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 [3H]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.

The isolated human umbilical vein as a bioassay for kinin-generating proteases: An in vitro model for therapeutic angioedema agents

“…As far as effects of kinins on B 2 R are concerned, icatibant may be the universal inhibitor of the vascular effects of these proteases. C1-inh, as expected, inhibited the contractile effect of apK+HK, but not that of KLK-1, itself previously shown to be sensitive to aprotinin [7] and to pefabloc SC in present experiments (Fig. 4B).…”

“…The experimental procedures have been recently reported [7]. Briefly, most experiments were based on a 3 hr-equilibration period protease and other agents.…”

Kallikreins – The melting pot of activity and function

“…Released into renal tubules, KLK1 modulates the activity of epithelial sodium channels, inhibits cortical collecting duct H + , K + -ATPase activity, and activates TRPV (Transient Receptor Potential Vanilloid) channels. Moreover, due to proteolytic activity of KLK-1 on HMWK and bradykinin release, the activated bradykinin-B2 receptor (B2R) mediates beneficial effects on the progression of type 2 diabetes and hypertension and ischemic renal injury in rodents [135]. …”

Species-specific pharmacology of maximakinin, an amphibian homologue of bradykinin: putative prodrug activity at the human B2receptor and peptidase resistance in rats

“…The construction of myc-tagged B 2 R expression vectors containing the human and rat receptor sequence has been recently reported, as well as the derivation of HEK 293a cell lines that stably express each one of these constructions (Charest-Morin et al, 2015). The HEK 293a cell line was originally obtained from Sigma-Aldrich.…”