARMACOLOGY

A Presenilin-1 Mutation Renders Neurons Vulnerable to Isoflurane Toxicity

Ge Liang, MD, Qiujun Wang, MD, Yujuan Li, MD, Baobin Kang, MD, Maryellen F. Eckenhoff, PhD, Roderic G. Eckenhoff, MD, and Huafeng Wei, MD, PhD

From the Department of Anesthesiology and Critical Care, University of Pennsylvania, Philadelphia, Pennsylvania.

Address correspondence and reprint requests to Huafeng Wei, MD, PhD, Department of Anesthesiology and Critical Care, University of Pennsylvania, 305 John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104. Address e-mail to weih{at}uphs.upenn.edu.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: Isoflurane, a commonly used inhaled anesthetic, induces apoptosis in rat pheochromocytoma neurosecretory cells (PC12) in a concentration- and time-dependent manner via an as yet unknown mechanism. We hypothesize that isoflurane induces apoptosis by causing abnormal calcium release from the endoplasmic reticulum (ER) via activation of inositol 1,4,5-trisphosphate (IP3) receptors. A presenilin-1 (PS1) mutation associated with familial Alzheimer’s disease was shown to increase the activity of IP3 receptors, and therefore may render cells vulnerable to isoflurane-induced cytotoxicity. Sevoflurane and desflurane have less ability to disrupt intracellular calcium homeostasis; and thus we predict they will cause less cytotoxicity.

METHODS: PC12 cells transfected with wild type, vector alone (Vector) or mutated PS1 (L286V) were treated with equivalent of 1 MAC of isoflurane, sevoflurane, and desflurane for 12 h. Mitochondria redox activity (MTT reduction) and lactate dehydrogenase release assays were performed to evaluate cell viability. Changes of calcium concentration in cytosolic space ([Ca2+]c) and production of reactive oxygen species (ROS) were determined after exposing different types of cells to various inhaled anesthetics. We also determined the effects of IP3 receptor antagonist xestospongin C on isoflurane-induced cytotoxicity and calcium release from the ER in L286V PC12 cells, and in rat primary cortical neurons.

RESULTS: Isoflurane at 1 MAC for 12 h induced cytotoxicity in L286V but not wild type or vector PC12 cells, and also caused greater and faster increase of peak [Ca2+]c in the L286V cells. Xestospongin C significantly attenuated isoflurane cytotoxicity in both L286V cells and primary cortical neurons and inhibited the calcium release from the ER in L286V cells. Isoflurane did not induce significant changes of ROS production in any type of PC12 cells. Sevoflurane and desflurane at equivalent exposure to isoflurane did not induce similar cytotoxicity or increase of peak [Ca2+]c in L286V PC12 cells.

CONCLUSION: Our results show that the L286V PS1 mutation augments the isoflurane-induced [Ca2+]c increase via calcium release from intracellular stores which, in turn, renders the cells vulnerable to isoflurane neurotoxicity. ROS production was not involved in isoflurane-induced neurotoxicity. Sevoflurane and desflurane, at equivalent exposure to isoflurane, did not induce a similar increase of [Ca2+]c or neurotoxicity in L286V PC12 cells.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Isoflurane causes cytotoxicity in different types of cells via an unknown mechanism.1–9 Also, isoflurane triggers widespread neuronal apoptosis in the developing rat brain, with subsequent persistent learning deficits.10 Isoflurane also caused cognitive dysfunction persisting for several weeks after treatment in adult and aged rats11,12 and mice.13 About 20% of patients older than 60-yr-of-age develop cognitive deficits after surgery,14 but the linkage to general anesthesia per se is not yet established. Although inhaled anesthetics are structurally and chemically similar, they appear to have different toxic potencies, for still unclear reasons.1 We have demonstrated previously that nonphysiological calcium release from the endoplasmic reticulum (ER) may be associated with isoflurane neurotoxicity because dantrolene, a ryanodine receptor (calcium release channel) antagonist that inhibits calcium release from the ER (and sarcoplasmic reticulum), significantly inhibited isoflurane cytotoxicity.1 The 1,4, 5-trisphosphate inositol (IP3)receptor (IP3R), another calcium release channel located on the ER membrane, may also play a important role in triggering apoptosis in neurons and in other cells by causing nonphysiological calcium release from the ER. This leads to depletion of ER calcium, increase of cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) calcium, all of which can contribute to cell death.15,16 The L286V or M146V presenilin-1 (PS1) mutation, a defect linked to familial Alzheimer’s disease (AD), is associated with increased ryanodine receptors17 or activity of the IP3 receptors18,19 respectively, which we predict will render cells more vulnerable to cytotoxicity induced by substances that activate ryanodine or IP3 receptors.

