title: Label-free versus conventional cellular assays: Functional investigations on the human histamine H1receptor.
abstract: A set of histamine H1receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred β-arrestin2 over β-arrestin1. The calcium and the impedimetric assay depended on Gqcoupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gqinhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gqand Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKbvalues. ECIS and calcium assays apparently only reflect Gqsignaling, whereas DMR and gene reporter assays appear to integrate both, Gqand Gimediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Lieb
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Littmann
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Plank
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Felixberger
authors.affiliation: Department of Chemistry, School of Science, University of Tokyo, Tokyo, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Department of Chemistry and Pharmacy, Molecular and Clinical Pharmacy, Friedrich-Alexander Universität Erlangen-Nürnberg, D-91058 Erlangen, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Schäfer
authors.affiliation: Bioprojet Biotech, 35762 Saint-Grégoire, France.
authors.forename: S
authors.initials: S
authors.lastname: Krief
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Elz
authors.affiliation: Department of Chemistry and Pharmacy, Molecular and Clinical Pharmacy, Friedrich-Alexander Universität Erlangen-Nürnberg, D-91058 Erlangen, Germany.
authors.forename: K
authors.initials: K
authors.lastname: Friedland
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany.
authors.forename: G
authors.initials: G
authors.lastname: Bernhardt
authors.affiliation: Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, D-93040 Regensburg, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Wegener
authors.affiliation: Department of Chemistry, School of Science, University of Tokyo, Tokyo, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ozawa
authors.affiliation: Institute of Pharmacy, University of Regensburg, D-93040 Regensburg, Germany. Electronic address: Armin.Buschauer@ur.de.
authors.forename: A
authors.initials: A
authors.lastname: Buschauer
mesh_terms: D002118:Calcium / Q000378:metabolism; D004353:Drug Evaluation, Preclinical; D017097:Electric Impedance; D019204:GTP-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D057809:HEK293 Cells; D006632:Histamine / Q000494:pharmacology; D017442:Histamine Agonists / Q000494:pharmacology*; D006634:Histamine H1 Antagonists / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D011869:Radioligand Assay; D011969:Receptors, Histamine H1 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D000071557:beta-Arrestins / Q000378:metabolism
chemical_list: D017442:Histamine Agonists; D006634:Histamine H1 Antagonists; D008024:Ligands; D011969:Receptors, Histamine H1; D000071557:beta-Arrestins; D006632:Histamine; D019204:GTP-Binding Proteins; D002118:Calcium
keywords: 2-(3-Trifluoromethylphenyl)histamine (PubChem CID: 5310982); Betahistine dihydrochloride (CID: 68643); Calcium assay; Clozapine (PubChem CID: 2818); Cyproheptadine hydrochloride (PubChem CID: 13770); Diphenhydramine hydrochloride (PubChem CID: 8980); Dynamic mass redistribution; FR900359 (PubChem CID: 14101198); Fexofenadine hydrochloride (PubChem CID: 63002); Gallein (PubChem CID: 73685); Histamine H(1) receptor; Histamine dihydrochloride (PubChem CID: 5818); Histaprodifen (PubChem CID: 10447834); Impedimetry; Levocetirizine dihydrochloride (PubChem CID: 9955977); Maprotiline hydrochloride (PubChem CID: 71478); Mepyramine maleate (PubChem CID: 5284451); Mirtazapine (PubChem CID: 4205); Reporter gene assay; [(3)H]mepyramine (PubChem CID: 656400); beta-Arrestin recruitment