Effects of Gaseous Anesthetics Nitrous Oxide and Xenon on Ligand-Gated Ion Channels Comparison with Isoﬂurane and Ethanol Tomohiro Yamakura, M.D., Ph.D.*, R

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Anesthesiology 2000; 93:1095–1101 © 2000 American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc. Effects of Gaseous Anesthetics Nitrous Oxide and Xenon on Ligand-gated Ion Channels Comparison with Isoﬂurane and Ethanol Tomohiro Yamakura, M.D., Ph.D.*, R. Adron Harris, Ph.D.§

Background: Ligand-gated ion channels are considered to be erties of these gaseous anesthetics have long been cen- potential general anesthetic targets. Although most general an- tral to molecular theories of anesthesia that seek to ␥ esthetics potentiate the function of -aminobutyric acid recep- explain how these simple and small molecules are able tor type A (GABAA), the gaseous anesthetics nitrous oxide and xenon are reported to have little effect on GABA receptors but to produce anesthesia. A Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/93/4/1095/401580/0000542-200010000-00034.pdf by guest on 26 September 2021 inhibit N-methyl-D-aspartate (NMDA) receptors. To define the During the past few decades, a consensus has emerged spectrum of effects of nitrous oxide and xenon on receptors that general anesthetics act on one or more superfamilies thought to be important in anesthesia, the authors tested these of ligand-gated ion channels that include ␥-aminobutyric anesthetics on a variety of recombinant brain receptors. acid type A (GABA ), glycine, nicotinic acetylcholine Methods: The glycine, GABA , GABA receptor type C (GABA ), A A C (nACh), 5-hydroxytryptamine (5-HT ), and glutamate re- NMDA, ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid 3 3 ceptors.1,2 Among the ligand-gated ion channels, the (AMPA), kainate, 5-hydroxytryptamine3 (5-HT3), and nicotinic acetylcholine (nACh) receptors were expressed in Xenopus oo- GABAA receptor is considered to be a prime target of cytes and effects of nitrous oxide and xenon, and as equipotent volatile and intravenous anesthetics.1,2 Recent studies sug- concentrations of isoflurane and ethanol, were studied using gest that nitrous oxide and xenon barely affect GABAA the two-electrode voltage clamp. receptors but inhibit N-methyl-D-aspartate (NMDA) recep- Results: Nitrous oxide (0.58 atmosphere [atm]) and xenon 3–6 (0.46 atm) exhibited similar effects on various receptors. Gly- tors. These findings suggest that molecular mechanisms of two gaseous anesthetics are different from those of cine and GABAA receptors were potentiated by gaseous anesthetics much less than by isoflurane, whereas nitrous oxide volatile and intravenous anesthetics. To confirm and inhibited GABAC receptors. Glutamate receptors were inhibited extend these findings, we selected nine recombinant by gaseous anesthetics more markedly than by isoflurane, but ligand-gated ion channels and determined the effects of less than by ethanol. NMDA receptors were the most sensitive nitrous oxide and xenon on these channels during sim- among glutamate receptors and were inhibited by nitrous oxide ilar experimental conditions. The choice of subunit com- by 31%. 5-HT3 receptors were slightly inhibited by nitrous oxide. The nACh receptors were inhibited by gaseous and volatile position of recombinant receptors was based on the anesthetics, but ethanol potentiated them. The sensitivity was predominance of the subunit distribution or combina- different between ␣4␤2 and ␣4␤4 nACh receptors; ␣4␤2 recep- tion in the central nervous system or the availability of tors were inhibited by nitrous oxide by 39%, whereas ␣4␤4 previous data for anesthetic sensitivity of the subunits. receptors were inhibited by 7%. The inhibition of NMDA and nACh receptors by nitrous oxide was noncompetitive and was slightly different depending on membrane potentials for NMDA receptors, but not for nACh receptors. Materials and Methods Conclusions: Nitrous oxide and xenon displayed a similar spectrum of receptor actions, but this spectrum is distinct from cDNA and cRNA Preparation ␣ that of isoflurane or ethanol. These results suggest that NMDA The cDNA encoding the human 1-glycine receptor receptors and nACh receptors composed of ␤2 subunits are subunit7 in the pBK-CMV N/B-200 vector; cDNAs of likely targets for nitrous oxide and xenon. (Key words: Electro- ␣ ␤ ␥ 8 human 1, 2, and 2S GABAA receptor subunits in physiology; excitatory amino acids; serotonin.) pBK-CMV N/B-200, pCDM8, and pCIS2 vectors, respec- ␳ 9 tively; cDNA of the human 1 GABAC receptor subunit AN anesthetic inorganic gas, nitrous oxide, is widely in pcDNA1 vector; cDNAs of the human NR1a and NR2A used for general anesthesia in combination with other NMDA receptor subunits10 in pcDNA1Amp vector; and 11 drugs. A more potent anesthetic inert gas, xenon, can be cDNA of NCB-20 5-HT3 receptor subunit in pBK-CMV used as the sole anesthetic agent. The anesthetic prop- N/B-200 vector were used for the nuclear injection. The cDNAs of the rat GluR1 and GluR2 ␣-amino-3-hydroxy- * Research Fellow, Institute for Cellular and Molecular Biology and Lecturer, 5-methyl-4-isoxazole propionic acid (AMPA) receptor Department of Anesthesiology, Niigata University School of Medicine, Niigata, subunits12 in the pBluescript SKϪ vector, cDNAs of the Japan. § Waggoner Professor of Molecular Biology. rat GluR6 and KA2 kainate receptor subunits13 in Received from the Waggoner Center for Alcohol and Addiction Research and Ϫ Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas. pGEM-HE and pBluescript SK vectors, respectively, and Submitted for publication March 30, 2000. Accepted for publication June 15, the rat nACh receptor subunit cDNAs14 in several ex- 2000. Support was provided by the National Institutes of Health (grant Nos. AA06399 and GM47818), Bethesda, Maryland. pression vectors (␣4 in pSP64 vector, ␤2 in pSP65 vec- Ϫ Address reprint requests to Dr. Harris: Institute for Cellular and Molecular tor, and ␤4 in pBluescript SK vector) were used for Biology, University of Texas, 2500 Speedway MBB 1.124, Austin, Texas 78712- 1095. Address electronic mail to: [email protected]. Individual article re- cRNA synthesis. In vitro transcripts were prepared using prints may be purchased through the Journal Web site, www.anesthesiology.org. the mRNA capping kit (Stratagene, La Jolla, CA).

