Regulation of membrane histamine H1-Receptor binding sites by guanine nucleotides,mono- and divalent cations

Agonist interaction with histamine H₁-receptor in [³H] mepyramine bovine aortic membranes labeled with [³H] mepyramine is selectively regulated by cations and guanine nucleotides. GTP and his nonhydrolisable analog Gpp(NH)p' markedly decrease histamine affinity for [³H] mepyramine binding sites. The effect of GTP is reversed in the presence of divalent cation, magnesium. Calcium and sodium ions have little effect on histamine binding whereas magnesium ions decrease the affinity of histamine for the radioantagonist binding sites about tenfold.GTP has little effect on [³H] mepyramine binding and the interaction of H₁-antagonist triprolidine with histamine H₁-receptors. The above results indicate that the effect of guanine nucleotides, mono and divalent cations involves the effect on membrane signal transducing mechanism probably GTP-binding protein(s) cation regulatory site(s) rather than receptor binding site directly.     

Histaminergic H1-receptors in smooth muscle and endothelium of bovine thoracic aorta

The vascular endothelium modulates relaxation and contraction of blood vessels. Since endothelial cells respond to a variety of vasoactive substances, it was suggested that specific cell membrane receptors exist on the endothelial cells which are responsible for the modulatory role of the endothelium on the blood vessels. We therefore investigated the localization and binding characteristics of histaminergic H₁-receptors in the vascular model system of the bovine thoracic aorta. Our earlier binding experiments showed that histaminergic H₁-receptor binding sites labelled with [³H]mepyramine are present on the vascular smooth muscle membranes of this tissue. In addition a small number of specific H₁-receptor binding sites also exist on the endothelial cells of this tissue with the following binding characteristics: B_max=34.6 fmol [³H]mepyramine/mg protein, K_D=2.13 nM. [³H]mepyramine binding is more effectively inhibited by H₁- than H₂-receptor agonists and antagonists. These results provide evidence for the existence of endothelial histaminergic H₁-receptor binding sites in addition to vascular smooth muscle H₁-receptors in the bovine thoracic aorta.     

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.     

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.     

H1 and H2 histamine receptors in human mammary carcinomas

We previously reported the presence of H₁ and H₂ histamine receptors in an experimental mammary carcinoma induced in rats. Experiments carried outin vivo andin vitro indicate that by acting on these receptors, histamine and antagonists modulate tumor growth. In the present study, binding experiments were performed with human breast cancer biopsies. All the tumors examined exhibited specific binding sites for histamine. Some of them showed the high and low affinity double site previously characterized in the experimental carcinoma. The high affinity site Kd=18±6 nM, 50±33 fm/mg, exhibits H₂ binding characteristics while the low affinity one, Kd=54±22 nM and 217±173 fm/mg, corresponds to an H₁ receptor, Approximately 30% of the tumors studied were negative for H₂ receptors while all of them showed specific binding for [³H]-mepyramine. These data suggest that histamine may regulate cell growth in a high proportion of human mammary carcinomas, offering possibilities for new therapeutic alternatives.     

Desensitization by histamine of H2 receptor activity in HGT-1 human cancerous gastric cells

Histamine produced a time-dependent (half-life: 20 min at 37°C), temperature-dependent (no effect at 20°C) and homologous desensitization of histamine H₂ receptor activity (H₂ R) in HGT-1 cells. Maximal and half-maximal desensitization were respectively observed at 10^–5 and 2×10^–7 M histamine. Decline of responsiveness in intact cells was related to a remarkable loss in histamine efficacy (from 15- to 2-fold stimulation in control and treated cells). The affinity of the H₂R for histamine (EC₅₀=10^–5 M) did not change during desensitization. Paradoxically, histamine treatment is associated with increased [³H] histamine binding capacity in intact HGT-1 cells, and no change in H₂ receptor antagonist binding ([³H]-tiodine and [³H]-SKF 93479). Desensitization process was preferentially mimicked by H₂ receptor agonists (impromidine > histamine > AET > PEA) and preferentially reversed by simultaneous addition of H₂ receptor antagonists (cimetidine > DPH). We suggest that the desensitization of H₂R activity by histamine presented here may be involved in the pathophysiological regulation and pharmacological control of gastric cell function in man.     

