Interactions between bradykinin and plasmin in the endothelial Ca²⁺ response

Plasmin is a fibrinolytic factor and a serine protease that activates protease-activated receptors (PARs) to produce endothelium-derived relaxing factors such as nitric oxide and prostacyclin. Nitric oxide and prostacyclin production is regulated, at least in part, by the intracellular Ca²⁺ concentration in various blood vessel types. Bradykinin and plasmin stimulate vascular endothelial cells and work simultaneously in pathophysiological conditions such as thrombosis and inflammation. Here, we explored the interactions between bradykinin and plasmin in the endothelial Ca²⁺ response using the fluorescent indicator, Fura-2/AM, in primary cultures of porcine aortic endothelial cells (PAECs). Plasmin (0.15-15 µg/ml) and bradykinin (0.1-10 nM) increased intracellular Ca²⁺ concentrations in PAECs in a dose-dependent manner, and the plasmin-induced endothelial Ca²⁺ response occurred only once. Bradykinin (0.1-10 nM) inhibited the plasmin-induced endothelial Ca²⁺ response in a dose-dependent manner, however, plasmin did not affect the bradykinin-induced endothelial Ca²⁺ response. Pretreatment with gabexate mesilate (GM, 100 µM), a serine protease inhibitor, that blocks plasmin's proteolytic activity, fully suppressed the plasmin-induced Ca²⁺ response. After washout of GM and the first plasmin, the second administration of plasmin caused Ca²⁺ increases. However, when the first plasmin-induced Ca²⁺ response was blocked by pretreatment with bradykinin, the second plasmin (15 µg/ml) application did not cause any Ca²⁺ response, even 30 min after the washout of the first plasmin and bradykinin. Our data suggested that bradykinin regulated the plasmin-induced endothelial Ca²⁺ response by inhibiting the pathway downstream of the PARs' N-terminus cleavage.