Burns K D, Inagami T, Harris R C
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee.
Am J Physiol. 1993 Apr;264(4 Pt 2):F645-54. doi: 10.1152/ajprenal.1993.264.4.F645.
The rabbit proximal tubule (PT) has been widely utilized to study the direct effects of angiotensin II (ANG II) on PT function. The purpose of the present study was to characterize the binding properties of PT ANG II receptors, using nonpeptide antagonists, and to clone a rabbit PT ANG II receptor. In rat and rabbit kidney cortical brush-border and basolateral membranes, specific binding of 125I-ANG II was inhibited by the AT1 ANG II-receptor antagonist DuP 753, but not by the AT2 antagonist PD 123319. Using a rabbit kidney cortex cDNA library, we isolated cDNA encoding an ANG II receptor, with an open-reading frame sharing a high degree of sequence homology to previously cloned AT1 ANG II receptors. In transfected COS-1 cells, this rabbit ANG II receptor had properties of the AT1 class. Northern analysis revealed high levels of mRNA expression for this receptor in rabbit kidney cortex and adrenal gland. Within the kidney, message was detected in primary cultures of rabbit PT cells, as well as in freshly isolated rabbit PT segments. Message was also present in cells of the mouse PT line, MCT, and in rat glomerular mesangial cells. Utilizing polymerase chain reaction (PCR) with primers derived from the 1st and 4th transmembrane domains of the rat AT1A ANG II receptor, a 279-bp DNA fragment was amplified from reverse-transcribed RNA from rabbit PT cells. This DNA encoded an amino acid sequence identical to that encoded by the rabbit kidney cDNA clone in the corresponding region and differed by a single base substitution. Southern analysis of rabbit genomic DNA restriction digests with the rabbit ANG II receptor probe revealed hybridization to a single band in each lane. These results indicate that an AT1 ANG II receptor is present in the PT and that a single gene codes for the AT1 receptor in rabbit. The clone isolated in the present study should provide a useful tool with which to study the regulation of the PT renin-angiotensin system.
兔近端小管（PT）已被广泛用于研究血管紧张素II（ANG II）对PT功能的直接影响。本研究的目的是使用非肽拮抗剂来表征PT ANG II受体的结合特性，并克隆兔PT ANG II受体。在大鼠和兔肾皮质刷状缘和基底外侧膜中，125I-ANG II的特异性结合被AT1 ANG II受体拮抗剂DuP 753抑制，但不被AT2拮抗剂PD 123319抑制。利用兔肾皮质cDNA文库，我们分离出编码ANG II受体的cDNA，其开放阅读框与先前克隆的AT1 ANG II受体具有高度的序列同源性。在转染的COS-1细胞中，这种兔ANG II受体具有AT1类的特性。Northern分析显示该受体在兔肾皮质和肾上腺中mRNA表达水平很高。在肾脏内，在兔PT细胞的原代培养物以及新鲜分离的兔PT节段中检测到了信息。在小鼠PT系MCT细胞和大鼠肾小球系膜细胞中也存在信息。利用从大鼠AT1A ANG II受体的第1和第4跨膜结构域衍生的引物进行聚合酶链反应（PCR），从兔PT细胞的逆转录RNA中扩增出一个279 bp的DNA片段。该DNA编码的氨基酸序列与兔肾cDNA克隆在相应区域编码的序列相同，仅相差一个单碱基替换。用兔ANG II受体探针进行兔基因组DNA限制性消化的Southern分析显示，每个泳道中均与一条带杂交。这些结果表明，PT中存在AT1 ANG II受体，并且兔中的AT1受体由单个基因编码。本研究中分离出的克隆应提供一个有用的工具，用于研究PT肾素-血管紧张素系统的调节。
