General anesthetics modulate members of the ligand-gated ion channel superfamily including γ-aminobutyric acidA, glycine, 5-hydroxytrip-tamine3 (5-HT3), and nicotinic acetylcholine (nACh) receptors, which are likely important for the clinical anesthetic state (1,2). Among these ligand-gated receptors, the neuronal nACh receptors have been receiving increased attention because these receptors, in cultured neurons or expressed in Xenopus oocytes, have been shown to be potently blocked by inhaled anesthetics such as halothane and isoflurane (2–6). The neuronal nACh receptors in the central nervous system (CNS) are found in both pre- and postsynaptic locations. The postsynaptic nACh receptors render the releasing ability of neurotransmitters including glutamate, norepinephrine, γ-aminobutyric acid, dopamine, serotonin, and ACh itself (7,8). Inhibitions of neuronal nACh receptors by general anesthetics may mediate the anesthetic action through modulations of the release of those neurotransmitters.
Molecular cloning studies have shown that the neuronal nACh receptors consist of various combinations of subunits. In combinations of those subunits, the subtypes of nACh receptors show distinct pharmacological properties. The (α7)5 receptor is α-bungarotoxin (α-BuTX) sensitive, whereas the (α4)2(β2)3 is α-BuTX insensitive (8,9). In addition to this pharmacological difference, isoflurane and propofol inhibit the (α4)2(β2)3 nACh receptor but do not affect the (α7)5 nACh receptor, suggesting that these two subtypes exhibit different pharmacological profiles to different types of general anesthetics (4). Several studies on the effects of general anesthetics at the (α7)5 nACh receptor have been performed. It remains unknown whether the gaseous anesthetics nitrous oxide (N2O) and xenon (Xe) can inhibit the (α7)5 nACh receptor, although it is reported that these gaseous anesthetics inhibit the (α4)2(β2)3 nACh receptor (10). The α7-containing nACh receptors presynaptically augment glutamate and ACh release, and the inhibitory effect of general anesthetics in these receptors may contribute to the anesthetic action through the suppression of the excitatory transmission (8).
We report the effects of two gaseous anesthetics, N2O and Xe, on the human (α7)5 nACh receptor expressed in Xenopus oocyte.
Human-cloned α7 nACh receptor subunit complimentary (c)DNA was kindly provided by Dr Jon Lindstrom at the University of Pennsylvania. The cDNA encoding the human α7 nACh receptor in the pMXT expression vector was linearized by a restriction enzyme, Xba I (TaKaRa, Osaka, Japan) to create template cDNA. Capped cRNA was synthesized in vitro from cDNA using SP6 RNA polymerase kit (SP6 mMESSAGE mMACHINE KIT™, Ambion, Austin, TX) by following the manufacturer’s recommended protocol. In accordance with a study protocol approved by the Animal Research Committee of Osaka University Medical School, a female Xenopus laevis was anesthetized on ice with 1% 3-aminobenzoic-ethyl ester (Tricaine; Sigma, St Louis, MO). Oocytes were harvested via a laparotomy incision and then defolliculated with collagenase type 1A (Sigma). Between 10 and 50 ng of cRNA was injected into each oocyte with a glass capillary using a Nanoject injector (Drummond Scientific, Broomall, PA). Prepared oocytes were incubated at 20°C in an incubation medium (in mM: NaCl 88, KCl 2, HEPES 10, MgCl2 1, NaHCO3 2.4, and CaCl2 2.4, with a pH value of 7.4) for 2–4 days until the start of the electrophysiological experiment.
Electrophysiological recordings were made using a two-electrode voltage-clamp technique. Oocytes placed in a 0.2-mL chamber were impaled with 1- to 5-MΩ electrodes filled with 3 M of KCl solution and voltage-clamped at −70 mV (CEZ-1250, Nihon Kohden, Tokyo, Japan). Extracellular solution (in mM: NaCl 88, KCl 2, HEPES 10, NaHCO3 2.4, and BaCl2 1.8, with a pH value of 7.4) was continuously superfused at 5–10 mL/min. N2O and Xe (99.995%) were introduced to the perfusate by bubbling. A 50-mL conical tube was filled with the solution, and anesthetic gases were continuously bubbled into the tube at a rate of 100 mL/min. Oxygen was added, and the concentrations of N2O and Xe were adjusted using precise flowmeters and monitored for N2O with a Datex Capnomac Ultima (Datex Instrumentarium Corp, Helsinki, Finland) and for Xe with a Xe meter (Riken, Tokyo, Japan). The concentrations of oxygen or nitrogen in the control solutions were adjusted to the same level as the oxygen concentration in the anesthetic solution. No changes of pH value in extracellular solutions containing the different gaseous anesthetics were detected. Anesthetic solutions were bubbled with the anesthetic-containing gas mixture for 30 min. To allow for time to equilibrium, anesthetic solutions were preapplied to oocytes before exposure to ACh (Sigma). Each drug application was separated by intervals of a few minutes and, to eliminate receptor desensitization, by longer intervals after the application of large concentrations. The presence of cumulative desensitization was excluded by confirming that, with a single oocyte from each sample, a small concentration of ACh induced the same response. The current induced by a 30% effective concentration (EC30) of ACh was used to compare the effects of anesthetics. The current itself at EC30 was steadily measured with little effect of desensitization, and it was appropriate to evaluate the inhibitory effects by N2O and Xe. The current was digitally recorded using AxoScope software (Axon Instruments, Foster City, CA) running on a personal computer. All electrophysiological experiments were performed at room temperature.
