Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor

Abstract

A set of histamine H₁ receptor (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 pEC₅₀ values, 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 G_q coupling of the H₁R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the G_q inhibitor 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, G_q and G_i, 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 pK_b values. ECIS and calcium assays apparently only reflect G_q signaling, whereas DMR and gene reporter assays appear to integrate both, G_q and G_i mediated 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.

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.