Correlation of histamine H1 receptor function and [3H]mepyramine binding in porcine tracheal tissue

In order to validate the use of [3H]mepyramine as a radioligand to label airway histamine H1 receptors, the results of radioligand binding experiments using porcine tracheal tissue membranes were compared with the results of physiologic studies measuring histamine-induced trachealis muscle contraction. Close agreement was found between histamine-induced [3H]mepyramine binding inhibition and histamine concentration-contraction-response curves. Close agreement was also found between the KD of mepyramine-induced [3H]mepyramine binding inhibition and the K beta of mepyramine antagonism of muscle contractions stimulated by 10(-4) M histamine. [3H]Mepyramine binding was found to be rapid, reversible, saturable and stereospecific. Only H1 agonists and antagonists displayed potent [3H]mepyramine binding inhibition in competition binding studies. The results fulfill criteria for histamine H1 receptor identification by radioligand binding with [3H]mepyramine.     

Thiomersal enhances the binding of histamine to the H1 receptor,but not histamine-stimulated inositol phosphate formation

Thiomersal (thimerosal) was a weak inhibitor of the binding of [(3)H]mepyramine to histamine H(1) receptors in guinea-pig cerebellar membranes (11 +/- 1% inhibition at 10 microM, 32 +/- 3% inhibition at 300 microM). However, in the concentration range 3-30 microM, thiomersal enhanced the binding of histamine to the H(1) receptor, as reflected by the displacement of curves of histamine inhibition of [(3)H]mepyramine binding to lower concentrations, without any change in the Hill coefficient. The ratio of the IC50 values (the concentration giving 50% inhibition) in the absence and presence of thiomersal increased from 1.8 with 3 microM to 3.6 with 30 microM thiomersal. There was no consistent effect of thiomersal at concentrations of 30 microM and below on curves of mepyramine inhibition of [(3)H]mepyramine binding. In the presence of 10 microM thiomersal histamine-induced accumulation of inositol phosphates in U373 MG astrocytoma cells was partially inhibited (37 +/- 8% inhibition of the maximum response), without any significant change in the EC50 (the concentration giving the half maximal response) for histamine. Thus although histamine binding was potentiated by thiomersal, there was no potentiation of an H(1) receptor-mediated functional response.     

The effect of histamine on cultured endothelial cells. A study of the mechanism of increased vascular permeability.

The role of endothelial cells in the histamine-induced vascular response was investigated using cultured endothelial cells obtained from human umbilical veins. A single population of histamine H1 receptors was detected in these cells by means of a [3H]mepyramine binding assay. Its Kd was 0.74 +/- 0.07 nM and Bmax was 41.4 +/- 8.68 fmol/mg protein. Actin filaments were distributed as dense bands at the margin of the cells and as sparse microfilament bundles traversing the center of the cells. Histamine caused a decrease in peripheral bands and an increase in longitudinal bands. The changes evoked by histamine were dose-dependent, related to the duration of incubation of the cells with histamine, and blocked by mepyramine, a histamine H1 receptor antagonist. The results suggest that the endothelial cells respond to histamine through the histamine H1 receptor. This may explain one of the mechanisms of histamine-induced vascular response including vascular permeability increase.     

Characteristics of the binding of [3H]-mepyramine to intact human U373 MG astrocytoma cells: evidence for histamine-induced H1-receptor internalisation.

