大鼠胃壁细胞胃泌素受体的放射配基结合法

目的　建立大鼠胃壁细胞胃泌素受体放射配基结合实验方法。方法　以12 5I [Leu15] gastrin17 I为标记配基 ,以大鼠胃壁细胞悬液为受体制备 ,反应体系总体积为 2 0 0 μl,37℃恒温水浴振荡孵育 30min ,以 49型玻璃纤维滤膜分离结合和游离配基。结果　胃泌素受体结合符合简单单位点结合模型 ,Scatchard作图求出胃泌素受体结合参数Bmax=4 46 0 4× 10 -12 mol·L-1,Kd=1 2 49× 10 -10 mol·L-1。单点法求出特异结合位点数 70 3 2sites·cell-1。细胞浓度在0 2 5× 10 9～ 2× 10 9cells·L-1范围内 ,与标记配基特异性结合容量成正比。同一样品特异结合测定方法变异系数为7 0 4%。竞争抑制实验证明12 5I Gastin与大鼠胃壁细胞结合特异性良好。结论　大鼠胃壁细胞胃泌素受体的放射配基结合实验方法符合受体结合实验方法学要求     

小鼠腹腔巨噬细胞PAF受体放射配基受体结合分析方法

目的　建立简便、快速的小鼠腹腔巨噬细胞PAF放射配基受体结合分析法 ,考察巨噬细胞表面PAF受体的特性。方法　以♂C5 7BL/ 6小鼠腹腔巨噬细胞为受体材料 ,[3 H] PAF为标记配基 ,非膜过滤法进行结合放射配基分离 ,液闪计数测定。结果　小鼠腹腔巨噬细胞PAF受体结合符合简单单位点受体结合模型 ,Scatchard分析求出的PAF受体结合参数Bmax=10 0 2fmol·1× 10 6cells-1,KD=3 2nmol·L-1。 [3 H] PAF与小鼠腹腔巨噬细胞PAF受体的结合呈现单一、饱和特点 ,并可被特异性PAF受体拮抗剂BN5 2 0 2 1竞争性抑制。结论　利用小鼠腹腔巨噬细胞贴壁特性 ,采用非滤过法处理细胞 ,能够在保持受体生理学活性的状态下 ,用于特异性PAF受体拮抗剂的筛选     

大鼠离体组织α1受体放射配基结合分析方法的研究

目的 建立大鼠下颌腺、肝脏、胸主动脉的α1受体放射配基结合实验的方法.方法 以3H-哌唑嗪为标记配基,♂ SD大鼠的下颌腺、肝脏和胸主动脉的细胞膜蛋白为受体材料,冰浴并玻璃纤维滤膜抽滤终止实验,液闪技术进行测定.结果 大鼠的下颌腺、肝脏和胸主动脉的α1受体符合简单单位点受体结合模型,用Scatchard作图求出α1受体的3种亚型受体的结合参数分别为:下颌腺Bmax＝ 155.9±1.67 pmol·L-1,KD ＝682.7±18.73 pmol·L-1；肝脏Bmax＝149.8±1.61 pmol·L-1,KD=381.0 ± 11.68 pmol·L-1；主动脉Bmax ＝146.1 ±2.37 pmol·L-1,KD＝ 338.2 ± 16.01 pmol·L-1.结论 大鼠下颌腺、肝脏和胸主动脉的α1受体放射配基结合实验方法符合受体结合实验方法学的要求.     

北京鸭红细胞膜β肾上腺素受体放射配基结合测定法

本文以[~3H]dihydroalprenolol([~3H]DHA)为放射配基。对北京鸭红细胞(RBC)膜β受体进行了系统研究。结果表明,[~3H]DHA与北京鸭RBC结合具有饱和性,肾上腺素能激动剂与[~3H]DHA竞争结合的强弱顺序与它们的生物活性一致,提示结合有结构特异性和立体特异性,[~3H]DHA与鸭RBC结合迅速、可逆。可见北京鸭RBC膜存在β受体,用于放射配基结合测定有取材方便,受体较丰富,膜蛋白得率高,[~3H]DHA非特异结合低和易于保存等优点,可以代替火鸡RBC。     

