SPECIAL TOPIC
Alveolar Epithelial Ion and Fluid Transport
cAMP regulation of Clminus and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion across rat fetal distal lung epithelial cells

Ahmed Lazrak1, Ulrich Thome2, Carpantanto Myles1, Janice Ware2, Lan Chen1, Charles J. Venglarik3, and Sadis Matalon1,2,3,4

Departments of 1 Anesthesiology, 2 Pediatrics, 3 Environmental Health Sciences, and 4 Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, Alabama 35233


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We isolated and cultured fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses and assayed for anion secretion in Ussing chambers. With symmetrical Ringer solutions, basal short-circuit currents (Isc) and transepithelial resistances were 7.9 ± 0.5 µA/cm2 and 1,018 ± 73 Omega  · cm2, respectively (means ± SE; n = 12). Apical amiloride (10 µM) inhibited basal Isc by ~50%. Subsequent addition of forskolin (10 µM) increased Isc from 3.9 ± 0.63 µA/cm2 to 7.51 ± 0.2 µA/cm2 (n = 12). Basolateral bumetanide (100 µM) decreased forskolin-stimulated Isc from 7.51 ± 0.2 µA/cm2 to 5.62 ± 0.53, whereas basolateral 4,4'-dinitrostilbene-2,2'-disulfonate (5 mM), an inhibitor of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, blocked the remaining Isc. Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl-- or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions; however, no response was seen using HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>- and Cl--free solutions. The forskolin-stimulated Isc was inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced Isc across monolayers having nystatin-permeablized basolateral membranes. Immunolocalization studies were consistent with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in FDLE cells. In aggregate, these findings indicate the presence of cAMP-activated Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion across rat FDLE cells mediated via CFTR.

short-circuit current; amiloride; nystatin; swelling-activated conductance; immunocytochemistry; adenosine 3',5'-cyclic monophosphate


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ADULT ALVEOLAR type II cells actively absorb Na+, with this process playing an important role in limiting the extent of alveolar edema following injury to the alveolar epithelium (15, 37). In contrast, the fetal alveolar epithelium in utero actively secretes Cl- into the developing alveolar space, driving the fluid secretion necessary for fetal lung growth (16, 22). Thus compared with fetal plasma, the fetal lung liquid contains a high concentration of Cl- and almost no protein (22). Agents that increase intracellular cAMP levels increase Cl- secretion across fetal lung cultures ex vivo (2), human fetal alveolar epithelial cell monolayers in vitro (16), and promote secretion of fetal lung liquid in fetal sheep in vivo (5).

However, the possible contribution of other ions, such as bicarbonate (HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>), to fetal transport, has not been previously elucidated. beta -Adrenergic stimulation of isolated adult Clara or immortalized Calu-3 human airway cells was shown to induce electrogenic transepithelial secretion of both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (12, 34). The influx of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> into the fetal fluid may be especially important because it may increase its pH which in turn may affect a number of enzymatic functions, production of pulmonary surfactant, and even the rate of production of fetal fluid.

Herein, we isolated fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses, cultured them on permeable supports until they formed resistive monolayers (36-48 h), mounted them in Ussing chambers, and measured short-circuit currents (Isc) before and after addition of forskolin, a substance known to increase intracellular cAMP levels. We then characterized 1) the contributions of Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> to baseline and forskolin-stimulated Isc across intact monolayers and 2) the apical membrane Cl- conductances following permeabilization of the basolateral membranes using the pore-forming antibiotic nystatin. Our results indicate the presence of significant basal and cAMP-activated anion currents across rat FDLE cells and show that both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> contribute to these currents by their movement through cystic fibrosis transmembrane conductance regulator (CFTR)-like channels.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FDLE cell isolation. The isolation procedure has been described previously (23). In brief, lungs of 17- to 19-day gestation fetal rats (term = 22 days) were digested in a solution containing 0.125% trypsin and 0.4 mg/ml DNase in Eagle's minimum essential medium (MEM) for 10 min. Digestion was stopped by the addition of MEM containing 10% fetal bovine serum (FBS). Cells were collected by centrifugation and resuspended in 15 ml of MEM containing 0.1% collagenase and DNase. This solution was incubated for 15 min at 37°C. The collagenase activity was neutralized by the addition of 15 ml of MEM containing 10% FBS. The cells were plated twice for 1.5 h to remove contaminating fibroblasts. The supernatant contained epithelial cells with >95% purity. Cells were counted and seeded on permeable Transwell culture inserts (Corning, NY) with 0.33 cm2 surface area and 0.4-µm pore size. They were then seeded at a density of 5 × 104 cells per filter, cultured in DMEM with 10% FBS and 1% penicillin/streptomycin (apical and basolateral vol: 500 and 1,000 µl, respectively), and exposed to 21% O2-5% CO2 mixture in a humidified incubator for 36-48 h. The transepithelial resistance (Rt) was monitored after ~36 h in culture using an epithelial voltohmmeter equipped with chopstick-style electrodes (World Precision Instruments, Sarasota, FL).

