title: Pharmacological effects of recombinant human tissue kallikrein on bradykinin B2 receptors.
abstract: Tissue kallikrein (KLK-1), a serine protease, initiates the release of bradykinin (BK)-related peptides from low-molecular weight kininogen. KLK-1 and the BK B2 receptor (B2R) mediate beneficial effects on the progression of type 2 diabetes and renal disease, but the precise role of KLK-1 independent of its kinin-forming activity remains unclear. We used DM199, a recombinant form of human KLK-1, along with the isolated human umbilical vein, a robust bioassay of the B2R, to address the previous claims that KLK-1 directly binds to and activates the human B2R, with possible receptor cleavage. DM199 (1-10 nmol/L) contracted the isolated vein via the B2R, but in a tachyphylactic, kinin-dependent manner, without desensitization of the tissue to exogenously added BK. In binding experiments with recombinant N-terminally tagged myc-B2Rs expressed in HEK 293a cells, DM199 displaced [(3)H]BK binding from the rabbit myc-B2R, but not from the human or rat myc-B2Rs. No evidence of myc-B2R degradation by immunoblot analysis was apparent following treatment of these 3 myc-B2R constructs with DM199 (30 min, ≤10 nmol/L). In HEK 293 cells stably expressing rabbit B2R-GFP, DM199 (11-108 pmol/L) elicited signaling-dependent endocytosis and reexpression, while a higher concentration (1.1 nmol/L) induced a partially irreversible endocytosis of the construct (microscopy), paralleled by the appearance of free GFP in cells (immunoblotting, indicative of incomplete receptor down-regulation). The pharmacology of DM199 at relevant concentrations (<10 nmol/L) is essentially based on the activity of locally generated kinins. Binding to and mild down-regulation of the B2R is possibly a species-dependent idiosyncratic response to DM199.
authors.affiliation: Centre de recherche en rhumatologie et immunologie, CHU de Québec Québec City, Québec, Canada, G1V 4G2.
authors.forename: Xavier
authors.initials: X
authors.lastname: Charest-Morin
authors.affiliation: DiaMedica Inc. One Carlson Parkway, Suite 124, Minneapolis, Minnesota, 55447.
authors.forename: Arvind
authors.initials: A
authors.lastname: Raghavan
authors.affiliation: DiaMedica Inc. One Carlson Parkway, Suite 124, Minneapolis, Minnesota, 55447.
authors.forename: Matthew L
authors.initials: ML
authors.lastname: Charles
authors.affiliation: DiaMedica Inc. One Carlson Parkway, Suite 124, Minneapolis, Minnesota, 55447.
authors.forename: Tadeusz
authors.initials: T
authors.lastname: Kolodka
authors.affiliation: Centre de recherche en rhumatologie et immunologie, CHU de Québec Québec City, Québec, Canada, G1V 4G2.
authors.forename: Johanne
authors.initials: J
authors.lastname: Bouthillier
authors.affiliation: Centre de recherche en rhumatologie et immunologie, CHU de Québec Québec City, Québec, Canada, G1V 4G2.
authors.forename: Mélissa
authors.initials: M
authors.lastname: Jean
authors.affiliation: DiaMedica Inc. One Carlson Parkway, Suite 124, Minneapolis, Minnesota, 55447.
authors.forename: Mark S
authors.initials: MS
authors.lastname: Robbins
authors.affiliation: Centre de recherche en rhumatologie et immunologie, CHU de Québec Québec City, Québec, Canada, G1V 4G2.
authors.forename: François
authors.initials: F
authors.lastname: Marceau
keywords: Bradykinin; bradykinin B2 receptor; human isolated umbilical vein; radioligand binding; receptor internalization; tissue kallikrein