UTP inhibits Na+ absorption
in wild-type and
F508 CFTR-expressing human bronchial
epithelia
Daniel C.
Devor1 and
Joseph M.
Pilewski1,2
Departments of 1 Physiology and
Cell Biology and 2 Medicine,
University of Pittsburgh, Pittsburgh, Pennsylvania 15261
 |
ABSTRACT |
Ca2+-mediated agonists,
including UTP, are being developed for therapeutic use in cystic
fibrosis (CF) based on their ability to modulate alternative
Cl
conductances. As CF is
also characterized by hyperabsorption of
Na+, we determined the effect of
mucosal UTP on transepithelial Na+
transport in primary cultures of human bronchial epithelia (HBE). In
symmetrical NaCl, UTP induced an initial increase in short-circuit current (Isc)
followed by a sustained inhibition. To differentiate between effects on
Na+ absorption and
Cl
secretion,
Isc was measured
in the absence of mucosal and serosal Cl
(INa). Again,
mucosal UTP induced an initial increase and then a sustained decrease
that reduced amiloride-sensitive
INa by 73%. The
Ca2+-dependent agonists histamine,
bradykinin, serosal UTP, and thapsigargin similarly induced sustained
inhibition (62-84%) of
INa. Mucosal UTP
induced similar sustained inhibition (half-maximal inhibitory concentration 296 nM) of
INa in primary
cultures of human CF airway homozygous for the
F508 mutation.
BAPTA-AM blunted UTP-dependent inhibition of
INa, but
inhibitors of protein kinase C (PKC) and phospholipase
A2 had no effect. Indeed, direct
activation of PKC by phorbol 12-myristate 13-acetate failed to inhibit
Na+ absorption. Apyrase, a tri-
and diphosphatase, did not reverse inhibitory effects of UTP on
INa, suggesting a
long-term inhibitory effect of UTP that is independent of receptor
occupancy. After establishment of a mucosa-to-serosa
K+ concentration gradient and
permeabilization of the mucosal membrane with nystatin, mucosal UTP
induced an initial increase in K+
current followed by a sustained inhibition. We conclude that increasing
cellular Ca2+ induces a long-term
inhibition of transepithelial Na+
transport across normal and CF HBE at least partly due to
downregulation of a basolateral membrane
K+ conductance. Thus UTP may have
a dual therapeutic effect in CF airway:
1) stimulation of a
Cl
secretory response and
2) inhibition of
Na+ transport.
cystic fibrosis; cystic fibrosis transmembrane conductance
regulator; epithelial sodium channel; uridine 5'-triphosphate; human airway
 |
INTRODUCTION |
CYSTIC FIBROSIS (CF) is characterized by a defect in
ion transport consisting of both a reduced or absent
Cl
secretory response to
cAMP-mediated agonists and a hyperabsorption of
Na+ (2). Recent evidence has
suggested that, in addition to functioning as a
Cl
channel, the CF
transmembrane conductance regulator (CFTR) regulates additional ion
conductive pathways, including the amiloride-sensitive epithelial
Na+ channel (ENaC; Ref. 35). CFTR
appears to act as a negative modulator of ENaC activity (35), which
likely explains the Na+
hyperabsorption observed in CF patients.
The human airway surface epithelium is a
Na+-absorbing tissue under basal
conditions. Cl
is at or
near its electrochemical equilibrium across the apical membrane in both
non-CF and CF epithelia (5, 41). Thus
Cl
secretory agonists fail
to stimulate a response in this basal, Na+ absorptive, state due to the
lack of driving force for
Cl
exit across the apical
membrane. Therefore, current therapeutic protocols for CF consist of
initially inhibiting the apical membrane Na+ conductance with amiloride.
This serves to hyperpolarize the apical membrane, thereby increasing
the electrochemical driving force for
Cl
exit across the apical
membrane. In contrast to cAMP-dependent agonists, increased
intracellular Ca2+ has been shown
to stimulate Cl
secretion
across CF airway (40). On the basis of this information, Ca2+-dependent agonists have been
proposed as therapeutically useful agents in CF therapy. In support of
this approach, Mason et al. (26) demonstrated that purine and
pyrimidine nucleotide triphosphates (ATP, UTP), acting at
P2U (P2Y2) receptors, are capable of
stimulating a Cl
secretory
response in CF tracheal epithelium via an increase in intracellular
Ca2+. Knowles et al. (19, 20) have
demonstrated that UTP induces a hyperpolarization of nasal potential
difference in both normal and CF patients that is consistent with
stimulation of Cl
secretion.
It is well known that increasing intracellular
Ca2+ in kidney epithelia inhibits
Na+ absorption via an inhibition
of apical membrane Na+ channels
(ENaC; Refs. 4, 25, 31). Indeed, such an inhibition of
Na+ transport was recently
reported in the cortical collecting duct of rabbit in response to
luminal UTP (22). Therefore, we determined whether, in addition to
stimulation of Cl
secretion, a second effect of UTP in the human airway might be the
inhibition of transepithelial Na+
absorption. We demonstrate that UTP, as well as bradykinin and histamine, inhibits transepithelial
Na+ absorption across primary
cultures of human bronchial epithelium (HBE) expressing both wild-type
(wt) and
F508 CFTR. These results suggest that amiloride
pretreatment may not be a prerequisite for mucosal UTP to have
therapeutic benefit in CF.
 |
METHODS |
Primary cultures of HBE.
HBE was obtained from excess pathological tissue remaining after lung
transplant under a protocol approved by the University of Pittsburgh
Investigational Review Board. Tissue expressing wt CFTR was obtained
following lung transplant for a variety of pathological conditions
including emphysema, primary pulmonary hypertension, pulmonary
fibrosis, and
1-antitrypsin
deficiency. All CF tissue employed in this study was shown to be
homozygous for the
F508 CFTR mutation by allele-specific
hybridization (performed at Genzyme, Framingham, MA). Bronchi of the
second to sixth generation were dissected, rinsed thoroughly, and
incubated overnight at 4°C in MEM containing 0.1% protease (type
XIV; Sigma Chemical, St. Louis, MO). The epithelial cells were isolated
by centrifugation and washed in MEM containing 5% fetal bovine serum
(FBS). After centrifugation, the cells were resuspended in serum-free
bronchial epithelial growth medium (Clonetics, San Diego, CA) and
plated into type VI human placental collagen (HPC; Sigma)-coated t-25 tissue culture flasks. When the cells reached 80-90% confluence, they were trypsinized, resuspended in MEM plus 5% FBS, and seeded onto
HPC-coated Costar Transwell filters (0.33 cm2) at a density of ~2 × 106
cells/cm2. After 24 h, the medium
was changed to DMEM/F-12 (1:1) plus 2% Ultroser G (BioSepra,
Villeneuve-la-Gavenne, France) and an air interface at the
apical membrane established. The medium bathing the basolateral surface
was changed every 48 h. Measurements of short-circuit current
(Isc) were
performed after ~10-20 additional days in culture.
