Muscarinic receptor M₃ mediates human gallbladder contraction through voltage-gated Ca²⁺ channels and Rho kinase

Abstract

Objective. Muscarinic receptors mediate contraction of the human gallbladder through unclear receptor subtypes. The aim of the present study was to characterize muscarinic acetylcholine receptors mediating contraction of the human gallbladder. Materials and methods. Contraction of human gallbladder muscle strips caused by agonists carbachol and muscarine was measured and the inhibition of carbachol-induced contraction by muscarinic receptor antagonists was evaluated. Reverse transcription polymerase chain reaction was performed to determine the existence of muscarinic receptor subtypes. Results. Carbachol and muscarine caused concentration-dependent contraction of gallbladder strips. Four receptor antagonists, including atropine, 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP), methoctramine, and pirenzepine, inhibited the carbachol-induced contraction. The relative inhibitory potency of these receptor antagonists was atropine > 4-DAMP > methoctramine > pirenzepine. The antagonist affinity estimates (pA₂ values) correlated with the known affinities at M₃, M₄, and M₅ muscarinic receptors. In addition, the M₄-selective antagonist muscarinic toxin 3 did not inhibit and the M₅-selective positive allosteric modulator VU0238429 did not potentiate carbachol-induced gallbladder contraction. This suggests that M₃ muscarinic receptors mediate the muscarinic response predominantly. The contractile response of carbachol was attenuated by the voltage-gated Ca²⁺ channel inhibitor nifedipine and Rho-kinase inhibitor H-1152, but not affected by protein kinase C inhibitor chelerythrine. This implies the involvement of voltage-gated Ca²⁺ channel and Rho kinase but not protein kinase C. Conclusions. These results suggest a major role of M₃ muscarinic receptors mediating the human gallbladder contraction through voltage-gated Ca²⁺ channels and Rho kinase. M₃-selective muscarinic receptor antagonists could be of therapeutic importance in the treatment of biliary motility disorders.

Key Words::

• contraction
• gallbladder
• human
• M₃ receptors
• muscarinic acetylcholine receptor
• Rho kinase

Acknowledgments

This research was supported by E-Da Hospital – National Taiwan University Hospital Joint Research Program (100-EDN02), National Science Council, Buddhist Tzu Chi General Hospital and E-Da Hospital. We thank Dr Jerry D, Gardner of Science for Organizations for the manuscript editing and Ling-Jung Chiu, Yu-Shyuan Wang, Mei-Ling Chen, Chien-Pin Wang, Cai-Jing Lee, and Chiung-Fang Hsu for their excellent technical assistance.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.