Voltage sensitivities and deactivation kinetics of histamine H3 and H4 receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by inhibitory autoreceptors. Likewise, receptor deactivation rates upon agonist removal have been implicated in autoreceptor function. Using G protein-coupled potassium (GIRK) channel activation in Xenopus oocytes as readout of receptor activity, we have investigated the voltage sensitivities and signaling kinetics of the hH₃⁴⁴⁵ and hH₃³⁶⁵ isoforms of the human histamine H₃ receptor, which functions as an inhibitory auto- and heteroreceptor in the nervous system. We have also investigated both the human and the mouse homologues of the related histamine H₄ receptor, which is expressed mainly on hematopoietic cells. We found that the hH₃⁴⁴⁵ receptor is the most sensitive to voltage, whereas the hH₃³⁶⁵ and H₄ receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH₃⁴⁴⁵ and hH₃³⁶⁵ isoforms, with the hH₃³⁶⁵ isoform being five to six-fold slower than the hH₃⁴⁴⁵ receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH₄ receptor. The differences in voltage sensitivities and deactivation kinetics between the hH₃⁴⁴⁵, hH₃³⁶⁵, and H₄ receptors might be relevant to their respective physiological roles.     

Identification of a histamine H4 receptor on human eosinophils--role in eosinophil chemotaxis

Eosinophils are recruited to sites of inflammation via the action of a number of chemical mediators, including PAF, leukotrienes, eotaxins, ECF-A and histamine. Although many of the cell-surface receptors for these mediators have been identified, histamine-driven chemotaxis has not been conclusively attributed to any of the three known histamine receptor subtypes, suggesting the possibility of a 4th histamine-responsive receptor on eosinophils. We have identified and cloned a novel G protein-coupled receptor (GPCR), termed Pfi-013, from an IL-5 stimulated eosinophil cDNA library which is homologous to the human histamine H3 receptor, both at the sequence and gene structure level. Expression data indicates that Pfi-013 is predominantly expressed in peripheral blood leukocytes, with lower expression levels in spleen, testis and colon. Ligand-binding studies using Pfi-013 expressed in HEK-293Galpha15 cells, demonstrates specific binding to histamine with a Kd of 3.28 +/- 0.76 nM and possesses a unique rank order of potency against known histaminergic compounds in a competitive ligand-binding assay (histamine > clobenpropit > iodophenpropit > thioperamide > R-alpha-methylhistamine > cimetidine > pyrilamine). We have therefore termed this receptor human histamine H4. Chemotaxis studies on isolated human eosinophils have confirmed that histamine is chemotactic and that agonists of the known histamine receptors (H1, H2, and H3) do not induce such a response. Furthermore, studies employing histamine-receptor antagonists have shown an inhibition of chemotaxis only by the H3 antagonists clobenpropit and thioperamide. Since these compounds are also antagonists of hH4 we postulate that the receptor mediating histaminergic chemotaxis is this novel histamine H4 receptor.     

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

The H(4)R (histamine H(4) receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H(4)R is primarily expressed in eosinophils and mast cells and has the highest homology with the H(3)R. The occurrence of at least twenty different hH(3)R (human H(3)R) isoforms led us to investigate the possible existence of H(4)R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H(4)R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H(4)R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H(4)R-ligand induced signalling or constitutive activity for these H(4)R splice variants. However, when co-expressed with full-length H(4)R [H(4)R((390)) (H(4)R isoform of 390 amino acids)], the H(4)R splice variants have a dominant negative effect on the surface expression of H(4)R((390)). We detected H(4)R((390))-H(4)R splice variant hetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H(4)R splice variants were detected in various cell types and expressed at similar levels to the full-length H(4)R((390)) mRNA in, for example, pre-monocytes. We conclude that the H(4)R splice variants described here have a dominant negative effect on H(4)R((390)) functionality, as they are able to retain H(4)R((390)) intracellularly and inactivate a population of H(4)R((390)), presumably via hetero-oligomerization.     

