Loss of CFTR Chloride Channels Alters Salt Absorption by Cystic Fibrosis Airway Epithelia In Vitro

Molecular Cell, Vol. 2, 397–403, September, 1998, Copyright 1998 by Cell Press

Loss of CFTR Chloride Channels Alters Salt Absorption by Cystic Fibrosis Airway Epithelia In Vitro Joseph Zabner,1,7 Jeffrey J. Smith,2,7 Philip H. Karp,1,3 Jonathan H. Widdicombe,5 and Michael J. Welsh1,3,6 1 Department of Internal Medicine 2 Department of Pediatrics 3 Department of Physiology and Biophysics 4 Howard Hughes Medical Institute University of Iowa College of Medicine Iowa City, Iowa 52242 5 Cardiovascular Research Institute University of California at San Francisco San Francisco, California 94143

Summary Cystic fibrosis (CF) is caused by the loss of functional CFTR Cl2 channels. However, it is not understood how this defect disrupts salt and liquid movement in the airway or whether it alters the NaCl concentration in the thin liquid film covering the airway surface. Using a new approach, we found that CF airway surface liquid had a higher NaCl concentration than normal. Both CF and non-CF epithelia absorbed salt and liquid; however, expression of CFTR Cl2 channels was required for maximal absorption. Thus, loss of CFTR elevates the salt concentration in CF airway surface liquid and in sweat by related mechanisms; the elevated NaCl concentration is due to a block in transcellular Cl2 movement. The high NaCl may predispose CF airways to bacterial infections by inhibiting endogenous antibacterial defenses.

Introduction In the genetic disease cystic fibrosis (CF), the loss of functional CFTR Cl 2 channels disrupts Cl2 transport across epithelia (Welsh et al., 1995). However, there is little knowledge of how loss of CFTR alters salt transport in CF airways, a major site of disease. Airway epithelial ion transport has most often been evaluated with large volumes of liquid on both surfaces and with transepithelial voltage held constant at zero, the short-circuit current (Isc) condition. Under these conditions, CFTRdependent current is due to Cl 2 secretion, and amiloride-sensitive current (Isc(Amil)) is due to Na1 absorption. In CF, disruption of Cl2 transport is measured as defective Cl 2 secretion, and increased activity of apical Na1 channels is measured as an increase in Isc(Amil) (Boucher et al., 1986; Stutts et al., 1995; Welsh et al., 1995). Under more physiologic conditions, the normal epithelium absorbs both Na1 and Cl2; however, the consequences of absent transcellular Cl 2 transport and increased Na1 channel activity in CF are uncertain. For example, in CF, 6

To whom correspondence should be addressed (e-mail: mjwelsh @blue.weeg.uiowa.edu). 7 These authors contributed equally to this work.

