二氢埃托啡对大鼠脑阿片受体的结合特性

本文在大鼠脑匀浆P_2膜上,观察了二氢埃托啡(DHE)对[~3H]纳洛酮,[~3H]DPDPE和[~3H]埃托啡(预先用30nmol/L吗啡和100nmol/L DADLE阻断μ和δ受体)与阿片受体结合的抑制强度。结果表明:DHE对[~3H]纳洛酮与阿片受体结合的抑制强度远远大于对[~3H]DPDPE和[~3H]埃托啡(预先阻断μ和δ受体后)。DHE对μ,δ和κ受体的相对亲和力之比为1951:2:1,提示DHE为μ受体相对选择性配体。     

[~3H]3-苯乙基-9β-甲氧基-9α-(间羟苯基)-3-氮杂二环[3,3,1]壬烷的制备及其与大鼠脑P_2膜阿片受体结合的特性(英文)

P—7521盐酸盐经溴化后,进行催化卤氚置换反应,得到>95％放化纯度的[~3H]P—7521。[~3H]P—7521可高亲和地结合大鼠脑P_2膜阿片受体,其K_(D1)和K_(D2)分别为0.030和0.75nmol/L。Na~+对[~3H]P—7521的特异结合无影响。[~3H]P—7521与阿片受体结合后的解离研究表明:约50％的结合在20min内很快解离,剩余的50％则解离极慢,4h时仍有35％的结合存在。[~3H]P—7521结合的膜经4次洗涤仍可测得对照组的87％的结合。[~3H]P—7521与阿片受体的结合是很牢固的。     

可乐定对苯二氮受体激动剂和反相激动剂与大鼠皮层受体结合特性的影响(英文)

本研究旨在探讨α_2受体激动剂可乐定对苯二氮(艹卓)受体激动剂抗焦虑和反相激动剂致焦虑作用影响的可能的分子机理。在10nmol/L至1μmol/L浓度范围内,可乐定对[~3H]氟硝安定与大鼠皮层相应受体低亲和位点结合无显著影响,但非竞争性拮抗其与高亲和位点的结合。在竞争取代实验中,激动剂安定和CL218 872均表现为双位点结合的亲和力,对低亲和位点无显著影响。反相激动剂DMCM竞争结合曲线亦具有双位点结合特性。可乐定可使这种双位点结合转变成三位点结合,出现一个超高亲和位点。可乐定对拮抗剂Ro15-1788竞争结合特性无显著影响。结果提示,α_2受体激动剂可乐定与受体结合可能导致与之相邻的苯二氮(艹卓)受体发生构象变化,这种构象变化有利于激动剂与受体结合,而不利于反相激动剂的结合。     

可乐定对苯二氮Zhuo受体激动剂和反相激动剂与大鼠皮层受体结合特性的影响

本研究旨在研讨α2受体激动剂可乐定对苯二氮Zhuo受体激动剂抗焦虑和反相激动剂致焦虑作用影响的可能的分子机理。在10nmol/L至1umol/L浓度范围内，可乐定对[^3H]氟硝安定与大鼠皮层相应受体低亲和位点结合无显著影响。但非竞争性拮抗其与高亲和位点的结合，在竞争取代实验中，激动剂安全和CL218872均表现为双位点结合的亲和力，对低亲和位点无显著影响，反相激动剂DMCM竞争结合曲线亦具有双位点结合特性，可乐定可使这种双位点结合转变成三位点结合，出现一个超高亲和位点，可乐定对拮抗剂Ro15-1788竞争结合特性无显著影响，结果提示，α2受体激动剂可乐定与受体结合可能导致与之相邻的苯二氮Zhuo受体发生构象变化，这种构象变化有利于激动剂与受体结合，而不利于反相激动剂的结合。     

牛大脑苯二氮受体的光亲和标记性质

本文报道牛大脑突触膜上苯二氮受体(BDZ-R)的光亲和标记性质。光亲和标记是特异性的,可饱和的,并可被苯二氮类药物抑制,其抑制常数K_1与可逆结合实验值相似,推测光亲和标记与可逆结合发生在BDZ-R的相同部位。已知光亲和标记与时间有关,本文采用254nm短波长紫外光源,25min为最佳光照时间,其光亲和标记效率最大为77％。光亲和标记与高压液相色谱技术联用,测得BDZ-R的亚基分子量为48000±1000。     