1. The kinetics of the binding of 5 nM [3H]-mepyramine to sites on intact human U373 MG astrocytoma cells, sensitive to inhibition by 2 microM pirdonium, were temperature-dependent. At 37 degrees C the half-time for association was 0.9 +/- 0.4 min and at 4 degrees C 19 +/- 3 min. Dissociation of bound [3H]-mepyramine was fast at 37 degrees C, t0.5 1.5 +/- 0.3 min, but at 6 degrees C dissociation initiated by dilution or addition of unlabelled mepyramine was negligible over 120 min. The very slow dissociation at 6 degrees C made it possible to reduce the level of pirdonium-insensitive binding from 56 +/- 5% to 39 +/- 5% by washing the cells in ice-cold medium before filtration. 2. The binding of [3H]-mepyramine sensitive to 2 microM temelastine, measured after 10 min equilibration at 37 degrees C, failed to saturate and was resolved into an hyperbola and an apparently linear component, whereas the fit to the binding of [3H]-mepyramine sensitive to 2 microM pirdonium was not significantly improved over that to an hyperbola. The mean Kd for the binding of [3H]-mepyramine to the saturable component, 2.5 +/- 0.4 nM, was in close agreement with the value of 3.5 nM for mepyramine derived from inhibition of histamine H1-receptor-mediated inositol phosphate formation in U373 MG cells. 3. Curves for the inhibition of the binding of 5 nM [3H]-mepyramine to U373 MG cells by histamine H1-receptor antagonists were biphasic and were fitted to a two site-model. Affinities calculated from the best-fit IC50 values for the high-affinity site correlated well with those expected for binding to H1-receptors. 4. The percentages of the high-affinity site in curves of the inhibition of [3H]-mepyramine binding to intact U373 MG cells by two tertiary amine antagonists, norpirdonium and 4-methyldiphenhydramine, 68 +/- 3 and 63 +/- 4%, were significantly greater than the percentages of the high-affinity site in the inhibition curves of their quaternary derivatives, 50 +/- 1 and 45 +/- 3%, respectively. Similarly, the percentage of the high-affinity site for unlabelled mepyramine, 65 +/- 7%, was greater than for the non-cell penetrant H1-antagonist temelastine, 42 +/- 5%. 5. Incubation of U373 MG cells with 100 microM histamine at 37 degrees C, followed by washing twice in ice-cold medium and then incubation with 1-15 nM [3H]-mepyramine for 120 min at 4 degrees C, resulted in a decrease in the binding of [3H]-mepyramine sensitive to 2 microM pirdonium, compared to control cells not exposed to histamine. The binding of [3H]-mepyramine in the absence of pirdonium was not altered by histamine pretreatment, whereas the level of the pirdonium-insensitive binding was significantly increased, except after 1 min exposure to histamine. The decreases in the pirdonium-sensitive binding after 5, 10 and 60 min incubation with 100 microM histamine were 41 +/- 6, 56 +/- 6 and 67 +/- 8%, respectively, but the decrease after 1 min incubation with histamine, 16 +/- 8%, was not statistically significant. 6. The results are consistent with the binding of [3H]-mepyramine to intact U373 MG cells being to both plasma membrane and intracellular histamine H1-receptors. The high-affinity binding sensitive to the non-cell penetrant quaternary compounds and to temelastine is thus to plasma membrane H1-receptors. On exposure to 100 microM histamine receptors are translocated to the intracellular pool, since the change in the high-affinity binding of [3H]-mepyramine is primarily in the level of the pirdonium-insensitive binding, rather than in the total binding.     

Histamine influences the expression of the interleukin-6 receptor on human lymphoid, monocytoid and hepatoma cell lines.

Based on our earlier data on the enhancing effect of histamine on the action of interleukin-6 (IL-6), we have studied the molecular mechanisms of these interactions. The effect of histamine was investigated on the binding of 125I-IL-6 by B lymphoma cell line CESS, monocytoid cell line U937 and hepatoma cell line HepG2. Histamine increases the IL-6 binding by CESS cells and inhibits that by U937 and HepG2 cells. Using H1 receptor (cetirizin and loderix) and H2 receptor (cimetidine and ranitidine) specific antagonists, an H1-dependent stimulation of IL-6 binding by CESS cells was found. In contrast, down-regulation of IL-6 binding by histamine was clearly mediated through H2 receptors. On U937 cells, using a monoclonal antibody reacting with the 80 kd chain of the human IL-6 receptor, and H2-receptor mediated inhibition of IL-6 receptor expression was found by FACS analysis.     

Histamine H1-receptors in the guinea-pig urinary bladder

Histamine H1-receptors were identified in the guinea-pig urinary bladder. Histamine (10(-8)-10(-3) M) produced a dose-dependent contraction which was not altered by either scopolamine or tetrodotoxin. Specific [3H]mepyramine binding to the urinary bladder was saturable and there was a single population of high affinity binding sites with an equilibrium dissociation constant (KD) of 0.77 nM and maximum binding capacity (Bmax) of 68.5 fmol/mg protein. Histamine-induced contraction and [3H]mepyramine binding were inhibited by triploridine, promethazine, d-chlorpheniramine and mepyramine but not by cimetidine. There was a good correlation between mechanical activity and [3H]mepyramine binding as shown by the inhibition affinity constant (Ki) of H1-antagonists. These results provide evidence that the histamine H1-receptor in the guinea-pig urinary bladder is involved in the histamine-induced contraction.     

