Characterization of histamine H-1 receptors on human mononuclear cells

Histamine H-1 receptors on peripheral human mononuclear cells were characterized by radioligand binding of the H-1 receptor antagonist [3H]pyrilamine to lymphocyte-rich preparations. Simultaneous computerized analyses of sixteen separate equilibrium-binding assays indicated the presence of two distinct classes of binding sites with dissociation constants (Kds) of 4 +/- 1 nM and 55 +/- 9 microM and binding capacities of 21 +/- 7 fmol and 117 +/- 15 pmol/million cells, respectively. Competition binding curves for displacement of [3H]pyrilamine binding by histamine receptor agonists and antagonists also indicated the presence of multiple binding sites for the H-1 receptor. Further, the ED50 values determined for histamine receptor agonists and antagonists were entirely consistent with the expected rank order of potency for interactions with H-1 receptors. Thus, human mononuclear cells have a large number of H-1 receptors that exhibit two distinct binding sites, and the Kds for these sites are within the range of histamine concentrations achieved either in physiologic states or after mast cell (or basophil) degranulation.     

Autoradiographic localization of binding sites for [3H]histamine and H1- and H2-antagonists on cultured neurones and glial cells

By means of autoradiography we have studied the cellular localization of binding of [3H]histamine and H1- and H2-antagonists in explant cultures of rat cerebellum, brain stem and spinal cord. In brain stem and spinal cord cultures, a relatively great number of neurones revealed binding sites for [3H]histamine and to a lesser extent also for the H1-antagonist [3H]pyrilamine and for the H2-antagonist [3H]tiotidine. In contrast, only a small number of labelled neurones was found in cerebellar cultures. The intensity of labelling was usually much stronger for [3H]histamine than for its antagonists, suggesting that binding sites for histamine might reflect both H1- and H2-receptors. Glial cells also showed binding sites for [3H]histamine and the H1- and H2-antagonists, the number of labelled astrocytes by these radioligands was, however, smaller than that observed with [3H]noradrenaline and alpha- and beta-adrenergic antagonists. It is suggested that in addition to alpha- and beta-adrenoceptors, glial cells also possess receptors for histamine.     

The interaction of azelastine with human lung histamine H1, beta, and muscarinic receptor-binding sites

Azelastine has previously been demonstrated to inhibit histamine release, to antagonize histamine-mediated responses, and to be a bronchodilator. To determine the mechanism by which azelastine has antihistaminic and bronchodilatory actions, we studied its interaction with relevant human lung receptors. We performed competitive radioligand binding assays and determined the affinity of azelastine for [3H]pyrilamine (histamine H1), [125I]pindolol (beta), and [3H]quinuclidinyl benzilate (muscarinic) binding sites. Azelastine had a relatively high affinity for histamine H1 receptors with IC50 values consistently as low or lower than values measured for other antihistamines. In contrast, azelastine had a very low affinity for both beta-receptors and muscarinic receptors with IC50 values greater than 2 logs greater than those determined for beta-agonists and muscarinic antagonists, respectively. Thus, bronchodilatory activity of azelastine does not appear to result from either beta-agonist or muscarinic-antagonist properties. Azelastine does, however, have the ability to inhibit the release of histamine and to bind to histamine H1 receptors, thereby effectively antagonizing histamine H1 receptor-mediated responses in the lung. These characteristics make azelastine a potentially very useful drug to treat allergic responses in the respiratory tract.     

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.     

Characterization and autoradiographic localization of histamine H1 receptors in human nasal turbinates.

To examine the localization of histamine H1 receptors (H1R) in human nasal mucosa, the autoradiographic distribution of H1R was studied in human nasal inferior turbinates. Cryostat sections were incubated with various concentration of [3H]pyrilamine in saturation-binding studies and with 1 nmol/L of [3H]pyrilamine for autoradiography. Nonspecific binding was determined by adding 2 mumol/L of pyrilamine. Scatchard analysis demonstrated high-affinity binding sites with a maximum binding capacity of H1R of 193 +/- 46 fmol/mg of protein, and dissociation constant was 0.6 +/- 0.1 nmol/L. Autoradiograms indicated H1R exist exclusively on the endothelium of vessels. No specific labeling could be observed in the submucosal glands or epithelium. These results extend and support our previous finding that histamine directly causes vascular permeability through H1R and stimulates nasal glandular secretion indirectly through reflexes.     

