SPECIAL TOPIC
Na+ transport in normal and CF human
bronchial epithelial cells is inhibited by BAY 39-9437
Robert J.
Bridges1,
Ben
B.
Newton2,
Joseph M.
Pilewski1,
Daniel C.
Devor1,
Christopher T.
Poll2, and
Rod L.
Hall2
1 Department of Cell Biology and Physiology, University of
Pittsburgh, Pittsburgh, Pennsylvania 15261; and 2 Bayer
Pharmaceutical Division, Slough SL2 4LY, United Kingdom
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ABSTRACT |
To test the hypothesis that
Na+ transport in human bronchial epithelial (HBE) cells is
regulated by a protease-mediated mechanism, we investigated the effects
of BAY 39-9437, a recombinant Kunitz-type serine protease inhibitor, on
amiloride-sensitive short-circuit current of normal [non-cystic
fibrosis (CF) cells] and CF HBE cells. Mucosal treatment of non-CF and
CF HBE cells with BAY 39-9437 decreased the short-circuit current, with
a half-life of ~45 min. At 90 min, BAY 39-9437 (470 nM) reduced
Na+ transport by ~70%. The inhibitory effect of BAY
39-9437 was concentration dependent, with a half-maximal inhibitory
concentration of ~25 nM. Na+ transport was restored to
control levels, with a half-life of ~15 min, on washout of BAY
39-9437. In addition, trypsin (1 µM) rapidly reversed the inhibitory
effect of BAY 39-9437. These data indicate that Na+
transport in HBE cells is activated by a BAY 39-9437-inhibitable, endogenously expressed serine protease. BAY 39-9437 inhibition of this
serine protease maybe of therapeutic potential for the treatment of
Na+ hyperabsorption in CF.
cystic fibrosis; Kunitz-type serine protease inhibitor; channel-activating protease; short-circuit current; primary cultures; epithelial sodium channel
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INTRODUCTION |
CYSTIC FIBROSIS (CF)
is characterized by abnormalities in anion secretion and
Na+ absorption in the airways (2, 3, 15, 22).
Although a great deal is known about the regulation of anion secretion, the underlying mechanisms regulating airway Na+ absorption
are poorly understood. Recent studies (6, 27, 28) have
demonstrated a novel extracellular serine protease-mediated signaling
pathway for the modulation of amiloride-sensitive epithelial Na+ channels (ENaCs). Amphibian and murine homologs of a
cation channel-activating protease (CAP1) have been identified and are
expressed in several epithelial tissues including kidney, prostate,
salivary glands, colon and lung. Xenopus CAP1 (xCAP1) and
murine CAP1 (mCAP1) share a 50% homology, whereas mCAP1 is 80%
homologous with human prostasin (28). Prostasin is also
expressed in epithelial tissues, including the lung (30,
31). Coexpression of xCAP1 (6, 27) or mCAP1 (28) with Xenopus, rat, or human ENaCs in
Xenopus oocytes caused a severalfold increase in the
amiloride-sensitive Na+ current. Aprotinin, a
bovine-derived serine protease inhibitor, blocked the activation of
ENaCs by CAP1. Aprotinin also inhibited the baseline Na+
transport in cultures of A6 cells, an amphibian renal cell line (27) and mpkCCDC14 cells, a murine renal cell
line (28).
The studies reported here were undertaken to test the hypothesis that
Na+ transport in human bronchial epithelial (HBE) cells is
regulated by a protease-mediated mechanism. To test this hypothesis, we first investigated the effects of several protease inhibitors for their
potential effects on the amiloride-sensitive short-circuit current
(Isc) in HBE cells. The results from these
initial studies demonstrated that aprotinin inhibited HBE cell
Na+ transport. In contrast, two other serine protease
inhibitors, soybean trypsin inhibitor (SBTI) and
1-antitrypsin (
1-AT), did not inhibit HBE
cell Na+ transport. The unique feature of aprotinin that
defines the protease-inhibitory site of this bovine protein is the
Kunitz domain (11, 12). A search for a human Kunitz-type
serine protease inhibitor was made and led to the development of the
recombinant protein BAY 39-9437. The studies reported here demonstrate
the inhibitory effects of BAY 39-9347 on non-CF and CF HBE cell
Na+ absorption.
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METHODS |
Primary cultures of HBE cells.
