title: Pharmacological characterization of a specific binding site for angiotensin IV in cultured porcine aortic endothelial cells.
abstract: This study demonstrated the existence of a specific binding site for angiotensin IV in porcine aortic endothelial cells. Non-equilibrium kinetic analyses at 37 degrees C allowed the calculation of a kinetic Kd of 0.44 nM. Pseudo-equilibrium saturation binding studies at 37 degrees C for 90 min indicated the presence of a single high-affinity site (Kd = 3.87 +/- 0.60 nM), saturable and abundant (Bmax = 9.64 +/- 1.44 pmol/mg protein). Competitive binding studies demonstrated the following rank order of effectiveness: angiotensin IV > angiotensin III > angiotensin II > angiotensin I > angiotensin II-(1-7), while 2-n-butyl-4-chloro-5-hydroxymethyl-1 [(2'-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl] imidazol (DuP 753: losartan), 1-(4-amino-3-methyl-phenyl) methyl-5-diphenylisoethyl-4,5,6,7-tetrahydro-1H-imidazo [4,5-C] pyridine-6-carboxylic acid (PD 123177) or nicotinic acid-Tyr-(N alpha -benzyl-oxycarbonyl-Arg) Lys-His-Pro-Ile-OH (CGP 42112A) were inactive at the concentration of 100 microM. This binding site is, therefore, distinct from angiotensin II receptors, AT1 and AT2. Addition of the divalent cations Mg2+, Mn2+ or Ca2+ to the incubation buffer resulted in 90-95% inhibition of the [125I]angiotensin IV-specific binding to porcine aortic endothelial cells. Furthermore, the chelator, EGTA, at 5 mM increased the number of binding sites (Bmax = 17.8 +/- 2.5 pmol/mg protein), with no change in affinity (Kd = 5.7 +/- 1.3 nM). Exposure of porcine aortic endothelial cell membranes to the non-hydrolyzable GTP analog, GTP gamma S, had no effect on [125I]angiotensin IV binding. The presence of a high concentration of binding sites for angiotensin IV in porcine aortic endothelial cells suggests that this peptide may play an important role in the modulation of the cardiovascular system.
authors.affiliation: Department of Cardiovascular Research, Synthélabo Recherche (LERS), Bagneux, France.
authors.forename: L
authors.initials: L
authors.lastname: Riva
authors.forename: A M
authors.initials: AM
authors.lastname: Galzin
mesh_terms: D000804:Angiotensin II / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001011:Aorta; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D002413:Cations, Divalent / Q000494:pharmacology; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D011485:Protein Binding / Q000187:drug effects; D013552:Swine
chemical_list: D002413:Cations, Divalent; D000804:Angiotensin II; C012877:angiotensin II, des-Asp(1)-des-Arg(2)-Ile(5)-; D004533:Egtazic Acid