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Anesth Analg 2008; 107:486-493
© 2008 International Anesthesia Research Society
doi: 10.1213/ane.0b013e31817b70c1
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ANESTHETIC PHARMACOLOGY

General Anesthetics Have Additive Actions on Three Ligand Gated Ion Channels

Andrew Jenkins, PhD*, Ingrid A. Lobo, PhD{dagger}, Diane Gong, PharmD{dagger}, James R. Trudell, PhD{ddagger}, Ken Solt, MD§, R. Adron Harris, PhD{dagger}, and Edmond I. Eger, II, MD||

From the *Department of Anesthesiology, Emory University, Atlanta, Georgia; {dagger}Waggoner Center for Alcohol and Addiction Research, Section of Neurobiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas; {ddagger}Department of Anesthesiology, Stanford University, Stanford, California; §Department of Anesthesia and Critical Care, Massachusetts General Hospital and Department of Anesthesia, Harvard Medical School, Boston Massachusetts; and ||Department of Anesthesia and Perioperative Care, University of CA, San Francisco, California.

Address correspondence and reprint requests to Dr. Andrew Jenkins, Department of Anesthesiology, Emory University School of Medicine, 1462 Clifton Rd NE Suite 420, Atlanta, GA 30322. Address e-mail to ajenki2{at}emory.edu.


   Abstract
Top
 Abstract
Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BACKGROUND: The purpose of this study was to determine whether pairs of compounds, including general anesthetics, could simultaneously modulate receptor function in a synergistic manner, thus demonstrating the existence of multiple intraprotein anesthetic binding sites.

METHODS: Using standard electrophysiologic methods, we measured the effects of at least one combination of benzene, isoflurane (ISO), halothane (HAL), chloroform, flunitrazepam, zinc, and pentobarbital on at least one of the following ligand gated ion channels: N-methyl-d-aspartate receptors, glycine receptors and {gamma}-aminobutyric acid type A receptors.

RESULTS: All drug-drug-receptor combinations were found to exhibit additive, not synergistic modulation. ISO with benzene additively depressed N-methyl-d-aspartate receptors function. ISO with HAL additively enhanced glycine receptors function, as did ISO with zinc. ISO with HAL additively enhanced {gamma}-aminobutyric acid type A receptors function as did all of the following: HAL with chloroform, pentobarbital with ISO, and flunitrazepam with ISO.

CONCLUSION: The simultaneous allosteric modulation of ligand gated ion channels by general anesthetics is entirely additive. Where pairs of general anesthetic drugs interact synergistically to produce general anesthesia, they must do so on systems more complex than a single receptor.


   Introduction
Top
 Abstract
 Introduction
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The precise molecular mechanisms of general anesthetic action are not yet fully understood. Many neuronal ion channels have been identified that are sensitive to several general anesthetic drugs,1 but the locations of drug binding sites on these receptors and the molecular events that follow anesthetic binding are still under investigation. An important question central to this endeavor is: Do receptors contain multiple anesthetic binding sites, or do all anesthetics modulate an individual receptor via action at a single site?

We have chosen to address this question in the present study by investigating the phenomenon known as "synergy." If two drugs, when applied at equi-effective concentrations, have significantly smaller effects on a target than a combination of the two drugs, applied at one-half of an equi-effective concentration, then the two drugs are said to be synergistic. This effect cannot occur if the two drugs both act at the same site. Synergism requires that the two drugs act at different sites. Therefore, the detection of a synergistic effect between two drugs on a given target would reveal the existence of multiple drug binding sites on a single receptor. However, when two drugs combine to give nonsynergistic effects, less can be said about the number of drug binding sites on the receptor. Additive and antagonistic (sub-additive or infra-additive) effects can both occur when drugs compete for the same site, or when they modulate a receptor via separate sites.

In this study, we sought to determine whether we could detect synergism between drugs with known or suspected separate binding sites. We also sought to determine if we could detect synergism between drugs with unknown binding sites, thereby defining multiple drug binding sites on the N-methyl-d-aspartate receptor (NMDAR), the glycine receptor (GlyR), and the {gamma}-aminobutyric acid type A receptor (GABAAR).


