Neutrophil elastase is a serine protease that is abundant in the airways of individuals with cystic fibrosis (CF), a genetic disease manifested by excessive airway Na(+) absorption and consequent depletion of the airway surface liquid layer. Although endogenous epithelium-derived serine proteases regulate epithelial Na(+) transport, the effects of neutrophil elastase on epithelial Na(+) transport and epithelial Na(+) channel (ENaC) activity are unknown. Low micromolar concentrations of human neutrophil elastase (hNE) applied to the apical surface of a human bronchial cell line (16HBE14o-/beta gamma) increased Na(+) transport about twofold. Similar effects were observed with trypsin, also a serine protease. Proteolytic inhibitors of hNE or trypsin selectively abolished the enzyme-induced increase of epithelial Na(+) transport. At the level of the single channel, submicromolar concentrations of hNE increased activity of near-silent ENaC approximately 108-fold in patches from NIH-3T3 cells expressing rat alpha-, beta-, and gamma-ENaC subunits. However, no enzyme effects were observed on basally active ENaCs. Trypsin exposure following hNE revealed no additional increase in amiloride-sensitive short-circuit current or in ENaC activity, suggesting these enzymes share a common mode of action for increasing Na(+) transport, likely through proteolytic activation of ENaC. The hNE-induced increase of near-silent ENaC activity in CF airways could contribute to Na(+) hyperabsorption, reduced airway surface liquid height, and dehydrated mucus culminating in inefficient mucociliary clearance.

.-The regulation of epithelial Na ϩ channel (ENaC) function is critical for normal salt and water balance. This regulation is achieved through cell surface insertion/retrieval of channels, by changes in channel open probability (P o), or through a combination of these processes. Epithelium-derived serine proteases, including channel activating protease (CAP) and prostasin, regulate epithelial Na ϩ transport, but the molecular mechanism is unknown. We tested the hypothesis that extracellular serine proteases activate a near-silent ENaC population resident in the plasma membrane. Single-channel events were recorded in outsideout patches from fibroblasts (NIH/3T3) stably expressing rat ␣-, ␤-, and ␥-subunits (rENaC), before and during exposure to trypsin, a serine protease homologous to CAP and prostasin. Under baseline conditions, near-silent patches were defined as having rENaC activity (NP o) Ͻ 0.03, where N is the number of channels. Within 1-5 min of 3 g/ml bath trypsin superfusion, NP o increased ϳ66-fold (n ϭ 7). In patches observed to contain a single functional channel, trypsin increased P o from 0.02 Ϯ 0.01 to 0.57 Ϯ 0.03 (n ϭ 3, mean Ϯ SE), resulting from the combination of an increased channel open time and decreased channel closed time. Catalytic activity was required for activation of near-silent ENaC. Channel conductance and the Na ϩ / Li ϩ current ratio with trypsin were similar to control values. Modulation of ENaC Po by endogenous epithelial serine proteases is a potentially important regulator of epithelial Na ϩ transport, distinct from the regulation achieved by hormone-induced plasma membrane insertion of channels. silent channels; protease; epithelial Na ϩ transport; cystic fibrosis; hypertension THE REGULATION of epithelial Na ϩ channel (ENaC) function is key to understanding the physiology of epithelial Na ϩ transport. The epithelium-derived serine proteases channel activating protease (CAP) and prostasin are expressed in tissues containing ENaC and augment amiloride-sensitive currents when coexpressed with ENaC in Xenopus oocytes (1,5,19,20). Moreover, aprotinin, a serine protease inhibitor, decreases the amiloride-sensitive short-circuit current in mouse cortical collecting duct and human bronchial epithelia (2, 20). These results suggest that proteases may regulate epithelial Na ϩ transport in vivo, but the precise functional role of endogenous protease activation of ENaC has remained elusive (16). Cell surface labeling of ENaC combined with amiloride-sensitive current measurements in oocytes provided evidence for an abundance of ENaC having very low channel activity (6). These "silent" channels were estimated to have a whole cell P o of ϳ0.02, much lower than the average P o reported with direct patch-clamp measurements (14, 15). Thus activation of silent channels by endogenous epithelium-derived serine proteases is a potentially important physiological regulatory mechanism of epithelial Na ϩ transport. Here, we functionally identify a near-silent ENaC population in the plasma membrane o...

Gadolinium (Gd3+) blocks cation-selective stretch-activated ion channels (SACs) and thereby inhibits a variety of physiological and pathophysiological processes. Gd3+ sensitivity has become a simple and widely used method for detecting the involvement of SACs, and, conversely, Gd3+insensitivity has been used to infer that processes are not dependent on SACs. The limitations of this approach are not adequately appreciated, however. Avid binding of Gd3+ to anions commonly present in physiological salt solutions and culture media, including phosphate- and bicarbonate-buffered solutions and EGTA in intracellular solutions, often is not taken into account. Failure to detect an effect of Gd3+ in such solutions may reflect the vanishingly low concentrations of free Gd3+ rather than the lack of a role for SACs. Moreover, certain SACs are insensitive to Gd3+, and Gd3+ also blocks other ion channels. Gd3+ remains a useful tool for studying SACs, but appropriate care must be taken in experimental design and interpretation to avoid both false negative and false positive conclusions.

Amiloride improves mucociliary clearance (MC) by blocking airway epithelial sodium channels (ENaC) and expanding airway surface liquid (ASL). However, the low potency and rapid absorption of amiloride by airway epithelia translated into a short duration of efficacy as an aerosolized therapy for cystic fibrosis (CF) patients. To improve ENaC blocker CF pharmacotherapy, a more potent and durable ENaC blocker tailored for aerosol delivery was synthesized. Parion compound N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-NЈ-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02) was tested for potency and reversibility of ENaC block, epithelial absorption and biotransformation, selectivity, durability of ASL expansion under isotonic and hypertonic conditions in canine and human CF bronchial epithelial cells, and drug dissociation on ENaC in Xenopus oocytes. Short-circuit current assessed compound potency and reversibility, patch-clamp recordings of ENaC current assessed drug off-rate (k off ), a gravimetric method and confocal microscopy measured mucosal water retention and ASL height, and drug absorption and biotransformation were assessed using liquid chromatography-mass spectrometry. Amiloride and 552-02 were tested in vivo for MC activity in sheep immediately and 4 to 6 h after aerosol dosing. Compared with amiloride, compound 552-02 was 60 to 100-fold more potent, it was 2 to 5-fold less reversible, it was slower at crossing the epithelium, and it exhibited a 170-fold slower k off value. 552-02 exhibited greater ASL expansion over 8 h in vitro, and it was more effective than amiloride at increasing MC immediately and 4 to 6 h after dosing. When combining hypertonic saline and 552-02, a synergistic effect on ASL expansion was measured in canine or CF bronchial epithelia. In summary, the preclinical data support the clinical use of 552-02 ϩ/Ϫ hypertonic saline for CF lung disease.The pulmonary disease in patients with cystic fibrosis (CF) reflects genetic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that produce defective epithelial ion transport. The CF airway epithelial ion transport abnormalities lead to a well described pathophysiological cascade that adversely affects the innate defense Article, publication date, and citation information can be found at

Studies in cystic fibrosis