Rapacuronium Augments Acetylcholine-induced Bronchoconstriction via  Positive Allosteric Interactions at the M3 Muscarinic Receptor

Clinical formulations of rapacuronium and vecuronium (Organon, West Orange, NJ) were used in these studies. Control vehicle solutions were prepared according to the manufacturer’s product insert. Methoctramine, tetrodotoxin, ranitidine, nifedipine, pyrilamine, N-vanillylnonanamide (capsaicin analog), and atropine were purchased from Sigma (St. Louis, MO). The peptide MEN10376 (NK² antagonist), sendide (NK¹ antagonist), BAY-u9773 (nonselective leukotriene antagonist), and MK571 (D4 leukotriene antagonist) were purchased from Biomol Research Laboratories (Plymouth Meeting, PA). [³H]N-methylscopolamine (³H-NMS; 81 Ci/mmol) was purchased from PerkinElmer (Boston, MA). [³H]-myo -inositol (20 Ci/mmol) was purchased from MP Biomedicals (Irvine, CA). All drugs were dissolved in distilled water except for nifedipine, which was dissolved in dimethyl sulfoxide such that the final concentration of dimethyl sulfoxide in the baths was 0.01%.

All studies were approved by the Institutional Animal Care and Use Committee (College of Physicians and Surgeons, Columbia University, New York, New York). Force measurements were performed on closed guinea pig tracheal rings suspended in organ baths as previously described. Briefly, Hartley male guinea pigs (approximately 400 g) were anesthetized with 50 mg intraperitoneal pentobarbital, and the tracheas were removed promptly and dissected into closed rings comprised of two cartilaginous rings from which mucosa, connective tissue, and epithelium were removed. Trachea were attached using silk threads to a fixed tissue hook in a 2-ml bath (Radnoti Glass Technology, Inc., Monrovia, CA) and a Grass (Grass Telefactor, West Warwick, RI) force transducer coupled to a computer via  BioPac hardware and Acqknowledge 7.3.3 software (Biopac Systems, Inc., Goleta, CA) for continuous digital recording of muscle force. Rings were equilibrated at 1 g isotonic force for 1 h with new Krebs-Henseleit buffer (including 10 μm indomethacin) (pH 7.4, 37°C) added every 15 min while gassed with 95% oxygen and 5% carbon dioxide.

Rings were subjected to (1) two cycles of acetylcholine-induced contractions (increasing cumulative concentrations of 0.1 μm–1.0 mm acetylcholine) followed by two washing protocols: washed only three times or washed nine times with Krebs-Henseleit buffer; (2) a single acetylcholine concentration (0.1 μm); or (3) no pretreatment. Cumulatively increasing concentrations of rapacuronium (1 nm–100 μm), vecuronium (10 nm–100 μm), rocuronium (10 nm–200 μm), or pancuronium (100 nm–100 μm) were then added to the organ baths. In the studies where rapacuronium caused an increase in baseline force, antagonists were added at the peak of contraction in an attempt to reverse the increase in baseline tone. The antagonists included atropine (1 μm), pyrilamine (10 μm; H¹ histamine receptor antagonist), nifedipine (10 μm; calcium channel blocker), sendide (30 μm; NK¹ receptor antagonist), MEN10376 (30 μm; NK² receptor antagonist), MK571 (10 μm; leukotriene D⁴ antagonist), or BAY-u9773 (10 μm; nonselective leukotriene antagonist) and were added to individual baths in an attempt to antagonize this rapacuronium-induced increase in baseline tone.

In a separate series of experiments, these same antagonists were added after the acetylcholine precontractions and washing but 30 min before the addition of cumulatively increasing concentrations of rapacuronium.