   METHODS
Top
 Abstract
 Introduction
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In this collaborative study, we performed four groups of electrophysiologic experiments in three different laboratories to detect synergistic modulation of ligand gated ion channels. All experiments were performed at 22°C to 24°C.

Receptor and Drug Selection
NMDARs are an important class of fast excitatory ligand gated channels found throughout the central nervous system. They have a complex pharmacology and harbor multiple binding sites for agonists, co-agonists, metal ions, drugs of abuse, and general anesthetics.2 Two NMDAR modulators, benzene and isoflurane (ISO), are thought to act via different binding sites3 and were therefore selected in this study since simultaneous modulation of the receptor at both sites could potentially produce a synergistic inhibition of NMDAR function.

GlyRs are fast inhibitory ligand gated channels found throughout the central nervous system that are positively modulated (potentiated) by zinc, many anesthetics and alcohols.4 Zinc is thought to allosterically enhance receptor function via an N-terminal binding site,5 and ISO is thought to stabilize the open state of the receptor by acting at an intrasubunit pocket defined by the four transmembrane segments of each subunit.6 The precise site of action of halothane (HAL) is unknown. Therefore, two pairs of compounds were selected for study: 1) ISO and zinc could potentially produce a synergistic potentiation of GlyR function via their different sites; and 2) ISO and HAL would interact synergistically if HAL has a separate and novel binding site from that of ISO.

GABAARs are the most common fast inhibitory ligand gated ion channels found in the central nervous system and are potentiated by a diverse group of sedative and hypnotic compounds including benzodiazepines and most general anesthetics. Benzodiazepines enhance receptor function via a well characterized N-terminal binding site7 whereas IV anesthetics, including pentobarbital (PB), are thought to interact with the transmembrane domain of the β subunit.8 Halogenated inhaled anesthetics are thought to act, and perhaps compete with, one another within a cavity defined by the transmembrane segments of the {alpha} subunit.6,9,10 The cavity has been hypothesized to be approximately 210 Å3 in size, thus accommodating either one ISO molecule, one HAL molecule or two chloroform molecules. A cavity of this size would also be able to simultaneously accommodate a molecule of HAL and chloroform at the same time which might bind more tightly than one HAL or two chloroform molecules. Therefore, the following four pairs of compounds were selected for study: ISO with flunitrazepam (FNZ), ISO with PB, ISO with HAL and HAL with chloroform. Because of their proposed different binding sites, ISO plus FNZ could potentially combine to synergistically potentiate GABAAR function, as could ISO with PB. Furthermore, ISO and HAL are thought to compete for the same site and could produce an additive or infra-additive effect. Finally, chloroform and HAL, which are also thought to bind to the same site,9 may combine to interact with different components of the binding cavity and produce a "superligand" that would produce greater potentiation and, therefore, synergy.

Oocytes—NMDAR Experiments
We have previously described in great detail the molecular biologic and electrophysiologic methods used to characterize the molecular pharmacology of NMDARs.11 Briefly, NR1 and NR2B subunit mRNAs were injected into Xenopus laevis oocytes at least 72 h before conducting 2-electrode voltage clamp experiments. Electrophysiologic solutions, electrodes, hardware and software were the same as previously published.12

ISO solutions were prepared by diluting a saturated solution using gas-tight syringes. Benzene solutions were prepared from a concentrated stock solution using gas-tight syringes.12,13

For each experiment, the oocyte was perfused for 30 s with buffer solution containing the agonist mixture [100 µM NMDA and 10 µM glycine (Gly)] to generate a control current. After at least 5 min of recovery, the oocyte was first perfused with buffer solution containing the test anesthetic for 30 s, and then perfused with buffer solution containing both the agonist mixture and the test anesthetic for 30 s. After another 5-min recovery period, the agonist mixture was again applied to the cell for 30 s to ensure reversibility of any anesthetic-induced change in current response. Peak current responses were recorded, and the magnitude of anesthetic-induced current inhibition was determined using the average of the two control experiments (before and after application of anesthetic).