Isc measurements.
Costar Transwell cell culture inserts were mounted in an Ussing chamber
(Jim's Instruments, Iowa City, IA), and the monolayers were
continuously short-circuited via an automatic voltage clamp (Dept. of
Bioengineering, University of Iowa, Iowa City, IA). Transepithelial
resistance was measured by periodically applying a 5-mV pulse, and the
resistance was calculated using 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. In
Cl
-free solutions, all
Cl
was replaced with
gluconate. The effects of UTP on basolateral membrane
K+ currents
(IK) were
assessed following permeabilization of the apical membrane with
nystatin (180 µg/ml) for 15-30 min and establishment of a
mucosa-to-serosa K+ concentration
gradient (11). For measurements of
IK, mucosal NaCl
was replaced with equimolar potassium gluconate, and serosal NaCl was
replaced with equimolar sodium gluconate.
Cl
was removed from these
solutions to prevent cell swelling that may be associated with the
limited Cl
permeability of
the nystatin pore. In all gluconate solutions, the
CaCl2 was increased to 4 mM to
compensate for the Ca2+-buffering
capacity of the gluconate anion. UTP was added to the indicated side of
the monolayer. Bumetanide, histamine, and bradykinin were added only to
the serosal bathing solution, whereas amiloride was added only to the
mucosal bathing solution. Changes in
Isc were
calculated as the difference in current between either the peak or
sustained phase of the response and the respective baseline value.
Chemicals.
Nystatin was a generous gift from Dr. S. Lucania (Bristol
Meyers-Squibb). RG-80267, a generous gift of Dr. Kim Barrett
(University of California, San Diego, CA), was made as a 1,000-fold
stock solution in DMSO. UTP, histamine, bradykinin, bisindolylmaleimide I, calphostin C, staurosporine, thapsigargin, forskolin, phorbol 12-myristate 13-acetate (PMA), and
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM were obtained from Calbiochem (La Jolla, CA). Bumetanide and apyrase were obtained from Sigma. Arachidonyl
trifluoromethyl ketone
(AACOCF3), palmitoyl
trifluoromethyl ketone
(PACOCF3), and
E-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (HELSS) were obtained from Biomol (Plymouth Meeting, PA)
and made up as 1,000-fold stock solutions in DMSO. Charybdotoxin (CTX) was obtained from Accurate Chemical and Scientific (Westbury, NY).
Forskolin was made up as a 1,000-fold stock solution in ethanol. Nystatin was made up as a 180 mg/ml stock solution in DMSO and sonicated for 30 s just before use. Cell culture media were obtained from GIBCO except as noted above.
Data analysis.
All data are presented as means ± SE;
n indicates the number of experiments.
The percent inhibition of Na+
current was calculated for each experiment under the assumption that
all of the current in the absence of
Cl
(wt CFTR HBE) or in
F508 HBE was due to Na+
absorption, such that %inhibition = (Ibaseline
IUTP)/(Ibaseline
Iamiloride),
where Ibaseline
is the baseline current and
IUTP and
Iamiloride are
the currents in presence of UTP and amiloride, respectively. Statistical analysis was performed on the
raw data using Student's t-tests. A
value of P < 0.05 was considered
statistically significant.
 |
RESULTS |
Effect of
Ca2+-mediated
agonists on HBE expressing wt CFTR.
The hyperabsorption of Na+ across
human CF airway may contribute to dehydration of airway secretions and
impairment of mucociliary clearance. This has led to clinical trials
designed to determine whether pharmacological inhibition of
Na+ transport would be
therapeutically beneficial in CF patients (14, 20, 39).
Ca2+-mediated agonists are known
negative modulators of Na+
absorption in both kidney (4, 25, 31) and colonic (38, 39) epithelia.
Therefore, we determined the effect of
Ca2+-mediated agonists on
Na+ absorption in primary cultures
of HBE. In a total of 25 filters expressing wt CFTR that were studied
in a symmetrical NaCl bath solution, the baseline
Isc averaged 26.0 ± 1.6 µA/cm2, with a
transepithelial potential difference of
19.5 ± 1.5 mV and a
transepithelial resistance of 888 ± 68
· cm2. The
effect of mucosal UTP (100 µM) on the basal
Isc response of
one representative HBE filter expressing wt CFTR is shown in Fig.
1,
left. We chose to use 100 µM UTP in these experiments because this concentration has been shown
to be maximally effective at modulating ion transport in human nasal
epithelium in vivo (19, 20). Addition of UTP to the mucosal compartment
resulted in a transient increase in
Isc followed by a
decrease to below baseline levels. Subsequent addition of the
Na+ channel blocker amiloride (10 µM) and the
Na+-K+-2Cl
cotransport inhibitor bumetanide (20 µM) reduced
Isc further. In
six experiments, UTP induced an initial increase in
Isc from 27.5 ± 4.1 to 36.2 ± 4.3 µA/cm2
(P < 0.001) followed by a sustained
inhibition to 21.2 ± 2.3 µA/cm2
(P < 0.01). The subsequent addition
of amiloride and bumetanide further reduced
Isc to 14.0 ± 1.9 and 7.3 ± 0.9 µA/cm2,
respectively. The initial increase in
Isc is likely due
to the stimulation of Na+
absorption, since Cl
is at
or below electrochemical equilibrium across the apical membrane in
human airway (41). Boucher and colleagues (5-7) have demonstrated
the existence of similar driving forces for Cl
across the apical
membrane of human airway in primary culture. Also, Clarke et al. (7)
previously demonstrated, using microelectrode techniques, that the
Ca2+-mediated agonist bradykinin
induces an initial transient increase in
Na+ absorption across human
airway, consistent with our results. However, as we have not directly
measured the electrochemical driving force for
Cl
across the apical
membrane of our cultures, we cannot rule out the possibility that a
portion of this initial increase is due to
Cl
secretion. Our results
suggest that, following the transient activation of
Na+ absorption, the effect of UTP
is inhibitory in nature. This was confirmed by comparing the
amiloride-sensitive Na+ current
(INa-amil) in
the absence or presence of mucosal UTP (Fig. 1,
right) on monolayers cultured from a
single patient and run on the same day. In 19 filters, amiloride
reduced Isc by
12.5 ± 1.4 µA/cm2.
In contrast, following mucosal UTP, amiloride reduced
Isc by only 6.2 ± 0.5 µA/cm2
(n = 6;
P < 0.01). This result is
consistent with an inhibition of active
Na+ absorption by mucosal UTP.

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Fig. 1.
