Voltage sensitivities and deactivation kinetics of histamine H(3) and H(4) 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 hH(3)(445) and hH(3)(365) isoforms of the human histamine H(3) 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 H(4) receptor, which is expressed mainly on hematopoietic cells. We found that the hH(3)(445) receptor is the most sensitive to voltage, whereas the hH(3)(365) and H(4) receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH(3)(445) and hH(3)(365) isoforms, with the hH(3)(365) isoform being five to six-fold slower than the hH(3)(445) receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH(4) receptor. The differences in voltage sensitivities and deactivation kinetics between the hH(3)(445), hH(3)(365), and H(4) receptors might be relevant to their respective physiological roles.     

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.     

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, 200 nM) abolished H₃R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5′-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca²⁺]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 [³⁵S]-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 [³H]-NMHA binding to intact cells. These results indicate that the hH₃R₄₄₅ undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

     

The Extracellular Loop 2 (ECL2) of the Human Histamine H4 Receptor Substantially Contributes to Ligand Binding and Constitutive Activity

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 Gα_i2 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.     

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.     

Rasagiline derivatives combined with histamine H3 receptor properties

The irreversible monoamine oxidase B (MAO B) inhibitor rasagiline has been described with multiple disease modifying effects in vitro on models of Parkinson’s disease. The combination of this established drug to recently developed histamine H₃ receptor (H₃R) antagonist elements gives new impetus to the design of multitargeting ligands. Surprisingly, the 5-substituted 3-piperidinopropyloxy rasagiline derivative 1 was more potent on both targets than its 6-substituted isomer. It showed nanomolar affinities at the desired targets (MAO B IC₅₀ = 256 nM; hH₃R K_i = 2.6 nM) with a high preference over monoamine oxidase A (MAO A) and negligible affinity at histamine H₁, H₄, dopamine D₂, D₃ receptors or acetyl-/butyrylcholinesterases.     

Alkyl derivatives of 1,3,5-triazine as histamine H4 receptor ligands

This study focuses on the design, synthesis, molecular modeling and biological evaluation of a novel group of alkyl-1,3,5-triazinyl-methylpiperazines. New compounds were synthesized and their affinities for human histamine H₄ receptor (hH₄R) were evaluated. Among them, 4-(cyclohexylmethyl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazin-2-amine (14) exhibited hH₄R affinity with a K_i of 160?nM and behaved as antagonist in functional assays: the cellular aequorin-based assay (IC₅₀?=?32?nM) and [³⁵S]GTPγS binding assay (pK_b?=?6.67). In addition, antinociceptive activity of 14 in vivo was observed in Formalin test (in mice) and in Carrageenan-induced acute inflammation test (in rats).     

Structural modifications and in vitro pharmacological evaluation of 4-pyridyl-piperazine derivatives as an active and selective histamine H3 receptor ligands

A novel series of 4-pyridylpiperazine derivatives with varying alkyl linker length and eastern part substituents proved to be potent histamine H₃ receptor (hH₃R) ligands in the nanomolar concentration range. While paying attention to their alkyl linker length, derivatives with a six methylene linker tend to be more potent than their five methylene homologues. Moreover, in the case of both phenoxyacetyl- and phenoxypropionyl- derivatives, an eight methylene linkers possess lower activity than their seven methylene homologues. However, in global analysis of collected data on the influence of alkyl linker length, a three methylene homologues appeared to be of highest hH₃R affinity among all described 4-pyridylpiperazine derivatives from our group up to date. In the case of biphenyl and benzophenone derivatives, compounds with para- substituted second aromatic ring were of higher affinity than their meta analogues. Interestingly, benzophenone derivative 18 showed the highest affinity among all tested compounds (hH₃R K_i = 3.12 nM). The likely protein-ligand interactions, responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at H₃R, as well as drug-like properties of selected ligands were evaluated using in vitro methods.     

Development of a selective and potent radioactive ligand for histamine H3 receptors: A compound potentially useful for receptor occupancy studies

Radioligands are powerful tools for examining the pharmacological profiles of chemical leads and thus facilitate drug discovery. In this study, we identified and characterized 3-([1,1,1-³H]methyl)-2-(4-{[3-(1-pyrrolidinyl)propyl]oxy} phenyl)-4(3H)-quinazolinone ([³H]1) as a potent and selective radioligand for histamine H₃ receptors. Radioligand [³H]1 exhibited appreciable specific signal in brain slices prepared from wild-type mice but not from histamine H₃ receptor-deficient mice, demonstrating the specificity and utility of [³H]1 as a selective histamine H₃ receptor radioligand for ex-vivo receptor occupancy assays.     