The effect of group selective reagentsN-ethylmaleimide and dithiothreitol on histamine H1-receptor binding sites in the vascular smooth muscle membranes

The chemical nature of the histamine H₁-receptors of beef aortic membranes has been elucidated by introducing two group selective reagents in the [³H]-mepyramine binding studies: dithiothreitol (DTT), a protein-disulphide group reducing reagent, andN-ethylmaleimide (NEM), a proteinthiol group alkylating agent.In the binding experiments, NEM independently inhibits [³H]-mepyramine binding. The inhibition is time and concentration dependent. DTT on the other hand potentiates the binding of the radioligand to its receptor and changes the affinity of histamine in competing for [³H]-mepyramine binding site. In the DTT-pretreated membranes (100 M), histamine shows a higher affinity for [³H]-mepyramine binding (K _i 0.35 M) than in the untreated membranes (K _i 3.7 M). Comparison of the pharmacological studies on the DTT-treated rabbit aortic strips and above binding studies, revealed a good correlation between the changes in the affinity of histamine for its receptor, when DTT was present. The results suggest an important role of the S-S and SH groups in the function of aortic histamine H₁-receptor.     

Binding of [3H]cimetidine to rat brain tissue

Binding of [³H]cimetidine to rat brain tissue was investigated, and a saturable binding with dissociation constant 0.22±0.05 M found. This binding is inhibited by a range of imidazole-derived histamine H₂-receptor antagonists, but not by a number of non-imidazole H₂-receptor antagonists. It is concluded that the [³H]cimetidine binding site in rat brain tissue that is labelled in these experiments is not the histamine H₂-receptor.     

Evidence for H2-receptor-mediated inhibition of histamine release from isolated rat mast cells

Impromidine, an H₂-receptor agonist, inhibited the release of histamine from isolated purified rat mast cells evoked by compound 48/80 and acetylcholine. Pyridilethyl-amine (PEA), an H₁-receptor agonist, on the other hand, was only slightly effective at very high concentrations. The inhibitory effect of impromidine was blocked by preincubating the cells with cimetidine, but not by chlorpheniramine.The existence of an H₂-mediated inhibitory feed-back regulation of histamine release was also suggested by the demonstration of specific binding sites for [³H]-cimetidine in rat mast cell membranes.     

Histamine H-1 binding site on human polymorphonuclear leukocytes

Neutrophilic polymorphonuclear leukocytes (PMN) infiltrate the sites of allergic reactions and may respond to histamine, one of the major mediators of allergy. In order to characterize histamine interactions with PMN, the binding of [³H]pyrilamine was studied. Human PMNs bind [³H]pyrilamine in a specific, saturable, and reversible fashion and demonstrate specificity (H-1 antagonists > histamine > H-2 antagonists) for the competitive binding agents studied. Human PMNs have a homogeneous population of H-1 receptors of moderate affinity (K _d=52 nM) in large number (265 × 10³/cell) which do not demonstrate cooperativity. Thus PMNs attracted to sites of allergic inflammation have H-1 binding sites which may respond to histamine stimulation.     

Sexual dimorphism modulates brain histamine functions at multiple sites

Sexual dimorphism has been demonstrated in rat brain and the ovarian steroids affect H₁ and H₂ histaminergic binding sites as demonstrated with [³H]-mepyramine and [³H]-histamine, respectively. The evaluation of histamine release, related to the presynaptic H₃ receptors, from cortical slice preparations reveals hyposensitive releasing activity in the female rat compared to the male, both after KCl-induced histamine release and after inhibition of histamine release by (R)-methylhistamine (H₃ selective agonist). Therefore, we may suggest a global hyposensitivity of the histaminergic neural system in female rats under the modulation of ovarian sexual steroids.     