平律复方抗实验性心律失常的作用及其机理研究

目的:研究平律复方(PL)抗实验性心律失常的作用,并初步探讨其作用机理。方法:采用氯仿、氯化钙以及心肌缺血再灌注三种心律失常动物模型,监测标准Ⅱ导联心电图;测定缺血再灌注大鼠血清肌酸激酶(CK)和乳酸脱氢酶(LDH)活性;放射配基受体结合法分析心肌组织血小板激活因子(plate-let activating factor,PAF)受体蛋白表达水平;RT-PCR法检测PAF受体mRNA表达水平;测定心肌脂质过氧化产物丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果:PL(ig,0.04、0.20、1.00 g.kg-1)对氯仿引起的小鼠心室纤颤有一定的保护作用;能减少氯化钙致大鼠室颤的发生率,降低死亡率;可显著降低心肌缺血再灌注大鼠心律失常的发生率,缩短持续时间,与模型对照组比较,PL小、中、大剂量组心肌CK和LDH分别降低了35.4%、51.8%、57.5%和22.4%、34.4%、38.4%;剂量依赖性下调心肌细胞PAF受体蛋白及mRNA表达水平,升高心肌SOD活性、降低MDA含量。结论:PL对氯仿、氯化钙以及心肌缺血再灌注诱发的心律失常均有较好的保护作用,其作用机制可能与下调心肌细胞PAF受体水平,抑制脂质过氧化有关。     

扩张型心肌病大鼠心脏左室内皮素受体及其亚型分析

目的 观察扩张型心肌病大鼠左室ETR及其亚型的变化.方法用125I-ET-1建立内皮素受体及其亚型的放射配基分析法,进行饱和结合试验,对扩张型心肌病大鼠左室心肌ETR及其亚型的含量进行分析.结果(1) 放射配基分析法检测ETR的基本实验条件为37 ℃孵育 60 min,膜蛋白投放量以20～40 μg 为佳; (2) ET-1及非选择性拮抗剂bosentan、选择性拮抗剂BQ123、BQ788等均能竞争抑制125I-ET-1与内皮素受体的结合,而去甲肾上腺素则不能抑制; (3) 正常大鼠左室内皮素受体数量为 (92.21±34.34) nmol·     

姜黄对胃泌素受体影响的研究

目的 研究姜黄郁金的活性成份对胃泌素受体的影响。方法 采用受体结合方法。结果 姜黄可能含有胃泌素受体激动剂或拮抗剂。其抑制活性强度有如下大致规律:乙酸乙酯部分>甲醇部分>乙醚部分>挥发油部分;块根>根茎。结论 对开发新的胃泌素受体作用特异性药物有重要意义。     

μ阿片受体及受体—配基相互作用的分子模拟

目的：构建μ阿片受体（μＯＲ）的三维结构模型并研究它与芬太尼衍生物的相互作用。方法：以细菌视紫红南为模板，模拟μＯＲ的三维结构。然后蒋芬太尼衍生物对接到μＯＲ的七个α螺旋束之内，并计算结合能，结果：（１）得以受体－配基作用模型．（２）模型中，基本结合位点可能是Ａｓｐ１７和Ｈｉｓ２９７，Ａｓｐ１４７与配基的正电性能铵基形成强的静电和氢键相互作用，这种作用在Ｈｉｓ２９７和配基的羰基Ｏ原子之间较弱，（３     

新兴的受体技术与新药筛选

在药物研究过程中 ,发现具有药物活性的物质是一个极为重要的过程。在人类文明的整个进程中 ,从神农尝百草的亲身体验到采用动物实验是一次巨大的飞跃。当前开发新药所面临的一个最大的难题在药物筛选方面。显然 ,药物筛选的模型越多越先进 ,发现新药的机率就越高 ,研制新药的速度也越快。从国外的发展来看 ,药物筛选模型和方法大致经过了 3个阶段[1] :(1 )经典的动物普筛方法 ,耗时长 ,浪费大 ,又容易漏筛 ,在实际应用中受到很大限制 ;(2 )酶筛选方法中应用各种与疾病有关的酶作为筛药工具 ,建立了各种酶抑制剂筛选模型。例如 ,以血管紧张…     