八肽胆囊收缩素对[~3H]埃托啡与大鼠脑阿片受体结合的抑制作用

本实验观察了CCK—8对[~3H]Et与大鼠脑阿片受体结合的影响.含硫CCK—8能抑制[~3H]Et与高亲和位点的结合,使K_D值增大(P<0.001),B_(max)减小(P<0.01),在10 fmol/L～Lμmol/L范围内呈量效关系.但对低亲和位点的结合没有影响.无硫CCK—8仅对[~3H]Et高亲和位点的K_D值有较小程度的增大(P<0.05),不影响B_(max)值.CCK—8(10 nmol/L)抑制[~3H]Et与阿片受体结合的作用能被CCK受体拮抗剂谷丙酰胺(1μmol/L)所阻断.结果提示,CCK—8可能通过激活CCK受体发挥对阿片受体的抑制作用。     

毛地黄黄酮和玄参黄酮抑制[~3H]地西泮和体外大鼠脑膜的结合(英文)

从阿拉伯艾蒿提取到两种苯并二氮杂受体的配基,毛地黄黄酮和玄参黄酮。两种化合物在体外可抑制[~3H]地西泮和大鼠皮层细胞膜的结合,IC_(50)值分别为1.3μmol·L~(-1)和23μmol·L~(-1)。两种化合物GABA比分别为1.1和1.2,都可少量增加[~(35)S]TBPS的结合,提示这种化合物是苯并二氮杂受体的拮抗剂或部分激动剂。     

葛根素和大豆甙元抑制[3H]氟硝西泮和体外大鼠脑膜的结合

从中药葛根中提取到两种苯二氮艹卓受体活性化合物:葛根素和大豆甙元。两种化合物在体外可抑制[3H]氟硝西泮和大鼠脑细胞膜的结合,IC₅₀值分别为18.46μmol·L^-1和15.43μmol·L^-1。大豆甙元还可抑制[³H]哌唑嗪和α1-肾上腺素受体的结合(IC₅₀值为89μmol·L^-1)。两种化合物的GABA比分别为1.11和1.12,提示两种黄酮化合物是苯二氮艹卓受体的拮抗剂或部分激动剂。Scatchardplot分析显示:两种化合物对[³H]氟硝西泮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。     

从阿拉伯艾蒿分离到两种对苯并二氮杂和α_1肾上腺素能受体有亲和力的黄酮化合物(英文)

干燥阿拉伯艾蒿的酒精提取物对体外[~3H]地西泮和大鼠大脑皮质细胞膜结合产生抑制作用,两种化合物被分离纯化,其化学结构为毛地黄黄酮和玄参黄酮(IC_(50)分别为1.3±s0.1和22.7±S2.5μmol·L~1),两种化合物在体外还能抑制[~3H]哌唑嗪和大鼠脑皮质细胞膜结合,但不能改变[~3H]蝇蕈碱乙酰胆碱受体,[~3H]蝇蕈醇,[~3H]SCH 23390和[~3H]螺哌隆和膜的结合,Scatchard plot分析提示两种化合物对[~3H]地西泮和膜结合的抑制作用是通过竟争性和非竞争性混合机理而实现的。     

[^3H]3—苯乙基—9β—甲氧基—9α—（间羟苯基）—3—氮杂二环[3，3，1]壬烷的制

P-7521盐酸盐经溴化后，进行催化卤氚置换反应，得到>95%放化纯度的[^3H]P-7521。[^3H]P-7521可高亲和地结合大鼠脑P2膜阿片受体，其KD1和KD2分别为0.030和0.75nmol/L。Na^ 对[^3H]P-7521的特异结合无影响。[^3H]P-7521与阿片受体结合后的解离研究表明，[^3H]P-7521与阿片受体结合后的解离研究表明：约50%的结合在20min内很快解离，剩余的50%则解离极慢，4h时仍有35%的结合存在，[^3H]P-7521结合的膜经4次洗涤仍可测得对照组的87%的结合。[^3H]P-7521与阿片受体的结合是很牢固的。     

Photoaffinity labeling of the brevetoxin receptor on sodium channels in rat brain synaptosomes.

