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.
Highlights
The histamine H4 receptor (H4R) [1,2,3,4,5] is preferably expressed on cells of hematopoietic origin such as eosinophils and mast cells and supposed to be involved in inflammatory diseases, e.g. asthma, and pruritis [6,7,8,9,10]
Optimization of the Assay Conditions In order to detect a Gai-mediated inhibitory effect on the adenylyl cyclase (AC) activity, the reporter gene assay was performed in the presence of the AC stimulator forskolin
Forskolin concentration-dependently increased the luciferase expression in HEK293T-SF-rH4R-His6–cyclic adenosine monophosphate (cAMP) responsive element (CRE)-Luc cells, which was inhibited by histamine (1) (Figure 3A) with pEC50 values of 6.8160.11, 6.5360.04, 6.2960.07 and 5.9160.04 (Figure 3B) at forskolin concentrations of 0.5, 1.0, 2.5 and 5 mM, respectively
Summary
The histamine H4 receptor (H4R) [1,2,3,4,5] is preferably expressed on cells of hematopoietic origin such as eosinophils and mast cells and supposed to be involved in inflammatory diseases, e.g. asthma, and pruritis [6,7,8,9,10]. Most of the studies confirmed the pro-inflammatory role of the H4R by blocking the H4R-mediated response with JNJ 7777120 (1-[(5chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine), which is reported to be equipotent as an antagonist at the human, mouse and rat H4R orthologs [18]. The overall amino acid identities of H4R species orthologs are remarkably low (human versus mouse and rat: ,70%) compared to other histamine receptor subtypes (H1R, H2R and H3R) [20]. In various in vitro assay systems the recombinantly expressed mouse and rat H4R revealed substantial species-dependent differences compared to the human receptor concerning affinity, potency and quality of action of pharmacological tools, compromising the predictive value with respect to translational animal models [20,21,22,23]. Biased signaling of the hH4R has been shown for other H4R ligands [27]
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