HBE cells were obtained from excess pathological tissue remaining after
lung transplantation under a protocol approved by the University of
Pittsburgh (Pittsburgh, PA) Investigational Review Board. Tissue
expressing wild-type CF transmembrane conductance regulator (CFTR) was
obtained after lung transplantation for a variety of pathological
conditions including bronchiectasis, emphysema, chronic obstructive
pulmonary disease (COPD), idiopathic pulmonary fibrosis, and
scleroderma. The CFTR genotype of the CF tissues was determined by
allele-specific hybridization (performed at Genzyme, Framingham, MA).
All cells were isolated from second through sixth generation bronchi in
both wild-type CFTR-expressing (non-CF) and CF HBE cells. The bronchi
were incubated overnight at 4°C in MEM containing 0.1% protease XIV,
0.01% deoxyribonuclease, and 1% fetal bovine serum (FBS). The
epithelial cells were removed from the underlying musculature by blunt
dissection, isolated by centrifugation, and washed in MEM containing
5% FBS. After centrifugation, the cells were resuspended in bronchial
epithelial growth medium (Clonetics, San Diego, CA). The cells
were then plated into human placental collagen-treated T-25
tissue culture flasks. On reaching 80-90% confluence, the cells
were trypsinized (0.1%), resuspended in MEM plus 5% FBS, and seeded
onto human placental collagen-coated Costar Transwell filters (0.33 cm2) at a density of ~2 × 106/cm2. After 24 h, the medium was
changed to DMEM-F-12 medium (1:1) plus 2% Ultroser G (BioSepra), and
an air interface at the apical membrane was established. The medium
bathing the basolateral surface was changed every 48 h.
Measurements of Isc were performed after ~10-20 additional days in culture.
Isc measurements.
Costar Transwell cell culture inserts were mounted in Costar Ussing
chambers, and the cultures were continuously short-circuited with an
automatic voltage clamp (Department of Bioengineering, University of
Iowa, Iowa City, IA). Transepithelial resistance was measured by
periodically applying a 2-mV bipolar pulse and calculated with Ohm's
law. The bath solution contained (in mM) 120 NaCl, 25 NaHCO3, 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, 1.2 CaCl2, and 10 glucose. The pH of this solution was 7.4 when
gassed with a mixture of 95% O2-5% CO2 at
37°C. The amiloride-sensitive Isc was taken as
a measure of net electrogenic Na+ transport.
Materials.
Aprotinin, SBTI,
1-AT, trypsin, and amiloride were from
Sigma and were dissolved in phosphate-buffered saline (PBS) at
1,000-fold the required experimental concentrations. BAY 39-9437 is a
170-amino acid human serine protease inhibitor (7, 17)
that was recombinantly expressed in Chinese hamster ovary (CHO) cells.
The secreted protein was chromatographically purified from the culture medium.
Data analysis.
All data are presented as means ± SE; n is the number
of experiments. Apparent inhibitory constant
(Ki) values were obtained with nonlinear
curve-fitting routines in SigmaPlot (Jandel Scientific, San Rafael,
CA). Statistical analysis was performed with Student's t-test. A value of P < 0.05 was considered significant.
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RESULTS |
The studies reported here were performed on HBE cultures with
cells derived from five patients expressing wild-type CFTR (non-CF cells) and seven CF patients (CF cells). Two of the non-CF patients were diagnosed with emphysema or COPD, one with idiopathic pulmonary fibrosis, one with fibrotic connective tissue disease, and one with
scleroderma. The CFTR genotype of the seven CF patients is given in
Table 1. All seven patients had at least
one
F508 CFTR allele and three were homozygous
F508 CFTR. In
total, we evaluated 128 non-CF HBE cultures and 167 CF HBE cultures
under Isc conditions. The baseline
Isc and transepithelial resistance of non-CF HBE cultures were 35.6 ± 0.90 µA/cm2 and 733 ± 14.7
· cm2, respectively. The
Isc and transepithelial resistance of the CF HBE
cultures were 42.4 ± 1.13 µA/cm2 and 684 ± 15.0
· cm2, respectively. As in a previous
study by Devor et al. (9), amiloride inhibited a
greater portion of the Isc in CF cells (82%) than in non-CF cells (70%). These results together with the higher Isc in CF cells versus non-CF cells demonstrate
a significant Na+ hyperabsorption by the CF HBE cultures.
