Allan C J, Halushka P V
Department of Cell and Molecular Pharmacology, Medical University of South Carolina, Charleston.
J Pharmacol Exp Ther. 1994 Aug;270(2):446-52.
Thromboxane A2 (TxA2) is the predominant eicosanoid metabolite produced by activated human blood-borne monocytes. This study was designed to characterize TxA2 receptors on human peripheral blood monocytes via identification of radioligand binding characteristics using the stable TxA2 mimetic [125I]-BOP ([1S,(1 alpha,2 beta(5Z),3 alpha(1E,3S*),4 alpha]-7-[3-hydroxy-4-(4'- iodophenoxy-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-he ptenoic acid) and via analysis of second messenger signal transduction pathways. Scatchard analysis of the binding of [125I]-BOP in human monocyte membranes revealed a single class of binding sites, Kd = 1.49 +/- 0.14 nM and Bmax = 696 +/- 113 fmol/mg membrane protein (n = 8). [125I]-BOP interacted specifically with a TxA2 receptor, as shown by competition binding studies with a range of TxA2 receptor agonists and antagonists that gave a rank order of potency of (-)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid > I-BOP = I-SAP > ONO11113 > SQ29548 > U46619 > (+)-9-chlorobenzyl-6-fluoro-1,2,3,4- tetrahydrocarbazol-1-yl acetic acid. I-BOP caused a dose-dependent increase in intracellular free calcium (EC50 = 7.14 +/- 1.1 nM, n = 4), which was attenuated by preincubation with the TxA2 receptor antagonists SQ29548 (1 microM) and (-)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid (100 nM). This study provides further evidence for a TxA2 receptor in monocytes and supports the potential for future characterization of TxA2 receptor subtypes using molecular techniques.
血栓素A2（TxA2）是活化的人血单核细胞产生的主要类二十烷酸代谢产物。本研究旨在通过使用稳定的TxA2模拟物[125I]-BOP（[1S,(1α,2β(5Z),3α(1E,3S*),4α]-7-[3-羟基-4-(4'-碘苯氧基-1-丁烯基)-7-氧杂双环-[2.2.1]庚烷-2-基]-5-庚烯酸）鉴定放射性配体结合特性以及分析第二信使信号转导途径，来表征人外周血单核细胞上的TxA2受体。对人单核细胞膜中[125I]-BOP结合进行的Scatchard分析显示存在一类结合位点，解离常数（Kd）= 1.49±0.14 nM，最大结合容量（Bmax）= 696±113 fmol/mg膜蛋白（n = 8）。[125I]-BOP与TxA2受体特异性相互作用，一系列TxA2受体激动剂和拮抗剂的竞争结合研究表明其效价顺序为（-）-9-氯苄基-6-氟-1,2,3,4-四氢咔唑-1-基乙酸＞I-BOP = I-SAP＞ONO11113＞SQ29548＞U46619＞（+）-9-氯苄基-6-氟-1,2,3,4-四氢咔唑-1-基乙酸。I-BOP引起细胞内游离钙浓度呈剂量依赖性增加（半数有效浓度（EC50）= 7.14±1.1 nM，n = 4），用TxA2受体拮抗剂SQ29548（1μM）和（-）-9-氯苄基-6-氟-1,2,3,4-四氢咔唑-1-基乙酸（100 nM）预孵育可使其减弱。本研究为单核细胞中存在TxA2受体提供了进一步证据，并支持未来使用分子技术对TxA2受体亚型进行表征的可能性。
