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Anesth Analg 2000;91:1289-1293
© 2000 International Anesthesia Research Society

GENERAL ARTICLES

The Anesthetic Potency of Propanol and Butanol Versus Propanethiol and Butanethiol in {alpha}1 Wild Type and {alpha}1(S267Q) Glycine Receptors

Maria Paola Mascia, PhD*, Diane H. Gong, BS{dagger}, Edmond I Eger, II, MD{dagger}, and R. Adron Harris, PhD*

*Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas; and {dagger}Department of Anesthesia and Perioperative Care, University of California, San Francisco, San Francisco, California

Address correspondence and reprint requests to Maria Paola Mascia, PhD, Institute for Cellular and Molecular Biology, University of Texas, 2500 Speedway MBB 1.124, Austin, TX 78712-1095. Address e-mail to mariapaola{at}mail.utexas.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Although similar in shape and size, and although differing only by substitution of a sulfur atom for an oxygen atom, propanethiol and butanethiol differ markedly from propanol and butanol in their in vivo potency and physical properties. Recent theories of narcosis suggest that anesthetics may act by enhancing the effect of inhibitory agonists, such as glycine, on their receptors. We tested whether propanol, butanol, propanethiol, and butanethiol enhance the effect of glycine on {alpha}1 glycine receptors expressed in Xenopus laevis oocytes in a manner that reflects the in vivo differences found for potencies. As anticipated, we found an immediate parallel between in vivo (rat minimum alveolar concentration of anesthetic required to eliminate movement in response to a noxious stimulus in 50% of subjects) and in vitro (recombinant receptor) effects. All four compounds enhanced the effect of glycine on wild type receptors, and the extent of enhancement for a given minimum alveolar concentration-multiple was approximately the same for all compounds. We also found that propanethiol, butanethiol, propanol, and butanol did not affect, or minimally affected, the action of glycine in anesthetic resistant mutants in which the amino acid serine at position 267 was replaced by glutamine [{alpha}1(S267Q)].

Implications: The in vivo potencies of propanethiol, butanethiol, propanol, and butanol correlate with their capacities to enhance the effect of glycine on {alpha}1 glycine receptors expressed in Xenopus laevis oocytes. These results support the notion that a protein mediates anesthetic action.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Although similar in shape and size, and differing only by substitution of a sulfur atom for an oxygen atom, alkanols differ from alkanethiols in their vivo potencies and physical properties (1). The alkanols are 30 times more potent than the corresponding alkanethiols, but are nearly equal in their lipophilicity. Such results run counter to the 100-year-old hypothesis offered by Meyer (2) and Overton (3) that potency correlates directly with lipophilicity. In the past two decades, the explanation for the action of anesthetics has shifted from one based on an interaction with lipid to one based on an interaction with proteins, particularly proteins forming receptors (4). Differences between the potencies and physical structures of alkanols and alkanethiols offer further tests of such interactions. The present and associated article (1) add to such tests, providing results consistent with the notion that the site of anesthetic action is amphipathic in character, having both polar and nonpolar components (5,6).

Inhaled anesthetics enhance the effect of the inhibitory neurotransmitters, {gamma}-amino butyric acid (GABA) and glycine, and this enhancement may, in part, underlie the production of anesthesia. The strychnine-sensitive glycine receptor is the main inhibitory receptor in the spinal cord and brainstem (7). These subcortical regions mediate one of the most important effects of anesthesia, suppression of movement in response to painful stimulation (8–10). The molecular basis for the enhancement of GABA and glycine receptors by volatile anesthetics is thought to result from effects on a site lying between transmembrane segment 2 (TM2) and transmembrane segment 3 (TM3) of the {alpha} subunit of the GABAA and glycine receptors (11,12). Two amino acid residues, Ser267 in TM2 and Ala288 in TM3, in the glycine {alpha} receptor subunit are essential for the modulation of the effect of glycine by inhaled anesthetics. Specific mutations of these residues can significantly reduce or completely abolish the potentiation of the glycine receptor function mediated by volatile anesthetics.