D2型多巴胺受体配基的研究进展

阐述了近几年D2型受体配基的研究状况，探讨了部分具有应用前景的化合物的构效关系。特别指出混合受体拮抗剂(如d2／d4、d2／5HTlA)和部分激动剂可能成为新的非经典抗精神失常药物，而兼有抗氧化作用的多巴胺受体激动剂则成为抗帕金森病药物研究的主要方向。     

Evaluation of drug-muscarinic receptor affinities using cell membrane chromatography and radioligand binding assay in guinea pig jejunum membrane

AIM: To study if cell membrane chromatography (CMC) could reflect drug-receptor interaction and evaluate the affinity and competitive binding to muscarinic acetylcholine receptor (mAChR). METHODS: The cell membrane stationary phase (CMSP) was prepared by immobilizing guinea pig jejunum cell membrane on the surface of a silica carrier, and was used for the rapid on-line chromatographic evaluation of ligand binding affinities to mAChR. The affinity to mAChR was also evaluated from radioligand binding assays (RBA) using the same jejunum membrane preparation. RESULTS: The capacity factor (k') profiles in guinea pig jejunum CMSP were: (-)QNB (15.4)>(+)QNB (11.5)>atropine (5.35)>pirenzepine (5.26)>4-DAMP (4.45)>AF-DX116 (4.18)>pilocarpine (3.93)>acetylcholine (1.31). These results compared with the affinity rank orders obtained from radioligand binding assays indicated that there was a positive correlation (r2= 0.8525, P<0.0001) between both data sets. CONCLUSION: The CMC method can be used to evaluate drug-receptor affinities for drug candidates.     

左旋咪唑对大鼠脑组织咪唑啉2受体-配体结合试验的影响

目的研究左旋咪唑(LMS)对大鼠脑组织咪唑啉2受体(I_2R)的影响。方法采用受体-配体竞争结合试验,观察LMS与受体的结合能力;采用受体-配体饱和试验,测定LMS处理大鼠和对照组大鼠脑内I_2R最大受体结合容量(B_(max))和平衡解离常数(K_d)变化。结果LMS抑制了放射性配基[~3H]idazoxan与大鼠脑组织内I_2R的结合,其半数抑制浓度(IC_(50))为4.04×10~(-7)mol·L~(-1)。LMS长期处理大鼠,脑内I_2R亲和力上调63%。结论LMS能与I_2R结合,长期应用LMS能通过刺激I_2R而改变了受体亲和力。     

MgCl2和Gpp（NH）p对腺苷A1受体激动剂和拮抗剂结合的作用比较

AIM: To investigate modulation of antagonist and agonist binding to adenosine A1 receptors by MgCI2 and 5'-guanylimidodiphosphate (Gpp(NH)p) using rat brain membranes and the A1 antagonist [^3H]-8-cyclopentyl-1,3-dipropylxanthine ([^3H]DPCPX) and the A1 agonist [^3H]-2-chloro-N^6-cyclopentyladenosine ([^3H]CCPA). METHODS:Parallel saturation and inhibition studies were performed using well-characterised radioligand binding assays and aBrandel Cell Harvester. RESULTS: MgCI2 produced a concentration-dependent decrease (44%), whereasGpp(NH)p increased [^3H]DPCPX binding (19%). In [^3H]DPCPX competition studies, agonist affinity was 1.5-14.6-fold higher and 4.6-10-fold lower in the presence of l0 mmol/L MgCl2 and l0μmol/L Gpp(NH)p respectively;antagonist affinity was unaffected. The decrease in agonist affinity with increasing Gpp(NH)p concentrations was due to a reduction in the proportion of binding to the high affinity receptor state. In contrast to [^3H]DPCPX, MgCl2produced a concentration-dependent increase (72%) and Gpp(NH)p a decrease (85%) in [^3H]CCPA binding.Using [^3H]CCPA, agonist affinities were 5-17-fold higher than those for [^3H]DPCPX, consistent with binding onlyto the high affinity receptor state. Agonist affinity was 1.3-10.5-fold higher and 2.4-4.7-fold lower on addingMgCl2 or Gpp(NH)p respectively; antagonist affinities were as for [^3H]DPCPX. CONCLUSION: The inconsistencies surrounding the effects of MgCl2 and guanine nucleotides on radioligand binding to adenosine A1 receptorswere systematically examined. The effects of MgCl2 and Gpp(NH)p on agonist binding to A1 receptors are consistent with their roles in stimulating GTP-hydrolysis at the G-protein α-subunit and in blocking formation of the highaffinity agonist-receptor-G protein complex.     

