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Anesth Analg 2007;104:1136-1144
© 2007 International Anesthesia Research Society
doi: 10.1213/01.ane.0000260615.12553.4e

ANESTHETIC PHARMACOLOGY

The Effects of General Anesthetics on P2X7 and P2Y Receptors in a Rat Microglial Cell Line

Mika Nakanishi, MD*, Takashi Mori, MD, PhD*, Kiyonobu Nishikawa, MD, PhD*, Makoto Sawada, PhD{dagger}, Miyuki Kuno, MD, PhD{ddagger}, and Akira Asada, MD, PhD*

From the *Department of Anesthesiology and Intensive Care Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan; {dagger}Department of Brain Science, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Aichi, Japan; and {ddagger}Department of Physiology, Graduate School of Medicine, Osaka City University, Osaka, Japan.

Address correspondence to Mika Nakanishi, MD, Department of Anesthesiology and Intensive Care Medicine, Graduate School of Medicine, Osaka City University, 1-5-7 Asahi-machi, Abeno-ku, Osaka 545-8586, Japan. Address e-mail to m1383144{at}med.osaka-cu.ac.jp.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: Microglial cells play important roles in coordinating the inflammatory brain responses to hypoxia and trauma. Ionotropic P2X receptors and metabotropic P2Y receptors (P2YRs) expressed in microglia can be activated by extracellular adenosine triphosphate (ATP) derived from damaged cells or astrocytes, and participate in the signaling pathways evoked in brain insult. Although several inhaled and IV anesthetics produce neuroprotective effects through neuronal mechanisms, little is known about how general anesthetics modulate microglial responses in the pathological state. We examined the effects of various general anesthetics on purinergic responses in a rat microglial cell line.

METHODS: Currents were consistently activated by applications of ATP via a U-tube system under the whole-cell configuration. ATP-induced nondesensitizing currents observed after several applications of ATP exhibited characteristics of P2X7 receptors. The P2YRs-mediated mobilization of intracellular Ca2+ was measured using a Ca2+-sensitive fluorescent dye (fura-2).

RESULTS: Inhaled anesthetics (sevoflurane, isoflurane, and halothane) at doses three times as high as minimum alveolar concentrations had no effect on the P2X7Rs-mediated currents. IV anesthetics (ketamine, propofol, and thiopental) enhanced the P2X7Rs-mediated currents reversibly. The potencies for activation of P2X7Rs were not correlated with the octanol/buffer partition coefficients. Thiopental, at low concentrations, slightly inhibited the P2X7Rs-mediated currents, suggesting its dual actions on P2X7Rs. The P2YRs-mediated mobilization of intracellular Ca2+ was not affected by any of the general anesthetics tested.

CONCLUSIONS: Our results suggest that IV anesthetics, particularly thiopental and propofol, may modulate microglial functions through P2X7Rs in pathological conditions.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Microglia, the principle immune cells of the central nervous system (CNS), play crucial roles in regulating inflammatory responses to ischemic brain damage, traumatic injury, and neurodegenerative diseases (1,2). Microglia activated during brain insult performs phagocytosis, migration, proliferation, and release of a number of biologically active substances (1). Microglial functions are basically neuroprotective (host defense, tissue repair), but over-stimulation of their immune reactions may result in acceleration of neuronal damage after brain insult (3,4). Therefore, the regulation of microglial functions are thought to provide new modalities in therapeutic intervention against CNS disorders where inflammatory activation of microglia is involved (2,5).

