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Epac1 mediates Protein Kinase A (PKA) independent mechanism of forskolin (FSK) stimulated Cl secretion in T84 cells
Author(s) -
Kazi Mirajul Hoque,
Rossum Damian B,
Chen Linxi,
Tse ChungMing
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a541-d
Subject(s) - forskolin , secretion , protein kinase c , protein kinase a , chemistry , bapta , ibmx , frequency shift keying , medicine , signal transduction , microbiology and biotechnology , endocrinology , stimulation , intracellular , biology , kinase , biochemistry , channel (broadcasting) , engineering , demodulation , electrical engineering
Intestinal Cl secretion is regulated by Ca 2+ and cAMP. It has been assumed that cAMP stimulated Cl secretion is mediated exclusively by PKA. However, FSK, the adenylate cyclase activator has been shown to also increase [Ca 2+ ]i by unknown mechanisms. Epac is a recent discovered protein activated by cAMP but its action is independent of PKA. Thus, we studied the role of Epac in FSK stimulated Cl secretion. By Western blot & RT‐PCR, Epac1 was found in T84 cells. Cells were grown on membranes for measurement of short‐circuit current (Isc) in Ussing chambers. FSK stimulated Isc (Cl secretion). Both the Ca‐dependent PKC inhibitor, GO6976 (5μM) (ΔIsc 18 ± 3μA vs 28 ± 5μA, P<0.05) and the PKA inhibitor H89 (1μM) (13 ±2μA vs 32 ±2μA, P<0.01) inhibited FSK stimulated Cl secretion, and their effects were additive. This suggested that FSK stimulated Cl secretion was both PKA dependent and independent. This PKA independent component was also inhibited by BAPTA/AM, an intracellular calcium chelator (ΔIsc 16±3μA vs 29±3μA; P<0.001) and by the PLC inhibitor U73122 (ΔIsc 20 ±2μA vs 32±4μA, p<0.01). Taken together, these results suggested that the PKA‐independent effect of FSK was PLC/Ca +2 /PKC dependent. Furthermore, FSK elevated [Ca +2 ]i (ΔCa 2+ ]i 42 ±15nM) which was inhibited by U73122. Epac agonist, 8pCPT‐2′‐O‐Me‐cAMP stimulated Cl secretion and this stimulation was not inhibited by H89 (1μM) but was inhibited by BAPTA/AM. Conclusions: our results suggest that cAMP mediated Cl secretion is caused by concurrent PKA dependent and Ca +2 dependent mechanisms. The latter is mediated through Epac‐PLC signaling. Epac mediates PKA independent mechanism of FSK stimulated Cl secretion.