Introduction

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The parasympathetic nerves play an important role in controlling the caliber of airways, and this neuronal mechanism is mediated through the release of ACh that binds to muscarinic receptors on airway smooth muscle. Indeed, muscarinic receptor blockers are useful in the treatment of airway constriction (Eglen and Watson, 1996). Muscarinic receptors have been classified as five subtypes, such as M₁, M₂, M₃, M₄ and m₅ (Caulfield, 1993; Doods et al., 1987; Eglen and Whiting, 1986; Hammer et al., 1980; Hulme et al., 1990), although a physiological significance of the m₅ gene product has not been identified. In the rabbit, Bloom et al. (1987b) found that PZ was more effective in inhibiting vagally mediated bronchoconstriction than in inhibiting vagally induced bradycardia, compared with the actions of atropine. However, they do not examine whether the dose of PZ that blocks vagally mediated bronchoconstriction inhibits ACh-induced bronchoconstriction. Administration of PZ at a relatively higher dose acts as a nonselective blocker for M₂ receptors in the heart as well as for M₃ receptors on airway smooth muscle (Eglen and Whiting, 1986; Maclagan and Faulkner, 1989; Watson et al., 1995). The study on guinea pig airways suggests that M₁ receptors may be neuronal receptors in the sympathetic nerves innervating the airway smooth muscle (Maclagan et al., 1989). However, sympathetic innervation of the airway smooth muscle is not found in the rabbit (Mann, 1971). On the basis of these observations, the function of M₁ receptors in the rabbit pulmonary parasympathetic and sympathetic nerve pathways remains uncertain.

The purposes of the present study were to define whether M₁ receptors function as the excitatory receptors in the parasympathetic nerve pathways in the trachea and lungs and, if so, whether this function is independent of M₂ receptors, M₃ receptors and beta adrenoreceptors. Because SARs are a good indicator of bronchoconstriction (Bartlett et al., 1976), the effects of vagal stimulation and ACh administration on SARs and total lung resistance (R_L) were compared before and after PZ in the presence and absence of propranolol, gallamine or 4-DAMP in anesthetized, artificially ventilated rabbits. To obtain further information on the mechanism of M₁ receptor actions on airway smooth muscle and parasympathetic ganglia, additional experiments were designed to examine whether atropine and hexamethonium inhibit vagally and ACh-induced bronchoconstrictions equally. The cut right vagus nerve was used for electrical stimulation, whereas SARs were recorded from the cut left vagus nerve. Experiments were performed in anesthetized, artificially ventilated rabbits.

	Materials and Methods

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Animal preparation. Thirty-four rabbits of either sex (2.5-3.5 kg) were anesthetized with urethane (1.0 g/kg intrapentoneal). The trachea and esophagus were retracted rostrally to obtain space for paraffin pool. The superior and recurrent laryngeal nerves were bilaterally identified and sectioned in advance. The femoral artery was cannulated for measurement of SAP with a pressure transducer. The femoral vein was also cannulated for administration of additional doses of urethane (0.1-0.2 g/kg/hr intravenous). A polyethylene catheter was also inserted into the right external jugular vein, and its tip was advanced into the right atrium for administration of drugs or a 0.9% NaCl solution. The rectal temperature was maintained at ~37°C with a heating pad. The vagus nerves were then exposed and sectioned. The animals were initially paralyzed with administration of suxamethonium (20 mg/kg intramuscular) and artificially ventilated with air. Then, additional doses of this muscular relaxant were maintained with a constant intravenous infusion at 10 µg/kg/min. The stroke volume and the frequency of the respirator were set at 10 ml/kg and 35 cycles/min, respectively. Tracheal CO₂ partial pressure ranged from 32 to 35 mm Hg.

Measurement of lung mechanics. Respiratory airflow () was measured by connecting the tracheal tube to a pneumotachograph and a differential pressure transducer. P_T was measured by connecting a polyethylene catheter inserted into the tracheal tube to a differential pressure transducer, in which one arm opened to the atmosphere. R_L was measured using the manual graphic method reported by Norlander et al. (1968).

