BACKGROUND: Streptococcus pneumoniae is a major etiologic agent of bacterial pneumonia. Autolysis and antibiotic-mediated lysis of pneumococci induce release of the pore-forming toxin, pneumolysin (PLY), their major virulence factor, which is a prominent cause of acute lung injury. PLY inhibits alveolar liquid clearance and severely compromises alveolar-capillary barrier function, leading to permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The alpha subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na(+) reabsorption across Na(+)-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-alpha and whether this subunit is involved in the regulation of their barrier function. METHODS: The presence of alpha, beta, and gamma subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-alpha in monolayer resistance of HL-MVEC was examined by depletion of this subunit by specific siRNA and by employing the TNF-derived TIP peptide, a specific activator that directly binds to ENaC-alpha. RESULTS: HL-MVEC express all three subunits of ENaC, as well as acid-sensing ion channel 1a (ASIC1a), which has the capacity to form hybrid non-selective cation channels with ENaC-alpha. Both TIP peptide, which specifically binds to ENaC-alpha, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-alpha depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-alpha and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin-dependent kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-alpha and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-alpha in both barrier function and liquid clearance. CONCLUSION: Our results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-alpha in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-alpha should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.
- Czikora, I.
- Alli, A. A.
- Sridhar, S.
- Matthay, M. A.
- Pillich, H.
- Hudel, M.
- Berisha, B.
- Gorshkov, B.
- Romero, M. J.
- Gonzales, J.
- Wu, G.
- Huo, Y.
- Su, Y.
- Verin, A. D.
- Fulton, D.
- Chakraborty, T.
- Eaton, D. C.
- Lucas, R.
Keywords
- Tnf
- endothelial barrier function
- epithelial sodium channel
- non-selective cation channel
- pneumolysin
- pneumonia
