Luciferase Reporter Gene Assay on Human, Murine and Rat Histamine H4 Receptor Orthologs: Correlations and Discrepancies between Distal and Proximal Readouts

Abstract

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. [32P]GTPase or [35S]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 [32P]GTPase or [35S]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 [32P]GTPase and [35S]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.

International Union of Basic and Clinical Pharmacology. XCVIII. Histamine Receptors

“…Partial sequences of other species orthologs have been identified but not published. Species differences in pharmacology (ligand binding and signaling efficacy) are profound, also depending on readout systems (Nordemann et al, 2013), and several molecular features for the observed species differences have been elucidated (Liu et al, 2001b;Lim et al, 2008Lim et al, , 2010Schnell et al, 2011).Whereas proximal readout systems such as [ ]GTPS binding assays show pronounced differences between species orthologs, more distal reporter gene assays revealed less pronounced differences. Species splice isoforms have yet to be reported.…”

[3H]UR‐DE257: Development of a Tritium‐Labeled Squaramide‐Type Selective Histamine H2 Receptor Antagonist

“…[52,53]) expressing the hH 2 R-G saS or the gpH 2 R-G saS and on the isolated spontaneously beating guinea pig right atrium [46,54] were performed as previously described, except that the incubation period of the guinea pig atrium in the presence of the antagonists was extended to 60 min. Binding data on recombinant histamine receptor subtypes and orthologues expressed in Sf9 cells (hH 1 R + RGS4; hH 2 R-G saS , gpH 2 R-G saS , rH 2 R-G saS ; hH 3 R + G ia2 + b 1 g 2 ; hH 4 R + G ia2 + b 1 g 2 ) or HEK293T CRE-Luc cells [55] (hH 2 R) using the following radioligands: H 1 R,[ …”

No Evidence for Histamine H4Receptor in Human Monocytes

“…As positive control we studied HEK293T cells stably expressing the hH 4 R (Nordemann et al, 2013). In these cells, the endogenous ligand HA and the selective H 4 R antagonist UR-PI376 (Igel et al, 2009) induced increases in [Ca 21 ] i that were abrogated by the selective H 4 R antagonist JNJ7777120 (Fig.…”

“…Most notably, Dijkstra et al (2007) reported that clobenpropit inhibits production of CC chemokine ligand 2 (CCL2) with a half-maximal effect of ∼1 pM. This value is at least 10,000-fold lower than the EC 50 values for clobenpropit reported in other publications for various parameters (Lim et al, 2005;Nordemann et al, 2013). The exceptionally high potency of clobenpropit in the study of Dijkstra et al (2007) remains unexplained.…”

“…Geneticin (G418) was obtained from InvivoGen (San Diego, CA). EGTA was purchased from Fluka (Deisenhofen, Germany (Nordemann et al, 2013). Cells were grown to 80% confluency at 37°C in a humidified atmosphere with 5% (v/v) CO 2 in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) bovine calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 2 mM L-glutamine.…”