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Anesth Analg 2005;101:1381-1388
© 2005 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000180190.05238.D5

ANESTHETIC PHARMACOLOGY

The Effects of Anesthetics and Ethanol on {alpha}2 Adrenoceptor Subtypes Expressed with G Protein-Coupled Inwardly Rectifying Potassium Channels in Xenopus Oocytes

Koji Hara, MD, PhD*{dagger}, Tomohiro Yamakura, MD, PhD{ddagger}, Takeyoshi Sata, MD, PhD{dagger}, and R. Adron Harris, PhD*

*Waggoner Center for Alcohol and Addiction Research and Institute for Cellular and Molecular Biology, University of Texas at Austin; {dagger}Department of Anesthesiology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu; and {ddagger}Division of Anesthesiology, Niigata University Graduate School of Medical and Dental Sciences, Japan

Address correspondence and reprint requests to Koji Hara, MD, PhD, Department of Anesthesiology, University of Occupational and Environmental Health, School of Medicine, 1-1, Iseigaoka, Yahatanishiku, Kitakyushu, 807-8555, Japan. Address e-mail to kojihara{at}med.uoeh-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
A wide range of physiological effects are mediated by {alpha}2-adrenoceptors (ARs) through their association with G protein-coupled inwardly rectifying potassium (GIRK) channels. Although {alpha}2-ARs are divided into three subtypes ({alpha}2A–C), a pharmacological distinction among the subtypes is difficult to establish because of the lack of a selective agonist and antagonist; therefore, little is known about the effects of anesthetics on the {alpha}2-AR subtypes. We expressed each subtype together with GIRK1/GIRK2 subunits in Xenopus oocytes and observed {alpha}2-AR-mediated GIRK1/GIRK2 currents to test the effects of ethanol, halothane, and several IV anesthetics at clinical concentrations. UK 14,304, a selective {alpha}2-AR agonist, evoked GIRK1/GIRK2 currents in every subtype. None of the IV anesthetics, which included pentobarbital, propofol, ketamine, and alphaxalone, influenced UK 14,304-evoked potassium currents in any of the receptor subtypes. Ethanol enhanced the UK 14,304-evoked potassium currents, whereas halothane inhibited the currents. However, these effects were not significantly different from those on the baseline-GIRK1/GIRK2 current, suggesting that neither ethanol nor halothane acts directly on the {alpha}2-AR subtypes. Although none of the drugs examined had any effect on the {alpha}2-ARs, the physiological actions of the {alpha}2-ARs mediated by the GIRK1/GIRK2 channels may be affected by ethanol and halothane.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
A wide range of physiological effects are mediated by {alpha}2-adrenoceptors (ARs), for example, cardiovascular, sedative, antinociceptive/anesthetic-sparing, and hypothermic effects (1,2). The {alpha}2-ARs belong to the superfamily of {gamma}-GTP binding protein (G protein) coupled receptors and have been divided into three subtypes ({alpha}2A, {alpha}2B, and a2C) on the basis of pharmacological and molecular cloning evidence (1,2). The three subtypes of {alpha}2-ARs are highly homologous, possessing a 50%–60% identity of amino acid sequences among the subtypes. They have similar pharmacological properties and signal transduction pathways mediated through the pertussis toxin-sensitive inhibitory G protein Gi/o. A previous study using genetically engineered mice with dysfunctional {alpha}2A-AR has established that the {alpha}2A-subtype mediates most of the physiological effects of {alpha}2-AR, which include sedative, analgesic, and anesthetic-sparing effects (3). The activation of the {alpha}2-ARs decreases neuronal excitation by opening G protein-coupled inwardly rectifying potassium (GIRK) channels, by inhibition of voltage-gated calcium channels, and by suppression of adenylate cyclase. The {alpha}2-AR agonists clonidine and dexmedetomidine are used in the treatment of patients with chronic pain and as adjuncts to general anesthesia. Despite the importance of {alpha}2-AR agonist use for anesthesia, there are few reports studying the effects of anesthetics on the {alpha}2-AR subtypes.