Chronotropic effect of histamine on cultured neonatal rat heart cells.

The positive chronotropic effect (PCE) of histamine in cultured neonatal rat heart cells was monitored using a microscopic method as well as an electro-optically recording device. The action potential frequency was also measured (by means of microelectrodes). An increase in PCE was noted when histamine (from 1 X 10(-6) M to 1 X 10(-5) M) was added to the cells. However, higher concentrations (from 1 X 10(-5) M to 1 X 10(-4) M) were less effective. The PCE of histamine was reduced by pretreating the cells with antihistaminic drugs. H1-blocking agents (promethazine and mepyramine) were more potent than H2-blocking drugs (metiamide and cimetidine). In addition, the PCE of histamine was abolished when the cells were in presence of high K+ medium (26 mEq) but contraction and action potential amplitudes were increased. Our results demonstrate that these cultures respond to histamine and that this response is abolished by antihistaminic drugs thus suggesting the H1 and/or H2 receptors may be present in the neonatal rat heart cell cultures.     

Biphasic response to histamine in rabbit penile dorsal artery

The effects of specific histamine agonists and antagonists on isolated rabbit penile dorsal artery segments were explored using in vitro isometric techniques. Histamine caused the constriction of both precontracted and resting segments. In precontracted arterial rings treated with the H1 receptor antagonist mepyramine, histamine evoked a vasodilatation, followed by contraction at higher concentrations. The vasoconstrictor effect of histamine and the H1 receptor agonist, 2-pyridylethylamine (PEA) on preparations under conditions of basal tone, was competitively antagonized by mepyramine (10(-9)-10(-8) M). The relaxant effect of histamine, unmasked by mepyramine, was abolished by cimetidine. Dimaprit, the H2 receptor agonist, provoked a relaxation of precontracted segments that was also competitively inhibited by cimetidine (10(-6)-10(-5) M). Selective H3 receptor activation with the agonist (R)alpha-methylhistamine (10(-10)-10(-4) M) produced no effect in penile dorsal artery. The biphasic response to histamine was unaffected by endothelium removal or the nitric oxide inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (3 x 10(-4) M) and its precursor, L-arginine (3 x 10(-4) M). Similarly, the cyclooxygenase inhibitor, indomethacin (3 x 10(-6) M) and a combination of Ca2+-activated K+ channel blockers apamin (5 x 10(-7) M) and charybdotoxin (10(-7) M) showed no effect on the histamine-induced relaxation or contraction. In conclusion, contraction, the predominant effect of histamine, is mediated by the activation of H1 receptors that mask the relaxant effect brought about by H2 receptors. Both these effects appear to be mediated by direct action on the smooth muscle, with no participation of nitric oxide or cyclooxygenase products or Ca2+-activated K+ channels.     

Eicosanoid release and mepyramine, LTC4 and LTD4 binding in passively sensitized human lung parenchyma in vitro

In vitro passive sensitization of human lung parenchyma with hyper-immune serum did not affect the release of prostaglandin D2 (PGD2) or leukotriene (LT)-like activity upon challenge with anti-IgE antibody with respect to control lung, despite a marked difference in IgE levels between control (C) and sensitized (S) tissue. Binding studies with [3H]LTC4, [3H]LTD4 and [3H]mepyramine (a histamine H1 antagonist) showed a statistically significant increase in the amount bound in sensitized vs control lung for [3H]mepyramine only. Contractile response to 5 x 10(-5) M histamine (H) in C and S lung parenchymal strips did not correlate with binding data. It is concluded that in vitro elevated IgE levels do not affect the interaction of sulfidopeptide leukotrienes with their putative receptors. As for the observed increase in [3H]mepyramine binding, this might not represent a true increase in histamine receptors on lung smooth muscle cells.     

