Human Histamine H1 Receptor Research Articles

The European Medicines Agency review of pitolisant for treatment of narcolepsy: summary of the scientific assessment by the Committee for Medicinal Products for Human Use

On 31 March 2016, the European Commission issued a decision for a marketing authorisation valid throughout the European Union (EU) for pitolisant (Wakix) for the treatment of narcolepsy with or without cataplexy in adults. Pitolisant is an antagonist/inverse agonist of the human histamine H3 receptor. The dose should be selected using an up-titration scheme depending on individual patient response and tolerance and should not exceed 36 mg/day.The main evidence of efficacy of pitolisant was based on two Phase III clinical trials. The improvement on excessive daytime sleepiness was shown against placebo in the Harmony I study (−3.33 points; 95% confidence interval (CI) [−5.83; −0.83]; p = 0.024) and in Harmony CTP (−3.41 points; 95% CI [−4.95; −1.87]; p < 0.0001). The daily cataplexy rate in Harmony I improved against placebo with a rate ratio (rR) of 0.38 whilst in the Harmony CTP the ratio of improvement on weekly cataplexy rate against placebo was 0.512. The most commonly reported adverse reactions were headache, insomnia and nausea. This article summarizes the scientific review leading to approval of pitolisant in the EU. The assessment report and product information are available on the European Medicines Agency website (http://www.ema.europa.eu).

The naturally occurring mutation Y197C does not affect the expression or signaling of the human histamine H3 receptor

There is evidence for genetic polymorphism within the human histamine H3 receptor (hH3R), and a Tyr to Cys exchange at position 197 (Y197C), located in the amino terminus of the fifth transmembrane domain, has been reported. In this work we compared the expression and the pharmacological and signaling properties of wild-type (hH3RWT) and mutant (hH3RY197C) receptors transiently expressed in CHO-K1 cells. The hH3RY197C cDNA was created by overlap extension PCR amplification. Receptor expression and affinity were assessed by N-α-[methyl-3H]-histamine binding to cell membranes and intact cells. Receptor function was evaluated by stimulation of [35S]-GTPγS binding to cell membranes and by inhibition of forskolin-induced cAMP accumulation in intact cells. The hH3RWT and hH3RY197C were expressed at similar levels (761±68 and 663±66fmol/mg protein for membranes, and 13,434±1533 and 15,894±1884 receptors per cell, respectively). There were no significant differences in the affinities for H3R agonists or antagonists/inverse agonists between the hH3RWT and hH3RY197C, and the H3R agonist RAMH was similarly efficacious and potent to stimulate [35S]-GTPγS binding and to inhibit forskolin-induced cAMP accumulation. These results indicate that the Y197C mutation does not affect the expression, ligand affinity or signaling of the human H3 receptor.

Ciproxifan, a histamine H3 receptor antagonist, reversibly inhibits monoamine oxidase A and B

Ciproxifan is a well-investigated histamine H3 receptor (H3R) inverse agonist/antagonist, showing an exclusively high species-specific affinity at rodent compared to human H3R. It is well studied as reference compound for H3R in rodent models for neurological diseases connected with neurotransmitter dysregulation, e.g. attention deficit hyperactivity disorder or Alzheimer’s disease. In a screening for potential monoamine oxidase A and B inhibition ciproxifan showed efficacy on both enzyme isoforms. Further characterization of ciproxifan revealed IC50 values in a micromolar concentration range for human and rat monoamine oxidases with slight preference for monoamine oxidase B in both species. The inhibition by ciproxifan was reversible for both human isoforms. Regarding inhibitory potency of ciproxifan on rat brain MAO, these findings should be considered, when using high doses in rat models for neurological diseases. As the H3R and monoamine oxidases are all capable of affecting neurotransmitter modulation in brain, we consider dual targeting ligands as interesting approach for treatment of neurological disorders. Since ciproxifan shows only moderate activity at human targets, further investigations in animals are not of primary interest. On the other hand, it may serve as starting point for the development of dual targeting ligands.

Docking-based Screening of Ficus religiosa Phytochemicals as Inhibitors of Human Histamine H2 Receptor.

