Single-Cell Transcriptomic Map of the Human and Mouse Bladders.

BACKGROUND: Having a comprehensive map of the cellular anatomy of the normal human bladder is vital to understanding the cellular origins of benign bladder disease and bladder cancer. METHODS: We used single-cell RNA sequencing (scRNA-seq) of 12,423 cells from healthy human bladder tissue samples taken from patients with bladder cancer and 12,884 cells from mouse bladders to classify bladder cell types and their underlying functions. RESULTS: We created a single-cell transcriptomic map of human and mouse bladders, including 16 clusters of human bladder cells and 15 clusters of mouse bladder cells. The homology and heterogeneity of human and mouse bladder cell types were compared and both conservative and heterogeneous aspects of human and mouse bladder evolution were identified. We also discovered two novel types of human bladder cells. One type is ADRA2A+ and HRH2+ interstitial cells which may be associated with nerve conduction and allergic reactions. The other type is TNNT1+ epithelial cells that may be involved with bladder emptying. We verify these TNNT1+ epithelial cells also occur in rat and mouse bladders. CONCLUSIONS: This transcriptomic map provides a resource for studying bladder cell types, specific cell markers, signaling receptors, and genes that will help us to learn more about the relationship between bladder cell types and diseases.

BODIPY(®) FL histamine as a new modality for quantitative detection of histamine receptor upregulation upon IgE sensitization in murine bone marrow-derived mast cells.

Flow cytometry is one of the most widely used methods for the qualitative and quantitative analysis of cell surface expressed proteins by making use of fluorescent specific antibodies. Lacking an antibody validated for flow cytometry, an alternative approach for labeling cell surface receptors is the use of fluorescently tagged ligands. In this study, histamine H4 receptor transfected Chinese hamster ovary cells and murine bone marrow-derived mast cells (mBMMCs) were selected for studying the possibility of staining individual histamine receptors using BODIPY(®) FL histamine and selective antagonists. Flow cytometric measurements and supporting calculations showed that BODIPY FL histamine is suitable tool for quantitating cell surface histamine receptors. The binding, and competitive inhibition of this fluorescent ligand were characterized, which were in good agreement with a semi-empirical model constructed from fundamental protein-binding relationships. Using this method it was shown for the first time that even though mature mBMMCs express H2R and H4R to the same extent, immunoglobulin E sensitization results in H4R upregulation only, while the surface expression of H2R remains unchanged.

Histamine receptors of the microvascular endothelium revealed in situ with a histamine-ferritin conjugate: characteristic high-affinity binding sites in venules.

Histamine covalently bound to glutaraldehyde-activated ferritin was prepared as either monomers or as small aggregates of approximately 0.05 to 0.15 micrometer Diam, suitable for electron microscopic detection of histamine cellular binding sites. The histamine-ferritin conjugates (MF) maintain the histamine capability to induce the opening of endothelial junctions in venules. To investigate the distribution of histamine receptors in the vascular endothelium, monomers or aggregates of MF were perfused in situ (mice), and various vascular beds, particularly that of the diaphragm, were fixed and processed for electron microscopy. The conjugate was preferentially bound on restricted areas of luminal endothelial cell plasmalemma especially in regions rich in filaments, and near the junctions between endothelial cells. The density of histamine binding sites was characteristically high in venules; it occurred to a much lesser extent in arterioles, veins, and muscular arteries whereas capillaries and aorta showed the lowest values. A similar distribution was obtained after perfusion of H1 or H2 receptor agonists coupled to ferritin (2-pyridylethylamine-ferritin [PF], or 4-methylhistamine-ferritin [MF], respectively). The binding specificity was assessed through control experiments with either native or activated ferritin or by competition with histamine. The findings suggest that histamine receptors are largely represented in the cell membrane of the vascular endothelium, particularly in venules. Experiments using specific H1 and H2 receptor agonists (PF and MF) and antagonists (mepyramine and cimetidine) indicate that the venular endothelium contains mainly H2 receptors.