salt and liquid absorption have been proposed to be increased, decreased, or equal to normal (Boucher et al., 1986; Jiang et al., 1993; Smith et al., 1994; Quinton, 1994; Welsh et al., 1995; Zhang et al., 1996). Likewise, estimates of airway surface liquid (ASL) ion concentrations have produced widely disparate results (Gilljam et al., 1989; Joris et al., 1993; Knowles et al., 1997). Limited knowledge of liquid and salt transport and ASL NaCl concentration ([NaCl]) in the airway are due to several factors. Model systems with air-covered apical surfaces that resemble in vivo airways have been difficult to develop and study. In addition, attempts to study CF in vivo are complicated by chronic airway infection and inflammation that could alter ion transport and ASL electrolyte composition from values that exist very early in the disease. Technical limitations are also a significant obstacle; the volume of ASL is very small and the process of removing and measuring ASL may change its composition. Touching the epithelium with a filter paper, an ion-selective electrode, or a bronchoscope as is required with current approaches could damage or irritate the epithelium, thereby altering ASL composition, ion transport, and gland secretion. For example, with an ASL depth of 20 mm, 1 cm2 of epithelium would be covered by 2 ml. Several studies used filter paper to collect ASL, although this method also draws liquid from the basolateral surface (Erjefa¨lt and Persson, 1990). One study collected 1–10 nl from distal trachea and found higher ASL [NaCl] in CF than non-CF (Joris et al., 1993). Three studies removed larger volumes of ASL; two found similar [NaCl] in non-CF and CF (Knowles et al., 1997; Hull et al., 1998), and we found inconsistent results between CF and non-CF nasal liquid (Smith et al., 1996). Additional studies using aspiration (Gilljam et al., 1989) or tracheas filled with liquid (Goldman et al., 1997) reported higher salt concentrations in CF. The current lack of understanding on how ASL composition is regulated is particularly frustrating because altered salt transport may be responsible for the clinical hallmark of CF airway disease, chronic infections. NonCF and CF ASL contain several antibacterial factors, including b-defensins and lysozyme (Fleming, 1922; Smith et al., 1996; Zhao et al., 1996; Goldman et al., 1997; McCray and Bentley, 1997). Activity of these factors is inhibited by high salt concentrations. Earlier work suggested that a high [NaCl] in CF inhibits ASL antibacterial factors, and this inhibition predisposes the CF airway to infection (Smith et al., 1996). A key feature of this hypothesis is the assumption that CF ASL has an abnormally high [NaCl]. Results NaCl Concentrations in ASL Because knowledge of the salt concentration at the air interface is critical to understanding CF airway disease, we developed a new radiotracer method to measure ASL [Na] and [Cl]. We studied primary cultures of human airway epithelia grown at the air-liquid interface under

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Figure 1. Na1 and Cl2 Concentrations in ASL (A–C) Comparison of [Na], [Cl], and volume in non-CF and CF ASL. n 5 15 for [Na], n 5 16 for [Cl], and n 5 31 for volume. Asterisk indicates p , 0.001. (D) Expression of CFTR corrected the ASL [Na] in CF epithelia. Treatment of CF epithelia with an adenovirus encoding CFTR increased bumetanide-sensitive Isc (Isc(Bumet)) to 8.3 6 1.1 mA 3 cm22 from 0.1 6 0.03 in CF epithelia treated with an adenovirus expressing b-galactosidase. n 5 6 non-CF, 6 CF, and 6 CF treated with Ad/CFTR. Asterisk indicates p , 0.01 compared to non-CF epithelia and double asterisk indicates p , 0.01 compared to CF epithelia treated with Ad/bGal. (E) Inhibiting Isc(Amil) increased ASL [Na] in non-CF epithelia. Epithelia were studied in the presence of benzamil in the medium or serum-free medium. Isc(Amil) was 20.3 6 2.01 mA 3 cm22 for control epithelia and 0.5 6 1.2 mA 3 cm22 for epithelia treated with serumfree medium. n 5 9 control, 9 benzamiltreated, and 9 serum-free epithelia. Asterisk indicates p , 0.01 compared to control epithelia.

conditions in which they differentiate, develop a ciliated apical surface, and retain transepithelial electrolyte transport by CFTR and Na1 channels (Yamaya et al., 1992; Zabner et al., 1996). To measure ASL [Na], we added 22Na to the basolateral medium together with 3 H2O. After the tracer content of ASL reached equilibrium, we removed ASL by rinsing the apical surface with 100 ml of medium. The basolateral medium was also sampled and its [Na] was measured. The ratio of 22 Na to 3H 2O in each compartment allowed us to calculate ASL [Na]. We performed similar experiments with 36Cl. There were two key methodological details. First, after isotopes were added, non-CF and CF epithelia were studied at the same time and sealed in the same chamber. Second, the sealed container was humidified with water that had the same specific activity of 3H 2O as the culture medium. In this way, water vapor over the surface of epithelia contained the same ratio of 3H 2O to H 2O as that in ASL and basolateral medium. Figures 1A and 1B show that in non-CF epithelia the ASL [Na] was 50 6 4 mM and [Cl] was 37 6 6 mM, concentrations lower than those in the basolateral medium where [Na] was 156 mM and [Cl] was 135 mM. ASL covering CF epithelia had [Na] and [Cl] that were increased compared to non-CF, even though the basolateral medium was identical. We also found that nonCF and CF epithelia had the same volume of ASL (Figure 1C). The measured volume corresponds to an average ASL depth of approximately 20 mm, in excellent agreement with microelectrode and microscopic measurements (Johnson et al., 1993; Wu et al., 1998). When we expressed wild-type CFTR in CF epithelia, the ASL [Na] decreased into the normal range (Figure 1D). Thus, the increased salt concentration in CF is due to the loss of CFTR function and not to some other factor. Conversely, when we inhibited Na1 channel activity with