Background:Ficus religiosa L. is generally known as Peepal and belongs to family Moraceae. The tree is a source of many compounds having high medicinal value. In gastrointestinal tract, histamine H2 receptors have key role in histamine-stimulated gastric acid secretion. Their over stimulation causes its excessive production which is responsible for gastric ulcer.Objective:This study aims to screen the range of phytochemicals present in F. religiosa for binding with human histamine H2 and identify therapeutics for a gastric ulcer from the plant.Materials and Methods:In this work, a 3D-structure of human histamine H2 receptor was modeled by using homology modeling and the predicted model was validated using PROCHECK. Docking studies were also performed to assess binding affinities between modeled receptor and 34 compounds. Molecular dynamics simulations were done to identify most stable receptor-ligand complexes. Absorption, distribution, metabolism, excretion, and screening was done to evaluate pharmacokinetic properties of compounds.Results:The results suggest that seven ligands, namely, germacrene, bergaptol, lanosterol, Ergost-5-en-3beta-ol, α-amyrin acetate, bergapten, and γ-cadinene showed better binding affinities.Conclusion:Among seven phytochemicals, lanosterol and α-amyrin acetate were found to have greater stability during simulation studies. These two compounds may be a suitable therapeutic agent against histamine H2 receptor.SUMMARY This study was performed to screen antiulcer compounds from F. religiosa. Molecular modeling, molecular docking and MD simulation studies were performed with selected phytochemicals from F. religiosa. The analysis suggests that Lanosterol and α-amyrin may be a suitable therapeutic agent against histamine H2 receptor. This study facilitates initiation of the herbal drug discovery process for the antiulcer activity. Abbreviations used: ADMET: Absorption, distribution, metabolism, excretion and toxicity, DOPE: Discrete Optimized Potential Energy, OPLS: Optimized potential for liquid simulations, RMSD: Root-mean-square deviation, HOA: Human oral absorption, MW: Molecular weight, SP: Standard-precision, XP: Extra-precision, GPCRs: G protein-coupled receptors, SASA: Solvent accessible surface area, Rg: Radius of gyration, NHB: Number of hydrogen bond

Asp73-dependent and -independent regulation of the affinity of ligands for human histamine H1 receptors by Na+

The affinity of ligands for G-protein-coupled receptors (GPCRs) is allosterically regulated by Na+ via a highly conserved aspartate residue (Asp2.50) in the second transmembrane domain of GPCRs. In the present study, we examined the Na+-mediated regulation of the affinity of ligands for Gq/11-protein-coupled human histamine H1 receptors in Chinese hamster ovary cells. The affinities of 3 agonists and 20 antihistamines were evaluated by their displacement curves against the binding of [3H]-mepyramine to membrane preparations in the presence or absence of 100mM NaCl. The affinities of most drugs including histamine, an agonist, and d-chlorpheniramine, a first-generation antihistamine, were reduced by NaCl, with the extent of NaCl-mediated changes varying widely between drugs. In contrast, the affinities of some second-generation antihistamines such as fexofenadine were increased by NaCl. These changes were retained in intact cells. The mutation of Asp2.50 (Asp73) to asparagine abrogated NaCl-induced reductions in affinities for histamine and d-chlorpheniramine, but not NaCl-induced increases in the affinity for fexofenadine. Quantitative structure-activity relationship (QSAR) analyses showed that these Na+-mediated changes were explained and predicted by a combination of the molecular energies and implicit solvation energies of the compounds. These results suggest that Na+ diversely regulates the affinity of ligands for H1 receptors from the extracellular sites of receptors via Asp73-dependent and -independent mechanisms in a manner that depends on the physicochemical properties of ligands. These results may contribute to a deeper understanding of the fundamental mechanisms by which the affinity of ligands for their receptors is allosterically regulated by Na+.

Isoform-Specific Biased Agonism of Histamine H3 Receptor Agonists.

