Fluid transport across cultured rat alveolar epithelial cells: a novel in vitro system

Xiaohui Fang, Yuanlin Song, Rachel Zemans, Jan Hirsch, and Michael A. Matthay

Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130

Submitted 30 May 2003 ; accepted in final form 19 February 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies have used fluid-instilled lungs to measure net alveolar fluid transport in intact animal and human lungs. However, intact lung studies have two limitations: the contribution of different distal lung epithelial cells cannot be studied separately, and the surface area for fluid absorption can only be approximated. Therefore, we developed a method to measure net vectorial fluid transport in cultured rat alveolar type II cells using an air-liquid interface. The cells were seeded on 0.4-µm microporous inserts in a Transwell system. At 96 h, the transmembrane electrical resistance reached a peak level (1,530 ± 115 {Omega}·cm2) with morphological evidence of tight junctions. We measured net fluid transport by placing 150 µl of culture medium containing 0.5 µCi of 131I-albumin on the apical side of the polarized cells. Protein permeability across the cell monolayer, as measured by labeled albumin, was 1.17 ± 0.34% over 24 h. The change in concentration of 131I-albumin in the apical fluid was used to determine the net fluid transported across the monolayer over 12 and 24 h. The net basal fluid transport was 0.84 µl·cm–2·h–1. cAMP stimulation with forskolin and IBMX increased fluid transport by 96%. Amiloride inhibited both the basal and stimulated fluid transport. Ouabain inhibited basal fluid transport by 93%. The cultured cells retained alveolar type II-like features based on morphologic studies, including ultrastructural imaging. In conclusion, this novel in vitro system can be used to measure net vectorial fluid transport across cultured, polarized alveolar epithelial cells.

transepithelial transport; lung


NEW INSIGHTS HAVE BEEN MADE into the mechanisms responsible for active reabsorption of edema fluid from the distal air spaces of the lung (6, 8, 3032, 34, 44). Many of the studies of fluid transport across the pulmonary epithelium have been conducted in intact animals and isolated lungs. These intact lung preparations have been indispensable for determining the physiology and clinical relevance of active ion transport across the distal pulmonary epithelial barrier. In most in vivo, in situ, and isolated perfused lung studies, a macromolecular method (e.g., albumin labeled with isotopic iodine or Evans blue) has been used to measure net fluid absorption from the distal air spaces (31, 32). A rise in the concentration of albumin in the fluid instilled into the distal air spaces reflects the fluid transport activity of the epithelial cells lining the distal air spaces.

Intact lung studies have two specific limitations. First, the contribution of different distal lung epithelial cell types (i.e., alveolar epithelial type I and II cells, Clara cells) cannot be studied separately. Second, the surface area for fluid absorption can only be roughly estimated. Prior in vitro methods for measuring lung epithelial fluid transport have utilized several methods, including dome formation in cultured epithelia, short-circuit current (Isc) in Ussing chambers, and ion uptake in suspended adult and fetal cells (17, 2729, 35). However, these methods cannot be used to quantify net vectorial fluid transport across cultured tight epithelial monolayers.

Therefore, the objective of this study was to develop a new method for measuring net fluid transport across cultured alveolar epithelial type II cells from rat lungs.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation and culture of rat alveolar type II cells. Alveolar epithelial type II cells were isolated from pathogen-free male Sprague-Dawley rats by enzymatic digestion and panning on plates coated with IgG to remove macrophages and leukocytes, as previously described (10, 16). Cell viability was >95% by trypan blue exclusion, and the yield was 15–25 x 106 cells per rat. Cell purity assessed by Papanicolous staining was >90%. Preparations containing <90% type II cells were discarded. The cells were prepared in culture medium DME H-21 containing 10% FBS (University of California-San Francisco cell culture facility) and were seeded at a density of 1.6 x 106 cells/cm2 in tissue culture-treated polycarbonate Transwell membranes with 0.4-µm pores and a surface area of 0.33 cm2 (Corning, NY) (Fig. 1). The culture medium containing 10% FBS was added to the lower compartment of the Transwell. The cells were maintained in a 37°C 5% CO2 incubator. After 48 h, the cells formed a confluent monolayer. The monolayer was washed with medium to remove unattached cells. After the confluent monolayer was achieved at 48 h, transmembrane electrical resistance was measured daily with an Ag-AgCl electrode. The electrode was sterilized with 70% alcohol for 10 min, rinsed twice with PBS, and placed in the DME H-21 for 1 h to reach electrical balance before measurement. Measurements were done before the culture medium was changed. After 120 h of growth, the cells in selected Transwells were counted. Cells were first washed twice with PBS solution, and then 0.1 ml and 0.5 ml of 0.25% trypsin were added to the upper and lower compartments of the Transwell, respectively. The Transwell was incubated at 37°C for 15–20 min, and the detached cells were collected and counted.