Effect of mucosal UTP (100 µM) on amiloride-blockable
Na+ current
(INa-amil) in
human bronchial epithelia (HBE) expressing wild-type (wt) cystic
fibrosis transmembrane conductance regulator (CFTR).
Left: UTP induced a transient increase
in short-circuit current
(Isc) followed
by a sustained decrease. Subsequent addition of amiloride (10 µM) and
bumetanide (20 µM; serosal addition) further reduced
Isc. Dashed line,
zero-current level. Right: average
INa-amil in absence [control
(Cont)] or presence of UTP.
INa-amil was
determined in separate experiments before addition of UTP (control) or
after stimulation by mucosal UTP. No. of experiments is indicated in
parentheses. * P < 0.01. All
experiments were carried out in presence of mucosal and serosal NaCl
(see METHODS).
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Although the above results suggest that UTP inhibits
Na+ transport across airway
epithelia, the interpretation of
Isc changes in
symmetrical NaCl is not straightforward. Any inhibition of apical
Na+ channels would be expected to
hyperpolarize the apical membrane, resulting in the establishment of a
favorable electrochemical gradient for
Cl
secretion. Because UTP
is known to activate a
Ca2+-dependent
Cl
channel, resulting in a
stimulation of Cl
secretion
(26), this will confound attempts to isolate the effects of UTP on
Na+ absorption. To overcome this,
we performed similar experiments in the absence of apical and
basolateral Cl
to study the
Na+ absorptive process in
isolation. As shown in Fig. 2,
left, addition of mucosal UTP (100 µM) produced a response similar to that seen in the presence of
Cl
, i.e., a transient
increase in Na+ absorption and
then a dramatic inhibition. Amiloride caused a further reduction in the
Isc measured in
the absence of mucosal and serosal
Cl
(INa). To
confirm that the reduction in
Isc was truly a
reduction in
INa-amil, we
inhibited the basal
Isc with
amiloride in the absence of UTP (Fig. 2,
right). As is apparent, the
postamiloride current level is similar in the two monolayers,
indicating that changes in
Isc reflect
changes in
INa-amil. In 15 HBE monolayers, the basal
INa averaged 13.9 ± 1.2 µA/cm2. Mucosal UTP
(100 µM) induced a transient increase in
INa to 16.9 ± 1.1 µA/cm2
(P < 0.001) that was followed by a
decline to 5.0 ± 0.5 µA/cm2.
The subsequent addition of amiloride (10 µM) further reduced INa to 2.7 ± 0.3 µA/cm2. In seven monolayers
from the same HBE culture, the baseline INa averaged 11.8 ± 1.0 µA/cm2, and this was
reduced to 3.0 ± 0.5 µA/cm2
by amiloride (10 µM). These results demonstrate that mucosal UTP
inhibits 76 ± 4% of the
INa-amil. Also,
these results confirm that UTP initially increases electrogenic
Na+ transport.

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Fig. 2.
Effect of mucosal UTP (100 µM) on
Isc in absence of
mucosal and serosal Cl (see
METHODS) in HBE expressing wt CFTR.
This current is referred to as
INa.
Left: mucosal UTP induced a transient
increase in INa
followed by a sustained inhibition. Subsequent addition of amiloride
(10 µM) further reduced
INa.
Right: effect of amiloride (10 µM)
on INa. Dashed
line, zero-current level.
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The above results demonstrate that mucosal UTP inhibits transepithelial
Na+ transport in human airway. We
next determined whether this effect was unique to mucosal UTP or
whether UTP added to the serosal membrane would have a similar effect.
The results of one representative experiment are shown in Fig.
3, top
left. In the absence of mucosal and
serosal Cl
, addition of UTP
to the serosal membrane induced a response that was qualitatively
similar to that seen with mucosal UTP, i.e., a transient increase in
Na+ absorption followed by a
dramatic inhibition. In six experiments, the baseline
INa averaged 23.2 ± 3.7 µA/cm2, and this
increased to a peak of 31.8 ± 3.9 µA/cm2
(P < 0.001) following stimulation
with serosal UTP. This increase was followed by a decrease in
INa to 6.6 ± 0.7 µA/cm2. After administration
of serosal UTP, amiloride further reduced INa to 1.5 ± 0.2 µA/cm2. Thus serosal UTP
inhibited INa by
an average of 74 ± 5%. These results indicate that the inhibition
of Na+ transport observed is not
restricted to mucosal UTP.

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Fig. 3.
Effect of Ca2+-mediated agonists
on INa in HBE
expressing wt CFTR. Addition of UTP (100 µM; top
left), histamine (30 µM; top
right), or bradykinin (100 nM;
bottom left) to serosal membrane of
HBE expressing wt CFTR resulted in a transient stimulation of
Na+ transport followed by a
sustained inhibition. Ca2+-ATPase
inhibitor thapsigargin similarly transiently increased
INa, followed by
a sustained inhibition (bottom
right). In each case, subsequent addition of
amiloride (10 µM) further reduced
INa. Dashed line,
zero-current level. All experiments were carried out in absence of
mucosal and serosal Cl .
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We next determined whether other
Ca2+-dependent agonists would
induce a similar inhibition of Na+
transport. The effects of two inflammatory mediators, histamine and
bradykinin, were evaluated. Both of these agonists have previously been
shown to induce a Ca2+-dependent
Cl
secretory response in
human airway following inhibition of
Na+ transport with amiloride (6,
7, 43-45). Both histamine (30 µM; Fig. 3,
top
right) and bradykinin (100 nM; Fig.
3, bottom left) induced an increase in
INa followed by
an inhibition, similar to what is observed following stimulation with
UTP. In three filters, histamine increased
INa from
19.1 ± 1.5 to 29.7 ± 1.6 µA/cm2
(P < 0.01) before a sustained
inhibition to 8.2 ± 1.1 µA/cm2. The subsequent addition
of amiloride further reduced
INa to 1.5 ± 0.4 µA/cm2, demonstrating that
histamine inhibited 62 ± 4% of the
INa-amil. Similarly, bradykinin increased
INa from 30.9 ± 1.5 to 43.2 ± 1.8 µA/cm2
(P < 0.01) before a sustained
inhibition to 12.0 ± 1.5 µA/cm2
(n = 3). Amiloride subsequently
reduced INa to
2.2 ± 0.5 µA/cm2,
demonstrating that bradykinin inhibits the
INa-amil by 65 ± 3%. These results indicate that the inhibition of
Na+ transport is not unique to
purinergic agonists. Rather, they suggest that any increase in
intracellular Ca2+ will inhibit
Na+ transport. This was confirmed
by utilizing the Ca2+-ATPase
inhibitor thapsigargin (1 µM) to elevate intracellular Ca2+. Thapsigargin initially
increased INa and
then caused a marked inhibition (Fig. 3,
bottom
right). In six experiments,
thapsigargin increased
INa from 25.2 ± 5.1 to 26.0 ± 5.1 µA/cm2
(P < 0.05) followed by a sustained
inhibition to 3.1 ± 1.3 µA/cm2. Amiloride further
reduced INa to
0.0 ± 1.3 µA/cm2, indicating
an 84 ± 3% inhibition of the
INa-amil.
