Muscle relaxants affect nicotinic and muscarinic receptors. Interaction of muscle relaxants with muscarinic receptors of human airways has been studied incompletely.
The effects of pipecuronium bromide (long-acting, nondepolarizing) and rocuronium bromide (intermediate-acting, nondepolarizing) on prejunctional and postjunctional muscarinic receptors were studied in 96 isolated human bronchial rings from 12 patients. Contractile isometric responses to electric field stimulation of pilocarpine-stimulated and nonstimulated M2 muscarinic receptors were compared before and after incubation with the two muscle relaxants. The effect on postjunctional muscarinic receptors was studied by comparing acetylcholine concentration-response curves before and after incubation with the two muscle relaxants.
Pipecuronium bromide, but not rocuronium bromide, inhibited pilocarpine-stimulated prejunctional M2 muscarinic receptors. Neither pipecuronium bromide nor rocuronium bromide had significant inhibitory effects on nonstimulated M2 muscarinic receptors and on postjunctional M3 muscarinic receptors.
The inhibitory effect of pipecuronium bromide on pilocarpine-stimulated prejunctional M2 muscarinic receptors occurred at clinical concentrations.
MUSCLE relaxants affect not only nicotinic receptors of neuromuscular junctions, but also muscarinic receptors of airways. 1,2There are several subtypes of muscarinic receptors in the airways. The M3muscarinic receptors, located on the surface of smooth muscle cells, initiate contraction if stimulated. Stimulation of M2muscarinic receptors, located on postganglionic nerve endings of cholinergic nerves, inhibits acetylcholine release. 3M2muscarinic receptors, located on the surface of smooth muscle cells, have several functions, including inhibition of adenylyl cyclase activity and relaxation in response to β2-adrenoceptor stimulation 4and inhibition of large-conductance calcium-activated potassium channels, thus contributing to contractile responses to metacholine. 5Interaction of muscle relaxants with muscarinic receptors of airways has been studied in anesthetized dogs 1and guinea pigs, 2but to our knowledge only the effect of gallamine has been studied in isolated human bronchial rings. 6Because the distribution and relative abundance of receptors may vary between species, 7,8the results of studies in animals cannot be extrapolated to human airways. Therefore, we decided to study the effect of two new muscle relaxants, the long-acting muscle relaxant pipecuronium bromide and the intermediate-acting agent rocuronium bromide (henceforth referred to as pipecuronium and rocuronium, respectively) on muscarinic receptors in human isolated bronchial rings.
Materials and Methods
Bronchi were obtained from 12 patients (aged 40–77 yr) who underwent operation for removal of lung cancer. All patients received general anesthesia for the surgical procedure (the choice of anesthetic drugs was made by attending anesthesiologists). Surgical specimens, remote from the cancerous lesions, were obtained from the surgical waste after tissue had been removed for microscopic examination. The specimens were immersed in chilled, aerated (95% O2and 5% CO2) physiologic salt solution (PSS) of the following composition: NaCl, 110.5 μM; KCl, 3.4 μM; CaCl2, 2.4 μM; MgSO4, 0.8 μM; KH2PO4, 1.2 μM; NaHCO3, 25.7 μM; and dextrose, 5.6 μM. They were transported to the laboratory and stored overnight in aerated PSS at 4°C. Eight bronchial rings (2–4 mm ID) were used from each patient.
Procedure
The bronchi were dissected from surrounding tissue without damaging the epithelium 9and cut into rings of 4 to 5 mm. The rings were suspended between stirrups in 25-ml water-jacketed tissue baths containing aerated PSS with propranolol 10−6M at 37°C. The lower stirrup was connected via a silk string to a stationary hook in the tissue bath; the upper stirrup was connected via a silk string to a force transducer (model FT 03 D; Grass Medical Instruments, Quincy, MA) mounted on a micromanipulator. Two platinum electrodes (1 × 4 cm) were placed on each side of the rings. The rings were stimulated by electric field stimulation (EFS). EFS was provided by a direct-current amplifier (Mayo Clinic, Section of Engineering, Rochester, MN) triggered by an electric stimulator (Model S 44; Grass Medical Instruments). Isometric forces were recorded continuously (TA 4000; Gould, Valley View, OH). The rings were stretched to a resting force of 1 ± 0.4 g, which corresponds to optimal length in human bronchi of this size. 9The lengths of the rings were not changed during the study.
