Free
Pain Medicine  |   April 2001
Blockade and Activation of the Human Neuronal Nicotinic Acetylcholine Receptors by Atracurium and Laudanosine
Author Affiliations & Notes
  • Florence Chiodini, Ph.D.
    *
  • Eric Charpantier, Ph.D.
  • Dominique Muller, M.D., Ph.D.
  • Edomer Tassonyi, M.D., Ph.D.
    §
  • Thomas Fuchs-Buder, M.D.
  • Daniel Bertrand, Ph.D.
    #
  • * Researcher Fellow, † Postdoctoral Fellow, # Professor of Physiology, Department of Physiology, ‡ Professor, Division of Neuropharmacology, Geneva Medical Centre. § Associate Professor, ∥ Chief Resident, Division of Anaesthesiology, Geneva University Hospitals, Division of Anaesthesiology Geneva University Hospitals. ∥ Current position: Klinik für Anaesthesiologie und Intensive Medicin, University of Saarland, HomburgSaar, Germany.
  • Received from the Department of Physiology and the Divisions of Neuropharmacology and Anaesthesiology, University Hospitals and Medical Center, Geneva, Switzerland.
Article Information
Pain Medicine
Pain Medicine   |   April 2001
Blockade and Activation of the Human Neuronal Nicotinic Acetylcholine Receptors by Atracurium and Laudanosine
Anesthesiology 4 2001, Vol.94, 643-651. doi:
Anesthesiology 4 2001, Vol.94, 643-651. doi:
THE benzylisoquinoline 1 derivative atracurium (ATR) is a widely used nondepolarizing neuromuscular blocking agent, with a relatively short half-life of 20–40 min, and a rapid elimination period. 2–4 Although well-tolerated, atracurium can cause different adverse reactions, 5,6 such as cardiovascular effects, that are thought to be mediated by the ganglionic neuronal nicotinic acetylcholine receptor (nAChR). 7 In addition, atracurium and its degradation product laudanosine were found in cerebrospinal fluid, indicating that this compound may cross, in some circumstances, the blood–brain barrier 8,9 and could, depending on the concentrations, affect brain function. In agreement with this hypothesis, it has been shown in animal models that, when administered at high concentration, neuromuscular blocking agents can trigger seizures. 10,11 Furthermore, atracurium application to hippocampal slices modifies synaptic transmission. 12 
The recent availability of complementary DNA (cDNA) coding for human neuronal nAChRs (reviewed in Bertrand and Changeux 13 and Lindstrom et al.  , 14) opens new possibilities for assessment of the possible effects of atracurium on these receptors. The aim of this study was to evaluate the effects of atracurium and laudanosine on the functional properties of the major brain and ganglionic human nAChRs reconstituted in Xenopus  oocytes. The α4β2 subunits, which are thought to constitute the major brain nicotinic receptors, were chosen as a model of central nAChRs. 13,14 Receptors corresponding to those found in ganglia were obtained by expression of α3β4 or α3β4α5 subunits. 15 In addition, we evaluated atracurium and laudanosine effects on the homomeric α7 nAChR that is expressed centrally and peripherally. 13,16–18 
Methods
Oocyte Preparation and cDNA Injection
Xenopus  oocytes were isolated and prepared as previously described. 19 Oocytes were intranuclearly injected with 2 ng cDNA. All subunits were injected with an equal concentration. Oocytes were kept separately in a 96-well microtiter plate (NUNC) at 18°C in Barth solution (88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO3, 10 mm HEPES, 0.82 mm MgSO47H2O, 0.33 mm Ca(NO3)2.4H2O, 0.41 mm CaCl2.6H2O, at pH 7.4 adjusted with NaOH, and supplemented with 20 μg/ml kanamycine, 100 U/ml penicillin, and 100 μg/ml streptomycine). Atracurium (Tracrium®) was purchased from Glaxo Wellcome (London, UK). All other drugs, including ACh, laudanosine and atropine were purchased from Sigma (Buchs, Switzerland).
Electrophysiology
Current recordings from oocytes were performed at 18°C, 2–4 days after cDNA injections. During the recording, cells were continuously superfused with original Ringer 2 (82.5 mm NaCl, 2.5 mm KCl, 5 mm HEPES, pH 7.4 adjusted with NaOH) with either Ca2+or Ba2+(2.5 mm). All drugs were diluted in an original Ringer 2 medium The flow rate was approximately 6 ml/min and the volume chamber was less than 100 μl. To prevent possible activation of endogenous muscarinic receptors, 0.5 μm atropine was added. Electrophysiologic recordings were performed with a two-electrode voltage clamp (GENECLAMP amplifier; Axon Instruments, Forster City, CA). Electrodes made from 1.2 mm borosilicate Q tubes were pulled using a BB CH PC puller (Mecanex, Nyon, Switzerland), and filled with 3 m KCl. Unless specified, cells were clamped at a holding potential of −100 mV and the current was measured at the peak current. All experiments were performed at 18°C. Current–voltage relations were determined by a linear voltage ramp. To best show open channel blockade, cells were first briefly held at +40 mV and the voltage was then ramped down within 500 ms to 100 mV. Current–voltage relation curves were obtained by reporting the current values measured every 4 mV. Subtraction of the currents determined in control conditions from those measured during ACh exposure allowed determination of the nAChR current–voltage relations in isolation.
Data Analysis
Concentration-response curves were adjusted using the empirical Hill equations; where Y is the fraction of activated current, EC50is concentration of half-activation, nH is the apparent cooperativity, and x is agonist concentration. where Y is the fraction of remaining current, IC50is concentration of half-inhibition, nH is the apparent cooperativity, and x is antagonist concentration.
Values indicated throughout the text are given with their respective standard deviations (SD).
Results
Evidence, including results obtained from biochemical and electrophysiologic studies, has shown that d  -tubocurarine produces multiple effects at the neuromuscular nAChR junction. Effects caused by this molecule are (1) competitive inhibition, 20,21 (2) open channel blockade, 22,23 and (3) direct activation of the receptor. 24–26 Therefore, when evaluating possible effects of the structurally related atracurium molecule on neuronal nAChRs, it is necessary to distinguish among these three modes of action.