The human histamine H3 receptor (hH3R) is subject to extensive gene splicing that gives rise to a large number of functional and nonfunctional isoforms. Despite the general acceptance that G protein-coupled receptors can adopt different ligand-induced conformations that give rise to biased signaling, this has not been studied for the H3R; further, it is unknown whether splice variants of the same receptor engender the same or differential biased signaling. Herein, we profiled the pharmacology of histamine receptor agonists at the two most abundant hH3R splice variants (hH3R445 and hH3R365) across seven signaling endpoints. Both isoforms engender biased signaling, notably for 4-[3-(benzyloxy)propyl]-1H-imidazole (proxyfan) [e.g., strong bias toward phosphorylation of glycogen synthase kinase 3β (GSK3β) via the full-length receptor] and its congener 3-(1H-imidazol-4-yl)propyl-(4-iodophenyl)-methyl ether (iodoproxyfan), which are strongly consistent with the former's designation as a "protean" agonist. The 80 amino acid IL3 deleted isoform hH3R365 is more permissive in its signaling than hH3R445: 2-(1H-imidazol-5-yl)ethyl imidothiocarbamate (imetit), proxyfan, and iodoproxyfan were all markedly biased away from calcium signaling, and principal component analysis of the full data set revealed divergent profiles for all five agonists. However, most interesting was the identification of differential biased signaling between the two isoforms. Strikingly, hH3R365 was completely unable to stimulate GSK3β phosphorylation, an endpoint robustly activated by the full-length receptor. To the best of our knowledge, this is the first quantitative example of differential biased signaling via isoforms of the same G protein-coupled receptor that are simultaneously expressed in vivo and gives rise to the possibility of selective pharmacological targeting of individual receptor splice variants.

Non-imidazole-based histamine H3 receptor antagonists with anticonvulsant activity in different seizure models in male adult rats.

A series of twelve novel non-imidazole-based ligands (3–14) was developed and evaluated for its in vitro binding properties at the human histamine H3 receptor (hH3R). The novel ligands were investigated for their in vivo protective effects in different seizure models in male adult rats. Among the H3R ligands (3–14) tested, ligand 14 showed significant and dose-dependent reduction in the duration of tonic hind limb extension in maximal electroshock (MES)-induced seizure model subsequent to acute systemic administration (5, 10, and 20 mg/kg, intraperitoneally), whereas ligands 4, 6, and 7 without appreciable protection in MES model were most promising in pentylenetetrazole (PTZ) model. Moreover, the protective effect observed for ligand 14 in MES model was lower than that observed for the reference drug phenytoin and was entirely abrogated when rats were co-administered with the brain-penetrant H1R antagonist pyrilamine (PYR) but not the brain-penetrant H2R antagonist zolantidine (ZOL), demonstrating that histaminergic neurotransmission by activation of postsynaptically located H1Rs seems to be involved in the protective action. On the contrary, PYR and ZOL failed to abrogate the full protection provided by 4 in PTZ model and the moderate protective effect by 14 in strychnine (STR) model. Moreover, the experimental and in silico estimation of properties such as metabolism was performed for five selected test compounds. Also, lipophilicity using planar reversed-phase thin-layer chromatography method was included for better understanding of the molecular properties of the tested compounds. Additionally, the absorption, distribution, metabolism, and elimination and toxicity parameters were evaluated for the most promising compounds 2, 4, 6, 7, and 14 utilizing in vitro methods. These interesting results highlight the potential of H3R ligands as new antiepileptic drugs or as adjuvants to available epilepsy medications.

Anticonvulsant effects of isomeric nonimidazole histamine H3 receptor antagonists.

