Characterization of Ion and Fluid Transport in Human Bronchioles

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1 Characterization of Ion and Fluid Transport in Human Bronchioles Sabine Blouquit, Hugues Morel, Jocelyne Hinnrasky, Emmanuel Naline, Edith Puchelle, and Thierry Chinet Laboratoire de Biologie et Pharmacologie des Epithéliums Respiratoires, Université Paris V, Boulogne; INSERM U514, Reims; and Institut de Pharmacologie, Paris, France The regulation of the volume and composition of airway surface liquid is achieved through epithelial ion transport processes. In humans, these processes have been characterized in proximal but not distal airways. Segments of human bronchioles were dissected from surgically removed lung pieces. The transmural potential difference of microperfused bronchioles was inhibited by luminal exposure to amiloride and increased when exposed to the Cl secretagogues forskolin and ATP in the presence of amiloride. Human bronchiolar epithelial cells were cultured on permeable supports and studied in Ussing chambers. They generated a short circuit current (Isc) that decreased in response to amiloride and increased in response to forskolin and to ATP in the presence of amiloride. In low-cl Kreb s Ringer bicarbonate, the baseline Isc and amilorideinduced decrease in Isc were not different, whereas the forskolin- and ATP-induced increases in Isc were smaller. Fluid transport measurement in excised bronchioles revealed a basal absorptive flow that was reduced by amiloride, whereas forskolin and ATP combined induced a secretory flow in the presence of amiloride. We conclude that human bronchioles actively absorb Na and fluid in unstimulated conditions and are capable of active Cl and fluid secretion when exposed to forskolin and to ATP. The mucosa of the proximal and distal airways is lined by a thin layer of liquid referred to as airway surface liquid (ASL). Maintenance of the proper depth and ion composition of this fluid is crucial for normal function of the mucociliary clearance (1, 2) and possibly for anti-infectious properties of airway mucosa (3). Epithelial ion transport processes regulate the volume and composition of the ASL, mainly through modulation of active Na absorption and active Cl secretion (1). In proximal airways numerous in vivo and in vitro studies have demonstrated that in the basal state the dominant ion transport is amiloride-sensitive Na absorption, which induces passive Cl absorption (1, 4). Active Cl secretion can be stimulated by various agents, including camp and ATP. Although these transepithelial Na and Cl movements are widely admitted, there is some controversy regarding the interrelationships between ion transport and fluid transport across the airway epithelium. One theory predicts that airway epithelial cells regulate the volume of the ASL rather than its NaCl concentration (5). The regulation of the volume of ASL is critical to optimize cilia (Received in original form April 2, 2002) Address correspondence to: Professeur Thierry Chinet, Laboratoire de Biologie et Pharmacologie des Epithéliums Respiratoires, Service de Pneumologie, Hôpital Ambroise Paré, 9 avenue Charles de Gaulle, Boulogne cedex, France. thierry.chinet@apr.ap-hop-paris.fr Abbreviations: aquaporin 3, AQP3; airway surface liquid, ASL; cystic fibrosis, CF; CF transmembrane conductance regulator, CFTR; Dulbecco s modified Eagle s medium, DMEM; short circuit current, Isc; Kreb s Ringer bicarbonate, KBR; phosphate-buffered saline, PBS; potential difference, PD; transmission electron microscopy, TEM. Am. J. Respir. Cell Mol. Biol. Vol. 27, pp , 2002 DOI: /rcmb.4869 Internet address: beating and mucus clearance. This theory is based in part on in vitro studies that have described a baseline fluid absorption across proximal airway epithelia. This fluid absorption is inhibited by amiloride, and may be reversed into net fluid secretion by forskolin and ATP (6 8). In support of this theory, several investigators have recently reported evidence for isotonic ASL and for relatively high water permeability of the airway epithelial layer (9, 10). An alternative and perhaps less widely admitted theory contends that airway epithelial cells regulate the NaCl concentration of the ASL, rather than its volume (11, 12). Indeed, some investigators have argued that the ASL has a low NaCl content, which implies a selective transepithelial absorption of NaCl but not water from ASL. This low NaCl content is postulated to activate defensins and therefore to provide antimicrobial defenses to airway surfaces. The crucial role of airway epithelial ion transport in maintaining healthy lungs is illustrated by the lung disease in cystic fibrosis (CF). Cystic fibrosis is a lethal autosomic recessive genetic disorder that results from mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) protein. These mutations lead to impairment of ion transport processes in various epithelia, including the airway epithelium. As a result, the volume and/or composition of the ASL is abnormal, leading to respiratory manifestations which include obstruction of the airways by hyperviscous secretions, chronic bacterial infection, and neutrophil-dominated inflammation, and leading progressively to fatal lung destruction (13). In distal airways, mucus plugging and obstruction are among the earliest features of CF lung disease, suggesting that regulation of ion and fluid transport across the epithelium of distal airways is important in maintaining normal lung physiology (14). Although distal airways account for a much larger part of the total surface area of airways and are a major site of pathology in diseases such as asthma, chronic bronchitis, and CF, ion and fluid transport processes have been more thoroughly characterized in proximal airways because of the relative inaccessibility of distal airways. A few studies of ion transport in distal airways have been performed in animals and most have used a microperfusion technique (15 20). Transport of fluid in distal airways has not been determined in animals or humans. As an initial attempt to study ion transport in human bronchioles, we used a microperfusion technique on excised segments of human bronchioles. We also established primary cultures of human bronchiolar epithelial cells and studied these preparations in Ussing-type chambers. In addition, we developed a system to measure fluid transport in segments of excised human bronchioles that had been opened lengthwise. We here report our findings regarding the first measurements of ion and fluid transport across human distal airway epithelium.

