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Meeting Abstracts  |   September 2006
Distinct Pharmacologic Properties of Neuromuscular Blocking Agents on Human Neuronal Nicotinic Acetylcholine Receptors: A Possible Explanation for the Train-of-four Fade
Author Affiliations & Notes
  • Malin Jonsson, M.D., Ph.D.
    *
  • David Gurley, M.S.
  • Michael Dabrowski, Ph.D.
  • Olof Larsson, Ph.D.
    §
  • Edwin C. Johnson, Ph.D.
    #
  • Lars I. Eriksson, M.D., Ph.D.
  • * Resident, ∥ Professor, Department of Anesthesiology and Intensive Care Medicine, Karolinska University Hospital and Karolinska Institutet. † Principal Scientist, Director, Target Biology, AstraZeneca Pharmaceuticals, Wilmington, Delaware. ‡ Senior Research Scientist, § Associate Professor, Molecular Pharmacology, AstraZeneca R&D, Södertälje, Sweden.
Article Information
Meeting Abstracts   |   September 2006
Distinct Pharmacologic Properties of Neuromuscular Blocking Agents on Human Neuronal Nicotinic Acetylcholine Receptors: A Possible Explanation for the Train-of-four Fade
Anesthesiology 9 2006, Vol.105, 521-533. doi:
Anesthesiology 9 2006, Vol.105, 521-533. doi:
NONDEPOLARIZING neuromuscular blocking agents (NMBAs) are extensively used in the practice of anesthesia and intensive care medicine to facilitate tracheal intubation and mechanical ventilation and to improve surgical conditions.
Although it is well established that nondepolarizing NMBAs block the postsynaptic α1β1ϵδ nicotinic acetylcholine receptor (nAChR) subtype at the muscle endplate, the effect on the presynaptic motor nerve ending has not been clarified (for a review, see Vizi and Lendvai1,2 and Bowman et al.  2). It is believed that the mechanism behind tetanic and train-of-four (TOF) fade during neuromuscular block by a nondepolarizing NMBA arise from an interaction with presynaptic cholinergic autoreceptors at the motor nerve ending.1,3 However, the affinity of nondepolarizing NMBAs to such presynaptic autoreceptors has not been investigated at the molecular level. Further, it has recently been shown that an inhibition of the presynaptic α3β2nAChR subtype at the motor nerve end4 induces tetanic fade.5 Based on this, it seems likely that the tetanic fade phenomenon seen during nondepolarizing neuromuscular block is due to an inhibition of the α3β2nAChR subtype.
The α1β1ϵδ and the α3β2nAChRs are members of the same neurotransmitter-gated ion channel superfamily. They are composed of five transmembrane subunits with a central cation pore, and the stoichiometry and identity of subunits determines each receptor's unique properties.6 To date, 17 nicotinic subunits have been cloned in vertebrates: the muscle α1, β1, δ, γ, and ϵ subunits and the neuronal α2–10and β2–4subunits.7 Although there are many potential combinations of neuronal nAChRs, only a few have as yet been found to be of biologic importance.8,9 The neuronal nAChRs are found both presynaptically and postsynaptically in neurons of the central (α4β2, α3β2, α7)9,10 and peripheral nervous system (α3β4, α3β2, α7)9,11,12 as well as in extraneuronal tissues and cells, such as keratinocytes, muscle, lymphocytes, macrophages, carotid bodies, and neurosecretory cells.6,7,13 
Interactions between NMBAs and neuronal nAChRs may cause serious cardiovascular and respiratory side effects. It has been shown that nondepolarizing NMBAs reduce hypoxic ventilatory response in partially paralyzed humans,14,15 and the mechanism behind this depression might be interference with nicotinic chemotransduction of the carotid bodies.16,17 At the molecular level, d-tubocurarine, pancuronium, atracurium, and its degradation product laudanosine have been shown to block neuronal nAChR subtypes expressed in Xenopus  oocytes.18–22 Interestingly, some reports indicate that NMBAs can act as partial agonists at α1β1γδ, α3β4, and α4β2nAChR subtypes21,23; however, other studies could not demonstrate any agonism by NMBAs.20,24 
The α7nAChR subtype plays a key role in the cholinergic reflex involved in inflammatory conditions such as sepsis,25,26 and it can be speculated whether NMBAs used in intensive care settings might interact with the inflammatory response to sepsis. Furthermore, although highly charged, NMBAs can under certain conditions cross the blood–brain barrier,27–29 thus having the potential to interact with central cholinergic receptors and the synaptic transmission30 and cause seizures.31,32 
Because most nondepolarizing NMBAs were developed before cloning and isolation of their target proteins, the precise modes of action have not been examined in detail. A better understanding of the molecular mechanisms of action of clinically used nondepolarizing NMBAs on human neuronal nAChR subtypes is needed. In addition, for future drug design, it is essential to define potential interactions with human neuronal nAChRs.
The aim of this study was therefore to investigate the potency and functional affinity of clinically used nondepolarizing NMBAs on acetylcholine-induced responses on human muscle and neuronal nAChRs heterologously expressed in Xenopus  oocytes. In addition, potential activation of nAChRs by nondepolarizing NMBAs was also investigated.
