Objective-To describe the in vitro effects of bethanechol on contractility of smooth muscle preparations from the small intestines of healthy cows and define the muscarinic receptor subtypes involved in mediating contraction.Sample Population-Tissue samples from the duodenum and jejunum collected immediately after slaughter of 40 healthy cows.Procedures-Cumulative concentration-response curves were determined for the muscarinic receptor agonist bethanechol with or without prior incubation with subtype-specific receptor antagonists in an organ bath. Effects of bethanechol and antagonists and the influence of intestinal location on basal tone, maximal amplitude (A max ), and area under the curve (AUC) were evaluated.Results-Bethanechol induced a significant, concentration-dependent increase in all preparations and variables. The effect of bethanechol was more pronounced in jejunal than in duodenal samples and in circular than in longitudinal preparations. Significant inhibition of the effects of bethanechol was observed after prior incubation with muscarinic receptor subtype M 3 antagonists (more commonly for basal tone than for A max and AUC). The M 2 receptor antagonists partly inhibited the response to bethanechol, especially for basal tone. The M 3 receptor antagonists were generally more potent than the M 2 receptor antagonists. In a protection experiment, an M 3 receptor antagonist was less potent than when used in combination with an M 2 receptor antagonist. Receptor antagonists for M 1 and M 4 did not affect contractility variables. Conclusions and ClinicalRelevance-Bethanechol acting on muscarinic receptor subtypes M 2 and M 3 may be of clinical use as a prokinetic drug for motility disorders of the duodenum and jejunum in dairy cows.The important role of the parasympathetic nervous system in physiologic processes of GI tract motility and in the pathophysiology of motility disorders has been described for various species. [1][2][3][4][5][6][7] In the cholinergic system, acetylcholine activates G-protein-coupled muscarinic receptors to cause smooth muscle contraction in several organs. 1,2 Five mAChR subtypes (M 1 to M 5 , respectively) have been cloned 8 and defined pharmacologically. 3,9 Address correspondence to Dr. Meylan.. NIH Public Access Author ManuscriptAm J Vet Res. Author manuscript; available in PMC 2009 September 7.Published in final edited form as: Am J Vet Res. 2007 March ; 68(3): 313-322. doi:10.2460/ajvr.68.3.313. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptThe M 2 and M 3 mAChR subtypes are the predominant receptors in the GI tract of several species, with a ratio for M 2 :M 3 of approximately 4:1. 1,10 However, the M 3 receptor subtype is considered to be of predominant importance in eliciting muscle contractions, and the exact role of the more abundant M 2 subtype remains unclear. 10,11 A study 12 in M 3 receptor knockout mice has emphasized the role of M 3 mAChRs for smooth muscle contraction because ileal contractile response to carbachol in vi...