Expression of the glycine receptor {alpha} subunit cDNA into Xenopus laevis oocytes generates homo-oligomeric, functional glycine receptors, which display a pharmacology similar to that of native glycine receptors (13). Therefore, the strychnine-sensitive glycine receptor expressed into Xenopus laevis oocytes provides a simple, but reliable model to test whether propanethiol, butanethiol, propanol, and butanol similarly enhance the glycine receptor function, whether Ser 267 in the {alpha}1 subunit of the glycine receptor influences such enhancement, and whether the results are consistent with in vivo data for anesthetic potency (1). Such results also might be of value to studies that simulate anesthetic actions at model sites (such as those in the glycine receptor) thought to mediate some portion of the effects of anesthetics.

The hypothesis for the present report was that the potency of alkanols and alkanethiols, in their capacity to enhance the action of glycine on glycine receptors, would parallel their in vivo potencies as defined by minimum alveolar concentration. This result would be consistent with an interaction with proteins as the basis for anesthesia. We also hypothesized that mutations of the amino acid residue at Ser267 would decrease or completely abolish the potentiation of the glycine receptor function by volatile alkanols and alkanethiols alike. This, too, would be consistent with an interaction with proteins as the basis for anesthesia and would suggest a specific protein receptor site as important to the mediation of anesthesia.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Adult female Xenopus laevis were obtained from Xenopus I (Ann Arbor, MI). Glycine was obtained from Bio-Rad Laboratories (Hercules, CA). 1-Propanethiol and 1-butanethiol were purchased from Aldrich Chemical Co (Milwaukee, WI), propanol from Fisher Scientific (Fair Lawn, NJ), and butanol from Sigma (St. Louis, MO). All compounds were reagent grade.

Human {alpha}1 and {alpha}1(S267Q) glycine receptor subunit cDNAs were subcloned into pCIS2 vector. Site-directed mutagenesis was performed as described by Ye et al. (14). Oocytes were prepared and cDNA microinjection performed as described elsewhere (15). Isolated oocytes were placed in modified Barth’s saline (MBS) containing (in mM) NaCl 88; KCl 1; HEPES 10; MgSO4 0.82; NaHCO3 2.4; CaCl2 0.91; and Ca(NO3)2 0.33 adjusted to pH 7.5. Glycine receptor subunit cDNAs [wild type {alpha}1 or {alpha}1(S267Q) 1 ng/30nL] were injected into the animal poles of oocytes by the "blind" method of Colman (16). The injected oocytes were cultured at 15°-19°C in sterile MBS supplemented with 10 mg/L streptomycin, 10,000 U/L penicillin, 50 mg/L gentamycin, 90 mg/L theophylline, and 220 mg/L pyruvate.

Measurements were made in oocytes 1 to 4 days after the injection. Each was placed in a rectangular chamber (approximately 0.1-mL volume) and perfused (2 mL/min) with MBS at 26°C, with or without test compounds, via a pump (Cole-Palmer Instrument Co., Chicago, IL) through 18-gauge polyethylene tubing (Clay Adams Co., Parsippany, NJ) that delivered drug solutions to the recording chamber. The animal poles of oocytes were impaled with two glass electrodes (0.5–1.0 M{Omega}) filled with 3M KCl and voltage clamped at –70 mV by using a Warner Oocyte Clamp OC-725C (Warner Instruments, Hamden, CT). A strip-chart recorder (Cole-Palmer Instrument Co., Vernon Ills, IL) continuously plotted the clamping currents. Glycine was dissolved in MBS and applied for 30 s, a period long enough to achieve a steady state. Propanol, propanethiol, butanol, or butanethiol were dissolved in MBS. Oocytes were perfused with these solutions for 1 min to allow for equilibration before a 30-s coapplication of glycine. A 5- to 20-min washout period was allowed between applications of test compounds. Chromatographic analysis established that approximately 50%–60% of propanethiol and 60%–70% of butanethiol was lost during perfusion. Little or no propanol or butanol was lost. Partial pressures given are final bath partial pressures. Because of variability in glycine concentration-response curves (16), with one exception, all experiments were performed by using glycine concentrations that produced peak currents equal to 5% (EC5) of the maximal current (produced by application of 1 mM glycine). Using this technique, we determined the potentiation produced by concurrent application of propanol, butanol, propanethiol, and butanethiol. For the one exception, we also measured the entire glycine dose-response relationship in the absence of any of the test compounds and in the presence of 0.15 atmospheres of propanethiol.

