Ligand-gated ion channels are considered to be potential general anesthetic targets. Although most general anesthetics potentiate the function of gamma-aminobutyric acid receptor type A (GABAA), the gaseous anesthetics nitrous oxide and xenon are reported to have little effect on GABAA receptors but inhibit N-methyl-d-aspartate (NMDA) receptors. To define the spectrum of effects of nitrous oxide and xenon on receptors thought to be important in anesthesia, the authors tested these anesthetics on a variety of recombinant brain receptors.
The glycine, GABAA, GABA receptor type C (GABAC), NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), kainate, 5-hydroxytryptamine3 (5-HT3), and nicotinic acetylcholine (nACh) receptors were expressed in Xenopus oocytes and effects of nitrous oxide and xenon, and as equipotent concentrations of isoflurane and ethanol, were studied using the two-electrode voltage clamp.
Nitrous oxide (0.58 atmosphere [atm]) and xenon (0.46 atm) exhibited similar effects on various receptors. Glycine and GABAA receptors were potentiated by gaseous anesthetics much less than by isoflurane, whereas nitrous oxide inhibited GABAC receptors. Glutamate receptors were inhibited by gaseous anesthetics more markedly than by isoflurane, but less than by ethanol. NMDA receptors were the most sensitive among glutamate receptors and were inhibited by nitrous oxide by 31%. 5-HT3 receptors were slightly inhibited by nitrous oxide. The nACh receptors were inhibited by gaseous and volatile anesthetics, but ethanol potentiated them. The sensitivity was different between alpha4beta2 and alpha4beta4 nACh receptors; alpha4beta2 receptors were inhibited by nitrous oxide by 39%, whereas alpha4beta4 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.
Nitrous oxide and xenon displayed a similar spectrum of receptor actions, but this spectrum is distinct from that of isoflurane or ethanol. These results suggest that NMDA receptors and nACh receptors composed of beta2 subunits are likely targets for nitrous oxide and xenon.
AN anesthetic inorganic gas, nitrous oxide, is widely used for general anesthesia in combination with other drugs. A more potent anesthetic inert gas, xenon, can be used as the sole anesthetic agent. The anesthetic properties of these gaseous anesthetics have long been central to molecular theories of anesthesia that seek to explain how these simple and small molecules are able to produce anesthesia.
During the past few decades, a consensus has emerged that general anesthetics act on one or more superfamilies of ligand-gated ion channels that include γ-aminobutyric acid type A (GABAA), glycine, nicotinic acetylcholine (nACh), 5-hydroxytryptamine3(5-HT3), and glutamate receptors. 1,2Among the ligand-gated ion channels, the GABAAreceptor is considered to be a prime target of volatile and intravenous anesthetics. 1,2Recent studies suggest that nitrous oxide and xenon barely affect GABAAreceptors but inhibit N -methyl-d-aspartate (NMDA) receptors. 3–6These findings suggest that molecular mechanisms of two gaseous anesthetics are different from those of volatile and intravenous anesthetics. To confirm and extend these findings, we selected nine recombinant ligand-gated ion channels and determined the effects of nitrous oxide and xenon on these channels during similar experimental conditions. The choice of subunit composition of recombinant receptors was based on the predominance of the subunit distribution or combination in the central nervous system or the availability of previous data for anesthetic sensitivity of the subunits.
Materials and Methods
cDNA and cRNA Preparation
The cDNA encoding the human α1-glycine receptor subunit 7in the pBK-CMV N/B-200 vector; cDNAs of human α1, β2, and γ2SGABAAreceptor subunits 8in pBK-CMV N/B-200, pCDM8, and pCIS2 vectors, respectively; cDNA of the human ρ1GABACreceptor subunit 9in pcDNA1 vector; cDNAs of the human NR1a and NR2A NMDA receptor subunits 10in pcDNA1Amp vector; and cDNA of NCB-20 5-HT3receptor subunit 11in pBK-CMV N/B-200 vector were used for the nuclear injection. The cDNAs of the rat GluR1 and GluR2 α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits 12in the pBluescript SK−vector, cDNAs of the rat GluR6 and KA2 kainate receptor subunits 13in pGEM-HE and pBluescript SK−vectors, respectively, and the rat nACh receptor subunit cDNAs 14in several expression vectors (α4 in pSP64 vector, β2 in pSP65 vector, and β4 in pBluescript SK−vector) were used for cRNA synthesis. In vitro transcripts were prepared using the mRNA capping kit (Stratagene, La Jolla, CA).
