title: Peptide and nonpeptide antagonist interaction with constitutively active human AT1 receptors.
abstract: Wild type human AT(1) receptors (WT-AT(1)) and mutant receptors, in which Asn(111) was replaced by glycine (N111G), alanine (N111A) and serine (N111S), or in which Asp(281) was replaced by alanine (D281A) or in which N111G and D281A replacements were combined, were transiently expressed in CHO-K1 cells. While the biphenyltetrazole compound candesartan dissociated slowly and behaved as an insurmountable antagonist for WT-AT(1), it dissociated swiftly and only produced a rightward shift of the angiotensin Ang II- and -IV dose-response curves for inositol phosphate (IP) accumulation in cells expressing N111G. [3H]candesartan competition binding yielded the same potency order of the related biphenyltetrazoles for WT-AT(1) and mutated receptors, i.e. candesartan>EXP3174>irbesartan>losartan. Affinities were equal for WT-AT(1) and D281A and 40- to 400-fold lower for all Asn(111) mutants. Mutations did not affect the affinity of the peptide antagonist [Sar(1)Ile(8)]Ang II (SARILE). Basal IP accumulation in cells with WT-AT(1) was not affected by any biphenyltetrazole antagonists and was increased by SARILE to 19% of the maximal Ang II stimulation. Basal IP accumulation was higher for cells expressing the Asn(111)-mutated receptors. For N111G, this accumulation was partially inhibited by all the biphenyltetrazoles upon long-term (18hr) exposure. In these cells SARILE produced the same maximal stimulation as Ang II. Asn(111)-mutated AT(1) receptors are thought to mimic the pre-activated state of the wild type receptor and comparing the efficacy and affinity of ligands for such mutated receptors facilitate the distinction of partial (SARILE) and inverse (biphenyltetrazoles) agonists from true antagonists.
authors.affiliation: Department of Molecular and Biochemical Pharmacology, Institute for Molecular Biology and Biotechnology, Vrije Universiteit Brussel (VUB), Pleinlaan 2, Belgium. tmleminh@vub.ac.be
authors.forename: Minh Tam
authors.initials: MT
authors.lastname: Le
authors.forename: Patrick M L
authors.initials: PM
authors.lastname: Vanderheyden
authors.forename: Márta
authors.initials: M
authors.lastname: Szaszák
authors.forename: László
authors.initials: L
authors.lastname: Hunyady
authors.forename: Veerle
authors.initials: V
authors.lastname: Kersemans
authors.forename: Georges
authors.initials: G
authors.lastname: Vauquelin
mesh_terms: D000409:Alanine; D019943:Amino Acid Substitution; D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology; D001224:Aspartic Acid; D001562:Benzimidazoles / Q000493:pharmacokinetics; D001713:Biphenyl Compounds; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D019808:Losartan / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D011869:Radioligand Assay; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013777:Tetrazoles / Q000493:pharmacokinetics / Q000494:pharmacology*; D014162:Transfection; D014316:Tritium
chemical_list: D000959:Antihypertensive Agents; D001562:Benzimidazoles; D001713:Biphenyl Compounds; D007093:Imidazoles; D044140:Receptor, Angiotensin, Type 1; D011945:Receptors, Angiotensin; D013777:Tetrazoles; D014316:Tritium; D001224:Aspartic Acid; C066026:losartan carboxylic acid; D019808:Losartan; D000409:Alanine; C081643:candesartan