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: Bmax = 34.6 fmol [3H]mepyramine/mg protein, KD = 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.     

Postsynaptic alpha 2-adrenoceptors predominate over alpha 1-adrenoceptors in canine tracheal smooth muscle and mediate neuronal and hormonal alpha-adrenergic contraction

Alpha-adrenoceptor subtypes in canine tracheal smooth muscle were investigated by radioligand binding and by in vitro responses of muscle strips to electrical field stimulation and exogenous alpha-agonists. [3H]Yohimbine identified a high density of alpha 2-receptors (51.4 +/- 4.9 fmoles/mg of protein; n = 5) in tracheal smooth muscle membranes, whereas [3H]prazosin revealed a low density of alpha 1-receptors (11.1 +/- 2.9 fmoles/mg of protein; n = 5). In peripheral lung membranes, however, alpha 1-receptors predominated (46.8 +/- 7.7 fmoles/mg of protein; n = 4) over alpha 2-receptors (4.1 +/- 1.5 fmoles/mg of protein; n = 4). After pretreatment with atropine and propranolol and precontraction with serotonin or histamine, the contractile response of tracheal smooth muscle to electrical field stimulation was partially inhibited by 0.3 microM prazosin (16%), potently inhibited by 0.3 microM yohimbine (89%), and abolished by a combination of the two drugs. The response to neuronally released norepinephrine is therefore mediated predominantly by alpha 2-receptors. The rank order of potency for adrenergic agonists was clonidine greater than norepinephrine greater than phenylephrine in both competition studies with [3H]yohimbine and in contraction studies, signifying a predominance of postsynaptic alpha 2-receptors. The contractile responses to exogenous norepinephrine, clonidine, and phenylephrine were only weakly inhibited by 0.3 microM prazosin but markedly inhibited by 0.3 microM yohimbine, with a Kb of 1.2 nM, which was similar to the Kd of [3H]yohimbine binding to airway smooth muscle membranes (2.7 nM).     

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.     

Histamine H1-agonist potentiation of adenosine-stimulated cyclic AMP accumulation in slices of guinea-pig cerebral cortex: comparison of response and binding parameters.

1 A range of histamine analogues have been examined as potentiators of the adenosine-stimulated accumulation of cyclic adenosine 3',5'-monophosphate (cyclic AMP) in slices of guinea-pig cerebral cortex. Dose-response curves were constructed for the 6 most active compounds and characterized in terms of the IC50, the slope and the maximum response attainable relative to that of histamine. 2 Histamine, 2-thiazolylethylamine and N alpha-methylhistamine produced a maximal or near maximal response. N alpha, N alpha-dimethylhistamine and 2-methylhistamine appear to be partial agonists. 3 The response to all the agonists was practically abolished by mepyramine 1 microM, indicating that the response is mediated largely or wholly via histamine H1-receptors. 4 The relative potencies of the agonists on cyclic AMP accumulation were in general similar to relative potencies in causing contraction of intestinal smooth muscle. The biggest difference was observed with N alpha-methylhistamine. 5 The histamine analogues were also examined as inhibitors of [3H]-mepyramine binding in homogenates of guinea-pig cerebral cortex. The inhibition curves were characterized in terms of IC50, the slope and the maximum percentage inhibition. This last value was compared with the inhibition produced by promethazine 2 microM. 6 For the 6 most potent agonists, the EC50 for cyclic AMP accumulation was compared with the IC50 against [3H]-mepyramine binding, corrected for inhibition of non-receptor binding and for competition with [3H]-mepyramine. With the possible exception of 2-pyridylethylamine, the values did not differ by more than a factor of 3.     

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.     

Vasomotor effect of histamine on pig and cattle coronary artery in vitro.

Vasomotor effects of histamine were examined in isolated coronary arteries from pigs and cattle. Histamine produced a concentration-dependent contraction in these arteries. These contractile responses were dose-dependently inhibited by diphenhydramine. The slopes of the Schild plots, however, were significantly lower than unity in both species. Cimetidine potentiated the histamine-induced contractions at relatively high doses of histamine (larger than 10(-5) M) in pig coronary arteries, but did not show a significant effect in cattle arteries. After the removal of endothelium, the Schild plot of diphenhydramine against histamine gave a straight line with a pA2 value of 7.80 and slope of 1.00 in pigs, confirming the competitive nature of the antagonism. In cattle, the slope was significantly lower than unity; however, in the presence of cimetidine, it was not significantly different from unity. Dimaprit did not contract the cattle coronary arteries with endothelium, but contracted them after the removal of endothelium. These results suggest that histamine-induced vasoconstriction in pig and cattle coronary arteries is mainly dependent on the H1-receptors in the smooth muscle cells, and that H1- and H2-receptors in the endothelial cells of pigs and H2-receptors in the smooth muscle cells of cattle modify the histamine-induced vasoconstrictions.     

