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NEUROPHARMACOLOGY

Site of Action of the General Anesthetic Propofol in Muscarinic M1 Receptor-Mediated Signal Transduction

Departments of Pharmacology (O.M., M.K., S.M., Y.D., K.T.) and Anesthesiology (Y.N., K.S.), Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

Received June 17, 2003; accepted August 14, 2003.

	Abstract

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Although a potential target site of general anesthetics is primarily the GABA _A receptor, a chloride ion channel, a previous study suggested that the intravenous general anesthetic propofol attenuates the M1 muscarinic acetylcholine receptor (M1 receptor)-mediated signal transduction. In the present study, we examined the target site of propofol in M1 receptor-mediated signal transduction. Two-electrode voltage-clamp method was used in Xenopus oocytes expressing both M1 receptors and associated G protein subunits (Gq). Propofol inhibited M1 receptor-mediated signal transduction in a dose-dependent manner (IC₅₀ = 50 nM). Injection of guanosine 5'-3-O-(thio)triphosphate (GTPS) into oocytes overexpressing Gq was used to investigate direct effects of propofol on G protein coupled with the M1 receptor. Propofol did not affect activation of Gq-mediated signal transduction with the intracellular injection of GTPS. We also studied effects of propofol on l-[N-methyl-³H]scopolamine methyl chloride ([³H]NMS) binding and M1 receptor-mediated signal transduction in mammalian cells expressing M1 receptor. Propofol inhibited the M1 receptor-mediated signal transduction but did not inhibit binding of [³H]NMS. Effects of propofol on Gs- and Gi/o-coupled signal transduction were investigated, using oocytes expressing the 2 adrenoceptor (2 receptor)/cystic fibrosis transmembrane conductance regulator or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K⁺ channels). Neither 2 receptor-mediated nor M2 receptor-mediated signal transduction was inhibited by a relatively high concentration of propofol (50 µM). These results indicate that propofol inhibits M1 receptor-mediated signal transduction by selectively disrupting interaction between the receptor and associated G protein.

It has been suggested that the muscarinic acetylcholine receptor and its signal transduction cascade also are targets for general anesthetics (Aronstam et al., 1986, for review, see Aronstam and Dennison, 1989). Using Xenopus laevis oocytes as an expression system, a well characterized system for the study of G protein-coupled receptors, effects of general anesthetics on a cloned M1 muscarinic receptor have been extensively studied. Several general anesthetics (enflurane, halothane, ketamine, isoflurane, and sevoflurane) attenuated M1 receptor-mediated signal transduction (Lin et al., 1993; Durieux, 1995a,b; Minami et al., 1997; Nietgen et al., 1998). However, mechanisms governing inhibition of M1 receptor-mediated signal transduction with general anesthetics are not so well understood.

We reported effects of propofol, which is now a widely used intravenous anesthetic, on M1 receptor-mediated signal transduction in oocytes (Nagase et al., 1999). Two-electrode voltage-clamp studies revealed that propofol inhibits the acetylcholine-induced Ca²⁺ activated Cl^– current in oocytes expressing M1 receptors. Propofol did not affect activation of the current by an intracellular injection of CaCl₂ or aluminum fluoride (AlF₄^–) (Nagase et al., 1999), thus indicating that the target site of propofol may be the upstream of G protein coupled with the M1 receptor, that is, binding site of the receptor or interaction site between the receptor and G protein. It is also debated whether propofol inhibits GDP-GTP exchange on G protein, because activation of G protein with AlF₄^– does not require the GDP-GTP exchange (Gilman, 1987).

We attempted to clarify mechanisms of inhibitory effects of propofol in M1 receptor-mediated signal transduction. We overexpressed subunits of Gq with M1 receptors in oocytes, to see whether propofol would affect the acetylcholine-induced Ca²⁺-activated Cl^– current in oocytes expressing the M1 receptor and Gq (M1 receptor/Gq). We also overexpressed only subunits of Gq in oocytes, and determined propofol affects activation of the current when giving an intracellular injection of GTPS. GTPS replaces GDP on subunits of G protein and activates G protein receptor independently (Gilman, 1987), and this enables one to study effects of propofol on GDP-GTP exchange on G protein. Using mammalian cells expressing the M1 receptor, we examined effects of propofol on binding of l-[N-methyl-³H]scopolamine methyl chloride ([³H]NMS). Finally, effects of propofol on Gs- and Gi/o-coupled signal transduction were studied, using oocytes expressing the 2 adrenoceptor (2 receptor)/cystic fibrosis transmembrane conductance regulator (CFTR, the Cl^– channel regulated by cAMP-dependent protein kinase) or oocytes expressing the M2 muscarinic acetylcholine receptor (M2 receptor)/Kir3.1 (a member of G protein-gated inwardly rectifying K⁺ channels).