H1-histamine receptors on human astrocytoma cells

The H1-histamine receptor antagonist [3H]mepyramine bound with high affinity (Kd = 3-5 nM) to membranes derived from 1321N1 human astrocytoma cells. The H1-receptor antagonists triprolidine and diphenhydramine inhibited [3H]mepyramine binding with Kj values of 1-5 nM, whereas the Kj of the H2-histamine receptor antagonist cimetidine was greater than 100 microM. Histamine also inhibited [3H]mepyramine binding to 1321N1 cell membranes, and the histamine inhibition curve was shifted to the right and steepened in the presence of 1 microM guanosine 5'-O-(3-thiotriphosphate). Treatment of 1321N1 cells with pertussis toxin had no effect on the capacity of histamine to inhibit [3H]mepyramine binding either in the absence or presence of guanosine 5'-O-(3-thiotriphosphate). Therefore, agonist-occupied histamine receptors in these cells apparently interact with a guanine nucleotide regulatory protein that is not the inhibitory guanine nucleotide regulatory protein of adenylate cyclase. Although adenylate cyclase activity was not affected by histamine in a cell-free preparation, incubation of 1321N1 cells with histamine resulted in an attenuation of cyclic AMP accumulation. Analysis of cyclic AMP degradation in the presence of histamine indicated that the effects of histamine on cyclic AMP accumulation are mediated through activation of phosphodiesterase. This idea was supported by the fact that the phosphodiesterase inhibitor 1-isobutyl 3-methylxanthine blocked attenuation of cyclic AMP accumulation by histamine in a noncompetitive manner. Histamine also markedly increased phosphoinositide breakdown and 45Ca2+ efflux in 1321N1 cells. These histamine-induced effects apparently are mediated through H1-receptors, since triprolidine, but not cimetidine, potently inhibited histamine action. As for histamine interaction with its receptor, pertussis toxin had no effect on histamine-induced phosphoinositide breakdown, 45Ca2+ efflux, or attenuation of cyclic AMP accumulation. Taken together, these data indicate that 1321N1 human astrocytoma cells are a useful model system for the study of H1-histamine receptors and the biochemical responses mediated through these receptors.     

Electrophysiological actions of phenytoin on N-methyl-D-aspartate receptor-mediated responses in rat hippocampus in vitro.

1. [3H]-bradykinin was used to characterize the bradykinin receptors associated with canine cultured tracheal smooth muscle cells (TSMCs). Receptor binding assay showed that TSMCs had specific, saturable, high-affinity binding sites for [3H]-bradykinin. 2. The specific [3H]-bradykinin binding increased linearly with increasing cell concentrations. The equilibrium for association of [3H]-bradykinin with the bradykinin receptors was attained within 2 h at 4 degrees C and 1 h at room temperature, respectively. 3. Analysis of binding isotherms yielded an apparent equilibrium dissociation constant (KD) of 2.5 +/- 0.3 nM and a maximum receptor density (Bmax) of 25.1 +/- 0.3 fmol mg-1 protein. The Hill coefficient for [3H]-bradykinin binding was 1.00 +/- 0.02. The association (K1) and dissociation (K-1) rate constants were (8.67 +/- 2.60) x 10(6) M-1 min-1 and 0.024 +/- 0.005 min-1, respectively. KD, calculated from the ratio of K-1 and K1 was 2.8 +/- 0.5 nM, a value close to that of KD calculated from Scatchard plots of binding isotherms. 4. The B1 receptor selective agonist, (des-Arg9-bradykinin, 0.1 nM-10 microM) and antagonist ([Leu8, des-Arg9]-bradykinin, 0.1 nM-10 microM) did not did not inhibit the [3H]-bradykinin binding to TSMCs, which excludes the presence of B1 receptors in canine TSMCs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intact cell binding for in vitro prediction of sedative and non-sedative histamine H1-receptor antagonists based on receptor internalization

