Urethane (ethyl carbamate) is a water-soluble compound whose molecular weight is 89.1 (Fig. 1) and has been widely used as an anesthetic in animal experiments. It is also a carcinogen, which precludes its use as a human anesthetic. A search of PubMed indicates that more than 100 studies are published each year using “urethane-anesthetized” animals. The advantages of urethane in animal anesthesia are that it can be administrated by several parenteral routes, produces a long-lasting steady level of surgical anesthesia, and has minimal effects on autonomic and cardiovascular systems (1,2). It is assumed that animals anesthetized with urethane represent similar physiologic and pharmacologic behaviors to those observed in unanesthetized animals. Indeed, the animals are used as clinical models in various investigations. Despite urethane’s importance in many investigations, little is known about its mechanism of action.
Recently, a consensus has emerged that anesthetics exert their effects via enhancement of inhibitory synaptic neurotransmission and/or via inhibition of excitatory neurotransmission. Particularly, anesthetics affect neurotransmitter-gated ion channels more than most other membrane proteins (3,4). Most anesthetics, including volatile anesthetics, barbiturates, propofol, and etomidate, markedly potentiate the function of the γ-aminobutyric acid typeA (GABAA) receptors (4–7). However, ketamine dramatically inhibits the channel function of N-methyl-d-aspartate (NMDA) receptors at a clinical concentration without substantial alteration of the function of GABA or other receptors (8,9). Neuronal nicotinic acetylcholine (nACh) receptors are inhibited by clinical concentrations of volatile and IV anesthetics (9–11), and this receptor is a possible target for anesthetics. In contrast to other injectable anesthetics, there are few studies of urethane’s actions, and the effects of urethane on GABAergic neurotransmission are not clear. Bowery and Dray (12) reported that urethane reversed the antagonistic effect of bicuculline on GABA-induced depolarization in the isolated rat superior cervical ganglion. However, other investigations indicate that urethane produces only minimal enhancement of GABAergic neurotransmission at a clinical concentration (13,14). Therefore, it is conceivable that there are other targets for urethane. This study was designed to determine whether urethane affects neurotransmitter-gated ion channels. Understanding its actions on multiple receptors may not only provide insight as to how urethane produces anesthesia, but may also help us to correctly interpret the data obtained from urethane-anesthetized animals.
In this study, we examined the effects of urethane on recombinant α1β2γ2S GABAA, α1 glycine, NR1a/NR2A NMDA, GluR1/GluR2 α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA), and α4β2 neuronal nACh receptors expressed in Xenopus oocytes. Subunit compositions of the recombinant receptors were chosen based on the predominance of subunit distribution in the central nervous system (CNS) (15).
This study was approved by the Animal Care and Use Committees of the University of Texas.
Xenopus laevis female frogs were purchased from Xenopus Express (Homosassa, FL). Urethane, glycine, l-glutamate, kainic acid, and pentobarbital sodium were obtained from Sigma (St. Louis, MO). GABA was obtained from Research Biochemical International (Natick, MA). 2,6-Diisopropilphenol (propofol) was obtained from Aldrich Chemical Co. (Milwaukee, WI).
The cDNA encoding human α1 glycine receptor subunit in the pBK-CMW N/B vector, the cDNAs of human α1, β2, and γ2S GABAA receptor subunits in pBK-CMV N/B, pCDM8, and pCIS2 vectors, respectively, and the cDNAs of human NR1a and NR2A NMDA receptor subunits in pcDNA Amp vector were used for the nuclear injection. The cDNAs of rat GluR1 and GluR2 AMPA receptor subunits in the pBluescript SK− vector, and the cDNAs of rat α4 and β2 nACh receptor subunits in pSP64 and pSP65 vectors, respectively, were used for cRNA synthesis. In vivo transcripts were prepared by using the mCAP™ Capping Kit (Stratagene, La Jolla, CA). The isolation of Xenopus laevis oocytes was conducted as described previously (16). Isolated oocytes were placed in modified Barth’s saline (MBS) containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.82 mM MgSO4, 0.91 mM CaCl2, 0.33 mM Ca(NO3)2, and 10 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) adjusted to pH 7.5. The α1 glycine receptor subunit cDNA (1 ng/30 nL), α1, β2, and γ2S GABAA receptor subunits’ cDNAs (2 ng/30 nL in a 1:1:2 molar ratio), or NR1a and NR2A NMDA receptor subunits’ cDNAs (1.5 ng/30 nL in a 1:1 molar ratio) were injected into the animal poles of oocytes by a blinded method (17). The GluR1 and GluR2 AMPA receptor subunits’ cRNAs (30 ng/30 nL in a 1:1 molar ratio) or the α4 and β2 nACh receptor subunits’ cRNAs (30 ng/30 nL in a 1:1 molar ratio) were injected into 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, 100,000 U/L penicillin, 50 mg/L gentamycin, 90 mg/L theophylline, and 220 mg/L pyruvate) and incubated at 15°–19°C. On 1 to 4 days after injection, the oocytes were used in electrophysiologic recording (18).