Piperidine variations in search for non-imidazole histamine H(3) receptor ligands

Synthesis and biological evaluation of the novel histamine H(3) receptor ligands is described. Two series of ethers (aliphatic and aromatic) have been prepared by four different methods. Compounds were evaluated for their affinities at recombinant human H(3) receptor stably expressed in CHO cells. The ethers show from low to moderate in vitro affinities in nanomolar concentration range. The most potent compound was the 1-[3-(4-tert-butylphenoxy)propyl]-4-piperidino-piperidine 16 (hH(3)R K(i)=100 nM). Several members of the new series investigated under in vivo conditions, proved to be inactive.     

Differential expression and signaling of the human histamine H3 receptor isoforms of 445 and 365 amino acids expressed in human neuroblastoma SH-SY5Y cells

In stably-transfected human neuroblastoma SH-SY5Y cells, we have compared the effect of activating two isoforms of 445 and 365 amino acids of the human histamine H₃ receptor (hH₃R₄₄₅ and hH₃R₃₆₅) on [³⁵S]-GTPγS binding, forskolin-induced cAMP formation, depolarization-induced increase in the intracellular concentration of Ca²⁺ ions ([Ca²⁺]i) and depolarization-evoked [^3?H]-dopamine release. Maximal specific binding (B_max) of [^3?H]-N-methyl-histamine to cell membranes was 953?±?204 and 555?±?140?fmol/mg protein for SH-SY5Y-hH₃R₄₄₅ and SH-SY5Y-hH₃R₃₆₅ cells, respectively, with similar dissociation constants (K_d, 0.86?nM and 0.81?nM). The mRNA of the hH₃R₃₆₅ isoform was 40.9?±?7.9% of the hH₃R₄₄₅ isoform. No differences in receptor affinity were found for the H₃R ligands histamine, immepip, (R)(-)-α-methylhistamine (RAMH), A-331440, clobenpropit and ciproxifan. Both the stimulation of [³⁵S]-GTPγS binding and the inhibition of forskolin-stimulated cAMP accumulation by the agonist RAMH were significantly larger in SH-SY5Y-hH₃R₄₄₅ cells ([³⁵S]-GTPγS binding, 158.1?±?7.5% versus 136.5?±?3.6% for SH-SY5Y-hH₃R₃₆₅ cells; cAMP accumulation, ?74.0?±?4.9% versus ?43.5?±?5.3%), with no significant effect on agonist potency. In contrast, there were no differences in the efficacy and potency of RAMH to inhibit [^3?H]-dopamine release evoked by 100?mM K⁺ (?18.9?±?3.0% and ?20.5?±?3.3%, for SH-SY5Y-hH₃R₄₄₅ and SH-SY5Y-hH₃R₃₆₅ cells), or the inhibition of depolarization-induced increase in [Ca²⁺]i (S2/S1 ratios: parental cells 0.967?±?0.069, SH-SY5Y-hH₃R₄₄₅ cells 0.639?±?0.049, SH-SY5Y-hH₃R₃₆₅ cells 0.737?±?0.045). These results indicate that in SH-SY5Y cells, hH₃R₄₄₅ and hH₃R₃₆₅ isoforms regulate in a differential manner the signaling pathways triggered by receptor activation.     

Mepyramine-JNJ7777120-hybrid compounds show high affinity to hH(1)R, but low affinity to hH(4)R

In literature, a synergism between histamine H(1) and H(4) receptor is discussed. Furthermore, it was shown, that the combined application of mepyramine, a H(1) antagonist and JNJ7777120, a H(4) receptor ligand leads to a synergistic effect in the acute murine asthma model. Thus, the aim of this study was to develop new hybrid ligands, containing one H(1) and one H(4) pharmacophor, connected by an appropriate spacer, in order to address both, H(1)R and H(4)R. Within this study, we synthesized nine hybrid compounds, which were pharmacologically characterized at hH(1)R and hH(4)R. The new compounds revealed (high) affinity to hH(1)R, but showed only low affinity to hH(4)R. Additionally, we performed molecular dynamic studies for some selected compounds at hH(1)R, in order to obtain information about the binding mode of these compounds on molecular level.     