Specific binding of [3H]mepyramine to histamine H1-receptors in vascular smooth muscle membranes

The antagonist-sensitive binding of [³H]mepyramine to beef aortic membranes was as expected for binding to histamine H₁-receptors. [³H]mepyramine binds rapidly and in saturable fashion to the specific receptor sites, specific binding reaching equilibrium in 3 min at 37°CScatchard's analysis of the binding data gave a dissociation constant of 3.0 nM for the radioligand-receptor complex and maximal number of binding sites: 31 fmol/mg protein. In the competition studies histamine H₁-antagonists are more potent inhibitors of radioligand binding than H₂-antagonist. They inhibit [³H]mepyramine binding in the following order: mepyramine >triprolidine     

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.     

Degranulation of polymorphonuclear leukocytes is induced by multivalent cross-linking of wheat germ agglutinin binding site(S) on cell membrane

Polymorphonuclear leukocyte (PMN) surface membrane glycoproteins very likely are involved in the phenomenon of stimulus-response coupling. Previously, we have shown that subagglutinating concentrations of the plant lectin, wheat germ agglutinin (WGA) specifically and irreversibly inhibitedN-formyl-methionyl-leucyl-phenyl-alanine (FMLP)-mediated PMN chemotaxis. WGA did not affect the binding of [³H]FMLP to its receptor on the PMN plasma membrane. We have examined the possibility that cross-linking of WGA binding sites may elicit PMN degranulation. We have found that multivalent, but not bivalent, cross-linking of WGA bound to PMNs elicits release of lysosomal constituents. This phenomenon was specific for WGA since it did not occur when concanavalin A (Con A), instead of WGA, was used. It is intriguing to speculate that WGA may attach to a physiologic receptor for FMLP on the PMN membrane and that redistribution (cross-iinking) of this receptor might be an early event in the activation of PMNs by FMLP.     

Platelet activating factor as a proinflammatory mediator in acetic-induced colitis in the rat

The nature of histamine receptors in peripheral tissues is still controversial. However, evidence of heterogeneous classes of binding sites for [³H]-mepyramine are reported in the literature. The aim of our study was, therefore, to investigate the nature of this heterogeneity by comparing [³H]-mepyramine study was, therefore, to investigate the nature of this heterogeneity by comparing [³H]-mepyramine binding in a central tissue (cerebellum) and in a peripheral tissue (lung) obtained from guinea pigs and to assess its dependence upon the temperature of incubation. The results revealed that the [³H]-mepyramine interaction in both tissues is temperature-dependent. At 25°C, the interaction between [³H]-mepyramine and the receptors was biphasic in the lung while only a single class of binding site was found in the cerebellum. At 0°C, [³H]-mepyramine interacted with three binding sites in the lung and two in the cerebellum. The behaviour of the reference compounds (clemastine, promethazine and histamine) also supported this temperature-dependence. Moreover, two new compounds (DF 11062 and DF 11113), synthesized in our laboratories and endowed with antihistamine activity, can differentiate between the low affinity site seen at 25°C in the lung and that seen in the cerebellum at 0°C.     

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.     

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.     

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.     

Specific binding of 1-[3H]-O-alkyl-2-acetyl-sn-glyceryl-3-phosphoryl choline (platelet-activating factor,PAF) by human polymorphonuclear neutrophils

1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF) is a potent activator of polymorphonuclear neutrophil (PMN) aggregation, exocytosis and chemotaxis. Specific desensitization of PMN to PAF suggests a receptor-mediated interaction. The binding of 1-[³H]-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (³H-PAF) to human PMN and platelets was analysed and compared. Binding was saturable at 0.6 nM and 0.1 nM for 2×10⁶ PMN and 5×10⁷ platelets, respectively. The time course of binding at 22°C and 37°C for both cell types reached the plateau at 2 min. The averageK _d was 45.0±1.7 nM (mean ±1 SD of 4 experiments) for PMN (27.391±1381 sites for PMN) and 20.1±6.3 nM (4 experiments) for platelets (1577±461 sites for platelets). The Scatchard plot analysis revealed two distinct binding sites both on PMN and platelets: a high affinity binding site and a non-saturable binding site.This work was supported by C.N.R. Rome grant no. 81.00089.04.     

Evidence for a nicotinic component to the actions of acetylcholine in cat visual cortex

Summary Radioligand binding assays, receptor autoradiography and iontophoresis have been used to look for evidence of a nicotinic component to the actions of acetylcholine in cat visual cortex. [³H]Nicotine bound to a uniform population of high affinity binding sites in cat primary visual cortex. This binding was inhibited by nicotine agonists and antagonists but not muscarinic antagonists. The concentration of nicotinic binding sites was about 10% of that of muscarinic binding sites measured with [³H]N-methylscopolamine. The muscarinic sites were resolved into M1 and M2 subtypes. Quantitative receptor autoradiography showed that there were muscarinic sites in all layers, although they were least abundant in layer IV of area 17. In contrast, the nicotinic sites were most concentrated in layer IV in area 17. The concentration of this labelling was reduced at the 17/18 border and also at the 18/19 border. Layer I of the cingulate and suprasylvian gyri were also labelled. Electrolytic lesions of the lateral geniculate nucleus (LGN) led to a loss of nicotinic binding sites in layer IV of area 17, indicating that these sites are most likely located on the LGN terminals. Iontophoresis of mecamylamine, a nicotinic antagonist, decreased evoked responses in visual cortex, providing evidence that the [³H]nicotine binding sites are functional receptors and suggesting that the release of acetylcholine onto these receptors on the LGN terminals facilitates the input of visual information into visual cortex.     

