Anesth Analg 2003;97:449-455
© 2003 International Anesthesia Research Society
ANESTHETIC PHARMACOLOGY
The Inhibitory Effects of Alphaxalone on M1 and M3 Muscarinic Receptors Expressed in Xenopus Oocytes
Munehiro Shiraishi, MD*,
Kouichiro Minami, MD PhD*,
Izumi Shibuya, PhD
,
Yasuhito Uezono, MD PhD
,
Junichi Ogata, MD*,
Takashi Okamoto, MD*,
Osamu Murasaki, MD PhD,
Muneshige Kaibara, MD PhD,
Yoichi Ueta, MD PhD, and
Akio Shigematsu, MD PhD*
Department of *Anesthesiology and
Physiology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu; and
Department of Pharmacology, Nagasaki University Graduate School of Biomedical Sciences, Japan
Address correspondence and reprint requests to Munehiro Shiraishi, MD, Department of Anesthesiology, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu 807-8555, Japan. Address e-mail to mshira{at}med.uoeh-u.ac.jp
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Abstract
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Alphaxalone is a neurosteroid anesthetic, but its mechanisms of action are not completely understood. Muscarinic receptors are involved in a variety of neuronal functions in the brain and autonomic nervous system, and much attention has been paid to them as targets of anesthetics. In this study, we investigated the effects of alphaxalone on M1 and M3 muscarinic receptors using the Xenopus oocyte expression system. Alphaxalone inhibited acetylcholine-induced currents in oocytes expressing M1 receptors at clinically relevant concentrations. Alphaxalone also suppressed acetylcholine-induced currents in oocytes expressing M3 receptors. The half-maximal inhibitory concentration values for the inhibition of M1- and M3-mediated currents were 1.8 ± 0.6 µM and 5.3 ± 1.0 µM, respectively. GF109203X, a selective protein kinase C inhibitor, had little effect on the inhibition of acetylcholine-induced currents by alphaxalone in oocytes expressing these receptors. Alphaxalone inhibited the specific binding of [3H]quinuclidinyl benzilate to oocytes expressing M1 or M3 receptors. These findings suggest that alphaxalone at clinically relevant concentrations inhibits the function of M1 and M3 receptors through a protein kinase C-independent mechanism by interfering with the [3H]quinuclidinyl benzilate binding sites on the receptors.
IMPLICATIONS: Alphaxalone, a neurosteroid anesthetic, inhibited the function of muscarinic M1 and M3 receptors and the specific binding of [3H]quinuclidinyl benzilate ([3H]QNB) to oocytes expressing these receptors. These findings suggest that alphaxalone inhibits these receptors by interfering with the QNB binding sites.
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Introduction
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Neurosteroids are synthesized from cholesterol in both the central and peripheral nervous systems (1) and have anesthetic activity (2). Alphaxalone (3
-hydroxy-5-pregnane-11, 20-dione) is an anesthetic steroid (3). Several electrophysiological studies have revealed potent and stereoselective effects of alphaxalone on 
-aminobutyric acid (GABA)A receptor-mediated responses in rat brain slices (4). This was further confirmed in experiments using dissociated neurons (4) and adrenal medullary chromaffin cells (5). Potentiation of the GABAA receptor function may be an anesthetic mechanism of alphaxalone. However, we recently reported that alphaxalone inhibits nicotinic acetylcholine (ACh) receptors in cultured bovine adrenal chromaffin cells (6). More recently, alphaxalone suppressed norepinephrine transporter function (7). Therefore, it is of interest to also study the pharmacological properties of alphaxalone on other receptors.
In the central nervous system, muscarinic signaling plays an important role in the level of consciousness, memory, and learning (8,9), and spinal muscarinic receptors are involved in antinociception (10). Molecular cloning studies have revealed five subtypes of muscarinic receptors (M1–M5) (11). All belong to the family of G-protein-coupled receptors, and M1 and M3 receptors are known to couple to Gq. Several investigators have shown that general anesthetics inhibit muscarine receptor functions. The volatile anesthetics halothane and isoflurane inhibit M1 and M3 muscarinic receptor function via enhancing protein kinase C (PKC) activity, and IV anesthetics and analgesics also inhibit the muscarinic receptors (12–17). Thus, both M1 and M3 are likely to be a target of the anesthetics and analgesics; however, the effects of alphaxalone on the muscarinic receptor functions have not yet been clarified. Therefore, there is a strong rationale for asking whether alphaxalone also affects the receptors.
