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The effect of local anesthetics on the inhibition of adult muscle-type nicotinic acetylcholine receptors by nondepolarizing muscle relaxants
European Journal of Pharmacology 630 (2010) 29–33
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
The effect of local anesthetics on the inhibition of adult muscle-type nicotinic acetylcholine receptors by nondepolarizing muscle relaxants Hong Wang, Ying Zhang, Shi-tong Li ⁎ Department of Anesthesiology, First People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Article history: Received 28 July 2009 Received in revised form 3 December 2009 Accepted 15 December 2009 Available online 4 January 2010 Keywords: Local anesthetic Nondepolarizing muscle relaxant Adult muscle-type nicotinic acetylcholine receptor Acetylcholine current Combined effect
a b s t r a c t The primary action of local anesthetics is to inhibit voltage-gated Na+ channels. However, local anesthetics also have an inhibitory effect on muscle-type nicotinic acetylcholine receptors. Because local anesthetics could increase the neuromuscular blockade produced by nondepolarizing muscle relaxants, we investigated the interaction of local anesthetics with nondepolarizing muscle relaxants at adult muscle-type nicotinic acetylcholine receptors. This study tested the effects of lidocaine and procaine, alone and in combination with vecuronium and cisatracurium, on adult muscle-type nicotinic acetylcholine receptors. The adult mouse muscle-type nicotinic acetylcholine receptor was expressed in HEK293 cells and activated with 10 µM acetylcholine. Currents were recorded using the whole-cell voltage-clamp technique. Adult muscle-type nicotinic acetylcholine receptors were potently inhibited by all the tested compounds. Although the potencies of procaine and lidocaine were statistically significantly different at adult muscle-type nicotinic acetylcholine receptors (50% inhibitory concentration values of 45.5 µM and 11.1 µM, respectively), procaine and lidocaine enhanced the inhibitory effect of nondepolarizing muscle relaxants at adult muscle-type nicotinic acetylcholine receptors to the same extent. The increased adult muscle-type nicotinic acetylcholine receptor inhibition produced when local anesthetics are combined with nondepolarizing muscle relaxants may contribute to the clinical enhancement of neuromuscular blockade by local anesthetics. © 2009 Elsevier B.V. All rights reserved.
1. Introduction It is well-known that local anesthetics also produce neuromuscular block and increase the neuromuscular responses to nondepolarizing muscle relaxants (Katz and Gissen, 1969; Usubiaga et al., 1967; Ellis et al., 1953). The voltage-gated sodium channel is considered to be the primary target of this effect of local anesthetics. However, previous studies have also shown that local anesthetics can noncompetitively inhibit muscle-type nicotinic acetylcholine receptor function at therapeutic doses (Neher and Steinbach, 1978; Gentry and Lukas, 2001), which may partly contribute to muscle-relaxing action of local anesthetics. Previous studies demonstrate preliminary administration of ineffective concentrations of local anesthetics significantly decreased the 50% inhibitory concentration values (IC50) of nondepolarizing muscle relaxants in nerve–muscle preparations (Matsuo et al., 1978). These findings suggest that local anesthetics powerfully enhance the neuromuscular blockade produced by nondepolarizing muscle relaxants at the neuromuscular junction. However, in these studies the
⁎ Corresponding author. Department of Anesthesiology, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200030, China. Tel.:+86 21 63240090 3022; fax:+86 21 63243749. E-mail address: [email protected] (ST. Li). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.12.028
interaction of local anesthetics with nondepolarizing muscle relaxants at muscle-type nicotinic acetylcholine receptors was not addressed. Consequently, to evaluate the interaction of local anesthetics and nondepolarizing muscle relaxants at the nicotinic acetylcholine receptor, we heterologously expressed adult muscle-type nicotinic acetylcholine receptors in HEK293 cells and observed the effects of two muscle relaxants (the aminosteroid vecuronium and the benzylisoquinolinium cisatracurium) in the presence of two local anesthetics (lidocaine or procaine). The aim was to determine directly whether local anesthetics could enhance the inhibitory effect of nondepolarizing muscle relaxants on adult muscle-type nicotinic acetylcholine receptors. 2. Materials and methods 2.1. Cell culture and transfection Expression plasmids Psp65α, Psp65β, Psp65δ, and Pbssk(+)ε, encoding complementary DNA coding sequences for the mouse muscle nicotinic acetylcholine receptor subunits α, β, δ, and ε, respectively, were provided by the Salk Institute in America. These plasmids were subcloned in pcDNA3.1+ (InvitrogenT Life Technologies, Carlsbad, CA, USA). Human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (InvitrogenTM, Grand Island, NY, USA) supplemented with 10% calf serum
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H. Wang et al. / European Journal of Pharmacology 630 (2010) 29–33
(InvitrogenTM, Grand Island, NY, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2/95% O2 incubator. HEK293 cells were transfected stably with LipofectamineTM 2000 according to the manufacturer's protocol (InvitrogenTM Life Technologies, Carlsbad, CA, USA). After transfection, the positive cell clones were selected with G418. The transfected cells were then incubated for 24 h before the recordings were made.
