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Anesth Analg 2000;91:1542-1549
© 2000 International Anesthesia Research Society

GENERAL ARTICLES

Nitrous Oxide and Xenon Increase the Efficacy of GABA at Recombinant Mammalian GABAA Receptors

Gerhard Hapfelmeier, MD*, Walter Zieglgänsberger, MD, PhD{dagger}, Rainer Haseneder*, Hajo Schneck, MD{ddagger}, and Eberhard Kochs, MD*

*Department of Anesthesiology, Klinikum rechts der Isar, Technische Universität München, München; {dagger}Max-Planck-Institute of Psychiatry, Munich; and {ddagger}Kreisklinik Ebersberg, Ebersberg, Germany

Address correspondence and reprint requests to Gerhard Hapfelmeier, MD, Klinik für Anaesthesiologie der Technischen Universität München, Klinikum rechts der Isar, Ismaninger Straße 22, D-81675 München, Germany. Address e-mail to hapfelmeier @mpipsykl.mpg.de.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
We investigated the interactions between recombinant gamma-aminobutyric acid receptor complex (GABAAR) and nitrous oxide (N2O) or xenon (Xe). Human embryonic kidney cells (HEK 293) were transfected with rat cDNA for {alpha}1ß2{gamma}2L or for {alpha}1ß2 recombinant GABAAR subunits. Patch clamp techniques were used in the whole-cell mode to evaluate the effect of N2O and Xe on GABA-induced currents. A piezo-driven "liquid filament switch" was used for fast application. Both N2O (100%, 29.2 mM) and Xe (100%, 3.9 mM) reversibly increased GABA-induced currents through the {alpha}1ß2{gamma}2L and the {alpha}1ß2 GABAAR channels. The potentiating effect of N2O or Xe on peak currents was prominent at small GABA concentrations (10-7 to 10-5 M). The addition of N2O or Xe increased the efficacy of GABA (10-7 to 10-3 M). Both N2O and Xe significantly decreased the risetime(10%–90%) of the currents elicited by small GABA concentrations. At the concentrations used, neither N2O nor Xe had an intrinsic effect. We conclude that, similar to other anesthetics, both N2O and Xe increase the efficacy of GABA at the GABAAR and enhance inhibitory GABAergic synaptic transmission. {abs}

Implications: The anesthetic gases nitrous oxide (laughing gas) and xenon increase the activity of a mammalian GABAA receptor. This receptor is held responsible for the inhibition of important actions of the human brain, e.g., maintenance of consciousness and awareness.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Anesthesia is considered the result of a shift in the balance between inhibition and excitation within a complex framework of signal flux in the brain. The respective mechanism(s) of action of anesthetic drugs has been subject to extensive research since the correlation between anesthetic potency and oil solubility was first observed (1–3). It is generally accepted that interactions of anesthetic substances with voltage-gated as well as with ligand-gated ion channels within the central nervous system play a major role in producing the anesthetic state.

Gamma-aminobutyric acid (GABA) binds to GABAA and GABAB receptors (4). Although both receptor types appear to be sensitive to volatile anesthetics (5), most experimental work focuses on the GABAA receptor complex (GABAAR) and its fast postsynaptic inhibition. Seventeen different subunits of the GABAAR have been cloned so far ({alpha}1–6, ß1–4, {gamma}1–4, {delta}, {rho}1–2). The pentameric receptor assembly 2{alpha}12ß2{gamma}2 predominates in the mammalian brain (4,6,7). The {gamma}2L (long) subtype of the {gamma}2 subunit contains eight more amino acids (8) and includes an additional intracellular phosphorylation site (6,9).

Current research suggests that compounds exhibiting anesthetic potency, such as ethanol (8), propofol (10–12), etomidate (13), barbiturates (14), steroids (15), volatile anesthetics (16), and benzodiazepines (17) increase the chloride (Cl-) influx through the GABAAR upon activation by GABA (for reviews see, e.g., Refs. 4 and 18). Of a series of halogenated compounds, only those exhibiting anesthetic potency in vivo were effective at the GABAAR in vitro and vice versa (19).

