Lin C, Stone R A, Wax M B
Department of Ophthalmology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084.
Invest Ophthalmol Vis Sci. 1990 Jan;31(1):147-52.
Angiotensin binding sites in membrane homogenates of rabbit iris/ciliary body and human nonpigmented ciliary epithelial (NPE) cells grown in culture were characterized using radioligand binding assays with (125I)-sarcosine1-isoleucine8-angiotensin II [(125I)-SARILE]. Scatchard analysis of the binding of (125I)-SARILE yielded linear plots with a Kd value of 0.55 +/- 0.1 nM and a Bmax of 98 +/- 23 fmol/mg protein in rabbit iris/ciliary body, and a Kd value of 0.63 +/- 0.1 nM, and a Bmax of 36.2 +/- 24 fmol/mg protein in NPE cells. Studies of the inhibition of the binding of (125I)-SARILE in rabbit iris/ciliary body were performed with a series of competing ligands, including the angiotensin receptor antagonist SARILE and the agonists angiotensin I, angiotensin II and angiotensin III. Inhibition curves for the antagonist resulted in Hill coefficients of approximately 1, consistent with the presence of a single class of binding sites with high affinity for (125I)-SARILE. Competition for the binding of (125I)-SARILE to binding sites with each of the agonists resulted in inhibition curves with Hill coefficients significantly less than 1 in the absence of GTP. However, in the presence of 100 microM GTP the Hill coefficients increased to approximately 1. The order of potencies of these agents was consistent with the pharmacologic profile of angiotensin II receptors. Thus rabbit iris/ciliary body homogenates, which include vascular tissue, contain a homogeneous population of angiotensin binding sites coupled to a guanine nucleotide binding protein. The presence of binding sites in cultured NPE cells indicates that at least some are located on the cells thought to be responsible for aqueous humor secretion.
采用放射性配体结合分析法，使用（125I）-肌氨酸1-异亮氨酸8-血管紧张素II [（125I）-SARILE]对兔虹膜/睫状体膜匀浆以及培养的人非色素睫状上皮（NPE）细胞中的血管紧张素结合位点进行了表征。对（125I）-SARILE结合的Scatchard分析在兔虹膜/睫状体中产生了线性图，其Kd值为0.55±0.1 nM，Bmax为98±23 fmol/mg蛋白质；在NPE细胞中Kd值为0.63±0.1 nM，Bmax为36.2±24 fmol/mg蛋白质。用一系列竞争性配体对兔虹膜/睫状体中（125I）-SARILE的结合抑制进行了研究，这些配体包括血管紧张素受体拮抗剂SARILE以及激动剂血管紧张素I、血管紧张素II和血管紧张素III。拮抗剂的抑制曲线产生的希尔系数约为1，这与存在对（125I）-SARILE具有高亲和力的单一类结合位点一致。在不存在GTP的情况下，（125I）-SARILE与各激动剂竞争结合位点产生的抑制曲线的希尔系数明显小于1。然而，在存在100μM GTP的情况下，希尔系数增加到约1。这些药物的效价顺序与血管紧张素II受体的药理学特征一致。因此，包含血管组织的兔虹膜/睫状体匀浆含有与鸟嘌呤核苷酸结合蛋白偶联的血管紧张素结合位点的同质群体。培养的NPE细胞中存在结合位点表明，至少有一些位于被认为负责房水分泌的细胞上。
