General Anesthetic Isoflurane Modulates Inositol 1,4,5-Trisphosphate Receptor Calcium Channel Opening

Abstract

Background

Pharmacological evidence suggests that inhalational general anesthetics induce neurodegeneration in vitro and in vivo through overactivation of inositol trisphosphate receptor (InsP₃R) Ca²⁺ release channels, but it is not clear whether these effects are due to direct modulation of channel activity by the anesthetics.

Methods

Using single-channel patch clamp electrophysiology, we examined the gating of rat recombinant type 3 inositol InsP₃R (InsP₃R-3) Ca²⁺-release channels in isolated nuclei (N = 3 to 15) from chicken lymphocytes modulated by isoflurane at clinically relevant concentrations in the absence and presence of physiological levels of the agonist InsP₃. We also examined the effects of isoflurane on InsP₃R-mediated Ca²⁺ release from the endoplasmic reticulum, and changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i).

Results

Clinically relevant concentrations (~ 1 Minimal Alveolar Concentration) of the commonly used general anesthetic, isoflurane, activated InsP₃R-3 channels with open probability similar to channels activated by 1 μM InsP₃ (P_o≈0.2). This isoflurane modulation of InsP₃R-3 P_o depended biphasically on [Ca²⁺]_i. Combination of isoflurane with subsaturating levels of InsP₃ in patch pipettes resulted in at least two-fold augmentations of InsP₃R-3 channel P_o compared to InsP₃ alone. These effects were not noted in the presence of saturating [InsP₃]. Application of isoflurane to DT40 cells resulted in a 30% amplification of InsP₃R-mediated [Ca²⁺]_i oscillations, whereas InsP₃-induced rise in [Ca²⁺]_i and cleaved caspase-3 activity were enhanced by nearly 2.5 fold.

Conclusion

These results suggest that the InsP₃R may be a direct molecular target of isoflurane and a player in the mechanisms of anesthetic-mediated pharmacological or neurotoxic effects.

Introduction

The inositol trisphosphate receptor (InsP₃R) is an intracellular Ca²⁺ release channel found mostly on the membrane of the endoplasmic reticulum (ER). Activation of InsP₃R by InsP₃ causes Ca²⁺ release from the ER lumen into the cytoplasm, where it acts as a second messenger to regulate many physiological processes such as cell survival and neurogenesis. ^1–4 InsP₃R over-activation also regulates some pathological processes ^1;5, especially apoptosis and neurodegeneration. ^6–8 InsP₃R channel activity can be regulated by a diverse array of interacting proteins, ⁴ including neurodegenerative-associated proteins such as Huntingtin-associated protein 1 and Alzheimer’s disease mutant presenilin 1 and presenilin 2. ^9;10

More than 260 million patients worldwide receive surgeries under general anesthesia each year. The diversity of the molecular mechanisms of general anesthetics is still not clear. Activation of InsP₃Rs may play important roles in anesthetic-mediated regulation of intracellular Ca²⁺ homeostasis and some physiological and pathological processes. ^11–14 General anesthetics, especially isoflurane, may precondition cells and provide cytoprotection by moderately activating InsP₃Rs and Ca²⁺ release from the ER. ¹² Isoflurane may also cause apoptosis and neurodegeneration by causing abnormal Ca²⁺ release from the ER via overactivation of InsP₃R. ^{3;11;13;15–17} Sensitized InsP₃R activity in Alzheimer’s and Huntington diseases appears to render neurons vulnerable to isoflurane-induced Ca²⁺ release from the ER and subsequent apoptosis. ^15;17;18 Isoflurane increases levels of β-site amyloid β precursor protein-cleaving enzyme ¹⁶ and aggregation of mutated Huntingtin proteins ¹⁷ via activation of InsP₃R. General anesthetics may cause neuronal apoptosis by disruption of intracellular Ca²⁺ homeostasis. ^18–21 Despite these strong associations of anesthetic exposure and InsP₃R activation in normal and pathological conditions, there is a lack of evidence to support direct activation of the InsP₃R by isoflurane.

In this study, we report for the first time that clinically relevant concentrations of isoflurane directly modulate the activity of the InsP₃R channel and sensitize the channel to basal levels of InsP₃, resulting in InsP₃R mediated Ca²⁺ release from the ER and amplification of InsP₃-induced [Ca²⁺]_i signals, and induction cell apoptosis. These results suggest that the InsP₃R may be one of the molecular mechanisms of anesthetic-mediated pharmacological and toxic effects in neurodegeneration.

