The activation of nicotinic acetylcholine receptors (nAChRs) expressed in the brain is exquisitely sensitive to inhibition by volatile anesthetics (1–4). The physiological mechanism of that inhibition is controversial. In a previous study, we found that isoflurane inhibition of α4β2 nAChRs was dependent on the activating agonist concentration. Inhibition of nAChR activation by isoflurane was more potent at small agonist concentrations, and a Schild analysis was suggestive of competitive inhibition (1). Although the structure of isoflurane makes a direct action at the agonist binding site unlikely, an allosteric action could cause an alteration in agonist affinity.
Volatile anesthetics alter agonist affinity at the evolutionarily related nAChRs from the electroplax organ of Torpedocalifornica in a biphasic manner, where small concentrations increase [3H]acetylcholine binding and larger concentrations decrease binding (5). Using a stopped-flow fluorescence assay, Raines and Zachariah (6) demonstrated that isoflurane increases the apparent agonist affinity of Torpedo nAChRs. To determine more directly whether volatile anesthetics alter agonist affinity at nAChRs expressed in the central nervous system, we studied the effect of the volatile anesthetics isoflurane and sevoflurane on equilibrium binding of epibatidine (a high-affinity nicotinic agonist) to whole mouse brain (7). We studied epibatidine binding and the anesthetic effects thereof in male and female brain separately because sex differences in the effects of nicotinic agonists have been reported in vivo and in vitro(8).
The Committee on Animal Research at Columbia University approved our study of epibatidine binding to brain tissue from male and female 6- to 8-wk-old Swiss Webster mice purchased from Harlan Labs (Indianapolis, IN). The animals were housed five to a cage and had continuous access to food and water during 12-h cycles of light and dark, before death. The animals were killed by decapitation, and the whole brain was harvested for preparation of the membrane fraction.
Brains from male and female mice were prepared separately. Brains were collected immediately after death into 20 volumes of iced Tris buffer, pH 7.4, and homogenized to slurry in groups of 10–20 brains. The resulting tissue was centrifuged at 20,000 rpm at 4°C for 30 min. The supernatant was then removed, and the pellet was resuspended in the original volume of iced Tris buffer. This procedure was repeated twice more. The protein concentration was then measured with a Lowry assay, with bovine serum albumin as the standard, and the tissue was aliquoted to 2 mg/mL for the binding assay.
In saturation binding experiments, all samples were incubated with 2 mg of membrane protein and 0.05 nM [3H]epibatidine (48 Ci/mmol; New England Nuclear, Boston, MA). To determine the effect of isoflurane and sevoflurane on epibatidine binding, samples were also incubated with either isoflurane or sevoflurane at concentrations between 0 and 2 mM. Volatile anesthetic concentrations were measured with gas chromatography from sample tubes processed in parallel without radioactivity. Nonspecific binding was measured at each anesthetic concentration with the addition of 300 μM nicotine, because specific binding is saturated at this concentration (9,10). The brain tissue was incubated as above in glass tubes sealed with Teflon tops to prevent anesthetic escape for 3 h at room temperature while being shaken gently. After the 3-h incubation, the assay mixtures were filtered and rapidly washed three times (∼4 mL per wash) by using a binding manifold (Brandel Laboratories, Inc., Gaithersburg, MD). The filters with mouse brain membranes were added to vials containing 1 mL of 5% sodium dodecyl sulfate and allowed to sit overnight before they were counted via liquid scintillation spectroscopy in 15 mL of scintillant. This procedure was initiated because our preliminary experiments suggested that the presence of anesthetic in the incubation media increased the speed of release of radioactivity from the filter into solution. With this method, there was no significant change in counts per minute when samples were counted multiple times. Specific binding was determined by subtracting nonspecific binding (typically 5%–10%) from total binding. Total [3H]epibatidine binding represented <10% of the total radioligand added.
Each observation was performed at least in triplicate. Membranes from groups of 10 to 20 mouse brains were pooled. Experiments were performed on samples from at least two different membrane preparations. The 50% inhibitory concentration (IC50) values and Hill coefficients were obtained by averaging values generated from nonlinear regression analyses (Prizm; GraphPad, San Diego, CA) of individual concentration-response curves.
All samples were incubated with 2 mg of membrane preparation in the absence and presence of 2.39 mM isoflurane and [3H]epibatidine at concentrations between 0.005 and 20 nM. Nicotine 300 μM was added at each concentration of epibatidine to determine specific binding. In all cases, the anesthetic was added last to prevent it from evaporating. Similar to competition binding experiments, the samples were then shaken at room temperature for 3 h in sealed tubes. The samples were processed with a Brandel cell harvester and rapidly washed three times with iced binding buffer. The filters were soaked in scintillation fluid with sodium dodecyl sulfate, as above, overnight and were counted the following day.
For competition binding experiments, the specific binding in the presence of anesthetic at each concentration was normalized to the specific binding in the absence of anesthetic. The results from the competition binding experiments were fit to the equationMATH where EC50 is the 50% effective concentration. The results from the saturation binding experiments are expressed as femtomoles per milligram of protein. Kd and Bmax values were determined from saturation binding assays by nonlinear regression fit of the data to a model incorporating a single class of noninteracting binding sites (Prizm). The results of a typical saturation binding experiment with and without isoflurane are displayed as a Scatchard plot. The data are fit to a linear equation for enhanced visualization.
Total binding to 0.05 nM [3H]epibatidine did not differ between male and female brain (4.7 ± 0.3 fmol/mg [female] versus 4.4 ± 0.2 fmol/mg [male]; Student’s t test, P > 0.05). Isoflurane inhibits the binding of epibatidine to mouse brain membranes (Fig. 1). Isoflurane inhibited the binding of 0.05 nM epibatidine to membranes from male mouse brain with a half-maximal effect at 0.58 ± 0.07 mM. The IC50 for inhibition of epibatidine binding to membranes from female brain was 1.62 ± 0.3 mM isoflurane.