The distribution of histamine H1-receptors in the rat brain: An autoradiographic study

The localization of histamine H₁-receptors in the rat brain was studied at the light microscopic level by quantitative autoradiography. Receptors were labeled in vitro with [³H]mepyramine. The autoradiograms revealed a widespread distribution of these receptors throughout the brain. Areas with high receptor concentrations are: the bed nucleus of the stria terminalis, polymorphic layer of the hilus of the area dentata, ventromedial nuclei of the hypothalamus, pontine nuclei and other nuclei in the pons, the nucleus tractus solitarii and the dorsal motor nucleus of the vagus nerve. Possible relationships of the distributions of histamine H₁-receptors with (a) specific anatomical systems, (b) the known distribution of histaminergic terminals and (c) the central pharmacological effects of histamine and antihistaminics are discussed.     

Influence of histamine H3-antagonists on human leukocytes

To determine the role of the histamine H₃-receptor on basophils, different specific H₃-antagonists were investigated. Incubation of washed leukocytes with N-acylated histamine-derivatives (N-ahd) induced elevated histamine levels. This process turned out to be dependent on dose, time and temperature, but independent of Ca²⁺ and Mg²⁺ ions. IgE-mediated histamine release was not modulated. [³H]-l-histidine was not decarboxylated into [³H]-histamine in spite of the observed histamine increase. Highly purified basophils did not show any histamine elevation but purified neutrophils and eosinophils were found to have increased histamine levels even after disintegration and subsequent incubation with N-ahd. It seems that the increased histamine levels result from the cleavage of the applied histamine amides. Other potent H₃-antagonists (e.g. thioperamide) neither produced increased histamine levels nor influenced IgE-mediated release from basophil leukocytes. The existence of H₃-receptors on human basophils therefore seems unlikely.This work was supported by Grant No. KI 622/1-1 from the Deutsche Forschungsgemeinschaft, Bonn, FRG.     

Unexpected partial H1-receptor agonism of imidazole-type histamine H3-receptor antagonists lacking a basic side chain

Objective and design:The putative partial H₁-receptor agonism of some H₃-receptor antagonists belonging to the proxifan series was characterized in a functional in-vitro assay using guinea-pig ileum. Methods:Whole segments of guinea-pig ileum were mounted in Tyrode’s solution under isotonic conditions in the presence of atropine (10^-7 M) and were cumulatively treated with histamine as an internal reference. After washout, the putative H₁-receptor agonists were added cumulatively to determine agonist potency (pEC₅₀) and intrinsic activity (E_max) relative to histamine. Maximal or supramaximal concentrations of partial agonists, or sufficient concentrations of H₁-receptor antagonists were incubated for 3–15 min prior to construction of a second concentration-effect curve to histamine in order to calculate partial agonist or antagonist affinity for the H₁ receptor (pK_P or pA₂ value, respectively). Results:Several analogues of FUB 372 displayed low H₁-receptor affinities (pA₂ or pKP 4.2–5.5) except for a methyl benzoate derivative (pA₂ = 6.81, Schild plot slope unity). FUB 372, four ortho-substituted derivatives (R = F, CH₃, OCH₃, CF₃), and ciproxifan were weak contractile agents (E _max 9–38%, pEC₅₀ 4.73–5.68, histamine: 6.70) susceptible to antagonism by the H₁-antihistaminergic drug mepyramine (2·10^-9–10^-7 M). Agonist potency and H₁-receptor affinity of these compounds did not correlate with the data of a set of H₁-histaminergic 2-phenylhistamines bearing the same substituents. Conclusions:A specific subset of proxifans related to FUB 372 and ciproxifan represent a unique type of H₁-receptor agonists lacking a basic side chain.     

Labelling of non-H1-receptor binding sites by [3H]-mepyramine on the rat liver plasma membrane

In the present study we characterized [³H]-mepyramine binding to rat liver plasma membranes. Binding of [³H]-mepyramine proved to be of high affinity (K _d =7.7±0.4 nM) and saturable, resulting in a B_max-value of 70.4±9.5 pmol/mg protein. However, displacement studies revealed that this binding site was different from other H₁-receptor systems. The two stereoisomers of chlorpheniramine were rather ineffective in displacing [³H]-mepyramine and showed a stercospecificity in favour of thel-isomer. Also several H₁-receptor agonists were not potent in displacing [³H]-mepyramine from rat liver plasma membranes. Morcover, the histamine metabolite imidazole-4-acetic acid was about as potent as the H₁-agonists, whereas imidazole was even more potent. These data strongly suggest that [³H]-mepyramine labels a non-H₁-receptor binding site on the rat liver plasma membrane.     

