pH-regulated chloride secretion in fetal lung epithelia

Carol J. Blaisdell, Rebecca D. Edmonds, Xi-Tao Wang, Sandra Guggino, and Pamela L. Zeitlin

Eudowood Division of Respiratory Sciences, Department of Pediatrics, and Department of Medicine, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The fetal lung actively transports chloride across the airway epithelium. ClC-2, a pH-activated chloride channel, is highly expressed in the fetal lung and is located on the apical surface of the developing respiratory epithelium. Our goal was to determine whether acidic pH could stimulate chloride secretion in fetal rat distal lung epithelial cells mounted in Ussing chambers. A series of acidic solutions stimulated equivalent short-circuit current (Ieq) from a baseline of 28 ± 4.8 (pH 7.4) to 70 ± 5 (pH 6.2), 114 ± 12.8 (pH 5.0), and 164 ± 19.2 (pH 3.8) µA/cm2. These changes in Ieq were inhibited by 1 mM cadmium chloride and did not result in large changes in [3H]mannitol paracellular flux. Immunofluorescent detection by confocal microscopy revealed that ClC-2 is expressed along the luminal surface of polarized fetal distal lung epithelial cells. These data suggest that the acidic environment of the fetal lung fluid could activate chloride channels contributing to fetal lung fluid production and that the changes in Ieq seen in these Ussing studies may be due to stimulation of ClC-2.

ClC-2; chloride channels; rat


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACTIVE CHLORIDE AND FLUID secretions across the airway epithelium are essential for normal lung morphogenesis to occur (2, 18). The molecular identity and regulation of the chloride channel(s) responsible for fetal lung chloride transport are largely unknown. Ion transport in the fetal lung is dominated by chloride secretion, whereas sodium absorption predominates in postnatal airways (9, 34). There are several chloride conductances that have been characterized in the mammalian lung, including the cAMP-dependent cystic fibrosis transmembrane conductance regulator (CFTR) (3), the outwardly rectifying chloride channel (12), the Ca2+-dependent chloride channel (10), the purinergic receptor-mediated Ca2+-activated chloride channel (8), and the voltage- and volume-regulated ClC family of chloride channels (15). CFTR is expressed in the fetal lung (20); however, chloride transport, fetal lung fluid secretion, and lung morphogenesis are independent of this cAMP-activated chloride channel (19). In the CFTR knockout mouse, the lungs are not severely affected because alternative Ca2+-activated chloride transport is elevated (24).

We have shown previously that a member of the ClC family of chloride channels (ClC-2) is abundantly expressed in the fetal lung and is downregulated at birth (22). We have speculated that it may contribute to fetal lung chloride secretion because it is immunolocalized along the luminal surface of developing airways (22). Overexpression of ClC-2 cDNA and antisense knockout in a cystic fibrosis (CF) airway epithelial cell line demonstrated that ClC-2 chloride currents can be activated by acidic pH (26). Because endogenous ClC-2 chloride currents have not been reported, we sought to identify pH-activated currents in primary polarized monolayers of fetal rat distal lung epithelial (FDLE) cells. We studied the electrophysiological responses of 18-day FDLE cells in modified Ussing chambers to pH 6.2, 5.0, and 3.8 on the luminal surface. We found that there are pH-activated, cadmium chloride (CdCl2)-inhibited chloride currents. These pH-stimulated short-circuit currents were not inhibited by diphenylamine-2-carboxylic acid (DPC), DIDS, or glibenclamide. In contrast to hypotonic cell swelling, acidic pH did not significantly increase [3H]mannitol flux rates. Because fetal lung fluid is normally acidic, with a pH of 6.3 (1), our data would suggest a regulatory mechanism for fetal lung chloride secretion. We also demonstrate by confocal microscopy that ClC-2 is abundantly expressed along the apical surface of these cultured lung epithelial cells. Because it is a pH-regulated channel, we speculate that ClC-2 contributes to fetal lung chloride secretion.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of primary FDLE cells. Sprague-Dawley rats were obtained from Harlan (Indianapolis, IN). Eighteen-day-gestation timed-pregnant dams were euthanized with carbon dioxide, and the fetuses were recovered by hysterectomy. Lungs from a single litter were pooled and placed in ice-cold Hanks' balanced salt solution (HBSS). Primary cells were isolated using a protocol modified from O'Brodovich et al. (23) as previously described (6). Tissue was rinsed three times with cold HBSS, trachea and bronchi were removed, and whole lung segments were minced to 1-mm3 pieces. Tissue was digested with 0.125% trypsin (GIBCO BRL, Gaithersburg, MD) and 20 µg/ml DNase (Worthington Biochemical; Freehold, NJ) in HBSS at 37°C, filtered through a 70-µm nylon cell strainer (Becton Dickinson; Franklin Lakes, NJ), and centrifuged to collect dissociated cells. Cells were resuspended and incubated in 0.1% collagenase and 20 µg/ml DNase in Ham's F-12 (Mediatech; Herndon, VA) for 20 min at 37°C and then pelleted. Isolated cells were resuspended in Ham's F-12 containing 10% fetal calf serum, penicillin, streptomycin, and Fungizone, and grown on an uncoated flask for 0.5 h and again for 1 h to enrich for epithelial cells. Cell viability was assessed by trypan blue exclusion using an inverted Olympus microscope and was greater than 90%.

