cAMP stimulates Na+ transport
in rat fetal pneumocyte: involvement of a PTK- but not a
PKA-dependent pathway
Naomi
Niisato,
Yasushi
Ito, and
Yoshinori
Marunaka
Lung and Cell Biology and Medical Research Council Group in Lung
Development, Department of Pediatrics and Institute of Medical
Science, The Hospital for Sick Children Research Institute, University
of Toronto Faculty of Medicine, Toronto, Ontario, Canada M5G 1X8
 |
ABSTRACT |
To study a cAMP-mediated signaling pathway in
the regulation of amiloride-sensitive
Na+ transport in rat fetal distal
lung epithelial cells, we measured an amiloride-sensitive short-circuit
current (Na+ transport).
Forskolin, which increases the cytosolic cAMP concentration, stimulated
the Na+ transport. Forskolin also
activated cAMP-dependent protein kinase (PKA). A
-adrenergic agonist
and cAMP mimicked the forskolin action. PKA inhibitors KT-5720, H-8,
and myristoylated PKA-inhibitory peptide amide-(14
22) did not
influence the forskolin action. These results suggest that forskolin
stimulates Na+ transport through a
PKA-independent pathway. Furthermore, forskolin increased tyrosine
phosphorylation of ~70- to 80-, ~97-, and ~110- to 120-kDa
proteins. Protein tyrosine kinase (PTK) inhibitors (tyrphostin A23 and
genistein) abolished the forskolin action. Moreover,
5-nitro-2-(3-phenylpropylamino)benzoate (a
Cl
-channel blocker)
prevented the stimulatory action of forskolin on
Na+ transport via abolishment of
the forskolin-induced cell shrinkage and tyrosine phosphorylation.
Based on these results, we conclude that forskolin (and cAMP)
stimulates Na+ transport in a
PTK-dependent but not a PKA-dependent pathway by causing cell
shrinkage, which activates PTK in rat fetal distal lung epithelial cells.
sodium transport; adenosine 3',5'-cyclic monophosphate; tyrosine phosphorylation; protein kinase A; protein tyrosine kinase; alveolar epithelium; amiloride
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INTRODUCTION |
THE FETAL LUNG FLUID secreted by lung epithelial cells
plays an important role in development, differentiation, and growth of
the fetal lung (2, 29). This fluid secretion depends on Cl
secretion from the
basolateral to the apical space (29). However, the fluid must be
cleared from the alveolar air space immediately at birth to allow
normal gas exchange. Catecholamines, circulating levels of which
increase during labor and delivery, induce clearance of the fluid by
stimulating amiloride-sensitive
Na+ transport in lung epithelial
cells (4, 32).
Ito et al. (11), Marunaka et al. (18), and Tohda et al.
(38) have previously shown that a
-adrenergic agonist,
the intracellular second messenger cAMP, stimulates an
amiloride-sensitive nonselective cation (NSC) channel and
amiloride-sensitive Na+ transport
in rat fetal distal lung epithelial (FDLE) cells. However, the
mechanism by which cAMP initiates activation of signal transduction pathways and how the pathway results in stimulation of
Na+ transport are still unknown.
Nakahari and Marunaka (25) have further reported that a
-adrenergic
agonist, forskolin, and cAMP cause cell shrinkage under isotonic
conditions by stimulating KCl release in rat FDLE cells. Recent reports
(31, 36) have shown that the change in cell volume causes an increase
in tyrosine phosphorylation, which is involved in the regulation of ion
transport and gene transcription. These studies provide a possibility
that in rat FDLE cells, cAMP-induced cell shrinkage might also cause an
increase in tyrosine phosphorylation, although cAMP-dependent signals
are generally converted to cAMP-dependent protein kinase (PKA)-mediated
signals. Our purpose in the present study was to explore the role of
cAMP in the stimulation of amiloride-sensitive Na+ transport in rat FDLE cells
with forskolin, which increases the cytosolic cAMP level by activating
adenylate cyclase. We reflect the fact that forskolin-stimulated
Na+ transport does not occur via
the conventional PKA-dependent pathway in rat FDLE cells.
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MATERIALS AND METHODS |
Cell preparation and culture. FDLE
cells were isolated from the fetuses of pregnant Wistar rats of 20-day
gestational age (term 22 days). Briefly, the rats were anesthetized
with inhalational ether, and the fetuses were removed from the uterus.
Their lungs were removed immediately, washed in cold Hanks' solution
(Mg2+ and
Ca2+ free) to remove erythrocytes,
and minced with scissors. The lung pieces were digested with trypsin
(0.125%) and DNase (0.002%) for 20 min at 37°C. The cell
suspension was filtered through a Nitex 100 filter (B. and S. H. Thompson, Scarborough, ON). The cells were then incubated with
collagenase (0.1%) and purified with a differential adhesion
technique. The purity of FDLE cells as fetal alveolar type II cells has
been established (35). The culture medium was MEM supplemented with
antibiotics and 10% fetal bovine serum. The cells were seeded at 3 × 105 cells/well onto Costar
wells (6.5-mm diameter, 6.5-mm Transwell filter, tissue culture-treated
Transwell) for short-circuit current (Isc)
measurement or at 5 × 106
cells/well onto translucent porous Nunc filter inserts (Nunc tissue
culture inserts) for single-channel current recording, Western
blotting, and measurement of PKA activity. Then they were cultured at
37°C in a 95% air-5% CO2
humidified incubator for 3 days.
Solution. The experimental solution
used in the present study contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES and
was stirred with air (pH 7.4).
Isc and conductance.
For measurement of
Isc, rat FDLE
cells were rinsed with the experimental solution and transferred to a
modified Ussing chamber (Jim's Instrument, Iowa City, IA) designed to
hold the filter cup.
