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TRANSLATIONAL PHYSIOLOGY
The Cystic Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina, Chapel Hill, North Carolina
Submitted 19 November 2004 ; accepted in final form 5 January 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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) 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 
108-fold in patches from NIH-3T3 cells expressing rat 
-, -, and -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. silent channels; cystic fibrosis; serine protease; elastase
Serine proteases endogenous to airway and renal epithelia regulate transepithelial Na+ absorption by increasing the amiloride-sensitive Na+ current (3, 10, 27, 38). Moreover, Kunitz-type protease inhibitors block epithelial serine protease activation of ENaC and have been suggested as a novel treatment for the Na+-hyperabsorption defect in CF airways (3). Serine protease-mediated regulation of epithelial Na+ transport likely results from proteolytic cleavage and activation of ENaC (5, 15).
Neutrophil elastase is also a serine protease and present in CF airways at concentrations often 
1 µM due to the unrelenting infection and inflammation of the airways (22). These enzyme concentrations are sufficient to overwhelm endogenous airway antiprotease activity (1) so that the majority of elastase is active (22, 29). The effects of neutrophil elastase on ENaC function, however, have not been reported.
We report that exposure to 0.02–1.5 µM of human neutrophil elastase (hNE) increases amiloride-sensitive Na+ transport in cultured human bronchial epithelia (16HBE14o-/) and increases the open probability of near-silent ENaC (5) present in excised patches from NIH-3T3 cells stably expressing rat -, -, and -ENaC subunits. The hNE-induced increase of ENaC activity in CF airways could contribute to excessive airway Na+ absorption, exacerbate the airway pathology, and contribute to disease progression.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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-ENaC subunit (24). To achieve the full complement of -, -, and -ENaC subunits necessary for efficient channel expression, cells were seeded at 1 x 102 cells/cm2 and exposed to retroviral vectors, pHIT-XIH-h and pHIT-XIN-h. pHIT-XIH and pHIT-XIN vectors were derived from the pHIT-LZ vector (18) by removing the lacZ gene and inserting a cassette containing a multiple cloning site, the poliovirus internal ribosome entry site, and either the hygromycin (pHIT-XIH) or neomycin (pHIT-XIN) drug selection genes. The cDNAs for the human - and -ENaC were cloned into the multiple cloning site region of these vectors to obtain pHIT-XIH-h and pHIT-XIN-h. MLV-based retroviral vectors, pseudotyped with the VSV-G envelope, were produced in 293T cells as described (18). Doubly drug-resistant clones were expanded and tested for protein expression (data not shown). Clones expressing both heterologous - and -ENaC subunits were evaluated for generation of amiloride-sensitive current, and clone 8 was designated as 16HBE14o-/ and used in the present study. 16HBE14o-/ cells were routinely maintained in MEM with 10 µM amiloride and seeded onto Costar Snapwell culture inserts (Corning) for study. After attaining confluence, cells were induced for 24 h before study with 1 mM Na-butyrate and 10–6 M dexamethasone.
NIH-3T3 cells were infected with retrovirus encoding cDNAs for rat -, -, and -ENaC subunits (rENaC) and grown in culture as previously described (5, 37). Cells were subcultured on 35-mm culture dishes and visually selected with an inverted microscope (Nikon) containing Hoffman modulation contrast optics for patch-clamp experiments.
Chemicals and drugs.
hNE, purified from CF sputum to >95%, was from Elastin Products (Owensville, MO). Amiloride, trypsin (type I, 10,800 U/mg, <4 U/mg chymotrypsin), aprotinin, and Igepal CA 630 were from Sigma-Aldrich (St. Louis, MO). Because of breakdown products and minor contaminants in commercial enzyme preparations, enzyme concentrations (20 nM–1.5 µM) are expressed in micrograms/ml (µg/ml). The neutrophil elastase inhibitor, MeOSuc-Ala-Ala-Pro-Val-chloromethylketone, was from Bachem (King of Prussia, PA). Amiloride, aprotinin, and all enzymes were dissolved directly in the bath solutions (see below). Neutrophil elastase inhibitor was first dissolved in dimethylsulfoxide (DMSO) and then added to the bathing solution. The final DMSO concentration was 0.1% (vol/vol). Igepal CA 630 (final dilution 5 x 10–6 vol/vol) was added to enzyme-containing solutions to reduce nonspecific binding to the bath chamber and superfusion apparatus (23).