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Expression in Oocytes (115 mM NaCl, 2.5 mM KCl, 0.18 mM CaCl2, and 10 mM The use of experimental animals was approved by the HEPES; pH 7.4) to reduce Ca2ϩ inhibition of currents. Animal Care and Use Committees of the University of The animal poles of oocytes were impaled with two Texas. Isolation of Xenopus laevis oocytes and microin- glass electrodes (0.5–10 megohms) filled with 3 M KCl jection of the cDNA or cRNA was performed as de- and voltage clamped at Ϫ70 mV, unless stated other- scribed previously.15 Isolated oocytes were placed in mod- wise, using a Warner Instruments model OC-725A (Ham- ified Barth’s saline (MBS) containing 88 mM NaCl, 1 mM KCl, den, CT) oocyte clamp. Glycine (for glycine receptors), M M M M ␥ 10 m HEPES, 0.82 m MgSO4, 2.4 m NaHCO3, 0.91 m -aminobutyric acid (GABAA receptors), L-glutamate plus ␣ CaCl2, and 0.33 mM Ca(NO3)2 adjusted to pH 7.5. The 1 glycine (NMDA receptors), kainate (AMPA and kainate ␳ glycine, 1 GABAC, and 5-HT3 receptor subunit cDNAs receptors), and acetylcholine (nACh receptors) were ␣ ␤ ␥ (0.5, 1.5, and 1.5 ng/30 nl, respectively); 1, 2, and 2S dissolved in Ringer’s solution and applied for 20 s; 5-hy- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/93/4/1095/401580/0000542-200010000-00034.pdf by guest on 26 September 2021 GABAA receptor subunit cDNAs (2.0 ng/30 nl in a 1:1:2 droxytriptamine (5-HT3) receptors and GABA (for molar ratio); and NR1a and NR2A subunit cDNAs (1.5 GABAC receptors) were applied for 30 s and 3 min, ng/30 nl in a 1:1 molar ratio) were injected into the respectively, to reach equilibrium state. For GluR6/KA2 16 animal poles of oocytes by a blind method. The GluR1 kainate receptors, 10 ␮M concanavalin A in MBS was and GluR2 subunit cRNAs (30 ng/30 nl in a 1:1 molar preapplied for 2 min to prevent desensitization. Unless ratio); GluR6 and KA2 subunit cRNAs (30 ng/30 nl in a stated otherwise, anesthetics were tested against median 1:1 molar ratio); and the ␣ and ␤ nACh receptor subunit effective concentrations (EC50s) of agonists, i.e., concen- cRNAs (10–50 ng/30 nl in a 1:1 molar ratio) were in- trations giving 50% of the maximal response calculated jected into the cytoplasm of oocytes. The injected oo- based on agonist dose–response curves (table 1) for cytes were singly placed in Corning cell wells (Corning NMDA, AMPA, kainate, 5-HT3, and nACh receptors. For Glass Works, Corning, NY) containing incubation me- glycine, GABAA, and GABAC receptors, anesthetic effects dium (sterile MBS supplemented with 10 mg/l strep- were evaluated against EC5 concentrations of agonists, tomycin, 10,000 U/l penicillin, 50 mg/l gentamicin, i.e., concentrations giving 5% of the maximal response 90 mg/l theophylline, and 220 mg/l pyruvate) and incu- obtainable in that oocyte. bated at 15–19°C. Two to 5 days after injection, oocytes The Ringer’s solution (20 ml) in a sealed vial was were used in electrophysiologic recording. bubbled with 100% gaseous anesthetics at a flow rate of approximately 250 ml/min for at least 10 min to provide Electrophysiologic Recording a saturated solution of anesthetic. The saturated solution