Isc and
conductance were measured with an amplifier (VCC-600, Physiologic
Instrument, San Diego, CA) (11, 27, 28). A positive current represents
a net flow of cation from the apical to the basolateral solution.
Transepithelial voltage was measured with a pair of calomel electrodes
that were immersed in a saturated KCl solution and bridged to the
modified Ussing chamber by a pair of polyethylene tubes filled with a
solution of 2% agarose in 2 M KCl.
Single-channel recordings.
Single-channel recordings and data analysis were performed with the
same methods as Marunaka et al. (18) have previously reported. Current
signals were digitized at a sampling rate of 5,000 Hz. To analyze the
channel kinetics, a 2,000-Hz low-pass Gaussian filter was used. A
500-Hz low-pass filter with a software Gaussian filter was used to
present the actual traces. Single-channel conductances were determined
by the slope of the current-voltage relationship around no applied potential via a patch pipette [i.e., pipette potential = 0 mV (the resting apical membrane potential)] in a cell-attached
configuration. Channel activity
(NPo, where
N is the number of channels and
Po is the open
probability) shown in the present study was measured as follows
where
i is the number of channels
simultaneously open, N is the maximum
number of channels simultaneously open in a patch, Ti is the time of
i channels simultaneously open, and TT is the total
recording time.
Western blotting. Rat FDLE cells were
lysed with lysis buffer (50 mM HEPES, 150 mM NaCl, 1.5 mM
MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 100 mM NaF, 10 mM pyrophosphate, 200 µM sodium orthovanadate, 250 µg/ml of leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, and 100 kallikrein inactivator units/ml of aprotinin, pH 7.4)
with and without forskolin stimulation. The cells were homogenized by
sonication and centrifuged at 12,000 g
for 10 min at 4°C to remove insoluble debris. The cell lysates
containing 25 µg of protein were boiled in SDS sample buffer
[60 mM Tris · HCl, 2% (wt/vol) SDS, and
5% (vol/vol) glycerol, pH 6.8] and then subjected to 7.5%
SDS-PAGE. After electrophoresis, the proteins were transferred to
nitrocellulose membranes. Nonspecific binding was blocked by incubation
in 5% (wt/vol) bovine serum albumin for 60 min. The membranes
were immunoblotted with a monoclonal anti-phosphotyrosine antibody,
PY99 (Santa Cruz Biotechnology, Santa Cruz, CA). After overnight
incubation at 4°C, the membrane was washed with Tris-buffered
saline containing 0.1% Tween 20 and incubated for 60 min at room
temperature with horseradish peroxidase-conjugated goat anti-mouse IgG.
After being washed in Tris-buffered saline-Tween 20, the blots were
developed with an enhanced chemiluminescence detection kit from
Amersham (Oakville, ON).
Measurement of PKA activity. To determine PKA
activity, rat FDLE cells were lysed with an extraction buffer (5 mM
EDTA and 50 mM Tris, pH 7.5). The cells were homogenized by sonication, and then the cellular debris was removed by centrifuging at 12,000 g for 5 min at 4°C. PKA activity
was measured with a commercially available PKA assay kit from GIBCO BRL
(Life Technologies, Grand Island, NY) under different assay conditions
(i.e., with and without PKA inhibitor and with and without cAMP) to
determine the total PKA-specific kinase activity and proportion of PKA
activity stimulated by forskolin. After preparation under four
different assay conditions (triplicates per each condition) in each
cell extraction, the lysate was incubated at room temperature for 15 min to allow a PKA inhibitor (9) to bind PKA. The heptapeptide
Leu-Arg-Arg-Ala-Ser-Leu-Gly ("Kemptide") was used as a specific
peptide substrate for PKA. Ten microliters of
32P per substrate solution
(20-25 µCi/ml of
[
-32P]ATP plus PKA
substrate) were added to each tube (final volume 40 µl). Each tube
was then placed at 30°C for 5 min. A sample of 20 µl was removed
from the tube and spotted onto the corresponding phosphocellulose disk.
After the sample was spotted onto the phosphocellulose disk, the disk
was washed in a tray containing an acid wash [1% (vol/vol)
phosphoric acid
(H3PO4)]
for 3-5 min twice and in H2O for 3-5 min twice. After the wash, the
32P incorporated in the peptide
was counted after the addition of scintillation fluid. Subtracting the
kinase activity without the addition of cAMP in the presence of the PKA
inhibitor (= the background kinase activity without PKA activity) from
the kinase activity with addition of cAMP in the absence of PKA
inhibitor (= the maximum PKA activity plus the background kinase
activity), we determined the maximum kinase activity of PKA. We also
determined the PKA-specific kinase activity in the tested cells,
subtracting the kinase activity with the addition of PKA inhibitor (=
the background kinase activity) from the kinase activity without any
addition of cAMP or PKA inhibitor (= the PKA activity plus the
background kinase activity). PKA activity is presented as the
percentage of the maximum PKA activity.
Cell volume. Cell volume was measured
with the same method reported in previous studies by Marunaka and
colleagues (19, 21) and Nakahari and Marunaka (25). The averaged value
measured for 2 min just before application of forskolin was used as the control. Cell shrinkage is shown as the percent change from the control
value. The method of cell volume estimation and its reliability have
been previously described in detail (5).
Chemicals. MEM medium and fetal bovine
serum were purchased from GIBCO BRL. Amiloride was purchased from Sigma
(St Louis, MO). Tyrphostin A23, genistein, AG-1295, KT-5720, H-8, and
myristoylated PKA inhibitory peptide amide-(14
22)
[Myr-PKI-(14
22)] were obtained from Calbiochem (San
Diego, CA). Lavendustin A was provided from BIOMOL Research
Laboratories (Plymouth Meeting, PA). Benzamil and
5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) were obtained from RBI
(Natick, MA). Tissue culture-treated Transwells were purchased from
Costar (Cambridge, MA). Nunc filters (Nunc tissue culture inserts) were
obtained from Nunc (Roskilde, Denmark). Other materials and chemicals
were purchased from Sigma, unless otherwise stated.