Fluorescent non-imidazole histamine H3 receptor ligands with nanomolar affinities

Omega-piperidinoalkanamine derivatives with fluorescent moieties (2-cyanoisoindol-1-yl, 7-nitrobenzofurazan-4-yl) have been synthesized starting from piperidine in three steps. The compounds display moderate to good histamine hH(3) receptor affinities with K(i) values ranging from 178 to 11nM. The new compounds may act as tools for identification and understanding of the binding site on the histamine H(3) receptor.     

Role of histamine H(3) receptors in the control of gastrointestinal motility. An overview.

Over the last few years, the biochemical and functional characterization of H(3) receptors has been a matter for extensive investigation, culminating in the cloning of the human, guinea pig and rat receptor protein from brain tissues. This discovery contributed to determine the distribution of receptors in the body and to define the molecular mechanisms which follow activation. The major breakthrough in the histamine H(3) receptor field came with the synthesis of selective and potent agonists and antagonists, which unravelled the function of this receptor subtype in the different tissues. As expected from the ubiquitous location of histamine in the body, histamine H(3) receptors have also been identified in virtually every tissue, although they are quantitatively less abundant than H(1) and H(2) receptors. Concerning the gastrointestinal tract, this new receptor subtype seems to have multiple cellular locations, which include neurons, enteric ganglia, paracrine and immune cells and, in some tissues, also smooth muscle cells. Therefore it might be regarded as a general regulatory system of different digestive functions, including motility. The effects mediated by histamine H(3)-receptors mainly reflect the presynaptic inhibition of the release of either excitatory or inhibitory neurotransmitters from the myenteric plexus. The molecular mechanism of presynaptic inhibition seems to involve a restriction of calcium entry into the nerve endings, but other mechanisms (reduction of cAMP), possibly associated to different H(3) receptor subtypes, may be involved. Despite the widespread distribution and the well defined inhibitory effects evoked in the majority of in vitro models of intestinal motility, no clear cut evidence of its involvement in the control of peristalsis could be provided. In vivo models of gastrointestinal transit, indeed, did not reveal a defined effect of histamine H(3) receptor ligands, even though the possibility of a central inhibition was pointed out in several studies. Therefore, it is not clear at the present what is the physiological meaning of the histamine H(3) receptor in the control of gastrointestinal motility and whether it could represent a potential target for novel therapeutic interventions in deranged motility, taking into account that human gastrointestinal tissues are apparently devoid of this receptor.     

The human histamine H2-receptor couples more efficiently to Sf9 insect cell Gs-proteins than to insect cell Gq-proteins: limitations of Sf9 cells for the analysis of receptor/Gq-protein coupling

The human histamine H2-receptor (hH2R) couples to Gs-proteins to activate adenylyl cyclase and to Gq-proteins to activate phospholipase C, but phospholipase C activation has not consistently been observed. The aim of this study was to compare coupling of hH2R to insect and mammalian Gs- and Gq-proteins in Spodoptera frugiperda (Sf9) cells. Interaction of hH2R with mammalian G proteins was assessed with coexpressed proteins or receptor-Galpha fusion proteins that enhance coupling efficiency. hH2R efficiently coupled to insect Gs-proteins to activate adenylyl cyclase. However, hH2R poorly coupled to insect Gq-proteins as assessed by the lack of enhancement of histamine-stimulated steady-state GTP hydrolysis by regulators of G protein signaling (RGS proteins). In contrast, RGS-proteins efficiently enhanced GTP hydrolysis stimulated by the human platelet-activating factor receptor (PAFR) and the histamine H1-receptor (H1R) from man and guinea pig. The measurement of intracellular free Ca2+ concentration was not useful for studying receptor/Gq-protein coupling. hH2R also efficiently interacted with mammalian Gs-proteins, specifically with fused Gsalpha as assessed by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-sensitive high-affinity agonist binding, agonist-stimulated [35S]GTPgammaS binding and adenylyl cyclase activation. In contrast, coupling of hH2R to coexpressed and fused mammalian Gqalpha was poor. However, our inability to reconstitute efficient coupling of PAFR and H1R to mammalian Gqalpha indicated that a large portion of the expressed G protein was functionally inactive. Taken together, our data show that hH2R couples more efficiently to insect cell Gs-proteins than to insect cell Gq-proteins. Unfortunately, there are significant limitations in the usefulness of Sf9 cells for comparing the coupling of receptors to mammalian Gs- and Gq-proteins and assessing Gq-mediated activation of effector systems.     