Materials and Methods

Cell Culture

DT40 chicken lymphocytes

DT40 cells lacking the genes for all 3 isoforms of InsP₃Rs (DT40-KO, RIKEN Cell Bank No. RCB 1467, Ibaraki, Japan) and DT40-KO cells stably over-expressing the rat type 3 InsP₃R (DT40-R3) ²² were used in this study. While the work was conducted in a chicken lymphocyte cell line, the channel studied is the rat recombinant InsP₃R channel. We elected to use InsP₃R-3 instead of other InsP₃R subtypes because InsP₃R-3 has been shown to have relatively higher opening probability activated by InsP₃R agonists and a useful in vitro model to examine the InsP₃R activation by its agonists. ²² Given the high level of primary sequence homology among mammalian InsP₃Rs, and the substantial similarities in the major features of the regulation of endogenous and recombinant InsP₃R channels by cytoplasmic InsP₃ and Ca²⁺ (biphasic activation and inhibition by Ca²⁺; and monotonic, saturable activation by InsP₃), ⁴ we expect that our findings that isoflurane affects Ca²⁺ and InsP₃ regulation of InsP₃R channels have relevance for similar regulations of neuronal InsP₃R channels, and thereby have relevance for Ca²⁺ homeostasis in neurons. Cells were maintained in suspension culture in Roswell Park Memorial Institute medium 1640 media containing 10% fetal calf serum, 1% chicken serum, penicillin (100 units/ml), streptomycin (100 μg/ml), and glutamine (2 mM) at 37°C in a 95% air and 5% CO₂ humidified incubator.

Electrophysiology

There is no way to directly observe single-channel activity of the InsP₃R in the native ER membrane since performing patch-clamp electrophysiology on the ER directly has not been achieved. Since the outer nuclear membrane is continuous with the ER membrane topologically, and since there is no evidence to suggest that the biochemical properties of the outer nuclear membrane are distinct from those of the rest of the ER, nuclear patch clamping of the ER membrane is a tool to record ER-localized ion channel activities, including the InsP₃R. ⁴ We have examined the effects of isoflurane on InsP₃Rs located on the outer nuclear membrane. Preparation of isolated nuclei from DT40 cells was performed as described. ^10;22–26 Cells were washed twice with PBS and suspended in a nuclear isolation solution containing 150 mM KCl, 250 mM sucrose, 1.5 mM 2-mercapoethanol, 10 mM Tris·HCl, 0.05 mM PMSF, and protease inhibitor mixture (Complete; Roche Molecular Biochemicals, Indianapolis, IN), adjusted to pH 7.3 with KOH. Isolated nuclei were placed in a recording chamber containing a standard bath solution: 140 mM KCl, 10 mM Hepes, and 0.5 mM BAPTA, ≈200 nM free-Ca²⁺, adjusted to pH 7.3 with KOH. During the nuclear isolation protocol and the introduction of nuclei onto the stage of the patch clamp microscope, any endogenous IP3 is washed away. Selected intact nuclei were patch-clamped and single channel activities were recorded in the on-nucleus configuration. Recording pipettes with resistances of 8–10 MΩ were used. The pipette solution contained 140 mM KCl, 0.5 mM ATP, 10 mM Hepes (pH 7.3), with varying concentrations of isoflurane, InsP₃ and Ca²⁺. The free Ca²⁺ concentration was varied by addition of an appropriate Ca²⁺ chelator, as previously described. ²⁷ Single-channel currents were amplified using an Axopatch-200B amplifier (Molecular Devices, Downingtown, PA), filtered at 1 kHz, and digitized at 5 kHz with an ITC-16 interface (Instrutech, Port Washington, NY) and Pulse+ Pulse Fit software (HEKA Electronik, Farmingdale, NY). All recordings were performed at room temperature with the pipette electrode at −40 mV relative to the reference bath electrode. Single channel-analyses were performed on recordings exhibiting only a single channel using QuB (University of Buffalo, Baffalo, New York) and Igor Pro (WaveMetrics, Lake Oswego, OR) software. Figures were generated using Igor Pro software and Adobe Illustrator (Adobe System Incorporated, San Jose, CA).