The Xenopus oocyte expression system has been widely used to study a multiplicity of brain receptors (18). Stimulation of muscarinic M1 or M3 receptors expressed in oocytes activates Ca2+-activated Cl- currents (13,15); activation of M1 or M3 receptor leads to Gq-protein mediated activation of phospholipase C, which causes formation of inositol-1,4,5-trisphosphate (IP3). IP3 releases Ca2+ from Ca2+ stores in the endoplasmic reticulum, and increased cytosolic Ca2+ triggers the opening of endogenous Ca2+-activated Cl- channels. This system has been well characterized and has proven to be useful for studying the effects of drugs acting on Gq-protein-coupled receptors.
The purpose of this study was to clarify the effects of alphaxalone on M1 and M3 receptor functions. To accomplish this, we examined the effects of alphaxalone on M1 and M3 receptors using the Xenopus oocyte expression system. Furthermore, we investigated the effects of alphaxalone on [3H]quinuclidinyl benzilate ([3H]QNB) binding using oocytes expressing M1 and M3 receptors.
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Methods
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Adult female Xenopus laevis were purchased from Seac Yoshitomi (Fukuoka, Japan). Alphaxalone, ACh, and atropine were purchased from Sigma (St Louis, MO). The Escherichia coli transformation kit was from Invitrogen (San Diego, CA). A kit from Qiagen (Chatworth, CA) was used to purify plasmid complimentary (c)DNA. Rat muscarinic M1 receptor cDNA was kindly provided by Dr. H. Lester (Caltech, Pasadena, CA), and cRNA for the M1 receptor was synthesized in vitro with T7 polymerase (Stratagene, La Jolla, CA) from cDNA linearized with Hind III; the rat M3 receptor cDNA was kindly provided by Dr. T.I. Bonner (National Institutes of Health, Bethesda, MD). In the M3 receptor coding region, the initiator codon and BstPI site at 277 in the open reading frame was amplified with polymerase chain reaction. It was ligated with the remaining coding region including stop codon with BstPI/ApaI site, and then the construct was subcloned into pCR3 vector (Invitrogen, Carlsbad, CA). The cRNA for the M3 receptor was synthesized in vitro with T7 polymerase (Stratagene) from cDNA linearized with ApaI. Bisindolylmaleimide I (GF109203X) was from Calbiochem (La Jolla, CA); HEPES was from Nacalai Tesque (Kyoto, Japan). Collagenase was from Nitta Zerachin (Osaka, Japan); [3H]QNB (48 Ci/mmol) was from Amersham (Buckinghamshire, UK).
Isolation and microinjection of Xenopus oocytes were performed as described by Minami et al (13). Xenopus oocytes were injected with 50 ng of rat cRNA encoding the M1 or M3 receptor, and electrophysiological recording was performed 2–5 days after the injection. Oocytes were placed in a 100-µL recording chamber and perfused with modified Barth’s saline (MBS) containing 88 mM of NaCl, 1 mM of KCl, 2.4 mM of NaHCO3, 10 mM of HEPES, 0.82 mM of MgSO4, 0.33 mM of Ca(NO3)2, and 0.91 mM of CaCl2 (pH value of 7.5 adjusted with NaOH) at a rate of 1.8 mL/min at room temperature. Recording electrodes (1–5 M
) filled with 3 M of KCl were inserted into the oocyte. A Warner Oocyte-clamp OC 725-C (Warner, Hamden, CT) was used to voltage clamp each oocyte at -70 mV. We measured the peak of the transient inward currents as ACh-induced currents because this component is dependent on ACh concentrations and is quite reproducible, as reported by Minami et al (13). Alphaxalone was preapplied for 2 min to allow a complete change of solution in the bath.
To study whether the inhibitory effects of alphaxalone on ACh-induced currents were modulated by PKC, oocytes expressing M1 or M3 receptors were exposed to the PKC inhibitor GF109203X (200 nM) (19) in MBS for 120 min. After exposure to GF109203X, oocytes were exposed to 1 µM of ACh, and the currents elicited were measured.
