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ANESTHETIC PHARMACOLOGY

Characterization of the Interactions Between Volatile Anesthetics and Neuromuscular Blockers at the Muscle Nicotinic Acetylcholine Receptor

Matthias Paul, MD DEAA^*, Ralf M. Fokt^*, Christoph H. Kindler, MD DEAA, Natalie C. J. Dipp, and C. Spencer Yost, MD^*

*Department of Anesthesia and Perioperative Care, University of California, San Francisco; Department of Anesthesia, Kantonsspital, Basel, Switzerland; and University of Cologne, Germany

Address correspondence and reprint requests to C. Spencer Yost, MD, Department of Anesthesia and Perioperative Care, University of California, 513 Parnassus Ave., Box 0542, San Francisco, CA 94143-0542. Address e-mail to spyost{at}itsa.ucsf.edu

	Abstract

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Volatile anesthetics enhance the neuromuscular blockade produced by nondepolarizing muscle relaxants (NDMRs). The neuromuscular junction is a postulated site of this interaction. We tested the hypothesis that volatile anesthetic enhancement of muscle relaxation is the result of combined drug effects on the nicotinic acetylcholine receptor. The adult mouse muscle nicotinic acetylcholine receptor (₂, ß, , ) was heterologously expressed in Xenopus laevis oocytes. Concentration-effect curves for the inhibition of acetylcholine-induced currents were established for vecuronium, d-tubocurarine, isoflurane, and sevoflurane. Subsequently, inhibitory effects of NDMRs were studied in the presence of the volatile anesthetics at a concentration equivalent to half the concentration producing a 50% inhibition alone. All individually tested compounds produced rapid and readily reversible concentration-dependent inhibition. The calculated 50% inhibitory concentration values were 9.9 nM (95% confidence interval [CI], 8.4–11.4 nM), 43.4 nM (95% CI, 33.6–53.3 nM), 897 µM (95% CI, 699–1150 µM), and 818 µM (95% CI, 685–1001 µM) for vecuronium, d-tubocurarine, isoflurane, and sevoflurane, respectively. Coapplication of either isoflurane or sevoflurane significantly enhanced the inhibitory effects of vecuronium and d-tubocurarine, especially so at small concentrations of NDMRs. Volatile anesthetics increase the potency of NDMRs, possibly by enhancing antagonist affinity at the receptor site. This effect may contribute to the clinically observable enhancement of neuromuscular blockade by volatile anesthetics.

IMPLICATIONS: Isoflurane and sevoflurane enhance the receptor blocking effects of nondepolarizing muscle relaxants on nicotinic acetylcholine receptors.

	Introduction

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Muscle relaxation produced by neuromuscular blockade is an important component of surgical anesthesia. Nondepolarizing muscle relaxants (NDMRs) block neuromuscular transmission by binding competitively at the agonist site on the muscle nicotinic acetylcholine receptor (nAChR), thereby producing skeletal muscle relaxation. Volatile anesthetics also produce muscle relaxation in their own right and enhance the neuromuscular blockade of NDMRs (1,2). In contrast, IV anesthetics do not exert as comparable a muscle-relaxing effect as volatile anesthetics (3).

Isoflurane has direct effects on the postsynaptic nAChR (4), and this may mediate its muscle-relaxing properties. However, the interaction of volatile anesthetics and NDMRs has not been defined at the receptor level. Previous in vitro studies have used nerve-muscle preparations to study these interactions. For example, a concentration-dependent decrease in the d-tubocurarine requirement in the presence of various anesthetics was shown with this model, leading to the conclusion that this effect may be explained by the actions of the anesthetics on the "chemosensitivity of the end-plate region" (5). In these studies the neuromuscular junction was left intact, leaving a dissection of the underlying molecular mechanisms unaddressed.

Thus, there are no published studies on the interaction of volatile anesthetics and NDMRs at the receptor level. We have used a model of junctional nAChR heterologously expressed in Xenopus laevis oocytes to characterize the effects of two muscle relaxants, the aminosteroid vecuronium and the benzylisoquinolinium d-tubocurarine, in the presence of two volatile anesthetics (isoflurane or sevoflurane). The aim of our study was to determine whether these two classes of drugs interact to produce an enhancement of neuromuscular blockade at the receptor site.