Measurement of cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i)

DT40-KO or DT40-R3 cells were seeded onto glass coverslips coated with 0.01% poly-L-ornithine for 1 hr before measurements. Cells were loaded with 2.5 μM Fura-2AM (Molecular Probes, Grand Island, NY) for 30 min at room temperature in Hanks’ balanced salt solution (HBSS, Sigma, Pittsburgh, PA) containing 1.8 mM CaCl₂ and 0.8 mM MgCl₂, pH 7.4. Coverslips were then placed in a sealed perfusion chamber (Warner Instruments, Hamden, CT) and continuously perfused at room temperature with HBSS. Fura-2AM was alternately excited at 340 and 380 nm, and emitted fluorescence (510 nm) was collected and recorded using a CCD-based imaging system running IPLab v3.7 software (Biovision Technologies, Exton, PA). The data are presented as the ratio of fluorescence intensities recorded at 340 nm and 380 nm excitations (F340/F380) during baseline and isoflurane application. F340/F380 ratios were recorded from > 30 cells in at least three separate experiments. For analyses of [Ca²⁺]_i oscillations, ratio images were collected from > 60 cells over 20 min in at least four separate experiments. The percentage of cells with obvious [Ca²⁺]_i responses (a single transient rise or sustained oscillations), peak [Ca²⁺]_i, area under the [Ca²⁺]_i versus time curve, and oscillation frequency were determined and analyzed as described previously. ²⁸ HBSS samples were collected from inflow and outflow tubes of the recording chamber to determine the anesthetic concentration to which the cells were exposed. High performance liquid chromatography measurements showed that isoflurane was consistently maintained at 0.4 mM (not shown). This concentration corresponds to a minimum alveolar concentration of ~1.

Detection of caspase-3 activity

Proteolytic activation of caspase-3 was measured in lysates from DT40 wild type (DT40-wt), DT40-KO and DT40-R3 cells following exposure to isoflurane using previously described protocols.¹³ Caspase-3 activity was measured using Ac-DEVD-AFC hydrolysis kit (Caspase-3, Calbiochem, Billerica, MA). Briefly, DT40-KO or DT40-R3 cells grown in six well plates were exposed to isoflurane (2.4%) for 24 h which resulted in 0.8 mM isoflurane in the culture medium²⁹ and reliably produced isoflurane toxicity in this anesthesia exposure cell model,¹³ harvested via trypsinization, and washed with phosphate buffered saline. The cell pellet was gently resuspended in CelLytic^™ M lysis buffer with protease inhibitor cocktail (Sigma). Lysate was centrifuged and the resultant supernatant was used for the assay. Ac-DEVD-AFC, the caspase substrate, was added at a final concentration of 50 μM and the samples were incubated for 45 min at 37°C. Ac-DEVD-AFC hydrolysis was monitored by fluorescence emission of the released AFC (excitation, 400 nm; emission, 500 nm) using a multi-wavelength excitation dual wavelength-emission flurorimeter (Delta RAM; photon Technology International, Edison, NJ). Under these excitation and emission conditions, Ac-DEVD-AFC hydrolysis produced a yellow-green fluorescence as suggested by the manufacturer (Calbiochem).

Analysis and Statistics

Data and statistically analyses were done with IGOR Pro software (Wavemetrics). All data are presented as mean ± SEM. Sample size for the electrophysiological and Ca²⁺ imaging experiments were used based on previous experiences ^27;30. Experimenters were not blinded to conditions. To avoid error introduced by subjectivity, all electrophysiological experiments in which nuclear membrane patches were successfully isolated were recorded and analyzed.

Statistical comparisons between data points were done using two-tailed tests. All statistically significant differences between data points stated were established using unpaired t-tests (with Bonferroni correction for multiple comparisons within a family of inferences). All lack of statistically significant differences between data points stated were established by ANOVA. Unless a more stringent P-value is stated, P < 0.05 was used for rejecting the null hypothesis.

