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Anesth Analg 2002;95:83-87
© 2002 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

Sensitivity of the {alpha}7 Nicotinic Acetylcholine Receptor to Isoflurane May Depend on Receptor Inactivation

Pamela Flood, MD, FACA, and Kristen M. Coates, BS

Department of Anesthesiology, Columbia University, New York

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


   Abstract
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 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
In previous studies, we demonstrated that nicotinic acetylcholine receptors (nAChRs) composed of the {alpha}7 subunit are unaffected by the co-application of isoflurane with agonists at concentrations up to 640 µM (two times the minimum alveolar anesthetic concentration). Modulation of {alpha}7-nAChR activity by isoflurane might have important behavioral ramifications because these receptors are expressed diffusely in the central and peripheral nervous systems and play pre- and postsynaptic roles in synaptic transmission. Here we have demonstrated that under some potentially physiologically relevant circumstances, the activation of {alpha}7 nAChRs may be inhibited by clinically relevant concentrations of isoflurane. We evaluated isoflurane inhibition of {alpha}7 nAChRs from chicks and humans expressed in Xenopus oocytes using two-electrode voltage clamp methodology. We determined the influence of time of preperfusion of isoflurane, agonist concentration, and membrane potential on inhibition by isoflurane. Both activation by a large concentration of agonist and isoflurane preperfusion increased inhibition. The half-maximal inhibitory concentration for isoflurane inhibition of chick {alpha}7 nAChR with isoflurane preperfusion and activation by 100 µM of acetylcholine was 938 ± 26, and when activated by 1 mM of acetylcholine, it was 408 ± 51 µM. The increase in inhibition with isoflurane preexposure and large agonist concentration raises the possibility that isoflurane interacts preferentially with a closed or closed-desensitized state of the channel.

IMPLICATIONS: Nicotinic receptors expressed in the brain have been considered a possible target for the actions of isoflurane. We studied the effect of isoflurane on {alpha}7 type nicotinic receptors expressed in Xenopus oocytes. We find that when activated by large concentrations of acetylcholine, {alpha}7 nicotinic receptors are inhibited by isoflurane at concentrations near MAC.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
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In 1997, we reported our findings that the activation of the chick {alpha}7 nicotinic acetylcholine receptor (nAChR) by 100 µM of acetylcholine (Ach) was not affected by isoflurane at two times its minimum alveolar anesthetic concentration (MAC) (640 µM) (1). During the same year, Zhang et al. (2) used the {alpha}7 nAChR as the volatile anesthetic sensitive receptor in an elegant experiment with a nAChR-5-hydroxytryptamine3 chimera. They reported approximately 60% inhibition of {alpha}7-nAChR activation by 5 mM of isoflurane and 5 mM of halothane. These findings were not in conflict because they reflected different anesthetic concentrations. In our studies, we sought to define the situations and concentration range within which isoflurane inhibits {alpha}7 nAChRs.

Nicotinic receptors containing the {alpha}7 subunit are expressed throughout the central and autonomic nervous systems where they are thought to play several physiological roles (3). Throughout the central and peripheral nervous systems, functional {alpha}7-containing nAChRs have been identified by numerous physiological studies where they are thought to modulate neurotransmitter release (4). nAChRs present on the presynaptic terminals of multiple neuronal types play a modulatory role in the release of glutamate, {gamma}-aminobutyric acid, dopamine, norepinephrine serotonin, and ACh itself. In addition to their presynaptic activity, {alpha}7-containing nAChRs are expressed in the somatodendritic domains of inhibitory interneurons in the hippocampus and somatosensory cortex where they perform a postsynaptic function to directly mediate fast synaptic transmission (5–10). Thus, {alpha}7-containing nAChRs are located in numerous sites in the central and peripheral nervous systems where they alter the intensity of neuronal excitability such that their inhibition by volatile anesthetics might be predicted to have behavioral consequences that are seen in clinical anesthesia.

Nicotinic receptors are composed of five nicotinic subunits. In contrast with most nicotinic receptors that are composed of both {alpha} and ß subunits, there is substantial evidence for the formation of homomeric {alpha}7 nAChRs in neurons and in heterologous systems (11,12). Although it has been more difficult to demonstrate in heterologous expression systems, there is also evidence that {alpha}7 can co-express with other nicotinic {alpha} and ß subunits in peripheral and central neurons (13,14). The unique feature of {alpha}7-containing nAChRs of interest to synaptic physiologists is their conductance of calcium ions. In heterologous systems, nAChRs composed entirely of {alpha}7 subunits have a sodium:calcium conductance ratio similar to N-methyl-D-aspartate receptors (15). Because calcium is an important second messenger involved in long- and short-term cellular changes, the potential inhibition of these receptors by isoflurane may be particularly important.