Isoflurane induces nonphysiological calcium release from ER via ryanodine receptors in neurons,20 although various subtypes of ryanodine receptors may react to anesthetics differently.21 However, desflurane has only minor effects22 and sevoflurane has either less23 or no effect,24,25 on Ca2+ release from the intracellular calcium stores. Consistent with these effects on calcium release, isoflurane, but not sevoflurane or desflurane at equivalent concentrations, triggers apoptosis in cultured cells and neurons,1,3,26 an effect partially inhibited by dantrolene.1 Further, isoflurane may have more specific effects on the underlying pathogenesis of neurodegenerative diseases. It enhances the production,8 aggregation and cytotoxicity2 of β-amyloid in cultured cells, and plaque load in transgenic animals.13 These observations might also be triggered by its upstream effects on calcium. We hypothesize that the PS1 mutation renders neurons more vulnerable to isoflurane neurotoxicity via augmented calcium release from the intracellular calcium stores via either ryanodine or IP3 receptors. In addition, because isoflurane-induced calcium release from the ER is transferred into mitochondria, it is possible that mitochondrial derived reactive oxygen species (ROS)27 contribute to the toxicity. Excessive ROS can induce apoptosis,28 but it is not clear if isoflurane has important effects on ROS production.

The aim of this study is to clarify whether the PS1 mutation enhances vulnerability to isoflurane-induced apoptosis, whether this effect is caused by ROS or calcium dysregulation, and whether vulnerability extends to other inhaled anesthetics.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultures
Rat pheochromacytoma cells (PC12) transfected with wild type PS1 (WT), vector alone control (Vector) and point mutated PS1 (L286V) were cultured as previously described.1,17,29 Briefly, cells were maintained in DMEM medium (Invitrogen Corporation, Grand Island, NY) supplemented with 10% heat-inactivated horse serum (Invitrogen Life Technologies, Carlsbad, CA), 5% fetal calf serum (Hyclone Laboratories, Logan, UT), 200 µg/mL G418 (Mediatech, Herrden, VA) and penicillin/streptomycin (Invtrogen Life Technologies, Carlsbad, CA). Monolayer cultures at a density of 0.3 x 105 cells/cm2 were incubated in plastic flasks precoated with 0.01% poly-l-ornithine (Sigma-Aldrich, St. Louis, MO) in a 95% air, 5% CO2 humidified atmosphere at 37°C. The culture medium was changed every 48 h. The transfection of the WT and mutant PS1 has been described and confirmed in detail previously.29,30 Before anesthetic exposures, all PC12 cells were transferred from culture flask into 24 well plates. Although of neurologic origin, PC12 cells may not accurately reflect mechanisms in normal neurons, so we used cultures of rat primary neurons to test whether isoflurane induces toxicity via actions on the IP3 receptor. The use of pregnant rats for primary cortical neuronal culture approved by the Institutional Animal Care and Use at the University of Pennsylvania. Primary cultures of cortical neurons were prepared from the dissociated cortices of rat fetus at embryonic day 16–18 essentially using a protocol previously described.1 Cortices were dissected from embryonic brain, and meninges were removed from the tissues. The cells were dissociated by trypsinization and trituration, followed by DNase treatment. The dissociated cells were resuspended in serum-free B27/neurobasal medium and were plated at a density of 1 x 105 cells/cm2 on poly-d-lysine-coated 96-well plates. Cultures were maintained in serum-free B27/neurobasal medium in a humidified atmosphere (5% CO2, 95% air) at 37°C. More than 95% of the cells present on day 5 in vitro differentiate into neurons, as characterized by the appearance of long neurites expressing neurofilament protein. Half of the medium was changed every fourth day. Mature neurons up to day 16 in vitro were used for the designed experiments.