Binding experiment of [3H]QNB to Xenopus oocytes were performed as described by Shiga et al (17). Xenopus oocytes were injected with 50 ng of rat cRNA encoding the M1 or M3 receptor, and QNB-binding experiments were performed 2–5 days after the injection. Oocytes were incubated for 60 min at 25°C with MBS (final volume 1 mL) containing [3H]QNB (0.5 nM) in the presence or absence of alphaxalone (0.1–100 µM). After incubation, oocytes were rapidly washed with 5 mL of ice-cold MBS four times under vacuum through the Whatman GF/C glass-fiber filters (Whatman, Maidstone, UK) to terminate the binding. The oocytes on the filters were placed in counting vials containing a scintillation cocktail. The radioactivity was counted in an Aloka LSC-3500E counter (Aloka, LSC-3500E, Tokyo, Japan). Specific binding of [3H]QNB was defined as the binding inhibited by 100 µM of atropine.
The results are expressed as percentages of control responses to minimize variability in receptor expression in oocytes. The control responses were measured before and after drug application. All values are presented as the mean ± SEM. The n values refer to the number of oocytes studied. Each experiment was performed with oocytes from at least two frogs. Statistical analyses were performed using a one-way analysis of variance. Estimation of half-maximal inhibitory concentration (IC50) value for concentration-response curves was performed using GraphPad Prism software (San Diego, CA).
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Results
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The effects of alphaxalone on ACh-induced Cl- currents of M1 were examined using ACh at the concentration of 1 µM, which is around the 50% effective concentration of ACh obtained in the same system (13,16). In Xenopus oocytes expressing cloned M1 receptors, 1 µM of ACh induced robust Ca2+-activated Cl- currents (2220 ± 257 nA; n = 22). Alphaxalone applied at 100 nM, 1 µM, and 10 µM inhibited ACh-induced currents in oocytes expressing M1 receptors to 90.0%, 74.6%, and 54.9%, respectively, of the control (Fig. 1). The IC50 of alphaxalone for the 1 µM of ACh-induced currents was 1.8 ± 0.6 µM in oocytes expressing M1 receptors (n = 8) (Fig. 1B).
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Figure 1. (A) Tracings obtained from a single oocyte expressing M1 receptors show the effect of alphaxalone on 1 µM of acetylcholine (ACh)-induced currents. ACh was applied for 20 s with or without 2-min treatment with 10 µM of alphaxalone. (B) Concentration-response relationship of alphaxalone inhibition of ACh-induced currents. Alphaxalone (100 nM–100 µM) was applied to oocytes for 2 min, and then 1 µM of ACh was applied for 20 s. Each data point represents the mean ± SEM of at least eight oocytes. *P < 0.05 and ***P < 0.001 compared with the control response using analysis of variance.
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We then examined ACh-induced currents in oocytes expressing M3 receptor. Maximal currents were observed with ACh at 100 µM, and the 50% effective concentration value for ACh was 1.0 ± 0.1 µM. Therefore, 1 µM was used to study the effects of alphaxalone on ACh-induced currents in oocytes expressing cloned M3 receptors. In oocytes expressing M3 receptors, 1 µM of ACh induced robust Ca2+-activated Cl- currents (2595 ± 288 nA; n = 47) (Fig. 2). Alphaxalone applied at 100 nM, 1 µM, and 10 µM also inhibited ACh-induced currents to 99.4%, 87.8%, and 65.0%, respectively, of the control (Fig. 2B). The IC50 of alphaxalone was 5.3 ± 1.0 µM (n = 9).
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Figure 2. (A) Tracings obtained from a single oocyte expressing M3 receptors show the effect of alphaxalone on 1 µM of acetylcholine (ACh)-induced currents. ACh was applied for 20 s with or without 2-min treatment with 10 µM of alphaxalone. (B) Concentration-response relationship of alphaxalone inhibition of ACh-induced currents. Alphaxalone (100 nM–100 µM) was applied to oocytes for 2 min, and then 1 µM of ACh was applied for 20 s. Each data point represents the mean ± SEM of nine oocytes. ***P < 0.001 compared with the control response using analysis of variance.
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Isoflurane inhibits M1 and M3 receptor functions via activation of PKC (13,15). Therefore, we examined the effect of alphaxalone on M1 and M3 receptor-mediated currents with oocytes that had been pretreated with the selective PKC inhibitor GF109203X, which has a Ki value for inhibition of PKC activity of 20 nM (19). Treatments of oocytes expressing M1 and M3 receptors with GF109203X (200 nM) for 120 min enhanced the currents induced by 1 µM of ACh to 231% ± 42% and 258% ± 28.0% (n = 5), respectively. The inhibitory effects of alphaxalone on ACh-induced currents on M1 and M3 receptors were still observed after pretreatment with GF109203X (200 nM), and the magnitude of the current inhibition by alphaxalone was not changed by GF109203X (Figs. 3 and 4). Alphaxalone (10 µM) inhibited the M1 and M3 receptors to 53% and 64% before treatment of GF109203X and to 56% and 68% after treatment of GF109203X, respectively (Figs. 3 and 4).
