Cloning of a rabbit kidney cortex AT1 angiotensin II receptor that is present in proximal tubule epithelium.
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.
Burns
K D
KD
Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee.
Inagami
T
T
Harris
R C
RC
D000595:Amino Acid Sequence; D000804:Angiotensin II / Q000037:antagonists & inhibitors; D057911:Angiotensin Receptor Antagonists; D000818:Animals; D001483:Base Sequence; D001713:Biphenyl Compounds / Q000494:pharmacology; D015152:Blotting, Northern; D015139:Blotting, Southern; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry / Q000648:ultrastructure; D007093:Imidazoles / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007687:Kidney Tubules, Proximal / Q000737:chemistry* / Q000166:cytology* / Q000378:metabolism; D019808:Losartan; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D011817:Rabbits; D011945:Receptors, Angiotensin / Q000032:analysis* / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013777:Tetrazoles / Q000494:pharmacology; D014162:Transfection
D057911:Angiotensin Receptor Antagonists; D001713:Biphenyl Compounds; D007093:Imidazoles; D007457:Iodine Radioisotopes; D011725:Pyridines; D011945:Receptors, Angiotensin; D013777:Tetrazoles; D000804:Angiotensin II; C073402:PD 123319; D004247:DNA; D019808:Losartan