Extracellular adenosine triphosphate (ATP) derived from injured cells or astrocytes is one of the most important key messengers for mediating pathological conditions of the CNS. Massive amounts of ATP are released from damaged tissue after ischemia and trauma, resulting in sustained elevation of ATP levels in the areas surrounding the injured zone (6–8). Microglia express various types of ionotropic (P2X: P2X4, P2X7) and metabotropic ATP receptors (P2Y: P2Y2, P2Y4, P2Y12) (5,9). The former receptors are nonselective cation channels, and are responsible for depolarization and Ca2+ influx (10), whereas the latter receptors, coupled with G-protein, activate phospholipase C, inositol-phosphate formation and cause the release of Ca2+ from intracellular stores (11). Activation of these P2 receptors leads to production of diverse microglial responses. P2X4 receptors (P2X4Rs) in spinal microglia have been shown to be involved in generating refractory pain and are recognized as a new target for the treatment of neuropathic pain (12). P2X7 receptors (P2X7Rs) are responsible for the release of interleukin-1ß, plasminogen, tumor necrosis factor-{alpha}, nitric oxide, and superoxide and, furthermore, apoptotic cell death (13–18). P2Y receptors (P2YRs) are related to microglial activation and motility (5), for instance, mitotic activity through activation of P2Y2 and P2Y4 receptors, and chemotaxis through P2Y12 receptors (9,19,20). Several recent studies have suggested that P2X7Rs and P2YRs participate in neuronal damage caused by ischemic and traumatic brain injury (21–24).

Much attention has been given to the neuroprotective effect of general anesthetics in ischemic brain damage (25). Several inhaled and IV anesthetics alleviate neuronal injury induced by focal cerebral ischemia or forebrain ischemia and reduce glutamate neurotoxicity. These neuroprotective effects have mostly been explained by actions of the anesthetics on neurons. However, the effect of general anesthetics on microglia-associated inflammatory processes remains to be defined. In the present study, we investigated the effects of various inhaled and IV anesthetics on P2X7Rs and P2YRs in a rat microglial cell line.


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Preparations
A rat microglial cell line (GMI-R1) (26,27) was used in the present study. GMI-R1, an immortalized microglial clone, was established from a rat primary microglial culture similarly as described for the mouse Ra2 cell line (26,27). The characteristics of GMI-R1 cells were considered almost similar to those of microglia in primary culture (28,29). GMI-R1 cells were cultured in Eagle’s MEM (Nissui Pharmac., Tokyo, Japan) supplemented with 10% fetal calf serum (Equitech-Bio, Ingram, TX), 0.5–2 ng/mL recombinant mouse GM-CSF (PeproTech, London, UK) containing 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.25 ng/mL amphotericin B at 37°C in a humidified atmosphere of 95% air and 5% CO2. The culture medium was changed every 3–4 d. For patch clamp experiments, cells were plated at a density of 1.0–2.0 x 105 cells/mL on coverslips and were cultured for 1–3 d. The procedure for culture of GMI-R1 rat microglial cell line was described elsewhere (26,27).

Electrophysiology
A whole-cell patch clamp technique was used to record ionic currents induced by ATP application through a U-tube system. Recording pipettes were pulled in two stages on a vertical pipette puller (Narishige PP-830, Tokyo, Japan). The pipette solution contained 130 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM EGTA, and 10 mM HEPES-KOH (290 mOs). The external solution contained 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 1 mM MgCl2, 10 mM d-glucose, and 10 mM HEPES-NaOH (290 mOs). The pH of the internal and external solutions was adjusted to 7.3 with KOH and NaOH, respectively. The pipette resistance was about 5 M{Omega}. The series resistance and the electrode capacitance were compensated and checked before and after pharmacological manipulations to ascertain the constant recording condition. The currents were recorded with a patch clamp amplifier (AXOPATCH 200A, Axon Instruments, Union City, CA) while a membrane potential was held at –60 mV. Current signals were filtered at 5 kHz, digitized at 1–10 kHz via AD converter (Digidata 1200, Axon Instruments), and analyzed using pCLAMP 9 software package (Axon Instruments). Glass coverslips bearing microglial cells were placed in a recording chamber (approximately 2 mL volume) where the external solution was perfused at a rate of 1–2 mL/min. ATP and general anesthetics were applied by a gravity-fed U-tube system placed within 30–50 µm of the target cell. The application times were controlled by computer-driven solenoid valves (General Valve, Fairfield, NJ). This device permitted us to exchange the solution around the cell within 50 ms. (30) To determine the effects of the general anesthetics on ATP-induced currents, all anesthetics were simultaneously applied with ATP from the U-tube onto the cells. All experiments were performed at room temperature (20°C–25°C).