2.2. Electrophysiology HEK293 cells were voltage-clamped using the whole-cell patchclamp technique (Hamill et al., 1981). Pipettes were pulled from borosilicate glass with a P-97 electrode puller (Sutter Instrument Co, Novato, CA, USA) and had a resistance of 2–3 MΩ. The pipette electrode was filled with the following solution (mM): CsCl 140, MgCl2 2, Hepes–CsOH 10, EGTA 0.5, and Na–ATP 4, pH 7.3. The external solution contained (mM) NaCl 140, KCl 2.5, CaCl2 2, MgCl2 2, Hepes– NaOH 10, and glucose 10, pH 7.3. Cells were voltage-clamped at −50 mV in the whole-cell configuration. All experiments were performed at room temperature (20–24 °C). Currents were measured with an EPC10 (HEKA Elektronik, Germany) amplifier and Patch-
Master software (HEKA Elektronik, Germany), sampled at 20 kHz, and stored on a computer. Acetylcholine, lidocaine hydrochloride, and procaine hydrochloride were purchased from Sigma (Sigma Chemical Co., Saint Louis, USA). Muscle relaxants were obtained in preparations for clinical use, namely vecuronium (NV. Organon, The Netherlands) and cisatracurium (Glaxosmithkline SpA, Italy). All drugs were dissolved in an external solution and applied by a gravity-driven perfusion system. Solutions and their dilutions to the experimental concentrations were prepared immediately before the experiments. The test solutions, containing either acetylcholine alone or in combination with various concentrations of nondepolarizing muscle relaxants or/and local anesthetics, were applied for 2 s to the HEK293 cells, and the peak current was determined. To determine the effect of the antagonist on the acetylcholineelicited current, the solution containing the nondepolarizing muscle relaxants or/and local anesthetics was perfused on HEK293 cells for 1 min prior to the application of acetylcholine in the presence of the antagonist. The washout time between each drug application was at least 60 s to minimize the amount of desensitization throughout the course of an experiment. Currents were acquired from five HEK293 cells. The control current in response to acetylcholine alone was repeated after washout of the antagonist. Taking the mean value of these two
Fig. 1. Concentration-dependent effects of vecuronium (VEC), cisatracurium (CISATR), procaine (PRO), and lidocaine (LIDO) on adult muscle-type nicotinic acetylcholine receptors expressed in HEK293 cells. Tracings represent raw currents observed during the application of acetylcholine (ACh) 10 µM for 2 s, either alone or in combination with various concentrations of vecuronium, cisatracurium, procaine, or lidocaine as indicated.
H. Wang et al. / European Journal of Pharmacology 630 (2010) 29–33
acetylcholine applications as the average control current, the antagonist response was calculated (percentage inhibition of average control current) using the following equation:
%inhibition = 100 ×
current in presence of antagonist 1− average control current
2.3. Statistical analysis Data analysis was performed off-line using Origin 8 (OriginLab, Northampton, MA, USA) and GraphPad Prism 4 (Graphpad software, Inc., San Diego, CA, USA). Concentration–response curves were fitted to the four-parameter logistic equation by nonlinear regression analysis, and IC50 values were determined. Results are expressed as means ± S.D. or as the 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 b 0.05 was considered significant. 3. Results Acetylcholine 10 µM was used to activate the adult muscle-type nicotinic acetylcholine receptors expressed in HEK293 cells because this concentration caused efficient acetylcholine currents without significant desensitization after repeated exposures. Vecuronium and cisatracurium reversibly inhibited acetylcholine-induced inward currents in a concentration-dependent fashion (Figs. 1 and 2). The adult muscle-type nicotinic acetylcholine receptor was equally sensitive to inhibition by the aminosteroid vecuronium (IC50 concentration, 11.5 nM; 95% CI, 8.7–15.0 nM) and the benzylisoquinolinium cisatracurium (IC50 concentration, 9.3 nM, 95% CI, 7.3–11.8 nM) (P N 0.05, unpaired two-tailed Student's t-test). The local anesthetics procaine and lidocaine also produced reversible, concentration-dependent inhibition of the currents induced by the application of acetylcholine (10 µM) (Figs. 1 and 2). Lidocaine (IC50 concentration, 11.1 µM; 95% CI, 5.0–24.8 µM) was significantly more potent than procaine (IC50 concentration, 45.5 µM; 95% CI, 36.0– 57.5 µM) in producing inhibition of acetylcholine-induced currents (P b 0.05, unpaired two-tailed Student's t-test).
Fig. 2. Concentration–response effects of vecuronium (A), cisatracurium (B), procaine (C), and lidocaine (D): inhibition of acetylcholine-induced currents in HEK293 cells expressing adult muscle-type nicotinic acetylcholine receptor. Data points show means ± S.D. (error bars) of five HEK293 cells. Error bars not visible are smaller than the symbols.