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 1. Side view of the Transwell system. Rat alveolar epithelial type II cells were seeded at a density of 1.6 x 106 cells/cm2 on the tissue culture-treated polycarbonate membrane with a 0.4-µm pore size and a surface area of 0.33 cm2. Culture medium (150 µl) containing 0.5 µCi 131-I albumin was placed in the upper compartment. Fluid in the lower compartment was at the same level as in the upper compartment to avoid a hydrostatic pressure gradient.

 
Rat alveolar epithelial monolayer permeability to protein. To determine whether the cultured monolayer was mostly impermeable to protein, we measured the protein flux across the rat alveolar epithelial cells by measuring the unidirectional flux of labeled 131I-albumin from the apical (upper compartment) to the basolateral side (lower compartment). The cells formed confluent monolayers 48 h after seeding, and the culture medium in both the upper and lower compartments of the Transwell was changed. At 72 h, the fluid in the upper compartment was removed with gentle aspiration, and the cells were then grown in an air-liquid interface. At 96 h, 150 µl of serum-free medium containing 0.5 µCi/ml 131I-albumin were pipetted on the top of the cells in a humidified tent, and the plate was returned to the 37°C 5% CO2 incubator. Twenty-four hours after 131I-albumin was pipetted in, all fluid in the upper and lower compartments of the Transwells was aspirated, and the total 131I was measured in the gamma counter (Packard MINAXI 5000 series). Trichloroacetic acid (20%) was added to the upper and lower compartments of selected Transwells to determine 131I binding to albumin as we have done before (33). There was <1.5% of free 131I.

Paracellular permeability of rat alveolar epithelial cell monolayer. [14C]mannitol was used to measure the paracellular permeability of the rat alveolar epithelial monolayer (n = 24 in four different preparations). Fluid containing 0.05 µCi of [14C]mannitol was pipetted into the upper compartment of the Transwell. The plate was incubated at 37°C. Samples were aspirated from both upper and lower compartments at 5 min, 12 h, and 24 h and counted in an LS 6500 Multipurpose scintillation counter (Beckman Coulter) for 40 min. [14C]mannitol permeability was expressed as µmol·cm–2·h–1 and percent change over 12 and 24 h.

Measurement of fluid transport across the rat alveolar epithelial monolayer with an isotope-labeled albumin method. To validate this method of measurement for the net fluid transport across a cell monolayer, we measured the 131I-albumin concentration change over time in empty Transwell membranes (no cells) that were sealed with parafilm to simulate an impermeable barrier. In this manner, we estimated the magnitude of 131I-albumin adsorption to the filter and the plastic walls of the Transwell, as well as the degree of evaporation, both of which are potential confounding factors in the estimation of fluid transport by this method.

Fluid in the upper and lower compartments of the Transwell was maintained at the same level to avoid a hydrostatic pressure gradient. 131I-albumin was used as a volume marker, as in our previous in vivo sheep, rat, mouse (13, 14, 32), and ex vivo human studies (38). The measurement of fluid transport from the apical to basolateral membranes of the epithelial cell monolayer was done 96 h after the isolation and plating of the cells, 24 h after an air-liquid interface had been achieved. Medium (150 µl) containing 0.5 µCi/ml 131I-albumin was pipetted into the apical chamber of the Transwell in the humidified tent. Five minutes after the 131I-albumin was added, 20 µl of the medium were aspirated as the initial sample. The 5-min period was allowed for adherence of protein to the chamber wall and potential initial dilution. The plate was then incubated for 12–24 h in the 37°C 5% CO2 incubator with 100% humidity. After either 12 or 24 h, 20 µl were aspirated from the upper compartment of the Transwell as the final sample. The samples were weighed and counted in the gamma counter. Fluid absorption was calculated as in our prior in vivo studies (31, 32, 41): Fluid absorption = [1 – (radioactivity in the initial sample/weight of initial sample)/(radioactivity in the final sample/weight of final sample)] x 100%. Forskolin and IBMX (10–5 M each, n = 36 wells) or amiloride (10–5 M, n = 24 wells) was added to the apical culture medium in selected studies. Ouabain (10–5 M, n = 12 wells) was added to the culture medium in the lower compartment in other studies.