Measurement of Intracellular Ca2+ Concentration
The intracellular Ca2+ concentration of single cells was determined with a digital fluorescence microscopy (Attofluor, Atto Bioscience, Rockville, MD) using a Ca2+-sensitive fluorescent dye, fura-2. Cells were loaded with the acetoxymethyl ester form of fura-2 (fura-2 AM, Dojindo, Kumamoto, Japan) (5 µM) for 15 min at 37°C in the following external solution: 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM d-glucose, 10 mM HEPES-NaOH, pH 7.4. After washing the dye, the ratios of fluorescent images (the emission wavelength ≥520 nm) excited at two wavelengths (340 and 380 nm) were measured every 5 s with 30–100 ms exposures. Data (80–120 pixels for each cell) for each illumination were averaged and plotted against time.

During the experimental session, the solution in the glass-bottom dish (2 mL volume) was continuously perfused by external solutions at a rate of 5 mL/min. The extracellular Ca2+ was reduced to approximately 100 nM by the mixture of Ca2+ and EGTA a few minutes before stimulation. The low Ca2+ solution had the following composition: 140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 1.5 mM EGTA, 10 mM d-glucose, 10 mM HEPES-NaOH, pH 7.4. ATP and/or anesthetics were dissolved in the low Ca2+ solution. At the end of each experiment, the Ca2+ responses were normalized by the maximal fluorescence ratio obtained by adding 10 µM ionomycin in the presence of 1 mM Ca2+. Ten to 30 cells were analyzed at the same time. Recordings were made at room temperature (20°C–25°C).

Materials and Solutions
ATP (Sigma-Aldrich, St. Louis, MO) was dissolved in the external solution at appropriate concentrations immediately before the experiments. The pH of ATP solution was adjusted to 7.4 by NaOH. Oxidized ATP (oATP) and Bz-ATP (all from Sigma-Aldrich) were dissolved in the external solution. Fura-2 stock solution was prepared by adding 50 µg of 75% dimethyl sulfoxide (DMSO) + 25% pluronic acid.

Saturated solutions of inhaled anesthetics (sevoflurane, isoflurane, halothane) were prepared by stirring each inhaled anesthetic in the external solution over 8 h in a sealed glass container with very little air space. In each experiment, inhaled anesthetics were prepared immediately before use by diluting the saturated solutions and were kept in air-free, closed glass bottles to prevent evaporation. The saturated concentrations of inhaled anesthetics were estimated to be 11.8 mM for sevoflurane, 15.3 mM for isoflurane, and 18 mM for halothane. To prevent evaporation of inhaled anesthetics, a gas-tight glass syringe and Teflon tube were used in the perfusion system (30,31).

Ketamine (Sigma-Aldrich) and thiopental (Sigma-Aldrich) were dissolved in the external solution and the pH of test solutions was adjusted to 7.3. Concentrated stock solutions of propofol (Sigma-Aldrich) and ionomycin (Sigma-Aldrich) were dissolved in DMSO. These stock solutions were diluted to appropriate concentrations with the external solution immediately before each application (<0.1% v/v DMSO). DMSO (<0.1%, v/v) had no effects on P2X7Rs-mediated currents and P2YRs-mediated Ca2+ mobilization.

Statistics
Data were expressed as the mean ± sem. For evaluation of general anesthetic action on P2X7Rs and P2YRs, analysis of variance or t-test was performed to assess the significance of differences. P < 0.05 was considered statistically significant.


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
ATP-Induced Currents in Rat Microglia
Most GMI-R1 cells were small round cells in medium supplemented with GM-CSF. These cells are considered to be in an activated state rather than a resting state, although they are somewhat different from microglia activated by a variety of substances such as lipopolysaccharide (28). GMI-R1 cells also express abundant voltage-gated proton channels, consistent with a characteristic of activated microglia in primary culture (29,32).

ATP induced an inward current at –60 mV in most GMI-R1 cells. Figure 1A shows representative currents induced by the first application of ATP from a U-tube onto each cell for 5 s. The ATP-induced current was increased in a concentration-dependent manner. The lowest effective concentration to elicit visible currents in almost half of the cells was 1 µM. ATP higher than 10 mM was not tested since it caused severe cell damage. ATP-induced currents seemed to consist of desensitizing and nondesensitizing components. Dose– responses of the peak amplitudes (circles) and nondesensitizing amplitudes (triangles) are plotted in Figure 1B. The amplitudes of nondesensitizing components were extrapolated by fitting the decay phase of the currents with single exponential function. We could not calculate the EC50 for each of the two components because of the lack of the maximum response. The currents induced by 100 µM and 1 mM ATP could well represent those two types of P2X receptors, one exhibiting desensitizing currents and the other, nondesensitizing currents.


