Boulay G, Servant G, Luong T T, Escher E, Guillemette G
Department of Pharmacology, Faculty of Medicine, University of Sherbrooke, Quebec, Canada.
Mol Pharmacol. 1992 Apr;41(4):809-15.
Angiotensin II (AII) is an important regulator of aldosterone secretion by adrenal glomerulosa cells. All interacts with a specific receptor coupled to a guanine nucleotide-binding protein that controls the activity of phospholipase C. Recently, novel All nonpeptide antagonists (DuP-753 and PD-123319) have been shown to discriminate between two subclasses of All receptors in many different tissues. Our studies confirmed that 125I-All specifically labeled two classes of binding sites for All in a membrane preparation of bovine adrenal glomerulosa cells. The first class (DuP-753 sensitive) represented approximately 85% of the total binding sites for All and possessed a high affinity (IC50 of 92.9 +/- 19.5 nM) for DuP-753. PD-123319 did not have any effect on 125I-All binding to this site. The second class of binding sites was more sensitive to PD-123319, with an IC50 of 6.9 +/- 3.7 nM, and had a much lower affinity for DuP-753 (IC50 around 10 microM). The two classes of receptors had different affinities for All. All showed an affinity around 2 nM for All type 1 receptor (AT1)(DuP-753 sensitive) and a higher affinity, around 0.3 nM, for All type 2 receptor (AT2) (PD-123319 sensitive). All-induced steroidogenesis was completely abolished in the presence of 3 microM DuP-753, indicating that this activity was mediated through a DuP-753-sensitive receptor. We also found that polyvinyl sulfate (PVS), a polyanion, could partly inhibit the binding of 125I-All to bovine adrenal glomerulosa cell membranes, with half-maximal efficiency at 17.3 +/- 8.2 nM. The inhibitory effect of PVS was selective for AT1. The inhibitory effect of PVS was due to a change in the affinity state of the receptor. Unexpectedly, PVS had no effect on All-induced steroidogenesis or on All binding to intact bovine adrenal glomerulosa cells. However, the inhibitory effect of PVS on All binding was recovered after permeabilization of cells. Direct interaction of polyanions with AT1 was suggested by the capacity of solubilized photoaffinity-labeled 125I-AT1 to adsorb to heparin-agarose gels. The adsorption of 125I-AT1 to heparin-agarose was inhibited by prior incubation of solubilized receptor with heparin or PVS. These results suggest that All-induced steroidogenesis is mediated by a DuP-753-sensitive receptor and that PVS decreases the affinity of this receptor by interacting with an intracellular domain (possibly the positively charged domain responsible for coupling with guanine nucleotide-binding proteins).
血管紧张素II（AII）是肾上腺球状带细胞醛固酮分泌的重要调节因子。AII与一种与鸟嘌呤核苷酸结合蛋白偶联的特异性受体相互作用，该蛋白控制磷脂酶C的活性。最近，新型AII非肽拮抗剂（DuP - 753和PD - 123319）已被证明在许多不同组织中可区分AII受体的两个亚类。我们的研究证实，125I - AII可特异性标记牛肾上腺球状带细胞膜制剂中AII的两类结合位点。第一类（对DuP - 753敏感）约占AII总结合位点的85%，对DuP - 753具有高亲和力（IC50为92.9±19.5 nM）。PD - 123319对125I - AII与该位点的结合没有任何影响。第二类结合位点对PD - 123319更敏感，IC50为6.9±3.7 nM，对DuP - 753的亲和力低得多（IC50约为10 μM）。这两类受体对AII具有不同的亲和力。AII对1型AII受体（AT1）（对DuP - 753敏感）的亲和力约为2 nM，对2型AII受体（AT2）（对PD - 123319敏感）的亲和力更高，约为0.3 nM。在存在3 μM DuP - 753的情况下，AII诱导的类固醇生成完全被消除，表明该活性是通过对DuP - 753敏感的受体介导的。我们还发现，聚阴离子聚乙烯硫酸盐（PVS）可部分抑制125I - AII与牛肾上腺球状带细胞膜的结合，半数最大抑制浓度为17.3±8.2 nM。PVS的抑制作用对AT1具有选择性。PVS的抑制作用是由于受体亲和力状态的改变。出乎意料的是，PVS对AII诱导的类固醇生成或对AII与完整牛肾上腺球状带细胞的结合没有影响。然而，细胞透化后PVS对AII结合的抑制作用得以恢复。溶解的光亲和标记的125I - AT1吸附到肝素 - 琼脂糖凝胶的能力表明聚阴离子与AT1存在直接相互作用。用肝素或PVS预先孵育溶解的受体可抑制125I - AT1对肝素 - 琼脂糖的吸附。这些结果表明，AII诱导的类固醇生成是由对DuP - 753敏感的受体介导的，并且PVS通过与细胞内结构域（可能是负责与鸟嘌呤核苷酸结合蛋白偶联的带正电结构域）相互作用降低了该受体的亲和力。