Anesthetic Exposures
All cells grown on 24-well plates were exposed to anesthetics in a gas-tight chamber inside the cell-culture incubator (Bellco Glass, Vineland, NJ), with 5%CO2/21%O2/balance N2 (AirGas East, Bellmawr, NJ) going through a calibrated agent-specific vaporizer, as described previously.1 Gas phase concentrations in the gas chamber were verified with infrared absorbance of the effluent gas, and constantly monitored and maintained at the designed concentration throughout experiments using an infrared Ohmeda 5330 agent monitor (Coast to Coast Medical, Fall River, MA). In a pilot study, the media was aspirated and extracted into hexane for high performance liquid chromatography (System Gold, Beckmam Coulter, Fullerton, CA) to verify the various anesthetic concentrations in the cell medium (in mM) are equivalent to the MAC concentration in the gas phase inside the gas chamber using the concentration correlation previously described.31

Cytotoxicity Assays
For the cytotoxicity assays, cells grown in 24-well plates were treated with different inhaled anesthetics as described previously,1 while control cells grown on separate 24-well plates were in the same incubator but not exposed to volatile anesthetics. Pilot studies demonstrated that the carrier gas alone did not significantly affect cell survival (data not shown). After the exposures, the plated cells were immediately used for lactate dehydrogenase (LDH) release and MTT (3-(4, 5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) reduction assays. The LDH release assay determined the cell plasma membrane integrity by measuring the degree of the LDH enzyme released from the cell into the culture medium, representing a relatively late stage of cell damage. The MTT reduction assays determined the cellular redox activity by measuring the mitochondrial dehydrogenase activity that reduces MTT, representing mitochondrial dysfunction, an earlier stage of damage. The amount of LDH released into the media after exposure to anesthetics was detected as described previously,1 using a LDH assay kit (Promega, Madison, WI). Briefly, 50 µL of media was mixed with 50 µL of substrate mix, and the assay plates incubated for 30 min at room temperature. The reaction was terminated with stop solution and the sample quantified spectrophotometrically at 490 nm with a plate reader (OPSYS MRTM Absorbance Reader, Dynex Technologies, Chantilly, VA). Background signal from the media was also measured and subtracted. Using a set of identically exposed cells, MTT reduction was determined using a quantitative colorimetric assay.1,32 MTT at 125 µg/mL (Sigma-Aldrich, St. Louis, MO) was added to the growth medium and the cells were incubated for 1 h at 37°C. The medium was then aspirated and the MTT reduction product, formazan, was dissolved in dimethyl sulfoxide and quantified spectrophotometrically at 570 nm. The results of both LDH release and MTT reduction assays were expressed as percentage of control.