Effects of Pipecuronium and Rocuronium on Postjunctional Muscarinic Receptors
Pipecuronium and rocuronium were gifts from Organon Technika (Turnhout, Belgium). Four rings from each of the 12 patients (48 rings total) were incubated with 10−6M tetrodotoxin for 30 min to block the effect of prejunctional stimulation of muscarinic receptors by acetylcholine. Acetylcholine concentration–response curves were then obtained by cumulatively increasing the concentration of acetylcholine from 10−9to 10−2M in half-log increments. After the acetylcholine concentration–response curves were completed, the rings were washed with PSS until the resting forces were reestablished. The rings were then reincubated with 10−6M tetrodotoxin for 30 min. Three rings from each of 6 of 12 patients (18 rings total) were incubated for 30 min with pipecuronium 10−7M (n = 6), 10−6M (n = 6), or 10−5M (n = 6). The remaining rings from each patient (six rings each) were not incubated with pipecuronium and served as controls. Complete sets of acetylcholine concentration–response curves were again obtained. The same procedure was used in 24 rings from the other six patients to study the effect of 10−7M (n = 6), 10−6M (n = 6), and 10−5M (n = 6) rocuronium.
Effects of Pipecuronium and Rocuronium on Nonstimulated M2Muscarinic Receptors
Four other rings from each of the 12 patients (48 rings total) were stimulated for 30 s at 3-min intervals by EFS (25 Hz, 25 V, 0.5 ms) until three reproducible contractions were observed. Three rings from each of 6 of the 12 patients (18 rings total) were then incubated for 30 min with pipecuronium 10−7M (n = 6), 10−6M (n = 6), or 10−5M (n = 6), and EFS was repeated. One ring from each patient (six rings total) was not incubated with pipecuronium and served as control. The same protocol was used in the 24 rings from the other six patients to study the effects of rocuronium, 10−7M (n = 6), 10−6M (n = 6), and 10−5M (n = 6).
Effects of Pipecuronium or Rocuronium on Pilocarpine-stimulated M2Muscarinic Receptors
Following the same procedure, the same 48 rings were incubated for 3 min with 10−9M pilocarpine, to stimulate M2muscarinic receptors. EFS (25 Hz, 25V, 0.5 ms), 30 s in duration, was then applied at 3-min intervals until the contractions became steady. The pilocarpine concentrations in the tissue bath were cumulatively increased in half-log increments up to a concentration of 10−4M after contractile responses to EFS became constant. After the study, all rings were blotted dry and weighed.
Data Analysis
Active contractile forces (total contractile force minus resting force) in response to EFS or acetylcholine were corrected for the effect of time by assuming the effect of time in control rings to be equal to that in rings incubated with muscle relaxants. 7Mean weights, maximal forces, and resting forces were compared by unpaired t tests. Contractile responses of nonstimulated M2muscarinic receptors before and after incubation with pipecuronium or rocuronium were compared by paired t tests.
Two-factor repeated-measure analysis of variance with the Newman–Keuls post hoc test was used for statistical analysis of the pilocarpine concentration–response curves and concentrations necessary for 50% inhibition of contraction (IC50).
Data were considered to be significantly different if P < 0.05. All data are reported as the mean ± SD.
Drugs
Pilocarpine hydrochloride, DL-propranolol hydrochloride, acetylcholine chloride, and tetrodotoxin were purchased from Sigma (Milan, Italy). All drugs were dissolved in distilled water before use, and fresh solutions were prepared daily.
Results
Resting forces of the rings in which the effect of pipecuronium was studied were not significantly different from rings in which rocuronium was studied (P = 0.09). The maximal forces were significantly different between the two groups (P < 0.03), but the difference in mean ring weights did not achieve statistical significance (P = 0.33).
