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1.
1. Ketamine has a number of effects that suggest that it may interact with α- and β-adrenoceptors. To date, the experimental evidence for this has been indirect and has been based on physiological studies using competitive blocking agents. In the present study we sought to determine from receptor binding studies whether ketamine binds directly to α- and β-adrenoceptors. 2. Membrane preparations o. α1- and β2-adrenergic binding sites were obtained from urinary bladder and urethrae of sheep. These binding sites were characterized by saturation analyses using [3H]-prazosin for α1-adrenoceptor binding sites and [125I]-cyanopindolol (CYP) for the β2-adrenoceptor binding sites. The receptors were further characterized by displacement studies using selective and non-selective antagonists. 3. Studies in which ketamine was used to displac. [3H]-prazosin revealed a Kd of 3.40±1.23× 10?3 mol/L for ketamine binding to ai-adrenoceptors. Displacement studies of [125I]-CYP by ketamine showed a Kd of 0.35±0.03× 10?3 mol/L for ketamine binding to β2-adrenoceptors. 4. We conclude that ketamine interacts directly with both ai- an. β2-adrenoceptors and that such interactions probably explain the reported effects of this agent on the vasculature and the bronchial tree. 相似文献
2.
David B. Wainscott D. A. Sasso J. David Kursar Melvyn Baez Virginia L. Lucaites D. L. Nelson 《Naunyn-Schmiedeberg's archives of pharmacology》1997,357(1):17-24
In previous reports, [3H]5-HT has been used to characterize the pharmacology of the rat and human 5-HT2B receptors. 5-HT, the native agonist for the 5-HT2B receptor, has a limitation in its usefulness as a radioligand since it is difficult to study the agonist low-affinity state
of a G protein-coupled receptor using an agonist radioligand. When using [3H]5-HT as a radioligand, rauwolscine was determined to have relatively high affinity for the human receptor (Ki human = 14.3 ± 1.2 nM, compared to Ki rat = 35.8 ± 3.8 nM). Since no known high affinity antagonist was available as a radioligand, these studies were performed
to characterize [3H]rauwolscine as a radioligand for the cloned human 5-HT2B receptor expressed in AV12 cells. When [3H]rauwolscine was initially tested for its usefulness as a radioligand, complex competition curves were obtained. After testing
several α2-adrenergic ligands, it was determined that there was a component of [3H]rauwolscine binding in the AV12 cell that was due to the presence of an endogenous α2-adrenergic receptor. The α2-adrenergic ligand efaroxan was found to block [3H]rauwolscine binding to the α2-adrenergic receptor without significantly affecting binding to the 5-HT2B receptor and was therefore included in all subsequent studies. In saturation studies at 37° C, [3H]rauwolscine labeled a single population of binding sites, Kd = 3.75 ± 0.23 nM. In simultaneous experiments using identical tissue samples, [3H]rauwolscine labeled 783 ± 10 fmol of 5-HT2B receptors/mg of protein, as compared to 733 ± 14 fmol of 5-HT2B receptors/mg of protein for [3H]5-HT binding. At 0° C, where the conditions for [3H]5-HT binding should label mostly the agonist high affinity state of the human 5-HT2B receptor, [3H]rauwolscine (Bmax = 951 ± 136 fmol/ mg), again labeled significantly more receptors than [3H]5-HT (Bmax = 615 ± 34 fmol/mg). The affinity of [3H]rauwolscine for the human 5-HT2B receptor at 0° C did not change, Kd = 4.93 ± 1.27 nM, while that for [3H]5-HT increased greatly (Kd at 37° C = 7.76 ± 1.06 nM; Kd at 0° C = 0.0735 ± 0.0081 nM). When using [3H]rauwolscine as the radioligand, competition curves for antagonist structures modeled to a single binding site, while agonist
competition typically resulted in curves that best fit a two site binding model. In addition, many of the compounds with antagonist
structures displayed higher affinity for the 5-HT2B receptor when [3H]rauwolscine was the radioligand. Typically, ∼ 85% of [3H]rauwolscine binding was specific binding. These studies display the usefulness of [3H]rauwolscine as an antagonist radioligand for the cloned human 5-HT2B receptor. This should provide a good tool for the study of both the agonist high- and low-affinity states of the human cloned
5-HT2B receptor.