Cell culture. Primary epithelial cells were seeded onto Transwells (Corning Costar; Cambridge, MA) at 1.0 × 106 cells/cm2 and grown at 37°C in defined fetal epithelial cell medium consisting of Ham's F-12, 7.5 µg/ml endothelial cell growth supplement, 5 µg/ml insulin, 7.5 ng/ml transferrin, 0.1 µM hydrocortisone, 15 µg/ml bovine pituitary extract (Collaborative Biomedical; Bedford, MA), and 20 ng/ml cholera toxin (List Biological Laboratories; Campbell, CA) as previously described (6). Keratinocyte growth factor (KGF) was added at 10 ng/ml unless otherwise indicated (Sigma; St. Louis, MO). Primary cells were fed the next morning and maintained in 5% CO2-95% air at 37°C in a Napco incubator.

Electrical characterization of monolayers. Cell monolayers grown on Transwells were polarized and had formed tight junctions (R > 300 Omega  · cm2) by 48 h when evaluated by a World Precision Instruments epithelial voltohmmeter (EVOM) (New Haven, CT). These were mounted in modified Ussing chambers (Diffusion Chamber System, Costar) and bathed in a nominally HCO-3-free Ringer solution of (in mM) 140 NaCl, 2.3 K2HPO4, 0.4 KH2PO4, 1.3 CaCl2, 1.2 MgCl2 · 6H2O, 10 HEPES, and 5 glucose (pH 7.4) as previously described (34). A heat block maintained the temperature of the bath at 37°C. Solutions were mixed by bubbling 5% CO2-95% air from a gas manifold into the bathing chambers on either side of the membranes. Silver-silver chloride electrodes were maintained in saturated KCl glass barrels that terminated in a ceramic tip. Transepithelial potential difference (Vte), short-circuit current (Isc), and transepithelial resistance (Rte) were determined with a DVC-1000 voltage-current clamp (World Precision Instruments, Sarasota, FL). The Vte and Isc of the Transwell polycarbonate membrane were 0 mV and 0 µA/cm2, respectively. Rte of the membrane alone was 30 Omega  · cm2 and was not subtracted from measurements. All measurements of Vte were referenced to the submucosal solution. To ensure that stable intact monolayers were present, monolayers with a resistance <150 Omega  · cm2 or with unstable bioelectric parameters over 10-20 min in Ringer solution were discarded. All measurements were corrected for minimal electrode potential difference offsets and fluid resistance.

During measurement of transepithelial electrical properties, open-circuit potential difference (Vte) was recorded continuously. At 1- to 5-min intervals, Vte was clamped to 0 and Isc was determined. In short-circuit conditions, a voltage pulse of 10 mV was applied. The change in current was used to calculate Rte. The equivalent short-circuit current (Ieq) was calculated by the equation Ieq Vte/Rte. Chloride-free current (IClf) is reported when current measurements were made in chloride-free conditions because short-circuit currents assume no chemical gradient. All data are expressed as means ± SE. Means were compared by Student's t-test for unpaired data. Differences were considered significant for P < 0.05.

Drugs and solutions. To increase the driving force for chloride secretion across the epithelium toward the lumen, the Ringer solution was replaced in the apical bath with a chloride-free, gluconate-substituted Ringer solution (in mM and pH 7.4): 142 sodium gluconate, 4 potassium gluconate, 2 calcium gluconate, and 5 HEPES. To lower apical pH, the chloride-free Ringer was adjusted with acetic acid to pH 6.2, 5.0, and 3.8.

