Coupling of M2 muscarinic receptors to Src activation in cultured canine colonic smooth muscle cells

The purpose of this study was to determine whether Src tyrosine kinases are one of the signaling intermediaries linking M-2 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 muM ACh results in a twofold increase in Src activity within 10 min but does not increase the activity of Fyn. Treatment with the M-2 antagonist AF-DX 116 (10 muM) blocks ACh-stimulated Src activation in primary CSMC cultures that express both M-2 and M-3 receptors and in first-passage CSMC cultures that express predominantly M-2 receptors. Alkylation of M-3 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 muM) or AF-DX 116 (10 muM) blocks ACh-stimulated ERK phosphorylation. Together these results indicate that M-2 receptors are coupled to Src tyrosine kinase and subsequent activation of ERK in cultured CSMC.