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Article: Studies of melatonin effects on epithelia using the human embryonic kidney-293 (HEK-293) cell line

TitleStudies of melatonin effects on epithelia using the human embryonic kidney-293 (HEK-293) cell line
Authors
Issue Date1997
PublisherThe Endocrine Society. The Journal's web site is located at http://endo.endojournals.org
Citation
Endocrinology, 1997, v. 138 n. 11, p. 4732-4739 How to Cite?
AbstractThe expression of melatonin receptors (MR) of the Mel(1a) subtype in basolateral membrane of guinea pig kidney proximal tubule suggests that melatonin plays a role in regulating epithelial functions. To investigate the cellular basis of melatonin action on epithelia, we sought to establish an appropriate in vitro culture model. Epithelial cell lines originating from kidneys of dog (MDCK), pig (LLC-PK1), opossum (OK), and human embryo (HEK- 293) were each tested for the presence of MR using 2-[125I]iodomelatonin (125I-MEL) as a radioligand. The HEK-293 cell line exhibited the highest specific 125I-MEL binding. By intermediate filament characterization, the HEK-293 cells were determined to be of epithelial origin. Binding of 125I- MEL in HEK-293 cells demonstrated saturability, reversibility, and high specificity with an equilibrium dissociation constant (K(d)) value of 23.8 ± 0.5 pM and a maximum number of binding sites (B(max)) value of 1.17 ± 0.11 fmol/mg protein (n = 5), which are comparable with the reported K(d) and B(max) values in human kidney cortex. Coincubation with GTPyS (10 μM) and pertussis toxin (100 ng/ml) provoked a marked decrease in binding affinity (K(d) was increased by a factor of 1.5-2.0), with no significant difference in B(max). Melatonin (1 μM) decreased the forskolin (10 μM) stimulated cAMP level by 50%. HEK-293 cells do not express dopamine D1A receptor. Following transient transfection of HEK-293 cells with human dopamine D1A receptor (hD1A-R), exposure of the cells to dopamine stimulated an increase in the level of cAMP. Similarly, transient transfection of HEK-293 cells with rat glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and PTH type 1 receptors, each resulted in an hormone inducible increase in cAMP levels. Surprisingly, only the stimulatory effect of dopamine could be inhibited by exposure to melatonin. The inhibitory effect of melatonin on dopamine D1-induced increase in cAMP was completely inhibited by pertussis toxin (100 ng/ml, 18 h). Immunoblot and immunocytochemical studies were carried out using two polyclonal antibodies raised against the extra and cytoplasmic domains of Mel receptor. Immunoblot studies using antibody against the cytoplasmic domain of Mel(1a) receptor confirmed the presence of a peptide blockable 37 kDa band in HEK-293 cells. Indirect immunofluorescent studies with both antibodies revealed staining predominantly at the cell surface, but staining with the antibody directed against the cytoplasmic domain required prior cell permeabilization. By RT- PCR, HEK-293 cells express both Mel(1a) and Mel(1b) messenger RNAs, but the messenger RNA level for Mel(1b) is several orders of magnitude lower than for Mel(1a). We conclude that HEK-293 cells express MR predominantly of the Mel(1a) subtype. Our evidence suggests that one of the ways that melatonin exerts its biological function is through modulation of cellular dopaminergic responses.
Persistent Identifierhttp://hdl.handle.net/10722/49299
ISSN
2015 Impact Factor: 4.159
2015 SCImago Journal Rankings: 2.363
ISI Accession Number ID
References

 