Received: 26 June 1997 / Accepted: 30 August 1997 相似文献
3.
《General pharmacology》1995,26(2):347-351
- 1.1. The aging changes of density of the α1-adrenoceptors in the kidney were evaluated with Wistar rats of several ages (8, 52 and 104 weeks old).
- 2.2. [3H]prazosin and [3H]YM617 (newly synthesized α1-blocker) were used for the ligand. The Bmax of [3H]prazosin was 74.0 ± 9.5 fmol/mg/protein in 8 week, 52.1 ± 7.3 fmol/mg protein in 52 week, and 31.3 ± 4.2 fmol/mg/protein in 104 week rats, and that of [3H]YM617 was 45.0 ± 6.6 fmol/mg/protein in 8 week, 32.4 ± 5.7 fmol/mg/protein in 52 week, and 19.3 ± 5.5 fmol/mg/protein in 104 week rats.
- 3.3. The Bmax of both ligands for 104 week rats was significantly decreased compared to 8 week rats, however, 52 week rats showed no decrease of Bmax for both ligands.
- 4.4. The Kd values showed no difference in these three age groups for both ligands.
- 5.5. Autoradiographic study supported the result above mentioned. Furthermore, the binding sites of α1-adrenoceptors were mainly in the cortex (vascular wall and peritubular area) and that α1-adrenoceptors were chiefly chlorethylclonidine dihydrochloride (CEC) insensitive.
4.
The imipramine binding sites located in the membrane of the rat hippocampus have been characterized in order to obtain a better understanding of their role in the treatment of mental depression. The binding of [3H]imipramine to hippocampal membranes has a Kd of 8.3 ± 1.5 nM, Bmax of 800 ± 60 fmol/mg protein and a Hill coefficient of 1.0. The number of specific binding sites for [3H]imipramine was rapidly and irreversibly reduced when the incubation temperature was raised from 0 to 37°C. Experiments using leupeptin and EGTA suggest that this temperature-dependent inactivation is due to a destruction of the binding sites by Ca2+-dependent proteases released upon disruption of the cells. The binding of [3H]imipramine to the hippocampal membrane is also sensitive to phospholipase A2 as well as pronase, suggesting the complexity of the regulation of this binding. The [3H]imipramine binding sites appear to have been solubilized from membranes by treatment with Triton X-100. However, these solubilized binding sites also display a high level of nonspecific binding of this ligand. Studies of tissue distribution and subcellular localization of the imipramine binding sites suggest that these sites are located in neurons. This possibility is strengthened by the finding that the high affinity imipramine binding sites are also detected in the membranes of neuroblastoma N4TG1 cells. 相似文献
5.