Responses to hypoionic medium alone were also examined by diluting the ionic strength of the Ringer solution to 60% while maintaining osmolality with sucrose and comparing this to hyposmotic Ringer diluted to 60% with water alone. Electrophysiological responses were measured every minute for 5 min, and pH remained stable for the duration of each exposure. The following stock solutions were prepared so that the final concentration of the vehicle was <1%: 100 mM CdCl2 dissolved in distilled water, 100 mM DIDS dissolved in DMSO, 300 mM DPC dissolved in DMSO, 10 mM glibenclamide dissolved in ethanol-DMSO, 100 µM ionomycin dissolved in 95% ethanol, 100 µM isoproterenol prepared in basolateral Ringer bath, and 1 mM propranolol dissolved in water. All drugs were obtained from Sigma with the exception of DPC (Aldrich; Milwaukee, WI) and isoproterenol (Elkin-Sinn; Cherry Hill, NJ). All drugs were added to the apical bath with the exception of isoproterenol and propranolol, which were added to the basolateral bath.

Paracellular flux of mannitol. Cells were grown on Transwells for 48 h. To measure paracellular flux, 5 µCi of [3H]mannitol and 5 mM unlabeled mannitol were added to Ringer solution in the basolateral compartment and 5 mM unlabeled mannitol was added to Ringer solution in the apical compartment. After incubation of the cells for 1 h at 37°C, 5-µl aliquots from the apical and basolateral compartments were collected for 10 min in symmetrical Ringer and again over 5 min following replacement of chloride-free solutions in the apical bath with pH 7.4, 6.2, 5.0, and 3.8. Radioactivities were measured, and flux rates were calculated in nanomoles per hour per square centimeter.

Confocal microscopy. Eighteen-day FDLE cells were seeded at 1.0 × 106 cells/cm2 onto 0.4-µm pore size polyethylene terephthalate track-etched membrane cell culture inserts (Becton Dickinson) and grown at 37°C in FDLE cell-defined medium as described in Isolation of primary FDLE cells. Cells were confluent after 48 h in culture and fixed in 4% paraformaldehyde-0.1 M PBS, pH 7.4, in parallel with cells used for electrophysiological measurements above. Cells were stored in PBS for up to 14 days at 4°C. All subsequent steps were performed at room temperature. Cells were washed in fresh PBS several times, permeabilized in 0.2% Triton X-PBS (vol/vol), and rinsed in PBS. Nonspecific sites were blocked with 5% goat or donkey serum in PBS before incubation overnight with anti-ClC-2 chicken serum diluted 1:100 in PBS. Cells were then rinsed with PBS several times and incubated with goat anti-chicken Texas red or donkey anti-chicken Cy3 (Jackson ImmunoResearch; West Grove, PA) diluted 1:100 in PBS. Cells were counterstained with Hoechst 33342 nuclear stain (Molecular Probes; Eugene, OR) diluted 1:3,000 in PBS, and mounted with Fluoromount (Southern Biotechnology Association; Birmingham, AL). Monolayers were examined using a Zeiss laser-scanning microscope (LSM 410).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

pH-stimulated IClf inhibition by CdCl2. Cell monolayers forming tight junctions by 48 h in culture (Rte >300 Omega  · cm2 by EVOM) were mounted in Ussing chambers. The basolateral and apical surfaces were bathed in balanced Ringer solution (described in Electrical characterization of monolayers) at 37°C. Recordings were made if Vte was hyperpolarized more than -1.0 mV and Rte more than 150 Omega  · cm2. Average baseline Ieq was 9.5 ± 1.2 µA/cm2 (n = 13). To enhance our ability to measure active chloride transport, Ringer was replaced with chloride-free (gluconate) solution in the apical bath. In a chloride-free apical bath, pH 7.4, the IClf increased on average to 28 ± 4.8 µA/cm2 (n = 13). Acidification of the luminal bath by replacement of the neutral chloride-free with acidic chloride-free solutions stimulated IClf to 70 ± 5.0 µA/cm2 by 1 min at pH 6.2 (P < 0.0001 compared with maximun IClf, pH 7.4) to 114 ± 12.8 µA/cm2 at pH 5.0 (P < 0.007 compared with maximun IClf, pH 6.2), and to 164 ± 19.2 µA/cm2 at pH 3.8 (P < 0.041 compared with maximun IClf at pH 5.0; n = 13, Fig. 1). The Ieq 1 min after replacing the chloride-free, pH 3.8 solution with Ringer was 88.35 ± 29.1 µA/cm2 (n = 9). The pH values of the apical bath after perfusion with solutions of pH 7.4, 6.2, 5.0, and 3.8 were 7.125 ± 0.03, 6.43 ± 0.04, 5.38 ± 0.045, and 4.1 ± 0.03, respectively (n = 4).