Phenytoin (PHT), valproic acid, and modern antiepileptic drugs (AEDs), eg, remacemide, loreclezole, and safinamide, are only effective within a maximum of 70%–80% of epileptic patients, and in many cases the clinical use of AEDs is restricted by their side effects. Therefore, a continuous need remains to discover innovative chemical entities for the development of active and safer AEDs. Ligands targeting central histamine H3 receptors (H3Rs) for epilepsy might be a promising therapeutic approach. To determine the potential of H3Rs ligands as new AEDs, we recently reported that no anticonvulsant effects were observed for the (S)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propanamide (1). In continuation of our research, we asked whether anticonvulsant differences in activities will be observed for its R-enantiomer, namely, (R)-2-(4-(3-(piperidin-1-yl)propoxy)benzylamino)propaneamide (2) and analogs thereof, in maximum electroshock (MES)-, pentylenetetrazole (PTZ)-, and strychnine (STR)-induced convulsion models in rats having PHT and valproic acid (VPA) as reference AEDs. Unlike the S-enantiomer (1), the results show that animals pretreated intraperitoneally (ip) with the R-enantiomer 2 (10 mg/kg) were moderately protected in MES and STR induced models, whereas proconvulsant effect was observed for the same ligand in PTZ-induced convulsion models. However, animals pretreated with intraperitoneal doses of 5, 10, or 15 mg/kg of structurally bulkier (R)-enantiomer (3), in which 3-piperidinopropan-1-ol in ligand 2 was replaced by (4-(3-(piperidin-1-yl)propoxy)phenyl)methanol, and its (S)-enantiomer (4) significantly and in a dose-dependent manner reduced convulsions or exhibited full protection in MES and PTZ convulsions model, respectively. Interestingly, the protective effects observed for the (R)-enantiomer (3) in MES model were significantly greater than those of the standard H3R inverse agonist/antagonist pitolisant, comparable with those observed for PHT, and reversed when rats were pretreated with the selective H3R agonist R-(α)-methyl-histamine. Comparisons of the observed antagonistic in vitro affinities among the ligands 1–6 revealed profound stereoselectivity at human H3Rs with varying preferences for this receptor subtype. Moreover, the in vivo anticonvulsant effects observed in this study for ligands 1–6 showed stereoselectivity in different convulsion models in male adult rats.

Histamine H3 receptor antagonist OUP-186 attenuates the proliferation of cultured human breast cancer cell lines

Histamine is involved in various physiological functions, including its neurotransmitter actions in the central nervous system and its action as a causative agent of inflammation, allergic reactions, and gastric acid secretions. Histamine expression and biosynthesis have been detected in breast cancer cells. It was recently suggested that the histamine H3 receptor (H3R) plays a role in the proliferation of breast cancer cells. We recently developed the non-imidazole H3R antagonist OUP-186 which exhibited a potent and selective human H3R antagonistic activity as well as no activity against the human histamine H4 receptor (H4R). In this study, we compared the effects of OUP-186 on the proliferation of estrogen receptor negative (ER−) breast cancer cells (MDA-MB-231) and ER+ breast cancer cells (MCF7) to the effects of clobenpropit (potent imidazole-containing H3R antagonist). OUP-186 and clobenpropit suppressed the proliferation of breast cancer cells. The IC50 values at 48 h for OUP-186 and clobenpropit were approximately 10 μM and 50 μM, respectively. Furthermore, OUP-186 potently induced cell death by activating caspase-3/7, whereas cell death was only slightly induced by clobenpropit. In addition, OUP-186 treatment blocked the proliferation increase triggered by 100 μM (R)-(-)-α-methylhistamine (H3R agonist). The use of 4-methylhistamine (H4R agonist) and JNJ10191584 (selective H4R antagonist) did not affect breast cancer proliferation. These results indicate that OUP-186 potently suppresses proliferation and induces caspase-dependent apoptotic death in both ER+ and ER-breast cancer cells.

Label-free versus conventional cellular assays: Functional investigations on the human histamine H1 receptor