In these experiments, we studied the effect of isoflurane on the activation of nAChRs composed of {alpha}7 subunits from chicks that we had studied previously and {alpha}7 nAChRs of human origin. We have studied the effects of agonist concentration, electrical gradient, and time of preexposure to isoflurane on the inhibition of the {alpha}7 nAChRs by isoflurane. We have found that the degree of isoflurane inhibition of the {alpha}7 nAChR is dependent on both agonist concentration and preexposure to an antagonist. When {alpha}7 nAChRs are pretreated with isoflurane and activated by large concentrations of ACh found at a traditional synapse (16), the half-maximal inhibitory concentration (IC50) of isoflurane inhibition occurs at concentrations near MAC.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Molecular Biology
The chick {alpha}7 complimentary DNA was in a PGH19 expression vector and the human {alpha}7 nAChR was in pMXT. The vectors were linearized and used as templates for run-off transcription from the T7 (chick) or SP6 (human) promoter using standard techniques (1).

Oocyte Extraction and Injection
Xenopus laevis oocytes were extracted from anesthetized women and placed in ND-96 medium (NaCl 96 mM, KCl 2 mM, MgCl2 1 mM, CaCl2 H2O 1.8 mM, HEPES 5 mM, Na-pyruvate 2.5 mM, theophylline 0.5 mM, and 10 mg/L of gentamicin and adjusted to a pH value of 7.5). The oocyte clusters were incubated in 0.2% collagenase (type IA, Sigma-Aldrich, St Louis, MO) in the ND-96 medium for defolliculation. Oocytes were agitated at 18.5°C for 4 h and afterwards were rinsed with Barth’s medium (NaCl 88 mM, KCl 1 mM, NaHCO3 2.4 mM, and HEPES 15 mM with a pH value of 7.6). The oocytes were left to recover for 24 h in a L-15 oocyte medium (Specialty Media, Phillipsburg, NJ) before an injection of complimentary RNA.

Approximately 10 ng of {alpha}7 nAChR complimentary RNA were injected into individual oocytes in volumes of ~10 nL using a manual injector (Nanoject, Drummond Scientific, Broomall, PA). The oocytes were incubated at 17°C for 5–7 days in the ND-96 medium before the electrophysiological recording.

Electrophysiology
Current recordings were made from whole oocytes at room temperature (19°C–23°C) using a Gene-Clamp 500 two-microelectrode voltage-clamp amplifier with an active ground circuit (Axon Instruments, Inc, Foster City, CA). The recording electrodes were pulled from glass capillary tubing (Drummond Scientific) to obtain a resistance between 1 and 5 M{Omega} and then filled with 3 M of KCl. The Ringer’s solution (115 mM of NaCl, 2.5 mM of KCl, 1.8 mM of BaCl2, 10 mM of HEPES, and 1 µM of atropine with a pH value of 7.4) used for recordings contained atropine to prevent muscarinic receptor stimulation and barium in place of calcium to avoid current amplification by calcium activated chloride currents. Oocytes were clamped at a holding potential of -60 mV unless otherwise indicated and held in a 125 µL cylindrical channel. All drugs were applied by perfusion at a flow rate of 4 mL/min. The oocyte was preexposed to isoflurane for 2 min unless otherwise noted and tested with a 2-s agonist application in the continued presence of isoflurane. Activation was complete during the application period. ACh and other chemicals used were obtained from Sigma-Aldrich. Isoflurane was obtained from Abbott Laboratories (North Chicago, IL). Isoflurane was made as a saturated solution and serially diluted to the appropriate concentration on the day of the experiment. All concentrations were verified with gas chromatography. To minimize the contribution of nAChR desensitization, 3 min passed between ACh applications. Using this time interval, steady-state recordings could be obtained in control experiments. A baseline control response to the agonist was measured before and after each agonist-antagonist co-application. Responses that did not return to within 80% of baseline values were rejected for analysis.

Inhibitory concentration-response relationships were constructed by calculating the current recorded in the presence of the antagonist as a percentage of that elicited by the agonist alone. The resulting data were fitted to a modified Hill equation, y = ymax / (1 + [x / EC50]n), where EC50 is the concentration of isoflurane that elicited 50% of the maximal response, ymax is the maximal current elicited by the agonist, and n is the Hill coefficient. Agonist dose-response curves were normalized to a saturating concentration of the agonist. The data points obtained at each concentration were averaged, and the calculated mean and SE were fit to a modified Hill equation. Clampex 7 (Axon Instruments) was used for data acquisition, and Microcal Origin 5.0 (Microcal, Northampton, MA) was used for graphics and statistical calculation. The response of current inhibition to membrane potential was fit with a linear equation and compared with the fit equation with a zero slope with an analysis of variance (ANOVA). P < 0.05 was considered significant, and data were represented as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Nicotinic {alpha}7 receptors can be inhibited by isoflurane. Inhibition of the chick nAChRs is significantly related to the activating agonist concentration (Fig. 1A). At near saturating agonist concentration (1 mM of ACh), inhibition by isoflurane is more potent then when the receptor is activated at a smaller agonist concentration. When the data are fit by a modified Hill equation, IC50 for inhibition is near MAC for isoflurane (408 ± 51 µM). When the chick {alpha}7 nAChR is activated by ACh at concentrations near its EC50, inhibition is less potent with an IC50 of 938 ± 26 µM. The inhibition by isoflurane is significantly different when the {alpha}7 nicotinic receptor is activated by saturating ACh as compared with an EC50 concentration of the agonist (ANOVA; P < 0.01).



