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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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 beta -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.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
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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
<IT>NP</IT><SUB>o</SUB> = <FR><NU><LIM><OP>∑</OP><LL><IT>i</IT>=1</LL><UL><IT>N</IT></UL></LIM> (<IT>i</IT> · <IT>T</IT><SUB><IT>i</IT></SUB>)</NU><DE><IT>T</IT><SUB>T</SUB></DE></FR>
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 [gamma -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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.

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).

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.

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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 Omega  · cm2 (n = 8 experiments); tyrphostin A23 (100 µM, 60 min), 1,787 ± 354 Omega  · 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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DISCUSSION
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