Abstract
The aims of this study were to assess whether ischemic preconditioning (PC) induces bradykinin (Bk) synthesis in bovine aortic endothelial cells (bAECs) and, if so, to explore the molecular mechanisms by which this peptide provides cytoprotection against hypoxia. PC was induced by exposing bAECs to three cycles of 15 min of hypoxia followed by 15 min of reoxygenation. Bk synthesis peaked in correspondence to the early and late phases of PC (10(-12) M and 10(-11) M, respectively) and was abolished by a selective tissue kallikrein inhibitor, aprotinin. Stimulation with exogenous Bk at concentrations of 10(-12) M and 10(-11) M reduced the cell death induced by 12 h of hypoxia by 50%. Pretreatment with HOE-140, a Bk receptor 2 (BKR2) inhibitor, in bAECs exposed to 12 h of hypoxia, abrogated the cytoprotective effect of early and late PC, whereas des-Arg-HOE-140, a Bk receptor 1 (BKR1) inhibitor, affected only the late PC. In addition, we found that PC evoked endocytosis and the recycling of BKR2 during both the early and late phases, and that inhibition of these pathways affected PC-mediated cytoprotection. Finally, we evaluated the activation of PKA and Akt in the presence or absence of BKR2 inhibitor. HOE-140 abrogated PKA and Akt activation during both early and late PC. Consistently, BKR2 inhibition abolished cross-talk between PKA and Akt in PC. In bAECs, Bk-synthesis evoked by PC mediates the protection against both apoptotic and necrotic hypoxia-induced cell death in an autocrine manner, by both BKR2- and BKR1-dependent mechanisms.