A set of histamine H1 receptor (H1R) agonists and antagonists was characterized in functional assays, using dynamic mass redistribution (DMR), electric cell-substrate impedance sensing (ECIS) and various signaling pathway specific readouts (Fura-2 and aequorin calcium assays, arrestin recruitment (luciferase fragment complementation) assay, luciferase gene reporter assay). Data were gained from genetically engineered HEK293T cells and compared with reference data from GTPase assays and radioligand binding. Histamine and the other H1R agonists gave different assay-related pEC50 values, however, the order of potency was maintained. In the luciferase fragment complementation assay, the H1R preferred β-arrestin2 over β-arrestin1. The calcium and the impedimetric assay depended on Gq coupling of the H1R, as demonstrated by complete inhibition of the histamine-induced signals in the presence of the Gq inhibitor FR900359 (UBO-QIC). Whereas partial inhibition by FR900359 was observed in DMR and the gene reporter assay, pertussis toxin substantially decreased the response in DMR, but increased the luciferase signal, reflecting the contribution of both, Gq and Gi, to signaling in these assays. For antagonists, the results from DMR were essentially compatible with those from conventional readouts, whereas the impedance-based data revealed a trend towards higher pKb values. ECIS and calcium assays apparently only reflect Gq signaling, whereas DMR and gene reporter assays appear to integrate both, Gq and Gi mediated signaling. The results confirm the value of the label-free methods, DMR and ECIS, for the characterization of H1R ligands. Both noninvasive techniques are complementary to each other, but cannot fully replace reductionist signaling pathway focused assays.

Histamine H3 receptor activation stimulates calcium mobilization in a subpopulation of rat striatal neurons in primary culture, but not in synaptosomes

The histamine H3 receptor (H3R) is abundantly expressed in the Central Nervous System where it regulates several functions pre and postsynaptically. H3Rs couple to Gαi/o proteins and trigger or modulate several intracellular signaling pathways, including the cAMP/PKA pathway and the opening of N- and P/Q-type voltage-gated Ca2+ channels. In transfected cells, activation of the human H3R of 445 amino acids (hH3R445) results in phospholipase C (PLC) stimulation and release of Ca2+ from intracellular stores. In this work we have studied whether H3R activation induces Ca2+ mobilization from intracellular stores in native systems, either isolated nerve terminals (synaptosomes) or neurons in primary culture. In rat striatal synaptosomes H3R activation induced inositol 1,4,5-trisphosphate (IP3) formation but failed to increase the intracellular calcium concentration ([Ca2+]i). In striatal primary cultures H3R activation resulted in IP3 formation and increased the [Ca2+]i in 18 out of 70 cells that responded with an elevation in the [Ca2+]i to membrane depolarization with KCl (100 mM) as evaluated by microfluorometry. Confocal microscopy studies corroborated the increase in [Ca2+]i induced by H3R activation in a fraction of those cells that were responsive to membrane depolarization. These results indicate that H3R activation stimulates the PLC/IP3/Ca2+ pathway but only in a subpopulation of striatal neurons.

Genetic variations within the promotor region of the human histamine H4 receptor gene in psoriasis patients

Environmental triggers and genetic factors are supposed to lead to complex gene expression changes in psoriasis and interact in the manifestation of the disease. The histamine H4 receptor (HRH4) is functionally expressed on Th17 cells and plasmacytoid dendritic cells (pDCs) which play a prominent role in the pathogenesis of psoriasis. On pDCs a higher basal expression level of the HRH4 in psoriasis patients compared to healthy controls has been detected. The functional relationship between predisposing genetic variations in the HRH4 gene and psoriasis is yet not known. The aim of the study was to evaluate a possible association between single nucleotide polymorphisms (SNPs) in the HRH4 gene primarily in the promotor region and incidence, severity as well as special clinical features (nail involvement, arthritis, palmoplantar location) of psoriasis.For this approach genomic DNA from 206 patients with psoriasis and 213 healthy controls of Caucasian origin was extracted and three SNPs in the promotor region and one SNP located in an intron of the HRH4 gene were analysed by PCR and pyrophosphate DNA-sequencing.The genotype distributions and allele frequencies between the different groups were compared by chi-square test. The analysis of association between HRH4 polymorphisms and psoriasis was assessed by odds ratio with 95% confidence interval.The genotype distributions and allele frequencies of the four SNPs in the HRH4 gene did not show obvious differences between the whole group of psoriasis patients and healthy controls. However, there were differences by trend in subgroup analysis: The mutant genotypes (A/G) of rs17203314 and (G/A) of rs615283 were more frequent in patients with severe psoriasis PASI≥30 (34.8% and 34.8%) when compared to the control groups (23.5% and 27.2%), respectively. The mutant G/A genotype of rs615283 was significantly more frequent in patients with moderate-to-severe psoriasis PASI≥10 when compared to mild psoriasis PASI<10 (33.3% vs 21.7%, p=0.022). For rs524149 and rs17797945 the wildtype CC genotype was more frequent by trend in moderately-to-severely affected patients with PASI≥10 (85.2% and 63.0%) when compared to the group with mild psoriasis PASI<10 (77.0% and 49.4%), respectively. Furthermore, a significant association of rs615283 with psoriasis palmoplantaris was detected.In conclusion our study suggests that genetic variations within the HRH4 gene might be associated with special clinical features of psoriasis. Further studies are needed in larger study populations to confirm the reported associations and investigate the functional relevance of the identified SNPs.

