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Front Immunol. 2017 Jul 21;8:842. doi: 10.3389/fimmu.2017.00842. eCollection 2017.

Epithelial Sodium Channel-α Mediates the Protective Effect of the TNF-Derived TIP Peptide in Pneumolysin-Induced Endothelial Barrier Dysfunction.

Author information

1
Vascular Biology Center, Medical College of Georgia, Augusta University, Augusta, GA, United States.
2
Department of Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL, United States.
3
Division of Nephrology, Hypertension, and Renal Transplantation, Department of Medicine, University of Florida College of Medicine, Gainesville, FL, United States.
4
Cardiovascular Research Institute, UCSF, San Francisco, CA, United States.
5
Institute for Medical Microbiology, Justus-Liebig University, Giessen, Germany.
6
Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta University, Augusta, GA, United States.
7
Department of Medicine, Medical College of Georgia, Augusta University, Augusta, GA, United States.
8
Department of Physiology, Emory University School of Medicine, Atlanta, GA, United States.

Abstract

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 α 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-α and whether this subunit is involved in the regulation of their barrier function.

METHODS:

The presence of α, β, and γ subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-α 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-α.

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-α. Both TIP peptide, which specifically binds to ENaC-α, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-α 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-α 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-α 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-α in both barrier function and liquid clearance.

CONCLUSION:

Our results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-α in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-α should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.

KEYWORDS:

TNF; endothelial barrier function; epithelial sodium channel; non-selective cation channel; pneumolysin; pneumonia

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