John A. Manderson Ian P. Hayward Elena Pak Sophie Horrigan Glenys E. Hanley Jennifer A. Stephenson† Lindsay Brown‡ Julie H. Campbell Gordon R. Campbell 《Clinical and experimental pharmacology & physiology》1995,22(12):912-918
1. This study has defined α1-adrenoceptors and their reactivity in rabbit aorta, following removal of the endothelium and formation of a myointimal thickening, and also in smooth muscle cells (SMC) in cell culture which had undergone serial passaging and changes in phenotype. 2. [3H]-prazosin binding to SMC from control aorta, vessels 2 weeks after endothelial denudation and sub-cultured SMC (passage 3–6) was specific (displaceable with 10 μmol/L phentolamine), and of high affinity to a single class of sites (Kd range: 71–114 pmol/L). The maximum binding density (Bmax) of α1-adrenoceptors on SMC from the neointima (11105 ± 771 sites/cell) was not significantly different to that of control medial SMC (14014 ± 2472 sites/cell). However, SMC cultured to passage 6, showed a 2-fold increase in Bmax (30227 ± 4349 sites/cell). 3. The production of inositol phosphates (IP1, IP2 and IP3) by SMC following 10μmol/L phenylephrine was assayed. Both freshly-dispersed aortic SMC and sub-cultured SMC were stimulated to produce increased inositol phosphates by the addition of phenylephrine which was completely inhibited by pre-incubation with 10 μmol/L phentolamine, suggesting that the stimulation was via α1-adrenoceptors. 4. Maximal contractile responses of isolated thoracic and abdominal aortic rings to KCl (100 mmol/L), 5-HT and phenylephrine were unchanged two weeks after endothelial denudation. However, phenylephrine was significantly less potent (2.7-fold) in both areas of the aorta, while the potency of 5-HT was significantly enhanced (2.7-fold) after endothelial denudation only in the abdominal aorta. 5. The decreased sensitivity of the rabbit aorta to α1-adrenoceptor agonists following endothelial denudation and the formation of a myointimal thickening is not due to changes in affinity or density of α1-adrenoceptors. However multiple passaging of SMC in culture leads to an increase in α1-adrenoceptor density. This change can be related to the altered cytodifferentiation of irreversible synthetic state SMC which are similar to those in atherosclerotic lesions. 相似文献
6.
7.
Adult female and male green monkey liver cytosol contains tritiated 17β-estradiol ([3H]E2) binding sites. The binding of [3H]E2 to these sites is reduced substantially by diethylstilbestrol. Unlike adult male rate liver cytosol, neither male nor female green monkey liver cytosol contains moderate affinity high capacity [3H]E2 binding sites. The green monkey liver cytosol [3H]E2 sites are precipitated by ammonium sulfate at 30% of saturation. The properties of the redissolved ammonium sulfate precipitated female and male [3H]E2 binding sites are similar: they have a high [3H]E2 affinity, a low [3H]E2 capacity, are estrogen specific, and are sensitive to sulfhydryl inactivation and protease digestion. Although the female and male sites are similar, there appears to be a 2- to 3-fold sex difference in the affinity (female Kd, 0.8 to 1.3 × 10?10 M; male Kd, 0.4 × 10?10M) and capacity (female, 2.3 to 3.7 fmoles/mg of liver; male, 1.2 fmoles/mg of liver)of the [3H]E2 binding sites. Accordingly, adult female and male green monkey liver cytosol contains putative estradiol receptors. 相似文献
8.
ATSUYUKI KOHARA MASAMICHI OKADA RIE TSUTSUMI KAZUSHIGE OHNO MASAYASU TAKAHASHI MASAO SHIMIZU-SASAMATA JUN-ICHI SHISHIKURA HIROSHI INAMI SHUICHI SAKAMOTO TOKIO YAMAGUCHI 《The Journal of pharmacy and pharmacology》1998,50(7):795-801
The in-vitro pharmacological properties of (2,3-dioxo-7-(1H-imidazol-***1-yl)-6-nitro-1,2,3,4-tetrahydro-1-quinoxalinyl)-acetic acid monohydrate, YM872, a novel and highly water-soluble α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor antagonist were investigated. YM872 is highly water soluble (83 mg mL?1 in Britton-Robinson buffer) compared with 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX), 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione hydrochloride (YM90K) or 6-cyano-7-nitroquinoxa-line-2,3-dione (CNQX). YM872 potently inhibits [3H]AMPA binding with a Ki (apparent equilibrium dissociation constant) value of 0.096 ± 0.0024 μM. However, YM872 had very low affinity for other ionotropic glutamate receptors, as measured by competition with [3H]kainate (high-affinity kainate binding site, concentration resulting in half the maximum inhibition (IC50) = 4.6 ± 0.14 μm), [3H]glutamate (N-methyl-D-aspartate (NMDA) receptor glutamate binding site, IC50 > 100 μM) and [3H]glycine (NMDA receptor glycine-binding site, IC50 > 100 μM). YM872 competitively antagonized kainate-induced currents in Xenopus laevis oocytes which express rat AMPA receptors, with a pA2 value of 6.97 ± 0.01. In rat hippocampal primary cultures, YM872 blocked a 20-μM AMPA-induced increase of intracellular Ca2+ concentration with an IC50 value of 0.82 ± 0.031 μM, and blocked 300-μM kainate-induced neurotoxicity with an IC50 value of 1.02 μM. These results show that YM872 is a potent and highly water-soluble AMPA antagonist with great potential for treatment of neurodegenerative disorders such as stroke. 相似文献
9.
