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Anesth Analg 2001;92:930-933
© 2001 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

Thiopental is a Competitive Inhibitor at the Human {alpha}7 Nicotinic Acetylcholine Receptor

Kristen Marie Coates, BS, Lawrence Edward Mather, PhD, FANZCA, FRCA, Raymond Johnson, MA, and Pamela Flood, MD, FACA

Department of Anesthesiology, Columbia University College of Physicians and Surgeons, New York, New York

Address correspondence to Dr. Pamela Flood, Department of Anesthesiology, Columbia University, 630 West 168th St., New York, NY 10032. Address e-mail to pdf3{at}columbia.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The nicotinic acetylcholine receptors (nAChRs) in the central nervous system may be a potential target for the anesthetic effects of thiopental. We evaluated the mechanism of action of thiopental on the human {alpha}7 nAChR by using 2-electrode voltage clamp methodology. Concentration response curves for agonist were prepared in the presence of 25–250 µM of thiopental. Inhibition by the S- and R-thiopental enantiomers was compared with inhibition by racemic thiopental. We found that thiopental acts as a competitive inhibitor at the human {alpha}7 nAChR. Inhibition is independent of membrane potential and the Ki(apparent) is 13 µM of thiopental. The clinical 50% effective concentration for thiopental in humans is 25 µM. Thus, with a Ki(apparent) of 13 µM, inhibition of the human {alpha}7 nAChR is within a clinically relevant range. The S- and R-enantiomers of thiopental cause inhibition indistinguishable from the inhibition caused by racemic thiopental. This discordance makes it unlikely that the {alpha}7 nAChR plays a role in loss of righting reflex induced by thiopental in mice, although nicotinic inhibition by thiopental may mediate other anesthetic effects and side effects.

Implications: Thereceptors for nicotine in the brain may be involved in the mechanism of general anesthetics. We have shown that a human receptor for nicotine is inhibited by the anesthetic barbiturate thiopental, at concentrations used clinically. The nicotinic receptor thus may mediate some of the actions of this drug.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The neuronal nicotinic acetylcholine receptors (nAChRs) may be a mediator of thiopental’s hypnotic and amnesic actions with clinically relevant concentrations (1–3). Thiopental inhibits peak current gated by acetylcholine (ACh) in chick nAChRs and PC12 cells, but the mechanism of that inhibition has not been studied in human recombinant nAChRs. To further evaluate the relevance of central nicotinic inhibition to thiopental anesthesia, we studied the concentration range and mechanism of thiopental inhibition of a human central nAChR ({alpha}7) response.

The {alpha}7 nAChRs are distributed diffusely in the central nervous system where they act both presynaptically to augment the release of other neurotransmitters as well as postsynaptically. The release of glutamate, {gamma}-aminobutyric acidA (GABA), dopamine, norepinephrine, and ACh itself can be regulated by nAChRs (4). Thus, inhibition of this control mechanism by thiopental could result in global changes in neurotransmitter release that may affect behavior. {alpha}7 containing nAChRs mediate excitatory input to inhibitory interneurons in the hippocampus and are thus a potential candidate for mediating amnesia caused by thiopental (5,6).

The S-thiopental enantiomer is approximately twice as potent as the R-thiopental enantiomer at inhibiting the righting reflex in mice in vivo (7,8). As a test of the potential involvement of thiopental inhibition of {alpha}7 activation in loss of righting reflex, we have tested racemic, S-, and R-thiopental on human {alpha}7 nAChRs. We demonstrate that a racemic mixture of thiopental competitively inhibits the activation of the {alpha}7 nAChR in a clinically relevant concentration range; however, there is no significant difference between the potency of the S- and R-thiopental enantiomers in their inhibition of the activation of the {alpha}7 nAChR.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
The human {alpha}7 nAChR subunit cRNA was prepared from the appropriate cDNA in a pMXT expression vector by using standard techniques (9). The human {alpha}7 nAChR clones were a gift from Dr. Jon Lindstrom at the University of Pennsylvania. The vector was linearized and used as a template for run-off transcription from the SP6 promoter. Xenopus laevis oocytes were surgically removed from the female and defolliculated with collagenase. After incubation overnight in L-15 oocyte medium, approximately 10 ng of {alpha}7 cRNA was injected per oocyte with a Nanoject Variable automatic injector (Drummond Scientific Co., Broomall, PA). The oocytes were incubated for 3–5 days in ND-96 medium (in mM: NaCl 96, KCl 2, MgCl2 1, CaCl2 1.8, HEPES 5, Na-pyruvate 2.5, theophylline 0.5, with gentamicin 100 mg/L) before physiological assay.

