Muscarinic receptor subtypes involved in carbacholinduced 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 M_2 or M_3 single receptor knockout (M_2KO, M_3KO), and M_2 and M_3 receptor double knockout mice (M_2/M_3KO). Carbachol (10 nM-100 μM) increased muscle tonus and phasic contractile activity of uterine strips of control mice in a concentration-dependent manner. The maximum carbacholinduced contractions (E_) 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), parafluoro- hexa hydro-sila-diphenidol (p-F-HHSiD), himbacine, methoctramine, pirenzepine, and tropicamide) inhibited carbachol-induced contractions in a competitive fashion. The pK_b values for these muscarinic receptor antagonists correlated well with the known pK_i values of these antagonists for the M_3 muscarinic receptor. In uterine strips isolated from mice treated with pertussis toxin (100 μg/kg, i.p. for 96 h), E_ values for carbachol were significantly decreased, but effective concentration that caused 50% of E_ values (EC_) remained unchanged. In uterine strips treated with 4-DAMP mustard (30 nM) and AF-DX116 (1 μM), 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 M_2 and M_3 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 M_2KO 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 M_3KO and M_2/M_3 double KO mice were virtually insensitive to carbachol. In conclusion, although both M_2 and M_3 muscarinic receptors were expressed in the mouse uterus, carbachol-induced contractile responses were predominantly mediated by the M_3 receptor. Activation of M_2 receptors alone did not cause uterine contractions; however, M_2 receptor activation enhanced M_3 receptor-mediated contractions in the mouse uterus