Modulation of gastric mucosal paracellular permeability by secretin: Role of src family tyrosine kinase

Abstract

Background: Previous studies found that physiological concentrations of secretin increase transepithelial resistance (TER) in gastric mucosa in a fashion synergistic with EGF, indicating that spacially limited receptors and distinct signal transduction pathways are involved in the regulation of paracellular permeability. Pretreatment with PP2, a selective inhibitor of sm family of tyrosine kinase, significantly attenuated the increase in TER activated by secretio, but not by EGF, suggesting the src family kinases mediates secretin action on paracelloiar permeability. Aim and Methods: In order to identify the mechanism(s) by which secretio receptors lead to src family kinase activation, we examined the stimulation of src Idnsse activity measured by the autophosphorylation of Tyr416 and the inhibition of the overall profile of cellular protein tyrosine phosphorylation in primary canine gastric epithelial cell monoiayors by PP2(5/~M) after secretin (lnM) activation. Results: Primary gastric epithelial cell cultures showed consistent amount of phosphorylation of src Tyr416 at the basal levels and the stimulation by secretin only caused a maximal 2-fold activation of src activity after 10 to 15 min. However, this activation was not blocked by pretreatment of PP2. When total lysates were blotted with tyrosine phosphorylation antibody (4G10), several tyrosine phosphoryluted proteins activated by secretin were blocked by pretreatment with PP2 in a dose-dependent manner. The secretin-activated tyrosine phosphorylations were not inhibited by pretreatment with PP3(inactive src kinase inhibitor), nor with AG1478, a specific EGFR tyrosine kinase inhibitor, suggesting that these tyrosine pbosphofylated proteins serve either as src-family kinases or as substrates for these kinases. Conclusion: Our results showed that secretin activated specific tyrosine phosphorylated proteins via src-family tyrosine kinases. This finding suggests a novel function of src-family tyrosine kinases in regulating gastric paracelluiar permeability.