Quantitative comparison of functional screening by measuring intracellular Ca2+ with radioligand binding at recombinant human dopamine receptors

The purpose of this study was to test whether screening at dopamine receptors performed with a recently described functional assay for G-protein coupled receptors (GPCRs) provides data that correlate significantly with radioligand binding data in the literature, thus possibly allowing researchers to replace radioligand binding with nonradioactive functional screening. Human dopamine receptors hD1 and hD2L (representing G_s [hD1] or G [hD2L] coupled GPCRs) were recombinantly expressed in human embryonic kidney (HEK293) cells. Cells were loaded with Oregon Green 488 BAPTA-1/AM and evenly distributed in 384 well plates. Seventeen test compounds were screened for agonistic activity by injection into the cell suspension and monitoringH of intracellular Ca²⁺ with a fluorescence microplate reader. Then, standard agonists (100nM SKF38393 for hD1, 30nM quinpirole for hD2L) were injected into wells preincubated with test compounds (screening for antagonism). Injection of various agonists resulted in a concentration-dependent increase in fluorescence. Further, preincubation of antagonists with dopamine receptor expressing cells inhibits concentration-dependent the agonist-induced increase in fluorescence. Calculated apparent functional K_i values correlate with radioligand binding data in the literature (r²=0.7796 for D1, r²=0.7743 for D2). The correlation between apparent functional K values and radioligand binding data for the 17 tested compounds suggests that screening of test compounds at dopamine receptors with the functional Ca²⁺ assay can replace radioligand binding studies. Furthermore, besides apparent K values, information about agonistic or antagonistic properties of a test compound can be obtained with the functional Ca²⁺ assay.     

EAE大鼠脑组织咪唑受体变化的研究

目的观察炎性脱髓鞘脑病动物模型(EAE)大鼠脑组织咪唑啉2受体(12R)的变化,探讨12R是否参与了炎性脱髓鞘白质脑病的病理过程。方法将大鼠分为EAE模型组及CFA对照组,EAE模型组通过免疫当天予四足垫皮下注射豚鼠脊髓匀浆(GPSCH)-CFA的方式免疫大鼠,后予腹腔注射NS0.4mL每12h一次,于发病高峰期处死。CFA对照组大鼠通过免疫当天予四足垫皮下注射NS-CFA的方式免疫大鼠,后予腹腔注射NS0.4mL每12h1次,连续19d;所有大鼠均予免疫0,48h腹股沟皮下注射百日咳菌苗0.1mL(1×109菌体)。两组大鼠脑组织12R进行饱和结合试验,检测其密度和亲和力变化。结果 CFA对照组大鼠没有发病,EAE组6只大鼠,发病4只。CFA对照组脑组织12R的Kd和Bmax值分别是3.367±0.94nM和162±22.19fmol·mg-1蛋白。EAE组大鼠脑组织12R的Kd和Bmax值分别是5.416±1.153nM和263.0±31.99fmol·mg-1蛋白;与CFA对照组相比,其受体密度明显上调(n=6,P<0.05),而亲和力明显下降(n=6,P<0.01)。结论炎性脱髓鞘脑病模型EAE大鼠脑内12R受体密度上调,亲和力降低。     

Preparation of high specific activity tritium‐labelled leukotriene B4 suitable for radioligand binding assay