Phosphoinositide hydrolysis mediated by histamine H1-receptors in rat brain cortex

Histamine stimulated the accumulation of [3H]inositol 1-phosphate in the presence of lithium in [3H]inositol-prelabelled slices from rat brain cortex in a concentration-dependent manner, with an EC50 value of 94.7 microM. High concentrations of antagonists of histamine H2 receptors, muscarinic receptors, alpha 1-adrenoceptors and serotonin receptors did not inhibit the effect. The histamine H1-receptor antagonists mepyramine, triprolidine, promethazine, d-chlorpheniramine and the tricyclic antidepressant doxepin inhibited the response with Ki values corresponding to an interaction with histamine H1-receptors. The EC50 for the response was about three times lower than the Ki value (approximately 300 microM) for the inhibition by histamine of [3H]mepyramine binding to membranes from rat brain cortex. Partial inactivation of H1-receptors with the alkylating antagonist phenoxybenzamine resulted in similar reductions in [3H]mepyramine binding sites and in the maximal histamine-induced [3H]inositol 1-phosphate accumulation, without affecting the KD for the radioligand or the EC50 for the response. The apparent dissociation constant for histamine calculated from these experiments (KA = 92.2 microM) was not different from the EC50 value. The present results indicate that histamine-stimulated phosphoinositide hydrolysis in rat brain cortex is mediated by H1-receptors and that no receptor reserve is present.     

Role of the endothelium in the vasodilator response of rat thoracic aorta to histamine

Despite their potent vasodilating action in vivo, acetylcholine and histamine often show a vasoconstricting action in vitro. As the endothelium has an important role in the vasodilating effect of acetylcholine, we investigated the possible role of the endothelium in the vasodilating effect of histamine in comparison to acetylcholine. Experiments were done on ring segments of rat thoracic aorta mounted for isometric tension measurements. We demonstrated that relaxation by histamine and acetylcholamine of pre-contracted rat aorta segments required the presence of endothelial cells. Acetylcholine acting on muscarinic receptors, and histamine acting on H₁-receptors seemed to initiate the production of mediator(s) from the endothelial cells, which leads to relaxation of the vascular smooth muscle cells. This production appeared to be depressed by ETYA and hydroquinone, and under hypoxic conditions.     

5-HT1B and 5-HT2 serotonin binding sites in cultured Wistar-Kyoto rat aortic smooth muscle cells

We studied [3H]serotonin [( 3H]5-HT) binding on cultured arterial smooth muscle cells from rat aorta. We found a high and low affinity binding site. Binding to the higher affinity site could be displaced by drugs in an order corresponding most closely with the 5-HT1B subtype, and high affinity [125I]iodo-cyanopindolol binding was also found. We found evidence for a 5-HT2 subtype using [3H]ketanserin binding, with similar results whether specific binding was determined using unlabelled ketanserin, methysergide or mianserin.     

The effect of dopamine D1 receptor stimulation on the up-regulation of histamine H3-receptors following destruction of the ascending dopaminergic neurones.

1. The binding of [3H]-(R)alpha-methylhistamine and [3H]-N alpha-methylhistamine to histamine H3-receptors, [3H]-SCH23390 to dopamine D1-receptors, and [3H]-YM09151-2 to dopamine D2-receptors was investigated by quantitative receptor autoradiography in the rat brain following 6-hydroxydopamine injection into the substantia nigra. 2. The levels of [3H]-(R)alpha-methylhistamine binding sites in the denervated striatum and substantia nigra were significantly higher than those in the contralateral side from 1 week to 12 weeks after nigral lesions. The H3-receptor binding was maximal at 3 weeks after nigral lesions and maintained until 12 weeks. 3. The increased number of histamine H3-receptors was decreased to the level of the contralateral side by chronic treatment with a selective dopamine D1 agonist, SKF38393, but not modified by a selective dopamine D2 agonist, quinpirole. 4. Dopamine D1- and D2-receptors in the striatum were similarly up-regulated after unilateral nigral lesion. On the other hand, the number of dopamine D2-receptors in the substantia nigra was markedly decreased after administration of 6-hydroxydopamine. 5. The treatment with (S)alpha-fluoromethylhistidine increased the H3-receptor binding in both the ipsilateral and contralateral sides. As a result, the magnitude of the ratio of the H3-receptor binding between ipsilateral and contralateral sides was partially attenuated by treatment with (S)-alpha-fluoromethylhistidine. 6. These results strongly suggest that the expression of histamine H3-receptors in the striatum and substantia nigra is influenced through D1-receptors by tonic nigrostriatal dopaminergic inputs.     