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Fig. 1.   Effect of low luminal pH on bioelectric measurements in polarized 18-day fetal rat distal lung epithelial (FDLE) cells cultured in presence (A) and absence (B) of keratinocyte growth factor (KGF). Cells were mounted in Ussing chambers and equivalent short-circuit current (Ieq), transepithelial potential difference (Vte), and transepithelial resistance (Rte) were measured in response to bilateral Ringer and apical chloride-free solutions (pH 7.4) as described in METHODS. Reductions in pH activated chloride-free current (IClf) at pH 6.2, 5.0, and 3.8 (final luminal pH 6.4, 5.4, and 4.1, respectively), and this was inhibited by 1 mM CdCl2 if cells were grown in KGF (A), but this inhibition was lost if cells were not exposed to KGF (B).

To examine the properties of the pH-stimulated IClf in fetal lung epithelial cells, 1 mM CdCl2, a ClC-2 chloride channel blocker (13, 26) was applied to the apical bath. Monolayers grown in the presence of KGF (which increases ClC-2 expression) (6) had a CdCl2-sensitive inhibition of pH-activated IClf (Fig. 1A) at pH 5.0 and 3.8 (P < 0.035 and P < 0.002, respectively), but CdCl2 inhibition was lost when KGF was excluded from the growth medium (Fig. 1B, P > 0.05 at each pH). In addition, KGF-treated cells maintained higher resistances more uniformly after 48 h. Thirteen of 13 monolayers (3 litters) grown in KGF had a Vte more negative than -1.0 and Rte > 150 Omega  · cm2, whereas only 6 of 13 untreated monolayers (3 litters) met these criteria. CdCl2 inhibited the pH-stimulated hyperpolarization of open-circuit Vte in KGF-treated cells (P > 0.05, Fig. 1A). Without KGF, CdCl2 had no effect on IClf, Vte, and Rte (P > 0.05, Fig. 1B).

Effects of drugs known to modulate chloride transport across fetal airway epithelia. We also examined the effect of three additional chloride channel inhibitors to characterize the pH-activated IClf in fetal airway epithelial cells. The maximal change in IClf at each acidic pH compared with maximal IClf measured in the chloride-replaced, pH 7.4 apical bath in control cells (no blocker) was compared with cells after the addition of each blocker (Fig. 2). DPC, which blocks CFTR (27) and is a partial blocker of ClC-2 (32), at 300 µM (5) did not inhibit pH-activated IClf (P > 0.05 by t-test, Fig. 2A). DIDS, which does not block ClC-2 (32) but does inhibit ClC-3 (33), ClC-5 (30), and Ca2+-dependent chloride channels (10), at 100 µM (5) also had no statistically significant inhibition of pH-stimulated currents (P > 0.05 by t-test, P = 0.08 at pH 3.8, Fig. 2B). Glibenclamide, which is an inhibitor of CFTR (28), at 100 µM also did not inhibit IClf with any pH change (P > 0.05 by t-test, Fig. 2C). Therefore, this pattern of response to the chloride channel blockers is most consistent with the pattern expected of ClC-2.


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Fig. 2.   Effect of apical chloride channel blockers on maximal pH stimulation of IClf from chloride-free Ringer, pH 7.4, baseline. There was no significant inhibitory effect (P > 0.05) with apical application of 300 µM diphenylamine-2-carboxylic acid (A, DPC), 100 µM DIDS (B), and 100 µM glibenclamide (C). n, No. of monolayers.

