To address conflicting reports concerning the number of angiotensin II (AII) receptor type 1 (AT1) coding loci in vertebrates, Southern blot analysis was used to determine the genomic representation of AT1 receptor genes in animals comprising a divergent evolutionary spectrum. The data demonstrate that the AT1 receptor gene is present as a single genomic copy in a broad spectrum of animals including human, monkey, dog, cow, rabbit, and chicken. In contrast, members of the rodent taxonomic order contain two genes in their genomes. These two genes may have arisen in rodents as a consequence of a gene duplication event that occurred during evolution following the branching of rodents from the mammalian phylogenetic tree. In order to investigate the properties of the human AT1 receptor in a pure cell system, the recombinant human AT1 receptor was stably expressed in mouse L cells. An isolated cell line, designated LhAT1-D6, was found to express abundant levels of recombinant receptor [430 +/- 15 fmol/mg] exhibiting high affinity [KD = 0.15 +/- 0.02 nM] for [125I][SAR1, Ile8] angiotensin II (SIA). The pharmacological profile of ligands competing for [125I] SIA binding to the expressed receptor was in accordance with that of the natural receptor. Radioligand binding of the expressed receptor was decreased in the presence of the non-hydrolyzable analog of GTP, guanosine 5'-(gamma-thio) triphosphate [GTP gamma S]. Angiotensin II evoked a rapid efflux of 45Ca2+ from LhAT1-D6 cells that was blocked by AT1 receptor specific antagonists. In addition, AII inhibited forskolin-stimulated cAMP accumulation in these cells which was blocked by the AT-1 antagonist. Thus, the LhAT1-D6 cell line provides a powerful tool to explore the human AT1 receptor regulation.
Calcitonin gene-related peptide (CGRP), a 37amino-acid peptide, is a member of a small family of peptides including amylin or islet amyloid polypeptide and salmon calcitonin . These related peptides have been shown to display similar effects on in vitro and in vivo carbohydrate metabolism . The present study was initiated to identify and characterize the binding sites for these peptides in lung and nucleus accumbens membranes prepared from pig and guinea pig. Both tissues in either species displayed high-affinity (2-['25 1]iodohistidyl'o )humanCGRPa ([' 25 1]hCGRPa) binding (IC5o = 0.4-7 .7 nM), which was displaced by hCGRP8 -37a with equally high affinity (IC5o = 0.4-7 .3 nM) . High-affinity binding for [1251 ]Bolton-Hunter human amylin (['25 1]BH-h-amylin) was also observed in these tissues Abbreviations used: [' 25 1]BH-h-amylin, [ . . . t]Bolton-Hunter human amylin ; cAMP, cyclic AMP ; CGRP, calcitonin gene-related peptide ; h-amylin, human amylin ; hCGRP, human calcitonin generelated peptide ; r-amylin, rat amylin ; sCT, salmon calcitonin ; VIP, vasoactive intestinal peptide .
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