Transepithelial transport studies. All experiments were conducted upon achievement of monolayer confluence within 36-48 h after isolation of FDLE cells. Confluent monolayers with Rt >= 1 kOmega · cm2 were mounted in modified Ussing chambers connected to a transepithelial voltage clamp (Physiological Instruments, San Diego, CA) that allowed continuous measurement of the Isc (10). Changes in Rt were monitored by imposing a 5-s voltage pulse (2 or 4 mV) across the monolayer every minute. Rt was calculated using Ohm's Law. The composition of the apical and basolateral bathing solutions was (in mM): 145 Na+, 5 K+, 125 Cl-, 1.2 Ca2+, 1.2 Mg2+, 25 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 3.3 H2PO<UP><SUB>4</SUB><SUP>−</SUP></UP>, 0.8 HPO<UP><SUB>4</SUB><SUP>2−</SUP></UP>, and 10 glucose (basolateral) or 10 mannitol (apical), pH 7.4. All solutions were gassed with 95% O2-5% CO2 using an airlift and warmed to 37°C.

Isc was allowed to stabilize before beginning each experiment (5-10 min). We then added 10 µM amiloride to the apical side of the monolayers to block Na+ absorption. Forskolin (10 µM) was added to both sides of the monolayer to evaluate possible effects of cAMP on transepithelial anion transport. To determine the dependence of Isc on Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, NaCl or NaHCO3 were replaced with equimolar amounts of sodium gluconate or Na+-HEPES, respectively. In a third set of ion substitution experiments, both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> were replaced with sodium gluconate and Na+-HEPES. In experiments conducted in the absence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, solutions in Ussing chambers were gassed with 100% O2 instead of 95% O2 and 5% CO2.

Bumetanide (100 µM) and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS; 5 mM), both instilled in the basolateral compartment, were used to inhibit the contribution of the Na+-K+-2Cl- and Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporters to the generation of anion currents (8). To avoid the generation of osmotic gradients, equal amounts of Na+ sulfate were added into the apical compartment. In an additional set of experiments, the Cl- channel blockers glibenclamide (200 µM) or DIDS (200 µM) were added into the apical compartments, and changes in Isc were recorded.

To evaluate the apical membrane Cl- conductance, monolayers were mounted in Ussing chambers under short-circuit conditions in the presence of either a basolateral to apical (125:5 mM) or an apical to basolateral (5:125 mM) Cl- gradient, and the pore-forming antibiotic nystatin (200 µM) was added into the basolateral compartment. Under these conditions, the basolateral membrane is eliminated as a barrier to the flow of monovalent ions, and Isc provides a direct measure of the apical membrane Cl- conductance. Spontaneously activating Isc were tested for sensitivity to increased extracellular osmolality, achieved by adding 10 or 30 mM sucrose into both compartments. Forskolin-stimulated Isc were tested for sensitivity to glibenclamide (3-300 µM), added to the apical compartments of the Ussing chambers.

CFTR immunolocalization. FDLE cells were grown on transparent cyclopore filters (Falcon). Monolayers were fixed in methanol at -20°C for 15 min followed by postfixation using formaldehyde (3%) in PBS for 20 min. Nonspecific protein binding was blocked with 1% (wt/vol) bovine serum albumin. Samples were treated with either a polyclonal antibody raised against the nucleotide binding domain-1 region of CFTR (a kind gift from Dr. D. Bedwell, Univ. of Alabama at Birmingham) or a monoclonal antibody against the COOH terminus of CFTR (Genzyme). The antibody raised against the NBD-1 region has been previously characterized and found to be specific for CFTR (4). Texas red X-labeled anti-mouse IgG and Oregon green-labeled anti-rabbit IgG (Molecular Probes) were used as secondary antibodies. Samples were counterstained with the nuclear dye bisbenzimide. In some cases, filters were cut and folded cell-side out during mounting to enable cross-sectional views using the technique developed by Tousson et al. (33). CFTR immunolocalization was assessed using a Lietz Orthoplan inverted epifluorescence microscope equipped with a step motor, filter wheel assembly (Ludl Electronics Products, Hawthorn, NY), and 83,000 filter set (Chroma Technology, Brattleboro, VT). Images were captured with a SenSys-cooled charge-coupled device high resolution digital camera (Photometrics, Tucson, AZ). Partial deconvolution of images was performed using IP Lab Spectrum software (Scanalytics, Fairfax, VA).