Regulation of ERK2 phosphorylation by histamine in splenocytes

Histamine is implicated in allergic disease and asthma and ERK1/2 is involved in allergic inflammation including Th2 differentiation and proliferation. This study was designed to study the effects of histamine on ERK1/2 phosphorylation in splenocytes. C57/BL6 splenocytes were treated with different concentrations of histamine (10^?4 to 10^?11 M). Histamine (10^?4 M) increased ERK2 phosphorylation. There was, however, no significant effect seen at other concentrations (10^?11 to 10^?6 M). Surprisingly, H1 receptor agonist β-histine (10^?5 M), H2 agonist amthamine (10^?5 M), H3 agonist methimepip (10^?6 M), and H4 agonist 4-methyl histamine (10^?6 M), all increased ERK2 phosphorylation. H1R antagonist pyrilamine (10^?6 M), H2R antagonist ranitidine (10^?5 M), H3/H4R antagonist thioperamide (10^?6 M), and H3R antagonist clobenpropit (10^?5 M) inhibited histamine-mediated ERK2 phosphorylation suggesting that all four histamine receptor subtypes played some role in this phosphorylation. Because tumor necrosis factor-α (TNF-α) causes phosphorylation of ERK1/2, we investigated whether histamine acted via secretion of TNF-α to affect ERK1/2 phosphorylation. As a consequence, TNF-α knockout mice were used and we found that there was inhibition of ERK1 and ERK2 phosphorylation by H2, H3, and H4 agonists. This was in contrast to the wild-type splenocytes where histamine augmented the phosphorylation of ERK2 via H2, H3, and H4 receptors. In TNF-α knockout mice histamine did not affect the phosphorylation of ERK2 via H1 receptors. The results suggested that histamine indirectly caused the ERK2 phosphorylation via its effects on the secretion of TNF-α and these effects were mediated via H1, H2, H3, and H4 receptors.     

Changes in histamine in the rat striatum following local injection of kainic acid

The influence of local administration of kainic acid on the metabolism of histamine in the striatum of the rat has been investigated. Intrastriatal injection of kainic acid (1 μg) resulted in a more than 100% increase of the striatal content of histamine, which persisted from two days after the lesion up to ten weeks. Using two independent methods this accumulation of histamine has been found to be associated with a neuronal compartment of the striatum: (a) the concentration of histamine in synaptosomes prepared from kainic acid lesioned striatum was higher than in synaptosomes derived from the untreated (contralateral) striatum; (b) electrolytic lesions of the medial forebrain bundle, in animals whose striatum had been lesioned with kainic acid, abolished the increase in histamine levels induced by the neurotoxin. Since the rate of disappearance of histamine from the tissue after inhibition of its synthesis by α-fluoromethylhistidine (20 mg/kg, i.p.) was reduced in the lesioned striatum, the kainic acid induced increase in striatal histamine level may be due to a decrease of histamine turnover.Specific binding of [³H]histamine and [³H]cimetidine was measured in the lesioned striatum, revealing a 40% and 20% decrease in binding sites, respectively, when compared with the untreated striatum; the affinity for either ligand was not changed in the lesioned tissue.The data demonstrate considerable neurochemical changes involving the neuronal histamine system in the striatum after destroying the interneurons and output neurons of this region with the convulsive neurotoxin kainic acid.     

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.     

Localization of the dopamine uptake complex using [H]N-[1-(2-benzo(b)thiophenyl)cyclohexyl]piperidine ([H]BTCP) in rat brain

[³H]N-[1-(2-Benzo(b)thiophenyl)cyclohexyl]piperidine ([³H]BTCP) is a novel phencyclidine derivative with considerable selectivity for dopamine uptake sites. [³H]BTCP was used to label dopamine uptake sites in vitro, in rat brain, and the regions containing these sites were visualized with an autoradiographic technique. The binding was found to be highest in the striatum, where > 90% of binding was specific. Furthermore, 6-hydroxydopamine lesions of the nigrostriatal pathway obliterated striatal [³H]BTCP binding ipsilaterally, whereas ibotenic acid injection into the caudate-putamen failed to significantly reduce [³H]BTCP binding in that structure. These results indicate that [³H]BTCP labels dopamine uptake sites in mammalian brain and that it can be employed for autoradiographic studies of this transport complex.     

