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 αβγ 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, γ but not α subunit of ENaC was proteolytically cleaved at ((177)GR↓KR(180)) by tcuPA. In summary, the underlying mechanisms of urokinase-mediated activation of ENaC include release of self-inhibition, proteolysis of γ 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.

The growth factor receptor/PI3K/AKT pathway is an important drug target in many cancers including Glioblastoma. AKT, a key node in the pathway, has 3 isoforms, AKT1, AKT2 and AKT3. Here we investigate their role in GBM. We find each activated, ser473 phosphorylated isoform is present in some GBMs but expression patterns vary. There is a direct relationship between human GBM patient outcome and both AKT1 and AKT2 mRNA levels, but an inverse relationship with AKT3 mRNA. Furthermore, AKT3 mRNA levels were high in a less aggressive GBM subtype. Overexpressing AKT3 improves survival in a rodent model of GBM and decreases colony forming efficiency, but not growth rate, in glioma cells. Silencing AKT3 slows cell cycle progression in one cell line and increases apoptosis in another. Our studies of AKT3 substrates indicate (1) silencing both AKT2 and AKT3 reduces GSK3 phosphorylation (2) only AKT2 silencing reduces S6 phosphorylation. Since S6 phosphorylation is a marker of mTORC1 activity this indicates that AKT2 activates mTORC1, but AKT3 does not. Our results indicate AKT isoforms have different roles and downstream substrates in GBM. Unexpectedly, they indicate AKT3 delays tumor progression. Therefore strategies that inhibit AKT3 may be unhelpful in some GBM patients.