We evaluated changes in the binding properties of sedative and non-sedative histamine H1-receptor antagonists induced by internalization of H1 receptors in intact human U373 MG astrocytoma cells. Internalization of H1 receptors was induced without their degradation by treatment with 0.1 mM histamine for 30 min at 37 degrees C, and then the intact cell binding assay was performed at 4 degrees C. The binding properties of [3H]mepyramine, a cell-penetrating radioligand for H1 receptors, were not changed by histamine pretreatment. Displacement curves for sedative H1-receptor antagonists (diphenhydramine, chlorpheniramine, promethazine, ketotifen, azelastine and oxatomide) against [3H]mepyramine binding were not changed by histamine pretreatment. In contrast, the displacement curves for non-sedative H1-receptor antagonists (mequitazine, bepotastine, olopatadine, epinastine, carebastine, desloratadine and fexofenadine) were changed by histamine pretreatment: two types of changes, i.e. a rightward shift in the monophasic curve or an increase in the proportion of the low affinity component of the biphasic curve, were prevented under hypertonic conditions, in which clathrin-mediated receptor internalization is known to be inhibited. Thus, internalization-mediated changes in the binding properties of H1-receptor antagonists were well correlated with their sedative and non-sedative behaviors, which might confirm their permeability through the biomembrane and possibly the blood brain barrier.     

Characterization of histamine type 1 receptors on natural suppressor lymphoid cells

Using the radioligand H1 antagonist [3H]pyrilamine, we have characterized the histamine type 1 receptor on cloned murine natural suppressor cells (NS). A single, specific binding site for [3H]pyrilamine exists on these cells. The binding was saturable and reversible by various specific H1 receptor antagonists. The rank order of potency for displacement of [3H]pyrilamine binding from the H1 receptor by H1 receptor antagonists was promethazine = pyrobutamine greater than pyrilamine greater than diphenhydramine greater than chlorpheniramine. The histamine type-2 agonists, impromidine and dimaprit, and antagonists, cimetidine and ranitidine, as well as selected non-histamine agonists, did not displace [3H]pyrilamine from its binding sites on the natural suppressor cells. The data indicate that the theoretical KD was 1.1 +/- 0.3 X 10(-7) M while the measured KD of binding for [3H]pyrilamine was 6.0 +/- 0.8 X 10(-8) M; the maximum binding was 4.13 nM and the number of binding sites/cell was 2.14 +/- 0.29 X 10(6) (N = 3).     

Effect of H1 and H2 agonists on the chemiluminescence of human blood mononuclear cells induced by phytohaemagglutinin

Previous results have shown a dose-dependent inhibition of the phytohaemagglutinin-elicited chemiluminescent response by histamine on human peripheral blood mononuclear cells (PBMs). The aim of the present experiments was to investigate the receptor specificity of this histamine action. The order of effectiveness of different histaminergic agonists was 4-methylhistamine greater than histamine = impromidine greater than dimaprit much greater than 2-methylhistamine greater than 2-pyridylethylamine. Cimetidine inhibited and mepyramine enhanced this effect of histaminergic agonists. The results are consistent with the view that histamine inhibits the chemiluminescent reaction of PBMs via H2 receptors. Histamine in low doses (10(-8) to 10(-10) M) stimulated the chemiluminescent reaction. Cimetidine enhanced the chemiluminescence-facilitatory action of histamine and unmasked that of 2-pyridylethylamine. It is concluded that histamine is a possible humoral modulator of PBM activity: it is inhibitory via H2 receptors.     

Solubilization, characterization and partial purification of [3H]mepyramine-binding protein, a possible histamine H1 receptor, from rat liver membrane

[3H]Mepyramine binding protein, a possible subtype of histamine H1 receptors, was solubilized from rat liver membrane with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and Tween 60 as detergents and glycerol as an enhancer of solubilization. The optimal concentration of CHAPS was 10 mM and that of glycerol was 20% or more (v/v). The molecular weight of the [3H]mepyramine binding protein-detergent complex was determined to be 670K by Sepharose CL-4B gel filtration and 800K by sucrose density gradient sedimentation. By target size analysis, the molecular weights of both the membrane-bound and solubilized [3H]mepyramine binding protein were determined to be 162K. These values are similar to those of other well-characterized H1-receptor proteins, though slightly different. Simultaneous computerized analysis of the data obtained by [3H]mepyramine binding to the solubilized [3H]mepyramine binding protein indicated the presence of a single binding site with a KD value of 19.0 +/- 5.6 nM and a binding capacity (Bmax) of 6.6 +/- 2.1 pmole/mg protein. The Ki value of cold mepyramine for [3H]mepyramine binding to the solubilized receptor was 20 +/- 4 nM, whereas those of diphenhydramine, d-chlorpheniramine and triprolidine were all 2.9 +/- 0.8 microM, or about 150 times that of mepyramine. These data on the molecular and binding characteristics of the solubilized protein reported here suggest that there is a subtype of histamine H1 receptor in rat liver membrane. The solubilized preparation retained 90% and 75% of its [3H]mepyramine binding activity after storage at -80 degrees C and 4 degrees C, respectively, for 20 days. The solubilized [3H]mepyramine binding protein was purified 30-fold by Sepharose CL-4B gel filtration, Bio Gel HTP hydroxylapatite, Octyl Sepharose 4B and hydroxylapatite HPLC column chromatographies.     