2 504 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL Materials and Methods Tissues and Cell Preparations Pieces of human lungs were obtained from 36 patients who underwent surgery, usually for lung cancer (34 smokers; 25 males; age: yr). The protocol was in accordance with the current French legislation. Lung pieces were transported within 3 h after surgery in Dulbecco s modified Eagle s medium (DMEM)/ Ham s F12 medium supplemented with penicillin/streptomycin (1 mg/ml), gentamycin (0.5 mg/ml), and amphotericin B (100 g/ml). Bronchioles were identified on the basis of absence of wall cartilage and outer diameter 1 mm. They were then carefully dissected free from the surrounding lung parenchyma with sharp curved scissors under a stereomicroscope (Olympus, Rungis, France). Immediately after dissection, they were placed in a dish containing phosphate-buffered saline (PBS) or culture medium and warmed to 37 C. Some fragments of excised bronchioles were selected for microperfusion studies or for fluid transport measurements and immediately processed for these studies. Other bronchioles were used to establish primary cultures of bronchiolar epithelial cells. Primary cultures were obtained using enzymatic isolation procedures derived from the method described by Yankaskas and coworkers (21). Bronchioles were incubated with 0.1% protease and 0.1% DNase in DMEM/Ham s F12 medium for 24 h at 4 C. After neutralization of these enzymes by fetal bovine serum (10%), cells were harvested by centrifugation (800 rpm for 5 min) and plated on homemade permeable collagen supports affixed to an orifice of 3.5- or 2-mm-diameter in polycarbonate cups at a density of 400,000 or 200,000 cells per cup, respectively. Cultures were grown to confluence at an air liquid interface at 37 C in a humidified atmosphere of 5% CO 2 in air. The culture medium consisted of DMEM/Ham s F12 medium supplemented with: insulin (5 g/ml), transferrin (7.5 g/ml), hydrocortisone (10 6 M), epithelial cell growth supplement (2 g/ml), EGF (25 ng/ml), triiodothyronine ( M), L-glutamine (1 mm), penicillin/streptomycin (100 g/ml), gentamycin (50 g/ml), and amphotericin B (5 g/ml). The culture medium was changed every other day. Histologic study Histologic examination was performed at the time of initial dissection for excised bronchioles and 5 d after plating for preparations of cultured epithelial cells. Preparations were fixed in 2.5% glutaraldehyde in PBS 0.1 M during 1 h, then rinsed with PBS, postfixed in 2% OsO 4, and dehydrated in graded series of ethanol. They were then embedded in Epon (Touzard et Matignon, Courtaboeuf, France). Sections 2 m thick were cut with an Ultracut E (Leica, Rueil Malmaison, France) and stained with toluidine blue. Primary bronchial cultures were also prepared for transmission electron microscopy (TEM) analysis. After Epon embedding, ultrathin sections (80 nm) were stained with uranyl acetate and lead citrate. Grids were observed using a JEOL 200 CX TEM (JEOL LTD, Tokyo, Japan) operating at 80 kv. Measurement of the Transmural Potential Difference in Microperfused Bronchioles We used a microperfusion technique derived from the one described by Al-Bazzaz and coworkers and by Ballard and coworkers (15, 16). A fragment of excised bronchiole (length: cm) was placed in a Petri dish filled with Kreb s Ringer bicarbonate (KBR) solution. Both ends of the bronchiole were tied with nylon suture 6 0 (Ethicon, Neuilly sur Seine, France) onto polyethylene microcatheters (diameter: m; Merck-Clevenot, Nogent sur Marne, France) under a stereomicroscope. The microcatheter upstream was then connected to a reservoir filled with regular KBR buffer or low-cl KBR, warmed to 37 C, and bubbled with 5%CO 2 /95%O 2. To avoid excessive hydrostatic pressure, the height of liquid in the reservoir never exceeded 10 cm H 2 O. The rate of perfusion was ml/h. The bronchiole connected to the catheters was then transferred to another Petri dish filled with KBR heated to 37 C and bubbled with 5% CO 2 / 95%O 2. The transmural potential difference (PD) of microperfused bronchioles was measured using two voltage-sensing electrodes filled with 3 M KCl-agar. The reference electrode was placed in the solution bathing the preparation and the measuring electrode was placed in the lumen of the downstream microcatheter. These electrodes were connected to a DVC1000 voltage clamp (WPI, Aston, UK). For experiments performed in low-cl conditions, tissue samples were incubated min in low-cl KBR before measurement of bioelectric variables. The following pharmacologic agents were studied: amiloride, an inhibitor of transepithelial Na transport; forskolin, an activator of the camp pathway; and ATP, which increases the intracellular Ca concentration in airway epithelial cells (22). The effects of the sequential additions of amiloride (10 5 M), forskolin (10 5 M), and ATP (10 4 M) in the perfusing solution were compared in regular KBR and in low-cl KBR. Ussing Chamber Studies At confluence, preparations of cultured cells were mounted in Ussing chambers and exposed on each surface to 10 ml of KBR solution gassed with 95%O 2 /5%CO 2. Experiments were conducted at 37 C. The short-circuit current (Isc) was monitored continuously using a DVC1000 voltage clamp (WPI, Aston, UK) and the PD was measured every 5 10 min. Voltage-sensing electrodes