We also measured the change in impermeant marker concentration in the lower compartment of selected Transwells to calculate fluid movement from the basolateral to apical surface to ensure that the calculated fluid transport is equal and opposite in the two directions. For measurement of fluid dilution in the lower compartment, 600 µl of medium containing 131I-albumin were pipetted into the lower compartment, and 150 µl of medium were pipetted into the upper compartment. Approximately 5 min after the 131I-albumin was added, 20 µl were aspirated from the lower compartment as the initial sample. After 24 h of incubation in the 37°C 5% CO2 incubator with 100% humidity, another 20 µl were aspirated from the lower compartment of the Transwell as the final sample. Fluid movement from the basolateral to apical side was calculated by the same method described in Measurement of fluid transport across the rat alveolar epithelial monolayer with an isotope-labeled albumin method (n = 18 in each group).

Cell markers for alveolar type II and alveolar type I phenotypes. Through the use of cell surface markers of alveolar type I (ATI) and type II (ATII) cells, other investigators have found that ~95% of the cells cultured as in these studies have an ATII phenotype (20, 32), with little conversion to an ATI phenotype in the first 96 h of culture. We addressed this issue in our culture conditions. Cells were seeded on 0.33-cm2 clear Transwell membranes for 48–120 h (with an air-liquid interface), and then cells were preincubated at 37°C in DMEM with Lysotrack green DND-26 (150 nmol/l, 30 min; Molecular Probes), which is a fluorescent dye to selectively stain lamellar bodies in primary culture ATII cells (1, 18, 19). Images were obtained with a Nikon inverted microscope (TE 2000-E) and Simple PCI Advance Image Capture system. Cells were also trypsinized from the membrane and stained with aquaporin-5 (AQP5) antibodies (Chemicon) to determine the relative numbers of ATI-like cells.

Ultrastructure of cultured rat ATII cells. Freshly isolated rat ATII cells were seeded in Transwell membranes at a density of 1.0x106/well. Then the cells were cultured under the same conditions as described in Isolation and culture of rat alveolar type II cells. At 120 h, the monolayers were fixed in 3% (wt/vol) Karnovsky fixative for 1 h at 0°C. In addition, cells on Transwell membranes were scraped off with a rubber police, and cell suspension was centrifuged briefly at 160 g. The resulting cell pellet was then fixed in 3% Karnovsky fixative for 1 h at 0°C. Both the cells from monolayer membranes and the pellet were postfixed 1 h in 1% veronal-buffered osmic acid. The cells were dehydrated in graded ethanols and/or propylene oxide. Then they were embedded in Epon or Araldite resins cured at 60°C. Thin sections were contrasted with saturated aqueous uranyl acetate and Reynolds lead citrate, then sections were screened with a JEOL 1200 EX transmission electron microscope operating at 80 kV.

Immunostaining of zonula occludens 1 protein in ATII cell monolayer. For detection of intercellular tight junctions, rat alveolar epithelial type II cells were seeded on Lab-Tek II chamber slides (Nalge Nunc International) at a density of 1.6 x 106 cells/cm2. At 120 h, the cell monolayer was washed twice with cold PBS and fixed in 4% paraformaldehyde for 30 min. The slides were then washed three times with PBS for 10 min in a gently shaking chamber at room temperature. The slides were quenched with NH4Cl and glycine for 10 min and then were permeabilized with PBS/fish skin gelatin (FSG)/saponin (SAP)/RNase for 30 min at 37°C. The slides were then incubated with primary rabbit polyclonal anti-zonula occludens 1 (ZO-1) protein antibody (Chemicon) for 1 h at 37°C. The slides were again washed four times with PBS/FSG/SAP/RNase for 10 min and then were incubated with a mixture of secondary rhodamine-labeled anti rabbit-IgG (Sigma) and 4',6-diamidino-2-phenylindole dihydrochloride (Molecular Probes) for 45 min. The slides were mounted with Vectashield mounting medium and covered with coverslips. Images were obtained by a Bio-Rad MRC-1024 laser scanning confocal microscopy system.