GIRK channels are opened by pertussis toxin-sensitive G proteins in a membrane-delimited manner in vivo and are activated by various G protein-coupled receptors, including M2-muscarinic, {alpha}2-adrenergic, D2-dopaminergic, {delta}-, {kappa}-, and µ-opioid, and {gamma}-aminobutyric acid type B (GABAB) receptors (4). In noradrenergic neurons, such as locus coeruleus and preganglionic sympathetic neurons, the GIRK channels underlie inhibitory postsynaptic potentials produced in response to the activation of the {alpha}2-ARs (5–7). Our study (8) demonstrated that the antinociceptive effect of clonidine was reduced in GIRK2 knockout mice. Although a precise distribution of the GIRK subtypes has not been determined, GIRK1/GIRK2 channels are highly abundant in the central nervous system (CNS) (9,10). Another study demonstrated that GIRK1/GIRK2 channels expressed in Xenopus laevis oocytes can elicit a potassium current (11). The physiological effects of the {alpha}2-ARs are mediated, at least in part, by the activation of GIRK channels, and thus expressing GIRK channels together with {alpha}2-ARs is regarded as a good tool for studying the effects of anesthetics on {alpha}2-ARs to mimic in vivo physiological effects.

Activation of Gq protein-coupled receptors, such as M1 muscarinic receptor, results in activation of phospholipase C, Ca2+ release from intracellular stores, and generation of Ca2+-dependent Cl– currents in the oocytes. However, Gi/o protein-coupled receptors, such as the {alpha}2-ARs, are unsuitable for electrophysiological analysis because agonist stimulations for the receptors do not generate measurable current. Furthermore, separating the {alpha}2-AR subtypes pharmacologically has been difficult because of the lack of a selective agonist and antagonist. Consequently, we are unaware of any published studies that investigated the effect of anesthetics on the {alpha}2-AR subtypes.

In the current study, we demonstrated that each subtype of {alpha}2-ARs could be coupled with GIRK channels in vitro. Using this system, we studied through electrophysiology the effects of various IV anesthetics, ethanol, and the volatile anesthetic halothane on the {alpha}2-ARs coupled with GIRK channels under the same condition. We chose the channels composed of GIRK1 and GIRK2 subunits (GIRK1/GIRK2) for studying the effects of the drugs on {alpha}2-AR subtypes using a two-electrode, voltage-clamp system.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Xenopus laevis female frogs were purchased from Xenopus Express (Homosassa, FL) and Seac Yoshitomi (Fukuoka, Japan). The three-aminobenzoic acid ethyl ester was obtained from Sigma (St. Louis, MO). XL-1 Blue cells and the mCAPTMRNA Capping kit were obtained from Stratagene (La Jolla, CA), and the QIAFilter Plasmid Maxi kit was obtained from Qiagen (Valencia, CA). Halothane was obtained from Halocarbon Laboratories (River Edge, NJ); 2,6-diisopropylphenol (propofol) was obtained from Aldrich Chemical Co. (Milwaukee, WI); alphaxalone was obtained from Research Biochemicals International (Natick, MA). Ethanol was obtained from Aaper Alcohol and Chemical (Shelbyville, KY); the pentobarbital sodium, ketamine hydrochloride, and other reagents were purchased from Sigma. Propofol and alphaxalone were first dissolved in dimethyl sulfoxide and then diluted in a high potassium (hK) solution. The largest final concentration of dimethyl sulfoxide (0.01%) did not affect the potassium current.