《Pulmonary pharmacology》1993,6(2):119-127
Summary: Muscarinic receptors present in guinea-pig lung were characterized using the M3 selective radioligand [3H]4-diphenylacetoxy-N-methyl-piperidine methiodide ([3H]4-DAMP). In saturation studies, [3H]4-DAMP identified two populations of binding sites with approximately 4% of the sites displaying high affinity (Kd=0.21 nM and Bmax= 10 fmol/mg prot.) while the remaining sites were low affinity ones (Kd=18.11 nM and Bmax=269 fmol/mg prot.). In competition studies with [3H]4-DAMP (0.35 nM), methoctramine and hexahydro-siladifenidol (HHSiD) identified 50 and 70% of high affinity binding sites displaying the pharmacological profile of the M2 and the M3 receptors, respectively. No evidence was found for high affinity [3H]pirenzepine binding sites in guinea-pig lung. However, pirenzepine/[3H]4-DAMP competition experiments suggested that pirenzepine recognized an equal proportion of [3H]4-DAMP binding sites with intermediate and low affinity binding constants. The intermediate affinity binding constant was inconsistent with the presence of M1 receptors and reflected more the presence of M4 or a mixture of M3 and M4 receptors. The low affinity pirenzepine binding sites may represent M2 receptors. These results provide further evidence for the occurrence of M2 and M3 receptors and suggest the presence of the M4 muscarinic receptor subtype in guinea-pig lung. 相似文献
10.
Silvia Gascn Mara Dierssen Frederic Mrmol Nuria M. Vivas Albert Badia 《The Journal of pharmacy and pharmacology》1993,45(10):907-909
Abstract— The effects of ageing on α1-adrenoceptor subtypes have been examined in heart ventricular muscle of young (2–3 months) and middle-aged (18 months) Sprague-Dawley rats. Radioligand binding studies with [3H]prazosin revealed an age-related loss of binding sites (Bmax 56·7 ± 1·93 fmol (mg protein)?1 age 2 months vs 31·7 ± 2·45 fmol (mg protein)?l age 18 months) not followed by changes in the dissociation constant value (Kd 0·16 ± 0·03 nm age 2 months and 0·10 ± 0·03 nm age 18 months). Competition curves with WB 4101 showed two distinct sites with different affinities, the proportion of sites with high affinity being similar for both age groups (22·2 ± 1·89% vs 17·8 ± 1·96% for animals aged 2 and 18 months, respectively). Agonist displacement curves of [3H]prazosin indicate the existence of two different affinity sites for the agonist, that are maintained regardless of the ageing process (Rhigh= 16·2 ± 1·54% and Rlow = 83·8 ± 1·89% in rats aged 2 months and Rhigh= 16·3 ± 3·23% and Rlow = 83·7 ± 3·95% in rats aged 18 months). The fractional inactivation of α1-adrenoceptors by chloroethylclonidine resulted in a loss of [3H]prazosin specific binding, and a percentage of 22·5 ± 0·95 and 22·6 ± 4·2 of remaining binding sites for the groups of 2 and 18 months of age, respectively. The percentage of chloroethylclonidine-insensitive [3H]prazosin binding sites was similar to those with high affinity for WB 4101. The present study confirms a decline of α1-adrenoceptors with increasing age and reveals that the equilibrium of the expression of the two existing subpopulations of the receptor is maintained during ageing. 相似文献
11.