Expression in Oocytes
The use of experimental animals was approved by the Animal Care and Use Committees of the University of Texas. Isolation of Xenopus laevis oocytes and microinjection of the cDNA or cRNA was performed as described previously. 15Isolated oocytes were placed in modified Barth’s saline (MBS) containing 88 mm NaCl, 1 mm KCl, 10 mm HEPES, 0.82 mm MgSO4, 2.4 mm NaHCO3, 0.91 mm CaCl2, and 0.33 mm Ca(NO3)2adjusted to pH 7.5. The α1glycine, ρ1GABAC, and 5-HT3receptor subunit cDNAs (0.5, 1.5, and 1.5 ng/30 nl, respectively); α1, β2, and γ2SGABAAreceptor subunit cDNAs (2.0 ng/30 nl in a 1:1:2 molar ratio); and NR1a and NR2A subunit cDNAs (1.5 ng/30 nl in a 1:1 molar ratio) were injected into the animal poles of oocytes by a blind method. 16The GluR1 and GluR2 subunit cRNAs (30 ng/30 nl in a 1:1 molar ratio); GluR6 and KA2 subunit cRNAs (30 ng/30 nl in a 1:1 molar ratio); and the α and β nACh receptor subunit cRNAs (10–50 ng/30 nl in a 1:1 molar ratio) were injected into the cytoplasm of oocytes. The injected oocytes were singly placed in Corning cell wells (Corning Glass Works, Corning, NY) containing incubation medium (sterile MBS supplemented with 10 mg/l streptomycin, 10,000 U/l penicillin, 50 mg/l gentamicin, 90 mg/l theophylline, and 220 mg/l pyruvate) and incubated at 15–19°C. Two to 5 days after injection, oocytes were used in electrophysiologic recording.
Oocytes expressing the glycine, GABAA, GABAC, AMPA, and kainate receptors were placed in a rectangular chamber (approximately 100 μl volume) and perfused (2 ml/min) with MBS. Oocytes expressing NMDA receptors were perfused with Ba2+Ringer’s solution to minimize the effects of secondarily activated Ca2+-dependent Cl−currents (115 mm NaCl, 2.5 mm KCl, 1.8 mm BaCl2, and 10 mm HEPES; pH 7.4) and those expressing nACh receptors were perfused with Ba2+Ringer’s solution containing 1 μm atropine sulfate. For the 5-HT3receptors, oocytes were perfused with low-Ca2+Ringer’s solution (115 mm NaCl, 2.5 mm KCl, 0.18 mm CaCl2, and 10 mm HEPES; pH 7.4) to reduce Ca2+inhibition of currents. The animal poles of oocytes were impaled with two glass electrodes (0.5–10 megohms) filled with 3 m KCl and voltage clamped at −70 mV, unless stated otherwise, using a Warner Instruments model OC-725A (Hamden, CT) oocyte clamp. Glycine (for glycine receptors), γ-aminobutyric acid (GABAAreceptors), l-glutamate plus glycine (NMDA receptors), kainate (AMPA and kainate receptors), and acetylcholine (nACh receptors) were dissolved in Ringer’s solution and applied for 20 s; 5-hydroxytriptamine (5-HT3) receptors and GABA (for GABACreceptors) were applied for 30 s and 3 min, respectively, to reach equilibrium state. For GluR6/KA2 kainate receptors, 10 μm concanavalin A in MBS was preapplied for 2 min to prevent desensitization. Unless stated otherwise, anesthetics were tested against median effective concentrations (EC50s) of agonists, i.e., concentrations giving 50% of the maximal response calculated based on agonist dose–response curves (table 1) for NMDA, AMPA, kainate, 5-HT3, and nACh receptors. For glycine, GABAA, and GABACreceptors, anesthetic effects were evaluated against EC5concentrations of agonists, i.e., concentrations giving 5% of the maximal response obtainable in that oocyte.