Sequential Application of Ligand and Structure Based Modeling Approaches to Index Chemicals for Their hH4R Antagonism

The human histamine H₄ receptor (hH₄R), a member of the G-protein coupled receptors (GPCR) family, is an increasingly attractive drug target. It plays a key role in many cell pathways and many hH₄R ligands are studied for the treatment of several inflammatory, allergic and autoimmune disorders, as well as for analgesic activity. Due to the challenging difficulties in the experimental elucidation of hH₄R structure, virtual screening campaigns are normally run on homology based models. However, a wealth of information about the chemical properties of GPCR ligands has also accumulated over the last few years and an appropriate combination of these ligand-based knowledge with structure-based molecular modeling studies emerges as a promising strategy for computer-assisted drug design. Here, two chemoinformatics techniques, the Intelligent Learning Engine (ILE) and Iterative Stochastic Elimination (ISE) approach, were used to index chemicals for their hH₄R bioactivity. An application of the prediction model on external test set composed of more than 160 hH₄R antagonists picked from the chEMBL database gave enrichment factor of 16.4. A virtual high throughput screening on ZINC database was carried out, picking ∼4000 chemicals highly indexed as H₄R antagonists'' candidates. Next, a series of 3D models of hH₄R were generated by molecular modeling and molecular dynamics simulations performed in fully atomistic lipid membranes. The efficacy of the hH₄R 3D models in discrimination between actives and non-actives were checked and the 3D model with the best performance was chosen for further docking studies performed on the focused library. The output of these docking studies was a consensus library of 11 highly active scored drug candidates. Our findings suggest that a sequential combination of ligand-based chemoinformatics approaches with structure-based ones has the potential to improve the success rate in discovering new biologically active GPCR drugs and increase the enrichment factors in a synergistic manner.     

Optimization and preclinical evaluation of novel histamine H3 receptor ligands: Acetyl and propionyl phenoxyalkyl piperazine derivatives

As a continuation of our search for novel histamine H₃ receptor ligands, a series of new acetyl and propionyl phenoxyalkylamine derivatives (2–25) was synthesized. Compounds with three to four carbon atoms alkyl chain spacer, composed of six various 4N-substituted piperazine moieties were evaluated for their binding properties at human histamine H₃ receptors (hH₃R). In vitro test results proved the 4-pyridylpiperazine moiety as crucial element for high hH₃R affinity (hH₃R K_i?=?5.2–115?nM). Moreover introduction of carbonyl group containing residues in the lipophilic part of molecules instead of branched alkyl substituents resulted in increased affinity in correlation to previously described series, whereas propionyl derivatives showed slightly higher affinities than those of acetyl (16 and 22vs.4 and 10; hH₃R K_i?=?5.2 and 15.4?nM vs. 10.2 and 115?nM, respectively). These findings were confirmed by molecular modelling studies, demonstrating multiple ligand-receptor interactions. Furthermore, pharmacological in vivo test results of compound 4 clearly indicate that it may affect the amount of calories consumed, thus act as an anorectic compound. Likewise, its protective action against hyperglycemia and the development of overweight has been shown. In order to estimate drug-likeness of compound 4, in silico and experimental evaluation of metabolic stability in human liver microsomes was performed.     

IDENTIFICATION OF A HISTAMINE H4 RECEPTOR ON HUMAN EOSINOPHILS—ROLE IN EOSINOPHIL CHEMOTAXIS

ABSTRACT

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-293Gα15 cells, demonstrates specific binding to histamine with a K_d 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-α-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.     

Indole- and benzothiophene-based histamine H3 antagonists

Previous research on histamine H₃ antagonists has led to the development of a pharmacophore model consisting of a central phenyl core flanked by two alkylamine groups. Recent investigation of the replacement of the central phenyl core with heteroaromatic fragments resulted in the preparation of novel 3,5-, 3,6- and 3,7-substituted indole and 3,5-substituted benzothiophene analogs that demonstrate good to excellent hH₃ affinities. Select analogs were profiled in a rat pharmacokinetic model.     

Assessment of [125I]WYE-230949 as a Novel Histamine H3 Receptor Radiopharmaceutical

Histamine H₃ receptor therapeutics have been proposed for several diseases such as schizophrenia, attention deficit hyperactivity disorder, Alzheimer''s disease and obesity. We set out to evaluate the novel compound, [¹²⁵I]WYE-230949, as a potential radionuclide imaging agent for the histamine H₃ receptor in brain. [¹²⁵I]WYE-230949 had a high in vitro affinity for the rat histamine H₃ receptor (K_d of 6.9 nM). The regional distribution of [¹²⁵I]WYE-230949 binding sites in rat brain, demonstrated by in vitro autoradiography, was consistent with the known distribution of the histamine H₃ receptor. Rat brain uptake of intravenously injected [¹²⁵I]WYE-230949 was low (0.11 %ID/g) and the ratio of specific: non-specific binding was less than 1.4, as determined by ex vivo autoradiography. In plasma, metabolism of [¹²⁵I]WYE-230949 into a less lipophilic species occurred, such that less than 38% of the parent compound remained 30 minutes after injection. Brain uptake and metabolism of [¹²⁵I]WYE-230949 were increased and specific binding was reduced in anaesthetised compared to conscious rats. [¹²⁵I]WYE230949 is not a potential radiotracer for imaging rat histamine H₃ receptors in vivo due to low brain uptake, in vivo metabolism of the parent compound and low specific binding.     