Histamine-induced ion secretion across rat distal colon: involvement of histamine H1 and H2 receptors.

The aim of the present study was to investigate the effect of histamine, a product of e.g. mast cells, on short-circuit current (I(sc)) across rat distal colon. Histamine concentration-dependently stimulated an increase in I(sc), which often was preceded by a transient negative current. Neither a release of neurotransmitters nor a release of prostaglandins contributed to the histamine response. The histamine-induced increase in I(sc) was blocked by the histamine H(1) antagonist, pyrilamine, but was resistant against the histamine H(2) antagonist, cimetidine. Conversely, the histamine H(1) agonist, TMPH (2-(3-trifluoromethylphenyl)histamine), exclusively evoked an increase in I(sc), whereas the histamine H(2) agonist, amthamine, evoked only a decrease in I(sc) suggesting that stimulation of different types of histamine receptors is responsible for the two phases of the response evoked by native histamine. Histamine induces the opening of glibenclamide-sensitive Cl(-) channels and of charybdotoxin-sensitive K(+) channels in the apical membrane as demonstrated by experiments at basolaterally depolarized epithelia. A further action site is the basolateral membrane, because histamine stimulates a charybdotoxin- and tetrapentylammonium-sensitive K(+) conductance in this membrane as observed in tissues, in which the apical membrane was permeabilized with an ionophore, nystatin. The increase in I(sc) evoked by histamine was blocked after depletion of intracellular Ca(2+) stores with cyclopiazonic acid and after blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors, suggesting a release of stored Ca(2+). This was confirmed by the observation that the histamine H(1) agonist TMPH induced an increase in the fura-2 ratio signal of epithelial cells within isolated colonic crypts. Consequently, the mediator histamine seems to stimulate both histamine H(1) and H(2) receptors, from which the former seems to be prominently involved in the induction of epithelial chloride secretion.

Characterization of a newly established human gastric cancer cell line HGT-1 bearing histamine H2-receptors.

A human gastric adenocarcinoma cell line, HGT-1, was established in vitro from the primary tumor of a 60-year-old patient. Histological examination of the tumor revealed a poorly differentiated adenocarcinoma. Primary tumor cells were cloned in soft agarose and gave rise to tumor colonies. The procedures enabling us to form a continuous cell line from the agarose colonies are described. The cultured cells grew as monolayers of closely apposed polygonal cells with a population-doubling time of 19.48 +/- 1.20 (S.E.) hr during exponential growth at passage 59. They had an epithelial morphology. Ultrastructural studies revealed the presence of microvilli and tight junctions. The HGT-1 cell line is tumorigenic in nude mice and has a hyperdiploid karyotype with a modal number of 57 chromosomes. It exhibits numerous marker chromosomes. These human gastric epithelial cells do not secrete mucus or carcinoembryonic antigen. They exhibit functional histamine H2-receptors mediating cellular cyclic adenosine 3':5'-monophosphate production and adenylate cyclase activation. In conclusion, the use of a soft-agarose clonogenic assay permitted us to develop a cancer cell line without the problems of fibroblastic cell contamination. The existence of histamine H2-receptors on gastric HGT-1 cells stresses the importance of this line as a model for studies of regulatory mechanisms involved in gastric secretion.

Effects of acute glucose overload on histamine H2 receptor-mediated Ca2+ mobilization in bovine cerebral endothelial cells.

