title: Lack of direct interaction between enalaprilat and the kinin B1 receptors.
abstract: It has been recently proposed that the second extracellular loop of the human bradykinin (BK) B1 receptor (B1R) contains a conserved HExxH motif also present in peptidases possessing a Zn2+ prosthetic group, such as angiotensin converting enzyme (ACE), and that ACE inhibitors directly activate B1R signaling in endothelial cells. However, the binding of ACE inhibitors to the B1Rs has never been directly evaluated. Information about binding of a radiolabeled inhibitor to natural or recombinant ACE in intact cells (physiologic ionic composition) was also collected. We used the tritiated form of an ACE inhibitor previously proposed to activate the B1R, enalaprilat, to address these questions using recombinant human B1Rs and naturally expressed or recombinant ACE. [3H]Lys-des-Arg9-BK bound to the human recombinant B1Rs with high affinity (KD 0.35 nM) in HEK 293a cells. [3H]Enalaprilat (0.25-10 nM) did not bind to cells expressing recombinant human B1R, but bound with a subnanomolar affinity to recombinant ACE or to naturally expressed ACE in human umbilical vein endothelial cells. The radioligand was further validated using a binding competition assay that involved unlabeled ACE inhibitors or their prodrug forms in endothelial cells. Membranes of HEK 293a cells that expressed B1Rs did not hydrolyze hippuryl-glycylglycine (an ACE substrate). Enalaprilat did not stimulate calcium signaling in HEK 293a cells that expressed B1Rs. A typical ACE inhibitor did not bind to nor stimulate the human B1Rs; nevertheless, several other indirect mechanisms could connect ACE inhibition to B1R stimulation in vivo.
authors.affiliation: Centre de recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Québec, QC, Canada G1V 4G2.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Morissette
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Couture
authors.forename: Anik
authors.initials: A
authors.lastname: Désormeaux
authors.forename: Albert
authors.initials: A
authors.lastname: Adam
authors.forename: François
authors.initials: F
authors.lastname: Marceau
mesh_terms: D000806:Angiotensin-Converting Enzyme Inhibitors / Q000378:metabolism*; D020013:Calcium Signaling; D002478:Cells, Cultured; D004347:Drug Interactions; D015773:Enalaprilat / Q000378:metabolism*; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D043783:Receptor, Bradykinin B1 / Q000378:metabolism*; D014471:Umbilical Veins / Q000378:metabolism
chemical_list: D000806:Angiotensin-Converting Enzyme Inhibitors; D043783:Receptor, Bradykinin B1; D015773:Enalaprilat