The Ringer’s solution (20 ml) in a sealed vial was bubbled with 100% gaseous anesthetics at a flow rate of approximately 250 ml/min for at least 10 min to provide a saturated solution of anesthetic. The saturated solution was pumped into a chamber via a roller pump (Core-Parmer Instrument Co., Chicago, IL) through 18-gauge polyethylene tubing. The concentrations of two bath samples for nitrous oxide were quantified by use of gas chromatography as previously described, 17and those were 0.6 atmosphere (atm) (12.6 mm) and 0.56 atm (11.7 mm); the average was 0.58 atm (12.2 mm). The concentration of a bath sample for xenon was 0.46 atm (2.0 mm). The isoflurane concentrations were calculated based on the loss of concentration from vial to bath (60%) previously obtained in our laboratory. Anesthetics were preapplied for 1 min before being coapplied with agonists. Initial studies using longer preapplication times indicated that preapplication for 1 min produced a maximal effect. Exposure to anesthetics alone produced no current response of receptors tested. A 5–10 min washout period was allowed between drug applications. Effects of anesthetics were expressed as the fraction of control responses that were evoked multiple times before and after anesthetic applications to take into account possible shifts in the control current throughout the experiment. Data were obtained from 4–15 oocytes taken from at least two different frogs. All experiments were performed at room temperature (23°C).
Xenopus laevis female frogs were purchased from Xenopus I (Ann Arbor, MI). Nitrous oxide, xenon, and nitrogen were obtained from Airgas-Southwest, Inc. (San Antonio, TX). Glycine, l-glutamate, kainate, 5-hydroxytryptamine (serotonin) hydrochloride, acetylcholine chloride, ketamine hydrochloride, concanavalin A type IV, and other reagents were purchased from Sigma Company (St. Louis, MO); isoflurane from Baxter Pharmaceutical Products (Liberty Corner, NJ); ethanol from Aaper Alcohol and Chemical Co. (Shelbyville, KY); GABA from Research Biochemicals International (Natick, MA).
Dose–response data for agonists were fitted to the equation
I/Imax= F/[1 + (EC50/A)n]
where I represents the current, Imaxthe maximal current in the absence of nitrous oxide, F the maximal relative response in the presence of nitrous oxide, EC50the agonist concentration for half-maximum response, A the concentration of agonists, and n the Hill coefficient. Parameter estimation was performed by nonlinear regression using GraphPad Prism software (GraphPad Software Inc., San Diego, CA). The results were statistically analyzed by use of t tests or one-way analysis of variance (ANOVA). P values < 0.05 were considered to be significant. Data are represented as mean ± SEM.
Effects of Gaseous Anesthetics on Ligand-gated Ion Channels
Nitrous oxide (0.58 atm) exerted differential effects on ligand-gated ion channels (fig. 1). Nitrous oxide slightly potentiated the current response of the α1β2γ2SGABAAreceptors in a reversible manner. Conversely, NMDA and neuronal nACh receptors were inhibited by nitrous oxide, also in a fully reversible manner. Furthermore, the extent of inhibition was different between α4β2 and α4β4 nACh receptors. Figure 2shows the effects of nitrous oxide, xenon, and nitrogen (control) on ligand-gated ion channels. Nitrous oxide (0.58 atm) and xenon (0.46 atm) exhibited similar effects on various receptors. The α1glycine and α1β2γ2SGABAAreceptors were potentiated by these gaseous anesthetics, and the glycine receptors were more sensitive than the GABAAreceptors. The NR1a/NR2A NMDA receptors were inhibited by nitrous oxide and xenon by 31 ± 2 and 34 ± 3%, respectively. The α4β2 nACh receptor was clearly inhibited by nitrous oxide and xenon (39 ± 1 and 41 ± 2%, respectively), whereas the α4β4 nACh receptor was only slightly inhibited by these gases (7 ± 2 and 13 ± 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. 18Using 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 bath concentrations of nitrous oxide and xenon (0.58 atm, 12.2 mm and 0.46 atm, 2.0 mm, respectively) used in these studies correspond to approximately 0.5 MAC. To compare the effects of gaseous anesthetics with those of volatile anesthetics and alcohols, we evaluated the effects of 0.5 MAC of isoflurane (150 μm1) and ethanol (90 mm for tadpoles 19) on ligand-gated ion channels (fig. 3). At these concentrations, the inhibitory glycine and GABAAreceptors were potentiated by nitrous oxide much less than by isoflurane. Conversely, nitrous oxide inhibited the ρ1GABACreceptors, which are known to be inhibited by volatile anesthetics and alcohols. 20Thus, the direction of effects (potentiation or inhibition) of nitrous oxide on the inhibitory receptors were similar to those of isoflurane and ethanol, but the effects were smaller. Three subtypes of glutamate receptors were moderately inhibited by nitrous oxide. Among the glutamate receptors, NMDA receptors were inhibited more than AMPA or kainate receptors (ANOVA followed by Scheffè multiple comparison tests, P < 0.001). Unlike the GABA and glycine receptors, effects of nitrous oxide on NMDA and AMPA receptors are more marked than those of isoflurane, but comparable with or less than those of ethanol. Kainate receptors, which were potentiated by isoflurane, were inhibited by nitrous oxide. The 5-HT3receptor was slightly inhibited by nitrous oxide, which was the opposite of the effects of isoflurane and ethanol on this receptor. Different sensitivity to nitrous oxide was observed between α4β2 and α4β4 nACh receptors, and the α4β2 receptor was one of the most sensitive receptors to gaseous anesthetics among ligand-gated ion channels tested. However, because the α4β2 receptors were also very sensitive to volatile anesthetics, effects of nitrous oxide were less prominent than those of 0.5 MAC isoflurane. The ethanol effects on nACh receptors were potentiation, which was the opposite of the effects of nitrous oxide and isoflurane.