[Ca2+]i and whole-cell membrane current were measured in microvascular endothelial cells from bovine brain. The effects of histamine on [Ca2+]i were examined, and the acute effect of changing extracellular glucose concentration on Ca2+ homeostasis was investigated. Application of 10 micromol/l histamine evoked an initially transient and then sustained increase in [Ca2+]i in normal Krebs solution, but only the transient component in Ca2+-free solution, thereby indicating that histamine mobilizes Ca2+ both from intracellular store sites and extracellular space. The effects of histamine on [Ca2+]i were inhibited by the H2 antagonists, ranitidine and cimetidine, but not by the H1 antagonist, pyrilamine. Incubation of the cells for 2 h in solutions containing low (1.1 and 2.3 mmol/l) or high (23 mmol/l) concentrations of glucose did not influence the resting level of [Ca2+]i. Treatment with low concentrations of glucose did not impair histamine-induced Ca2+ mobilization. On the other hand, when histamine was applied to the cells pretreated with 23 mmol/l glucose, it failed to mobilize Ca2+ from both intracellular store sites and extracellular space. The effect of histamine was mimicked by dibutyryl cyclic AMP, but glucose overload failed to inhibit this, suggesting that glucose overload inhibits H2 receptor-mediated cyclic AMP production. Glucose overload-induced impairment of histamine action was reversed by pretreatment with staurosporine and calphostin C and mimicked by phorbol-12,13-dibutyrate, thereby suggesting the involvement of protein kinase C in the high glucose-induced inhibition of Ca2+ mobilization. Whole-cell membrane current measurement showed that there was no difference in the membrane currents between control and high glucose-treated cells. These results indicate that in bovine brain microvascular endothelial cells, histamine induces Ca2+ release from intracellular store sites and subsequent entry from the extracellular space through the activation of H2 receptors. Glucose overload acutely inhibits histamine-induced Ca2+ mobilization by the activation of protein kinase C.

Histaminergic regulation of antibody-dependent cellular cytotoxicity of granulocytes, monocytes, and natural killer cells.

We investigated effects of the biogenic diamine histamine on antibody-dependent cellular cytotoxicity (ADCC) against autologous anti-D-coated red blood cells mediated by human granulocytes, monocytes, and natural killer (NK) cells. Effector cells were separated from peripheral blood by countercurrent centrifugal elutriation. ADCC of monocytes and neutrophilic granulocytes was suppressed by histamine. ADCC of enriched CD3-/56+ NK cells was unchanged by histamine. ADCC of NK cells was effectively inhibited by elutriated monocytes or neutrophils. Histamine completely reversed the inhibition of NK cell-mediated ADCC induced by monocytes and partly reversed the inhibition induced by neutrophils; thereby, histamine augmented ADCC of NK cells in the presence of monocytes or neutrophils. The indirect effect of histamine on ADCC of NK cells and the effect of histamine on ADCC of monocytes/neutrophils were completely antagonized by the specific H2 receptor (H2R) blocker ranitidine. We conclude that activation of H2R suppresses ADCC reactivity of monocytes/neutrophils and, concomitantly, promotes ADCC reactivity of NK cells by abrogating a phagocyte-derived, suppressive signal.

H1 and H2 histamine receptors are absent on Langerhans cells and present on dermal dendritic cells.

Human monocyte-derived dendritic cells (MoDC) have both histamine H1 and H2 receptors and can induce CD86 expression by histamine. Nevertheless, it has not been reported whether human epidermal Langerhans cells (LC) have histamine receptors or not. In this study, using RT-PCR, we investigated the expression of H1 and H2 receptor mRNA on DC with the features of LC (LC-like DC) that were generated in vitro from peripheral blood monocytes, LC derived from CD34+ hematopoietic progenitor cells, and LC obtained from human epidermis. We compared the histamine-induced CD86 expression among these cells. In contrast to MoDC, LC and LC-like DC did not express H1 or H2 receptors. In addition, they could not augment the CD86 expression by histamine. Interestingly, when transforming growth factor-beta1 (TGF-beta1) was added to the culture of MoDC, the expression of H1 and H2 receptors and the histamine-induced CD86 expression were abrogated in a concentration-dependent fashion. Finally, in the assessment of the cell surface expression of histamine receptors using fluorescence-labeled histamine, histamine could bind to MoDC and dermal dendritic cells obtained from the skin, whereas there was no specific binding of histamine to LC-like DC or LC obtained from the skin. These data suggest that LC do not express either H1 or H2 receptors, mainly because of the effect of TGF-beta1. This made a striking contrast with the expression of the functional H1 and H2 receptors on MoDC and dermal dendritic cells.