consisted of 3 M KCl-agar bridges, and the reference electrode was placed at the basolateral side. Current-passing bridges consisted of KBR-agar bridges. Transepithelial resistance (R) was determined by clamping the PD to 2 mv at 10-s intervals, recording the deflection in Isc, and applying Ohm s law. Cell preparations were allowed to equilibrate until stabilization of bioelectric variables took place, which required min. Basal bioelectric activity was monitored for 10 min before addition of drugs. Pharmacologic agents were added to the apical and/or basolateral bathing solutions and bioelectric activity was monitored for 5 15 min thereafter. Amiloride, forskolin, and ATP were added sequentially. Forskolin (10 5 M) was added to the apical and basolateral baths, whereas amiloride (10 5 M) and ATP (10 4 M) were added to the apical bath only. Changes in R and Isc were calculated as the variations between the values measured immediately before the addition of reagents and the values corresponding to the plateau phase after the addition of amiloride and forskolin, and corresponding to the maximal change after the addition of ATP. For experiments performed in low-cl KBR, cell cultures were incubated min in low-cl KBR before measurement of bioelectric parameters. The effects of the sequential additions of amiloride, forskolin, and ATP on cell cultures were compared in regular KBR and in low-cl KBR. Measurement of Fluid Movement across Bronchiolar Mucosa Fluid movements across bronchiolar mucosa were measured using homemade devices that comprised two nested polycarbonate supports (3.8- and 4-cm diameter dishes; Dutscher, Brumath, France). A rectangular hole (width: mm, length: 4 5 mm) was drilled in the middle of these supports. The two orifices were superimposed when the polycarbonate supports were nested. Each segment of bronchiole was cut open lengthwise and laid over the orifice of the lower polycarbonate support with its lumi-

3 Blouquit, Morel, Hinnrasky, et al.: Ion and Fluid Transport in Human Bronchioles 505 nal side on top. A small quantity of silicone (Rotisilon; Carl Roth, Karlsruhe, Germany) was deposited around the bronchiole to avoid fluid leaks. The second polycarbonate support was then fitted to the first one with its orifice facing the luminal side of the tissue, thus maintaining the opened bronchiole in place. The whole system was then placed in a 6-cm diameter polycarbonate dish filled with 8.5 ml DMEM/Ham s F12 medium, leveled to bathe the serosal side of the bronchiole. Fifty microliters of DMEM/Ham s F12 medium were then deposited on the luminal surface of the bronchiole across the orifice of the upper polycarbonate support. After incubation during 2 h at 37 C in a humidified atmosphere of 5% CO 2 in air, the luminal liquid was retrieved using a preweighed filter paper. For sampling, the filter paper was held using forceps at the edges of the orifice of the upper polycarbonate support. Care was taken to avoid touching the surface of the tissue to prevent mucosal damage. The filter paper was then weighed immediately using an electronic balance (Sartorius; OSI, Elancourt, France) to determine the quantity of liquid retrieved. This experimental procedure was repeated three times: first without any drug in the 50- l luminal liquid, then with amiloride (10 5 M) in the luminal liquid, and then with amiloride (10 5 M), forskolin (10 5 M), and ATP (10 4 M) in the luminal liquid. To correct for evaporative water loss, a second 50- l drop of DMEM/Ham s F12 medium was deposited on the surface of the upper polycarbonate support 1 cm distant from the orifice during each experiment. It was retrieved using a filter paper and its weight was measured less than 1 min after processing the liquid bathing the luminal side of the bronchiole. To correct for potential leaks of fluid across the tissue preparation each bronchiolar preparation was left mounted in the system, exposed on both sides to ethanol (70%) during 1 min at the end of the experimental sequence, and was then washed on both sides with DMEM/ Ham s F12 medium. A 50- l drop of culture medium was then deposited on the mucosa, and the serosal side was bathed with DMEM/Ham s F12 medium. The preparation was left in the incubator during 2 h, after which the luminal liquid was sampled by using a filter paper and the weight of the liquid measured. The change in volume of the luminal liquid during each experimental procedure was corrected for evaporative water loss and for leaks across the tissue preparation and was expressed in l/h/cm 2. Solutions and Drugs The composition of the regular KBR solution was: 120 mm NaCl, 0.7 mm Na 2 HPO 4, 1.5 mm NaH 2 PO 4, 2 mm CaCl 2, 0.5 mm MgCl 2, 0.45 mm KCl, 15 mm NaHCO 3, and 1 mm glucose (ph 7.3). In ion substitution experiments, all but 4 6 mm Cl was replaced with gluconate. The composition of PBS was: 140 mm NaCl, 4 mm KCl, 0.5 mm Na 2 HPO 4, 0.15 mm KH 2 PO 4, ph 7.4. All chemicals were purchased from Sigma (Saint Quentin Fallavier, France). Statistical Analysis Results are expressed as means standard error of the mean. Comparisons were made using the unpaired or paired Student t test as appropriate. A P 0.05 was considered to be significant. Results Histology Histologic sections of excised bronchioles and of cultured bronchiolar epithelial cells are shown in Figure 