Results

Activation of InsP₃R by isoflurane

Previous studies indicated that InsP₃Rs are involved in isoflurane-mediated increases in [Ca²⁺]_i in PC12 cells, DT40 chicken B lymphocytes, Huntington’s striatal neuronal cell lines and primary cortical neurons. ^11;13;16;31 To test whether isoflurane might directly modulate the activity of the InsP₃R, we measured single InsP₃R channel currents in native ER membranes by nuclear patch clamp electrophysiology. ^{4;10;23;24;26;28} Homo-tetrameric InsP₃R-3 channel activities were recorded by patch-clamping outer membranes of nuclei isolated from DT40-KO cells stably expressing the rat type 3 InsP₃R. ^10;22;32 This isoform has ligand regulation and permeation properties similar to other InsP₃R isoforms, but with robust gating that provides sensitive detection of modulation of channel activity in patch clamp electrophysiology. InsP₃R-3 channel activity was observed with 2 μM Ca²⁺ and either 1 or 10 μM InsP₃ in the pipette solution (fig. 1A–C) in ≥ 90% of patches (table 1; 18/20 and 31/34 for 1 and 10 μM InsP₃, respectively). Surprisingly, with 400 μM isoflurane in the pipette solution, InsP₃R channel activity could be elicited in the absence of InsP₃ (fig. 1D), albeit with low open probability (P_o) and in < 20% of patches (table 1; 15/85). The channels activated by isoflurane were identified as InsP₃R by their sensitivity to the InsP₃R antagonists heparin (100 μg/ml) or xestospongin C (1 μM) (fig. 1E and F; table 1). The mean open probability, open duration (t_o) and closed time (t_c) of the InsP₃R-3 channels activated by 400 μM isoflurane were similar to those of channels activated by subsaturating 1 μM InsP₃ (fig. 1G–I), suggesting that isoflurane activates InsP₃R-3 channels with similar kinetics to the endogenous agonist InsP₃ but with lower efficacy. Interestingly, the concentration dependence of the modification of InsP₃R-3 channel activity by isoflurane was biphasic: at low [isoflurane] (< 400 μM), increases in [isoflurane] increased channel P_o, but channel P_o decreased as [isoflurane] was increased beyond 400 μM. (fig. 2). Thus, InsP₃R-3 channels are activated within a narrow range of [isoflurane]. This is very different from InsP₃ activation of the channel, in which channel P_o increases with [InsP₃] until the saturating [InsP₃] is reached. Beyond that, further increase in [InsP₃] does not enhance channel activity any more. ³²

Figure 1. Isoflurane activates single inositol trisphosphate receptor (InsP₃R) channel gating.

(A–F) Typical single-channel current traces in membrane patches from nuclei isolated from DT40-KO cells stably expressing rate type 3 InsP₃R. Concentrations of InsP₃, isoflurane and InsP₃R inhibitors used are as indicated. In this and all subsequent figures, currents shown were recorded at room temperature with applied potential (V_app) = −40 mV, and arrows indicate the closed channel current level. Concentration of free Ca²⁺ ([Ca²⁺]_i) = 2 μM in all the experiments unless stated otherwise. (G–I) Bar graphs showing InsP₃R gating characteristics: mean open probability (P_o), open duration (t_o) and closed time (t_c), respectively, in various inositol trisphosphate (InsP₃) or isoflurane (ISO) concentrations. Error bars indicate standard error of the mean, and number of experiments analyzed is tabulated. ** and * indicate P (t-test with Bonferroni correction) < 0.005 and 0.05, respectively, for the quantities connected by the brackets.

Table 1.

Channel Detection Rate (Pd) of Isoflurane-activated Type 3 InsP₃R

	Isoflurane (0.4 mM)	InsP3 (μM)	Heparin (100 μg/ml)	Xestospongin C (μM)	Channel Detection Rate (Pd)	Patches (n)
Type-3 InsP3R	−	0	−	−	0/14(0)	14
	+	0	−	−	15/85(0.18)	85
	−	1	−	−	18/20(0.9)	20
	−	10	−	−	31/34(0.92)	34
	+	−	+	+	0/40(0)	40
	+	−	−	+	0/40(0)	40
InsP3R Knockout	−	10	−	−	0/10(0)	10
	+	0	−	−	0/40(0)	40

InsP₃R – inositol 1,4,5-trisphosphate receptors.

Figure 2. Dependence of inositol trisphosphate receptor (InsP₃R) channel activity on isoflurane concentration.

(A–D) Typical single-channel current traces of type 3 (InsP₃R-3) channel activity in various isoflurane concentrations as indicated. Concentration of free Ca²⁺ ([Ca²⁺]_i) = 2 μM. See figure 1D for single InsP₃R channel current trace in 400 μM isoflurane. (E) [isoflurane] dependence of InsP₃R-3 channel open probability P_o. Error bars indicate standard error of the mean, and number of experiments analyzed is tabulated.