GIRK1 and GIRK2 subunit complimentary (c)DNAs were subcloned into the pBluescript II KS– vector, which was kindly provided by Dr. Michel Ladunski (Institut de Pharmacologie Moleculaire et Cellulaire, Valbonne, France). Human {alpha}2-AR subtypes 2A, 2B, and 2C cDNAs in a pBC expression vector were kindly given to us by Dr. Robert J. Lefkowitz (Department of Medicine, Duke University Medical Center, Durham, NC). NcoI-SalI fragments coding the {alpha}2A- and {alpha}2B-ARs were ligated and subcloned into pGEM-T Easy Vector. For the {alpha}2C-AR, the authors introduced an NcoI site upstream and an NdeI site downstream of the coding region and subcloned the fragment into the NcoI and NdeI sites of pBluescript II KS–. XL-1 Blue cells were transformed with the cDNAs, and amplified plasmid was purified with a QIAFilter Plasmid Maxi kit. The {alpha}2A- and {alpha}2B-AR cDNAs were linearized with SalI, and {alpha}2C-ARs were linearized with NdeI. The cRNAs were prepared using an mCAPTMRNA Capping kit.

The use of animals and experiments was approved by the Animal Care and Use Committees of University of Texas and the Ethics Committee of Animal Care and Experimentation by University of Occupational and Environmental Health in Japan. The surgical procedure was performed on frogs after they were anesthetized in water containing 3-aminobenzoic acid ethyl ester (240 mg/200 mL of water). The isolation of Xenopus oocytes was performed, as described previously (12). Isolated oocytes were placed in modified Barth's saline (MBS) containing 88 mM of NaCl, 1 mM of KCl, 2.4 mM of NaHCO3, 0.82 mM of MgSO4, 0.91 mM of CaCl2, 0.33 mM of Ca(NO3)2, and 10 mM of HEPES buffer adjusted to a pH value of 7.5. The oocytes were injected with cRNA (30 nL/oocyte) encoding a combination of GIRK channels and {alpha}2-AR subtype in a 1:1 ratio and were individually placed in Corning cell wells (Corning Glass Works, Corning, NY) containing incubation medium (sterile MBS supplemented with 10 mg/L of streptomycin, 100,000 U/L of penicillin, 50 mg/L of gentamicin, 90 mg/L of theophylline, and 220 mg/L of pyruvate). The injected oocytes were incubated at 15°C–19°C for 2–3 days and were then used for electrophysiological recording (12).

Oocytes co-expressing GIRK1/GIRK2 channels and each of the {alpha}2-AR subtypes were placed in a rectangular chamber (~100-µL volume) and perfused (2 mL/min) with MBS. The effects of IV anesthetics including pentobarbital, propofol, ketamine, and alphaxalone were studied using a two-electrode voltage-clamp system, as reported previously (11). The animal poles of oocytes were impaled with 2 glass electrodes (0.5–10 M{Omega}) filled with 3 M of KCl, and the oocytes were voltage-clamped at –80 mV using a Warner Instruments model OC-752B oocyte clamp (Hamden, CT). The oocytes were initially bathed in MBS and then changed to a hK solution containing 2 mM of NaCl, 96 mM of KCl, 1 mM of MgCl2, 1.5 mM of CaCl2, and 5 mM of HEPES buffer adjusted to a pH value of 7.5. Because the baseline current obtained in physiological buffer (MBS) was small, the hK solution was used to reverse the driving force of the channel and to provide a large inward current, as previously reported (13). After stable responses were established, UK 14,304, a selective full-agonist of {alpha}2-AR, was applied in the hK solution for 15 s before returning to the hK solution. All drugs dissolved in hK solution were preapplied for 1 min before being co-applied with UK 14,304. The effects of the drugs were expressed as the fraction of control responses, which were calculated by averaging the control responses before and after the application of the drugs.

