The intrinsic activity of ligands was referred to the maximal response to histamine (HA), defined as ?=?1 (full agonist). 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 usually highly sensitive and reliable. Regarding discrepancies compared to data from functional assays such as [32P]GTPase and [35S]GTPS binding, the readout may reflect multifactorial causes downstream from G-protein activation, e.g. activation/amplification of or cross-talk between different signaling pathways. Introduction The histamine H4 receptor (H4R) C is usually 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 C. To investigate the (patho)physiological role Rabbit polyclonal to ZC3H8 of the H4R translational, animal models for allergic asthma and allergic contact dermatitis in mice C or rat models for acute inflammation and conjunctivitis ,  were used. Most of the studies confirmed the pro-inflammatory role of the H4R by blocking the H4R-mediated response with JNJ 7777120 (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine), which is usually reported to be equipotent as an antagonist at the human, mouse and rat H4R orthologs . However, there are also controversial reports. The administration of the H4R agonist 5(4)-methylhistamine was benefical in a murine asthma model , and JNJ 7777120 increased the ocular histamine concentration in a rat conjunctivitis model  (for a recent review cf. Neumann et al. ). Furthermore, 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) . Although relatively small differences in the sequence of histamine receptor species orthologs can result in different potencies and efficacies of individual ligands, the discrepancies are exceptionally high in case of the H4R . 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 C. For example, in comparison to the human H4R, UR-PI294 (N1-[3-(1H-imidazol-4-yl)propyl]-N2-propionylguanidine) and UR-PI376 (2-cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[(2-phenylthio)ethyl]guanidine) ,  displayed considerably lower potencies and efficacies (UR-PI376) in the [32P]GTPase and [35S]GTPS binding assays on membrane preparations of Sf9 insect cells expressing the mouse or rat H4R C 87 . Most strikingly, JNJ 7777120 exhibited stimulatory effects at the mouse and rat H4R in C 87 functional assays on Sf9 cell membranes . Moreover, the use of JNJ 7777120 as standard antagonist in animal models was questioned due to stimulation of G-protein impartial -arrestin recruitment . Biased signaling of the hH4R has also been shown for other H4R ligands . The aforementioned controversial findings underline the necessity to evaluate pharmacological tools at the H4R species orthologs of interest using different assay systems. For this purpose, a cAMP response element (CRE) controlled luciferase reporter gene assay in HEK293T cells, stably expressing the human, the mouse or the rat H4R, was established. The H4R is usually Gi/o-coupled and reduces forskolin stimulated cyclic adenosine monophosphate (cAMP) formation after agonist binding . The optimal concentration of forskolin used for pre-stimulation depends on the cell type  and should correspond to the EC50 of forskolin in the assay system . Therefore, the potency of forskolin was decided, and the effect of the phosphodiesterase (PDE) inhibitor isobutylmethylxanthine (IBMX) was evaluated to optimize the sensitivity of the procedure. Due to the delayed onset of gene expression, incubation periods of four to six hours are required , increasing the risk of agonist mediated receptor desensitization, which can lead to a decrease in agonist potencies . Therefore, the time course of the luciferase expression was determined to find the minimum C 87 incubation period required for appropriate signal strength. For validation, potencies and efficacies of 23 selected H4R ligands, comprising agonists, inverse agonists and antagonists, were determined (Physique 1). Open in a separate window Physique 1 Chemical structures of the examined H4R.