Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.

The investigation of the (patho)physiological role of the histamine H4 receptor (H4R) and its validation as a possible drug target in translational animal models are compromised by distinct species-dependent discrepancies regarding potencies and receptor subtype selectivities of the pharmacological...

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Autores principales:	Uwe Nordemann, David Wifling, David Schnell, Günther Bernhardt, Holger Stark, Roland Seifert, Armin Buschauer
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/fc716c87b4f8424da737494f98cce70f
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spelling	oai:doaj.org-article:fc716c87b4f8424da737494f98cce70f2021-11-18T08:57:18ZLuciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.1932-620310.1371/journal.pone.0073961https://doaj.org/article/fc716c87b4f8424da737494f98cce70f2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24023919/?tool=EBIhttps://doaj.org/toc/1932-6203The investigation of the (patho)physiological role of the histamine H4 receptor (H4R) and its validation as a possible drug target in translational animal models are compromised by distinct species-dependent discrepancies regarding potencies and receptor subtype selectivities of the pharmacological tools. Such differences were extremely pronounced in case of proximal readouts, e. g. [(32)P]GTPase or [(35)S]GTPγS binding assays. To improve the predictability of in vitro investigations, the aim of this study was to establish a reporter gene assay for human, murine and rat H4Rs, using bioluminescence as a more distal readout. For this purpose a cAMP responsive element (CRE) controlled luciferase reporter gene assay was established in HEK293T cells, stably expressing the human (h), the mouse (m) or the rat (r) H4R. The potencies and efficacies of 23 selected ligands (agonists, inverse agonists and antagonists) were determined and compared with the results obtained from proximal readouts. The potencies of the examined ligands at the human H4R were consistent with reported data from [(32)P]GTPase or [(35)S]GTPγS binding assays, despite a tendency toward increased intrinsic efficacies of partial agonists. The differences in potencies of individual agonists at the three H4R orthologs were generally less pronounced compared to more proximal readouts. In conclusion, the established reporter gene assay is highly sensitive and reliable. Regarding discrepancies compared to data from functional assays such as [(32)P]GTPase and [(35)S]GTPγS binding, the readout may reflect multifactorial causes downstream from G-protein activation, e.g. activation/amplification of or cross-talk between different signaling pathways.Uwe NordemannDavid WiflingDavid SchnellGünther BernhardtHolger StarkRoland SeifertArmin BuschauerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 9, p e73961 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Uwe Nordemann David Wifling David Schnell Günther Bernhardt Holger Stark Roland Seifert Armin Buschauer Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.
description	The investigation of the (patho)physiological role of the histamine H4 receptor (H4R) and its validation as a possible drug target in translational animal models are compromised by distinct species-dependent discrepancies regarding potencies and receptor subtype selectivities of the pharmacological tools. Such differences were extremely pronounced in case of proximal readouts, e. g. [(32)P]GTPase or [(35)S]GTPγS binding assays. To improve the predictability of in vitro investigations, the aim of this study was to establish a reporter gene assay for human, murine and rat H4Rs, using bioluminescence as a more distal readout. For this purpose a cAMP responsive element (CRE) controlled luciferase reporter gene assay was established in HEK293T cells, stably expressing the human (h), the mouse (m) or the rat (r) H4R. The potencies and efficacies of 23 selected ligands (agonists, inverse agonists and antagonists) were determined and compared with the results obtained from proximal readouts. The potencies of the examined ligands at the human H4R were consistent with reported data from [(32)P]GTPase or [(35)S]GTPγS binding assays, despite a tendency toward increased intrinsic efficacies of partial agonists. The differences in potencies of individual agonists at the three H4R orthologs were generally less pronounced compared to more proximal readouts. In conclusion, the established reporter gene assay is highly sensitive and reliable. Regarding discrepancies compared to data from functional assays such as [(32)P]GTPase and [(35)S]GTPγS binding, the readout may reflect multifactorial causes downstream from G-protein activation, e.g. activation/amplification of or cross-talk between different signaling pathways.
format	article
author	Uwe Nordemann David Wifling David Schnell Günther Bernhardt Holger Stark Roland Seifert Armin Buschauer
author_facet	Uwe Nordemann David Wifling David Schnell Günther Bernhardt Holger Stark Roland Seifert Armin Buschauer
author_sort	Uwe Nordemann
title	Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.
title_short	Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.
title_full	Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.
title_fullStr	Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.
title_full_unstemmed	Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.
title_sort	luciferase reporter gene assay on human, murine and rat histamine h4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/fc716c87b4f8424da737494f98cce70f
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Luciferase reporter gene assay on human, murine and rat histamine H4 receptor orthologs: correlations and discrepancies between distal and proximal readouts.

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