Potentiating effects of volatile anesthetics have also been reported for the inhibitory glycine receptor (20), whereas excitatory synaptic transmission, mediated via the activation of N-methyl-D-aspartate (NMDA) and non-NMDA receptors, is depressed by volatile anesthetics (21).

Recently, xenon (Xe) has gained interest as an anesthetic (22). With a minimum alveolar anesthetic concentration (MAC) value of 71% in humans, it is more potent than nitrous oxide (N2O). Either gas blocks excitatory NMDA receptors in rat hippocampal neurons (23,24). We studied the effect of N2O and Xe on recombinant {alpha}1ß2{gamma}2L and {alpha}1ß2 GABAAR.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Human embryonic kidney cells (HEK 293) (supplied by DSMZ, Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany) were maintained in minimum essential medium, supplemented with 10% fetal calf serum, 4 mM L-glutamine, 100 U/mL penicillin, and 100 U/mL streptomycin. Cultures were incubated in an atmosphere of 5% CO2, 95% air, 100% relative humidity, and 37°C. The cells were harvested three times per week by resuspending in phosphate-buffered saline containing trypsin (100 µg/mL). After washing by centrifugation and resuspending in fresh medium, cells were seeded with 20%–40% confluency in 20 x 100 mm culture dishes.

Transfection was performed 48 h after culturing by using an electroporation system (Biotechnologies and Experimental Research, Inc., San Diego, CA). Cells were cotransfected with plasmids (PPK 235) containing cDNAs for rat {alpha}1, ß2, and {gamma}2L or {alpha}1 and ß2 GABAA receptor subunits, respectively. cDNA for green fluorescent protein (GFP) as an expression marker was cotransfected. After harvesting and resuspending, the cells were suspended in a buffer used for transfection (distilled H2O with 50 mM K2HPO4 x 3H2O, 20 mM K-acetate, 25 mM MgSO4 x 7H2O, pH 7.35). Plasmids containing cDNAs for the GABAA receptor subunits (5 µg for each subunit) and for GFP (10 µg) were added to the cell suspension. Electroporation was performed at 250–400 V, (adjusted to the success of the previous transfection), and 1mF with a pulse time of 30–45 ms. Transfected cells were replaced in 10 x 35 mm culture dishes with supplemented medium and incubated (5% CO2, 95% air, and 100% relative humidity, 37°C) for 12–18 h before the experiments. Five to thirty percent of the cells expressed GFP, and more than 95% of the green fluorescing cells yielded a GABA-induced current. GFP is dissolved in the cytoplasm. Kinetics of GABA-induced currents in HEK 293 cells with cotransfected GFP did not differ from those in HEK 293 cells without GFP.

Before each experiment, the medium was replaced by extracellular solution (162 mM NaCl, 5.3 mM KCl, 0.67 mM NaHPO4, 0.22 mM KH2PO4, 2 mM CaCl2, 15 mM HEPES, 5.6 mM glucose, pH 7.4 adjusted with NaOH). For all patch clamp experiments, cells were used in the 10 x 35 mm culture dishes at 20°–23°C. The patch clamp technique was used to measure GABA-mediated Cl- currents under whole-cell voltage-clamp conditions at –30 mV holding potential. The recording chamber was perfused with extracellular solution (2 mL/min). Borosilicate glass pipettes (GC150TF-10TM; Clark Electromedical Instruments, Pangbourne Reading, UK) were pulled, using a two-step puller (Zeitz Instruments, Augsburg, Germany), and heat polished. The resulting tip diameters were 0.2–1 µm, and electrical resistance was 4–9 M{Omega}. For recording, the pipettes were filled with pseudo-intracellular solution (140 mM KCl, 11 mM EGTA, 10 mM HEPES, 10 mM glucose, 2 mM MgCl2, 1 mM CaCl2, pH 7.3 adjusted with KOH). The GABA-induced currents were recorded with an Axopatch 200BTM patch-clamp amplifier, digitized with a sampling rate of 20 kHz, using a digidata 1200TM A/D converter, and low-pass filtered at a cutoff frequency of 5 kHz, performed with pClamp 6.0TM software (all from Axon Instruments, Inc., Foster City, CA).

