Chiu A T, McCall D E, Roscoe W A
Du Pont Merck Pharmaceutical Company, Wilmington, DE 19880-0400.
Biochem Biophys Res Commun. 1992 Nov 16;188(3):1030-9. doi: 10.1016/0006-291x(92)91335-n.
[125I]EXP985 is the first nonpeptide radioligand with high specific activity for the AT1 angiotensin receptor. The biochemical and pharmacological profiles of this ligand were determined using either ligand-receptor binding techniques in rat adrenal cortical microsomes or cellular Ca2+ mobilization in rat smooth muscle cells. Specific binding with 0.1 nM [125I]EXP985 increased slowly with time reaching an equilibrium at 60 min of incubation (22 degrees C). Scatchard analysis of the inhibition/binding data revealed a single class of binding sites having a Kd of 1.49 +/- 0.06 nM and a Bmax of 3.6 +/- 0.1 pmol/mg protein. These sites were saturable and the ligand-receptor complex dissociated with a t1/2 of 58 min. The binding was inhibited by Ang peptides with the following order of potency and IC50 (nM): Ang II (3.7) > Ang III (69) > Ang I (3650), and by the nonpeptide AT1 receptor antagonist, losartan, with an IC50 of 3.2 nM. PD123177, an AT2 selective antagonist, showed minimal inhibitory effect. Specific binding of [125I]EXP985 was found on rat aortic smooth cells. Ang II-induced Ca2+ mobilization in these cells was blocked by EXP985 in a noncompetitive manner. These data show that [125I]EXP985 (or its unlabeled) is a potent and highly specific radioligand or noncompetitive antagonist which represents a novel tool to further our understanding of the biochemistry of AT1 receptors.
[125I]EXP985是首个对血管紧张素AT1受体具有高比活性的非肽类放射性配体。使用大鼠肾上腺皮质微粒体中的配体-受体结合技术或大鼠平滑肌细胞中的细胞Ca2+动员来确定该配体的生化和药理学特性。与0.1 nM [125I]EXP985的特异性结合随时间缓慢增加，在孵育60分钟（22摄氏度）时达到平衡。对抑制/结合数据进行Scatchard分析，结果显示存在一类结合位点，其解离常数（Kd）为1.49±0.06 nM，最大结合容量（Bmax）为3.6±0.1 pmol/mg蛋白质。这些位点具有饱和性，配体-受体复合物的解离半衰期（t1/2）为58分钟。血管紧张素肽按以下效力顺序和半数抑制浓度（IC50，nM）抑制该结合：血管紧张素II（3.7）＞血管紧张素III（69）＞血管紧张素I（3650），非肽类AT1受体拮抗剂氯沙坦的IC50为3.2 nM。AT2选择性拮抗剂PD123177的抑制作用极小。在大鼠主动脉平滑肌细胞上发现了[125I]EXP985的特异性结合。EXP985以非竞争性方式阻断这些细胞中血管紧张素II诱导的Ca2+动员。这些数据表明，[125I]EXP985（或其未标记形式）是一种强效且高度特异性的放射性配体或非竞争性拮抗剂，是进一步了解AT1受体生物化学的新型工具。