[Ca²⁺]_i dependence of isoflurane-induced InsP₃R channel activity

Gating of InsP₃Rs is under complex allosteric regulation by InsP₃ and [Ca²⁺]_i. In general, InsP₃R channel activity is biphasically regulated by [Ca²⁺]_i with maximum channel P_o observed over a broad range of [Ca²⁺]_i in the presence of saturating (10 μM) InsP₃. In the presence of subsaturating [InsP₃], sensitivity of the channel to inhibition by high [Ca²⁺] is enhanced, resulting in a narrower P_o versus [Ca²⁺]_i dependence. ^4;32 Thus, we investigated the [InsP₃] and [Ca²⁺]_i dependencies of isoflurane-activated InsP₃R channel activity. At low (0.4 or 0.9 μM) or high (6 and 10 μM) [Ca²⁺]_i, 400μM isoflurane-activated channel P_o was substantially lower (P_o < 0.006) than that observed in 2 μM Ca²⁺_i, (P_o = 0.2; fig. 3), resulting in a narrow biphasic [Ca²⁺]_i dependence.

Figure 3. Ca²⁺ dependence of isoflurane-activated type 3 inositol trisphosphate receptor (InsP₃R-3) channel.

(A) Typical single channel current traces of InsP₃R channels activated by 400 μM isoflurane in free Ca²⁺ concentration ([Ca²⁺]_i) of 900 nM. (B) [Ca²⁺]_i dependence of InsP₃R-3 channel open probability (P_o) in 400 μM isoflurane. Error bars indicate standard error of the mean, and number of experiments analyzed is tabulated. Note the logarithmic channel P_o axis. Low channel P_o (<0.01, marked with §) were observed in all [Ca²⁺]_i examined other than 2 μM. There is no statistically significant difference among these low channels P_o (P > 0.05, ANOVA test). These channel P_o were so low that the number of active channel(s) (N) in the membrane patch cannot be accurately determined. Thus, the data plotted are effectively the NP_o value, which may be an over-estimate of the actual P_o value.

Isoflurane modulates InsP₃R channel sensitivity to InsP₃

Although isoflurane activated InsP₃R-3 channel activity in the absence of InsP₃ in the pipette solution, the sensitivity of isoflurane activation of the channel to the competitive inhibitor heparin suggests that isoflurane may activate the InsP₃R-3 by sensitizing the channel to low [InsP₃] generated locally in the patched nuclear membrane. To test this, we recorded InsP₃R channel activity in DT40-R3 cells with 400 μM isoflurane, 2 μM Ca²⁺ and a range of [InsP₃] in the pipette solution. As expected, InsP₃ enhanced channel P_o in a dose-dependent manner (fig. 4A–D). At very low (0.1 and 0.5 μM) [InsP₃], 400 μM isoflurane potentiated channel activity to a level greater than either agonist alone (fig. 4E). In contrast, at higher (1 μM) [InsP₃], 400 μM isoflurane no longer potentiated channel activity significantly (fig. 4E). In saturating (10 μM) [InsP₃], 400 μM isoflurane did not enhance channel activity measurably (fig. 4E). These results suggest that isoflurane increases the functional sensitivity of the InsP₃R-3 to InsP₃ only at low, subsaturating [InsP₃] (<0.1 μM).

Figure 4. Effects of 400 μM isoflurane on the activity of inositol trisphosphate receptor (InsP₃R) channel in various [InsP₃].

(A–D) Typical single-channel current traces of InsP₃R channels in various [InsP₃] in the absence and presence of 400 μM isoflurane, as indicated. See figure 1A–C for InsP₃R channel current traces in 0, 1, and 10 μM InsP₃ with no isoflurane, and figure 1D for InsP₃R channel current trace in 400 μM isoflurane only. (E) Statistically similar InsP₃R channel activities (P > 0.05, ANOVA test) were observed in 100 nM ≤ [InsP₃] ≤ 1 μM in the absence of isoflurane. With 400 μM isoflurane, statistically similar InsP₃R channel activities (P > 0.05, ANOVA test) were also observed in 0 ≤ [InsP₃] ≤ 1 μM. Importantly, 400 μM isoflurane significantly increased InsP₃R channel activity at low [InsP₃] (0, 100, and 500 nM). Error bars indicate standard error of the mean, and number of experiments analyzed is tabulated. ** and * indicate P (t-test) < 0.005 and 0.05, respectively, for the quantities connected by the brackets. InsP₃R channel P_o in saturating 10 μM InsP₃ were significantly higher (P < 0.05, t-test with Bonferronic correction) than those in subsaturating [InsP₃], in the presence or absence of 400 μM isoflurane. However, InsP₃R channel activity in saturating (10 μM) InsP₃ was not enhanced by 400 μM isoflurane (P > 0.05, t-test).

