Amine dependence of proliferative activity in two transplantable lines of mouse colonic carcinoma

Serotonin, histamine and their antagonists have previously been shown to influence both the cell proliferation rate and the volumetric growth rate of colonic tumours. Of these earlier studies, those on cell proliferation could not distinguish between direct effects on tumour cells and indirect effects on the host, whereas those on the volumetric growth rate of tumours, whilst suggesting an outcome related to the individual properties of the tumour rather than the host, could not distinguish between influences on cell gain, cell loss or stromal changes. In an attempt to distinguish between these possibilities the current experiments on the mitotic rate in two lines of transplantable mouse colonic carcinoma were undertaken. One line of tumour proved to be sensitive to inhibition by a histamine H2 receptor antagonist and a dopamine D2 antagonist but resistant to serotonin antagonists; the inhibition by histamine antagonists was surmountable by co-administration of histamine. The other line proved to be highly sensitive to the inhibitory effects of serotonin antagonist and less so to antagonists of the other two amines and in this case the effect of serotonin antagonists was surmountable by serotonin. These results suggest that the variations between different colonic tumours in the response to amine antagonists is due to differences in the extent of inhibition of cell proliferation rather than differences in cell loss or stromal effects. Thus it appears likely that amine antagonists are able to directly interfere with the proliferation of some colonic tumour cells.     

The influence of histamine at various concentrations on the cell cycle state of hematopoietic stem cells (CFU-s)

The influence of histamine at various concentrations on the cell cycle state of hematopoietic stem cells (CFU-s) was investigated. CFU-s were triggered from the G0 state into the S phase of the cell cycle by in vitro treatment of mouse bone marrow cells with high concentrations of histamine. This effect could be antagonized by a histamine H2 receptor blocking agent. When bone marrow cells were treated with a histamine H1 receptor antagonist prior to histamine treatment, low concentrations of histamine also triggered the entrance of CFU-s into the DNA synthetic phase. Our findings further suggest the existence of histamine H1 and H2 receptors on the surface of CFU-s cells and the antagonistic effect of these two histamine receptor subtypes on the cell cycle state of CFU-s. Our results also suggest that histamine may participate in regulating the proliferation of hematopoietic stem cells in vivo during immune or inflammatory responses.     

Different h2 receptor antihistamines dissimilarly retard the growth of xenografted human melanoma cells in immunodeficient mice

Melanoma cells and tissues contain considerable amounts of histamine and express histamine receptors, suggesting the existence of autocrine and paracrine regulation by histamine. Our previous in vitro results suggested that histamine elevates melanoma cell growth through the H2 receptor. In this work we show that in vivo tumour proliferation in immunodeficient mice xenotransplanted with a human melanoma cell line is diminished by cimetidine, an H2 receptor antagonist, if combined with a tamoxifen derivate acting on cytochrome p450 molecules (DPPE). Ranitidine, another H2 receptor antagonist, has a weaker inhibitory effect, the kinetics and mechanism of which is probably dissimilar to that of the cimetidine/DPPE mixture.     

Histamine as a growth factor and chemoattractant for human carcinoma and melanoma cells: action through Ca2(+)-mobilizing H1 receptors

Histamine receptors are present on the surface of various normal and tumor-derived cell types, where their biological function is incompletely understood. Here we report that histamine not only stimulates cell proliferation under serum-free conditions, but also is chemotactic for human carcinoma (Hela and A431) and melanoma (A875) cells expressing H1 type receptors. Histamine was found to be a potent activator of phospholipase C, leading to polyphosphoinositide hydrolysis and subsequent intracellular Ca2+ mobilization. In addition, histamine also causes the protein kinase C-mediated activation of Na+/H+ exchange, as evidenced by an amiloride-sensitive rise in cytoplasmic pH. All histamine-induced responses, including chemotaxis and DNA synthesis, are completely inhibited by the H1 receptor antagonist pyrilamine, but not by cimetidine, an inhibitor of histamine H2 type receptors. Our results suggest that histamine may have a previously unrecognized role in the migration and proliferation of cells expressing H1 receptors.     

Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways

It is well established that histamine modulates cell proliferation through the activation of the histamine H1 receptor (H1R), a G protein-coupled receptor (GPCR) that is known to couple to phospholipase C (PLC) activation via Gq. In the present study, we aimed to determine whether H1R activation modulates Rho GTPases, well-known effectors of Gq/G₁₁-coupled receptors, and whether such modulation influences cell proliferation. Experiments were carried out in CHO cells stably expressing H1R (CHO-H1R). By using pull-down assays, we found that both histamine and a selective H1R agonist activated Rac and RhoA in a time- and dose-dependent manner without significant changes in the activation of Cdc42. Histamine response was abolished by the H1R antagonist mepyramine, RGS2 and the PLC inhibitor U73122, suggesting that Rac and RhoA activation is mediated by H1R via Gq coupling to PLC stimulation. Histamine caused a marked activation of serum response factor activity via the H1R, as determined with a serum-responsive element (SRE) luciferase reporter, and this response was inhibited by RhoA inactivation with C3 toxin. Histamine also caused a significant activation of JNK which was inhibited by expression of the Rac-GAP β2-chimaerin. On the other hand, H1R-induced ERK1/2 activation was inhibited by U73122 but not affected by C3 or β2-chimaerin, suggesting that ERK1/2 activation was dependent on PLC and independent of RhoA or Rac. [³H]-Thymidine incorporation assays showed that both histamine and the H1R agonist inhibited cell proliferation in a dose-dependent manner and that the effect was independent of RhoA but partially dependent on JNK and Rac. Our results reveal that functional coupling of the H1R to Gq-PLC leads to the activation of RhoA and Rac small GTPases and suggest distinct roles for Rho GTPases in the control of cell proliferation by histamine.     

Inhibition of human skin fibroblast proliferation by histamine and phorbol esters is mediated by protein kinase C

The proliferation of human skin fibroblasts in culture was examined using a [3H]thymidine incorporation assay. Histamine inhibited thymidine incorporation with an IC50 of about 0.2 microM. This effect was blocked by the H1 receptor antagonist mepyramine but not by the H2 receptor antagonist cimetidine. Protein kinase C activators, including several phorbol esters and mezerine, also inhibited thymidine incorporation. The IC50 for beta-phorbol 12,13-didecanoate was less than 0.1 nM. The alpha-isomer of this compound was inactive. Long-term treatment of cells with the beta-isomer eliminated the ability of both histamine and phorbol ester to inhibit thymidine incorporation, presumably due to downregulation of protein kinase C. Our results suggest that histamine H1 receptors are linked to activation of protein kinase C and that activation of this enzyme leads to an inhibition of cell proliferation.     

Histamine H2 receptor activity during the differentiation of the human monocytic-like cell line U-937. Comparison with prostaglandins and isoproterenol

Histamine H2 receptor activity (cAMP generation) has been characterized in U-937 cells before and after retinoic acid-induced differentiation into monocyte-/macrophage-like cells. The differentiation is associated with a decreased capacity of U-937 monocytes to generate cAMP under basal conditions or after cell surface receptor stimulation by histamine, isoproterenol and PGE1. In contrast, the potencies of the hormones are unchanged during monocytic maturation (EC50 values = 3.2-4.6 X 10(-6) M histamine, 4.6-7 X 10(-9) M isoproterenol, 2-4.6 X 10(-6) M PGE1). The data support the view that histamine and cAMP-inducing agents may control the proliferation and differentiation of normal and leukemic cells committed to monocytic maturation in man. They also raise the possibility that normal human monocytes also possess functional H2 receptors and that histamine may be implicated in the regulation of monocyte/macrophage functions.     

GM-CSF in association with IL-1 triggers day-8 CFU-S into cell cycle: role of histamine.

Our recent evidence for the requirement of endogeneous histamine in IL-3-induced proliferation of day-8 CFU-S has prompted us to investigate whether or not GM-CSF, which shares with IL-3 the ability to stimulate bone marrow histamine synthesis, could also affect the cell cycle status of CFU-S via this mediator. We show herein that recombinant GM-CSF alone fails to trigger day-8 CFU-S into S phase, but supports their survival. However, in the same experimental conditions, GM-CSF in combination with IL-1 induces a CFU-S proliferation similar to that obtained in response to IL-3, while IL-1 by itself has no effect on this biological activity. We further provide evidence that this phenomenon is completely abolished: i) by preventing GM-CSF-induced histamine synthesis by alpha-FMH, the specific inhibitor of histidine decarboxylase (HDC), or ii) by blocking the binding sites of H2 histamine receptors with their specific antagonist oxmetidine. Similar results are obtained when progenitor-enriched bone marrow cells are used instead of the unfractionated population. In addition, we provide an argument in support of a histamine receptor modulation by GM-CSF that could explain the lack of effect of factor-induced histamine on day-8 CFU-S cell cycling. Indeed, the entry of these progenitors into S phase that is normally promoted by dimaprit, a specific histamine H2 receptor agonist, is abolished by a preincubation with GM-CSF. Taken together, our data support the conclusion that IL-1 makes CFU-S sensitive to GM-CSF-induced endogeneous histamine that will trigger them into cell cycle, while GM-CSF alone has no such effect on this biological activity.     

