Original Article

Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 377, Issue 4, pp 503-513

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

  • Takio KitazawaAffiliated withDepartment of Pharmacology, School of Veterinary Medicine, Rakuno Gakuen University Email author 
  • , Ryuichi HiramaAffiliated withDepartment of Pharmacology, School of Veterinary Medicine, Rakuno Gakuen University
  • , Kozue MasunagaAffiliated withDepartment of Pharmacology, School of Veterinary Medicine, Rakuno Gakuen University
  • , Tatsuro NakamuraAffiliated withDepartment of Pharmacology, School of Veterinary Medicine, Rakuno Gakuen University
  • , Koichi AsakawaAffiliated withDepartment of Pharmacology, School of Veterinary Medicine, Rakuno Gakuen University
  • , Jinshan CaoAffiliated withDepartment of Pharmacology, School of Veterinary Medicine, Rakuno Gakuen University
  • , Hiroki TeraokaAffiliated withDepartment of Toxicology, School of Veterinary Medicine, Rakuno Gakuen University
  • , Toshihiro UnnoAffiliated withLaboratory of Pharmacology, Faculty of Applied Biological Science, Gifu University
  • , Sei-ichi KomoriAffiliated withLaboratory of Pharmacology, Faculty of Applied Biological Science, Gifu University
    • , Masahisa YamadaAffiliated withYamada Research Unit, RIKEN Brain Science Institute
    • , Jürgen WessAffiliated withLaboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases
    • , Tetsuro TaneikeAffiliated withDepartment of Pharmacology, School of Veterinary Medicine, Rakuno Gakuen University

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Abstract

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 μM) increased muscle tonus and phasic contractile activity of uterine strips of control mice in a concentration-dependent manner. The maximum carbachol-induced contractions (E max) 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 pK b values for these muscarinic receptor antagonists correlated well with the known pK i values of these antagonists for the M3 muscarinic receptor. In uterine strips isolated from mice treated with pertussis toxin (100 μg/kg, i.p. for 96 h), E max values for carbachol were significantly decreased, but effective concentration that caused 50% of E max values (EC50) 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 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.

Keywords

Mouse uterus M2 receptor M3 receptor Carbachol Contraction