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Article: Real-time evaluation of human secretin receptor activity using cytosensor microphysiometry

TitleReal-time evaluation of human secretin receptor activity using cytosensor microphysiometry
Authors
KeywordscAMP
Cytosensor microphysiometry
Human secretin receptor
Issue Date1999
PublisherJohn Wiley & Sons, Inc. The Journal's web site is located at http://www3.interscience.wiley.com/cgi-bin/jhome/35503
Citation
Journal Of Cellular Biochemistry, 1999, v. 72 n. 4, p. 517-527 How to Cite?
AbstractHuman secretin receptor is a G protein-coupled receptor thai is functionally linked to the cAMP second messenger system by stimulation of adenylate cyclase. To functionally characterize the receptor and evaluate its signal transduction pathway, the full-length human secretin receptor cDNA was subcloned into the mammalian expression vector pRc/CMV and expressed in cultured CHO cells. Intracellular cAMP accumulation of the stably transfected cells was measured by a radioimmunoassay (RIA), while the extracellular acidification rate was measured by the Cytosensor microphysiometer. Human secretin and biotinylated human secretin were equipotent in both assays in a dose-dependent manner. The EC50 values of stimulating the intracellular cAMP accumulation and the extracellular acidification rate were 0.2-0.5 nM and 0.1 nM, respectively, indicating that microphysiometry is more sensitive than the cAMP assay in monitoring ligand stimulation of the human secretin receptor. The secretin-stimulated response could be mimicked by forskolin and augmented by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, indicating that the extracellular acidification response is positively correlated with intracellular cAMP level. The response could be abolished by the protein kinase A inhibitor H-89, suggesting that protein kinase A plays an essential role in the intracellular signaling of the receptor. Upon repeated stimulation by the ligand, the peak acidification responses did not change significantly at both physiological (0.03 nM and 3 nM) and pharmacological (0.3 μM) concentrations of human secretin, suggesting that the human secretin receptor did not exhibit robust homologous desensitization.
Persistent Identifierhttp://hdl.handle.net/10722/84830
ISSN
2015 Impact Factor: 3.446
2015 SCImago Journal Rankings: 1.520
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorNg, SSMen_HK
dc.contributor.authorPang, RTKen_HK
dc.contributor.authorChow, BKCen_HK
dc.contributor.authorCheng, CHKen_HK
dc.date.accessioned2010-09-06T08:57:37Z-
dc.date.available2010-09-06T08:57:37Z-
dc.date.issued1999en_HK
dc.identifier.citationJournal Of Cellular Biochemistry, 1999, v. 72 n. 4, p. 517-527en_HK
dc.identifier.issn0730-2312en_HK
dc.identifier.urihttp://hdl.handle.net/10722/84830-
dc.description.abstractHuman secretin receptor is a G protein-coupled receptor thai is functionally linked to the cAMP second messenger system by stimulation of adenylate cyclase. To functionally characterize the receptor and evaluate its signal transduction pathway, the full-length human secretin receptor cDNA was subcloned into the mammalian expression vector pRc/CMV and expressed in cultured CHO cells. Intracellular cAMP accumulation of the stably transfected cells was measured by a radioimmunoassay (RIA), while the extracellular acidification rate was measured by the Cytosensor microphysiometer. Human secretin and biotinylated human secretin were equipotent in both assays in a dose-dependent manner. The EC50 values of stimulating the intracellular cAMP accumulation and the extracellular acidification rate were 0.2-0.5 nM and 0.1 nM, respectively, indicating that microphysiometry is more sensitive than the cAMP assay in monitoring ligand stimulation of the human secretin receptor. The secretin-stimulated response could be mimicked by forskolin and augmented by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, indicating that the extracellular acidification response is positively correlated with intracellular cAMP level. The response could be abolished by the protein kinase A inhibitor H-89, suggesting that protein kinase A plays an essential role in the intracellular signaling of the receptor. Upon repeated stimulation by the ligand, the peak acidification responses did not change significantly at both physiological (0.03 nM and 3 nM) and pharmacological (0.3 μM) concentrations of human secretin, suggesting that the human secretin receptor did not exhibit robust homologous desensitization.en_HK
dc.languageengen_HK
dc.publisherJohn Wiley & Sons, Inc. The Journal's web site is located at http://www3.interscience.wiley.com/cgi-bin/jhome/35503en_HK
dc.relation.ispartofJournal of Cellular Biochemistryen_HK
dc.rightsJournal of Cellular Biochemistry. Copyright © John Wiley & Sons, Inc.en_HK
dc.subjectcAMPen_HK
dc.subjectCytosensor microphysiometryen_HK
dc.subjectHuman secretin receptoren_HK
dc.titleReal-time evaluation of human secretin receptor activity using cytosensor microphysiometryen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0730-2312&volume=72&spage=517&epage=527&date=1999&atitle=Real-Time+Evaluation+of+Human+Secretin+Receptor+Activity+Using+Cytosensor+Microphysiometryen_HK
dc.identifier.emailNg, SSM: ssmng@hku.hken_HK
dc.identifier.emailPang, RTK: rtkpang@hku.hken_HK
dc.identifier.emailChow, BKC: bkcc@hku.hken_HK
dc.identifier.authorityNg, SSM=rp00767en_HK
dc.identifier.authorityPang, RTK=rp01761en_HK
dc.identifier.authorityChow, BKC=rp00681en_HK
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1002/(SICI)1097-4644(19990315)72:4<517::AID-JCB7>3.0.CO;2-1en_HK
dc.identifier.pmid10022611-
dc.identifier.scopuseid_2-s2.0-0033559278en_HK
dc.identifier.hkuros40391en_HK
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0033559278&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume72en_HK
dc.identifier.issue4en_HK
dc.identifier.spage517en_HK
dc.identifier.epage527en_HK
dc.identifier.isiWOS:000078362700007-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridNg, SSM=7403358718en_HK
dc.identifier.scopusauthoridPang, RTK=7004376636en_HK
dc.identifier.scopusauthoridChow, BKC=7102826193en_HK
dc.identifier.scopusauthoridCheng, CHK=7404798014en_HK

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