The GIRK1/GIRK2 channels are mainly distributed in the CNS (9,10). Our previous work (11) has shown that the GIRK1/GIRK2 channels are not affected by IV anesthetics. Accordingly, we co-expressed the GIRK1/GIRK2 channels with the {alpha}2-ARs to study the effects of the drugs. For studying the effect of ethanol, solutions of 50, 100, and 200 mM of ethanol were initially assessed for effects on the baseline GIRK currents. Next, ethanol was tested on UK 14,304-evoked GIRK currents. For testing the volatile anesthetic halothane, 250, 500, and 1000 µM (1, 2, and 4 mean alveolar anesthetic concentration) of halothane solutions were initially assessed for their effects on baseline GIRK currents. The concentrations of halothane in the chamber were quantified by gas chromatography. Approximately the half-maximal effective concentration (EC50) of UK 14,304 in each subtype was applied to test the effects of the anesthetics and ethanol. Data are represented as mean ± sem. Statistical analysis was performed by one-way analysis of variance for multiple comparisons and by unpaired t-test for comparisons between two groups. Differences were considered statistically significant at P < 0.05. The values of the EC50 and the Hill coefficient were calculated by nonlinear regression using GraphPad Prism software version 3.0 (GraphPad Inc., San Diego, CA). All experiments were performed at room temperature (23°C).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The application of UK 14,304 to the oocytes co-expressing the GIRK1/GIRK2 with each {alpha}2-AR subtype gave consistent {alpha}2-AR subtype-mediated potassium currents (Fig. 1). Initial studies using longer application times of UK 14,304 indicated that 15 s yielded a consistent and mostly maximal effect. The oocytes were exposed to various concentrations of UK 14,304 ({alpha}2A, 0.01 nM–1 µM; {alpha}2B, 0.1 nM–10 µM; {alpha}2C, 1 nM–100 µM) to obtain the concentration-response relationship. UK 14,304 increased {alpha}2-AR-mediated potassium current in a concentration-dependent manner (Fig. 2). The EC50 values of UK 14,304 for {alpha}2A, {alpha}2B, and {alpha}2C were 3.4 ± 0.3 nM, 89 ± 3 nM, and 212 ± 6 nM, respectively, and the Hill coefficients were 1.0 ± 0.1 for all the subtypes.



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  		Figure 1. Representative tracing of G protein-coupled inwardly rectifying potassium (GIRK) current and UK 14,304-evoked GIRK current in the oocyte expressing the {alpha}2A-adrenoceptor (AR) and GIRK1/GIRK2 channel. The oocyte was bathed in modified Barth's saline (MBS) and then changed to a high potassium (hK) solution. After a stable baseline current was established, UK 14,304 was repeatedly applied for 15 s with consistent responses being observed.

 


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  		Figure 2. The UK 14,304 concentration-response relationship in oocytes expressing each of the {alpha}2-adrenoceptor (AR) subtypes and G protein-coupled inwardly rectifying potassium (GIRK)1/GIRK2 channels. UK 14,304-evoked potassium currents were observed for all the subunits in a concentration-dependent manner. Data are represented as mean ± sem.

 

To obtain a control response, UK 14,304 was applied repeatedly until a consistent response was observed. After the control currents were obtained, concentrations of 2- and 4-times the EC50 of each IV anesthetic, including propofol, ketamine, pentobarbital, and alphaxalone, were applied in a hK solution for 1 min before coadministration of the anesthetic and UK 14,304 for 15 s. None of the anesthetics affected the baseline potassium current, which is consistent with our previous results (11). Similarly, the anesthetics tested did not significantly affect UK 14,304-evoked potassium currents in the oocytes co-expressing the GIRK1/GIRK2 with each of the {alpha}2-AR subtypes (Fig. 3).



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  		Figure 3. The effects of IV anesthetics on UK 14,304-evoked potassium currents. (A) UK 14,304 in the high potassium (hK) solution was applied to oocytes expressing the {alpha}2A-adrenoceptor (AR) and G protein-coupled inwardly rectifying potassium (GIRK)1/GIRK2 channel for 15 s. After returning to the baseline current, IV anesthetics were preapplied for 1 min before being co-applied with UK 14,304 for 15 s. (B) Effects of IV anesthetics on the UK 14,304-evoked GIRK1/GIRK2 currents. Anesthetics were applied at concentrations corresponding to 2- and 4-times the half-maximal effective concentration (EC50) of the anesthetic, i.e., pentobarbital (100 and 200 µM) (14), propofol (2 and 4 µM) (14), ketamine (20 and 40 µM) (15), and alphaxalone (10 and 20 µM) (15). None of the anesthetics that were tested showed any effect at clinical concentrations. Data are represented as mean ± sem.

