M3 muscarinic receptors mediate positive inotropic responses in mouse atria: a study with muscarinic receptor knockout mice.

The potential functional roles of M(3) muscarinic receptors in mouse atria were examined by pharmacological and molecular biological techniques, using wild-type mice, muscarinic M(2) or M(3) receptor single knockout (M(2)KO, M(3)KO), and M(2) and M(3) muscarinic receptor double knockout mice (M(2)/M(3)KO). Real-time quantitative reverse transcriptase-polymerase chain reaction analysis showed that the M(2) receptor mRNA was expressed predominantly in mouse atria but that the M(1), M(3), M(4), and M(5) receptor subtypes were also expressed at low levels. Carbachol (10 nM-30 microM) decreased the spontaneous beating frequency of right atria isolated from wild-type mice. Studies with subtype-preferring antagonists and atria from M(2)KO mice confirmed that this activity is mediated by the M(2) receptor subtype. In left atria from wild-type mice, carbachol decreased the amplitude of electrical field stimulation-evoked contractions (negative inotropic action), but this inhibition was transient and was followed by a gradual increase in contraction amplitude (positive inotropic response). In atria from M(3)KO mice, the transient negative inotropic action of carbachol changed to a sustained negative inotropic action. In contrast, in atria from M(2)KO mice, carbachol showed only positive inotropic activity. In atria from M(2)/M(3) double KO mice, carbachol was devoid of any inotropic activity. These observations, complemented by functional studies with subtype-preferring antagonists, convincingly demonstrate that atrial M(3) muscarinic receptors mediate positive inotropic effects in mouse atria. Physiologically, this activity may serve to dampen the inhibitory effects of M(2) receptor activation on atrial contractility.

Muscarinic receptor subtypes involved in carbachol-induced contraction of mouse uterine smooth muscle.

Functional muscarinic acetylcholine receptors present in the mouse uterus were characterized by pharmacological and molecular biological studies using control (DDY and wild-type) mice, muscarinic M2 or M3 single receptor knockout (M2KO, M3KO), and M2 and M3 receptor double knockout mice (M2/M3KO). Carbachol (10 nM-100 microM) increased muscle tonus and phasic contractile activity of uterine strips of control mice in a concentration-dependent manner. The maximum carbachol-induced contractions (Emax) differed between cervical and ovarian regions of the uterus. The stage of the estrous cycle had no significant effect on carbachol concentration-response relationships. Tetrodotoxin did not decrease carbachol-induced contractions, but the muscarinic receptor antagonists (11-[[2-[(diethylaminomethyl)-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b[2,3-b][1,4]benzodiazepin6-one (AF-DX116), N-[2-[2-[(dipropylamino)methyl]-1-piperidinyl]ethyl]-5,6-dihydro-6-oxo-11H-pyrido[2,3-b][1,4] benzodiazepine-11-carboxamide (AF-DX384), 4-diphenylacetoxy-N-methyl-piperidine(4-DAMP), para-fluoro-hexa hydro-sila-diphenidol (p-F-HHSiD), himbacine, methoctramine, pirenzepine, and tropicamide) inhibited carbachol-induced contractions in a competitive fashion. The pKb values for these muscarinic receptor antagonists correlated well with the known pKi values of these antagonists for the M3 muscarinic receptor. In uterine strips isolated from mice treated with pertussis toxin (100 microg/kg, i.p. for 96 h), Emax values for carbachol were significantly decreased, but effective concentration that caused 50% of Emax values (EC50) remained unchanged. In uterine strips treated with 4-DAMP mustard (30 nM) and AF-DX116 (1 microM), followed by subsequent washout of AF-DX116, neither carbachol nor N,N,N,-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium iodide (oxotremorine-M) caused any contractile responses. Both M2 and M3 muscarinic receptor messenger RNAs were detected in the mouse uterus via reverse transcription polymerase chain reaction. Carbachol also caused contraction of uterine strips isolated from M2KO mice, but the concentration-response curve was shifted to the right and downward compared with that for the corresponding wild-type mice. On the other hand, uterine strips isolated from M3KO and M2/M3 double KO mice were virtually insensitive to carbachol. In conclusion, although both M2 and M3 muscarinic receptors were expressed in the mouse uterus, carbachol-induced contractile responses were predominantly mediated by the M3 receptor. Activation of M2 receptors alone did not cause uterine contractions; however, M2 receptor activation enhanced M3 receptor-mediated contractions in the mouse uterus.