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To determined the combined effects of local anesthetics and nondepolarizing muscle relaxants, test compounds were coapplied to adult muscle-type nicotinic acetylcholine receptors expressed in HEK293 cells. An enhancement of inhibition of acetylcholine-elicited currents with vecuronium or cisatracurium by 0.5 × IC50 of either lidocaine (∼5.5 µM) or procaine (∼23 µM) was observed. Both the local anesthetics increased the inhibitory effects of all three concentrations of vecuronium and cisatracurium on nicotinic acetylcholine receptors. For both local anesthetics, the enhancement was synergistic with the smallest dose of vecuronium (0.1 nM) or cisatracurium (0.1 nM), and was less intense as vecuronium or cisatracurium was used in larger concentrations. Compared with procaine, there was an equipotent enhancement by lidocaine for the three concentrations of vecuronium or cisatracurium (Fig. 3). Representative tracings for vecuronium and lidocaine are shown in Fig. 4. 4. Discussion A previous study demonstrated that combinations of ineffective concentrations of nondepolarizing muscle relaxants with ineffective concentrations of local anesthetics caused a greater than 90% neuromuscular blockade in a rat phrenic nerve–hemidiaphragm preparation (Matsuo et al., 1978). This interaction of local anesthetics was attributed to the blockade of the Na+ channels (e.g., with procaine) (Cassuto et al., 2006) or both the Na+ and the K+ channels (e.g., with lidocaine) (Maeno et al., 1971). However, this explanation ignored the contribution of the action of the local anesthetics at the nicotinic acetylcholine receptor. Our results demonstrated that lidocaine and procaine can
Fig. 3. Graphic representation of the enhancement of the percentage inhibition of acetylcholine-induced (10 µM) currents with vecuronium (A) and cisatracurium (B) by procaine and lidocaine. Data were presented as means ± S.D. Three concentrations of vecuronium (0.1, 5.5, and 11 nM; Panel A) or cisatracurium (0.1, 4.5, and 9 nM; Panel B) were applied alone (white bars) and in combination with 0.5 × 50% inhibitory concentration (IC50) of either procaine (23 µM; greyish color bars) or 0.5 × IC50 of lidocaine (5.5 µM; hatched bars). Procaine or lidocaine significantly increased the inhibition of acetylcholine-induced current with vecuronium and cisatracurium (*P b 0.01 using one-way analysis of variance followed by Tukey's test).
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Fig. 4. Inhibition of acetylcholine (ACh)-induced currents of adult muscle-type nicotinic acetylcholine receptors expressed in HEK293 cells by vecuronium, alone and in combination with lidocaine. Tracings represent raw currents observed during the application of acetylcholine (10 µM) for 2 s, either alone or in combination with one of three concentrations of vecuronium (0.1, 5.5, 11 nM) with or without lidocaine (5.5 µM) as indicated.
significantly enhance inhibition of vecuronium and cisatracurium at adult muscle-type nicotinic acetylcholine receptors. Furthermore, lidocaine and procaine enhanced inhibition of acetylcholine-induced currents when the smallest concentration of vecuronium or cisatracurium was used, in a synergistic fashion. In this study, lidocaine and procaine produced concentrationdependent inhibition of nicotinic acetylcholine receptor-mediated inward currents. The IC50 concentration of procaine producing adult muscle-type nicotinic acetylcholine receptor blockade is similar to that reported previously (Yost and Dodson, 1993). There is substantial evidence that local anesthetics bind to and inhibit nicotinic acetylcholine receptors via noncompetitive mechanisms in which they bind to a regulatory site on the nicotinic acetylcholine receptor, inhibit channel opening, and decrease the concentration of nicotinic acetylcholine receptors in the open state by slowly switching them to their inactive form. Alternatively, local anesthetics may also bind to open channels with subsequent inhibition of ion flux (Arias, 1999; Gentry and Lukas, 2001). Consistent with previous studies (Demazumder and Dilger, 2001; Paul et al., 2002a,b), our results also showed that vecuronium and cisatracurium potently inhibited adult muscle-type nicotinic acetylcholine receptors. The muscle-type nicotinic acetylcholine receptor is a ligand-gated ion channel that consists of four different subunits assembled in a pentameric structure to create a central ion-conducting pore (Changeux et al., 1990; Paul et al., 2002a,b). The acetylcholine binding
sites are located at the interface of the α–γ and α–δ subunits in the extracellular domain of the receptor (Sine and Claudio, 1991). Nondepolarizing muscle relaxants act in a competitive manner by binding to the acetylcholine binding sites on nicotinic acetylcholine receptors (Garland et al., 1988). We found that concentrations of 0.5 × IC50 lidocaine and procaine caused effective enhancement of the nondepolarizing muscle relaxant-induced inhibition, which accords with the idea that these two classes of drugs interact with different regions of the nicotinic acetylcholine receptor and local anesthetics bind to a regulatory site on the nicotinic acetylcholine receptor to cause allosterism of receptor (Karpen and Hess, 1986; Niu et al., 1995; Niu and Hess, 1993), which may lead to an increase in the affinity of nondepolarizing muscle relaxants for