INTRODUCTION

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THE ION TRANSPORT PROPERTIES of airway epithelia have been extensively characterized in numerous mammalian species (for review see Refs. 4 and 27). Na⁺ is typically absorbed across the apical membrane of this barrier through amiloride-sensitive epithelial Na⁺ channels (ENaC) and is actively extruded across the basolateral membrane by the Na⁺-K⁺-ATPase. Many species also possess the capability to actively secrete Cl across the airway epithelium. This mechanism typically involves entry of Cl across the basolateral membrane of the epithelial cells by Na⁺-K⁺-2Cl cotransport and exit across the apical membrane through anion channels, such as the cystic fibrosis transmembrane conductance regulator protein (CFTR) or Ca²⁺-activated Cl channels. In most species, active Na⁺ absorption quantitatively exceeds Cl secretion (4, 6). It has long been assumed that ions of opposite charge to the dominant actively transported species move across this barrier through the paracellular pathway in response to the transepithelial voltage gradient. The osmotic gradient created by the net transepithelial flow of ions thus provides the driving force for liquid movement across the airway epithelium.

Because of the inherent difficulties associated with measurement of water movement, the direction and magnitude of liquid flux across airway epithelia are often inferred from the bioelectric properties or ion fluxes across airway epithelia. Only a few studies report measurements of liquid volume flux that result from active ion transport across the native epithelium. In ferret trachea, absorptive liquid flux has been measured (18). Sheep trachea absorbs liquid by an amiloride-sensitive process, suggesting that it is driven by active Na⁺ transport (21). Canine tracheal epithelium exhibits a small basal secretion of liquid (28), whereas no measurable liquid flux occurs across bovine tracheal epithelium (8). Cultures of canine airway epithelial cells, grown on cylindrical biofibers, and human airway epithelial cells, grown on planar support, absorb liquid by an amiloride-inhibitable process (12, 14).

Several recent studies report that the concentrations of Na⁺ and Cl in airway surface liquid (ASL) are substantially lower (<90 mM) than in plasma or interstitial liquid (1, 7, 10, 16, 17). If it is assumed that no other ions or solutes contribute significantly to the luminal solution osmolality, these low Na⁺ and Cl concentrations imply that ASL is substantially hyposmotic to extracellular liquid. Maintenance of a hyposmotic ASL requires that the airway epithelium exhibits a relatively low water permeability, permitting the absorption of ions, but not water, across this barrier. This view is reinforced by studies showing that Na⁺ and Cl concentrations in ASL, collected in vivo, are higher in CF patients than in normal subjects (10, 16), suggesting that the CFTR could play an important role in absorption of Cl and maintenance of low-Cl ASL in normal airways. According to this paradigm, Cl moves transcellularly through the CFTR during absorption with the paracellular pathway being relatively impermeable to ions and water. This model seems in disagreement, however, with observations of liquid transport across airway epithelia (see above) and with reports that the hydraulic conductivity of airway epithelia is relatively high (8, 9, 19, 28).

The present study was designed to determine whether significant penetration of water occurs across porcine airway epithelium and to clarify the roles of Na⁺ and Cl transport in mediating water movement across this barrier. We report evidence that 1) significant liquid flow occurs across the intact tracheal epithelial barrier in response to osmotic gradients, 2) liquid absorption is driven by a Na⁺-dependent, amiloride-sensitive pathway, and 3) liquid absorption is unaffected by inhibitors that block the CFTR and other luminal Cl channels.

	METHODS

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Airway excision. Eighty-seven young pigs (~10-20 kg, 7-8 wk old), obtained from local vendors, were sedated with an intramuscular injection of ketamine (80 mg) and xylazine (4 mg) and killed with an intravenous overdose of pentobarbital sodium. Tracheas, 31-57 mm long, were excised and placed in Krebs-Ringer bicarbonate solution (KRB) at room temperature (25°C). The KRB bath containing the tracheas was then gradually warmed (0.1-0.2°C/min) from room temperature to 37°C.