Effects of atracurium on the Central or Ganglionic nAChRs
To determine atracurium effects in steady state conditions, this compound was pre- and coapplied with ACh. As shown in figure 1A, the application of 10 μm atracurium markedly inhibits the ACh-evoked current at the α4β2 nAChR. This effect was reversible within 1 min of washout. A comparable inhibition of ACh-evoked current was observed at 4 μm atracurium for the ganglionic α3β4 and α3β4α5 nAChRs and at 10 μm atracurium for the α7 receptor. Full recovery was observed within 2 min. For each of these receptor subtypes, the ACh concentration test pulse was adjusted to near their respective EC50. A small inward deflection of the current was observed during atracurium application alone on the α4β2 and α3β4 receptors. To evaluate further this putative receptor activation, currents evoked by low ACh concentrations were compared with those evoked by either atracurium or laudanosine alone (fig. 1B). Increasing the drug concentrations greater than those shown always resulted in a smaller current, indicating that these compounds act as inhibitors at relatively higher concentrations. The comparison of the currents evoked by ACh, atracurium, and laudanosine on a log–log scale, highlights the differences in sensitivity of the α4β2 and α3β4 receptors to these three substances, with ACh always being the most effective agonist. As shown in figure 1B, data are well-fitted by straight lines and yielded respective slope values of 0.73 and 0.78 for ACh and atracurium on the α4β2 and 1.22, 0.6, and 0.31 for ACh, laudanosine, and atracurium on the α3β4. Offset values were 2.72 and 2.3 for ACh and atracurium on the α4β2 and 1.5, 0.56, and −0.02 for ACh, laudanosine, and atracurium on the α3β4. All correlation factors were superior to 0.95. As expected for a receptor with a lower affinity, all responses on the α3β4 are shifted to the right. Therefore, the relative agonist sensitivities are ACh greater than atracurium on α4β2 receptors and ACh greater than laudanosine greater than atracurium on α3β4 receptors. Laudanosine alone, however, evoked no detectable current at α4β2. Similarly, no currents could be recorded in response to atracurium or laudanosine exposure at the homomeric α7 receptors (data not shown). Given the paucity of α5 expression and the absence of distinguishable atracurium effects, no attempts were made to characterize the activation of this receptor subtype.
Fig. 1. Blockade and activation of neuronal nAChRs. (  A  )  ACh-evoked currents recorded in α4β2, α3β4, α3β4α5, and α7 receptors expressing oocytes in control conditions (left  ), during atracurium exposure (middle  ), and after recovery (right  ). Dashed lines indicate the atracurium application time, whereas bars indicate the ACh application. (B, Left  )  Activation of the α4β2 and α3β4 neuronal nicotinic acetylcholine receptors (nAChRs) by low concentrations of ACh, atracurium, or laudanosine. (Right  ) Plots on a log–log scale ACh- (squares), atracurium- (triangles), and laudanosine (circles)–evoked currents.
Fig. 1. Blockade and activation of neuronal nAChRs. (  A  ) 
	ACh-evoked currents recorded in α4β2, α3β4, α3β4α5, and α7 receptors expressing oocytes in control conditions (left 
	), during atracurium exposure (middle 
	), and after recovery (right 
	). Dashed lines indicate the atracurium application time, whereas bars indicate the ACh application. (B, Left  ) 
	Activation of the α4β2 and α3β4 neuronal nicotinic acetylcholine receptors (nAChRs) by low concentrations of ACh, atracurium, or laudanosine. (Right 
	) Plots on a log–log scale ACh- (squares), atracurium- (triangles), and laudanosine (circles)–evoked currents.
Fig. 1. Blockade and activation of neuronal nAChRs. (  A  )  ACh-evoked currents recorded in α4β2, α3β4, α3β4α5, and α7 receptors expressing oocytes in control conditions (left  ), during atracurium exposure (middle  ), and after recovery (right  ). Dashed lines indicate the atracurium application time, whereas bars indicate the ACh application. (B, Left  )  Activation of the α4β2 and α3β4 neuronal nicotinic acetylcholine receptors (nAChRs) by low concentrations of ACh, atracurium, or laudanosine. (Right  ) Plots on a log–log scale ACh- (squares), atracurium- (triangles), and laudanosine (circles)–evoked currents.
×
Dual Blockade Mechanisms of Atracurium
To further characterize the atracurium inhibition, we first evaluated the atracurium concentration–response inhibition curve at a fixed ACh test condition (fig. 2and table 1). Afterward, atracurium was kept at a constant concentration while the concentration of ACh was progressively increased. As shown in figure 2A, IC50of the α4β2 nAChR was observed at 1.5 μm atracurium, for 0.1 μm ACh test pulse. Increasing the ACh concentration decreased the IC50value, suggesting that atracurium and ACh may compete for the same binding site at α4β2. The competitive nature of atracurium blockade at the α4β2 nAChR was further confirmed with the evaluation of how atracurium altered the ACh concentration–response relation (fig. 2B). For each cell, data were normalized to the saturating current recorded at maximal ACh concentration (1 mm) in control conditions. The graph shows that 10 μm atracurium caused a shift of the concentration–response curve toward higher concentrations (table 1), without affecting the maximal evoked current.
Fig. 2. Mode of action of atracurium inhibition. (Inset  ) Typical currents recorded for three atracurium concentrations are superimposed. Current amplitudes were scaled with respect to the ACh concentration–response curve. Currents in each cell were normalized to the value measured at 1 mm ACh and plotted on a semilogarithmic scale. The number of cells measured in each condition is indicated in table 1. Continuous lines through the data point are the best fits obtained with the Hill equations (equation 1) for the activation and (equation 2) for the inhibition (table 1).
