Interaction of Nondepolarizing Muscle Relaxants with M²and M³Muscarinic Receptors in Guinea Pig Lung and Heart

Dunkin-Hartley guinea pigs weighing 350–450 g were used. Guinea pigs were handled in accordance with the standards established by the United States Animal Welfare Acts set forth in the National Institutes of Health guidelines and the Policy and Procedures Manual published by the Johns Hopkins University School of Hygiene and Public Health Animal Care and Use Committee.

The guinea pigs were anesthetized with urethane (1.5 g/kg) injected intraperitoneally. The carotid artery was cannulated and connected to a Spectromed DTX pressure transducer (Spectromed, Oxnard, CA) for monitoring heart rate and blood pressure. Both jugular veins were cannulated for the administration of drugs. Both vagus nerves were cut and the distal ends were placed on shielded electrodes immersed in a pool of liquid paraffin. The animal's body temperature was maintained at 37 degrees C using a heating blanket. The animals were treated with guanethedine (10 mg/kg intravenous) to deplete norepinephrine, paralyzed with succinylcholine (infused at 10 micro gram *symbol* kg sup -1 *symbol* min sup -1), and their lungs artificially ventilated via a tracheal cannula using a positive pressure, constant volume animal ventilator (Harvard Apparatus Co., South Natick, MA; tidal volume 2.5–3.5 ml, 100–120 breaths/min). In this preparation, succinylcholine has been shown previously to have no effect on baseline pulmonary inflation pressure (Ppi), heart rate, or blood pressure [5]and no effect on vagally induced increases in Ppi. [5]Pulmonary inflation pressure was measured at the trachea with a Spectromed DTX pressure transducer. All signals were displayed on a Grass polygraph (Grass Instruments, Quincy, MA). Partial pressure of oxygen and partial pressure of carbon dioxide were measured from arterial blood samples at the beginning and the end of each experiment (Corning 170 pH/blood gas analyzer; Corning Glass, Medfield, MA).

Basal Ppi was produced by positive pressure ventilation of the guinea pigs' lungs. Bronchoconstriction was measured as an increase in Ppi over the basal pressure produced by the ventilator. [10]The sensitivity of the method was increased by taking the output Ppi signal from the driver to the input of the preamplifier of a second channel on the polygraph. Thus, baseline Ppi was recorded on one channel and increases in Ppi above the baseline were recorded on a second channel at a greater sensitivity. With this method, increases in Ppi as small as 2–3 mmH²O could be amplified and recorded accurately.

Electrical stimulation of both vagus nerves (15 Hz, 0.2 ms, 45 pulses/train) produced increases in Ppi and bradycardia. The voltage was selected within a range of 5–30 V to yield similar increases in Ppi between animals. The nerves were stimulated at 1-min intervals, and at regular intervals, ACh (1 or 2 micro gram/kg) was administered intravenously to monitor the function of the postjunctional muscarinic receptors on the heart and airway smooth muscle.

In separate experiments, cumulative doses of pancuronium (0.01–3.0 mg/kg), mivacurium (0.01–5.0 mg/kg), pipecuronium (0.01–3.0 mg/kg), and doxacurium (0.01–1.0 mg/kg) were administered to guinea pigs. The doses used were based on the ED⁹⁵(effective dose, 95% of subjects) of each drug for neuromuscular relaxation. All drugs were administered intravenously at 5-min intervals and the effects were measured immediately after administration. The effects of each muscle relaxant on pulmonary and cardiac M²muscarinic receptor function were tested by comparing the magnitude of vagally induced bronchoconstriction and bradycardia before and after each dose of the drug. The effect of each drug on postjunctional M³muscarinic receptors was also tested by measuring ACh-induced bronchoconstriction and bradycardia responses before and after each dose of the muscle relaxant. Results are expressed as the ratio of the maximum bronchoconstriction (or bradycardia) after a particular dose of a muscle relaxant to the response before that dose of drug.

At the end of each experiment, intravenous atropine (1 mg/kg), was given to determine whether responses were mediated via muscarinic receptors. In the animals given mivacurium, the histamine-1 receptor antagonist pyrilamine (5 mg/kg intravenous) was administered after the atropine to determine whether the remaining airway and/or cardiac changes seen were related to the release of histamine by mivacurium.

The drugs used in these experiments were: urethane, guanethidine, succinylcholine, pyrilamine, pancuronium, atropine, and ACh, all purchased from Sigma Chemical (St. Louis, MO). Pipecuronium was a gift from Organon (West Orange, NJ) and doxacurium and mivacurium were gifts from Burroughs Wellcome (Research Triangle Park, NC). All drugs were dissolved and diluted in 0.9% NaCl.