To determine whether rapacuronium was increasing baseline tone by activation of nonadrenergic, noncholinergic (NANC) nerves, N-vanillylnonanamide (10 μm; capsaicin analog) was added first to activate and deplete NANC contractile tachykinins. After N-vanillylnonanamide–induced force had returned to baseline (60–90 min), rapacuronium was added to determine whether force increased despite depletion of NANC neurotransmitters. To confirm adequate depletion of contractile NANC neurotransmitters, control tissues received a repeated dose of N-vanillylnonanamide.

The M3 muscarinic receptor classically couples to the stimulation of inositol phosphate synthesis, and this assay was thus used as a measure of M3 receptor function in response to acetylcholine in the absence and presence of rapacuronium. Synthesis of total ³H-inositol phosphates was measured in Chinese hamster ovary (CHO) cells stably expressing the human M3 muscarinic receptor (a kind gift from Tom I. Bonner, Ph.D., Senior Investigator, Laboratory of Genetics, National Institutes of Health, Bethesda, Maryland)23,,24 grown to confluence in 24-well tissue culture plates. Briefly, after overnight loading with ³H-myo -inositol (10 μCi/ml, 20 Ci/mmol) in inositol-free and serum-free Dulbecco’s modified Eagle’s medium, plates were washed three times (37°C, 500 μl Hanks balanced salt solution with 10 mm LiCl). Incubation of cells in a final volume of 300 μl at 37°C for 30 min was performed in the absence and presence of 1 μm acetylcholine. Parallel wells were incubated with 1 μm acetylcholine plus a range of concentrations (0.1–10 μm) of rapacuronium. Reactions were terminated, and total [³H]inositol phosphates recovered by chromatography as described. To confirm that the effects of rapacuronium (and acetylcholine) on inositol phosphate synthesis was via  the M3 muscarinic receptor, two types of control experiments were performed. The M3 muscarinic receptor expressed in CHO-M3 cells was inactivated by alkylation with 1 μm 4-DAMP mustard for 1 h before exposure to acetylcholine or rapacuronium. In a separate cell line that expresses the human M2 but not the M3 muscarinic receptor (CHO-M2; a kind gift from Tom I. Bonner, Ph.D.). cells were exposed to acetylcholine and rapacuronium as above before the measurement of inositol phosphate synthesis.

Allosteric effects are defined by the ability of a substance (e.g. , muscle relaxant) to delay the dissociation of a ligand from a receptor.26,,27,,28 We determined whether clinically relevant concentrations of several muscle relaxants (rapacuronium, vecuronium, rocuronium, and gallamine) had allosteric interactions at the M3 muscarinic receptor by slowing the atropine-induced dissociation of ³H-NMS from M3 muscarinic receptors. CHO-M3 membranes (0.2–0.3 mg/ml) were incubated in triplicate in buffer (20 mm HEPES, 100 mm NaCl, 10 mm MgCl², pH 7.4) with 0.88 nm ³H-NMS (two times the K^d for these membranes) for 70 min at 32°C. Dissociation of ³H-NMS was induced at time 0 min by the simultaneous addition of 1 μm atropine with or without muscle relaxants. The reaction was terminated at 5 min (3 ml cold wash buffer [50 mm Tris-HCl in 0.9% NaCl, pH 7.4]) and collected on glass fiber filters (GF/B) presoaked in 0.1% polyethylenimine using a cell harvester (Brandell, Gaithersburg, MD). Filters were washed three times with 3 ml cold wash buffer. Filters were immersed in 5 ml Econo scintillation fluid, stored overnight, and counted (Beckman Liquid Scintillation 5000 TD; Beckman, Fullerton, CA).16,,20,,29,,30 

Smooth muscle force was continuously recorded using Acqknowledge 3.7.3 software. Statistical analysis was determined using GraphPad Instat 3.01 software (GraphPad Software, Inc., San Diego, CA) by two-way analysis of variance with Bonferroni multiple comparison post test. Statistical significance was defined by a P  value less than 0.05. In all experiments, n represents the number of individual tracheal rings studied or the independent number of experiments for radioligand dissociation studies or inositol phosphate assays.