Effects of protease inhibitors on Na+ transport across
HBE cultures.
In the first series of experiments, we investigated the effects of
several protease inhibitors for their potential effects on
Na+ transport across HBE cultures. A baseline
Isc was first measured after a 20-min
equilibration period, at which time the protease inhibitor was added to
the mucosal bath. After 90 min, the Isc was
again recorded, and amiloride (10 µM) was then added to the mucosal
bath. After an additional 5 min, the Isc was
again recorded. Figure 1 summarizes
the results of the effects of several protease inhibitors on
the Isc across CF cells with the above protocol. Control cultures were given an equivalent volume of PBS and studied in
parallel with the protease-treated cultures. The
Isc of the PBS-treated cultures was stable over
a 90-min period. Amiloride caused an inhibition of 34 ± 3.8 µA/cm2 (n = 29) in the PBS-treated
cultures. In contrast, aprotinin reduced the Isc
from a baseline value of 37 ± 2.9 µA/cm2 to a value
of 15 ± 3.5 µA/cm2 (n = 9) after 90 min and amiloride caused a further inhibition of 9.3 ± 3.9 µA/cm2. Thus aprotinin caused a significant inhibition in
the amiloride-sensitive Isc compared with the
PBS-treated control cultures (P < 0.001). SBTI and
1-AT did not inhibit the Isc or
alter the amiloride-sensitive current (Fig. 1). Similar results were
obtained with cells from non-CF patients.

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Fig. 1.
Effects of various protease inhibitors on cystic fibrosis
(CF) human bronchial epithelial (HBE) cell Na+ transport.
The short-circuit current (Isc) of CF epithelial
cultures under continuous short-circuit conditions was measured at
time 0 (baseline), 90 min after the addition of PBS or
protease inhibitor to the mucosal solution (T90min), and after the
addition of amiloride. Drug concentrations were 1 µM aprotinin, 10 µM soybean trypsin inhibitor (STBI), 1 µM
1-antitrypsin ( 1-AT), and 10 µM
amiloride. Values are means ± SE for 29 PBS-treated cultures and
9 cultures for each of the inhibitors. * P < 0.001 for
aprotinin-treated vs. PBS-treated control cultures.
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These results demonstrate that Na+ transport in HBE cells
is regulated by an aprotinin-sensitive mechanism as previously shown in
studies on renal epithelial cells and ENaC expression studies in
Xenopus oocytes (6, 27, 28). Aprotinin is a
serine protease inhibitor of bovine origin and a potent inhibitor of
trypsin (11, 12). Although SBTI and
1-AT
are also serine protease inhibitors, only aprotinin contains a Kunitz
domain, the protease-inhibitory active site. Reasoning that the Kunitz
domain was the essential feature of the inhibitory effect of aprotinin
on Na+ transport, we searched for a human Kunitz-type
protease inhibitor. BAY 39-9437 is such a protein and is further
described in DISCUSSION. In BAY 39-9437 inhibition of
HBE Na+ transport, we document the inhibitory
effects of BAY 39-9437 on Na+ transport across non-CF and
CF HBE cell cultures.
BAY 39-9437 inhibition of HBE Na+ transport.
Figures 2 and
3 illustrate the time-dependent
inhibition of Isc caused by BAY 39-9437 in
non-CF and CF cells, respectively. BAY 39-9437 (470 nM) caused the
Isc to decrease, with a half-life (t1/2) of ~45 min, whereas the
PBS-treated control cells showed little or no change in
Isc over a 90-min period. After 90 min,
amiloride caused a greater inhibition of the 90-min
Isc in the PBS-treated control cells compared
with the BAY 39-9437-treated cells. Figure
4 summarizes the results of 25-38
experiments performed as illustrated in Figs. 2 and 3. Over a
90-min period, BAY 39-9437 (470 nM) inhibited a significant portion of
the amiloride-sensitive Isc in both non-CF
(68 ± 3.3%) and CF cells (72 ± 3.9%) compared with the
time-dependent changes in the PBS-treated control cells run in parallel
(P < 0.001). The inhibition in Isc
by BAY 39-9437 was observed with cells from all five non-CF patients
and all seven CF patients.

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Fig. 2.
