Functional muscarinic m3 receptor expressed in gastric cancer cells stimulates tyrosine phosphorylation and MAP kinase

Human gastric cancer cells were used to examine the trophic effect of the muscarinic m3 receptor subtype. Expression of the m3 receptor was detected in five of night cell lines examined, MKN-1, 7, 28, 74, and TMK-1 cells. An increase in intracellular Ca(2+) in response to carbachol was observed in more than 90% of TMK-1 cells, allowing us to use these cells in the following experiments. Western blot analysis showed that carbachol predominantly phosphorylated tyrosine in a 100-kDa protein. While mitogen-activated protein (MAP) kinase activity in the presence of 100 mu M carbachol or 10ng/ml transforming growth factor (TGF)a was augmented to 15- to 60-fold of the baseline level for 5 min, the activation was transient. Pretreatment of the cells with 1 mu M phorbol 12-myristate U-acetate abolished carbacol-induced MAP kinase activation, whereas no suppression was observed in the presence of 500 nM Calphostin C (Kyowa Medex, Tokyo Japan), a specific protein kinase C inhibitor. No DNA synthesis or cell proliferation was observed in the presence of carbachol. These results indicate that stimulation of the m3 subtype leads to tyrosine phosphorylation and MAP kinase activation, but is unlikely to have trophic effects in gastric mucosal cells.