Plasminogen activator inhibitor 1 (PAI-1) level is extremely elevated in the edematous fluid of acutely injured lungs and pleurae. Elevated PAI-1 specifically inactivates pulmonary urokinase-type (uPA) and tissue-type plasminogen activators (tPA). We hypothesized that plasminogen activation and fibrinolysis may alter epithelial sodium channel (ENaC) activity, a key player in clearing edematous fluid. Two-chain urokinase (tcuPA) has been found to strongly stimulate heterologous human Î±Î²Î³ ENaC activity in a dose- and time-dependent manner. This activity of tcuPA was completely ablated by PAI-1. Furthermore, a mutation (S195A) of the active site of the enzyme also prevented ENaC activation. By comparison, three truncation mutants of the amino-terminal fragment of tcuPA still activated ENaC. uPA enzymatic activity was positively correlated with ENaC current amplitude prior to reaching the maximal level. In sharp contrast to uPA, neither single-chain tPA nor derivatives, including two-chain tPA and tenecteplase, affected ENaC activity. Furthermore, Î³ but not Î± subunit of ENaC was proteolytically cleaved at (177GRâ†“KR180) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of Î³ ENaC, incremental increase in opening rate, and activation of closed (electrically \"silent\") channels. This study for the first time demonstrates multifaceted mechanisms for uPA-mediated up-regulation of ENaC, which form the cellular and molecular rationale for the beneficial effects of urokinase in mitigating mortal pulmonary edema and pleural effusions.
Journal of Biological Chemistry
Digital Object Identifier (DOI)
Ji, Hong Long; Zhao, Runzhen; Komissarov, Andrey A.; Chang, Yongchang; Liu, Yongfeng; and Matthay, Michael A., "Proteolytic Regulation Of Epithelial Sodium Channels By Urokinase Plasminogen Activator: Cutting Edge And Cleavage Sites" (2015). Neurobiology. 64.