Isoflurane modulates InsP₃R-mediated [Ca²⁺]_i signaling and apoptosis

Our single channel recordings indicate that isoflurane at clinically relevant concentrations potentiates the activity of the InsP₃R-3 in low levels of [InsP₃] that can exist in un-stimulated cells in basal conditions. To determine whether this effect influences intracellular Ca²⁺ signaling, we measured [Ca²⁺]_i in individual DT40-KO or DT40-R3 cells kept in complete growth medium. Application of 400 μM isoflurane resulted in a significant transient increases in [Ca²⁺]_i in the DT40-R3 cells that were absent in DT40-KO cells lacking InsP₃R expression (fig. 5A–C).

Figure 5. Isoflurane amplifies cytosolic Ca²⁺ signals in DT40 cells by sensitizing inositol trisphosphate receptor (InsP₃R) activation and induces cell apoptosis.

(A) Representative single cell Ca²⁺ responses to 400 μM isoflurane (Iso) in DT-40 cells with triple knock out of InsP₃R (DT40-KO) or DT40-KO transfected with rat recombinant type 3 InsP₃R (DT40-R3) cells. Arrows mark the addition of iso to the complete growth medium. (B) Peak amplitudes and (C) area under curve (AUC) for DT40-KO and DT40-R3 cells responding to isoflurane. (D) Representative single cell Ca²⁺ responses and oscillations induced by addition (marked by arrows) of 50 ng/ml anti-IgM antibody (α-IgM) or 50 ng/ml α-IgM plus isoflurane in Dulbecco’s Modified Eagle Medium. (E) Fraction of cells with various kind of response to stimulation by α-IgM or α-IgM+iso in the medium. (F) AUC of cells with single Ca²⁺ peak response triggered by α-IgM or α-IgM+iso. (G) AUC of cells with Ca²⁺ oscillations in response to stimulation by α-IgM or α-IgM+iso. (H) Isoflurane significantly elevated caspase-3 activity only in DT40 wild type (DT40-wt) cells or in DT40 expressing only InsP₃R (DT40-R3), but not in DT40 cells with triple knock out of InsP₃R (DT40-KO). All calcium measurement data are summary of at least 141 cells (N≥141, fig. 5 B and C) or 212 cells (N≥212, fig. 5 E, F, and G) from 4 separate experiments. The N values for caspase-3 activity are the average from three separate experiments (N = 3). Error bars indicate standard error of the mean, and number of experiments analyzed is tabulated. *, **, and *** indicate significant difference (P < 0.05, P < 0.01 and P < 0.001, respectively, t test).* indicate significant difference (P < 0.05, t-test) between quantities connected by the brackets in the bar graphs.

To further study the potentiating effects of isoflurane on intracellular Ca²⁺ signaling in weakly stimulated cells, the cells were perfused with complete growth medium containing a low concentration (50 ng/ml) of anti-IgM antibody to weakly stimulate the B-cell receptor to generate low, but higher than basal, levels of InsP₃. ³³ 82 ± 4% of DT40-R3 cells responded to anti-IgM antibody with either a single transient elevation [Ca²⁺]_i or with [Ca²⁺]_i oscillations (fig. 5D and E). Whereas the addition of 400 μM isoflurane did not change the percentage of cells responding to anti-IgM antibody (fig. 5D and E), it enhanced the percentage of cells that responded with a single prolonged [Ca²⁺]_i peak (fig. 5E), consistent with a stronger InsP₃R response. Because of the variable responses of cells (oscillations vs. single-peak responses), we quantified the [Ca²⁺]_i responses in all cells by the areas under the [Ca²⁺]_i-versus-time curves (AUC). The presence of isoflurane significantly increased the AUC for cells responding with a single-peak transient (fig. 5F; P < 0.001) or with [Ca²⁺]_i oscillations (fig. 5G; P < 0.01). These results suggest that isoflurane, at a clinically used concentration, potentiates low level InsP₃-mediated [Ca²⁺]_i signaling.

