Abstract

Muscarinic receptors play a major role in gallbladder function, although the muscarinic receptor(s) mediating smooth muscle contractility is unclear. This study compared smooth muscle contractile responses to carbamylcholine (10⁻⁷-10⁻³ M) in isolated gallbladder from wild-type and M₂, M₃, and M₄ receptor knockout mice. Carbamylcholine-induced contraction in gallbladder was associated with tachyphylaxis and the release of a cyclooxygenase product because indomethacin (10⁻⁶ M) inhibited carbamylcholine-induced contraction. The M₃ receptor was the major muscarinic receptor involved in contraction because carbamylcholine-induced contractility was inhibited in gallbladder from M₃ receptor knockout mice. Furthermore, the muscarinic receptor antagonists 11-[[[2-diethylamino-O-methyl]-1-piperidinyl]acetyl]-5,11-dihydrol-6H-pyridol[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) and pirenzepine dextrally shifted contraction to carbamylcholine in gallbladder from wild-type, M₂, and M₄ receptor knockout mice, with affinities consistent with M₃ receptor interaction. In addition, maximal contraction to carbamylcholine was reduced in gallbladder from M₂receptor knockout mice and affinities for AF-DX 116 and pirenzepine in gallbladder from M₃ receptor knockout mice were consistent with their affinities at M₂ receptors. In M₄receptor knockout mice, contraction to carbamylcholine was dextrally shifted, although the affinities for AF-DX 116 and pirenzepine in gallbladder from M₂ or M₃ knockout mice were not similar to their affinities at M₄ receptors. The M₄ receptor may serve as an accessory protein necessary for optimal potency of M₂ and M₃ receptor-mediated responses. Thus, muscarinic receptor knockout mice provided direct and unambiguous evidence that M₃, and to a lesser extent, M₂ receptors are the predominant muscarinic receptors mediating gallbladder contractility, and M₄ receptors appear necessary for optimal potency of carbamylcholine in gallbladder contraction.