Yang Wenqi, Tuniki Venugopal Raju, Anjaiah Siddam, Falck J R, Hillard Cecilia J, Campbell William B
Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA.
J Pharmacol Exp Ther. 2008 Mar;324(3):1019-27. doi: 10.1124/jpet.107.129577. Epub 2008 Jan 2.
Epoxyeicosatrienoic acids (EETs) are important regulators of vascular tone and homeostasis. Whether they initiate signaling through membrane receptors is unclear. We developed 20-iodo-14,15-epoxyeicosa-8(Z)-enoic acid (20-I-14,15-EE8ZE), a radiolabeled EET agonist, to characterize EET binding to membranes of U937 cells. 20-I-14,15-EE8ZE stimulated cAMP production in U937 cells with similar potency, but it decreased efficacy compared with 11,12-EET. Maximum cAMP production increased 4.2-fold, with an EC(50) value of 9 muM. Like 14,15-EET, 20-I-14,15-EE8ZE relaxed bovine coronary arteries, with a similar EC(50) value. Both 20-I-14,15-EE8ZE agonist activities were blocked by the EET antagonist 14,15-epoxyeicosa-5(Z)enoic acid (14,15-EE5ZE). Specific 20-(125)I-14,15-EE8ZE binding to U937 membranes reached equilibrium within 10 min and remained unchanged for 30 min at 4 degrees C. The binding was saturable, reversible, and exhibited K(D) and B(max) values of 11.8 +/- 1.1 nM and 5.8 +/- 0.2 pmol/mg protein, respectively. Pretreatment of the membranes with guanosine 5'-O-(3-thio)triphosphate reduced the B(max) in a concentration-related manner. 20-(125)I-14,15-EE8ZE binding was inhibited by eicosanoids with potency order of 11,12-EET >14,15-EE5ZE approximately 14,15-EET >> 15-hydroxyeicosatetraenoic acid > 14,15-EET-thiirane >14,15-dihydroxyeicosatrienoic acid. This order is in agreement with the efficacy and potency of cAMP production. In summary, 20-(125)I-14,15-EE8ZE is a radiolabeled EET agonist that is useful to study binding and metabolism. Using this radioligand, we have identified a specific high-affinity and high-abundance EET binding site in U937 cell membranes. This binding site could represent a specific EET receptor, which is probably a G protein-coupled receptor.
环氧二十碳三烯酸（EETs）是血管张力和体内平衡的重要调节因子。它们是否通过膜受体启动信号传导尚不清楚。我们开发了一种放射性标记的EET激动剂20-碘-14,15-环氧二十碳-8（Z）-烯酸（20-I-14,15-EE8ZE），以表征EET与U937细胞膜的结合情况。20-I-14,15-EE8ZE刺激U937细胞中cAMP的产生，其效力相似，但与11,12-EET相比，其效能降低。cAMP产生的最大值增加了4.2倍，EC（50）值为9μM。与14,15-EET一样，20-I-14,15-EE8ZE可舒张牛冠状动脉，EC（50）值相似。20-I-14,15-EE8ZE的两种激动剂活性均被EET拮抗剂14,15-环氧二十碳-5（Z）烯酸（14,15-EE5ZE）阻断。20-（125）I-14,15-EE8ZE与U937细胞膜的特异性结合在10分钟内达到平衡，并在4℃下30分钟内保持不变。这种结合是可饱和的、可逆的，K（D）和B（max）值分别为11.8±1.1 nM和5.8±0.2 pmol/mg蛋白质。用鸟苷5'-O-（3-硫代）三磷酸预处理细胞膜以浓度相关的方式降低了B（max）。20-（125）I-14,15-EE8ZE的结合被类花生酸抑制，其效力顺序为11,12-EET>14,15-EE5ZE≈14,15-EET>>15-羟基二十碳四烯酸>14,15-EET-硫杂环丙烷>14,15-二羟基二十碳三烯酸。该顺序与cAMP产生的效能和效力一致。总之，20-（125）I-14,15-EE8ZE是一种放射性标记的EET激动剂，可用于研究结合和代谢。使用这种放射性配体，我们在U937细胞膜中鉴定出一个特异性的高亲和力和高丰度的EET结合位点。该结合位点可能代表一种特异性的EET受体，可能是一种G蛋白偶联受体。