	Methods

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All experimental procedures involving the X. laevis were approved by the Committee on Animal Research of the University of California, San Francisco. Briefly, oocytes were harvested and prepared as previously described (6). Within 16 h after harvesting, mature oocytes were chosen for cytoplasmic injection of diluted aliquots of complementary RNA encoding the , ß, , and subunits of the adult nAChR (₂ß) by using an automated microinjector (Nanoject; Drummond Scientific, Broomall, PA). Thereafter, the oocytes were maintained for 4 to 7 days at 18°C in modified Barth’s solution with HEPES [MBSH; composition in mM: 88 NaCl, 1 KCl, 10 HEPES, 7 NHCO₃, 1 CaCl₂, 1 Ca(NO₃)₂, pH adjusted to 7.4] to which 50 mg/mL of gentamycin, 2.5 mM sodium pyruvate, 5% heat-inactivated horse serum, and 5 mM theophylline were added. The expression plasmids pSP1, pGEMß, and pSP, encoding complementary DNA coding sequences for the mouse muscle nAChR subunits , ß, and , were kindly provided by Drs. John Forsayeth and Zach Hall (Department of Physiology, University of California, San Francisco), and expression plasmid pSP was provided by Dr. Paul Gardner (Department of Biochemistry, Dartmouth Medical School, Hanover, NH). These plasmids contain an SP6 promoter 5' to the translation start codon that allows in vitro synthesis of the RNA that directs the translation of each subunit.

Current recordings from oocytes were performed at room temperature (20°C–22°C). A single defolliculated oocyte was placed in a continuous-flow recording chamber (25-µL volume) and superfused with 3–5 mL/min of MBSH containing 0.5 µM atropine sulfate. The oocytes were impaled with two glass electrodes filled with 3 M KCl (resistances, 0.4–2.5 M) and voltage-clamped at a holding potential of -60 mV (Axoclamp 2A; Axon Instruments, Foster City, CA). Signals were filtered with an eight-pole low-pass Bessel filter (Frequency Devices, Haverhill, MA) set at a 40-Hz cutoff before sampling at 100 Hz. The resulting signals were digitized and stored on a Power Macintosh 7100 (Apple Computer, Cupertino, CA) by using data acquisition software (MacLab; ADInstruments, Milford, MA).

Acetylcholine (ACh) and atropine were purchased from Sigma (St. Louis, MO), vecuronium and isoflurane from Baxter Healthcare Corp. (Deerfield, IL), and d-tubocurarine and sevoflurane from Abbott Laboratories (Chicago, IL). All drugs were dissolved in MBSH. Solutions and their dilutions to the experimental concentrations were prepared immediately before use. Saturated stock solutions of isoflurane and sevoflurane were prepared by adding 50 mL of volatile anesthetic to an airtight glass bottle with 400 mL of MBSH. An equilibration period of 24 h at room temperature was allowed. Aliquots were taken from the bottle through a needle with its tip in a fixed position close to the phase of volatile anesthetic to ensure a constant concentration. Volatile anesthetics were applied by using an airtight perfusion system, including glass syringes and Teflon tubing. Their concentrations in the recording chamber were determined by gas chromatography (GowMac Series 750; GowMac, Bethlehem, PA) from samples taken at the outlet of the perfusion system.

An agonist concentration of 10 µM ACh was used for all experiments. This concentration was close to the 50% effective concentration previously determined for this receptor subtype (6); it ensured robust baseline responses and diminished receptor desensitization due to repetitive ACh application.

Test solutions containing either ACh alone or ACh in combination with various concentrations of a muscle relaxant were applied for 20 s; the peak current was taken as a measure of receptor activity. Volatile anesthetics were preapplied for 1 min before coapplication of ACh or ACh plus NDMR (7). Longer preexposure times were tested in preliminary experiments and showed no additional effect. Preapplication of anesthetics on their own usually caused no or only a minimal change of the resting current.

Control responses to ACh alone were determined before and after each application of antagonist, and the mean value of these two ACh applications was taken as the "average control current," to which the antagonist response was normalized by using the following equation:

equation

Washout times of 60 s after the NDMR application and 150 s after the volatile anesthetic exposure allowed a satisfactory receptor recovery in most cases (>90% of the previous control current). For the combination experiments, three concentrations of vecuronium and d-tubocurarine (IC₅, 0.5 x IC₅₀, and IC₅₀, i.e., concentration achieving a 50% inhibition of control currents) were tested in the presence of 0.5 x IC₅₀ of isoflurane or sevoflurane. Each data point represents measurements from five to eight oocytes, and for each experiment, oocytes from at least two different batches were used.

The concentration-response relations for each muscle relaxant and volatile anesthetic were fitted to the following equation by using GraphPad Prism software Version 3.0a for Macintosh (GraphPad Software, San Diego, CA):

equation

where Y is the fraction of remaining current, nH is the apparent cooperativity, and x is the antagonist concentration.