Brevetoxin, a neurotoxin isolated from the marine dinoflagellate Ptychodiscus brevis, has been derivatized into a photoaffinity probe by carbodiimide linkage to p-azidobenzoic acid. Rosenthal analysis of a tritiated p-azidobenzoate brevetoxin derivative indicates that specific binding of the toxin occurs at two distinct and separate sites, with Kd and Bmax values of 0.21 nM and 2.12 pmol/mg of protein for the high affinity site and 50.7 nM and 91.5 pmol/mg of protein for the low affinity site, respectively. Binding of tritiated photoaffinity probe to the high affinity/low capacity site can be displaced in a competitive manner by native brevetoxin (Kd = 1.9 nM), demonstrating a specific competitive interaction with the receptor site. Rat brain synaptosomes, covalently labeled with the brevetoxin photoaffinity probe, were subjected to detergent solubilization. The covalently labeled membrane protein was estimated to have a Stokes radius of 55 +/- 3 A. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific labeling of a 260-kDa protein. Treatment with 2-mercaptoethanol and neuraminidase resulted in retention of brevetoxin binding to this high molecular weight protein. The affinity-purified membrane protein-brevetoxin photoaffinity probe complex was specifically recognized by a sodium channel antibody directed against the intracellular side of transmembrane segment IS6. The sodium channel alpha subunit is implicated as the specific site of brevetoxin interaction.     

Binding of [3H]dihydrotetrabenazine and [125I]azidoiodoketanserin photoaffinity labeling of the monoamine transporter of platelet 5-HT organelles

The carrier for 5-hydroxytryptamine (5-HT) of the 5-HT storage organelles of blood platelets was characterized by [3H]dihydrotetrabenazine binding and [125I]azidoiodokentanserin photoaffinity labeling. [3H]Dihydrotetrabenazine bound with high affinity to membrane preparations from different animal species. The [3H]dihydrotetrabenazine Bmax value was about 10-fold higher in rabbit (9.4 +/- 1.3 pmol/mg protein) than in human, rat and guinea-pig preparations (Bmax values = 1.1 +/- 0.2, 1.2 +/- 0.1 and 0.52 +/- 0.06 pmol/mg protein, respectively). After rabbit platelet subcellular fractionation, [3H]dihydrotetrabenazine binding was highly enriched in the fraction corresponding to pure 5-HT organelles, whereas ligand binding was much lower in the other subcellular fractions. Conversely, [3H]paroxetine binding sites were more concentrated in the lower density fractions, with no binding to the 5-HT granules. In competition experiments, [3H]dihydrotetrabenazine binding to human platelet membranes and rabbit platelet 5-HT organelles was markedly inhibited by the benzo[a]quinolizine derivatives, tetrabenazine and Ro 4-1284, and by ketanserin. In isolated rabbit platelet 5-HT organelles, reserpine showed a relatively high IC50 (930 nM), but the presence of ATP increased its potency about 10-fold. Paroxetine, methysergide and carrier substrates had little or no effect. After photoaffinity labeling of rabbit 5-HT granules with [125I]azidoiodoketanserin, the radioactivity was incorporated into several polypeptides. The presence of Ro 4-1284, reserpine and ketanserin prevented the labeling of a polypeptide of 85 kDa. The data obtained suggest that this protein represents a component of the granular carrier which binds [3H]dihydrotetrabenazine.     

125I]iodoclonazepam, a specific high affinity radioligand for the identification of BZ1 and BZ2 sites in rat brain.

Binding of the radioligand [125I]iodoclonazepam to three different areas of rat brain (cerebellum, hippocampus and striatum) has been characterised. In all three regions binding is rapid, saturable and of high affinity (cerebellum Bmax = 1.49 +/- 0.3 pmol/mg of protein, Kd = 0.39 +/- 0.06 nM; hippocampus Bmax = 1.5 +/- 0.14 pmol/mg of protein, Kd = 0.38 +/- 0.6 nM and striatum Bmax = 0.53 +/- 0.1 pmol/mg of protein, Kd = 0.34 +/- 0.03 nm, n = 3). In all regions only one population of sites was apparent. However, competition for [125I]iodoclonazepam sites by a series of benzodiazepine agonists and antagonists showed some regional differences. The BZ1 selective compounds zolpidem and CL218,872 showed a 4.3-fold and 5.2-fold selectivity for cerebellar binding sites respectively. We conclude that [125I]iodoclonazepam is a novel, high affinity ligand which recognises both BZ1 and BZ2 classes of receptor and should be a useful addition to the panel of benzodiazepine ligands currently available.     

