Figure 1.
Successful generation of HEK293 cells stably expressing mH1R and mH4R.
HEK293 cells were transfected with plasmids encoding the myc-tagged mH1R, the Flag-tagged mH4R, or the empty vectors (e.v.) as indicated and clones thereof were generated by limiting dilution technique. Stable expression of the transgenes was anlayzed by Western blot (A) and flow cytometry (B) using myc- and Flag-specific reagents as indicated. Presented are data from a single clone of each cell line, which has been chosen for further analyses. Arrows in (A) indicate the recombinantly expressed proteins.
Figure 2.
Mobilization of [Ca2+]i by histamine is mediated by H1R and H4R.
HEK293 mH1R cells (A+D), HEK293 mH4R cells (B+E), and empty vector-transfected HEK293 cells (C) were labelled with Fura 2-AM and stimulated with different concentrations of histamine (HA) (A–C) or with a constant HA concentration in combination with DMSO or varying concentrations of mepyramine (MEP) or JNJ7777120 (JNJ) (D+E). Changes in [Ca2+]i were determined by fluorescence measurements at an emission wavelength of 508 nm and excitation wavelengths of 340 nm and 380 nm. Data shown are means ± SD (n = 2–3, each consisting of 2 replicates).
Figure 3.
Intracellular cAMP concentrations are enhanced by histamine via the H1R and reduced by HA via the H4R.
HEK293 mH1R cells (A), HEK293 mH4R cells (B), and empty vector-transfected HEK293 cells (C) cells were stimulated with forskolin (Forsk) and histamine (HA) in the absence or presence of mepyramine (MEP) or JNJ7777120 (JNJ) for 10 min. Intracellular cAMP was extracted from the cells and the concentration was determined using HPLC/MS/MS and calculated in relation to the protein concentration. The cAMP-concentration after stimulation with forskolin (H1R: 186±57 pmol/mg protein; H4R: 309±111 pmol/mg protein) was set to 100 % and other values were calculated on this base. Data shown are means ± SD (n = 2–5, each consisting of 3 replicates; *: p≤0.05; **: p≤0.01; ***: p≤0.005; ns: not significant).
Figure 4.
Pertussis toxin selectively blocks signaling of the mH4R.
HEK293 mH1R cells (A+C) and HEK293 mH4R cells (B+D) were incubated in the presence or absence of 50 ng/ml pertussistoxin (PTX) for 18 h. Changes in [Ca2+]i were determined as described in Figure 2 after stimulation with 100 µM histamine (HA) or 10 µM ATP as indicated on the x-axis (A+B). Intracellular cAMP concentrations were analyzed as described in Figure 3 after stimulation for 10 min with 100 µM forskolin (Forsk) and histamine (HA) (C+D). Forskolin-induced cAMP concentrations were 2385±160 pmol/mg protein in HEK293 H1R and 2164±436 pmol/mg protein in HEK 293 H4R cells, respectively. Data shown are means ± SD (n = 2, each consisting of 2–3 replicates; *: p≤0.05; **: p≤0.01; ***: p≤0.005; ns: not significant).
Figure 5.
Histamine induces phosphorylation of MAP-kinases via H1R and H4R.
HEK293 empty vector cells, HEK293 mH1R cells, and HEK293 mH4R cells were incubated with or without 10 µM HA for 5 min and lysed afterwards. Phosphorylation of different serine/threonine kinases in these lysates were detected by MAPK array. Phosphorylation intensity was quantified by analysis of the pixel density of every single spot (A). Shown are the differences of the densities of corresponding spots obtained using lysates of cells with and without HA stimulation. Data shown are means ± SD (n = 2, each measured in 2 replicates; *: p≤0.05; **: p≤0.01; ***: p≤0.005). In B, exemplarily the spots of ERK1 and ERK2 after histamine induction in the cells as indicated on the left, and quantification control spots (ctr.) are demonstrated.
Figure 6.
Histamine regulates EGR-1 mRNA expression.
HEK293 mH1R cells (A) and HEK293 mH4R cells (B) were incubated without any inhibitor (Ø) or with 50 µM PD 980598 (PD) for 1 h, with 2 µM SB 203580 (SB) for 30 min, or 25 µM KG-501 (KG) for 20 min and then incubated with or without histamine for 4 h. Total RNA was extracted from the cells and reverse transcribed into cDNA. Expression of EGR-1 was determined by real time PCR using TagMan probes. Data shown are means ± SD of the relative EGR-1 expression in relation to unstimulated cells (basal) (n = 3, each consisting of 2 replicates; **: p≤0.01; ***: p≤0.005; ns: not significant).