illustrates a representative force recording of a tracheal ring exposed to two cycles of cumulatively increasing acetylcholine concentrations followed by three washings and then exposed to 1–100 μm rapacuronium, resulting in increased force that returned to baseline after extensive washing (nine times). After extensive washing (i.e. , removing residual acetylcholine), this tracheal ring was unresponsive to another rapacuronium dose response. illustrates the failure of rapacuronium (1–100 μm) to induce contraction in the absence of a precontraction with acetylcholine; however, the contractile effect of rapacuronium subsequently occurs after the same ring is subjected to two cycles of cumulatively increasing acetylcholine concentrations followed by three washes. To further confirm that subthreshold acetylcholine concentrations were necessary for the effect of rapacuronium, we added a low concentration of acetylcholine (0.1 μm) to the baths. This concentration alone had no effect on muscle force but allowed for the dose-dependent potentiation of muscle force of rapacuronium (). Although we did not measure the concentration of acetylcholine in the organ baths, both methods resulted in approximately the same percent increase in baseline force when rapacuronium was added. Rapacuronium (10–100 μm) significantly increased baseline muscle force in the presence (but not in the absence) of subthreshold acetylcholine (P  < 0.001; n = 5; ). Similarly, vecuronium (5–100 μm) significantly increased baseline force in the presence of subthreshold acetylcholine but only at vecuronium concentrations above those achieved clinically (P  < 0.001; n = 5; ). Conversely, pancuronium (2–100 μm) significantly decreased baseline force within clinically achieved concentrations in the presence of subthreshold acetylcholine concentrations (P  < 0.001; n = 4; ). High concentrations of rocuronium (50–100 μm) also increased baseline force in the presence of subthreshold concentrations of acetylcholine, but this contraction was qualitatively different from the other relaxants studied in that it spontaneously and rapidly returned to baseline (data not shown). Taken together, these results suggest that rapacuronium, vecuronium, and rocuronium can increase baseline force under conditions where subthreshold concentrations of acetylcholine are present and are consistent with a positive allosteric effect at M3 muscarinic receptors. Most significantly, however, rapacuronium was the only muscle relaxant studied to increase force within clinically relevant concentrations.

In an attempt to confirm that this rapacuronium-induced increase in resting force in the presence of low acetylcholine concentrations involved muscarinic receptors and did not involve other classic contractile agonists in airway smooth muscle, a series of inhibitors was used either before the addition of rapacuronium to the baths or after the rapacuronium-induced force had reached a peak. 3,,4 illustrate representative force recordings of tracheal rings prepared as above with two acetylcholine dose responses followed by three washes, pretreatment with an inhibitor, and then the addition of rapacuronium. Only atropine prevented () or reversed () the rapacuronium-induced increase in baseline force. Pretreatment of tracheal rings with pyrilamine (H1 histamine receptor antagonist; ), sendide plus MEN10376 (neurokinin receptor 1 and 2 antagonists; ), MK-571 (leukotriene LTD4 antagonist; ), BAY-υ9773 (nonselective leukotriene antagonist; ), or nifedipine (L-type calcium channel blocker; ) had no effect on the subsequent muscle contraction induced by rapacuronium. Furthermore, preactivation and depletion of NANC neurotransmitters by a capsaicin analog did not prevent the subsequent increase in baseline tone elicited by rapacuronium (data not shown). The functional studies in tracheal rings suggesting a positive allosteric effect of rapacuronium at muscarinic receptors led us to perform biochemical (inositol phosphate) and radioligand (dissociation rates) assays in vitro  using human recombinant M3 muscarinic receptors expressed in CHO cells.