Materials and Methods
Clones
The human nAChR subunits α1, α3–4, α7, β1, β2, β4, δ, and ϵ were cloned from a human complementary DNA (cDNA) library. GenBank (Bethesda, MD) access numbers for the cDNA nucleotide sequences are as follows: NM 000079 (α1), HSU62432 (α3), L35901 (α4), Y08420 (α7), NM 000747 (β1), Y08415 (β2), NM 000750 (β4), NM 000751 (δ), and NM 000080 (ϵ). The cDNAs were subcloned into different expression vectors, pKGem (AstraZeneca, Wilmington, DE) (α1, α3, β1, β2, δ and ϵ), pBluescript II SK (−) (Stratagene, La Jolla, CA) (α7), and pBSTA (University of California, Irvine, CA) (α4and β4). Messenger RNA (mRNA) was transcribed in vitro  using the mMessage mMachine® T7 kit (Ambion, Austin, TX) and analyzed using a bioanalyzer (Agilent Technologies, Palo Alto, CA).
Xenopus  Oocyte Injection
The study was approved by the local animal ethics committee at Karolinska Institutet, Stockholm, Sweden. Preparation and injection of oocytes and the electrophysiologic recordings were conducted as previously described.33 Briefly, Xenopus laevis  oocytes were isolated by partial ovariectomy from frogs anesthetized with 0.2% Tricaine (Sigma, St. Louis, MO). The ovaries were mechanically dissected to smaller lumps and digested in OR-2 buffer (82.5 mm NaCl, 2 mm KCl, 1 mm MgCl2, 5 mm HEPES, pH adjusted to 7.5 with NaOH) containing 1.5 mg/ml collagenase (type 1A; Sigma) for 90 min to remove the follicular epithelia from the oocytes. After 1–24 h, the oocytes were injected with 0.2–18 ng mRNA in a total volume of 30–40 nl/oocyte. Multiple subunit combinations were injected at a 1:1 ratio (α1β1ϵδ or αxβy), except for α4β2, where the injection ratio was 1:9. The oocytes were maintained in Leibovitz L-15 medium (Sigma) diluted 1:1 with Millipore filtered double distilled H2O (Billerica, MA) and 80 μg/ml gentamicin, 100 U/ml penicillin, and 100 μg/ml streptomycin added. Oocytes were incubated at 18°–19°C for 2–7 days after injection before being studied.
Electrophysiologic Recordings
All recordings were performed at room temperature (20°–22°C). During recording, the oocytes were continuously perfused with ND-96 (96.0 mm NaCl, 2.0 mm KCl, 1.8 mm CaCl2, 1.0 mm MgCl2, 5.0 mm HEPES, pH 7.4 adjusted with NaOH). Oocyte recordings were performed using an integrated system that provides automated impalement of up to eight oocytes, studied in parallel with two-electrode voltage clamp, and current measurements were automatically coordinated with fluid delivery throughout the experiment (OpusXpress 6000A; Molecular Devices, Union City, CA). Electrodes were made from 1.5-mm borosilicate tubes (World Precision Instruments Inc., Sarasota, FL) and filled with 3 m KCl (0.5–2.5 MΩ resistance). The oocytes were voltage clamped at −60 mV, because it has previously been shown that inhibition of nAChRs by nondepolarizing NMBAs is voltage independent at holding voltages from −100 to −40 mV.20,23 
Protocol
Oocytes were continuously perfused with ND-96 at a rate of 2 ml/min in a 150-μl chamber. Drugs were delivered from a 96-well plate using disposable tips and administered at a rate of 2 ml/min for the first 2 s, and thereafter at 1 ml/min. Concentration–response curves for acetylcholine were constructed, before and after the addition of 10 μm antagonist in each oocyte, for the neuronal nAChRs. To determine whether nondepolarizing NMBAs activate and furthermore inhibit acetylcholine-induced currents, nondepolarizing NMBAs were applied for 55 s before a 20-s coapplication of both antagonist and acetylcholine. Two different concentrations of acetylcholine were applied on each neuronal receptor subtype. The concentrations 1 and 10 μm (for α4β2), 50 and 300 μm (for α3β2and α3β4), and 30 and 100 μm (for α7) were chosen to represent concentrations below and above the EC50for each receptor subtype. The muscle nAChR (α1β1ϵδ) was used as a reference, and therefore only one acetylcholine concentration (10 μm) was studied. Between each drug application, there was a 6-min washout period to allow clearance of the drugs and to avoid desensitization of the channels. Before and after each concentration–response experiment, three control responses were recorded at EC50acetylcholine concentration to exclude desensitization. Experiments were rejected if the postcontrol response was less than 80% of the precontrol response. To adjust for the level of channel expression, the responses in acetylcholine concentration–response experiments were normalized to the peak response in each individual oocyte. For inhibition experiments, responses in each oocyte were normalized to the mean of the second and third acetylcholine precontrols.
Drugs
Acetylcholine and d-tubocurarine were purchased from Sigma. Atracurium and cisatracurium were provided by GlaxoSmithKline (Barnard Castle Durham, United Kingdom). Mivacurium (Mivacron®) was purchased from GlaxoSmithKline (Mölndal, Sweden). Org NC 97 (pancuronium), Org NC 45 (vecuronium), and rocuronium were provided by Organon (BH Oss, The Netherlands). Chemicals used in buffers were purchased from Sigma unless otherwise stated. Stock solution of 1mm acetylcholine in ND-96 was prepared and frozen. Nondepolarizing NMBAs were prepared fresh each day and stored at +4°C. All drugs were then diluted in ND-96 immediately before use.