	Materials and Methods

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Materials
Rat m1 receptor, rat Kir3.1, and human 2 receptor cDNAs were obtained from Dr. Lester (California Institute of Technology, Pasadena, CA). Rat Gq cDNA was obtained from Dr. Aragay (California Institute of Technology). Human CFTR cDNA was obtained from Dr. Riordan (Children's Hospital, Toronto, Canada) and human m2 receptor cDNA was from Dr. Peralta (Harvard University, Cambridge, MA). Acetylcholine (ACh), isoproterenol, and GTPS was purchased from Sigma-Aldrich (St. Louis, MO). l-[N-methyl-³H]Scopolamine methyl chloride (80 Ci/mmol) was obtained from PerkinElmer Life Sciences (Boston, MA).

Propofol (10 mg/ml) (containing 100 mg/ml soybean oil, 12 mg/ml egg lecithin, and 22.5 mg/ml glycerin) was obtained from AstraZeneca (Osaka, Japan). Vehicle solution was prepared using Intralipid (Pharmacia AB, Stockholm, Sweden) (containing 100 mg/ml soybean oil, 12 mg/ml egg lecithin, and 25 mg/ml glycerin).

Methods
Electrophysiological Studies Using Xenopus Oocytes. Guidelines for Institutional Animal Care and Utilization were followed during the experiments. Oocyte preparation and two-electrode voltage-clamp method have been described elsewhere (Nagase et al., 1999). Female X. laevis toads were immersed in ice until unresponsive to painful stimulus. Stage V to VI oocytes were isolated and defolliculated by gentle shaking at 23°C for 90 min in ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, and 10 mM HEPES, pH 7.4) containing 0.5 mg/ml collagenase (Yakult, Tokyo, Japan). cRNAs were synthesized in vitro using MEGAscript kits (Ambion, Austin, TX). cRNAs (5 ng), unless otherwise mentioned, were injected into oocytes using Picospritzer II (General Calve Co., Fairfield, NJ), and then the oocytes were incubated at 19°C in modified Barth's solution 88 mM (NaCl, 1 mM KCl, 2.2 mM NaHCO₃, 0.82 mM MgSO₄, 0.41 mM CaCl₂, and 10 mM HEPES, pH 7.4) containing 2.5 mM sodium pyruvate and 100 µg/ml gentamicin. Electrophysiological measurements were made between 2 and 4 days after cRNAs injection, using a two-electrode voltage-clamp amplifier (TEV-200; Dagan, Minneapolis, MN).

The oocytes were clamped at –60 mV and superfused with bath solution: ND96 with 1 mM CaCl₂, or high potassium solution (96 mM KCl, 2 mM NaCl, 1 mM CaCl₂, 1 mM MgCl₂, and 5 mM HEPES, pH 7.4). The bath had a volume of 150 µl and the flow rate was 2 ml/min. Selected concentrations of chemical compounds (ACh, propofol, Intralipid, and isoproterenol) were dissolved in the bath solution and superfused. To obtain the ACh concentration-response curve in oocytes expressing the M1 receptor, selected concentrations of ACh in the bath solution were applied for 15 s after 15-min equilibrium periods from the start of voltage clamp. At 30 min after the initial application of ACh, effects of propofol on the ACh-induced current were observed in the presence of selected concentrations of propofol for 10 min. In case of oocytes expressing 2 receptor or M2 receptor, effects of propofol on the isoproterenol-induced current or the AChinduced current were also observed in the presence of propofol for 10 min at 30 min after the initial application of isoproterenol or ACh.

Under two-electrode voltage clamp, oocytes were injected with GTPS (30 mM, 5 nl) loaded into an injection glass pipette using a pressure injector, Picospritzer II.