benzamil, an amiloride analog (Kleyman and Cragoe, 1988), the ASL [Na] increased (Figure 1E). We also incubated epithelia in serum-free medium for 2 days before the start of the study; this maneuver inhibits Na1 current without altering CFTR-dependent current (see below, unpublished data, and Zabner et al., 1997). Figure 1E shows that this intervention also increased the ASL [Na]. These data indicate that both CFTR Cl2 channels and Na1 channels are required to maintain ASL [NaCl] in the normal range. Relationship between Isc(Amil) and Liquid Absorption At first inspection, the high [NaCl] in CF ASL may seem inconsistent with reports that the Isc(Amil) is increased across CF epithelia (Boucher et al., 1986). However, Isc(Amil) only measures the rate of active transepithelial Na1 transport in the absence of any transepithelial ion or voltage gradients; thus, there is no requirement for Cl2 to balance charge. To examine the relationship between Isc(Amil) and liquid absorption, we varied Isc(Amil) by incubating epithelia without serum, with serum, or with serum plus cAMP agonists (20 hr). These treatments resulted in low, intermediate, and high values of Isc(Amil) but had minimal effects on transcellular Cl 2 transport (Figure 2A). We then placed 60 ml (a depth of z1 mm) of isosmotic liquid on the apical surface and measured liquid absorption. In non-CF epithelia, as Isc(Amil) increased liquid absorption increased (Figure 2B). In contrast, in CF epithelia, as Isc(Amil) increased liquid absorption increased initially but then plateaued. Thus, at high values of Isc(Amil), CF epithelia showed a defect in liquid absorption. This result indicates that CFTR Cl 2 channels are required for maximal liquid absorption. To test this conclusion further, we expressed CFTR in CF epithelia and measured electrical properties and liquid absorption. CFTR increased Cl2 transport as measured in an Ussing

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Figure 2. Effect of Modifying Isc(Amil) on Rate of Liquid Absorption (A) Amiloride-sensitive and bumetanide-sensitive Isc (indices of transcellular Na1 and Cl2 transport, respectively) in non-CF and CF epithelia. Before study, airway epithelia were cultured either in the absence of serum for 4 days, in the presence of serum (Basal), or in the presence of serum plus cAMP agonists (10 mM forskolin and 100 mM IBMX) for 20 hr. cAMP agonists were present during the study. Data are from epithelia shown in (B). For each individual experiment (each point in [B]), 3 epithelia were used to measure electrical properties; bars show mean 6 SEM from the epithelia studied. (B) Relationship between Isc(Amil) and liquid absorption for non-CF (left) and CF (right) epithelia. Prior to study, epithelia were cultured under basal conditions (closed circles), 20 hr of cAMP agonists (open circles), or serumfree medium (diamonds). Each point represents mean 6 SEM for Isc(Amil) (n 5 3) and liquid absorption (n 5 4–8); some SEM bars are hidden by symbols.