To determine whether cAMP- or Ca2+-mediated chloride secretion participated in or could augment pH-stimulated IClf, the effects of the beta -adrenergic receptor blocker propranolol, beta -agonist isoproterenol, and Ca2+ agonist ionomycin were examined in FDLE cells mounted in Ussing chambers. The maximal IClf of FDLE monolayers bathed with apical chloride-free solutions was plotted for control cells vs. treated cells at pH 7.4, 6.2, 5.0, and 3.8 (Fig. 3). Preincubation of monolayers with basolateral 1 µM propranolol did not influence pH-stimulated IClf, suggesting that cAMP-mediated pathways were not important to this type of current activation, even though beta -adrenergic receptors are present in the fetal lung (4). Similarly, stimulation of cAMP with 100 µM isoproterenol did not augment baseline (pH 7.4) or acidic (pH 6.2, 5.0, 3.8) IClf (Fig. 3). Maximal activation of intracellular Ca2+ with 1 µM ionomycin in the apical bath failed to significantly enhance IClf stimulated by low pH (P > 0.05 compared with control, Fig. 3). Therefore the pH-activated IClf measured in these experiments does not reflect a major contribution by the cAMP- or Ca2+-mediated signaling pathways.


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Fig. 3.   cAMP- and Ca2+-mediated effects on pH-stimulated IClf. Drugs were applied as described in METHODS. There was no significant inhibition of IClf with basolateral 1 µM propranolol or significant stimulation of IClf with 100 µM isoproterenol or 1 µM ionomycin (P > 0.05; control, n = 5; propranolol, n = 4; isoproterenol, n = 4; and ionomycin, n = 3).

Volume-activated Ieq is not inhibited by CdCl2. To examine the effects of hypoionic medium independently of hyposmotic stress on fetal distal lung epithelial cells, electrophysiological measurements were made in Ussing chambers with symmetrical Ringer by replacement of the apical bath with hypoionic-isosmotic Ringer, followed by hypoionic-hyposomotic Ringer (Fig. 4). Ieq increased from 7.4 ± 1.3 µA/cm2 in Ringer (measured, 283 mosM) to 20.1 ± 3.2 µA/cm2 when exposed to 60% hypoionic-isosmotic apical bath solution (measured, 265 mosM) and to 56.1 ± 14.5 µA/cm2 when exposed to 60% hypotonic Ringer solution (measured, 162 mosM; Fig. 4). Resistance decreased from a baseline of 498 ± 56.5 Omega  · cm2 to 116 ± 22.8 Omega  · cm2 in hypotonic Ringer, similar to results from acidic pH-exposed monolayers. Ieq, Vte, and Rte in response to hypoionic and hyposmotic apical solutions were not significantly affected by 1 mM CdCl2 (P > 0.05).


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Fig. 4.   Electrophysiological responses of polarized 18-day FDLE cells exposed to changes in ionic strength and 60% hypotonicity in apical bath. Ieq was stimulated significantly by hypotonicity (P > 0.05 for control, n = 9, vs. CdCl2, n = 9) and Rte was significantly reduced. These effects were not inhibited by CdCl2.

Paracellular flux of mannitol. The decreases in resistance identified in acidic pH-exposed monolayers raises the concern that increases in Ieq may be due to paracellular chloride transport. We compared [3H]mannitol flux rates in monolayers exposed to increasingly acidic pH and those exposed to hypotonic stress (50% Ringer) under similar conditions as studied in Ussing chambers (Fig. 5). Hypotonicity induced significantly higher flux rates (36.7 ± 22 nM · h-1 · cm-2) than did acidic pH (8.5 ± 1.5 nM · h-1 · cm-2; Fig. 5, P = 0.002) over the same period of time, suggesting that increases in Ieq and decreases in Rte in response to acidic extracellular pH have a smaller component of paracellular chloride transport than does hypotonic stress. Fourfold increases in mannitol flux at pH 3.8 compared with Ringer suggests there may be both transcellular and paracellular flux in response to extreme acidic luminal pH, but this did not reach statistical significance (P = 0.07).


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Fig. 5.   Effect of acidic and hypotonic solutions on paracellular flux of mannitol in 18-day FDLE cells grown in presence of KGF on permeable supports. Paracellular flux of mannitol was monitored over 10 min in Ringer and over 5 min in chloride-replaced solutions (pH 7.4, 6.2, 5.0, and 3.8) as used in Ussing chamber experiments (n = 6). Apical surface of cells was exposed to 50% hypotonic Ringer over similar time course (30 min, n = 6). Hypotonicity induced greater paracellular flux of mannitol (P < 0.05 at each time point) than low pH. Acidic pH of 3.8 induced more paracellular flux of mannitol compared with control Ringer but this did not reach statistical significance (P = 0.07).