Chemical-Space-Based de Novo Design Method To Generate Drug-Like Molecules.

To discover drug compounds in chemical space containing an enormous number of compounds, a structure generator is required to produce virtual drug-like chemical structures. The de novo design algorithm for exploring chemical space (DAECS) visualizes the activity distribution on a two-dimensional plane corresponding to chemical space and generates structures in a target area on a plane selected by the user. In this study, we modify the DAECS to enable the user to select a target area to consider properties other than activity and improve the diversity of the generated structures by visualizing the drug-likeness distribution and the activity distribution, generating structures by substructure-based structural changes, including addition, deletion, and substitution of substructures, as well as the slight structural changes used in the DAECS. Through case studies using ligand data for the human adrenergic alpha2A receptor and the human histamine H1 receptor, the modified DAECS can generate high diversity drug-like structures, and the usefulness of the modification of the DAECS is verified.

C-terminal of human histamine H1 receptors regulates their agonist-induced clathrin-mediated internalization and G-protein signaling.

It has been suggested that the agonist-induced internalization of G-protein-coupled receptors from the cell surface into intracellular compartments regulates cellular responsiveness. We previously reported that Gq/11 -protein-coupled human histamine H1 receptors internalized via clathrin-dependent mechanisms upon stimulation with histamine. However, the molecular determinants of H1 receptors responsible for agonist-induced internalization remain unclear. In this study, we evaluated the roles of the intracellular C-terminal of human histamine H1 receptors tagged with hemagglutinin (HA) at the N-terminal in histamine-induced internalization in Chinese hamster ovary cells. The histamine-induced internalization was evaluated by the receptor binding assay with [3 H]mepyramine and confocal immunofluorescence microscopy with an anti-HA antibody. We found that histamine-induced internalization was inhibited under hypertonic conditions or by pitstop, a clathrin terminal domain inhibitor, but not by filipin or nystatin, disruptors of the caveolar structure and function. The histamine-induced internalization was also inhibited by truncation of a single amino acid, Ser487, located at the end of the intracellular C-terminal of H1 receptors, but not by its mutation to alanine. In contrast, the receptor-G-protein coupling, which was evaluated by histamine-induced accumulation of [3 H]inositol phosphates, was potentiated by truncation of Ser487, but was lost by its mutation to alanine. These results suggest that the intracellular C-terminal of human H1 receptors, which only comprises 17 amino acids (Cys471-Ser487), plays crucial roles in both clathrin-dependent internalization of H1 receptors and G-protein signaling, in which truncation of Ser487 and its mutation to alanine are revealed to result in biased signaling toward activation of G-proteins and clathrin-mediated internalization, respectively.

Efficient Approaches to S-alkyl-N-alkylisothioureas and Application to Novel Histamine H3R Antagonists.

S-Alkyl-N-alkylisothiourea compounds, which contain various cyclic amines, were synthesized using 3-phenylpropionyl isothiocyanate (PPI) to discover novel non-imidazole histamine H3 receptor (H3R) antagonists. The synthetic route was improved remarkably by using 2-nitrophenylacetyl isothiocyanate (NPAI). Among the synthesized compounds, N-[4-(4-chlorophenyl)butyl]-S-[3-piperidin-1-yl)propyl]isothiourea (1k, OUP-186) exhibited potent and selective antagonism against human H3R but not human H4R, in vitro. Of particular interest, they did not show antagonism for the histamine release in rat brain microdialysis in vivo, suggesting species-selective differences in antagonist affinities. Furthermore, in silico docking studies of OUP-186 and its C2-homolog (OUP-181) in human/rat H3Rs suggested that the structural difference of antagonist-docking sites between human and rat H3Rs was attributable to the Ala122/Val122 mutation.