Cell Culture, Radioligand Binding Assay, and Measurement of Intracellular Ca²⁺. cDNA for the rat M1 receptor was inserted into the pGEM1 vector. We subcloned the complete coding region of the cDNA for the rat M1 receptor into pcDNA 3.1/Hygro(–) (Invitrogen, Carlsbad, CA). Chinese hamster ovary (CHO) cells were grown in Ham's F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum and penicillin (200 U/ml)-streptomycin (200 mg/ml). Human kidney (293T) cells were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum and penicillin (200 U/ml)-streptomycin (200 µg/ml), and then transfected with plasmid DNA containing the M1 gene using an Effectene transfection reagent (QIAGEN GmbH, Hilden, Germany) (Kaibara et al., 2002). For the radiobinding assay, CHO cells stably expressing the M1 receptor, established by hygromycin B selection (500 µg/ml), the cells were cultured in 10-cm dishes and were then passed into 24-well culture plates. The cells in 24-well culture plates were washed twice with phosphate-buffered saline and then with 300 pM [³H]NMS in the presence or absence of atropine or propofol at room temperature for 3 h. After incubation, these cells were washed three times with phosphate-buffered saline and then dissolved into 1% SDS buffer. Subsequently, radioactivity was measured in a liquid scintillation counter (LSC-5000; Aloka, Tokyo, Japan).

The intracellular Ca²⁺ concentration was measured using Fura-2 essentially as described previously (Chen et al., 1994). Briefly, transiently transfected 293T cells grown on glass coverslips were loaded with 5 mM fura-2/AM (Dojindo, Kumamoto, Japan) for 20 min in Earle's balanced salt solution in the presence of 0.4 mM CaCl₂. After two washes with the Ca²⁺ contained Earle's balanced salt solution, the coverslip was positioned in a quartz cuvette containing 3.5 ml of the Ca²⁺ contained Earle's balanced salt solution at a 45° angle to both excitation and emission light paths. The fura-2 fluorescence was determined at 37°C using an RF-5000 spectrofluorophotometer (Shimadzu, Kyoto, Japan) operating at an emission wavelength of 505 nm with excitation wavelength of 340 and 380 nM. R is the ratio (F1/F2) of the fluorescence of excitation 340 nm/emission 505 (F1) to that of excitation 380/emission 505 (F2). R_cont is the ratio obtained 45 s before an application of ACh, and R_ACh is the ratio obtained 45 s after the application of ACh.

Data Analysis. Data are shown as mean ± S.E.M. for the indicated number of experiments. Log concentration-response curves were analyzed by nonlinear regression using a commercially available program (Kaleidagraph 3.5; Synergy Software, Reading, PA). All statistics were generated using StatView 5.0 (SAS Institute, Cary, NC), and p values less than 0.05 were considered statistically significant.

	Results

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Effects of Propofol on ACh Responses in Xenopus Oocytes Expressing M1 Receptor and Gq. In our series of studies, defolliculated oocytes not injected with cRNA for the M1 receptor, application of ACh induced no significant current (Matsumoto et al., 1998; Nagase et al., 1999). As shown in Fig. 1, A and B, when 2.5 ng of cRNA for Gq was coinjected with 5 ng of cRNA for the M1 receptor in each oocyte, the ACh response was small, the mean peak size being 13 ± 7 nA (Fig. 1C). Decrease in the injection of cRNA (1 ng) for Gq increased the response of ACh, the mean peak size being 583 ± 108 nA (Fig. 1C). The measured resting membrane potentials were –23.7 ± 1.7 mV for 2.5 ng of Gq and –42.0 ± 1.9 mV for 1 ng of Gq, these values being significantly different (p < 0.0001). We determined the concentration-response relationship for ACh in oocytes injected with 1 ng of cRNA for Gq and 5 ng of cRNA for the M1 receptor. As shown in Fig. 1D, the response of ACh was concentration-dependent (EC₅₀ = 87 nM). To determine whether the expressed receptor and G protein desensitized after repeated agonist applications, we induced two consecutive Ca²⁺-activated Cl^– currents with ACh (100 nM) in the same oocyte at 30-min intervals. The mean peak sizes for the two applications were 396 ± 57 and 358 ± 74 nA. No statistically significant difference between the means of the two response sizes was found (n = 14; p = 0.535; paired t test).