Statistical analysis. Results are expressed as means ± SE. Statistical significance among means was determined by Student's t-test (2 samples) or ANOVA followed by the Bonferroni modification of the t-test, corrected for multiple comparisons.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cultured rat FDLE cells demonstrate cAMP-stimulated Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion. After 36-48 h in culture, monolayers of rat FDLE cells were mounted in Ussing chambers and bathed on both sides with standard Ringer solutions. The monolayers generated a basal Isc of 7.9 ± 0.54 (means ± SE; n = 12) µA/cm2 and had an Rt of 1,018 ± 73 Omega  · cm2 (means ± SE; n = 12). A representative trace illustrating the effects of amiloride and forskolin on Isc across rat FDLE monolayers is shown in Fig. 1. Approximately 50% of the basal Isc was inhibited after addition of apical amiloride (10 µM). This result is consistent with the presence of electrogenic Na+ absorption via an amiloride-sensitive epithelial Na+ channel, most likely ENaC. On average, amiloride reduced the Isc from 7.9 ± 0.54 to 3.9 ± 0.63 µA/cm2 (n = 12; P < 0.01). Subsequent addition of forskolin (10 µM) to both sides of the monolayer significantly increased Isc on average from 3.9 ± 0.63 to 7.51 ± 0.2 µA/cm2 (n = 12), presumably by stimulating anion secretion.


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Fig. 1.   Representative trace showing the short-circuit current (Isc) response across fetal distal lung epithelial (FDLE) cells bathed in the presence of Ringer solution. Rat FDLE cells were isolated, grown on filters, and mounted in Ussing chambers as described in MATERIALS AND METHODS. Amiloride (Amil; 10 µM) was added to apical bathing solution of the monolayers at the time indicated to abolish the Isc due to active Na+ absorption. Forskolin (Forsk; 10 µM) was then added to both sides of the monolayer to increase intracellular cAMP. Bumetanide (Bumet; 100 µM) and 4,4'-dinitrostilbene-2,2'-disulfonate (DNDS; 5 mM) were added to the basolateral compartment. These experiments were repeated 6 times each with similar results.

Next we investigated the possible contribution of Cl- vs. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion to the basal and forskolin-stimulated Isc across FDLE cells using inhibitors. Specifically, Cl- secretion across airway cells is driven by a basolateral bumetanide-sensitive Na+-K+-Cl- cotransporter while HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion is mediated by a basolateral Na-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter that is inhibited by DNDS but not bumetanide (8). As shown in Fig. 1, addition of basolateral bumetanide (100 µM) decreased a component of the forskolin-stimulated Isc. On average Isc was reduced from 7.51 ± 0.2 µA/cm2 to 5.62 ± 0.53 (n = 6). Figure 2 further demonstrates the presence of significant forskolin-stimulated Isc after pretreatment of monolayers with bumetanide. The bumetanide-insensitive current was reduced to 3.1 ± 0.25 (n = 12) upon addition of DNDS (5 mM) to the basolateral bathing solution (Figs. 1 and 2). Although this value was lower than the amiloride-insensitive component of Isc, the difference was not statistically significant. Together, these data provide pharmacological evidence for cAMP-stimulated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl- secretion across cultured rat FDLE cells.


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Fig. 2.   Representative trace showing the Isc response across FDLE cells bathed in the presence of Ringer solution. Amiloride (10 µM) was added to apical bathing solution of the monolayers at the time indicated to abolish the Isc due to active Na+ absorption. Bumetanide (100 µM) was then added to the basolateral compartment before addition of forskolin (10 µM). Once a stable Isc was achieved, DNDS (5 mM) was added to the basolateral compartment. These experiments were repeated 6 times, each with similar results.

