Tissue kallikrein (KLK‐1), a serine protease, initiates the release of bradykinin (BK)‐related peptides from low‐molecular weight kininogen.KLK‐1 and theBKB₂receptor (B₂R) mediate beneficial effects on the progression of type 2 diabetes and renal disease, but the precise role ofKLK‐1 independent of its kinin‐forming activity remains unclear. We usedDM199, a recombinant form of humanKLK‐1, along with the isolated human umbilical vein, a robust bioassay of the B₂R, to address the previous claims thatKLK‐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 addedBK. In binding experiments with recombinant N‐terminally tagged myc‐B₂Rs expressed inHEK293a cells,DM199 displaced [³H]BKbinding 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 withDM199 (30 min, ≤10 nmol/L). InHEK293 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 freeGFPin cells (immunoblotting, indicative of incomplete receptor down‐regulation). The pharmacology ofDM199 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.

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