Temperature. All experiments were
performed at 37°C, unless otherwise indicated.
Data presentation. All data are means ± SE. Where SE bars are not visible, they are smaller than the
symbol. Unpaired Student's t-test,
ANOVA, and Duncan's multiple range comparison test were used for
statistical analysis as appropriate, and a
P value < 0.05 was considered significant.
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RESULTS |
Forskolin action on amiloride-sensitive and -insensitive
Isc.
Forskolin (5 µM, basolateral application) increased
Isc, most of
which was sensitive to amiloride (10 µM, apical application; Fig.
1A).
Amiloride diminished the basal
Isc (Fig.
1B). In the presence of 10 µM
amiloride, forskolin failed to increase
Isc (Fig.
1B). The concentration of amiloride
showing half-maximum inhibition
(IC50) was <1 µM; 0.1 µM
amiloride showed 19 ± 1% inhibition (n = 6 experiments) and 1 µM amiloride showed 63 ± 3% inhibition (n = 6 experiments). Furthermore,
benzamil (10 µM), a more specific blocker of the
Na+ channel than the
Na+/H+
exchanger (12), inhibited the
Isc in a manner
identical to 10 µM amiloride (data not shown). Furthermore, a study
(30) has shown that the inhibitory action of amiloride on the
Isc is not due to
blockade of the
Na+/H+
exchanger but to blockade of the
Na+ channel. Therefore, the
inhibitory action of 10 µM amiloride is due to blockade of the
Na+ channel. Similar to results
reported in previous studies by Ito et al. (11) and Nakahari and
Marunaka (24, 25), a
-agonist (terbutaline), dibutyryl cAMP, and
8-bromo-cAMP mimicked the forskolin action (data not shown). These
results suggest that in rat FDLE cells forskolin increases
amiloride-sensitive Na+ transport
via a cAMP-dependent pathway.

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Fig. 1.
Effect of forskolin (5 µM, basolateral application) on short-circuit
current (Isc).
A:
Isc increased
gradually with time after application (arrows) of forskolin. Amiloride
(10 µM, apical application) diminished increased
Isc.
B: amiloride was added before
application of forskolin. Amiloride diminished basal
Isc. In presence
of amiloride, forskolin failed to increase
Isc.
C: forskolin increased
amiloride-sensitive
Isc
(n = 4 experiments). Base, baseline.
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Action of forskolin on single amiloride-blockable
channels. A previous study by Marunaka (16) indicated
that rat FDLE cells have two types of amiloride-blockable channels that
have permeability to Na+:
1) one is an NSC channel with a
single-channel conductance of 28 pS and
2) the other is a highly
Na+-selective channel with a
single-channel conductance of 12 pS. However, it is unknown which
channel contributes to the forskolin-induced amiloride-sensitive
Isc. Therefore,
we applied the single-channel current recording technique to clarify
this point. Characteristics of the 28-pS NSC channel were obtained from
cell-attached patches formed on rat FDLE cells under unstimulated and
forskolin (5 µM, 30 min)-stimulated conditions (Fig.
2). Under the unstimulated condition (control), we observed a little activity of the channel (Fig.
2A,
top). However, after application of
forskolin (5 µM), we observed channels with high activity (Fig.
2A,
bottom). As shown in Fig.
2B, the current-voltage relationship
was shifted leftward ~20 mV by application of forskolin; however,
single-channel conductance was not affected by forskolin application.
The ion selectivity of the channel was not altered by forskolin (data not shown; cf. Ref. 18); therefore, the shift of the current-voltage relationship means that forskolin induced depolarization of the apical
membrane, which may be caused by activation of the 28-pS NSC channel as
shown in Fig. 2C. These observations
suggest that the 28-pS NSC channel responds to forskolin, contributing
to the forskolin-induced stimulation of amiloride-sensitive
Isc. We also studied the effect of forskolin on the characteristics of the 12-pS
Na+ channel. Characteristics of
the 12-pS Na+ channel were
obtained from cell-attached patches formed on rat FDLE cells under
unstimulated and forskolin (5 µM, 30 min)-stimulated conditions (Fig.
3). Figure
3A shows the single-channel currents without (top) and with
(bottom) forskolin application. The
current-voltage relationship was shifted leftward ~20 mV by forskolin
(Fig. 3B). However, unlike the 28-pS
NSC channel, we did not observe any significant forskolin-induced
changes in the activity
(NPo) of the
12-pS Na+ channel (Fig.
3C). The ion selectivity of the
12-pS Na+ channel was not altered
by forskolin (data not shown). These observations suggest that the
Na+ channel does not respond to
forskolin and the forskolin-induced stimulation of amiloride-sensitive
Isc shown in Fig.
1A is not mediated through an
increase in activity of the 12-pS
Na+ channel. Taken together, these
experimental observations indicate that forskolin would stimulate the
amiloride-sensitive
Isc via activation of the 28-pS NSC channel without any effects on the 12-pS
Na+ channel. The forskolin-induced
leftward shift of the current-voltage relationship of the 28-pS NSC and
12-pS Na+ channels would be due to
depolarization of the apical membrane, which would be caused by
activation of the 28-pS NSC channel. Furthermore, it is also suggested
that the 12-pS Na+ channel
predominantly contributes to the basal amiloride-sensitive Na+ current and the 28-pS NSC
channel significantly contributes to the amiloride-sensitive
Na+ current under the
forskolin-stimulated condition.

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Fig. 2.