Oocytes expressing the glycine, GABAA, GABAC, AMPA, was pumped into a chamber via a roller pump (Core- and kainate receptors were placed in a rectangular Parmer Instrument Co., Chicago, IL) through 18-gauge chamber (approximately 100 ␮l volume) and perfused (2 polyethylene tubing. The concentrations of two bath sam- ml/min) with MBS. Oocytes expressing NMDA receptors ples for nitrous oxide were quantiﬁed by use of gas chro- were perfused with Ba2ϩ Ringer’s solution to minimize matography as previously described,17 and those were 0.6 ϩ Ϫ the effects of secondarily activated Ca2 -dependent Cl atmosphere (atm) (12.6 mM) and 0.56 atm (11.7 mM); the

currents (115 mM NaCl, 2.5 mM KCl, 1.8 mM BaCl2, and average was 0.58 atm (12.2 mM). The concentration of a 10 mM HEPES; pH 7.4) and those expressing nACh re- bath sample for xenon was 0.46 atm (2.0 mM). The ceptors were perfused with Ba2ϩ Ringer’s solution con- isoﬂurane concentrations were calculated based on the ␮ taining 1 M atropine sulfate. For the 5-HT3 receptors, loss of concentration from vial to bath (60%) previously oocytes were perfused with low-Ca2ϩ Ringer’s solution obtained in our laboratory. Anesthetics were preapplied

Table 1. EC50 (Concentrations Giving 50% of the Maximal Response) and Hill Coefﬁcient Values for Various Ligand-gated Ion Channels Expressed in Xenopus Oocytes

␮ Receptors Agonists EC50 ( M) Hill coefﬁcient n ␣ Ϯ Ϯ 1 glycine Glycine 206 18 2.1 0.1 5 ␣ ␤ ␥ Ϯ Ϯ 1 2 2S GABAA GABA 94 3 1.2 0.04 6 ␳ Ϯ Ϯ 1 GABAC GABA 1.8 0.2 1.7 0.1 4 NR1a/NR2A Glutamate 2.1 Ϯ 0.3 1.3 Ϯ 0.1 5 GluR1/GluR2 Kainate 91 Ϯ 4 1.4 Ϯ 0.1 6 GluR6/KA2 Kainate 0.8 Ϯ 0.1 1.5 Ϯ 0.1 4 Ϯ Ϯ 5-HT3 5-HT 1.8 0.2 2.1 0.1 5 ␣ ␤ Ϯ Ϯ 4 2 nACh ACh 2.6 0.2 1.1 0.04 6 ␣ ␤ Ϯ Ϯ 4 4 nACh ACh 9.3 1.1 1.3 0.03 5

Values are mean Ϯ SEM. The dose–response relations of NR1a/NR2A receptors for L-glutamate were examined in the presence of 10 ␮M glycine. n ϭ number of oocytes included in each group. GABA ϭ ␥-aminobutyric acid; ACh ϭ acethylcholine; 5-HT ϭ 5-hydroxytriptamine.