1. Light microscopy examination of excised bronchioles revealed intact mucosal epithelium with ciliated cells that constituted the majority of cells, and mucus-secreting cells (goblet cells) accounting for 10 20% of cells (Figure 1A). The submucosal connective tissue contained no gland and no cartilage. Primary cultures were confluent and well-differentiated, with ciliated cells and nonciliated granular cells (Figure 1B). TEM observations showed that the cultured bronchiolar cells were wellpolarized and exhibited tight junctions (Figures 1C and 1D). Potential Difference of Microperfused Human Bronchioles The transmural PD of microperfused bronchioles was measured in 16 preparations from 14 patients (age: yr). Basal PD was mv (Table 1). Addition of amiloride in the perfusate significantly inhibited the PD by 43%. Sequential additions of forskolin and ATP to the perfusing solution in the presence of amiloride increased the transmural PD by 36 and 18%, respectively. The role of anion transport was examined by measuring the transmural PD with low-cl KBR in the bath and in the perfusate. Under these conditions, the baseline PD and the amiloride-induced decrease in PD were not significantly different from the values measured in regular KBR. In contrast, neither forskolin nor ATP induced a significant change in PD in low-cl KBR. Ussing Chamber Studies Twenty-four preparations of cultured bronchiolar epithelial cells obtained from 11 patients (age: yr) were studied in Ussing chambers, 5 11 d after plating. Twelve preparations were studied in regular KBR. Their basal bioelectric properties at steady state were PD: mv; Isc: A/cm 2 ; R: cm 2 (Table 2). Addition of the Na-channel blocker amiloride caused a significant decrease in Isc (Figure 2). Addition of the -adrenergic agonist forskolin induced a significant increase in Isc. Subsequent addition of ATP resulted in a significant increase in Isc. When expressed as percent change in Isc, the effects of amiloride, forskolin, and ATP were a 46 6% decrease, a 31 7% increase, and a % increase in Isc, respectively. To evaluate the relative contribution of Cl transport, 12 additional preparations of cultured bronchiolar epithelial cells were studied in low-cl KBR. As compared with experiments performed with regular KBR, there was no significant difference in basal PD, but a significant increase in R (Table 2). Basal Isc was lower in low-cl KBR, but the difference was not statistically significant. The amilorideinduced decrease in Isc was not significantly different from that measured in preparations bathed in regular KBR. In contrast, the forskolin- and ATP-induced increases in Isc were significantly lower in low-cl KBR as compared with control conditions (P 0.02 and P 0.001, respectively). When expressed as percent change in Isc, the amilorideinduced decrease in Isc was similar in low-cl KBR and in regular KBR (respectively 40 11% and 46 6%, NS). In contrast, in low-cl KBR the percent changes in Isc after addition of forskolin and ATP were significantly lower: 5 4% in low-cl KBR versus 31 7% in regular KBR for forskolin (P 0.001) and 29 9% in low-cl KBR versus % in regular KBR for ATP (P 0.05) (Figure 2). Fluid Movement across Human Bronchiolar Mucosa Nineteen segments of human bronchioles were obtained from 12 patients (age: yr). At baseline we detected a de-

4 506 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL crease in the volume of the luminal fluid. After correction for evaporative water loss and for leaks, this decrease was l/h/cm 2 and was significantly different from zero (P 0.05). It was also significantly different from the change in volume measured in the same tissues after they had been exposed to ethanol ( l/h/cm 2 ; P 0.05). These results indicate a significant absorption of fluid across human bronchiolar mucosa in the basal state. When amiloride was added to the luminal fluid, the decrease in fluid volume was reduced to l/h/cm 2 (P 0.05 compared with basal state). Addition of forskolin and ATP on top of amiloride in the luminal fluid resulted in a large increase in volume of the luminal fluid that was l/h/cm 2 (P compared with values measured in the basal state and in the presence of amiloride only). Discussion The distal airways provide most of the surface area of the conducting airways and are therefore likely to play a major role in the regulation of the volume and composition of airway surface liquid. Until recently, direct assessment of airway epithelial transport has been limited to proximal airways because these tissues are more readily accessible for measurement of bioelectric properties and radioisotopic ion fluxes, and because primary cultures of proximal airway epithelial cells suitable for electrophysiologic studies have been established for over 15 yr (21). In contrast, the assessment of the fluid and ion transport properties of distal airways has been greatly limited because these structures are small and anatomically complex. The present study examines the bioelectric properties of human bronchiolar epithelium and provides new information on secretory and absorptive processes in the peripheral lung. To characterize electrogenic transepithelial ion transport in human distal airways, we first turned to the technique of microperfusion of excised segments of bronchioles a technique that has been