Experimental protocol. The caudal end of each trachea was tied onto a cylindrical acrylic plug. The cranial end was tied onto another acrylic cannula that contained an 8-mm hollow bore through which solutions of various compositions could be instilled and withdrawn from the lumen. Each trachea was suspended in a warm (37°C) 600-ml KRB bath that was continuously gassed with 95% O₂-5% CO₂ to maintain tissue oxygenation and solution pH (Fig. 1). To assess the magnitude of liquid volume flux (J_V) in response to osmotic gradients, the lumen of the tracheas was filled with one of the following solutions: 1) 280 mM (isosmotic) sucrose, 2) 220 mM (hyposmotic) sucrose, or 3) 100 mM (hyposmotic) sucrose. To examine the influence of Na⁺ and Cl transport on basal liquid flux, the lumen was filled with 1) normal KRB, 2) Cl-free KRB, 3) Na⁺-free KRB, 4) normal KRB + 100 µM amiloride, an inhibitor of ENaC, 5) KRB + 100 µM diphenylamine-2-carboxylic acid (DPC), a relatively nonselective arylaminobenzoate Cl channel blocker, or 6) KRB + 300 µM 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB), a congener of DPC that reportedly expresses selectivity for the CFTR (15). The adventitial surface of the airways was exposed at all times to normal KRB. All luminal solutions contained 0.1% blue dextran as a volume marker. The luminal solution was frequently mixed by rapidly withdrawing about one-half of the instillate and reinstilling it. The instillate was sampled at regular intervals during the exposure period, which varied from 6 to 60 min.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Schematic diagram of apparatus for measurement of liquid flux across porcine tracheal epithelium. Liquid containing the blue dextran volume probe is instilled through the small bore in the upper cannula and sampled at regular intervals. Liquid flow into or out of the luminal compartment is determined by changes in luminal blue dextran concentration.

Analysis. Blue dextran absorbance of standards and samples was measured at 620 nm using a spectrophotometer (model DU 65, Beckman). The blue dextran concentration was determined from standard curves and plotted against time to determine the rate of change. From the linear slope of these data, J_V into or out of the luminal compartment was calculated and normalized to luminal surface area, estimated from the tissue dimensions.

Solution composition and drugs. KRB contained (in mM) 112 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, and 11.6 glucose in aqueous solution. To make Cl-free KRB, Cl was replaced with equimolar gluconate. For Na⁺-free KRB, Na⁺ was replaced with equimolar choline. Solution osmolalities, measured with a vapor pressure osmometer (model 5500, Wescor), were as follows: 286 ± 2 mosmol/kg (KRB), 289 ± 6 mosmol/kg (280 mM sucrose), 239 ± 1 mosmol/kg (220 mM sucrose), and 78 ± 2 mosmol/kg (100 mM sucrose). Sucrose was purchased from J. T. Baker Chemical, DPC (as N-phenylanthranilic acid) from Aldrich Chemical, and NPPB from Calbiochem; all other chemicals were purchased from Sigma Chemical.

Statistics. Values are means ± SE. Groups were compared by ANOVA. Multiple comparisons were made by Tukey's test unless comparisons were limited to a common control, in which case Dunnett's test was used. Differences were considered significant when P < 0.05. Each experiment was performed with a single trachea taken from one animal. The number of tracheas in each group is indicated by n. Four data points (1 from each of 4 treatments), which differed from the mean values of each group by >2 SD, were considered statistical outliers and excluded from analysis.