We determined means and standard deviations or standard errors of the mean. A two-way analysis of variance was applied to some of the studies in oocytes. We accepted P < 0.05 as indicative of significance.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
As with other anesthetics, the dose-response relationship for the response to glycine shifted to the left during application of 0.15 atmospheres of propanethiol (Fig. 1). The control EC50 (concentration producing 50% of the maximum effect) is 169 ± 53 mM glycine (mean ± SD), whereas that for the application in the presence of propanethiol is 106 ± 24 mM glycine. The associated Hill coefficients are 2.2 and 2.5, respectively. Both propanol and propanethiol potentiated the effect of glycine on wild type receptors expressed in oocytes (Fig. 2). The highest test partial pressures of both propanol and propanethiol were lethal to one or more oocytes. Potentiation was significant at 0.5 and 1 minimum alveolar concentration but was most apparent at greater partial pressures. Propanethiol also significantly potentiated the effect of glycine on the mutant glycine {alpha}1(S267Q), but the effect was slight, particularly relative to that found in the wild type. Propanol did not potentiate, in fact inhibited, the effect of glycine on {alpha}1(S267Q).



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  		Figure 1. The presence of 0.15 atmospheres of propanethiol enhanced the action of glycine (i.e., shifted the dose-response relationship to the left) at all glycine concentrations between 1% and 100% of the maximal effective concentration of glycine. The control EC50 (concentration producing 50% of the maximum effect) was 154 mM glycine, whereas that for the application in the presence of propanethiol was 103 mM glycine. The maximum glycine concentration was 1000 mM. The associated Hill coefficients were 2.2 and 2.5, respectively. Control values (open circles, dashed lines) and values for 0.15 atmospheres propanethiol (open triangles, continuous line) are given as mean and SE.

 


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  		Figure 2. Propanol and propanethiol potentiated currents evoked by an EC5 (concentration producing 5% of the maximum effect) concentration of glycine in oocytes expressing {alpha}1 wild type glycine receptors. Propanol inhibited and propanethiol only slightly potentiated the effect of glycine in the mutant {alpha}1(S267Q) receptor. Data for each point are the mean and SE of results for five to six oocytes except for the highest partial pressures where one or more oocytes died in the course of measurement. The partial pressures given in the figure can be converted to mM by the application of the solubility data given in the accompanying article (1). Thus, for propanol, 0.01 atmospheres (i.e., a 1% concentration at 1 atmosphere) equals a solution concentration of approximately 1000 mM. For propanethiol, the concentration is approximately 1.06 mM. MAC = minimum alveolar concentration.

 
Butanol and butanethiol also produced a concentration-dependent potentiation of the effect of glycine on wild type receptors (Fig. 3). The highest test partial pressure of butanol was lethal to two of five oocytes. Mutation of serine to glutamine at residue 267 abolished potentiation by either butanol or butanethiol. As with propanol, higher concentrations of butanol inhibited the effect of glycine on {alpha}1(S267Q).



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  		Figure 3. Butanol and butanethiol potentiated currents evoked by an EC5 (concentration producing 5% of the maximum effect) of glycine in oocytes expressing {alpha}1 wild type glycine receptors. Butanol inhibited and butanethiol did not affect the glycine response in the mutant {alpha}1(S267Q) receptor. Data for each point are the mean and SE of results for three to nine oocytes except that at the highest partial pressure of butanol two of five test oocytes died in the course of measurement. The partial pressures given in the figure can be converted to mM by the application of the solubility data given in the accompanying article (1). Thus, for butanol, 0.01 atmospheres (i.e., a 1% concentration at 1 atmosphere) equals a solution concentration of approximately 664 mM. For butanethiol, the concentration is approximately 0.885 mM. MAC = minimum alveolar concentration.