Measurement of slowly adapting pulmonary stretch receptors. The peripheral end of the cut left vagus nerve was desheathed. Then, a thin filament containing afferent nerve fibers was separated and placed on a unipolar silver electrode. In a pool of warm liquid paraffin (37-38°C), the small filament was split until single-unit activity of SARs had been electrically isolated. Afferent impulses of SARs were identified, on the basis of their characteristic firing patterns during inflation, as follows. (1) They fired during both inflation and deflation (low-threshold SARs). (2) The increases in SAR discharge frequency were proportional to the stroke volume of the respirator. (3) The discharge of SARs continued as long as the tracheal tube was occluded in a hyperinflated condition. The SAR activity was amplified by a preamplifier, selected by a window discriminator for counting numbers of the impulses, monitored on an oscilloscope and recorded on a polygraph. The location of the receptors recorded was determined using a small balloon catheter (Matsumoto et al., 1996). When the tip of the catheter reached the carina, the balloon was inflated and pulled rostrally. If the receptors were located below the carina, they were not stimulated by pulling the inflated balloon catheter. Thirty-four SARs located below the carina were obtained in 34 rabbits.

Vagal stimulation. The peripheral end of the cut right vagus nerve was desheathed in a paraffin pool and placed on the stimulating electrodes. To prevent electrical noise, the stimulating electrodes were covered with a rubber sheet. The vagus nerve was stimulated at 5 to 20 Hz, 13 V, and 0.2 msec for ~30 sec.

Experimental design. Five different types of experiments were performed. In 6 different SARs in 6 rabbits, the effects of vagal stimulation (5, 10 and 20 Hz) and ACh injection (1 and 3 µg/kg) on SAR activity and R_L were determined. The same experiments were repeated with 3 min of PZ at each dose (3, 10 and 30 µg/kg). Similarly, the same experimental maneuvers as described above were repeated in other series of experiments using propranolol (1 mg/kg)-treated animals (n = 6). The effectiveness of beta adrenoreceptor blockade seen after propranolol administration was assessed by the presence of a further reduction of SAP during vagal stimulation at 20 Hz. When the propranolol action was reduced, additional doses (0.3-0.4 mg/kg) of this beta adrenoreceptor blocker were administered as needed. In 12 different SARs in 12 rabbits, the responses of SAR activity and R_L to vagal stimulation (5-20 Hz) and ACh administration (1 and 3 µg/kg) were examined before and after gallamine (n = 6) and 4-DAMP (n = 6) with different doses (3, 10 and 30 µg/kg). Finally, changes in SAR activity and R_L in response to vagal stimulation (5-20 Hz) and ACh injection (1 and 3 µg/kg) were compared before and after atropine (2 mg/kg, n = 5) and C₆ (20 mg/kg, n = 5) in 10 different SARs in 10 rabbits. The absence of PZ action was confirmed by restoring the SAR and R_L responses to vagal stimulation and ACh injection. After each experiment on vagal stimulation and ACh injection, lung compliance was restored to the control by inflating the lungs for several respiratory cycles with a volume of 30 ml/kg.

Drugs. PZ (Sigma Chemical, St. Louis, MO; 10 mg/ml), ACh (Daiichiseiyaku Tokyo, Japan; 100 mg/ml), propranolol (Sumitomoseiyaku Tokyo, Japan; 10 mg/ml), gallamine (Sigma; 10 mg/ml), 4-DAMP (Funakoshi Tokyo, Japan; 10 mg/ml) and atropine (Sigma; 10 mg/ml) were dissolved and diluted with distilled water.

Data analysis. During control conditions, the impulses of SARs and the values of P_T and were measured over several respiratory cycles, and R_L was calculated. The average activities of SARs during inflation and deflation were expressed as impulses/sec. The average value of R_L was expressed as cm H₂O/liter/sec. The SAR responses to vagal stimulation (5, 10 and 20 Hz) were measured for the last three ventilatory cycles in the stimulation, and the average activities of receptors during inflation and deflation were expressed as impulses/sec. The SAR responses to ACh injection (1 and 3 µg/kg) were calculated by counting all action potentials between the onset of increased activity and recovery to the control level, and the average activities of receptors during inflation and deflation were expressed as impulses/sec. Similarly, the average values of R_L were expressed as percent change from the control. The two measured parameters (SAR activity and R_L) were expressed as mean ± S.E.M. of a number (n) of observations. Statistical significance of the effects of PZ in the presence and absence of propranolol, gallamine, 4-DAMP, atropine and C₆ on the responses of SAR activities and R_L to vagal stimulation and ACh injection was initially calculated using a one-way analysis of variance for repeated measurements. Then, the data were analyzed by means of the modified t statistics and further assessed by Bonferroni's test for one comparison (k = 1) to the control. In addition, two-sample t test with Welch's correction was used to determine the differences between propranolol (1 mg/kg)-untreated and -treated animals in the responses of SAR activities and R_L to vagal stimulation (5-20 Hz) and ACh injection (1 and 3 µg/kg) before and after PZ at each dose (3, 10 and 30 µg/kg). A value of P < .05 was considered statistically significant.