Measurement of Cytosolic Calcium Concentration ([Ca2+]c)
[Ca2+]c was determined using fura-2 fluorescence (Molecular probe, Eugene, OR) with a photometer coupled to an Olympus 1 x 70 inverted microscope and the IPLab v3.7 imaging Processing and Analysis software (Biovision Technologies, Exton, PA www.BioVis.com.) with the measurement of F340/F380 ratio. The protocol to determine the F340/F380 ratio was similar to that previously described with some modifications.33 Briefly, PC12 cells were grown on 25 mm round glass cover slips, coated with 0.01% poly-L-ornithine, in DMEM medium at a density of 1 x 105 cells, 4 days before the calcium measurement experiments. On the day of the calcium measurements, the cells were first washed 3x with Krebs-Ringer buffer (HEPES 10 mM, NaCl 145 mM, KCl 5 mM, NaHPO4 0.5 mM, Glucose 10 mM, MgSO4 1 mM, CaCl2 1 mM, pH 7.4). They were then loaded with 2.5 µM fura-2/am (Molecular Probes, Eugene, OR) in Krebs-Ringer buffer for 30 min at room temperature, washed 3x with Krebs-Ringer buffer. The cells were then placed in a sealed chamber (Warner Instrument, Hamden, CT) connected with multiple inflow infusion tubes and one outflow tube, which provided constant flow to the chamber. The cells were washed with Krebs-Ringer buffer through one inflow tube for the baseline measurement and then exposed to different volatile anesthetics via a separate inflow infusion tubes driven by a syringe pump (Braintree Scientific, Braintree, MA). The concentrations of isoflurane, sevoflurane, and desflurane in the Krebs-Ringer buffer were approximately 0.4 mM, 0.46 mM, and 0.66 mM respectively, each corresponding to approximately 1.3 MAC.31 Samples of the volatile anesthetics in both the inflow and outflow tubes were collected and their concentrations measured by high performance liquid chromatography (System Gold, Beckmam Coulter, Fullerton, CA) to confirm constant anesthetic concentrations. The fluorescence signals were measured with excitation at 340 and 380 alternatively and emission at 510 nM for a period up to 18 min for each treatment. The F340/F380 ratio, which correlated to the level of cytosolic calcium concentration, was constantly determined after exposing cells to various volatile anesthetics. The results of the F340/F380 ratio were averaged from a minimum of 30 cells in at least three separate experiments. The peak cytosolic calcium after treatment of various inhaled anesthetics was measured at the time point of highest F340/F380 ratio and this peak level could appear at different time after starting various treatments.

Measurement of ROS
ROS production was measured as previously described with minor modifications.32 Briefly, cells growing in 24-well plates at density of 2 x 105/well with or without treatment of isoflurane at 2 MAC for 24 h were loaded with 5 µM DCFH-DA (Molecular Probe, Eugene, OR) diluted in PBS buffer, and incubated for 45 min at 37°C. DCFH-DA is permeable to the cells and can be hydrolyzed by cellular esterase to form 2',7'-dichlorofluorescin (DCFH). Oxidation of DCFH by hydrogen peroxide and hydroxyl radicals yields a highly fluorescent product, 2',7'-dichlorofluorescein (DCF). The intensity of DCF fluorescence was determined in triple by using a Chameleon multilabel plate reader (Hidex Personal Life Science, Hidex Oy, Finland), with an excitation wavelength of 450 nM and an emission wavelength of 535 nM. The background fluorescence intensity in the control wells without DCFH-DA was subtracted in all cases. Fluorescence intensities expressed as a percentage of control were compared among all experimental groups.

Statistical Analysis
Results for effects of xestospongin c on isoflurane-induced calcium release from ER in L286V cells were analyzed with unpaired two-tail t-test. All other data were analyzed by one-way ANOVA followed by Newman-Keuls multiple comparison tests. P < 0.05 was considered as statistical significance.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Isoflurane, but not Sevoflurane or Desflurane at Equipotent Concentrations, Induced Cytotoxicity in PC12 Cells Transfected with the PS1 Mutation
Our prior study1 demonstrated that the cytotoxic effect of isoflurane was concentration- and time-dependent and that a minimal exposure of 2 MAC for 24 h was needed to induce cytotoxicity in WT PC12 cells. Thus, in PC12 cells transfected with the PS1 mutation, we examined the effect of isoflurane at an exposure regimen lower than 2 MAC for 24 h and compared with WT or vector controls. As shown in Figure 1A, only 1.2% isoflurane (approximately 1 MAC) for 12 h caused a significant reduction of MTT (early cell damage) and an increase of LDH release (late cell damage) in the L286V, but not in WT or vector control PC12 cells. An exposure regimen of isoflurane at <1.2% for 12 h did not induce cytotoxicity in any type of PC12 cells (data not shown). In contrast, neither sevoflurane (1 MAC, 2% for 12 h) nor desflurane (1 MAC, 6.6% for 12 h) caused significant cytotoxicity in any of the PC12 cells (Figs. 1B and C).