Rapacuronium (0.1–1 μm) accentuated the acetylcholine-induced increase in inositol phosphate synthesis in CHO cells expressing the human M3 muscarinic receptor consistent with a positive allosteric effect (P  < 0.05 compared with acetylcholine alone; n = 6; ). Rapacuronium alone had no effect on inositol phosphate synthesis. Higher concentrations of rapacuronium (5–10 μm) decreased inositol phosphate synthesis likely due to either an apparent classic orthosteric antagonist effect or steric hindrance of the orthosteric site by the allosteric binding of rapacuronium on the M3 muscarinic receptor (). Inactivation of the M3 muscarinic receptor by alkylation (1 h of 1 μm 4-DAMP mustard) blocked the ability of acetylcholine or acetylcholine plus rapacuronium to increase inositol phosphate synthesis (). To further confirm that rapacuronium’s potentiation of acetylcholine-induced inositol phosphate synthesis was mediated via  the effect of both acetylcholine and rapacuronium at the M3 muscarinic receptor, these assays were repeated in a CHO cell line devoid of M3 muscarinic receptors (but expressing human M2 muscarinic receptors, which do not couple to inositol phosphate synthesis). Neither rapacuronium nor acetylcholine increased inositol phosphate synthesis in this human M2 muscarinic receptor–expressing cell line (0.5 μm rapacuronium, 93.8 ± 5.3% of control; 1 μm acetylcholine, 99.2 ± 5.1% of control).

Further evidence for the allosteric effects of muscle relaxants at M3 muscarinic receptors was provided by dissociation kinetic radioligand binding studies. Rapacuronium (50–100 μm) significantly inhibited atropine induced ³H-NMS dissociation from the CHO-M3 muscarinic receptors (P  < 0.01; n = 6; ) within clinically relevant concentrations. Gallamine, a muscle relaxant with known allosteric interactions with muscarinic receptors (positive control), significantly inhibited ³H-NMS dissociation at 100 μm (P  < 0.001; n = 6; ). Similarly, vecuronium (50–100 μm) (P  < 0.001; n = 6; ) and rocuronium (P  < 0.05; n = 5; ) significantly inhibited atropine-induced ³H-NMS dissociation; however, the concentrations required are above those concentrations achieved with routine clinical use of these relaxants (peak serum concentrations: vecuronium, 3.2 μm; rocuronium, 4.7 μm).

These data, along with previously published data, demonstrate that rapacuronium has at least two detrimental effects on airway muscarinic receptors leading to bronchospasm. Rapacuronium exhibits a positive allosteric effect at M3 muscarinic receptors, which potentiated the contractile effect of acetylcholine at airway muscarinic receptors. This detrimental airway effect is in addition to and is additive to the previously described preferential antagonism of presynaptic M2 muscarinic receptors described for rapacuronium.

Muscarinic receptors on airway parasympathetic nerves are of the M2 subtype and normally function in an inhibitory autofeedback mode to prevent the further release of acetylcholine. Muscarinic receptors on the airway smooth muscle are of the M2 and M3 subtypes, which function to inhibit relaxation and facilitate contraction, respectively. Irritation of the well-innervated upper trachea (e.g. , by the introduction of an endotracheal tube) initiates a neural reflex that results in the release of acetylcholine from parasympathetic nerves that acts on M2 and M3 muscarinic receptors in airway smooth muscle, resulting in bronchoconstriction. Normally, the release of acetylcholine is terminated by activation of the inhibitory autofeedback receptors on the parasympathetic nerves. The introduction of a large intubating dose of a muscle relaxant with the properties of rapacuronium during this period of parasympathetic nerve activation creates a scenario where negative feedback inhibition of acetylcholine release is blocked (antagonism of neural M2 muscarinic receptor) coincident with augmentation of the contractile effect of acetylcholine at the muscle M3 muscarinic receptor (positive allosteric effect at M3 muscarinic receptor). This dual detrimental effect of rapacuronium at airway muscarinic receptors within clinically relevant concentrations is unique among the muscle relaxants we have studied.10,,11 

Increased baseline force of tracheal rings seen when rapacuronium was added in the presence of subthreshold acetylcholine (i.e. , after an acetylcholine dose response with minimal washing or after a single small dose of acetylcholine) suggests that rapacuronium increases airway tone by a positive allosteric effect at the M3 muscarinic receptor. Two clinical studies are also consistent with this mechanism. Increased peak airway inflating pressures or a decrease in maximal expiratory flow occurred with the administration of rapacuronium during steady state anesthesia, a period not associated with heightened activity of airway parasympathetic nerves.