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for 1 min before being coapplied with agonists. Initial relative response in the presence of nitrous oxide, EC50 studies using longer preapplication times indicated that the agonist concentration for half-maximum response, A preapplication for 1 min produced a maximal effect. the concentration of agonists, and n the Hill coefficient. Exposure to anesthetics alone produced no current re- Parameter estimation was performed by nonlinear response of receptors tested. A 5–10 min washout period gression using GraphPad Prism software (GraphPad Soft- was allowed between drug applications. Effects of anes- ware Inc., San Diego, CA). The results were statistically thetics were expressed as the fraction of control re- analyzed by use of t tests or one-way analysis of variance sponses that were evoked multiple times before and (ANOVA). P values Ͻ 0.05 were considered to be signif- after anesthetic applications to take into account possi- icant. Data are represented as mean Ϯ SEM. ble shifts in the control current throughout the experi- ment. Data were obtained from 4–15 oocytes taken from Results at least two different frogs. All experiments were per- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/93/4/1095/401580/0000542-200010000-00034.pdf by guest on 26 September 2021 formed at room temperature (23°C). Effects of Gaseous Anesthetics on Ligand-gated Ion Channels Compounds Nitrous oxide (0.58 atm) exerted differential effects on Xenopus laevis female frogs were purchased from ligand-gated ion channels (fig. 1). Nitrous oxide slightly ␣ ␤ ␥ Xenopus I (Ann Arbor, MI). Nitrous oxide, xenon, and potentiated the current response of the 1 2 2S GABAA nitrogen were obtained from Airgas-Southwest, Inc. (San receptors in a reversible manner. Conversely, NMDA and Antonio, TX). Glycine, L-glutamate, kainate, 5-hydroxytryp- neuronal nACh receptors were inhibited by nitrous ox- tamine (serotonin) hydrochloride, acetylcholine chloride, ide, also in a fully reversible manner. Furthermore, the ketamine hydrochloride, concanavalin A type IV, and extent of inhibition was different between ␣4␤2 and other reagents were purchased from Sigma Company ␣4␤4 nACh receptors. Figure 2 shows the effects of (St. Louis, MO); isoflurane from Baxter Pharmaceutical nitrous oxide, xenon, and nitrogen (control) on ligand- Products (Liberty Corner, NJ); ethanol from Aaper Alco- gated ion channels. Nitrous oxide (0.58 atm) and xenon hol and Chemical Co. (Shelbyville, KY); GABA from (0.46 atm) exhibited similar effects on various receptors. ␣ ␣ ␤ ␥ Research Biochemicals International (Natick, MA). The 1 glycine and 1 2 2S GABAA receptors were potentiated by these gaseous anesthetics, and the glycine

Statistical Analysis receptors were more sensitive than the GABAA recep- Dose–response data for agonists were ﬁtted to the tors. The NR1a/NR2A NMDA receptors were inhibited equation by nitrous oxide and xenon by 31 Ϯ 2 and 34 Ϯ 3%, ϭ ϩ n ␣ ␤ I/Imax F/[1 (EC50/A) ] respectively. The 4 2 nACh receptor was clearly inhib- Ϯ Ϯ where I represents the current, Imax the maximal cur- ited by nitrous oxide and xenon (39 1 and 41 2%, rent in the absence of nitrous oxide, F the maximal respectively), whereas the ␣4␤4 nACh receptor was

Fig. 1. Representative tracings of current responses of ligand-gated ion channels before (left), during (middle), and after (right) perfusion of 0.58 atm nitrous oxide (N2O). The current responses of the ␣ ␤ ␥ 1 2 2S GABAA receptor were evoked by 10 ␮M GABA; the NR1a/NR2A receptor by 2 ␮ML-glutamate plus 10 ␮M glycine (Glu/ Gly); the ␣4␤2 nACh receptor by 3 ␮M acetylcholine (ACh); the ␣4␤4 nACh receptor by 10 ␮M acetylcholine. Inward current is downward. The period of treatment with drugs is indicated by bars.

