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Loss of vagally mediated bradycardia and bronchoconstriction in mice lacking M₂ or M₃ muscarinic acetylcholine receptors¹

JOHN T. FISHER^*,2, SANDRA G. VINCENT^*, JESUS GOMEZA, MASAHISA YAMADA and JÜRGEN WESS

^* Departments of Physiology, Medicine and Pediatrics, Queen’s University, Kingston, Ontario, Canada; and
Laboratory of Bioorganic Chemistry, National Institutes of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland, USA

²Correspondence: Department of Physiology, Queen’s University, Kingston, Ontario, Canada K7L 3N6. E-mail: fisherjt{at}post.queensu.ca

SPECIFIC AIMS

Muscarinic acetylcholine receptors (mAChRs) play a fundamental role in regulating cardiac and pulmonary function via the vagus nerve; activation of cardiac vagal efferents leads to the release of ACh, which reduces heart rate and ACh released from pulmonary vagal efferents causes bronchoconstriction via airway smooth muscle contraction. We used mutant mice lacking functional M₂ or M₃ mAChRs (M2^–/– and M3^–/– mice, respectively) to explore the roles of M₂ and M₃ receptors in cardiac and pulmonary function in vivo.

PRINCIPAL FINDINGS

1. Absence of the M₂ receptor abolishes the bradycardia response to vagal stimulation or muscarinic agonists
Mice were anesthetized, paralyzed, and ventilated before activation of vagal efferents to the heart and lungs by supramaximal stimulation for 10 s at various stimulus frequencies (5–20 pulses/s). Animals were then exposed to increasing doses of the muscarinic agonist methacholine (MCh; 5–250 µg/kg) injected i.v. Heart rate decreased in a frequency-dependent manner during vagal stimulation (Fig. 1 , upper panel) and in a dose-dependent manner to MCh (Fig. 1 , lower panel) that was similar for M2^+/+, M3^+/+, and M3^–/– mice. In contrast, M2^–/– displayed no decrease in heart to either stimulus (Fig. 1 , upper and lower panels). Our data support a critical role for M₂ receptors in the in vivo decrease in heart rate due to acetylcholine release from cardiac vagal innervation.

View larger version (23K): [in this window] [in a new window]   Figure 1. Upper panel: heart rate (HR) response of M2 and M3 mAChR mutant (open symbols) and WT (filled symbols) mice during vagal stimulation. Vagal stimulation caused frequency-dependent bradycardic responses in M2+/+, M3+/+, and M3–/– mice, which did not differ significantly among the three groups. In contrast, M2–/– mice displayed no HR response to vagal stimulation (P<0.001 for M2–/– vs. other groups). Data are shown as means ± SE (n=6/genotype). Lower panel: heart rate (HR) response of M2 and M3 mAChR mutant (open symbols) and WT (filled symbols) mice after administration of MCh. M2–/– and M3–/–mice and the corresponding WT control animals (M2+/+ and M3+/+ mice) received i.v. injections of increasing doses of the muscarinic agonist, MCh (5–250 µg/kg). MCh caused dose-dependent bradycardic responses in M2+/+, M3+/+, and M3–/– mice, which did not differ significantly among the three groups. In contrast, M2–/– mice displayed no HR response to MCh administration (P<0.001 for M2–/– vs. other groups). Data are given as means ± SE (n=7–11 per genotype).

2. Absence of the M₂ receptor increases the bronchoconstrictor response to vagal stimulation
Airway bronchoconstrictor responses were estimated from the time-integrated change in peak airway pressure, denoted as airway pressure time index (APTI). APTI is a measure of total respiratory impedance that accurately reflects bronchoconstrictor responses measured by standard respiratory mechanics. Vagal stimulation or MCh caused a frequency- and dose-dependent increase, respectively, in the APTI for M2^+/+and M3^+/+ mice (Fig. 2 ). However, the M2^–/– genotype displayed an enhanced bronchoconstrictor response to vagal stimulation compared with the M2^+/+and M3^+/+ groups (Fig. 2 , upper panel), consistent with the hypothesis that M₂ autoinhibitory receptors limit the release of ACh from vagal nerve endings and provide a physiological brake to ongoing bronchoconstriction.