Identification of the ligand-binding subunit of the human 5-hydroxytryptamine1A receptor with N-(p-azido-m-[125I] iodophenethyl)spiperone, a high affinity radioiodinated photoaffinity probe

The ligand-binding subunit of the human 5-hydroxytryptamine1A (5-HT1A) receptor transiently expressed in COS-7 cells and of the native human 5-HT1A receptor derived from hippocampus and frontal cortex were identified by photoaffinity labeling with N-(p-azido-m-[125I]iodophenethyl)spiperone [( 125I]N3-NAPS), previously characterized as a high affinity radioiodinated D2-dopamine receptor probe. The identity of the ligand-binding subunit was confirmed by immunoprecipitation with an antipeptide rabbit antiserum, JWR21, raised against a synthetic peptide derived from the predicted amino acid sequence of the putative third intracellular loop of the human 5-HT1A receptor. In transiently transfected COS-7 cells expressing 14 +/- 3 pmol/mg of protein human 5-HT1A receptors, a single broad 75-kDa band was photoaffinity labeled by [125I]N3-NAPS. This band displayed the expected pharmacology of the 5-HT1A receptor, as evidenced by the ability of a series of competing ligands to block [125I]N3-NAPS photoincorporation. Moreover, antiserum JWR21 specifically and quantitatively immunoprecipitated the 75-kDa photoaffinity-labeled band from a soluble extract of the transfected COS-7 cell membranes, further confirming its identity. Finally, utilizing a combination of photoaffinity labeling and immunoprecipitation, the native ligand-binding subunit of 62-64 kDa was identified in human hippocampus and frontal cortex. The availability of the high specific activity, high affinity, photoaffinity ligand [125I]N3-NAPS and of a potent immunoprecipitating antiserum (JWR21) should greatly facilitate the biochemical characterization of the human 5-HT1A receptor.     

Radioreceptor study of [3H]-olanzapine specific binding in the rat frontal cortex and striatum

The direct radioreceptor assay was used to elucidate the interactions of clinically employed atypical antipsychotic drug olanzapine with the specific binding sites in rat brain membranes. Tritium-labeled olanzapine was obtained by isotope exchange method. HPLC and mass spectrometry assays confirmed the identity of [3H]-olanzapine to the initial compound. The specific [3H]-olanzapine binding sites of the two types were identified in frontal cortex by means of the method of Scatchard's plots. High affinity sites showed steady-state dissociation binding constant (KD) of 1.7 nM, which is indicative of olanzapine affinity to 5-HT-2a and histamine receptors. However, according to available literature data, the density of these receptors is several times smaller than that determined in our study (maximum number of binding sites corresponded to B(max) = 1.4 pmol/mg protein), thus allowing an assumption about the existence of yet unknown binding sites of the antipsychotic drug under consideration. Moderate affinity binding sites of olanzapine in frontal cortex (KD= 68.5 nM, B(max) = 10.8 pmol/mg protein) apparently correspond to dopamine, muscarinic, and alpha-1-adrenoceptors. [3H]olanzapine binding sites of the two types were also determined in striatum. The moderate-affinity sites of the receptor interaction had KD = 18.0 nM and B(max) = 13.0 pmol/mg protein. These values in fact correspond to D2 receptors, whose density prevails in this cortical region. The low-affinity binding sites of olanzapine in striatum had KD = 117.0 nM and B(max) = 32.5 pmol/mg protein.     

3H]opipramol labels a novel binding site and sigma receptors in rat brain membranes.

Opipramol (OP), a clinically effective antidepressant with a tricyclic structure, is inactive as an inhibitor of biogenic amine uptake. [3H]Opipramol binds saturably to rat brain membranes (apparent KD = 4 nM, Bmax = 3 pmol/mg of protein). [3H]Opipramol binding can be differentiated into haloperidol-sensitive and -resistant components, with Ki values for haloperidol of 1 nM (Bmax = 1 pmol/mg of protein) and 350 nM (Bmax = 1.9 pmol/mg of protein), respectively. The drug specificity of the haloperidol-sensitive component is the same as that of sigma receptors labeled with (+)-[3H]3-(3-hydroxyphenyl)-N-(1-propyl)piperdine. The haloperidol-resistant component does not correspond to any known neurotransmitter receptor or uptake recognition site. It displays high affinity for phenothiazines and related structures such as perphenazine, clopenthixol, and flupenthixol, whose potencies are comparable to that of opipramol. Because certain of these drugs are more potent at the haloperidol-resistant opipramol site than in exerting any other action, it is possible that this opipramol-selective site may mediate their therapeutic effects.     