In subsequent studies, we tested the anion dependency of Isc across rat FDLE cells. Specifically, we replaced bath Cl- and/or HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> with large organic anions (e.g., gluconate and HEPES). We found that the basal Isc was not affected by replacement with a HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solution (Fig. 3), whereas it was reduced by Cl--free solutions (Fig. 4). However, in both cases, forskolin elicited a current, which on average was lower than that observed with normal Ringer (Figs. 3 and 4). As shown in Fig. 4, in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions, pretreatment with bumetanide before forskolin abolished the forskolin-induced increase in Isc. Figure 4 also shows that addition of bumetanide after forskolin totally decreased the forskolin-induced Isc. Together, Figs. 3 and 4 show that rat FDLE cells possess two components of cAMP-stimulated current that depend on both HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl-, respectively.


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Fig. 3.   Representative trace showing the Isc response across FDLE cells bathed in the presence of Cl--free solutions (NaCl was replaced with an equimolar amount of sodium gluconate). Amiloride (10 µM) was added to apical bathing solution of the monolayers at the time indicated to abolish the Isc due to active Na+ absorption. Once a stable baseline was achieved, forskolin (10 µM) was added to both compartments. Subsequent addition of the loop-diuretic bumetanide (100 µM) did not alter the Isc. These experiments were repeated 4 times, each with similar results.



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Fig. 4.   Representative traces showing the Isc responses across FDLE cells bathed in the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free solutions. NaHCO3 was replaced with an equimolar amount of sodium-HEPES. Top: bumetanide (100 µM) was added to the basolateral compartment before addition of forskolin (10 µM). As expected, forskolin did not increase Isc. Bottom: addition of forskolin increased Isc to a peak value, which then returned to a stable baseline. The difference between the peak and plateau may be due to transient release of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> from the cells. Addition of bumetanide totally inhibited the Isc. These experiments were repeated 4 times, each with similar results.

Figure 5 shows that forskolin had little effect on Isc when both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> were replaced with gluconate and HEPES, respectively. The transient increase in Isc was likely due to secretion of residual intracellular Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. These data indicate that the forskolin response depends on both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Figure 6 summarizes and compares results from the pharmacological and ion substitution experiments demonstrating the presence of cAMP-stimulated HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and Cl- secretion in FDLE cells.


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Fig. 5.   Representative trace showing the Isc response across FDLE cells bathed in the presence of both Cl-- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-free media. Addition of forskolin (10 µM) resulted in only a transient increase of Isc, most likely due to the movement of intracellular Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> or to any residual Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> left in the chambers. The Isc returned to its baseline value within 5 min. These experiments were repeated 5 times, each with similar results.



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Fig. 6.   Mean values (± 1 SE) are shown for the baseline Isc, response to amiloride (10 µM), forskolin (10 µM), bumetanide (100 µM), and DNDS (5 mM). All values of Isc following addition of amiloride were significantly different from their corresponding baselines. * Significantly different (P < 0.05) compared with the corresponding amiloride values; #significantly different (P < 0.05) compared with the corresponding forskolin values; +significantly different (P < 0.05) compared with the corresponding bumetanide values.

To test the possibility that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> entered FDLE cells as CO2, which was then converted to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> by the action of carbonic anhydrase, we added 100 µM acetazolamide (a carbonic anhydrase inhibitor) to both the apical and basolateral compartments of FDLE monolayers bathed in Cl--free solutions after the addition of 10 µM forskolin. Acetazolamide did not alter Isc (mean Delta Isc = -0.04 µA/cm2; n = 9). These data suggest that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> does not enter the cells as CO2.

Defining the apical membrane Cl- conductance functionally. Initially, we compared the effects of the Cl- channel blockers glibenclamide (200 µM both sides) and DIDS (200 µM apical) on intact monolayers to identify the apical membrane ion channel(s) mediating anion secretion across FDLE cells. Monolayers were bathed in symmetrical normal Ringer solutions and pretreated with a Cl- channel blocker. Glibenclamide markedly attenuated the Isc response to forskolin (Delta Isc) from 3.8 ± 0.6 (n = 12) to 0.8 ± 0.1 µA/cm2 (n = 11; means ± SE; P < 0.01). This value was not different from the response seen in the absence of both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (Fig. 6). In contrast, apical DIDS had no significant effect on forskolin-induced Isc (Delta Isc = 4.4 ± 0.4; n = 11). These findings are suggestive of the presence of CFTR.

