Enhancement of Bradykinin and Resensitization of Its B 2 Receptor

Abstract

—We studied the enhancement of the effects of bradykinin B ₂ receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B ₂ receptor were assessed by measuring [ ³ H]arachidonic acid release and [Ca ²⁺ ] _i mobilization in Chinese hamster ovary cells transfected to express human ACE and B ₂ receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca ²⁺ ] _i mobilization) by agents such as enalaprilat (1 μmol/L). Enalaprilat was inactive in the absence of ACE expression. La ³⁺ (100 μmol/L) inhibited the apparent resensitization, probably by blocking the entry of extracellular calcium. Enalaprilat resensitized the receptor via ACE to release arachidonic acid by bradykinin at a lower concentration (5 nmol/L) than required to mobilize [Ca ²⁺ ] _i (1 μmol/L). Monoclonal antibodies inhibiting the ACE N-domain active center and polyclonal antiserum potentiated bradykinin. The snake venom peptide BPP5a and metabolites of angiotensin and bradykinin (angiotensin-[1–9], angiotensin-[1–7], bradykinin-[1–8]; 1 μmol/L) enhanced arachidonic acid release by bradykinin. Angiotensin-(1–9) and -(1–7) also resensitized the receptor. Enalaprilat potentiated the bradykinin effect in cells expressing a mutant ACE with a single N-domain active site. Agents that reacted with a single active site, on the N-domain or on the C-domain, potentiated bradykinin not by blocking its inactivation but by inducing crosstalk between ACE and the receptor. Enalaprilat enhanced signaling via ACE by Gα _i in lower concentration than by Gα _q -coupled receptor.