Unless otherwise specified, results are expressed as mean ± SEM or 95% confidence interval (CI). Statistical significance was assessed with unpaired two-tailed Student’s t-tests or one-way analysis of variance followed by Tukey’s test. P < 0.05 was considered significant.

	Results

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Injection of Xenopus oocytes with complementary RNA coding for subunits of the junctional nAChR resulted in the expression of robust ACh-activated membrane currents as described previously for this model (6). Vecuronium and d-tubocurarine reversibly inhibited whole cell currents induced by the application of ACh (10 µM) in a concentration-dependent manner. As we reported previously, the aminosteroid vecuronium (IC₅₀ concentration, 9.9 nM; 95% CI, 8.4–11.7 nM) was more potent than the benzylisoquinolinium d-tubocurarine (IC₅₀ concentration, 43.4 nM; 95% CI, 33.6–56.2 nM) (8).

The volatile anesthetics isoflurane and sevoflurane also inhibited ACh-induced currents in a concentration-dependent fashion (Fig. 1). Fitting the concentration responses of the volatile anesthetics to the Hill equation yielded IC₅₀ values of 897 µM (95% CI, 699–1150 µM) and 818 µM (95% CI, 685–1001 µM) and Hill coefficients of 1.55 ± 0.24 and 1.57 ± 0.33 for isoflurane and sevoflurane, respectively. The two anesthetics were equipotent regarding their ability to block the nAChR currents (P > 0.05). Their IC₅₀ concentrations were approximately five orders of magnitude larger than those determined for the NDMRs.

View larger version (14K): [in this window] [in a new window]   Figure 1. Concentration-response curves of isoflurane and sevoflurane for inhibition of acetylcholine-induced (10 µM) currents in Xenopus oocytes expressing mouse adult-type muscle nicotinic acetylcholine receptors. Data points are mean peak currents ± SD (error bars) of five to eight oocytes. Error bars not visible are smaller than symbols. The lines are unweighted least-squares fits of the mean peak currents to a Hill equation (Equation 2).

View larger version (32K): [in this window] [in a new window]   Figure 2. Graphic representation of the enhancement of percentage inhibition of acetylcholine-induced (10 µM) currents with vecuronium (A) and d-tubocurarine (B) by isoflurane and sevoflurane. Three concentrations of vecuronium (0.1, 5, and 10 nM; Panel A) or d-tubocurarine (1, 21.5, and 43 nM; Panel B) were administered alone (white bars) and in the presence of 0.5 x 50% inhibitory concentration (IC50) of either isoflurane (450 µM; black bars) or 0.5 x IC50 of sevoflurane (410 µM; hatched bars). The presence of isoflurane or sevoflurane enhanced the current-blocking effect of vecuronium and d-tubocurarine significantly (*P < 0.001). For the smallest concentrations of vecuronium (0.1 nM) and d-tubocurarine (1 nM), the enhancing effect of both volatile anesthetics was more than additive. Enhancement by sevoflurane compared with isoflurane was different only for the largest concentrations of nondepolarizing muscle relaxants (vecuronium 10 nM and d-tubocurarine 43 nM, respectively) (#P < 0.05).

View larger version (19K): [in this window] [in a new window]   Figure 3. Inhibition of acetylcholine (Ach)-induced currents of the adult-type muscle nicotinic acetylcholine receptors expressed in Xenopus oocytes by vecuronium, alone and in combination with isoflurane. Tracings represent raw currents observed during the application of ACh (10 µM) for 20 s, either alone (as control) or in combination with one of three concentrations of vecuronium (0.1, 5, and 10 nM) with or without isoflurane (450 µM, preapplied for 1 min) as indicated.

	Discussion

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In this study we have determined the effects of two volatile anesthetics, alone and in combination with NDMRs, on the function of the adult muscle-type nAChR. The IC₅₀ concentrations of isoflurane and sevoflurane producing block of the adult nAChR are similar to those reported for the fetal nAChR studied previously (7). Compared with vecuronium or d-tubo-curarine, isoflurane and sevoflurane were much less potent, with IC₅₀ concentrations that were several orders of magnitude larger. However, isoflurane and sevoflurane strongly enhanced the inhibitory effects of vecuronium and d-tubocurarine at concentrations that occur within the clinical range. Both volatile anesthetics enhanced the blocking effects of vecuronium and d-tubocurarine to a similar degree. Furthermore, at the smallest concentration of NDMRs tested, isoflurane and sevoflurane enhanced the AChR inhibition in a synergistic manner. These results identify the receptor site as a target for the observed combined effect of NDMRs and volatile anesthetics and are of clinical importance, because both classes of drugs are frequently administered together during general anesthesia.