Synthesis of novel histamine H4 receptor antagonists

This letter describes the discovery and synthesis of a series of octahydropyrrolo[3,4-c]pyrrole based selective histamine hH4 receptor antagonists. The amidine compound 20 was found to be a potent and selective histamine H4 receptor antagonist with moderate clearance and a high volume of distribution.     

Synthesis and structure-activity relationships of indole and benzimidazole piperazines as histamine H(4) receptor antagonists

We describe the structure-activity relationships for a series of ligands structurally related to the recently identified (5-chloro-1H-indol-2-yl)-(4-methyl-piperazin-1-yl)-methanone (1) as histamine H(4) receptor (H(4)R) antagonists. Furthermore, we identified related benzimidazoles as novel lead compounds for the H(4)R. The ligands have been evaluated by radioligand displacement studies and functional assays for their interaction with both the human histamine H(3) and H(4) receptors and exhibit pK(i) values up to 7.5 at the human H(4)R.     

The rat H3 receptor: gene organization and multiple isoforms

Genomic DNA analysis revealed that the coding region of the rat histamine H3 receptor comprises three exons interrupted by two introns of approximately 1 kb each. Several H3 receptor mRNA variants were identified by PCR and cDNA cloning and sequencing. Four variants generated by pseudo-intron retention/deletion at the level of the third intracellular loop were designated H3(445), H3(413), H3(410), and H3(397), according to the length of their deduced amino acid sequence and display differential tissue expression. When expressed in CHO-K1 or Cos-1 cells, the H3(445), H3(413), and H3(397) were found to generate specific 125I iodoproxyfan binding of similar pharmacological profile. In addition, we identified two short variants, termed H3(nf1) and H3(nf2), which correspond to frame shift and stop codon interposition, respectively, and are presumably nonfunctional, among which H3(nf2) displays brain expression similar to that of the longer isoforms.     

Synthesis and evaluation of a new series of 1'-cyclobutyl-6-(4-piperidyloxy)spiro[benzopyran-2,4'-piperidine] derivatives as high affinity and selective histamine-3 receptor (H3R) antagonists

A novel class of 1'-cyclobutyl-6-(4-piperidyloxy)spiro[benzopyran-2,4'-piperidine] derivatives with low nanomolar affinity for the human and rat histamine-3 receptors (H(3)Rs) are described. The spirobenzopyran piperidine ether analogs demonstrated excellent H(3)R affinity and selectivity against histamine receptor subtypes (H(1)R, H(2)R, and H(4)R), were stable in liver microsomes, and had selectivity against CYP P450 enzymes. Compounds 10, 13, 15, and 16 demonstrated high H(3)R affinity, in vitro liver microsomal stability, selectivity against CYP isoforms, moreover, these ether analogs exhibited acceptable iv pharmacokinetic (PK) properties but had poor oral exposure in rat.     