Atropine- and scopolamine-resistant subtypes of muscarinic receptors in the rabbit aorta

Tritiated acetylcholine ([3H]ACh) binds specifically to different muscarinic binding sites in the rabbit aorta. The binding of [3H]ACh to endothelial membranes was displacable by nanomolar concentrations of scopolamine but only by micromolar concentrations of atropine and homatropine. The reverse was true for smooth muscle membranes, i.e. [3H]ACh binding was displacable by nanomolar concentrations of atropine and homatropine but only by micromolar concentrations of scopolamine. Pirenzepine, AF-DX 116 and 4-diphenyl-acetoxy-N-methylpiperidine methobromide (4-DAMP) displaced the binding of [3H]ACh from both tissues in the nano- to micromolar range. Our findings indicate that endothelial receptors are characterised by a high affinity for scopolamine, which possesses a scopine base, and that muscle binding sites have a high affinity for antagonists possessing a tropine base (atropine, homatropine). Both the endothelial and smooth muscle binding sites have a low affinity for AF-DX 116, indicating that they are not of the cardiac type.     

Preclinical pharmacology of bilastine, a new selective histamine H1 receptor antagonist: receptor selectivity and in vitro antihistaminic activity

OBJECTIVE: This study aimed to establish the receptor selectivity and antihistaminic activity of bilastine, a new selective antihistamine receptor antagonist. DESIGN AND METHODS: In vitro experiments were conducted using a receptor binding screening panel and guinea-pig and rat tissues. Antihistaminic activity was determined using H1 receptor binding studies and in vitro H1 antagonism studies conducted in guinea-pig tissues and human cell lines. Receptor selectivity was established using a receptor binding screening panel and a receptor antagonism screening conducted in guinea-pig, rat and rabbit tissues. Inhibition of inflammatory mediators was determined through the Schultz-Dale reaction in sensitised guinea-pig ileum. RESULTS: Bilastine binds to histamine H1-receptors as indicated by its displacement of [3H]-pyrilamine from H1-receptors expressed in guinea-pig cerebellum and human embryonic kidney (HEK) cell lines. The studies conducted on guinea-pig smooth muscle demonstrated the capability of bilastine to antagonise H1-receptors. Bilastine is selective for histamine H1-receptors as shown in receptor-binding screening conducted to determine the binding capacity of bilastine to 30 different receptors. The specificity of its H1-receptor antagonistic activity was also demonstrated in a series of in vitro experiments conducted on guinea-pig and rat tissues. The results of these studies confirmed the lack of significant antagonism against serotonin, bradykinin, leukotriene D4, calcium, muscarinic M3-receptors, alpha1-adrenoceptors, beta2-adrenoceptors, and H2- and H3-receptors. The results of the in vitro Schultz-Dale reaction demonstrated that bilastine also has anti-inflammatory activity. CONCLUSIONS: These preclinical studies provide evidence that bilastine has H1- antihistamine activity, with high specificity for H1-receptors, and poor or no affinity for other receptors. Bilastine has also been shown to have anti-inflammatory properties.     

Synthesis and binding assays of H3-receptor ligands.

The preparation of a representative group of derivatives of the known H3-antagonist thioperamide is described. Binding affinity for histamine H3-receptors of thioperamide and its derivatives, which were obtained by substitution on the imidazole ring, was measured on rat brain cortex synaptosomes. Competitive binding assays were performed with two different labelled ligands, the physiological agonist [3H]histamine ([3H]HA) and the potent H3-agonist N alpha-[3H]methyl-histamine ([3]NAMHA). We observed a remarkable difference in Ki values obtained versus the two labelled ligands, both for thioperamide and its derivatives. In particular, 5-methylthioperamide showed a considerable selectivity for the system recognized by [3H]NAMHA, being about 100 times more potent versus this system than versus the system recognized by [3H]HA. On the basis of these observations, we suggest that it is necessary to consider this difference in evaluating the affinity of new compounds for the H3-receptors.     

Adenosine transporters in vascular smooth muscle and endothelium: Multiple [3H]Nitrobenzylthioinosine binding sites in human umbilical vein endothelium