nicotinic acetylcholine receptors. In this study, mouse nicotinic acetylcholine receptor expressed in HEK293 cells was more sensitive to blockade by lidocaine and procaine than human nicotinic acetylcholine receptor expressed in the TE671/RD cell line (Gentry and Lukas, 2001). Differences in species, expression systems (cell environments), experimental approach (electrophysiological or ion flux assays), or perhaps degrees of nicotinic acetylcholine receptor desensitization (Ryan and Baenziger, 1999) could account for the different IC50 values observed in different studies (Yost and Dodson, 1993). In clinical practice, between 40 and 80 mM of lidocaine is used; therefore, a level below 1 mM is considered to be a clinically acceptable concentration (Onizuka et al., 2008). However, in the central nervous system, 0.1 mM lidocaine is a toxic dose that may induce convulsions in the rat (Kim et al., 1996; Takahashi et al., 2006; Yokoyama et al., 1995). In humans, the dose of local anesthetics inducing convulsion is approximate 18–26 μg/mL (about 0.1 mM) (Onizuka et al., 2008). Thus, from this study we conclude that lidocaine and procaine at concentrations within the clinical range can increase the inhibiting effects of vecuronium and cisatracurium on nicotinic acetylcholine receptors. During the course of study, we used 10 µM acetylcholine as an agonist concentration. This concentration can ensure sufficient acetylcholine current responses and minimize nicotinic acetylcholine receptor desensitization owing to repetitive application of acetylcholine (Paul et al., 2002a,b). Because the antagonistic effects of nondepolarizing muscle relaxants are independent of holding voltages ranging from −100 to − 40 mV (Garland et al, 1988; Paul et al., 2002a,b), HEK293 cells were clamped at −50 mV to minimize the potentiation of local anesthetics inhibition of transient acetylcholine-evoked currents by more negative potential (Arias, 1999; Yost and Dodson, 1993). In conclusion, our results demonstrate that the presence of clinically relevant concentrations of lidocaine and procaine can significantly enhanced the inhibitory effect of nondepolarizing muscle relaxants on nicotinic acetylcholine receptors. These findings indicate that a combined effect of the two classes of drugs on adult muscle-type nicotinic acetylcholine receptors can partly explain how local anesthetics enhance nondepolarizing muscle relaxant-induced neuromuscular blockade. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30571796). We thank Dr Liu Xin-qiu (Department of Neurobiology, School of Medicine, Shanghai Jiaotong University) for technical assistance and strong support. References Arias, H.R., 1999. Role of local anesthetics on both cholinergic and serotonergic ionotropic receptors. Neurosci. Biobehav. Rev. 23, 817–843. Cassuto, J., Sinclair, R., Bonderovic, M., 2006. Anti-inflammatory properties of local anesthetics and their present and potential clinical implications. Acta. Anaethesiol. Scand. 50, 265–282.
H. Wang et al. / European Journal of Pharmacology 630 (2010) 29–33 Changeux, J.P., Benoit, P., Bessis, A., Cartaud, J., Devillers-Thiery, A., Fontaine, B., Galzi, J.L., Klarsfeld, A., Laufer, R., Mulle, C., 1990. The acetylcholine receptor: functional architecture and regulation. Adv. Second Messenger Phosphoprotein Res. 24, 15–19. Demazumder, D., Dilger, J.P., 2001. The kinetics of competitive antagonism by cisatracurium of embryonic and adult nicotinic acetylcholine receptors. Mol. Pharmacol. 60, 797–807. Ellis, C.H., Wnuck, A.L., Debeer, E.J., Folds, F.F., 1953. Modifying actions of procaine on the myoneural blocking actions of succinylcholine, decamethonium and dtubocurarine in dogs and cats. Am. J. Physiol. 174, 277–282. Garland, C.M., Foreman, R.C., Chad, J.E., Holden-Dye, L., Walker, R.J., 1988. The actions of muscle relaxants at nicotinic acetylcholine receptor isoforms. Eur. J. Pharmacol. 357, 83–92. Gentry, C.L., Lukas, R.J., 2001. Local anesthetics noncompetitively inhibit function of four distinct nicotinic acetylcholine receptor subtypes. J. Pharmacol. Exp. Ther. 299, 1038–1048. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Siqworth, F.j., 1981. Improved patchclamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391, 85–100. Karpen, J.W., Hess, G.P., 1986. Cocaine, phencyclidine, and procaine inhibition of the acetylcholine receptor: characterization of the binding site by stopped-flow measurements of receptor-controlled ion flux in membrane vesicles. Biochemistry 25, 17777–17785. Katz, R.L., Gissen, A.J., 1969. Effects of intravenous and intra-arterial procaine and lidocaine on neuromuscular transmission in man. Acta. Anaesth. Scand. 36, 103–113 Suppl. Kim, Y.J., Mcfarlane, C., Warner, D.S., Baker, M.T., Choi, W.W., Dexter, F., 1996a. The effect of plasma and brain magnesium concentrations on lidocaine-induced seizures in the rat. Anesth. Analg. 83, 1223–1228. Maeno, T., Edwards, C., Hashimura, S., 1971. Difference in effects of end-plate potentials between procaine and lidocaine as revealed by voltage-clamp experiments. J. Neurophysiol. 34, 32–46. Matsuo, S., Rao, D.B., Chaudry, I., Foldes, F.F., 1978. Interaction of muscle relaxants and local anesthetics at the neuromuscular junction. Anesth. Analg. 57, 580–587. Neher, E., Steinbach, J.H., 1978. Local anesthetics transiently block currents through single acetylcholine-receptor channels. J. Physiol. 277, 153–176.
