Published Online
on February 3, 2003

A more recent version of this article appeared on March 1, 2003

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Submitted on June 27, 2002
Revised on August 8, 2002

Tissue Kallikrein Actions at the Rabbit Natural or Recombinant Kinin B₂ Receptors

Steeve Houle; Giuseppe Molinaro; Albert Adam; and François Marceau^*

From the Centre Hospitalier Universitaire de Québec, Centre de recherche du Pavillon l'Hôtel-Dieu de Québec (S.H., F.M.), and the Faculté de Pharmacie, Université de Montréal, Montréal (G.M., A.A.), Québec, Canada.

^* To whom correspondence should be addressed. E-mail: francois.marceau{at}crhdq.ulaval.ca.

Abstract--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 µmol/L, but not 100 nmol/L, and promoted B₂R-GFP endocytosis in HEK 293 cells (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 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 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 µmol/L) can not only initiate the degradation of rabbit B₂ receptors but also exert nonreceptor-mediated effects.

Key words: receptors, bradykinin • kallikrein • rabbits • fluorescence

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