It is well established that the nAChR is a ligand-gated ion channel that consists of four different subunits assembled in a pentameric structure to create a central ion-conducting pore (9). The adult form (-nAChR) is composed of ₂ß subunits. Two binding sites for agonists or specific antagonists, such as NDMRs, have been identified and are located at the interface of the - and - subunits in the extracellular domain of the receptor (10). Four transmembrane domains anchor each receptor subunit in the postsynaptic membrane, with one of these (termed M2) providing the lining for the aqueous ion-conducting pathway. Mutational analyses of the M2 region have identified an isoflurane binding site within the pore of the channel (11). Patch-clamp studies, focusing on single ion channel effects, have shown that isoflurane inhibited ion flow through the nAChR pore, resulting in open channel blockade (4).

Our findings of a concentration-dependent synergistic interaction between NDMRs and volatile anesthetics are in accordance with the idea that these drugs interact with different regions of the nAChR. They also suggest that a change in the ACh binding site, leading to an increase in the apparent agonist affinity (12), may also extend to drugs that act as competitive antagonists at the ACh site. In our study we have shown that the functional effect of antagonists on ion channel function itself is potentiated in the presence of isoflurane or sevoflurane. We predict that binding studies performed with NDMRs would also show an increase in their apparent affinities in the presence of volatile anesthetics.

To compare the NDMR-enhancing effects of isoflurane with those of sevoflurane, we applied equipotent concentrations of the volatile anesthetics with respect to their nAChR-blocking ("peripheral") ability, as determined in our experiments, rather than at equipotent minimum alveolar anesthetic concentrations (MACs). The applied concentrations of isoflurane (450 µM) and sevoflurane (410 µM) during these experiments were within clinically used limits and correspond to 1.7 MAC of isoflurane and 1.3 MAC of sevoflurane in aqueous solution (13). Under these conditions, both volatile anesthetics enhanced the NDMR effects to a similar degree. These findings are in accordance with studies in patients, in which concentrations of 1 MAC isoflurane and sevoflurane, achieving similar aqueous concentrations of 270 and 300 µM, were compared and found to have a similar potency in augmenting a vecuronium-induced neuromuscular blockade (14). In vitro studies using a nerve-muscle model have also reported that volatile anesthetics with different MAC values were equipotent in their abilities to depress end-plate depolarization at similar aqueous concentrations (15).

Therefore, our results suggest that the degree of enhancement of neuromuscular blockade by volatile anesthetics depends on their actual aqueous concentration and that their muscle-relaxing properties do not parallel their anesthetic potencies expressed as MAC. We suggest that the clinically used vapors are approximately equipotent in inhibiting neuromuscular transmission via the nAChR and that the greater clinical muscle-relaxing effect produced by less potent anesthetics (i.e., desflurane versus isoflurane) is mainly caused by their larger aqueous concentrations, if both anesthetics are administered at equipotent MAC concentrations. This conclusion is supported by a volunteer study which demonstrated that the infusion requirements to maintain 85% twitch depression during anesthesia with 1.25 MAC of volatile anesthetic were 20% less for desflurane compared with isoflurane (16). A decrease of the vapor concentration from 0.75 to 0.25 MAC increased twitch tension by 46% for desflurane but only 25% for isoflurane. Because desflurane is less potent than isoflurane (MAC values 6% vs 1%), a reduction from 0.75 to 0.25 MAC represents a much larger reduction in the actual aqueous concentration for desflurane.

Although the oocyte model allowed us to study drug effects on the pure receptor, some limitations of our model need to be considered. Throughout the study we used an agonist concentration of 10 µM ACh; this concentration is smaller than the estimated transient peak concentrations within the neuromuscular junction, but it is close to the 50% effective concentration described for this receptor subtype (6). A holding voltage of -60 mV was applied, because the antagonistic effects of NDMRs are independent of holding voltages ranging from -100 to -40 mV (17). Our experiments were performed at room temperature, whereas the expressed receptor is derived from a homeothermic animal (mouse). However, we have previously reported that the open probability and single-channel conductance of adult mouse nAChR expressed in Xenopus oocytes are the same as reported for native receptors at physiologic temperature (18). Finally, the close sequence homology and functional similarity with human nAChRs justified the use of mouse receptors (19).