Effect of 5 µM forskolin (30-min application) on 28-pS nonselective
cation (NSC) channel. A:
single-channel currents obtained from cell-attached patches without
(top) and with
(bottom) forskolin at no applied
pipette potential. -c, Closed level of channel.
B: current-voltage relationships
obtained from cell-attached patches without (control) and with
forskolin (n = 40 experiments).
Vp, pipette
potential. When
Vp was 40 mV, apical membrane was clamped to a voltage more positive by 40 mV
than apical resting membrane potential.
C: channel activity
(NPo, where
N is no. of channels and
Po is open
probability) obtained from cell-attached patches without (control) and
with forskolin at no applied
Vp
(n = 40 experiments).
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Fig. 3.
Effect of 5 µM forskolin (30-min application) on 12-pS
Na+ channel.
A: single-channel currents obtained
from cell-attached patches without
(top) and with
(bottom) forskolin at no applied
Vp.
B: current-voltage relationships
obtained from cell-attached patches without (control) and with
forskolin (n = 20 experiments). When
Vp was 40 mV, apical membrane was clamped to a voltage more positive by 40 mV
than apical resting membrane potential.
C:
NPo obtained from
cell-attached patches without and with forskolin at no applied
Vp
(n = 20 experiments).
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Effects of PKA inhibitors on forskolin
action. Because cAMP-dependent signalings are generally
mediated through PKA activation, we measured PKA activity in
unstimulated and forskolin-stimulated cells. Stimulation with forskolin
(5 µM, 30 min) increased PKA activity approximately threefold (from
11.7 ± 3.5 to 34.2 ± 8.5%; n = 3 experiments; P < 0.025). To
study whether forskolin stimulates Na+ transport through a
PKA-mediated signaling pathway, we next examined the effects of PKA
inhibitors on the action of forskolin. KT-5720 is a specific PKA
inhibitor (6). Bilateral pretreatment with KT-5720 (0.5 µM, 2 h)
decreased basal
Isc (Fig.
4, A and
B), suggesting that KT-5720 applied
in the present study (0.5 µM, 2 h) was effective in rat FDLE cells.
The amiloride-insensitive
Isc, which was
defined as the residual
Isc after
application of 10 µM amiloride, was not affected by KT-5720 treatment
(Fig. 4B; compare the amount of
residual Isc
after application of 10 µM amiloride). Namely, KT-5720 diminished the
basal amiloride-sensitive
Isc (Fig.
4C) but did not affect the
amiloride-insensitive
Isc (Fig.
4B). Although KT-5720 diminished the
basal amiloride-sensitive
Isc (Fig.
4C, Base), forskolin still stimulated
the amiloride-sensitive
Isc even in
KT-5720-treated rat FDLE cells (Fig.
4C; compare Base and Forskolin with
KT-5720-treated cells). The forskolin-induced amiloride-sensitive
Isc was not
affected by KT-5720 (Fig. 5). These
observations suggest that KT-5720 decreases basal amiloride-sensitive Na+ transport, but the action of
forskolin is not blocked by KT-5720.

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Fig. 4.
Effect of KT-5720 [an inhibitor of cAMP-dependent protein kinase
(PKA)] on unstimulated and forskolin-stimulated
Isc.
A: treatment with KT-5720 (0.5 µM, 2 h pretreatment and presence during
Isc measurement,
bilateral application) decreased basal
Isc. However,
even in KT-5720-treated cells, forskolin increased
Isc. Amiloride
(10 µM, apical application) blocked most of forskolin-stimulated
Isc to an
identical level irrespective of KT-5720 treatment. Arrows, time of
application; ( ), without; (+), with.
B: amiloride (10 µM, apical
application) was added before application of forskolin. Amiloride
diminished basal
Isc to an
identical level irrespective of KT-5720 treatment. In presence of
amiloride, forskolin failed to increase
Isc irrespective
of KT-5720 treatment. Treatment with KT-5720 had no significant effects
on amiloride-insensitive
Isc irrespective
of forskolin stimulation. C: basal and
forskolin-stimulated amiloride-sensitive
Isc
(n = 4 experiments). KT-5720 decreased
basal and forskolin-stimulated amiloride-sensitive
Isc. However,
amount of KT-5720-induced decrease in basal and forskolin-stimulated
amiloride-sensitive
Isc was identical
(compare control values with those with KT-5720 treatment). Namely,
forskolin-induced increase in amiloride-sensitive
Isc was not
affected by KT-5720 treatment (cf. Fig. 5).
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Fig. 5.
Effects of KT-5720, H-8 and myristoylated PKA-inhibitory peptide
amide-(14 22) [Myr-PKI-(14 22); inhibitors of PKA] on
forskolin-induced amiloride-sensitive
Isc
(n = 4 experiments). Bilateral
application of KT-5720 (0.5 µM, 2 h pretreatment), H-8 (10 µM, 2 h
pretreatment), or Myr-PKI-(14 22) (3 µM, 4 h pretreatment) had no
significant effects on forskolin-induced increase in
amiloride-sensitive
Isc. Each
inhibitor was also present in bathing solution during time period when
Isc was
measured.
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Single-channel data indicate that the 12-pS
Na+ channel predominantly
contributes to basal amiloride-sensitive
Na+ transport as described above.