Guido V. Marinetti Stephen I. Rosenfeld Patricia A. Thiem John J. Condemi John P. Leddy 《Biochemical pharmacology》1983,32(13):2033-2043
Owing to the large differences in reported values for β-adrenergic receptor numbers and binding affinity in normal leukocytes, we undertook a systematic re-examination of the binding of two widely used beta antagonists, (-)-[3H]dihydroalprenolol (DHA) and (HYP), to intact normal mononuclear (MN) leukocytes and polymorphonuclear (PMN) leukocytes and membrane preparations. Assays were conducted in the presence and absence of chloroquine, which has been proposed recently to eliminate ligand uptake into a non-receptor cell compartment such as lysosomes. The binding curves relating radioligand concentration to specific sitesper intact cell were biphasic. At high (10–24 nM) (-)-DHA ligand concentration in the absence of chloroquine, a large number (20,000–60,000 sites/cell) of low affinity (Kd 12–15 nM) stereospecific binding sites were detected in both cell types. This class of binding sites was eliminated by 10 ,μM chloroquine not only in PMN cells but also in the lysome-poor MN cells (? 90% lymphocytes), leaving 2000–3000 specific high affinity (-)-DHA sites/cell. In the absence of chloroquine, comparably low numbers of specific high affinity binding sites/cell were also obtained by the use of appropriately low concentrations of (-)-DHA or (±)-HYP (800 pM or less). However, even at these low radioligand concentrations chosen to measure high affinity specific binding, the addition of 10 μM chloroquine produced a moderate reduction in the number of sites/cell, without a detectable change in the apparent Kd. Mean (± S.E.M.) site numbers obtained in the presence of chloroquine were: 1331 ± 100 sites/MN cell and 1135 ±129 sites/PMN cell (Kd 143–153 pM) using (-)-DHA; and 1487 ± 210 sites/MN cell and 1065 ± 69 sites/PMN cell [avg. Kd(±) 224–274 pM] using (±)-HYP. Chloroquine had no effect on agonist-stimulated cAMP production but produced an apparent increase in the effectiveness of (-)-propranolol as an inhibitor of DHA binding. Competition studies on the binding of DHA and HYP with zinterol and practolol confirmed that the receptor was of the β2-subtype for both MN and PMN cells. The detection of a moderately larger number of high affinity binding sites at saturation (Scatchard analysis) by (±)-HYP than by (-)-DHA was a consistent finding with either intact cells or membranes, with or without chloroquine. The possible overestimation of receptor numbers by a racemic ligand such as (±)-HYP is discussed and leads us to favor the use of a pure stereoisomer such as (-)-DHA. A system employing 800 pM (-)-[3H]DHA, 1 ,μM (-)-propranolol and 10, μM chloroquine with intact MN and PMN cells yielded reproducible and plausible results. Our values for β-adrenergic receptor numbers of intact MN and PMN cells and membranes are compared to others in the literature. 相似文献
12.