Evidence for the existence of a histamine H2-receptor in the mouse thyroid.

The existence of a histamine H2-receptor in the thyroid was investigated. Histamine in vitro stimulated the formation of cyclic AMP and colloid droplet formation in mouse thyroid lobes. Stimulation by histamine of cyclic AMP formation in mouse thyroid lobes was significantly inhibited by metiamide, a histamine H2-receptor antagonist. 4-Methylhistamine, a histamine H2-receptor agonist, markedly stimulated cyclic AMP formation, whereas 2-methylhistamine, a histamine H1-receptor agonist, was ineffective. The stimulation by 4-methylhistamine of cyclic AMP formation was markedly inhibited by metiamide, but not by chlorpheniramine, a histamine H1-receptor antagonist. In contrast, metiamide did not affect cyclic AMP formation induced either by TSH or by the long-acting thyroid stimulator. Therefore, it is suggested that there exists a histamine H2-receptor in the membranes of the thyroid follicular cells which facilitate thyroid hormone secretion via the adenylate cyclase-cyclic AMP system.

Histamine and the hypothalamus.

The chemical tools that could be used to examine the function of histamine in the brain are considered together with the evidence linking histamine specifically with the hypothalamus. The distribution of histamine and the enzymes responsible for its synthesis and metabolism is consistent with there being both mast cells and histaminergic nerve terminals within the hypothalamus. Iontophoresis, mepyramine binding and histamine-stimulated adenylate cyclase studies suggest that both histamine H1- and H2- receptors are present in the hypothalamus. In addition, intracerebroventricularly injected histamine receptor agonists and antagonists affect many functions associated with the hypothalamus such as cardiovascular control, food intake, body temperature control, and pituitary hormones whose release is mediated via the hypothalamus, such as corticotropin, growth hormone, thyroid stimulating hormone, prolactin, gonadotropins and vasopressin. However, only in the case of thyroliberin release, prolactin release, body fluid control and blood pressure control is there evidence yet that such effects are mediated via histamine receptors actually in the hypothalamus. The effects of enzyme inhibitors suggest endogenous histamine may be involved in the physiological control of thyroid stimulating hormone, growth hormone and blood pressure, and the effects of receptor antagonists support a role for endogenous histamine in prolactin control. Otherwise, there is little evidence for a physiological role for endogenous, as against exogenous, histamine whether it be from histaminergic terminals or mast cells. In addition, few studies have tried to distinguish possible effects on presynaptic receptors, postsynaptic receptors, hypothalamic blood vessels or the hypophyseal portal blood vessels. It is concluded that although there is good evidence now linking histamine and the hypothalamus more specific studies are required, for instance using microinjection or in vitro techniques and the more specific chemical tools now available, to enable a clearer understanding of the physiological role of histamine in the hypothalamus.

Characterization of vascular histamine receptors in the rat.

1 In the rat the decrease in blood pressure caused by histamine involves activation of both H1- and H2-receptors. Since arterial pressure measurements alone do not permit the separation of responses into cardiac and vascular components, the following experiments were undertaken to study vascular histamine receptors. 2 Vascular responses were studied in the autoperfused hindquarters of anaesthetized rats. Intra-arterial histamine caused vasodilatation which was only partially attenuated by treatment with mepyramine, an H1-receptor antagonist. Treatment with metiamide, the H2-receptor antagonist, did not affect vasodilatation caused by histamine but did attenuate vasodilatation which persisted after mepyramine. 3 Intra-arterial 4-methylhistamine, an H2-receptor agonist, caused vasodilatation which was reduced by metiamide. The H1-receptor agonist, 2-(2-pyridyl)ethylamine also caused vasodilatation which was blocked by mepyramine. 4 It is concluded that in the rat, histamine causes vasodilatation mediated by both H1- and H2-receptors.

H2-receptors in the human ocular surface.