Cells were patched by using a microscope combined with a fluorescence device (filter frequency 460 nm) for GFP (AxiovertTM; Zeiss, Oberkochem, Germany). After formation of a giga seal, cell membrane rupture resulted in the whole-cell configuration. Cell membrane capacitance and serial resistance were compensated by the patch-clamp amplifier. Nonspecific linear leak current was negligible.

An ultrafast exchange technique was used to apply GABA alone, or combined with the drug under investigation to the whole-cell patches (25). The drugs were administered to the cell via a "liquid filament," i.e., a tiny jet of solution, discharged from a borosilicate glass tube (inner diameter 0.15 mm) inside the recording chamber, which was perfused by extracellular solution. The recording chamber and glass tube were connected to a piezo crystal-driven device (Minitranslator P-249.20TM; Physik Instrumente, Waldbronn, Germany) that, upon activation, shifts the tube upward by 20 µm to immerse the cell (Fig. 1) This technique allows for a complete exchange of solutions in the vicinity of the cell held in the whole-cell mode within 1 ms. The "liquid filament" consisted of extracellular solution containing indicated concentrations of the agonist GABA alone (controls), or with the respective agent (test solution). The test solution was applied to the whole-cell patch in pulses of 1.5 s. An interval of 10 s between the pulses allowed full recovery of the GABAAR channels from a desensitized state. Each current trace was averaged from at least three stable currents, recorded from the 1.5-s pulses of agonist application. A manual valve was used to switch between test solution and control within approximately 10 s. For every GABA concentration, several separate patches, as indicated in the respective legend, were used for measuring baseline (GABA alone), test (GABA plus gas), and reversibility (GABA alone).



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  		Figure 1. The "liquid filament" application system for fast exchange of solutions. The tube, releasing a fine jet containing the test solution, is moved vertically by a piezo element to immerse the whole-cell patch. This technique allows for a complete exchange of solutions in the vicinity of the whole-cell patch within 1 ms (25). Test solution and background solution form a laminar flow system.

 
The respective gas (N2O, Xe; helium, oxygen as controls) was bubbled through a 10-mL vial containing the control solution with the respective concentration of GABA. A continuous flow of 20 mL gas/min was applied for a minimum of 3.5 min. The bubbling container was sealed with a rubber top, punctured with a drain tube as an escape hole. A closed glass syringe served as solution reservoir. The reservoirs containing the test solutions with the dissolved gases were tightly sealed to prevent evaporation and used within 20 s. No change of pH was observed after addition of the agents to the control solution. GABA was obtained from Sigma Chemicals (St. Louis, MO), xenon (class 2, 4a; UN 2036) from Messer Griesheim GmbH (Krefeld, Germany), N2O, helium, and oxygen from Linde AG (Höllriegelskreuth, Germany).

MAC values in humans for N2O and for Xe are 1.04 atm (26) or 0.71 atm (27), respectively. Using published solubility coefficients for 37°C (28), MAC equivalents for dissolved N2O or Xe were calculated to 20.6 or 2.9 mM, respectively.

The solubility of N2O and of Xe in the extracellular solution used in our experiments was measured at 20°C, 1 atm, by using a volumetric technique for determination of the solubility of gases in fluids (29), as modified by Dr. Karl-Heinz Meister (Linde AG, Höllriegelskreuth, Germany). This technique replaces and measures the soluted aliquot of a given gas in a closed space to correct for the resulting difference in gas pressure. For N2O (Xe), saturation (>95%) was achieved within 2.5 ± 0.3 min (3.1 ± 0.3 min). Solubility coefficients were 0.654 ± 0.010 (0.088 ± 0.005) or 29.2 ± 0.4 mM (3.9 ± 0.2 mM), respectively, in the extracellular solution at 20°C, 1 atm.

For comparison, published values for solubility in H2O at 20°C are 0.6788 for N2O and 0.1178 for Xe (28). Saturation of extracellular solution with N2O was found complete within 2 min by Dzoljic and van Duijn (30); no major desaturation occurred within 2 min of exposure to air.