chamber but had little effect on Isc(Amil) (Figure 3A). Importantly, expression of CFTR increased liquid absorption (Figure 3A). Figure 3B shows that inhibition of Na1 transport with benzamil inhibited liquid absorption in both non-CF and CF epithelia. This result is consistent with data in Figure 2B showing that when Isc(Amil) is near zero liquid absorption is inhibited. These findings indicate that transcellular pathways both for Na1 and for Cl2 are required for normal liquid absorption. Transepithelial Cl2 Transport When we measured liquid absorption as described above, absorption was isosmotic; after 4 hr, the measured osmolality of the apical liquid was 331 6 3 mOsm (n 5 8) in non-CF epithelia, 332 6 1 mOsm in CF (n 5 9), and 332 6 1 mOsm in the basolateral medium. These results are consistent with the high water permeability of airway epithelia (Folkesson et al., 1996). When taken together with the ability of non-CF epithelia to absorb more liquid, these findings suggested that non-CF epithelia should have a higher rate of salt absorption. To test this directly, we measured unidirectional 36 Cl fluxes in epithelia treated chronically with cAMP agonists. Consistent with the liquid absorption measurements, net Cl2 absorption was greater in non-CF than in CF epithelia (Figure 4). Discussion The data show that in an in vitro model, CF epithelia have a defect in liquid and salt absorption that is corrected by expression of CFTR. This abnormality leads to an elevated [NaCl] in ASL. Thus, both normal sweat duct and normal airway epithelia can lower the luminal [NaCl], and in CF this process is defective. The consequences of losing CFTR function are most readily appreciated in the sweat gland (Quinton, 1990).

In the normal sweat gland duct, apical Na1 channels and CFTR Cl 2 channels absorb salt across the duct epithelium. Because liquid cannot follow through the water-impermeable epithelium, the [NaCl] in the duct lumen falls. In CF, the lack of CFTR blocks transcellular absorption of Cl2. The demands of electroneutrality also prevent absorption of Na1. As a result, the sweat [NaCl] is high, a diagnostic hallmark of the disease. How does CFTR influence liquid absorption and [NaCl] in airway epithelia? First, consider CF epithelia. Active Na1 absorption occurs through the cell, with Na1 entering through amiloride-sensitive, apical Na1 channels and exiting via the basolateral membrane Na-K-ATPase (Welsh et al., 1995). Because the apical membrane of CF epithelia is Cl2 impermeable, Cl2 absorption must be primarily through the paracellular pathway in response to the transepithelial voltage. However, paracellular pathways (Bradley and Purcell, 1982; Schneeberger and Lynch, 1992) usually have a Na1 to Cl2 selectivity greater than 1. Thus, the relative paracellular Na1 to Cl 2 selectivity will determine, in part, the rate of liquid absorption and the transepithelial [NaCl] gradient that can be maintained. In contrast to CF, in normal epithelia the presence of CFTR in the apical membrane and an incompletely defined Cl2 channel in the basolateral membrane (Willumsen et al., 1989) provide an additional transcellular Cl2-selective pathway that allows for more liquid absorption when apical volume is large and a lower ASL [NaCl] when the ASL volume is small. The ability of sweat duct epithelia to reduce apical [NaCl] is explained by its water impermeability; water cannot osmotically follow salt absorption and therefore the apical [NaCl] falls to low values. In contrast, airway epithelia have a significant water permeability (Folkesson et al., 1996). How then can airway epithelia generate transepithelial ion concentration gradients? There must be some force to counter osmotic pressure generated

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Figure 4. Cl 2 Fluxes across Non-CF and CF Epithelia Open squares indicate basolateral to apical flux and closed squares apical to basolateral flux. Lines are linear least-squares fit. Net absorption (dashed line) is difference between the two unidirectional fluxes. Net absorption was greater in non-CF than CF epithelia, p , 0.0001. n 5 12–18. In some cases, SEM bars are hidden by symbols.

