Tissue Kallikrein Actions at the Rabbit Natural or Recombinant Kinin B 2 Receptors

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Abstract
We have examined whether exogenous human tissue kallikrein exerts pharmacological actions via the bradykinin B 2 receptor; specifically, whether the protease can bind to, cleave, internalize, and/or activate a fusion protein composed of the rabbit B 2 receptor conjugated to the green fluorescent protein (B 2 R-GFP). The enzyme partially digested the fusion protein at 1 μmol/L, but not 100 nmol/L, and promoted B 2 R-GFP endocytosis in HEK 293 cells (≥50 nmol/L). Trypsin and endoproteinase Lys-C, but not plasma kallikrein, also cleaved B 2 R-GFP. Phospholipase A 2 was activated by 50 nmol/L tissue kallikrein in HEK 293 cells expressing B 2 R-GFP, and this was mediated by the receptor, as shown by the effect of a B 2 receptor antagonist and by the lack of response in untransfected cells. However, 500 nmol/L kallikrein elicited a strong receptor-independent activation of phospholipase A 2 . Tissue kallikrein competed for [ 3 H]bradykinin binding to B 2 R-GFP only at 1 μmol/L. A simulation involving kallikrein treatment of HEK 293 cells, pretreated or not with human plasma, evidenced the formation of immunoreactive bradykinin. The enzyme (50 nmol/L) contracted the rabbit isolated jugular vein via its endogenous B 2 receptors, but the effect was tachyphylactic, and there was no cross-desensitization with bradykinin effects. Aprotinin prevented all pharmacological responses to tissue kallikrein, indicating that the enzyme activity is required for its effect. The local generation of kinins is a plausible mechanism for the pharmacological effects of lower concentrations of tissue kallikrein (50 to 100 nmol/L); higher levels (0.5 to 1 μmol/L) can not only initiate the degradation of rabbit B 2 receptors but also exert nonreceptor-mediated effects.