Ten normal human volunteers participated in a two-part study of H2-receptor activity in the ocular surface. Dimethylaminopropylisothiourea (trivial name, dimaprit dihydrochloride), a highly selective H2-receptor agonist, produced vasodilation without itch. Pretreatment with the H2-receptor antagonist, cimetidine, significantly blocked the vasodilatory effect of dimethylaminopropylisothiourea, whereas pretreatment with the H1-receptor antagonist, antazoline phosphate, did not. We conclude that H2-receptors are present in the human ocular surface.

Three histamine receptors (H1, H2 and H3) visualized in the brain of human and non-human primates.

The distribution of histamine H1, H2 and H3 receptors in postmortem human and rhesus monkey brain was examined using receptor autoradiography. [125I]Iodobolpyramine, [125I]iodoaminopotentine and [3H](R) alpha-methylhistamine were used as ligands to label H1, H2 and H3 receptors respectively. The 3 receptor subtypes were identified in the human and monkey brains. Each receptor presented comparable distribution in the two primate brains. H1 and H2 receptors were particularly enriched in the caudate and putamen and observed in other brain areas such as the neocortex and hippocampus. H3-receptors were found to predominate in the basal ganglia where the highest densities were localized in the two segments of the globus pallidus. They were also observed in the hippocampus and cortical areas. The distribution of these 3 histamine receptors in the primate brain suggests the involvement of histaminergic mechanism in the functions of many brain areas. In particular, H2 and H3 receptors could play a role in the regulation of the basal ganglia functions in primates.

Occurrence of H2-inhibitory histamine receptors in chicken ileum.

Metiamide (H2-histamine receptor antagonist) blocked histamine-induced relaxations and significantly potentiated contractile responses to histamine on isolated spiral strips of chicken ileum. This investigation showed the presence of H2-inhibitory receptors in chicken ileum.

Histamine H1- and H2-receptors in the mesenteric vascular bed of the pig.

In order to study whether both histamine H1- and H2-receptors are present in the pig mesenteric vascular bed, natural histamine, 2-(2-pyridyl) ethylamine and 4-methylhistamine, as well as mepyramine and metiamide, were infused directly into the superior mesenteric artery. The results indicate that histamine H1- and H2-receptors, both inducing vasodilation, are present in the mesenteric circulation of the pig. Jejunal motility proved to be influenced by H1-receptor stimulation only.

Identification of endothelial H1, vascular H2 and cardiac presynaptic H3 receptors in the pithed rat.

In pithed and vagotomized rats the effects of the H3 receptor agonist R-(-)-alpha-methylhistamine, the H1 receptor agonist 2-(2-thiazolyl)ethylamine and the H2 receptor agonist dimaprit on basal diastolic blood pressure, basal heart rate and the electrically induced rise in heart rate were examined. Basal diastolic blood pressure was not altered by low, but increased by high doses of R-(-)-alpha-methylhistamine; the latter effect was not affected by selective H1, H2 or H3 receptor antagonists and by prazosin, but was attenuated by rauwolscine. Rauwolscine also unmasked a vasodepressor response to R-(-)-alpha-methylhistamine not affected by the H3 receptor antagonist thioperamide, but counteracted by the H1 receptor antagonist dimetindene or the H2 receptor antagonist ranitidine. The vasodepressor responses to 2-(2-thiazolyl)ethylamine and dimaprit were antagonized by dimetindene and ranitidine, respectively. The vasodepressor response to 2-(2-thiazolyl)ethylamine was not altered by indomethacin, but reduced by an inhibitor of endothelial nitric oxide synthase, N omega-nitro-L-arginine methyl ester (which, by itself, markedly increased blood pressure). Both drug tools did not alter the effect of dimaprit. Basal heart rate was not affected by 2-(2-thiazolyl)ethylamine (examined after administration of propranolol), dimaprit and R-(-)-alpha-methylhistamine. The electrically induced increase in heart rate (studied in animals which had received rauwolscine) was decreased by R-(-)-alpha-methylhistamine but not affected by 2-(2-thiazolyl)ethylamine and dimaprit. The effect of R-(-)-alpha-methylhistamine was abolished by thioperamide. R-(-)-alpha-methylhistamine did not influence the increase in heart rate produced by isoprenaline.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of histamine receptors in isolated pig basilar artery by functional and radioligand binding studies.