Cultured vascular smooth muscle and endothelial cells may be useful models for studying the cardiovascular adenosine transport system and metabolism. We have quantified the nucleoside transporter elements of cultured primate vascular smooth muscle, bovine aortic endothelial, and human umbilical vein endothelial cells by radioligand binding and by using membrane preparations of these cells and the nucleoside transporter probe nitrobenzylthioinosine ([³H]NBMPR), a potent and tightly bound inhibitor of nucleoside transport. The binding was rapid, reversible, saturable, and site-specific. Scatchard analysis of the saturation data showed that [³H]NBMPR bound to high and low affinity binding sites in human umbilical vein endothelial cell membranes with apparent binding affinities (K_D) of 0.093±0.01 nM 1.92±0.1 nM, and binding site densities (Bmax values) of 13.48±1.2 and 69+5.3 fmol/mg protein, respectively. In contrast, the binding to primate vascular smooth muscle and bovine aortic endothelial cell membranes occurred to an apparently high affinity single class of binding sites at which the K_D was 1.4±0.3nM and 0.28±0.05nM, respectively, and which had Bmax values of 1,977±163 and 1,284±267 fmol/mg protein, respectively. Scatchard analysis of the binding inhibition by dipyridamole showed a mixed type inhibition, while NBMPR inhibited the binding competitively. Several recognized nucleoside transport inhibitors and vasodilators inhibited the binding with an order of potency similar to that observed for the inhibition of [³H]NBMPR binding to guinea pig cardiac membranes.     

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 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.     

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.     

Solubilization and characterization of the pyrilamine-binding protein from cultured smooth muscle cells

The cultured smooth muscle cell line DDT1MF-2 expresses a large number (9.7 x 10(6) receptors/cell) of functional histamine H1-type receptors [J. Cell. Physiol. 134:367-375 1988]. Two different binding assays, gel filtration and polyethylene glycol precipitation, indicated that the [3H]pyrilamine binding activity was solubilized by 1% digitonin with binding characteristics similar to those of intact cells. The solubilized proteins were then purified by sequential gel filtration, chromatofocusing, and reverse phase high pressure liquid chromatography. The calculated molecular weight of this purified pyrilamine-binding protein was 38-40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]Pyrilamine binding to these 38-40-kDa proteins indicated a single class of binding site with a Kd of 288 nM, which is equivalent to that of intact cells and digitonin-solubilized proteins. The computer analysis Scatfit also indicated that one molecule of [3H]pyrilamine bound to one molecule of purified protein. Furthermore, a polyclonal antibody raised against the purified protein recognized the 38-40-kDa band by Western blotting techniques, specifically bound to the cell surface of DDT1MF-2 cells, and inhibited [3H]pyrilamine binding to these cells in a dose-dependent manner. These data strongly suggest that the purified 38-40-kDa protein is part of an antagonist binding domain on the histamine H1 receptor on DDT1MF-2 cells.     

Zolantidine (SK&F 95282) is a potent selective brain-penetrating histamine H2-receptor antagonist.

1. The novel benzthiazole derivative zolantidine (SK&F 95282) is a potent antagonist of histamine at H2-receptors in guinea-pig atrium and rat uterus. Only apparent pA2 values of 7.46 and 7.26 respectively could be calculated since the slopes of the Schild plots were significantly less than unity. 2. Zolantidine is equally potent as an antagonist at histamine H2-receptors in guinea-pig brain. The compound inhibited histamine stimulated adenylate cyclase (pKi 7.3) and dimaprit stimulated adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation (approx pA2 7.63), and competed with [3H]-tiotidine binding (pKi 7.17). 3. Zolantidine is at least 30 fold more potent at H2-receptors than at other peripheral and central receptors investigated. 4. Infusion of zolantidine into rats produces a brain concentration greater than the plateau blood concentration (brain/blood ratio 1.45). 5. Zolantidine is thus characterized as a potent selective brain-penetrating H2-receptor antagonist, and will be a valuable pharmacological tool for investigating possible physiological and pathological roles for histamine in the central nervous system.     

An autoradiographic study of muscarinic cholinoceptors in blood vessels: no localization on vascular endothelium

In vitro labelling and autoradiographic techniques were used to examine the localization of [3H]quinuclidinyl benzilate ([3H]QNB) and [125I]4-iodo-QNB ([125I]4IQNB) to slide-mounted sections of rabbit aorta and pulmonary artery, cat aorta, pulmonary and superior mesenteric arteries. These vessels all respond to acetylcholine (ACh) with endothelium-dependent relaxation, yet there was no evidence for endothelium-related binding of either [3H]QNB or [125I]4IQNB. Muscarinic receptors were localized over the medial smooth muscle and, in the rabbit pulmonary artery, the density of binding increased towards the adventitia. Binding of either radioligand to sections of rabbit pulmonary artery was not affected by the muscarinic M1 receptor antagonist pirenzepine (20 nM) but was markedly reduced by the muscarinic M2 antagonist 4DAMP (4-diphenylacetoxy-N-methyl-piperidine methobromide) (1 nM). This study provides evidence for muscarinic receptors located directly on smooth muscle cells, indicating that endothelium-dependent relaxation to ACh results from an indirect mechanism involving smooth muscle muscarinic receptors.