The ACh-induced currents in the (α7)5 receptor are relatively small and quite easy to be desensitized, especially in large concentrations and long applications of ACh.
The concentrations of volatile and gaseous anesthetics in experimental solutions were quantified by gas chromatography/mass spectrometry/selected ion monitoring (Trace™ GC2000 and GCQ™ plus, ThermoQuest, CE Instruments, Austin, TX) with the headspace sampling technique described in our previous report (11). Because the extracellular solution used in the present study has almost the same osmolarity as the previous solution and electrophysiological experiments were performed at the same temperature, the data described in the previous study were adopted as the concentrations of N2O and Xe dissolved in solution. Accordingly, concentrations of anesthetic solutions were 12.9 ± 0.4 mM, 26 ± 0.4 mM, and 37.0 ± 0.4 mM for 35%, 70%, and 100% of N2O and 1.6 ± 0.1 mM, 3.5 ± 0.1 mM, and 4.7 ± 0.1 mM for 35%, 70%, and 100% of Xe, respectively, at 25°C (n ≧ 3) (11).
Peak amplitudes of the current elicited by the drugs were measured directly from digital recordings stored in AxoScope. To obtain the concen-tration-response curve for ACh-induced currents, observed peak amplitudes were normalized and plotted, and the data were then fitted to the following Hill equation using Sigmaplot software (Jandel Scientific, San Rafael, CA):MATH where I is the peak current at a given concentration of ACh, Imax is the maximum current, EC50 is the concentration of ACh eliciting a half-maximum response, and n denotes the Hill coefficient. Statistical analyses were performed using one-way analysis of variance followed by Tukey-Kramer test for multiple comparisons between different concentrations of anesthetics and Student’s t-test for comparison between different membrane potentials. A P value <0.05 was considered to indicate a significant difference. All data were expressed as mean ± sem (n ≧ 5).
The peaks of ACh-induced currents were concentration-dependent, and the concentration-response curve for ACh fit the Hill equation well, with an EC50 of 220 ± 40 μM and a Hill coefficient of 0.98 ± 0.14. Applications of N2O and Xe themselves without ACh produced no detectable current (data not shown). N2O and Xe reversibly inhibited ACh-induced currents of the (α7)5 nACh receptor expressed in oocytes. Inhibition of 100 μM (EC30) of ACh-induced current by N2O was 9.7 ± 0.9 at 35%, 19 ± 1 at 70%, and 29 ± 1 at 100% (Fig. 1A) and by Xe was 22 ± 3 at 35%, 31 ± 1 at 70%, and 47 ± 1 at 100% (Fig. 1B). Inhibitory actions of both N2O and Xe were concentration dependent (P < 0.01). Figure 2 shows the concentration-response relations in the presence and absence of 100% N2O (Fig. 2A) and Xe (Fig. 2B). Both N2O and Xe reduced the ACh-induced maximal response without changing the EC50 values (220 ± 40 μM in the absence of anesthetics and 200 ± 40 and 200 ± 20 μM in the presence of 100% N2O and 100% Xe, respectively;P > 0.05). These data suggest that the inhibitory actions of these anesthetics are noncompetitive. The inhibitory effects of N2O and Xe were also examined at various membrane potentials (Fig. 3). The effects of 100% N2O (Fig. 3A) and 100% Xe (Fig. 3B) on the ACh-induced currents were not significantly different at membrane potentials ranging from −90 mV to −10 mV (P > 0.05). To exclude the involvement of small oxygen concentration under 100% gaseous anesthetics, effects of the solution bubbled with 100% nitrogen were evaluated. Perfusion of nitrogen produced no measurable current at the (α7)5 nACh receptor (data not shown), suggesting that the observed effects of N2O and Xe in their large concentrations are not caused by displacement of oxygen from the solutions.
