Selective disappearance of histamine H2-receptor activity in the human gastric cancer cell line HGT-1 after short-term or chronic treatment by histamine or its H2-antagonists

Homologous loss of histamine H₂-receptor activity (cAMP generation) was observed after short-term (10–20 min) or chronic treatment (6 days) of cultured HGT-1 cells with histamine (desensitization) or the H₂-receptor antagonist SKF 93479. This inactivation process was not observed when HGT-1 cells were exposed to the classical H₂-antihistamine cimetidine.The data show: (1) that the compound SKF 93479 has a very prolonged inhibitory action on histamine receptor activity, suggesting an irreversible interaction between the antagonist and the receptor; (2) that cimetidine is a reversible H₂-receptor antagonist which can be removed without changing the the efficacy and the potency of histamine on gastric cells; (3) that the H₂-receptor antagonists cimetidine and SKF 93479 specifically block histamine H₂-receptor activity in HGT-1 cells since cAMP generation induced by other hormones such as vasoactive intestinal peptide (VIP), glucagon or gastric inhibitory peptide (GIP) was unchanged after treatment; (4) the first evidence for time-dependent (half-life: 20 min) desensitization of gastric H₂-receptors.     

Relationship between3H-histamine uptake and H2-receptors in the human promyelocytic leukemia cell line HL-60

Association of³H-histamine (³H-H) with HL-60 cells was a time- and temperature-dependent process. At 37°C, the association of³H-H (50 fmole/10⁶ cells) was maximal and constant 60 min after the addition of HL-60 cells, while there is no significant radioactivity associated with cells incubated at 4°C during 120 min incubation. Under these conditions, the cell-associated radioactivity remains unchanged, even after a washout period and addition of large excess (10^–3 M) of histamine, indicating an irreversible interaction between histamine and HL-60 cells. The interaction of³H-H with HL-60 cells depends on cell viability, cell concentration and was reduced by various metabolic inhibitors such as iodacetamide, dinitrophenol, sodium azide and ouabain. Histamine uptake by HL-60 cells was inhibited by a series of H₁, H₂ histamine agonists (impromidine, histamine, 4-MH, AET, PEA) and antagonists (cimetidine, oxmetidine, ranitidine, diphenhydramine), according to the following order of potencies: 1>H, 4-MH>AET>PEA and H, C>DPH. Among the histamine analogs tested (acidN-acetyl histamine, imidazole, acid imidazole acetic and histidine), only theN-acetyl histamine acid was able to inhibit³H-H uptake by HL-60 cells. Three antidepressant drugs (amitriptyline, imipramine, clomipramine) also antagonize³H-H uptake by HL-60 cells, but this effect was only observed at toxic concentrations (10^–5 M and above) and was related to a loss of the cell viability. It was concluded that the promyelocytic leukemia cells HL-60 take up histamine by a specific energy-dependent mechanism, preferentially inhibitable by histamine H₂-receptor agonists and antagonists (H₂>H₁). Therefore, the uptake of³H-H by HL-60 cells accounts for an H₂-receptor-mediated process, consistent with the pharmacological specificity of the histamine H₂-receptor linked to the cAMP generating system in this cell line.     

Cardiac electrophysiological effects of histamine,H1 and H2 agonists

The effects of histamine, H₁ and H₂ agonists on the transmembrane action potentials (APs) of electrically driven left auricle of guinea-pig were studied in Tyrode and in 25 mM K⁺ Tyrode solution. Under physiological conditions, histamine (10^–7–10^–5 M) increased the amplitude and the plateau phase of AP. The H₁ agonist pyridylethylamine (PEA; 10^–5–10^–4 M) exerted similar effects as histamine while the H₂-agonist impromidine (10^–8–10^–7 M) decreased the duration of AP. PEA restored the abolished electromechanical activity in high K⁺ (25 mM)-depolarized atria. These slow APs were unaffected by the -adrenergic blocker pindolol (5×10^–7 M) but were abolished by the H₁ antagonist mepyramine (10^–5 M). Neither dimaprit nor impromidine was able to induce slow AP. Our results suggest that in guinea-pig left atria histamine enhances the slow inward Ca²⁺ current mediated by H₁ receptors.     

