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

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

Research output: Contribution to journalArticle

48 Citations (Scopus)

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 1 2010

Fingerprint

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

All Science Journal Classification (ASJC) codes

  • Physiology

Cite this

Hoque, Kazi Mirajul ; Woodward, Owen M. ; Van Rossum, Damian B. ; Zachos, Nicholas C. ; 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.
@article{1460e2d545614147b24aaf79d8d0cce8,
title = "Epac1 mediates protein kinase A-independent mechanism of forskolinactivated intestinal chloride secretion",
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 {\`i}M). 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.",
author = "Hoque, {Kazi Mirajul} and Woodward, {Owen M.} and {Van Rossum}, {Damian B.} and Zachos, {Nicholas C.} and Linxi Chen and Leung, {George P.H.} and Guggino, {William B.} and Guggino, {Sandra E.} and Tse, {Chung Ming}",
year = "2010",
month = "1",
day = "1",
doi = "10.1085/jgp.200910339",
language = "English (US)",
volume = "135",
pages = "43--58",
journal = "Journal of General Physiology",
issn = "0022-1295",
publisher = "Rockefeller University Press",
number = "1",

}

Hoque, KM, Woodward, OM, Van Rossum, DB, Zachos, NC, Chen, L, Leung, GPH, Guggino, WB, Guggino, SE & Tse, CM 2010, 'Epac1 mediates protein kinase A-independent mechanism of forskolinactivated intestinal chloride secretion', Journal of General Physiology, vol. 135, no. 1, pp. 43-58. https://doi.org/10.1085/jgp.200910339

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

In: Journal of General Physiology, Vol. 135, No. 1, 01.01.2010, p. 43-58.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Epac1 mediates protein kinase A-independent mechanism of forskolinactivated intestinal chloride secretion

AU - Hoque, Kazi Mirajul

AU - Woodward, Owen M.

AU - Van Rossum, Damian B.

AU - Zachos, Nicholas C.

AU - Chen, Linxi

AU - Leung, George P.H.

AU - Guggino, William B.

AU - Guggino, Sandra E.

AU - Tse, Chung Ming

PY - 2010/1/1

Y1 - 2010/1/1

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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=73549089880&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=73549089880&partnerID=8YFLogxK

U2 - 10.1085/jgp.200910339

DO - 10.1085/jgp.200910339

M3 - Article

C2 - 20038525

AN - SCOPUS:73549089880

VL - 135

SP - 43

EP - 58

JO - Journal of General Physiology

JF - Journal of General Physiology

SN - 0022-1295

IS - 1

ER -