Figure 121
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  		Figure 1. Isoflurane, but not sevoflurane or desflurane, induced cytotoxicity in PC12 cells transfected with the mutated presenilin-1 (L286V). L286V, vector alone (Vector) or wild type presenilin-1 (WT) were exposed to equivalent of 1 MAC of isoflurane (1.2%, Fig. 1A), sevoflurane (2%, Fig. 1B) and desflurane (6.6%, Fig. 1C) for 12 h. MTT reduction and LDH release assays were done immediately after treatments. Data represent mean ± sd of at least 12 repeats from a minimum of three separate experiments. **P < 0.01, ***P < 0.001 compared with its own corresponding control without isoflurane treatment.

 

Effects of Volatile Anesthetics on Intracellular Calcium Homeostasis
[Ca2+]c was determined by measuring the ratio of F340/F380 during exposure of the three different types of PC12 cells to isoflurane, sevoflurane, or desflurane. Our pilot study confirmed that the F340/F380 ratio baseline did not change significantly, even after continuously exposing these cells for 18 min to the excitation UV light, nor did the cell viability change significantly, as determined by a trypan blue assay (data not shown). In the presence of extracellular calcium, isoflurane at about 1.3 MAC (0.4 mM) immediately increased the peak ratio of F340/F380 in the L286V cells, which was significantly higher than in WT or vector control PC12 cells (Figs. 2A and B). In addition, the time to reach peak ratio of F340/F380 in L286V cells was significantly shorter than in WT or L286V PC12 cells (Figs. 2A and C). These results suggested an association between isoflurane-induced cytotoxicity and its greater ability to disrupt intracellular calcium homeostasis in L286V PC12 cells.


Figure 221
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  		Figure 2. Isoflurane increased the mean peak F340/F380 ratio more significantly in PC12 cells transfected with mutated presenilin-1 (L286V) than in wild type (WT) or vector controls. Data represent mean ± sd from minimum of 30 neurons of at least three separate experiments. (A) Averaged typical response of F340/F380 induced by 0.4 mM isoflurane (1.3 MAC) in three different types of PC12 cells. (B) Comparison of the peak increase of F340/F380induced by 0.4 mM isoflurane. ***P < 0.001 compared with vector or WT control. (C) Isoflurane increased faster peak increase of F340/F380 ratio in L286V than in WT or Vector PC12 cells. ***P < 0.001 compared with L286V and WT; +++P < 0.001 compared with L286V.

 

To determine whether calcium changes might underlie the differences in cytotoxic potency of the volatile anesthetics in PS1 mutated PC12 cells, we compared changes in [Ca2+]c after exposing L286V PC12 cells to 1.3 MAC equivalent concentrations of isoflurane (0.4 mM), sevoflurane (0.46 mM) and desflurane (0.66 mM). Isoflurane caused a significantly larger (Figs. 3A and B) and more rapid (Figs. 3A and C) increase in the peak F340/F380 ratio than either sevoflurane or desflurane in the presence of extracellular calcium in PS1 mutated PC12 cells. These results suggested an association between greater cytotoxicity and greater disturbance of intracellular calcium homeostasis by isoflurane in PS1 mutated PC12 cells, and contrast with the lack of change induced by either sevoflurane or desflurane.


Figure 321
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  		Figure 3. Isoflurane increased significantly greater and faster mean peak F340/F380 ratio than sevoflurane or desflurane in PC12 cells transfected with mutated presenilin-1 (L286V). Data represent mean ± sd from a minimum of 30 neurons of at least three separate experiments. (A) Averaged typical response of F340/F380 induced by equivalent of 1.3 MAC isoflurane (ISO, 0.4 mM), sevoflurane (SEVO, 0.46 mM), and desflurane (DES, 0.66 mM) in L286V PC12 cells. (B) ISO increased greater peak F340/F380 as percentage over its own baseline than SEVO or DES in L286V PC12 cells. ***P < 0.001 compared with SEVO or DES. (C) ISO induced faster peak increase of F340/F380 ratio than SEVO or DES in L286V PC12 cells. +++P < 0.001 compared with ISO; ***P < 0.001 compared with both ISO and SEVO.