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Niu, L., Hess, G.P., 1993. An acetylcholine receptor regulatory site in BC3H-1 cell: characterized by laser-pulse photolysis in the microsecond-to-millisecond time region. Biochemistry 32, 3831–3835. Niu, L., Abood, L.G., Hess, G.P., 1995. Cocaine: mechanism of inhibition of a muscle acetylcholine receptor studied by laser-pulse photolysis technique. Proc. Natl Acad. Sci. USA 92, 12008–12012. Onizuka, S., Kasaba, T., Takasaki, M., 2008. The effect of lidocaine on cholinergic neurotransmission in an identified reconstructed synapse. Anesth. Analg. 107, 1236–1242. Paul, M., Fokt, R.M., Kindler, C.H., Dipp, N.C., Yost, C.S., 2002a. Characterization of the interactions between volatile anesthetics and neuromuscular blockers at the muscle nicotinic acetylcholine receptor. Anesth. Analg. 95, 362–367. Paul, M., Kindler, C.H., Fokt, R.M., Dresser, M.J., Dipp, N.C., Yost, C.S., 2002b. The potency of new muscle relaxants on recombinant muscle-type acetylcholine receptors. Anesth. Analg. 94, 597–603. Ryan, S., Baenziger, J., 1999. A structure-based approach to nicotinic receptor pharmacology. Mol. Pharmacol. 55, 348–355. Sine, S.M., Claudio, T., 1991. Gamma- and delta-subunits regulate the affinity and the cooperativity of ligand binding to the acetylcholine receptor. J. Biol. Chem. 266, 19369–19377. Takahashi, R., Oda, Y., Tanaka, K., Morishima, H.O., Inoue, K., Asada, A., 2006. Epinephrine increases the extracellular lidocaine concentration in the brain: a possible mechanism for increased central nervous system toxicity. Anesthesiology 105, 984–989. Usubiaga, J.E., Wikinski, J.A., Morales, R.L., Usubiaga, L.E., 1967. Interaction of intravenously administered procaine, lidocaine and succinylcholine in anesthetized subjects. Anesth. Analg. 46, 39–45. Yokoyama, M., Hirakawa, M., Goto, H., 1995. Effect of vasoconstrictive agents added to lidocaine on intravenous lidocaine-induced convulsions in rats. Anesthesiology 82, 574–580. Yost, C.S., Dodson, B.A., 1993. Inhibition of the nicotinic acetylcholine receptor by barbiturates and by procaine: do they act at different sites? Cell. Mol. Neurobiol. 13, 159–172.
Contents lists available at ScienceDirect
European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / e j p h a r
Molecular and Cellular Pharmacology
The effect of local anesthetics on the inhibition of adult muscle-type nicotinic acetylcholine receptors by nondepolarizing muscle relaxants Hong Wang, Ying Zhang, Shi-tong Li ⁎ Department of Anesthesiology, First People's Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China
a r t i c l e
i n f o
Article history: Received 28 July 2009 Received in revised form 3 December 2009 Accepted 15 December 2009 Available online 4 January 2010 Keywords: Local anesthetic Nondepolarizing muscle relaxant Adult muscle-type nicotinic acetylcholine receptor Acetylcholine current Combined effect
a b s t r a c t The primary action of local anesthetics is to inhibit voltage-gated Na+ channels. However, local anesthetics also have an inhibitory effect on muscle-type nicotinic acetylcholine receptors. Because local anesthetics could increase the neuromuscular blockade produced by nondepolarizing muscle relaxants, we investigated the interaction of local anesthetics with nondepolarizing muscle relaxants at adult muscle-type nicotinic acetylcholine receptors. This study tested the effects of lidocaine and procaine, alone and in combination with vecuronium and cisatracurium, on adult muscle-type nicotinic acetylcholine receptors. The adult mouse muscle-type nicotinic acetylcholine receptor was expressed in HEK293 cells and activated with 10 µM acetylcholine. Currents were recorded using the whole-cell voltage-clamp technique. Adult muscle-type nicotinic acetylcholine receptors were potently inhibited by all the tested compounds. Although the potencies of procaine and lidocaine were statistically significantly different at adult muscle-type nicotinic acetylcholine receptors (50% inhibitory concentration values of 45.5 µM and 11.1 µM, respectively), procaine and lidocaine enhanced the inhibitory effect of nondepolarizing muscle relaxants at adult muscle-type nicotinic acetylcholine receptors to the same extent. The increased adult muscle-type nicotinic acetylcholine receptor inhibition produced when local anesthetics are combined with nondepolarizing muscle relaxants may contribute to the clinical enhancement of neuromuscular blockade by local anesthetics. © 2009 Elsevier B.V. All rights reserved.