Statistical analysis. Results are expressed as the means ± SE of values from at least three separate rat experiments, each done with six wells. Comparisons between two groups were made by an unpaired, two-tailed t-test. Comparisons between more than two groups were made by a one-way analysis of variance and with Tukey's post hoc test. P < 0.05 was taken as statistically significant.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transmembrane electrical resistance and phenotype of cultured ATII cells. Approximately 48 h after plating, the cells developed measurable transmembrane electrical resistance, which peaked at 96 h (Fig. 2A). Because daily measurements may alter the cells and underestimate the transmembrane electrical resistance, we also measured the resistance only at 120 h in some Transwells (n = 24). In these Transwells, the peak transmembrane electrical resistance reached was 1,530 ± 115 {Omega}·cm2. Tight junctions were also evident by visualization of ZO-1 protein in the cultured monolayers 120 h after plating (Fig. 2B). Therefore, the 24-h period between 96 and 120 h after initial plating of the cells was selected for the fluid transport experiments.



View larger version (22K):
[in this window]
[in a new window]
 
Fig. 2. A: time course of transmembrane electrical resistance after cells were plated. The y-axis is transmembrane electrical resistance in {Omega}·cm2. Data are presented as means ± SE. B: immunostaining of zonula occludens (ZO)-1 protein in a cultured monolayer of rat alveolar epithelial type II cells (red). Cell nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (blue).

 
To define the phenotype of cultured ATII cells, cells were grown on permeable Transwell membrane (with an air-liquid interface). Cells were trypsinized from the membrane and stained with AQP5 antibody. Less than 5% of the cells stained positive for AQP5 during the time (96–120 h) in which the fluid transport studies were done. At both 48 h and 120 h, the monolayer showed that the cells had lamellar bodies (Fig. 3, A and B). There was no difference between the numbers of ATII cells from 48–120 h for cells grown on Transwell membrane. Electron microscopy of the cells showed that they contained lamellar bodies and microvilli (Fig. 3C).



View larger version (64K):
[in this window]
[in a new window]
 
Fig. 3. Rat alveolar epithelial type II cells grown on Transwell membranes with an air-liquid interface for 120 h. A: phase contrast of rat alveolar epithelial type II cell monolayer. B: staining of lamellar bodies with Lysotrack green DND-26 of the monolayer (19). C: higher-magnification views of cells showing alveolar epithelial type II cells with typical lamellar bodies and microvilli. Top: cell on the membrane. Bottom: cell scrapped off from the membrane (bar, 1 µm).

 
Protein and mannitol flux across the rat alveolar epithelial type II cell monolayer. Flux of the bound 131I-albumin across the cell monolayer from the apical to the basolateral surface was 1.17 ± 0.34% over 24 h. Transwells with protein permeability >3% were discarded. Approximately 5% of the monolayers were not used for this reason. [14C]mannitol flux measured across the alveolar epithelial monolayer at 12 and 24 h was 8.1 x 10–6 and 14.4 x 10–6 µmol·cm–2·h–1 (5.4% over 12 h and 9.6% over 24 h), respectively (Fig. 4).



View larger version (9K):
[in this window]
[in a new window]
 
Fig. 4. [14C]mannitol flux across the rat alveolar epithelial monolayer over 12 and 24 h. The y-axis is the [14C]mannitol flux in 10–6 µmol·cm–2·h–1. Experiments were done in 4 different cell preparations (n = 24 in each time point). *P < 0.05, data as means ± SE.