We describe a method of preparation of high specific activity tritium‐labelled leukotriene (LT) B₄ from [5,6,8,9,11,12,14,15‐³H] arachidonic acid (AA; 6.66 TBq/mmol) utilizing a LTB₄‐synthesizing enzyme system from rat basophilic leukemia (RBL‐1) cells. It was shown that both cyclooxygenase inhibitor indomethacin and adenosine 5′‐triphosphate induced [³H] AA transformation to [³H] LTB₄. In optimized conditions up to 15% of total radioactivity of the incubation mixture was present in [³H] LTB₄. A separation of [³H] LTB₄ from other labelled C_20:4 products was achieved by a three‐step reverse phase‐high‐performance liquid chromatography in methanol‐ and acetonitrile‐based solvent systems. [³H] LTB₄ was confirmed to be identical to the naturally occurring LTB₄ by a radioligand binding assay using a culture of HF1 cells that express a BLT₁ receptor. Copyright © 2008 John Wiley & Sons, Ltd.     

Pharmacological properties of adenosine receptors and adenosine binding proteins

Two major subtypes of adenosine receptors occur in different tissues which have been distinguished by pharmacological and biochemical criteria. The A₁ adenosine receptor has a high-affinity for adenosine and mediates inhibition of adenylyl cyclase, whereas the A₂ adenosine receptor usually has a lower affinity and mediates stimulation of the enzyme. Furthermore, evidence has been obtained that A₁ receptors increase the conductance of receptor-regulated potassium channels, induce inactivation of calcium channels, and modulate the breakdown of phosphoinositides by phospholipase C. Selective agonists and antagonists have been developed for both receptor subtypes. In addition, both adenosine receptors have extensively been characterized by radioligand binding studies. Suitable radioligands for the A₁ receptor are the agonist [³H]2-chloro-N⁶-cyclopentyladenoisine (CCPA) and the antagonist [³H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX)and for the A_2a receptor [³H]2-[p-(carboxyethyl)phenethylamino]- 5′-N-carboxamidoadenosine (CGS 21860). Furthermore, photoaffinity ligands were developed from adenosine derivatives, which can be covalently incorporated into the binding unit of both receptor subtypes. With this approach, it has been shown that the A₁ receptor has an apparent molecular weight of approximately 36 kDa and the A_2a receptor of 45 kDa. A second approach to elucidate the structure of adenosine receptors involves the purification of receptor protein by affinity chromatography. With this procedure, cerebral A₁ receptors have been purified to apparent homogeneity. More recently, the structure of receptor subtypes has been elucidated by cloning the receptors from a cDNA library. Furthermore, a novel adenosine binding with [³H] 5′ -N-ethylcarboxamidoadenosine ([³H]NECA). The pharmacological profile of this NECA-binding protein has been determined in competition experiments with adenosine receptor ligands. It can be distinguished from that of A_2a adenosine receptors and other adenosine binding proteins. We propose the name A_x for this unique adenosine binding protein. © 1993 Wiley-Liss, Inc.     

Experimental design and estimation of parameters in complex radioligand binding systems

A computer was used to simulate data from typical radioligand binding experiments with a 2-site competitive-allosteric model of a receptor. The 4 equilibrium parameters of this model cannot be estimated by fitting the model to equilibrium data. Data from simulations of association experiments give satisfactory estimates of the 9 competitive-allosteric model parameters. From the kinetic parameters, equilibrium constants may be calculated. Combining data from equilibrium simulations with data from association simulations provided estimates of the model parameters with smaller standard deviations. A further improvement in design was shown possible by including simulated experiments in which receptor was preincubated with inhibitor before adding ligand. This improvement was documented using Monte Carlo replications of parameter estimates using competing experimental designs. Replications also revealed certain biases in the parameter estimates and could provide a means of estimating those biases when parameter estimates are made using experimental rather than simulated data. Simulations offer a powerful tool in planning experiments designed to estimate kinetic parameters of a receptor system. This is especially true with complex systems that may require pooling data from different kinds of experiments in order to estimate the kinetic parameters.