Singh G, Hussain J F, MacKinnon A, Brown C M, Kendall D A, Wilson V G
Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Nottingham, UK.
Naunyn Schmiedebergs Arch Pharmacol. 1995 Jan;351(1):17-26. doi: 10.1007/BF00169059.
In the present study we have prepared crude, methanolic extracts of bovine lung and bovine brain and, using radioligand binding assays in conjunction with a number of simple chromatographic techniques, provided evidence for the presence of a non-catecholamine 'clonidine-displacing substance' (CDS). The level of CDS in lung extracts (9 units/g wet weight n = 11) is approximately 3 times that in the brain extracts. Furthermore, the effect of the crude, methanolic extracts are selective for non-adrenoceptor, imidazoline (labelled by [3H]-idazoxan) and alpha 2-adrenoceptor binding sites (labelled by [3H]-clonidine); both extracts are 5-10-fold more potent displacers of ligand binding to alpha 2-adrenoceptors compared with binding to opiate receptors (labelled by [3H]-etorphine) and practically inactive against alpha 1-adrenoceptor and muscarinic binding sites (labelled by [3H]-prazosin and [3H]-quinuclidinyl benzilate, respectively). With the exception of the non-adrenoceptor, imidazoline binding assay, which used rat kidney membranes labelled by [3H]-idazoxan in the presence of the alpha 2-adrenoceptor antagonist RS-15385-197, all radioreceptor assays involved bovine cerebral cortex membranes. Although the extracts contain catecholamines (brain only), histamine (lung only) and monovalent cations (both), which have the potential to interfere with the radioligand binding assays, their concentrations were too low to account for the effects observed. Preliminary attempts at purification of the extracts revealed that CDS activities from the two tissues are similar, i.e., practically insoluble in organic solvents at room temperature, not affected by either Sep-Pak C18 column or anion exchange resins but retained (along with the monovalent cations) by cation exchange resin. However, following chromatographic separation on a Biogel P2 column, the CDS-containing eluates are cation-free and exhibit qualitatively similar elution profiles. Future experiments will involve further purification of 'clonidine-displacing substance' to characterize its interaction with alpha 2-adrenoceptor binding sites in greater detail and establish whether it has biological activity consistent with the properties implied by its effects in radioligand binding assays.
在本研究中，我们制备了牛肺和牛脑的粗甲醇提取物，并通过放射性配体结合测定法结合一些简单的色谱技术，为一种非儿茶酚胺类“可乐定置换物质”（CDS）的存在提供了证据。肺提取物中CDS的水平（9单位/克湿重，n = 11）约为脑提取物中的3倍。此外，粗甲醇提取物的作用对非肾上腺素能受体、咪唑啉（由[3H]-伊达唑胺标记）和α2-肾上腺素能受体结合位点（由[3H]-可乐定标记）具有选择性；与结合阿片受体（由[3H]-埃托啡标记）相比，两种提取物对配体与α2-肾上腺素能受体结合的置换能力要强5-10倍，而对α1-肾上腺素能受体和毒蕈碱结合位点（分别由[3H]-哌唑嗪和[3H]-奎宁环基苯甲酸酯标记）几乎无活性。除了非肾上腺素能受体咪唑啉结合测定法（在α2-肾上腺素能受体拮抗剂RS-15385-197存在下，使用由[3H]-伊达唑胺标记的大鼠肾膜）外，所有放射性受体测定均涉及牛大脑皮层膜。尽管提取物中含有儿茶酚胺（仅脑中有）、组胺（仅肺中有）和单价阳离子（两者都有），它们有可能干扰放射性配体结合测定，但它们的浓度过低，无法解释所观察到的效应。对提取物进行纯化的初步尝试表明，两种组织中的CDS活性相似，即在室温下几乎不溶于有机溶剂，不受Sep-Pak C18柱或阴离子交换树脂的影响，但可被阳离子交换树脂保留（与单价阳离子一起）。然而，在Biogel P2柱上进行色谱分离后，含CDS的洗脱液不含阳离子，并且呈现出定性相似的洗脱曲线。未来的实验将包括进一步纯化“可乐定置换物质”，以更详细地表征其与α2-肾上腺素能受体结合位点的相互作用，并确定它是否具有与其在放射性配体结合测定中的效应所暗示的特性相符的生物活性。