Histamine activates suppressor cellsin vitro using a coculture technique

Aliquots of human blood mononuclear cells were culturedin vitro in the absence or presence of varying concentrations of histamine (10^–3–10^–8 M) for 24 hr, mitomycin treated, washed, and cocultured with autologous indicator cells that were then stimulated by PHA. The degree of suppression of thymidine uptake was measured in the presence of histamine-activated cells. The results demonstrated that histamine, in a dose-dependent fashion (10^–3 and 10^–4 M), significantly suppressed the PHA proliferative response. A minimum of 2 hr was required to activate cells by histamine to express suppressor function. While initial studies were carried out with a 1:1 suppressor:indicator cell ratio, it was found that maximum suppression resulted with lower rations (1:2 or 1:5) in some experiments, and was dependent somewhat on the concentration of histamine used. That the suppressor cells expressed histamine receptors was shown by the finding that histamine-Sepharose columns (but not control columns) depleted the histamine-reactive cells. In addition, Con A-reactive suppressor cells also adhered to histamine columns. Based on experiments with H₁ and H₂ antagonists and agonists, the histamine-responsive cell was apparently activated through its histamine type-2 receptor.     

Histamine induced elevation of cyclic AMP phosphodiesterase activity in human monocytes

We have previously reported histamine desensitization of human blood mononuclear leukocytes resulting in reduced cAMP responses to -adrenergic agonists, histamine and prostaglandin E₁. This heterologous desensitization occurred at low, micromolar histamine concentrations and was accompanied by elevation of cAMP-phosphodiesterase (PDE) activity in these cells. We have now investigated the activity of PDE in the lymphocyte and monocyte fractions of mononuclear leukocytes to determine the site of histamine effect.PDE activity per cell was higher in monocytes (0.075±0.070 units) than lymphocytes (0.026±0.08) units). Monocytes responded to 10^–6 M histamine stimulation with a much greater increase in PDE activity (0.354±0.1 units) than did lymphocytes (0.047±0.015 units). Histamine receptor studies, using thiazolylethylamine and chlorpheniramine as H₁-agonist and antagonist respectively and dimaprit and cimetidine as H₂-agonists and antagonists respectively, indicated that the histamine stimulation of PDE activity is mediated predominantly through H₁ histamine receptor in the monocytes and the H₂ receptor in the lymphocytes. Previously histamine had been thought to increase cyclic AMP by acting on H₂ receptors to activate adenylate cyclase. Our studies show that stimulation of H₁ or H₂ receptors by low histamine concentration can cause the opposite effect i.e. increased catabolism and a net reduction in cAMP levels. The localization of this effect predominantly to monocytes indicates a potentially important mechanism for histamine action on immune regulation.     

Histaminergic receptors modulate the coronary vascular response in isolated guinea pig hearts. Role of nitric oxide

Objective: The effects of histamine and of histamine receptor agonists and antagonists on the coronary outflow and on the generation of nitric oxide (NO) were evaluated on isolated guinea pig hearts.Methods: Isolated guinea pig hearts were perfused for 50 min in a Langendorff apparatus with histamine (10^–7– 10^–8 M), in the absence or in the presence of N^G-monome-thyl-L-arginine (L-NMMA, 10^–4 M), a NO synthase inhibitor and of triprolidine (310^–8 M) and cimetidine (10^–7 M), H₁ receptor and H₂ receptor antagonists, and with trifluoromethyl-phenylhistamine (TFMPH, 10^–7 M) and dimaprit (10^–7 M), H₁ and H₂ receptor agonists. The effects of (R)--methylhistamine (10^–7 M), a H₃ receptor agonist and of FUB 181 (10^–7 M), a H₃ receptor antagonist, were studied in the presence of bradykinin (10^–7 M).Results: Histamine increases the coronary outflow and the generation of NO in a concentration-dependent fashion. The effects were completely abolished by blocking NO-synthase (NOS) with L-NMMA (10 ^–4 M). The effects were also abolished by cimetidine (10 ^–7 M), H ₂ receptor antagonist, and only scarcely affected by triprolidine (310 ^–8 M), H ₁ receptor antagonist. The effects were reproduced by dimaprit (10 ^–7 M), H ₂ receptor agonist, and only scarcely by TFMPH (10 ^–7 M), a selective H ₁ receptor agonist. Bradykinin (10 ^–7 M) produces a sustained coronary dilation paralleled by a marked increase in the generation of NO; the effects were significantly reduced by L-NMMA. The stimulation of H ₃ receptors by (R)--methylhistamine (10 ^–7 M) significantly reduced both effects, which reverted to normal with FUB 181 (10 ^–7 M), an H ₃ receptor antagonist.Conclusion: These results suggest that, in isolated guinea pig hearts, histamine produces coronary dilation through an H ₂ /H ₃ -dependent mechanism involving the generation of nitric oxide.Received 3 December 2002; returned for revision 8 April 2003; accepted by M. Parnham 26 May 2003     