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Figure 3. (A) Tracings obtained from a single oocyte show the effect of alphaxalone on 1 µM of acetylcholine (ACh)-induced currents in oocytes expressing M1 receptors before and after treatment with GF109203X, a selective protein kinase C (PKC) inhibitor. Oocytes were incubated with 200 nM of GF109203X for 2 h and were then stimulated by ACh in the presence of alphaxalone (10 µM). (B) Summary results for the effects of alphaxalone (10 µM) on 1 µM of ACh-induced currents with or without GF109203X (200 nM) pretreatment. Values are the mean ± SEM of five oocytes.
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Figure 4. (A) Tracings obtained from a single oocyte show the effect of alphaxalone on 1 µM of acetylcholine (ACh)-induced currents in oocytes expressing M3 receptors before and after treatment with GF109203X. Oocytes were incubated with 200 nM of GF109203X for 2 h and were then stimulated by ACh in the presence of alphaxalone (10 µM). (B) The effects of alphaxalone (10 µM) on 1 µM of ACh-induced currents with or without GF109203X (200 nM) pretreatment. Values are the mean ± SEM of seven oocytes.
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We reported that the analgesic tramadol inhibits the M1 receptor function and M3 receptor function by acting at the atropine-binding site (16,17). To study whether alphaxalone affects ACh binding to muscarinic receptors, we examined the effects of alphaxalone on the binding of [3H]QNB to oocytes expressing M1 and M3 receptors. The total binding of M1 and M3 receptors were 525 ± 23 cpm and 615 ± 17 cpm, respectively, and nonspecific binding of M1 and M3 receptors were 400 ± 33 cpm and 323 ± 35 cpm, respectively. This large nonspecific binding might be caused by a small expression of muscarinic receptors in contrast with the surface of an oocyte. Another possibility to be considered is that the residual radio ligand could not be washed out completely during the washing step once the ligand had penetrated the oocyte. The number of binding sites of M1 and M3 were 2 and 2.6 fmol/oocyte, respectively. Alphaxalone applied at 0.1, 1, and 10 µM concentration-dependently inhibited [3H]QNB binding to oocytes expressing M1 receptors to 88%, 69%, and 47%, respectively, of the control value. The IC50 of alphaxalone for the binding of [3H]QNB to oocytes expressing M1 receptor was 2.6 ± 1.9 µM (n = 4). Alphaxalone applied at 0.1, 1, and 10 µM also concentration-dependently inhibited [3H]QNB binding to oocytes expressing M3 receptors to 96%, 73%, and 61%, respectively, of the control value (Fig. 5). The IC50 of alphaxalone for the binding of [3H]QNB to oocytes expressing M3 receptors was 4.5 ± 0.6 µM (n = 4).
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Figure 5. Oocytes expressing M1 or M3 receptors were incubated with [3H]quinuclidinyl benzilate (QNB) (0.5 nM) and various concentrations of alphaxalone (100 nM–100 µM) for 60 min at 25°C. The radioactivity in oocytes after atropine (10 µM) treatment was taken as the total specific binding. The data shown are the mean ± SEM of four experiments. *P < 0.05 and ***P < 0.001 compared with control responses.
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Discussion
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In this study, alphaxalone inhibited the ACh-induced responses in both M1 and M3 receptors expressed in Xenopus oocytes. This is the first report to provide direct evidence for inhibition by a neurosteroid of muscarinic receptor functions at the cellular and molecular levels. Sear and Prys-Roberts (20) described the effects of a number of infusion regimens and their associated total drug concentrations. The infusion rates varied between 13.5 and 67.8 µg · kg-1 · min-1, with total drug plasma concentrations of 1.9–3.9 µg/mL (5.7–11.7 µM). It was reported that alphaxalone bound plasma protein, and the quoted plasma protein binding for alphaxalone is approximately 40% in humans and between 35% and 44% in rats (21). From this evidence, this equates to a free drug concentration of 3.5–7.5 µM at clinical concentrations. The IC50 concentrations of alphaxalone that were estimated in our study varied from 1.8 µM to 5.3 µM. Therefore, it is possible to say that alphaxalone inhibits muscarinic M1 and M3 receptors at clinically relevant concentrations. Our results are consistent with the findings that alphaxalone suppressed the secretion of catecholamines stimulated with muscarinic agonists in the dog adrenal medulla (22).