Fig. 2. Mode of action of atracurium inhibition. (Inset 
	) Typical currents recorded for three atracurium concentrations are superimposed. Current amplitudes were scaled with respect to the ACh concentration–response curve. Currents in each cell were normalized to the value measured at 1 mm ACh and plotted on a semilogarithmic scale. The number of cells measured in each condition is indicated in table 1. Continuous lines through the data point are the best fits obtained with the Hill equations (equation 1) for the activation and (equation 2) for the inhibition (table 1).
Fig. 2. Mode of action of atracurium inhibition. (Inset  ) Typical currents recorded for three atracurium concentrations are superimposed. Current amplitudes were scaled with respect to the ACh concentration–response curve. Currents in each cell were normalized to the value measured at 1 mm ACh and plotted on a semilogarithmic scale. The number of cells measured in each condition is indicated in table 1. Continuous lines through the data point are the best fits obtained with the Hill equations (equation 1) for the activation and (equation 2) for the inhibition (table 1).
×
Table 1. Effects of Atracurium at α4β2, α3β4, α3β4α5, and α7 nAChRs
Image not available
Table 1. Effects of Atracurium at α4β2, α3β4, α3β4α5, and α7 nAChRs
×
As shown in figures 2C and 2D,a different pattern of inhibition was observed at the ganglionic α3β4 receptor. First, inhibition was independent of the ACh concentration (fig. 2C). Second, atracurium caused only a small decrease on the ACh sensitivity, and blockade could not be relieved by increasing the ACh concentration. Because it is known that α5 receptor contributes to a fraction of ganglionic receptors, 15 the effects of atracurium were assessed after coinjection of α3, β4, and α5 subunits. As shown in table 1, injection of this subunit caused no detectable changes in atracurium affinity. No differences could be observed on the ACh concentration–response profile either (data not shown). These results suggest that atracurium may act on the ganglionic receptor as an open channel blocker.
Quantification of the atracurium inhibition at the α7 receptor with three ACh test pulse conditions indicates that, as for the α4β2 nAChR, the IC50progressively shifted toward the lower sensitivities as the agonist concentration was increased (fig. 2E, table 1). The IC50dependency of the ACh concentration is indicative of the competitive mode of action of atracurium. This hypothesis was further reinforced by the observation that atracurium inhibition is fully overcome by an increase of the ACh concentration (fig. 2F). Because α7 is highly permeable to Ca2+(its response may be contaminated by calcium-dependent chloride activation), atracurium concentration–response inhibitions were measured in a Ba2+-containing medium, a condition that is known to reduce chloride activation. 27 Substitution of extracellular calcium by barium caused a small shift to the left of the IC50and slightly increased the EC50(300–430 μm, data not show). This indicates that, even when present, chloride contamination plays a minor role in the atracurium blockade. The lower calcium permeability of α4β2 or α3β4 receptors would imply that calcium-dependent chloride contamination might also be neglected for these subtypes. Therefore, all further experiments were performed during normal divalent cation conditions.
Effects of Laudanosine on the Central or Ganglionic nAChRs
To isolate the effects of atracurium from those of laudanosine, experiments were performed using pure laudanosine. When the same experimental protocols as those presented in figure 2were used, we found that laudanosine also inhibits the α4β2, α3β4, and α7 receptors (fig. 3, table 2).
Table 2. Effects of Laudanosine on α4β2, α3β4, and α7 nAChRs
Image not available
Table 2. Effects of Laudanosine on α4β2, α3β4, and α7 nAChRs
×
Measurement of the fraction of ACh current inhibition at α4β2 as a function of agonist concentration showed that laudanosine blockade was only partially removed by increasing the ACh concentration (fig. 3A). As for atracurium, adequate curve fitting was obtained with the Hill equations (equation 1), providing addition of a scaling factor of 0.65 to account for the laudanosine insurmountable blockade. These data show that, in contrast to atracurium, the mechanism of laudanosine blockade on the α4β2 receptor is competitive, but this compound acts also in a noncompetitive manner for at least 35% of the blockade. Typical ACh-evoked currents recorded in control and during coapplication of laudanosine, are shown in figure 3B. Contrarily to atracurium, laudanosine alone caused no detectable signal. The small rebound observed at the end of the ACh–laudanosine application is compatible with mechanisms of open channel blockade. Concentration–response inhibition measured with a 0.1-μm ACh test pulse yielded an IC50of 9.4 μm (table 2). Here we used low ACh concentration to avoid rapid desensitization of this receptor.
Fig. 3. Laudanosine is a competitive inhibitor and open channel blocker on neuronal nicotinic acetylcholine receptors (nAChRs). Concentration–response curves to ACh were determined for oocytes expressing the α4β2 (A  ), α3β4 (  C  ), and α7 (E  ) nAChR in controls (open squares) and during pre- (5 s) and coapplication of a fixed concentration laudanosine (filled symbols). Currents measured in at least 6 cells (see also table 2) were averaged and normalized to the maximal value recorded in control conditions. Typical currents recorded in an oocyte expressing the α4β2 (B  ) and α3β4 (D  ) are shown. Responses evoked by a 5 s ACh application in control (left  ) or during laudanosine exposure (100 μm, right  ) are superimposed. Note the important rebound at the end of the coapplication of ACh and laudanosine. (F  )  α7 nAChR current–voltage relations recorded first in control conditions (open squares) and then during exposure to laudanosine (30 μm; filled circles). Current–voltage relation was determined using a voltage ramp as described in Methods. The α7 receptor was activated by 100 μm ACh application.
Fig. 3. Laudanosine is a competitive inhibitor and open channel blocker on neuronal nicotinic acetylcholine receptors (nAChRs). Concentration–response curves to ACh were determined for oocytes expressing the α4β2 (A 
	), α3β4 (  C 
	), and α7 (E 
	) nAChR in controls (open squares) and during pre- (5 s) and coapplication of a fixed concentration laudanosine (filled symbols). Currents measured in at least 6 cells (see also table 2) were averaged and normalized to the maximal value recorded in control conditions. Typical currents recorded in an oocyte expressing the α4β2 (B 
	) and α3β4 (D 
	) are shown. Responses evoked by a 5 s ACh application in control (left 
	) or during laudanosine exposure (100 μm, right 
	) are superimposed. Note the important rebound at the end of the coapplication of ACh and laudanosine. (F  ) 
	α7 nAChR current–voltage relations recorded first in control conditions (open squares) and then during exposure to laudanosine (30 μm; filled circles). Current–voltage relation was determined using a voltage ramp as described in Methods. The α7 receptor was activated by 100 μm ACh application.