Effect of UTP on HBE homozygous for the
F508 CFTR
mutation.
The above results demonstrate that UTP inhibits
Na+ transport in human airway
expressing wt CFTR. We next determined whether UTP would similarly
inhibit Na+ transport in HBE
expressing the
F508 CFTR mutation. We evaluated a total of 60 filters using symmetrical NaCl bath solutions. The basal
Isc,
transepithelial potential difference, and transepithelial resistance
averaged 30.1 ± 1.4 µA/cm2,
15.2 ± 0.9 mV, and 539 ± 24
· cm2,
respectively. In contrast to wt CFTR-expressing HBE, for which ~50%
of this basal Isc
is sensitive to block by amiloride, nearly 90% of the basal current of
F508 CFTR-expressing HBE is amiloride sensitive (unpublished
observations). Thus our primary HBE cultures exhibit the characteristic
hyperabsorption of Na+ associated
with CF. The effect of mucosal UTP (100 µM) on
Isc in one
representative monolayer expressing
F508 CFTR is shown in Fig.
4, left. UTP induced a response
qualitatively identical to that observed in wt CFTR-expressing HBE.
That is, UTP induced an initial increase in
Isc followed by a
sustained inhibition. Addition of amiloride (10 µM) further reduced
Isc. In eight
experiments, UTP increased
Isc from 38.2 ± 5.5 to 46.4 ± 3.7 µA/cm2
(P < 0.01) followed by a decline to
14.0 ± 3.0 µA/cm2. The
subsequent addition of amiloride further reduced
Isc to 5.2 ± 1.3 µA/cm2. Addition of
bumetanide had no effect on the small remaining Isc, suggesting
that the inhibition of
Isc is unrelated
to any Cl
secretory current
evoked by UTP. If it is assumed that
Isc is a purely
Na+ absorptive current, mucosal
UTP inhibits 75 ± 2% of the amiloride-blockable current. This is
similar to the results obtained in wt CFTR-expressing HBE. These
results suggest that mucosal UTP inhibits transepithelial Na+ absorption in human CF airway.
To confirm this observation, we evaluated the effect of mucosal
UTP (100 µM) on Na+ transport
across CF airway in the absence of mucosal and serosal Cl
. The results of one
representative experiment are shown in Fig. 4, right. Similar
to our previous results, UTP induced an initial increase in
INa that was
followed by a sustained inhibition. This initial transient response to
UTP appears to have a shorter duration in CF HBE than in wt
CFTR-expressing HBE (compare Figs. 1 and 2 with Figs. 4, 7, and 8),
although this was not further analyzed. The subsequent addition of
amiloride further reduced
INa. In 12 experiments, UTP increased
INa from 12.0 ± 0.9 to 15.2 ± 1.2 µA/cm2
(P < 0.001) followed by a sustained
decrease to 3.4 ± 0.8 µA/cm2. Amiloride further
reduced INa to
1.1 ± 0.3 µA/cm2, indicating
that UTP inhibited 80 ± 4% of the amiloride-blockable current.

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Fig. 4.
Effect of mucosal UTP (100 µM) on
Na+ transport in HBE homozygous
for F508 mutation either in presence
(left) or absence
(right) of mucosal and serosal NaCl.
Under both conditions, mucosal UTP induced a transient increase in
Na+ transport followed by a
sustained inhibition. Subsequent addition of amiloride (10 µM)
further reduced Na+ current.
Dashed line, zero-current level.
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The demonstration of similar changes in
INa in the
absence of mucosal and serosal
Cl
indicates that our
primary CF airway cultures can be studied using a standard NaCl bath
solution on both the serosal and mucosal membranes. Because this
provides more stable baseline currents, the remainder of our studies on
CF epithelia were carried out using standard bath solutions (120 NaCl). To determine the half-maximal inhibitory concentration
(Ki) for UTP
on Na+ transport in CF airway,
each filter was challenged with one concentration of UTP and then with
amiloride, to avoid difficulties associated with downregulation of the
purinergic receptor. The data were fitted to a Michaelis-Menten
function with an apparent
Ki of 296 nM
(Fig. 5).

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Fig. 5.
Concentration-response curve for UTP-dependent inhibition of
Na+ transport in F508/ F508
HBE. Na+ current after UTP
stimulation
(INa-UTP) minus
Na+ current before UTP stimulation
(INa-Cont) was
normalized to
INa-Cont minus
INa-amil (10 µM
amiloride). Data (means ± SE of 4 or 5 individual experiments) were
fitted to a Michaelis-Menten function with an apparent half-maximal
inhibitory concentration of 296 nM. Experiments were carried out in
presence of mucosal and serosal NaCl. C, control (0 UTP).
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We also determined whether serosal UTP and other
Ca2+-mediated agonists would
inhibit Na+ absorption across CF
airway in a manner similar to that of mucosal UTP. In four experiments,
serosal UTP (100 µM) increased
INa from 33.4 ± 2.1 to 38.6 ± 2.2 µA/cm2
(P < 0.001) before a sustained
inhibition to 11.3 ± 0.9 µA/cm2. The subsequent addition
of amiloride further reduced
INa to 2.5 ± 0.2 µA/cm2, indicating that
serosal UTP inhibits 72 ± 1% of the
Na+ current across human CF
airway. We next determined whether an additional
Ca2+-mediated agonist, bradykinin
(100 nM), would similarly inhibit Na+ transport in
F508 CFTR HBE.
In three filters, bradykinin increased INa from 66.3 ± 14.4 to 72.1 ± 13.9 µA/cm2
(P < 0.01) before a sustained
inhibition to 25.6 ± 8.1 µA/cm2. Amiloride further
reduced INa to
5.5 ± 1.0 µA/cm2, indicating
that bradykinin inhibits 69 ± 4% of the
Na+ current across
F508
CFTR-expressing HBE. These results indicate that, similar to the
situation in wt CFTR-expressing airway, the inhibition of
Na+ transport is not restricted to
mucosal UTP. They suggest instead that
Ca2+-mediated agonists in general
are capable of inhibiting Na+
transport across human CF airway.