The effects of heavy metal ions (Cd2+, Hg2+, Pb2+, Bi3+) on histamine release from human adenoidal and cutaneous mast cells

The influence of lead (Pb[CH₃COO]₂), mercury (HgCl₂), cadmium (CdSO₄) and bismuth (BiO[ClO₄]) on the spontaneous and stimulated histamine release from human adenoidal and cutaneous mast cells was tested in the concentration range 10^–8–10^–4 M. Lead displayed a bell shaped dose-response relationship in adenoidal mast cells with a maximum at 10^–6 M whereas in cutaneous cells only the spontaneous release was slightly enhanced at 10^–4 M. Mercury induced a presumably toxic histamine release in adenoidal and cutaneous mast cells at 10^–4 M. Cadmium increased the histamine release in adenoidal cells at 10^–4 M but in cutaneous cells only the stimulated release (10^–8–10^–5 M) was affected. Bismuth inhibited the histamine release at 10^–4 M in the adenoidal mast cells only. In conclusion, human adenoidal and cutaneous mast cells are affected differently by metal ions.     

The influence of H1-, H2- and H3-receptors on the spontaneous and ConA induced histamine release from human adenoidal mast cells

The effects of the H₃-agonist R--methylhistamine (R--MeHA) and the H₃-antagonist thioperamide on the spontaneous and concanavalin A (ConA) induced histamine release from human mast cells were tested and compared with the effect of some H₁- and H₂-receptor active substances. R--MeHA (10^–9–10^–7 M) exerted no effect on histamine release whereas thioperamide increased the spontaneous release at 10^–6–10^–4 M but inhibited the ConA induced release in a narrow concentration range (10^–6–10^–5 M). This enhancement might be taken as an indication of the existence of H₃-receptor dependent autoregulation although presently other mechanism cannot be excluded.     

Binding of [3H]ICIA 5165, an H2-receptor antagonist to guinea pig gastric mucosa

ICIA 5165, 2-guanidino-4-[4-(2-cyano-3-methylguanidino)butyl] thiazole, a selective histamine H₂-receptor antagonist was radiolabelled with tritium to a specific activity of 50.8 Ci/mmoll for use in binding studies. Radiolabelling did not impair bioactivity.Binding characteristics of [³H]ICIA 5165 to guinea pig gastric mucosa were determined. Ligand binding was rapid, reaching equilibrium within five minutes at 0°C, reversible and saturable. Specific [³H]ICIA 5165 binding had an equilibrium dissociation constant of 1.29×10^–8 M, determined by Scatchard plot analysis, and of 1.02×10^–8 M, calculated from the ratio of the dissociation to association rate constants. A Hill number, n_H, of 1.02 was determined for the specific binding component. Specific binding of [³H]ICIA 5165 to gastric mucosal supernatant was not inhibited by methapyrilene, diphenhydramine, mepyramine, d-chlorpheniramine or I-chlorpheniramine (all at 10^–7 M), or by atropine or propranolol (both at 10^–6 M). Specific [³H]ICIA 5165 binding was inhibited in a concentration dependent manner by non-radioactive ICIA 5165 and tiotidine, as well as by a variety of other agents, with H₂ agonist or H₂ antagonist properties. In competition experiments, however, difficulties encountered in accurately defining the degree of specific binding indicate some reservation should be observed in interpreting these results.     

