Epac1 mediates protein kinase A-independent mechanism of forskolinactivated intestinal chloride secretion

Kazi Mirajul Hoque, Owen M. Woodward, Damian B. Van Rossum, Nicholas Zachos, Linxi Chen, George P H Leung, William B Guggino, Sandra E. Guggino, Chung Ming Tse

Research output: Contribution to journalArticle

Abstract

Intestinal Cl- secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl- secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Cl - secretion. FSK-stimulated Cl- secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 μM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N, N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 ìM). Both FSK and the Epac activator 8-pCPT-2′-O-Me-cAMP (50 μM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl- secretion in intact or basolateral membrane-permeabilized T84 cells and mouse ileal sheets. The 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 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2′-O-Me-cAMP on Cl - secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2′-O-Me- cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl- conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl ->Br->I- permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl- secretion, which is carried by a novel, previously undescribed Cl- channel.

Original languageEnglish (US)
Pages (from-to)43-58
Number of pages16
JournalJournal of General Physiology
Volume135
Issue number1
DOIs
StatePublished - Jan 2010

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Intestinal Secretions
Cyclic AMP-Dependent Protein Kinases
Cyclic AMP
Chlorides
Colforsin
Cystic Fibrosis
Proteins
ras Proteins
Ethane
Glyburide
Type C Phospholipases
Protein Kinase Inhibitors
Chelating Agents
Cricetulus
Small Intestine
Ovary
Permeability
Esters
Calcium

ASJC Scopus subject areas

  • Physiology

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Epac1 mediates protein kinase A-independent mechanism of forskolinactivated intestinal chloride secretion. / Hoque, Kazi Mirajul; Woodward, Owen M.; Van Rossum, Damian B.; Zachos, Nicholas; Chen, Linxi; Leung, George P H; Guggino, William B; Guggino, Sandra E.; Tse, Chung Ming.

In: Journal of General Physiology, Vol. 135, No. 1, 01.2010, p. 43-58.

Research output: Contribution to journalArticle

Hoque, Kazi Mirajul ; Woodward, Owen M. ; Van Rossum, Damian B. ; Zachos, Nicholas ; Chen, Linxi ; Leung, George P H ; Guggino, William B ; Guggino, Sandra E. ; Tse, Chung Ming. / Epac1 mediates protein kinase A-independent mechanism of forskolinactivated intestinal chloride secretion. In: Journal of General Physiology. 2010 ; Vol. 135, No. 1. pp. 43-58.
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AU - Guggino, William B

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AU - Tse, Chung Ming

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N2 - Intestinal Cl- secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl- secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Cl - secretion. FSK-stimulated Cl- secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 μM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N, N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 ìM). Both FSK and the Epac activator 8-pCPT-2′-O-Me-cAMP (50 μM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl- secretion in intact or basolateral membrane-permeabilized T84 cells and mouse ileal sheets. The 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 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2′-O-Me-cAMP on Cl - secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2′-O-Me- cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl- conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl ->Br->I- permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl- secretion, which is carried by a novel, previously undescribed Cl- channel.

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