Dimeric carbamoylguanidine-type histamine H2 receptor ligands: A new class of potent and selective agonists.

The bioisosteric replacement of the acylguanidine moieties in dimeric histamine H2 receptor (H2R) agonists by carbamoylguanidine groups resulted in compounds with retained potencies and intrinsic activities, but considerably improved stability against hydrolytic cleavage. These compounds achieved up to 2500 times the potency of histamine when studied in [(35)S]GTPγS assays on recombinant human and guinea pig H2R. Unlike 3-(imidazol-4-yl)propyl substituted carbamoylguanidines, the corresponding 2-amino-4-methylthiazoles revealed selectivity over histamine receptor subtypes H1R, H3R and H4R in radioligand competition binding studies. H2R binding studies with three fluorescent compounds and one tritium-labeled ligand, synthesized from a chain-branched precursor, failed due to pronounced cellular accumulation and high non-specific binding. However, the dimeric H2R agonists proved to be useful pharmacological tools for functional studies on native cells, as demonstrated for selected compounds by cAMP accumulation and inhibition of fMLP-stimulated generation of reactive oxygen species in human monocytes.

Histamine type 2 receptor expression on peripheral blood regulatory lymphocytes in patients with allergic rhinitis treated with specific immunotherapy.

BACKGROUND: Both histamine H1- and H2-receptors (H2R) were found on regulatory T (Treg) cells; however, there is a paucity of information regarding the role of H2R in Treg function. This study aimed to investigate the effects of natural allergen stimulation and specific immunotherapy (SIT) on H2R expression in Treg cells in patients with allergic rhinitis (AR). METHODS: In this prospective, double-blind, placebo-controlled study 41 patients with AR were screened for 1 year and treated with SIT (n = 21) or placebo (n = 20) for the next 2 years. Fifteen healthy subjects were included as a control. Subsets of Treg cells that expressed H2R were assessed annually in the blood by flow cytometry: before, at the height of the pollen season, and after, at the end of the pollen season. In addition, total nasal symptom score, the use of rescue medication, and nasal eosinophilia were evaluated. RESULTS: Treg cells of AR patients slightly up-regulate H2R out of the pollen season. Natural allergen stimulation results in prompt up-regulation of H2R within these cells. SIT significantly decreased the number of Treg cells with increased expression of H2R in the blood exclusively at the height of pollen season, which, however, had no impact on the expression of H2R in Treg cells. SIT improved significantly the symptom score, rescue medication use, and decreased nasal eosinophilia. CONCLUSION: Natural pollen exposure results in up-regulation of H2R in Treg cells. Immunotherapy might transiently decrease the number of Treg-H2R(+) cells in the blood, which may be associated with their migration to the peripheral tissues. This study was part of the clinical trial registered in www.clinicaltrials.gov.

Effects of Se deficiency on serum histamine concentration and the expression of histamine H2 receptor in the jejunum of chickens.

UNLABELLED: The present study was designed to investigate the influence of Se deficiency on serum histamine concentration and the expression of histamine receptor in the jejunum of chickens. Forty neonatal chickens were randomly divided into two groups. Experimental chickens were fed a low-Se diet (0.034 mg/kg), whereas chickens in the control group were fed a diet with a Se level of 0.229 mg/kg. Ten chickens were sacrificed on days 30, 45, 60 and 75. Blood and jejunum samples were collected. Histamine concentration in the jejunum was measured by ELISA, the jejunal mast cell (MC) ultrastructure was studied by transmission electron microscopy, and the expression level of histamine H2 receptor (H2R) mRNA in the jejunum was examined using real-time PCR. RESULTS: The jejunal histamine concentration in chickens fed the low-Se diet was significantly higher than that in the control group (P < 0.01). Se deficiency induced degranulation of MC in the jejunum of chickens in the low-Se diet group; their cytoplasm was filled with fused granules and vacuoles. The expression level of jejunal H2R mRNA in chickens fed the low-Se diet was also significantly higher than that in the control group (P < 0.01). The results obtained suggest that Se deficiency stimulates MC degranulation and release of histamine, binding H2R promotes both regulation of digestion and cell proliferation while protects the jejunum from injury induced by Se deficiency.

Enhanced antinociceptive effects of morphine in histamine H2 receptor gene knockout mice.