Heterologous, PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells.

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H3 receptor of 445 amino acids (hH3R445) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH3R445. In CHO-K1 cells stably transfected with the hH3R445 direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200nM) abolished H3R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5'-triphosphate, 10μM) increased the free calcium intracellular concentration ([Ca(2+)]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H3R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H3R-mediated stimulation of [(35)S]-GTPγS binding to membranes from CHO-K1-hH3R445 cells by 39.7 and 54.2%, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H3Rs (-30.4 and -45.1%) as evaluated by [(3)H]-NMHA binding to intact cells. These results indicate that the hH3R445 undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

PI3K pathway is involved in ERK signaling cascade activation by histamine H2R agonist in HEK293T cells

BackgroundHistamine, through histamine H2 receptor (H2R), modulates different biological processes, involving the modulation of PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways. Many evidences have demonstrated the existence and importance of the crossregulation between these two signaling pathways. The aim of the present work was to determine the molecular mechanisms leading to PI3K and ERK pathways modulation induced by the H2R agonist amthamine and to evaluate the possible interplay between them. MethodsPhosphorylation levels of ERK and Akt were examined by Western blot in HEK293T cells expressing the human H2R, in the presence of H2R agonist and dominant negative mutants or pharmacological inhibitors of different proteins/pathways. Transcriptional activity assays were assessed to determine SRE activity. Amthamine-mediated cellular proliferation was investigated in MA-10A cells in the presence of PI3K inhibitor. ResultsH2R agonist inhibits PI3K/Akt/mTOR and stimulates Ras/MEK/ERK pathways. Moreover, PI3K/Akt/mTOR signaling inhibition is necessary to achieve H2R mediated ERK activation. In the presence of a constitutive active mutant of Akt, amthamine is not able to mediate ERK activation. This crosstalk affects classical ERK downstream targets such as Elk1 phosphorylation and the transcriptional activity of the SRE, classically associated to proliferation. We further demonstrate that amthamine-induced proliferation in Leydig MA-10 tumor cells, is enhanced by LY294002, a PI3K inhibitor. ConclusionsThese results describe a crosstalk between PI3K/AKT/mTOR and Ras/MEK/ERK pathways induced by H2R stimulation with implications in cell proliferation. General significanceThis work indicates that the modulation of PI3K/AKT/mTOR pathway by H2R in turn regulates Ras/MEK/ERK activation conditioning the proliferative capacity of the cells.

Label-free analysis of GPCR-stimulation: The critical impact of cell adhesion

Label-free cell-based assays have been attracting growing attention in drug research. Optical approaches based on evanescent electric fields (e.g. EPIC, RWG/DMR, SPR) and electrochemical impedance analysis (ECIS, xCELLigence) are by far the most widespread techniques for such purposes. We compared three label-free approaches (ECIS, RWG/DMR and SPR) with respect to the activation of the human histamine H1 receptor (H1R) expressed by U-373 MG glioblastoma and genetically engineered HEK 293T cells. HEK 293T cells were either expressing the hH1R alone or in combination with the adhesion protein hMSR1. The β2-adrenergic receptor (β2-AR) expressed by bovine aortic endothelial cells (BAEC) served as a second cell model. Reduced cell adhesion to the surface of the sensing devices affected both, the optical and the impedance-based readout, but became much more obvious in case of RWG- or SPR-based assays. By contrast, the co-expression of hH1R and hMSR1 in HEK 293T cells strongly enhanced the signal compared to hH1R expression alone. As the sensitivity of the optical readouts is confined to a distance of 100–200nm from the surface, depending on the wavelength of the incident light, this observation is in accordance with tighter adhesion of the co-transfectants, inducing a shorter distance between the cell membrane and the substrate. Combining ECIS and SPR, allowing for simultaneous registration of both signals for a single cell population, provided a direct correlation of both readouts, when H1R or β2-AR stimulation was investigated for the same cell populations. Cell adhesion was found to have a critical impact on the results of label-free cell monitoring, in particular when techniques based on evanescent electric fields are applied.