Ca2+‐dependent down‐regulation of human histamine H1 receptors in Chinese hamster ovary cells

G_q/11 protein‐coupled human histamine H₁ receptors in Chinese hamster ovary cells stimulated with histamine undergo clathrin‐dependent endocytosis followed by proteasome/lysosome‐mediated down‐regulation. In this study, we evaluated the effects of a sustained increase in intracellular Ca²⁺ concentrations induced by a receptor‐bypassed stimulation with ionomycin, a Ca²⁺ ionophore, on the endocytosis and down‐regulation of H₁ receptors in Chinese hamster ovary cells. All cellular and cell‐surface H₁ receptors were detected by the binding of [³H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H₁ receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine‐ and pirdonium‐sensitive binding sites of [³H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca²⁺ and partially by a ubiquitin‐activating enzyme inhibitor (UBEI‐41), but were not affected by inhibitors of calmodulin (W‐7 or calmidazolium) and protein kinase C (chelerythrine or GF109203X). These ionomycin‐induced changes were also not affected by inhibitors of receptor endocytosis via clathrin (hypertonic sucrose) and caveolae/lipid rafts (filipin or nystatin) or by inhibitors of lysosomes (E‐64, leupeptin, chloroquine, or NH₄Cl), proteasomes (lactacystin or MG‐132), and a Ca²⁺‐dependent non‐lysosomal cysteine protease (calpain) (MDL28170). Since H₁ receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H₁ receptors, even after the ionomycin treatment, H₁ receptors appeared to exist in a form to which [³H]mepyramine was unable to bind. These results suggest that H₁ receptors are apparently down‐regulated by a sustained increase in intracellular Ca²⁺ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.

     

Potential utility of histamine H3 receptor antagonist pharmacophore in antipsychotics

Histamine H₃ receptor (H₃R) antagonists have some antipsychotic properties although the clear molecular mechanism is still unknown. As actually the most effective and less side effective antipsychotics are drugs with multiple targets we have designed typical and atypical neuroleptics with an additional histamine H₃ pharmacophore. The 4-(3-piperidinopropoxy)phenyl pharmacophore moiety has been linked to amitriptyline, maprotiline, chlorpromazine, chlorprothixene, fluphenazine, and clozapine. Amide, amine and ester elements have been used generally to maintain or slightly shift affinity at dopamine D₂-like receptors (D₂ and D₃), to decrease affinity at histamine H₁ receptors, and to obtain H₃R ligands with low nanomolar or subnanomolar affinity. Change of effects at D₁-like receptors (D₁ and D₅) were heterogeneous. With these newly profiled compounds different antipsychotic properties might be achieved.     

Guanosine 5′-(γ-[35S]Thio)triphosphate Autoradiography Allows Selective Detection of Histamine H3 Receptor-Dependent G Protein Activation in Rat Brain Tissue Sections

Abstract: Histamine elicits its biological effects via three distinct G protein-coupled receptors, termed H₁, H₂, and H₃. We have used guanosine 5′-(γ-[³⁵S]thio)triphosphate (GTPγ[³⁵S]) autoradiography to localize histamine receptor-dependent G protein activation in rat brain tissue sections. Initial studies revealed that in basal conditions, adenosine was present in tissue sections in sufficient concentrations to generate an adenosine A₁ receptor-dependent GTPγ[³⁵S] signal in several brain regions. All further incubations therefore contained 8-cyclopentyl-1,3-dipropylxanthine (10 µM), a selective A₁ receptor antagonist. Histamine elicited dose-dependent increments in GTPγ[³⁵S] binding to discrete anatomical structures, most notably the caudate putamen, cerebral cortex, and substantia nigra. The overall anatomical pattern of the histamine-evoked binding response closely reflects the known distribution of H₃ binding sites and was faithfully mimicked by N^α-methylhistamine, (R)-α-methylhistamine, and immepip, three H₃-selective agonists. In all regions examined, the GTPγ[³⁵S] signal was reversed with thioperamide and clobenpropit, two potent H₃-selective antagonists, whereas mepyramine, a specific H₁ antagonist, and cimetidine, a prototypic H₂ antagonist, proved ineffective. These data indicate that in rat brain tissue sections, GTPγ[³⁵S] autoradiography selectively detects H₃ receptor-dependent signaling in response to histamine stimulation. As the existing evidence suggests that GTPγ[³⁵S] autoradiography preferentially reveals responses to G_i/o-coupled receptors, our data indicate that most, if not all, central H₃ binding sites represent functional receptors coupling to G_i/o, the inhibitory class of G proteins. Besides allowing more detailed studies on H₃ receptor signaling within anatomically restricted regions of the CNS, GTPγ[³⁵S] autoradiography offers a novel approach for functional in vitro screening of H₃ ligands.     