 

We further tested if calcium release from ER IP3 receptors contributed to the isoflurane-induced cytotoxicity and increase of [Ca2+]c in L286V PS1 PC12 cells as well as in primary cortical neurons as demonstrated previously.1 Pretreatment with a potent IP3 receptor antagonist xestospongin C34,35 for 30 min nearly abolished the LDH release induced by 2.4% isoflurane for 24 h in both primary cortical neurons and in PS1 mutated PC12 cells (Fig. 4A). In the absence of extracellular calcium, calcium release from intracellular stores contributes to most isoflurane-induced [Ca2+]c increase in L286V cells (compare Figs. 2A and B, Figs. 3A and B with Figs. 4B and C), which again was nearly abolished by xestospongin C (Figs. 4B and C). No measurements of [Ca2+]c in cerebral cortical neurons were performed due to the technical difficulty.


Figure 421
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  		Figure 4. The IP3 R antagonist xestospongin C inhibited isoflurane-induced cytotoxicity and calcium release from the endoplasmic reticulum (ER). (A) Pretreatment of xestospongin C (Xc, 100 nM) for 30 min abolished the LDH release (late cell damage) induced by 2.4% isoflurane (ISO) for 24 h in both aged rat cerebral cortical neurons (CC DIV16) and L286V PC12 cells. Data represent mean ± sd from minimal 12 repeats (n > 12) of three separate experiments. **P < 0.01 compared with control, ++P < 0.01, +++P < 0.001 compared with ISO treatment alone. (B) and (C) In the absence of extracellular calcium, IP3 R antagonist Xc at 1 µM pretreatment for 30 min nearly abolished isoflurane-triggered peak increase of [Ca2+]c, representative of calcium release from the ER in this situation. Data represent mean ± sd from minimum 40 cells (n > 40) of three separate experiments. Tracing reveals averaged isoflurane-evoked increase of [Ca2+]C with or without pretreatment of Xc in the absence of extracellular calcium in L286V PC12 cells (B). Xc (1 µM) pretreatment for 30 min significantly inhibited isoflurane-induced peak increase of [Ca2+]C in the absence of extracellular calcium in L286V PC12 cells (C). ***P < 0.001 compared with ISO treatment alone without Xc pretreatment.

 

Isoflurane did not Significantly Change ROS Production
To test whether ROS contributes to isoflurane-induced cytotoxicity, we measured ROS immediately after isoflurane exposure in the different types of PC12 cells. Because our previous study1 demonstrated that 2 MAC isoflurane for 24 h was needed to induce cytotoxicity in WT PC12 cells, we used this exposure regimen for our ROS studies. Isoflurane did not cause a significant increase in ROS production in WT, vector or L286V PC12 cells compared with their nonanesthetic controls (Fig. 5). We did not measure ROS production after exposure to sevoflurane or desflurane because these two inhaled anesthetics did not induce cytotoxicity in either the current or in previous experimental models.1


Figure 521
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  		Figure 5. Isoflurane did not significantly change the reactive oxygen species (ROS) in PC12 cells transfected with mutated presenilin-1 (L286V), wild type presenilin-1 (WT) or vector control (Vector). The ROS were measured immediately after treatment of three types of PC12 cells with 2 MAC isoflurane for 24 h and were expressed as percentage of control (no anesthetic treatment). Data represent mean ± sd from at least 16 repeats of minimum three separate experiments.

 


   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We have demonstrated that isoflurane, but not sevoflurane and desflurane at equipotent concentrations, induced cytotoxicity in PC12 cells transfected with the L286V PS1 mutation, when compared with WT or vector control PC12 cells. Further, isoflurane induced a larger and more rapid increase in the peak [Ca2+]c in L286V PC12 cells than in the WT or vector control cells. Our studies implicated the ER IP3 receptors as the source of most calcium release from the ER. In contrast, sevoflurane and desflurane caused less calcium release. Finally, we showed that isoflurane had no effect on ROS production in any of the PC12 cells.