Effects of Pipecuronium and Rocuronium on Postjunctional Muscarinic Receptors
Pipecuronium (10−7–10−5M) and rocuronium (10−7–10−5M) had no significant effects on acetylcholine concentration–response curves (P > 0.07). Data for pipecuronium are shown in figure 1.
Fig. 1. Acetylcholine concentration–response curves for control bronchial rings and bronchial rings incubated with 10−5M (n = 6) pipecuronium. There was no significant difference between the curves ( P = 0.82), suggesting that pipecuronium had no effect on postjunctional M3muscarinic receptors.
Fig. 1. Acetylcholine concentration–response curves for control bronchial rings and bronchial rings incubated with 10−5M (n = 6) pipecuronium. There was no significant difference between the curves ( P = 0.82), suggesting that pipecuronium had no effect on postjunctional M3muscarinic receptors.
Effects of Pipecuronium and Rocuronium on Nonstimulated M2Muscarinic Receptors
Pipecuronium (10−7to 10−5M) and rocuronium (10−7to 10−5M) had no significant effects on contractile responses to EFS (table 1).
Effects of Pipecuronium and Rocuronium on Pilocarpine-stimulated M2Muscarinic Receptors
Pilocarpine reduced significantly contractile responses to EFS in a concentration-dependent manner (P < 0.0001)(fig. 2). Contractile responses to EFS were increased significantly after incubation with 10−7M pipecuronium at pilocarpine concentrations of 3 × 10−6and 10−5M (P < 0.05), with 10−6M pipecuronium at 10−7to 3 × 10−6M pilocarpine (P < 0.03), and with 10−5M pipecuronium at 10−7M to 3 × 10−5M pilocarpine concentrations (P < 0.01). The IC50s of the pilocarpine concentration–response curves were significantly reduced by pipecuronium 10−6and 10−5M (P = 0.02 and P = 0.0004, respectively), but not with 10−7M pipecuronium (P = 0.46). Conversely, rocuronium (10−7to 10−5M) had no significant effect (P > 0.05) on the contractile responses to EFS at any pilocarpine concentration and did not significantly (P > 0.15) reduce the IC50(table 2).
Fig. 2. Pilocarpine concentration–response curves for control bronchial rings and bronchial rings incubated with pipecuronium 10−7M (n = 6), 10−6M (n = 6), or 10−5M (n = 6). With pipecuronium 10−7M, contractile responses to EFS were significantly increased ( P < 0.05) at 3 × 10−6and 10−5M pilocarpine, with pipecuronium 10−6M at pilocarpine concentrations ranging from 10−7to 3 × 10−6M ( P < 0.03), and with pipecuronium 10−5M at pilocarpine concentrations ranging from 10−7to 3 × 10−5M ( P < 0.01). For clarity only standard deviations for control measurements and for pipecuronium 10−5M are shown.
Fig. 2. Pilocarpine concentration–response curves for control bronchial rings and bronchial rings incubated with pipecuronium 10−7M (n = 6), 10−6M (n = 6), or 10−5M (n = 6). With pipecuronium 10−7M, contractile responses to EFS were significantly increased ( P < 0.05) at 3 × 10−6and 10−5M pilocarpine, with pipecuronium 10−6M at pilocarpine concentrations ranging from 10−7to 3 × 10−6M ( P < 0.03), and with pipecuronium 10−5M at pilocarpine concentrations ranging from 10−7to 3 × 10−5M ( P < 0.01). For clarity only standard deviations for control measurements and for pipecuronium 10−5M are shown.
No significant (P > 0.14) differences in increases in resting forces between control rings and rings incubated with pipecuronium 10−7or 10−6M occurred (fig. 3). However, with 10−5M pipecuronium, increases in resting forces were significantly smaller than those in control rings at pilocarpine concentrations larger than 3 × 10−7M (P < 0.002).