Whole oocyte currents were recorded by using a Gene-Clamp 500 two-microelectrode voltage-clamp amplifier with an active ground (Axon Instruments, Inc., Foster City, CA). The voltage, current, and active ground electrodes were filled with a 3 M KCl solution, such that voltage and current electrodes had a resistance of 1–5 M{Omega}. Extracellular recording solution consisted of (mM): 115 NaCl, 2.5 KCl, 1.8 BaCl2 10 HEPES, 0.001 atropine, pH 7.4. Experiments were performed at room temperature (20°–24°C). Racemic thiopental (Sigma, St. Louis, MO) was prepared daily as a 1-mM stock solution in external recording solution. Thiopental enantiomers were resolved with a chiral column by LM. Enantiomers were prepared as a 100-µM solution. Test solutions were prepared by serial dilution. Concentrations of racemic thiopental were measured by high-pressure liquid chromatography and found to be within 10% of the calculated concentration. Thiopental enantiomers were available in small quantity, thus they were diluted in buffer to a target concentration for experimentation, and each sample was measured by high-pressure liquid chromatography. The mea-sured value was used in all figures.

Oocytes were held at a membrane potential of -60 mV (except where indicated), and peak current was measured in response to ACh with and without thiopental. The drug solutions were placed in a closed syringe at the time of application and injected into a closed loop with a volume of 1/8 mL. The drug containing solution replaced the bath perfusate, when activated by a manual switch, in a specially prepared recording dish with a 125-µL cylindrical channel. Oocytes were perfused with thiopental for 30 s before agonist application. Agonist was applied for an approximately 2-s pulse of known volume and concentration. Perfusate was run continually at approximately 4 mL per minute. Five minutes were allowed to elapse between repeated agonist application in all experiments to allow recovery from desensitization. A 5-min interval provides stable peak current values in control experiments (data not shown). A baseline control response to ACh was measured before and after each ACh-anesthetic coapplication. Each anesthetic response was expressed relative to this internal control (n >= 5 for each data point).

Concentration response curves were prepared by plotting the fraction of current remaining after the coapplication of ACh and varying concentrations of thiopental as compared with the current response to ACh alone. A Hill equation, 100/(1+[x/IC50]n), was fitted to the data where IC50 is the dose at which 50% of available receptors are inhibited and n is the Hill coefficient. The data were analyzed by the method of Schild such that the normalized current was plotted against ACh concentration in the presence of varying concentrations of thiopental from 25 to 250 µM, and data were presented as mean ± SEM (10). The Schild method allows calculation of a Ki(apparent) and analysis of the mechanism of inhibition without the requirement for true saturation of currents (10). All fitting algorithms and graphs were produced with Microcal Origins 5.0 software (Microcal Software Inc., Northamton, MA). Responses were acquired online by using pClamp 7 (Axon Instruments, Foster City, CA), low pass filtered at 5 kHz, digitized (Digidata 1200 interface; Axon Instruments) by using pClamp7.