Cardiac sodium channels (hH1) are intrinsically more sensitive to block by lidocaine than are skeletal muscle (mu 1) channels

When lidocaine is given systemically, cardiac Na channels are blocked preferentially over those in skeletal muscle and nerve. This apparent increased affinity is commonly assumed to arise solely from the fact that cardiac Na channels spend a large fraction of their time in the inactivated state, which exhibits a high affinity for local anesthetics. The oocyte expression system was used to compare systematically the sensitivities of skeletal (mu 1-beta 1) and cardiac (hH1-beta 1) Na channels to block by lidocaine, under conditions in which the only difference was the choice of alpha subunit. To check for differences in tonic block, Na currents were elicited after 3 min of exposure to various lidocaine concentrations at -100 mV, a potential at which both hH1-beta 1 and mu 1-beta 1 channels were fully reprimed. Surprisingly, hH1-beta 1 Na channels were threefold more sensitive to rested-state block by lidocaine (402 +/- 36 microM, n = 4-22) than were mu 1-beta 1 Na channels (1,168 +/- 34 microM, n = 7-19). In contrast, the inactivated state binding affinities determined at partially depolarized holding potentials (h infinity approximately 0.2) were similar (Kd = 16 +/- 1 microM, n = 3-9 for hH1-beta 1 and 12 +/- 2 microM, n = 4-11 for mu 1-beta 1). Lidocaine produced more use- dependent block of peak hH1-beta 1 Na current elicited by trains of short-(10 ms) or long- (1 s) duration step depolarizations (0.5 Hz, -20 mV) than of mu 1-beta 1 Na current. During exposure to lidocaine, hH1- beta 1 channels recover from inactivation at -100 mV after a prolonged delay (20 ms), while mu 1-beta 1 channels begin repriming immediately. The overall time course of recovery from inactivation in the presence of lidocaine is much slower in hH1-beta 1 than in mu 1-beta 1 channels. These unexpected findings suggest that structural differences in the alpha subunits impart intrinsically different lidocaine sensitivities to the two isoforms. The differences in steady state affinities and in repriming kinetics are both in the correct direction to help explain the increased potency of cardiac Na channel block by local anesthetics.     

Identification and Characterization of ZEL-H16 as a Novel Agonist of the Histamine H(3) Receptor

The histamine H3 receptor (H3R) has been recognized as a promising target for the treatment of various central and peripheral nervous system diseases. In this study, a non-imidazole compound, ZEL-H16, was identified as a novel histamine H3 receptor agonist. ZEL-H16 was found to bind to human H3R with a Ki value of approximately 2.07 nM and 4.36 nM to rat H3R. Further characterization indicated that ZEL-H16 behaved as a partial agonist on the inhibition of forskolin-stimulated cAMP accumulation (the efficacy was 60% of that of histamine) and activation of ERK1/2 signaling (the efficacy was 50% of that of histamine) at H3 receptors, but acted as a full agonist just like histamin in the guinea-pig ileum contraction assay. These effects were blocked by pertussis toxin and H3 receptor specific antagonist thioperamide. ZEL-H16 showed no agonist or antagonist activities at the cloned human histamine H1, H2, and H4 receptors and other biogenic amine GPCRs in the CRE-driven reporter assay. Furthermore, our present data demonstrated that treatment of ZEL-H16 resulted in intensive H3 receptor internalization and delayed recycling to the cell surface as compared to that of control with treatment of histamine. Thus, ZEL-H16 is a novel and potent nonimidazole agonist of H3R, which might serve as a pharmacological tool for future investigations or as possible therapeutic agent of H3R.     