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Fig. 2. Differential effects of nitrous oxide and xenon on ligand- Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/93/4/1095/401580/0000542-200010000-00034.pdf by guest on 26 September 2021 gated ion channels. The effects of nitrous oxide (0.58 atm) and ␣ xenon (0.46 atm) on the current responses of 1 glycine (Gly) ␣ ␤ ␥ and 1 2 2S GABAA receptors to EC5 (concentrations giving 5% of the maximal response) concentrations of glycine and GABA, responses of NR1a/2A NMDA receptors ;(15–8 ؍ respectively (n responses of ;(15–11 ؍ to 2 ␮ML-glutamate plus 10 ␮M glycine (n ␣4␤2 and ␣4␤4 nACh receptors to 3 and 10 ␮M acetylcholine, Effects of 100% nitrogen are also .(15–5 ؍ respectively (n .(6–4 ؍ shown as a control for hypoxia (n

only slightly inhibited by these gases (7 Ϯ 2 and 13 Ϯ 1%, respectively). To rule out the involvement of low oxygen concentrations (hypoxia), effects of the solution bubbled with 100% nitrogen were evaluated. Perfusion of nitrogen produced no measurable effect on these receptors, suggesting that the observed effects of nitrous oxide and xenon are not caused by displacement of oxygen from the solutions.

Comparison with Isoflurane and Ethanol The minimum alveolar concentration (MAC) values of nitrous oxide are 1.01 atm for humans and 1.5 atm for mice, whereas those of xenon are 0.71 atm for humans and 0.95 atm for mice.18 Using our open perfusion system, it is not possible to achieve 1 MAC for the gaseous anesthetics, and we tested them at the maximal concentrations that could be obtained in our apparatus. The Fig. 3. Comparison of the effects of nitrous oxide, isoflurane and ethanol on ligand-gated ion channels. The effects of nitrous bath concentrations of nitrous oxide and xenon (0.58 atm, oxide (0.58 atm), isoflurane (150 ␮M), and ethanol (90 mM)on ␣ ␣ ␤ ␥ ␳ 12.2 mM and 0.46 atm, 2.0 mM, respectively) used in these the current responses of 1 glycine (Gly), 1 2 2S GABAA, and 1 GABA receptors to EC (concentrations giving 5% of the max- studies correspond to approximately 0.5 MAC. To compare C 5 imal response) concentrations of glycine and GABA, respec- responses of NR1a/2A NMDA receptors to 2 ;(15–6 ؍ the effects of gaseous anesthetics with those of volatile tively (n responses of ;(15–6 ؍ anesthetics and alcohols, we evaluated the effects of ␮ML-glutamate plus 10 ␮M glycine (n ␮ 1 GluR1/GluR2 AMPA and GluR6/KA2 kainate (KA) receptors to ؍ MAC of isoflurane (150 M ) and ethanol (90 mM for ␮ 0.5 19 100 and 1 M kainate, respectively (n 6–9); responses of 5-HT3 responses of ␣4␤2 and ␣4␤4 ;(10–8 ؍ tadpoles ) on ligand-gated ion channels (fig. 3). At these receptors to 2 ␮M 5-HT (n ؍ ␮ concentrations, the inhibitory glycine and GABAA recep- nACh receptors to 3 and 10 M acetylcholine, respectively (n tors were potentiated by nitrous oxide much less than by 8–15). ␳ isoflurane. Conversely, nitrous oxide inhibited the 1 comparison tests, P Ͻ 0.001). Unlike the GABA and GABAC receptors, which are known to be inhibited by volatile anesthetics and alcohols.20 Thus, the direction of glycine receptors, effects of nitrous oxide on NMDA and effects (potentiation or inhibition) of nitrous oxide on AMPA receptors are more marked than those of isoflu- the inhibitory receptors were similar to those of isoflurane, but comparable with or less than those of ethanol. rane and ethanol, but the effects were smaller. Three Kainate receptors, which were potentiated by isoflu- subtypes of glutamate receptors were moderately inhib- rane, were inhibited by nitrous oxide. The 5-HT3 recep- ited by nitrous oxide. Among the glutamate receptors, tor was slightly inhibited by nitrous oxide, which was NMDA receptors were inhibited more than AMPA or the opposite of the effects of isoflurane and ethanol on kainate receptors (ANOVA followed by Scheffe` multiple this receptor. Different sensitivity to nitrous oxide was

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Fig. 4. Effects of nitrous oxide on the agonist dose–response curves of ligand- ؍ gated ion channels (n 5). (A) The EC50 (concentrations giving 50% of the maxi- ␣ ␤ ␥ mal response) values of 1 2 2S GABAA receptors for GABA before and during treatment with nitrous oxide (0.58 atm) were 100 ؎ 8 and 77 ؎ 5 ␮M, and Hill ؎ coefﬁcient values were 1.1 ؎ 0.1 and 1.2