We have previously shown that antinociceptive effects of morphine are enhanced in histamine H1 receptor gene knockout mice. In the present study, involvement of supraspinal histamine H2 receptor in antinociception by morphine was examined using histamine H2 receptor gene knockout (H2KO) mice and histamine H2 receptor antagonists. Antinociception was evaluated by assays for thermal (hot-plate, tail-flick and paw-withdrawal tests), mechanical (tail-pressure test) and chemical (formalin and capsaicin tests) stimuli. Thresholds for pain perception in H2KO mice were higher than wild-type mice. Antinociceptive effects of intracerebroventricularly administered morphine were enhanced in the H2KO mice compared to wild-type mice. Intracerebroventricular co-administration of morphine and cimetidine produced significant antinociceptive effects in the wild-type mice when compared to morphine or cimetidine alone. Furthermore, zolantidine, a selective and hydrophobic H2 receptor antagonist, enhanced the effects of morphine in all nociceptive assays examined. These results suggest that histamine exerts inhibitory effects on morphine-induced antinociception through H2 receptors at the supraspinal level. Our present and previous studies suggest that H1 and H2 receptors cooperatively function to modulate pain perception in the central nervous system.

Site specific therapy: an integrative approach to treating melanoma.

There have been many proposed theories for effectively treating melanoma, especially through the regulation of histamine. Histamine has been proven to be a major regulator of the immune system's T-helper cell subset balance and major shifts in this balance towards TH2 cytokines have contributed to diseases such as asthma, lupus and cancer. Histamine also causes suppression of interferon-induced proteins needed for anti-tumor response and activates T-suppressor cell function in cancers such as squamous cell carcinoma and melanoma. Scientific evidence has suggested the possibility of an anthistamine approach as treatment to these diseases and for melanoma, there has been great promise. This is due to the fact that melanotic cells have been elucidated to express histamine receptors and as a result, regulation of histamine could occur specifically at the site of these epidermal growths. Another factor to consider is how effective an inflammatory response can be when combined with regulation of histamine. Inflammation is a very powerful tool against pathogenic environments by causing cytokine recruitment and migration of dendritic cells to infected sites. Adequate stimulation of an inflammatory response at the specific site of any cancerous region would greatly weaken its evasive mechanisms. However, there are no reports showing high efficacy utilizing the benefits of regulating inflammation and histamine that could cause TH1 subset levels to predominate, down-regulate T-suppressor cells, up-regulate interferon-induced proteins and properly sustain migration of dendritic cells concurrently. These benefits have been proven in separate instances for a range of diseases but have not been assessed as a combined modality for melanoma therapy. Therefore successful melanoma treatment should integrate these principles involving: the use of H2 antagonists for preventing the negative effects of histamine, monoclonal antibodies to ensure an effective dendritic cell response, and routine pro-inflammatory induction at the specific site of the melanotic tissue to ensure recognition of the cancer that has evaded immunity.

The laminar histamine receptor system in human prefrontal cortex suggests multiple levels of histaminergic regulation.

Human prefrontal cortex is essential for high brain functions and its activity is modulated by multiple neurotransmitters, including histamine. However, the histamine receptors in this brain area have not been systematically studied so far. In situ hybridization and receptor binding autoradiography were employed to map and quantify the mRNA expression and receptor binding of three of the four histamine receptors (H(1), H(2), H(3)). mRNA expression and receptor binding of these three histamine receptors displayed characteristic laminar distribution patterns. Both H(1) and H(3) receptor mRNAs were mainly expressed in the deeper layers (H(1) in laminae V and VI; H(3) in lamina V), where most of the corticothalamic projections originate, whereas H(2) receptor mRNA was primarily expressed in the superficial layer II. Receptor ligand binding of these three histamine receptors displayed relatively even distribution patterns throughout the gray matter. However, higher densities of H(1) and H(3) receptor radioligand binding sites were seen in the middle layers III and IV that receive abundant thalamic inputs and where some of the apical dendrites of the deep-layer pyramidal neurons terminate, whereas higher density of H(2) receptor radioligand binding sites was seen in the superficial layers I-III. The results, together with data on histaminergic regulation of thalamic oscillations suggest that histamine regulates both cortico-cortical and thalamo-cortical circuits. As histamine receptors are also abundant in thalamus, histamine may be involved also in human diseases of the thalamocortical system.