Data Analysis and Statistics
Off-line analyses were made using Clampfit 9.2 (Molecular Devices). Changes in currents were studied both as peak and net charge responses (area under the curve); however, for the α7subtype, only net charge analysis was used, as previously described.33–35 The baseline current immediately before drug application was subtracted from the response, and the analysis region for peak and net charge analysis was 20 s, i.e.  , during the time of agonist application. Concentration–response relations for acetylcholine were fitted by nonlinear regression (Prism 4.0; GraphPad, San Diego, CA) to the four-parameter logistic equation
where Y is the normalized response, x is the logarithm of concentration, and EC50is the logarithm of the concentration of agonist eliciting half-maximal response. When NMBA-induced inhibition was studied, the same equation was used, and EC50was replaced by IC50, which is the concentration of antagonist eliciting half-maximal inhibition, Bottom = 0, Top = 1. Unless otherwise stated, data are given as mean ± SEM or 95% confidence interval (CI). Differences between fitted curves were analyzed using an F test, followed by a t  test (Prism 4.0). A P  value of less than 0.05 was considered significant.
Results
Acetylcholine Concentration–Response Relations for Muscular and Neuronal nAChRs
Acetylcholine produced a concentration-dependent inward current in voltage clamped oocytes injected with mRNA encoding muscle- and neuronal-type nAChRs, whereas uninjected oocytes did not respond to acetylcholine (data not shown). The responses to acetylcholine in terms of kinetics and EC50values at the nAChR subtypes were consistent with previous reports18,33,36 (fig. 1and table 1), thus confirming the receptor expression model. However, kinetics can also be determined using net charge analysis. Unfortunately, there is a lack of published data for comparison of net charge in human nAChRs, except for the α7nAChR subtype.35 As shown in figure 1, at α3β4and α4β2nAChR concentration–response relations based on net charge analysis correlate well with peak currents, with almost identical EC50and Hill coefficients (1). However, the α7subtype nAChR displays unique properties, with very fast desensitization kinetics (fig. 1A), which gives a different concentration–response relation depending on whether peak response or net charge was measured (1). At the α3β2subtype, which has an initial rapid desensitization, there was a small difference between the EC50values.
Fig. 1. Effect of 10 μm nondepolarizing neuromuscular blocking agents (NMBAs) on acetylcholine (ACh)–mediated responses in voltage clamped (−60 mV)  Xenopus  oocytes expressing human neuronal nAChRs. (  A  ) Representative traces showing the inhibitory effect of 10 μm rocuronium (Roc) on the α3β2, α3β4, α4β2, and α7receptor subtypes (displayed in the same order). (  B  ) Responses in each oocyte were normalized to the maximal acetylcholine peak current and maximal net charge (α7) in each oocyte, giving the concentration–response curves. Each  symbol  represents mean ± SEM of 3–14 oocytes. When no  error bars  are seen, they are smaller than the  symbols. 
Fig. 1. Effect of 10 μm nondepolarizing neuromuscular blocking agents (NMBAs) on acetylcholine (ACh)–mediated responses in voltage clamped (−60 mV)  Xenopus  oocytes expressing human neuronal nAChRs. (  A  ) Representative traces showing the inhibitory effect of 10 μm rocuronium (Roc) on the α3β2, α3β4, α4β2, and α7receptor subtypes (displayed in the same order). (  B  ) Responses in each oocyte were normalized to the maximal acetylcholine peak current and maximal net charge (α7) in each oocyte, giving the concentration–response curves. Each  symbol  represents mean ± SEM of 3–14 oocytes. When no  error bars  are seen, they are smaller than the  symbols. 
Fig. 1. Effect of 10 μm nondepolarizing neuromuscular blocking agents (NMBAs) on acetylcholine (ACh)–mediated responses in voltage clamped (−60 mV)  Xenopus  oocytes expressing human neuronal nAChRs. (  A  ) Representative traces showing the inhibitory effect of 10 μm rocuronium (Roc) on the α3β2, α3β4, α4β2, and α7receptor subtypes (displayed in the same order). (  B  ) Responses in each oocyte were normalized to the maximal acetylcholine peak current and maximal net charge (α7) in each oocyte, giving the concentration–response curves. Each  symbol  represents mean ± SEM of 3–14 oocytes. When no  error bars  are seen, they are smaller than the  symbols. 
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Table 1. Pharmacologic Properties of NMBAs on Human Neuronal nAChRs Expressed in  Xenopus  Oocytes and Activated by Acetylcholine 
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Table 1. Pharmacologic Properties of NMBAs on Human Neuronal nAChRs Expressed in  Xenopus  Oocytes and Activated by Acetylcholine 
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Inhibition of Muscle nAChRs by Nondepolarizing NMBAs
Because the adult muscle (α1β1ϵδ) nAChR is the clinical target for nondepolarizing NMBAs, this receptor subtype was used as reference in the oocyte setup. Atracurium, cisatracurium, d-tubocurarine, mivacurium, pancuronium, rocuronium, and vecuronium all concentration-dependently inhibited 10 μm acetylcholine–induced currents in oocytes expressing the human α1β1ϵδ nAChR (fig. 2and table 2). The IC50values were in the nanomolar range and were comparable with a similar study investigating nondepolarizing NMBAs, using mouse cRNA.37 
Fig. 2. Nondepolarizing neuromuscular blocking agents (NMBAs) concentration-dependently inhibit the 10 μm acetylcholine (ACh)–induced current response in  Xenopus  oocytes expressing the human muscle (α1β1ϵδ) nicotinic acetylcholine receptor. (  A  ) Representative traces from one oocyte. (  B  ) Current responses in each oocyte were normalized to the 10 μm acetylcholine control responses in each oocyte as described in the Materials and Methods. Each  symbol  represents mean ± SEM of 4–7 oocytes. When no  error bars  are seen, they are smaller than the  symbols  . Roc = rocuronium. 