We next evaluated the apical plasma membrane Cl- conductances by permeabilizing the basolateral membrane with nystatin (200 µg/ml) in the presence of transepithelial Cl- gradients. Initially, we measured the Isc arising from a 125 mM basolateral to a 5 mM apical Cl- "secretory" gradient. The representative current tracing in Fig. 7A shows that basolateral nystatin addition produced a small initial decrease in Isc, followed by a large spontaneous increase in Isc. On average, the Isc increased by 24.5 ± 0.9 µA/cm2 (means ± SE; n = 10). The subsequent addition of forskolin increased the Isc further, by 29.6 ± 2.3 µA/cm2 (means ± SE; n = 10). Eliminating the transepithelial Cl- gradient by increasing Cl- concentration in the apical bath to 125 mM caused the Isc to drop rapidly (Fig. 7A).


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Fig. 7.   Evaluation of the apical membrane Cl- conductance following nystatin treatment of the basolateral membranes in the presence of a 125:5 basolateral-to-apical Cl- gradient (i.e., secretory). A: representative current trace. Nystatin (200 µM) was added to the basolateral solution at the time indicated to increase the anion permeability of the basolateral membrane. Subsequently, forskolin (10 µM) was added to both compartments. Finally, we eliminated the Cl- gradient by adding 120 mM Cl- to the apical bath. These experiments were repeated 10 times with similar results. B: comparison of the nystatin-induced Isc (Delta Isc-nystatin) in the absence and presence of sucrose, added into both the apical and basolateral compartments, just before the addition of forskolin. Data represent means ± SE; number of measurements were control: n = 5; 10 mM sucrose: n = 5; 30 mM: n = 7; * P < 0.05 compared with 0 mM.

Previous studies have shown that addition of polyene antibiotics such as nystatin or amphotericin B to bathing solutions containing Cl- can cause cells to swell and thus activate plasma membrane conductances (9). Therefore, we added sucrose to both bath solutions to determine whether the nystatin-induced current across FDLE cells was swelling mediated. These data are summarized in Fig. 7B. These results show that 10 mM sucrose reduced the nystatin-activated current by >50%, whereas 30 mM sucrose abolished it. We were unable to increase Cl- conductance in intact monolayers either by fourfold decreases in bath osmolality or by isoosmotic urea (data not shown).

When the Cl- gradient was directed in the absorptive direction (i.e., 125 mM apical to 5 mM basolateral), we observed a small increase in Isc (3.7 ± 0.2 µA/cm2) following nystatin treatment and no swelling-activated conductance, which was the expected result since the basolateral solution contained the impermeant anion gluconate (9). A representative current tracing is shown in Fig. 8A. The subsequent addition of forskolin to the bathing solutions significantly increased the Isc without altering the conductance of the monolayers (Fig. 8). A typical response of the forskolin-induced Isc to glibenclamide is shown in Fig. 9, while the dose-response relationship to glibenclamide is shown in Fig. 10. Glibenclamide inhibited the forskolin-induced Isc with an IC50 of ~25 µM. Maximal inhibition (~85%) was seen at nearly 100 µM. Apical DIDS (200 µM) did not alter the forskolin-induced Isc in permeabilized monolayers (data not shown). Glibenclamide has been shown to block CFTR with an inhibition constant of ~30 µM (29). However, it can also block outwardly rectifying chloride channels (26). Fortunately, disulfonic stilbenes can be used to distinguish between these two possibilities. Extracellular DIDS blocks outwardly rectifying chloride channels (35). DIDS can also block CFTR but only from the cytosolic side (13). Thus the inhibition constant for glibenclamide observed in this study (25 µM) and lack of effect of apical DIDS provide functional evidence that a cAMP-activated glibenclamide-sensitive channel, like CFTR, mediates anion secretion across the apical membranes of FDLE cells.


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Fig. 8.   A: representative current trace from an FDLE monolayer bathed with an "absorptive" Cl- gradient (i.e., 5 mM basolateral to 125 mM apical). All other conditions are as in Fig. 7. Notice the lack of increase in Isc following the addition of nystatin into the basolateral compartment. B: comparison of the magnitudes of the nystatin- and forskolin-induced Isc in the presence of secretory (basolateral right-arrow apical) vs. absorptive (apical right-arrow basolateral) Cl- gradients. Data are means ± SE; n = 10. * P < 0.001 from the corresponding value obtained with the opposite gradient.