In summary, our results indicate that an increase in intracellular
Ca2+ underlies the inhibition of
Na+ current observed. To evaluate
this possibility further, we incubated the HBE monolayers in the
cell-permeant Ca2+ chelator
BAPTA-AM (50 µM for 1 h) before determining the effect of mucosal UTP
on Na+ transport. In 11 monolayers, the baseline
Isc averaged 35.2 ± 1.6 µA/cm2. Mucosal UTP
(100 µM) induced only a small increase in
Isc to 36.5 ± 1.8 µA/cm2
(P < 0.02) followed by a sustained
inhibition to 24.8 ± 1.4 µA/cm2. The subsequent addition
of amiloride further reduced
Isc to 2.4 ± 0.3 µA/cm2. Thus mucosal UTP
inhibited only 31 ± 3% of the amiloride-blockable current in the
presence of BAPTA-AM. Because the total amiloride-sensitive Isc was not
different in the absence (33.0 ± 4.3 µA/cm2,
n = 8) or presence (32.8 ± 1.5 µA/cm2,
n = 11) of BAPTA-AM, we directly
compared the UTP-dependent portion of these currents (control, 24.3 ± 2.6 µA/cm2; BAPTA-AM, 10.3 ± 1.3 µA/cm2). These
results demonstrate that incubating the HBE monolayers in BAPTA-AM
significantly attenuated the UTP-dependent inhibition of
Na+ absorption
(P < 0.0001). It should be noted
that BAPTA-AM also significantly attenuated the initial increase in
Isc induced by mucosal UTP (P < 0.01), consistent
with this being due to stimulation of
Na+ transport via a
Ca2+-mediated process.
Effects of second messenger modulators on the UTP-dependent
inhibition of
Na+ transport.
Our results with thapsigargin suggest that an increase in
Ca2+ alone is sufficient to
inhibit Na+ transport. However, a
second possibility is that the increase in intracellular
Ca2+ by UTP increases additional
second messengers that may be important in the observed inhibition of
Na+ transport. Boucher and
colleagues previously demonstrated that UTP induces an increase in both
protein kinase C (PKC) (3) and arachidonic acid (23) in human airway
epithelia, and these second messengers are known to modulate
Na+ transport in kidney epithelia
(12, 14). Therefore, we evaluated the effects of inhibitors of PKC and
phospholipase A2
(PLA2) on the UTP-dependent
inhibition of Na+ transport in wt
CFTR-expressing HBE in the absence of
Cl
and
F508
CFTR-expressing HBE in standard NaCl bath solutions. In the presence of
the cytosolic PLA2 inhibitor
AACOCF3 (100 µM; 15-min
pretreatment), mucosal UTP inhibited 76 ± 2%
(n = 4) of the
INa-amil in
F508 CFTR-expressing HBE. Similarly, in the presence of a different
cytosolic PLA2 inhibitor,
PACOCF3 (100 µM; 15-min pretreatment), and a secreted PLA2
inhibitor, HELSS (2 µM; 15-min pretreatment), UTP inhibited 77 (n = 2) and 90 ± 1%
(n = 3) of INa,
respectively. An alternate means of generating arachidonic acid is via
the conversion of diacylglycerol (DAG) via DAG lipase. Mucosal UTP
inhibited 74% of the
INa-amil in the
presence of the DAG lipase inhibitor RG-80267 (50 µM; 15-min
pretreatment; n = 2). These results
suggest that arachidonic acid does not mediate the UTP-dependent
inhibition of Na+ transport observed.
The effects of the PKC inhibitors staurosporine (100 nM),
bisindolylmaleimide I (200 nM), and myristolated PKC (amino acids 19-27 fragment; 10 µM) were evaluated in wt
CFTR-expressing HBE. After incubation of the monolayers in
staurosporine (15 min), bisindolylmaleimide I (15 min), or myristolated
PKC (25 min), mucosal UTP inhibited 84 ± 3 (n = 3), 79 ± 4 (n = 3), and 71 ± 6% (n = 4) of
INa,
respectively. The effects of the PKC inhibitors bisindolylmaleimide I
(1 µM) and calphostin C (2 µM) were also evaluated in
F508
CFTR-expressing HBE. After incubation of the monolayers in
bisindolylmaleimide I (15 min) or calphostin C (15 min), mucosal UTP
inhibited 72 ± 1 (n = 3) and
77 ± 1% (n = 3) of the
INa-amil,
respectively. These results suggest that PKC is not involved in the
UTP-dependent inhibition of Na+
transport across HBE.
Our results with the PKC inhibitors were somewhat surprising, since
Eaton and colleagues (21, 25) previously demonstrated that the
Ca2+-dependent inhibition of
Na+ transport in kidney epithelia
is dependent on PKC. Therefore, we directly evaluated the effects of
the PKC agonist PMA on Na+
transport across HBE expressing
F508 CFTR. The results of one experiment are shown in Fig. 6,
left. PMA (100 nM) had no effect on
Isc, whereas the
subsequent addition of amiloride inhibited Isc as expected.
In six monolayers, the baseline
Isc averaged 29.0 ± 2.6 µA/cm2; this was
unaffected by PMA, whereas amiloride reduced
Isc to 7.8 ± 1.6 µA/cm2. As a positive
control, we evaluated the effect of PMA on
Cl
secretion after
amiloride in wt CFTR-expressing HBE (Fig. 6, right). Hanrahan and colleagues (36)
previously demonstrated that PKC activates CFTR in excised patches, and
we have demonstrated that PMA stimulates
Cl
secretion across T84
cells (9). After inhibition of Na+
absorption by amiloride, PMA (100 nM) stimulated a bumetanide-sensitive Cl
secretory response, as
expected. In six experiments, amiloride reduced
Isc from 38.4 ± 3.0 to 11.9 ± 2.1 µA/cm2. Addition of PMA
increased Isc an
average of 3.8 ± 0.7 µA/cm2
(P < 0.01). These results
demonstrate that direct activation of PKC has no effect on
Na+ transport across HBE and
confirm our PKC inhibitor studies described above.

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Fig. 6.
Effect of protein kinase C activator phorbol 12-myristate 13-acetate
(PMA; 100 nM) on Na+ absorption
across F508 CFTR-expressing HBE
(left) and
Cl secretion across wt
CFTR-expressing HBE (right).
Addition of PMA had no effect on
Na+ absorption across F508 CFTR
HBE, whereas subsequent addition of amiloride (10 µM) inhibited
Isc as expected
(left). After inhibition of
Na+ absorption with amiloride (10 µM), PMA stimulated a bumetanide (Bumet)-sensitive increase in
Cl secretion across HBE
expressing wt CFTR (right).
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Because Ca2+-dependent agonists
often modulate tyrosine kinase activity, we determined whether the
protein tyrosine kinase inhibitors genistein (50 µM) and lavendustin
A (5 µM) would modulate the effect of mucosal UTP on
Na+ transport in HBE expressing
F508 CFTR. Genistein induced a sustained increase in
Na+ transport, as shown in Fig.
