title: Distinct binding properties of the AT(1) receptor antagonist [(3)H]candesartan to intact cells and membrane preparations.
abstract: [(3)H]-2-Ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H-benzimidazoline-7-carboxylic acid ([(3)H]candesartan), a non-peptide angiotensin II type 1 receptor (AT(1) receptor) antagonist bound with high affinity and specificity to intact adherent human AT(1) receptor transfected Chinese hamster ovary cells. The binding characteristics were preserved when cells were suspended, but the dissociation was 3-4-fold faster and the affinity 2-fold lower, while examining [(3)H]candesartan binding to cell membranes. These data suggested the role of the intracellular organisation of living CHO-hAT(1) cells in antagonist-AT(1) receptor interactions. Yet, a specific role of microtubule or actin filaments of the cytoskeleton, receptor phosphorylation by Protein Kinase C, membrane polarity, cytoplasmic components like ATP and the need of an intact cell membrane could be excluded. The potential effect of protease degradation or receptor oxidation during the membrane preparation was also unlikely. The dissociation rate and the equilibrium dissociation constant of [(3)H]candesartan increased with the temperature for both intact cells and membranes. Thermodynamic studies suggested that the bonds between candesartan and the hAT(1) receptor may be of different nature in intact CHO-hAT(1) cells and membranes thereof. Whereas the binding was almost completely enthalpy-driven on intact cells, there was a mixed contribution of both enthalpy and entropy on membranes.
authors.affiliation: Department of Molecular and Biochemical Pharmacology, Free University of Brussels (VUB), 65 Paardenstraat, B-1640 Sint-Genesius Rode, Belgium. ffierens@vub.ac.be
authors.forename: Frederik L P
authors.initials: FL
authors.lastname: Fierens
authors.forename: Patrick M L
authors.initials: PM
authors.lastname: Vanderheyden
authors.forename: Chantal
authors.initials: C
authors.lastname: Roggeman
authors.forename: Pascal
authors.initials: P
authors.lastname: Vande Gucht
authors.forename: Jean Paul
authors.initials: JP
authors.lastname: De Backer
authors.forename: Georges
authors.initials: G
authors.lastname: Vauquelin
mesh_terms: D057911:Angiotensin Receptor Antagonists; D000818:Animals; D001562:Benzimidazoles / Q000494:pharmacology*; D001665:Binding Sites; D001713:Biphenyl Compounds; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D044140:Receptor, Angiotensin, Type 1; D013777:Tetrazoles / Q000494:pharmacology*; D013816:Thermodynamics; D014316:Tritium
chemical_list: D057911:Angiotensin Receptor Antagonists; D001562:Benzimidazoles; D001713:Biphenyl Compounds; D044140:Receptor, Angiotensin, Type 1; D013777:Tetrazoles; D014316:Tritium; C081643:candesartan