Deficiency in C3b receptors on neutrophils of patients with chronic granulomatous disease and hyperimmunoglobulin-E recurrent infection (Job's) syndrome

C3b receptor (CR1) expression by neutrophils (PMNs) and erythro-cytes (Es) from patients with chronic granulomatous disease (CGD) or with hyper-IgE, frequent infection (Job's) syndrome was compared with that of control subjects. The control subjects consisted of one group of patients with infections and a second group of normal, healthy individuals. Three quantitative assays were used: rosette formation with C3b-coated cellular intermediates (EAC43b), binding of radiolabeled monoclonal anti-CRl ([¹²⁵I]anti-CRl) to PMN surfaces, and binding of the antibody to nonidet P-40 (NP-40) extracts of PMNs and Es in an immunoradiometric assay. Rosette formation by the PMNs of five male CGD patients was about 50% of that of paired normal control subjects, whereas the rosette formation of three female CGD patients was similar to that of the control subjects. Surface binding of [¹²⁵I]anti-CRl to PMNs of 10 CGD patients was about half that of the normal subjects (mean percent binding was 2.33% for the CGD patients vs. 3.86% for the normal subjects, giving a difference of -1.53 ± 0.22%,P < 0,001 by the paired-samplet test). The degree of PMN binding was similarly low for both the male and the female CGD patients. Conversely, the binding of anti-CRl to the PMNs of 11 infected control patients appeared to be similar to that of the normal subjects (4.51% for the patient vs. 4.21% for the paired normal subjects). The infected control group originally included four Job's syndrome patients, and when this subgroup was analyzed separately, their PMNs were shown to bind significantly less anti-CRl than did the PMNs of the normal subjects (P < 0.01 by the paired-samplet test). In contrast, the other infected control patients showed higher-than-normal levels of anti-CR 1 binding (P < 0.05). When compared to that of the normal subjects, the total CR1 quantitated in PMN extracts was also lower than normal in CGD patients (P < 0.01 and in the PMN extracts of eight Job's syndrome patients tested (P < 0.01). The PMNs of the other infected control subjects were not significantly different from those of the normal subjects in total CRI expression. Extracts of Es from Job's syndrome patients also had fewer than normal CR1 (P < 0.02). On the other hand, CR1 levels in E extracts from the CGD patients and the other control patients were similar to those in the normal control subjects. Quantitations of C 3, C4, and factor B were normal in CGD. Significant levels of immune complexes were detected in serum samples from several CGD and Job's syndrome patients. However, the level of immune complexes did not correlate significantly with the CR1 deficit in CGD or Job's syndrome patients. Thus, in CGD patients, CRI expression of PMNs is below normal, while that of Es is normal; in Job's syndrome patients, both the PMN and the E CRI expression are below normal. These abnormalities do not appear to result from the frequent infections that occur in these diseases since the other infected control patients exhibit an above-normal amount of surface PMN CR1.     

Characterization of subtypes of gamma-aminobutyric acid receptors in an Ascaris muscle preparation by binding assay and binding of PF1022A,a new anthelmintic,on the receptors

 We examined the effect of PF1022A, one of the gabergic anthelmintics newly developed in Japan, on gamma-aminobutyric acid (GABA) receptors using a radioligand binding technique in isolated membrane preparations of the nematode Ascaris suum. Membrane protein was prepared from the homogenate of somatic muscle cells after ultracentrifugation. In addition to the basic binding of [2,3-³H-(N)]-GABA, the radioligand [methyl-³H]-bicuculline is used to identify the GABA_A receptor, whereas [butyl-4-³H]-baclofen is employed for GABA_B receptor sites. The dissociation constants (K _d values) and the maximal numbers of binding sites (B_max values) from Scatchard plotting for GABA receptors are close to those obtained in mammalian brain. PF1022A displaced in a concentration-dependent way the binding of [2,3-³H(N)]-GABA and [methyl-³H]-bicuculline as did other specific gabergic agents. In addition, PF1022A decreased the binding of [butyl-4-³H]-baclofen at a higher concentration, although this binding did not represent GABA_B sites. In a comparison of the inhibition constants (K_i values) of PF1022A with those of other agents, it is conclusive that PF1022A bound with GABA receptors. A direct effect of PF1022A on GABA receptors can thus be postulated. Received: 10 March 1995 / Accepted: 27 June 1995