Fig. 2. Nondepolarizing neuromuscular blocking agents (NMBAs) concentration-dependently inhibit the 10 μm acetylcholine (ACh)–induced current response in  Xenopus  oocytes expressing the human muscle (α1β1ϵδ) nicotinic acetylcholine receptor. (  A  ) Representative traces from one oocyte. (  B  ) Current responses in each oocyte were normalized to the 10 μm acetylcholine control responses in each oocyte as described in the Materials and Methods. Each  symbol  represents mean ± SEM of 4–7 oocytes. When no  error bars  are seen, they are smaller than the  symbols  . Roc = rocuronium. 
Fig. 2. Nondepolarizing neuromuscular blocking agents (NMBAs) concentration-dependently inhibit the 10 μm acetylcholine (ACh)–induced current response in  Xenopus  oocytes expressing the human muscle (α1β1ϵδ) nicotinic acetylcholine receptor. (  A  ) Representative traces from one oocyte. (  B  ) Current responses in each oocyte were normalized to the 10 μm acetylcholine control responses in each oocyte as described in the Materials and Methods. Each  symbol  represents mean ± SEM of 4–7 oocytes. When no  error bars  are seen, they are smaller than the  symbols  . Roc = rocuronium. 
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Table 2. Pharmacologic Properties of NMBAs as Inhibitors of Acetylcholine-induced Activation of Human Neuronal nAChRs Expressed in  Xenopus  Oocytes 
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Table 2. Pharmacologic Properties of NMBAs as Inhibitors of Acetylcholine-induced Activation of Human Neuronal nAChRs Expressed in  Xenopus  Oocytes 
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Neuronal nAChRs Are Inhibited by Nondepolarizing NMBAs
Atracurium, cisatracurium, d-tubocurarine, mivacurium, pancuronium, rocuronium, and vecuronium reversibly and concentration-dependently inhibited all of the neuronal nAChR subtypes tested (i.e.  , α3β2, α3β4, α4β2, and α7) with IC50values in the micromolar range (figs. 3 and 4, table 2, and 2).
Fig. 3. Representative traces of vecuronium-induced inhibition of acetylcholine (ACh) currents in  Xenopus  oocytes expressing the human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor subtypes. Vecuronium (Vec) was preapplied for 55 s before a 20-s coapplication with acetylcholine as indicated with the  horizontal bars  . For each receptor subtype, traces shown in each row are from the same oocyte. 
Fig. 3. Representative traces of vecuronium-induced inhibition of acetylcholine (ACh) currents in  Xenopus  oocytes expressing the human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor subtypes. Vecuronium (Vec) was preapplied for 55 s before a 20-s coapplication with acetylcholine as indicated with the  horizontal bars  . For each receptor subtype, traces shown in each row are from the same oocyte. 
Fig. 3. Representative traces of vecuronium-induced inhibition of acetylcholine (ACh) currents in  Xenopus  oocytes expressing the human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor subtypes. Vecuronium (Vec) was preapplied for 55 s before a 20-s coapplication with acetylcholine as indicated with the  horizontal bars  . For each receptor subtype, traces shown in each row are from the same oocyte. 
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Fig. 4. Concentration–response curves of the inhibition of acetylcholine (ACh)–mediated response by nondepolarizing neuromuscular blocking agents (NMBAs) in human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor expressed in  Xenopus  oocytes. For each receptor subtype, two acetylcholine concentrations were applied, one lower and one higher than the EC50: 50 and 300 μm for α3β2and α3β4, 1 and 10 μm for α4β2, and 30 and 100 μm for α7. Control acetylcholine peak current or net charge (α7) responses in each oocyte were normalized to the acetylcholine response with respective nondepolarizing NMBA added, yielding the concentration–response relations. For each receptor subtype, 4–11 oocytes were studied. Data are presented as mean ± SEM. When no  error bars  are seen, they are smaller than the  symbols. 
Fig. 4. Concentration–response curves of the inhibition of acetylcholine (ACh)–mediated response by nondepolarizing neuromuscular blocking agents (NMBAs) in human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor expressed in  Xenopus  oocytes. For each receptor subtype, two acetylcholine concentrations were applied, one lower and one higher than the EC50: 50 and 300 μm for α3β2and α3β4, 1 and 10 μm for α4β2, and 30 and 100 μm for α7. Control acetylcholine peak current or net charge (α7) responses in each oocyte were normalized to the acetylcholine response with respective nondepolarizing NMBA added, yielding the concentration–response relations. For each receptor subtype, 4–11 oocytes were studied. Data are presented as mean ± SEM. When no  error bars  are seen, they are smaller than the  symbols. 
Fig. 4. Concentration–response curves of the inhibition of acetylcholine (ACh)–mediated response by nondepolarizing neuromuscular blocking agents (NMBAs) in human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor expressed in  Xenopus  oocytes. For each receptor subtype, two acetylcholine concentrations were applied, one lower and one higher than the EC50: 50 and 300 μm for α3β2and α3β4, 1 and 10 μm for α4β2, and 30 and 100 μm for α7. Control acetylcholine peak current or net charge (α7) responses in each oocyte were normalized to the acetylcholine response with respective nondepolarizing NMBA added, yielding the concentration–response relations. For each receptor subtype, 4–11 oocytes were studied. Data are presented as mean ± SEM. When no  error bars  are seen, they are smaller than the  symbols. 