Localization of ClC-2 in polarized fetal lung epithelia. ClC-2 channels are expressed along the luminal surface of developing fetal lung (22) and are regulated by pH (16, 26). Because acidic pH stimulated IClf in 18-day FDLE cells mounted in Ussing chambers, we examined ClC-2 protein expression in polarized FDLE cells using an anti-COOH-terminal ClC-2 serum (21, 22). Confocal microscopy revealed that ClC-2 is abundantly expressed in primary fetal lung cells. ClC-2 expression is predominant along the cell membrane as demonstrated in red in Fig. 6a (X-Y plane), but there is no membrane staining in a comparable section with Texas red secondary antibody alone (Fig. 6b). The Z plane shows that ClC-2 immunoreactivity is concentrated on the apical surface of polarized FDLE cells (Fig. 6c) and not visible on the basolateral surface. Figure 6d with secondary Texas red antibody alone shown also in the Z plane demonstrates nonspecific staining only. Figure 6e also shows ClC-2 expression in the cell membrane using a Cy3 secondary antibody. Competition of the primary antibody with a ClC-2 COOH-terminal fusion protein (22) eliminated staining of the luminal membrane (Fig. 6f). The specificity of this ClC-2 antibody has been reported previously (22). ClC-2 expression was not uniform in all cells examined as might be expected in nonclonal, primary epithelial cells.


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Fig. 6.   Confocal microscopy demonstrating ClC-2 immunoreactivity (shown in red) in membrane of polarized FDLE cells; green is Hoescht 33342 nuclear counterstain. a: X-Y plane, ClC-2 polyclonal antibody with Texas red secondary antibody. b: X-Y plane, Texas red secondary antibody alone. c: Z plane, apical localization of ClC-2 with no basolateral membrane staining. d: Z plane, Texas red secondary antibody alone. e: X-Y plane with ClC-2 polyclonal antibody and Cy3 secondary antibody. f: X-Y plane demonstrating competition of ClC-2 membrane staining with ClC-2 fusion protein, Cy3 secondary antibody. Bar, 10 µm.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

When cultured on a porous substrate, FDLE cell monolayers exhibit the spontaneous bioelectric properties of an epithelium with active ion transport (5, 23, 25). Of these bioelectric properties, the equivalent current is of particular interest because it provides an index of the transport properties of the epithelium. In the presence of chemical gradients such as low chloride on the apical side of an epithelium, most of the transport of ions measured in the IClf will be due to movement of chloride across the epithelium. Our results suggest that the acidic environment of the fetal lung fluid (1) could have an important role in regulating ion transport across the developing respiratory epithelium by activating ClC-2.

We have demonstrated that acidic luminal fluid activates chloride currents in polarized primary fetal lung epithelial cells. This activation is immediate and is stable over 5 min. Exposure of these primary cells to KGF was essential for CdCl2 inhibition of pH-activated chloride secretion. Currents could be activated by low pH in KGF-free cultures but were not inhibited by CdCl2, suggesting another pH-dependent pathway. KGF is important for cell differentiation (11) and enhancement of active ion transport (7). In this study, primary fetal lung epithelial cell tight junctions were not as stable when assembled in KGF-free medium. We have previously shown that KGF increases the expression of ClC-2 mRNA and protein in FDLE cells (6), and, therefore, the CdCl2 responsiveness of KGF-treated cells suggests that ClC-2 may contribute to chloride secretion. Cadmium-sensitive pH-activated changes in Ieq were likely largely transcellular because paracellular flux of [3H]mannitol was significantly less in response to acidic luminal pH than to hypotonic stress.

