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.     

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.     

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.     

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.     

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.     

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.     

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.     

[3H]Adenosine binding to rat mast cells — pharmacologic and functional characterization

Rat serosal mast cell adenosine receptors were characterized by [³H]adenosine binding to cell membrane particulates, and functional changes in mast cell mediator release and cyclic AMP levels were assessed, utilizing various adenosine analogs. [³H]adenosine binding to sonicated mast cell membrane preparations at 0°C in the presence of deoxycoformycin is linear with initial cell number, rapid and reversible. The cells display 16,400±1600 high affinity [³H]adenosine binding sites/cell, equivalent to 118 fmol bound/mg protein, with an equilibrium dissociation constant of 27.97±3.0 nM. Competition studies reveal that adenosine>2-chloroadenosine>NECA>l-PIA>d-PIA in competing for [³H]adenosine binding sites and that aminophylline and cromolyn sodium also bind to the putative receptor. Adenosine and its analogs, NECA, andl-PIA, appear to activate adenylate cyclase in resting mast cells by raising cyclic AMP, suggesting anR _a cell surface adenosine receptor subtype; these same analogs potentiate mast cellb-hexosaminidase release stimulated by specific antigen. The identification of rat mast cell [³H]adenosine binding sites whose stimulation augments resting cell cyclic AMP levels and antigen-induced mediator release suggests that these receptors may be important in the biochemical mechanisms of allergic diseases. The ability to assess the number and affinity of mast cell adenosine receptors will enable one to monitor receptor alterations during pharmacologic manipulation and in disease states.     

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     

Mast cell receptors controlling histamine release: Influences on the mode of action of drugs used in the treatment of adverse drug reactions

Summary In drug-induced allergic diseases of the immediate type (anaphylactic and anaphylactoid reactions), the primary target cells are tissue mast cells, which discharge their granular content upon interaction with different secretagogues (immunological releasers; histamine liberators) on specific plasma membrane receptors.Experiments are reviewed here which report that IgE-mediated histamine release from mast cells, and the secretion of histamine induced by non-immunological secretagogues (dextran; compound 48/80; acetylcholine) are blocked by beta-adrenoceptor and H₂-receptor agonists, their inhibiting effect being surmountable by beta-adrenoceptor blocking drugs and by anti-H₂-antihistamines. Specific radioligands ([³H]-dihydroalprenolol; [³H]-cimetidine) binding to rat mast cell membranes points to the possibility that inhibition of histamine release is brought about by the activation of mast cell beta-adrenoceptors and H₂-receptors.Drugs used in therapy of anaphylactic or anaphylactoid reactions may act either on tissue receptors, competing with released mediators, or by inhibiting the release of allergic mediators from mast cells, on activation of specific receptors located in mast cell plasma membranes.     

Dihydropyridine receptors in transverse tubules from normal and dystrophic chicken skeletal muscle

Summary Calcium overload is a fundamental pathogenic event associated with chronic muscle degeneration in muscular dystrophies. The possibility that l-type voltage-dependent calcium channels were involved in the etiology of chicken muscular dystrophy was investigated by studying the dihydropyridine receptors in transverse tubule membranes isolated from skeletal muscle of normal (line 412) and dystrophic (line 413) chickens. The yield of T-tubular protein from dystrophic muscle was considerably increased compared with that from normal muscle (2.51±0.18 vs 1.04±0.31 mg protein × 100 g muscle^–1). The binding of the calcium channel antagonist (+) [³H]PN200-110 to the dihydropyridine receptor in transverse tubule preparations was relatively slow, markedly affected by temperature and required divalent cations. (+) [³H]PN200-110 equilibrium binding assays revealed a single class of high-affinity sites and showed that maximum binding capacity (B_max) (3.17±0.47 for normal and 3.51±0.52 pmol × mg protein^–1 for dystrophic transverse tubules) and dissociation constant (K_d) (0.32±0.07 and 0.26±0.09 nm, respectively) were not significantly different in normal and dystrophic membranes. Kinetic studies indicated that normal and dystrophic transverse tubules did not differ significantly in association (2.54×10⁶ and 2.27×10⁶ m ^–1 s^–1, respectively) and dissociation (8.5×10^–4 and 9.3×10^–4 s^–1, respectively) rate constants. Since dissociation kinetics for both preparations were monoexponential under all the experimental conditions employed, no low-affinity binding sites for (+) [³H]PN200-110 could be detected in chicken transverse tubules membranes. However, immunoblot assay, using a monoclonal antibody, revealed that dystrophic transverse tubules as compared with normal membranes were enriched twofold with the ₁-subunit of the dihydropyridine receptor. Therefore, although dihydropyridine-binding sites were not altered in transverse tubule membranes from dystrophic chicken skeletal muscle, both the increased yield in T-tubule vesicles and the enhanced immunodetection of the ₁-subunit of the dihydropyridine receptor, suggest that total content in dihydropyridine receptor is higher in dystrophic than in normal muscle.     