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All the nondepolarizing NMBAs except mivacurium showed similar affinity at the α3β2nAChR subtype, with IC50values of 3–20 μm after activation by 50 μm acetylcholine and 1–62 μm at 300 μm acetylcholine. Vecuronium and d-tubocurarine were most potent as inhibitors at the α3β2subtype independent of acetylcholine concentration, whereas mivacurium had the lowest potency of all the nondepolarizing NMBAs and did not reduce the 300-μm acetylcholine response at concentrations lower than 100 μm. For atracurium, cisatracurium, pancuronium, and rocuronium, an increase from 50 to 300 μm acetylcholine slightly increased the IC50(not significant), suggesting a competitive inhibition or higher affinity to the closed channel. Nondepolarizing NMBAs tended to increase the acetylcholine EC50for the α3β2nAChR, although the effect was not statistically significant. The peak acetylcholine current was not reduced by 10 μm nondepolarizing NMBAs (fig. 1). Therefore, the inhibition induced by NMBAs at the α3β2receptor seems mainly to be competitive, except for d-tubocurarine and vecuronium, where there is a noncompetitive component.
All of the nondepolarizing NMBAs concentration-dependently inhibited the α3β4nAChR subtype, with IC50values from 2 to 20 μm and from 0.3 to 2 μm for 50 and 300 μm acetylcholine, respectively. There was no component of competitive inhibition by the NMBAs at this receptor because the IC50values were unchanged, or even lower, at the higher acetylcholine concentration used (fig. 1and table 2). Furthermore, addition of the NMBAs to the acetylcholine concentration–response relations both increased the EC50(table 1) and reduced peak acetylcholine responses in presence of a NMBA (P  < 0.05), independent of concentration. Therefore, the NMBAs seem to inhibit the α3β4nAChR subtype in a noncompetitive way. All NMBAs showed higher affinity for the closed channel, except cisatracurium and mivacurium.
By contrast, the α4β2nAChR subtype was competitively blocked by most of the NMBAs. That is, increasing the concentrations of acetylcholine from 1 to 10 μm increased the IC50from 1–13 μm to 5–67 μm. In addition, NMBAs generally right-shifted the acetylcholine concentration–response relations without reducing the peak response at the α4β2nAChR (fig. 1), further suggesting a competitive mode of inhibition. However, 10 μm rocuronium significantly reduced the peak response to all concentrations of acetylcholine tested (fig. 1), and thus, its inhibition is noncompetitive. Further, rocuronium seemed to desensitize the α4β2receptor because the control responses after both the acetylcholine concentration–response experiment with rocuronium and after the rocuronium inhibition experiment with 10 μm acetylcholine did not return to 80% of the control response in most of the series, which were therefore excluded (see Material and Methods, Protocol). Interestingly, rocuronium inhibition experiments with 1 μm acetylcholine did not show this pattern. Rocuronium therefore inhibits the α3β4and α4β2subtypes noncompetitively and with variable state dependency.38 
All NMBAs concentration-dependently right-shifted the acetylcholine concentration–response curve for the α7nAChR subtype, with increased EC50values (table 1), but did not reduce peak responses (not significant). In general, the inhibition of 30 and 100 μm acetylcholine at the α7nAChR subtype by NMBAs was concentration dependent (table 2), suggesting that NMBAs inhibit the α7nAChR subtype in a competitive manner. However, for rocuronium, the IC50decreased with increased acetylcholine concentration (table 2), indicating a noncompetitive component in the action of rocuronium at the α7nAChR subtype.
Nondepolarizing Neuromuscular Blocking Agents Do Not Activate Human nAChRs
Application of 1 nm to 100 μm atracurium, cisatracurium, d-tubocurarine, mivacurium, pancuronium, rocuronium, or vecuronium to oocytes expressing human muscle (α1β1ϵδ) or neuronal (α3β2, α3β4, α4β2, and α7) nAChRs did not result in receptor activation (data not shown).
Discussion
This study shows that nondepolarizing NMBAs inhibit neuronal nAChRs and that the inhibitory mechanism differs between individual receptor subtypes and NMBAs. In addition, we found no evidence that the nAChR subtypes tested were activated by any of the nondepolarizing NMBAs.
All the nondepolarizing NMBAs reversibly and concentration-dependently inhibited the neuronal α3β2, α3β4, α4β2, and α7nAChR subtypes in the micromolar range. Hence, the block occurs at clinically relevant concentrations.39 In general, nondepolarizing NMBAs block the α4β2, α3β2, and α7subtypes in a competitive manner; the exception is the α3β4subtype, where the block seems noncompetitive. However, the nondepolarizing NMBAs have individual action profiles on different receptors. Mivacurium had rather low potency at the α3β2nAChR, whereas the other NMBAs showed similar IC50values across the nAChRs tested.
Nondepolarizing NMBAs have higher functional affinity for the α1β1ϵδ nAChR subtype than for the neuronal nAChR subtypes, as determined by current amplitude measurements. For the muscle nAChR, the IC50values (nanomolar range) presented here as well as in other studies using the Xenopus  oocyte expression system20,23,37 contrast with the micromolar concentrations of NMBAs needed to reduce the nerve-evoked twitch by 50% in isolated rat nerve-muscle preparations40 and in the clinical setting.39 This apparent discrepancy probably reflects the large safety factor in the neuromuscular transmission, where approximately 75% of the receptors must be occupied by a nondepolarizing NMBA before there is any reduction in twitch tension, and 90% occupancy is required for full paralysis.41 This safety factor has not been reported for the neuronal nAChRs as far as we know.