The increase in muscle force by rapacuronium in the presence of subthreshold acetylcholine in the organ baths was inhibited by atropine but was unaffected by antagonists of histamine, neurokinin, or leukotriene receptors or by inhibition of calcium channels or by previous depletion of NANC neurotransmitters. Taken together, these findings suggest that the mechanism of the effect of rapacuronium on baseline force required acetylcholine and involved muscarinic receptors. These findings again are consistent with a positive allosteric effect of rapacuronium on M3 muscarinic receptors that allows acetylcholine to have an increased effect as an agonist at its orthosteric site.

Neuromuscular blocking agents are known to have the potential for both orthosteric9,,11 and allosteric interactions with muscarinic receptors.15,,16,,33,,34 Gallamine and alcuronium are the most extensively characterized muscle relaxants in terms of muscarinic allosteric interactions, and gallamine serves as the prototypical muscarinic allosteric ligand. Similarly, alcuronium has been shown to enhance binding of muscarinic antagonists , but neither gallamine nor alcuronium has been shown to enhance the binding of agonists  at the muscarinic receptor.37,,38 In our previous studies, gallamine displayed antagonist affinities for the M2 versus  M3 muscarinic receptors that were similar to the antagonist affinity profile seen with rapacuronium (i.e. , M2 > M3), but clinical bronchospasm was not a widely reported problem with gallamine. One interpretation of these findings that may explain the different clinical experiences with these two drugs is that positive allosteric effects at the M3 muscarinic receptor (by rapacuronium) has more deleterious effects in the airway than antagonism of prejunctional M2 muscarinic receptors (by gallamine or rapacuronium). Alternatively, the combination of prejunctional M2 antagonism and postjunctional M3 positive allosterism may synergize to account for the detrimental airway effects seen with rapacuronium. Indeed, rapacuronium is the only muscle relaxant studied that within clinically relevant concentrations displays both M2 muscarinic receptor antagonism and M3 muscarinic receptor positive allosteric effects. Although M1 muscarinic receptors have been described in the parasympathetic ganglia of some species, they were not relevant to the current study, which was performed without activation of the cholinergic nerves in the tracheal rings (i.e. , without electrical field stimulation).

Interestingly vecuronium, another commonly used muscle relaxant, had a similar increase in baseline force in the organ baths (), but this effect occurred at much greater concentrations than those achieved clinically. Likewise, rocuronium led to an increase in baseline force; however, it was not sustained, and it only occurred at concentrations of rocuronium above those achieved clinically. Conversely, pancuronium potently decreased the baseline force, demonstrating either classic orthosteric antagonism or strong negative cooperativity at the airway smooth muscle M3 muscarinic receptor.

These functional studies in the organ baths suggesting allosteric interactions led us to perform biochemical (i.e. , inositol phosphate) and radioligand (i.e. , dissociation kinetic) studies to further characterize the interaction of rapacuronium on the allosteric site on recombinantly overexpressed human M3 muscarinic receptors in CHO cells. Low concentrations of rapacuronium alone had no effect on inositol phosphate synthesis but enhanced acetylcholine-induced increases in inositol phosphate synthesis via  the M3 muscarinic receptor, whereas higher concentrations of rapacuronium decreased acetylcholine-induced inositol phosphate synthesis. This biphasic effect of rapacuronium on acetylcholine-induced inositol phosphate synthesis mimicked the effects on force in intact airway smooth muscle, suggesting that at low concentrations (but still within clinically achieved ranges), rapacuronium has a positive allosteric effect at M3 muscarinic receptors, whereas at high concentrations (above those achieved clinically), rapacuronium can antagonize M3 muscarinic receptors via  blockade of the orthosteric site. The selectivity of this effect for the M3 muscarinic receptor was further confirmed by inactivation of the M3 muscarinic receptor by alkylation with 4-DAMP mustard, which resulted in complete attenuation of the increase in inositol phosphate synthesis seen with acetylcholine with or without rapacuronium. Acetylcholine or rapacuronium also had no effect on inositol phosphate synthesis in another cell line (CHO-M2) that expresses the human M2 muscarinic receptor.