	Results

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Effect of PZ in the presence and absence of propranolol on the responses of SARs and R_L to vagal stimulation and ACh injection. Electrical stimulation of the right vagus nerve (5 to 20 Hz, 13 V, 0.2 msec) caused an increase in SAR activity during both inflation and deflation, and the response was associated with an increase in P_T and a decrease in . The changes in SAR activity, P_T and induced by vagal stimulation became more pronounced as the stimulus frequencies were increased (fig. 1, A-C). In the same animal shown in figure 1, administration of ACh (1 and 3 µg/kg) produced the increases in SAR activity and P_T and a decrease in , and these changes were more pronounced by increasing the dose of ACh (fig. 2, A and B). Before PZ administration, the inspiratory and expiratory discharges of SARs were 57.8 ± 1.8 and 32.1 ± 1.4 impulses/sec, respectively, and baseline R_L was 23.4 ± 3.1 cm H₂O/liter/sec. The bronchoconstrictor responses (measured as significant increases in SAR activity and R_L) to vagal stimulation (5-20 Hz) and ACh injection (1 and 3 µg/kg) were frequency and dose dependent, respectively, and those responses obtained were not significantly altered by administration of PZ, a specific M₁ receptor antagonist, at 3 µg/kg. The bronchoconstrictor responses to vagal stimulation were significantly inhibited by PZ administration at 10 µg/kg, whereas the M₁ receptor antagonist at this dose had no significant effect on ACh-induced bronchoconstriction. When the dose of PZ was increased to 30 µg/kg, it did not significantly alter ACh-induced bronchoconstriction (fig. 3A). To test whether beta adrenoreceptors modify the functional role of M₁ receptors in evoking vagally mediated bronchoconstriction, animals were pretreated with 1 mg/kg propranolol (fig. 3B). The inspiratory discharge of SARs under control conditions was 55.8 ± 1.9, and baseline R_L was 24.5 ± 3.5 cm H₂O/liter/sec. PZ at 3 µg/kg had no effect on either vagally mediated bronchoconstriction or ACh-induced bronchoconstriction. The bronchoconstrictor responses to vagal stimulation (5-20 Hz) were significantly inhibited by pretreatment with PZ (10 µg/kg), which did not significantly alter ACh-induced bronchoconstriction. The effects of PZ on the vagally and ACh-induced bronchoconstrictions were made by comparing untreated and propranolol-treated rabbits (fig. 3, A and B). Propranolol treatment did not cause any significant change on the average discharges of SAR activity in inflation and the values of R_L but augmented those responses after ACh injection. The dose-related inhibitory effects of PZ on the increases in SAR activity during inflation and R_L induced by vagal stimulation in the absence of propranolol were similar to those in the presence of beta adrenoreceptor blockers. The responses of inspiratory SARs and R_L to ACh injection in the absence and presence of propranolol were not significantly influenced by PZ at any dose. Furthermore, SAR activity during deflation was weaker but showed essentially the same responses to drugs as SAR activity during inflation.

View larger version (56K): [in this window] [in a new window]   Fig. 1.   Effects of vagal stimulation on PT, , SAR activity and SAP. , Period of vagal stimulation. A, Vagal stimulation at 5 Hz. B, Vagal stimulation at 10 Hz. C, Vagal stimulation at 20 Hz.

View larger version (61K): [in this window] [in a new window]   Fig. 2.   Effects of ACh injection on PT, , SAR activity and SAP. , Administration of ACh. A, injection at 1 µg/kg. B, ACh injection at 3 µg/kg.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Changes in SAR activity during inflation and RL in response to vagal stimulation (VS) and ACh injection before and after PZ in propranolol-untreated (A) and -treated (B) rabbits. , VS (5 Hz); , VS (10 Hz); , VS (20 Hz); , ACh (1 µg/kg); , ACh (3 µg/kg). 0, Control before PZ; 1, after PZ (1 µg/kg); 3, after PZ (3 µg/kg); 10, after PZ (10 µg/kg). Vertical bars are mean ± S.E.M. (n = 6). *P < .05 for significant difference from control values. P < .05 for significant difference form PZ effects. **P < .05 for significance from propranolol (1 mg/kg) effects.