7. After genistein, mucosal UTP increased Na+ transport before a sustained
inhibition. Addition of amiloride further reduced
Na+ absorption. In seven filters,
genistein increased
Isc from 32.9 ± 4.6 to 43.0 ± 5.8 µA/cm2
(P < 0.001). The subsequent addition
of mucosal UTP decreased Isc to 16.4 ± 1.7 µA/cm2. Amiloride further
reduced Isc to
3.4 ± 1.0 µA/cm2,
demonstrating that UTP inhibited 56 ± 5% of the
amiloride-dependent Na+ transport.
This large increase in
Isc is unlikely
to be due to a genistein-induced
Cl
secretory response,
since we have demonstrated that genistein increases
Cl
current by only ~2
µA/cm2 in
F508
CFTR-expressing HBE after amiloride (10). The presence of amiloride
would enhance the electrochemical driving force for Cl
secretion in the
experiments of Ref. 10 relative to those reported here. In four
additional experiments, the effect of lavendustin A on
Na+ transport was evaluated.
Lavendustin A had no effect on
Isc, although the
subsequent addition of genistein further increased Isc from 26.6 ± 2.9 to 37.0 ± 4.8 µA/cm2
(P < 0.01). These results suggest
that the effect of genistein on
Na+ absorption is independent of
tyrosine kinase inhibition and further indicate that the UTP-dependent
inhibition of Na+ transport is not
due to the activation of a tyrosine kinase.

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Fig. 7.
Effect of genistein on Na+ current
in F508/ F508 HBE. Addition of genistein (50 µM; mucosal and
serosal addition) induced a sustained increase in
Na+ current. Subsequent addition
of mucosal UTP (100 µM) induced a transient increase in
Na+ current followed by a
sustained inhibition. Amiloride (10 µM) further reduced
Na+ current. Dashed line,
zero-current level.
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An additional possibility is that the UTP-dependent increase in
intracellular Ca2+ inhibits
adenylyl cyclase in the cell. Increased cAMP is well known to activate
Na+ transport, and increased
Ca2+ potently downregulates
adenylyl cyclase (8). Therefore, we determined whether exogenous cAMP
could either prevent or reverse the effect of mucosal UTP on
Na+ transport in
F508
CFTR-expressing HBE. We utilized the cell-permeant cAMP analog
8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) to bypass the adenylyl cyclase. The results of one representative experiment are shown in Fig.
8. Addition of CPT-cAMP (500 µM) stimulated a small increase in
Na+ transport. The subsequent
addition of mucosal UTP (100 µM) resulted in an increase in
Na+ transport followed by a
sustained inhibition. Addition of amiloride further reduced
Isc. In six
filters, CPT-cAMP increased
Isc from 32.5 ± 6.7 to 38.8 ± 6.8 µA/cm2
(P < 0.001). Mucosal UTP increased
Isc to 44.6 ± 8.2 µA/cm2
(P < 0.05) before a sustained
inhibition to 19.5 ± 3.6 µA/cm2. The addition of
amiloride further reduced
Isc to 4.4 ± 0.4 µA/cm2; thus UTP inhibited
48 ± 6% of the amiloride-sensitive current. This demonstrates that
cAMP stimulates Na+ transport in
CF airway and suggests that exogenous cAMP partially prevents the
effects of UTP on Na+ transport.

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Fig. 8.
Effect of CPT-cAMP and apyrase on
Na+ current in F508/ F508
HBE. CPT-cAMP (500 µM) induced a sustained increase in
Na+ current. Addition of mucosal
UTP (100 µM) induced a further transient increase followed by a
sustained decrease in Na+ current.
Addition of tri- and diphosphatase (apyrase; 5 U/ml; mucosal addition)
failed to induce a recovery of Na+
current. Amiloride (10 µM) further reduced
Na+ current. Dashed line,
zero-current level.
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To determine whether cAMP could reverse the effect of UTP on
Na+ transport, the monolayer was
first challenged with mucosal UTP, and then CPT-cAMP was added during
the sustained inhibitory phase. UTP increased
Isc from 20.7 ± 0.8 to 30.4 ± 1.2 µA/cm2
(P < 0.001) before a sustained
inhibition to 6.7 ± 0.4 µA/cm2
(n = 4). Addition of CPT-cAMP
increased Isc to
11.0 ± 1.1 µA/cm2
(P < 0.05), and this was inhibited
by amiloride (4.1 ± 0.9 µA/cm2). Bumetanide reduced
Isc to only 3.2 ± 1.0 µA/cm2, confirming
that CPT-cAMP was not increasing
Cl
secretion across the CF
airway. These results demonstrate that cAMP partially reverses the
inhibitory effect of mucosal UTP.
Effect of apyrase on the UTP-dependent inhibition of
Na+ transport.
We next determined whether the inhibitory effect of UTP was dependent
on the continued presence of the agonist. To accomplish this, apyrase,
which cleaves both tri- and diphosphate nucleotides, thus removing UTP
from the mucosal solution, was added during the sustained inhibitory
phase of the UTP response. As shown in Fig. 8, apyrase failed to induce
a recovery of current; it increased Isc by only
1.1 ± 0.5 µA/cm2
(n = 4). In the absence of CPT-cAMP,
mucosal UTP induced a sustained inhibition of
Na+ transport from 31.8 ± 6.8 to 9.2 ± 2.6 µA/cm2, and
subsequent addition of apyrase increased
Isc slightly to 13.0 ± 3.3 µA/cm2
(n = 7;
P < 0.05). Amiloride reduced
Isc to 2.5 ± 0.9 µA/cm2. To ensure that
apyrase was removing UTP from the mucosal solution, apyrase was added
before UTP in parallel filters. In the presence of apyrase, mucosal UTP
had no effect on Na+ transport,
but the subsequent addition of serosal UTP inhibited Na+ transport as described above
(data not shown). Collectively, these results indicate either that
mucosal UTP generates an inhibitory second messenger that remains
active despite the removal of the agonist or that
Na+ channels have been removed
from the membrane and are no longer available for activation despite
the removal of UTP.
Effect of mucosal UTP on
IK.
Modulation of Na+ transport across
epithelia is typically associated with parallel changes in apical and
basolateral membrane conductances. Therefore, we determined the effect
of mucosal UTP on the
IK after
establishment of a mucosa-to-serosa
K+ concentration gradient and
permeabilization of the mucosal membrane with nystatin (see
Isc
measurements). As shown in Fig.
