Figure 1.
Chemical structures of the examined H4R ligands.
Agonists (1–17), antagonists/inverse agonists (18–23) at the human H4R.
Figure 2.
Stimulation of luciferase activity by forskolin.
(A) Representative time course of the luciferase expression in HEK293T-CRE-Luc cells, stably expressing the CRE-controlled luciferase, upon stimulation with 10 µM of forskolin. The luciferase activity was determined after the indicated incubation periods (mean values ± SEM; n = 9). (B) Representative “bell-shaped” concentration-response curve obtained with HEK293T-SF-hH4R-His6-CRE-Luc cells, stably expressing the hH4R and the CRE-controlled luciferase. (C) Concentration response curves covering the ascending region of the signal obtained with different transfectants.
Figure 3.
Inhibition of luciferase activity by histamine in rH4R expressing cells.
Gαi/o mediated inhibition of forskolin (0.5 µM–5.0 µM) stimulated luciferase activities by histamine (HA) in HEK293T-SF-rH4R-His6-CRE-Luc cells, stably expressing the rH4R and the CRE-controlled luciferase. (A) Representative luciferase reporter gene with RLU values as readout. (B) Normalized inhibition of forskolin stimulated luciferase activity (100%) by histamine (HA), with the maximum inhibitory effect of which set at 0%. Data points shown are the mean ± SEM of at least three independent experiments performed in triplicate.
Figure 4.
Effect of histamine and thioperamide on the luciferase activity in hH4R expressing cells.
Concentration-response curves of histamine (HA) and thioperamide (THIO) on HEK293T-SF-hH4R-His6-CRE-Luc cells, stably co-expressing the CRE-controlled luciferase and the hH4R. The cells were pre-stimulated with 500 nM of forskolin alone or in combination with IBMX (50 µM). The effect of forskolin or that of forskolin plus IBMX was defined as 100% luciferase activity. Data points shown are the mean ± SEM of two independent experiments performed in triplicate.
Table 1.
Potencies and efficacies of H4R ligands in the luciferase reporter gene assay at the hH4R, the mH4R and the rH4R.
Table 2.
Reference data of H4R ligands determined in the [35S]GTPγS binding assay at the hH4R, the mH4R and the rH4R and reported in literature.
Figure 5.
Effect of selected standard ligands on H4R orthologs.
(A) Potencies and efficacies of histamine (HA), thioperamide (THIO), UR-PI294 and JNJ 7777120 at the hH4R, (B) the mH4R and (C) the rH4R (agonist mode). (D) Reversal of the HA (100–150 nM) mediated inhibition of the forskolin-stimulated luciferase activity by JNJ 7777120 at the hH4R and the mH4R (antagonist mode), in the luciferase reporter gene assay in HEK293T cells. Reaction mixtures contained ligands at the concentrations indicated on the abscissa to achieve saturated concentration response curves. Data points shown are the mean ± SEM of at least three independent experiments performed in triplicate. Data points connected by dashed lines reflect H4R-independent increase in luciferase activity at high ligand concentrations. The corresponding values were therefore excluded from non-linear correlations (D).
Figure 6.
H4R-independent cellular effects of selected ligand.
Representative H4R-independent increase in the forskolin (1 µM) stimulated luciferase activity by ciproxyfan (CIP), proxyfan (PRO), JNJ 7777120 and thioperamide (THIO) in HEK293T-CRE-Luc cells, stably expressing the CRE-controlled luciferase and devoid of the H4R.
Figure 7.
Inhibition of the response to histamine and clozapine by JNJ7777120.
Concentration response curves of histamine (A) and clozapine (B) alone and in the presence of JNJ7777120 at increasing concentrations, determined on hH4R expressing HEK293T-CRE-Luc cells in the luciferase reporter gene assay, and corresponding Schild plots (C). The pA2 values determined for JNJ 7777120 from Schild regression were 8.39 (slope: 0.83±0.02) and 8.17 (slope: 0.45±0.01) versus histamine and clozapine, respectively. Data points shown are the mean ± SEM of at least three (histamine) or five (clozapine) independent experiments performed in triplicate.
Figure 8.
Comparison of distal and proximal readouts.
Correlation between agonist potencies in the luciferase reporter gene assay and the [35S]GTPγS assay at the (A) hH4R (slope: 0.90±0.20; r2 = 0.80), (B) mH4R (slope: 1.431±0.23; r2 = 0.95) and (C) rH4R (slope: 1.171±0.28, r2 = 0.85).