A safe and effective ultra-short-acting nondepolarizing neuromuscular blocking agent is required to block nicotinic receptors to facilitate intubation. Rapacuronium, which sought to fulfill these criteria, was withdrawn from clinical use due to a high incidence of bronchospasm resulting in death. Understanding the mechanism by which rapacuronium induces fatal bronchospasm is imperative so that newly synthesized neuromuscular blocking agents that share this mechanism will not be introduced clinically. Selective inhibition of M2 muscarinic receptors by muscle relaxants during periods of parasympathetic nerve stimulation (e.g., intubation) can result in the massive release of acetylcholine to act on unopposed M3 muscarinic receptors in airway smooth muscle, thereby facilitating bronchoconstriction.
Competitive radioligand binding determined the binding affinities of rapacuronium, vecuronium, cisatracurium, methoctramine (selective M2 antagonist), and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP; selective M3 antagonist) for M2 and M3 muscarinic receptors.
Rapacuronium competitively displaced 3H-QNB from the M2 muscarinic receptors but not from the M3 muscarinic receptors within clinically relevant concentrations. Fifty percent inhibitory concentrations (mean +/- SE) for rapacuronium were as follows: M2 muscarinic receptor, 5.10 +/- 1.5 microm (n = 6); M3 muscarinic receptor, 77.9 +/- 11 microm (n = 8). Cisatracurium and vecuronium competitively displaced 3H-QNB from both M2 and M3 muscarinic receptors but had affinities at greater than clinically achieved concentrations for these relaxants.
Rapacuronium in clinically significant doses has a higher affinity for M2 muscarinic receptors as compared with M3 muscarinic receptors. A potential mechanism by which rapacuronium may potentiate bronchoconstriction is by blockade of M2 muscarinic receptors on prejunctional parasympathetic nerves, leading to increased release of acetylcholine and thereby resulting in M3 muscarinic receptor-mediated airway smooth muscle constriction.
NEUROMUSCULAR blocking agents are needed to facilitate tracheal intubation and to maintain muscle relaxation during many surgical procedures. Due to the numerous undesirable side effects of succinylcholine, the search has continued for a nondepolarizing muscle relaxant that can rapidly achieve optimal intubation conditions and that has a rapid termination of action in the event of difficulties in managing the airway.
Rapacuronium, a nondepolarizing muscle relaxant to be used in large doses during intubation as a substitute for succinylcholine, was introduced and then subsequently withdrawn from clinical practice because of a high incidence of bronchospasm 1and at least five fatalities 2that have been attributed to irreversible bronchoconstriction. However, the mechanism(s) by which rapacuronium potentiates bronchoconstriction are unknown. Defining this potential mechanism for muscle relaxant–induced bronchospasm is critical, as additional nondepolarizing muscle relaxants will continue to be introduced into clinical practice to replace succinylcholine.
Bronchospasm during induction of general anesthesia is a potentially life-threatening event. Histamine release by drugs is one known risk factor for bronchospasm. Instrumentation of the well-innervated upper trachea initiates an irritant reflex that results in the release of acetylcholine from parasympathetic nerves that act on M2 and M3 muscarinic receptors in airway smooth muscle, resulting in bronchoconstriction. Normally the release of acetylcholine is terminated by acetylcholine acting on M2 muscarinic auto-feedback receptors present in the presynaptic terminals of postganglionic parasympathetic nerves. However, nondepolarizing muscle relaxants are known to antagonize muscarinic receptors. 3Moreover, nondepolarizing muscle relaxants have different affinities for subtypes of muscarinic receptors. 3Agents that have a higher affinity for the M2 muscarinic receptor than the M3 muscarinic receptor can block these presynaptic parasympathetic M2 receptors, allowing for the augmented release of acetylcholine to act on unopposed M3 muscarinic receptors in airway smooth muscle and thereby resulting in enhanced bronchoconstriction. 4Therefore, the characteristics of a muscle relaxant that together could potentiate vagally induced bronchoconstriction include (1) a higher affinity for M2 versus M3 muscarinic receptors, (2) an affinity for M2 muscarinic receptors but not for M3 muscarinic receptors within a clinically obtained concentration range, and (3) use of large doses of this muscle relaxant during a period of heightened parasympathetic tone (e.g. , intubation). Therefore, we questioned whether rapacuronium exhibits a higher affinity for M2 versus M3 muscarinic receptors, which would increase its potential to precipitate bronchospasm during anesthesia.