Molecular basis for the interaction of histamine with the histamine H2 receptor.

We undertook these studies to characterize the molecular basis of the interaction of histamine with the H2 receptor. Key areas of homology in the structures of the histamine H2 and beta 2 adrenergic receptor suggested specific transmembrane amino acids that might be important for binding of histamine. A third transmembrane aspartic acid of the histamine receptor (Asp98), thought to serve as a counter anion that interacts with the cationic amine moiety of histamine, was mutated to Asn98, and the mutated receptor was expressed in Hepa cells. Removal of the negatively charged amino acid abolished both binding of the H2 receptor antagonist [methyl-3H]tiotidine and histamine stimulated increases in cellular cAMP content. Mutation of a fifth transmembrane aspartic acid (Asp186) to Ala186 or Asn186 by itself or in conjunction with mutation of another fifth transmembrane amino acid (Thr190 to Ala190) resulted in a loss of [methyl-3H] tiotidine binding, although the generation of cAMP in response to histamine was maintained. The histamine receptor with only a Thr190 to Ala190 or Cys190 mutation retained the ability to bind [methyl-3H]tiotidine, but both the affinity and efficacy of binding were reduced. These data lead us to propose a model for histamine binding in which Asp98 is essential for histamine binding and action, Asp186 defines H2 selectivity, and Thr190 is important in establishing the kinetics of histamine binding, but is not essential for H2 selectivity.     

Selective regulation of H1 histamine receptor signaling by G protein-coupled receptor kinase 2 in uterine smooth muscle cells

Histamine stimulates uterine contraction; however, little is known regarding the mechanism or regulation of uterine histamine receptor signaling. Here we investigated the regulation of Galpha(q/11)-coupled histamine receptor signaling in human myometrial smooth muscle cells using the inositol 1,4,5-trisphosphate biosensor pleckstrin homology domain of phospholipase-delta1 tagged to enhanced green fluorescent protein and the Ca(2+)-sensitive dye Fluo-4. Histamine addition caused concentration-dependent increases in inositol 1,4,5-trisphosphate and [Ca(2+)](i) in the ULTR human uterine smooth muscle cell line and primary human myometrial cells. These effects were completely inhibited by the H(1) histamine receptor antagonist, diphenhydramine, and were unaffected by the H(2) histamine receptor antagonist, cimetidine. ULTR and primary myometrial cells were transfected with either dominant-negative G protein-coupled receptor kinases (GRKs) or small interfering RNAs targeting specific GRKs to assess the roles of this protein kinase family in H(1) histamine receptor desensitization. Dominant-negative GRK2, but not GRK5 or GRK6, prevented H(1) histamine receptor desensitization. Similarly, transfection with short interfering RNAs (that each caused >70% depletion of the targeted GRK) for GRK2, but not GRK3 or GRK6, also prevented H(1) histamine receptor desensitization. Our data suggest that histamine stimulates phospholipase C-signaling in myometrial smooth muscle cells through H(1) histamine receptors and that GRK2 recruitment is a key mechanism in the regulation of H(1) histamine receptor signaling in human uterine smooth muscle. These data provide insights into the in situ regulation of this receptor subtype and may inform pathophysiological functioning in preterm labor and other conditions involving uterine smooth muscle dysregulation.     

Molecular cloning of the human histamine H2 receptor.

We utilized the technique of polymerase chain reaction with oligonucleotide primers based upon the nucleotide sequence of the canine H2 histamine receptor gene which we recently isolated to clone its human homologue. Transfection of a construct of this gene in Colo-320 DM cells led to the expression of a receptor that bound to [methyl-3H] tiotidine and was linked to 3',5'cyclic adenosine monophosphate (cAMP) generation in response to histamine. Both cAMP generation and [methyl-3H] tiotidine binding were inhibited with the H2 histamine receptor selective antagonist cimetidine but not diphenhydramine or thioperamide which are, respectively, H1 and H3 histamine receptor antagonists. These data confirm that we have successfully cloned a novel gene encoding the human H2 histamine receptor.     