Oocytes—GlyR and GABAAR Experiments
We have previously described in great detail the methods used to characterize the function of Gly and GABAA receptors.14 Briefly, GlyR {alpha}1 or GABAAR {alpha}1:β2:{gamma}2s subunit cDNAs were injected into Xenopus laevis oocyte nuclei at least 72 h before conducting 2-electrode voltage clamp experiments. Electrophysiologic solutions, electrodes, hardware, software and sources of chemicals were identical to our previously published work.15

GABA 1 mM or Gly was applied for 20 s to test the maximal response, and lower concentrations were applied for 30 s to reach a peak response for that concentration. After determining the maximal current, the EC5–10 of GABA or Gly was determined for each expressing oocyte, where EC5 is the effective concentration that elicits 5% of the maximal response and EC10 is the effective concentration that elicits 10% of the maximal response. After 10 min, the anesthetic solutions were applied as a 1-min preincubation in extracellular saline alone followed by a 30-s co-application of the anesthetic in an EC5–10 solution of GABA or Gly. After a 10-min washout, a second GABA or Gly EC5–10 test pulse was applied. Potentiation by drugs was calculated by dividing the drug-induced current by the average EC5–10 GABA- or Gly-induced currents applied 10 min before and after each drug application.

Concentrations of anesthetics were chosen that elicited approximately 100% potentiation (i.e., a doubling) of the EC5–10 GABA- and Gly-induced response. For GABA experiments, these concentrations were 120 µM ISO, 114 µM HAL, 15 µM sodium PB, and 0.5 µM FNZ. For Gly experiments, the concentrations were 75 µM ISO, 62.5 µM HAL, 0.1 µM zinc. Drugs were dissolved in solution immediately before application to the oocytes. For each experiment, ISO was paired with one of the other three anesthetics, and recordings were made in a single cell as follows 1) potentiation by [ISO] was determined at the full concentration (for 100% potentiation), 2) potentiation by the full concentration of the paired anesthetic e.g.: [HAL] was determined, 3) potentiation by half the concentration of [ISO]/2 was determined, 4) potentiation by half the concentration of the paired anesthetic [HAL]/2 was determined), and 5) finally, the potentiation by mixed solutions of concentrations of the drugs in steps 3 and 4 were tested ([ISO]/2+[HAL]/2). The Student's paired t-test was used to compare the potentiation measured in step 5 with step 1 and step 2. The same procedure was used to examine additivity between ISO and sodium PB on GABAARs, between ISO and FNZ on GABAARs, between ISO and HAL on GlyRs and between ISO and zinc on GlyRs.

GABAAR Patch Clamp Experiments
We have previously published the detailed methods used to measure the effects of general anesthetic drugs on the concentration-effect relationship of GABAARs.16 Briefly, human embryonic kidney 293 cells transiently expressing human {alpha}1, β2 and {gamma}2s subunits were whole cell patch clamped at –60 mV and superfused with 18 different solutions containing 0.3 to 1000 µM GABA and a combination of 0 to 10 minimum alveolar anesthetic concentration (MAC) HAL and 0 to 10 MAC chloroform. For each GABA exposure, the peak current amplitudes were measured. Stock solutions of GABA were diluted in extracellular solutions shortly before use. HAL and chloroform solutions were prepared by injection of liquid anesthetic with a gas-tight syringe as previously described.16

In all experiments, the concentrations of the selected drugs were chosen to give the desired modulations as described, but were also chosen to be in the clinically relevant range. Aqueous human MACs for ISO, HAL and chloroform were taken to be 310 µM, 220 µM and 900 µM respectively and the human EC50 for PB anesthesia was taken to be 50 µM.17,18 All concentrations less than four times these values can be considered to be clinically relevant and all concentrations above this can be assumed to be toxic and are used here simply to complete concentration response relationships.

Analysis
Concentration—response relationships were fit to the Hill equation:



Formula 1

where I is the peak current of each response, Imax is the maximum response elicited, C50 is the concentration eliciting half maximal effect (IC50 for the blockade of NMDARs—the concentration that elicits 50% inhibition, and EC50 for the activation of GABAARs—the effective concentration of GABA that elicits 50% of maximal activation), [D] is the concentration of the ligand and n is the Hill coefficient.