View larger version (13K): [in this window] [in a new window]   Fig. 1. ACh (100 nM)-induced currents in oocytes expressing the M1 receptor/Gq. Representative current traces in oocytes injected with cRNAs for the M1 receptor (5 ng) and Gq (2.5 ng) (A) and with cRNAs for the M1 receptor (5 ng) and Gq (1 ng) (B). C, ACh (100 nM) induced small currents in the oocytes injected with cRNAs for M1 receptor (5 ng) and Gq (2.5 ng). Data represent the mean ± S.E.M. of experiments for each concentration (n = 6 for 2.5 ng, n = 9 for 1 ng). D, concentration-response relationships for ACh-induced currents in oocytes injected with cRNAs for the M1 receptor (5 ng) and Gq (1 ng). The EC50 value was 87 nM. Data represent the mean ± S.E.M. of independent experiments for each concentration of ACh (n = 4 for 10 nM and 10 µM, n = 9 for 100 nM, n = 6 for 1 µM). Each oocyte was given only one concentration of ACh.

In a previous study, effects of propofol on ACh-induced M1 receptor-mediated signal transduction were studied using a 10-min application of propofol in oocytes expressing the M1 receptor (Nagase et al., 1999), because a relatively long period of application of propofol was required for inhibition. To obtain comparable data, we applied propofol for 10 min in the present study, although a 2-min application of propofol (100 nM) completely inhibited the activation of current with ACh (data not shown). As shown in Fig. 2, propofol inhibited activation of the current with ACh in oocytes expressing the M1 receptor/Gq in a dose-dependent manner (IC₅₀ = 50.0 nM). Intralipid at the concentration of 1.6 x 10^–3% (v/v), which was composed of soybean oil, egg lecithin, and glycerin at similar concentrations of 1 µM propofol, did not affect activation of the current (the mean peak sizes for control versus Intralipid, 515 ± 100 versus 506 ± 93 nA; n = 6).

View larger version (12K): [in this window] [in a new window]   Fig. 2. Effects of propofol on induction of current by ACh in oocytes injected with cRNAs for the M1 receptor (5 ng) and Gq (1 ng). A, application of propofol (1 µM) for 10 min before the second application of ACh (100 nM) inhibits activation of the current. The third application of ACh 60 min after the second one induces a similar magnitude of current to the initial application of ACh. B, propofol inhibited the current activated by ACh in a dose-dependent manner. The IC50 value was 50 nM. Data represent the mean ± S.E.M. of independent experiments for each concentration of propofol (n = 3 for 100 pM and 10 µM, n = 6 for 10 nM, 100 nM, and 1 µM).

Effects of Propofol on Induction of Current by Injection of GTPS into Oocytes Expressing Gq. As previously reported, oocytes overexpressing Gq are a useful system for studying the functional role of Gq in regulating cellular events (Kaibara et al., 2001). To study effects of propofol on GDP-GTP exchange on Gq, we used oocytes expressing Gq. Intracellular injection of GTPS, a nonhydrolyzable analog of GTP, produced a long-lasting inward current. Each oocyte was given only one injection of GTPS, because a second injection of GTPS did not elicit a significant current at 1 h after the initial injection. As shown in Fig. 3, propofol (1 µM) did not affect the induction of current with the injection of GTPS (the mean peak sizes for control versus propofol, 167 ± 21 versus 152 ± 20 nA). The activation and deactivation time courses of currents induced by GTPS were not affected by propofol. The measured resting membrane potential of oocytes injected with cRNA (5 ng) for Gq was –40.9 ± 1.3 mV (n = 14).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Effects of propofol on the induction of currents by intracellular injection of GTPS (30 mM, 5 nl) into oocytes expressing Gq. A, representative current trace is induced by an injection of GTPS. B, representative current trace is induced by an injection of GTPS in the presence of propofol (50 µM) in an oocyte expressing Gq. C, propofol did not inhibit the activation of the current by the intracellular injection of GTPS in oocytes expressing Gq. Data represent the mean ± S.E.M. (n = 9 for control and n = 5 for propofol).

Effects of Propofol on [³H]NMS Binding and M1 Receptor-Mediated Signal Transduction in Mammalian Cells. To obtain further information on the site of inhibitory action of propofol, we studied effects of propofol on [³H]NMS binding to the M1 receptor. As shown in Fig. 4, [³H]NMS binding to CHO cells stably expressing M1 receptors was displaced with atropine in a dose-dependent manner (IC₅₀ = 1.2 nM). Propofol (1 nM–100 µM) did not affect the [³H]NMS binding.