View larger version (21K): [in this window] [in a new window]   Figure 2. Upper panel: bronchoconstrictor response of M2 and M3 mAChR mutant (open symbols) and WT (filled symbols) mice after vagal stimulation. Airway pressure time-index (APTI) responses were measured during vagal efferent stimulation. Vagal stimulation caused frequency-dependent increases in APTI in M2+/+ and M3+/+ mice. Strikingly, these responses were significantly enhanced in M2–/– mice (P<0.001 for M2–/– vs. other groups). In contrast, M3–/– mice were devoid of APTI responses at all stimulus frequencies (P<0.001 for M3–/– vs. other groups). Data are given as means ± SE (n=6/genotype). Lower panel: bronchoconstrictor response of M2 and M3 mAChR mutant (open symbols) and WT (filled symbols) mice after administration of MCh. M2–/– and M3–/–mice and the corresponding WT control animals (M2+/+ and M3+/+ mice) received i.v. injections of increasing doses of MCh (5–250 µg/kg). MCh caused dose-dependent increases in APTI in M2+/+ and M3+/+ mice that did not differ significantly in magnitude between the two WT control groups. M2–/– mice displayed an increased responsiveness to MCh compared with M2+/+ mice (P=0.025). M3–/– mice were devoid of bronchoconstrictor responses to MCh, even at the highest MCh dose tested (P<0.001 for M3–/– vs. other groups). Data are given as means ± SE (n=7–11/genotype).

3. Absence of the M₃ receptor abolishes the bronchoconstrictor response to vagal stimulation or muscarinic agonists
Mice lacking the M₃ receptor protein failed to display the frequency- and dose-dependent increase in APTI observed for M2^+/+, M3^+/+ and M2^–/– mice during vagal stimulation or MCh injection (Fig. 2 ). These data implicate the M₃ receptor as the primary muscarinic receptor mediating bronchoconstrictor responses in vivo.

CONCLUSIONS AND SIGNIFICANCE

The presence of multiple mAChR subtypes in heart and lung, combined with the lack of mAChR subtype-selective ligands, has complicated the task of identifying the mAChR subtypes mediating cardiac and pulmonary responses to ACh. The activity of vagal efferent nerves plays a predominant role in the physiologic regulation of heart rate. By acting on mAChRs located in the sinoatrial node, ACh released from cardiac vagal nerve endings triggers a chronotropic effect by reducing cardiac beat frequency. We found that vagally induced bradycardic responses were abolished in M₂ receptor-deficient mice in vivo (Fig. 1 ). Similarly, the muscarinic agonist MCh was devoid of bradycardic activity in M2^–/– mice (Fig. 1 ). These observations indicate unambiguously that vagally/ACh-mediated regulation of heart rate is exclusively mediated by M₂ receptors in vivo. On the basis of pharmacological studies, it has been proposed that cardiac M₃ receptors may also mediate bradycardic responses. However, we found that M3^–/– mice showed unaltered bradycardic responses after vagal stimulation or MCh administration in vivo (Fig. 1 ). Stengel and co-workers recently showed that carbachol-mediated negative chronotropic effects in vitro remained unchanged in isolated spontaneously beating atria derived from M3^–/– mice. These observations, together with the absence of muscarinic bradycardic activity in M2^–/– mice of the present study, clearly indicate that M₃ receptors do not play a significant role in the regulation of heart rate in vivo. Since inappropriate vagal tone is thought to contribute to the development of various forms of life-threatening cardiac arrhythmias, our observation that vagus-dependent regulation of heart rate is mediated exclusively via cardiac M₂ receptors is of considerable clinical significance.

Systemic administration of ACh and other muscarinic agonists causes pronounced reductions in blood pressure, generally assumed to be due to a generalized vasodilation caused by activation of mAChRs located on vascular endothelial cells. In the present study, MCh caused dose-dependent decreases in MAP in WT mice, as expected. In M3^–/– mice, the magnitude of these hypotonic responses was reduced by 20% compared with M3^+/+ mice. The remaining hypotension in the M3^–/– mice presumably reflects the bradycardia mediated by intact M₂ receptors.