Effects of gallamine on the responses of SARs and R_L to vagal stimulation and ACh injection. As shown in figure 4, gallamine (3-30 µg/kg) did not significantly alter either vagally mediated bronchoconstriction or ACh-induced bronchoconstriction.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Change in SAR activities during inflation and deflation and RL in response to vagal stimulation (VS) and ACh injection before and after gallamine. , VS (5 Hz); , VS (10 Hz); , VS (20 Hz); , ACh (1 µg/kg); , ACh (3 µg/kg). 0, Control before gallamine; 1, after gallamine (1 µg/kg); 3, after gallamine (3 µg/kg); 10, after gallamine (10 µg/kg). Vertical bars are mean ± S.E.M. (n = 6). *P < .05 for significant difference from control values.

Effects of 4-DAMP on the responses of SARs and R_L to vagal stimulation and ACh injection. At the dose that inhibited the SAR and R_L responses to ACh injection, 4-DAMP (3 µg/kg) augmented vagally mediated bronchoconstriction. At doses larger than 3 µg/kg, 4-DAMP dose-dependently inhibited both vagally and ACh-induced bronchoconstriction (fig. 5).

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Changes in SAR activities during inflation and deflation and RL in response to vagal stimulation (VS) and ACh injection before and after 4-DAMP. , VS (5 Hz); , VS (10 Hz); , VS (20 Hz); , ACh (1 µg/kg); , ACh (3 µg/kg). 0, Control before 4-DAMP; 1, after 4-DAMP (1 µg/kg); 3, after 4-DAMP (3 µg/kg); 10, after 4-DAMP (10 µg/kg). Vertical bars are mean ± S.E.M. (n = 6). *P < .05 for significant difference from control values. P < .05 for significant difference from PZ effects.

Effects of atropine and C₆ on the responses of SARs and R_L to vagal stimulation and ACh injection. The bronchoconstrictor responses to vagal stimulation (5-20 Hz) and ACh injection (1 and 3 µg/kg) were completely blocked by administration of atropine at 2 mg/kg (fig. 6). As shown in figure 7, pretreatment with C₆ at 20 mg/kg abolished vagally mediated bronchoconstriction but had no significant effect on ACh-induced bronchoconstriction.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Changes in SAR activities during inflation and deflation and RL in response to vagal stimulation (VS) and ACh injection before and after atropine. , VS (5 Hz); , VS (10 Hz); , VS (20 Hz); , ACh (1 µg/kg); , ACh (3 µg/kg). Vertical bars a re mean ± S.E.M. (n = 5). *P < .05 for significant difference from control values. P < .05 for significant difference from atropine effects.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Change in SAR activities during inflation and deflation and RL in response to vagal stimulation (VS) and ACh injection before and after hexamethonium (C6). , VS (5 Hz); , VS (10 Hz); , VS (20 Hz); , ACh (1 µg/kg); , ACh (3 µg/kg). Vertical bars are mean ± S.E.M. (n = 5). *P < .05 for significant difference from control values. P < .05 for significant difference from C6 effects.

	Discussion

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The majority of muscarinic receptors in the rabbit peripheral lung are of the type with high affinity for PZ, which is thought to be a putative M₁ receptor subtype (Bloom et al., 1987a). Other muscarinic receptors (e.g., M₂ and M₄ subtypes) are also found in the peripheral lung tissues of the rabbit (Dörje et al., 1991; Lazareno et al., 1990), but the function and location of M₄ receptors remain unclarified. PZ is 22-fold more potent than atropine, a nonselective M₁ and M₃ receptor antagonist, in inhibiting the vagus nerve stimulation-induced contraction in the rabbit main stem bronchial ring with intact vagal innervation (Bloom et al., 1988). In the experiments to determine the inhibition of preganglionic (vagus nerve stimulation) and postganglionic (field stimulation) contractions of the rabbit isolated bronchus/trachea by different muscarinic receptor antagonists, PZ can differentiate >10-fold between M₁ and M₃ receptors in comparison with the inhibitory potency of other muscarinic receptor blockers such as telenzepine and (+)-biperiden, but this M₁ receptor blocker is not more potent than atropine in inhibiting vagally stimulated contraction compared with field-stimulated contraction (Eltze and Galvent, 1994). In an in vivo preparation, PZ can block the bronchoconstrictor response to vagal stimulation in the rabbit (Bloom et al., 1987b) and dog (Beck et al., 1987), but these studies do not describe the exact dose of PZ, which has little action on ACh-induced bronchoconstriction. In the rabbit, Maclagan and Faulkner (1989) reported that the dose of PZ of >1 µM/kg (400 µg/kg) was equipotent in inhibiting vagally and ACh-induced bronchoconstrictions. They concluded that there is no evidence to support the presence of a function of M₁ receptors in the rabbit lung. From these observations, the functional role of M₁ receptors in modifying vagally stimulated contraction of airway smooth muscle remains to be determined.