0.1, respectively. (B) The EC50 values of NR1a/NR2A NMDA receptors for L-glutamate in the presence of 10 ␮M glycine before and during treatment with nitrous oxide were 2.3 ؎ 0.3 and 2.4 ؎ 0.4 ␮M, and Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/93/4/1095/401580/0000542-200010000-00034.pdf by guest on 26 September 2021 Hill coefficient values were 1.2 ؎ 0.1 and ؎ 1.2 0.2, respectively. (C) The EC50 values of ␣4␤2 nACh receptors for acetylcholine before and during treatment with nitrous oxide were 2.5 ؎ 0.4 and 2.4 ؎ 0.5 ␮M, and Hill coefficient values were 1.1 ؎ 0.1 and ؎ 1.0 0.1, respectively. (D) The EC50 values of ␣4␤4 nACh receptors for acetylcholine before and during treatment with nitrous oxide were 8.3 ؎ 1.6 and 8.6 ؎ 1.6 ␮M, and Hill coefficient values were 1.3 ؎ 0.1 and .respectively ,0.1 ؎ 1.2 observed between ␣4␤2 and ␣4␤4 nACh receptors, and Effects of the Membrane Potential on Inhibition by the ␣4␤2 receptor was one of the most sensitive recep- Nitrous Oxide tors to gaseous anesthetics among ligand-gated ion chan- We further determined whether the inhibition of nels tested. However, because the ␣4␤2 receptors were NMDA and nACh receptors by nitrous oxide is depen- also very sensitive to volatile anesthetics, effects of ni- dent on the membrane potential (fig. 5). For the com- trous oxide were less prominent than those of 0.5 MAC parison, inhibition by ketamine, a channel blocker of isoflurane. The ethanol effects on nACh receptors were NMDA and nACh receptors21 was also evaluated. The potentiation, which was the opposite of the effects of extent of inhibition of NMDA receptors by nitrous oxide nitrous oxide and isoflurane. was larger at a membrane potential of Ϫ100 mV than at Ϫ30 mV (t tests, P Ͻ 0.001). However, the difference Effects of Nitrous Oxide on the Agonist between Ϫ100 and Ϫ30 mV was much less than that of Dose–Response Relations 1 ␮M ketamine. Ketamine (50 ␮M) also exhibited the To characterize the actions of gaseous anesthetics, we different extent of inhibition of ␣4␤2 nACh receptors evaluated the effects of nitrous oxide on the agonist between Ϫ100 and Ϫ30 mV (t tests, P Ͻ 0.001). Con- dose–response relations of receptors (fig. 4). Nitrous versely, the extent of inhibition of ␣4␤2 nACh receptors oxide slightly shifted the GABA dose–response curve of by nitrous oxide was not significantly different between Ϫ Ϫ Ͼ GABAA receptors to the left, without affecting the max- 100 and 30 mV (t tests, P 0.70). ␣ ␤ ␥ imal responses. The EC50 value for GABA of the 1 2 2S receptor during treatment with nitrous oxide was lower than that of the control (77 Ϯ 5 and 100 Ϯ 8 ␮M, Discussion respectively; t tests, P Ͻ 0.05). Nitrous oxide reduced the maximal responses of NMDA receptors to L-gluta- In the current investigation, we demonstrated the ef- Ϯ mate without changing the EC50 values (2.3 0.3 and fects of gaseous anesthetics nitrous oxide and xenon on 2.4 Ϯ 0.4 ␮M, respectively; t tests, P Ͼ 0.71), and nitrous various ligand-gated ion channels during similar experi- oxide also inhibited the maximal responses to coagonist mental conditions. Nitrous oxide and xenon had a simi- glycine without changing the EC50 values (data not lar pattern of action on the ligand-gated ion channels, shown). Similarly, the maximal responses of ␣4␤2 and but the pattern was different from that of isoflurane or ␣4␤4 nACh receptors were reduced by nitrous oxide, ethanol. though to different extents, whereas the EC50 values The effects of gaseous anesthetics on the inhibitory were not affected (fig. 4). These results suggest that receptors were smaller than those of isoflurane. These nitrous oxide increases the apparent affinity for agonist results are consistent with previous reports that show of GABAA receptors, whereas nitrous oxide inhibits the that GABAA receptors are weakly potentiated by nitrous NMDA and nACh receptors in a noncompetitive manner. oxide3,5,22 and are little affected by xenon.4,6 Nitrous