For GlyR and GABAAR potentiation experiments, if the effect of each drug when applied alone (the first two modulations) were both significantly less than the combination (the final modulation), then the two drugs were said to be synergistic. If the effect of the drug combination was less than that of each drug when applied alone, then the two drugs were said to be infra-additive. Drug pairs that were in neither of these categories were said to be additive. Significance between different experimental conditions was assessed using a Student's t-test. A similar criterion was applied to the modulation of the NMDAR. ISO and benzene would be considered to be synergistic if the response to the drug combination was significantly less than both drugs when each was applied alone.

By adapting the method of Minto et al.,19 we have previously used a response surface method to interpret the modulation of receptor function by a pair of general anesthetic drugs. Briefly, the reduction of GABA EC50 that underpins the potentiation by a pair of general anesthetics can be fitted to a response surface. The midpoint of this surface [described here as C50, previously described as U50({theta})] and slope [described here as n and previously described as {gamma}({theta})] can be approximated by a pair of parabolas, the curvatures of which are an indication of synergy or infra-additivity.16,19


   RESULTS
Top
 Abstract
 Introduction
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NMDA Receptors
ISO and benzene both inhibited NMDA receptor-mediated currents. Figure 1a shows a typical set of current traces demonstrating the inhibition of NMDA receptor-mediated current by a mixture of 1200 µM ISO and 170 µM benzene. In this oocyte, the anesthetic mixture inhibited NMDA receptor-mediated current by 51.3%. When applied individually, both ISO and benzene inhibited NMDA receptor function in a concentration-dependent fashion, as shown in Figure 1b. For ISO, the IC50 was 2400 ± 300 µM and the Hill coefficient –1.0 ± 0.1. For benzene, the IC50 was 340 ± 40 µM and the Hill coefficient was –0.96 ± 0.09.


Figure 123
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  		Figure 1. Inhibition of N-methyl-d-aspartate (NMDA) receptor function by isoflurane and benzene. a) 2-electrode voltage clamp recordings of NMDA/glycine responses (100 µM NMDA and 10 µM glycine) in Xenopus laevis oocytes before during and after the application of 1 mM isoflurane + 150 µM benzene. Bars above the current traces indicate the duration of agonists application (black) and the duration of the modulator application (gray). Calibration bars indicate the amplitude and duration of the responses. b) Normalized concentration response relationship for the inhibition of NMDA receptor function by isoflurane and benzene. Symbols represent the mean ± sem inhibition of responses to 100 µM NMDA and 10 µM glycine determined from six cells. The IC50s and Hill coefficients for isoflurane and benzene were 2400 ± 300 µM; –1.0 ± 0.1 and 340 ± 40 µM; –0.96 ± 0.09, respectively. c) Inhibition of NMDA receptor function by a combination of isoflurane and benzene did not differ for 300 µM benzene and a 50/50 mixture (1000 µM isoflurane + 150 µM benzene). Isoflurane (2000 µM) alone was slightly more effective as an inhibitor than the 50/50 mixture. *P < 0.05.

 

We then tested whether ISO and benzene have additive inhibitory effects on NMDA receptor-mediated currents by determining NMDA receptor inhibition by an anesthetic mixture containing 1200 µM ISO and 170 µM benzene (i.e., a mixture containing one-half the IC50 concentration of each drug). The experiment shown in Figure 1a was repeated in six different oocytes yielding an average inhibition of 56 ± 5% (Fig. 1c), suggesting that ISO and benzene have additive inhibitory effects on human NR1/NR2B NMDA receptors.

Glycine Receptors
ISO and HAL both potentiated GlyR function. This enhancement is shown in Figure 2a where applications of 37.5 µM, 75 µM ISO ([ISO]/2, [ISO]), 31.2 µM, 62.5 µM HAL ([HAL]/2, [HAL]) and a combination of 37.5 µM ISO and 31.2 µM HAL ([ISO]/2+[HAL]/2) all enhanced the amplitude of EC5–10 responses to Gly. Figure 2b shows that when the two drugs were applied at half this concentration (37.5 µM ISO and 31.2 µM HAL) the amplitude of the modulation was indistinguishable from the modulation produced by 75 µM ISO or 62.5 µM HAL suggesting purely additive actions of ISO and HAL.