We next studied how alphaxalone inhibited the M1 and M3 receptor-mediated currents. There have been several reports that PKC plays an important role in the inhibition of M1 and M3 receptors by anesthetics. For example, we reported that halothane, F3 (1-chloro-1,2,2-trifluorocyclobutane), and ethanol inhibited M1 receptor-mediated responses via a PKC-dependent mechanism (13). More recently, Do et al. (15) reported that PKC activation with phorbol-12-myrisate-13-acetate potently suppressed ACh-induced currents in oocytes expressing M3 receptors, indicating that PKC activation is involved in the inhibition of the M3 function. However, in our present study, the PKC inhibitor GF109203X did not change the inhibitory effects of alphaxalone on M1- or M3-mediated currents, suggesting that the inhibitory effects of alphaxalone on these responses were independent of PKC, and therefore the mechanism of inhibition of the muscarinic function by alphaxalone is quite different from inhibition by the other anesthetics.
We further studied the effects of alphaxalone on [3H]QNB binding to oocytes expressing M1 or M3 receptors. Alphaxalone inhibited the specific binding of [3H]QNB to both muscarinic receptors in a concentration-dependent manner, and the concentration ranges and IC50 for the inhibition of binding were similar to those for the inhibition of M1- or M3-mediated currents. However, the inhibition curves level off at approximately 50% of [3H]QNB binding. These results indicated that alphaxalone would inhibit M1 and M3 receptors in a noncompetitive manner. It is reported that a submaximal inhibition of muscarinic radioligand binding is seen with muscarinic allosteric drugs, which affect the kinetics of radioligand (23). Alphaxalone may inhibit the M1 and M3 by its allosteric interaction on these receptors. However, the ACh-induced current in oocytes expressing M1 or M3 receptors is the result of a multistep signaling pathway involving the muscarinic receptor subtype, a Gq-protein, phospholipase C, endoplasmic reticulum, and Ca2+-activated Cl- channels. It is reported that halothane does not alter the action of angiotensin II receptors, although they have the same signaling steps as M1 and M3, suggesting the signaling pathway might not be the target of the anesthetic action (9). In our preliminary experiments, pentobarbital, ketamine, and propofol had no effects on substance P receptors, which also share the same signaling steps as M1 and M3 receptors. However, it may be that alphaxalone has additional targets within the signaling pathway because local anesthetics inhibit the G protein-coupled receptor signaling by interference with Gq protein function (24). More study is necessary to clarify the mechanisms of action of alphaxalone on the function of M1 and M3 receptors.
Muscarinic receptors in brain have been considered to play an important role in memory and consciousness (9), and many investigators have shown the inhibitory effects of volatile and IV anesthetics on recombinant muscarinic receptors (12–17). Moreover, inhibition of the muscarinic pathway induced by administering scopolamine to rats decreased the minimal alveolar anesthetic concentration of inhaled anesthetics (25). However, more recently, Eger et al. (26) concluded that acetylcholine receptors, muscarinic as well as nicotinic receptors, do not play a role as mediators of immobilization by inhaled anesthetics. Although this report provides strong rationale for understanding the relationship between the immobility caused by anesthetics and ACh receptors, there are possibilities that other anesthetic effects (e.g., amnesia, hypnosis, and unconsciousness) are involved in the inhibition of ACh receptors. In the present study, it is not clear whether the inhibition of M1 and M3 receptors by alphaxalone influences its anesthetic potency. We suggest the pharmacological properties of alphaxalone revealed in the study might affect the autonomic nervous systems via inhibition of the receptor function. This hypothesis could be tested by measuring blood pressure, circulating noradrenaline, intestine constriction, or glandular secretion in whole-animal experiments.
In conclusion, alphaxalone, at clinically relevant concentrations, inhibits the function of M1 and M3 muscarinic receptors by, at least in part, interfering with the QNB binding sites on the receptors. Our findings may help to understand neuronal actions of alphaxalone other than the enhancement of GABAA receptors.
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Acknowledgments
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Supported, in part, by Grants-in-Aid (Nos. 11671532, 10770778, 12770851, 11770878, 12671515, 12671516, 12770849, 14657399, 14704040, and 14571479) from the Ministry of Education, Science, and Culture of Japan, by a UOEH Research Grant for Promotion of Occupational Health, by the Japan Research Foundation for Clinical Pharmacology, and Uehara Memorial Foundation.
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Accepted for publication March 13, 2003.
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Abstract
Introduction