applied by Al-Bazzaz and coworkers and by Ballard and coworkers to sheep and pig bronchioles (15, 16). Al-Bazzaz and coworkers provided evidence for amiloride-sensitive Na absorption and camp-activated Cl secretion in isolated perfused segments of sheep bronchioles (15, 23). Amiloride inhibited the resting transepithelial PD by 65%, suggesting that Na absorption accounts for a major part of the basal ion transport activity. Ballard and coworkers used a similar technique to study excised segments of porcine bronchioles (16). Ion substitution and drug studies suggested that porcine proximal bronchioles absorbed Na and were capable of Cl secretion. To further characterize active ion transport in bronchioles, they used cable analysis to measure PD, Isc, and R in microperfused excised airways from pigs (17). Luminal amiloride reduced the basal PD and Isc by 37 and 41%, suggesting that active Na absorption accounts Figure 1. Light micrographs of an excised human bronchiole (A) (bar: 80 m) and of a primary culture of surface bronchiolar epithelial cells grown on a collagen support (CS) to confluence at air liquid interface (B) (bar: 9 m). Transmission electron microscopy (TEM) micrograph of primary culture of human bronchiolar epithelial cells showing the well-differentiated bronchiolar epithelium with ciliated cells, nonciliated cells, and secretory cells (C) (bar: 2 m). Inset: Apical intercellular tight junctional (TJ) complex are present between adjacent cells (D) (bar: 0.4 m).

5 Blouquit, Morel, Hinnrasky, et al.: Ion and Fluid Transport in Human Bronchioles 507 TABLE 1 Transmural potential difference of microperfused segments of human bronchioles Basal PD (mv) PD Amiloride (mv) PD Forskolin (mv) PD ATP (mv) Regular KBR (n 10) * * * Low-Cl KBR (n 6) * Definition of abbreviations: PD, transmural potential difference; PD, changes in PD. Amiloride, forskolin, and ATP were sequentially added in the perfusate as described in MATERIALS AND METHODS. Measurements were obtained in regular KBR and in low-cl KBR. *Significantly different from zero (P 0.05). Significantly different from values obtained in regular KBR (P 0.005). for a significant part of the resting Isc in proximal porcine bronchioles. They then provided evidence for stimulation of Cl secretion by isoproterenol a -adrenergic agonist that increases camp levels in airway epithelial cells and by luminal ATP (19). However, the bronchi used in the latter study had an average external diameter of 3.6 mm, and these results may therefore apply more to bronchi than to bronchioles. Recently, Inglis and coworkers (20) studied the effects of luminal nucleotides on ion transport in microperfused porcine distal bronchi. The average outer diameter of these bronchi was 3.7 mm. The authors provided evidence that luminal UTP inhibits Na absorption by a Ca-dependent mechanism and also stimulates Cl secretion by a Ca-independent mechanism. Animal studies have therefore provided evidence for amiloride-sensitive Na absorption and forskolin- and triphosphate nucleotide activated Cl secretion. In addition, they agree that active Na absorption accounts for a significant part if not most of the resting ionic flow. Using a similar technique, we measured the basal transepithelial PD of human excised bronchioles and examined the effects of amiloride, forskolin, and ATP in regular KBR and low-cl KBR on the transepithelial PD. We found that microperfused human bronchioles generate a significant transepithelial PD, which indicates active transepithelial ion transport. The amiloride-induced inhibition in the basal PD suggests that Na absorption contributes significantly to the basal ionic flow. In contrast, the absence of significant change in basal PD when preparations were studied in low-cl KBR suggests that Cl transport does not contribute significantly to the basal ionic flow across the bronchiolar epithelium. Subsequent additions of forskolin and ATP raised the transepithelial PD in regular KBR. The fact that these increases were observed in the presence of the Na channel blocker amiloride, and that they were inhibited when the preparations were bathed in low-cl KBR, suggests that forskolin and ATP activate Cl secretion. Thus, this study indicates that human bronchiolar mucosa exhibits some characteristics of both Cl secretion and Na absorption and that Na absorption is a major contributor to the basal ionic flow. Overall, our results are consistent with those obtained in pig and sheep bronchioles. However, the basal PD in microperfused human bronchioles was lower than the PD measured in microperfused pig and sheep bronchioles (15, 16). Several mechanisms may contribute to this low basal PD. Although we exercised extreme care when dissecting the airways from the lung, it is possible that some airways were damaged during dissection. This is especially true because these tissues are very small and do not possess rigid walls. In addition, characterization of ion transport in human airways is difficult and poses unique problems not encountered in studies of airways from laboratory animals. Human airways are not routinely resected as rapidly as tissue from experimental animals. Excised airways from humans were studied 1 3 h after surgical excision, in contrast to animal studies, in which preparations are processed immediately after excision. It is