Much evidence has been accumulated to indicate that neuronal nACh receptors directly participate in the control of neuronal functions in the CNS (12,13). These receptors are both postsynaptically and presynaptically located in the CNS, where they act by modulating transmitter release (8). The (α4)2(β2)3 receptor, which is the most prevalent neuronal nACh receptor subtype in the CNS, possesses the pharmacological properties to be α-BuTX insensitive and sensitive to general anesthetics such as halothane, isoflurane, sevoflurane, and propofol (3–5). The homomeric (α7)5 receptor is less abundant than the (α4)2(β2)3 receptor but distributes diffusely in the brain (14). The ACh receptors containing the (α7)5 subunit are α-BuTX sensitive and highly permeable to Ca2+. Autoradiographic studies indicate that α-BuTX sites in some lesions of the brain might be presynaptic in origin (8). The (α7)5 receptor may contribute to enhancing the excitatory transmission through the presynaptic augmentation of glutamate and ACh releases (8). Thus, even if the inhibition of this receptor at clinical concentrations by anesthetics is relatively small, it could cause a significant alteration in the overall function of the neural network via presynaptic mechanism and play a role in the clinical anesthetic state.
The effects of volatile and IV anesthetics on the (α7)5 ACh receptor have also been examined and compared with those on other heteromeric neuronal nACh receptors, e.g., (α4)2(β2)3 receptors. It is noteworthy that the (α7)5 and (α4)2(β2)3 receptors have different sensitivities to those anesthetics. Halothane inhibits both receptors, and its effect on the (α4)2(β2)3 receptor is much greater (5). However, isoflurane and propofol potently inhibit the (α4)2(β2)3 receptor, but they have no effect, even at large concentrations, on the (α7)5 nACh receptor (4). Thiopental and ketamine inhibit both receptors equally at clinically relevant concentrations (15,16). Although ethanol potentiates the (α4)2(β2)3 receptor, it inhibits the (α7)5 receptor (17). As for gaseous anesthetics that are thought to inhibit the postsynaptic N-methyl-d-aspartate receptor, Yamakura and Harris (10) showed that N2O and Xe clearly inhibited the (α4)2(β2)3 receptor and slightly inhibited the (α4)2(β4)3 receptor. They further identified a single amino acid residue (valine at position 253 in the β2 subunit or phenylalanine at position 255 in the β4 subunit) near the middle of the second transmembrane segment that determines gaseous anesthetic sensitivity (18). However, no information on the effects of gaseous anesthetics at the (α7)5 nACh receptor is available.
In this study, we demonstrate that, at clinically relevant concentrations, both N2O and Xe inhibit the human (α7)5 nACh receptor in a concentration-dependent manner. The inhibitory mechanism of these two gaseous anesthetics at the (α7)5 nACh receptor was noncompetitive and not dependent on membrane potential. These findings are similar to a previous study that showed that N2O and Xe inhibit the rat (α4)2(β2)3 and (α4)2(β4)3 nACh receptors in a noncompetitive way (10). By contrast, the inhibitions of the (α4)2(β2)3 receptors by the volatile anesthetic isoflurane and the IV anesthetic propofol are both competitive (4). These observations suggest the mechanisms of the inhibition by gaseous anesthetics, N2O and Xe, are similar, but other classes of general anesthetics such as isoflurane and propofol can act differently in the homomeric (α7)5 nACh receptor and the heteromeric α4β2 receptor. According to our previous investigation on the effects of N2O and Xe on the homomeric human 5-HT3A receptor (11), which has a highly homologous sequence with the (α7)5 nACh receptor (19), N2O and Xe inhibit the 5-HT3 receptor at clinical concentrations, and the inhibitory effects are both competitive for 5-HT and independent of membrane potential. The inhibitory mechanism of N2O and Xe at the homomeric 5-HT3 receptor seems to be different regardless of the great similarity of the sequence to nACh receptors (19). Molecular manipulation of sequences in these receptor subunits by chimeric analysis or with site-directed mutagenesis will reveal a more detailed mechanism including the site of action for general anesthetics in the receptor.
We found that Xe inhibited ACh-induced (α7)5 nACh receptor currents (47% at 100% Xe) more than N2O (29% for 100% N2O). This finding is similar to those on other ligand-gated ion channels (10,11), which have shown that the inhibition by Xe is greater in the N-methyl-d-aspartate receptor, the heteromeric nACh receptors, and the 5-HT3 receptor than that by N2O. In addition, our results for the (α7)5 nACh receptor are consistent with these in vitro findings. The rank order of potency on N2O and Xe at the (α7)5 nACh is in line with the in vivo human data for the minimum alveolar anesthetic concentration and the analgesic assessment in clinical research. The human minimum alveolar anesthetic concentration of N2O was reported to be 105%(20) and 70% for Xe (21). In multimodal experimental pain testing and assessment technique with healthy young volunteers, Petersen-Felix et al. (22) concluded that the analgesic potencies of Xe were 1.5 times larger than those of N2O (22).