Fig. 3. Laudanosine is a competitive inhibitor and open channel blocker on neuronal nicotinic acetylcholine receptors (nAChRs). Concentration–response curves to ACh were determined for oocytes expressing the α4β2 (A  ), α3β4 (  C  ), and α7 (E  ) nAChR in controls (open squares) and during pre- (5 s) and coapplication of a fixed concentration laudanosine (filled symbols). Currents measured in at least 6 cells (see also table 2) were averaged and normalized to the maximal value recorded in control conditions. Typical currents recorded in an oocyte expressing the α4β2 (B  ) and α3β4 (D  ) are shown. Responses evoked by a 5 s ACh application in control (left  ) or during laudanosine exposure (100 μm, right  ) are superimposed. Note the important rebound at the end of the coapplication of ACh and laudanosine. (F  )  α7 nAChR current–voltage relations recorded first in control conditions (open squares) and then during exposure to laudanosine (30 μm; filled circles). Current–voltage relation was determined using a voltage ramp as described in Methods. The α7 receptor was activated by 100 μm ACh application.
×
A difference in the mode of action between atracurium and laudanosine was also identified at the ganglionic α3β4 nAChR (figs. 3C and D). Figure 3Cshows the dual mode of blockade caused by laudanosine with a shift in the ACh EC50(table 2) and an insurmountable blockade. Another difference was the inward currents observed during the prepulse of laudanosine alone (figs. 1B and 3D). An important rebound of current was observed at the end of the ACh–laudanosine application (fig. 3D). This rebound was observed in every cell tested. Concentration–response curves to laudanosine yielded an IC50of approximately 38 μm for an ACh test pulse of 50 μm (table 2).
A dual mode of action of laudanosine was also observed on the α7 receptor but with a smaller fraction of insurmountable blockade (fig. 3E). The Hill equation 1was used with a scaling factor of 0.85, introduced into the curve fitting to account for this small fraction of blockade. The ACh EC50increased from 80 to 240 μm during exposure to 30 μm laudanosine (table 2). It is well-documented that when charged molecules enter and block the ionic pores of a ligand-gated channel, its fraction of blockade will depend on the transmembrane potential. 28,29 Therefore, if laudanosine causes a blockade by steric hindrance in the channel pore, its inhibition may be voltage dependent. Typical current–voltage relations recorded in control and during laudanosine exposure showed a marked voltage dependency of laudanosine blockade (fig. 3F). The small rebound observed at the end of the ACh–laudanosine application is coherent with a mechanism of open channel blockade. Determination of the concentration–response inhibition profile with an ACh test pulse of a 100 μm yielded an IC50of 18.3 μm (table 2).
Discussion
Recent advances in molecular biology and DNA cloning have identified the nAChR subtypes expressed in various regions of the central and peripheral nervous system (reviewed in Bertrand and Changeux 13 and Lindstrom et al  . 14). Central nicotinic receptors mainly contain the α4 and β2 subunits, whereas ganglionic receptors result from the assembly of α3 and β4. Coimmunoprecipitation experiments have shown that a fraction of ganglionic receptors also contain the α5 subunit. 15 Finally, it has been shown that the homomeric α7 receptor is expressed centrally and peripherally. To evaluate the possible interaction between neuronal nAChRs and atracurium and its first degradation product laudanosine, these two substances were applied alone or with ACh on Xenopus  oocytes expressing the human α4β2, α3β4, α3β4α5, and α7 receptors.
Incubation with atracurium or laudanosine caused a marked inhibition of these four receptor subtypes, with IC50s in the micromolar range (tables 1 and 2). Blockade of the ACh-evoked current was fast, and complete recovery was obtained within 1 min, indicating rapid onset and offset kinetics. In addition, as expected from data obtained on muscle nAChRs, atracurium and laudanosine effects are multiple: (1) competitive inhibition, (2) open channel blockade, and (3) activation of the receptor. The latter effect was observed only for the central α4β2 and ganglionic α3β4 receptors. It is noteworthy to recall that the α5 subunit does not contribute to the pharmacologic profile of the ganglionic receptor. 30 Therefore, it is not surprising that injection of this subunit caused no detectable changes in atracurium sensitivity of the α3β4 receptor.
One of the difficulties in the study of the effects caused by atracurium is the instability of this product. It is well-documented that one molecule of atracurium quickly degrades into two laudanosine molecules. The Hoffman degradation of atracurium is independent of biologic processes and therefore precludes an evaluation of the effects of atracurium alone. A few percent contamination of laudanosine in the atracurium solution cannot be excluded. A comparison of the atracurium and laudanosine effects is mandatory to understand their respective contribution. As shown in figures 2 and 3, where atracurium and laudanosine caused a comparable inhibition, a marked difference between these two compounds was observed when comparing the fraction of current they can activate. The major brain α4β2 nAChR was activated by low atracurium concentrations but was unresponsive to laudanosine (data not shown). In contrast, the ganglionic α3β4 nAChR was activated by laudanosine but almost unresponsive to atracurium. Plots of the ACh, atracurium, and laudanosine currents evoked by low concentrations of these three compounds on a log–log scale show that neuronal nAChRs are at least five to fifty times more sensitive to ACh than to curaremimetics. At higher concentrations, atracurium and laudanosine inhibit the ACh-evoked currents. In the absence of significant differences between the α3β4 and the α3β4α5 receptor profiles and given the paucity of expression of these latter receptor subtypes, activation experiments were therefore not performed.