therefore possible that some tissue alteration occurred during this delay, for instance because of hypoxia. However, although the basal PD was quite low in excised human bronchioles, it was inhibited by amiloride and increased in response to forskolin and ATP, implying that active ion processes were preserved. Moreover, the transepithelial PD in primary cultures of bronchiolar epithelial cells in Ussing chambers was very close to that measured in microperfused excised bronchioles. Due to their small size, epithelial bioelectric properties were studied by previous investigators in bronchioles mainly as perfused tubes. However, the measurement of transmural voltage alone provides little information about TABLE 2 Bioelectric properties of cultured human bronchiolar epithelial cells BASELINE AMILORIDE FORSKOLIN ATP PD (mv ) Isc ( A/cm 2 ) R (. cm 2 ) Isc ( A/cm 2 ) R (. cm 2 ) Isc ( A/cm 2 ) R (. cm 2 ) Isc ( A/cm 2 ) R (. cm 2 ) Regular KBR (n 12) * * * * Low-Cl KBR (n 12) * * Definition of abbreviations: PD, transmural potential difference; Isc, short circuit current; R, transepithelial resistance; Isc, changes in Isc; R, changes in transepithelial resistance. Primary cultures of human bronchiolar epithelial cells were studied in regular KBR and in low-cl KBR in Ussing chambers. Amiloride, forskolin, and ATP were added sequentially and PD, Isc, and R were measured before and after addition of each drug. *Statistically significant difference from zero (P 0.05). Statistically significant difference between preparations studied in low-cl KBR and in regular KBR (P 0.05).

6 508 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL Figure 2. Typical Isc tracing from a human bronchiolar epithelial cell culture bathed in regular KBR, showing the effects of addition of amiloride, forskolin, and ATP (A). Changes in Isc ( Isc) induced by sequential additions of amiloride, forskolin, and ATP in bronchiolar epithelial cell cultures (B). Bronchiolar cultures were studied in regular KBR bath (n 12; filled bars) and in low- Cl KBR bath (n 12; open bars). *Statistically significant difference between regular KBR and low-cl KBR (P 0.05). ion transport processes and, as acknowledged by the authors, the cable analysis method causes R and Isc to be in some error (17). We turned, therefore, to the Ussing chamber technique to better characterize the bioelectric properties of human bronchiolar epithelial cells. In trying to mount open excised bronchioles in Ussing-type chambers, we encountered technical difficulties because of their small size, and consequently decided to culture bronchiolar epithelial cells on permeable supports that fit in our homemade Ussing-type chambers. We developed a method for the isolation and culture of epithelial cells from human bronchioles derived from that described by Yankaskas and coworkers in nasal polyps (21). Cultured cells from human bronchioles were epithelial in nature, as shown by the presence of tight junctions, retained morphologic features similar to those of freshly excised tissue, and formed electrogenic barriers. The bioelectric variables of these preparations were then measured in Ussing chambers. In the basal state, the PD in cultured bronchiolar epithelial cells was close to that measured in microperfused preparations. The transepithelial resistance was low, indicating that this tissue may be very leaky. Addition of amiloride induced an 40% decrease in Isc in both regular and low-cl KBR. These results indicate that active transepithelial Na absorption accounts for a substantial fraction of the basal Isc. When added in the presence of amiloride, forskolin and ATP significantly increased Isc by 31 and 159%, respectively. Responses to these secretagogues were observed in the presence of amiloride and were significantly reduced when Cl was removed from the bathing solutions, indicating that forskolin and ATP stimulate active Cl secretion. Low-Cl conditions did not totally inhibit the effects of forskolin and ATP on Isc. This response could be attributed to the presence of a low but significant amount of Cl ions in the low-cl KBR and to the possibility that other anions such as HCO 3 were secreted by the airway epithelial cells under low-cl conditions as described in human cultured proximal airways and in porcine small bronchi (19, 24). PD and Isc increased in response to ATP after pretreatment with forskolin, indicating an additive effect of forskolin and ATP on Cl secretion. The patterns of increase in Isc in response to forskolin and ATP were obviously very dissimilar as shown in Figure 2, suggesting that the mechanisms of stimulation of Cl secretion by forskolin and ATP differ as observed in proximal airways (1, 4). Overall, our results are consistent with amiloride-sensitive Na absorption and forskolin- and ATP-activated Cl secretion in human bronchioles. Data from cultured cells provide important information. However, enzyme exposure and culture conditions may cause morphologic and functional changes. For example, culturing airway epithelial cells with exposure of the apical surface to air rather than culture medium dramatically modifies their ion transport properties with higher baseline Isc and higher responses to amiloride and camp (25). Therefore, data from cultured cells need to be verified and related to functions observed in intact