Materials and Methods
Rapacuronium and vecuronium were purchased from Organon (West Orange, NJ), cisatracurium was purchased from GlaxoSmithKline (Research Triangle Park, NC), and methoctramine and 4-diphenylacetoxy-N -methylpiperidine methiodide (4-DAMP) were purchased from Sigma (St. Louis, MO). The muscarinic receptor antagonist 3H-QNB was purchased from Amersham Life Science (Arlington Heights, IL). All drugs were dissolved in deionized water. Chinese hamster ovary (CHO) cells stably transfected with complementary DNA encoding the rat M3 muscarinic receptor were purchased from the American Type Culture Collection (Rockville, MD). CHO cells stably transfected with complementary DNA encoding the human M2 muscarinic receptor were provided by Norman Lee, Ph.D. (The Institute for Genome Research, Rockville, Maryland).
Chinese hamster ovary cells stably transfected with either the M2 or the M3 muscarinic receptor were grown in Dulbecco's modified essential medium (DMEM) containing 10% fetal bovine serum and antibiotic agents (100 U/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, 0.25 μg/ml amphotericin B, and 100 U/ml nystatin). Cells were cultured in T500 flasks (500 cm2) at 37°C in a humidified atmosphere of 5% carbon dioxide and 95% air and were harvested at confluence.
Preparation of Chinese Hamster Ovary Cell Membranes
At confluence, culture media were removed from the flasks. The cells were incubated in lysis buffer (10 mm HEPES, 2 mm EDTA, 100 μm phenyl-methane-sulfonyl fluoride; pH, 8.0) at 37°C in a carbon dioxide incubator until detached (20–40 min). Lysed cells were centrifuged at 48,000 g (Sorvall RC-5B with SS-34 rotor; Sorvall, Newton, CT) for 20 min at 4°C. Cold HEPES buffer (100 mm; pH, 7.4) was used to resuspend the pellet after the removal of the supernatant. The lysates were washed two additional times, and the final pellet was resuspended in 6 ml HEPES buffer at 2–5 mg/ml and stored at −70°C until used for radioligand binding assays.
Competitive Radioligand Binding Assays
Twenty-five micrograms of CHO cell membrane protein was incubated in triplicate tubes with 3H-QNB (0.18 nm) and muscle relaxant of increasing concentrations (10−9.5− 10−3m) in binding buffer (40 mm KH2PO4, 160 mm K2HPO4in 50 mm NaCl; pH, 7.4). All radioligand experiments were incubated for 2 h at room temperature in a final volume of 0.25 ml. Preliminary experiments confirmed that the 2-h incubation period was adequate to achieve equilibration binding. All binding experiments were terminated by filtration through GF/B glass fiber filters and were washed three times with 5 ml cold NaCl, 0.9%, using a cell harvester (Brandell, Gaithersburg, MD). Filters were immersed in 5 ml Econo scintillation fluid, stored overnight, and counted in a scintillation counter (Beckman LS 5000 TD; Beckman, Fullerton, CA) with an efficiency of 45–50%. Methoctramine, a selective M2 muscarinic receptor antagonist, and 4-DAMP, a selective M3 muscarinic receptor antagonist, also were used in increasing concentrations (10−9.5− 10−3.5m) to confirm the M2 and M3 muscarinic receptor expression in our CHO cell preparations. The chosen radioligand concentration (0.18 nm) for the competition experiments was 3.6 times the equilibration constant (Kd) of the M2 muscarinic receptor and 1 times the Kdof the M3 muscarinic receptor. The competitive displacement of 3H-QNB by increasing concentrations of muscle relaxants was analyzed by nonlinear regression. A reiterative curve-fitting program, Prism 3.0 (Graph Pad, San Diego, CA), was used to calculate the relative binding affinity (50% inhibitory concentration [IC50]) values using a four-parameter logistic equation (log scale) with the slope factor set to −1.
Comparisons between IC50values for the M2 versus the M3 muscarinic receptor for each muscle relaxant were made using unpaired, two-tailed Student t test.
All of the muscle relaxants studied (rapacuronium, cisatracurium, and vecuronium) displaced 3H-QNB from M2 and M3 muscarinic receptors in a dose-dependent manner (figs. 1–3). In competitive binding experiments using muscle relaxants and 3H-QNB, the IC50values of each muscle relaxant at the M2 and M3 muscarinic receptors were determined by nonlinear regression analysis (table 1). There is a wide variation in the affinity of muscle relaxants for both M2 and M3 muscarinic receptors, and, in general, the affinity for the M3 muscarinic receptor is lower than the affinity for the M2 muscarinic receptor.