	RESULTS

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When isosmotic (280 mM) sucrose solution, nominally Na⁺ and Cl free, was placed in the tracheal lumen, a small secretion of 7.7 ± 2.2 nl · cm² · s¹ (n = 6) was observed (Fig. 2). However, when the lumen was filled with liquid that was hyposmotic to the KRB bath, a substantial and significant absorption of liquid occurred. Luminal 220 mM sucrose, which produced a measured inwardly directed osmotic gradient of 47 ± 1 mosmol/kg between the luminal solution and adventitial KRB bath, induced absorption of 34.5 ± 12 nl · cm² · s¹ (n = 7). A much larger osmotic gradient of 202 ± 3 mosmol/kg, produced by placing 100 mM sucrose solution in the luminal space, induced a similar absorptive J_V of 38.1 ± 7.3 nl · cm² · s¹ (n = 7).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Transepithelial liquid movement in response to osmotic gradients. Ordinate, rate of liquid flux (JV) into or out of the luminal compartment (positive values denote absorption; negative values indicate secretion); abscissa, measured osmotic gradient between the luminal instillate and the adventitial bath (positive values indicate an absorptive osmotic gradient). The lumen was filled with solutions containing 280 mM (isosmotic) sucrose (n = 6), 220 mM (hyposmotic) sucrose (n = 7), or 100 mM (hyposmotic) sucrose (n = 7). The adventitial surface of all tissues was bathed with normal Krebs-Ringer bicarbonate solution. *Significantly different (P < 0.05) from 280 mM (isosmotic) sucrose instillates.

When the lumen and the adventitial surfaces of the tracheas were bathed with normal KRB, absorptive J_V from the lumen was 10.2 ± 1.1 nl · cm² · s¹ (n = 34; Fig. 3). Addition of 100 µM amiloride, an ENaC inhibitor, abolished the absorptive J_V (0.9 ± 1.3 nl · cm² · s¹, n = 7). Replacement of the luminal KRB with Na⁺-free KRB also caused a significant reduction in the absorptive J_V (2.3 ± 0.7 nl · cm² · s¹, n = 8).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Effects of luminal ion substitution and channel inhibitors on liquid absorption rates across porcine tracheal epithelium. Ordinate, JV when the luminal solution consisted of normal Krebs-Ringer bicarbonate solution (KRB, n = 34), normal KRB + 100 µM amiloride (n = 7), Na+-free KRB (n = 8), normal KRB + 100 µM diphenylamine-2-carboxylic acid (DPC, n = 6), normal KRB + 300 µM 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, n = 6), or Cl-free KRB (n = 6). Positive values denote liquid absorption; negative values denote liquid secretion. *Significantly different (P < 0.05) from normal KRB instillate.

To determine whether liquid absorption was dependent on apical membrane Cl channels, the effects of two anion channel blockers were assessed. Luminal addition of 100 µM DPC, a blocker of broad specificity that should inhibit all classes of Cl channels likely to be present in airway epithelia including the CFTR, did not affect the absorptive J_V (8.2 ± 2.1 nl · cm² · s¹, n = 6; Fig. 3). Similarly, addition of 300 µM NPPB, an anion channel inhibitor that has greater selectivity for the CFTR than DPC, to the instillate had no significant effect on J_V (7.8 ± 1.6 nl · cm² · s¹, n = 6). In contrast to the anion channel blockers, luminal Cl-free KRB significantly reduced the absorptive J_V by ~60% (4.1 ± 1.8 nl · cm² · s¹, n = 6; Fig. 3).

	DISCUSSION

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This study demonstrates that water flows across native airway epithelium in response to imposed osmotic gradients. Additionally, conditions expected to inhibit transcellular Na⁺ absorption (amiloride and luminal Na⁺-free KRB) were shown to block liquid absorption, while conditions expected to block Cl absorption through transcellular Cl channels (luminal DPC or NPPB) did not. The simple interpretation of these results is that intact porcine tracheal epithelium is indeed permeable to water and that liquid absorption is normally driven by active transcellular Na⁺ absorption. Insensitivity of liquid absorption to the anion channel inhibitors suggests that Cl is absorbed across the barrier by a channel-independent pathway.

These data are consistent with a variety of studies showing that airway epithelium exhibits relatively high permeabilities to water. The hydraulic conductivities for dog trachea (28) and bovine trachea (8) fall between values reported for Necturus proximal tubule (11) and human jejunum (24), tissues that are considered to be highly conductive to water and incapable of maintaining significant osmotic gradients in vivo. This relatively large water permeability exists despite the absence of an identifiable aquaporin from the apical membrane of the epithelium (20) suggesting that the absence of these structures does not allow predictions of water impermeability. Indeed, physiological rates of liquid absorption across mouse pulmonary epithelium appear to be insensitive to aquaporin deletion (26). We conclude that porcine tracheal epithelium exhibits a relatively high water permeability and is unlikely to support appreciable osmotic gradients in the steady state.

