Falcone R C, Orzechowski R F, Aharony D
Philadelphia College of Pharmacy and Science, Dept. of Pharmacology and Toxicology, PA 19104.
Prostaglandins. 1990 Mar;39(3):241-58. doi: 10.1016/0090-6980(90)90044-v.
Peptido-leukotrienes (LTs) elicit myocardial depression in several mammalian species, and radioligand binding assays with 3H-LTC4 and 3H-LTD4 have provided evidence of putative receptor sites on guinea pig cardiac ventricular membranes (GPCVM). Our objective was to characterize specific binding of 3H-ICI 198,615, a potent and selective LTD4 antagonist, to the 155,000 X g pellet of GPCVM. 3H-ICI 198,615 (0.01-3.8 nM) showed high specific binding (85-90% of total), which was protein dependent, saturable (Bmax = 4914 +/- 706 fmol/mg protein, n = 3), of high affinity (Kd = 4.3 +/- 0.8 nM, n = 3) and without cooperativity. Equilibrium binding was achieved by 20 minutes and could be rapidly reversed by addition of excess unlabeled ICI 198,615 or FPL55712. Competition studies with 3H-ICI 198,615 against several LTD4 antagonists produced an order of potency: ICI 198,615 much greater than SKF102922 greater than FPL55712 greater than or equal to LY171883. Addition of divalent cations caused a concentration dependent decrease in specific binding apparently due to a reduction in affinity. Binding was not influenced by the guanine nucleotide analogs GTP gamma S and Gpp(NH)p, EDTA, or a multitude of diverse non-LT receptor agonists and antagonists. These data provide evidence supporting the existence of specific and high affinity binding sites for 3H-ICI 198,615 in GPCVM.
肽白三烯（LTs）在几种哺乳动物中会引起心肌抑制，并且使用3H-LTC4和3H-LTD4进行的放射性配体结合测定已提供证据表明豚鼠心室肌膜（GPCVM）上存在假定的受体位点。我们的目标是表征强效选择性LTD4拮抗剂3H-ICI 198,615与GPCVM的155,000×g沉淀的特异性结合。3H-ICI 198,615（0.01 - 3.8 nM）显示出高特异性结合（占总量的85 - 90%），其依赖于蛋白质，具有饱和性（Bmax = 4914±706 fmol/mg蛋白质，n = 3），高亲和力（Kd = 4.3±0.8 nM，n = 3）且无协同性。平衡结合在20分钟内达到，并且通过添加过量未标记的ICI 198,615或FPL55712可迅速逆转。用3H-ICI 198,615与几种LTD4拮抗剂进行的竞争研究产生了如下效价顺序：ICI 198,615远大于SKF102922大于FPL55712大于或等于LY171883。添加二价阳离子导致特异性结合呈浓度依赖性降低，这显然是由于亲和力降低所致。结合不受鸟嘌呤核苷酸类似物GTPγS和Gpp(NH)p、EDTA或多种不同的非LT受体激动剂和拮抗剂的影响。这些数据提供了支持在GPCVM中存在3H-ICI 198,615特异性高亲和力结合位点的证据。