Figure 223
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  		Figure 2. Potentiation of glycine receptor function by isoflurane (ISO), halothane (HAL) and zinc. a) 2-electrode voltage clamp recordings of glycine responses EC5–10 responses in Xenopus laevis oocytes before, during and after the application of ISO and/or HAL. Bars above the current traces indicate the duration of agonist application. Gray represents the duration of the anesthetic (75 µM ISO or 62.5 µM HAL) application and black represents the duration of the anesthetic (37.5 µM ISO and/or 31.2 µM HAL). Calibration bars indicate the amplitude and duration of the responses. b) Potentiation of glycine receptor function by ([ISO] = 75 µM) and ([HAL] = 62.5 µM) did not differ from [ISO]/2+[HAL]/2. c) Potentiation of glycine receptor function by a combination of ([ISO] = 75 µM) and zinc ([Zinc] = 0.1 µM): There was no significant difference between the potentiation by [ISO] and [ISO]/2+[Zinc]/2, but [ISO]/ 2+[Zinc]/2 was slightly more effective as a potentiator than [Zinc] alone.*P < 0.05.

 

Because zinc also enhances GlyR function, we examined whether combinations of 50 nM zinc and 37.5 µM ISO synergistically modulated GlyR function. We found no significant difference in the modulation by [ISO] and [ISO]/2+[Zinc]/2 (Fig. 2c). However, the potentiation by [ISO]/2+[Zinc]/2 versus [Zinc] did differ significantly.

GABAA Receptors
The methodology used in the GlyR experiments was repeated with GABAA receptors, using ISO in combination with either HAL or FNZ. Figure 3a shows the modulation of responses to EC5–10 concentrations of GABA by ISO and HAL. In all cases, the anesthetic applications potentiated receptor function. The enhancements of receptor function by [ISO]/ 2+[HAL]/2 or [ISO]/2+[FNZ]/2 did not differ from that produced by [ISO] (Figs. 3b and c), indicating additive effects of ISO with HAL and FNZ. For combinations of ISO and PB, the effects of [ISO] and [ISO]/2+[PB]/2 were indistinguishable, but there was a small but significant difference for [PB] and [ISO]/2+[PB]/2 (Fig. 3d).


Figure 323
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  		Figure 3. Potentiation of {gamma}-aminobutyric acid (GABAA) receptor function by isoflurane (ISO), halothane (HAL), flunitrazepam (FNZ) and pentobarbital (PB). a) 2-electrode voltage clamp recordings of GABA responses EC5–10 responses in Xenopus laevis oocytes before during and after the application of ISO and/or HAL. Bars above the current races indicate the duration of agonist application. The gray bars above these represent the duration of 120 µM ISO or 114 µM HAL application; and the black bars indicate the duration of 60 µM ISO and/or 57 µM HAL. b) Potentiation of GABAA receptor function by a combination of [ISO] = 120 µM, [HAL] = 114 µM. The potentiation by [ISO] or [HAL] did not differ significantly from that by [ISO]/2+[HAL]/2. c) Potentiation of GABA receptor function by a combination of [ISO] = 120 µM, [FNZ] = 500 nM. There was no significant difference between the potentiation by [ISO] and [FNZ] and [ISO]/2+[FNZ]/2. d) Potentiation of GABA receptor function by a combination of [ISO] = 120 µM, [PB] = 15 µM. [PB] did not differ from potentiation by [ISO] = 120 µM and [ISO]/2+[PB]/2, but [ISO]/2+[PB]/2 was slightly more effective as potentiator than [PB] alone. **P = 0.007.