Histamine and hepatic glutathione in the mouse

A number of histamine receptor agonists and antagonists were utilized to study the effects of histamine on hepatocellular reduced glutathione (GSH) concentrations and the potential role of histamine as a mediator of morphine-induced hepatic GSH depression. Administration of histamine, the H1-histamine receptor agonist thiazolylethylamine, the H2-histamine receptor agonist impromidine, or the histamine-releasing substance compound 48/80 resulted in no significant change in hepatic GSH concentrations. The H1-histamine receptor antagonist chlorpheniramine and the H2-histamine receptor antagonist ranitidine were also without significant effect on hepatic GSH and did not antagonize morphine-induced GSH depression. These observations indicate that histamine release following morphine administration does not play a significant role in the subsequent depletion of hepatic GSH.     

Effects of H1 and H2 receptor antagonists on Tetrahymena.

In Tetrahymena pyriformis the phagocytotic rate increases in response to histamine, but neither the H1 antagonist phenindamine nor the H2 antagonist metiamide stimulate phagocytosis. The H1 antagonist counteracts the effect of histamine, whereas the H2 antagonist does not. The histamine receptor of Tetrahymena is of H1-type, since it cannot distinguish between histamine and antagonists which are closely related to it chemically. It does, however, distinguish between histamine and the chemically unrelated H1 antagonist, phenindamine. The H2 antagonist does not interact with the receptor.     

New role for histamine in interleukin-3-induced proliferation of hematopoietic stem cells

This study investigated the effect of histamine generated by murine bone marrow cells in response to IL-3 on one particular biological activity of this growth factor, i.e., triggering of cells forming colonies in spleen (CFU-S) into S phase. Evidence is provided that i) IL-3-induced day-8 CFU-S cell cycling, evaluated by hydroxy-urea suicide, is completely abrogated when the binding of histamine to its H2 receptors is blocked by the specific antagonist oxmetidine, whereas cetirizine, a H1 receptor antagonist, is ineffective; and ii) the entry of day-8 CFU-S into S phase in response to IL-3 is likewise abolished when the histamine synthesis promoted by the growth factor is prevented by alpha-fluoromethylhistidine, a specific inhibitor of the histamine-forming enzyme, histidine decarboxylase. Similar results are obtained with both drugs, when a progenitor-enriched bone marrow population is used instead of total cells. Furthermore, i.v. injection of recombinant (r)IL-3 results within 2 hr in a substantial increase in bone marrow cell histamine synthesis together with triggering of day-8 CFU-S into cycle, the latter being completely abolished by a simultaneous injection of the H2 histamine receptor antagonist oxmetidine. Thus, our findings support the notion that both in vitro and in vivo the proliferation of early CFU-S in response to IL-3 is modulated by histamine via its H2 receptors. This conclusion is also consistent with the observation that dimaprit, a specific agonist of these receptors not only enhances the sensitivity of day-8 CFU-S to HU after a 2 hr incubation with bone marrow cells but also increases, to the same extent as IL-3, the number of colonies formed in irradiated spleens after a 5 hr pretreatment.     

Histamine protects against NMDA-induced necrosis in cultured cortical neurons through H receptor/cyclic AMP/protein kinase A and H receptor/GABA release pathways

Using histamine and the H3 receptor antagonist thioperamide, the roles of histamine receptors in NMDA-induced necrosis were investigated in rat cultured cortical neurons. Within 3 h of intense NMDA insult, most neurons died by necrosis. Histamine reversed the neurotoxicity in a concentration-dependent manner and showed peak protection at a concentration of 10(-7) m. This protection was antagonized by the H2 receptor antagonists cimetidine and zolantidine but not by the H1 receptor antagonists pyrilamine and diphenhydramine. In addition, the selective H2 receptor agonist amthamine mimicked the protection by histamine. This action was prevented by cimetidine but not by pyrilamine. 8-Bromo-cAMP also mimicked the effect of histamine. In contrast, both the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purine-6-amine and the cAMP-dependent protein kinase inhibitor N-[2-(p-bromocinnamylamino) ethyl]-5-isoquinolinesulfonamide reversed the protection by histamine. Thioperamide also attenuated NMDA-induced excitotoxicity, which was reversed by the H3 receptor agonist (R)-alpha-methylhistamine but not by pyrilamine and cimetidine. In addition, the protection by thioperamide was inhibited by the GABA(A) receptor antagonists picrotoxin and bicuculline. Further study demonstrated that the protection by thioperamide was due to increased GABA release in NMDA-stimulated samples. These results indicate that not only the H2 receptor/cAMP/cAMP-dependent protein kinase pathway but also the H3 receptor/GABA release pathway can attenuate NMDA-induced neurotoxicity.     