Figure 3. Effect of Expressing CFTR and Inhibiting Na1 Transport on Liquid Absorption (A) Expression of CFTR in CF epithelia increases the rate of liquid absorption. CF epithelia were treated with adenovirus expressing b-galactosidase (Ad/bGal) or CFTR (Ad/CFTR) before study. All epithelia were treated with cAMP agonists for 20 hr before study as well as during study. Asterisks indicate p , 0.001; in each group n 5 6 for Isc and 21 for absorption. Note that expression of CFTR increased liquid absorption even though Isc(Bumet) increased to values only 1/6 those in non-CF epithelia studied under similar conditions (compare with Figure 2A). (B) Inhibition of Na1 transport with benzamil inhibits liquid absorption. Epithelia were studied under basal conditions (closed circles) or following 20 hr treatment with cAMP agonists (open circles). Benzamil (1 mM) was included in apical and basolateral solutions as indicated. Each data point is mean from 4–6 epithelia; in some cases SEM bars are hidden by symbols. Basal Isc was 39.7 6 3.1 mA 3 cm22 in non-CF epithelia and 61.3 6 19.0 mA 3 cm22 in CF epithelia; following treatment with benzamil, Isc decreased to 8.6 6 1.4 mA 3 cm22 in non-CF and 20.2 6 0.4 mA 3 cm22 in CF epithelia.

by the ion concentration difference. Impermeable osmolytes in the small volume of ASL might be responsible. In addition, the air–liquid interface introduces surface tensions not present in liquid-covered epithelia. As suggested by Widdicombe and Widdicombe (1995), capillary forces might oppose a transepithelial osmotic pressure. Once osmotically driven absorption lowers ASL to the tips of the cilia, the closely packed cilia and microvilli could generate a large surface tension countering the osmotic pressure and thereby preventing further liquid absorption. Thus, the level of ASL would not fall below the tips of cilia. In addition, an apical mucus gel (Basbaum and Finkbeiner, 1989) could hold water in the lumen; as liquid is absorbed, capillary pressure would increase and counter an ionic osmotic pressure. With ASL volume effectively prevented from decreasing to zero by one or a combination of these forces, active epithelial transport could reduce the ASL [NaCl]. These considerations are consistent with our finding that nonCF and CF epithelia have the same volume of ASL.

The data show that the lack of a cellular Cl2 conductive pathway is responsible for the increased ASL [NaCl] in CF. Thus, while active amiloride-sensitive Na1 transport is necessary to generate a low ASL [NaCl] and liquid absorption, in the absence of CFTR, Isc(Amil) is not directly related to the rate of liquid absorption. Perhaps the simplest way to think about this is to consider that an epithelium could have a significant Isc(Amil), but if there were no cellular or paracellular pathways for Cl 2 flow then there would be no net transepithelial movement of Cl2, Na1, or H2 O. In other words, the demands of electroneutrality only permit net Na1 movement if there is also a pathway for anion movement. Our approach to measuring ASL [NaCl] has several advantages: non-CF and CF epithelia were studied simultaneously under identical conditions; we measured the ratio of tracers after rapidly collecting ASL so that we minimized the chance that altered epithelial transport or ASL composition would influence our results; there was no infection or inflammation that could secondarily alter ASL ion concentrations; we studied well-differentiated, ciliated epithelia; and the epithelial surface was covered with air and a thin film of liquid throughout the study. A limitation of this study is that it is done in vitro. This model does not have submucosal glands, which are present in vivo in large airways. In this respect, our model may more closely resemble the distal airways, which lack submucosal glands but which are an important site of disease in CF. Although at present it is not clear how to measure ASL ion composition in vivo without altering either ion transport or the collected sample, our in vitro measures should provide a reasonably accurate reflection of conditions at the apical surface. Our results may also explain discrepant findings in earlier reports. Previous work suggested that liquid absorption by non-CF epithelia was less than or similar to CF epithelia (Jiang et al., 1993; Smith et al., 1994). These earlier studies by our laboratories were done at a time when airway epithelia were being cultured under conditions that did not produce well differentiated, ciliated epithelia, and the epithelia did not have a substantial Isc(Amil). Our data show that only in epithelia with a high Isc(Amil) is liquid absorption greater in non-CF epithelia. Thus, an important conclusion is that depending on absolute