KT-5720 induced significant decreases in 12-pS
Na+ channel activity under basal
conditions [control, 0.23 ± 0.15 (n = 20 experiments);
KT-5720, 0.12 ± 0.09 (n = 20 experiments); P < 0.005] and
forskolin-stimulated conditions [control, 0.35 ± 0.31 (n = 20 experiments); KT-5720, 0.16 ± 0.11 (n = 20 experiments); P < 0.01]. On the other hand,
KT-5720 did not significantly affect the basal activity of the 28-pS
NSC channel [control, 0.02 ± 0.03 (n = 40 experiments); KT-5720, 0.02 ± 0.02 (n = 40 experiments); not
significant] or the activity of the forskolin-stimulated 28-pS NSC channel [control, 2.54 ± 1.37 (n = 40 experiments); KT-5720, 2.30 ± 1.12 (n = 40 experiments); not
significant]. These observations suggest that
1) KT-5720 would diminish basal
amiloride-sensitive Na+ transport
by influencing the 12-pS Na+
channel activity without any effects on the 28-pS NSC channel activity
and 2) forskolin-induced stimulation
even in KT-5720-treated cells is due to activation of the 28-pS NSC channel.
We further applied other types of PKA inhibitors, H-8 (10) and
Myr-PKI-(14
22) (9). Myr-PKI-(14
22) is a heat-stable peptide
sequence (14
22) myristoylated at the
NH2 terminus, enhancing its
membrane permeability, and this peptide is a highly specific inhibitor
of PKA (9). Pretreatment with H-8 (10 µM, 2 h) or Myr-PKI-(14
22) (3 µM, 4 h) decreased basal amiloride-sensitive Isc similar to
that of KT-5720. Although these inhibitors decreased basal
amiloride-sensitive
Isc, they had no
inhibitory effects on the forskolin-induced amiloride-sensitive
Isc (Fig. 5).
These results strongly suggest that the forskolin-activated PKA may not
be involved in the forskolin regulation of the amiloride-sensitive Na+ transport.
Effects of PTK inhibitors on the action of
forskolin. A previous study by Nakahari and Marunaka
(25) has demonstrated that forskolin induces cell shrinkage by
stimulating KCl release in rat FDLE cells and this action is mimicked
by cAMP. On the other hand, several reports (e.g., Refs. 36, 37) have
shown that cell volume change causes a rapid increase in tyrosine
phosphorylation and that, in some cases, tyrosine phosphorylation is
involved in regulation of ion transport (e.g., Ref. 15). Using PTK
inhibitors, therefore, we next studied whether PTK is involved in the
forskolin stimulation of Na+
transport in rat FDLE cells. Tyrphostin A23 (100 µM, bilateral application) decreased basal
Isc gradually
with time (Fig. 6). In the cells treated
with 100 µM tyrphostin A23 for 30 min, forskolin failed to increase
Isc (Figs.
6A and
7A).
Tyrphostin A23 diminished the forskolin-stimulated amiloride-sensitive
Isc dose
dependently (Fig. 6B). However,
tyrphostin A23 (100 µM, 30-60 min) did not affect the basal
activity of the 28-pS NSC channel [control, 0.02 ± 0.03 (n = 40 experiments); tyrphostin A23,
0.01 ± 0.02 (n = 30 experiments);
not significant]. On the other hand, forskolin did not increase
the activity of the 28-pS NSC channel in tyrphostin A23-treated cells
[control, 0.01 ± 0.02 (n = 30 experiments); forskolin, 0.02 ± 0.02 (n = 25 experiments); not significant]. Furthermore, we tested whether
tyrphostin A23 was toxic to the 28-pS NSC channel. Exposure of the
28-pS NSC channel to high
Ca2+ (1 mM) in excised
inside-out patches obtained from tyrphostin A23 (100 µM, 60 min)-treated cells increased its activity to a level identical with
that of the control cells [without tyrphostin A23; control, 0.35 ± 0.12 (n = 15 experiments);
tyrphostin A23, 0.31 ± 0.10 (n = 10 experiments); not
significant]. This observation suggests that treatment with
tyrphostin A23 was not toxic to the channel. These observations suggest
that the failure of forskolin to stimulate amiloride-sensitive
Isc in rat FDLE
cells with tyrphostin A23 treatment is not due to abolishment of the
basal activity of the 28-pS NSC channel but that the stimulatory action
of forskolin requires a tyrphostin A23-sensitive factor that is not the
28-pS NSC channel itself.

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Fig. 6.
Effect of tyrphostin A23 [an inhibitor of protein tyrosine kinase
(PTK); 100 µM, bilateral application] on unstimulated and
forskolin-stimulated
Isc.
A: tyrphostin A23 (TY23; 100 µM)
diminished basal
Isc. In cells
with TY23 treatment, forskolin (5 µM) failed to increase
Isc. However,
some amount of amiloride-sensitive
Isc was still
observed even in cells with TY23 treatment. This observation suggests
that failure of forskolin to stimulate
Isc was not due
to abolishment of amiloride-sensitive channels but that one of the
forskolin signaling pathways was blocked by TY23, resulting in blockade
of stimulatory action of forskolin on amiloride-sensitive
Isc
(n = 4 experiments).
B: dose-dependent effects of
tyrphostin A23 on forskolin-stimulated amiloride-sensitive
Isc
(n = 4 experiments).
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Fig. 7.
Effects of PTK inhibitors TY23 and genistein (Gen) on
amiloride-sensitive
Isc
(A) and conductance
(B). TY23 and Gen were bilaterally
applied to rat fetal distal lung epithelial (FDLE) cells 30 min before
addition of forskolin (5 µM) and were also present in bathing
solution during time period when
Isc was measured
(n = 4 experiments).
A: TY23 and Gen abolished
forskolin-induced amiloride-sensitive
Isc.
B: TY23 and Gen markedly diminished
forskolin-stimulated amiloride-sensitive conductance.
|
|
Another PTK inhibitor, genistein (200 µM, 30 min, bilateral
treatment), also abolished the action of forskolin (Fig.
