Abstract

Peripheral muscarinic receptors play key roles in the control of heart rate and smooth muscle activity. In this study, bradycardic and smooth muscle contractile responses to the muscarinic agonist carbamylcholine were compared in isolated tissues from M₂ and M₄ muscarinic receptor knockout mice and their wild-type littermates. Carbamylcholine (1 × 10⁻⁸-3 × 10⁻⁵ M) produced similar concentration-dependent bradycardia in spontaneously beating atria from M₄ receptor knockout and wild-type control mice. In contrast, carbamylcholine did not produce bradycardia in atria derived from M₂ receptor knockout mice, whereas such atria were responsive to adenosine-induced bradycardia. Carbamylcholine-induced contractile responses were similar in stomach fundus, urinary bladder, and tracheal preparations from M₄ receptor knockout mice and their wild-type littermates for each tissue (−logEC₅₀ values ranging from 6.20 ± 0.10 to 6.76 ± 0.08), suggesting that M₄ receptors do not participate in smooth muscle contraction in these tissues. In contrast, ∼2-fold higher carbamylcholine concentration was required for contraction of stomach fundus, urinary bladder, and trachea from M₂ receptor knockout mice (−logEC₅₀ = 6.39 ± 0.05, 6.07 ± 0.06, and 6.27 ± 0.12, respectively) than from wild-type littermates (−logEC₅₀ = 6.68 ± 0.07, 6.27 ± 0.07, and 6.56 ± 0.06, respectively). Furthermore, the affinity of the M₂ “selective” receptor antagonist AF-DX116 in inhibiting carbamylcholine-induced smooth muscle contraction was significantly reduced in M₂ receptor knockout mice compared with tissues from wild-type littermates. Collectively, these results provide direct and unambiguous evidence that M₂ receptors mediate muscarinic receptor-induced bradycardia and play a role in smooth muscle contractility, whereas M₄ receptors are not involved in stomach fundus, urinary bladder, or tracheal contractility.