Characterization of the AT4 receptor in bovine kidney cells

Rajash Handa, J. W. Harding, S. M. Simasko

Research output: Contribution to journalArticle

2 Citations (Scopus)

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)
JournalFASEB Journal
Volume11
Issue number3
StatePublished - 1997
Externally publishedYes

Fingerprint

kidney cells
Kidney
receptors
cattle
Binding Sites
binding sites
antagonists
Losartan
Fura-2
GTP-Binding Proteins
calcium
Disulfides
Isotherms
AT4 receptor
Assays
Proteins
G-proteins
sulfides
Tissue
agonists

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

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

Characterization of the AT4 receptor in bovine kidney cells. / Handa, Rajash; Harding, J. W.; Simasko, S. M.

In: FASEB Journal, Vol. 11, No. 3, 1997.

Research output: Contribution to journalArticle

Handa, R, Harding, JW & Simasko, SM 1997, 'Characterization of the AT4 receptor in bovine kidney cells', FASEB Journal, vol. 11, no. 3.
Handa, Rajash ; Harding, J. W. ; Simasko, S. M. / Characterization of the AT4 receptor in bovine kidney cells. In: FASEB Journal. 1997 ; Vol. 11, No. 3.
@article{073e6ce2ab7b4fee9702dd5f19e1c11f,
title = "Characterization of the AT4 receptor in bovine kidney cells",
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.",
author = "Rajash Handa and Harding, {J. W.} and Simasko, {S. M.}",
year = "1997",
language = "English (US)",
volume = "11",
journal = "FASEB Journal",
issn = "0892-6638",
publisher = "FASEB",
number = "3",

}

TY - JOUR

T1 - Characterization of the AT4 receptor in bovine kidney cells

AU - Handa, Rajash

AU - Harding, J. W.

AU - Simasko, S. M.

PY - 1997

Y1 - 1997

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=17344372410&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=17344372410&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:17344372410

VL - 11

JO - FASEB Journal

JF - FASEB Journal

SN - 0892-6638

IS - 3

ER -