Conformational Restriction and Enantioseparation Increase Potency and Selectivity of Cyanoguanidine-Type Histamine H4 Receptor Agonists.

2-Cyano-1-[4-(1H-imidazol-4-yl)butyl]-3-[2-(phenylsulfanyl)ethyl]guanidine (UR-PI376, 1) is a potent and selective agonist of the human histamine H4 receptor (hH4R). To gain information on the active conformation, we synthesized analogues of 1 with a cyclopentane-1,3-diyl linker. Affinities and functional activities were determined at recombinant hHxR (x: 1-4) subtypes on Sf9 cell membranes (radioligand binding, [(35)S]GTPγS, or GTPase assays) and in part in luciferase assays on human or mouse H4R (HEK-293 cells). The most potent H4R agonists among 14 racemates were separated by chiral HPLC, yielding eight enantiomerically pure compounds. Configurations were assigned based on X-ray structures of intermediates and a stereocontrolled synthetic pathway. (+)-2-Cyano-1-{[trans-(1S,3S)-3-(1H-imidazol-4-yl)cyclopentyl]methyl}-3-[2-(phenylsulfanyl)ethyl]guanidine ((1S,3S)-UR-RG98, 39a) was the most potent H4R agonist in this series (EC50 11 nM; H4R vs H3R, >100-fold selectivity; H1R, H2R, negligible activities), whereas the optical antipode proved to be an H4R antagonist ([(35)S]GTPγS assay). MD simulations confirmed differential stabilization of the active and inactive H4R state by the enantiomers.

Agonist‐induced activation of histamine H3 receptor signals to extracellular signal‐regulated kinases 1 and 2 through PKC‐, PLD‐, and EGFR‐dependent mechanisms

The histamine H3 receptor (H3R), abundantly expressed in the central and the peripheral nervous system, has been recognized as a promising target for the treatment of various important CNS diseases including narcolepsy, Alzheimer's disease, and attention deficit hyperactivity disorder. The H3R acts via Gi/o -proteins to inhibit adenylate cyclase activity and modulate MAPK activity. However, the underlying molecular mechanisms for H3R mediation of the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) remain to be elucidated. In this study, using HEK293 cells stably expressing human H3R and mouse primary cortical neurons endogenously expressing mouse H3R, we found that the H3R-mediated activation of ERK1/2 was significantly blocked by both the pertussis toxin and the MEK1/2 inhibitor U0126. Upon stimulation by H3R agonist histamine or imetit, H3R was shown to rapidly induce ERK1/2 phosphorylation via PLC/PKC-, PLDs-, and epidermal growth factor receptor (EGFR) transactivation-dependent pathways. Furthermore, it was also indicated that while the βγ-subunits play a key role in H3R-activated ERK1/2 phosphorylation, β-arrestins were not required for ERK1/2 activation. In addition, when the cultured mouse cortical neurons were exposed to oxygen and glucose deprivation conditions (OGD), imetit exhibited neuroprotective properties through the H3R. Treatment of cells with the inhibitor UO126 abolished these protective effects. This suggests a possible neuroprotective role of the H3R-mediated ERK1/2 pathway under hypoxia conditions. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the H3R-mediated activation of ERK1/2. Histamine H3 receptors are abundantly expressed in the brain and play important roles in various CNS physiological functions. However, the underlying mechanisms for H3R-induced activation of extracellular signal-regulated kinase (ERK)1/2 remain largely unknown. Here, we provide evidence that upon activation by an agonist, H3Rs trigger ERK1/2 activation via phospholipase C/protein kinase C (PLC/PKC)-, phospholipase D (PLD)s-, and matrix metallopeptidase/epidermal growth factor receptor (MMP/EGFR) transactivation-dependent pathways. Moreover, we demonstrate that H3Rs exhibit a neuroprotective effect on the cultured mouse cortical neurons under hypoxia conditions through the ERK1/2 pathway.