Plasma levels of atracurium range between 0.5 and 5.1 μg/ml (0.4 and 4.1 μm, respectively), with the lowest values found during surgical anaesthesia and the highest during intensive care conditions. The degradation of one atracurium molecule into two laudanosine molecules implies that a plasma concentration as high as 8 μm can be reached for the latter compound. Considering these data, it was important to determine whether these compounds can be responsible for adverse effects by direct action on neuronal nicotinic receptors.
The major central nicotinic receptor α4β2 was blocked by atracurium and laudanosine with an IC50of 1.4 and 9.4 μm, respectively, when stimulated with 0.1 μm ACh (tables 1 and 2). The ACh EC50was shifted toward higher concentration in the presence of a constant atracurium concentration (figs. 2B and 3B). The atracurium blockade was fully reversible by increasing the ACh concentration, whereas an insurmountable block of approximately 35% persisted with laudanosine. This indicates differences in the mode of action, with these two compounds atracurium inducing a purely competitive blockade and laudanosine a mixed competitive and noncompetitive action. These data are in agreement with previous findings that showed that d  -tubocurarine, a related chemical structure, is a competitive inhibitor on the chick α4β2 nAChR with an IC50in the micromolar range. 31 Evidence for open channel blockade is clearly seen with the rebounds observed at the end of ACh and laudanosine coapplication on the α4β2 receptor (fig. 3B). The difference observed in EC50for the α4β2 receptor between tables 1 and 2is attributable to the use of different oocyte batches. A recent report showed that the neuronal nAChR concentration–response curves are best fitted using the two Hill equations. 32 However, because of technical limitations, the number of points collected was restricted and does not allow for further conclusion.
Muscle relaxant drugs have been described to have adverse effects and, in the worst cases, can trigger seizures in vitro  or in the animal model. 10,11 Here we report that atracurium and laudanosine can block the major brain nicotinic receptor at concentrations that can be present in the plasma of patients. Recently mutations on the α4 subunit have been shown to induce autosomal dominant nocturnal epilepsy (reviewed in Steinlein 33). It follows that a modification of the α4β2 receptor activity could be the origin of seizures. Even if these receptors are exclusively expressed in the brain and thus protected by the blood–brain barrier, it has been already shown that atracurium and laudanosine can be found in cerebrospinal fluid. 8,9 This suggests that part of atracurium adverse effects may be caused through its action on the α4β2 receptor.
A different mode of action was observed at the ganglionic α3β4 nAChR with atracurium and laudanosine, causing a noncompetitive blockade. Note that, in agreement with the absence of effects of α5 on ganglionic receptor pharmacology, the addition of α5 caused no detectable changes on the action of atracurium. The ganglionic α3β4 receptor was inhibited by atracurium and laudanosine with an IC50of 3.2 and 38.4 μm, respectively. In agreement with a noncompetitive blockade, half-inhibition was independent on the agonist test pulse concentration. Moreover, the addition of a constant inhibitor concentration induced a slight shift of the concentration–response curve toward higher ACh concentrations, but the maximal response measured in the presence of an antagonist was reduced by at least 35%. In addition, for laudanosine, an important rebound indicative of an open channel blockade was observed at the end of the test pulse. Results obtained with atracurium on the α3β4 receptor suggest that this compound blocks these receptors by noncompetitive blockade. Finally, the atracurium IC50of 3.2 μm observed in our conditions (table 1) is compatible with the 3 μM dissociation constant reported for d  -tubocurarine blockade on rat ganglia. 34 
Recalling that the blood–brain barrier does not isolate autonomic ganglia, it is conceivable that concentrations of atracurium and laudanosine comparable with plasma levels may be reached in their environment. Our data are in agreement with previous hypotheses that adverse cardiovascular effects may be attributed to the direct action of atracurium or laudanosine on cardiac ganglia. 7 
At the homomeric α7 receptor, atracurium acts as a competitive inhibitor. This was shown by the shift in the IC50of the atracurium blockade in function of the ACh test pulse concentration and by the full relief of inhibition observed at saturating ACh. Therefore, as for the α4β2 and α3β4, it can be concluded that the effects caused by laudanosine contaminant on α7 are negligible. Previous studies of the blockade caused by d  -tubocurarine have shown that this compound acts as a noncompetitive blocker on the chick receptor and that 0.5 μm was already sufficient to reduce by 40% the ACh evoked current. 35 Although initially different, these results and ours are not contradictory. The difference in the mode of action between atracurium and d  -tubocurarine may be attributed to the difference in size between these two molecules. Moreover, experiments performed with the desensitized open L247T receptor have shown the dual mode of action of d  -tubocurarine, with activation and blockade of this mutant. 36 Although it was proposed that, in some cases, perfusion conditions might affect the α7 responses in Xenopus  oocytes, 37 we think that the agreement between our results and those obtained with the same cDNA expressed in human embryonic kidney cells is indicative of adequate experimental conditions. Therefore, no attempts were made to compensate ACh concentration–response curves, and raw data are presented herein.
The α7 receptor is expressed in both the central and the peripheral nervous systems (reviewed in Bertrand and Changeux 13 and Lindstrom et al  . 14), but also in nonneuronal cells such as the embryonic skeletal muscle cells. However, α7 has never been found in adult innervated muscle. 38–40 According to these findings, we can conclude that muscular effect observed during atracurium treatment cannot be caused by interaction with α7 receptors. The atracurium and laudanosine IC50values on this receptor were 3.1 and 18.3 μm, respectively. These data confirmed that atracurium and laudanosine in therapeutic conditions could block α7 receptors. Therefore, we cannot exclude that side effects observed during atracurium administration could be caused by a direct effect on these receptors.
In conclusion, we have shown that atracurium and laudanosine interact with neuronal nicotinic ACh receptors at concentrations that can be present in clinical conditions.
The authors thank Sonia Bertrand for her perfect technical assistance during the preparation of this manuscript; Dr. Carole Yamate-Poitry for the continuous discussion; and Bruno Buisson, Isabelle Favre, Logos Curtis, and Yann Villiger for their critical reading of the manuscript. All are affiliated with the Department of Physiology, Centre Médical Universitaire, Geneva, Switzerland.
