Muscarinic receptor subtypes involved in regulation of colonic motility in mice: Functional studies using muscarinic receptor-deficient mice

Although muscarinic M_2 and M_3 receptors are known to be important for regulation of gastric and small intestinal motility, muscarinic receptor subtypes regulating colonic function remain to be investigated. The aim of this study was to characterize muscarinic receptors involved in regulation of colonic contractility. M_2 and/or M_3 receptor knockout (KO) and wild-type mice were used in in vivo (defecation, colonic propulsion) and in vitro (contraction) experiments. Amount of feces was significantly decreased in M_3R-KO and M_2/M_3R-KO mice but not in M_2R-KO mice. Ranking of colonic propulsion was wild-type = M_2R-KO > M_3R-KO > M_2/M_3R-KO. In vitro, the amplitude of migrating motor complexes in M_2R-KO, M_3R-KO and M_2/M_3R-KO mice was significantly lower than that in wild-type mice. Carbachol caused concentration-dependent contraction of the proximal colon and distal colon from wild-type mice. In M_2R-KO mice, the concentration-contraction curves shifted to the right and downward. In contrast, carbachol caused non-sustained contraction and relaxation in M_3R-KO mice depending on its concentration. Carbachol did not cause contraction but instead caused relaxation of colonic strips from M_2/M_3R-KO mice. 4-[[[(3-chlorophenyl)amino]carbonyl]oxy]-N,N,N-trimethyl-2-butyn-1-aminium chloride (McN-A-343) caused a non-sustained contraction of colonic strips from wild-type mice, and this contraction was changed to a sustained contraction by tetrodotoxin, pirenzepine and L-nitroarginine methylester (L-NAME). In the colon of M_2/M_3R-KO mice, McN-A-343 caused only relaxation, which was decreased by tetrodotoxin, pirenzepine and L-NAME. In conclusion, M_1, M_2 and M_3 receptors regulate colonic motility of the mouse. M_2 and M_3 receptors mediate cholinergic contraction, but M_1 receptors on inhibitory nitrergic nerves counteract muscarinic contraction

M3 Muscarinic Receptors Mediate Positive Inotropic Responses in Mouse Atria: A Study with Muscarinic Receptor Knockout Mice

The potential functional roles of M3 muscarinic receptors in mouse atria were examined by pharmacological and molecular biological techniques, using wild-type mice, muscarinic M2 or M3 receptor single knockout (M2KO, M3KO), and M2 and M3 muscarinic receptor double knockout mice (M2/M3KO). Real-time quantitative reverse transcriptase-polymerase chain reaction analysis showed that the M2 receptor mRNA was expressed predominantly in mouse atria but that the M1, M3, M4, and M5 receptor subtypes were also expressed at low levels. Carbachol (10 nM–30 μM) decreased the spontaneous beating frequency of right atria isolated from wild-type mice. Studies with subtype-preferring antagonists and atria from M2KO mice confirmed that this activity is mediated by the M2 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 M3KO mice, the transient negative inotropic action of carbachol changed to a sustained negative inotropic action. In contrast, in atria from M2KO mice, carbachol showed only positive inotropic activity. In atria from M2/M3 double KO mice, carbachol was devoid of any inotropic activity. These observations, complemented by functional studies with subtype-preferring antagonists, convincingly demonstrate that atrial M3 muscarinic receptors mediate positive inotropic effects in mouse atria. Physiologically, this activity may serve to dampen the inhibitory effects of M2 receptor activation on atrial contractility

Functional roles of muscarinic M2 and M3 receptors in the mouse stomach motility : Studies with muscarinic receptor knockout mice

Functional roles of muscarinic acetylcholine receptors in the regulation of mouse stomach motility were examined using mice genetically lacking muscarinic M(2) receptor and/or M(3) receptor and their corresponding wild-type (WT) mice. Single application of carbachol (1 nM-30 microM) produced concentration-dependent contraction in antral and fundus strips from muscarinic M(2) receptor knockout (M(2)R-KO) and M(3) receptor knockout (M(3)R-KO) mice but not in those from M(2) and M(3) receptors double knockout (M(2)/M(3)R-KO) mice. A comparison of the concentration-response curves with those for WT mice showed a significant decrease in the negative logarithm of EC(50) (pEC(50)) value (M(2)R-KO) or amplitude of maximum contraction (M(3)R-KO) in the muscarinic receptor-deficient mice. The tonic phase of carbachol-induced contraction was decreased in gastric strips from M(3)R-KO mice. Antagonistic affinity for 4-diphenylacetoxy-N-methyl-piperidine (4-DAMP) or 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX116) indicated that the contractile responses in M(2)R-KO and M(3)R-KO mice were mediated by muscarinic M(3) and M(2) receptors, respectively. Electrical field stimulation (EFS, 0.5-32 Hz) elicited frequency-dependent contraction in physostigmine- and N(omega)-nitro-L-arginine methylester (l-NAME)-treated fundic and antral strips from M(2)R-KO and M(3)R-KO mice, but the cholinergic contractile components decreased significantly compared with those in WT mice. In gastric strips from M(2)/M(3)R-KO mice, cholinergic contractions elicited by EFS were not observed but atropine-resistant contractions were more conspicuous than those in gastric strips from WT mice. Gastric emptying in WT mice and that in M(2)/M(3)R-KO mice were comparable, suggesting that motor function of the stomach in the KO mice did not differ from that in the WT mice. The results indicate that both muscarinic M(2) and M(3) receptors but not other subtypes mediate carbachol- or EFS-induced contraction in the mouse stomach but that the contribution of each receptor to concentration-response relationships is distinguishable. Although there was impairment of nerve-mediated cholinergic responses in the stomach of KO mice, gastric emptying in KO mice was the same as that in WT mice probably due to the compensatory enhancement of the non-cholinergic contraction pathway

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