Effect of histamine on the hippocampal neurons in guinea-pigs

Histamine produced an excitatory effect on CA3 pyramidal cells in hippocampal slices of guinea-pigs. The amplitude of population spikes and EPSPs was augmented by histamine at concentrations of 10^–7 to 10^–6 M. Cimetidine but not pyrilamine prevented the stimulatory effect. The histamine effect was mimicked by dimaprit but not by 2-pyridylethylamine. It is concluded that histamine has a facilitatory influence on the hippocampal excitatory system via H₂ receptor.     

Structural requirements of imidazole compounds to be inhibitors or activators of histamine methyltransferase: Investigation of histamine analogues and H2-receptor antagonists

The influence of imidazole compounds (histamine analogues and H₂-receptor antagonists) and of the specific histamine H₂-receptor agonist dimaprit on histamine methyltransferase (HMT) from pig gastric mucosa was investigated.By their effect on HMT two groups of substituted imidazole compounds could be differentiated. Some were pure inhibitors of the enzyme, whereas others were inhibitors only in high concentrations (>10^–3 M) and activators of the enzyme in low concentrations. The strongest inhibitor of all the histamine analogues tested was 2-methylhistamine (I.D.₅₀=0.6×10^–4 M), whereas the strongest activation was exerted by 4-[(2-amino-ethylmercapto)-methyl]-5-methylimidazole (57% increase of enzymic activity at 10^–4 M concentration). From the group of histamine H₂-receptor antagonists only burimamide was an inhibitor (I.D.₅₀=1.6×10^–4 M) whereas metiamide and cimetidine belonged to the strongest activators of the enzyme (179% enzymic activity at 10^–4 M concentration of metiamide).The strongest activator of all the substances tested in this series of compounds, however, was the non-imidazole compound dimaprit, which increased enzymic activity by 86% in as small a concentration as 10^–5 M.For substituted imidazole ring systems an attempt is made to evaluate the structural requirements of the single compound to classify it as a pure inhibitor or a concentration-dependent inhibitor/activator of HMT.Dedicated to Prof. Dr Dr E. Werle () on the occasion of his 75th birthday.Supported by grant Lo 199/7 from Deutsche Forschungsgemeinschaft.Presented at the Sixth Annual Meeting of the European Branch of the Histamine Club, held in London, April 20–22, 1977.     

A radioimmunoassay for the histamine H2-antagonist,tiotidine

A specific and sensitive radioimmunoassay (RIA) for the histamine H₂-antagonist, tiotidine, has been developed. The assay is based upon competition of tiotidine with [³H]-tiotidine for antibody (Ab) obtained from immunized rabbits. The immunogen used was a glutaraldehyde coupled conjugate of the tiotidine derivative (ICI 147,655) and bovine serum albumin (BSA). Displacement of [³H]-tiotidine by unlabeled tiotidine was competitive over a concentration range of 10–1000 fmol. The Ab was 100-fold less sensitive to the pharmacologically inactive metabolite of tiotidine (ICI 129,585) and did not cross-react with histamine, cimetidine or phenylguanidine. In dogs given an i.v dose of tiotidine, plasma levels of the antagonist, as measured by the RIA, were correlated with inhibition of histamine-induced gastric acid secretion. Tiotidine (0.3 or 0.6 mol kg^–1) caused a dose-dependent and transient decrease in acid secretion at plasma concentrations of 10^–6 to 10^–8 M. Other potential analytical and research uses of H₂-antagonist radioimmunoassays are discussed.     