7A). In the case of genistein, we
also obtained a result similar to that with tyrphostin A23; i.e.,
genistein (200 µM, 30 min) did not affect the basal activity of the
28-pS NSC channel [control, 0.02 ± 0.03 (n = 40 experiments); genistein, 0.02 ± 0.02 (n = 20 experiments); not
significant]. However, forskolin did not increase the activity of
the 28-pS NSC channel in genistein-treated cells [control, 0.02 ± 0.02 (n = 20 experiments);
forskolin, 0.03 ± 0.02 (n = 20 experiments); not significant]. Furthermore, exposure of the
28-pS NSC channel to high Ca2+ (1 mM) in excised inside-out patches obtained from genistein-treated cells
increased its activity to a level identical to that of control cells
without genistein treatment [control, 0.35 ± 0.12 (n = 15 experiments); genistein, 0.30 ± 0.16 (n = 10 experiments); not significant]. This observation suggests that
the genistein treatment was not toxic to the channel. Taken together,
these results suggest that a PTK-dependent pathway may be involved in
the forskolin stimulation of Na+
transport in rat FDLE cells.
We further measured amiloride-sensitive conductance. The PTK inhibitors
tyrphostin A23 and genistein markedly diminished forskolin-stimulated amiloride-sensitive conductance (Fig.
7B). However, other PTK inhibitors,
lavendustin A [10 µM for 30-60 min; an inhibitor of epidermal growth factor receptor kinase (33)] or AG-1295
[50 µM for 30-60 min; an inhibitor of platelet-derived
growth factor receptor kinase (13)] had no significant effects on
the forskolin-stimulated Isc (data not shown).
Taken together, these results obtained from measurements of
Isc, conductance,
and single-channel current suggest that forskolin stimulates
Na+ transport by increasing the
28-pS NSC channel activity via a pathway dependent on PTK (maybe a
nonreceptor type).
Action of forskolin on phosphotyrosine and effects of
PTK inhibitor. Furthermore, we examined whether
forskolin increases tyrosine phosphorylation. Western blotting data
with a monoclonal anti-phosphotyrosine antibody (PY99) show that
forskolin increased phosphorylation of tyrosine residues of ~70- to
80-, ~97-, and ~110- to 120-kDa proteins (Fig.
8). A PTK inhibitor, tyrphostin A23,
abolished the action of forskolin on tyrosine phosphorylation (Fig. 8).
These results suggest that forskolin would activate PTK, leading to
tyrosine phosphorylation and supporting an idea that activated PTK may
be involved in the forskolin stimulation of
Na+ transport in rat FDLE cells.

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Fig. 8.
Effects of forskolin and TY23 on phosphorylation of tyrosine.
Left: typical result of action of
forskolin (5 µM) without TY23 on phosphorylation of tyrosine.
Forskolin increased phosphorylation of tyrosine residues of ~70- to
80-, ~97-, and ~110- to 120-kDa proteins. Nos. on left,
molecular-mass markers in kDa. Right:
typical result of action of TY23 (100 µM) on forskolin-induced
increase in phosphorylation of tyrosine. TY23 was applied to rat FDLE
cells 30 min before addition of forskolin and was also present in
bathing solution after addition of forskolin. TY23 abolished
forskolin-increased tyrosine phosphorylation. Time, time period after
application of forskolin.
|
|
Effects of a Cl
-channel blocker,
NPPB, on action of forskolin.
Although these observations suggest that forskolin would activate PTK,
it is still unclear how forskolin activates PTK. Recent studies (36,
37) suggest that membrane tension and/or cell shrinkage induces
tyrosine phosphorylation. As previously reported (25), cAMP and
forskolin induce cell shrinkage in rat FDLE cells by stimulating KCl
efflux via K+ and
Cl
channels. Therefore, we
considered a possibility that the forskolin-induced cell shrinkage
would be involved in the signaling of forskolin to the stimulation of
Na+ transport via activation of
PTK. To study the role of forskolin-induced cell shrinkage in its
signaling, we applied a
Cl
-channel blocker that was
expected to block forskolin-induced cell shrinkage. If
forskolin-induced cell shrinkage is involved in forskolin signaling to
stimulation of Na+ transport and a
Cl
-channel blocker
abolishes the forskolin-induced cell shrinkage, a
Cl
-channel blocker should
abolish the stimulatory action of forskolin on the amiloride-sensitive
Isc. Accordingly,
we studied the effect of NPPB, a
Cl
-channel blocker, on the
forskolin-induced changes in cell volume and
Isc. NPPB (100 µM) abolished the action of forskolin on cell volume and
amiloride-sensitive
Isc (Fig.
9). If forskolin stimulates phosphotyrosine
via cell shrinkage, NPPB should also block the stimulatory action of
forskolin on phosphotyrosine. Therefore, we studied whether NPPB blocks
the action of forskolin on phosphotyrosine. The stimulatory action of
forskolin on phosphotyrosine was also abolished by NPPB (Fig.
10). These observations suggest that NPPB abolishes the action of forskolin on amiloride-sensitive
Na+ transport by preventing
activation of PTK via blockade of cell shrinkage through its inhibitory
action on Cl
channels
(conductances), which are required for cell shrinkage.

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Fig. 9.
Effects of 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB) on
forskolin-induced cell shrinkage (A)
and amiloride-sensitive
Isc
(B). NPPB (100 µM) was applied to
rat FDLE cells 5 min before addition of forskolin (5 µM) and was also
present in bathing solution during time period when cell volume and
Isc were measured
(n = 6 experiments). Forskolin-induced
cell shrinkage and amiloride-sensitive
Isc were measured
30 min after application of forskolin without and with NPPB
application. NPPB abolished effects of forskolin on cell shrinkage and
amiloride-sensitive
Isc.
|
|

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Fig. 10.
Effects of NPPB on forskolin-induced phosphorylation of tyrosine.
