M(2) and M(4) receptor knockout mice: muscarinic receptor function in cardiac and smooth muscle in vitro.

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 M(2) and M(4) muscarinic receptor knockout mice and their wild-type littermates. Carbamylcholine (1 x 10(-8)-3 x 10(-5) M) produced similar concentration-dependent bradycardia in spontaneously beating atria from M(4) receptor knockout and wild-type control mice. In contrast, carbamylcholine did not produce bradycardia in atria derived from M(2) 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 M(4) receptor knockout mice and their wild-type littermates for each tissue (-logEC(50) values ranging from 6.20 +/- 0.10 to 6.76 +/- 0.08), suggesting that M(4) receptors do not participate in smooth muscle contraction in these tissues. In contrast, approximately 2-fold higher carbamylcholine concentration was required for contraction of stomach fundus, urinary bladder, and trachea from M(2) receptor knockout mice (-logEC(50) = 6.39 +/- 0.05, 6.07 +/- 0.06, and 6.27 +/- 0.12, respectively) than from wild-type littermates (-logEC(50) = 6.68 +/- 0.07, 6.27 +/- 0.07, and 6.56 +/- 0.06, respectively). Furthermore, the affinity of the M(2) "selective" receptor antagonist AF-DX116 in inhibiting carbamylcholine-induced smooth muscle contraction was significantly reduced in M(2) receptor knockout mice compared with tissues from wild-type littermates. Collectively, these results provide direct and unambiguous evidence that M(2) receptors mediate muscarinic receptor-induced bradycardia and play a role in smooth muscle contractility, whereas M(4) receptors are not involved in stomach fundus, urinary bladder, or tracheal contractility.