A postsynaptic inhibitory histamine H2-receptor in the mouse isolated vas deferens

The effect of histamine on adrenergic neurotransmission in the mouse isolated vas deferens preparation was investigated. Concentrations of histamine ranging from 0.2 to 650 M depressed, in a dose-related manner, not only the contractile response elicited by field stimulation but also the response caused by the addition of exogenous noradrenaline and acetylcholine. However, the release of [³H]-NA evoked by field stimulation or by high K⁺ remained unchanged in the presence of these concentrations of histamine. The inhibitory effect of histamine on the contractile responses caused by various stimuli was reduced or completely antagonized by cimetidine, a histamine H₂-receptor antagonist but not by mepyramine, a conventional antihistamine. The inhibitory effect of histamine was found to be inversely proportional to both the Ca²⁺ concentration in the bathing medium and to the frequency of field stimulation. Further, the inhibitory effect of histamine was markedly reduced when Mg²⁺ was omitted from the bathing medium. It is concluded that the mouse vas deferens preparation contains a post-junctional inhibitory H₂-receptor. The stimulation of H₂-receptors by histamine inhibits the contractile response of the vas deferens, possibly by decreasing the availability of Ca²⁺ required for contraction by depressing the influx of Ca²⁺.     

H1-receptor dependent increase in platelet aggregation is mediated by intracellular calcium

The influence of histamine on human platelet function was studied by measuring the uptake of [³H]-histamine and by evaluating the effect of histamine on Ca²⁺ influx and on intracellular Ca²⁺ levels. The uptake of histamine by platelets is significantly reduced by mepyramine and unaffected by cimetidine. Histamine was found to increase both the [⁴⁵Ca]²⁺ uptake by resting and stimulated platelets, and the intracellular Ca²⁺ levels in Fura 2-AM loaded platelets. H₁-antihistaminics significantly reduced these processes, suggesting a role for histamine, which in platelets promotes aggregation by acting through H₁-receptors modulating intracellular Ca²⁺ levels.     

Evidence for heterogenous glycine domains but conserved multiple states of the excitatory amino acid recognition site of the NMDA receptor: regional binding studies with [3H]glycine and [3H]L-glutamate

Summary The possible heterogeneity of the agonist and glycine sites of the N-methyl-D-aspartate (NMDA) receptor-complex was examined using receptor binding techniques. Binding of [³H]L-glutamate ([³H]GLU) and [³H]glycine to synaptic membranes of cerebral and cerebellar cortices, and membranes of a granule cell preparation of rat cerebellum, was characterized. [³H]Glycine always labelled a single population of sites; densities of binding sites (B_max) in cortical, cerebellar and granule membranes were 3.1, 0.87 and 3.6 pmol/mg protein, respectively. Dissociation constants (K_d) in the same three preparations were 0.13, 0.31 and 1.9 M, respectively. In competition studies, D-cycloserine, but not D-serine and 7-chlorokynurenate, showed varying potency between the membrane preparations, and analysis of variance (ANOVA) revealed a significant interaction between ligands and membrane fractions. Binding of [³H]GLU was saturable and to a single population of sites: K_d 0.5–0.9 M and B_max 3.2–3.6 pmol/mg protein. In all three membrane preparations the rank order of potency of NMDA agonists as inhibitors of the binding of [³H]GLU was always L-aspartate>L-cysteate>L-cysteinesulphinate>L-serine-O-sulphate>ibotenate>L-homocysteate. NMDA, quinolinate and competitive NMDA antagonists were only weak inhibitors of the binding of [³H]GLU and never fully inhibited specific binding. Other subtype-selective excitatory amino acids were very weak or ineffective inhibitors of binding. Binding of NMDA agonists was better described by a two site model whereby the proportion of high affinity sites did not vary significantly across the three membrane preparations. Although the binding of [³H]GLU was relatively insensitive to NMDA itself and competitive NMDA antagonists, binding may be to a recognition site for NMDA-like agonists, since they fully inhibited specific binding. This excitatory amino acid recognition for NMDA agonists was conserved in the three membrane preparations. In cortical and granule membranes the B_max values for the binding of [³H]GLU and [³H]glycine had a stoichiometry of 1:1, whilst in cerebellar synaptic membranes this ratio was 4:1. Receptor autoradiography of NMDA-related [³H]GLU and [³H]glycine binding in tissue sections failed to reveal any differential labelling patterns in cerebral cortex and cerebellum. In the cerebellum, densities of silver grains found with both [³H]ligands were concentrated in the granule cell layer relative to the molecular layer, but the differences detected in membrane binding studies were not observed in cerebellum. Our findings suggest the existence of three types of heterogeneity for the glycine domain of the NMDA receptor: (1) differing affinities for glycine, (2) differing pharmacological profiles, and (3) differing stoichiometry in relation to the putative NMDA-like agonist site. Our evidence supports an hypothesis for the existence of multiple glycine domains which might differentially modulate NMDA-mediated neurotransmission.     

