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 (I sc) and transepithelial resistances were 7.9 ± 0.5 μA/cm2 and 1,018 ± 73 Ω · cm2, respectively (means ± SE;n = 12). Apical amiloride (10 μM) inhibited basalI sc by ∼50%. Subsequent addition of forskolin (10 μM) increased I sc from 3.9 ± 0.63 μA/cm2 to 7.51 ± 0.2 μA/cm2(n = 12). Basolateral bumetanide (100 μM) decreased forskolin-stimulated I sc 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 secretion, blocked the remainingI sc. Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl−- or HCO -free solutions; however, no response was seen using HCO - and Cl−-free solutions. The forskolin-stimulated I sc was inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced I sc 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 secretion across rat FDLE cells mediated via CFTR.
- short-circuit current
- swelling-activated conductance
- adenosine 3′,5′-cyclic monophosphate
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 ), to fetal transport, has not been previously elucidated. β-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 (12, 34). The influx of HCO 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 (I sc) before and after addition of forskolin, a substance known to increase intracellular cAMP levels. We then characterized 1) the contributions of Cl− and HCO to baseline and forskolin-stimulatedI sc 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 contribute to these currents by their movement through cystic fibrosis transmembrane conductance regulator (CFTR)-like channels.
MATERIALS AND METHODS
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 (R t) 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 R t ≥1 kΩ · cm2 were mounted in modified Ussing chambers connected to a transepithelial voltage clamp (Physiological Instruments, San Diego, CA) that allowed continuous measurement of the I sc(10). Changes in R t were monitored by imposing a 5-s voltage pulse (2 or 4 mV) across the monolayer every minute. R t 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 , 3.3 H2PO , 0.8 HPO , 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.
I sc 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 I sc on Cl− and HCO , NaCl or NaHCO3were replaced with equimolar amounts of sodium gluconate or Na+-HEPES, respectively. In a third set of ion substitution experiments, both Cl− and HCO were replaced with sodium gluconate and Na+-HEPES. In experiments conducted in the absence of HCO , solutions in Ussing chambers were gassed with 100% O2instead 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 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 inI sc 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, andI sc provides a direct measure of the apical membrane Cl− conductance. Spontaneously activatingI sc were tested for sensitivity to increased extracellular osmolality, achieved by adding 10 or 30 mM sucrose into both compartments. Forskolin-stimulated I sc were tested for sensitivity to glibenclamide (3–300 μM), added to the apical compartments of the Ussing chambers.
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).
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 thet-test, corrected for multiple comparisons.
Cultured rat FDLE cells demonstrate cAMP-stimulated Cl− and HCO 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 basalI sc of 7.9 ± 0.54 (means ± SE;n = 12) μA/cm2 and had anR t of 1,018 ± 73 Ω · cm2 (means ± SE; n = 12). A representative trace illustrating the effects of amiloride and forskolin on I sc across rat FDLE monolayers is shown in Fig. 1. Approximately 50% of the basal I sc 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 theI sc 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 I sc on average from 3.9 ± 0.63 to 7.51 ± 0.2 μA/cm2(n = 12), presumably by stimulating anion secretion.
Next we investigated the possible contribution of Cl− vs. HCO secretion to the basal and forskolin-stimulatedI sc across FDLE cells using inhibitors. Specifically, Cl− secretion across airway cells is driven by a basolateral bumetanide-sensitive Na+-K+-Cl− cotransporter while HCO secretion is mediated by a basolateral Na-HCO 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 I sc. On averageI sc 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 I scafter 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 ofI sc, the difference was not statistically significant. Together, these data provide pharmacological evidence for cAMP-stimulated HCO and Cl− secretion across cultured rat FDLE cells.
In subsequent studies, we tested the anion dependency ofI sc across rat FDLE cells. Specifically, we replaced bath Cl− and/or HCO with large organic anions (e.g., gluconate and HEPES). We found that the basalI sc was not affected by replacement with a HCO -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 -free solutions, pretreatment with bumetanide before forskolin abolished the forskolin-induced increase in I sc. Figure 4 also shows that addition of bumetanide after forskolin totally decreased the forskolin-induced I sc. Together, Figs. 3 and 4show that rat FDLE cells possess two components of cAMP-stimulated current that depend on both HCO and Cl−, respectively.
Figure 5 shows that forskolin had little effect on I sc when both Cl− and HCO were replaced with gluconate and HEPES, respectively. The transient increase in I sc was likely due to secretion of residual intracellular Cl− and HCO . These data indicate that the forskolin response depends on both Cl− and HCO . Figure6 summarizes and compares results from the pharmacological and ion substitution experiments demonstrating the presence of cAMP-stimulated HCO and Cl−secretion in FDLE cells.
To test the possibility that HCO entered FDLE cells as CO2, which was then converted to HCO 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 I sc (mean ΔI sc = −0.04 μA/cm2;n = 9). These data suggest that HCO 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 theI sc response to forskolin (ΔI sc) 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 (Fig. 6). In contrast, apical DIDS had no significant effect on forskolin-inducedI sc (ΔI sc = 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 I scarising from a 125 mM basolateral to a 5 mM apical Cl−“secretory” gradient. The representative current tracing in Fig.7 A shows that basolateral nystatin addition produced a small initial decrease inI sc, followed by a large spontaneous increase inI sc. On average, the I scincreased by 24.5 ± 0.9 μA/cm2 (means ± SE;n = 10). The subsequent addition of forskolin increased the I sc 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 theI sc to drop rapidly (Fig. 7 A).
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. 7 B. 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 I sc (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.8 A. The subsequent addition of forskolin to the bathing solutions significantly increased theI sc without altering the conductance of the monolayers (Fig. 8). A typical response of the forskolin-inducedI sc to glibenclamide is shown in Fig.9, while the dose-response relationship to glibenclamide is shown in Fig. 10. Glibenclamide inhibited the forskolin-inducedI sc with an IC50 of ∼25 μM. Maximal inhibition (∼85%) was seen at nearly 100 μM. Apical DIDS (200 μM) did not alter the forskolin-inducedI sc 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.
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. Figure12 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.
The main conclusions of these studies are 1) baselineI sc 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 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 exhibitedI sc that were almost completely inhibited by amiloride. Inhibitors of Na+-K+-2Cl− cotransporter, Na+-glucose cotransporters, or Cl− channels had no significant effect on I sc (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 I scdue to HCO secretion (30) and a forskolin response consisting of a transient increase inI sc due, in part, to Cl−, followed by a sustained increase due mostly to HCO secretion (8). Secretion of both Cl− and HCO 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 current following stimulation of rat FDLE cells with β-agonists. An increase in HCO 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 I sc and acetazolamide pretreatment of FDLE cells, bathed in Cl−-free solutions, had no effect on I sc suggests that HCO entered the cells via the Na+-HCO cotransporter (8,12). However, DNDS has been shown to have several other nonspecific effects that may influence the entry of HCO , 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 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 secretion, consistent with the notion that HCO 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 ΔF508-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 I sc 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.
We thank G. Davis for valuable assistance in isolating and culturing fetal lung epithelial cells.
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:).
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- Copyright © 2002 the American Physiological Society