Figure 124
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  		Figure 1. (A) ATP-induced currents in rat microglia. Current traces induced by the first application of ATP through a U-tube for 5 s are shown. The holding potential was –60 mV. ATP-induced currents seemed to consist of desensitizing and nondesensitizing components. (B) ATP concentration– response curves for the peak (circles) and the steady-state currents (triangles). The peak amplitudes were obtained from currents induced by the first applications of ATP in each cell. The amplitudes of nondesensitizing component (steady-state currents) were estimated from fitting the decay phase of the currents with single exponential function. Data are given as mean ± sem.

 

The ATP-induced desensitizing currents were further characterized. At a concentration of 100 µM, currents desensitized rapidly (Fig. 1A). The currents were observed in cells pretreated with the P2X7Rs antagonist, oATP (2 mM), for 1–2 h (10). Thus the desensitized currents seem to be mediated by P2X4Rs.

At 1 mM ATP, the currents comprised desensitizing and nondesensitizing currents as well (Fig. 1A, right). After repetitive (3–6 times) applications of 1 mM ATP, the desensitizing component disappeared and only the nondesensitizing component remained in all cells tested (Fig. 2A). Applications of ATP at an interval of 2 min ensured a stable response of the nondesensitizing currents. The nondesensitizing currents were also activated by Bz-ATP, a specific agonist of P2X7Rs (Fig. 2B) and blocked by oATP, an antagonist of P2XRs (Fig. 2C) (n = 5), indicating that they were mediated mostly via P2X7Rs.


Figure 224
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  		Figure 2. (A) Isolation of P2X7 receptor-mediated currents. The currents (–60 mV) induced by the first application of ATP (1 mM) comprise two types of currents; a desensitizing component and a nondesensitizing component. After repetitive applications of 1-mM ATP for three to six times, the desensitizing component disappeared and only the nondesensitizing component remained in all cells tested. (B) The nondesensitizing currents were also activated with Bz-ATP (300 µM), a specific agonist of P2X7Rs. (C) The currents were almost completely blocked after 1 h treatment with oATP (200 µM). Current traces shown in B and C were recorded in the same cell.

 

The electrophysiological features of P2XRs in the microglial cell line were similar to those reported in rat microglia in primary culture (10). In later experiments, we focused on investigating the effects of general anesthetics on P2X7R-mediated currents, because P2X7Rs are thought to be important in the process of microglial inflammatory responses during prolonged exposure to ATP in pathological conditions such as ischemic brain damage, traumatic injury, and neurodegenerative diseases (5,17,21,24).

Effects of General Anesthetics on P2X7Rs
The effects of general anesthetics on ATP-induced currents via P2X7Rs were examined after P2X4Rs were desensitized. First we tested inhaled anesthetics (sevoflurane, isoflurane). These anesthetics did not generate currents when applied alone (n = 5) (Fig. 3A). The P2X7Rs-mediated currents were unchanged by coapplication of ATP and anesthetics at doses three times as high as the minimum alveolar concentrations (990 µM for sevoflurane, 840 µM for isoflurane, and 690 µM for halothane) (Fig. 3B). P2X7Rs-mediated current was not affected even when isoflurane was preperfused in the bath for 2 min (Fig. 3C, right). As with coapplication, preperfusion of isoflurane (840 µM) had no effect on P2X7Rs (97% ± 12% for coapplication and 94% ± 7% for preperfusion, n = 3). Figure 3D summarizes the effects of anesthetics on ATP-induced currents. Even 10 minimum alveolar concentrations of sevoflurane (3300 µM) was ineffective.