Left: typical result of action of
forskolin without NPPB on phosphorylation of tyrosine. Forskolin
increased phosphorylation of tyrosine residues of ~70- to 80-, ~97-, and ~110- to 120-kDa proteins. Nos. on left,
molecular-mass markers in kDa. Right:
typical result of NPPB (100 µM) action on forskolin-induced increase
in phosphorylation of tyrosine. NPPB was applied to rat FDLE cells 5 min before addition of forskolin (5 µM) and was also present in
bathing solution after addition of forskolin. NPPB abolished action of
forskolin on tyrosine phosphorylation. Time, time period after
application of forskolin.
|
|
 |
DISCUSSION |
Forskolin acts on cells via an increase in the cytosolic cAMP level by
activating adenylate cyclase, and the cAMP-dependent signal is, in
general, thought to be converted to PKA-mediated signaling pathways
that cause specific cellular responses. Interestingly, the present
study strongly suggests that 1)
forskolin signals are converted to a PTK-mediated signal and
2) the PTK-mediated signal is
involved in forskolin-stimulated
Na+ transport in rat FDLE cells.
Action of PKA inhibitors. PKA
inhibitors decreased basal amiloride-sensitive
Isc, suggesting
that the PKA inhibitor used in the present study functions in rat FDLE
cells. On the other hand, forskolin increased PKA activity
approximately threefold. However, the PKA inhibitor failed to block the
action of forskolin on the amiloride-sensitive
Isc, although the
PKA inhibitor was effective at least on basal amiloride-sensitive
Na+ transport. These observations
suggest that the regulation of Na+
transport by forskolin is not mediated through activation of PKA.
In general, the inactive form of PKA consists of four subunits: two
regulatory subunits and two catalytic subunits. When cAMP binds to the
regulatory subunits of inactive PKA, the active catalytic subunits are
separately released and phosphorylate serine or threonine residues of
their substrates. Myr-PKI-(14
22) is a potent, competitive PKA
inhibitor (9). This potent synthetic peptide inhibitor of PKA binds to
the catalytic subunit of PKA, leading to inhibition of its activity.
The concentration showing the half-maximum inhibitory effect
(Ki) of
Myr-PKI-(14
22) on PKA is 36 nM (9, 22). H-8 is a highly active
inhibitor of PKA
(Ki = 1.2 µM)
and one of the reversible, competitive types of PKA inhibitors against
ATP (10). KT-5720 is the same type of PKA inhibitor as H-8, with a
Ki of 56 nM
(6). We used three kinds of PKA inhibitors to study the role of PKA in the regulation of
Na+ transport by forskolin (cAMP)
in rat FDLE cells. These PKA inhibitors functioned in rat FDLE cells;
nevertheless, they failed to block the action of forskolin on
Na+ transport. These observations
suggest that forskolin stimulates Na+ transport in a PKA-independent
pathway. On the other hand, PTK inhibitors abolished the action of
forskolin on tyrosine phosphorylation and the amiloride-sensitive
Isc and
conductance. Although we should still consider the possibility that the
PKA inhibitor used in the present study cannot abolish the activity of
all PKA isoforms, it is strongly suggested that forskolin stimulates
Na+ transport through a
PTK-mediated but not a PKA-dependent pathway.
Effects of inhibition of the
Na+,K+
pump on the Isc.
Amiloride-sensitive transepithelial
Na+ transport is mediated through
two steps: 1) an entry step of
Na+ through amiloride-sensitive
Na+-permeable channels at the
apical membrane and 2) an extrusion step of Na+ by the
Na+,K+
pump at the basolateral membrane (for a review, see Ref. 17). If either
of these two steps is blocked, the amiloride-sensitive transepithelial
Na+ transport is abolished. In the
present study, we show that the PTK inhibitors tyrphostin A23 and
genistein abolished the stimulatory action of forskolin on
amiloride-sensitive Na+ transport.
Therefore, abolishment of the action of forskolin on
amiloride-sensitive Na+ transport
by tyrphostin A23 or genistein might be mediated through inhibition of
Na+,K+
pump activity but not through inhibition of the amiloride-sensitive Na+ channel. However, the
forskolin-stimulated amiloride-sensitive conductance, which means
activity of amiloride-sensitive channels, was diminished by tyrphostin
A23 or genistein. This observation suggests that the stimulatory action
of forskolin on the activity of amiloride-sensitive
Na+ channels is blocked by
tyrphostin A23 or genistein. Accordingly, we conclude that the PTK
inhibitors tyrphostin A23 and genistein abolish the stimulatory action
of forskolin on amiloride-sensitive Na+ transport through diminution
of the stimulatory action on amiloride-sensitive Na+-permeable (NSC) channel
activity that contributes to the forskolin-induced increase in
amiloride-sensitive Na+ transport.
Furthermore, when ouabain is applied, amiloride-sensitive Na+ transport is abolished due to
blockade of the
Na+,K+
pump as described above. However, in this case, we did not detect any
significant change in amiloride-sensitive conductance, unlike with
tyrphostin A23. Moreover, even in tyrphostin A23-treated rat FDLE
cells, the transepithelial resistance was still kept high
[control, 1,664 ± 354
· cm2
(n = 8 experiments); tyrphostin A23
(100 µM, 60 min), 1,787 ± 354
· cm2
(n = 8 experiments); not
significant], suggesting that the FDLE cells can keep tight
junctions well and that the action of tyrphostin A23 is not simply due
to cell damage that makes epithelial cells leaky (low resistances).