Characterization of an adenylate cyclase system sensitive to histamine H2-receptor excitation in cells from dog gastric mucosa

A method of preparing viable cells from dog gastric mucosa is described. Cyclic AMP in these cells is elevated by histamine and 4-methyl histamine but 2-methyl histamine is only a weak agonist. The effects on cyclic AMP levels are inhibited competitively by metiamide and burimamide which give apparent K_Bvalues of 3.5×10^–7 M and 2.3×10^–6 M, respectively. These values are similar to those reported for other histamine H₂-receptor systems. The H₁-receptor antagonists, mepyramine and chlorpheniramine, have no inhibitory effect on the histamine induced elevation of cyclic AMP: promethazine inhibits the system but not by a competitive mechanism. It is concluded that the histamine stimulated adenylate cyclase system is probably located in the parietal cell component.     

The effects of food additives and aflatoxin B1 on histamine release from human mast cells

The effects of the food additives tartrazine, biphenyl, sorbic acid and the mycotoxin contaminant aflatoxin B₁ were studied in mechanically isolated human adenoidal mast cells. Tartrazine inhibited the spontaneous histamine release in the concentration range of 10^–9 to 10^–5 M and the concanavalin A (Con A)-induced histamine release dose-dependently at 10^–11–10^–5 M [10.3%–31.6%]. Biphenyl [10^–9–10^–6 M] neither influenced the spontaneous nor the stimulated histamine release. Sorbic acid [10^–7–10^–4 M] slightly inhibited the Con A-induced release at the highest concentration tested. Aflatoxin B₁ [10^–10–10^–7 M] did not influence mediator release after a preincubation time of 5 min. Extension of the preincubation period inhibited the histamine release slightly. In summary, none of the tested substances enhanced histamine release from human adenoidal mast cells. Tartrazine even had an inhibitory effect.     

Effects of dimethindene maleate (Fenistil?) on histaminic, muscarinic and serotoninergic receptor systems

The effects of dimethindene maleate (DM) on histamine H₁, muscarinic and serotonin receptor systems were studied, using ligand binding studies (guinea-pig cerebral cortex membranes) and functional studies (guinea-pig ileum). DM was very potent at histamine H₁ receptors (K_i=1.5×10^–9 M, pA₂=9.33) using either method. DM showed a lower affinity for muscarinic receptors and a very low affinity for serotonin receptors. The only discrepancy found between receptor binding studies and functional tests concerned the activity of DM on muscarinic receptors where the K_i for binding studies using³H-pirenzepine was 6.4 ×10^–8 M, but the pA₂ for the carbachol-stimulated ileum was 6.7. As the guinea-pig ileum possesses a rather low level of M₁ muscarinic receptors when compared to M₂ and M₃ muscarinic receptors, the difference observed indicates that DM is more potent at M₁ than other muscarinic receptors.     

Characterization of vascular histamine H1-receptors: Multiple affinity states of aortic histamine H1-receptor

We have shown that [³H]mepyramine labels histamine H₁-receptor-binding sites in bovine aortic membranes. Further characterization of H₁-receptors in this tissue was done by the interaction of an unlabelled histamine receptor agonist or antagonist, with the radioantagonist [³H]mepyramine-binding sites. The competition-binding assays have uncovered differences in the characteristics of the agonist/receptor interaction not shared by antagonists. Agonists interact in the heterogeneous manner with the radioantagonist-labelled sites, showing shallow competition curves with then _H 0.50–0.72, whereas antagonists were devoid of this effect (steeper slopes of the inhibition curvesn _H1). The results suggest the presence in this tissue of multiple affinity states of histamine H₁-receptor, differentiated by high and low affinity for agonists and the same affinity for antagonists.     

Biochemical and pharmacological characterization of histamine-mediated up-regulation of human platelet serotonin uptake. Evidence for a subclass of histamine H2 receptors (H2h) highly sensitive to H2 receptor antagonists