Specific binding of a phenyl-pyridazinium derivative endowed with GABAA receptor antagonist activity to rat brain

SR 95531 has been shown to be a potent, selective, reversible and competitive GABAA antagonist. In the present study we report that (3H)SR 95531 binds with high affinity and in a specific and saturable manner to rat brain membranes. Scatchard analysis revealed two binding sites (KD: 6 nM; Bmax: 0.24 pmol/mg protein and KD: 38 nM; Bmax: 0.66 pmol/mg protein). Only GABA ligands were effective displacers of (3H)SR 95531. The respective IC50 values obtained with these compounds suggests that (3H)SR 95531 labels the GABA receptor in its antagonist conformation.     

Characteristics of inositol 1,4,5-trisphosphate binding to rat cerebellar and bovine adrenal cortical membranes: evidence for the heterogeneity of binding sites

The equilibrium and kinetic binding characteristics of D-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) have been studied in membrane preparations of rat cerebellum and bovine adrenal cortex. Saturation analysis of isotopic dilution binding data demonstrated apparent KD values for Ins(1,4,5)P3 binding of 23 +/- 5 nM and 3.0 +/- 1.3 nM for cerebellar and adrenal cortical preparations, respectively, with approximately 20-fold greater receptor density present in the cerebellar preparation (Bmax: 10.2 +/- 2.5 pmol/mg protein). Kinetic analysis confirmed the equilibrium binding-derived KD value for cerebellum (KD: 39.9 nM), but revealed a second, very high affinity site (KD: 0.06 nM) to be present in adrenal cortex. The affinity differences between the investigated preparations was also observed with respect to the IC50 values obtained for inhibition of specific [3H]Ins(1,4,5)P3 binding by a number of inositol polyphosphate analogues including D-inositol 2,4,5-trisphosphate, DL-inositol 1,4,5-trisphosphorothioate and L-Ins(1,4,5)P3. In contrast, the Ins(1,4,5)P3-receptor antagonist heparin displayed greater potency for the cerebellar (IC50: 16.5 +/- 6.2 micrograms . ml-1) compared to the adrenal cortical preparation (IC50: 51.0 +/- 6.1 micrograms . ml-1). The apparent differences between the Ins(1,4,5)P3 receptors characterized in the two tissue preparations are discussed.     

Localization of 5-HT3 receptor binding sites in human dorsal vagal complex

Binding of the 5-HT3 receptor ligand [3H]BRL 43694 was investigated in the human medulla oblongata using in vitro autoradiography. High levels of saturable, displaceable binding (Bmax 1.88 pmol/mg protein, Kd 1.21 nM) were seen in the dorsal vagal complex but in no other medullary region. The results provide evidence for the existence of 5-HT3 receptor binding sites in a brain region involved in the control of vomiting in man.     

Autoradiographic study of peripheral benzodiazepine receptors in animal brain tumor models and human gliomas.

In vitro binding of [3H]PK-11195 (1-(2-chlorophenyl)-N-methyl-(1- methylpropyl)-3-isoquinoline carboxamide) in rodent AA ascites and C6 glioma as well as in human gliomas was investigated. The Bmax (mean +/- S.D.) of AA ascites tumor and C6 glioma is 1.39 +/- 0.15 pmol/mg tissue and 4.50 +/- 0.76 pmol/mg tissue, respectively. This Bmax is 9 and 30 times, respectively, higher than the one found in the rat cortex (0.15 +/- 0.03 pmol/mg tissue). A Bmax of 1.26 +/- 0.24 pmol/mg tissue and 0.64 +/- 0.08 pmol/mg tissue was found in human malignant and low grade gliomas respectively. This Bmax value should be compared to 0.35 +/- 0.04 pmol/mg tissue found in the normal human cortex. There are significant (P less than 0.05) differences between Bmax in tumors and normal cortex. There was no significant difference in KD between the malignant and low grade gliomas. C6 glioma has a KD significantly greater than rat cortex. In some cases of human low grade gliomas, kinetic measurements suggested the presence of two affinity receptor sites. However, at this time, heterogeneity of the tissue cannot be excluded as being at least in part a source of this.