Singer, Cherie A., Sa Vang, and William T. Gerthoffer. Coupling of M2 muscarinic receptors to Src activation in cultured canine colonic smooth muscle cells. Am J Physiol Gastrointest Liver Physiol 282: G61–G68, 2002; 10.1152/ ajpgi.00100.2002.—The purpose of this study was to determine whether Src tyrosine kinases are one of the signaling intermediaries linking M2 receptor stimulation to extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) in cultures of canine colonic smooth muscle cells (CSMC). RT-PCR studies demonstrate expression of multiple Src tyrosine kinases, including Src, Fyn, and Yes, in CSMC. Muscarinic stimulation of CSMC with 10 µM ACh results in a twofold increase in Src activity within 10 min but does not increase the activity of Fyn. Treatment with the M2 antagonist AF-DX 116 (10 µM) blocks ACh-stimulated Src activation in primary CSMC cultures that express both M2 and M3 receptors and in first-passage CSMC cultures that express predominantly M2 receptors. Alkylation of M3 receptors with 100 nM N,N-dimethyl-4-piperidinyl diphenylacetate mustard has no effect on Src activity. Treatment with the pyrazolopyrimidine Src inhibitor PP1 (10 µM) or AF-DX 116 (10 µM) blocks ACh-stimulated ERK phosphorylation. Together these results indicate that M2 receptors are coupled to Src tyrosine kinase and subsequent activation of ERK in cultured CSMC.