Structural and functional characterization of gastric mucosa and central nervous system in histamine H2 receptor-null mice.

To examine the physiological role of the histamine H(2) receptor, histamine H(2) receptor-null mice were generated by homologous recombination. Histamine H(2) receptor-null mice, which developed normally and were fertile and healthy into adulthood, exhibited markedly enlarged stomachs and marked hypergastrinemia. The former was due to hyperplasia of gastric gland cells (small-sized parietal cells, enterochromaffin-like cells and mucous neck cells which were rich in mucin), but not of gastric surface mucous cells, which were not increased in number as compared with those in wild-type mice despite the marked hypergastrinemia. Basal gastric pH was slightly but significantly higher in histamine H(2) receptor-null mice. Although carbachol but not gastrin induced in vivo gastric acid production in histamine H(2) receptor-null mice, gastric pH was elevated by both muscarinic M(3) and gastrin antagonists. Thus, both gastrin and muscarinic receptors appear to be directly involved in maintaining gastric pH in histamine H(2) receptor-null mice. Interestingly, gastric glands from wild-type mice treated with an extremely high dose of subcutaneous lansoprazole (10 mg/kg body weight) for 3 months were very similar to those from histamine H(2) receptor-null mice. Except for hyperplasia of gastric surface mucous cells, the findings for gastric glands from lansoprazole-treated wild-type mice were almost identical to those from gastric glands from histamine H(2) receptor-null mice. Therefore, it is possible that the abnormal gastric glands in histamine H(2) receptor-null mice are secondary to the severe impairment of gastric acid production, induced by the histamine H(2) receptor disruption causing marked hypergastrinemia. Analyses of the central nervous system (CNS) of histamine H(2) receptor-null mice revealed these mice to be different from wild-type mice in terms of spontaneous locomotor activity and higher thresholds for electrically induced convulsions. Taken together, these results suggest that (1) gastrin receptors are functional in parietal cells in histamine H(2) receptor-null mice, (2) abnormal gastric glands in histamine H(2) receptor-null mice may be secondary to severe impairment of gastric acid production and secretion and (3) histamine H(2) receptors are functional in the central nervous system.

The histaminergic system in human thalamus: correlation of innervation to receptor expression.

The mRNA expression of three histamine receptors (H1, H2 and H3) and H1 and H3 receptor binding were mapped and quantified in normal human thalamus by in situ hybridization and receptor binding autoradiography, respectively. Immunohistochemistry was applied to study the distribution of histaminergic fibres and terminals in the normal human thalamus. mRNAs for all three histamine receptors were detected mainly in the dorsal thalamus, but the expression intensities were different. Briefly, H1 and H3 receptor mRNAs were relatively enriched in the anterior, medial, and part of the lateral nuclei regions; whereas the expression level was much lower in the ventral and posterior parts of the thalamus, and the reticular nucleus. H2 receptor mRNA displayed in general very low expression intensity with slightly higher expression level in the anterior and lateropolar regions. H1 receptor binding was mainly detected in the mediodorsal, ventroposterolateral nuclei, and the pulvinar. H3 receptor binding was detected mainly in the dorsal thalamus, predominantly the periventricular, mediodorsal, and posterior regions. Very high or high histaminergic fibre densities were observed in the midline nuclear region and other nuclei next to the third ventricle, ventroposterior lateral nucleus and medial geniculate nucleus. In most of the core structures of the thalamus, the fibre density was very low or absent. The results suggest that histamine in human brain regulates tactile and proprioceptory thalamocortical functions through multiple receptors. Also, other, e.g. visual areas and those not making cortical connections expressed histamine receptors and contained histaminergic nerve fibres.

Detailed mapping of the histamine H2 receptor and its gene transcripts in guinea-pig brain.