9, after inhibition of apical
Na+ channels with amiloride,
permeabilization of the mucosal membrane with nystatin revealed a
K+ current
(IK) across the
basolateral membrane. The subsequent addition of mucosal UTP induced an
increase in IK
followed by a sustained inhibition. Addition of
Ba2+ (10 mM) further inhibited
IK. In a total of
17 filters, nystatin increased
IK from 2.8 ± 0.9 to 58.6 ± 5.8 µA/cm2. In
13 of these filters, the subsequent addition of mucosal UTP increased
IK to 79.8 ± 8.9 µA/cm2
(P < 0.001) before a sustained
decrease to 40.0 ± 3.9 µA/cm2
(P < 0.001). As expected for a
K+ conductance, in nine of these
filters, addition of Ba2+ further
reduced IK to
18.6 ± 3.4 µA/cm2
(P < 0.001); quinine similarly
inhibited IK
(data not shown). These results suggest that the basal
IK represents the
K+ conductance associated with
Na+ absorption across human airway
epithelia. In support of this, both
Ba2+ and quinine inhibit
Na+ absorption across CF HBE
(unpublished observations). To determine whether the increase in
IK is due to
activation of a Ca2+-dependent
K+ channel, we evaluated the
effect of CTX (50 nM) on the initial increase in
IK induced by
mucosal UTP in the additional four filters. CTX completely inhibited
the transient increase in
IK
(n = 4), indicating that this increase
is due to the activation of a
Ca2+-dependent
K+ channel.

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Fig. 9.
Effect of mucosal UTP on basolateral membrane
K+ current
(IK) in
F508/ F508 HBE. After establishment of a mucosa-to-serosa
K+ gradient, inhibition of
Na+ transport with amiloride (10 µM), and permeabilization of mucosal membrane with nystatin (Nyst;
see METHODS), mucosal UTP (100 µM)
induced a transient increase in
IK followed by an
inhibition. Subsequent addition of
Ba2+ (10 mM; serosal addition)
further inhibited
IK. Dashed line,
zero-current level.
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 |
DISCUSSION |
Both a defective cAMP-dependent
Cl
secretion and a
hyperabsorption of Na+ across
airway epithelia characterize CF. However,
Ca2+-dependent
Cl
secretion remains intact
across CF airway epithelia (40); this has led to the evaluation of
Ca2+-dependent agonists as
potential therapeutics in CF (19, 20). Because
Cl
has been shown to be at
electrochemical equilibrium across the apical membrane of airway
epithelia (41), clinical trials have been conducted in the presence of
the Na+ channel blocker amiloride
to hyperpolarize the apical membrane and thereby increase the driving
force for Cl
secretion.
However, Ca2+-dependent agonists
have previously been shown to inhibit
Na+ absorption in other epithelia,
including kidney (4, 21, 22, 24, 25, 31) and colon (39). We therefore
speculated that Ca2+-mediated
agonists would similarly inhibit transepithelial
Na+ absorption across human airway
epithelia, obviating the requirement for exogenous
Na+ channel inhibition. Indeed, if
Ca2+-mediated agonists are in fact
Cl
secretagogues, then
inhibition of Na+ absorption, and
the resultant hyperpolarization of the apical membrane, would be a
prerequisite to inducing Cl
secretion by these agonists. We demonstrate that
Ca2+-dependent agonists are
capable of dramatically inhibiting
Na+ transport across HBE and that
this occurs in both wt and
F508 CFTR-expressing monolayers.
The role of basolateral membrane
K+ channels in
Na+ transport.
We demonstrate that Ca2+-mediated
agonists initially stimulate Na+
absorption across human airway before a sustained inhibition. Although
we have been unable to electrically isolate the apical membrane
Na+ conductance, we have succeeded
in obtaining information with regard to the regulation of the
basolateral membrane K+
conductances involved in modulating
Na+ absorption. We hypothesize
that the initial increase in Na+
absorption is due to the activation of a
Ca2+-dependent basolateral
membrane K+ channel that results
in a hyperpolarization and increased driving force for
Na+ entry across the apical
membrane. Incubation of our cultures in the cell-permeant
Ca2+ chelator BAPTA-AM resulted in
an attenuation of this initial increase in
Na+ absorption, consistent with
our hypothesis. Also, addition of agonists (UTP, histamine, or
bradykinin) to the serosal membrane resulted in a greater increase in
Isc than did
mucosal UTP. Clarke et al. (5-7) demonstrated, using
microelectrode techniques, that Ca2+-dependent agonists activated
a basolateral K+ conductance that
results in a hyperpolarization of human nasal epithelium. Indeed,
activation of this basolateral K+
conductance was greater when agonist was added to the serosal membrane
than when it was added to the mucosal side (5). Finally, in
permeabilized monolayers, we demonstrate that mucosal UTP activates a
basolateral membrane, CTX-sensitive
K+ conductance (Fig. 9). We (10)
and others (28) previously characterized this CTX-sensitive
K+ channel in human airway
epithelia. We conclude that activation of this basolateral membrane
K+ conductance results in the
initial stimulation of Na+
absorption across human airway.
Apical Na+ entry is considered the
rate-limiting step in Na+
absorption. Abundant experimental data employing kidney and colonic epithelia demonstrate that increasing cellular
Ca2+ results in an inhibition of
Na+ conductance (4, 14, 21, 22,
24, 25, 31, 39). Thus, although there is little doubt that inhibition
of Na+ conductance is important in
the observed UTP-dependent inhibition of
Isc, we
demonstrate that mucosal UTP also inhibits a basolateral IK (Fig. 9).
Indeed, inhibition of this K+
conductance is sufficient to account for a large portion of the observed inhibition of Na+
absorption. Unfortunately, the nature of this inhibited basolateral K+ channel is not known at the
single-channel level. Dawson and colleagues (38, 39) demonstrated
previously that the Ca2+-dependent
agonist carbachol inhibited Na+
absorption across the turtle colon. Similar to our results, these authors (38) demonstrated that carbachol induced a profound inhibition
of the basolateral
IK in turtle
colon. In addition to this effect on the
K+ conductance, it was also
demonstrated that carbachol inhibited the apical membrane
Na+ conductance in these cells
(39), consistent with the concept of cross talk between apical and
basolateral membranes. Also, Harvey (14) demonstrated that the
basolateral ATP-sensitive K+
channel of frog principal cells is inhibited by
Ca2+ with an affinity similar to
inhibition of the Na+ channel,
suggesting a parallel downregulation of both membranes. Thus it is
impossible to separate the relative contributions of these two
membranes to the overall inhibitory response observed because both are
regulated in parallel to maintain cellular homeostasis. A complete
understanding of the
Ca2+-dependent inhibition of
Na+ transport across human airway
will require an analysis of this basolateral membrane
K+ conductance at the
single-channel level, including its regulation by
Ca2+.
Second messenger regulation of
Na+ transport
across human airway.