Sevoflurane also reduced [3H]epibatidine binding to membranes from both male and female mouse brains (Fig. 2). The IC50 value for inhibition of epibatidine binding to membranes from male brains is 0.77 ± 0.05 mM sevoflurane, and the IC50 for inhibition of binding to membranes from female brains is 0.77 ± 0.04 mM.
The data from a typical saturation binding experiment with and without isoflurane 2.39 mM are displayed as a Scatchard plot in Figure 3. Bmax and Kd were calculated with a nonlinear regression. Bmax was 23.6 ± 0.6 fmol/mg and was not significantly changed in the presence of isoflurane at 22.3 ± 0.5 fmol/mg. The Kd for epibatidine was increased in the presence of isoflurane from 0.37 ± 0.04 nM to 0.46 ± 0.90 nM.
Both isoflurane and sevoflurane decrease the affinity of epibatidine for its receptor in a clear, concentration-dependent manner. However, the reduction in agonist affinity is unlikely to be related to the inhibition of activation that occurs at far smaller concentrations of isoflurane and sevoflurane (1,2). The IC50 for inhibition of activation of the α4β2 nAChR expressed in Xenopus oocytes was 85 μM isoflurane. At this isoflurane concentration, which induces a half-maximal inhibition of effect, there is no significant change in agonist binding (Fig. 1, A and B). Thus we can conclude that the volatile anesthetics isoflurane and sevoflurane have at least two actions: one with a half maximal effect at small, subanesthetic concentrations, and a second with lower affinity that affects agonist binding. We were unable to detect an enhancement of agonist affinity by small anesthetic concentrations as seen in experiments on nicotinic receptors from Torpedo electroplax (5,6).
Although there is recent evidence that nicotinic receptor inhibition by volatile anesthetics does not cause immobility or hypnosis (11), there is evidence that nicotinic inhibition by volatile anesthetics has nociceptive consequences (12). Sex differences have been documented in nicotine’s analgesic effects and in isoflurane’s pronociceptive actions (8) (P. Flood, unpublished observations). Therefore, we studied epibatidine binding and the effects of isoflurane and sevoflurane on epibatidine binding to brain tissue derived from male and female animals. We determined that brains from male and female animals bind equivalently to a small concentration of epibatidine and that there is no statistical difference in the effect of isoflurane or sevoflurane on agonist binding to male and female brain tissue. However, because we did not study binding to a full range of agonist concentration in female tissue, total epibatidine sites may differ between the sexes. Sex differences relating to anesthetic action on nicotinic receptors may be due to differences in regulation of receptors by hormones or other substances not present in our preparation.
The volatile anesthetics isoflurane and sevoflurane inhibit agonist binding to nAChRs, but the relevant concentrations are larger by several orders of magnitude than those that result in diminished function. At anesthetic concentrations that cause half-maximal reduction in nAChR function, there is no significant difference in agonist binding. As such, the inhibition of nAChR activation by isoflurane and sevoflurane must occur downstream of agonist binding, likely affecting the translation of binding energy to channel gating.
1. Flood P, Ramirez-Latorre J, Role L. α4β2
neuronal nicotinic acetylcholine receptors in the central nervous system are inhibited by isoflurane and propofol, but α 7-type nicotinic acetylcholine receptors are unaffected. Anesthesiology 1997; 86: 859–65.
2. Violet JM, Downie DL, Nakisa RC, et al. Differential sensitivities of mammalian neuronal and muscle nicotinic acetylcholine receptors to general anesthetics. Anesthesiology 1997; 86: 866–74.
3. Cardoso RA, Yamakura T, Brozowski SJ, et al. Human neuronal nicotinic acetylcholine receptors expressed in Xenopus
oocytes predict efficacy of halogenated compounds that disobey the Meyer-Overton rule. Anesthesiology 1999; 91: 1370–7.
4. Mori T, Zhao X, Zuo Y, et al. Modulation of neuronal nicotinic acetylcholine receptors by halothane in rat cortical neurons. Mol Pharmacol 2001; 59: 732–43.
5. Firestone LL, Sauter JF, Braswell LM, et al. Actions of general anesthetics on acetylcholine receptor-rich membranes from Torpedocalifornica
. Anesthesiology 1986; 64: 694–702.
6. Raines DE, Zachariah VT. Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor. Anesthesiology 1999; 90: 135–46.
7. Perry DC, Kellar KJ. [3
H]Epibatidine labels nicotinic receptors in rat brain: an autoradiographic study. J Pharmacol Exp Ther 1995; 275: 1030–4.
8. Damaj MI. Influence of gender and sex hormones on nicotine acute pharmacological effects in mice. J Pharmacol Exp Ther 2001; 296: 132–40.
9. Court JA, Perry EK, Spurden D, et al. Comparison of the binding of nicotinic agonists to receptors from human and rat cerebral cortex and from chick brain (α4β2
) transfected into mouse fibroblasts with ion channel activity. Brain Res 1994; 667: 118–22.
10. Whiteaker P, Jimenez M, McIntosh JM, et al. Identification of a novel nicotinic binding site in mouse brain using 125
I-epibatidine. Br J Pharmacol 2000; 131: 729–39.
11. Eger EI II, Zhang Y, Laster M, et al. Acetylcholine receptors do not mediate the immobilization produced by inhaled anesthetics. Anesth Analg 2002; 94: 1500–4.
12. Flood P, Sonner JM, Gong D, et al. Isoflurane hyperalgesia is modulated by nicotinic inhibition. Anesthesiology 2002; 97: 192–8.