Prostaglandin E(2) (PGE(2)) plays an important role in the regulation of duodenal bicarbonate (HCO(3)(-)) secretion, but its signaling pathway(s) are not fully understood. In the present study, we investigated the signaling pathways involved in PGE(2)-mediated duodenal HCO(3)(-) secretion. Murine duodenal mucosal HCO(3)(-) secretion was examined in vitro in Ussing chambers by pH-stat titration in the presence of a variety of signal transduction modulators. Phosphatidylinositol 3-kinase (PI3K) activity was measured by immunoprecipitation of PI3K and ELISA, and Akt phosphorylation was measured by Western analysis with anti-phospho-Akt and anti-Akt antibodies. PGE(2)-stimulated duodenal HCO(3)(-) secretion was reduced by the cAMP-dependent signaling pathway inhibitors MDL-12330A and KT-5720 by 23% and 20%, respectively; the Ca(2+)-influx inhibitor verapamil by 26%; and the calmodulin antagonist W-13 by 24%; whereas the PI3K inhibitors wortmannin and LY-294002 reduced PGE(2)-stimulated HCO(3)(-) secretion by 51% and 47%, respectively. Neither the MAPK inhibitor PD-98059 nor the tyrosine kinase inhibitor genistein altered PGE(2)-stimulated HCO(3)(-) secretion. PGE(2) application caused a rapid and concentration-dependent increase in duodenal mucosal PI3K activity and Akt phosphorylation. These results demonstrated that PGE(2) activates PI3K in duodenal mucosa and stimulates duodenal HCO(3)(-) secretion via cAMP-, Ca(2+)-, and PI3K-dependent signaling pathways.