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  		Figure 1. Isoflurane inhibits chick and human {alpha}7 nicotinic acetylcholine receptors (nAChRs). (A) Concentration response relationship for isoflurane inhibition of the activation of the chick {alpha}7 nAChR by 1 mM of acetylcholine (ACh): the half-maximal inhibitory concentration (IC50) was 408 ± 51 µM (Hill coefficient, 1.37 ± 0.27; n = 5–12). When the {alpha}7 nAChRs were activated by 100 µM of ACh, the IC50 for isoflurane was 938 ± 26 µM (Hill coefficient, 2.64 ± 0.18; n = 5–7). (B) Concentration response relationship for isoflurane inhibition of the activation of the human {alpha}7 nAChR by 1 mM of ACh: the IC50 was 671 ± 70 µM (Hill coefficient, 1.55 ± 0.21; n = 4–8). When the {alpha}7 nAChRs were activated by 100 µM of ACh, the IC50 for isoflurane was 614 ± 21 µM (Hill coefficient, 2.11 ± 0.15; n = 4–6).

 
Isoflurane inhibition of activation of the human {alpha}7 nAChR is less dependent on agonist concentration (Fig. 1B). The IC50 for inhibition of activation by 1 mM of ACh is 671 ± 70 µM of isoflurane. The IC50 for inhibition of activation by 200 µM of ACh (near EC50 agonist concentration) is not significantly different at 614 ± 21 µM of isoflurane (ANOVA; P > 0.05).

Isoflurane 320 µM reduces the maximal activation by ACh at the chick {alpha}7 nAChR to 71% ± 10% (Fig. 2A). The EC50 is not significantly changed. The Hill coefficient is decreased from 2.7% ± 0.3% to 1.4% ± 0.6%. The maximal ACh activation at the human {alpha}7 nAChR is decreased to 64.8% ± 2.2% (Fig. 2B).



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  		Figure 2. Acetylcholine (ACh) activation in the presence and absence of 320 µM of isoflurane. (A) Concentration response relationship for ACh activation of the chick {alpha}7 nicotinic acetylcholine receptor (nAChR) with and without 320 µM of isoflurane. The 50% effective concentration (EC50) for ACh without isoflurane was 182 ± 7 µM (Hill coefficient, 2.67 ± 0.30; n = 3–6). With isoflurane, the EC50 for ACh was 198 ± 63 µM (Hill coefficient, 1.38 ± 0.60; n = 5–8). The maximal activation was reduced to 71.2% ± 9.6%. (B) Concentration response relationship for ACh activation of the human {alpha}7 nAChR with and without 320 µM of isoflurane. The EC50 for ACh without isoflurane was 349 ± 52 µM (Hill coefficient, 1.19 ± 0.18; n = 6–10). With isoflurane, the EC50 for ACh was 156 ± 16 µM (Hill coefficient, 2.22 ± 0.46; n = 4–5). The maximal activation was reduced to 64.8% ± 2.2%.

 
After the measurement of baseline current, ACh was applied every 3 min in the continued presence of isoflurane. Inhibition of the human nAChR by isoflurane is not increased by repeated agonist applications (Fig. 3A). Nor is inhibition by isoflurane dependent on membrane potential. The percentage of current remaining after the application of 600 µM of isoflurane is displayed at holding potentials from -20 to -100 mV (Fig. 3B). When the data are fit with a linear regression, the slope is not significantly different from 0 mV (ANOVA; P > 0.05). To determine the time course for the effect of isoflurane preapplication, isoflurane was applied for periods between 0 and 120 s before co-application of ACh and isoflurane. Preapplication of isoflurane increased inhibition of nAChR activation with maximal activity at 30 s (Fig. 3C).