Although Ca2+-dependent agonists
are well-known negative modulators of
Na+ absorption in the kidney, this
inhibition appears to be indirect, involving an activation of PKC (21,
25). In addition, other second messengers associated with
Ca2+-dependent agonists (e.g.,
oxidative metabolites of arachidonic acid) have been shown to modulate
ENaC activity during patch-clamp recordings (12). Boucher and
colleagues (3, 23) previously demonstrated that luminal purinergic
agonists increase intracellular Ca2+, PKC, and arachidonic acid in
human airway epithelia. In contrast to previous reports from kidney
epithelia (12, 21, 25), we demonstrate that inhibitors of PKC and
PLA2 have no effect on the
UTP-dependent inhibition of Na+
transport across HBE. Indeed, we demonstrate that the direct activation
of PKC by PMA has no effect on Na+
transport (Fig. 6). Together, these results argue against a role for
PKC in the observed inhibition of
Na+ transport by UTP. We also
demonstrate that the Ca2+-ATPase
inhibitor thapsigargin similarly inhibits
Na+ transport across HBE.
Thapsigargin fails to induce the accumulation of inositol phosphates in
both human airway (23) and colonic (18) epithelia, further suggesting
that PKC accumulation is not involved in the observed inhibitory
response. Finally, we demonstrate that incubation of HBE in the
cell-permeant Ca2+ chelator
BAPTA-AM results in a significant attenuation of the UTP-dependent
inhibition of Na+ transport.
Similar to our results, Yamaya et al. (45, 46) were unable to
completely suppress the response in HBE to
Ca2+-mediated agonists in the
presence of BAPTA-AM. On the basis of our results with both
thapsigargin and BAPTA-AM, we speculate that
Ca2+ itself may act as a negative
modulator of Na+ transport across
human airway. In this regard, Rotin and colleagues (17) recently
demonstrated that ENaC, heterologously expressed in MDCK cells, could
be directly inhibited by Ca2+ in
excised, inside-out patches. However, previous reports demonstrated that increasing Ca2+ at the
cytoplasmic face of ENaC excised from rat and rabbit cortical collecting tubule cells does not affect the channel open probability (14, 25). Thus the mechanism for
Ca2+ regulation of ENaC remains to
be determined.
Our results demonstrate that UTP induces a long-term inhibition of
Na+ transport that is independent
of the continued presence of agonist (Fig. 8). One explanation for
these results is that UTP generates a second messenger that has
long-term inhibitory effects on
Na+ transport. Barrett and
colleagues (37) recently demonstrated, in the T84 cell line, that the
generation of inositol 3,4,5,6-tetrakisphosphate by muscarinic agonists
produces a long-term inhibitory effect on
Cl
secretion. An additional
possibility is that increased cellular Ca2+ causes the retrieval of
Na+ channels from the apical
membrane and thus long-term inhibition. Wilkinson and Dawson (39)
concluded, employing fluctuation analysis, that increased
Ca2+ resulted in a decrease in
apical Na+ channel density in
turtle colon. Recently, Rotin and colleagues (32-34) characterized
the ubiquitin-ligase Nedd4 as an important negative modulator of ENaC.
Nedd4 was shown to translocate to the apical membrane of MDCK cells
following an increase in cellular Ca2+. These authors speculated
that Nedd4 may be important in the endocytosis of ENaC from the apical
membrane of epithelia. Interestingly, an increase in cAMP was able to
partially reverse the effect of UTP on
Na+ absorption (Fig. 8). cAMP is
known to increase apical membrane Na+ channel density (12). Further
studies are required to directly determine whether UTP modulates
Na+ channel density, open probability, or both in HBE and
the potential role of Nedd4 in this process.
We also evaluated the effect of the tyrosine kinase inhibitor genistein
on the UTP-dependent inhibition of
Na+ transport. Surprisingly,
genistein alone stimulated Na+
transport across human CF airway (Fig. 7). In contrast, the
structurally unrelated tyrosine kinase inhibitor lavendustin A failed
to modulate Na+ transport,
suggesting that the effect of genistein is unrelated to its known
tyrosine kinase inhibitory activity. Despite this stimulation of
Na+ absorption, genistein failed
to modulate the inhibitory effect of UTP. Our results with genistein
are opposite to what has been reported in A6 kidney epithelia.
Matsumoto et al. (27) demonstrated that genistein inhibited
Na+ transport across A6 cells,
suggesting that a tonic tyrosine kinase activity modulated
Na+ transport.
In conclusion, we demonstrate that
Ca2+-mediated agonists
dramatically inhibit Na+ transport
across human airway epithelia expressing both wt and
F508 CFTR.
Indeed, the Ki
for UTP-dependent inhibition of
Na+ absorption is similar to the
half-maximal stimulatory concentration previously reported for the
stimulation of Cl
secretion
in primary HBE cultures (46). This inhibition of Na+ transport is directly
dependent on a rise in intracellular
Ca2+, but it is independent of
activation of either the PKC or
PLA2 second messenger cascades.
These results may help explain the observation that UTP increased
mucociliary clearance in normal volunteers and that this effect was not
potentiated by amiloride (30). However, it should be noted that in CF
patients a combination of amiloride plus UTP was required to increase
mucociliary clearance (1), although, as indicated by the authors of
that study, a greater number of subjects need to be
studied to rule out effects of either agent alone. We also demonstrate
that genistein stimulates Na+
absorption across CF epithelia. Because genistein and its analogs have
been shown to stimulate Cl
secretion across epithelia (15, 29) and are being evaluated as
Cl
secretory agonists in
human nasal epithelia (13, 16), this potentially confounding
stimulation of Na+ absorption
warrants further investigation. On the basis of these results, we
predict that amiloride may not be required in combination with
Ca2+-mediated agonists to
stimulate Cl
secretion
across human CF airway.
Ca2+-mediated agonists have a dual
therapeutic role in CF: 1)
inhibition of Na+ absorption and
2) stimulation of
Cl
secretion.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the excellent secretarial skills of
Michele Dobransky, the technical assistance of Cheng Zhang Shi and Joe
Latoche in both tissue culture and Ussing chamber experiments, the
assistance of Drs. Jan Manzetti and Robert Keenan (University of
Pittsburgh Medical Center lung transplant program) in obtaining human
lung tissue, and the help of Mark Gerardi (Genzyme) in facilitating genotype analysis.
 |
FOOTNOTES |
This work was supported by Cystic Fibrosis Foundation Grants DEVOR960
and Q933 and a Cystic Fibrosis Research Development Program Center grant.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. C. Devor,
Dept. of Cell Biology and Physiology, S312 BST, 3500 Terrace St.,
University of Pittsburgh, Pittsburgh, PA 15261 (E-mail:
dd2+{at}pitt.edu).
Received 13 October 1998; accepted in final form 13 January 1999.
 |
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