Effects of Nitrous Oxide on the Agonist Dose–Response Relations
To characterize the actions of gaseous anesthetics, we evaluated the effects of nitrous oxide on the agonist dose–response relations of receptors (fig. 4). Nitrous oxide slightly shifted the GABA dose–response curve of GABAAreceptors to the left, without affecting the maximal responses. The EC50value for GABA of the α1β2γ2Sreceptor during treatment with nitrous oxide was lower than that of the control (77 ± 5 and 100 ± 8 μm, respectively;t tests, P < 0.05). Nitrous oxide reduced the maximal responses of NMDA receptors to l-glutamate without changing the EC50values (2.3 ± 0.3 and 2.4 ± 0.4 μm, respectively;t tests, P > 0.71), and nitrous oxide also inhibited the maximal responses to coagonist glycine without changing the EC50values (data not shown). Similarly, the maximal responses of α4β2 and α4β4 nACh receptors were reduced by nitrous oxide, though to different extents, whereas the EC50values were not affected (fig. 4). These results suggest that nitrous oxide increases the apparent affinity for agonist of GABAAreceptors, whereas nitrous oxide inhibits the NMDA and nACh receptors in a noncompetitive manner.
Effects of the Membrane Potential on Inhibition by Nitrous Oxide
We further determined whether the inhibition of NMDA and nACh receptors by nitrous oxide is dependent on the membrane potential (fig. 5). For the comparison, inhibition by ketamine, a channel blocker of NMDA and nACh receptors 21was also evaluated. The extent of inhibition of NMDA receptors by nitrous oxide was larger at a membrane potential of −100 mV than at −30 mV (t tests, P < 0.001). However, the difference between −100 and −30 mV was much less than that of 1 μm ketamine. Ketamine (50 μm) also exhibited the different extent of inhibition of α4β2 nACh receptors between −100 and −30 mV (t tests, P < 0.001). Conversely, the extent of inhibition of α4β2 nACh receptors by nitrous oxide was not significantly different between −100 and −30 mV (t tests, P > 0.70).
In the current investigation, we demonstrated the effects of gaseous anesthetics nitrous oxide and xenon on various ligand-gated ion channels during similar experimental conditions. Nitrous oxide and xenon had a similar pattern of action on the ligand-gated ion channels, but the pattern was different from that of isoflurane or ethanol.
The effects of gaseous anesthetics on the inhibitory receptors were smaller than those of isoflurane. These results are consistent with previous reports that show that GABAAreceptors are weakly potentiated by nitrous oxide 3,5,22and are little affected by xenon. 4,6Nitrous oxide also potentiated the glycine receptor slightly, as previously reported, 23but inhibited ρ1GABACreceptors, suggesting that gaseous anesthetics discriminate between these homologous receptor subunits as do volatile anesthetics and alcohols. 24Therefore, although the sensitivity is different between gaseous and volatile anesthetics, some mechanisms of actions of gaseous anesthetics on the inhibitory receptors might be shared with those of volatile anesthetics and alcohols.
Unlike the inhibitory receptors, effects of nitrous oxide on glutamate receptors were more prominent than those of isoflurane, confirming previous studies that showed that NMDA receptors are inhibited by nitrous oxide and xenon, 3–6and that non-NMDA receptors are also inhibited mildly by nitrous oxide. 5Specifically, we found that recombinant NMDA receptors were inhibited moderately by nitrous oxide and xenon, and AMPA and kainate receptors were also inhibited by nitrous oxide, though by a lesser degree than NMDA receptors. Kainate receptors are potentiated by volatile anesthetics, and a transmembrane site is critical for the volatile anesthetic enhancement. 25Because nitrous oxide inhibited kainate receptors and AMPA receptors, the critical site for volatile anesthetics might not be involved in the action of gaseous anesthetics.