View larger version (13K): [in this window] [in a new window]   Fig. 4. Effects of propofol on [3H]NMS binding in CHO cells expressing the M1 receptor. Atropine (filled circles) inhibited the binding in a dose-dependent manner, the IC50 value was 1.2 nM. Data represent the mean ± S.E.M. Propofol (open circles) (1 nM–100 µM) did not inhibit the binding. Data represent the mean ± S.E.M. of three separate experiments, each run in duplicate.

We then determined whether propofol affects M1 receptor-mediated signal transduction in mammalian cells. We investigated the effects of propofol on intracellular Ca²⁺ responses to ACh (100 nM) in human cells (293T) transiently expressing M1 receptors. As shown in Fig. 5, in transfected cells, ACh increased intracellular Ca²⁺ concentrations (R_ACh/R_cont = 1.32 ± 0.05). In untransfected cells, application of ACh did not elicit a significant intracellular Ca²⁺ response (R_ACh/R_cont = 0.97 ± 0.02). In transfected cells, atropine at a concentration of 10 µM attenuated ACh-induced intracellular Ca²⁺ responses (R_ACh/R_cont = 0.99 ± 0.03). These results indicate that the M1 receptor transiently expressed in the cells is involved in the ACh-induced intracellular Ca²⁺ response. The ACh-induced intracellular Ca²⁺ responses were attenuated by propofol (100 nM) (R_ACh/R_cont = 1.0 ± 0.02).

View larger version (25K): [in this window] [in a new window]   Fig. 5. A, representative traces of intracellular Ca2+ ([Ca2+]i) responses to ACh (100 nM) in 293T cells untransfected, transfected the M1 receptor gene. The fluorescence trace is the ratio (R) of excitation 340 nM, emission 505 nM over excitation 380, emission 505 (F1/F2). B, ACh increased [Ca2+]i in the cells expressing the M1 receptor (n = 5, *, p < 0.004, paired t test). Propofol (100 nM) and atropine (10 µM) inhibited the effect of ACh. Data represent the mean ± S.E.M. (n = 5 for untransfected, n = 4 for propofol and atropine).

Effects of Propofol on Gi- and Gs-Mediated Signal Transduction. We also investigated effects of propofol on signal transduction via Gi or Gs in oocytes. To investigate the effect on Gi-mediated signal transduction, we used oocytes expressing the M2 receptor/Kir3.1. To study effects on Gs-mediated signal transduction, we used oocytes expressing the 2 receptor/CFTR. Propofol was used at concentration of 50 µM, with which M1 receptor-mediated signal transduction is almost inhibited. As shown in Fig. 6, propofol did not inhibit Gi-mediated (the mean peak sizes for control versus propofol, 170 ± 63 versus 152 ± 49 nA) or Gs-mediated signal transduction (the mean peak sizes for control versus propofol, 1149 ± 221 versus 1072 ± 146 nA).

View larger version (18K): [in this window] [in a new window]   Fig. 6. Effects of propofol on oocytes expressing the M2 receptor/Kir3.1, the 2 receptor/CFTR. A, representative current trace recorded in high potassium bath solution in an oocyte expressing the M2 receptor/Kir3.1. Second application of ACh (100 nM) in the presence of propofol (50 µM) induced a similar amplitude of current as the initial application. cRNA (0.2 ng) for Kir3.1 was injected. B, representative current trace in an oocyte expressing the 2 receptor/CFTR. Second application of isoproterenol (ISO) (100 nM) in the presence of propofol (50 µM) induced a similar amplitude of current as the initial application. C, propofol (50 µM) did not inhibit M2 receptor-mediated (n = 5) or 2 receptor-mediated (n = 5) signal transduction. Data represent the mean ± S.E.M.

	Discussion

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Our findings limited the target site of propofol in inhibition of M1 receptor-mediated signal transduction. Propofol did not inhibit [³H]NMS binding to M1 receptors stably expressed in CHO cells, thus indicating that the target site is not a ligand binding site of the receptor. Propofol did not affect activation of the current by the injection of GTPS, indicating that GDP-GTPS exchange on Gq and its downstream of the signal transduction are not target sites of propofol. A possible target site of propofol in the inhibition of the M1 receptor-mediated signal transduction exists between the ligand binding site of the receptor and GDP/GTP binding site of Gq. This possibility is consistent with previous data that 1) propofol did not inhibit [³H]NMS binding to M1, M2, and M3 receptors expressed in CHO cells (Hirota et al., 2002); and 2) propofol did not affect activation of the Cl^– current by an intracellular injection of AlF₄^– (Nagase et al., 1999).