Regulation of Norepinephrine Release from Isolated Bovine Irides by Histamine

In the present study, we investigated the effect of histamine on sympathetic neurotransmission from isolated, superfused bovine irides. We also studied the pharmacology of prejunctional histamine receptors that regulate the release of norepinephrine (NE) from this tissue. The effect of exogenous histamine and various histamine receptor agonists was examined on the release of [³H]-norepinephrine ([³H]NE) triggered by electrical field stimulation using the Superfusion Method. Histamine receptor agonists caused a concentration-dependent inhibition of field-stimulated [³H]NE overflow with the following rank order of potency: imetit > histamine > R-α-methylhistamine. In all cases, the inhibitory action of histamine receptor agonists was attenuated at high concentrations of these compounds. The histamine receptor antagonists, clobenpropit (H₃-antagonist/H₄-agonist) and thioperamide (H₃-antagonist) blocked the inhibitory response elicited by R-α-methylhistamine and imetit, respectively. Inhibitory effects of R-α-methylhistamine and clonidine were not additive suggesting that prejunctional H₃- and α₂-adrenoceptors coexist at neurotransmitter release sites. We conclude that histamine produces an inhibitory action on sympathetic neurotransmission in the bovine iris, an effect mimicked by selective H₃-receptor agonists and blocked by H₃-antagonists.     

Activation and deactivation rates of recombinant GABA(A) receptor channels are dependent on alpha-subunit isoform.

The role of subunit composition in determining intrinsic maximum activation and deactivation kinetics of GABA(A) receptor channels is unknown. We used rapid ligand application (100-micros solution exchange) to examine the effects of alpha-subunit composition on GABA-evoked activation and deactivation rates. HEK 293 cells were transfected with human cDNAs encoding alpha1beta1gamma2- or alpha2beta1gamma2-subunits. Channel kinetics were similar across different transfections of the same subunits and reproducible across several GABA applications in the same patch. Current rise to peak was at least twice as fast for alpha2beta1gamma2 receptors than for alpha1beta1gamma2 receptors (reflected in 10-90% rise times of 0.5 versus 1.0 ms, respectively), and deactivation was six to seven times slower (long time constants of 208 ms versus 31 ms) after saturating GABA applications. Thus alpha-subunit composition determined activation and deactivation kinetics of GABA(A) receptor channels and is therefore likely to influence the kinetics and efficacy of inhibitory postsynaptic currents.     

Cloning and expression of the mouse histamine H3 receptor: evidence for multiple isoforms

The existence of mouse H3-receptor isoforms was investigated by PCR analysis and cDNA cloning. Splicing mechanisms previously reported in various species are conserved in the mouse. The retention/deletion of a fragment in the third intracellular loop of the mouse receptor leads to the existence of three isoforms designated mH(3(445)), mH(3(413)) and mH(3(397)) according to the length of their deduced amino acid sequence. PCR analysis showed that mouse H3-receptor isoforms display different expression patterns in the brain. Following expression in Cos-1 cells, [125I]iodoproxyfan binding indicated similar pharmacological profiles of the mH(3(445)), mH(3(413)) and mH(3(397)) isoforms. The pharmacological profile of the mouse H3 receptor is more similar to the rat receptor than to the human receptor, although some differences were also observed between the mouse and rat receptors. For example, the potency of thioperamide and ciproxifan is slightly higher at the mouse receptor than at the rat receptor but 40-100-fold higher than at the human receptor. In situ hybridization histochemistry showed that the distribution of H3-receptor mRNAs in the mouse brain is rather similar to that previously reported in the rat brain. However, the autoradiographic and cellular expression patterns observed in several brain areas such as the thalamus or hippocampus reveal important differences between the two species.     

Ether derivatives of 3-piperidinopropan-1-ol as non-imidazole histamine H3 receptor antagonists

A series of aliphatic and aromatic ether derivatives of 3-piperidinopropan-1-ol has been prepared by four different methods. The ethers obtained were evaluated for their affinities at recombinant human histamine H3 receptor, stably expressed in CHO-K1 or HEK 293 cells. All compounds investigated show from moderate to high in vitro affinities in the nanomolar concentration range. Selected compounds were investigated under in vivo conditions after oral administration to mice. Some proved to be highly potent and orally available histamine H3 receptor antagonists. The most potent antagonists in this series have been in vitro the 4-(1,1-dimethylpropyl)phenyl ether 19 (hH3R K(i) = 8.4 nM) and in vivo the simple ethyl ether 2 (ED50 = 1.0mg/kg).