Histamine modulation of eosinophil migration.

Preincubation of eosinophils with 10(-5) M or higher concentrations of histamine inhibited the eosinophil chemotactic response to endotoxin-activated serum whether by using the nucleopore filter assay and counting the cells migrating through the filter, or by using the Zigmond-Hirsch assay and counting the cells at each 10-mum interval. When the H2-receptor sites on the eosinophils were blocked by metiamide, the inhibitory capacity of histamine was prevented. Preincubation of eosinophils with 10(-6) M histamine increased the number of responding eosinophils to endotoxin-activated serum and this enhancement was blocked by an H1-receptor antagonist. Isoproteronol and aminophylline inhibited eosinophil movement and increasing concentrations of dibutryl cyclic AMP inhibited eosinophil migration. Concentrations of histamine that consistently resulted in inhibition of eosinophil movement stimulated an increase in cyclic AMP that was prevented by blocking the H2-receptor but not the H1-receptor. Thus, histamine-dependent inhibition of the eosinophil chemotactic response to other agents is mediated through the H2-receptor and is associated with an increase in the intracellular level of cyclic AMP whereas histamine dependent enhancement of eosinophil migration to other agents appears to be mediated through the H1-receptor. Eosinophils behave as a heterogeneous population as assessed by the ability of histamine to augment or inhibit cell migration. This may reflect differences in H1 to H2 receptor density or cell responsiveness to receptor stimulation. The chemoattractant activity of histamine itself is not influenced by H1 or H2 receptor antagonists, thus it is possible that an eosinophil has a third type of histamine receptor.     

Histamine elevates the expression of Ets-1, a protooncogen in human melanoma cell lines through H2 receptor

Histamine is known to act, at least in part, as a growth factor for several cell types, and as production of this biogen amine has been found to accelerate the rate of tissue proliferation in wound repair, embryogenesis and malignant growth. Abundant experimental and clinical data suggest that histamine augments in vivo tumour cell proliferation via histamine H2 receptors (H2R). Here, we report that exogenously added histamine stimulates Ets-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) synthesis in human melanoma cells. Involvement of histamine receptors in the histamine induced ets-1 expression has been also studied. Our data show that these newly recognized actions of histamine are mediated by the H2R. Modification of local protooncogen Ets-1 level is likely being involved in the regulation of melanoma growth.     

Histamine and the heart

Histamine has been known as a cardiac stimulant for over 70 years. Work in our laboratory over the past decade has established that histamine receptors exist in the hearts of various species. The type of histamine receptor varies not only between species but also in the various regions of the heart. In the guinea pig heart H1 receptors are found in left atria and ventricles while H2 receptors are found in right atria and are the predominant histamine receptor in the ventricles. Rabbit atria contain both H1 and H2 receptors while the ventricles appear to possess only H1. Rat and cat heart do not seem to have histamine receptors and the positive inotropic and chronotropic effects elicited by histamine in cardiac preparations of these species are due to the release of noradrenaline. Dog heart contains H1 receptors while human heart has H2 receptors. In all cases H2 receptors are associated with adenylate cyclase and stimulation of such receptors results in an increase in cyclic AMP levels. H1 receptors are not associated with cyclic nucleotides in the heart. There are certain similarities between beta-adrenergic and H2-histaminergic receptors as well as between alpha-adrenergic and H1-histaminergic receptors. Stimulation of either histamine receptor must result in an increase in the free calcium ion concentration in the cardiac cell but the mechanisms involved are obviously different.     

Switch of histamine receptor expression from H2 to H1 during differentiation of monocytes into macrophages

It is known that histamine suppresses gene expression and synthesis of tumor necrosis factor alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in human peripheral blood mononuclear monocytes (HPM) or alveolar macrophages via histamine H2 receptors. We investigated the effect of histamine and differentiation in macrophages on the expression and secretion of TNF-alpha, TNF-alpha-converting enzyme (TACE), and histamine H1 and H2 receptors by use of a leukemia cell line, U937, and HPM. Differentiation of U937 and HPM cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the H1 receptor expression and rather suppressed the H2 receptor, resulting in up-regulation of the histamine-induced expression and secretion of TNF-alpha, modulated via TACE. Therefore, histamine failed to inhibit up-regulated expression of TNF-alpha induced by LPS in macrophages. The switch from H2 to H1 receptors during differentiation in the monocyte/macrophage lineage could participate in the pathogenic processes of atherosclerosis and inflammatory reactions in the arterial wall.