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Fig. 9.   Representative trace showing a dose-response inhibition of the forskolin-induced Isc by glibenclamide (3-300 µM) added at the times shown by the arrows in the apical compartment. The monolayer was bathed with an absorptive Cl- gradient (i.e., 5 mM basolateral to 125 mM apical). Changes in conductance (mS) of the monolayer following addition of apical amiloride (10 µM), basolateral nystatin (200 µM), and forskolin (10 µM) into both compartments are also shown. These experiments were repeated 5 times each with similar results.



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Fig. 10.   Dose-response relations for glibenclamide inhibition of the forskolin-induced Isc across permeabilized monolayers in the presence of an absorptive Cl- gradient (i.e., 5 mM basolateral to 125 mM apical) as described in Fig. 9. Data are means ± SE (n = 9). The line represents the best fit using regression. IC50 for the forskolin-induced current was 26 µM.

Immunocytochemical localization of CFTR in rat FDLE cells. Having obtained functional evidence for a CFTR-like channel in rat FDLE cells, we then used antibodies raised against CFTR for immunostaining. Staining consistent with CFTR was observed in rat FDLE cells using two different anti-CFTR antibodies (Figs. 11 and 12). Figure 11 compares cross-sectional views of rat FDLE that were stained with or without a monoclonal antibody raised against the COOH terminus of CFTR. In this case, the secondary antibody was Texas red X-labeled goat anti-mouse IgG. Although there was some slight background staining associated with both protocols, the overall pattern of staining was consistent with the presence of significant levels of CFTR protein in FDLE cells. Figure 12 shows a typical en face view of a rat FDLE monolayer stained with a polyclonal antibody raised against the first nucleotide binding fold of CFTR. A companion monolayer was treated with rabbit IgG to serve as a control. Oregon green-labeled goat anti-rabbit IgG was used as a secondary antibody, and the nuclei were stained with bisbenzimide.


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Fig. 11.   Photomicrographs of FDLE cells treated with a monoclonal anti-cystic fibrosis transmembrane conductance regulator (CFTR) antibody raised against the first nucleotide binding fold of CFTR (top) and nonantibody-treated control cells (bottom). Both monolayers were exposed to Texas red X-labeled secondary antibodies and counterstained with the nuclear dye bisbenzimide. The cross-sectional view was obtained by folding the filters and viewing the edge using confocal microscopy as previously described (33).



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Fig. 12.   Photomicrographs of FDLE cells treated with a monoclonal anti-CFTR antibody raised against the COOH terminus of CFTR. The secondary antibody was Texas red X-labeled goat anti-mouse IgG. The staining is consistent with the presence of significant levels of CFTR in FDLE cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The main conclusions of these studies are 1) baseline Isc across intact 19-day FDLE monolayers is mediated partly by Na+ absorption and partly by an unidentified pathway; 2) incubation of amiloride-treated FDLE monolayers with forskolin, an agent that increases cAMP levels, evokes a sustained increase in anion secretion, consisting of a combination of Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> current; 3) in the presence of a secretory Cl- gradient, permeabilization of the basolateral membrane with nystatin activates a "swelling" conductance; 4) in the presence of an absorptive Cl- gradient, permeabilization of the basolateral membrane with nystatin reveals a cAMP-stimulated glibenclamide-sensitive apical membrane anion conductance similar to CFTR; and 5) immunostaining provides further evidence for the expression of CFTR-like channels in rat FDLE cells.

Surprisingly, in a previous study, FDLE cells from 18- to 21-day gestation rat fetuses cultured on porous supports in the presence of serum and mounted in Ussing chambers exhibited Isc that were almost completely inhibited by amiloride. Inhibitors of Na+-K+-2Cl- cotransporter, Na+-glucose cotransporters, or Cl- channels had no significant effect on Isc (24, 28). On the basis of these findings, it was concluded that near-term FDLE cells actively transport Na+ through amiloride-sensitive pathways, similar to adult alveolar type II cells, but have little or no Cl- secretion. The presence of Na+ absorption and the lack of Cl- secretion were attributed to the fact that FDLE cells are cultured in room air (21%), which promotes expression of the various subunits of the ENaC protein and downregulates Cl- transporters in these cells (27). Thus differences in gestational age (17-19 days in our studies vs. 18-20 days in the previously mentioned studies) as well as differences in oxygenation due to the depth of the air-liquid interface may account for these differences. However, testing these possibilities is beyond the scope of the present study.