 

Finally, in order to understand the effect of HAL and chloroform on GABAAR function, a full concentration response surface was constructed for the actions of these two anesthetics on the GABA concentration response relationship. Application of one MAC HAL mixed with one MAC chloroform enhanced currents elicited by low (<10 µM) concentrations of GABA, but decreased the amplitudes of responses to higher (>10 µM) concentrations (Fig. 4a). Figures 4b–e highlight these effects for each drug when applied alone. Both HAL and chloroform decreased the GABA EC50 in a dose-dependent and saturable manner (Figs. 4b and c) and both HAL and chloroform decreased the maximal current elicited by GABA, with chloroform being more effective than HAL (Figs. 4d and e). Using the methods described, the effects of 17 HAL-chloroform combinations on the fractional change in GABA EC50 were determined and a response surface fitted to the data. The functions C50 and n [previously defined as U50({theta}) and {gamma}({theta})]16,19 did not differ significantly from unity across the surface, indicating that HAL and chloroform were additive in their ability to enhance GABAAR function (Figs. 4f and g).


Figure 423
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  		Figure 4. Responses in human embryonic kidney-293 cells voltage clamped at –60mV expressing {alpha}1β2{gamma}2s {gamma}- aminobutyric acid (GABAA) receptor subunits to 0.3 to 1000 µM GABA are modulated by 220 µM halothane and 900 µM chloroform (1 MAC of each). a) Halothane and chloroform enhance the amplitude of currents when applied together at lower (<10 µM) but depressed at greater concentrations of GABA. Calibration bars indicate the amplitude and duration of the responses. b) Halothane enhances GABAA receptor function by reducing the GABA EC50. c) Cloroform enhances GABAA receptor function by reducing GABA EC50. d) Halothane reduces maximal GABAA receptor function. e) Chloroform reduces maximal GABAA receptor function. f) & g) Halothane and chloroform are additive in their ability to decrease GABA EC50. C50 and n were determined using the methods described and plotted in the range 0 < {theta} ≤ 1. Both C50 and n did not significantly deviate from unity indicating that there is no significant synergism or antagonism between the two anesthetics in the reduction of GABAAR EC50.

 


   DISCUSSION
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we tested the ability of general anesthetic combinations to modulate three neuronal receptors, anticipating that the detection of synergy would demonstrate that the two drugs under examination acted via dissimilar sites. The three receptors investigated are prototypic allosteric proteins; the binding of a ligand at one site alters the binding of a ligand at another site on the same protein. Therefore, we were interested to see if there was allosteric linkage between these proposed separate modulatory sites. A priori, we expected a certain degree of synergism since for some combinations there are published data that suggest that our drug pairs bind to different amino acids on the receptor.7,20,21 However, in all three preparations tested, synergistic receptor modulation by drug was not detected. That is to say, the modulation by two drugs in combination did not differ significantly from both compounds when each was applied alone. In some cases, we did see one of the comparisons differ modestly but significantly, but in no case did we see both comparisons differ.

One can argue that absence of synergism implies a unified common site of action as the only mechanism of action.22 However, as we have demonstrated here, two compounds that likely bind at different locations on a receptor do not necessarily produce synergism. Thus, our additive data would imply that, rather than there being a unitary site of action, there seems instead to be a missing allosteric link between the two intramolecular targets. Moreover, this supports the absence of an imaginary mechanism whereby infinitesimally small molecular effects can be amplified to generate a significant response.23 However, an interesting consideration is the allosteric and highly synergistic interaction between the general anesthestic and the neurotransmitter molecule. ISO, HAL, PB and chloroform all enhance Gly and GABA-activated responses at concentrations at which they are without intrinsic efficacy. However, they are agonists in their own right at much higher concentrations. In light of this, one prediction we can make is that maybe for these drugs, maximal synergism has been reached with potentiation alone, with nothing left to be done by the second drug.