epithelia. In this respect, it is important to point out that data obtained using the Ussing chamber technique on cultured bronchiolar epithelial cells are very consistent with data obtained on microperfused segments of excised human bronchioles. To our knowledge, the only assessment of bioelectric properties of bronchiolar epithelial cells in Ussing chambers has been done in cultured rabbit Clara cells and in excised sheep bronchioles. Van Scott and coworkers obtained confluent preparations highly enriched in Clara cells and suitable for studies of transepithelial ion transport (26). Bioelectric studies and radioisotopic flux measurements showed that rabbit Clara cells actively absorb Na in the basal state. No net movement of Cl was detected under basal conditions, but active Cl secretion was induced by luminal amiloride and stimulated by activators of the camp pathway and by extracellular triphosphate nucleotides (26 28). However, in human lung, Clara cells represent only a small percentage of bronchiolar epithelial cells ( 0.4% in nonterminal bronchioles, 11% in terminal bronchioles, and 22% in respiratory bronchioles) (29), and other cell types might exhibit other ion transport properties. Al-Bazzaz and coworkers examined the bioelectric properties of sheep distal airways (inner diameter 1 mm) in a miniature Ussingtype chamber (18). In their findings, the transepithelial PD, Isc, and R were close to the values we measured in cultured human bronchiolar cells. Amiloride inhibited the baseline Isc by 51%. The calcium ionophore A increased Isc in the presence of amiloride not, however, after pretreatment with bumetanide, suggesting that raising intracellular Ca stimulated secretion of Cl or another anion. In contrast to our data, Al-Bazzaz and coworkers found no significant effect of isoproterenol or dibutyryl camp on PD or Isc. Importantly, in their study, sheep tracheal mucosa also failed to respond to camp-activating agents, which is different from human proximal airway mucosa (4, 18).

7 Blouquit, Morel, Hinnrasky, et al.: Ion and Fluid Transport in Human Bronchioles 509 The sensitivity of Na absorption to amiloride suggests that amiloride-sensitive epithelial Na channels (ENaC) are present in the apical membrane of human bronchiolar epithelial cells. In human bronchioles, Gaillard and coworkers did detect expression of ENaC subunits by immunohistochemistry in ciliated and Clara cells (30). Our preparations also responded to agonists that modulate activity of two Cl paths known to be active in human proximal airway epithelium. The first Cl path is activated by agents that raise camp, and the second is activated by extracellular nucleotides (4). As in proximal airways, the camp-activated Cl path is likely associated with CFTR, which is expressed by human bronchiolar epithelial cells (31). The molecular basis of the ATP-activated Cl path in human proximal airways is less well known and may involve more than one mechanism (including a direct effect of Cl channels or an increase in intracellular Ca activity), and more than one type of Cl channel (32, 33). Expression of Cl channels other than CFTR has been reported in human bronchioles, including members of the ClCN family (34). We found no study on expression of purinergic receptors in human distal airways. However, results obtained in pig distal bronchi and cultured rabbit Clara cells indicate that the purinergic receptors involved in the stimulation of Cl secretion belong to the P 2 Y type and presumably the P 2 Y 2 subclass (20, 27). Water transport across permeable epithelia is considered to be secondary to transepithelial osmotic gradients produced by active ion transport. By secreting or absorbing ions, airway epithelium could regulate fluid transport across the mucosa and thereby control the volume and composition of the airway surface fluid. We decided to test the hypothesis that in human bronchioles, transepithelial ion transport was associated with water transport. Excised bronchioles exhibited baseline fluid absorption, driven presumably by active absorption of Na, because fluid absorption was inhibited by amiloride. Due to the small number and fragile nature of these preparations and the length of duration of fluid transport experiments, we did not measure the effects of forskolin on the one hand and ATP on the other hand on fluid transport. Our goal was to ensure that transepithelial Cl secretion was stimulated and to assess the resulting effect on fluid transport. Stimulation of Cl secretion by forskolin and ATP in the presence of amiloride reversed the baseline fluid absorption to a large net fluid secretion. Thus, fluid movements across excised bronchiolar mucosa paralleled ion movements as determined in microperfused excised bronchioles and in cultured bronchiolar epithelial cells. Active transport of liquid across human proximal airway epithelia has been measured by a variety of techniques. For example, a double-sided capacitance probe technique revealed an 5 l/h/cm 2 baseline liquid absorption across tracheal epithelial cultures (6). Amiloride decreased liquid absorption, whereas activation of the camp pathway or exposure to UTP caused a significant secretion. Another approach involved adding 100 l of culture medium to the mucosal surface of human nasal epithelial cell cultures, then covering it