Evidence for ligand-specific conformations of the histamine H(2)-receptor in human eosinophils and neutrophils.

The histamine H(2)-receptor (H(2)R) couples to G(S)-proteins and induces adenylyl cyclase-mediated cAMP accumulation. In human neutrophils and eosinophils, the H(2)R reduces chemotactic peptide-stimulated superoxide anion (O(2)(-)) formation. However, pharmacological characterization of the H(2)R in these cells is far from being complete. The aim of this study was to provide a comprehensive profiling of the H(2)R in neutrophils and eosinophils. Histamine inhibited O(2)(-) formation in human neutrophils more effectively than in eosinophils. H(2)R agonists mimicked the effects of histamine and H(2)R antagonists blocked the effects of histamine. We noticed multiple discrepancies in the potencies and efficacies of H(2)R agonists with respect to cAMP accumulation and inhibition of O(2)(-) formation in both cell types. There were also differences in the antagonist profiles between cAMP accumulation and inhibition of O(2)(-) formation in neutrophils. Moreover, the pharmacological profile of the recombinant H(2)R did not match the H(2)R profile in native cells. The H(2)R sequence identified in human neutrophils corresponds to the published H(2)R sequence, excluding the exclusive expression of a new H(2)R isoform as explanation for the differences. Very likely, the differences between ligands are explained by the existence of ligand-specific receptor conformations with unique affinities, potencies and efficacies. Thus, our data provide evidence for the notion that the concept of ligand-specific receptor conformations can be extended from recombinant systems to native cells.

The bivalent ligand approach leads to highly potent and selective acylguanidine-type histamine H2 receptor agonists.

Bivalent histamine H(2) receptor (H(2)R) agonists were synthesized by connecting pharmacophoric 3-(2-amino-4-methylthiazol-5-yl)-, 3-(2-aminothiazol-5-yl)-, 3-(imidazol-4-yl)-, or 3-(1,2,4-triazol-5-yl)propylguanidine moieties by N(G)-acylation with alkanedioic acids of various chain lengths. The compounds were investigated for H(2)R agonism in GTPase and [(35)S]GTPγS binding assays at guinea pig (gp) and human (h) H(2)R-Gsα(S) fusion proteins including various H(2)R mutants, at the isolated gp right atrium, and in GTPase assays for activity on recombinant H(1), H(3), and H(4) receptors. The bivalent ligands are H(2)R partial or full agonists, up to 2 orders of magnitude more potent than monovalent acylguanidines and, with octanedioyl or decanedioyl spacers, up to 4000 times more potent than histamine at the gpH(2)R. In contrast to their imidazole analogues, the aminothiazoles are highly selective for H(2)R vs other HR subtypes. Compounds with (theoretically) sufficient spacer length (20 CH(2) groups) to simultaneously occupy two orthosteric binding sites in H(2)R dimers are nearly inactive, whereas the highest potency resides in compounds with considerably shorter spacers. Thus, there is no evidence for interaction with H(2)R dimers. The high agonistic potency may result from interaction with an accessory binding site at the same receptor protomer.

Role of the second and third extracellular loops of the histamine H(4) receptor in receptor activation.

The histamine H(4) receptor subtype (H(4)R) belongs to the class 1 of G protein-coupled receptors and is involved in inflammatory and immunological processes. The aim of this study was to elucidate the importance of extracellular regions for the large species differences between human (h) and canine (c) H(4)R. Therefore, chimeric receptors were generated by replacing corresponding domains of the hH(4)R with canine N-terminus (h(cNT)H(4)R) and three canine extracellular loops, respectively (h(cE1)H(4)R, h(cE2)H(4)R and h(cE3)H(4)R). Wild type and chimeric H(4) receptors were expressed in Sf9 insect cells and subsequently characterized in [(3)H]histamine-binding experiments and in steady-state GTPase activity assays, where standard H(4)R ligands histamine, 5-methylhistamine, thioperamide, 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) and clozapine were examined. The exchange of N-terminus or first extracellular loop did not influence hH(4)R pharmacology. The effect of altered second extracellular loop (E2-loop) and third extracellular loop (E3-loop) was rather ligand specific than agonist/inverse agonist specific. At h(cE3)H(4)R, the potency of histamine and 5-methylhistamine significantly decreased. The efficacy of the inverse agonist thioperamide was strongly reduced at h(cE2)H(4)R and h(cE3)H(4)R. Surprisingly, JNJ7777120 as weak inverse agonist at hH(4)R exhibited partial agonistic activity at h(cE2)H(4)R and h(cE3)H(4)R. Molecular dynamic simulations suggest that the E2- and E3-loops are independently of each other involved in the partial/inverse agonism of JNJ7777120 and that E2- as well as E3-loop do not directly interact with JNJ7777120 in the binding pocket. In conclusion, our study indicates an involvement of the E2- and E3-loops in H(4)R activation process after binding of some but not all examined ligands.