Methoctramine, a selective M2 muscarinic receptor antagonist, and 4-DAMP, a selective M3 muscarinic receptor antagonist, also were used in competitive binding experiments to confirm specific expression of M2 and M3 muscarinic receptors in our CHO cell membranes and to allow a comparison between the affinities of the muscle relaxants and classic M2 and M3 antagonists. As expected, methoctramine had a 26-fold higher affinity for receptors expressed in CHO-M2 cells as compared with receptors expressed in CHO-M3 cells (fig. 4), whereas 4-DAMP had a fourfold higher affinity for receptors expressed in CHO-M3 cells as compared with receptors expressed in CHO-M2 cells (fig. 5;table 1). These differential affinities are consistent with those previously reported for the M2 and M3 muscarinic receptor subtypes expressed in CHO cells. 5,6
The primary finding of this study is that although rapacuronium was found to be an antagonist at both M2 and M3 muscarinic receptors, it had an approximately 15-fold greater affinity for blocking the M2 versus the M3 muscarinic receptor. Moreover, the IC50of rapacuronium for the M2 muscarinic receptor, but not for the M3 muscarinic receptor, was within a clinically relevant concentration range (fig. 1;table 1). 7–10Thus, a mechanism that could account for severe bronchospasm resulting from a large intubating dose of rapacuronium is preferential blockade of presynaptic M2 muscarinic receptors on parasympathetic nerves. This would result in the enhanced release of acetylcholine from activated parasympathetic nerves by airway irritant receptors, leading to potent activation of M3 muscarinic receptors on airway smooth muscle and thereby producing bronchoconstriction.
In human airways, parasympathetic innervation predominates in the more central airways, and parasympathetic tone is heightened by the presence of endotracheal tubes or other irritating substances introduced into the airway, 11mediating rapid reflex changes in airway caliber. Irritant receptors are found just beneath the tight junctions of the epithelial lining of the airway. The afferent and efferent connections of these receptors travel in the vagus nerve. Acetylcholine administered exogenously or released from parasympathetic postganglionic nerves induces airway constriction by activating muscarinic receptors on airway smooth muscle. 12Airway smooth muscle expresses both M2 and M3 muscarinic receptors. In airway smooth muscle, M3 muscarinic receptors initiate contraction, while M2 muscarinic receptors inhibit relaxation. The massive increase in acetylcholine release and binding of acetylcholine to M3 muscarinic receptors on the muscle overrides the inhibition of M2 muscarinic receptors on the muscle itself in every model studied, and the net effect is constriction. 13Conversely, blockade of M3 muscarinic receptors on the airway smooth muscle inhibits both vagally induced and exogenously administered acetylcholine-induced airway constriction. Studies in animal models show that pancuronium and gallamine, at clinically relevant concentrations, can potentiate bronchospasm in the setting of vagally induced acetylcholine release. 14,15Antagonism of presynaptic M2 muscarinic receptors was thought to be the mechanism of action.
The clinical experience with rapacuronium has been unique because of the frequency and severity of pulmonary complications. In premarketing studies, broncho-spasm was reported in 3.2% of patients who received rapacuronium compared with 2.1% who received succinylcholine. Subsequent independent studies documented an incidence of bronchospasm of 10.7% with rapacuronium versus 4.1% with succinylcholine. 12During its short period of clinical use, a number of cases of severe bronchospasm were reported. 1,16–19The bronchospasm was so severe in these cases that no end-tidal carbon dioxide was detected. The mechanism of the bronchospasm was unclear from these accounts but had the following characteristics.
Bronchospasm most frequently occurred after intubation but also occurred in the absence of intubation.
Bronchospasm lasted 8–15 min.
Attempted treatments included positive pressure ventilation, nebulized albuterol, increased concentrations of volatile anesthetics, antihistamines, and epinephrine; however, no one treatment seemed to be effective, and if the bronchospasm resolved, it appeared to do so on its own.
A higher incidence of bronchospasm occurred in patients with reactive airway disease.
Only one patient had erythema, possibly denoting histamine release. 16–18
Although most cases of bronchospasm were associated with intubation, at least one of the reported cases occurred in the absence of intubation, and two recent studies demonstrated an increase in peak airway inflating pressures 20or a decrease in maximal expiratory flow 21with the administration of rapacuronium during steady state anesthesia in intubated patients. This suggests that, during general anesthesia, rapacuronium is still capable of facilitating acetylcholine release from postganglionic parasympathetic nerves, leading to increases in airway tone but not overt bronchospasm. Alternatively, rapacuronium may increase airway tone by other mechanisms, such as histamine release. However, neither of these two sets of investigators reported other signs of histamine release, and both proposed selective M2 muscarinic receptor antagonism as a mechanism to account for their findings. 20,21
If M2 muscarinic receptor inhibition with rapacuronium is an important mechanism in eliciting broncho-spasm, other muscle relaxants with a similar profile should also induce bronchospasm during anesthesia. Gallamine has a profile similar to that of rapacuronium, yet bronchospasm seldom was reported with its use. 22Gallamine is the most selective M2 muscarinic receptor antagonist available, 3but it is no longer used as a muscle relaxant because of its inhibition of cardiac M2 muscarinic receptors, which induces tachycardia. Gallamine was not used frequently for intubation due to its slow onset of action. It is plausible that if intraoperative studies had been performed with gallamine using the same techniques as those used to measure airway tone with rapacuronium, similar results would have been found.