with mineral oil to prevent evaporation and, finally, measuring the volume of fluid remaining after 24 h (7). The rates of liquid absorption were times smaller than with the first method, but the authors observed that amiloride inhibited fluid absorption, whereas camp agonists in the presence of amiloride induced a net fluid secretion. A third technique consisted of adding 100 l of culture medium to the mucosal surface of human nasal epithelial cell cultures, retrieving the mucosal solution 2 h later and weighing this volume (8). Baseline fluid absorption reached l/h/cm 2. It was inhibited by amiloride, but reversed to net fluid secretion by exposure to luminal ATP or UTP. As compared with our data, reported rates of fluid transport across human proximal airway epithelia are obviously lower, especially the basal fluid absorption and the secretion induced by Cl secretagogues. This may reflect differences in techniques of fluid transport measurement or a true difference in fluid transport between airway regions. Under the latter hypothesis, because the basal Isc and the amiloride-sensitive component of Isc are similar in cultured epithelial cells from proximal airways and from bronchioles (1, 4), our results could indicate that proximal and distal airways differ in nonelectrogenic ion transport processes or that epithelial water permeability is higher in distal than in proximal airways. It is unclear whether electroneutral ion transport comprises a significant fraction of total net ion transport in any region of the pulmonary epithelium. Radioisotopic ion flux studies will be necessary to determine the existence and relative contribution of electroneutral transport to net active ion flux across bronchiolar epithelium. On the other hand, the consistently low transepithelial R of cultured bronchiolar epithelial cells suggests that this tissue may be very leaky to accommodate a high degree of water permeability. In support of this hypothesis, high levels of expression of aquaporin 3 (AQP3) have been demonstrated in the apical membrane of cuboidal cells in human bronchiolar epithelium by in situ hybridization and immunofluorescence (35). This level of AQP3 expression was postulated to confer a significant fluid transport capacity to this epithelium. Our data are also consistent with functional studies reporting a relatively high osmotic water permeability ( cm/s at 23 C) in microperfused guinea pig distal airway mucosa as measured using fluorescent probes (36). Thus, the epithelium of human distal airways is probably much leakier that the epithelium of human proximal airways, resulting in a larger transport of fluid in distal airways compared with proximal airways. Our results have several implications in airway physiology. Proximal bronchioles provide a transition from distal lung spaces to cartilaginous bronchi. In these aglandular airways, ASL is postulated to be transported axially from peripheral to central lung regions (5, 37). This cephalad movement implies that absorption of fluid must occur as ASL converges onto gradually narrowing airway surfaces. Studies of transepithelial ion and fluid transport across large airways have found that net absorption of ion and fluid occurs under basal, unstimulated conditions. However, some investigators have argued that ions, but not fluid, are absorbed across the proximal airway mucosa in the basal state leading to a hypotonic ASL (38). Our study confirms that fluid is absorbed in the proximal segments of human bronchioles in the basal state. Furthermore, if se-

8 510 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL cretion of fluid does exist in distal lung regions, our results indicate that the shift from fluid secretion to fluid absorption lies in the very distal part of the bronchial tree. In addition to clearing excess liquid from the air spaces, ion transport in the bronchiolar epithelium most likely regulates the depth of surface liquid, because fluid secretion occurred under appropriate stimulation. In summary, we report that the mucosa of human proximal bronchioles absorbs liquid by an Na-dependent active transport in the basal condition and is capable of active Cl and fluid secretions when exposed to forskolin and to ATP. Data presented in this report also show the feasibility of establishing primary cultures of human bronchiolar epithelial cells. These results may help to explain the complex regulation of ion and fluid transport in normal human airways, and to shed some light on the roles of these processes in the development of lung diseases such as cystic fibrosis. Acknowledgments: The authors thank the surgeons in the Department of Thoracic Surgery at the Clinique du Val d Or at Saint Cloud, France for providing us with human lung tissues. The authors also thank Dr. Sherif Gabriel (University of North Carolina, Chapel Hill, NC) for critical review of the manuscript and Mrs. Cecily Lyle for help with the English language. This work was supported by grants from the Association Vaincre la Mucoviscidose. References 1. Boucher, R. C Human airway ion transport (Part 1). Am. J. Respir. Crit. Care Med. 150: Widdicombe, J. H., and J. G. Widdicombe Regulation of human airway surface liquid. Respir. Physiol. 99: Smith, J. J., S. M. Travis, E. P. Greenberg, and M. J. 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