 
Fluid transport across cultured rat alveolar epithelial type II cells. Measurement of the 131I-albumin concentration changein the setting of the impermeable, parafilm-sealed Transwell membrane revealed that total radioactivity in both the upper and lower compartments after 24 h was almost equal to the total radioactivity in the initial sample. The adherence of albumin to the chamber wall in the upper compartment at the initial 5 min was 0.05%. The evaporative loss of fluid from the upper compartment was 0.6 ± 0.4%, which we determined by comparing final and initial 131I-albumin concentrations in the upper compartment of empty Transwells during the 24-h period.

When bidirectional fluid movement was measured, fluid transport from the apical to the basolateral surface was 0.84 µl·cm–2·h–1, whereas fluid movement from the basolateral to apical surface was –0.82 µl·cm–2·h–1. cAMP stimulation with forskolin and IBMX increased fluid transport from the apical to basal side to 1.65 µl·cm–2·h–1 and, likewise, further diluted the fluid in the lower compartment (Fig. 5).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 5. Comparison of the rates of fluid movement in opposite direction in the Transwells. Experiments were carried out under basal and forskolin + IBMX-stimulated conditions. Experiments were done in 4 different preparations (n = 18 in each group). The y-axis is fluid movement in µmol·cm–2·h–1. *P < 0.05 compared with basal, data as means ± SE.

 
Basal fluid transport rate was 0.84 µl·cm–2·h–1 over 24 h. Forskolin and IBMX significantly stimulated fluid transport to 1.65 µl·cm–2·h–1 at 24 h. Fluid transport volume at 12 h was 3.1 µl for control and 7.3 µl for forskolin plus IBMX, suggesting a nearly linear relationship between the volume and the time of transport.

Effect of sodium channel and Na+-K+-ATPase inhibitors on vectorial fluid transport. Amiloride inhibited the basal rate of fluid transport from the apical to the basolateral side by 65% (0.29 µl·cm–2·h–1). Ouabain, which acts on basolateral Na+-K+-ATPase, inhibited fluid transport by 93% (Fig. 6A). cAMP stimulation with forskolin and IBMX was inhibited by 68% with amiloride (Fig. 6B).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6. Fluid transport across rat alveolar epithelial type II cells over 24 h. Culture medium containing 0.5 µCi 131I-albumin was pipetted onto the upper compartment of the Transwell. Fluid transport was measured as described in methods. A: effect of amiloride and ouabain on basal fluid transport. B: effect of amiloride on forskolin + IBMX-stimulated transport. As indicated, the fluid in the upper compartment of the Transwell contained forskolin + IBMX (10–5 M each), amiloride (10–5M), ouabain (10–5 M), and different combinations of these substances. Experiments were done in 12 different cell preparations. *P < 0.05, compared with control; **P < 0.05, compared with forskolin + IBMX group. Data are means ± SE.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our present understanding of active ion transport, barrier resistance, and fluid transport across the alveolar epithelium has been derived from both in vivo and in vitro studies. Insights into the specific mechanisms of alveolar epithelial cell transport have often derived from in vitro experimental design because there is more precise control of experimental variables. One of the earliest studies of the isolated alveolar epithelium used amphibian lung (6, 15). A few years later, rat ATII cells were harvested and plated on both porous and nonporous substrata (17, 28). On plastic culture dishes, numerous domes were formed, indicating that the cells exhibited active transport and formed occluding junctions. Amiloride and ouabain inhibited dome formation, whereas terbutaline stimulated dome formation, indicating vectorial sodium-dependent fluid transport from the apical to the basolateral surface. More detailed information regarding ion transport across ATII cells was obtained by culturing alveolar epithelial cells on porous supports, mounting them in Ussing chambers, and measuring Isc and ion flux under voltage-clamp conditions (3, 23, 24, 26, 31, 42). The Isc in an ATII cell monolayer corresponded closely to net sodium absorption. However, these methods cannot provide a quantitative measurement of net fluid transport.

Fluid absorption can be detected by making use of capacitance measurement (46). A double-sided capacitance microprobe technique can measure very small quantities of fluid transport in airway epithelia (21, 45). However, this technique is complicated, and removing probes while changing to fresh bathing solution in the chamber might alter the experimental results. Therefore, on the basis of previous experiments, we developed an improved method to quantify net fluid transport across cultured ATII cells by measuring the change in concentration of a nearly impermeant marker over time.

