File Download
  Links for fulltext
     (May Require Subscription)
Supplementary

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

TitleEpac1 mediates protein kinase A-independent mechanism of forskolinactivated intestinal chloride secretion
Authors
Issue Date2010
PublisherRockefeller University Press. The Journal's web site is located at www.jgp.org/
Citation
Journal Of General Physiology, 2010, v. 135 n. 1, p. 43-58 How to Cite?
AbstractIntestinal 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. © 2010 Hoque et al.
Persistent Identifierhttp://hdl.handle.net/10722/124000
ISSN
2015 Impact Factor: 4.511
2015 SCImago Journal Rankings: 3.061
PubMed Central ID
ISI Accession Number ID
Funding AgencyGrant Number
Johns Hopkins Center for Global Health80019485
National Institutes of Health/NIDDK Hopkins Digestive Disease Basic Research DevelopmentR24DK064399
Cystic Fibrosis FoundationR025-CR07
Funding Information:

This work was supported by the Johns Hopkins Center for Global Health (grant 80019485 to C.-M. Tse) and the National Institutes of Health/NIDDK Hopkins Digestive Disease Basic Research Development (grant R24DK064399; mouse core to S. E. Guggino and C.-M. Tse). The Cystic Fibrosis Foundation supported S. E. Guggino (grant R025-CR07).

References

 

DC FieldValueLanguage
dc.contributor.authorHoque, KMen_HK
dc.contributor.authorWoodward, OMen_HK
dc.contributor.authorVan Rossum, DBen_HK
dc.contributor.authorZachos, NCen_HK
dc.contributor.authorChen, Len_HK
dc.contributor.authorLeung, GPHen_HK
dc.contributor.authorGuggino, WBen_HK
dc.contributor.authorGuggino, SEen_HK
dc.contributor.authorTse, CMen_HK
dc.date.accessioned2010-10-19T03:22:10Z-
dc.date.available2010-10-19T03:22:10Z-
dc.date.issued2010en_HK
dc.identifier.citationJournal Of General Physiology, 2010, v. 135 n. 1, p. 43-58en_HK
dc.identifier.issn0022-1295en_HK
dc.identifier.urihttp://hdl.handle.net/10722/124000-
dc.description.abstractIntestinal 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. © 2010 Hoque et al.en_HK
dc.languageeng-
dc.publisherRockefeller University Press. The Journal's web site is located at www.jgp.org/en_HK
dc.relation.ispartofJournal of General Physiologyen_HK
dc.subject.meshChlorine - metabolism-
dc.subject.meshCyclic AMP - administration and dosage-
dc.subject.meshCyclic AMP-Dependent Protein Kinases - metabolism-
dc.subject.meshForskolin - administration and dosage-
dc.subject.meshGuanine Nucleotide Exchange Factors - metabolism-
dc.titleEpac1 mediates protein kinase A-independent mechanism of forskolinactivated intestinal chloride secretionen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0022-1295&volume=135&issue=1&spage=43&epage=58&date=2010&atitle=Epac1+mediates+protein+kinase+A-independent+mechanism+of+forskolin-activated+intestinal+chloride+secretion-
dc.identifier.emailLeung, GPH: gphleung@hkucc.hku.hken_HK
dc.identifier.authorityLeung, GPH=rp00234en_HK
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.1085/jgp.200910339en_HK
dc.identifier.pmid20038525-
dc.identifier.pmcidPMC2806414-
dc.identifier.scopuseid_2-s2.0-73549089880en_HK
dc.identifier.hkuros172611-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-73549089880&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume135en_HK
dc.identifier.issue1en_HK
dc.identifier.spage43en_HK
dc.identifier.epage58en_HK
dc.identifier.isiWOS:000273099700005-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridHoque, KM=20436385200en_HK
dc.identifier.scopusauthoridWoodward, OM=13406220900en_HK
dc.identifier.scopusauthoridVan Rossum, DB=7004262055en_HK
dc.identifier.scopusauthoridZachos, NC=7801576219en_HK
dc.identifier.scopusauthoridChen, L=24471094500en_HK
dc.identifier.scopusauthoridLeung, GPH=35963668200en_HK
dc.identifier.scopusauthoridGuggino, WB=35448882600en_HK
dc.identifier.scopusauthoridGuggino, SE=7006776836en_HK
dc.identifier.scopusauthoridTse, CM=7103295076en_HK

Export via OAI-PMH Interface in XML Formats


OR


Export to Other Non-XML Formats