Dopamine D2-like receptors on human peripheral blood lymphocytes:: a radioligand binding assay and immunocytochemical study
Dopamine D2-like receptors on human peripheral blood lymphocytes:: a radioligand binding assay and immunocytochemical study
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DOI：
10.1046/j.1365-2680.1999.00135.x
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发表时间：
1999-06-01
期刊：
影响因子：
--
通讯作者：
Tayebati, SK
中科院分区：
文献类型：
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作者：
Amenta, F;Bronzetti, E;Tayebati, SK
1应用放射配基结合分析和分子生物学技术研究了外周血淋巴细胞表达多巴胺D-1样受体和D-2样受体。多巴胺D-2样受体的人外周血淋巴细胞表达的放射性配体结合分析，可能提供一种快速的技术，用于评估受体的变化，其特征在于参与多巴胺能系统的疾病。然而，放射性配体结合测定技术测量多巴胺D-2样受体的适用性受到质疑。2鉴于分子生物学和放射性配体结合测定技术测定多巴胺D-2样受体的数据之间的差异，我们使用多巴胺受体激动剂7-[H-3]-羟基-N，N-二-正丙基-2-氨基四氢萘（[H-3]-7-OH-DPAT）和两种拮抗剂（[H-3]-螺哌隆和[H-3]-奈莫那必利）。3饱和曲线分析显示所有化合物与人外周血淋巴细胞的结合呈浓度依赖性。不同放射性配体的解离常数（K-d）值平均在0.15和0.40 nM之间。结合位点的最大密度（B-max）较低，范围为[H-3]-螺哌隆为4.15 +/- 0.05 fmol/10（6）个细胞，[H-3]-7-OH-DPAT为8.66 +/- 0.04 fmol/10（6）个细胞。[H-3]-螺哌隆和[H-3]-奈莫必利与人外周血淋巴细胞的结合显示，使用放射性配体浓度可获得最高的特异性：非特异性结合比，药理学特征与多巴胺D-2样受体的标记一致。使用较高的放射性配体浓度导致可置换性和可表征性结合较差。5在细胞离心器离心的外周血淋巴细胞中检测多巴胺D-2、D-3和D-4受体免疫反应性，显示多巴胺D-3和D-4，但不显示D-2受体免疫染色。6上述发现表明与分子生物学研究一致，人外周血淋巴细胞表达的多巴胺D-2样受体属于D-3和D-4受体亚型。如果遵循受控的实验条件，则使用多巴胺D-2样受体激动剂和拮抗剂作为放射性配体可检测这些受体。标准化的免疫细胞化学技术检测人外周血淋巴细胞多巴胺受体可能有助于澄清其在淋巴细胞功能或作为外周标志物的多巴胺能系统的状态。
1 Peripheral blood lymphocytes express dopamine D-1-like and D-2-like receptors which were investigated using radioligand binding assay and molecular biology techniques. Analysis of dopamine D-2-like receptors expressed by human peripheral blood lymphocytes with radioligand binding assay may offer a rapid technique for assessing receptor changes in disorders characterized by involvement of the dopaminergic system. However, the suitability of radioligand binding assay techniques to measure dopamine D-2-like receptors is questioned.2 In view of the discrepancy between data of dopamine D-2-like receptor determination with molecular biology and radioligand binding assay techniques, we have assayed dopamine D-2-like receptors expressed by human peripheral blood lymphocytes using as radioligands the dopamine receptor agonist 7-[H-3]-hydroxy-N,N-di-n-propyl-2-aminotetraline ([H-3]-7-OH-DPAT) and two antagonists ([H-3]-spiperone and [H-3]-nemonapride).3 Analysis of saturation curves revealed a concentration-dependent binding of all compounds to human peripheral blood lymphocytes. Dissociation constant (K-d) values averaged between 0.15 and 0.40 nM for different radioligands. The maximum density of binding sites (B-max) was low, ranging from 4.15 +/- 0.05 fmol/10(6) cells with [H-3]-spiperone and 8.66 +/- 0.04 fmol/10(6) cells with [H-3]-7-OH-DPAT.4 Displacement curves of [H-3]-7-OH-DPAT, [H-3]-spiperone and [H-3]-nemonapride binding to human peripheral blood lymphocytes revealed, using radioligand concentrations giving the highest specific:non-specific binding ratio, a pharmacological profile consistent with the labelling of dopamine D-2-like receptors. The use of higher radioligand concentrations resulted in a poorly displaceable and characterizable binding.5 Detection of dopamine D-2, D-3 and D-4 receptor immunoreactivity in cytospin centrifuged peripheral blood lymphocytes revealed dopamine D-3 and D-4 but not D-2 receptor immunostaining.6 The above findings indicate in agreement with molecular biology studies, that dopamine D-2-like receptors expressed by human peripheral blood lymphocytes belong to the D-3 and D-4 receptor subtypes. These receptors are detectable using either dopamine D-2-like receptor agonists and antagonists as radioligands if controlled experimental conditions are followed. The standardisation of immunocytochemical techniques for detecting human peripheral blood lymphocyte dopamine receptors may contribute to clarify their role in lymphocyte function or as a peripheral marker of the status of the dopaminergic system.