 
Some of the effects summarized in the preceding two figures are illustrated in Figures 4 and 5. The tracings provide glycine (G)-induced Cl– currents in Xenopus laevis oocytes expressing {alpha}1 wild type glycine receptor subunits (Fig. 4) and show the enhanced current responses to an EC5 concentration of glycine in the presence of 0.001 atmospheres of propanol (P-OH, upper panel), or 0.038 atmospheres of propanethiol (P-SH, lower panel). In contrast (Fig. 5), 0.0005 atmospheres of butanol (B-OH, upper panel), or 0.015 atmospheres of butanethiol (B-SH, lower panel) did not enhance receptor Cl– current response to an EC5 concentration of glycine in {alpha}1 (S267Q) receptors. All test anesthetics were preapplied for 1 min before being co-applied with glycine for 30 s. The test anesthetics enhanced the effect of glycine on wild type receptors and had no effect or depressed the effect of glycine on the {alpha}1S267Q receptor. None of the test anesthetics increased Cl– current in the absence of glycine.



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  		Figure 4. These representative tracings of glycine (G)-induced Cl- currents in Xenopus laevis oocytes expressing {alpha}1 wild type glycine receptor subunits show the enhanced receptor Cl- current response to an EC5 (concentration producing 5% of the maximum effect) of glycine in the presence of 0.001 atmospheres of propanol (P-OH, upper panel) or 0.038 atmospheres of propanethiol (P-SH, lower panel). Propanol or propanethiol were preapplied for 1 min before being co-applied with glycine for 30 s.

 


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  		Figure 5. These representative tracings of glycine (G)-induced Cl- currents in Xenopus laevis oocytes expressing {alpha}1 (S267Q) glycine receptor subunits show the absent or decreased receptor Cl- current response to an EC5 (concentration producing 5% of the maximum effect) concentration of glycine in the presence of 0.0005 atmospheres of butanol (B-OH, upper panel) or 0.015 atmospheres of butanethiol (B-SH, lower panel). Butanol or butanethiol were preapplied for 1 min. before being co-applied with glycine for 30 s.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Our results are quantitatively consistent with the in vivo findings reported in the accompanying article by Zhang et al. (1). For wild type receptors, the potencies of propanol and butanol exceeded those of propanethiol and butanethiol by a factor of 30 to 60, approximately the same factor as found in rats in vivo. Thus, these results are consistent with the possibility that glycine receptors may mediate a portion of the capacity of volatile anesthetics to suppress movement in response to a noxious stimulus. The finding that propanethiol and butanethiol do not potentiate the glycine receptor function in the TM2 mutant {alpha}1(S267Q) is consistent with previous studies showing that although this mutation does not change the sensitivity to glycine [EC50 was 211 ± 53 µM (mean ± SD) in wild type and 188 ± 10 µM in {alpha}1(S267Q) receptors; data not shown], the potentiation of glycine action by volatile anesthetics is decreased or abolished (11).

Propanol and butanol modestly inhibited glycine receptor function in the mutated receptor. This result agrees with our recent finding that substitution of Ser 267 with larger amino acids, such as glutamine, produces inhibition of glycine receptor function by ethanol (14). That is, the volume of the amino acid at the 267 residue in the {alpha} subunit of the glycine receptor crucially influences the effect of alkanols on glycine receptor function. Our explanation for this "volume-dependent" change in the alkanol effect (potentiation in the wild type, but inhibition in the TM2 mutant) is that mutation of serine 267 to the larger glutamine decreases the ability of alkanols to bind to and to stabilize the closed state of the glycine receptor versus the open state, thereby inhibiting the action of glycine (14). These observations are consistent with the notion that anesthesia is produced by an action of anesthetics on protein receptor structure.

Both the potency and physical characteristics of the test compounds are consistent with mediation of an anesthetic effect by anesthetic interactions with amino acids lining a pocket adjacent to the amino acid at the 267 residue. Again, such observations are consistent with the notion that anesthesia is produced by an action of anesthetics on protein receptor structure.