Action of various types of PTK inhibitors on the
action of forskolin. In the present study, we show that
the PTK inhibitors tyrphostin A23 and genistein abolished the action of
forskolin on Na+ transport but
that other PTK inhibitors, lavendustin A and AG-1295, did not influence
the action of forskolin on Na+
transport. Lavendustin A and AG-1295 are more specific inhibitors of
PTK involved in epidermal growth factor and platelet-derived growth
factor receptors, respectively (13, 33) than tyrphostin A23 (14) or
genistein (1). A previous report by Hagiwara et al. (8) indicates that
although lavendustin A (10 µM, the same concentration as that used in
the present study) had no significant effects on basal
Isc, lavendustin
A diminished the action of insulin on
Na+ transport. That report (8)
suggested that lavendustin A at the concentration of 10 µM is
effective in rat FDLE cells. Namely, lavendustin A is effective in rat
FDLE cells; nevertheless, lavendustin A does not affect the action of
forskolin on Na+ transport.
AG-1295 showed an effect similar to that of lavendustin A, although
AG-1295 slightly showed an inhibitory action on basal Isc. These
observations suggest that a nonreceptor type of PTK might be involved
in the signaling of forskolin to the stimulation of
Na+ transport. Although we have to
perform further experiments to determine which type of PTK is involved
in forskolin signaling, the present study suggests a role for PTK in
the forskolin-induced regulation of
Na+ transport as the first step in
the study.
To more directly test our hypothesis, we may apply pure PTK to the
cytosolic surface of the patch membrane containing the 28-pS NSC
channel. As indicated in a previous report by Ito et al. (11), the
stimulatory action of forskolin (or cAMP) on the 28-pS NSC channel is
due to recruitment of the 28-pS channel and/or regulatory proteins to
the apical membrane from the intracellular store site. Therefore, we
could not test whether PTK mimics the effect of forskolin (or cAMP) on
the recruitment of the 28-pS NSC channel and/or regulatory proteins to
the apical membrane from the intracellular store site in cell-free
excised inside-out patches. However, to reach a complete conclusion
indicated in the present study, we have to perform further experiments
to determine which type of PTK is involved in the signaling pathway of
cAMP and whether the channel itself is phosphorylated in tyrosine.
Role of forskolin-induced cell volume in PTK
activity. In rat FDLE cells, cAMP and forskolin cause
cell shrinkage by stimulating KCl efflux under isosmotic conditions
(e.g., Refs. 25, 26). Several studies have reported that cell shrinkage
or swelling by the change in extracellular osmolality affects ion
channel and/or transporter activity by activating kinases and/or
phosphatases (15, 31) that regulate ion channels and/or transporters
(20, 27). However, it is still unknown how the change in cell volume activates membrane-bound kinases and/or phosphatases. A recent study
(36) has reported that chlorpromazine, which is known to cause membrane
deformation, can mimic hyposmotic stress-increased tyrosine
phosphorylation in cardiac myocytes, suggesting that the change in
membrane tension may play a key role in the activation of PTK. In
Chinese hamster ovary cells, hyperosmotic stress-caused cell shrinkage
strongly induces tyrosine phosphorylation (37), indicating that the
initial signal is a change in cell volume but not in the intra- or
extracellular osmotic concentration. On the other hand, several reports
(e.g., Refs. 7, 15) have indicated that cytosolic
Cl
plays an important role
in the regulation of ion transport by activating protein kinases. Cell
shrinkage causes a decrease in cytosolic
Cl
concentration (for a
review, see Ref. 17), which plays an important role in the regulation
of ion transport (3, 23-25, 34, 38). Therefore, it is expected
that a decrease in cytosolic
Cl
concentration or cell
volume caused by forskolin may activate protein kinases, including
PTKs, which regulate Na+ transport
in rat FDLE cells. This PTK-mediated pathway is a novel signaling
pathway to regulate Na+ transport
by forskolin (and cAMP) in rat FDLE cells.
In conclusion, the present study in rat FDLE cells indicates that
1) forskolin activates a PTK by
causing cell shrinkage in a cAMP-dependent but not a PKA-dependent
pathway, 2) the activated PTK
stimulates the 28-pS amiloride-blockable NSC channel, and 3) this stimulation of the NSC
channel causes an increase in amiloride-sensitive Na+ transport (Fig.
11).

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Fig. 11.
A hypothesized scheme of forskolin-induced regulatory pathway of
amiloride-blockable Na+ transport
(channels) through a PTK-dependent pathway. (+), Stimulatory effect;
( ), inhibitory effect; PKA, cAMP-dependent protein kinase:
PKA#, PKA with basal activity;
PKA*, forskolin-activated PKA;
PTK#, PTK with basal activity;
PTK*, forskolin-activated PTK. Na+
channel is fully activated by
PKA#. PKA* does not activate
Na+ channel anymore; i.e.,
forskolin does not activate Na+
channel. Furthermore, Na+ channel
is also fully activated by PTK#;
therefore Na+ channel is not
activated anymore by PTK*. NSC channel is activated by forskolin. This
activation is not mediated through a PKA-dependent pathway but through
a PTK-dependent pathway. Stimulatory action on NSC channel is mediated
through PTK* caused by cell shrinkage, which is induced by
forskolin-stimulated KCl efflux.
|
|
 |
ACKNOWLEDGEMENTS |
This work was supported by Grants-in-Aid from the Medical Research
Council of Canada (Group Grant), the Kidney Foundation of Canada, and
the Ontario Thoracic Society (Block Term Grant) to Y. Marunaka.
 |
FOOTNOTES |
N. Niisato and Y. Ito are Research Fellows of the Hospital for Sick
Children (Toronto, ON) and were supported by Research Fellowships from
The Research Training Center, The Hospital for Sick Children Research
Institute. Y. Marunaka is a recipient of Scholar Award of the Medical
Research Council of Canada and Premier's Research Excellence Award.
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: Y. Marunaka, Lung Biology, Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail:
marunaka{at}sickkids.on.ca).
Received 23 December 1998; accepted in final form 24 May 1999.
 |
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