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values of Isc(Amil) (as shown by the curves in Figure 2B), CF epithelia may have a lower, similar, or higher rate of liquid absorption than non-CF. The physiologic significance of this in different airway regions is not known. Finding that the ASL [NaCl] is elevated in CF supports a mechanism by which dysfunction of the CFTR Cl2 channel may contribute directly to the pathogenesis of airway infections; the high [NaCl] may inhibit antibacterial factors in ASL (Smith et al., 1996; Goldman et al., 1997; McCray and Bentley, 1997). The data also suggest that interventions that increase apical membrane Cl2 conductance could have a beneficial effect; such maneuvers might include, for example, activation of other apical Cl 2 channels with agents such as inhaled nucleotides (Knowles et al., 1991), enhancing mutant CFTR function (Rubenstein and Zeitlin, 1998), incorporation of exogenous Cl 2 channels into the apical membrane (ElEtri and Cuppoletti, 1996), or, as we show here, gene transfer of CFTR (Flotte et al., 1993; Hyde et al., 1993; Crystal, 1995). Experimental Procedures Epithelial Culture Airway epithelial cells were isolated from nasal, tracheal, and bronchial tissue obtained from 7 CF and 12 non-CF people. Cells were seeded onto collagen-coated, semi-permeable membranes (0.6 cm2 Millicel-HA; Millipore, Bedford, MA) and grown at the air–liquid interface as previously described (Yamaya et al., 1992; Smith et al., 1996; Zabner et al., 1996). Culture medium, a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (DME/F12), was supplemented with 2% Ultroser G (BioSepra; Villeneuve, France) and initially with 100 mU/ml penicillin, 100 mg/ml streptomycin, 50 mg/ml gentamicin, 15 mg/ml colimycin, 125 mg/ml ceftazidime, and 2 mg/ml fluconazole. Basolateral culture medium was changed every 2–4 days. All epithelia were studied at least 14 days after seeding when they had differentiated. All epithelia were evaluated with scanning electron microscopy for the development of a ciliated apical surface. Measurement of Transepithelial Electrical Properties For measurement of transepithelial electrical properties, epithelia were mounted in Ussing chambers and studied as previously described (Smith et al., 1994; Zabner et al., 1996). Epithelia were bathed in symmetrical solutions containing (in mM): NaCl 135, K 2HPO4 2.4, KH2 PO4 0.6, CaCl2 1.2, MgCl 2 1.2, dextrose 10, and HEPES 5 (at pH 7.2, 378C), and gassed with 100% O2. Isc(Amil) is the decrease in current after apical addition of 10 mM amiloride or benzamil. Bumetanide-sensitive Isc (Isc(Bumet)) is the decrease in current after basolateral addition of 100 mM bumetanide to epithelia studied in the presence of apical 10 mM amiloride and basolateral cAMP agonists (10 mM forskolin plus 100 mM 3-isobutyl 1-methylxanthine, IBMX); Isc(Bumet) is a measure of the transepithelial Cl2 transport pathway that includes CFTR. Measurement of ASL [NaCl] The basolateral medium (500 ml) of epithelia was spiked with 2.5 3 104 cpm of 3H2 O and 22Na or 36Cl and then placed in a sealed chamber containing a water-saturated atmosphere of 5% CO2 in air. Water used for chamber humidification was labeled with the same specific activity of 3H2O to ensure that at equilibrium the ratio of labeled to unlabeled water would be identical in the water vapor, culture medium, and ASL. Non-CF and CF epithelia were always studied at the same time in the same chamber. Preliminary studies indicated that the tracer content of ASL had reached equilibrium by 24 hr. After incubation at 378C for 48 hr, ASL was collected by rapidly rinsing the apical surface with 100 ml medium. The ratio of 22Na or 36Cl to 3H2 O was determined by liquid scintillation. Aliquots of submucosal solution were also collected for measurement of 22Na,

Figure 5. Effect of Rinse Duration on 3H2O Movement Each data point indicates n 5 18 from 3 different experiments. Lines are linear least-squares fit of the data.

