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.     

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.     

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.     

Effects of calcium channel modulators on the proliferation of mouse spleen lymphocytesin vitro

The effects of nimodipine (voltage-dependent calcium channel blocker), CGP 28392 and BAY K 8644 (novel dihydropyridine derivatives that are considered as calcium entry stimulators) on the spontaneous proliferation of mouse spleen lymphocytes were studiedin vitro. [³H]-thymidine incorporation into DNA of lymphocytes was used as an sensitive index of the cell proliferation. It has been found that nimodipine (10^–4 M–10^–6 M) significantly inhibited the [³H]-thymidine uptake in a dose dependent fashion with ED₅₀ value of 2.4×10^–5 M. Unexpectedly, CGP 28392 (10^–4 M–10^–7 M) acts as a calcium entry blocker and produces a strong inhibitory effect on lymphocyte proliferation (ED₅₀–2×10^–5 M). BAY K 8644 at a high concentration (10^–4 M) also has an inhibitory effect but at a lower concentration (10^–6 M–10^–10 M) significantly increased [³H]-thymidine uptake and abolished the inhibitory effect of nimodipine. This effect of nimodipine was also reversed by 5×10^–3 M calcium chloride. These findings indicate that calcium channel modulators can regulate the proliferation of mouse spleen lymphocytesin vitro.     

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.     

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.     

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.     

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     

Biochemical effects of luxabendazole onTrichinella spiralis

Biochemical changes produced by luxabendazole in muscle-stageTrichinella spiralis larvae consisted of a decrease in free glucose and glycogen levels (46.71% and 35.66%, respectively) after in vivo treatment, slight in vitro inhibition of fumarate reductase activity (24.15%) and, finally, inhibition of [³H]-colchicine-tubulin binding, which was found to be of a competitive nature, with an inhibition constant (K_i) of 0.9×10^–7 M. In a parallel study, luxabendazole did not appear to be inhibitory to [³H]-colchicine binding to pig-brain tubulin.     

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.     

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.     

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.     

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.     

Action of histamine and of some H2-antagonists on gastric secretion ‘in vitro’

The effect of histamine and of some H₂-antagonists on isolated gastric mucosal preparation from immature (14–18 days) rats, was investigated. Basal secretion varied, in our experimental conditions, between 1.06 and 3.54 mol cm^–2 h^–1, reaching higher values (approximately 4.6 mol cm^–2 h^–1) only in a small percentage of animals (10%). Histamine exerted a concentration-dependent stimulation of acid secretion in concentrations varying between 2×10^–6 and 1.6×10^–4 M. the response to histamine was competitively antagonized by ranitidine (pA₂ value=6.78) and by 4(5)-(4-isopropylaminomethyleniminophenyl) imidazole (compound marked DA 4577) (pA₂ value=7.37). Oxmetidine acted as a competitive antagonist only for concentrations as low as 10^–8 M; higher concentrations (10^–7 and 10^–6 M) determined a non-competitive inhibition. Ranitidine and compound marked DA 4577 did not affect basal secretion up to concentrations of 3×10^–4 M. On the contrary oxmetidine exerted a concentration-dependent inhibition starting from 10^–5 M. Since in our experimental conditions the role of calcium ions in the regulation of basal secretion could not be established, the mechanism of action of oxmetidine was not completely clarified, even if an interference in the utilization of calcium ions may be suggested. In any case it is deemed of interest that this H₂-antagonist was the only compound capable of inducing a reversible complete inhibition of basal acid secretion (only KSCN, in very high concentrations, had a similar behaviour).     

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.     

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.     

Ranitidine but not famotidine releases acetylcholine from the guinea pig myenteric plexus

The effects of the histamine H2-receptor antagonists ranitidine and famotidine on acetylcholine release have been studied in the guinea pig myenteric plexus longitudinal muscle preparation incubated with [³H]-choline. Ranitidine (3×10^–5–3×10^–4 M) dose-dependently increased the resting release of acetylcholine and that evoked by electrical stimulation. The effect was present only in strips perfused with 10^–5 M physostigmine. The effect of ranitidinc was inhibited by tetrodotoxin and hexamethonium. Famotidine (10^–5–3×10^–4 M) was totally ineffective in modifying both the resting release and that evoked by field stimulation. Ranitidine did not antagonize the inhibitory effect of oxotremorine, which specifically activates negative feedback mechanisms via presynaptic muscarinic receptors.     

Mechanism of action of H2-antagonists on histamine-or dimaprit-stimulated H2-receptors of spontaneously beating guinea-pig atrium

Cimetidine, ranitidine and famotidine are antagonists of the histamine H₂-receptors on the spontaneously beating right atrium of the guinea pig. When analyzed by the classical Schild method theirpA ₂-values are respectively: 6.3, 6.8 and 7.7 with dimaprit as agonist and 5.8, 6.5 and 7.7 with histamine as agonist. Radiolignad-displacement studies with [³H]-tiotidine as radioligand resulted inpK _d values for cimetidine, ranitidine and famotidine of 6.3; 6.9 and 8.2 respectively.In dimaprit-stimulated atria all antagonists added at concentrations above theirK _d values depressed the maximal increase in frequency. In the presence of histamine this effect was much less pronounced and only visible at concentrations of ranitidine and famotidine around 10 ·K _d.The rightward shift of the curves as well as the decrease inE _max are reversible but the dissociation constants of the antagonists are small (less than 10^–3 s^–1).The spontaneously beating right atrium showed receptor reserve for histamine and virtually no receptor reserve for dimaprit.The results have been interpreted in a model in which H₂-antagonists act mainly by competing with the agonist for the histamine receptor site but have in addition a distinct affinity for a secondary site on the receptor. Occupation of this site by the antagonist prevents building up of the stimulus elicited by the agonist and thus decreases theE _max. In systems with receptor reserve (histamine) the effect of antagonist binding to the secondary binding site is seen only at high concentration of antagonist while in absence of receptor reserve (dimaprit) the depression ofE _max is directly visible.Simulations of the model show that the affinity of this secondary binding site is 50- (famotidine) to 100-(cimetidine and ranitidine) fold lower than for the agonist binding site.     

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.     

Differential inhibition by nedocromil sodium of superoxide generation elicited by platelet activating factor in human neutrophils

Nedocromil sodium (10^–10–10^–9 M) produced a dose-related inhibition of superoxide anion generation induced by platelet activating factor (PAF) in human polymorphonuclear leukocytes (PMNs). At a higher concentration (3×10^–7 M), nedocromil sodium significantly inhibited superoxide generation elicited by N-formyl-methionyl-leucylphenylalanine, but was unable to block the response to phorbol dibutyrate. Nedocromil sodium (10^–11–10^–5 M) enhanced PAF-stimulated lysozyme release in a non-concentration-dependent manner, and was completely ineffective in depressing PAF-induced release of [³H]arachidonic acid and the rise in cytosolic Ca²⁺. The preferential inhibitory effects of nedocromil sodium on PAF-induced activation of superoxide generation may provide insight into the therapeutic action of this drug as an anti-asthmatic agent.