Histamine induces interleukin-6 expression in the human synovial sarcoma cell line (SW982) through the H1 receptor

Methods The effect of histamine on inositol phosphate generation and interleukin-6 (IL-6) release from the synovial sarcoma cell line SW982 was investigated. Results SW982 cells express functional H₁ and H₂ receptors. The H₁ receptor antagonist [³H]-mepyramine binds to membranes from SW982 cells with high affinity and the binding was potently blocked by H₁ antagonists. Histamine potently stimulated phosphoinositide (PI) hydrolysis and Ca²⁺ mobilization with EC₅₀ of 4.0 ± 0.8 μM and 1.3 ± 0.6 μM respectively and these activities were blocked by the H₁ selective antagonist mepyramine. Histamine (EC₅₀ = 1.8 ± 1.1 μM) stimulated the release of IL-6 that was attenuated by selective H₁ antagonists. The PKC inhibitor, GF1090203X, blocked the histamine stimulated IL-6 release. The H₂ selective antagonist, cimetidine, had no significant effect on histamine-induced PI turnover, Ca²⁺ mobilization and IL-6 release. Conclusion We conclude that histamine stimulates IL-6 release from SW982 cells by binding to the H₁ receptor and this is coupled to the PI/PKC signal transduction pathway. Development of an H₁ antagonist that inhibits the release of IL-6 from synoviocytes may be beneficial for the treatment of inflammatory joint disease. Received 15 September 2005; returned for revision 12 December 2005; accepted by A. Falus 13 April 2006     

Kinetic and pharmacological properties of [3H]-histamine transport into cultured type 1 astrocytes from neonatal rats

Objective and design:  Astrocytes actively participate in the inactivation of neurotransmitters. In this work we elucidated the contribution of astrocytes in clearance of histamine, a process which has not yet been fully clarified. Methods:  The characteristics of [³H]-histamine uptake were determined in cultured neonatal rat type 1 astrocytes and histamine-N-methyl-transferase expression was determined using RT-PCR. Results:  These cells transport [³H]-histamine in a time- and concentration-dependent manner. The histamine clearance by astrocytes was described by a mathematical model including two processes: electrodiffusion and active transport. A further analysis of kinetic parameters of a carrier-operated transport revealed a single transport system with Michaelis constant (K_m) of 3.5 ± 0.8 μM and a maximal uptake rate (V_max) of 7.9 ± 0.3 pmol/mg protein/min. From drugs tested amitriptyline, desipramine, mepyramine and cimetidine significantly decreased [³H]-histamine uptake. Taken-up histamine could be metabolically degraded in cultured astrocytes, since they express mRNA for enzyme histamine-N-methyltransferase. Conclusions:  Astrocytes participate in the clearance of extracellular histamine by electrodiffusion and active transport by a yet not identified carrier. Taken up histamine can be converted to tele-methylhistamine within astrocytes thus indicating the involvement of astrocytes not only in clearance but also in the inactivation of histamine. Received 24 November 2007; returned for revision 4 January 2008; received from final revision 22 May 2008; accepted by A. Falus 3 November 2008