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Cl, and 3H2 O, and for measurement of [Na] and [Cl] by flame photometry and chloridometry, respectively. From these measurements, we determined the 3 H2O activity per ml of water, the cpm of 22 Na per mole of Na1, and the cpm of 36Cl per mole of Cl 2 in the basolateral medium. ASL volume was calculated from the ASL 3H2 O collected divided by the ratio of 3 H2O activity per ml of basolateral medium. Na1 content was calculated from the ASL 22Na collected divided by the ratio of 22Na per mole of Na1 in the basolateral medium. ASL [Na] was calculated from the Na1 content divided by the volume of ASL. [Cl] was determined in a similar manner. Incomplete collection of ASL would not alter the calculated [Na] and [Cl] because concentrations are calculated from the ratio of 22 Na or 36Cl to 3H2 O in the single sample of ASL removed by rinsing. However, incomplete collection could cause an underestimate of ASL volume. ASL was removed with a 1 sec rinse of the apical surface. Movement of 3H2 O from basolateral to apical solution through the water-permeable epithelium during the rinse could cause an underestimate of ion concentrations. To examine the extent of 3 H2O movement during the rinse, we varied the rinse time (Figure 5). Linear extrapolation to time zero indicates that we could have underestimated [Na] and [Cl] by at most 10%. Measurement of Liquid Absorption and Cl2 Flux Liquid absorption was measured using methods similar to those previously described (Mangoo Karim et al., 1989) and modified for airway epithelia (Smith et al., 1994). Only epithelia that had a transepithelial electrical resistance $800 v 3 cm2 (EVOM; World Precision Instruments, Sarasota, FL) were used. At the start of the experiment, basolateral solution was replaced with 500 ml fresh medium containing 10 mM forskolin and 100 mM IBMX; all epithelia were treated with cAMP agonists during measurement of liquid absorption to maximally activate CFTR Cl2 channels. To the apical surface we applied 60 ml of saline containing (in mM): NaCl 137.8, KCl 4, NaHCO 3 29, CaCl2 1.2, MgCl2 0.6, and NaH2 PO4 1; osmolality of the submucosal solution was adjusted to equal that of mucosal solution using a vapor pressure osmometer (Wescor Inc., Logan, UT). After incubation for 4 hr, apical solutions were collected under mineral oil and their volume measured as previously described (Smith et al., 1994). Methods to measure Cl2 flux were similar to those used to assay liquid absorption. Saline (250 ml as described above) was applied to the apical surface. The basolateral surface was bathed in 750 ml of isosmolar medium. To measure basolateral to apical and apical to basolateral flux, 36Cl at 2 3 105 cpm/ml was added to the appropriate solution. Ten ml samples were collected from apical or basolateral solutions at indicated times. Radioactivity was measured by liquid scintillation. Interventions Serum-Free Medium Epithelia were cultured in the absence of serum for 4 days. Incubation in serum-free medium inhibits Isc(Amil) without altering CFTR function (see Figure 2A and unpublished data). For studies of liquid absorption, incubation in serum-free medium began 2 days before the start of the 48 hr study period.

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Chronic cAMP Agonists cAMP agonists (10 mM forskolin and 100 mM IBMX) were added to the medium for 20 hr before study. Chronic treatment with cAMP agonists increases Isc(Amil) (Figure 2A; unpublished data) and is similar to addition of cholera toxin to the culture medium (Yankaskas et al., 1985). During all studies of liquid absorption, cAMP agonists were present during the study to maximally activate CFTR. Benzamil Benzamil (10 mM, Research Biochemicals International, Natick, MA) was added to the basolateral solution rather than the apical surface to avoid adding liquid to or disturbing the ASL. Benzamil is more potent than amiloride at inhibiting the Na1 channel in airway epithelia (Kleyman and Cragoe, 1988; Stutts et al., 1995). When benzamil is added to the basolateral (or apical) solution of epithelia studied in Ussing chambers, it rapidly blocks amiloride-sensitive current (data not shown). Adenovirus CF epithelia were treated with a recombinant adenovirus expressing CFTR (Ad2/CFTR-16; Genzyme) or b-galactosidase Ad2/bGal-16) as previously described (Zabner et al., 1996). Epithelia were incubated with 50 MOI for 12 hr on days 3, 7, and 10 after seeding.