In this study, both vagal stimulation and ACh injection caused the increases in inspiratory and expiratory discharges of SARs, and these changes were frequency and dose dependent. Enhancement of both inspiratory and expiratory SAR discharges is thought to be mediated through activation of the receptors that are mechanically connected in parallel with the smooth muscle in the central and upper airways (Miserocchi and Sant'Ambrogio, 1974). The receptors in this study are therefore considered to be located in the conducting airways.

The increases in SAR discharges during vagal stimulation and after ACh injection corresponded to an increase in R_L in both propranolol-treated and -untreated animals. Because the change in R_L is a good indicator of central airway constriction (Blaber et al., 1985), vagus nerve-stimulated and ACh-induced SAR increases may reflect contractions of the smooth muscle in the relatively larger airways. The analysis of the SAR activity recorded from the cut left vagus nerve and the change in R_L or dynamic lung compliance provide the method to quantify the bronchoconstriction evoked by electrical stimulation of the cut right vagus nerve in anesthetized, artificially ventilated rabbits with bilateral vagotomy (Matsumoto, 1996).

Pretreatment with propranolol facilitates the bronchoconstrictor responses to vagal stimulation and ACh injection in guinea pigs (Maclagan et al., 1989), although no such effect is observed in the rabbit (Maclagan and Faulkner, 1989). Before PZ administration, pretreatment with propranolol significantly potentiated ACh-induced bronchoconstriction (the increases in SAR activity during inflation and R_L) but had no significant effect on vagally induced bronchoconstriction. The difference in propranolol action in this study was confirmed by evidence showing that the airway smooth muscle of the rabbit has no sympathetic innervation (Mann, 1971). Augmentation of ACh-induced bronchoconstriction in the presence of beta adrenoreceptor antagonists may therefore depend on the magnitude of blockade of circulating catecholamines from the adrenal gland.

The classic view of neurotransmission through autonomic ganglia is believed to be mediated by nicotinic receptors only, which are responsible for the fast EPSP and are blocked by C₆. Nevertheless, muscarinic receptors are also involved in the mediation of ganglionic transmission represented by a slow EPSP and a slow inhibitory postsynaptic potential in both parasympathetic and sympathetic ganglia (Brown and Constanti, 1980; Gallagher et al., 1982; Kilbinger and Wessler, 1980; North et al., 1985; Suzuki and Volle, 1979). Bloom et al. (1988) investigated the effectiveness of PZ as an antagonist acting on vagus nerve- and field-stimulated contractions of the rabbit main stem bronchus and found that C₆ blocked vagally mediated contractions but did not effect any significant change on field stimulation. The similarity of the C₆ effect was found in the present study. It seems reasonable to speculate that C₆ blocks nicotinic receptors in the ganglionic transmission of the parasympathetic pathway. Furthermore, the minimum dose of PZ, which elicited a significant inhibition of vagally mediated bronchoconstriction, was 10 µg/kg, and PZ at this dose had no significant effect on ACh-induced bronchoconstriction. Even when the dose of PZ increased to 30 µg/kg, this M₁ receptor blocker did not significantly influence the SAR and R_L responses to ACh injection. When considering these results together, it seems reasonable to speculate that M₁ receptors on neurotransmission of the tracheal ganglia may have a facilitatory effect on nicotinic receptors. The lack of a dose-related effect of PZ (3 µg/kg) is probably due to the dose being insufficient to inhibit the fast EPSP and slow EPSP. However, in the present study, the effect of field stimulation on the responses of SARs and R_L before and after PZ treatment was not examined. The possibility that M₁ receptors exert their effect at the level of the parasympathetic nerve terminals cannot therefore be ruled out, as suggested by Bloom et al. (1988).

PZ selectively inhibits the synaptic transmission in the rabbit superior cervical ganglion by blocking the slow EPSP (Ashe and Yarosh, 1984). In addition, [³H]PZ selectively binds with high affinity to muscarinic receptors in the rabbit sympathetic ganglia (Bloom et al., 1987a). However, in this study, the similarity of dose-related effects of PZ in the absence and presence of propranolol was found on both vagally and ACh-induced bronchoconstrictions. The results could provide little support to the concept that M₁ receptors that facilitate the sympathetic transmission function in the airway of the rabbit.