In other studies, significant levels of Cl- secretion occur across rat and human FDLE cells cultured in serum-free media (1, 3, 16). Because the fetal lung secretes Cl- in utero (6), one would expect that FDLE cells would also be capable of Cl- secretion as observed herein. It is interesting to note that agents increasing intracellular cAMP have also been shown to activate Cl- channels in the apical membranes of adult alveolar type II cells (11, 20).

Our results are consistent with the recently proposed model for anion secretion across Calu-3, a human airway serous cell line (8, 12). Calu-3 cells also have a basal Isc due to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (30) and a forskolin response consisting of a transient increase in Isc due, in part, to Cl-, followed by a sustained increase due mostly to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion (8). Secretion of both Cl- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> has been reported in a variety of secretory epithelia such as duodenum, pancreas (21), bile duct (36), and Clara cells (34). Marunaka et al. (14) also identified the presence of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> current following stimulation of rat FDLE cells with beta -agonists. An increase in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> flux may increase the pH of the fetal fluid, which may have important consequences both in the volume of secreted fluid and other homeostatic functions of the alveolar epithelium, such as surfactant secretion and reabsorption.

The fact that DNDS inhibited the bumetanide-insensitive fraction of the forskolin-induced Isc and acetazolamide pretreatment of FDLE cells, bathed in Cl--free solutions, had no effect on Isc suggests that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> entered the cells via the Na+-HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> cotransporter (8, 12). However, DNDS has been shown to have several other nonspecific effects that may influence the entry of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, including blocking K+ channels. Quantifying the precise effect of basolateral DNDS on rat FDLE cells and testing several alternative possibilities is beyond the scope of the present study.

We have identified two distinct anion conductive pathways in the apical membranes of FDLE cells: a cAMP-activated, DIDS-insensitive conductance, likely CFTR; and a cAMP-independent, DIDS-sensitive, swelling-activated conductance. Immunocytochemical studies also indicate the presence of CFTR at the apical membranes of FDLE cells in agreement with our functional data and earlier studies (16).

Previous studies suggest that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> ions may be secreted across the apical membranes through CFTR. For example, incubation of normal but not cystic fibrosis human airway epithelial cells with forskolin led to HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion, consistent with the notion that HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was secreted through CFTR (31). Also, Poulsen et al. (25) demonstrated the existence of 10-pS Cl- channels in forskolin-treated fibroblasts transfected with wild-type CFTR but not Delta F508-CFTR. Our observations are consistent with this precept (see below).

The swelling conductance was activated by the pore-forming antibiotic nystatin since the drug-induced pores are permeable mostly to small univalent cations and less to anions (selectivity ratio ~7:1). Hence, addition of nystatin to NaCl- or KCl-containing solutions is expected to cause cell swelling that can be prevented by addition of impermeant anions such as gluconate or sulfate into the basolateral compartment (9). This explains why we observed a significant increase in Isc across FDLE cells when the Cl- gradient was oriented from the basolateral to the apical side of monolayers and not when the gradient was oriented in the opposite direction. The ability of 30 mM sucrose to abolish the nystatin activation lends further credence to our hypothesis that this is a swelling-activated conductance, although we cannot exclude the contribution of Ca2+-activated Cl- conductances and the voltage-sensitive Cl- channels. This is consistent with the observation of both mRNA and protein expression of CIC-2, a voltage- and volume-activated Cl- channel (32), in 19-day fetal rat lung, with levels decreasing significantly after birth (19), in agreement with our functional measurements.

A number of previous studies have reported anatomically normal lungs in newborn and older human infants with cystic fibrosis, although they lacked functional CFTR (7, 17). Thus the swelling-activated conductance identified in the FDLE cells may be activated by another unidentified mechanism and play an important role in fluid secretion in utero under conditions in which normal CFTR may be lacking.


    ACKNOWLEDGEMENTS

We thank G. Davis for valuable assistance in isolating and culturing fetal lung epithelial cells.


    FOOTNOTES

This work was supported in part by National Institutes of Health Grants HL-31197, HL-51173, and P30-DK-54781.

Address for reprint requests and other correspondence: S. Matalon, Dept. of Anesthesiology, Univ. of Alabama at Birmingham, 619 19th St. S., THT 940, Birmingham, AL 35249 (E-mail: Sadis.Matalon{at}ccc.uab.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00370.2001

Received 19 September 2001; accepted in final form 27 November 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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