Oocytes expressing GABAA, glycine or AMPA receptors were placed in a rectangular chamber (∼100-μL volume) and perfused (2 mL/min) with MBS. Oocytes expressing NMDA receptors were perfused with Ba2+ Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM BaCl2, and 10 mM HEPES adjusted to pH7.4) to minimize the effects of secondarily activated Ca2+-dependent Cl− currents and oocytes expressing nACh receptors were perfused with Ba2+ Ringer’s solution containing 1 μM atropine sulfate. The animal poles of oocytes were impaled with two glass electrodes (0.5–10 MΩ) filled with 3 M KCl, and the oocytes were voltage-clamped at −70 mV by using a Warner Instruments model OC-752B (Hamden, CT) oocyte clamp. Glycine, GABA, or kainic acid (for AMPA receptors) dissolved in MBS was applied to the oocytes for 20 or 30 s to reach a maximal response. Likewise, l-glutamate plus 10 μM glycine (for NMDA receptors) or ACh dissolved in Ba2+ Ringer’s solution was applied to the oocytes for 20 s. To study the effects of concentrations of urethane, for GABAA or glycine receptors, the experiments were performed at EC5 of agonist that produced 5% of the maximal currents produced by 1 mM glycine or GABA. For NMDA, AMPA, or nACh receptors, the experiments were performed at the half-maximal effective concentration (EC50) of agonist. All agonists were repeatedly applied until a consistent response was observed. Then, urethane dissolved in MBS or Ba2+ Ringer’s solution was preapplied for 1 min before being coapplied with agonists. Initial studies using longer preapplication times indicated that preapplication for 1 min yielded a maximal effect. A 5- to 10-min washout period was allowed between drug applications. The effects of urethane were expressed as the fraction of control responses, which were calculated by averaging the control responses before and after anesthetics application. To address the mechanism of urethane’s actions on the receptors, we further examined the effects of 100 mM urethane on the maximal response to agonists. Based on the concentration-response relations studied in our previous work (19), which was performed under the same conditions as the current study, we tested 300 μM GABA, 300 μM glycine, 100 μM l-glutamate plus 10 μM glycine, or 1 mM kainic acid for each receptor to obtain maximal response. In regard to rat α4β2 nACh receptor, we performed a concentration-response study with varying concentrations (0.1 μM–1 mM) of ACh, and 1 mM ACh was used to obtain maximal response. To compare urethane with other anesthetics, parallel experiments using the anesthetic EC50(3) of pentobarbital, and propofol on glycine, NMDA, and/or AMPA receptors were conducted in the same conditions. Data were obtained from 6 to 12 oocytes taken from at least three different frogs. The values of the EC50 and the half-maximal inhibitory concentration of urethane were calculated by nonlinear regression using GraphPad Prism software (GraphPad Inc., San Diego, CA). Data were represented as means ± sem. All experiments were performed at room temperature (23°C). Statistical analysis was conducted by one-way analysis of variance for multiple comparisons and unpaired t-test for comparisons between two groups. Differences were considered as statistically significant at P value < 0.05.