The stimulatory effect of histamine: H (1.2 to 3-fold increase) on serotonin (5-HT) uptake by human platelets was observed after a 5 min incubation period in the presence of 2.5×10^–7 M histamine, followed by subsequent 5 min incubation of the platelets with 10^–7 M [³H] 5-HT. Methyl, ethyl and acetyl substituents in the side chain of H mimiked the stimulatory effect of H. In contrast, H analogs methylated at the position N-1 of the imidazole ring of H, as well as imidazole and histidine inhibited platelet 5-HT uptake. The cAMP-inducing agents forskolin and theophylline have no effect on 5-HT uptake when they are tested alone or in combinations with H. In contrast, the cGMP-inducing agent sodium nitroprusside (10^–7 M–10^–6 M) stimulated and potentiated H-mediated up-regulation of 5-HT uptake. Histamine H₂ receptor agonists and antagonists are more potent than drugs acting on H₁ receptors (H₂>H₁). However, the inhibition constants Ki are not consistent with those determined for typical H₁, H₂, H₃ receptors characterized in other tissues. This findings provide further evidence for the existence of multiple forms of H receptors and suggest the involvement of a subpopulation of H₂ receptors, highly sensitive to H₂ receptors antagonists (H_2h), mediating 5-HT uptake in human platelets.     

The source and action of histamine in the isolated guinea-pig gallbladder

We have investigated the effects of histamine on motility of the gallbladder and characterized the receptor types involved. Histamine and the histamine H₁-receptor agonist, 2-thiazolylethylamine (2-TEA) contracted the isolated guinea-pig gallbladder strip in a dose dependent manner. The contractile response to histamine was shifted to the right by the H₁-receptor antagonist, mepyramine. In pre-contracted gallbladder strips, the H₂-receptor agonist dimaprit reduced the tension generated in a dose dependent fashion. The histamine H₂-receptor antagonist, ranitidine shifted the histamine concentration effect curve to the left and attenuated the dose dependent relaxations elicited at high concentrations. The histamine H₃-receptor agonist, (R)--methylhistamine (RMHA) elicited dose dependent contraction of the tissue which was significantly inhibited in the presence of mepyramine. The effects of electrical field stimulation (EFS) on the strips were not significantly altered by the presence of RMHA (10^–10–10^–7 M) indicating little pre-synaptic H₃ activity in this tissue. Histamine immunoreactivity (IR) was detected in gallbladder whole mount preparations of the mucosa and the muscularis/serosa. The histamine IR appeared cell bound in cells of varying morphological characteristics but no IR was detected in nerve fibres or cell bodies (ganglia). Alcian blue staining was consistent with the distribution of histamine IR cells as mast cells. The results indicate that histamine is distributed in the guinea-pig gallbladder and it can regulate contractile activity via activation of H₁ and H₂ but not H₃ receptors.     

Effect of histamine on the T-cell colony formation of PHA-stimulated cells

The effect of histamine on T-cell colony formation was studied in human peripheral blood mononuclear cells. Histamine inhibited dose-dependently (10^–4–10^–6 M) the colony formation of PHA-stimulated T-cells. The inhibition was similar in normal controls and rheumatoid arthritis (RA) patients in spite of the fact that in RA the colony formation was significantly lower than in the normal controls. No increase of colony formation was observed at low concentrations (less than 10^–7 M). Impromidine was less effective than histamine, and pyridylethylamine (PEA) was inactive. Cimetidine counteracted the effect of histamine while chlorpheniramine did not. The results show that colony formation may be inhibited through H₂-receptors. This action may be of importance in cellular interactions in tissues with high local histamine concentrations.     

Modulation of the release of histamine and arachidonic acid metabolites from human basophils and mast cells by auranofin

In these experiments the effects of pharmacological concentrations of auranofin, a new absorbable gold compound, were assessed on the release of histamine and peptide leukotriene C₄ (LTC₄) from human basophils and lung mast cells. Auranofin, at pharmacological concentrations, inhibitedin vitro histamine and LTC₄ release from human basophils induced by anti-IgE. Inhibition began at about 3×10^–7 M and was maximum at 10^–5 M. We also evaluated the effect of auranofin on the release of histamine and LTC₄ induced by anti-IgE from mast cells purified from human lung. Auranofin (3×10^–7 to 10^–5 M) dose-dependently inhibited the release of histamine and LTC₄ from human lung mast cells. Thus pharmacological concentrations of auranofin cause dose-related inhibition of histamine release andde novo synthesis of LTC₄ by human basophils and lung mast cells.Supported in part by grants from the C.N.R. (83.00430.04, 84.01756.04) and (85.00491.04) and M.P.I. (Rome, Italy).