References
Fuchs-Buder T: New muscle relaxants. Update on mivacurium, rocuronium and cis-atracurium [German]. Anaesthesist 1997; 46: 350–9Fuchs-Buder, T
Boyd AH, Eastwood NB, Parker CJ, Hunter JM: Comparison of the pharmacodynamics and pharmacokinetics of an infusion of cis-atracurium (51W89) or atracurium in critically ill patients undergoing mechanical ventilation in an intensive therapy unit. Br J Anaesth 1996; 76: 382–8Boyd, AH Eastwood, NB Parker, CJ Hunter, JM
Grigore AM, Brusco L Jr, Kuroda M, Koorn R: Laudanosine and atracurium concentrations in a patient receiving long-term atracurium infusion. Crit Care Med 1998; 26: 180–3Grigore, AM Brusco, L Kuroda, M Koorn, R
Hughes R, Chapple DJ: The pharmacology of atracurium: A new competitive neuromuscular blocking agent. Br J Anaesth 1981; 53: 31–44Hughes, R Chapple, DJ
Goldberg M, Rosenberg H: New muscle relaxants in outpatient anesthesiology. Dent Clin North Am 1987; 31: 117–29Goldberg, M Rosenberg, H
Foldes FF, Nagashima H, Boros M, Tassonyi E, Fitzal S, Agoston S: Muscular relaxation with atracurium, vecuronium and duador under balanced anaesthesia. Br J Anaesth 1983; 55: 97S–103SFoldes, FF Nagashima, H Boros, M Tassonyi, E Fitzal, S Agoston, S
Ostergaard D, Engbaek J, Viby-Mogensen J: Adverse reactions and interactions of the neuromuscular blocking drugs. Med Toxicol Adverse Drug Exp 1989; 4: 351–68Ostergaard, D Engbaek, J Viby-Mogensen, J
Fahey MR, Canfell PC, Taboada T, Hosobuchi Y, Miller RD: Cerebrospinal fluid concentrations of laudanosine after administration of atracurium. Br J Anaesth 1990; 64: 105–6Fahey, MR Canfell, PC Taboada, T Hosobuchi, Y Miller, RD
Eddleston JM, Harper NJ, Pollard BJ, Edwards D, Gwinnutt CL: Concentrations of atracurium and laudanosine in cerebrospinal fluid and plasma during intracranial surgery. Br J Anaesth 1989; 63: 525–30Eddleston, JM Harper, NJ Pollard, BJ Edwards, D Gwinnutt, CL
Cardone C, Szenohradszky J, Yost S, Bickler PE: Activation of brain acetylcholine receptors by neuromuscular blocking drugs. A possible mechanism of neurotoxicity. A nesthesiology 1994; 80: 1155–61;Cardone, C Szenohradszky, J Yost, S Bickler, PE
Szenohradszky J, Trevor AJ, Bickler P, Caldwell JE, Sharma ML, Rampil IJ, Miller RD: Central nervous system effects of intrathecal muscle relaxants in rats. Anesth Analg 1993; 76: 1304–9Szenohradszky, J Trevor, AJ Bickler, P Caldwell, JE Sharma, ML Rampil, IJ Miller, RD
Chiodini FC, Tassonyi E, Fuchs-Buder T, Fathi M, Bertrand D, Muller D: Effects of neuromuscular blocking agents on excitatory transmission and γ-aminobutyric acid A-mediated inhibition in the rat hippocampal slice. A nesthesiology 1998; 88: 1003–13Chiodini, FC Tassonyi, E Fuchs-Buder, T Fathi, M Bertrand, D Muller, D
Bertrand D, Changeux JP: Nicotinic receptor: An allosteric protein specialized for intercellular communication. Semin Neurosci 1995; 7: 75–90Bertrand, D Changeux, JP
Lindstrom J, Anand R, Peng X, Gerzanich V, Wang F, Li Y: Neuronal nicotinic receptor subtypes. Ann N Y Acad Sci 1995; 757: 100–16Lindstrom, J Anand, R Peng, X Gerzanich, V Wang, F Li, Y
Conroy WG, Berg DK: Neurons can maintain multiple classes of nicotinic acetylcholine receptors distinguished by different subunit compositions. J Biol Chem 1995; 270: 4424–31Conroy, WG Berg, DK
Seguela P, Wadiche J, Dineley-Miller K, Dani JA, Patrick JW: Molecular cloning, functional properties, and distribution of rat brain alpha 7: A nicotinic cation channel highly permeable to calcium. J Neurosci 1993; 13: 596–604Seguela, P Wadiche, J Dineley-Miller, K Dani, JA Patrick, JW
Albuquerque EX, Pereira EF, Alkondon M, Schrattenholz A, Maelicke A: Nicotinic acetylcholine receptors on hippocampal neurons: distribution on the neuronal surface and modulation of receptor activity. J Recept Signal Transduct Res 1997; 12: 243–66Albuquerque, EX Pereira, EF Alkondon, M Schrattenholz, A Maelicke, A
Couturier S, Bertrand D, Matter JM, Hernandez MC, Bertrand S, Millar N, Valera S, Barkas T, Ballivet M: A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX. Neuron 1990; 5: 847–56Couturier, S Bertrand, D Matter, JM Hernandez, MC Bertrand, S Millar, N Valera, S Barkas, T Ballivet, M
Bertrand D, Cooper E, Valera S, Rungger D, Ballivet M: Electrophysiology of neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes following nuclear injection of genes or cDNA, Methods in Neuroscience. Edited by Conn M. New York, Academic Press, 1991, pp 174–93
Del Castillo J, Katz B: A study of curare action with an electrical micro-method. Proc Royal Soc (Lond) 1957; B146: 339–56Del Castillo, J Katz, B
Adams PR: Drug blockade of open end-plate channels. J Physiol (Lond) 1976; 260: 531–52Adams, PR
Colquhoun D, Sheridan RE: The effect of tubocurarine competition on the kinetics of agonist action on the nicotinic receptor. Br J Pharmacol 1982; 75: 77–86Colquhoun, D Sheridan, RE