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between muscle and neuronal nACh receptors,27,28 there might be a conserved molecular determinant of the sensitivity to gaseous anesthetics. The inhibition extent of NMDA receptors by nitrous oxide was slightly different between membrane potentials tested in the manner described previously,5 whereas ketamine exhibited strong voltage dependence. Con- versely, inhibition of nACh receptors by nitrous oxide was not different within the range of membrane potentials tested, although ketamine inhibition of ␣4␤2 nACh receptors was different between membrane potentials

tested. These results suggest that mechanisms of actionDownloaded from http://pubs.asahq.org/anesthesiology/article-pdf/93/4/1095/401580/0000542-200010000-00034.pdf by guest on 26 September 2021 of nitrous oxide are different between NMDA and nACh receptors and also suggest that nitrous oxide and ket- Fig. 5. Effects of nitrous oxide and ketamine on NMDA and amine have different mechanisms of action on nACh .A) receptors) .(5 ؍ nACh receptors at different membrane potentials (n Inhibition of NR1a/NR2A NMDA receptors by nitrous oxide The neuronal nACh receptors, especially those com- (0.58 atm) and ketamine (1 ␮M) was evaluated at membrane potentials of ؊100 and ؊30 mV. (B) Inhibition of the ␣4␤2 posed of the ␤2 subunit, are affected by many general nACh receptors by nitrous oxide (0.58 atm) and ketamine anesthetics at clinically relevant concentrations. Isoflu- ␮ ؊ ؊ (50 M) at membrane potentials of 100 and 30 mV. rane and 1-chloro-1,2,2-trifluorocyclobutane (F3), but not the nonimmobilizer 1,2-dichlorohexafluorocyclobu- oxide also potentiated the glycine receptor slightly, as tane (F6), are reported to potently inhibit neuronal nACh 23 ␳ 15,29 previously reported, but inhibited 1 GABAC receptors, receptors, and nACh receptors composed of the ␤2 suggesting that gaseous anesthetics discriminate be- subunit are more sensitive to volatile anesthetics than tween these homologous receptor subunits as do volatile those of the ␤4 subunit.30 Fluorinated alcohols that have 24 anesthetics and alcohols. Therefore, although the sen- in vivo anesthetic effects31 inhibit nACh receptors,32 sitivity is different between gaseous and volatile anes- 33 but do not potentiate GABAA receptors. Therefore, thetics, some mechanisms of actions of gaseous anes- gaseous anesthetics provide other examples of anesthet- thetics on the inhibitory receptors might be shared with ics that only slightly affect GABAA receptors, but inhibit those of volatile anesthetics and alcohols. nACh receptors composed of the ␤2 subunit. These Unlike the inhibitory receptors, effects of nitrous ox- results, together with a predominant distribution of the ide on glutamate receptors were more prominent than ␣4 and ␤2 subunits in the central nervous system,27,34 those of isoflurane, confirming previous studies that suggest that neuronal nACh receptors composed of the showed that NMDA receptors are inhibited by nitrous ␤2 subunit could be potential anesthetic targets. How- 3–6 oxide and xenon, and that non-NMDA receptors are ever, observations argue against nACh receptors as uni- 5 also inhibited mildly by nitrous oxide. Specifically, we versal anesthetic targets. Short-chain alcohols (e.g., eth- found that recombinant NMDA receptors were inhibited anol) have anesthetic effects and enhance GABAA moderately by nitrous oxide and xenon, and AMPA and receptor function35 but do not inhibit ␣4␤2 nACh recep- kainate receptors were also inhibited by nitrous oxide, tors; rather, they enhance receptor function.36 Clinically though by a lesser degree than NMDA receptors. Kainate relevant concentrations of propofol potentiate GABAA receptors are potentiated by volatile anesthetics, and a receptors,37 but propofol inhibits ␣4␤2 receptors only at transmembrane site is critical for the volatile anesthetic concentrations much higher than relevant concentra- enhancement.25 Because nitrous oxide inhibited kainate tions.29 Dissociative anesthetics ketamine and dizo- receptors and AMPA receptors, the critical site for vola- cilpine inhibit nACh receptors composed of the ␤2 sub- tile anesthetics might not be involved in the action of unit at concentrations higher than relevant anesthetic gaseous anesthetics. concentrations.38 Thus, the nACh receptor composed of The sensitivity of neuronal nACh receptors to gaseous the ␤2 subunit is an attractive target for some anesthet- anesthetics was different between ␣4␤2 and ␣4␤4 re- ics, but it is unlikely that an effect on this single receptor ceptors, and the ␣4␤2 receptor was one of the most is involved in producing general anesthesia. sensitive receptors to gaseous anesthetics among ligand- Unlike volatile anesthetics, nitrous oxide and xenon are gated ion channels tested. Interestingly, muscle nACh potent analgesics at subanesthetic concentrations.39,40 Be- receptors (composed of the ␣1, ␤1, ␥, and ␦ subunits) in cause activation, not inhibition, of the nACh receptors is 41 BC3H1 cells are also sensitive to nitrous oxide. At 0.80 suggested to elicit antinociception, it is unlikely that atm, nitrous oxide reduces the channel open-time of inhibitory effects of gaseous anesthetics on ␣4␤2 nACh muscle nACh receptors by half.26 Therefore, although receptors are responsible for the analgesic actions. How- physiologic and pharmacologic properties are distinct ever, inhibition of glutamate receptors is considered to