Effect of BAY 39-9437 on Na+ transport in non-CF HBE
cells. PBS (A) or BAY 39-9437 (470 nM; B) was
added to the mucosal side, and Isc was monitored
for 90 min before the addition of amiloride (10 µM) to the mucosal
solution. Vertical deflections are the current response to 2-mV bipolar
pulses.
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Fig. 3.
Effect of BAY 39-9437 on Na+ transport in CF HBE cells.
PBS (A) or BAY 39-9437 (470 nM; B) was added to
the mucosal side, and Isc was monitored for 90 min before the addition of amiloride (10 µM) to the mucosal solution.
Vertical deflections are the current response to 2-mV bipolar pulses.
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Fig. 4.
Effects of PBS and BAY 39-9437 on non-CF (A) and CF
(B) HBE cell Na+ transport. Studies were
performed as in Fig. 2. Drug concentrations were 470 nM BAY 39-9437 and
10 µM amiloride. Values are means ± SE; n = 25-35 filters. * P < 0.001 for BAY 39-9437-treated
vs. PBS-treated control cells.
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The effects of BAY 39-9437 on Isc were
concentration dependent in all patients as illustrated in Fig.
5 for studies on CF cells from
patient 4. The values shown in Fig. 5 are the percent change
in Isc at 90 min after correction for the
amiloride-insensitive Isc. BAY 39-9437 at 4.7 nM
did not significantly alter the Isc. However, at
16 nM, BAY 39-9437 caused a significant inhibition in the
Isc, suggesting that the minimal effective
concentration falls between 4.7 and 16 nM. The half-maximal effective
concentration (K1/2) for the studies shown
in Fig. 5 was 25 ± 7.9 nM and a maximal inhibition of 87 ± 6.4% when fit to a simple Michaelis-Menten inhibition function. The
inhibition of Isc by BAY 39-9437 in non-CF cells
from a patient diagnosed with emphysema or COPD was also concentration
dependent, with a very similar K1/2 of
24 ± 8.5 nM but with a lower maximal inhibition of 54 ± 2.8%. The lower maximal inhibition in non-CF cells compared with CF
cells may reflect the contribution of anion secretion to the
Isc in non-CF cells.

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Fig. 5.
Concentration-dependent effects of BAY 39-9437 on
Na+ transport in CF HBE cells. Values are percent change in
Isc at 90 min corrected for the
amiloride-insensitive current; n = 5 filters/group. BAY
39-9437 was added to the mucosal solution at time 0 at the
indicated concentrations, and amiloride (10 µM) was added at 90 min.
Solid line, fit to a simple Michaelis-Menten inhibition function. The
PBS-treated control cells for this series of experiments had a mean
baseline Isc of 30.4 ± 2.8 µA/cm2 that was at 107 ± 7% at 90 min.
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The inhibitory effects of BAY 39-9437 were completely reversed by the
addition of trypsin to the mucosal solution (Fig.
6B). In the experiment shown
in Fig. 6, BAY 39-9437 (470 nM) decreased the
Isc from a baseline value of 66 µA/cm2 to a value of 26 µA/cm2 over a
90-min period. Amiloride (10 µM) further inhibited the Isc to a value of 6 µA/cm2. An
exchange of the mucosal bath with BAY 39-9437 and amiloride-free buffer
restored the Isc to the preamiloride
Isc value. The addition of trypsin (1 µM) to
the mucosal bath caused a rapid increase in the
Isc to the pre-BAY 39-9437 (baseline) level of
70 µA/cm2, and this current was nearly completely
inhibited by amiloride. These results are representative of nine
similar experiments with CF cells in which trypsin increased the
Isc to 93 ± 7% of the pre-BAY 39-9437 level. The addition of trypsin to the mucosal bath of PBS-treated cells
had little or no effect on the Isc (Fig. 6A). Similar results were obtained with PBS- and BAY
39-9437-treated non-CF cells (data not shown).

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Fig. 6.
Time course and reversal by trypsin of the inhibitory effect of BAY
39-9437 (B) on Na+ transport in CF HBE cells.
A: PBS treatment. Drug concentrations were 470 nM BAY
39-9437, 10 µM amiloride (Amil), and 1 µM trypsin, and drugs were
added to the mucosal solution at the indicated times (arrows). After
the 1st addition of amiloride, the mucosal bath was exchanged
with a 20× volume (100 ml) of amiloride- and BAY 39-9437-free buffer.