All data were expressed as mean+/-SEM. Control responses to vagal stimulation or ACh between groups of guinea pigs were compared using Student's t test for unpaired samples. Dose-response curves were analyzed by analysis of variance with repeated measures. A P value of less than 0.05 was considered significant. Appropriate ED⁵⁰'s (effective dose, 50% of subjects) were obtained from Figure 1, Figure 2, Figure 3, and Figure 4.

At the beginning of each experiment, baseline pulmonary inflation pressure (range 9.6–11.5 cmH²O), heart rate (range 260–300 beats/min) and blood pressure (systolic range 43–50 mmHg; diastolic range 21–26 mmHg) were not different between groups. None of the drug treatments, except mivacurium, had any effect on these parameters.

Electrical stimulation of the vagus nerves (15 Hz, 0.2 ms, 5–40 V, 45 pulses/train) caused an increase in Ppi. Intravenous injection of ACh (1 or 2 micro gram/kg) also caused an increase in Ppi. Atropine (1 mg/kg intravenous) given at the end of each experiment blocked increases in Ppi induced by both electrical stimulation of the vagus nerves and intravenous ACh, indicating that these responses are mediated by muscarinic receptors. In the heart, vagal stimulation and intravenously administered ACh caused bradycardia (measured as a decrease in heart rate) via stimulation of M²muscarinic receptors on cardiac muscle.

Pancuronium at doses of 0.01–1.0 mg/kg potentiated the vagally induced increase in Ppi (P = 0.05). However, at the highest concentration used, 3.0 mg/kg, pancuronium inhibited vagally induced increases in Ppi by 40%(Figure 1, left). This inhibition is probably a result of the large, coincident blockade of M³muscarinic receptors because at this dose, increases in Ppi induced by intravenous ACh (and therefore mediated solely by M³receptors) are almost completely inhibited (open triangles). At all doses used, pancuronium caused an inhibition of ACh-induced increase in Ppi (P = 0.009). This effect of pancuronium on M³receptors was dose-dependent with a maximal inhibition (at 3 mg *symbol* kg sup -1 pancuronium) of 74%. In the heart, pancuronium (0.01–3.0 mg *symbol* kg sup -1 intravenous) inhibited both vagally induced (P = 0.0001) and ACh-induced (P = 0.0001) bradycardia (Figure 1, right). This effect of pancuronium in the heart was dose-related. After administration of 1 mg/kg or more of pancuronium, no bradycardia could be elicited either by vagal stimulation or by intravenous ACh. Atropine (1 mg/kg) abolished the vagally induced and ACh-induced increase in Ppi and bradycardia provoked by pancuronium.

Mivacurium, at doses of 1–5 mg/kg, increased baseline Ppi (Table 1). Pyrilamine, but not atropine, prevented the increase in baseline Ppi (Table 1), indicating that this increase was mediated via histamine release, rather than via a muscarinic receptor. In separate experiments, mivacurium (0.01–1.0 mg/kg) inhibited both vagally induced and ACh-induced increases in Ppi (Figure 2, left). Larger doses of mivacurium (1 and 3 mg/kg) reversed the inhibition of vagally induced, but not ACh-induced increases in Ppi (Figure 2, left). Vagally induced changes in Ppi were not measured at doses larger than 3 mg/kg because of large postjunctional effects of atracurium at these doses. In the heart, mivacurium inhibited both vagally induced (P = 0.0001) and ACh-induced (P = 0.0002) bradycardia similarly in a dose-related manner (Figure 2, right).

Pipecuronium (0.01–3.0 mg/kg) potentiated vagally induced increases in Ppi (P = 0.02) but did not alter ACh-induced increases in Ppi (Figure 3, left). The maximum effect, a 2.5-fold potentiation, was obtained in response to 1.0 mg/kg pipecuronium. Both vagally (P = 0.0001) and ACh-induced (P = 0.0001) bradycardia were inhibited in a dose-related fashion by pipecuronium (Figure 3, right). Atropine (1 mg/kg) abolished vagally induced and ACh-induced increases in Ppi and bradycardia provoked by pipecuronium. Doxacurium (0.01–1.0 mg/kg) had no significant effect on either vagally induced or ACh-induced increases in Ppi or bradycardia (Figure 4).

The relative order of potency for the M²receptor was pancuronium > pipecuronium > mivacurium > doxacurium (Table 2). The relative order of potency for the M³receptor was pancuronium > mivacurium. Pipecuronium and doxacurium had no effect on M³muscarinic receptors at the doses used (Table 2).