Bufler J, Wilhelm R, Parnas H, Franke C, Dudel J: Open channel and competitive block of the embryonic form of the nicotinic receptor of mouse myotubes by (+)-tubocurarine. J Physiol (Lond) 1996; 495: 83–95Bufler, J Wilhelm, R Parnas, H Franke, C Dudel, J
Trautmann A: Curare can open and block ionic channels associated with cholinergic receptors. Nature 1982; 298: 272–5Trautmann, A
Steinbach JH, Chen Q: Antagonist and partial agonist actions of d-tubocurarine at mammalian muscle acetylcholine receptors. J Neurosci 1995; 15: 230–40Steinbach, JH Chen, Q
Fletcher GH, Steinbach JH: Ability of nondepolarizing neuromuscular blocking drugs to act as partial agonists at fetal and adult mouse muscle nicotinic receptors. Mol Pharmacol 1996; 49: 938–47Fletcher, GH Steinbach, JH
Galzi JL, Devillers-Thiery A, Hussy N, Bertrand S, Changeux JP, Bertrand D: Mutations in the channel domain of a neuronal nicotinic receptor convert ion selectivity from cationic to anionic. Nature 1992; 359: 500–5Galzi, JL Devillers-Thiery, A Hussy, N Bertrand, S Changeux, JP Bertrand, D
Woodhull AM: Ionic blockage of sodium channels in nerve. J Gen Physiol 1973; 61: 687–708Woodhull, AM
Buisson B, Bertrand D: Open-channel blockers at the human alpha4beta2 neuronal nicotinic acetylcholine receptor. Mol Pharmacol 1998; 53: 555–63Buisson, B Bertrand, D
Wang F, Gerzanich V, Wells GB, Anand R, Peng X, Keyser K, Lindstrom J: Assembly of human neuronal nicotinic receptor alpha5 subunits with alpha3, beta2, and beta4 subunits. J Biol Chem 1996; 271: 17656–65Wang, F Gerzanich, V Wells, GB Anand, R Peng, X Keyser, K Lindstrom, J
Bertrand D, Ballivet M, Rungger D: Activation and blocking of neuronal nicotinic acetylcholine receptor reconstituted in Xenopus oocytes. Proc Natl Acad Sci U S A 1990; 87: 1993–7Bertrand, D Ballivet, M Rungger, D
Covernton PJO, Connolly JG: Multiple components in the agonist concentration-response relationships of neuronal nicotinic acetylcholine receptors. J Neurosci Meth 2000; 96: 63–70Covernton, PJO Connolly, JG
Steinlein OK: Neuronal nicotinic receptors in human epilepsy [In Process Citation]. Eur J Pharmacol 2000; 393: 243–7Steinlein, OK
Fieber LA, Adams DJ: Acetylcholine-evoked currents in cultured neurones dissociated from rat parasympathetic cardiac ganglia. J Physiol (Lond) 1991; 434: 215–37Fieber, LA Adams, DJ
Bertrand D, Bertrand S, Ballivet M: Pharmacological properties of the homomeric alpha 7 receptor. Neurosci Lett 1992; 146: 87–90Bertrand, D Bertrand, S Ballivet, M
Bertrand D, Devillers-Thiery A, Revah F, Galzi JL, Hussy N, Mulle C, Bertrand S, Ballivet M, Changeux JP: Unconventional pharmacology of a neuronal nicotinic receptor mutated in the channel domain. Proc Natl Acad Sci U S A 1992; 89: 1261–5Bertrand, D Devillers-Thiery, A Revah, F Galzi, JL Hussy, N Mulle, C Bertrand, S Ballivet, M Changeux, JP
Papke RL, Thinschmidt JS: The correction of alpha7 nicotinic acetylcholine receptor concentration-response relationships in Xenopus oocytes. Neurosci Lett 1998; 256: 163–6Papke, RL Thinschmidt, JS
Corriveau RA, Romano SJ, Conroy WG, Oliva L, Berg DK: Expression of neuronal acetylcholine receptor genes in vertebrate skeletal muscle during development. J Neurosci 1995; 15: 1372–83Corriveau, RA Romano, SJ Conroy, WG Oliva, L Berg, DK
Fischer U, Reinhardt S, Albuquerque EX, Maelicke A: Expression of functional alpha7 nicotinic acetylcholine receptor during mammalian muscle development and denervation. Eur J Neurosci 1999; 11: 2856–64Fischer, U Reinhardt, S Albuquerque, EX Maelicke, A
Romano SJ, Pugh PC, McIntosh JM, Berg DK: Neuronal-type acetylcholine receptors and regulation of alpha 7 gene expression in vertebrate skeletal muscle. J Neurobiol 1997; 32: 69–80Romano, SJ Pugh, PC McIntosh, JM Berg, DK
Fig. 1. Blockade and activation of neuronal nAChRs. (  A  )  ACh-evoked currents recorded in α4β2, α3β4, α3β4α5, and α7 receptors expressing oocytes in control conditions (left  ), during atracurium exposure (middle  ), and after recovery (right  ). Dashed lines indicate the atracurium application time, whereas bars indicate the ACh application. (B, Left  )  Activation of the α4β2 and α3β4 neuronal nicotinic acetylcholine receptors (nAChRs) by low concentrations of ACh, atracurium, or laudanosine. (Right  ) Plots on a log–log scale ACh- (squares), atracurium- (triangles), and laudanosine (circles)–evoked currents.
Fig. 1. Blockade and activation of neuronal nAChRs. (  A  ) 
	ACh-evoked currents recorded in α4β2, α3β4, α3β4α5, and α7 receptors expressing oocytes in control conditions (left 
	), during atracurium exposure (middle 
	), and after recovery (right 
	). Dashed lines indicate the atracurium application time, whereas bars indicate the ACh application. (B, Left  ) 
	Activation of the α4β2 and α3β4 neuronal nicotinic acetylcholine receptors (nAChRs) by low concentrations of ACh, atracurium, or laudanosine. (Right 
	) Plots on a log–log scale ACh- (squares), atracurium- (triangles), and laudanosine (circles)–evoked currents.
