A Novel Neutrophil Elastase Inhibitor Prevents Elastase Activation and Surface Cleavage of the Epithelial Sodium Channel Expressed in Xenopus laevis Oocytes

Michael Harris,Dmitri Firsov,Grégoire Vuagniaux,M Jackson Stutts,Bernard C Rossier

doi:10.1074/jbc.m605125200

Abstract

The amiloride-sensitive epithelial sodium channel (ENaC) constitutes a limiting step in sodium reabsorption across distal airway epithelium and controlling mucociliary clearance. ENaC is activated by serine proteases secreted in the extracellular milieu. In cystic fibrosis lungs, high concentrations of secreted neutrophil elastase (NE) are observed. hNE could activate ENaC and contribute to further decreased mucociliary clearance. The aims of this study were (i) to test the ability of an engineered human neutrophil elastase inhibitor (EPI-hNE4) to specifically inhibit the elastase activation of ENaC-mediated amiloride-sensitive currents (I(Na)) and (ii) to examine the effect of elastase on cell surface expression of ENaC and its cleavage pattern (exogenous proteolysis). Oocytes were exposed to hNE (10-100 microg/ml) and/or trypsin (10 microg/ml) for 2-5 min in the presence or absence of EPI-hNE4 (0.7 microm). hNE activated I(Na) 3.6-fold (p < 0.001) relative to non-treated hENaC-injected oocytes. EPI-hNE4 fully inhibited hNE-activated I(Na) but had no effect on trypsin- or prostasin-activated I(Na). The co-activation of I(Na) by hNE and trypsin was not additive. Biotinylation experiments revealed that cell surface gamma ENaC (but not alpha or beta ENaC) exposed to hNE for 2 min was cleaved (as a 67-kDa fragment) and correlated with increased I(Na). The elastase-induced exogenous proteolysis pattern is distinct from the endogenous proteolysis pattern induced upon preferential assembly, suggesting a causal relationship between gamma ENaC cleavage and ENaC activation, taking place at the plasma membrane.

Highlights

Clearance [2], indicating ENaC as a major driving force for fluid reabsorption from the alveolar space at the time of birth
Elastase-induced ENaC Activation and Cleavage at the Cell Surface: Evidence for Specific Exogenous Proteolysis—Recent evidence clearly indicates that ENaC activation can be directly induced by exposing membrane patches to external trypsin [17] or elastase [17]
We took advantage of the very rapid ENaC activation by external application of hNE, which can be correlated with the pattern of proteolysis at the cell surface in the same oocytes

Summary

Introduction

Clearance [2], indicating ENaC as a major driving force for fluid reabsorption from the alveolar space at the time of birth. Mall et al [11] have recently established that increased ENaC expression and activity in airways of transgenic mice may cause per se a “cystic fibrosis-like lung disease” characterized by decreased mucociliary clearance and tissue inflammation even in the absence of microbial infection. Together, these findings suggest that overactivity of ENaC contributes significantly to the CF phenotype. Numerous studies have shown that several serine proteases are capable of increasing ENaC-mediated INa in multiple cell types [12], including the in vitro and in vivo rodent lung [13,14,15]. We have examined the effectiveness of one of these compounds, EPI-hNE4, an engineered hNE inhibitor that was designed to be a specific, high affinity drug, resistant to oxidative and proteolytic inactivation

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Conclusion