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In contrast to the corresponding mouse and rat orthologs, the human histamine H4 receptor (hH4R) shows extraordinarily high constitutive activity. In the extracellular loop (ECL), replacement of F169 by V as in the mouse H4R significantly reduced constitutive activity. Stabilization of the inactive state was even more pronounced for a double mutant, in which, in addition to F169V, S179 in the ligand binding site was replaced by M. To study the role of the FF motif in ECL2, we generated the hH4R-F168A mutant. The receptor was co-expressed in Sf9 insect cells with the G-protein subunits Gαi2 and Gβ1γ2, and the membranes were studied in [3H]histamine binding and functional [35S]GTPγS assays. The potency of various ligands at the hH4R-F168A mutant decreased compared to the wild-type hH4R, for example by 30- and more than 100-fold in case of the H4R agonist UR-PI376 and histamine, respectively. The high constitutive activity of the hH4R was completely lost in the hH4R-F168A mutant, as reflected by neutral antagonism of thioperamide, a full inverse agonist at the wild-type hH4R. By analogy, JNJ7777120 was a partial inverse agonist at the hH4R, but a partial agonist at the hH4R-F168A mutant, again demonstrating the decrease in constitutive activity due to F168A mutation. Thus, F168 was proven to play a key role not only in ligand binding and potency, but also in the high constitutive activity of the hH4R.

The alkylating agent bendamustine is approved for the treatment of hematopoietic malignancies such as non-Hodgkin lymphoma, chronic lymphocytic leukemia and multiple myeloma. As preliminary data on recently disclosed bendamustine esters suggested increased cytotoxicity, we investigated representative derivatives in more detail. Especially basic esters, which are positively charged under physiological conditions, were in the crystal violet and the MTT assay up to approximately 100 times more effective than bendamustine, paralleled by a higher fraction of early apoptotic cancer cells and increased expression of p53. Analytical studies performed with bendamustine and representative esters revealed pronounced cellular accumulation of the derivatives compared to the parent compound. In particular, the pyrrolidinoethyl ester showed a high enrichment in tumor cells and inhibition of OCT1- and OCT3-mediated transport processes, suggesting organic cation transporters to be involved. However, this hypothesis was not supported by the differential expression of OCT1 (SLC22A1) and OCT3 (SLC22A3), comparing a panel of human cancer cells. Bendamustine esters proved to be considerably more potent cytotoxic agents than the parent compound against a broad panel of human cancer cell types, including hematologic and solid malignancies (e.g. malignant melanoma, colorectal carcinoma and lung cancer), which are resistant to bendamustine. Interestingly, spontaneously immortalized human keratinocytes, as a model of "normal" cells, were by far less sensitive than tumor cells against the most potent bendamustine esters.

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

In neutrophils, activation of the β2-adrenergic receptor (β2AR), a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various β2AR ligands on adenosine-3',5'-cyclic monophosphate (cAMP) accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP)-induced superoxide anion (O2(•-)) production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling) in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2(•-) formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β2AR agonists were generally more potent in inhibiting fMLP-induced O2(•-) production than in stimulating cAMP accumulation. (-)-Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2(•-) production. Moreover, (-)-adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2(•-) assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2(•-) production after β2AR-stimulation although cAMP-increasing substances inhibited O2(•-) production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β2AR inhibits O2(•-) production in a cAMP-independent manner.

The neuropeptide Y (NPY) Y(1) receptor (Y(1)R) has been suggested as a tumor marker for in vivo imaging and as a therapeutic target. In view of the assumed link between estrogen receptor (ER) and Y(1)R in mammary carcinoma and with respect to the development of new diagnostic tools, we investigated the Y(1)R protein expression in human MCF-7 cell variants differing in ER content and sensitivity against antiestrogens. ER and Y(1)R expression were quantified by radioligand binding using [(3)H]-17β-estradiol and the Y(1)R selective antagonist [(3)H]-UR-MK114, respectively. The latter was used for cellular binding studies and for autoradiography of MCF-7 xenografts. The fluorescent ligands Cy5-pNPY (universal Y(1)R, Y(2)R and Y(5)R agonist) and UR-MK22 (selective Y(1)R antagonist), as well as the selective antagonists BIBP3226 (Y(1)R), BIIE0246 (Y(2)R) and CGP71683 (Y(5)R) were used to identify the NPY receptor subtype(s) by confocal microscopy. Y(1)R functionality was determined by mobilization of intracellular Ca(2+). Sensitivity of MCF-7 cells against antiestrogen 4-hydroxytamoxifen correlated directly with the ER content. The exclusive expression of Y(1)Rs was confirmed by confocal microscopy. The Y(1)R protein was up-regulated (100%) by 17β-estradiol (EC(50) 20 pM) and the predominant role of ERα was demonstrated by using the ERα-selective agonist "propylpyrazole triol". 17β-Estradiol-induced over-expression of functional Y(1)R protein was reverted by the antiestrogen fulvestrant (IC(50) 5 nM) in vitro. Furthermore, tamoxifen treatment of nude mice resulted in an almost total loss of Y(1)Rs in MCF-7 xenografts. In conclusion, the value of the Y(1)R as a target for therapy and imaging in breast cancer patients may be compromised due to Y(1)R down-regulation induced by hormonal (antiestrogen) treatment.