Roger G. Evans John M. Haynes 《Clinical and experimental pharmacology & physiology》1994,21(8):649-658
1. We characterized the binding of [3H]-rauwolscine, [3H]-p-aminoclonidine and [3H]-idazoxan in a dog kidney membrane preparation. Our aim was to determine the pharmacological nature of the α2-adrenoceptor- and imidazoline-preferring binding sites in this organ. 2. [3H]-Rauwolscine bound to an apparent single site with an affinity (KD) of 2.2 nmol/ L and a maximum density (Bmax) of 58.5 fmol/mg protein, when 10 μmol/L idazoxan defined non-specific binding. However displacement studies demonstrated that a number of compounds, including prazosin, inhibited [3H]-rauwolscine binding in a complex manner consistent with displacement from two distinct binding sites. The majority (69%) of the [3H]-rauwolscine binding sites had a relatively low affinity for prazosin (KI= 398 nmol/L), while the remainder had a relatively high affinity for prazosin (KI= 7.9 nmol/ L). 3. [3H]-p-Aminoclonidine bound to an apparent single site (KD= 5.2 nmol/L; Bmax= 72.4 fmol/mg protein), when 10 μmol/L phentolamine defined non-specific binding. When 1 μmol/L of the potent and selective α2-adrenoceptor antagonist 2-methoxyidazoxan was included in the incubate, no specific binding was detected. We therefore conclude that under the conditions of this experiment [3H]-p-aminoclonidine binds only to α2-adrenoceptors in the dog kidney. 4. [3H]-Idazoxan bound to two sites, with a higher (KD= 0.95 nmol/L; Bmax= 43.9 fmol/mg protein) and lower (KD= 9.1 nmol/L; Bmax= 93.8 fmol/mg protein) affinity, respectively, when 1 mmol/L phentolamine defined non-specific binding. When 10 μmol/ L GTPγS was included in the incubate, the low affinity site was unaffected but the maximum binding at the higher affinity site was reduced by 79%. 2-Methoxyidazoxan displaced [3H]-idazoxan in a monophasic manner and with low potency (IG50=11.5 μmol/L). Yohimbine, efaroxan, clonidine, rilmenidine, guanabenz and idazoxan itself displaced [3H]-idazoxan in a complex manner; the slope of the displacement curves being less than unity. 5. We conclude that the dog kidney contains a heterogeneous population of α2-adrenoceptors that can be labelled either with [3H]-rauwolscine or [3H]-p-aminoclonidine. The dog kidney also contains a heterogeneous population of non-adrenoceptor imidazoline-preferring binding sites of the I2-subtype, that can be labelled with [3H]-idazoxan. The binding site for which [3H]-idazoxan has the highest affinity appears to be coupled to a guanine nucleotide binding regulatory protein. 相似文献
13.
Drug inhibition against [3H]paroxetine binding to rat cortex and human putamen was investigated in saturation experiments. The addition of 5-HT, imipramine, citalopram and clomipramine all produced changes in apparent binding affinity (Kd) without changes in the number of binding sites (Bmax). These data suggest that there is no heterogeneity of specific [3H]paroxetine binding, supporting a single site model of the 5-HT uptake site and antidepressant binding site. 相似文献
14.
J. Fichna J.‐C. Do‐Rego M. Mirowski J. Costentin A. Janecka 《Chemical biology & drug design》2005,65(4):459-464
Abstract: Endomorphin‐2 (Tyr‐Pro‐Phe‐Phe‐NH2) binds with high affinity and selectivity to the μ‐opioid receptor. In the present study, [125I]endomorphin‐2 has been used to characterize μ‐opioid‐binding sites on transplantable mouse mammary adenocarcinoma cells. Cold saturation experiments performed with [125I]endomorphin‐2 (1 nm ) show biphasic binding curves in Scatchard coordinates. One component represents high affinity and low capacity (Kd = 18.79 ± 1.13 nm , Bmax = 635 ± 24 fmol/mg protein) and the other shows low affinity and higher capacity (Kd = 7.67 ± 0.81 μm , Bmax = 157 ± 13 pmol/mg protein) binding sites. The rank order of agonists competing for the [125I]endomorphin‐2 binding site was [d ‐1‐Nal3]morphiceptin > endomorphin‐2 ? [d ‐Phe3]morphiceptin > morphiceptin > [d ‐1‐Nal3]endomorphin‐2, indicating binding of these peptides to μ‐opioid receptors. The uptake of 131I‐labeled peptides administered intraperitoneally to tumor‐bearing mice was also investigated. The highest accumulation in the tumor was observed for [d ‐1‐Nal3]morphiceptin, which reached the value of 8.19 ± 1.14% dose/g tissue. 相似文献
15.