The binding of doxepin to histamine H1-receptors in guinea-pig and rat brain

The affinity constant for doxepin obtained from inhibition of histamine-induced contraction of guinea-pig intestinal smooth muscle at 30 degrees C was 2.6 +/- 0.18 X 10(10)M-1. The slope of a Schild plot was not significantly different from unity. The affinity constant of doxepin did not vary markedly with temperature. At 37 degrees C it was 3.75 +/- 0.02 X 10(10)M-1 and at 25 degrees C 2.1 X 10(10)M-1. Doxepin was a competitive inhibitor of [3H]-mepyramine binding to guinea-pig cerebellar homogenates. The affinity constant derived for doxepin at 30 degrees C was 1.12 +/- 0.45 X 10(10)M-1. Hill coefficients for curves of doxepin or mepyramine inhibition of [3H]-mepyramine binding in guinea-pig cerebellum, cerebral cortex and hippocampus did not differ significantly from unity. The mean affinity of mepyramine for histamine H1-receptors in rat brain homogenates at 30 degrees C was 3.5 X 10(8)M-1. Hill coefficients for curves of doxepin or mepyramine inhibition of [3H]-mepyramine binding to homogenates of rat cerebral cortex or rat whole brain were near unity. These studies provide no evidence that doxepin binds preferentially to a sub-class of histamine H1-receptors in rat brain.     

Characterization of histamine receptors modulating inotropic and biochemical activities in rabbit left atria.

The experiments were performed to identify histamine H1- and H2-receptors in rabbit left atrium and to characterize the pharmacological properties mediated by the respective subtypes of histamine receptors. High-affinity saturable binding to the left atrial membranes was obtained for [3H]mepyramine, yielding a maximum binding capacity (Bmax) of 96 fmol/mg of protein and an equilibrium dissociation constant (KD) of 3.8 nM and also for [3H]tiotidine, yielding a Bmax of 126 fmol/mg of protein and a KD of 14.7 nM. In isolated left atrium, histamine produced a concentration-dependent positive inotropic effect, an effect which was competitively antagonized by cimetidine but not altered by chlorpheniramine. Schild analysis showed that the pA2 value for cimetidine was 6.55 and the slope was not significantly different from unity. An excellent correlation was found between the increase in force of contraction and cyclic AMP in the presence of histamine, suggesting that the positive inotropic effect of histamine in rabbit left atrium is dependent on an increased level of intracellular cyclic AMP through stimulation of histamine H2-receptors. Histamine also produced concentration-dependent stimulation of phosphoinositide hydrolysis as measured by [3H]inositol monophosphate accumulation. The phosphoinositide response to histamine was blocked by chlorpheniramine and mepyramine but not by cimetidine. The data indicate that histamine H1-receptors, in addition to histamine H2-receptors, are present in the rabbit left atrium. Although this tissue lacks an inotropic response to histamine H1-receptor stimulation, the histamine H1-receptors interact with histamine to mediate the stimulation of phosphoinositide hydrolysis.     

Desensitization of histamine H1 receptor-mediated inositol phosphate accumulation in guinea pig cerebral cortex slices.

