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Role of a non‐CFTR chloride channel in intestinal epithelial chloride secretion
Author(s) -
Kazi Mirajul Hoque,
Woodward Owen M,
Guggino Sandra E,
Tse ChungMing
Publication year - 2010
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.24.1_supplement.1014.1
Subject(s) - cystic fibrosis transmembrane conductance regulator , forskolin , chloride channel , protein kinase a , secretion , apical membrane , chemistry , microbiology and biotechnology , ibmx , ussing chamber , medicine , endocrinology , biophysics , biology , kinase , biochemistry , receptor , membrane , gene
The cystic fibrosis transmembrane regulator (CFTR) is the major Cl channel found in the apical membrane of intestinal epithelial cells. Protein kinase A (PKA) phosphorylates and opens CFTR. E xchange p rotein directly a ctivated by c AMP (Epac) upon binding of cAMP, activates PKA‐independent signaling via small G proteins. We tested the hypothesis that Epac are involved in cAMP‐stimulated Cl secretion. Human intestinal T84 cells and mouse intestine were used for short circuit current (Isc) measurement in response to agonist stimulated Cl secretion. Epac activator, 8‐pCPT‐2′‐O‐Me‐cAMP, elevated [Ca 2+ ]i, activated Rap2 protein and induced Cl secretion in intact and basolateral membrane permeabilized T84 cells and mouse ileum. These effects of 8‐pCPT‐2′‐O‐Me‐cAMP were completely abolished by BAPTA‐AM, but not by H89. In contrast, T84 cells with silenced Epac1 protein had reduced Isc response to forskolin which was completely inhibited by H89 but not by the phospholipase C inhibitor, U73122. The lack of effect of 8‐pCPT‐2′‐O‐Me‐cAMP either to CFTRinhi‐172, glibenclamide or on whole cell patch clamp recording of Cl current in CHO cells transiently expressing the human CFTR, implicates that CFTR channels are not involved. Biophysical characterization of the Epac1‐dependent Cl conductance in Ussing chambers suggests that Epac mediated Cl conductance was hyperpolarization‐activated, inward rectifying, and displays a Cl − >Br − >I − permeability sequence. These results lead us to conclude that the Epac‐Rap‐PLC‐[Ca 2+ ]i signaling pathway is involved in cAMP‐stimulated Cl secretion through non‐CFTR Cl channel.