Figure 324
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  		Figure 3. Effects of inhaled anesthetics on P2X7Rs-mediated currents. (A) Sevoflurane and isoflurane themselves induced no currents (n = 5). (B) Representative traces of the effects of inhaled anesthetics on P2X7Rs-mediated currents. The currents were induced by 1 mM ATP. Inhaled anesthetics were coapplied with ATP via a U-tube. (C) P2X7Rs-mediated current was not affected even when isoflurane (840 µM) was preperfused in the bath for 2 min (right). (D) Sevoflurane, isoflurane, and halothane had no effect on P2X7Rs-mediated currents at doses up to three times as high as the minimum alveolar concentration (MAC) in all cells tested (n = 14, 5, and 5, respectively). The holding potential was –60 mV. Data are given as mean ± sem.

 

Next, the effects of IV anesthetics such as ketamine, propofol, and thiopental on P2X7Rs were examined. Although these anesthetics alone induced no currents (n = 5) (Fig. 4A), they enhanced the ATP-induced currents in a dose-dependent manner (Figs. 4B and 5). The enhancement of P2X7Rs was reversible after washing with anesthetic-free solutions (Fig. 4B, right column). Figure 4C shows that preperfusion of propofol (300 µM) produced no additional effect on P2X7Rs-mediated currents as compared with coapplication. There were no differences in the effect of ketamine, propofol, and thiopental on P2X7Rs-mediated currents between the two application methods, preperfusion and coapplication. The enhancing effects of preperfusion and coapplication as assessed by the percentage of control were 155% ± 16% and 162% ± 22% for 1 mM ketamine, 402% ± 170% and 397% ± 160% for 300 µM propofol, and 182% ± 7% and 174% ± 13% for 300 µM thiopental (n = 3, each drug). Significant enhancement was observed at 300 µM for ketamine (Fig. 5, top), 10 µM for propofol (middle), and 100 µM thiopental (bottom) (paired t-test). From the dose– response relationships, the effective concentrations to increase P2X7Rs-mediated currents to 125% and 150% of the control (EC125 and EC150), respectively, were 570 and 1790 µM for ketamine, 40 and 60 µM for propofol, and 76 and 160 µM for thiopental. The potencies for activation of P2X7Rs were correlated neither with potencies in inducing general anesthesia nor with the octanol/buffer partition coefficients (33,34). The enhancing effects of ketamine and propofol were compared between two concentrations of ATP (300 µM and 1 mM). Ketamine-induced enhancement at 300 µM ATP (Fig. 5A, inset) was not significantly different from that at 1 mM ATP. The enhancement by propofol at 300 µM ATP was also almost the same as that at 1 mM ATP (122% ± 10% with 30 µM propofol, 205% ± 34% with 100 µM propofol, n = 3 each). Thus, the effects of these IV anesthetics were not significantly different between the two doses of ATP. It should be noted that thiopental at low concentrations (3 and 10 µM) decreased, rather than increased, the P2X7Rs-mediated currents. The reduction was slight but significant (13% ± 5% reduction with 3 µM, P < 0.05, n = 12; 13% ± 6% reduction with 10 µM, P < 0.05, n = 12).


Figure 424
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  		Figure 4. Effects of IV anesthetics on P2X7Rs-mediated currents. (A) Ketamine, propofol, and thiopental themselves had no effect on the currents (–60 mV). (B) Ketamine, propofol, and thiopental increased the P2X7Rs-mediated currents in a dose-dependent manner. ATP was 1 mM. These effects were reversible after washout of the anesthetics. (C) Traces show preperfusion of propofol produced no additional effect on P2X7Rs-mediated currents as compared with coapplication.

 

Figure 524
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  		Figure 5. Dose–response relationships of IV anesthetics for P2X7Rs-mediated currents induced by 1 mM ATP. Ordinate represents the current amplitude as percentages of the control. Ketamine (A), propofol (B), and thiopental (C) enhanced the P2X7Rs-mediated currents at concentrations higher than 300, 10, and 100 µM, respectively. Ketamine-induced enhancement at 300 µM ATP was not significantly different from that at 1 mM ATP (A, inset). Thiopental reduced the currents at lower concentrations (C, inset). Data are means ± sem. Asterisks indicate significant difference from the control (*P < 0.05, ***P < 0.005) (paired t-test).