In our study, we observed J_V of 34-38 nl · cm² · s¹ with 220 or 100 mM sucrose in the luminal space. We cannot state with certainty why such widely disparate osmotic gradients produce similar absorptive J_V. The nonlinearity of these responses could be due to the skewness of the data at the 40 mosmol/kg gradient. If two data points with unusually high J_V values were omitted, the mean J_V response at this gradient would be much closer to a linear line between 100 and 0 mosmol/kg. Alternatively, the nonlinearity might result from unstirred layers, which certainly dissipate the solute gradients across the epithelial barrier by an unpredictable amount (13).

Our results are in conflict with the theory that ASL is normally hyposmotic to extracellular liquid. For this situation to occur in the steady state, the epithelial barrier must have a very low water permeability. If it is assumed that the ASL in vivo is 30 µm thick with an osmolality of 100 mosmol/kg (~50 mM Na⁺ and 50 mM Cl), at the approximate linear rate of water absorption that we observed (40 nl · cm² · s¹), we project that the ASL would become isosmotic with the submucosal solutions in <1 min. This projection corroborates our preliminary studies with porcine small bronchi, where the 100 mM sucrose instillate became isosmolar with the KRB bath solution within 5 min (unpublished observations). Therefore, we must conclude that substantial osmotic gradients such as these cannot be maintained in ASL for prolonged periods.

Reports from other studies that ASL in vivo contains lower concentrations of ions than interstitial liquid could be due to several possible factors. One possibility is that the liquid collected from airway surfaces represents transient deviations from the steady-state conditions. Knowles and co-workers (17) reported that salt concentrations in ASL from human nasal and tracheal epithelia were similar to those in plasma, whereas the concentrations in bronchial ASL were significantly lower. When patients were given chili peppers to eat, Na⁺ and Cl concentrations in nasal ASL fell significantly. The authors concluded that glandular nasal secretions, which were stimulated by the chili peppers, were hyposmotic. They suggested the possibility that collection of the bronchial ASL by bronchoscopy evoked hyposmotic submucosal gland secretions that could have transiently affected ASL osmolality. However, findings that feline tracheal submucosal glands secrete liquid with ionic composition similar to that of plasma (22) argue against this hypothesis. Another possibility is that the water permeability in the airway epithelium is substantially lower in humans than in pigs or the other surrogate species from the studies mentioned above. We are unaware of studies reporting water permeability properties of human intact airway epithelium. However, Matsui and co-workers (19) report that the osmotic water permeability was ~10 times greater in cultured human bronchial epithelium than in Madin-Darby canine kidney cells, a renal cell line with relatively low water permeability, and showed that the bronchial epithelial cells rapidly changed shape when the apical or basolateral membrane surfaces were exposed to hyperosmotic solutions. A third explanation relates to the forces of capillarity that possibly form between the cilia when ASL depth approaches the height of the cilia. Because capillary forces may be great enough to prevent complete absorption of ASL (29), it is plausible that absorption of ions, but not water, can occur from this solution. Empirical evidence in support of this hypothesis, however, is lacking. A fourth possibility is that another as yet unidentified osmolyte is present in ASL. This was suggested by Zabner and co-workers (30), who reported that the ASL in cultures of surface epithelium were isosmotic (331 mosM) with the basolateral solutions, even though Na⁺ and Cl concentrations in the ASL were as low as 50 and 37 mM, respectively. They also reported that liquid absorption occurred under these conditions, observations that are consistent with water-permeable epithelia (30). We observed that the basal rate of liquid absorption was unaffected by the arylaminobenzoates DPC and NPPB, which should block not only CFTR but also other classes of anion channels likely to be present in the apical membrane of airway epithelia, including the Ca²⁺-activated Cl channel and the outwardly rectifying Cl channel (23). These results suggest that absorbed Cl does not cross the apical membrane through Cl-selective channels. We cannot discount the possibility that CFTR becomes downregulated in our preparation, but if this is true, persistence of absorption under these conditions also argues against the absolute requirement of apical membrane channels for Cl absorption. If porcine tracheal epithelium accurately reflects the properties of native human airway epithelium, it is difficult to reconcile how absorption of Cl is disrupted by the presence of defective CFTR in CF. A possible explanation for the variable responses and findings could be related to differences in tight junctional resistances that exist between experimental preparations. Zabner and co-workers report evidence for transcellular Cl absorption across cell monolayers with electrical resistances >800  · cm², values that are greater than the 70-500  · cm² resistances reported for intact airways from a variety of mammalian species (27). Uyekubo and co-workers (25) also report evidence for transcellular Cl absorption across cultures of bovine and human airway epithelia that exhibit resistances of 430 ± 100 and 710 ± 290  · cm², respectively. From our previous experiments (2), we estimate that the electrical resistance of excised pig trachea is ~115  · cm². Therefore, the ability to transcellularly transport Cl could possibly be related to the relative leakiness of the epithelial tight junctions. In cultured airway epithelia, in which electrical resistances are high, the paracellular pathway may serve as a substantial barrier to absorptive Cl flow; therefore, the transcellular route for Cl movement may be preferred. In native airway epithelia, where the tight junctions are comparatively leaky, Cl may favor the low-resistance paracellular pathway. This notion could explain why NPPB inhibits liquid absorption across cultures of bovine and human airway epithelial cells (25) but not, as shown in the present study, across intact porcine airway epithelium. This argument cannot account for reports that the Cl concentration in ASL from CF patients exceeds that in ASL from normal subjects (10, 16). However, findings that Na⁺ and Cl concentrations in ASL are different between CF patients and normal subjects have been disputed by some researchers. Knowles and associates (17) report no differences in ASL salt concentration between these groups, a finding that is more consistent with the results of the present study, which suggest that apical membrane Cl channels are not required for absorption of this anion.

