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 B2 receptor; specifically, whether the protease can bind to, cleave, internalize, and/or activate a fusion protein composed of the rabbit B2 receptor conjugated to the green fluorescent protein (B2R-GFP). The enzyme partially digested the fusion protein at 1 micromol/L, but not 100 nmol/L, and promoted B2R-GFP endocytosis in HEK 293 cells (> or =50 nmol/L). Trypsin and endoproteinase Lys-C, but not plasma kallikrein, also cleaved B2R-GFP. Phospholipase A2 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 B2 receptor antagonist and by the lack of response in untransfected cells. However, 500 nmol/L kallikrein elicited a strong receptor-independent activation of phospholipase A2. Tissue kallikrein competed for [3H]bradykinin binding to B2R-GFP only at 1 micromol/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 B2 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 micromol/L) can not only initiate the degradation of rabbit B2 receptors but also exert nonreceptor-mediated effects.
Houle
Steeve
S
Centre Hospitalier Universitaire de Québec, Québec, Canada.
Molinaro
Giuseppe
G
Adam
Albert
A
Marceau
François
F
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001667:Binding, Competitive; D001920:Bradykinin / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D007601:Jugular Veins / Q000187:drug effects / Q000502:physiology; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D009119:Muscle Contraction / Q000187:drug effects; D010446:Peptide Fragments / Q000276:immunology; D011817:Rabbits; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000378:metabolism; D020840:Tissue Kallikreins / Q000378:metabolism / Q000494:pharmacology*
D008164:Luminescent Proteins; D010446:Peptide Fragments; D043782:Receptor, Bradykinin B2; D018002:Receptors, Bradykinin; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D016718:Arachidonic Acid; D020840:Tissue Kallikreins; D001920:Bradykinin