As shown by several investigations with recombinant α1β2γ2S GABAA or α1 glycine receptors, inward chloride currents were observed in response to the applications of agonists (Fig. 2). For the experiments testing glutamate or nACh receptors, oocytes expressing NR1a/NR2A NMDA, GluR1/GluR2 AMPA, or α4β2 nACh receptors yielded inward cation currents (Figs. 3,4). Control currents in GABAA and glycine receptors in response to EC5 of agonists were 870 ± 90 nA and 960 ± 110 nA, respectively. Control currents in NMDA, AMPA, and nACh receptors in response to EC50 of agonists were 2200 ± 350 nA, 260 ± 40 nA, and 1600 ± 250 nA, respectively. Urethane (10–300 mM) significantly potentiated the current responses of both GABAA and glycine receptors in a reversible and concentration-dependent manner (Figs. 2,5). The urethane concentration-response curves for both receptors were sigmoid-shaped, and nonlinear regression analysis yielded the EC50 values for GABAA and glycine receptors of 64 mM and 46 mM, respectively, and the Hill coefficient for GABAA and glycine receptors of 1.5 and 1.4, respectively. At a concentration of 10 mM, urethane enhanced the currents of GABAA and glycine receptors by 23% ± 4% and 33% ± 4%, respectively. At concentrations of agonists that produce maximal responses (EC100), the enhancing effects of urethane (100 mM) were almost abolished in both receptors (GABAA receptor: EC5 246% ± 43% and EC100 12% ± 3%, P < 0.05; glycine receptor: EC5 276% ± 29% and EC100 19% ± 4%, P < 0.05), suggesting that urethane increases apparent affinity for agonist with little or no increase in the maximal response.
Conversely, urethane (10–300 mM) inhibited the responses of NMDA and AMPA receptors (Fig. 6) and these effects were reversible (Fig. 3). The urethane EC50 values for NMDA and AMPA receptors were 70 mM and 34 mM, respectively, and the Hill coefficient for NMDA and AMPA receptors were 1.2 and 1.3, respectively. At 10 mM, urethane suppressed the currents of NMDA and AMPA receptors by 10% ± 3%, and 18% ± 2%, respectively. Even at EC100 of agonists, the inhibitory effects of urethane (100 mM) were not changed (NMDA receptor: EC50 42% ± 2% and EC100 38% ± 2%; AMPA receptor: EC50 14% ± 3% and EC100 11% ± 3%), indicating noncompetitive inhibition of these receptors by urethane. For the α4β2 ACh receptor, the ACh EC50 value was 60 ± 3 μM and the Hill coefficient was 0.9 ± 0.1. Urethane (10–300 mM) potentiated the function of the nACh receptor as was seen for GABA and glycine receptors (Fig. 4). The urethane EC50 value and the Hill coefficient for the α4β2 nACh receptor were 114 mM and 1.5, respectively. At a concentration of 10 mM, urethane enhanced the currents of this receptor by 15% ± 3%. At EC100 of ACh, urethane (100 mM) enhanced to a similar extent as compared with EC50 (EC50: 148% ± 21% and EC100: 212% ± 33%). Urethane (up to 300 mM) on its own produced no current in any receptor studied (Figs. 2,3,4). In parallel experiments, pentobarbital (50 μM), slightly potentiated glycine-induced chloride current by 17% ± 3% (P < 0.05 compared with 10 mM urethane or control value), and it slightly inhibited NMDA receptors (−9% ± 2%, P < 0.05 compared with control value). Propofol (1 μM) did not affect NMDA receptors (−3% ± 2%, P < 0.05 compared with 10 mM urethane).
Recently, studies of anesthetic mechanisms have shifted from the interaction of anesthetics with lipid-bilayer of the plasma membrane to the interaction with channel proteins, in particular, neurotransmitter-gated ion channels. GABAA receptors are thought to be a primary target of anesthetics because most volatile and nonvolatile anesthetics augment the channel activity at clinical concentrations. Glycine receptors are the main inhibitory receptors in the spinal cord and brainstem, and volatile anesthetics enhance the function of these receptors. Glutamate plays a major role in synaptic excitation in the CNS and is critical for information storage in memory and learning (20). With respect to anesthesia, NMDA receptors mediate nociceptive neurotransmission in the CNS and both NMDA and AMPA receptors are important for memory. More recently, nACh receptors were proposed as targets for anesthetics, because volatile anesthetics and some IV anesthetics, such as thiopental, inhibit the function of nACh receptors (9–11).