Because the selectivity of PZ is lost at higher doses (Eglen and Whiting, 1986), it is important to confirm the selectivity of PZ compared with the actions of M₂ and M₃ receptor antagonists at the same dosages for PZ treatment. Nerve endings are thought to be autoreceptors inhibiting further release of a neurotransmitter via a negative presynaptic feedback mechanism (Starke et al., 1989). The existence of inhibitory M₂ receptors that are considered to be located on the parasympathetic ganglia and nerve terminals has been demonstrated in the airways and lungs of several mammalian species because administration of gallamine potentiates vagally mediated bronchoconstriction (Blaber et al., 1985; Faulkner et al., 1986; Fryer and Maclagan, 1984; Ito and Yoshitomi, 1988). Maclagan and Faulkner (1989), however, reported that administration of gallamine ranging from 0.089 to 8.9 mg/kg did not enhance the bronchoconstrictor response to vagal stimulation at 30 Hz in the rabbit, but in the same species, Matsumoto et al. (1995) found that gallamine (1-10 mg/kg) augmented an increase of P_T induced by vagal stimulation at 10 Hz in a dose-dependent manner. In this study, gallamine at smaller doses (3-30 µg/kg) had no significant effect on the responses of SAR activity and R_L to vagal stimulation (5-20 Hz) and ACh injection (1 and 3 µg/kg), suggesting that the dosage of gallamine used in this study was not sufficient to antagonize M₂ receptors. Although 4-DAMP is known to selectively inhibit M₃ receptors in in vivo and radioligand experiments (Doods et al., 1987; Michel et al., 1989), this M₃ receptor blocker, ranging 10-30 µg/kg, inhibited both vagally and ACh-induced bronchoconstrictions in a dose-dependent manner. Administration of 4-DAMP at 3 µg/kg augmented vagally mediated bronchoconstriction but significantly suppressed ACh-induced bronchoconstriction. Similar effects of atropine at the lower doses (50 pM to 1 nM/kg; 0.3-6.8 µg/kg) are found in guinea pigs (Fryer and Maclagan, 1987). The appearance of a paradoxical effect of 4-DAMP on vagally mediated bronchoconstriction would occur as a result of the blockade of the presynaptic M₂ autoreceptors because there is evidence that atropine dose-dependently inhibits contractions of the rabbit trachea induced by field stimulation but at the same time enhances the release of ACh via an autoregulatory feedback system (Loenders et al., 1992). Fryer and Maclagan (1987) suggested that the magnitude of augmentation of vagally mediated bronchoconstriction in guinea pigs depends on the balance between their M₂ and M₃ receptor-blocking activities. The inhibitory effect of 4-DAMP at 3 µg/kg on ACh-induced bronchoconstriction is responsible for its potent antagonistic action of M₃ receptors on airway smooth muscle. Administration of 4-DAMP over 3 µg/kg significantly attenuated but did not abolish the SAR and R_L responses to vagal stimulation and ACh injection. In other experiments, atropine at 2 mg/kg completely blocked both vagally and ACh-induced bronchoconstrictions. As a result, blockade of the autoreceptors by 4-DAMP (10 and 30 µg/kg) would result in an increase in ACh output, and this effect modifies both vagally and ACh-induced bronchoconstrictions. Considering these results, taken together, it is most likely that the inhibitory action of PZ on vagally mediated bronchoconstriction in in vivo experiments is related to blockade of excitatory M₁ receptors in the airways.

The Hering-Breuer inflation reflex that terminates inspiration and prolongs expiration is mediated by the SAR activity capable of regulating the depth and rate of breathing in anesthetized animals (Coleridge and Coleridge, 1986). The excitatory mechanism of M₁ receptors on vagally mediated bronchoconstriction would therefore be expected to strengthen the Hering-Breuer inflation reflex, but augmentation of the SAR activity is known to reduce the activity of vagal efferent fibers innervating the airway smooth muscle (Widdicombe and Nadel, 1963). Thus, further excitation of SARs corresponding to M₁ receptor activation would impair the mechanism that optimizes the conflicting influences of dead space and airway resistance on alveolar ventilation, and such an effect would act as an inhibitory action on vagally mediated bronchoconstriction.