Short-circuit current and patch-clamp ionic solutions. The short-circuit current (Isc) bath solution contained (in mM): 115 NaCl, 1.2 CaCl2, 1.2 MgCl2, 25 NaHCO3, 2.4 K2HPO4, 0.8 KH2PO4, and 5.2 glucose and was gassed with 95:5% O2/CO2 to achieve pH 7.4. The 3T3 cell bath solution contained (in mM): 150 Li+-aspartate, 2 MgCl2, 1 CaCl2, and 5 HEPES, titrated to pH 7.30 with LiOH. The 3T3 cell pipette solution contained (in mM): 120 Tris-aspartate, 20 NaCl, 3 MgATP, 0.2 Na2GTP, 0.1 CaCl2, 1 EGTA, and 5 HEPES titrated to pH 7.10 with NaOH.
Isc and patch-clamp measurements.
For Isc measurements, culture inserts were mounted in Ussing chambers (Physiologic Instruments, San Diego, CA) and voltage clamped to 0-mV. Isc was digitally acquired at 20 Hz with commercial data acquisition software (Physiologic Instruments). Transepithelial resistance was monitored with 10-mV voltage steps, lasting 500 ms, and applied at 0.12 Hz. Enzymes and inhibitors were added to the apical bath chamber once Isc reached a stable value (within 10–20 min). Epithelial Na+ transport was measured as the difference in Isc recorded before and after amiloride addition (100 µM, apical) at the end of experiments. The maximum rate of change in Isc (dI/dtmax) following enzyme addition was used to assess reaction velocity with and without inhibitors. Experiments were performed at 37°C. For patch-clamp measurements, outside-out patches configured from 3T3 cells were studied as previously described (5). Briefly, patch-clamp signals (EPC-7; List, Darmstadt, Germany) were filtered at 0.1 kHz (–3 dB, Bessel), digitized at 1 kHz (16-bit, ITC, Instrutech), and acquired with a personal computer running HEKA-PULSE acquisition software (Bruxton, Seattle, WA). Patch pipettes (borosilicate Warner Instruments, Hamden, CT) were fabricated from thin-walled glass using a three-stage pull routine (DMZ Universal Puller, Zeitz Instruments). Pipette resistance with the indicated patch solutions was 11.0 ± 0.1 m
(n = 85). Single channel activity was recorded from –100-mV to 0-mV membrane potential (Vm) with flowing bath conditions. Exchange of bath solution, reported as the 10–90% exchange time, was 69.7 ± 7.5 ms (means ± SE, n = 6) and achieved using a Fast-step solution exchanger (Warner Instruments). Bath amiloride inhibition of single channels was used to confirm ENaC identity and patch configuration. Channels observed as amiloride insensitive were not included for analysis. Vm was not adjusted for an 2-mV (n = 3) diffusion potential measured between pipette and bath solutions. Experiments were performed at 23°C.
Data analysis. Channel transitions were measured before and during protease exposure from the same patch as previously described (5). Briefly, idealized records were constructed from channel openings meeting the half-amplitude threshold criterion. Unitary current values were obtained from amplitude histograms, binned into 0.05- to 0.1-pA intervals, and fitted with a Gaussian function at each Vm. For assessment of enzyme effects on channel activity, the average channel activity, defined as the product of NPo, where N is the number of cell surface-resident channels and Po, the open probability, was analyzed by integrating the area under the Gaussian curves fitted to the all-points current-amplitude histogram and normalized to the peak area of the baseline current level (i.e., closed channel current level) (9). Data analysis was performed with single channel analysis software (TAC and TACFit, Bruxton) and custom written programs in Sigma Plot version 8.0.
