title: Coupling of M(2) muscarinic receptors to Src activation in cultured canine colonic smooth muscle cells.
abstract: 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 microM 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 microM) 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 microM) or AF-DX 116 (10 microM) 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.
authors.affiliation: Department of Pharmacology, University of Nevada School of Medicine, Reno, Nevada 89557-0046, USA. cas@med.unr.edu
authors.forename: Cherie A
authors.initials: CA
authors.lastname: Singer
authors.forename: Sa
authors.initials: S
authors.lastname: Vang
authors.forename: William T
authors.initials: WT
authors.lastname: Gerthoffer
mesh_terms: D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology / Q000201:enzymology; D004285:Dogs; D005260:Female; D015971:Gene Expression Regulation, Enzymologic; D019833:Genistein / Q000494:pharmacology; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009130:Muscle, Smooth / Q000166:cytology* / Q000201:enzymology; D010276:Parasympatholytics / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D010890:Pirenzepine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051076:Proto-Oncogene Proteins c-fyn; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000235:genetics* / Q000378:metabolism*; D013722:Teprotide / Q000494:pharmacology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*
chemical_list: C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D018727:Muscarinic Antagonists; D010276:Parasympatholytics; D010880:Piperidines; D011518:Proto-Oncogene Proteins; D011720:Pyrazoles; D011743:Pyrimidines; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; D010890:Pirenzepine; D001285:Atropine; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D013722:Teprotide; D019833:Genistein; D051076:Proto-Oncogene Proteins c-fyn; D019061:src-Family Kinases; D020928:Mitogen-Activated Protein Kinases; C048593:otenzepad