A. R. Hosseini P. R. King W. J. Louis A. L. Gundlach 《Naunyn-Schmiedeberg's archives of pharmacology》1996,355(1):131-138
2-(2-Benzofuranyl)-2-imidazoline (2-BFI) has recently been characterised as a selective ligand for the I2-type of imidazoline-receptor binding site(s) (I2-RBS). The present studies determined the relative levels of specific [3H]2-BFI binding to membrane homogenates of brain and kidney from rat, guinea pig and rabbit and identified the pharmacological characteristics of [3H]2-BFI binding sites in rabbit kidney membranes. Rabbit kidney membranes had the highest relative density of specific [3H]2-BFI binding of all tissues studied (2000?fmol/mg protein). Rabbit brain and guinea pig kidney had moderate levels of specific [3H]2-BFI binding (350–500?fmol/mg protein), while rat kidney and guinea pig and rat brain displayed much lower densities of binding (40–65?fmol/mg protein). Studies of [3H]2-BFI binding kinetics in rabbit kidney homogenates revealed binding to two distinct sites with K d values of 0.10?±?0.01?nmol/l and 1.00?±?0.36?nmol/l respectively. Equilibrium saturation studies were also consistent with the presence of two binding sites – [3H]2-BFI (0.01–20?nmol/l) bound to sites with affinities of 0.10?± 0.01?nmol/l and 0.92?±?0.13?nmol/l and binding densities of 470?±?80 and 840?±?60?fmol/mg protein (n=3), representing 36 and 64% respectively. Drug inhibition studies revealed that l-adrenaline; α1-adrenoceptor drugs (prazosin, l-phenylephrine) and α2-adrenoceptor drugs (rauwolscine, methoxyidazoxan, 2-(2,4-(O-methoxyphenyl)-piperazin-1-yl)-ethyl-4,4-dimethyl-1,3-(2H,4H)-isoquinolindione (ARC-239) had extremely low affinities for [3H]2-BFI binding sites (IC50?≥?10?μmol/l). Putative I1-RBS compounds, p-aminoclonidine, moxonidine, imidazole-4-acetic acid and cimetidine, inhibited [3H]2-BFI binding to rabbit renal membranes with low to very low affinities (K i values 3 to ≥100?μmol/l), suggesting [3H]2-BFI does not label I1-RBS in rabbit kidney membranes. I2-RBS compounds – 2-(4,5-dihydroimidaz-2-yl)-quinoline (BU224), 2-(4,5-dihydroimidaz-2-yl)-quinoxaline (BU239), idazoxan and cirazoline – potently inhibited [3H]2-BFI binding (K i values 0.37–1.6?nmol/l), confirming the labelling of I2-RBS. Inhibition of [3H]2-BFI binding by certain compounds was consistent with their interaction with two binding site populations – for example (drug, K i values) guanabenz, 0.65?nmol/l and 0.17?μmol/l; naphazoline, 0.94?nmol/l and 2.8?μmol/l; amiloride, 76?nmol/l and 26?μmol/l rilmenidine, 150?nmol/l and 50?μmol/l; and clonidine, 230?nmol/l and 70?μmol/l. The high affinity of amiloride for a high proportion (85%) of the binding is consistent with the presence of the I2A-subtype of I-RBS in rabbit kidney. These results demonstrate that [3H]2-BFI is a highly selective and high affinity radioligand for I2-RBS which should be useful for the further characterisation of these sites in mammalian tissues. 相似文献
16.
用标记的血小板活化因子拮抗剂[3H]WEB 2086,在培养的牛脑前动脉平滑肌细胞上鉴定了血小板活化因子受体。结果表明在25℃时该细胞上存在两种与配基具有不同亲和力的受体结合位点,其中Kd-1=22.8±5.0 nmol·L-1,Kd-2=186+20.5 nmol·L-1;Bmax-1=2.1±0.3 pmol/104细胞,Bmax-2=12.1±1-5 pmol/106细胞。蝙蝠葛碱和粉防己碱均能抑制[3H]WEB2086与上述细胞的结合。 相似文献
17.