Statistics. All results are reported as means ± SE, with n = number of Isc or patch recordings, unless otherwise stated. Comparison of dI/dtmax was performed using an unpaired t-test. Comparison of more than two treatment groups for Isc and patch-clamp experiments was performed with Tukey's test following one-way ANOVA. For patch-clamp experiments, comparisons of channel activity were made on log-transformed NPo data before and after serine protease exposure. A P value < 0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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. This cell line exhibited an easily resolved increase in the Isc response to serine protease exposure. Figure 1A shows a representative recording. The basal current was recorded for 40 min, and then trypsin (10 µg/ml) was applied to the apical cell surface. As shown, trypsin induced a rapid increase in the Isc, which peaked and then declined to a steady-state value that was sustained approximately twofold above the baseline current level. Figure 1B shows a representative recording before and during exposure to hNE. Enzyme (10 µg/ml) applied to the apical cell surface also resulted in a rapid increase in current having a comparable profile and time course as trypsin. Moreover, subsequent trypsin application to the apical surface showed no additive effects on the steady-state current level. Summary data from these experiments are shown in Fig. 1C. Both hNE and trypsin significantly increased Isc over the baseline current, but the combination of hNE and trypsin effects was not additive.
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To assess whether catalytic activity was necessary for the serine protease-induced increase of amiloride-sensitive short-circuit current (Isc-amil), serine protease in the presence of excess inhibitor was studied. Aprotinin is a potent trypsin inhibitor [Ki 0.06 pM (26)] containing a Kunitz domain, which appears necessary for the marked inhibition of the Isc-amil in primary cultures of airway epithelia (3). However, hNE has a relatively low affinity for Kunitz-containing inhibitors [aprotinin Ki 3.5 µM (26)]. Figure 2A shows a representative Isc-amil recording of the trypsin and hNE response in the presence of aprotinin (15 µM). No discernable effects of aprotinin alone were observed on the Isc-amil, as shown. However, aprotinin completely abolished the trypsin-induced Isc-amil. Subsequent hNE exposure resulted in an 1.6-fold increase in steady-state Isc-amil. Summary data from these experiments are shown in Fig. 2B. hNE increased the steady-state Isc-amil 2.5-fold with aprotinin, which was comparable, albeit larger than the fold increase observed in the absence of the inhibitor.
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52%) in aprotinin compared with elastase alone (hNE + aprotinin dI/dtmax = 1.2 ± 0.2 µA·cm–2·min–1 vs. elastase dI/dtmax = 2.5 ± 0.2 µA·cm–2·min–1; n range, 11–20) as expected for enzyme inhibition. Also, the referenced Ki of aprotinin for hNE (26) was reported using ionic solutions quite different from the physiological salt solution used in our experiments. Factors such as ionic strength and pH affect Ki. Thus it is possible that hNE is even less sensitive than the indicated Ki with aprotinin in a physiological salt solution (pH 7.4). Thus the residual uninhibited elastase activity was likely responsible for the comparable fold increase of the hNE-induced Isc-amil at steady state. Figure 2C shows a representative Isc-amil recording before and during exposure to neutrophil elastase inhibitor, a peptidyl chloromethyl ketone-containing inhibitor that is devoid of a Kunitz domain (36). Apical application of the inhibitor (25 µM) abolished the hNE-induced increase in Isc-amil, but trypsin retained the ability to augment the Isc-amil, as shown (Fig. 2C). Figure 2D summarizes the serine protease effects on Isc-amil with the elastase inhibitor.
Because trypsin showed no additive effects on the hNE-induced increase in Isc-amil, it was possible that hNE proteolytically cleaved and inactivated trypsin. However, after inhibiting hNE with elastase inhibitor following Isc-amil stimulation, trypsin was still unable to further stimulate the current above that achieved with hNE (hNE = 11.9 ± 0.9 µA/cm2 vs. trypsin = 10.6 ± 0.7 µA/cm2, n = 6). Taken together, these results strongly suggest hNE and trypsin catalytic activity are required for the serine protease-induced increase in Isc-amil. Importantly, hNE is relatively resistant to the Kunitz-type serine protease inhibitors that potently inhibit trypsin and epithelium-derived serine proteases implicated in the endogenous activation of ENaC (3, 10, 38).