Ca2+ regulation in neurons is complex. Both calcium flux from the extracellular space and release from intracellular stores may increase [Ca2+]c.16 Ryanodine and IP3 receptors, two calcium release channels on ER membrane, are both calcium-activated calcium release channels. Calcium release from the ER via activation of ryanodine receptors can activate IP3 receptors and vice versa.36 In this study, isoflurane induced nearly the same degree of increase of [Ca2+]c in the absence of extracellular calcium, suggesting a major role of calcium release from intracellular calcium stores (compare Fig. 4C with Figs. 2B and 3B). In addition, activation of ER IP3 receptors explains most of isoflurane-induced calcium release, because of inhibition by xestospongin c. Further, our results suggest that the mechanism for increased toxicity is disruption of the intracellular calcium homeostasis via non-physiological calcium release from the ER, a mechanism also thought to play a role in the pathogenesis of AD.15,19,37,38

Calcium influx is unlikely to explain isoflurane effects, both for the reasons stated above and because isoflurane has been shown to decrease calcium influx via inhibition of voltage-gated calcium channel,39,40 N-methyl-d-aspartate receptors,41 or the Na+-Ca2+ exchange system.42 However, isoflurane has also been shown to inhibit calcium ATPase on plasma membrane (PMCA) in different types of cells, including neurons,43,44 which may decrease intracellular Ca2+ clearance after increase of [Ca2+]c and therefore contribute to sustained increase of [Ca2+]c. It seemed that there was no sustained increase of [Ca2+]c after isoflurane exposure in the presence (Figs. 2A and 3A) or absence (Fig. 4B) of extracellular calcium, although we did not determine if isoflurane inhibited PMCA and contributed the increased [Ca2+]c in this study.

The enhancement in cytosolic calcium levels by isoflurane in cells carrying the PS1 L286V mutation is consistent with the higher degree of neurotoxicity observed in these same cells after exposure to isoflurane. Our previous study suggested that isoflurane cytotoxicity is concentration and time-dependent.1 In comparison to our previous study in WT PC12 cells,1 the L286V PS1 mutation significantly decreased the isoflurane concentration and exposure time necessary to cause cytotoxicity. Although this may indicate a generalized vulnerability to stressors, similar exposures to other anesthetics did not elicit cytotoxicity. These data indicate that a known risk factor for early AD increases cell vulnerability to clinical concentrations of isoflurane. A recent study suggested that PS1 mutation reduced passive calcium release from the ER and therefore increased intracellular calcium stores, thus causing greater calcium release from the ER and increase of cytosolic calcium concentration upon activation of calcium release channels on the ER membrane by their agonists.45 Although this study only examined one such mutation, many other PS1 mutations have been shown to contribute to cell apoptosis,46–48 and it is not clear from this study whether those PS1 mutations also render cells vulnerable to isoflurane cytotoxicity.

Our results are also consistent with a recent study showing that isoflurane induced apoptosis, along with an increase in amyloid β (Aβ) production and an alteration of amyloid precursor protein processing in a human neuroglioma cell line.8 Aβ has been shown to augment calcium release from the ER via either the ryanodine or IP3 receptors,49 and thus an increase in Aβ production by anesthetics provides another, indirect mechanism by which anesthetics might enhance ER calcium release. However, the xestospongin C experiments in both rat cerebral cortical neurons and PC12 cells make it more likely that the anesthetic is acting directly on the IP3 receptor to enhance calcium release.1,20

Another important finding is the differential potency of the three anesthetics for inducing cytotoxicity in cells carrying the PS1 L286V mutation. These results are consistent with our studies and others demonstrating similar differences in potency among these three anesthetics for DNA damage and cytotoxicity.1,3,6,26 The mechanisms for the different potency for induction of apoptosis among volatile anesthetics are not clear, but may relate to calcium regulation. In L286V cells, the differences in calcium transients among the anesthetics were clear, consistent with previous studies22–25 and consistent with the hypothesis that cytotoxicity occurs via calcium dysregulation. Regardless of the precise mechanisms, our results suggest that volatile anesthetics should not be considered identical when they are administered to patients with risk factors for, or symptoms consistent with, AD.