 

To obtain the current–voltage (I–V) relationship, voltage ramps ranging from –100 to +100 mV were applied before and during application of ATP (Fig. 6, left). The data presented in Figure 6 are "pure " ATP (with or without thiopental) effects, derived by subtracting the control currents (first ramp) from the currents in the presence of ATP (second ramp). The I–V curve in the presence of 1 mM ATP (dotted line) had the reversal potential of approximately 0 mV, since P2X7Rs are nonselective cationic channels. Thiopental at 1 mM (continuous line), as well as 1 mM ketamine and 300 µM propofol (data not shown), increased both inward and outward conductances without changes in the reversal potential.


Figure 624
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  		Figure 6. The current–voltage (I–V) relationship for P2X7Rs-mediated currents in the absence and presence of thiopental. Voltage ramps from –100 to +100 mV were applied before (first ramp) and during the ATP application (second ramp) (left). I–V relationship of ATP-induced currents was obtained from the subtracted currents (right). Coapplication of 1-mM thiopental with 1-mM ATP increased both inward and outward currents without changes in the reversal potential.

 

Effects of General Anesthetics on P2YRs
ATP increases the concentration of intracellular Ca2+ in microglia because of Ca2+ influx via P2XRs and Ca2+ release from the intracellular Ca2+ stores via P2Y receptors (10,35). To examine the effects of general anesthetics on P2YRs, the Ca2+ responses were measured in a low Ca2+ (100 nM) medium which avoided the P2XR-mediated Ca2+ influx. In each experiment, the standard external solution was replaced by the low Ca2+ solution a few minutes before application of anesthetics and/or ATP. The intracellular Ca2+ level was increased by application of 100 µM ATP and then it slowly decreased to the baseline level in the continuous presence of ATP (Fig. 7A). A second application of ATP 10 min later induced a considerably smaller response than the initial response (data not shown) (35), and therefore only the data obtained from the first application of test solution were analyzed. At the end of each experiment, the medium was switched back to the Ca2+ (1 mM) containing standard solution. The data from different preparations were normalized to the maximum increase determined by addition of 20 µM ionomycin, a Ca2+-ionophore.


Figure 724
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  		Figure 7. ATP-induced increase in intracellular Ca2+ concentration. Intracellular Ca2+ level was measured in a low Ca2+ solution (100 nM). ATP was applied for approximately 2 min. (A) A rapid transient increase in intracellular Ca2+ produced by application of ATP (100 µM). At the end of each experiment, the medium was switched back to the Ca2+ (1 mM) containing standard solution with 20 µM ionomycin for calibration of ATP response. (B) ATP-induced Ca2+ mobilization in the presence of 100 µM ketamine. Anesthetics were pre-applied for approximately 2 min before application of ATP. (C) Effects of general anesthetics on ATP-induced increase in intracellular Ca2+ level. There was no significant difference between ATP-induced Ca2+ increases in control and those with general anesthetics. Data are given as mean ± sem.

 

General anesthetics (330–990 µM sevoflurane, 10–100 µM ketamine, 30–300 µM propofol, and 30–100 µM thiopental) were pre-applied for 2 min before ATP (Fig. 7B). Anesthetics alone did not induce Ca2+ mobilization except for an extremely high concentration (300 µM) of propofol which increased the Ca2+ concentration (data not shown). None of the inhaled and IV anesthetics tested affected the peak response (Fig. 7C) and the time course (Figs. 7A and B) of the ATP-induced Ca2+ transients.


   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
To the best of our knowledge, this is the first report investigating the effects of general anesthetics on ATP-induced responses in microglia. We assessed the actions of both inhaled and IV anesthetics on P2X7Rs and P2YRs using whole-cell clamp recording and single cell fluorometry of intracellular Ca2+. P2YRs-mediated Ca2+ responses were not affected by all general anesthetics tested. IV anesthetics (ketamine, propofol, and thiopental), however, enhanced P2X7Rs-mediated currents reversibly in a dose-dependent manner, whereas inhaled anesthetics had no effect on the P2X7Rs response.

In the present study, the ATP-induced nondesensitizing currents were characterized by activation with Bz-ATP (an agonist for P2X7Rs), blockade with oATP (an antagonist for P2X7Rs), the reversal potential closed to approximately 0 mV, and flow in both inward and outward directions. These properties were consistent with those of P2X7Rs in rat microglia in primary culture (10).