An alternative mechanism to account for rapacuronium-induced bronchospasm is the release of histamine from mast cells lining the vessels into which the drug is injected. Histamine release has been described following the administration of several nondepolarizing muscle relaxants, including curare, atracurium, and mivacurium. 23–26However, in a study of 47 adult patients receiving rapacuronium during elective general anesthesia, 7 developed bronchospasm without increases in plasma histamine levels. 19Therefore, it does not appear that histamine release can account for rapacuronium-induced bronchospasm. Although β2-adrenoceptor antagonism is another potential mechanism precipitating bronchospasm, there is also no current evidence suggesting that rapacuronium or any other clinically used muscle relaxants behave as β2-adrenoceptor antagonists.
In the present study, we measured the affinity of muscle relaxants for M2 and M3 muscarinic receptors in CHO cells stably transfected to express pure populations of these receptor subtypes. These cells are a widely used model in which to study binding affinities of receptor agonists or antagonists of the muscarinic receptor family. 5,6,27–30Radioligand binding studies in cells expressing a pure population of receptors eliminate the confounding effects of multiple receptor subtypes competing with differential affinities for the same ligand. This occurs in tissues that express a mixed population of muscarinic receptors, such as airway smooth muscle. 31Furthermore, CHO cells expressing muscarinic receptors are often used as a standard for defining an unknown receptor subtype in a tissue. 29
Muscle relaxants can be grouped into four different categories according to their muscarinic receptor–blocking capabilities in clinical concentration ranges. The first group is composed of muscle relaxants that, at clinically relevant concentrations, bind to the M2 muscarinic receptor with a much greater affinity than to the M3 muscarinic receptor and, thus, could potentiate bronchoconstriction during a period of parasympathetic stimulation (e.g. , rapacuronium, gallamine, cisatracurium). In the current study, cisatracurium also was found to have a higher affinity for the M2 versus the M3 receptor. However, unlike rapacuronium, the clinical use of cisatracurium has not resulted in a large number of reports of isolated bronchospasm. This may be due to the fact that the M2 affinity of cisatracurium is to the far right of the reported concentration range, whereas that of rapacuronium falls to the left of even the lowest reported concentration range. Most reports of bronchospasm following the administration of cisatracurium have been associated with an anaphylactoid reaction, 32–34which likely involves histamine release. Muscle relaxants that block both M2 and M3 muscarinic receptors at clinically relevant concentrations comprise the second group. Pancuronium and rocuronium are also potent M2 muscarinic antagonists, 3but their use has not been associated with bronchospasm because both are also potent antagonists for the M3 muscarinic receptor, particularly at clinically used doses. Muscle relaxants in the third group only have affinities for the M3 muscarinic receptor (e.g. , mivacurium) and, thus, lack the potential to produce bronchospasm via this reflex; in fact, these muscle relaxants may protect against the effect of acetylcholine on M3 muscarinic receptors on airway smooth muscle. However, their ability to release histamine in animals 15,26and in humans 25may overwhelm any potential protective effects on M3 muscarinic receptors and may account for the clinical reports of bronchospasm associated with these muscle relaxants. 35The majority of muscle relaxants are in the fourth and final category, having affinities for both M2 and M3 muscarinic receptors but only at concentrations higher than those typically achieved with routine clinical dosing. Vecuronium, pipecuronium, and doxacurium are examples of such relaxants. 3
Despite the removal of rapacuronium from clinical practice, it is important to understand the mechanism by which it apparently contributed to fatal bronchospasm. All of the muscle relaxants developed to date have affinities for muscarinic receptors, and it is likely that the next generation of muscle relaxants that are introduced to replace succinylcholine also will have affinities for muscarinic receptors. Therefore, if the higher affinity of rapacuronium for M2 versus M3 muscarinic receptors accounts for induced bronchospasm, it seems prudent that all newly designed muscle relaxants be evaluated for their potential to selectively inhibit M2 muscarinic receptors at concentrations used clinically—especially if the intended use of the muscle relaxant is to facilitate tracheal intubation, which requires large doses coinciding with the activation of vagal reflexes by intubation of the trachea.