To minimize evaporative losses, the studies were done in a tent in which 5% CO2 water vapor at 37°C was continuously circulated to maintain humidity at 90–100%. This procedure should have the same effect as coating the apical solution with mineral oil (43). The monolayer was relatively protein impermeable, since 131I-albumin flux was only 1.17 ± 0.34% over 24 h. Paracellular permeability as measured by [14C]mannitol transport was only 14.4 x 10–6 µmol·cm–2·h–1 over 24 h. Tight junctions formed by the alveolar epithelial type II cell monolayer at 120 h were also demonstrated by the staining for the ZO-1 protein in cell monolayers grown on glass slides. It was not technically feasible to do the ZO-1 imaging studies on the Transwell filter. The impermeability of the monolayer to protein validates this macromolecular approach to measuring net fluid transport, as in our in vivo studies (31, 32).

The transmembrane electrical resistance across the cultured monolayers rapidly increased after 48 h. The peak resistance level was at 96 h and was maintained for another 48–72 h. Therefore, we selected the 24-h period between 96 and 120 h after the plating of cells as the experimental time frame for the fluid transport studies. The relationship between the volume of transported fluid and time was linear, suggesting a constant rate of transport. These data provide evidence that this in vitro system is suitable for measuring net fluid transport in rat alveolar epithelial cell monolayers.

Basal fluid transport in rat ATII cells was 0.84 µl·cm–2·h–1. cAMP stimulation increased fluid transport by ~96% over baseline. The inhibition of cAMP-stimulated fluid transport by amiloride suggests that functional epithelial sodium channels (ENaC) were present (20, 31). As expected, ouabain inhibited ~90% of fluid transport, which is consistent with the results of our previous studies in the in situ sheep lung and human lungs (39, 40).

An issue common to any study of cultured alveolar epithelial cells is the definition of the phenotype, since there is ample evidence that ATII cells gradually transition to an ATI-like phenotype after several days in culture (12). Early experiments established that ATII cells plated on plastic culture dishes rapidly lose characteristics that are associated with a differentiated type II cell phenotype (9, 11). Most surfactant protein expression is downregulated (25), and some transport proteins, such as Na-phosphate, and Na+-K+-2Cl cotransport vanish (4, 5). After 7 days in culture, rat ATII cells demonstrate decreased expression of the {alpha}1-subunit and increased expression of the {alpha}2-subunit of Na+-K+-ATPase, as shown by both mRNA and protein levels (37). In addition, after several days in culture, ATII cells demonstrate decreased expression of surfactant protein C. As they lose their ATII characteristics, cultured alveolar cells develop an ATI-like phenotype, as shown by increased expression of AQP5 and {alpha}2-Na+-K+-ATPase after 7 days in culture (36). The precise definition of the phenotype of cultured alveolar epithelial cells is particularly important in the setting of the ongoing uncertainty about the relative contributions of ATI and ATII cells to fluid transport. Although considerable evidence has indicated that the ATII cell plays the principal role in edema clearance, recent data have suggested that the ATI cell may also contribute significantly to clearance. Freshly isolated ATI cells contain all three subunits of ENaC, as well as the {alpha}1- and {beta}1-subunits of Na+-K+-ATPase, suggesting that ATI cells possess the machinery to contribute significantly to the maintenance of alveolar fluid balance (2, 22, 36).

In the experiments presented here, the cells were studied between 96 and 120 h, a time period when the cells are known to have features of both types I and II cells (6, 31). However, recent work indicates that growth of cultured cells in an air-liquid interface preserves the ATII phenotype, including the expression of highly selective sodium channels (20). In this study, the cells were grown in an air-liquid interface for 24 h before the transport studies. Immunostaining of the cell monolayer growing at 120 h with Lysotrack green DND-26 showed that most of the cells still had lamellar bodies, a typical characteristic of ATII cells. Electron microscopy also confirmed that the cells studied here maintained an ATII phenotype. Therefore, we conclude that the rates of fluid transport measured probably reflect primarily the transport properties of ATII cells.