In conclusion, we have quantitated the potency of two widely used volatile anesthetics for inhibiting the adult form of the muscle-type nAChR. Our findings show that inhibition of the nAChR by NDMRs can be greatly enhanced by the presence of clinically relevant concentrations of isoflurane or sevoflurane. These findings suggest that the enhancement of NDMR-induced neuromuscular blockade by volatile anesthetics can be explained in part by a combined effect of these drugs on the nAChR.

	Acknowledgments

 
Supported by National Institutes of Health Grant GM-58149 (CSY).

The authors thank Beth Sampson and Frank Shen for excellent technical assistance, Diane Gong for her help with gas chromatography, and Dr. Charles E. McCulloch (Department of Epidemiology and Biostatistics, University of California, San Francisco) for statistical advice.

	Footnotes

 
Presented in part at the American Society of Anesthesiologists, New Orleans, LA, October 13–17, 2001.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Waud BE, Waud DR. Comparison of the effects of general anesthetics on the end-plate of skeletal muscle. Anesthesiology 1975; 43: 540–7.[Web of Science][Medline]
2. Miller RD, Way WL, Dolan WM, et al. Comparative neuromuscular effects of pancuronium, gallamine, and succinylcholine during forane and halothane anesthesia in man. Anesthesiology 1971; 35: 509–14.[Web of Science][Medline]
3. Suzuki T, Munakata K, Watanabe N, et al. Augmentation of vecuronium-induced neuromuscular block during sevoflurane anaesthesia: comparison with balanced anaesthesia using propofol or midazolam. Br J Anaesth 1999; 83: 485–7.[Abstract/Free Full Text]
4. Dilger JP, Vidal AM, Mody HI, Liu Y. Evidence for direct actions of general anesthetics on an ion channel protein: a new look at a unified mechanism of action. Anesthesiology 1994; 81: 431–42.[Web of Science][Medline]
5. Waud BE. Decrease in dose requirement of d-tubocurarine by volatile anesthetics. Anesthesiology 1979; 51: 298–302.[Web of Science][Medline]
6. Kindler CH, Verotta D, Gray AT, et al. Additive inhibition of nicotinic acetylcholine receptors by corticosteroids and the neuromuscular blocking drug vecuronium. Anesthesiology 2000; 92: 821–32.[Web of Science][Medline]
7. 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.[Web of Science][Medline]
8. Paul M, Kindler CH, Fokt RM, et al. Potency of new muscle relaxants on recombinant muscle-type acetylcholine receptors. Anesth Analg 2001; 94: 597–603.[Abstract/Free Full Text]
9. Changeux JP, Benoit P, Bessis A, et al. The acetylcholine receptor: functional architecture and regulation. Adv Second Messenger Phosphoprotein Res 1990; 24: 15–9.[Web of Science][Medline]
10. Sine SM, Claudio T. Gamma- and delta-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor. J Biol Chem 1991; 266: 19369–77.[Abstract/Free Full Text]
11. Forman SA, Miller KW, Yellen G. A discrete site for general anesthetics on a postsynaptic receptor. Mol Pharmacol 1995; 48: 574–81.[Abstract]
12. Raines DE, Zachariah VT. Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor. Anesthesiology 1999; 90: 135–46.[Web of Science][Medline]
13. Franks NP, Lieb WR. Temperature dependence of the potency of volatile general anesthetics: implications for in vitro experiments. Anesthesiology 1996; 84: 716–20.[Web of Science][Medline]
14. Kurahashi K, Maruta H. The effect of sevoflurane and isoflurane on the neuromuscular block produced by vecuronium continuous infusion. Anesth Analg 1996; 82: 942–7.[Abstract]
15. Waud BE, Waud DR. Effects of volatile anesthetics on directly and indirectly stimulated skeletal muscle. Anesthesiology 1979; 50: 103–10.[Web of Science][Medline]
16. Wright PM, Hart P, Lau M, et al. The magnitude and time course of vecuronium potentiation by desflurane versus isoflurane. Anesthesiology 1995; 82: 404–11.[Web of Science][Medline]
17. Garland CM, Foreman RC, Chad JE, et al. The actions of muscle relaxants at nicotinic acetylcholine receptor isoforms. Eur J Pharmacol 1998; 357: 83–92.[Web of Science][Medline]
18. Yost CS, Winegar BD. Potency of agonists and competitive antagonists on adult- and fetal-type nicotinic acetylcholine receptors. Cell Mol Neurobiol 1997; 17: 35–50.[Web of Science][Medline]
19. Boulter J, Luyten W, Evans K, et al. Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor. J Neurosci 1985; 5: 2545–52.[Abstract]

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