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Article: Real-time analysis of the activities of GnRH and GnRH analogs in αT3-1 cells by the Cytosensor microphysiometer

TitleReal-time analysis of the activities of GnRH and GnRH analogs in αT3-1 cells by the Cytosensor microphysiometer
Authors
Issue Date2000
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, 2000, v. 80 n. 3, p. 304-312 How to Cite?
AbstractGonadotropin-releasing hormone (GnRH), acting via the GnRH receptor, elicited rapid extracellular acidification responses in mouse gonadotrope-derived αT3-1 cells as measured by the Cytosensor microphysiometer, which indirectly monitors cellular metabolic rates. GnRH increased the extracellular acidification rate of the cells in a dose-dependent manner (EC50 = 1.81 ± 0.24 nM). The GnRH-stimulated acidification rate could be attenuated by protein kinase C (PKC) down-regulation, extracellular Ca2+ depletion, and the voltage-sensitive Ca2+ channel (VSCC) blocker nifedipine, indicating that the acidification response is activated by both Ca2+ and PKC-mediated pathways. Upon continuous exposure to 100 nM GnRH or periodic stimulation by 10 nM GnRH at 40 min intervals, homologous desensitization was more pronounced in the absence of extracellular Ca2+, suggesting that desensitization of GnRH activity may be mediated via depletion of intracellular Ca2+ stores. We have also compared the potency of eight GnRH analogs on αT3-1 cells. No acidification response was detected for GnRH free acid, consistent with the idea that the C-terminal amide is a critical structural determinant for GnRH activity. Replacement of Gly-NH2 at the C-terminus by N-ethylamide dramatically reduced the EC50 value, suggesting that substitution of the Gly-NH2 moiety by N-ethylamide increases the potency of GnRH analogs. Substitution of Gly at position 6 by D-Trp significantly reduced the EC50 value, whereas D-Lys at the same position slightly increased the EC50 value, implying that either an aromatic amino acid or a non-basic amino acid at position 6 may be essential for potent GnRH agonists. In summary, our results demonstrate that the Cytosensor microphysiometer can be used to evaluate the actions of GnRH and GnRH analogs in αT3-1 cells in a real-time and noninvasive manner. This silicon-based microphysiometric system should provide new information on the structure-function studies of GnRH and is an invaluable tool for the screening of new GnRH agonists and antagonists in the future. © 2001 Wiley-Liss, Inc.
Persistent Identifierhttp://hdl.handle.net/10722/178718
ISSN
2015 Impact Factor: 3.446
2015 SCImago Journal Rankings: 1.520
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorNg, SSMen_US
dc.contributor.authorYu, KLen_US
dc.contributor.authorYung, WHen_US
dc.contributor.authorChow, BKCen_US
dc.date.accessioned2012-12-19T09:49:19Z-
dc.date.available2012-12-19T09:49:19Z-
dc.date.issued2000en_US
dc.identifier.citationJournal Of Cellular Biochemistry, 2000, v. 80 n. 3, p. 304-312en_US
dc.identifier.issn0730-2312en_US
dc.identifier.urihttp://hdl.handle.net/10722/178718-
dc.description.abstractGonadotropin-releasing hormone (GnRH), acting via the GnRH receptor, elicited rapid extracellular acidification responses in mouse gonadotrope-derived αT3-1 cells as measured by the Cytosensor microphysiometer, which indirectly monitors cellular metabolic rates. GnRH increased the extracellular acidification rate of the cells in a dose-dependent manner (EC50 = 1.81 ± 0.24 nM). The GnRH-stimulated acidification rate could be attenuated by protein kinase C (PKC) down-regulation, extracellular Ca2+ depletion, and the voltage-sensitive Ca2+ channel (VSCC) blocker nifedipine, indicating that the acidification response is activated by both Ca2+ and PKC-mediated pathways. Upon continuous exposure to 100 nM GnRH or periodic stimulation by 10 nM GnRH at 40 min intervals, homologous desensitization was more pronounced in the absence of extracellular Ca2+, suggesting that desensitization of GnRH activity may be mediated via depletion of intracellular Ca2+ stores. We have also compared the potency of eight GnRH analogs on αT3-1 cells. No acidification response was detected for GnRH free acid, consistent with the idea that the C-terminal amide is a critical structural determinant for GnRH activity. Replacement of Gly-NH2 at the C-terminus by N-ethylamide dramatically reduced the EC50 value, suggesting that substitution of the Gly-NH2 moiety by N-ethylamide increases the potency of GnRH analogs. Substitution of Gly at position 6 by D-Trp significantly reduced the EC50 value, whereas D-Lys at the same position slightly increased the EC50 value, implying that either an aromatic amino acid or a non-basic amino acid at position 6 may be essential for potent GnRH agonists. In summary, our results demonstrate that the Cytosensor microphysiometer can be used to evaluate the actions of GnRH and GnRH analogs in αT3-1 cells in a real-time and noninvasive manner. This silicon-based microphysiometric system should provide new information on the structure-function studies of GnRH and is an invaluable tool for the screening of new GnRH agonists and antagonists in the future. © 2001 Wiley-Liss, Inc.en_US
dc.languageengen_US
dc.publisherJohn Wiley & Sons, Inc. The Journal's web site is located at http://www3.interscience.wiley.com/cgi-bin/jhome/35503en_US
dc.relation.ispartofJournal of Cellular Biochemistryen_US
dc.rightsJournal of Cellular Biochemistry. Copyright © John Wiley & Sons, Inc.-
dc.subject.meshAmino Acid Sequenceen_US
dc.subject.meshAnimalsen_US
dc.subject.meshBiosensing Techniquesen_US
dc.subject.meshCell Lineen_US
dc.subject.meshGonadotropin-Releasing Hormone - Analogs & Derivatives - Physiologyen_US
dc.subject.meshHydrogen-Ion Concentrationen_US
dc.subject.meshStructure-Activity Relationshipen_US
dc.titleReal-time analysis of the activities of GnRH and GnRH analogs in αT3-1 cells by the Cytosensor microphysiometeren_US
dc.typeArticleen_US
dc.identifier.emailNg, SSM: ssmng@hku.hken_US
dc.identifier.emailChow, BKC: bkcc@hku.hken_US
dc.identifier.authorityNg, SSM=rp00767en_US
dc.identifier.authorityChow, BKC=rp00681en_US
dc.description.naturelink_to_subscribed_fulltexten_US
dc.identifier.doi10.1002/1097-4644(20010301)80:3<304::AID-JCB20>3.0.CO;2-Hen_US
dc.identifier.pmid11135359-
dc.identifier.scopuseid_2-s2.0-0034479734en_US
dc.identifier.hkuros56554-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0034479734&selection=ref&src=s&origin=recordpageen_US
dc.identifier.volume80en_US
dc.identifier.issue3en_US
dc.identifier.spage304en_US
dc.identifier.epage312en_US
dc.identifier.isiWOS:000166756000002-
dc.publisher.placeUnited Statesen_US
dc.identifier.scopusauthoridNg, SSM=7403358718en_US
dc.identifier.scopusauthoridYu, KL=7403385265en_US
dc.identifier.scopusauthoridYung, WH=7103137893en_US
dc.identifier.scopusauthoridChow, BKC=7102826193en_US

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