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 language||English (US)|
|State||Published - Dec 1 1997|
ASJC Scopus subject areas
- Molecular Biology