When lipid bilayers are fused with gastric parietal cell membrane vesicles (17), chloride currents have characteristics of ClC-2. The pH activation of ClC-2 has been demonstrated also in whole cell patch-clamp studies of CF bronchial epithelial cells transfected with ClC-2 cDNA (26) and by single-channel analysis in Xenopus oocytes (16). With extracellular acidic pH, ClC-2 currents can be activated at resting potentials (16), whereas initial descriptions of ClC-2 suggested that nonphysiological hyperpolarization or cell swelling was required for activation (32). Previous studies have demonstrated that CdCl2 can inhibit ClC-2-like currents (13, 26) similar to the CdCl2-sensitive pH-activated currents found in this study. DPC partially inhibits ClC-2 (32) in single-channel studies but had no effect on pH-activated currents in polarized fetal lung cells in this study. DIDS is a chloride channel blocker of other ClC family members (30, 33) but does not inhibit ClC-2 (32), consistent with this study, which suggests that ClC homologs do not contribute to pH-activated currents. Glibenclamide blocks CFTR (28) but had no effect in this study, suggesting that CFTR does not play a significant role in pH-activated chloride currents in FDLE cells. There is some controversy about the protein kinase A (PKA) effects on ClC-2 (14, 16, 17). In lipid bilayers, rabbit ClC-2 currents are enhanced by the addition of the catalytic subunit of PKA to the cytosolic side of the membrane (17); however, in the oocyte, the cAMP agonist forskolin had no effect on inwardly rectifying rabbit ClC-2 currents (14). Our studies in rat cells did not uncover any contribution to pH activation by cAMP-dependent pathways. Isoproterenol did not enhance, nor did propranolol inhibit, acidic pH stimulation of Isc. In addition, pH-activated currents were not further stimulated by the Ca2+ ionophore ionomycin.

We have shown previously that ClC-2 is most abundantly expressed along the luminal surface of fetal lung airways (22). Confocal microscopy confirmed the hypothesis that ClC-2 is also expressed along the apical surface of polarized cultured respiratory epithelia, such as FDLE cells examined in Ussing chambers. In vivo measurements of alveolar pH in the developing fetal lamb have shown this fluid to be acidic (pH 6.3). Although extremely acidic lung fluid may be nonphysiological, we have demonstrated the potential for pH-activated currents with luminal pH as low as 5.0 and 3.8, which is not due to significant disruption of tight junctions as demonstrated by mannitol flux experiments. Our Isc experiments in polarized fetal distal lung epithelia demonstrate pH-activated, CdCl2-sensitive chloride currents, which is consistent with the inhibition of ClC-2 currents by CdCl2 by single-channel patch-clamp analysis (26). Cadmium sensitivity appears on treatment with KGF, and ClC-2 expression is increased by KGF (6). These findings along with the apical localization of ClC-2 by confocal microscopy strongly suggest that ClC-2 contributes to the pH-sensitive currents in the developing lung. This would be important for normal lung development to occur because it is dependent on chloride and fluid secretion. There has been little information about the molecular identity of fetal lung chloride channels responsible for normal lung morphogenesis. CFTR is not essential for normal lung development because CF individuals are born with normal lung morphology (31), the CFTR knockout mouse has normal lungs at birth (29), and lung cysts from fetal CF lungs have non-CFTR-dependent chloride and fluid secretion (19).

Strategies to circumvent the defects in chloride secretion due to mutations in CFTR might include regulation of alternative chloride channels in respiratory epithelia. ClC-2 is an attractive candidate because the distribution of ClC-2 overlaps with CFTR in the lung. Jordt and Jentsch (16) have identified two putative pH-sensing regions of ClC-2. Deletions of the amino-terminal domain of ClC-2 and mutations in the cytoplasmic loop between domains D7 and D8 constitutively open ClC-2 and sensitivity to pH is abolished (16). Pharmacological agents that mimic pH activation of ClC-2 could be designed to provide therapeutic alternatives for the treatment of diseases of ion transport such as CF.

This study is the first to demonstrate endogenous ClC-2-like currents in airway epithelia and suggests that the fetal lung may be an appropriate model to study ClC-2 modulation. Further investigation of gating properties of ClC-2 and study of its regulation will be important for the development of new therapies to treat CF lung disease.


    ACKNOWLEDGEMENTS

We appreciate the review of this manuscript by Dr. William B. Guggino.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant KO8-HL-03469 (C. J. Blaisdell); Cystic Fibrosis Foundation, Zeitli96PO (P. L. Zeitlin); and Eudowood Foundation support to the Eudowood Division of Pediatric Respiratory Sciences.

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 and present address of C. J. Blaisdell: University of Maryland, Bressler 10-021, 655 W. Baltimore St., Baltimore, MD 21201 (E-mail: cblaisdell{at}som.umaryland.edu).

Received 11 May 1999; accepted in final form 19 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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