Isoflurane may be both neurotoxic and neuroprotective, depending on the concentration and duration of exposure. A transient and moderate increase of cytosolic calcium by isoflurane may provide cytoprotection through upregulation of host preconditioning responses,50,51 but prolonged exposure to higher concentrations of isoflurane may maintain the IP3 receptors in an open state, in turn increasing [Ca2+]c and [Ca2+]m, depleting ER calcium, and ultimately leading to cell damage.15,16

Isoflurane-induced increase of ROS (primarily superoxide) in cardiac cells has been proposed as one of the mechanisms for cardioprotection by isoflurane preconditioning.52 Enhanced ROS production is also one of the proposed mechanisms in neurodegeneration in AD.53 We did not detect significant changes in ROS after isoflurane treatment in any of the PC12 cells in this study. However, DCFH, the dye we used to detect ROS, is readily oxidized by H2O2 or hydroxyl radical, but is relatively insensitive to superoxide itself,54 so we cannot strictly conclude that isoflurane had no effect on superoxide generation. In addition, we cannot eliminate the possibility that isoflurane may have effects on oxidative stress through increased generation of reactive nitric species. We did not determine the effects of sevoflurane or desflurane on ROS production because these two inhaled anesthetics did not induce similar cell damage at equipotent concentrations and duration as isoflurane. However, it is still possible that these two inhaled anesthetics may affect ROS production, although such an effect on ROS production seems unlikely to explain the decreased potency for sevoflurane or desflurane to elevate cytosolic calcium concentration and to induce cell damage than isoflurane.

Isoflurane neurotoxicity has been characterized by apoptosis in PC12 cells, primary neuron cultures and rat brain,1,10 so the cytotoxicity observed in this study with MTT reduction and LDH release assays may have occurred via apoptotic pathways. While this study only focused on the L286V PS1 mutation, there are other contributors to neurodegeneration in AD. These include tau, amyloid precursor protein, various secretases, ApoE and perhaps heat shock proteins and ferritins. Many of these may be modulated by calcium.38,55,56 While our measurements of intracellular calcium were limited to the cytosolic compartment, secondary or primary alterations in Ca2+ may be occurring in the ER and mitochondria. More studies are needed to clarify the effects of volatile anesthetics on calcium dynamics in these organelles.

In summary, a PS1 mutation associated with familial AD renders PC12 cells more vulnerable to isoflurane, but not sevoflurane or desflurane, cytotoxicity. Calcium dysregulation rather than changes in ROS production may underlie the anesthetic cytotoxic effects. This study calls for further investigation into the cytotoxic effects of isoflurane in animal models and in patients with risk factors for, or symptoms of AD, so that anesthesiologists can make informed decisions about the use of volatile anesthetics in vulnerable populations.


   ACKNOWLEDGMENTS
 
The authors thank Drs. Mark P. Mattson and Sic L. Chan from the Laboratory of Neurosciences, National Institute on Aging Intramural Research Program, 5600 Nathan Shock Drive, Baltimore, Maryland 21224 for providing us PC12 cells transfected with PS1 mutation. We thank Dr. Qingcheng Meng from the Department of Anesthesiology for his assistance measuring concentrations in culture medium and the intellectual assistance and support by Dr. Randall Pittman from the Department of Pharmacology, University of Pennsylvania.


   Footnotes
 
Accepted for publication October 11, 2007.

Supported by National Institute of General Medical Science (NIGMS) K08 grant (1-K08-GM-073224-01, to H.W.), partially supported by March of Dimes Birth Defects Foundation Research Grant (12-FY05-62, to H.W.) and the Research Fund at the Department of Anesthesiology and Critical Care, University of Pennsylvania (to H.W.).

Conflict of Interest: None of the authors declares a conflict of interest.

Baobin Kang is currently at Mississippi Functional Genomics Network, University of Southern Mississippi, 118 College Drive #5018, Hattiesburg, MS 39406.


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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