Vertical deflections are the current responses to 2-mV pulses.
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In a second series of experiments designed to investigate the
reversible inhibition of Na+ transport by BAY 39-9437, CF
cells were incubated overnight with 25 µl of PBS or PBS plus 470 nM
BAY 39-9437 on the apical surface. The cells were then washed and
placed in Ussing chambers, and the Isc was
monitored. Overnight treatment with BAY 39-9437 caused a similar degree
of inhibition in Na+ transport as observed after a 90-min
treatment compared with that in PBS-treated cells (PBS treated:
50.8 ± 2.6 µA/cm2, n = 25; BAY
39-9437 treated: 15.5 ± 2.8 µA/cm2,
n = 14). However, in contrast to the PBS-treated cells,
the BAY 39-9437-treated cells showed a steady rise in
Isc over a 45-min period to a new higher plateau
value that was nearly equal to the initial Isc
of the PBS-treated cells (Fig. 7). The
addition of trypsin (1 µM) to the mucosal bath caused little change
in the Isc of PBS-treated cells (<2.5 ± 1.3 µA/cm2; n = 9) as previously shown in
Fig. 6. In contrast, trypsin caused a rapid rise in the
Isc of BAY 39-9437-treated cells, and the magnitude of this increase was greater in cells left for 15 min compared with cells left for 45 min (Fig. 7, B and
C, respectively). The results shown in Fig. 7 are
representative of six similar experiments. Trypsin added at 15 min
increased the Isc by 27 ± 60 µA/cm2 and at 45 min by only 12 ± 4.2 µA/cm2 in the BAY 39-9437-treated cells. These results
demonstrate that the effects of BAY 39-9437 are reversible after a
wash, with a t1/2 of ~15 min, and suggest
that the activity of an endogenous protease is preserved after BAY
39-9437 treatment and washout.

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Fig. 7.
Effects of overnight treatment with BAY 39-9437 on
Na+ transport in CF cells. Cells were treated with 25 µl
of PBS (A) or PBS plus BAY 39-9437 (470 nM; B and
C) on the apical surface for 18 h. The cells were
washed with 50 ml of warm gassed inhibitor-free buffer and mounted in
Ussing chambers, and Isc was measured as
described in METHODS. Trypsin (1 µM) and amiloride (10 µM) were added to the mucosal bath at 15 (A and
B) and 45 (C) min. Results are representative of
6 similar experiments.
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DISCUSSION |
BAY 39-9437 is a recombinant human serine protease inhibitor. The
natural protein is a 252-amino acid protein composed of a signal
peptide, two protease-binding Kunitz domains, a transmembrane domain,
and an intracellular domain. This protein was originally identified and
isolated from the placenta and is referred to as placental bikunin
(7, 17). Northern blot analysis demonstrated the
expression of placental bikunin in pancreas, kidney, brain, heart, and
lung. The gene for placental bikunin was mapped to chromosome 19q13
(17), and it is noteworthy that a CF modifier gene has
been mapped to this same locus (33). Loss of function mutations in placental bikunin are expected to increase the severity of
CFTR disease, causing mutations, whereas an increase in the expression
or activity of placental bikunin should diminish the severity of CFTR
disease, causing mutations. The recombinant product BAY 39-9437 is a
protein of 170 amino acid residues. BAY 39-9437 was expressed in CHO
cells as a secreted form of placental bikunin lacking the transmembrane
and intracellular domains such that it is composed of the signal
peptide and the two extracellular Kunitz domains. Both placental
bikunin and BAY 39-9437 inhibit trypsin (Ki = 0.01 nM), plasmin (Ki = 0.1 nM), and
kallikrein (Ki = 0.3 µM) at a 2:1
enzyme-to-inhibitor binding stoichiometry (7, 17).
However, placental bikunin and BAY 39-9437 do not inhibit urokinase,
tissue plasminogen activator, or elastase at concentrations up to 1 µM (7, 17).