Histamine receptors in pig basilar arteries were investigated in vitro by radioligand binding assays and by measuring the contractile and relaxant responses to histamine. Histamine and 2-pyridylethylamine (H1-agonist) induced concentration-dependent contractions, whereas impromidine (H2-agonist) induced concentration-dependent relaxations. These responses were independent of the presence of endothelial cells. Diphenhydramine (H1-antagonist) partially reversed the histamine-induced contractions to relaxations. Cimetidine (H2-antagonist) potentiated the contraction in a concentration-dependent manner. In the presence of cimetidine, the pEC50 value of histamine for the contraction was 6.30, and diphenhydramine competitively antagonized the histamine-induced contractions (pA2, 7.77). In the presence of diphenhydramine, the pEC50 value of histamine for the relaxation was 5.93, and cimetidine competitively antagonized the histamine-induced relaxations (pA2, 6.62). In the binding studies, the Kd value of [3H]mepyramine was 2.1 nM and the Bmax value was 95.6 fmol/mg protein. A competition experiment with diphenhydramine showed that the pKi value (7.51) was similar to the pA2 value. The Kd value for [3H]cimetidine was 126.0 nM and the Bmax value was 459.8 fmol/mg protein. The pKd (6.90) for [3H]cimetidine was similar to the pA2 for cimetidine. The Hill coefficients for these experiments were not significantly different from unity. The present findings indicate that the number of H1-receptors, in terms of the Bmax value for [3H]mepyramine, is smaller than that of H2-receptors, in terms of the Bmax value for [3H]cimetidine. However, the contractile response to histamine is predominantly mediated through stimulation of H1-receptors on vascular smooth muscle cells in pig basilar artery.

Immunohistochemical localization of histamine receptors in rat cochlea.

OBJECTIVE: Histamine may have physiologic functions in the inner ear. The locations of histamine receptors, however, have not yet been identified in the mammalian cochlea. The aim of this study was to investigate the localization of histamine receptor subtypes (H1, H2, and H3 receptors) in rat cochlea. METHODS: Immunohistochemistry was performed with antibodies specific for each of the histamine receptors (H1, H2, and H3). To identify the type I and II spiral ganglion cells in the cochlea, some cryostat sections were double stained with antibodies to both a histamine receptor and neurofilament 200 kD, which predominantly stains type II spiral ganglion cells in the cochlea. RESULTS: All H1, H2, and H3 receptor immunoreactive staining was limited to the spiral ganglion cells of the cochlea. Spiral ganglion cells with positive immunoreactivity to the neurofilament 200 kD antibody were stained only slightly by histamine H1, H2, and H3 receptor antibodies, indicating that histamine receptor immunoreactivity is specific to type I ganglion cells. CONCLUSIONS: These findings indicate that histamine receptors are present in the cochlea and support the hypothesis that histamine plays a physiologic role in the cochlea.

Histamine receptors in the guinea-pig duodenum.

Guinea-pig duodenum contracted by histamine or by acetylcholine was relaxed dose-dependently by a series of H2-receptor selective agonists namely dimaprit, impromidine, clonidine and tolazoline. This relaxation was not neurally mediated since it was not modified by tetrodotoxin nor was it exerted through sympathetic receptors because it was not modified by pretreatment with propranolol or phentolamine. Apparently it was connected with the H2-receptor stimulation more than to peculiarities of the single compounds. However a series of H2-blocker (metiamide, cimetidine, ranitidine or oxmetidine) were unable to counteract the effect of the H2-agonists or the relaxant effect of histamine in the presence of chlorpheniramine. This peculiar situation seems to indicate the existence of anomalous H2-receptors, susceptible to the action of the agonists by not to that of the antagonists.