title: A novel neutrophil elastase inhibitor prevents elastase activation and surface cleavage of the epithelial sodium channel expressed in Xenopus laevis oocytes.
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.
authors.affiliation: Département de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Harris
authors.forename: Dmitri
authors.initials: D
authors.lastname: Firsov
authors.forename: Grégoire
authors.initials: G
authors.lastname: Vuagniaux
authors.forename: M Jackson
authors.initials: MJ
authors.lastname: Stutts
authors.forename: Bernard C
authors.initials: BC
authors.lastname: Rossier
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D053503:Epithelial Sodium Channels / Q000378:metabolism / Q000502:physiology*; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors*; D009865:Oocytes / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000494:pharmacology*; D015202:Protein Engineering; D018075:RNA, Complementary / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry; D013499:Surface Properties; D014357:Trypsin / Q000737:chemistry; D014982:Xenopus laevis; D000099271:Prostasin
chemical_list: D004791:Enzyme Inhibitors; D053503:Epithelial Sodium Channels; D019272:Leukocyte Elastase; D010455:Peptides; D018075:RNA, Complementary; D012697:Serine Endopeptidases; D014357:Trypsin; D000099271:Prostasin; C512995:depelestat