In conclusion, these results are of interest to theories of anesthesia because anesthetic actions at a single receptor immediately parallel their in vivo actions. In addition, mutation of a single amino acid in this receptor markedly alters the action of both alkanethiols and conventional anesthetics (including alkanols), a finding consistent with a common site of action for chemically diverse anesthetics. Production of animals carrying mutations of the glycine receptor as reported in this study would allow a more specific test of the importance of this receptor as a mediator of the capacity of anesthetics to suppress movement in response to noxious stimulus.


   Acknowledgments
 
This work was supported, in part, by National Institutes of Health Grant 1P01GM47818–06.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Zhang Y, Trudell JR, Mascia MP, et al. Anesthetic potencies of alkanethiols for rats: relevance to theories of narcosis. Anesth Analg 2000; 91: .
  2. Meyer HH. Theorie der Alkoholnarkose. Arch Exptl Pathol Pharmakol 1899; 42: 109–18.
  3. Overton E. Studien ü ber die narkose, zugleich ein beitrag zur allgemeinen pharmakologie. Jena: Gustav Fischer, 1901: 1–195.
  4. Franks NP, Lieb WR. Do general anaesthetics act by competitive binding to specific receptors? Nature 1984; 310: 599–601.[Medline]
  5. Abraham MH, Lieb WR, Franks NP. Role of hydrogen bonding in general anesthesia. J Pharm Sci 1991; 80: 719–24.[Web of Science][Medline]
  6. Eger EI II, Koblin DD, Harris A, et al. Hypothesis: inhaled anesthetics produce immobility and amnesia by different mechanisms at different sites. Anesth Analg 1997; 84: 915–8.[Web of Science][Medline]
  7. Betz H. Glycine receptors: heterogenous and widespread in the mammalian brain. Trends Neurosci 1991; 14: 458–61.[Web of Science][Medline]
  8. Rampil IJ, Mason P, Singh H. Anesthetic potency (MAC) is independent of forebrain structures in the rat. Anesthesiology 1993; 78: 707–12.[Web of Science][Medline]
  9. Antognini JF, Schwartz K. Exaggerated anesthetic requirements in the preferentially anesthetized brain. Anesthesiology 1993; 79: 1244–9.[Web of Science][Medline]
  10. Rampil IJ. Anesthetic potency is not altered after hypothermic spinal cord transection in rats. Anesthesiology 1994; 80: 606–10.[Web of Science][Medline]
  11. Mihic SJ, Ye Q, Wick MJ, et al. Sites of alcohol and volatile anaesthetic action on GABAA and glycine receptors. Nature 1997; 389: 385–9.[Medline]
  12. Wick MJ, Mihic SJ, Ueno S, et al. Mutations of GABA and glycine receptors change alcohol cutoff: evidence for an alcohol receptor? Proc Natl Acad Sci USA 1998; 95: 6504–9.[Abstract/Free Full Text]
  13. Taleb O, Betz H. Expression of the human glycine receptor {alpha}1subunit in Xenopus oocytes: apparent affinities of agonists increase at high receptor density. EMBO J 1994; 13: 1318–24.[Web of Science][Medline]
  14. Ye Q, Koltchine VV, Mihic SJ, et al. Enhancement of glycine receptor function by ethanol is inversely correlated with molecular volume at position alpha267. Biol Chem 1998; 273: 3314–9.[Abstract/Free Full Text]
  15. Lin LH, Chen LL, Zirrolli JA, Harris RA. General anesthetics potentiate gamma-aminobutyric acid actions on gamma-aminobutyric acid alpha receptors expressed by Xenopus oocytes: lack of involvement of intracellular calcium. J Pharmacol Exp Ther 1992; 263: 569–78.[Abstract/Free Full Text]
  16. Colman A. Expression of exogenous DNA in Xenopus oocytes. In: Hames BD, Higgins SJ, eds. Transcription and translation: a practical approach. Washington, DC: Oxford Press, 1984: 49–59.
Accepted for publication June 15, 2000.




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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins with the assistance of Stanford University Libraries' HighWire Press®. Copyright 2006 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press