   Results
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 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Thiopental, at a concentration of 25 µM, inhibited the macroscopic current induced by ACh activation of the human {alpha}7 nAChR by 52.5% ± 3.5% ( Fig. 1 inset). Inhibition was readily reversible with washout of thiopental. This inhibition was dependent on both thiopental and ACh concentration (Fig. 1). Half-maximal inhibition was achieved with 147 ± 11 µM of thiopental with ACh at 50% effective concentration (EC50) (200 µM). The Hill number was 1.2 ± 0.1. When 100 µM of ACh was used as the agonist, the IC50 for inhibition was reduced to 40.6 ± 5.0 µM of thiopental with a Hill number of 1.0 ± 0.13. Thiopental was a less potent inhibitor at larger concentrations of ACh ( Figs. 1 and 2a). When the {alpha}7 nAChR was activated by a saturating concentration of ACh (1 mM), the IC50 for inhibition by thiopental was 285 ± 30 µM of thiopental and the Hill number was 1.2 ± 0.2.



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  		Figure 1. Thiopental inhibits the human {alpha}7 nicotinic acetylcholine receptor. Twenty-five micromolars of thiopental inhibits the peak current activated by 100 µM of acetylcholine (ACh) (inset). Concentration response relationships for inhibition by thiopental of current generated at several concentrations of ACh. Concentration response curves are fit by a Hill type equation with the form: y = 100/(1 + (IC50/X)n). For 1 mM of ACh, IC50 = 285 ± 30 µM of thiopental and n = 1.2 ± 0.2. When ACh is 200 µM, IC50 is 147 ± 11 µM of thiopental, n is 1.1 ± 0.1. When the concentration of ACh is 100 µM, IC50 is 41 ± 5 µM of thiopental and n = 1.0 ± 0.13.

 


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  		Figure 2. Thiopental is a competitive antagonist at the human {alpha}7 nicotinic acetylcholine (ACh) receptor. a, A Schild plot demonstrates the normalized current at log [ACh], in the presence of thiopental concentrations of 0, 25, 50, and 250 µM. b, The relationship between (r-1) (Schild’s dose ratio) and log [thiopental]. Linear regression provides a good fit of the data, P < 0.02. Ki(apparent) is 13 µM of thiopental.

 
When the data for inhibition of the human {alpha}7 nAChR were evaluated using the method of Schild (10), the Ki(apparent) was found to be 13 µM of thiopental, and the relationship between the dose ratio and the thiopental concentration was linear (P < 0.05), suggesting competitive inhibition (Fig. 2b). Inhibition of the ACh response is not dependent on membrane holding potential. When the percentage of current inhibition by 110 µM of thiopental was measured at voltages between -80 mV and -40 mV, the slope was not significantly different to 0 (P > 0.05) ( Fig. 3). Inhibition of the ACh response by both the S- and R-enantiomers of thiopental was not significant from that by the racemic thiopental ( Fig. 4).



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  		Figure 3. Inhibition by thiopental is independent of membrane holding potential. Peak current gated by 200 µM of acetylcholine (50% effective concentration) in the presence of 110 µM of thiopental is plotted versus membrane holding potential. Linear regression results in the equation y = mx + b. Slope (m) is not significantly different from 0, P > 0.05.

 


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  		Figure 4. Inhibition by stereoisomers of thiopental is not significantly different than that by the racemic mixture. The response of the human {alpha}7 nicotinic acetylcholine receptor to acetylcholine at its 50% effective concentration was measured in the presence of varying concentrations of racemic, S-, and R-thiopental. The response to racemic thiopental is fit to a Hill equation with 95% confidence intervals. Filled squares are data from racemic thiopental, hollow up-triangles are from S-thiopental, and hollow-down triangles are from R-thiopental.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
We have demonstrated that the human {alpha}7 nAChR was inhibited by thiopental in a clinically relevant concentration range. At large concentrations of ACh that would be found at a synapse at the neuromuscular junction, thiopental inhibition of agonist response is approximately 15%. This inhibition is small, but could cause a significant alteration in the function of a neural network. Many nAChRs in the central nervous system function as presynaptic receptors; however, their synaptic morphology is not well known (11). It is not known, for example, whether presynaptic nicotinic receptors are activated at tight synaptic junctions or by smaller, environmental concentrations of ACh. When the nAChR is activated with 100 µM of ACh, thiopental at its clinical EC50 (25 µM) inhibits the response by 52% ± 3.5%. The clinical EC50 concentration taken from the literature is an estimate of free aqueous concentrations of thiopental and corrected for protein binding (12). At smaller concentrations of ACh, thiopental at its clinical EC50 causes more significant inhibition of activation. Indeed, the Ki(apparent) for thiopental inhibition of {alpha}7 nAChR activation is 13 µM, approximately half of the clinical EC50.