1. Histamine stimulated the production of [3H]-inositol phosphates in untreated (control) guinea-pig cerebral cortex slices with a best-fit EC50 of 17 +/- 4 microM, and a best-fit maximum response of 385 +/- 23% over basal accumulation. 2. Histamine pretreatment desensitized guinea-pig cortex slices to a subsequent challenge with histamine, which was observed as a reduction in the best-fit maximum response to 182 +/- 32% over basal accumulation. 3. The time-course for the histamine-induced production of [3H]-inositol phosphates was approximately linear over 90 min of stimulation in both control and histamine pretreated slices. The rate of production in pretreated slices was significantly slowed compared to control, such that by 90 min of histamine stimulation the desensitized slices produced 2.8 times the basal [3H]-inositol phosphate accumulation compared to 5.3 fold the basal [3H]-inositol phosphate accumulation in the control slices. 4. Displacement of [3H]-mepyramine binding to homogenates of guinea-pig cerebral cortex by mepyramine and histamine revealed that histamine pretreatment did not alter the apparent affinity of the H1 receptor for histamine (control Kd = 6.3 +/- 0.7 microM, desensitized Kd = 7.9 +/- 1.6 microM) or mepyramine (control Kd = 3.4 +/- 0.8 nM, desensitized Kd = 3.4 +/- 1.3 nM), nor was there any reduction in the calculated maximum number of [3H]-mepyramine binding sites (control Bmax = 192 +/- 31 fmol mg-1 protein, desensitized Bmax = 220 +/- 50 fmol mg-1 protein).(ABSTRACT TRUNCATED AT 250 WORDS)     

Testing the specificity of allosteric modulators of muscarinic receptors in phylogenetically closely related histamine H1-receptors

Gallamine, alcuronium and W84 (hexane-1,6-bis[dimethyl-3'-phthalimidopropyl-ammonium bromide]) are prototype allosteric modulators of the G-protein coupled muscarinic acetylcholine receptor family, especially of the M2-subtype. In order to probe the specificity of muscarinic allosteric modulation, we checked whether these agents interact with histamine H1-receptors which have a high homology with muscarinic receptors. Binding experiments (38 mM Na2HPO4, 12 mM KH2PO4, pH 7.5) were performed with the H1-receptor antagonist [3H]mepyramine ([3H]MEP) in guinea pig cerebellar homogenates. For the sake of comparison, binding of [3H]N-methylscopolamine ([3H]NMS) at muscarinic M2-receptors was measured in porcine cardiac homogenates under identical conditions. The modulators retarded [3H]NMS dissociation (t1/2 control=1.3 min) concentration-dependently indicating their allosteric action with half-maximum effects for gallamine at EC50,discs=27 microM, for alcuronium at EC50,diss=53 nM, and for W84 at EC50,diss=170 nM. In contrast, [3H]MEP dissociation from H1-receptors (t1/2,control=2.6 min) remained unchanged up to concentrations of 1 mM of the modulators. Equilibrium binding of [3H]NMS (KD=0.46 nM, Bmax=98 fmol/mg protein) was inhibited by gallamine, elevated by alcuronium and left almost unchanged by W84, indicating negative, positive and nearly neutral cooperativity, respectively, with the radioligand. The ternary complex model of allosteric actions yielded the equilibrium dissociation constants K(A) for the binding of the allosteric modulators to free M2-receptors: K(A,gallamine)=100 nM, K(A,alcuronium)=450 nM, K(A,W84)=69 nM. In H1-receptors, more than 1,000-fold higher concentrations than in M2-receptors were required to elicit an effect on the binding of [3H]MEP (KD=1.2 nM, Bmax=205 fmol/mg protein). Half-maximal reduction was observed at 10 mM for gallamine, 1 mM for alcuronium and 92 microM for W84. In conclusion, the muscarinic modulators have little effect on the histamine H1-receptors.     

Characterization of 5-HT3 receptors in intact N1E-115 neuroblastoma cells

The highly selective 5-HT3 receptor antagonist [3H]GR65630 has been used to characterize 5-HT3 receptors in intact N1E-115 neuroblastoma cells. Equilibrium binding analysis demonstrated high-affinity binding to a single class of receptors with a Kd of 0.69 (+/- 0.12) nM and Bmax of 31.4 (+/- 11.4) fmol/10(5) cells, equivalent to approximately 200,000 sites per cell. Specific binding was displaced by low concentrations of 5-HT3-selective ligands, and by the nicotinic antagonist d-tubocurarine.