1. Introduction It is well-known that local anesthetics also produce neuromuscular block and increase the neuromuscular responses to nondepolarizing muscle relaxants (Katz and Gissen, 1969; Usubiaga et al., 1967; Ellis et al., 1953). The voltage-gated sodium channel is considered to be the primary target of this effect of local anesthetics. However, previous studies have also shown that local anesthetics can noncompetitively inhibit muscle-type nicotinic acetylcholine receptor function at therapeutic doses (Neher and Steinbach, 1978; Gentry and Lukas, 2001), which may partly contribute to muscle-relaxing action of local anesthetics. Previous studies demonstrate preliminary administration of ineffective concentrations of local anesthetics significantly decreased the 50% inhibitory concentration values (IC50) of nondepolarizing muscle relaxants in nerve–muscle preparations (Matsuo et al., 1978). These findings suggest that local anesthetics powerfully enhance the neuromuscular blockade produced by nondepolarizing muscle relaxants at the neuromuscular junction. However, in these studies the
⁎ Corresponding author. Department of Anesthesiology, First People's Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200030, China. Tel.:+86 21 63240090 3022; fax:+86 21 63243749. E-mail address: [email protected] (ST. Li). 0014-2999/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2009.12.028
interaction of local anesthetics with nondepolarizing muscle relaxants at muscle-type nicotinic acetylcholine receptors was not addressed. Consequently, to evaluate the interaction of local anesthetics and nondepolarizing muscle relaxants at the nicotinic acetylcholine receptor, we heterologously expressed adult muscle-type nicotinic acetylcholine receptors in HEK293 cells and observed the effects of two muscle relaxants (the aminosteroid vecuronium and the benzylisoquinolinium cisatracurium) in the presence of two local anesthetics (lidocaine or procaine). The aim was to determine directly whether local anesthetics could enhance the inhibitory effect of nondepolarizing muscle relaxants on adult muscle-type nicotinic acetylcholine receptors. 2. Materials and methods 2.1. Cell culture and transfection Expression plasmids Psp65α, Psp65β, Psp65δ, and Pbssk(+)ε, encoding complementary DNA coding sequences for the mouse muscle nicotinic acetylcholine receptor subunits α, β, δ, and ε, respectively, were provided by the Salk Institute in America. These plasmids were subcloned in pcDNA3.1+ (InvitrogenT Life Technologies, Carlsbad, CA, USA). Human embryonic kidney 293 (HEK293) cells were cultured in Dulbecco's modified Eagle's medium (InvitrogenTM, Grand Island, NY, USA) supplemented with 10% calf serum
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H. Wang et al. / European Journal of Pharmacology 630 (2010) 29–33
(InvitrogenTM, Grand Island, NY, USA), 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2/95% O2 incubator. HEK293 cells were transfected stably with LipofectamineTM 2000 according to the manufacturer's protocol (InvitrogenTM Life Technologies, Carlsbad, CA, USA). After transfection, the positive cell clones were selected with G418. The transfected cells were then incubated for 24 h before the recordings were made.
2.2. Electrophysiology HEK293 cells were voltage-clamped using the whole-cell patchclamp technique (Hamill et al., 1981). Pipettes were pulled from borosilicate glass with a P-97 electrode puller (Sutter Instrument Co, Novato, CA, USA) and had a resistance of 2–3 MΩ. The pipette electrode was filled with the following solution (mM): CsCl 140, MgCl2 2, Hepes–CsOH 10, EGTA 0.5, and Na–ATP 4, pH 7.3. The external solution contained (mM) NaCl 140, KCl 2.5, CaCl2 2, MgCl2 2, Hepes– NaOH 10, and glucose 10, pH 7.3. Cells were voltage-clamped at −50 mV in the whole-cell configuration. All experiments were performed at room temperature (20–24 °C). Currents were measured with an EPC10 (HEKA Elektronik, Germany) amplifier and Patch-
Master software (HEKA Elektronik, Germany), sampled at 20 kHz, and stored on a computer. Acetylcholine, lidocaine hydrochloride, and procaine hydrochloride were purchased from Sigma (Sigma Chemical Co., Saint Louis, USA). Muscle relaxants were obtained in preparations for clinical use, namely vecuronium (NV. Organon, The Netherlands) and cisatracurium (Glaxosmithkline SpA, Italy). All drugs were dissolved in an external solution and applied by a gravity-driven perfusion system. Solutions and their dilutions to the experimental concentrations were prepared immediately before the experiments. The test solutions, containing either acetylcholine alone or in combination with various concentrations of nondepolarizing muscle relaxants or/and local anesthetics, were applied for 2 s to the HEK293 cells, and the peak current was determined. To determine the effect of the antagonist on the acetylcholineelicited current, the solution containing the nondepolarizing muscle relaxants or/and local anesthetics was perfused on HEK293 cells for 1 min prior to the application of acetylcholine in the presence of the antagonist. The washout time between each drug application was at least 60 s to minimize the amount of desensitization throughout the course of an experiment. Currents were acquired from five HEK293 cells. The control current in response to acetylcholine alone was repeated after washout of the antagonist. Taking the mean value of these two
Fig. 1. Concentration-dependent effects of vecuronium (VEC), cisatracurium (CISATR), procaine (PRO), and lidocaine (LIDO) on adult muscle-type nicotinic acetylcholine receptors expressed in HEK293 cells. Tracings represent raw currents observed during the application of acetylcholine (ACh) 10 µM for 2 s, either alone or in combination with various concentrations of vecuronium, cisatracurium, procaine, or lidocaine as indicated.
