Characterization and function of the bovine kidney epithelial angiotensin receptor subtype 4 using angiotensin IV and divalinal angiotensin IV as receptor ligands

Rajash Handa, Joseph W. Harding, Steven M. Simasko

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

125I-Angiotensin (Ang) IV and 125l-divalinal Ang IV [AT receptor subtype 4 (AT4)] receptor agonist and putative antagonist, respectively] were used to characterize the AT4 receptor in Mardin-Darby bovine kidney epithelial cells (MDBK cell line). Both 125I-Ang IV and 125I-divalinal Ang IV bound to a single high-affinity site (K(D) = 1.37 and 1.01 nM, respectively) and to a comparable density of binding sites (B(max) = 1335 and 1407 fmol/mg protein, respectively). Competition of either radiolabeled ligand with several Ang related peptides demonstrated similar displacement affinities in the following affinity order: Ang IV = divalinal Ang IV > Ang III > Ang II > losartan = PD 123177. Guanosine-5'-O-(3-thio)triphosphate or sulfhydryl reducing agents did not affect the binding of either radiolabeled ligand. Brief exposure of MDBK cells to Ang IV or divalinal Ang IV (0.1 nM to 1 μM) caused a concentration-dependent rise in intracellular calcium concentration levels with a reduced calcium response observed with Ang IV at micromolar concentrations. These results indicate that Ang IV and divalinal Ang IV bind with high affinity to the same receptor and that the MDBK AT4 receptor is not coupled to a classic G protein, nor are sulfhydryl bonds important in regulation of receptor affinity. The MDBK AT4 receptor appears to be pharmacologically similar to that described in nonrenal tissues. Functional studies suggest that AT4 receptor activation can increase intracellular calcium concentration levels in MDBK cells and that divalinal Ang IV possesses agonist activity with respect to this particular intracellular signaling system.

Original languageEnglish (US)
Pages (from-to)1242-1249
Number of pages8
JournalJournal of Pharmacology and Experimental Therapeutics
Volume291
Issue number3
StatePublished - Dec 1999
Externally publishedYes

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Angiotensin Receptors
Ligands
Kidney
Calcium
Angiotensin III
Guanosine 5'-O-(3-Thiotriphosphate)
Losartan
Reducing Agents
Angiotensins
GTP-Binding Proteins
Angiotensin II
AT4 receptor
divalinal-angiotensin IV
des-Asp(1)-des-Arg(2)-Ile(5)-angiotensin II
Epithelial Cells
Binding Sites
Cell Line
Peptides
Proteins

ASJC Scopus subject areas

  • Pharmacology

Cite this

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abstract = "125I-Angiotensin (Ang) IV and 125l-divalinal Ang IV [AT receptor subtype 4 (AT4)] receptor agonist and putative antagonist, respectively] were used to characterize the AT4 receptor in Mardin-Darby bovine kidney epithelial cells (MDBK cell line). Both 125I-Ang IV and 125I-divalinal Ang IV bound to a single high-affinity site (K(D) = 1.37 and 1.01 nM, respectively) and to a comparable density of binding sites (B(max) = 1335 and 1407 fmol/mg protein, respectively). Competition of either radiolabeled ligand with several Ang related peptides demonstrated similar displacement affinities in the following affinity order: Ang IV = divalinal Ang IV > Ang III > Ang II > losartan = PD 123177. Guanosine-5'-O-(3-thio)triphosphate or sulfhydryl reducing agents did not affect the binding of either radiolabeled ligand. Brief exposure of MDBK cells to Ang IV or divalinal Ang IV (0.1 nM to 1 μM) caused a concentration-dependent rise in intracellular calcium concentration levels with a reduced calcium response observed with Ang IV at micromolar concentrations. These results indicate that Ang IV and divalinal Ang IV bind with high affinity to the same receptor and that the MDBK AT4 receptor is not coupled to a classic G protein, nor are sulfhydryl bonds important in regulation of receptor affinity. The MDBK AT4 receptor appears to be pharmacologically similar to that described in nonrenal tissues. Functional studies suggest that AT4 receptor activation can increase intracellular calcium concentration levels in MDBK cells and that divalinal Ang IV possesses agonist activity with respect to this particular intracellular signaling system.",
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N2 - 125I-Angiotensin (Ang) IV and 125l-divalinal Ang IV [AT receptor subtype 4 (AT4)] receptor agonist and putative antagonist, respectively] were used to characterize the AT4 receptor in Mardin-Darby bovine kidney epithelial cells (MDBK cell line). Both 125I-Ang IV and 125I-divalinal Ang IV bound to a single high-affinity site (K(D) = 1.37 and 1.01 nM, respectively) and to a comparable density of binding sites (B(max) = 1335 and 1407 fmol/mg protein, respectively). Competition of either radiolabeled ligand with several Ang related peptides demonstrated similar displacement affinities in the following affinity order: Ang IV = divalinal Ang IV > Ang III > Ang II > losartan = PD 123177. Guanosine-5'-O-(3-thio)triphosphate or sulfhydryl reducing agents did not affect the binding of either radiolabeled ligand. Brief exposure of MDBK cells to Ang IV or divalinal Ang IV (0.1 nM to 1 μM) caused a concentration-dependent rise in intracellular calcium concentration levels with a reduced calcium response observed with Ang IV at micromolar concentrations. These results indicate that Ang IV and divalinal Ang IV bind with high affinity to the same receptor and that the MDBK AT4 receptor is not coupled to a classic G protein, nor are sulfhydryl bonds important in regulation of receptor affinity. The MDBK AT4 receptor appears to be pharmacologically similar to that described in nonrenal tissues. Functional studies suggest that AT4 receptor activation can increase intracellular calcium concentration levels in MDBK cells and that divalinal Ang IV possesses agonist activity with respect to this particular intracellular signaling system.

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