The studies reported here demonstrate that BAY 39-9437 is a potent
inhibitor of electrogenic Na+ transport in HBE cells. The
Na+ transport inhibitory effects of BAY 39-9437 develop
slowly, with a t1/2 of ~45 min (Figs. 2,
3, and 6), and are reversible on washing, with a
t1/2 of ~15 min (Fig. 7), or develop
rapidly with the addition of trypsin (Figs. 6 and 7). The inhibitory
effects of BAY 39-9437 on Na+ transport were concentration
dependent and well described by a simple Michaelis-Menten inhibition
function, with a K1/2 of ~25 nM in both
non-CF and CF cells. These results do not preclude the possibility that
both Kunitz domains participate in the inhibition of Na+
transport. However, they do suggest that there is no cooperativity between the two domains if indeed both Kunitz domains participate. BAY
39-9437 (470 nM) caused approximately the same degree of inhibition in
the amiloride-sensitive Isc in non-CF (68%) and
CF (72%) cells. The inhibitory effect of BAY 39-9437 was nearly
completely reversed after a wash with an inhibitor-free solution.
Isc increased, with a
t1/2 of ~15 min, after removal of BAY
39-9437, suggesting that the activity of the ENaC-activating protease
was preserved after BAY 39-9437 treatment. The inhibition of
Na+ transport by BAY 39-9437 could also be rapidly reversed
by the addition of trypsin to the mucosal bath. Mucosal trypsin had no effect on the baseline Isc or the 90-min
Isc in PBS-treated non-CF or CF HBE cells. Only
after inhibition with BAY 39-9437 was the stimulation of an
amiloride-sensitive Isc with trypsin observed.
Our working hypothesis for the protease-mediated modulation and
inhibition of HBE Na+ transport by BAY 39-9437 is
illustrated in Fig. 8. As originally suggested for the modulation of ENaC in renal epithelia (6, 27,
28), we propose that there is a CAP1-like protease in the apical
membrane of HBE cells. ENaC is inserted into the apical membrane as an
inactive or partially active channel where it is activated by an apical
membrane CAP1-like protease. Active ENaC remains in the apical membrane
for a t1/2 of ~45 min when it is then
retrieved, probably ubiquitinated, and degraded (25). The
addition of a CAP1 inhibitor such as BAY 39-9437 prevents the
activation of ENaC by CAP1. The addition of an exogenous protease such
as trypsin can circumvent the inhibition of CAP1 and cause the
activation of ENaC.

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Fig. 8.
Working hypothesis for the protease-mediated
regulation of Na+ transport in HBE cells. Inactive
epithelial Na+ channel (ENaC) is inserted into the apical
membrane where it is activated by an apical membrane, extracellular BAY
39-9437-inhibitable protease. Active ENaC is removed from the apical
membrane with a half-life of 45 min. Retrieved ENaC is probably
ubiquitinated and then degraded. Proteolytic activation of ENaC can be
blocked by extracellular BAY 39-9437 without altering the insertion of
new inactive ENaC, which can subsequently be activated by the addition
of exogenous trypsin. The substrate of the channel-activating protease
or the exogenous trypsin is unknown and may be ENaC or some regulatory
protein closely associated with ENaC.
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The molecular identity of the HBE cell CAP1-like protease remains to be
established, but one possible candidate is prostasin (30-32). In support of this notion, prostasin shares
an 80% homology with mCAP1 and 50% homology with xCAP1. Moreover,
prostasin was shown to be inhibited by aprotinin but not by SBTI
(30), results consistent with the inhibition of
Na+ transport by aprotinin but not by SBTI (Fig. 1). The
prostatin gene (PRSS8) has been localized to chromosome 16p11.2
(21) and is another potential gene that could act as a CF
modifier gene. Prostasin is synthesized by the cell as a preproenzyme
and may remain on the plasma membrane or be released from the cell
surface as a secreted protein (30, 31). The results shown
in Fig. 7 suggest that the protease responsible for the activation of ENaC in HBE cells is not readily washed off and thus may remain as a
membrane-anchored protein. The enzymes involved in the synthesis of
prostasin and other CAP1-like proteases are unknown. The substrate for
the CAP1-like proteases is also unknown. Initial experiments suggest
that ENaC may not be the CAP1 substrate (6, 27). However,
it remains possible that ENaC is a protease-activated channel. If ENaC
is not the CAP1 substrate, it will be important to identify the
substrate as well as the signal transduction cascade that leads to the
activation of ENaC. Given the emergence of protease-activated receptors
(PARs) that are G protein coupled to intracellular signal transduction
cascades (8), one may speculate that CAP1 acts on a novel
PAR to cause the activation of ENaC. Clearly, additional studies are
necessary to determine whether ENaC is a protease-activated channel or
to identify the CAP1-activated PAR involved in the regulation of ENaC.