Prolonged exposure to isoflurane is associated with widespread cell death in diverse in vivo and in vitro systems. ^13;16 Because activation of InsP₃R-mediated Ca²⁺ signaling has been linked to apoptosis, ^34;35 and considering the effects of isoflurane on InsP₃R gating observed here, we assessed the role of this effect in isoflurane-mediated apoptosis. Caspases are a family of cysteine proteases that play crucial roles in apoptosis. Activation of caspase-3 is a central event in the progression of programmed cell death. We therefore monitored caspase-3 cleavage as a measure of apoptosis. In agreement with our previous observations of cells expressing endogenous InsP₃R, ^13;16 isoflurane triggered apoptosis in the DT40-R3 cells, but not in the InsP₃R deficient cells (fig. 5H). These observations support a role for exaggerated activation of InsP₃R in isoflurane-induced apoptosis, consistent with previous observations. ^11;13;16

Discussion

We have, for the first time, demonstrated that isoflurane at clinically relevant concentrations modulates the activity of the InsP₃R Ca²⁺ release channel at the single channel level. The modulating effects of isoflurane on the InsP₃R regulate Ca²⁺ release from the ER that results in exaggerated [Ca²⁺]_i signaling. We have also demonstrated, for the first time at the molecular level, that isoflurane causes Ca²⁺ release from the ER via this activation of InsP₃R, and can therefore affect intracellular Ca²⁺ homeostasis, regulation of cytosolic Ca²⁺ oscillations and cell survival. These results provide novel insights into possible molecular mechanisms of anesthesia-mediated effects on neurodegeneration and cognitive function.

Inhalational anesthetic modulate InsP₃R activation

It was previously demonstrated that both isoflurane and halothane may elevate [Ca²⁺]_i primarily by inducing Ca²⁺ release from the intracellular Ca²⁺ stores in neurons. ³⁶ Our results provide the first evidence that an inhalational anesthetic can modulate activation of InsP₃R channels. Interestingly, isoflurane activates InsP₃R channels with a biphasic dose response with optimal concentrations around 0.4 – 1mM, close to clinically used concentrations for general anesthesia. This activation of InsP₃R channels by isoflurane showed a strong Ca²⁺ concentration dependence, with the optimum [Ca²⁺]_i of 2 μM, qualitatively similar to the [Ca²⁺] dependence of InsP₃-activated channel activity. The InsP₃ competitive antagonist, heparin, blocked the ability of isoflurane to activate InsP₃Rs. Whether isoflurane requires InsP₃ for it to activate the channel, or isoflurane activation is direct but also sensitive to heparin inhibition is unclear. The former may be likely since isoflurane (0.4 mM) potentiated activation of the InsP₃R by low concentrations of InsP₃, but failed to further enhance InsP₃R-3 activity at saturating [InsP₃]. Nevertheless, further studies are needed to investigate possible biochemical interactions between isoflurane and the InsP₃R. Together, these results establish the InsP₃R as a novel target of isoflurane and perhaps other inhalational or intravenous general anesthetics.

Isoflurane enhances Ca²⁺ signals and cell apoptosis via activation of InsP₃R

The biphasic effects of [Ca²⁺]_i on InsP₃R activation play important roles on intracellular Ca²⁺ oscillations, waves, and spreading of global Ca²⁺ signals. ³⁷ Our finding that isoflurane activates InsP₃R-3 channels raised the possibility that it could enhance [Ca²⁺]_i signals. Application of 400 μM isoflurane resulted in a transient increase in [Ca²⁺]_i in DT-40 cells expressing InsP₃R-3 but not in DT-40 cells lacking the channels. In addition, [Ca²⁺]_i signals generated by IgM stimulation of DT40-R3 cells, both single-peaks and oscillations, were more prominent in the presence of isoflurane. (fig. 5F and G).