Data were analyzed offline with AxoGraph 3TM software (Axon Instruments). Peak currents and risetimes(10%–90%) were analyzed by using automated detection algorithms. All data were presented as means ± SD. Differences (P < 0.05 was considered as significant) between agent and control were estimated by using the Student’s paired t-test.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Recombinant {alpha}1ß2{gamma}2L GABAAR channels, transiently expressed in HEK 293 cells, were studied by ultrafast application of GABA to the whole-cell patches at –30 mV clamp potential. The GABA-induced currents were reversed at 0 mV, corresponding to the equilibrium of Cl- under our adjusted experimental conditions, and were blocked by the competitive antagonist bicuculline. No intrinsic effect of 29.2 mM N2O or 3.9 mM Xe, respectively, was seen in {alpha}1ß2{gamma}2L or {alpha}1ß2 GABAAR in the absence of the agonist GABA (data not shown).

The test solution (see Fig. 1) containing concentrations of GABA as indicated, together with N2O (29.2 mM) or Xe (3.9 mM), respectively, was applied to the whole-cell patches. The application of 10-5 M GABA (EC16±4.1; i.e., this concentration induced 16% ± 4.1% of the maximum GABA response, elicited by >10-3 M GABA at this receptor) alone induced a peak current of –219 pA with a risetime(10%–90%) of 203 ms. Application of 10-5 M GABA, together with 29.2 mM N2O, to the same whole-cell patch increased the peak current to –367 pA, and decreased the risetime(10%–90%) to 133 ms. Upon washout of N2O, the peak current returned to –249 pA and the risetime(10%–90%) to 183 ms (Fig. 2). AFigure 2B illustrates the effect of Xe on the GABA-induced current. In this example, 10-6 M GABA (EC3.1±1.9) alone induced a peak current of –95 pA with a risetime(10%–90%) of 174 ms. The application of 10-6 M GABA, combined with 3.9 mM Xe, increased the peak current to –133 pA and decreased the risetime(10%–90%) to 137 ms. Upon washout of Xe, both effects were fully reversible.



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  		Figure 2. Currents flowing through {alpha}1ß2{gamma}2L gamma-aminobutyric acid receptor (GABAAR) channels within one patch. A, Application of 10-5 M GABA with or without 29.2 mM nitrous oxide (N2O). B, Application of 10-6 M GABA with or without 3.9 mM xenon (Xe). Each trace represents the average from 4–5 recordings.

 
Additional tests were performed with preexposure of the cells. The background solution (see Fig. 1) containing extracellular buffer with 3.9 mM Xe was preapplied to the whole-cell patch for 4 min before the coapplication of 10-5 M GABA and 3.9 mM Xe (test solution). Figure 3 shows traces from one representative patch. The preapplication of Xe had no additional effect on the Xe-induced increase in the current amplitude elicited by 10-5 M GABA: Xe significantly increased the GABA-induced current by 55% ± 12% or 51% ± 16% with or without preapplication of Xe. Furthermore, the time constant {tau} of deactivation, representing channel closing and agonist unbinding, was measured. In the presence of Xe (Fig. 3C), the time constant {tau} significantly and reversibly increased from 65 ± 13 ms (control, Fig 3A) to 105 ± 16 ms, suggesting a decreased rate of unbinding of GABA from the receptor under the influence of Xe. The results of identical protocols with N2O were similar: {tau} increased from 58 ± 15 ms to 99 ± 12 ms in the continued presence of N2O (Table 1).



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  		Figure 3. A, Control: 10-5 M gamma-aminobutyric acid (GABA) was applied to one whole-cell patch ({alpha}1ß2{gamma}2L GABAAR). B, 3.9 mM xenon (Xe) was coapplied with 10-5 M GABA. C, Preincubation with 3.9 mM Xe for 4 min. The time constant of channel deactivation ({tau}) is increased in the presence of Xe, indicating a decreased rate of unbinding of GABA. D, Washout of Xe. One representative of three separate experiments is shown.