Xenopus oocyte expression studies (6, 27) suggest that CAP1 activation of ENaC involves an increase in the channel open probability and not an increase in the number of channels,
results that are consistent with the model shown in Fig. 8. Prolonged
overnight incubation with aprotinin or BAY 39-9437 did not completely
inhibit the amiloride-sensitive Isc in HBE cells
(Fig. 7). These results suggest that ENaC may be inserted into the
apical membrane as a partially active channel, that is, a channel with
a finite but low open probability. CAP1-mediated activation then leads
to an increase in the channel open probability. Fluctuation analysis to
obtain estimates of channel density and open probability could be
used to investigate the mechanisms involved in the protease-mediated
modulation of ENaC activity.1
Although controversy abounds in the CF research community regarding the
composition and volume of the airway surface liquid in normal and CF
airways (29), there is unanimous agreement that improving
the clearance of mucus in CF patients would be of major therapeutic
benefit. Indeed, impaired mucociliary clearance is a clinical feature
of many airway diseases including CF (20, 23). The result
of impaired mucociliary clearance is mucus retention and accumulation
that appear to contribute to the severity of the disease. The
inhibition of Na+ transport is anticipated to improve
mucociliary clearance. Support for this logic is seen in another
genetic disease, pseudohypoaldosteronism, caused by loss of function
mutations in ENaC (5, 15, 26). These patients display a
fourfold increase in mucociliary transport compared with control
patients (14). Several attempts have been made with
amiloride to show the therapeutic benefit of inhibiting Na+
transport in CF patients (4, 13, 16). However, the results of these trials have been disappointing. The weak affinity of amiloride
for ENaC (Ki = 300 nM) and the rapid
clearance of this low molecular mass compound from the airways
(1, 18, 19) are thought to be important reasons for the
poor efficacy of amiloride. The in vitro studies reported here indicate
that BAY 39-9437 has a 10-fold improved potency for the inhibition of
ENaC compared with amiloride (25 vs. 300 nM). Moreover, as a 21-kDa
recombinant protein, BAY 39-9437 is expected to have a longer residence
time in the airways compared with that of amiloride. Thus if BAY
39-9437 can be delivered to the airways, it should inhibit
Na+ transport and thereby improve mucociliary clearance.
Animal studies reported by Newton et al. (21) document a
decrease in the tracheal transepithelial potential difference caused by
the local instillation of BAY 39-9437, results consistent with the
inhibition of Na+ transport. Most importantly, BAY 39-9437 caused a twofold increase in tracheal mucociliary clearance. Thus we
are optimistic that the human recombinant serine protease inhibitor BAY
39-4937 may be of therapeutic benefit in the treatment of CF.
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ACKNOWLEDGEMENTS |
We acknowledge the excellent technical assistance of Matt Green and
Joe Latoche and the secretarial assistance of Michele Dobransky. This
work has also benefited from the many helpful discussions with
colleagues at the Bayer Research Facility at Stoke Court (Slough, UK)
and the Cystic Fibrosis (CF) Research Center at the University of
Pittsburgh. This work was inspired by two young CF children, Maria and
Antonia Hug.
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FOOTNOTES |
This work was supported by the Bayer Pharmaceutical Company.
Original submission in response to a special call for papers on
"CFTR Trafficking and Signaling in Respiratory Epithelium."
Address for reprint requests and other correspondence: R. J. Bridges, Dept. of Cell Biology and Physiology, Univ. of Pittsburgh, 3500 Terrace St., S310 Biomedical Science Tower, Pittsburgh, PA 15261 (E-mail: bbridges+{at}pitt.edu).
1
An alternative hypothesis that has not been
thoroughly addressed by the present or previous studies is the possible
direct inhibition of ENaC by the Kunitz-type protease inhibitors. A
number of studies (e.g., Refs. 10, 24) have demonstrated
the inhibition of K+ channels by Kunitz-type protease
inhibitors, and this possibility remains to be formally excluded for
the action of aprotinin and BAY 39-9437 on ENaC.
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.
Received 2 November 2000; accepted in final form 9 February 2001.
 |
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