Characterization of coexistent histamine H1- and H2-receptor binding sites in the purified guinea pig myocardial membranes from ventricles

In the present work, we identified and characterised histamine H1-and H2-receptors in highly purified myocardial membranes isolated from female guinea pig ventricles. We determined the binding parameters for the interactions of³H-mepyramine with the histamine H1-receptor binding site and³H-tiotidine with the histamine H2-receptor binding site. Binding of both ligands in our study was saturable, reversible and of high affinity. Scatchard's analysis of the specific³H-mepyramine binding revealed the existence of high and low affinity binding sites with apparent K_D values of 0.4 nM and 4.5 nM, respectively. The density of binding sites (B_max) was 100 fmol/mg protein for the high and 466 fmol/mg protein for the low affinity binding site.³H-tiotidine binds to a single population of binding sites with a K_D of 1.0 nM and a B_max of 27 fmol/mg protein. These data suggest that both histamine H1-and H2-receptors coexist in the guinea pig myocardium with a significantly higher prevalence of the histamine H1-receptor population.     

Autoinhibition of histamine release by H3 receptors in rat brain cortex depends on stimulation frequency

Rat cortical slices preloaded with [³H]histidine released [³H]histamine upon electrical stimulation or after depolarization with elevated K⁺ levels. The release was dependent on the presence of Ca²⁺, suggesting a neurosecretory process. Histamine has been shown to inhibit its own release mediated by an autoreceptor belonging to the H₃-receptor subclass. In this study we have investigated the autoinhibition using different electrical field stimulation conditions (1, 10, 20 and 33.3 Hz). Applying electrical stimulation, the inhibition of [³H]histamine release by histamine is decreased when the stimulation frequency is elevated. When stimulated with 1 Hz histamine is able to block [³H]histamine release completely, with ap(EC₅₀) of 8.1±0.1. At higher frequencies histamine still blocks [³H]histamine release completely, but with a lowerp(EC₅₀).     

Binding of [3H]sulpiride to purified rat striatal synaptic membranes

Previous results showing that sulpiride, unlike classical neuroleptics, does not block the effects of dopamine on the dopamine-sensitive adenylate cyclase have led to the concept of dopaminergic D₁ and D₂ receptors. It has been suggested that sulpiride is a specific antagonist of D₂ receptors and [³H]sulpiride has been used as a specific ligand for these. This report examines the binding of [³H]sulpiride to purified synaptic membranes from rat striata. There appears to be a single saturable binding component with an affinity constant of 7 nm and a maximum binding capacity of 240 fmol/mg protein. This binding site appears to be dopaminergic in origin since it is present in high concentration in the striatum and nucleus accumbens but in low concentration in nori-dopaminergic regions of the brain. Our results show that sulpiride binding is potently inhibited by classical neuroleptics and benzamides, whilst dopamine agonists have much weaker activity. The binding site shows stereochemical specificity for cis-flupenthixol, cis-clopenthixol, (+)-butaclamol and s-(?)sulpiride.The rank order of potency of the dopamine antagonists is not consistent with a specific benzamide binding site since the classical neuroleptics were very potent inhibitors of binding. In particular, the thioxanthines, reputedly good inhibitors of the dopamine-sensitive adenylate cyclase, were amongst the most potent inhibitors of [³H]sulpiride binding.Thus it is clear that these results are not in accord with the present concept and classification of dopaminergic D₁ and D₁ receptors.     

[S]tert.-butylbicyclophosphorothionate and avermectin bind to different sites associated with the γ-aminobutyric acid-benzodiazepine receptor complex

Low concentrations of avermectin B₁a (AVM) stimulated the specific high affinity binding of [³⁵S]tert.-butylbicyclophosphorothionate ([³⁵S]TBPT) to membranes from rat cerebral cortex in the absence or presence of chloride or bromide ions. In contrast, TBPT either weakly stimulates or does not significantly influence the specific high affinity binding of [³H]AVM to the same membranes in the absence or presence of chloride ions, respectively. These results indicate that [³H]AVM and [³⁵S]TBPT bind to different but closely associated binding sites.     