N-Substituted piperazine derivatives as potential multitarget agents acting on histamine H3 receptor and cancer resistance proteins

Taking into account that multidrug resistance (MDR) is the main cause for chemotherapeutic failure in cancer treatment, the ability of novel histamine H₃ receptor ligands to reverse the cancer MDR was evaluated, using the ABCB1 efflux pump inhibition assay in mouse MDR T-lymphoma cells. The most active compounds displayed significant cytotoxic and antiproliferative effects as well as a very potent MDR efflux pump inhibitory action, 3–5-fold stronger than that of reference inhibitor verapamil. Although these compounds possess weak antagonistic properties against histamine H₃ receptors, they are valuable pharmacological tools in the search for novel anticancer molecules. Furthermore, for the most active compounds, an insight into mechanisms of action using either, the luminescent Pgp-Glo™ Assay in vitro or docking studies to human Pgp, was performed.     

Discovery of novel steroidal histamine H3 receptor antagonists/inverse agonists

Emerging from an HTS campaign, novel steroid-based histamine H₃ receptor antagonists were identified and characterized. Structural moieties of the hit compounds were combined to improve binding affinities which resulted in compound 4 as lead molecule. During the lead optimization due to the versatile modifications of diamino steroid derivatives, several in vitro potent compounds with subnanomolar binding affinities to histamine H₃ receptors were found. The unfavorable binding to rat muscarinic receptors was successfully reduced by tuning the basicity. Compound 20 showed significant in vivo activity in the rat dipsogenia model and could serve as a pharmacological tool in the future.     

Histamine-induced suppressor factor (HSF): further studies on the nature of the stimulus and the cell which produces it.

Guinea pig lymphocytes are stimulated by histamine to produce a soluble factor with immunosuppressive properties. This factor, termed histamine-induced suppressor factor or HSF, abrogates the production of migration inhibitory factor (MIF) and proliferative response to specific antigen. In the present study we have determined the lymphocyte subpopulation which elaborates HSF, the lymphoid tissue source, the kinetics of its generation in relation to immunization, and the nature of the histamine receptor involved in modification of the release of HSF. HSF activity could be detected in populations of cells from spleen and lymph nodes prior to active immunization of the donor, but not in cells from the donor's blood or thymus. Following immunization with ortho-chloro benzoyl-bovine γ-globulin in complete Freund's adjuvant (CFA), more HSF activity was detected in cells from the donor's spleen and lymph nodes. The peak response was seen 2 weeks postimmunization when significant amounts of HSF also were made by cells from the blood and thymus. Concentrations of T-cell-enriched and B-cell-enriched populations were tested for their ability to make HSF. We found that T-cell-enriched, but not B-cell-enriched populations, made significant amounts of HSF. Cells from the lymph nodes of immunized donors were chromatographed over affinity columns made of insolubilized conjugates of histamine with albumin. The nonretained cells were unable to generate HSF, whereas HSF activity was detected in the cells that were retained by the columns. This finding strongly suggests that the HSF-producing cells have receptors for histamine. Cells from CFA-immune lymph nodes were incubated with H₁ (2-methyl histamine) and H₂ (4-methyl histamine) agonists to determine their relative potency and, therefore, the nature of the histamine receptors on these cells that were modifying HSF release. Although both agonists could induce generation of HSF when high concentrations (10^?3M) were used, only the H₂ agonist stimulated production or release of HSF at lower concentrations (10^?5M). These HSF-producing cells appear to be selectively sensitive to H₂ agonists and likely have a predominance of H₂ receptors. Allergic mediators other than histamine were studied to determine their ability to allow elaboration of HSF-like activity from CFA-immune lymph node cells. Serotonin (10^?3M), slow-reacting substance of anaphylaxis (100 units/ml), eosinophil chemotactic factor (tetrapeptide; 10^?5M), and prostaglandin E₁ (10^?4M) were unable to induce HSF-like activity in lymph node cells from donors immunized with CFA. Furthermore, other agents which raise intracellular levels of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) such as isoproterenol and cholera toxin, as well as the dibutyryl form of cyclic AMP itself, were also unable to generate HSF-like activity. Thus, histamine is unique among the allergic mediators in stimulating elaboration of the suppressive substance. These findings also suggest that the ability of histamine to stimulate HSF may not reside in the conventional pathway linked to cAMP accumulation, but rather to an as yet undefined pathway of cell activation. A model is presented which further implicates histamine as a modulator of cellular immune reactions.