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 (KD) 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 KD 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. 相似文献
18.
Milt Titeler G.R. Van Loon Philip Seeman G.M. Brown 《European journal of pharmacology》1981,72(1):143-144
Using conditions which revealed high affinity binding sites for [3H]ADTN (KD of 1.6 nM), [3H]apomorphine (KD of 3.5 nM) and [3H] dopamine (KD of 1.5 nM) in the calf caudate nucleus, no such high affinity sites could be detected in the calf anterior pituitary gland. In contrast, high affinity binding sites for [3H]-spiperone were found in both the caudate and anterior pituitary. The apparent absence of high affinity sites for these [3H]-catecholamine ligands (in the 1–10 nM region) and the presence of the [3H]spiperone binding site in the anterior pituitary is compatible with the view that the pituitary [3H] neuroleptic receptor with its low affinity for dopamine, may be the physiological site of dopamine action in the pituitary. The data, therefore, support the presence of D2- but not D3-dopamine receptor sites in the calf anterior pituitary. 相似文献
19.
从远志中分离鉴定出一种多巴胺受体活性化合物 总被引:3,自引:0,他引:3
从中药远志中提出一种多巴胺受体的配基,四氢非州防己胺。此化合物在体外可抑制[3H]SCH23390和[3H]螺哌隆与大鼠纹状体膜的结合,IC50值分别为0.75±0.08μmol·L-1和0.92±0.10μmol·L-1。它在体外还能抑制[3H]哌唑嗪和大鼠脑皮质细胞膜结合(IC50值为46μmol·L-1),但不能改变[3H]QNB及[3H]muscimol对膜的结合。Scatchandplot分析显示此化合物对[3H]SCH23390和[3H]螺哌隆与膜结合的抑制作用是通过竞争性与非竞争性混合机制而实现的。 相似文献
20.
Subclassification of alpha 1-adrenoceptor recognition sites by urapidil derivatives and other selective antagonists 总被引:22,自引:20,他引:2
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1. The affinities of urapidil derivatives and other antagonists for alpha 1-adrenoceptors labelled by [3H]-prazosin were determined on membranes of six different rat tissues. 2. Urapidil and its 5-acetyl-, 5-formyl- and 5-methyl-derivative displaced [3H]-prazosin from alpha 1-adrenoceptor binding sites in a concentration-dependent manner which varied with tissue. IC50 values were lower in vas deferens, hippocampus and cerebral cortex than in heart, liver and spleen. For 5-methyl-urapidil, binding to two distinct sites could be demonstrated with mean K1 values of about 0.6 and 45 nM. Saturation binding studies with [3H]-prazosin in the presence of 5-methyl-urapidil indicated a competitive type of interaction between 5-methyl-urapidil and [3H]-prazosin. 3. The proportion of [3H]-prazosin binding sites with high affinity for 5-methyl-urapidil was 58% in vas deferens, 69% in hippocampus, 41% in cerebral cortex and 23% in myocardium. In liver and spleen virtually no high affinity sites were found. These values were in good agreement with the percentages of binding sites with high affinities for WB-4101 and phentolamine, indicating that all these antagonists bind to the same subtype of alpha 1-recognition sites, whereas other alpha-antagonists like BE 2254, yohimbine and unlabelled prazosin did not discriminate between two binding sites. 4. Preincubating membranes of the cerebral cortex with chloroethylclonidine preferentially inactivated [3H]-prazosin binding sites with low affinity for 5-methyl-urapidil. 5. The antagonist potencies of 5-methyl-urapidil and WB-4101 against alpha 1- adrenoceptor-mediated contractile responses were higher in vas deferens than in myocardium. The alpha 1-mediated effects in vas deferens but not in the heart were highly susceptible to nitrendipine. 6. Using 5-methyl-urapidil, the existence of two distinct alpha 1-adrenoceptor recognition sites could be demonstrated which correspond to the proposed alpha 1A- and alpha 1B-subtypes.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献