General anesthetic synergism is common for many drug pairs acting on different target receptors.22,24,25 The results presented here suggest that when synergism for one of the drug pairs tested in this study occurs in an animal, it must do so as a result of action at different receptors in different parts of the neuronal circuitry that underpin the response being measured. A companion study to this report describes how the amplitudes of simple activating and inactivating stimuli can be integrated within neuronal circuits to give very different combinatorial effects.22 It is noteworthy that similar methods have been used before26 and could be used extensively in the future to predict the temporal effects of simultaneously enhancing hyperpolarizing and inhibiting depolarizing inputs within neuronal circuits. The results of such models would be of great interest to the present debate. However, by comparing the molecular studies shown here with the results of animal studies,24 in which quantal responses were being measured with the full spectrum of potential target sites present, and the findings of a meta-analysis,25 it is now apparent that general anesthetic drugs often have different effects at different sites, yet these effects simply add up linearly in the circuitry within which they are embedded. This appears to be especially true for the inhaled anesthetics.

In summary, synergism was not observed in the modulation of NMDARs, GlyRs or GABAARs by pairs of general anesthetics. Our results are in agreement with recent mutagenesis experiments that indicate that ISO, FNZ and PB all have separate sites of action, yet these drugs share a converging mechanism of action, namely the prolongation of the open time of ligand gated ion channels.


   ACKNOWLEDGMENTS
 
The authors thank Meagan A. Jenkins, Carrie Williams, Robert S. Harris, Peter Sebel, Jay Johansen, M. Bruce MacIver, Jan Hendrickx, Pamela Flood, and Steve Shafer for helpful discussions.


   Footnotes
 
Accepted for publication March 25, 2008.

Supported by NIH GM073959 (A.J.), NIH GM04718 (E.I.E. & R.A.H.) and institutional and/or departmental resources.

Dr. Eger is a paid consultant to Baxter Healthcare Corp.