We previously showed in in vitro and in vivo models that exposure to isoflurane for prolonged durations significantly induced apoptosis that required activation of InsP₃Rs.^11;13 Our results here suggest that this is medicated at least in part by isoflurane activation of InsP₃R channel gating. The effects of isoflurane on changes in [Ca²⁺]i are in good agreement with the conclusions reached from the single channel recordings of the effects of isoflurane on InsP₃R activity observed in the current study. It has long been known that anesthetics including halothane activate the other major ER Ca²⁺ release channels, the RyR channel complex,³⁸ and this is thought to be the basis for malignant hyperthermia. ³⁹ Like InsP₃Rs, RyRs are expressed throughout the nervous system and play important roles in both normal cell functions ⁴⁰ and in various neurodegenerative diseases. ^2;41–43 Our previous study indicated a role of RyR activation in isoflurane-induced apoptosis in neuronal tissue cultures. ²⁹ Both InsP₃R and RyR contribute to regulation of intracellular Ca²⁺ homeostasis and may interact with each other through Ca²⁺-induced Ca²⁺ release in a common pathway in normal neuronal function and neurodegeneration. Excessive Ca²⁺ release from the ER via these release channels could cause Ca²⁺ overload in mitochondria and depletion of ER Ca²⁺, both of which can contribute to apoptosis. ^6;34;44 In addition, mitochondrial Ca²⁺ overload causes cytochrome C release, activating caspase-3, which can cleave the InsP₃R, resulting in a constituent Ca²⁺ leak from the ER. ⁴⁵ Thus, excessive or prolonged activation of InsP₃R by isoflurane may set in motion a cascade of events resulting in apoptosis in different tissue culture cells including neurons, ^{3;11;13;15–17} and in developing brains.¹⁶ Our data suggest that the InsP₃R may represent an ideal target for prevention of the harmful side effects of inhalational anesthetics. Inhibition of excessive activation of the InsP₃R may ameliorate or prevent anesthesia-mediated neurodegeneration as demonstrated in animal models. ¹⁶ This is especially relevant in pediatric ^46;47 and aged patients, ⁴⁸ who appear to be the most vulnerable to the harmful side effects of anesthetics. Unfortunately, there is no good pharmacology for the InsP₃R. Although heparin is an InsP₃R antagonist, its poor penetration across cell membranes limits its use for protection against anesthetic neurotoxicity in animals or patients. It should be noted that anesthetics have also been shown to be protective against various stresses, also by activation of InsP₃Rs in different tissue culture models. ^{3;12;14;49;50} As demonstrated here (fig. 6), mild or moderate activation of InsP₃R and moderate Ca²⁺ release from the ER by isoflurane provides cytoprotection, possibly via physiological Ca²⁺ uptake into mitochondria and stimulation of ATP production, ²⁵ or by activation of AKT and MAPK/ERK cytoprotective pathways. ^12;14;50 Nevertheless, it is prudent to minimize the use of general anesthetics as much as possible, so that their beneficial effects can be utilized and the harmful effects be minimized.

Figure 6. Modulation of inositol trisphosphate (InsP₃)receptor (InsP₃R) activity by isoflurane and the effects on cell survival.

Isoflurane can activate InsP₃R or potentiate the activation of InsP₃R by endogenous agonist InsP₃ generated by various extracellular stimulation including antibody for IgM (IgM Ab). These effects can be inhibited by InsP₃R antagonists xestospongin and heparin. Moderate activation of InsP₃R by isoflurane at low concentration for short duration cause moderate Ca²⁺ release from the endoplasmic reticulum (ER) and elevation of cytosolic Ca²⁺, which in turn, inhibit cell death by inducing endogenous cytoprotective mechanisms (left side), such as activation of phosphatidylinositol-4,5-bisphosphate 3-kinase(PI3K)- protein kinase B (AKT) and microtubule-associated protein kinase (MAPK)/extracellular-signal-regulated kinases (ERK) pathways. Excessive activation of InsP₃R by isoflurane at high concentration for prolonged duration cause excessive Ca²⁺ release from the endoplasmic reticulum and abnormal elevation of cytosolic Ca²⁺, resulting in induction of cell death via apoptosis directly (right side), through rerelease of cytochrome C (Cyc C) from mitochondria into cytosolic space. ATP = adenosine triphosphate.

In summary, our results indicate that the commonly used inhalational anesthetic isoflurane modulates gating of the InsP₃R Ca²⁺ release channel, enhancing ER Ca²⁺ release and contributing to isoflurane-mediated apoptosis. These results suggest that InsP₃Rs are molecular targets of general anesthetics and that these receptors may provide the basis for some pharmacologic effects of general anesthetics and therapeutic interventions for anesthesia-mediated cell death by apoptosis.