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 Kitazawa
  • Ryuichi Hirama
  • Kozue Masunaga
  • Tatsuro Nakamura
  • Koichi Asakawa
  • Jinshan Cao
  • Hiroki Teraoka
  • Toshihiro Unno
  • Sei-ichi Komori
  • Masahisa Yamada
  • Jürgen Wess
  • Tetsuro Taneike
Original Article

DOI: 10.1007/s00210-007-0223-1

Cite this article as:
Kitazawa, T., Hirama, R., Masunaga, K. et al. Naunyn-Schmied Arch Pharmacol (2008) 377: 503. doi:10.1007/s00210-007-0223-1

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 (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 μg/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 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 uterusM2 receptorM3 receptorCarbacholContraction

Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Takio Kitazawa
    • 1
  • Ryuichi Hirama
    • 1
  • Kozue Masunaga
    • 1
  • Tatsuro Nakamura
    • 1
  • Koichi Asakawa
    • 1
  • Jinshan Cao
    • 1
  • Hiroki Teraoka
    • 2
  • Toshihiro Unno
    • 3
  • Sei-ichi Komori
    • 3
  • Masahisa Yamada
    • 4
  • Jürgen Wess
    • 5
  • Tetsuro Taneike
    • 1
  1. 1.Department of Pharmacology, School of Veterinary MedicineRakuno Gakuen UniversityEbetsuJapan
  2. 2.Department of Toxicology, School of Veterinary MedicineRakuno Gakuen UniversityEbetsuJapan
  3. 3.Laboratory of Pharmacology, Faculty of Applied Biological ScienceGifu UniversityGifuJapan
  4. 4.Yamada Research UnitRIKEN Brain Science InstituteSaitamaJapan
  5. 5.Laboratory of Bioorganic ChemistryNational Institute of Diabetes, Digestive and Kidney DiseasesBethesdaUSA