Cloning of a rabbit kidney cortex AT1 angiotensin II receptor that is present in proximal tubule epithelium.
Cloning of a rabbit kidney cortex AT1 angiotensin II receptor that is present in proximal tubule epithelium.
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近端小管上皮中存在的兔肾皮质 AT1 血管紧张素 II 受体的克隆。
DOI：
10.1152/ajprenal.1993.264.4.f645
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Harris,RC
中科院分区：
文献类型：
--
作者：
Burns,KD;Inagami,T;Harris,RC
兔近端小管（PT）被广泛用于研究血管紧张素II（ANG II）对PT功能的直接影响。本研究的目的是表征PT ANG II受体的结合特性，使用非肽类拮抗剂，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，特异性结合的125 I-ANG II抑制AT 1 ANG II受体拮抗剂DuP 753，但不AT 2拮抗剂PD 123319。使用兔肾皮质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AT 1 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AT 1类的特性。北方分析表明，高水平的表达，该受体在兔肾皮质和肾上腺。在肾脏内，在兔PT细胞的原代培养物以及新鲜分离的兔PT节段中检测到消息。信使也存在于小鼠PT系、MCT和大鼠肾小球系膜细胞中。利用聚合酶链反应（PCR）与引物来自第1和第4跨膜结构域的大鼠AT 1A ANG II受体，一个279 bp的DNA片段扩增从逆转录RNA从兔PT细胞。该DNA编码的氨基酸序列与兔肾cDNA克隆在相应区域编码的氨基酸序列相同，不同之处在于单个碱基取代。用兔ANG II受体探针对兔基因组DNA限制性内切酶进行Southern分析，结果显示每条泳道中均与单一条带杂交。这些结果表明，AT 1 ANG II受体存在于PT和一个单一的基因编码的AT 1受体在兔。本研究中分离的克隆为研究PT肾素-血管紧张素系统的调节提供了有用的工具。
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.