Vagal nerve stimulation in animals is an accepted model to study drug effects on bronchoconstriction. [8,9]The increase in pulmonary inflation pressure over the basal inflation pressure produced by the ventilator was used as our measure of bronchoconstriction, [10,11]because increases in pulmonary inflation pressure during vagal stimulation reflect primarily an increase in lung resistance with little change in dynamic compliance. [12,13]Cholinergic efferent nerve fibers of the parasympathetic nervous system pass down the vagus nerve and synapse in the smooth muscle of the airway wall. In the human lung, the parasympathetic nervous system regulates airway caliber, both in the baseline state and after mechanical, chemical, or physical stimulation, via release of ACh onto postjunctional muscarinic M³receptors of airway smooth muscle. However, previous studies from this laboratory have demonstrated the presence of prejunctional M²receptors, which inhibit ACh release, thus limiting vagally induced bronchoconstriction. [5]Thus, blockade of M³receptors inhibits bronchoconstriction induced by either vagal nerve stimulation or intravenous ACh, whereas blockade of M²receptors potentiates bronchoconstriction induced by vagal nerve stimulation. The net effect of a muscarinic antagonist on bronchoconstriction provoked by vagal nerve stimulation depends on its relative potency as an M²or M³antagonist. M², but not M sub 3, antagonists inhibit bradycardia induced by either vagal nerve stimulation or intravenous ACh.

Our current study demonstrates that pancuronium is a potent antagonist for both M²and M³receptors, thus confirming previous studies in guinea pigs [8]and dogs. [9]In the lungs, the M²antagonist effect must predominate between 0.03 mg/kg and 1 mg/kg, resulting in potentiation of vagally induced bronchoconstriction. At clinical doses for humans, 0.05–0.1 mg/kg, [14]the potentiation would be small (1.2-fold;Figure 1), assuming that the dose-response relationship of the human and guinea pig airway are similar. At larger doses, the M³effect predominates and the potentiation is reversed. Pancuronium also inhibited vagally mediated bradycardia consistent with its M²-blocking properties.

Our findings that mivacurium inhibited bradycardia and bronchoconstriction (measured as an increase in Ppi), induced by either intravenous ACh or vagal nerve stimulation, suggest that mivacurium blocks both M²and M³receptors. It appears to be less potent than pancuronium as an M²antagonist. Thus, mivacurium does not potentiate vagally induced bronchoconstriction at doses between 0.03 and 0.3 mg/kg. The potentiation of vagally induced, but not ACh-induced, bronchoconstriction at 1- and 3-mg/kg doses could be caused by blockade of M²receptors. However, it is more likely caused by enhanced ACh release by histamine, [15,16]because this effect occurred at doses at which histamine was released [17]and was prevented by atropine. The increase in baseline airway tone at these same concentrations is likely a result of the direct effects of histamine on H¹receptors on airway smooth muscle, because this effect occurred at doses at which histamine is released [17]and was prevented by pyrilamine, an H¹receptor antagonist.

Our findings that pipecuronium potentiated bronchoconstriction induced by vagal stimulation, with no effect on bronchoconstriction induced by intravenous ACh, and inhibited bradycardia, suggest that pipecuronium is selective for M²muscarinic receptors. Pipecuronium, however, is less potent than pancuronium as an M²antagonist, only producing significant potentiation of vagally induced bronchoconstriction at doses greater than 0.3 mg/kg. Because the concentrations of pipecuronium used clinically are tenfold less, it is unlikely that pipecuronium will have any clinically relevant effects on muscarinic receptors. Our results with doxacurium showing neither potentiation nor inhibition of bronchoconstriction or bradycardia suggest that doxacurium has no effect on either M²or M³receptors at doses up to 1 mg/kg.

ACh release from vagal nerve stimulation may be inhibited by prejunctional beta-adrenoceptors. [13]Thus, inhibition of these receptors by pancuronium and pipecuronium could explain our results. However, all animals were pretreated with guanethidine to deplete norepinephrine, which rules out the possibility that pancuronium and pipecuronium potentiated bronchoconstriction by inhibiting prejunctional beta adrenoceptors.

M²and M³antagonist activity by the neuromuscular blockers studied generally occurred at concentrations that were higher than the ED⁹⁵for the nicotinic receptor on skeletal muscle, except for pancuronium (Table 2). Although pancuronium is a potent antagonist of the M²receptor, it is also a potent antagonist of M³receptors in the clinical range, inhibiting bronchoconstriction. Thus, the net effect of pancuronium on any potential irritant-induced bronchoconstriction would be expected to be small. Mivacurium is a more potent M³than M²antagonist, and should not potentiate irritant-induced bronchoconstriction in the clinical range. In contrast, pipecuronium is not an antagonist for M³receptors but does inhibit M²receptor function. However, this occurs only at doses larger than are used clinically.

In summary, the most important findings of this study are: mivacurium is a muscarinic antagonist with similar potencies for both M sub 2 and M³receptors; pipecuronium is an antagonist for M²but not M³muscarinic receptors; and doxacurium has no effect on either M²or M³receptors. Although pipecuronium is an M²receptor antagonist and can potentiate reflex-induced bronchoconstriction, this effect occurs only at doses that are larger than those used clinically. If these studies in the guinea pig are relevant to the clinical situation in humans, pipecuronium, mivacurium, and doxacurium, in concentrations in the clinical range, should have few adverse effects on airway tone and reactivity in humans.