In addition to inhibiting nAChRs, nondepolarizing NMBAs have also been described as partial agonists at both muscle and neuronal nAChR subtypes.21,23,32 Atracurium-induced activation of the α4β2nAChR subtype occurs at very low concentrations, even lower than those required for inhibition.21 However, the reports are contradictory, because Garland et al.  20 were unable to show any activation of the α1β1γδ or α1β1ϵδ nAChR subtype induced by d-tubocurarine or pancuronium. Here, we did not observe activation of the nAChRs by any of the seven nondepolarizing NMBAs studied. One possible explanation for this discrepancy might be that our study, in contrast to the previous one,21 did not use atropine in the perfusion buffer. A low concentration of atropine (i.e.  , 0.5 μm) has commonly been used to prevent activation of putative endogenous muscarinic receptors at the epithelial layer of the Xenopus  oocyte surface.42,43 However, during recent years, it has become clear that there is no endogenous surface expression of muscarinic receptors in Xenopus  oocytes themselves,44 and furthermore, it has been shown that atropine can both inhibit and activate nAChR expressed in the Xenopus  oocyte.44 Notably, the α4β2subtype is activated by atropine; therefore, we suggest that the activation of the α4β2subtype previously attributed to atracurium might instead have been elicited by atropine. For the muscle-type nAChR, we could not record any activation of the α1β1ϵδ nAChR subtype, thus confirming observations reported in previous studies.20,23,24 
Many of the nondepolarizing NMBAs tested have breakdown products with potential to block muscle nAChRs39; however, most of the nondepolarizing NMBAs are not degraded in vitro  . Atracurium and cisatracurium can to some extent undergo spontaneous degradation to laudanosine by Hofmann reaction in vitro  ,45 depending mainly on temperature and pH, and although we controlled these parameters, we cannot exclude some contamination of laudanosine. Laudanosine has been shown to block the neuronal α3β4, α4β2, and α7in a noncompetitive manner with IC50values of 8–38 μm.21,22 Because both atracurium and cisatracurium inhibit the α4β2and α7nAChRs in a competitive way, we consider it unlikely that there is any substantial involvement of laudanosine under our experimental conditions.
To date, only one study investigating the effect of clinically used nondepolarizing NMBAs on nAChRs has used human DNA21; all of the others have used rat and mouse DNA.20,24,37 Although there is more than 80% homology between the human and rodent nAChR subunit DNA,6 a small difference in amino acid sequence can cause significant changes in biophysical and pharmacologic properties of the receptors.18 Therefore, we believe that our study directly comparing the effect of the clinically used NMBAs at the human receptors in the same system adds to the current knowledge on basic pharmacologic properties of nondepolarizing NMBAs.
The mechanisms of the block of neuromuscular transmission by nondepolarizing NMBAs are likely dual; the most important is a postsynaptic block at the α1β1ϵδ nAChR subtype, but there is also an inhibition of presynaptic cholinergic receptors.2,3,46 It has become evident that the tetanic and TOF fade phenomena induced by nondepolarizing relaxants most likely arise from a block of the presynaptic α3β2nicotinic receptor.1,2,5 Interestingly, recent data indicate that adenosine and adenosine triphosphate interacting with purinergic receptors also are important in mobilization and release of acetylcholine from the motor nerve ending.47,48 Here, we can for the first time show that clinically used nondepolarizing NMBAs inhibit the α3β2nAChR subtype in the micromolar concentration range, thus providing a molecular explanation for the tetanic and TOF fade seen during neuromuscular blockade by nondepolarizing NMBAs. However, mivacurium, which had a lower affinity for the α3β2nAChR, nonetheless elicited fade, indicated that a block of the α3β2nAChR is probably not the only mechanism behind tetanic and TOF fade. The reduction in peak tension and TOF fade seen during neuromuscular monitoring are likely caused by two separate events,1–3,46 namely the presynaptic and the postsynaptic inhibition by nondepolarizing NMBAs. This also explains the clinical observations that nondepolarizing NMBAs differ in the degree of twitch reduction versus  tetanic and TOF fade.49 Furthermore, animal studies of the twitch tension and tetanic fade clearly show that these events are due to two separate mechanisms. Hexamethonium produced a complete tetanic fade without any twitch depression; pancuronium produced tetanic fade in doses that also produced pronounced twitch depression; α-bungarotoxin did not produce tetanic fade but elicited a pronounced twitch depression.50 Comparing this study to our results, it is clear that nondepolarizing NMBAs have a much higher affinity for the α1β1ϵδ nAChR subtype compared with the α3β2subtype, but still, the IC50values for the α3β2nAChR subtype are in a clinically relevant range and furthermore correspond roughly to the concentrations that produce a 50% neuromuscular block in in vitro  animal experiments (1.68–12.3 μm).40,50 In addition, we have recently shown that succinylcholine, which does not produce tetanic or TOF fade at normal dosage, does not block the α3β2nAChR subtype at clinically relevant concentrations.33 The fact that nondepolarizing NMBAs do block the α3β2nAChR subtype in clinically relevant concentrations, and the fact that succinylcholine does not, strongly support the concept that the clinically observed tetanic and TOF fade are due to a block of the presynaptic α3β2nAChR subtype.
Based on our results, nondepolarizing NMBAs have the potential to inhibit neuronal nAChRs present in peripheral autonomic ganglia.
