Tissue kallikrein actions at the rabbit natural or recombinant kinin B2 receptors

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 (B2R-GFP). The enzyme partially digested the fusion protein at 1 mumol/L, but not 100 nmol/L, and promoted B2R-GFP endocytosis in HEK 293 cells (greater than or equal to50 nmol/L). Trypsin and endoproteinase Lys-C, but not plasma kallikrein, also cleaved B2R-GFP. Phospholipase A(2) was activated by 50 nmol/L tissue kallikrein in HEK 293 cells expressing B2R-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 [H-3]bradykinin binding to B2R-GFP only at 1 mumol/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 mumol/L) can not only initiate the degradation of rabbit B-2 receptors but also exert nonreceptor-mediated effects.