Characterization of the AT4 receptor in bovine kidney cells

R. K. Handa, J. W. Harding, S. M. Simasko

Research output: Contribution to journalArticle

2 Scopus citations

Abstract

125I-Ang IV (= Ang-(3-8), AT4 receptor agonist) and 125I-Divalinal Ang IV (putative AT4 receptor antagonist) were used to characterize the AT4 receptor in cultured Madin-Darby bovine kidney (MDBK) cells. These cells did not bind Ang II, yet demonstrated a high density of Ang IV binding sites. Saturation binding isotherms showed high affinity binding and a similar number of binding sites for each radioligand (125I-Ang IV Kd = 2 nM, Bmax = 1009 fmol/mg protein, 125I-Divalinal-Ang IV Kd = 1 nM, Bmax = 1048 fmol/mg protein). Competition assays with either radioiigand displayed a one-binding site model with a relative binding affinity order of Ang IV Dualinal Ang IV > Ang III > Ang II > Ang-(1-7) > Sar1, Ile8-Ang II > losartan - PD123177. GTPγS or dithiotreitol did not affect 125I-Ang IV binding, suggesting that the AT4 receptor is not G protein-linked and that disulphide bridges are not important in legulating receptor affinity. Brief exposure of cells to 01-10 nM Ang IV caused a transient increase in [Ca2+]i (fura-2 measurements), but not at higher μM concentrations. Thus, AT4 receptors are highly expressed in bovine kidney cells with binding pioperties similar to that reported in non-epithelial tissue, and activate at least one signaling pathway that involves a change in [Ca2+]i. Also, we provide evidence that the putative AT4 receptor antagonist, Divalinal Ang IV, binds to the same receptor as Ang IV.

Original languageEnglish (US)
Pages (from-to)A86
JournalFASEB Journal
Volume11
Issue number3
StatePublished - Dec 1 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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    Handa, R. K., Harding, J. W., & Simasko, S. M. (1997). Characterization of the AT4 receptor in bovine kidney cells. FASEB Journal, 11(3), A86.