It has previously been shown that nondepolarizing NMBAs reduce hypoxic ventilatory response in humans14,15 and furthermore impair both the hypoxic and nicotine-induced carotid body chemoreceptor response.16,17,51,52 Neuronal nAChRs have been found to be present and functional in the carotid body and its afferent system.53–55 We believe that the affinity of NMBAs to the human neuronal subtypes of nAChRs is a key component behind the interaction between nondepolarizing NMBAs and regulation of breathing during hypoxia. We also speculate whether the block of the α7nAChR subtype may have an impact on the cholinergic inflammatory reflex mediated via  the vagus nerve25,26 and thus on outcome for patients with inflammatory conditions such as sepsis.
In summary, neuronal nAChRs are widespread in the central and peripheral nervous system, as well as in extraneuronal tissues, and a block of these receptors by nondepolarizing NMBAs might interfere with important vital functions.
We conclude that nondepolarizing NMBAs concentration-dependently inhibit human neuronal nAChRs expressed in Xenopus  oocytes and that the inhibition mechanisms vary between different receptor subtypes and NMBAs. The inhibition of the presynaptic α3β2nAChR subtype at the motor nerve end provides a possible molecular explanation for the tetanic and TOF fade seen during a nondepolarizing neuromuscular block.
The authors thank Professor Bertil Fredholm, M.D., Ph.D. (Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden), for advice and constructive criticism.
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Appendix 1: Pharmacologic Properties of Human Neuronal nAChRs Activated by Acetylcholine and with the Addition of NMBAs 
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Appendix 1: Pharmacologic Properties of Human Neuronal nAChRs Activated by Acetylcholine and with the Addition of NMBAs 
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Appendix 2: Pharmacologic Properties of NMBAs as Inhibitors of Acetylcholine-induced Activation of Human Neuronal nAChRs Expressed in  Xenopus  Oocytes 
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Appendix 2: Pharmacologic Properties of NMBAs as Inhibitors of Acetylcholine-induced Activation of Human Neuronal nAChRs Expressed in  Xenopus  Oocytes 
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Appendix 2: Continued 
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Appendix 2: Continued 
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Fig. 1. Effect of 10 μm nondepolarizing neuromuscular blocking agents (NMBAs) on acetylcholine (ACh)–mediated responses in voltage clamped (−60 mV)  Xenopus  oocytes expressing human neuronal nAChRs. (  A  ) Representative traces showing the inhibitory effect of 10 μm rocuronium (Roc) on the α3β2, α3β4, α4β2, and α7receptor subtypes (displayed in the same order). (  B  ) Responses in each oocyte were normalized to the maximal acetylcholine peak current and maximal net charge (α7) in each oocyte, giving the concentration–response curves. Each  symbol  represents mean ± SEM of 3–14 oocytes. When no  error bars  are seen, they are smaller than the  symbols. 
Fig. 1. Effect of 10 μm nondepolarizing neuromuscular blocking agents (NMBAs) on acetylcholine (ACh)–mediated responses in voltage clamped (−60 mV)  Xenopus  oocytes expressing human neuronal nAChRs. (  A  ) Representative traces showing the inhibitory effect of 10 μm rocuronium (Roc) on the α3β2, α3β4, α4β2, and α7receptor subtypes (displayed in the same order). (  B  ) Responses in each oocyte were normalized to the maximal acetylcholine peak current and maximal net charge (α7) in each oocyte, giving the concentration–response curves. Each  symbol  represents mean ± SEM of 3–14 oocytes. When no  error bars  are seen, they are smaller than the  symbols. 
Fig. 1. Effect of 10 μm nondepolarizing neuromuscular blocking agents (NMBAs) on acetylcholine (ACh)–mediated responses in voltage clamped (−60 mV)  Xenopus  oocytes expressing human neuronal nAChRs. (  A  ) Representative traces showing the inhibitory effect of 10 μm rocuronium (Roc) on the α3β2, α3β4, α4β2, and α7receptor subtypes (displayed in the same order). (  B  ) Responses in each oocyte were normalized to the maximal acetylcholine peak current and maximal net charge (α7) in each oocyte, giving the concentration–response curves. Each  symbol  represents mean ± SEM of 3–14 oocytes. When no  error bars  are seen, they are smaller than the  symbols. 
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Fig. 2. Nondepolarizing neuromuscular blocking agents (NMBAs) concentration-dependently inhibit the 10 μm acetylcholine (ACh)–induced current response in  Xenopus  oocytes expressing the human muscle (α1β1ϵδ) nicotinic acetylcholine receptor. (  A  ) Representative traces from one oocyte. (  B  ) Current responses in each oocyte were normalized to the 10 μm acetylcholine control responses in each oocyte as described in the Materials and Methods. Each  symbol  represents mean ± SEM of 4–7 oocytes. When no  error bars  are seen, they are smaller than the  symbols  . Roc = rocuronium. 
Fig. 2. Nondepolarizing neuromuscular blocking agents (NMBAs) concentration-dependently inhibit the 10 μm acetylcholine (ACh)–induced current response in  Xenopus  oocytes expressing the human muscle (α1β1ϵδ) nicotinic acetylcholine receptor. (  A  ) Representative traces from one oocyte. (  B  ) Current responses in each oocyte were normalized to the 10 μm acetylcholine control responses in each oocyte as described in the Materials and Methods. Each  symbol  represents mean ± SEM of 4–7 oocytes. When no  error bars  are seen, they are smaller than the  symbols  . Roc = rocuronium. 