Fig. 1. Blockade and activation of neuronal nAChRs. (  A  )  ACh-evoked currents recorded in α4β2, α3β4, α3β4α5, and α7 receptors expressing oocytes in control conditions (left  ), during atracurium exposure (middle  ), and after recovery (right  ). Dashed lines indicate the atracurium application time, whereas bars indicate the ACh application. (B, Left  )  Activation of the α4β2 and α3β4 neuronal nicotinic acetylcholine receptors (nAChRs) by low concentrations of ACh, atracurium, or laudanosine. (Right  ) Plots on a log–log scale ACh- (squares), atracurium- (triangles), and laudanosine (circles)–evoked currents.
×
Fig. 2. Mode of action of atracurium inhibition. (Inset  ) Typical currents recorded for three atracurium concentrations are superimposed. Current amplitudes were scaled with respect to the ACh concentration–response curve. Currents in each cell were normalized to the value measured at 1 mm ACh and plotted on a semilogarithmic scale. The number of cells measured in each condition is indicated in table 1. Continuous lines through the data point are the best fits obtained with the Hill equations (equation 1) for the activation and (equation 2) for the inhibition (table 1).
Fig. 2. Mode of action of atracurium inhibition. (Inset 
	) Typical currents recorded for three atracurium concentrations are superimposed. Current amplitudes were scaled with respect to the ACh concentration–response curve. Currents in each cell were normalized to the value measured at 1 mm ACh and plotted on a semilogarithmic scale. The number of cells measured in each condition is indicated in table 1. Continuous lines through the data point are the best fits obtained with the Hill equations (equation 1) for the activation and (equation 2) for the inhibition (table 1).
Fig. 2. Mode of action of atracurium inhibition. (Inset  ) Typical currents recorded for three atracurium concentrations are superimposed. Current amplitudes were scaled with respect to the ACh concentration–response curve. Currents in each cell were normalized to the value measured at 1 mm ACh and plotted on a semilogarithmic scale. The number of cells measured in each condition is indicated in table 1. Continuous lines through the data point are the best fits obtained with the Hill equations (equation 1) for the activation and (equation 2) for the inhibition (table 1).
×
Fig. 3. Laudanosine is a competitive inhibitor and open channel blocker on neuronal nicotinic acetylcholine receptors (nAChRs). Concentration–response curves to ACh were determined for oocytes expressing the α4β2 (A  ), α3β4 (  C  ), and α7 (E  ) nAChR in controls (open squares) and during pre- (5 s) and coapplication of a fixed concentration laudanosine (filled symbols). Currents measured in at least 6 cells (see also table 2) were averaged and normalized to the maximal value recorded in control conditions. Typical currents recorded in an oocyte expressing the α4β2 (B  ) and α3β4 (D  ) are shown. Responses evoked by a 5 s ACh application in control (left  ) or during laudanosine exposure (100 μm, right  ) are superimposed. Note the important rebound at the end of the coapplication of ACh and laudanosine. (F  )  α7 nAChR current–voltage relations recorded first in control conditions (open squares) and then during exposure to laudanosine (30 μm; filled circles). Current–voltage relation was determined using a voltage ramp as described in Methods. The α7 receptor was activated by 100 μm ACh application.
Fig. 3. Laudanosine is a competitive inhibitor and open channel blocker on neuronal nicotinic acetylcholine receptors (nAChRs). Concentration–response curves to ACh were determined for oocytes expressing the α4β2 (A 
	), α3β4 (  C 
	), and α7 (E 
	) nAChR in controls (open squares) and during pre- (5 s) and coapplication of a fixed concentration laudanosine (filled symbols). Currents measured in at least 6 cells (see also table 2) were averaged and normalized to the maximal value recorded in control conditions. Typical currents recorded in an oocyte expressing the α4β2 (B 
	) and α3β4 (D 
	) are shown. Responses evoked by a 5 s ACh application in control (left 
	) or during laudanosine exposure (100 μm, right 
	) are superimposed. Note the important rebound at the end of the coapplication of ACh and laudanosine. (F  ) 
	α7 nAChR current–voltage relations recorded first in control conditions (open squares) and then during exposure to laudanosine (30 μm; filled circles). Current–voltage relation was determined using a voltage ramp as described in Methods. The α7 receptor was activated by 100 μm ACh application.
Fig. 3. Laudanosine is a competitive inhibitor and open channel blocker on neuronal nicotinic acetylcholine receptors (nAChRs). Concentration–response curves to ACh were determined for oocytes expressing the α4β2 (A  ), α3β4 (  C  ), and α7 (E  ) nAChR in controls (open squares) and during pre- (5 s) and coapplication of a fixed concentration laudanosine (filled symbols). Currents measured in at least 6 cells (see also table 2) were averaged and normalized to the maximal value recorded in control conditions. Typical currents recorded in an oocyte expressing the α4β2 (B  ) and α3β4 (D  ) are shown. Responses evoked by a 5 s ACh application in control (left  ) or during laudanosine exposure (100 μm, right  ) are superimposed. Note the important rebound at the end of the coapplication of ACh and laudanosine. (F  )  α7 nAChR current–voltage relations recorded first in control conditions (open squares) and then during exposure to laudanosine (30 μm; filled circles). Current–voltage relation was determined using a voltage ramp as described in Methods. The α7 receptor was activated by 100 μm ACh application.
×
Table 1. Effects of Atracurium at α4β2, α3β4, α3β4α5, and α7 nAChRs
Image not available
Table 1. Effects of Atracurium at α4β2, α3β4, α3β4α5, and α7 nAChRs
×
Table 2. Effects of Laudanosine on α4β2, α3β4, and α7 nAChRs
Image not available
Table 2. Effects of Laudanosine on α4β2, α3β4, and α7 nAChRs
×