DC FieldValueLanguage
dc.contributor.authorChan, CWYen_HK
dc.contributor.authorSong, Yen_HK
dc.contributor.authorAilenberg, Men_HK
dc.contributor.authorWheeler, Men_HK
dc.contributor.authorPang, SFen_HK
dc.contributor.authorBrown, GMen_HK
dc.contributor.authorSilverman, Men_HK
dc.date.accessioned2008-06-12T06:38:50Z-
dc.date.available2008-06-12T06:38:50Z-
dc.date.issued1997en_HK
dc.identifier.citationEndocrinology, 1997, v. 138 n. 11, p. 4732-4739en_HK
dc.identifier.issn0013-7227en_HK
dc.identifier.urihttp://hdl.handle.net/10722/49299-
dc.description.abstractThe expression of melatonin receptors (MR) of the Mel(1a) subtype in basolateral membrane of guinea pig kidney proximal tubule suggests that melatonin plays a role in regulating epithelial functions. To investigate the cellular basis of melatonin action on epithelia, we sought to establish an appropriate in vitro culture model. Epithelial cell lines originating from kidneys of dog (MDCK), pig (LLC-PK1), opossum (OK), and human embryo (HEK- 293) were each tested for the presence of MR using 2-[125I]iodomelatonin (125I-MEL) as a radioligand. The HEK-293 cell line exhibited the highest specific 125I-MEL binding. By intermediate filament characterization, the HEK-293 cells were determined to be of epithelial origin. Binding of 125I- MEL in HEK-293 cells demonstrated saturability, reversibility, and high specificity with an equilibrium dissociation constant (K(d)) value of 23.8 ± 0.5 pM and a maximum number of binding sites (B(max)) value of 1.17 ± 0.11 fmol/mg protein (n = 5), which are comparable with the reported K(d) and B(max) values in human kidney cortex. Coincubation with GTPyS (10 μM) and pertussis toxin (100 ng/ml) provoked a marked decrease in binding affinity (K(d) was increased by a factor of 1.5-2.0), with no significant difference in B(max). Melatonin (1 μM) decreased the forskolin (10 μM) stimulated cAMP level by 50%. HEK-293 cells do not express dopamine D1A receptor. Following transient transfection of HEK-293 cells with human dopamine D1A receptor (hD1A-R), exposure of the cells to dopamine stimulated an increase in the level of cAMP. Similarly, transient transfection of HEK-293 cells with rat glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and PTH type 1 receptors, each resulted in an hormone inducible increase in cAMP levels. Surprisingly, only the stimulatory effect of dopamine could be inhibited by exposure to melatonin. The inhibitory effect of melatonin on dopamine D1-induced increase in cAMP was completely inhibited by pertussis toxin (100 ng/ml, 18 h). Immunoblot and immunocytochemical studies were carried out using two polyclonal antibodies raised against the extra and cytoplasmic domains of Mel receptor. Immunoblot studies using antibody against the cytoplasmic domain of Mel(1a) receptor confirmed the presence of a peptide blockable 37 kDa band in HEK-293 cells. Indirect immunofluorescent studies with both antibodies revealed staining predominantly at the cell surface, but staining with the antibody directed against the cytoplasmic domain required prior cell permeabilization. By RT- PCR, HEK-293 cells express both Mel(1a) and Mel(1b) messenger RNAs, but the messenger RNA level for Mel(1b) is several orders of magnitude lower than for Mel(1a). We conclude that HEK-293 cells express MR predominantly of the Mel(1a) subtype. Our evidence suggests that one of the ways that melatonin exerts its biological function is through modulation of cellular dopaminergic responses.en_HK
dc.format.extent418 bytes-
dc.format.mimetypetext/html-
dc.languageengen_HK
dc.publisherThe Endocrine Society. The Journal's web site is located at http://endo.endojournals.orgen_HK
dc.relation.ispartofEndocrinologyen_HK
dc.rightsEndocrinology. Copyright © The Endocrine Society.en_HK
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.subject.meshKidney - cytology - drug effects - embryologyen_HK
dc.subject.meshMelatonin - metabolism - pharmacologyen_HK
dc.subject.meshEpithelial Cells - drug effects - metabolismen_HK
dc.subject.meshImmunoblottingen_HK
dc.subject.meshImmunohistochemistryen_HK
dc.titleStudies of melatonin effects on epithelia using the human embryonic kidney-293 (HEK-293) cell lineen_HK
dc.typeArticleen_HK
dc.identifier.openurlhttp://library.hku.hk:4550/resserv?sid=HKU:IR&issn=0013-7227&volume=138&issue=11&spage=4732&epage=4739&date=1997&atitle=Studies+of+melatonin+effects+on+epithelia+using+the+human+embryonic+kidney-293+(HEK-293)+cell+lineen_HK
dc.identifier.emailChan, CWY:camchan@hku.hken_HK
dc.identifier.authorityChan, CWY=rp01311en_HK
dc.description.naturepublished_or_final_versionen_HK
dc.identifier.doi10.1210/en.138.11.4732en_HK
dc.identifier.pmid9348200-
dc.identifier.scopuseid_2-s2.0-0030831096en_HK
dc.identifier.hkuros38155-
dc.relation.referenceshttp://www.scopus.com/mlt/select.url?eid=2-s2.0-0030831096&selection=ref&src=s&origin=recordpageen_HK
dc.identifier.volume138en_HK
dc.identifier.issue11en_HK
dc.identifier.spage4732en_HK
dc.identifier.epage4739en_HK
dc.identifier.isiWOS:A1997YB47900028-
dc.publisher.placeUnited Statesen_HK
dc.identifier.scopusauthoridChan, CWY=12240386600en_HK
dc.identifier.scopusauthoridSong, Y=7404920869en_HK
dc.identifier.scopusauthoridAilenberg, M=6602758253en_HK
dc.identifier.scopusauthoridWheeler, M=7403129168en_HK
dc.identifier.scopusauthoridPang, SF=7402528719en_HK
dc.identifier.scopusauthoridBrown, GM=35493704500en_HK
dc.identifier.scopusauthoridSilverman, M=7403299191en_HK

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