A radioligand dissociation kinetic assay evaluates whether an agent has an allosteric interaction with a receptor. Allosteric agents inhibit the rate of dissociation of a radioligand induced by an orthosteric competitive ligand (e.g. , atropine or acetylcholine for the M3 muscarinic receptor). Rapacuronium at clinically significant doses inhibited atropine-induced dissociation of ³H-NMS, confirming that rapacuronium interacts with the M3 muscarinic receptor in an allosteric manner. Vecuronium and rocuronium also exhibited allosteric interactions with the M3 muscarinic receptor; however, this only occurred at concentrations of vecuronium or rocuronium above those achieved with clinical use.

Another common mechanism associated with the potential of muscle relaxants for bronchoconstriction is histamine release. Histamine release is described after administration of several nondepolarizing muscle relaxants, including curare, atracurium, and mivacurium.39,,40,,41,,42 However, this seems to be an unlikely mechanism for rapacuronium because two studies of increased airway tone during the maintenance phase of general anesthesia attributed their results to selective M2 muscarinic receptor antagonism,31,,32 and in a study of seven adult patients who had development of bronchospasm, histamine concentrations did not increase while the patients were receiving rapacuronium. Our current study is consistent with this in that pyrilamine pretreatment had no effect on rapacuronium-induced increases in muscle force, ruling out release of endogenous histamine from cells within the tracheal rings.

The search for a muscle relaxant with rapid onset and offset has led to the clinical introduction of relaxants with low potency, requiring larger doses to achieve a targeted clinical effect. In the current study, rapacuronium and vecuronium both demonstrated similar increases in baseline force at similar concentrations. However, because rapacuronium is a much less potent agent, its clinically recommended intubation dose was 10–20 times greater (1.5–2.5 mg/kg) than the clinically recommended dose of vecuronium (0.1 mg/kg). Therefore, this allosteric effect at M3 muscarinic receptors is not unique to rapacuronium, because many muscle relaxants act allosterically with muscarinic receptors. The detrimental consequences of rapacuronium at airway muscarinic receptors are most likely due to the requirement of large clinical doses and the achievement of clinical concentrations that allow for positive cooperativity at the airway smooth muscle M3 muscarinic receptor and antagonism of the M2 muscarinic receptor.

In summary, rapacuronium has dual detrimental and unique effects at airway muscarinic receptors. Not only does rapacuronium block M2 muscarinic receptors, potentiating vagal induced acetylcholine release,10,,11 but rapacuronium also binds to an allosteric site on the M3 muscarinic receptor, resulting in an enhancement of acetylcholine activation of this receptor. Both of these mechanisms lead to increased smooth muscle tone and would be additive in their ability to provoke bronchospasm. Despite the removal of rapacuronium from clinical practice, it is important to understand the mechanism by which rapacuronium apparently contributed to fatal bronchospasm. All muscle relaxants developed to date have affinity for muscarinic receptors, and it is likely that the next generation of muscle relaxants that are developed to attempt to replace succinylcholine will also have affinities for muscarinic receptors. We have defined two muscarinic receptor–mediated effects of rapacuronium that likely contribute to its potential to induce bronchospasm. It is therefore prudent that all newly designed muscle relaxants should be evaluated for these properties at concentrations likely to be achieved clinically.