Background & Aims: 

The exchange factor directly activated by cAMP (Epac) is a newly discovered direct target for cAMP and cAMP has been shown to stimulate intestinal Cl^- secretion. Thus, we studied the role of Epac in FSK stimulated Cl^- secretion in T84 cells.

Methods: 

T84 cell monolayers grown on transwell insert were studied in Ussing chamber under voltage clamp condition for the measurement of agonist stimulated anion secretion. The Ca⁺² sensitive fluorescence dye, Fura-2 was used for [Ca²⁺]_i measurement. Activation of Rap2 was determined by EZ-Dectect^TM Rap activation kit (Pierce).

Results: 

FSK stimulated Cl^- secretion was inhibited by PKC inhibitor, 5mM GO6976 and by the PKA inhibitor H-89 (1mM). Complete inhibition of Cl^- secretion was obtained by GO6976 plus H-89, suggesting that FSK stimulated Cl^- secretion was both PKA dependent and independent. FSK elevated [Ca²⁺]_i and FSK stimulated Cl^- secretion was inhibited by BAPTA/ AM, an intracellular calcium chelator, and by the PLC inhibitor U73122, confirming the role of Ca²⁺in FSK stimulated Cl^- secretion. By western blot and RT-PCR, Epac1 was found expressed in T84 cells. The Epac agonist, 8pCPT-2'-O-Me-cAMP stimulated Cl^- secretion, and this was inhibited by BAPTA/AM but not by H-89. It has been shown that Rap2 activates PLCe/ Ca²⁺signaling. FSK and 8pCPT-2'-O-Me-cAMP activated Rap2 and this suggests that Epac1 and Rap2 linked cAMP into PLC/Ca²⁺signaling in this secretory process.

Conclusion: 

We concluded that cAMP mediated epithelial Cl^- secretion was caused by the sum of PKA dependent and independent events, the latter was mediated through Epac1-Rap2-PLC-Ca²⁺signaling.