 

As previously demonstrated (16), ethanol enhanced the baseline GIRK1/GIRK2 currents per se in a concentration-dependent manner. UK 14,304-evoked potassium currents were also enhanced by ethanol to a degree essentially similar to that of baseline currents (Fig. 4). Conversely, the volatile anesthetic halothane inhibited the baseline currents at 1, 2, and 4 mean alveolar anesthetic concentration (Fig. 5). Halothane also inhibited UK 14,304-evoked currents, but the degree of the inhibition was not significantly different from that of the baseline currents. There were no significant differences among {alpha}2-AR subtypes in these experiments.



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  		Figure 4. The effect of ethanol on UK 14,304-evoked potassium currents. (A) Representative tracing of UK 14,304-evoked potassium currents in the oocytes expressing the {alpha}2A-adrenoceptor (AR) and G protein-coupled inwardly rectifying potassium (GIRK)1/GIRK2 channel. Ethanol (100 mM) enhanced the baseline potassium currents and UK 14,304-evoked potassium currents. (B) Ethanol (50, 100, and 200 mM) augmented the UK 14,304-evoked potassium currents and baseline potassium currents in a concentration-dependent manner. The percent potentiation was calculated from the following equations: percent potentiation for baseline currents = 100(b/a – 1), and percent enhancement for UK 14,304-evoked currents = 100(d/c – 1). Data are represented as mean ± sem. These values were not statistically different from each other. N.S. indicates no significance.

 


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  		Figure 5. Effect of halothane on UK 14,304-evoked potassium currents. (A) Representative tracing of UK 14,304-evoked potassium currents in the oocytes expressing the {alpha}2A-adrenoceptor (AR) and G protein-coupled inwardly rectifying potassium (GIRK)1/GIRK2 channel. Halothane (500 µM) inhibits the baseline potassium currents and UK 14,304-evoked potassium currents. (B) Halothane at 250 µM, 500 µM, and 1 mM, corresponding to 1, 2, and 4 mean alveolar anesthetic concentration, respectively (14), suppressed the UK 14,304-evoked potassium currents and baseline potassium currents in a concentration-dependent manner. The percent change for the UK 14,304-evoked currents, which was calculated from 100(1 – d/c), was essentially similar to that for the baseline currents calculated from 100(1 – b/a). Data are represented as mean ± sem. N.S. indicates no significance.

 


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We demonstrated in vitro coupling of {alpha}2-ARs and GIRK channels in this study. The physiological effects of {alpha}2-ARs are partly mediated through GIRK channels in the CNS. Therefore, the assay system used in this study is a good model for mimicking in vivo physiological conditions to study the effects of anesthetics on {alpha}2-ARs. Additionally, we are unaware of any published reports that compare the effects of anesthetics on {alpha}2-AR subtypes. There are no selective agonists and antagonists for the {alpha}2-AR subtypes, making the pharmacological separation of the subtypes difficult.

The physiological effects of the {alpha}2-ARs are associated with the production of some anesthetic qualities, such as sedation and analgesia. Our previous work (8) demonstrated that an analgesic effect of clonidine is mediated by the GIRK2 subtype, which suggests that anesthetic actions of {alpha}2-ARs are at least partially mediated by GIRK channels. Another study showed that the GIRK1/GIRK2 channels per se are insensitive to IV anesthetics at clinical concentrations (11). We used the GIRK1/GIRK2 subunits distributed predominantly in the CNS to test anesthetics and ethanol and found that the IV anesthetics had no effect on any {alpha}2-AR subtypes coupled with the GIRK1/GIRK2 channels. These results indicate that the IV anesthetics tested are unlikely to act directly on any of the {alpha}2-AR subtypes. Next, we tested the effects of ethanol on UK 14,304-evoked GIRK1/GIRK2 currents. As shown by the previous study (16), ethanol enhanced baseline GIRK1/GIRK2 currents in a concentration-dependent manner. Similarly, ethanol increased UK 14,304-evoked currents to a degree similar to baseline currents. Last, we tested a volatile anesthetic halothane on the GIRK1/GIRK2-coupled {alpha}2-AR subtypes. Halothane inhibited not only the baseline GIRK1/GIRK2 current, but also the UK 14,304-evoked currents in a concentration-dependent manner. The degree of the inhibition was not significantly different between the baseline currents and the UK 14,304-evoked currents.