In conclusion, this is the first report on the recombinant human (α7)5 nACh receptor to show that the gaseous anesthetics N2O and Xe at a clinical concentration reduced ACh-induced currents, both acting in a noncompetitive manner without voltage-dependency. The (α7)5 nACh receptor may play a role in the mechanism of general anesthesia induced by N2O and Xe.
The authors thank Dr Jon Lindstrom at the University of Pennsylvania for providing the human α7 subunit clone and Mr Kazuro Nakano, chief technician at the Central Laboratory for Research and Education of Osaka University Medical School, for determining the concentration of N2O and Xe.
1. Franks NP, Lieb WR. Molecular and cellular mechanisms of general anesthesia. Nature 1994; 367: 607–14.
2. Harris RA, Mihic SJ, Dildy-Mayfield JE, et al. Actions of anesthetics on ligand-gated ion channels: role of receptor subunit composition. FASEB J 1995; 9: 1454–62.
3. McKenzie D, Franks NP, Lieb WR. Actions of general anaesthetics on a neuronal nicotinic acetylcholine receptor in isolated identified neurons of Lymnaea stagnalis
. Br J Pharmacol 1995; 115: 275–82.
4. Flood P, Ramirez-Latorre J, Role L. α4
neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but α7
-type nicotinic acetylcholine receptors are unaffected. Anesthesiology 1997; 86: 859–65.
5. Mori T, Zhao X, Zuo Y, et al. Modulation of neuronal nicotinic acetylcholine receptors by halothane in rat cortical neurons. Mol Pharmacol 2001; 59: 732–43.
6. Coates KM, Mather LE, Johnson RJ, Flood P. Thiopental is a competitive inhibitor at the human α7
nicotinic acetylcholine receptor. Anesth Analg 2001; 92: 930–3.
7. McGehee DS, Heath M, Gelber S, et al. Nicotine activation of presynaptic receptors on CNS neurons potentiates fast excitatory synaptic transmission. Science 1995; 22: 1692–7.
8. Role LW, Berg D. Nicotinic receptors in the development and modulation of CNS synapse. Neuron 1996; 16: 1077–85.
9. Whiting PJ, Schoepfer R, Conroy WG, et al. Expression of nicotinic acetylcholine receptor subtypes in brain and retina. Brain Res Mol Brain Res 1991; 10: 61–70.
10. Yamakura T, Harris RA. Effects of gaseous anesthetics nitrous oxide and xenon on ligand-gated ion channels. Anesthesiology 2000; 93: 1095–101.
11. Suzuki T, Koyama H, Sugimoto M, et al. The diverse actions of volatile and gaseous anesthetics on human-cloned 5-hydroxytryptamine3
receptors expressed in Xenopus
oocytes. Anesthesiology 2002; 96: 699–704.
12. McGehee DS, Role LW. Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu Rev Physiol 1995; 57: 521–46.
13. Colquhoun LM, Patrick JW. Pharmacology of neuronal nicotinic acetylcholine receptor subtypes. Adv Pharmacol 1997; 39: 191–220.
14. Court JA, Martin-Ruiz C, Graham A, et al. Nicotinic receptors in human brain: topography and pathology. J Chem Neuroanat 2000; 20: 281–98.
15. Downie DL, Franks NP, Lieb WR. Effects of thiopental and its optical isomers on nicotinic acetylcholine receptors. Anesthesiology 2000; 93: 774–83.
16. Coates KM, Flood P. Ketamine and its preservative, benzethonium chloride, both inhibit human recombinant alpha7 and alpha4beta2 neuronal nicotinic acetylcholine receptors in Xenopus
oocytes. Br J Pharmacol 2001; 134: 871–9.
17. Cardoso RA, Brozowski SJ, Chavez-Noriega LE, et al. Effects of ethanol on recombinant human neuronal nicotinic acetylcholine receptors expressed in Xenopus
oocytes. J Pharmacol Exp Ther 1999; 289: 774–80.
18. Yamakura T, Borghese C, Harris RA. A transmembrane site determines sensitivity of neuronal nicotinic acetylcholine receptors to general anesthetics. J Biol Chem 2000; 275: 40879–86.
19. Ortells MO, Lunt GG. Evolutionary history of the ligand-gated ion-channel superfamily of receptors. Trends Neurosci 1995; 18: 121–7.
20. Hornbein TF, Eger EI II, Winter PM, et al. The minimum alveolar concentration of nitrous oxide. Anesth Analg 1982; 61: 553–6.
21. Cullen SC, Eger EI II, Cullen BF, et al. Observations on the anesthetic effect of the combination of xenon and halothane. Anesthesiology 1969; 31: 305–9.
22. Petersen-Felix S, Luginbühl M, Schnider TW, et al. Comparison of the analgesic potency of xenon and nitrous oxide in humans evaluated by experimental pain. Br J Anaesth 1998; 81: 742–7.