Carbachol stimulates transactivation of epidermal growth factor receptor and mitogen-activated protein kinase in T84 cells -: Implications for carbachol-stimulated chloride secretion

We have examined the role of tyrosine phosphorylation in regulation of calcium-dependent chloride secretion across T-84 colonic epithelial cells. The calcium-mediated agonist carbachol (CCh, 100 mu M) stimulated a time dependent increase in tyrosine phosphorylation of a range of proteins (with molecular masses ranging up to 180 kDa) in T-84 cells. The tyrosine kinase inhibitor, genistein (5 mu M), significantly potentiated chloride secretory responses to CCh, indicating a role for CCh-stimulated tyrosine phosphorylation in negative regulation of CCh-stimulated secretory responses. Further studies revealed that CCh stimulated an increase in both phosphorylation and activity of the extracellular signal-regulated kinase (ERK) isoforms of mitogen-activated protein kinase, Chloride secretory responses to CCh were also potentiated by the mitogen-activated protein kinase inhibitor, PD98059 (20 mu M), Phosphorylation of ERK in response to CCh was mimicked by the protein kinase C (PKC) activator, phorbol myristate acetate (100 nM), but was not altered by the PKC inhibitor GF 109203X (1 mu M). ERK phosphorylation was also induced by epidermal growth factor (EGF) (100 ng/ml), Immunoprecipitation/Western blot studies revealed that CCh stimulated tyrosine phosphorylation of the EGF receptor (EGFr) and increased co immunoprecipitation of the adapter proteins, Shc and Grb2, with the EGFr, An inhibitor of EGFr phosphorylation, tyrphostin AG1478 (1 mu M), reversed CCh-stimulated phosphorylation of both EGFr and ERK, Tyrphostin AG1478 also potentiated chloride secretory responses to CCh, We conclude that CCh activates ERK in T-84 cells via a mechanism involving transactivation of the EGFr, and that this pathway constitutes an inhibitory signaling pathway by which chloride secretory responses to CCh may be negatively regulated.