H. Wang et al. / European Journal of Pharmacology 630 (2010) 29–33
acetylcholine applications as the average control current, the antagonist response was calculated (percentage inhibition of average control current) using the following equation:
%inhibition = 100 ×
current in presence of antagonist 1− average control current
2.3. Statistical analysis Data analysis was performed off-line using Origin 8 (OriginLab, Northampton, MA, USA) and GraphPad Prism 4 (Graphpad software, Inc., San Diego, CA, USA). Concentration–response curves were fitted to the four-parameter logistic equation by nonlinear regression analysis, and IC50 values were determined. Results are expressed as means ± S.D. or as the 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 b 0.05 was considered significant. 3. Results Acetylcholine 10 µM was used to activate the adult muscle-type nicotinic acetylcholine receptors expressed in HEK293 cells because this concentration caused efficient acetylcholine currents without significant desensitization after repeated exposures. Vecuronium and cisatracurium reversibly inhibited acetylcholine-induced inward currents in a concentration-dependent fashion (Figs. 1 and 2). The adult muscle-type nicotinic acetylcholine receptor was equally sensitive to inhibition by the aminosteroid vecuronium (IC50 concentration, 11.5 nM; 95% CI, 8.7–15.0 nM) and the benzylisoquinolinium cisatracurium (IC50 concentration, 9.3 nM, 95% CI, 7.3–11.8 nM) (P N 0.05, unpaired two-tailed Student's t-test). The local anesthetics procaine and lidocaine also produced reversible, concentration-dependent inhibition of the currents induced by the application of acetylcholine (10 µM) (Figs. 1 and 2). Lidocaine (IC50 concentration, 11.1 µM; 95% CI, 5.0–24.8 µM) was significantly more potent than procaine (IC50 concentration, 45.5 µM; 95% CI, 36.0– 57.5 µM) in producing inhibition of acetylcholine-induced currents (P b 0.05, unpaired two-tailed Student's t-test).
Fig. 2. Concentration–response effects of vecuronium (A), cisatracurium (B), procaine (C), and lidocaine (D): inhibition of acetylcholine-induced currents in HEK293 cells expressing adult muscle-type nicotinic acetylcholine receptor. Data points show means ± S.D. (error bars) of five HEK293 cells. Error bars not visible are smaller than the symbols.
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To determined the combined effects of local anesthetics and nondepolarizing muscle relaxants, test compounds were coapplied to adult muscle-type nicotinic acetylcholine receptors expressed in HEK293 cells. An enhancement of inhibition of acetylcholine-elicited currents with vecuronium or cisatracurium by 0.5 × IC50 of either lidocaine (∼5.5 µM) or procaine (∼23 µM) was observed. Both the local anesthetics increased the inhibitory effects of all three concentrations of vecuronium and cisatracurium on nicotinic acetylcholine receptors. For both local anesthetics, the enhancement was synergistic with the smallest dose of vecuronium (0.1 nM) or cisatracurium (0.1 nM), and was less intense as vecuronium or cisatracurium was used in larger concentrations. Compared with procaine, there was an equipotent enhancement by lidocaine for the three concentrations of vecuronium or cisatracurium (Fig. 3). Representative tracings for vecuronium and lidocaine are shown in Fig. 4. 4. Discussion A previous study demonstrated that combinations of ineffective concentrations of nondepolarizing muscle relaxants with ineffective concentrations of local anesthetics caused a greater than 90% neuromuscular blockade in a rat phrenic nerve–hemidiaphragm preparation (Matsuo et al., 1978). This interaction of local anesthetics was attributed to the blockade of the Na+ channels (e.g., with procaine) (Cassuto et al., 2006) or both the Na+ and the K+ channels (e.g., with lidocaine) (Maeno et al., 1971). However, this explanation ignored the contribution of the action of the local anesthetics at the nicotinic acetylcholine receptor. Our results demonstrated that lidocaine and procaine can
Fig. 3. Graphic representation of the enhancement of the percentage inhibition of acetylcholine-induced (10 µM) currents with vecuronium (A) and cisatracurium (B) by procaine and lidocaine. Data were presented as means ± S.D. Three concentrations of vecuronium (0.1, 5.5, and 11 nM; Panel A) or cisatracurium (0.1, 4.5, and 9 nM; Panel B) were applied alone (white bars) and in combination with 0.5 × 50% inhibitory concentration (IC50) of either procaine (23 µM; greyish color bars) or 0.5 × IC50 of lidocaine (5.5 µM; hatched bars). Procaine or lidocaine significantly increased the inhibition of acetylcholine-induced current with vecuronium and cisatracurium (*P b 0.01 using one-way analysis of variance followed by Tukey's test).