The potencies of IV anesthetics for enhancing P2X7Rs were not correlated with the octanol/buffer partition coefficients (approximately 60 for ketamine, approximately 390 for thiopental, and approximately 4000 for propofol). This finding may suggest that the actions of these anesthetics on P2X7Rs are not explained by nonspecific hydrophobic interactions. Further study is required to elucidate the mechanisms of the enhancement of microglia P2X7Rs by IV anesthetics. Interestingly, thiopental at low concentrations (3 and 10 µM) slightly decreased the P2X7Rs-mediated currents (by approximately 13%), suggesting dual actions of thiopental on P2X7Rs.

Clinically relevant concentrations of ketamine, propofol, and thiopental range from 2 to 7 µM, from 0.4 to 3 µM, and from 40 to 80 µM, respectively (33). The peak plasma concentrations increased up to 60 µM with ketamine, 56 µM with propofol, and 120 µM with thiopental after bolus administration (36,37). Therefore, IV anesthetics, in particular propofol and thiopental, have a potential to modulate the microglial P2X7R-mediated response at concentrations which can be reached after bolus injection.

ATP is important in mediating interactions among various cell types in the brain, including synaptic transmission and inflammatory glial activation (5,38). Microglial P2X 7Rs are involved in modulating neuronal damage caused by oxygen/glucose deprivation and focal cerebral ischemia (23,24). The activation of microglia P2X7Rs has been suggested to contribute to neuronal damage in certain neurodegenerative diseases, such as Alzheimer’s disease (17,39). Thus, enhancement of microglial P2X7Rs by anesthetics could aggravate the pathological condition. Our results suggest that care should be taken when large doses of propofol and thiopental are administered in patients with brain infarction, traumatic brain injury, and certain neurodegenerative diseases. Further study is needed to clarify the role and mechanisms of P2X7Rs enhancement by IV anesthetics in the pathological condition of the brain.

An in vivo study (22) using transcranial two-photon microscopy elegantly demonstrated that extracellular ATP released from damaged tissues and astrocytes induced microglial migration through stimulation of P2YRs. In our study, inhaled and IV anesthetics had no effect on the P2YRs-mediated mobilization of intracellular Ca2+, suggesting that these anesthetics do not affect P2YRs-related microglial activation and motility.

P2XRs have been found in cells in the whole body; P2X1Rs in smooth muscle, P2X2–5Rs in neurons, P2X5Rs in heart, thymus, and skeletal muscle (40). P2X7Rs are found in most cells of the immune system and share limited homology with other members. Microglia are macrophage-lineage phagocytic cells. Functional properties of macrophage P2X7Rs are similar to those of microglia and mediate biological processes, such as the release of cytokine, and the killing of pathogenic bacteria (41). Although pharmacological properties of macrophage P2X7Rs may not be necessarily the same as those of microglia, our results suggest the possibility that propofol and thiopental may affect macrophage function via P2X7Rs modulation.

The action of several inhaled and IV anesthetics on P2X receptors has been reported in a variety of cells. Sevoflurane (0.5 mM) apparently inhibited P2XRs-mediated currents in locus coeruleus neurons in rats (42). Propofol at relatively higher concentrations (50, 100 µM) slightly inhibited P2XRs in vagus nerve neurons (43) whereas it enhanced P2X4Rs expressed in HEK cells (44). At clinically relevant concentrations, thiopental and propofol had no effect on P2XRs in PC12 cells (45,46). Our experiments showed that IV anesthetics augmented P2X7Rs-mediated currents. These various effects of anesthetics may be attributed at least partly to involvement of different types of P2XRs among cells.

In summary, it is suggested that IV anesthetics, propofol and thiopental in particular, may modulate microglial immunological responses through P2X7Rs in pathological conditions.


   ACKNOWLEDGMENTS
 
The authors thank Junko Kawawaki for technical assistance.


   Footnotes
 
Accepted for publication January 25, 2007.

Supported, in part, by the Japanese Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan grant 15790827.

Reprint requests to Takashi Mori, MD, Department of Anesthesiology and Intensive Care Medicine, Graduate School of Medicine, Osaka City University, 1-5-7 Asahi-machi, Abeno-ku, Osaka 545-8586, Japan. Address e-mail to m1377033{at}med.osaka-cu.ac.jp.


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2007 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press