One of the advantages of our new in vitro system is that the surface area is controlled and we can study one cell type, in this case type II cells. In our in vitro studies, the rate of transport by the cultured ATII cells was 0.84 µl·cm–2·h–1. How does this rate relate to the anticipated in vivo transport properties of type II cells in the intact rat lung? Interestingly, on the basis of several studies we have done in anesthetized, ventilated rats (31), the net basal fluid clearance rate was 27.4 ± 6.6% (mean ± SD) over 1 h in rat lungs (instillate in both lungs). In these studies, ~75% of the alveolar surface has been covered with the instillate. The total basal, unstimulated transport rate estimated in the in vivo setting is 0.18 µl·cm–2·h–1, which was lower than in these in vitro studies. This calculation suggests that the in vitro fluid transport rates in this study are not low. In addition, this calculation provides some interesting additional information that may have value in estimating the relative contribution of type II and type I cells in the intact lung. If the basal in vitro transport rate for the type II cells in this study approximates their in vivo capacity, then type II cells transport at a rate that is four- to fivefold faster than would be expected by their surface area alone. This is certainly a tenable possibility, since type II cells are metabolically very active and have more easily detectable concentrations of sodium transport proteins than the nearby type I cells. However, this calculation also indicates that type II cells probably do not account for all of the alveolar fluid transport. If we assume that the type II cells occupy 3.7% of the rat lung surface area as previously published (7), then type II cells could account for 17% of basal fluid transport in the intact rat lung. There would still be 83% of fluid transport to be accounted for, which could be generated by type I cells and perhaps distal airway epithelial cells. By this calculation the type I cells, which occupy 96.3% of the alveolar surface, would account for most of the remaining fluid transport, which would be at a lower rate per surface area than type II cells. Therefore, these data support the hypothesis, based on other recent data (2, 22, 36, 37), that type I cells may contribute to fluid transport, although the contribution per cell based on surface area would be less than that of the type II cell.

In summary, these studies demonstrate that an in vitro system can be adapted to provide a useful new method for the measurement of net vectorial fluid transport across alveolar epithelial cell monolayers. Although we used radioactively labeled albumin as the impermeant indicator, it should also be possible to use a fluorescently labeled protein in this model. The system should also be useful for studies of drug absorption and protein transport by isolated alveolar and distal airway cells.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grants HL-51854 and HL-51856 (M. A. Matthay) and was also supported in part by an American Lung Association grant (X. Fang).