Interestingly, urethane potentiated the function of an nACh receptor. Plasma concentrations of urethane during surgical anesthesia in mammals are estimated to be equal to or larger than 10 mM (1). Tonner et al. (21) reported that the EC50 of urethane for loss of righting reflex of tadpoles was 16.4 mM. In this study, we assumed that the anesthetic EC50 of urethane is 10 mM and this concentration enhanced the functions of α1β2γ2S GABAA and α1 glycine receptors by 23% and 33%, respectively. However, this concentration inhibited the functions of NR1a/NR2A NMDA and GluR1/GluR2 AMPA receptors by 10% and 18%, respectively. Our results suggest that an anesthetic concentration of urethane can modulate the activities of all receptors tested.
It is useful to compare the effects of urethane with other anesthetics. In our previous studies, pentobarbital (50 μM) and propofol (1 μM) enhanced the function of GABAA receptors by more than 100%(5,6). However, these drugs have only small effects on glycine receptors [pentobarbital, +17%; propofol, approximately +10%(22)]. In the course of our study, we found little effect of pentobarbital or propofol on NMDA receptors (pentobarbital −9%, propofol −3%). Other laboratories reported that pentobarbital significantly inhibited AMPA receptors [−50%(23)], but propofol did not affect these receptors at all (24). Ketamine is a noncompetitive inhibitor of the NMDA receptor, and reduces NMDA receptor function more than 80% at 10 μM (8), the anesthetic EC50, but has no effect on GABAA, glycine, and AMPA receptors (9,15). Volatile anesthetics such as halothane and isoflurane potentiate both GABAA and glycine receptors more than 100% at the anesthetic EC50(19,25). These anesthetics have minimal effects on AMPA receptors composed of GluR1 and GluR2 subunits (15). In regard to the effect on the nACh receptor, urethane is similar to ethanol, but different from other anesthetics. Urethane (10 mM) enhanced the function of the nACh receptor by 15%. Halothane, isoflurane, ketamine, and thiopental, a barbiturate-like pentobarbital, inhibit 50% or more at their anesthetic EC50(9–11). Thus, urethane has a spectrum of action on ion channels, which is distinct from other anesthetics. Gaseous, volatile, and injectable anesthetics seem to have either enhancement of GABAergic or inhibition of glutamatergic neurotransmission as a primary action. In contrast, urethane affects both inhibitory and excitatory systems and the magnitude of the change is less than is seen with anesthetics that are more selective for one system (e.g., ketamine and NMDA receptor, propofol and GABAA receptor). The only compound with a spectrum of action similar to urethane is ethanol. It also produces modest enhancement of glycine, GABAA and nACh receptor functions, and inhibition of AMPA and NMDA receptors (26). Thus, it is possible that anesthesia can be achieved by marked changes in the inhibitory or excitatory system (most injectable and volatile anesthetics) or by modest changes in both systems (urethane and ethanol).
The modest effects on multiple neurotransmitter-gated ion channels at concentrations close to the anesthetic EC50 may make urethane suitable for maintaining anesthesia during electrophysiologic recording. However, we should consider that urethane exerts marked effects on the channels above the concentration required for surgical anesthesia and may significantly alter several neurotransmitter systems in the CNS. Thus, the assumption that the responses produced by physiologic or pharmacologic manipulations in the urethane-anesthetized animal are the same as those that would be produced in the awake animal may not be valid in all cases.
We thank Dr. Paul J. Whiting for kindly providing GABAA and NMDA receptor subunits’ cDNAs, Dr. Heinrich Betz for glycine receptor subunit cDNA, Dr. Stephen Heinemann for AMPA receptor subunits’ cDNAs, and Dr. Charles W. Luetje for nACh receptor subunits. We also thank Dr. Henry Lester for prompting us to study urethane and Dr. Edmond I Eger II for helpful advice.
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