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H. Wang et al. / European Journal of Pharmacology 630 (2010) 29–33
Fig. 4. Inhibition of acetylcholine (ACh)-induced currents of adult muscle-type nicotinic acetylcholine receptors expressed in HEK293 cells by vecuronium, alone and in combination with lidocaine. Tracings represent raw currents observed during the application of acetylcholine (10 µM) for 2 s, either alone or in combination with one of three concentrations of vecuronium (0.1, 5.5, 11 nM) with or without lidocaine (5.5 µM) as indicated.
significantly enhance inhibition of vecuronium and cisatracurium at adult muscle-type nicotinic acetylcholine receptors. Furthermore, lidocaine and procaine enhanced inhibition of acetylcholine-induced currents when the smallest concentration of vecuronium or cisatracurium was used, in a synergistic fashion. In this study, lidocaine and procaine produced concentrationdependent inhibition of nicotinic acetylcholine receptor-mediated inward currents. The IC50 concentration of procaine producing adult muscle-type nicotinic acetylcholine receptor blockade is similar to that reported previously (Yost and Dodson, 1993). There is substantial evidence that local anesthetics bind to and inhibit nicotinic acetylcholine receptors via noncompetitive mechanisms in which they bind to a regulatory site on the nicotinic acetylcholine receptor, inhibit channel opening, and decrease the concentration of nicotinic acetylcholine receptors in the open state by slowly switching them to their inactive form. Alternatively, local anesthetics may also bind to open channels with subsequent inhibition of ion flux (Arias, 1999; Gentry and Lukas, 2001). Consistent with previous studies (Demazumder and Dilger, 2001; Paul et al., 2002a,b), our results also showed that vecuronium and cisatracurium potently inhibited adult muscle-type nicotinic acetylcholine receptors. The muscle-type nicotinic acetylcholine receptor is a ligand-gated ion channel that consists of four different subunits assembled in a pentameric structure to create a central ion-conducting pore (Changeux et al., 1990; Paul et al., 2002a,b). The acetylcholine binding
sites are located at the interface of the α–γ and α–δ subunits in the extracellular domain of the receptor (Sine and Claudio, 1991). Nondepolarizing muscle relaxants act in a competitive manner by binding to the acetylcholine binding sites on nicotinic acetylcholine receptors (Garland et al., 1988). We found that concentrations of 0.5 × IC50 lidocaine and procaine caused effective enhancement of the nondepolarizing muscle relaxant-induced inhibition, which accords with the idea that these two classes of drugs interact with different regions of the nicotinic acetylcholine receptor and local anesthetics bind to a regulatory site on the nicotinic acetylcholine receptor to cause allosterism of receptor (Karpen and Hess, 1986; Niu et al., 1995; Niu and Hess, 1993), which may lead to an increase in the affinity of nondepolarizing muscle relaxants for nicotinic acetylcholine receptors. In this study, mouse nicotinic acetylcholine receptor expressed in HEK293 cells was more sensitive to blockade by lidocaine and procaine than human nicotinic acetylcholine receptor expressed in the TE671/RD cell line (Gentry and Lukas, 2001). Differences in species, expression systems (cell environments), experimental approach (electrophysiological or ion flux assays), or perhaps degrees of nicotinic acetylcholine receptor desensitization (Ryan and Baenziger, 1999) could account for the different IC50 values observed in different studies (Yost and Dodson, 1993). In clinical practice, between 40 and 80 mM of lidocaine is used; therefore, a level below 1 mM is considered to be a clinically acceptable concentration (Onizuka et al., 2008). However, in the central nervous system, 0.1 mM lidocaine is a toxic dose that may induce convulsions in the rat (Kim et al., 1996; Takahashi et al., 2006; Yokoyama et al., 1995). In humans, the dose of local anesthetics inducing convulsion is approximate 18–26 μg/mL (about 0.1 mM) (Onizuka et al., 2008). Thus, from this study we conclude that lidocaine and procaine at concentrations within the clinical range can increase the inhibiting effects of vecuronium and cisatracurium on nicotinic acetylcholine receptors. During the course of study, we used 10 µM acetylcholine as an agonist concentration. This concentration can ensure sufficient acetylcholine current responses and minimize nicotinic acetylcholine receptor desensitization owing to repetitive application of acetylcholine (Paul et al., 2002a,b). Because the antagonistic effects of nondepolarizing muscle relaxants are independent of holding voltages ranging from −100 to − 40 mV (Garland et al, 1988; Paul et al., 2002a,b), HEK293 cells were clamped at −50 mV to minimize the potentiation of local anesthetics inhibition of transient acetylcholine-evoked currents by more negative potential (Arias, 1999; Yost and Dodson, 1993). In conclusion, our results demonstrate that the presence of clinically relevant concentrations of lidocaine and procaine can significantly enhanced the inhibitory effect of nondepolarizing muscle relaxants on nicotinic acetylcholine receptors. These findings indicate that a combined effect of the two classes of drugs on adult muscle-type nicotinic acetylcholine receptors can partly explain how local anesthetics enhance nondepolarizing muscle relaxant-induced neuromuscular blockade. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30571796). We thank Dr Liu Xin-qiu (Department of Neurobiology, School of Medicine, Shanghai Jiaotong University) for technical assistance and strong support. References Arias, H.R., 1999. Role of local anesthetics on both cholinergic and serotonergic ionotropic receptors. Neurosci. Biobehav. Rev. 23, 817–843. Cassuto, J., Sinclair, R., Bonderovic, M., 2006. Anti-inflammatory properties of local anesthetics and their present and potential clinical implications. Acta. Anaethesiol. Scand. 50, 265–282.
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