Gq protein-coupled receptors, which include muscarinic acetylcholine, 5-hydroxytryptamine, and metabotropic glutamate receptors, were shown to be affected by anesthetics and ethanol (17–20). Gq protein-coupled receptors may be plausible targets of the drugs. The results of this study suggest that, at clinical concentrations, neither the anesthetics used in this study nor ethanol have a direct effect on the molecules of the {alpha}2-AR subtypes or on the transport of the G protein ß{gamma} subunit to the GIRK channels expressed in the oocytes. It might be speculated that Gi/o protein-coupled receptors are relatively insensitive to anesthetics and ethanol, as compared with Gq protein-coupled receptors. However, it is presumed that the physiological effects of the endogenous {alpha}2-AR agonists, that is, catecholamines acting through the GIRK channels, may be affected by ethanol and halothane. Ethanol may augment endogenous agonist-evoked GIRK1/GIRK2 current, which may partially explain the antinociceptive effect of ethanol. We (8) demonstrated that the antinociceptive effects of ethanol are reduced in GIRK2-null mutant mice. Furthermore, Mao and Abdel-Rahman (21) demonstrated in rats that ethanol synergistically increased the analgesic effect of clonidine.

The absence of any effect of volatile anesthetics on {alpha}2-ARs was shown in another assay system by Pentyala et al. (22), who demonstrated that volatile anesthetics, including halothane, did not influence {alpha}2-adrenergic signaling in isolated platelets. Halothane may inhibit endogenous {alpha}2-AR agonist-evoked GIRK1/GIRK2 current in vivo. GIRK channels are unlikely to contribute to the anesthetic-sparing effect of {alpha}2-AR agonist. Currently, the clinical roles of the inhibitory effect of halothane on GIRK1/GIRK2 channels are not clear; however, they might be associated with some side effects, such as tachycardia, or an excitement during anesthesia.

The results of this study also imply that the physiological effects of other receptors coupled with the GIRK1/GIRK2 channels, such as µ-opioid, cannabinoid, and GABAB receptors, may be modulated by ethanol and halothane in the CNS. Antinociceptive effects associated with these receptors have been eliminated in GIRK2 knockout or mutant mice (8,23), which indicates that the GIRK2 subunits are involved in antinociceptive effects through various neuronal systems.

Other GIRK subunits were not addressed in the present study. Specifically, GIRK1/GIRK4 subunits dominantly exist and couple with M2 receptors in the heart (24). Our previous study (11) demonstrated that GIRK1/GIRK4 subunits are insensitive to anesthetic, and further experiments testing the effects of anesthetics on GIRK1/GIRK4 channels coupled with M2 receptors would be beneficial to understand the drugs' actions on cardiac function in detail.

The present study showed that all {alpha}2-AR subtypes can be coupled with GIRK channels in vitro in a Xenopus oocyte expression system. The {alpha}2-AR, a member of the G protein-coupled receptor family, was found to be an unlikely target of both ethanol and the anesthetics used in this study. Some clinically significant aspects of ethanol and volatile anesthetics may arise through the modulation of the effects of {alpha}2-ARs via GIRK channels.

The authors thank Dr. Michel Ladunski for kindly providing cDNAs of GIRK1 subunit and GIRK2 subunit and Dr. Robert J. Lefkowitz for the generous gift of cDNAs of {alpha}2-AR subtypes.


   Footnotes
 
Supported, in part, by Grants-in-Aid for Research from the Ministry of Education, Science and Culture of Japan, No. 15790842.

Accepted for publication April 20, 2005.


   References
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 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 with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press