 

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  		Table 1. Influence of Xe (N2O) on the Time Constant of Current Decay, {tau}, upon Receptor Deactivation
 
Both N2O (29.2 mM) and Xe (3.9 mM) significantly increased the currents induced by small concentrations of GABA (10-7 to 10-5 M; Fig. 4, A and B, left). The acceleration of the risetimes(10%–90%) at small GABA concentrations suggests an increased rate of GABA binding (Fig. 4, A and B, right). These results point to an increased efficacy of GABA at the {alpha}1ß2{gamma}2L GABAAR under the influence of N2O or Xe. In control experiments, helium was without effect on current amplitudes, or on risetimes(10%–90%), at any GABA concentration tested (10-7 to 10-3 M; data not shown).



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  		Figure 4. Effects of 29.2 mM nitrous oxide (N2O) (A) or 3.9 mM xenon (Xe) (B), respectively, on gamma-aminobutyric acid (GABA)-induced currents through {alpha}1ß2{gamma}2L GABAAR channels in whole-cell patches (n = 5–9 [5–8] for N2O [Xe] for every GABA concentration, means ± SD, *P < 0.05). The potentiating effect of 29.2 mM N2O or 3.9 mM Xe on peak currents is prominent at smaller concentrations of GABA (10-7 to 10-5 M). The relative increase in the current amplitude is shown. Within each patch, the control current, elicited by GABA alone (concentration as indicated), is set for 1.0 (left). Corresponding risetimes(10%–90%) are reduced by 29.2 mM N2O or 3.9 mM Xe at smaller concentrations of GABA (right). *P < 0.05 versus control.

 
The effects of N2O and of Xe on the currents induced by 10-5 M GABA were compared with the effects of 100% oxygen, prepared and applied identically. Application of the test solution (10-5 M GABA), containing 29.2 mM N2O (3.9 mM Xe), increased the current through the {alpha}1ß2{gamma}2L GABAAR channels to 169% (139%) (10-5 M GABA alone = 100%; Fig. 5A), and decreased the risetime(10%–90%) from 171 to 118 ms (from 170 to 119 ms) (Fig. 5B). The application of pulses of extracellular solution saturated with oxygen was without effect on peak current or risetime(10%–90%) (Fig. 5, A and B).



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  		Figure 5. Effect of saturated solutions of nitrous oxide (N2O), xenon (Xe), or oxygen (O2) on currents through {alpha}1ß2{gamma}2L gamma-aminobutyric acid receptor (GABAAR) channels elicited by GABA (10-5 M). A, Relative changes in the current amplitude (within each patch, the control current, elicited by GABA [10-5 M] alone, is set for 1.0). B, Absolute changes in the risetime(10%–90%) (open bars are controls). Bars represent means ± SD from 16, 7, and 6 separate patches for N2O, Xe, and O2, respectively. *P < 0.05 versus control.

 
Transfection with plasmids containing inserts encoding for {alpha}1 and ß2 subunits also resulted in a functional GABAAR channel complex, as reported previously (31). Though this receptor differs in several kinetic properties from the {alpha}1ß2{gamma}2L GABAAR (32), the effects of N2O and of Xe were similar: at this {alpha}1ß2 GABAAR, 29.2 mM N2O (3.9 mM Xe) increased the peak current by 88% (40%) and decreased the risetime(10%–90%) by 30% (37%) (Table 2, Fig. 6).


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  		Table 2. The Effect of Xe (N2O) on Amplitude and Risetime(10%–90%) of GABA (10-5 M)-Evoked Currents Through {alpha}1ß2 GABAAR
 


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  		Figure 6. Effect of 29.2 mM nitrous oxide (N2O) or 3.9 mM xenon (Xe) on currents through the {alpha}1ß2 gamma-aminobutyric acid receptor (GABAAR) elicited by 10-5 M GABA. A, Raw current traces. B, Relative changes in the current amplitude (within each patch, the control current, elicited by GABA [10-5 M] alone, is set for 1.0). C, Absolute changes in the risetime(10%–90%) (open bars are controls). Bars represent means ± SD from 7 (4) separate patches for N2O (Xe). *P < 0.05 versus control.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our experiments clearly demonstrate that N2O and Xe reversibly increase the efficacy of GABA at recombinant mammalian GABAAR channels, expressed in HEK 293 cells. Both gases enhanced the GABA-induced Cl- influx and decreased the risetimes(10%–90%) of the currents. The latter finding suggests that N2O and Xe increase the rate of GABA binding.

