De León H, Garcia R
Laboratory of Experimental Hypertension and Vasoactive Peptides, Clinical Research Institute of Montreal, Quebec, Canada.
Regul Pept. 1995 Dec 7;60(1):1-8. doi: 10.1016/0167-0115(95)00112-1.
Endothelin (ET) increases renal vascular resistance by constriction of post- and preglomerular vessels in the rat. However, ET receptor subtypes in renal microvessels have not been clearly defined. Radioligand binding experiments were performed in isolated arcuate and interlobular arteries as well as branching afferent arterioles to characterize 125I-ET-1 binding sites. Competitive inhibition assays were performed with ET-1, ET-2, ET-3, sarafotoxin 6b (S6b), BQ-123 (a preferential ETA receptor antagonist) and 4-Ala-ET-1 (a preferential ETB receptor agonist). Saturation data revealed a single class of high affinity binding sites with a kd of 0.31 +/- 0.03 nM and a Bmax of 1336 +/- 181 fmol/mg protein. Competitive inhibition of 125I-ET-1 binding showed that all assayed compounds displaced 125I-ET-1 in a dose-related manner. ET-1 displaced 100% of 125I-ET-1 binding, displaying monophasic curves with Bmax and kd values of 1369 +/- 170 fmol/mg protein and 0.35 +/- 0.04 nM, respectively. ET-2's displacement curves were similar to those of ET-1. ET-3 and S6b inhibited 100% of 125I-ET-1 binding in a biphasic manner, suggesting these peptides bind both high and low affinity sites. BQ-123 displaced about 50% of 125I-ET-1 in a monophasic manner, indicating a single high affinity binding site. 4-Ala-ET-1 displaced 125I-ET-1 in a clearly biphasic manner with an almost equal proportion of high and low affinity binding sites. Our results suggest that both ETA and ETB receptors are expressed in rat renal preglomerular vessels in almost equal proportions. However, the characteristics of competitive inhibition of 125I-ET-1 binding by several agents cannot be fully explained by a two-receptor model.
内皮素（ET）通过收缩大鼠肾小球后和肾小球前血管来增加肾血管阻力。然而，肾微血管中的ET受体亚型尚未明确界定。在分离的弓形动脉、小叶间动脉以及分支的入球小动脉中进行放射性配体结合实验，以表征125I-ET-1结合位点。用ET-1、ET-2、ET-3、萨拉毒素6b（S6b）、BQ-123（一种选择性ETA受体拮抗剂）和4-丙氨酸-ET-1（一种选择性ETB受体激动剂）进行竞争性抑制试验。饱和数据显示存在一类单一的高亲和力结合位点，解离常数（kd）为0.31±0.03 nM，最大结合容量（Bmax）为1336±181 fmol/mg蛋白质。125I-ET-1结合的竞争性抑制表明，所有测定的化合物均以剂量相关的方式取代125I-ET-1。ET-1取代了100%的125I-ET-1结合，呈现单相曲线，Bmax和kd值分别为1369±170 fmol/mg蛋白质和0.35±0.04 nM。ET-2的取代曲线与ET-1相似。ET-3和S6b以双相方式抑制100%的125I-ET-1结合，表明这些肽同时结合高亲和力和低亲和力位点。BQ-123以单相方式取代约50%的125I-ET-1，表明存在一个单一的高亲和力结合位点。4-丙氨酸-ET-1以明显的双相方式取代125I-ET-1，高亲和力和低亲和力结合位点的比例几乎相等。我们的结果表明，ETA和ETB受体在大鼠肾小球前血管中的表达比例几乎相等。然而，几种试剂对125I-ET-1结合的竞争性抑制特征不能完全用双受体模型来解释。