The IC₅₀ value of propofol on the signal transduction in oocytes expressing the M1 receptor/Gq was 50 nM. Such being lower than the IC₅₀ value obtained in oocytes expressing only the M1 receptor (IC₅₀ = 5.9 µM) (Nagase et al., 1999). These IC₅₀ values are comparable with the concentration of propofol (1.7–16.8 µM), which potentiates amplitude of the membrane current elicited by GABA via the GABA_A receptor. Our data indicate that propofol also inhibits M1 receptor-mediated signal transduction in mammalian cells, because the M1 receptor-mediated intracellular Ca²⁺ response in 293T cells was inhibited by propofol. Clinically, the relevant blood concentration during intravenous infusion of propofol is considered to be around 5 µM (Shafer et al., 1988). The IC₅₀ values observed in the present study (50 nM) and our previous study (5.9 µM) are certainly within the clinically relevant range. It is well recognized that, in Xenopus oocytes, the expressed receptors use many types of G protein, such as Gq, G₁₁, Gi, and Go, to couple to phosphoinositide phospholipase C (Blitzer et al., 1993). It might be argued that the results obtained with the oocyte expression system do not mimic the action of propofol in mammalian systems. However, the difference in IC₅₀ values between oocytes expressing the M1 receptor/Gq and the M1 receptor alone indicates functional coupling of the M1 receptor and mammalian Gq in oocytes. The difference also indicates that G proteins other than Gq are involved in M1 receptor-mediated signal transduction in oocytes expressing the M1 receptor alone. The finding that propofol affects neither 2 receptor-mediated nor M2 receptor-mediated signal transduction supports the specific effects of propofol on the M1 receptor/Gq. Injection of GTPS into oocytes activates all kinds of G proteins in oocytes. If activation of the current with the injection of GTPS results from nonspecific activation of G proteins, it is difficult to examine effects of propofol on Gq involved in M1 receptor-mediated signal transduction. We previously showed that stable induction of the current with the intracellular injection of GTPS requires overexpression of Gq, indicating that the heterologously expressed Gq provides a useful system for studying functional roles of Gq (Kaibara et al., 2001).

In oocytes injected with both cRNA for the M1 receptor and a relatively large amount of cRNA for Gq, ACh evoked a very small current. This result was similar to that previously observed in oocytes expressing the thyrotropin-releasing hormone receptor and subunits of the Gq family G₁₆ (Quick et al., 1996). They suggested that overexpressed free subunits activated phospholipase C and desensitized the pathway. In our present study, in oocytes injected with relatively large amounts of cRNAs for Gq alone, an intracellular injection of GTPS could induce the Ca²⁺ activated Cl^– current. Our results suggest that receptors may stimulate desensitization of the signal transduction with overexpressed subunits of Gq. It is worth noting that the resting membrane potential of oocytes injected with both cRNA for M1 receptor and relatively large amounts of cRNA for Gq was significantly depolarized.

In summary, our results reveal unique anesthetic actions of propofol and are consistent with the proposal of involvement of inhibitory effects of anesthetics in M1 receptor-mediated signal transduction. General anesthetics at high enough levels can act nonspecifically on a variety of neuronal sites. Propofol, at relatively high concentrations, also interacts with the nicotinic acetylcholine receptor (Wachtel and Wegrzynowicz, 1992), the potassium channel (Magnelli et al., 1992), the sodium channel (Frenkel and Urban, 1991), and the calcium channel (Olcese et al., 1994), other than the GABA_A receptor (Hales and Lambert, 1991; for review, see Trapani et al., 2000). The selective and sensitive effect of propofol on the interaction between the M1 receptor and associated G protein indicates the clinical significance of involvement of muscarinic signal transduction processes in actions of anesthetics.

	Footnotes

 
This study was supported by a grant-in-aid from the Ministry of Education, Science, Technology, Sports and Culture of Japan.

DOI: 10.1124/jpet.103.055772.

ABBREVIATIONS: GTPS, guanosine 5'-O-(3-thio)triphosphate; [³H]NMS, l-[N-methyl-³H]scopolamine methyl chloride; CFTR, cystic fibrosis transmembrane conductance regulator; ACh, acetylcholine; CHO, Chinese hamster ovary.

Address correspondence to: Dr. Muneshige Kaibara, Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan. E-mail: mkaibara{at}alpha.med.nagasaki-u.ac.jp

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