Fig. 3. No significant differences in increases of resting forces with pilocarpine between control rings and rings incubated with pipecuronium 10−7or 10−6M were found ( P > 0.14). The increase in resting force, however, was significantly smaller in rings incubated with 10−5M pipecuronium than in control rings at pilocarpine concentrations > 3 × 10−7M ( P < 0.002).
Fig. 3. No significant differences in increases of resting forces with pilocarpine between control rings and rings incubated with pipecuronium 10−7or 10−6M were found ( P > 0.14). The increase in resting force, however, was significantly smaller in rings incubated with 10−5M pipecuronium than in control rings at pilocarpine concentrations > 3 × 10−7M ( P < 0.002).
Discussion
The two important findings of this study are that pipecuronium had an inhibitory effect on pilocarpine-stimulated prejunctional M2muscarinic receptors, but no effect on nonstimulated prejunctional M2- or on postjunctional M3muscarinic receptors. Rocuronium had neither pre- nor postjunctional inhibitory effects on muscarinic receptors.
Limitations
One must be careful in extrapolating these results obtained in isolated human bronchi to humans in vivo . First, only bronchi with internal diameters of 2–4 mm were studied. The diameters of the studied bronchi may be important because relative abundance of receptors may vary among airway generations. 7,8Second, in intact subjects, the response of airways to muscle relaxants may be modulated by circulating hormones and humoral substances carried in the blood. This may be of particular importance in those muscle relaxants releasing histamine from mast cells. Furthermore, the response of airway smooth muscles may be altered by stimuli from the central nervous system.
Stimulation of muscarinic receptors in airways may result in synthesis and release of prostaglandins, 10which in turn inhibit release of acetylcholine from postganglionic prejunctional cholinergic fibers, thus reducing contractile response to EFS. 11To inhibit the reduction in contractile response, synthesis and release of prostaglandins can be experimentally antagonized by incubation with indomethacin. 7,12But indomethacin can inhibit prejunctional M2muscarinic receptor function in guinea pigs 13; therefore, we decided not to incubate the bronchial rings with indomethacin.
Low concentrations of pilocarpine selectively stimulate prejunctional M2muscarinic receptors, 6,9with no change in resting force. At higher concentrations postjunctional muscarinic M2- and M3muscarinic receptors also are stimulated, 6,9resulting in an increase in resting force. EFS stimulates not only cholinergic nerves, but also excitatory and inhibitory nonadrenergic noncholinergic (i-NANC) nerves. Human airways have few excitatory nonadrenergic noncholinergic nerves, 14making it unlikely that pipecuronium enhanced contractile responses by stimulation of excitatory nonadrenergic noncholinergic nerves. But human airways have i-NANC nerves. 15Inhibition of i-NANC nerves by pipecuronium could contribute to the increased contractile responses to EFS. To exclude this possibility, we determined in eight bronchial rings from two additional patients the effect of 10−7to 10−5M pipecuronium on i-NANC nerve stimulation and found no consistent effect, suggesting that inhibition of i-NANC nerves by pipecuronium did not contribute to the increased contractile responses to EFS.
All patients received a general anesthetic. To remove the anesthetic drugs from the tissue, all bronchi were stored overnight in 100 ml aerated PSS, and they were washed repeatedly for 2 h with PSS on the day of the study before measurements were begun. One cannot exclude the possibility that the drugs were not washed out completely.