The mechanism of thiopental inhibition of the human {alpha}7 nAChR is competitive and not dependent on membrane holding potential. These findings differ from those reported for thiopental inhibition of PC12 cell activation by ACh (1). Andoh et al. (1) found that thiopental inhibition of nicotinic activation of PC12 cells is both use-dependent and voltage dependent. Subunit composition may underlie the difference in mechanism of inhibition. The nAChRs from PC12 cells are predominantly heteromeric and contain {alpha}3 and ß4 ± {alpha}5 and {alpha}7 (13). It is not unprecedented that an anesthetic has a different mechanism of action on homomeric and heteromeric nAChRs, because heteromeric nAChRs are potently inhibited by volatile anesthetics and the {alpha}7 nAChRs are insensitive (14,15).

In vivo studies of an anesthetic behavior in mice show stereospecificity. The S-thiopental isomer is approximately twice as potent as the R-thiopental isomer at inhibiting the righting reflex in mice (7,8) and in producing electroencephalogram evidence of hypnosis in rats (16). However, in our physiological assay of thiopental activity at the human {alpha}7 nAChR, neither of the thiopental enantiomers caused significantly different inhibition from that caused by the racemic thiopental or to each other. Thus, inhibition of the nicotinic response by thiopental is not likely to play a major role in the inhibition of the righting reflex in the mouse. In contrast, enantiomers of thiopental, and other sedative barbiturates potentiate the GABAA response with the same order of potency as the in vivo (17,18). These results suggest that potentiation of GABA response may be involved in barbiturate inhibition of righting reflex in the mouse. Although our studies demonstrate that thiopental inhibition of the {alpha}7 nAChR is unlikely to underlie loss of righting reflex, it may mediate other behaviors induced by thiopental. The stereospecificity of other aspects of thiopental-induced behavior such as amnesia, immobility, and hypnosis has not been studied. If thiopental inhibition of the nicotinic response were to underlie any of these behaviors, no difference in the in vivo potency of the enantiomers would be anticipated.


   Acknowledgments
 
Supported by GM 000695 to PF.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Andoh T, Furuya R, Oka K, et al. Differential effects of thiopental on neuronal nicotinic acetylcholine receptors and P2X purinergic receptors in PC12 cells. Anesthesiology 1997; 87: 1199–209.[ISI][Medline]
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  6. Alkondon M, Pereira EF, Eisenberg HM, Albuquerque EX. Choline and selective antagonists identify two subtypes of nicotinic acetylcholine receptors that modulate GABA release from CA1 interneurons in rat hippocampal slices. J Neurosci 1999; 19: 2693–705.[Abstract/Free Full Text]
  7. Christensen HD, Lee IS. Anesthetic potency and acute toxicity of optically active disubstituted barbituric acids. Toxicol Appl Pharmacol 1973; 26: 495–503.[ISI][Medline]
  8. Haley TJ, Gidley JT. Pharmacological comparison of R(+), S(-) and racemic thiopentone in mice. Eur J Pharmacol 1976; 36: 211–4.[ISI][Medline]
  9. Anand RC, Schoepfer R, Whiting P, et al. Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes have a pentameric quaternary structure. J Biol Chem 1995; 266: 11192–8.[Abstract/Free Full Text]
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Accepted for publication December 7, 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