The sensitivity of neuronal nACh receptors to gaseous anesthetics was different between α4β2 and α4β4 receptors, and the α4β2 receptor was one of the most sensitive receptors to gaseous anesthetics among ligand-gated ion channels tested. Interestingly, muscle nACh receptors (composed of the α1, β1, γ, and δ subunits) in BC3H1 cells are also sensitive to nitrous oxide. At 0.80 atm, nitrous oxide reduces the channel open-time of muscle nACh receptors by half. 26Therefore, although physiologic and pharmacologic properties are distinct between muscle and neuronal nACh receptors, 27,28there 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, 5whereas ketamine exhibited strong voltage dependence. Conversely, inhibition of nACh receptors by nitrous oxide was not different within the range of membrane potentials tested, although ketamine inhibition of α4β2 nACh receptors was different between membrane potentials tested. These results suggest that mechanisms of action of nitrous oxide are different between NMDA and nACh receptors and also suggest that nitrous oxide and ketamine have different mechanisms of action on nACh receptors.
The neuronal nACh receptors, especially those composed of the β2 subunit, are affected by many general anesthetics at clinically relevant concentrations. Isoflurane and 1-chloro-1,2,2-trifluorocyclobutane (F3), but not the nonimmobilizer 1,2-dichlorohexafluorocyclobutane (F6), are reported to potently inhibit neuronal nACh receptors, 15,29and nACh receptors composed of the β2 subunit are more sensitive to volatile anesthetics than those of the β4 subunit. 30Fluorinated alcohols that have in vivo anesthetic effects 31inhibit nACh receptors, 32but do not potentiate GABAAreceptors. 33Therefore, gaseous anesthetics provide other examples of anesthetics that only slightly affect GABAAreceptors, but inhibit nACh receptors composed of the β2 subunit. These results, together with a predominant distribution of the α4 and β2 subunits in the central nervous system, 27,34suggest that neuronal nACh receptors composed of the β2 subunit could be potential anesthetic targets. However, observations argue against nACh receptors as universal anesthetic targets. Short-chain alcohols (e.g. , ethanol) have anesthetic effects and enhance GABAAreceptor function 35but do not inhibit α4β2 nACh receptors; rather, they enhance receptor function. 36Clinically relevant concentrations of propofol potentiate GABAAreceptors, 37but propofol inhibits α4β2 receptors only at concentrations much higher than relevant concentrations. 29Dissociative anesthetics ketamine and dizocilpine inhibit nACh receptors composed of the β2 subunit at concentrations higher than relevant anesthetic concentrations. 38Thus, the nACh receptor composed of the β2 subunit is an attractive target for some anesthetics, but it is unlikely that an effect on this single receptor is involved in producing general anesthesia.
Unlike volatile anesthetics, nitrous oxide and xenon are potent analgesics at subanesthetic concentrations. 39,40Because activation, not inhibition, of the nACh receptors is suggested to elicit antinociception, 41it is unlikely that inhibitory effects of gaseous anesthetics on α4β2 nACh receptors are responsible for the analgesic actions. However, inhibition of glutamate receptors is considered to be involved in antinociception, 42and gaseous anesthetics inhibited the glutamate receptors, including NMDA receptors. Therefore, it is possible that analgesic effects of gaseous anesthetics result from inhibition of glutamate receptors.
In conclusion, nitrous oxide and xenon had a similar pattern of action on ligand-gated ion channels, but the pattern is different from that of isoflurane or ethanol. In addition to NMDA receptors, nACh receptors composed of the β2 subunit were found to be sensitive to these gaseous anesthetics. The different pattern of action of gaseous anesthetics on various ligand-gated ion channels may underlie the distinct anesthetic states.
The authors thank Professor Edmond I Eger II, M.D., Department of Anesthesia and Perioperative Care, University of California, San Francisco, for helpful discussions and Michael Laster, D.V.M., and Diane Gong, B.S., Department of Anesthesia and Perioperative Care, University of California, San Francisco, for technical assistance. The authors thank the laboratories cited herein for kindly providing cDNA.