Fig. 2. Nondepolarizing neuromuscular blocking agents (NMBAs) concentration-dependently inhibit the 10 μm acetylcholine (ACh)–induced current response in  Xenopus  oocytes expressing the human muscle (α1β1ϵδ) nicotinic acetylcholine receptor. (  A  ) Representative traces from one oocyte. (  B  ) Current responses in each oocyte were normalized to the 10 μm acetylcholine control responses in each oocyte as described in the Materials and Methods. Each  symbol  represents mean ± SEM of 4–7 oocytes. When no  error bars  are seen, they are smaller than the  symbols  . Roc = rocuronium. 
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Fig. 3. Representative traces of vecuronium-induced inhibition of acetylcholine (ACh) currents in  Xenopus  oocytes expressing the human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor subtypes. Vecuronium (Vec) was preapplied for 55 s before a 20-s coapplication with acetylcholine as indicated with the  horizontal bars  . For each receptor subtype, traces shown in each row are from the same oocyte. 
Fig. 3. Representative traces of vecuronium-induced inhibition of acetylcholine (ACh) currents in  Xenopus  oocytes expressing the human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor subtypes. Vecuronium (Vec) was preapplied for 55 s before a 20-s coapplication with acetylcholine as indicated with the  horizontal bars  . For each receptor subtype, traces shown in each row are from the same oocyte. 
Fig. 3. Representative traces of vecuronium-induced inhibition of acetylcholine (ACh) currents in  Xenopus  oocytes expressing the human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor subtypes. Vecuronium (Vec) was preapplied for 55 s before a 20-s coapplication with acetylcholine as indicated with the  horizontal bars  . For each receptor subtype, traces shown in each row are from the same oocyte. 
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Fig. 4. Concentration–response curves of the inhibition of acetylcholine (ACh)–mediated response by nondepolarizing neuromuscular blocking agents (NMBAs) in human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor expressed in  Xenopus  oocytes. For each receptor subtype, two acetylcholine concentrations were applied, one lower and one higher than the EC50: 50 and 300 μm for α3β2and α3β4, 1 and 10 μm for α4β2, and 30 and 100 μm for α7. Control acetylcholine peak current or net charge (α7) responses in each oocyte were normalized to the acetylcholine response with respective nondepolarizing NMBA added, yielding the concentration–response relations. For each receptor subtype, 4–11 oocytes were studied. Data are presented as mean ± SEM. When no  error bars  are seen, they are smaller than the  symbols. 
Fig. 4. Concentration–response curves of the inhibition of acetylcholine (ACh)–mediated response by nondepolarizing neuromuscular blocking agents (NMBAs) in human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor expressed in  Xenopus  oocytes. For each receptor subtype, two acetylcholine concentrations were applied, one lower and one higher than the EC50: 50 and 300 μm for α3β2and α3β4, 1 and 10 μm for α4β2, and 30 and 100 μm for α7. Control acetylcholine peak current or net charge (α7) responses in each oocyte were normalized to the acetylcholine response with respective nondepolarizing NMBA added, yielding the concentration–response relations. For each receptor subtype, 4–11 oocytes were studied. Data are presented as mean ± SEM. When no  error bars  are seen, they are smaller than the  symbols. 
Fig. 4. Concentration–response curves of the inhibition of acetylcholine (ACh)–mediated response by nondepolarizing neuromuscular blocking agents (NMBAs) in human α3β2, α3β4, α4β2, and α7nicotinic acetylcholine receptor expressed in  Xenopus  oocytes. For each receptor subtype, two acetylcholine concentrations were applied, one lower and one higher than the EC50: 50 and 300 μm for α3β2and α3β4, 1 and 10 μm for α4β2, and 30 and 100 μm for α7. Control acetylcholine peak current or net charge (α7) responses in each oocyte were normalized to the acetylcholine response with respective nondepolarizing NMBA added, yielding the concentration–response relations. For each receptor subtype, 4–11 oocytes were studied. Data are presented as mean ± SEM. When no  error bars  are seen, they are smaller than the  symbols. 
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Table 1. Pharmacologic Properties of NMBAs on Human Neuronal nAChRs Expressed in  Xenopus  Oocytes and Activated by Acetylcholine 
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Table 1. Pharmacologic Properties of NMBAs on Human Neuronal nAChRs Expressed in  Xenopus  Oocytes and Activated by Acetylcholine 
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Table 2. Pharmacologic Properties of NMBAs as Inhibitors of Acetylcholine-induced Activation of Human Neuronal nAChRs Expressed in  Xenopus  Oocytes 
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Table 2. Pharmacologic Properties of NMBAs as Inhibitors of Acetylcholine-induced Activation of Human Neuronal nAChRs Expressed in  Xenopus  Oocytes 
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Appendix 1: Pharmacologic Properties of Human Neuronal nAChRs Activated by Acetylcholine and with the Addition of NMBAs 
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Appendix 1: Pharmacologic Properties of Human Neuronal nAChRs Activated by Acetylcholine and with the Addition of NMBAs 
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Appendix 2: Pharmacologic Properties of NMBAs as Inhibitors of Acetylcholine-induced Activation of Human Neuronal nAChRs Expressed in  Xenopus  Oocytes 
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Appendix 2: Pharmacologic Properties of NMBAs as Inhibitors of Acetylcholine-induced Activation of Human Neuronal nAChRs Expressed in  Xenopus  Oocytes 
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Appendix 2: Continued 
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Appendix 2: Continued 
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