Despite differences in substrate specificity, hNE and trypsin could share a common mechanism to augment transepithelial Na+ absorption. At the single channel level, trypsin increases the Po of the near-silent ENaC without altering pore conductance, ion selectivity, or the ability of the pore to be inhibited with amiloride (5). To test whether the hNE-induced increase in Isc-amil could also be explained by increasing the Po of near-silent ENaC, outside-out patches from 3T3 cells stably expressing rat -, -, -ENaC subunits were studied. Selected consecutive traces of a patch recording are shown in Fig. 3A. The characteristic brief and infrequent openings of near-silent ENaC activity were observed during the baseline recording period (downward deflections, sweeps 12, 13). However, within 15 s of bath-applied hNE (1 µg/ml, sweep 14), a robust increase in channel activity was observed (sweeps 14–18). At least four channels were open simultaneously in some instances during enzyme exposure, and the patch never reverted to the near-silent ENaC activity observed in control recordings (i.e., sweeps 12, 13) for the lifetime of the patch (10 min). In other instances (n = 2), extensive washout of hNE from the bath for 20 min did not restore near-silent ENaC activity (not shown). Bath application of amiloride (10 µM, sweep 18) reversibly inhibited channel activity and confirmed ENaC identity and patch configuration. An average 0.04 channels were open during the 5-min control recording (e.g., sweep 13). After a 5-min hNE exposure, 2.8 channels were open, on average, reflecting an 70-fold increase in channel activity over baseline activity.
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100-fold difference in channel activity. The basally active ENaC population was characterized as having 1.9 channels open on average, whereas the near-silent ENaC population was characterized as having 0.02 open channels. Channel activity measured in the latter population was indistinguishable from the near-silent ENaC Po value previously reported in this expression system (5). hNE increased near-silent ENaC activity 108-fold over baseline, but neither hNE nor trypsin significantly increased basally active ENaC activity (Fig. 3D). Thus, consistent with the lack of response with trypsin following hNE in Isc-amil measurements, trypsin showed no increased channel activity following patch exposure to hNE. No consistent effects on the passive properties of serine protease-exposed channels were evident compared with basally active or near-silent ENaC (Fig. 4). The unitary conductance with Li+ as the charge carrier was 9.4 pS (Fig. 4) and within the range of values previously reported for ENaC (17, 20).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Our measurements of channel gating (e.g., Po) reveal two functionally distinct ENaC populations, a near-silent ENaC pool and a basally active ENaC pool. hNE increased near-silent ENaC activity 100-fold by increasing Po of membrane-resident channels, whereas no enzyme effects were observed on basally active ENaC activity. We previously reported that trypsin increased the Po of near-silent ENaC (5). In the present study, both macroscopic current and single channel assays demonstrate trypsin effects on Isc-amil and channel activity are not additive to the hNE response. Taken together, these results suggest that both hNE and trypsin target the near-silent ENaC pool as substrate and liberate as product, active channels with functional properties indistinguishable from basally active ENaC activity.
The serine protease-induced activation of near-silent ENaC likely results from direct proteolytic cleavage of a limited number of sites in the channel complex. This is because in single channel measurements the functional properties of serine protease-exposed channels were indistinguishable from basally active ENaC including channel gating, unitary conductance, or inhibition with amiloride. Moreover, channel activity of basally active ENaC did not increase after exposure to hNE or trypsin, suggesting this channel population was already cleaved by endogenous protease(s).
As previously reported by us and others, G protein inhibition failed to block the trypsin-induced increase of near-silent ENaC activity (5) and amiloride-sensitive current (6). Whereas elastase inhibits G protein-coupled protease-activated receptors (PARs, e.g., PAR1 and PAR3), together, thrombin and trypsin activate all PAR subtypes identified (25), but of these enzymes, only trypsin and elastase increased amiloride-sensitive currents (R. A. Caldwell, unpublished observations). Thus, it is unlikely that hNE and trypsin stimulation of PARs in excised patches indirectly links proteolytic activity with near-silent ENaC activation. On the other hand, identifying the trypsin and hNE cleavage sites, especially in view of their location(s) relative to the furin cleavage sites in ENaC (15) will be important for probing how these and furin sites are involved in ENaC gating.
ENaC activity and susceptibility to proteolysis at the cell surface appear to vary considerably depending on tissue and culture conditions. For instance, in primary cultures of human bronchial epithelia (HBE) (3) and nasal epithelia (10), no discernable trypsin response was observed without prior aprotinin exposure. Whereas aprotinin markedly inhibited the basal Isc-amil in primary HBE cultures (70%), we did not detect any effects of aprotinin at similar concentrations on this current in 16HBE140-/ but observed a near doubling of the current with trypsin or hNE. Using a CF nasal cell line (JME/CF15), Tong et al. (38) observed a significant trypsin-induced increase (18%) in Isc-amil without aprotinin pretreatment. However, up to 80% of Isc-amil in JME/CF15 was inhibited with aprotinin and small interfering RNAs directed against the endogenous epithelial serine protease, prostasin (38).