The rate of liquid absorption was significantly reduced when Cl-free KRB was placed in the lumen but was unaffected by anion channel blockers. Several explanations are possible for this finding. One is that the replacement anion, gluconate, does not permeate apical membrane Cl channels and thereby blocks the transcellular pathway for Cl absorption. This explanation is unlikely because of the failure of the anion channel blockers DPC and NPPB to inhibit absorption. In porcine bronchi, both of these agents, at the same concentrations used in the present study, abolish acetylcholine-induced Cl, HCO, and liquid secretion from submucosal glands, a process that is likely mediated by the CFTR (3). Although we cannot rule out the possible contribution of a DPC- and NPPB-insensitive anion channel to airway liquid absorption, the data do not support a role for the CFTR. A second possibility is that Cl is absorbed across the apical membrane by a channel-independent mechanism such as an anion exchanger. We are unaware of any studies showing that such a pathway plays a significant role in ion and liquid absorption across airway epithelia. A third possibility is that luminal Cl-free solution creates a gradient for Cl efflux across the apical membrane, thus depolarizing this barrier and reducing the driving force for Na⁺ influx and absorption. Evidence for this response has been reported for rabbit trachea by Boucher and Gatzy (5). Finally, it is possible that the paracellular pathway exhibits greater permeability for Cl than for gluconate. Our observation that secretion of liquid was observed when Na⁺ and Cl were replaced with isotonic sucrose is most consistent with the last two hypotheses, which could both account for the small net secretion of ions and liquid under these conditions.

In conclusion, we report that the porcine tracheal epithelium is highly conductive to water. Under basal conditions, this tissue absorbs liquid by Na⁺-dependent active transport, whereas apical membrane Cl channels appear to play no measurable role in Cl absorption. We are hopeful that these results shed some light on the roles of these processes in the development of CF lung disease.