   REFERENCES
Top
 Abstract
 Introduction
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hemmings HC Jr, Akabas MH, Goldstein PA, Trudell JR, Orser BA, Harrison NL. Emerging molecular mechanisms of general anesthetic action. Trends Pharmacol Sci 2005;26:503–10[Medline]
  2. Harris RA, Mihic SJ, Dildy-Mayfield JE, Machu TK. Actions of anesthetics on ligand-gated ion channels: role of receptor subunit composition. Faseb J 1995;9:1454–62[Abstract]
  3. Ogata J, Shiraishi M, Namba T, Smothers CT, Woodward JJ, Harris RA. Effects of anesthetics on mutant N-methyl-d-aspartate receptors expressed in Xenopus oocytes. J Pharmacol Exp Ther 2006;318:434–43[Abstract/Free Full Text]
  4. Laube B, Kuhse J, Rundstrom N, Kirsch J, Schmieden V, Betz H. Modulation by zinc ions of native rat and recombinant human inhibitory glycine receptors. J Physiol 1995;483(Pt 3):613–19[Abstract/Free Full Text]
  5. Miller PS, Da Silva HM, Smart TG. Molecular basis for zinc potentiation at strychnine-sensitive glycine receptors. J Biol Chem 2005;280:37877–84[Abstract/Free Full Text]
  6. Mihic SJ, Ye Q, Wick MJ, Koltchine VV, Krasowski MD, Finn SE, Mascia MP, Valenzuela CF, Hanson KK, Greenblatt EP, Harris RA, Harrison NL. Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors. Nature 1997;389:385–9[Medline]
  7. Rudolph U, Crestani F, Benke D, Brunig I, Benson JA, Fritschy JM, Martin JR, Bluethmann H, Mohler H. Benzodiazepine actions mediated by specific gamma-aminobutyric acid(A) receptor subtypes. Nature 1999;401:796–800[Medline]
  8. Amin J. A single hydrophobic residue confers barbiturate sensitivity to gamma-aminobutyric acid type C receptor. Mol Pharmacol 1999;55:411–23[Abstract/Free Full Text]
  9. Jenkins A, Greenblatt EP, Faulkner HJ, Bertaccini E, Light A, Lin A, Andreasen A, Viner A, Trudell JR, Harrison NL. Evidence for a common binding cavity for three general anesthetics within the GABAA receptor. J Neurosci 2001;21:RC136[Abstract/Free Full Text]
  10. Jenkins A, Andreasen A, Trudell JR, Harrison NL. Tryptophan scanning mutagenesis in TM4 of the GABA(A) receptor alpha 1 subunit: implications for modulation by inhaled anesthetics and ion channel structure. Neuropharmacology 2002;43:669–78[Web of Science][Medline]
  11. Raines DE, Claycomb RJ, Scheller M, Forman SA. Nonhalogenated alkane anesthetics fail to potentiate agonist actions on two ligand-gated ion channels. Anesthesiology 2001;95:470–7[Web of Science][Medline]
  12. Solt K, Eger EI II, Raines DE. Differential modulation of human N-methyl-d-aspartate receptors by structurally diverse general anesthetics. Anesth Analg 2006;102:1407–11[Abstract/Free Full Text]
  13. Kelly EW, Solt K, Raines DE. Volatile aromatic anesthetics variably impact human gamma-aminobutyric acid type A receptor function. Anesth Analg 2007;105:1287–92[Abstract/Free Full Text]
  14. Mascia MP, Gong DH, Eger EI II, Harris RA. The anesthetic potency of propanol and butanol versus propanethiol and butanethiol in alpha1 wild type and alpha1(S267Q) glycine receptors. Anesth Analg 2000;91:1289–93[Abstract/Free Full Text]
  15. Lobo IA, Trudell JR, Harris RA. Accessibility to residues in transmembrane segment four of the glycine receptor. Neuropharmacology 2006;50:174–81[Web of Science][Medline]
  16. Sebel LE, Richardson JE, Singh SP, Bell SV, Jenkins A. Additive effects of sevoflurane and propofol on gamma-aminobutyric acid receptor function. Anesthesiology 2006;104:1176–83[Web of Science][Medline]
  17. Franks NP, Lieb WR. Molecular and cellular mechanisms of general anaesthesia. Nature 1994;367:607–14[Medline]
  18. Krasowski MD, Harrison NL. General anaesthetic actions on ligandgated ion channels. Cell Mol Life Sci 1999;55:1278–1303[Web of Science][Medline]
  19. Minto CF, Schnider TW, Short TG, Gregg KM, Gentilini A, Shafer SL. Response surface model for anesthetic drug interactions. Anesthesiology 2000;92:1603–16[Web of Science][Medline]
  20. Dalziel JE, Cox GB, Gage PW, Birnir B. Mutant human alpha(1)beta(1)(T262Q) GABA(A) receptors are directly activated but not modulated by pentobarbital. Eur J Pharmacol 1999;385:283–6[Web of Science][Medline]
  21. Harvey RJ, Thomas P, James CH, Wilderspin A, Smart TG. Identification of an inhibitory Zn2+ binding site on the human glycine receptor alpha1 subunit. J Physiol 1999;520(Pt 1):53–64[Abstract/Free Full Text]
  22. Shafer SL, Hendrickx JFA, Flood P, Sonner J, Eger EI II. Additivity versus synergy: a theoretical analysis of implications for anesthetic mechanisms. Anesth Analg 2008;107:507–24[Abstract/Free Full Text]
  23. Eger EI II, Fisher DM, Dilger JP, Sonner JM, Evers A, Franks NP, Harris RA, Kendig JJ, Lieb WR, Yamakura T. Relevant concentrations of inhaled anesthetics for in vitro studies of anesthetic mechanisms. Anesthesiology 2001;94:915–21[Web of Science][Medline]
  24. Eger EI II, Tang M, Liao M, Laster M, Solt K, Flood P, Jenkins A, Raines DE, Hendrickx JFA, Shafer SL, Yasumasa T, Sonner JM. Inhaled anesthetics do not combine to produce synergistic effects regarding minimum alveolar anesthetic concentration in rats. Anesth Analg 2008;107:479–85[Abstract/Free Full Text]
  25. Hendrickx JFA, Eger EI II, Sonner J, Shafer SL. Is synergy the rule? A review of anesthetic interactions producing hypnosis and immobility. Anesth Analg 2008;107:494–506[Abstract/Free Full Text]
  26. Dutton RC, Zhang Y, Stabernack CR, Laster MJ, Sonner JM, Eger EI II. Temporal summation governs part of the minimum alveolar concentration of ISO anesthesia. Anesthesiology 2003;98:1372–7[Web of Science][Medline]



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