Effects of Pipecuronium and Rocuronium on Postjunctional Muscarinic Receptors
Pipecuronium, 10−7to 10−5M, and rocuronium, 10−7to 10−5M, had no significant effects on acetylcholine concentration–response curves. Because the bronchial rings used for acetylcholine concentration–response curves were incubated with tetrodotoxin to interrupt neuronal conduction, only postjunctional effects of acetylcholine could contribute to the contractile response. The unchanged acetylcholine concentration–response curves therefore suggest that pipecuronium and rocuronium did not inhibit postjunctional muscarinic receptors. In canine isolated trachealis muscle, the specific M3antagonist 4-diphenylacetoxy-N -methylpiperidine (4-DAMP) methiode attenuates the response to acetylcholine, suggesting that postjunctional M3muscarinic receptors primarily mediate the contractile response to acetylcholine. 16Also, the characteristics of the antagonist effect of (PA2) 4-DAMP methiode on acetylcholine is consistent with M3receptors mediating contractile responses to acetylcholine. 16By contrast gallamine, a specific M2-receptor agonist, does not alter the contractile response to acetylcholine in canine isolated trachealis muscle, 16suggesting that postjunctional M3- and not M2muscarinic receptors mediate contractile responses to acetylcholine. Assuming human airways respond similarly, the data of this study suggest that pipecuronium and rocuronium had no effect on postjunctional M3muscarinic receptors.
But postjunctional M2muscarinic receptors can also contribute to contractile responses. 4If pilocarpine stimulated postjunctional M2muscarinic receptors, inhibition of postjunctional M2muscarinic receptors by pipecuronium should reduce the resting force in response to pilocarpine. The increase in resting forces with pilocarpine was significantly less after incubation with 10−5M pipecuronium than in control rings, suggesting an inhibitory effect on postjunctional M2muscarinic receptors by this large dose of pipecuronium.
Effects of Pipecuronium and Rocuronium on Nonstimulated M2Muscarinic Receptors
No consistent or convincing evidence for functional prejunctional M2muscarinic receptors in human airways using nonstimulated M2muscarinic receptors has been published. 6,8,17We also did not find consistent or significant increases in contractile responses to EFS in bronchial rings incubated with pipecuronium.
However, functional prejunctional M2muscarinic receptors have been shown in human isolated bronchi using pilocarpine-stimulated M2muscarinic receptors. 6,9More recently, measurement of acetylcholine release in response to vagus nerve stimulation before and after incubation with M2muscarinic antagonists 3has provided more direct evidence for functioning prejunctional M2muscarinic receptors in human airways.
Effects of Pipecuronium and Rocuronium on Pilocarpine-stimulated M2Muscarinic Receptors
Low concentrations of pilocarpine (10−9to 3 × 10−7M) selectively stimulated M2muscarinic receptors. Contractile responses to EFS were increased by pipecuronium 10−6and 10−5M at low concentrations of pilocarpine, suggesting that pipecuronium inhibited prejunctional M2muscarinic receptors. Inhibition of prejunctional M2-receptors has also been shown with gallamine in isolated human bronchi. 6Rocuronium did not increase contractile responses to EFS at the three tested concentrations, suggesting that it had no inhibitory effects on prejunctional M2muscarinic receptors.
The conclusion of an inhibitory effect of pipecuronium on prejunctional M2muscarinic receptors agrees with observations by Okanlami et al. 2in intact guinea pigs. These authors, however, suggested that the M2-inhibitory effect occurred at doses larger than used clinically. However, plasma concentrations of pipecuronium as high as 1.3 × 10−6M occur in humans after bolus injections of 0.07 mg/kg, 18suggesting that pipecuronium may exert inhibitory effects on prejunctional M2muscarinic receptors during clinical practice.
In an elegant recent study, Hou et al. 19determined the binding affinities of muscle relaxants in cells with either pure M2- or M3muscarinic receptor populations. These authors found a higher binding affinity for rocuronium (IC50= 3.0 μM) than for pipecuronium (IC50= 5.8 μM) for M2muscarinic receptors. These results appear to be inconsistent with the results of the current study, which found no effect of rocuronium on M2muscarinic receptors. However, in the study by Hou et al. 19no statistical analyses for the binding affinities were included, and differences in binding affinities between pipecuronium and rocuronium were small compared with differences between pancuronium and pipecuronium or pancuronium and rocuronium.
The authors thank Professors Carla Carli and Roberto Giua, Drs. Luca Anselmi and Maurizio Chiaramondia, and Mauro Zampini for providing the tissue; Drs. Vito Brusasco and David Warner for valuable discussions and for reviewing the manuscript; and Janet Beckman for her consistently outstanding secretarial work.