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  		Figure 3. The effect of agonist application, voltage, and preperfusion time on the inhibition of {alpha}7-nicotinic acetylcholine receptor (nAChR) activation by isoflurane. (A) Representative traces showing activation of {alpha}7 nAChR by 1 mM of ACh and response to repeated pulses of 1 mM of ACh in the presence of 320 mM of isoflurane. There is no difference in percent inhibition with repeated agonist application (t-test; P > 0.05; n = 5). (B) Percent of inhibition by 600 µM of isoflurane at different holding potentials. The linear regression (solid line) has a slope not significantly different from zero (t-test; P > 0.05; n = 3–5). (C) The effect of different isoflurane (640 µM) preapplication times on the percent inhibition of chick {alpha}7-nAChR activation by 1 mM of ACh. Activation is complete at 30 s (n = 3–8).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Isoflurane at its clinical EC50 concentration reduces the maximal activation by ACh at both chick and human nAChRs composed of the {alpha}7 subunit when the receptor is preexposed (Fig. 1, A and B). In our previous experiments with chick {alpha}7 nAChRs, we found that when activated by 100 µM of ACh, 640 µM of isoflurane co-applied with the agonist had no effect on activation. Here we confirm that the same isoflurane concentration has no effect when co-applied with the agonist (1 mM of ACh), but with 30 seconds of exposure to isoflurane before the agonist application, inhibition becomes significant (Fig. 3C). In a study that reported on the effect of thiopental on nAChRs, Downie et al. (17) argued that preperfusion of an anesthetic modeled more physiological conditions and, in fact, produced different results. With preexposure to isoflurane, chick {alpha}7 nAChRs (activated by 100 µM of ACh) are indeed inhibited by isoflurane but with an EC50 concentration of 938 ± 26 µM.

Agonist concentration also affects the degree of isoflurane inhibition. Although chick and human {alpha}7 nAChRs are different with respect to relevant agonist concentrations (Figs. 1 and 2), both are more potently affected by isoflurane when activated at large agonist concentrations (Fig. 2). When the chick {alpha}7 nAChR is activated by 1 mM of ACh, the EC50 for inhibition is reduced to a clinically relevant concentration just above the MAC value for isoflurane (Fig. 1A). Enhanced potency of isoflurane for inhibition of the {alpha}7 nAChR with preexposure could occur for one of several reasons. It may take time for isoflurane to equilibrate at its inhibitory site of action, perhaps because it is located at an energetically inaccessible location. As the {alpha}7 nAChR undergoes very infrequent unliganded openings, this site may be preferentially reached in a closed or desensitized state of the channel. The fact that activation by large agonist concentrations makes the isoflurane inhibition more potent supports this supposition because a larger proportion of receptors will be in a desensitized state when larger agonist concentrations are used for activation. We cannot exclude the possibility that isoflurane is not acting directly on the receptor but through a secondary modulatory system. It is possible that exposure of another target to isoflurane results in modification of the nAChR to decrease the number of nAChRs available for activation by ACh. However, the change would have to be quite temporary because the inhibition was readily reversible within a three-minute washout of isoflurane. There was no dependence of blockade on membrane potential, as would be expected, because isoflurane is uncharged. However, if calcium influx mediated the change, voltage dependence would be expected because the relative calcium conductance is dependent on membrane potential (18).

We conclude that under conditions of isoflurane preexposure and activation by large agonist concentrations, the {alpha}7 nAChR is inhibited by clinically relevant concentrations of isoflurane. Although the oocyte model system may not mimic native physiology, conditions of anesthetic pre and continual exposure and large agonist concentrations may be particularly relevant for the consideration of in vivo synaptic effects.


   Acknowledgments
 
Supported, in part, by GM00695 to P.F.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Flood P, Ramirez-Latorre J, Role L. Alpha 4 beta 2 neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but alpha 7-type nicotinic acetylcholine receptors are unaffected. Anesthesiology 1997; 86: 859–65.[Web of Science][Medline]
  2. Zhang LO, Stewart M, Peoples RR, Weight FF. Volatile general anaesthetic actions on recombinant nACh alpha 7, 5-HT3 and chimeric nACh alpha 7–5-HT3 receptors expressed in Xenopus oocytes. Br J Pharmacol 1997; 120: 353–5.[Web of Science][Medline]
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  5. Frazier CJ, Buhler AV, Weiner JL, Dunwiddie TV. Synaptic potentials mediated via alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in rat hippocampal interneurons. J Neurosci 1998; 18: 8228–35.[Abstract/Free Full Text]
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  7. Albuquerque EX, Pereira EF, Braga MF, Alkondon M. Contribution of nicotinic receptors to the function of synapses in the central nervous system: the action of choline as a selective agonist of alpha 7 receptors. J Physiol Paris 1998; 92: 309–16.[Web of Science][Medline]
  8. Jones S, Yakel JL. Functional nicotinic ACh receptors on interneurons in the rat hippocampus. J Physiol 1997; 504: 603–10.[Abstract/Free Full Text]
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Accepted for publication March 6, 2002.




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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 2002 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press