An enhancement of the Cl- influx through the GABAAR channels, triggered by IV and volatile anesthetics, has been reported previously (13,33). Xe, however, was recently reported to have virtually no effect on the GABAAR in cultured rat hippocampal neurons (24,34). The latter study focuses on the effect of Xe on inhibitory and excitatory postsynaptic currents in cultured "microislands." Thus, the subunit composition of the GABAAR studied is unknown and may be different from the one used in our experiments. It is obvious from recent studies that only minor changes in the ß subunit (35,36) or the presence of additional {delta} or {theta} subunits (37,38) notably affect receptor response to neuroactive drugs. Moreover, the GABAAR used in this study (expressed in HEK 293 cells) responds differently to anesthetics, when expressed in Xenopus oocytes (unpublished data from our laboratory). This fact can be explained by the influence of the host cell on the subunit composition of the receptor (39). N2O enhances the GABA-induced currents in native rat hippocampal neurons (30); the results compare well with the results from this study. The authors (30) discuss an increase in Cl- conductance through the GABAAR channel caused by phosphorylation. Phosphorylation increased the response of the {alpha}1ß1{gamma}2L GABAAR to GABA. This increase occurred only if both the ß1 and the {gamma}2L subunit were present (6). Intracellular phosphorylation has been reported for the {gamma}2L, as well as for the ß1 subunit (9,40). According to our results, the effect of N2O or Xe is independent of the {gamma}2L subunit. The effect of N2O or Xe appeared after milliseconds of agonist application, which is apparently too fast to suggest an effect mediated by intracellular phosphorylation.

Our results clearly indicate that both N2O and Xe increase the GABA-induced current at this GABAAR and accelerate the risetime(10%–90%) (see Fig. 4). An increase in the affinity of GABA would explain both the accelerated risetime(10%–90%) and the slower current decay in the presence of N2O or Xe (see Fig. 3C), as reflected by an increase in the time constant of deactivation, {tau} (Table 1).

N2O and Xe are clinically used anesthetics with well defined MAC values [Xe: MAChuman 0.71 atm (27); N2O: MAChuman 1.04 atm (26)]. To explain the interactions between molecules of noble or diatomic gases and proteins, Trudell et al. (41) performed computer simulations. The authors demonstrated that polarizability alters electron distribution and, thus, exerts weak forces between the gas molecule and the protein. Obviously, the size of the molecule correlates with polarizability, and correlates closely with the immobilizing potency (41). Immobilizing gases (argon, krypton, Xe, and nitrogen), or nonimmobilizing gases (helium, neon, and hydrogen), seem to be distinguishable from each other by a "favorable" or "unfavorable" balance between binding energies and the repulsive entropy contribution (3,41). In our study, helium exhibited no effect on the GABAAR, as did the "immobilizers" Xe and N2O.

In conclusion, our data demonstrate that N2O and Xe enhance GABA-induced Cl- currents through recombinant GABAAR ({alpha}1ß2{gamma}2L and {alpha}1ß2). The reduced risetimes(10%–90%), together with an increased efficacy of GABA, suggest an increase in the affinity for the ligand. The effect of N2O or Xe can clearly be attributed to the gases, because any effects caused by the varying content of oxygen on the GABAAR were thoroughly controlled. The effect on GABAergic ion flux is obviously independent of the presence of {gamma}2L.


   Acknowledgments
 
This work was supported in part by the Deutsche Forschungsgemeinschaft (Grant Schn-514/2–2) and by the Dr.-Ing. Leonhard Lorenz-Stiftung, München, Az. 376/97.

We gratefully thank Dr. Karl-Heinz Meister (Linde AG, Höllriegelskreuth, Germany) for the measurements of the solubility of N2O and Xe. We thank Monika Hammel for expert technical assistance, Prof. Dr. B. Urban and Dr. M. Barann for their critical review of the manuscript, and K. W. Miller, PhD, Boston, MA, for his comments.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication August 10, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2000 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press