Oocytes from Xenopus laevis contain an intrinsic σ2-like binding site

In preparation for expression studies for rat brain σ-binding sites, Xenopus oocytes were tested for the presence of [³H]di-o-tolylguanidine (DTG)-binding sites. Native oocytes were found to contain two intrinsic [³H]DTG-binding sites, a high-affinity site (K_d = 32 ± 6 nM, B_max of 45.7 ± 19 pmol/mg protein) and a low-affinity binding site (K_d = 1.3 ± 0.7 μM, B_max of 3.2 ± 0.7 nmol/mg protein). In a series of radioligand-binding-displacement studies, the high-affinity binding sites were found to have a binding profile which has a similar K_d to that of the mammalian σ₂-binding site (32 vs. 38 nM). Comparison of the IC₅₀ values for inhibition of [³H]DTG binding in rat liver and oocytes for DTG, haloperidol (HAL), (−)-pentazocine, (+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP), (+(-pentazocine and Zn²⁺, showed similarity in rank (r² = 0.913) but a 7-fold lower potency in oocytes. These results suggest that the high-affinity [³H]DTG-binding site in oocytes represents a σ₂-like binding site.     

Quantitative localization of Na-K pump sites in the frog sacculus

Summary The Na-K pump site distribution within the macula, perimacula, and wall epithelia of the sacculus in the frog inner ear was examined with quantitative [³H]ouabain autoradiography. Excised tissue was incubated for 10–30 min (23 ° C) in micromolar concentrations of high specific activity [³H]ouabain (14–70 Ci mT^–1, 5–15 Ci mmor^–1), washed for 30 min (4 ° C), then rapidly frozen (–175 ° C) and processed for light and electron microscope autoradiography. Control experiments based on (1) high K⁺ (50 mm) in the incubation and (2) low specific activity [³H]Jouabain (1 mM, 0.013–0.025 Ci mmol^–1) indicated negligible nonspecific binding of the [³H]ouabain.Measurable levels of specific [³H]ouabain binding occurred in all saccular regions examined. Binding was localized to the basolateral cell membranes with no detectable binding to the apical membranes. [³H]ouabain binding across the apical-basal axis of the saccule macular epithelium was nonuniform. Binding was low in the apical region, rose to a peak in the middle two-thirds, and then fell again close to the basement membrane. Electron microscope autoradiography suggested that this peak was due to ouabain binding to nerve terminals. Denervation of the sacculus eliminated the peak in [³H]ouabain binding and quantitative grain density analysis revealed that 45% of the Na-K pumps within the saccule macula were located on the nerve terminals.Na-K pump site density per unit volume was estimated by quantitative grain density analysis and the following values were obtained (sites m^–3 × 10³, means ± S.E.M.): saccule macula, 1.9 ± 0.2; saccule perimacula, 1.1 ± 0.1; saccule wall, 2.3 ± 0.3. Stereological analysis of conventionally fixed tissue was used to estimate overall plasma membrane surface area per unit volume (S _v). Na-K pump site densities per unit membrane area for the various regions were calculated by combining the autoradiographical and Stereological data. The following values were obtained (sites m^–2 ± 25%): saccule macula, 2500; saccule perimacula, 2500. Values for individual cells within the macula (sites m^–2 ± 25%) were: hair cells, 3000; nerve terminals, 3000; supporting cells, 1500.     

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.     

[3H]Strychnine binding suggests glycine receptors in the ventral tegmental area of rat brain

[³H]Strychnine binds to membranes prepared from the ventral tegmental area of rat brain. At 4°C the binding is rapid and saturable, and can be displaced by strychnine, glycine and their analogues. Scatchard analysis showed that the K_D for [³H]strychnine binding was 6.0 nM and that the density of binding sites was 17.9 pmol/g wet wt. tissue. Hill plots of the binding data showed that there were no cooperative site interactions associated with binding. [³H]Strychnine binding in the ventral tegmental area is of similar affinity to that to spinal cord membranes, although the density of binding sites is only half that in spinal cord membranes. Drug displacement studies demonstrated that [³H]strychnine binding to membranes of ventral tegmentum was similar to that seen in spinal cord. The results indicate that glycine receptors are present in the ventral tegmentum area.