Autoradiographic studies of the distribution of the histamine H2 receptor and its messenger RNAs were performed on serial frontal and a few sagittal sections of guinea-pig brain using [(125)I]iodoaminopotentidine for radioligand binding and a 33P-labelled complementary RNA probe for in situ hybridization, respectively. Both probes were validated by assessing non-specific labelling using non-radioactive competing H2 receptor ligands and a sense probe for binding sites and gene transcripts, respectively. In some areas, e.g., cerebral cortex, hippocampal complex or cerebellum, such studies were completed by identification of neurons expressing the H2 receptor messenger RNAs on emulsion-dipped sections. Nissl-stained sections from comparable levels were used to localize brain structures. In many brain areas, the distribution of the H2 receptor and its messenger RNAs appeared to parallel that known for histaminergic axons. For instance. high levels of both H2 receptor markers were detected in striatal and limbic areas known to receive abundant histaminergic projections. In contrast, in septum, hypothalamic, pontine and several thalamic nuclei, a comparatively low density of both H2 receptor markers was detected, suggesting that histamine actions in these areas are mediated by H1 and/or H3 receptors. Generally, the distribution of H2 receptor messenger RNA correlates well with that of [(125)I]iodoaminopotentidine binding sites, although some differences were observed. In a few regions (e.g., substantia nigra, locus coeruleus) high or moderate densities of binding sites were accompanied by a much more restricted expression of H2 receptor transcripts. Conversely, the mammillary region and the pontine nucleus exhibited higher levels of hybridization than of binding sites. In hippocampus, cerebral and cerebellar cortex there was a selective localization of the H2 receptor messenger RNA in the granule cells of dentate gyrus, pyramidal cells of the Ammon's horn and cerebral cortex, and Purkinje cells of cerebellum, whereas [(125)I]iodoaminopotentidine binding sites were located in layers where the dendritic trees of these messenger RNA-expressing neurons extend. The same discrepancy between messenger RNAs and binding sites suggests that striatonigral endings are endowed with the H2 receptor. The histamine H1 and H2 receptors both appear to be present in several brain areas, in some cases in a way suggesting their potential co-expression by the same neuronal populations, e.g., in granule and pyramidal cells in the hippocampal formation. This co-expression accounts for synergic responses, e.g., on cAMP generation, previously observed upon co-stimulation of both receptor subtypes. The widespread distribution of the H2 receptor, namely in thalamic nuclei or in telencephalic areas such as most layers of the cerebral cortex, together with its excitatory role previously established in electrophysiological studies, support its alleged function in mediating the histamine-driven control of arousal mechanisms. In addition, the detection of H2 receptor expression in brainstem areas from which other monoaminergic pathways involved in the control of states of sleep and wakefulness emanate, e.g., several raphe nuclei, locus coeruleus or substantia innominata, suggests possible interrelationships between all of these systems with highly divergent projections to the thalamus and telencephalon. The present mapping of the H2 receptor and its gene transcripts should facilitate neurochemical, neurophysiological and behavioural studies aimed at clarifying the role of histaminergic systems in brain.

G proteins of the Gq family couple the H2 histamine receptor to phospholipase C.

In several cell systems histamine has been shown to stimulate both adenylyl cyclase and phospholipase C through activation of a G protein-coupled H2 receptor. To analyze the bifurcating signal emanating from the activated H2 receptor and to identify the G proteins involved, H1 and H2 histamine receptors were functionally expressed in baculovirus-infected insect cells. Histamine challenge lead to concentration-dependent cAMP formation and Ca2+ mobilization in Sf9 cells infected with a virus encoding the H2 receptor, whereas H1 receptor stimulation only resulted in pronounced phospholipase C activation. To analyze the G protein coupling pattern of histamine receptors, activated G proteins were labeled with [alpha-32P]GTP azidoanilide and identified by selective immunoprecipitation. In insect cell membranes expressing H1 histamine receptors, histamine led to incorporation of the label into alpha q-like proteins, whereas activation of the H2 receptor resulted in labeling of alpha q- and alpha s-like G protein alpha-subunits. In COS cells transfected with H2 receptor complementary DNA, histamine caused concentration-dependent accumulation of cAMP and inositol phosphates; the latter effect was insensitive to pertussis toxin treatment. Histamine stimulation led to a pronounced increase in inositol phosphate production when complementary DNAs coding for alpha q, alpha 11, alpha 14, or alpha 15 G protein alpha-subunits were cotransfected. This increase was specific for Gq family members, as overexpression of alpha 12 or alpha s did not enhance histamine-stimulated phospholipase C activation. In membranes of guinea pig heart, addition of [alpha-32P]GTP azidoanilide resulted in labeling of alpha q and alpha 11 via the activated H1 and also via H2 receptors. These data demonstrate that dual signaling of the activated H2 histamine receptor is mediated by coupling of the receptor to Gs and Gq family members.