Joris, L., Dab, I., and Quinton, P.M. (1993). Elemental composition of human airway surface fluid in healthy and diseased airways. Am. Rev. Respir. Dis. 148, 1633–1637.

Acknowledgments

Kleyman, T.R., and Cragoe, E.J., Jr. (1988). Amiloride and its analogs as tools in the study of ion transport. J. Membr. Biol. 105, 1–21.

We thank Pary Weber, Jan Launspach, Tom Moninger, Tony Thompson, and Theresa Mayhew for excellent assistance. We especially appreciate the help of James Abbenhaus, James Flynn, E. L. Grandon, Denzel Hartshorn, Mike McCubbin, Thomas Pasic, William Portilla, Mary Prudinsky, Warren Regelman, Mary Schroth, and their associates, and the Iowa Statewide Organ Procurement Organization. We thank Genzyme and the University of Iowa Gene Transfer Vector Core (supported in part by the Carver Charitable Trust) for adenovirus vectors. This work was supported by the National Institutes of Health (HL42385, M. J. W., and HL42368, J. H. W.), the Cystic Fibrosis Foundation, and the Howard Hughes Medical Institute. J. Z. is supported by the Carver Charitable Trust. M. J. W. is an Investigator of the Howard Hughes Medical Institute.

Goldman, M.J., Anderson, G.M., Stolzenberg, E.D., Kari, U.P., Zasloff, M., and Wilson, J.M. (1997). Human b-defensin-1 is a saltsensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88, 2–9. Hull, J., Skinner, W., Robertson, C., and Phelan, P. (1998). Elemental content of airway surface liquid from infants with cystic fibrosis. Am. J. Respir. Crit. Care. Med. 157, 10–14. Hyde, S.C., Gill, D.R., Higgins, C.F., Trezise, A.E., MacVinish, L.J., Cuthbert, A.W., Ratcliff, R., Evans, M.J., and Colledge, W.H. (1993). Correction of the ion transport defect in cystic fibrosis transgenic mice by gene therapy. Nature 362, 250–255. Jiang, C., Finkbeiner, W.E., Widdicombe, J.H., McCray, P.B., Jr., and Miller, S.S. (1993). Altered fluid transport across airway epithelium in cystic fibrosis. Science 262, 424–427. Johnson, L.G., Dickman, K.G., Moore, K.L., Mandel, L.J., and Boucher, R.C. (1993). Enhanced Na1 transport in an air-liquid interface culture system. Am. J. Physiol. 264, L560-L565.

Knowles, M.R., Clarke, L.L., and Boucher, R.C. (1991). Activation by extracellular nucleotides of chloride secretion in the airway epithelia of patients with cystic fibrosis. New Engl. J. Med. 325, 533–538. Knowles, M.R., Robinson, J.M., Wood, R.E., Pue, C.A., Mentz, W.M., Wager, G.C., Gatzy, J.T., and Boucher, R.C. (1997). Ion composition of airway surface liquid of patients with cystic fibrosis as compared with normal and disease-control subjects. J. Clin. Invest. 100, 2588– 2595. Mangoo Karim, R., Uchic, M.E., Grant, M., Shumate, W.A., Calvet, J.P., Park, C.H., and Grantham, J.J. (1989). Renal epithelial fluid secretion and cyst growth: the role of cyclic AMP. FASEB J. 3, 2629–2632. McCray, P.B., and Bentley, L. (1997). Human airway epithelia express a b-defensin. Am. J. Respir. Cell Mol. Biol. 16, 343–349.

Received May 13, 1998; revised July 2, 1998.

Quinton, P.M. (1990). Cystic fibrosis: a disease in electrolyte transport. FASEB J. 4, 2709–2717.

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