Anesthesiology, V 93, No 4, Oct 2000 GASEOUS ANESTHETICS AND LIGAND-GATED ION CHANNELS 1101 be involved in antinociception,42 and gaseous anesthet- tion and Translation: A Practical Approach. Edited by Hames BD, Higgins SJ. Washington DC, Oxford Press, 1984, pp 49–69 ics inhibited the glutamate receptors, including NMDA 17. Dildy-Mayfield JE, Eger EI II, Harris RA: Anesthetics produce subunit- receptors. Therefore, it is possible that analgesic effects selective actions on glutamate receptors. J Pharmacol Exp Ther 1996; 276: 1058–65 of gaseous anesthetics result from inhibition of gluta- 18. Firestone LL, Miller JC, Miller KW: Tables of physical and pharmacological mate receptors. properties of anesthetics, Molecular and Cellular Mechanisms of Anesthetics. Edited by Roth SH, Miller KW. New York, Plenum Press, 1986, pp 455–70 In conclusion, nitrous oxide and xenon had a similar 19. Alifimoff JK, Firestone LL, Miller KW: Anaesthetic potencies of primary pattern of action on ligand-gated ion channels, but the alkanols: Implications for the molecular dimensions of the anaesthetic site. Br J Pharmacol 1989; 96:9–16 pattern is different from that of isoflurane or ethanol. In ␳ 20. Mihic SJ, Harris RA: Inhibition of 1 receptor GABAergic currents by addition to NMDA receptors, nACh receptors composed alcohols and volatile anesthetics. J Pharmacol Exp Ther 1996; 277:411–6 ␤ 21. Lodge D, Anis NA, Burton NR: Effects of optical isomers of ketamine on of the 2 subunit were found to be sensitive to these excitation of cat and rat spinal neurones by amino acids and acetylcholine. gaseous anesthetics. The different pattern of action of Neurosci Lett 1982; 29:281–6 22. Dzoljic M, Van Duijn B: Nitrous oxide-induced enhancement of ␥-aminobu- gaseous anesthetics on various ligand-gated ion channels Downloaded from http://pubs.asahq.org/anesthesiology/article-pdf/93/4/1095/401580/0000542-200010000-00034.pdf by guest on 26 September 2021 tyric acidA-mediated chloride currents in acutely dissociated hippocampal neu- may underlie the distinct anesthetic states. rons. ANESTHESIOLOGY 1998; 88:473–80 23. Daniels S, Roberts RJ: Post-synaptic inhibitory mechanisms of anaesthesia; glycine receptors. Toxicol Lett 1998; 71–6, 100–101 The authors thank Professor Edmond I Eger II, M.D., Department of Anesthesia 24. Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, Mascia and Perioperative Care, University of California, San Francisco, for helpful dis- MP, Valenzuela CF, Hanson KK, Greenblatt EP, Harris RA, Harrison NL: Sites of cussions and Michael Laster, D.V.M., and Diane Gong, B.S., Department of alcohol and volatile anaesthetic action on GABAA and glycine receptors. Nature Anesthesia and Perioperative Care, University of California, San Francisco, for 1997; 389:385–9 technical assistance. The authors thank the laboratories cited herein for kindly 25. Minami K, Wick MJ, Stern-Bach Y, Dildy-Mayfield JE, Brozowski SJ, Gonza- providing cDNA. les EL, Trudell JR, Harris RA: Sites of volatile anesthetic action on kainate (glutamate receptor 6) receptors. J Biol Chem 1998; 273:8248–55 26. Wachtel RE: Relative potencies of volatile anesthetics in altering the

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Anesthesiology, V 93, No 4, Oct 2000