Whereas cleavage of - and/or -ENaC subunits (30) correlates with expression of amiloride-sensitive current (11, 15), it is of physiological and potential therapeutic importance to know in what environment does cleavage occur. The aprotinin sensitivity of basal Isc-amil is consistent with a model of near-silent ENaC emerging onto the plasma membrane and then becoming proteolytically activated, but proteolytic activation of ENaC by aprotinin-insensitive proteases [i.e., CAP3, furin (32, 39)] is thought to occur intracellularly (16). In view of recent biochemical evidence for two plasma membrane-resident ENaC populations, consisting of furin-cleaved and uncleaved channel complexes (16), our functional characterization of basally active and near-silent ENaC populations in patches from 3T3 cells is consistent with both intracellular (e.g., furin mediated) and extracellular proteolytic activation of ENaC. Interestingly, we observed basally active or near-silent channel populations, but not both in the same patch (Fig. 3C). We speculate this could occur in one of two ways: first, by mutual exclusion. That is, a cell consists of a homogenous population of only near-silent (e.g., uncleaved) or basally active channels. Alternatively, a cell consists of mixed populations of near-silent and basally active channels, but these populations are restricted to distinct regions [e.g., within vs. outside of lipid rafts (14), but see Ref. 13] of the cell surface membrane. Limited functional data support the latter scenario. In a voltage-ramp protocol on an outside-out macropatch configured from a 3T3 cell, a 44-pS amiloride-sensitive conductance was observed to increase 3.5-fold following brief trypsin exposure (R. A. Caldwell, unpublished observation). The susceptibility of ENaC to activation by extracellularly applied serine protease varies with cell type and culture conditions and could reflect a balance between endogenous proteases and their inhibitors (1, 19), biosynthetic pathways (16), and channel recycling (34). Future studies are needed to address these important questions.
Bridges et al. (3) reported that out of several serine protease inhibitors tested in primary cultures of HBE, only inhibitors containing the naturally occurring Kunitz domains inhibited Isc-amil. The Bayer pharmaceutical compound BAY 39-9437 is also a Kunitz-containing serine protease inhibitor similar to placental bikunin and is considered a potential therapeutic agent for CF lung disease that would act by ameliorating the Na+ hyperabsorption in CF airways. However, CF airways disease is caused by chronic infection, which is accompanied by marked influx of neutrophils and is characterized by an imbalance between hNE and endogenous antiprotease activity (1). Indeed, the excessive hNE concentrations present in CF airways overwhelm endogenous protease inhibitors (e.g., 1-antiprotease, secretory leukoprotease inhibitor) (21). Active hNE in airways will have adverse effects including increased lung permeability (12), reduced mucociliary clearance (33, 35), and ultimately bronchiectasis resulting from the loss of lung structural integrity (4). The reported insensitivity of hNE activity to placental bikunin and BAY 39-9437 (3, 8), combined with results presented here, suggests that Kunitz inhibitors will not effectively inhibit the micromolar concentrations of elastase activity present in CF airways. This may limit their potential utility to early stage CF.
Excessive hNE levels in the CF lung has received attention for the enzyme's harmful contribution to inflammation and structural damage (21). However, in the early stages of CF airway disease, before the loss of lung structural integrity, hNE activation of near-silent ENaC is expected to exacerbate the airway epithelial Na+ absorption and contribute to the depletion of the ASL layer and the subsequent pathological sequelae. Thus blockers of ENaC having long duration of action may be therapeutically efficacious for ameliorating effects of hNE stimulation of ENaC as well as the Na+ hyperabsorption in CF airways. Additionally, development of potent small-molecular-weight hNE inhibitors holds promise for ameliorating CF lung disease (7), but maximum benefit may come in the form of combination therapy with hNE and ENaC inhibitors.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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), basally active ENaC (Ba-ENaC, 
), neutrophil elastase (hNE, 1–5 µg/ml, 
), and trypsin (Tryp, 3 µg/ml, 
) were similar. Slope conductance for Tryp-modified channels, measured between –60 and –100 mV with Li+ as the charge carrier, was 9.4 pS; i values from 3–8 outside-out patch recordings at different membrane voltages (Vmembrane) are plotted. SE bars are shown when they exceed symbol size.