Role of Epac in cAMP mediated PKA independent mechanism of intestinal Cl‐secretion

Abstract

cAMP is a major regulator of Cl−secretion. With the recognition that the exchange protein directly activated by cAMP (Epac) transduces cAMP signaling cascades independent of protein kinase A (PKA), we test the hypothesis that both PKA and Epac are involved in forskolin (FSK) stimulated Cl− secretion. FSK is an adenylate cyclase activator. T84 cells were used for short circuit current (Isc) measurement in the Ussing chamber. FSK stimulated Isc was completely inhibited by the additive effects of the PKA inhibitor, H89 (1μM) and the [Ca2+]i chelator, BAPTA‐AM (25μM), suggesting the presence of both PKA dependent and independent mechanisms. Epac1 was found to be expressed in T84 cells. Both FSK and the Epac selective agonist, 8‐pCPT‐2‘‐O‐Me‐cAMP (8‐CPT, 50μM), elevated [Ca2+]i. Activation of Epac1 by 8‐CPT induced Isc in intact T84 cells and in cells permeabilized with nystatin at the basolateral membrane. This effect of 8‐CPT was completely abolished by BAPTA‐AM, but not by H89 (1μM) and the CFTR specific blocker, cftr‐172, suggesting that 8‐CPT stimulated non‐CFTR channel(s) via elevation of [Ca2+]i. FSK stimulated Isc was inhibited by the PLC inhibitor U73122. Both FSK and 8‐CPT increased the amount of activated (GTP‐bound) Rap proteins, which have been shown to activate PLCε to mobilize [Ca2+]i. Our findings in T84 cells were further validated in mouse ileum. 8‐CPT stimulated Isc in mouse ileal sheet and this stimulation was not inhibited by H89. We conclude that PKA and Epac1 contribute to FSK stimulated Cl− secretion in intestine via PKA dependent and Ca2+ dependent pathways, the latter mediated by Epac1‐Rap2‐PLC‐Ca2+ signaling involving non‐CFTR channel(s).