A novel C-terminal alpha ENaC inhibitory peptide is released following extracellular protease activity in bronchiolar H441 epithelial cells.

Oliver Mace, Deborah Baines

St Georges University of London, London, UK

It is important for alveolar epithelial cells, which are exposed from the luminal domain to an epithelial fluid layer environment to sense and regulate the amount of fluid lining the lungs to maintain efficient gas exchange. The ability of the alveolar mucosa to sense the fluid layer lining the mucosa is therefore essential to its function. The epithelial sodium channel (ENaC) mediates a rate-limiting step in amiloride-sensitive transepithelial Na+ absorption in alveolar epithelial cells including Clara cells, and type I and type II epithelial cells, and regulates the fluid volume in the lung lumen (O’Brodovich et al., 2007).

Recent studies suggest proteases such as furin and prostasin play a role in ENaC regulation (Sheng et al., 2006; Kleyman et al., 2006). In the lung, the underlying mechanisms are not well understood.

Here we used trypsin (1 μM), the trypsin inhibitor aprotinin (30 μM), and the cell permeable, potent and highly specific furin inhibitor, decanoyl-Arg-Val-Lys-Arg (40 μM), to investigate protease activation of ENaC in the Clara-like H441 epithelial cell line. Resistive H441 monolayers, cultured at air interface for 7 days, were mounted in Ussing chambers in a physiological salt solution.

In vitro studies using the Clara-like H441 cell line showed that amiloride-sensitive transepithelial Na+ transport was augmented by expansion of the apical fluid volume. Addition of 100 μl PBS (vehicle) increased amiloride-sensitive short circuit currents (ISC-AMIL) by 77.3 ± 16.4 % (P < 0.05 c.f. air interface control). Addition of trypsin to the apical epithelial surface of H441 monolayers with expanded apical fluid volume augmented ISC-AMIL by a further 17.3 ± 6.1 % (P < 0.05) whilst inhibition of extracellular protease activity with aprotinin reduced ISC-AMIL by 78 ± 11.4 % (P < 0.05). The subsequent addition of trypsin to the Ussing chamber augmented ISC-AMIL by 67.9 ± 9.6 % (P < 0.05). The apical addition of decanoyl-Arg-Val-Lys-Arg to monolayers with expanded surface volume caused an 89.8 ± 11.2 % (P < 0.05) diminution of ISC-AMIL. Western blotting of biotinylated apical alpha ENaC showed that activation of ENaC by surface volume expansion caused the appearance of an 18 kDa C-terminal alpha ENaC fragment that has not been previously reported. This 18 kDa C-terminal alpha ENaC fragment disappeared in the presence of aprotinin, but not Decanoyl-Arg-Val-Lys-Arg, and reappeared following subsequent trypsin treatment. The 18 kDa C-terminal alpha ENaC fragment appears necessary for extracellular protease, but not furin-mediated, ENaC activation.

Thus in H441 epithelial monolayers, apical fluid volume expansion stimulates ENaC activity via a mechanism involving extracellular proteolysis. It is tempting to speculate that the 18 kDa C-terminal alpha ENaC fragment is a novel alpha ENaC inhibitory peptide.

Kleyman TR et al. (2006) Regulation of ENaCs by proteases: An increasingly complex story. Kidney International 70: 1391-1392

O’Brodovich H et al. (2007) Amiloride-insensitive Na+ and fluid absorption in the mammalian distal lung. Am J Physiol Lung Cell Mol Physiol. 294: L401-L408

Sheng S et al. (2006) Furin cleavage activates the epithelial Na+ channel by relieving Na+ self-inhibition. Am J Physiol Renal Physiol 290: F1488-F1496