Expression and functional properties of canine, rat, and murine histamine H4 receptors in Sf9 insect cells.

The histamine H4 receptor (H4R) is expressed on cells of the immune system including eosinophils, dendritic cells, and T cells and plays an important role in the pathogenesis of bronchial asthma, atopic dermatitis, and pruritus. Analysis of the H4R in these diseases depends on the use of animal models. However, there are substantial pharmacological differences between various H4R species orthologs. The purpose of this study was to analyze the pharmacological properties of canine, rat, and murine H4R in comparison to human H4R expressed in Sf9 insect cells. Only hH4R and cH4R exhibited a sufficiently high [³H]histamine affinity for radioligand binding studies. Generally, cH4R exhibited lower ligand-affinities than hH4R. Similarly, in high-affinity GTPase studies, ligands were more potent at hH4R than at other H4R species orthologs. Unlike the other H4R species orthologs, hH4R exhibited high agonist-independent (constitutive) activity. Most strikingly, the prototypical H4R antagonist (1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine) (JNJ7777120) exhibited partial agonistic activity at cH4R, rH4R, and mH4R, whereas at hH4R, JNJ7777120 was a partial inverse agonist. H4R agonists from the class of N ( G )-acylated imidazolylpropylguanidines and cyanoguanidines exhibited substantial differences in terms of affinity, potency, and efficacy among H4R species orthologs, too. The species-dependent pharmacological profiles are not due to the highly variable amino acid sequence position 341. Finally, H4R species orthologs differ from each other in terms of regulation by NaCl. Collectively, there are profound pharmacological differences between H4R species orthologs. Most importantly, caution must be exerted when interpreting pharmacological effects of "the prototypical H4R antagonist" JNJ7777120 as H4R antagonism.

Paradoxical stimulatory effects of the "standard" histamine H4-receptor antagonist JNJ7777120: the H4 receptor joins the club of 7 transmembrane domain receptors exhibiting functional selectivity.

The histamine H(4) receptor (H(4)R) is expressed in several cell types of the immune system and is assumed to play an important pro-inflammatory role in various diseases, including bronchial asthma, atopic dermatitis, and pruritus. Accordingly, H(4)R antagonists have been suggested to provide valuable drugs for the treatment of these diseases. Over the past decade, the indole derivative 1-[(5-chloro-1H-indol-2-yl)carbonyl]-4-methylpiperazine (JNJ7777120) has become the "standard" H(4)R antagonist and has been extensively used to assess the pathophysiological role of the H(4)R. However, the situation has now become more complicated by recent data (p. 749 and Naunyn Schmiedebergs Arch Pharmacol doi: 10.1007/s00210-011-0612-3) showing that JNJ7777120 can also activate ß-arrestin in a supposedly G(i)-protein-independent (pertussis toxin-insensitive) manner and that at certain H(4)R species orthologs, JNJ7777120 exhibits partial agonist efficacy with respect to G(i)-protein activation (steady-state high-affinity GTPase activity). These novel findings can be explained within the concept of functional selectivity or biased signaling, assuming unique ligand-specific receptor conformations with distinct signal transduction capabilities. Thus, great caution must be exerted when interpreting in vivo effects of JNJ7777120 as H(4)R antagonism. We discuss future directions to get out of the current dilemma in which there is no "standard" H(4)R antagonist available to the scientific community.