Efferent vagal nerve activity plays a fundamental role in regulating pulmonary function. ACh released from postganglionic parasympathetic nerves causes bronchoconstriction by interacting with mAChRs located on airway smooth muscle cells, and both M₂ and M₃ mAChRs have been shown to be expressed in airway smooth muscle cells. We found that vagally or MCh-mediated bronchoconstrictor (APTI) responses were abolished in M3^–/– mice in vivo (Fig. 2 ). A recent in vitro study showed that carbachol retained considerable contractile activity in isolated tracheal smooth muscle preparations from M3^–/– mice, suggesting that non-M₃ mAChRs (perhaps M₂ receptors) contribute to tracheal smooth muscle contraction in vitro. However, the results of the present study suggest that non-M₃ smooth muscle mAChRs do not play a significant role in muscarinic bronchoconstrictor responses in vivo. Our in vivo findings are consistent with the concept that bronchoconstrictor responses are dominated by bronchial rather than tracheal smooth muscle activity.

Although bronchoconstrictor (APTI) responses were abolished in M3^–/– mice, M2^–/– mice showed enhanced APTI responses to vagal stimulation (Fig. 2 ). This observation is consistent with the notion that acetylcholine-mediated activation of prejunctional M₂ mAChRs located on pulmonary parasympathetic nerve endings inhibits acetylcholine release, reducing the magnitude of the bronchoconstriction response in control mice. Loss of function of these inhibitory pulmonary M₂ autoreceptors has been shown to occur in animal models of airway hyperreactivity and some patients with asthma.

Our findings provide a physiologic basis for the pharmacotherapy of lung diseases such as chronic obstructive pulmonary disease (COPD) or acute asthma, which are associated with an increase in pulmonary vagal tone. Our data suggest that pharmacologic blockade of M₃ receptors should prevent clinically relevant bronchoconstrictor responses triggered by excessive vagal activation (Fig. 3 ). In agreement with previous pharmacologic studies, our results indicate that pharmacologic blockade of pulmonary M₂ receptors should be avoided so as to prevent counteracting bronchoconstrictor responses. The development of highly selective M₃ receptor antagonists to treat COPD and asthma therefore represents a highly desirable goal.

View larger version (44K): [in this window] [in a new window]   Figure 3. Schematic diagram of the role of M2 and M3 receptors in the vagally mediated decrease in heart rate (bradycardia) and magnitude of bronchoconstriction in mice. Our data implicate M2 and M3 receptors as playing a causal role in the decrease in heart rate and presence of bronchoconstriction respectively in response to acetylcholine. M2–/– mice displayed no decrease in heart rate during vagal stimulation or in response to the muscarinic agonist methacholine (MCh). M3–/– mice displayed no bronchoconstrictor responses to vagal stimulation or MCh. In contrast, M2–/– mice possessed an enhanced bronchoconstriction during vagal stimulation due to loss of the prejunctional autoinhibitory function of M2 receptors on vagal efferents to airway smooth muscle. The prejunctional autoinhibitory M2 receptors provide a brake to ongoing bronchoconstriction.

In summary, results of the present study answer questions that have been associated with mAChR biology since Einthoven’s classic observations of more than a century ago. Our data provide unambiguous evidence of the primary roles for M₂ and M₃ muscarinic receptors in vagally mediated bradycardia and bronchoconstriction respectively (Fig. 3 ). The results reported here should be of therapeutically relevant, since altered cardiac or pulmonary vagal tone is involved in a number of pathophysiological conditions, including cardiac arrhythmias, COPD, and asthma.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0648fje; doi: 10.1096/fj.03-0648fje

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This Article Full Text (PDF) All Versions of this Article: 18/6/711 03-0648fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by FISHER, J. T. Articles by WESS, J. Search for Related Content PubMed PubMed Citation Articles by FISHER, J. T. Articles by WESS, J.