    ACKNOWLEDGMENTS
 
We thank Vebeke Peterson for generous help with electron microscopy.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. A. Matthay, Cardiovascular Research Inst., 513 Parnassus Ave., Box 0130, UCSF, San Francisco, CA 94143-0130 (E-mail: mmatt{at}itsa.ucsf.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ashino Y, Ying X, Dobbs LG, and Bhattacharya J. [Ca2+]i oscillations regulate type II cell exocytosis in the pulmonary alveolus. Am J Physiol Lung Cell Mol Physiol 279: L5–L13, 2000.[Abstract/Free Full Text]
  2. Borok Z, Liebler JM, Lubman RL, Foster MJ, Zhou B, Li X, Zabski SM, Kim KJ, and Crandall ED. Na transport proteins are expressed by rat alveolar epithelial type I cells. Am J Physiol Lung Cell Mol Physiol 282: L599–L608, 2002.[Abstract/Free Full Text]
  3. Clerici C. Sodium transport in alveolar epithelial cells: modulation by O2 tension. Kidney Int Suppl 65: S79–S83, 1998.[Medline]
  4. Clerici C, Couette S, Loiseau A, Herman P, and Amiel C. Evidence for Na-K-Cl cotransport in alveolar epithelial cells: effect of phorbol ester and osmotic stress. J Membr Biol 147: 295–304, 1995.[ISI][Medline]
  5. Clerici C, Soler P, and Saumon G. Sodium-dependent phosphate and alanine transports but sodium-independent hexose transport in type II alveolar epithelial cells in primary culture. Biochim Biophys Acta 1063: 27–35, 1991.[ISI][Medline]
  6. Crandall ED and Matthay MA. Alveolar epithelial transport. Basic science to clinical medicine. Am J Respir Crit Care Med 163: 1021–1029, 2001.[Free Full Text]
  7. Crapo JD, Young SL, Fram EK, Pinkerton KE, Barry BE, and Crapo RO. Morphometric characteristics of cells in the alveolar region of mammalian lungs. Am Rev Respir Dis 128: S42–S46, 1983.[ISI][Medline]
  8. Dada LA, Chandel NS, Ridge KM, Pedemonte C, Bertorello AM, and Sznajder JI. Hypoxia-induced endocytosis of Na,K-ATPase in alveolar epithelial cells is mediated by mitochondrial reactive oxygen species and PKC-zeta. J Clin Invest 111: 1057–1064, 2003.[Abstract/Free Full Text]
  9. Diglio CA and Kikkawa Y. The type II epithelial cells of the lung. IV. Adaption and behavior of isolated type II cells in culture. Lab Invest 37: 622–631, 1977.[ISI][Medline]
  10. Dobbs LG. Isolation and culture of alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 258: L134–L147, 1990.[Abstract/Free Full Text]
  11. Dobbs LG, Williams MC, and Brandt AE. Changes in biochemical characteristics and pattern of lectin binding of alveolar type II cells with time in culture. Biochim Biophys Acta 846: 155–166, 1985.[ISI][Medline]
  12. Filippatos GS, Hughes WF, Qiao R, Sznajder JI, and Uhal BD. Mechanisms of liquid flux across pulmonary alveolar epithelial cell monolayers. In Vitro Cell Dev Biol Anim 33: 195–200, 1997.[Medline]
  13. Frank JA, Gutierrez JA, Jones KD, Allen L, Dobbs L, and Matthay MA. Low tidal volume reduces epithelial and endothelial injury in acid-injured rat lungs. Am J Respir Crit Care Med 165: 242–249, 2002.[Abstract/Free Full Text]
  14. Fukuda N, Folkesson HG, and Matthay MA. Relationship of interstitial fluid volume to alveolar fluid clearance in mice: ventilated vs. in situ studies. J Appl Physiol 89: 672–679, 2000.[Abstract/Free Full Text]
  15. Gatzy JT. Ion transport across the excised bullfrog lung. Am J Physiol 228: 1162–1171, 1975.[Abstract/Free Full Text]
  16. Geiser T, Jarreau PH, Atabai K, and Matthay MA. Interleukin-1{beta} augments in vitro alveolar epithelial repair. Am J Physiol Lung Cell Mol Physiol 279: L1184–L1190, 2000.[Abstract/Free Full Text]
  17. Goodman BE and Crandall ED. Dome formation in primary cultured monolayers of alveolar epithelial cells. Am J Physiol Cell Physiol 243: C96–C100, 1982.[Abstract/Free Full Text]
  18. Haller T, Dietl P, Pfaller K, Frick M, Mair N, Paulmichl M, Hess MW, Furst J, and Maly K. Fusion pore expansion is a slow, discontinuous, and Ca2+-dependent process regulating secretion from alveolar type II cells. J Cell Biol 155: 279–289, 2001.[Abstract/Free Full Text]
  19. Haller T, Ortmayr J, Friedrich F, Volkl H, and Dietl P. Dynamics of surfactant release in alveolar type II cells. Proc Natl Acad Sci USA 95: 1579–1584, 1998.[Abstract/Free Full Text]
  20. Jain L, Chen XJ, Ramosevac S, Brown LA, and Eaton DC. Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions. Am J Physiol Lung Cell Mol Physiol 280: L646–L658, 2001.[Abstract/Free Full Text]
  21. Jiang C, Finkbeiner WE, Widdicombe JH, McCray PB Jr, and Miller SS. Altered fluid transport across airway epithelium in cystic fibrosis. Science 262: 424–427, 1993.[ISI][Medline]
  22. Johnson MD, Widdicombe JH, Allen L, Barbry P, and Dobbs LG. Alveolar epithelial type I cells contain transport proteins and transport sodium, supporting an active role for type I cells in regulation of lung liquid homeostasis. Proc Natl Acad Sci USA 99: 1966–1971, 2002.[Abstract/Free Full Text]
  23. Kim KJ, Borok Z, and Crandall ED. A useful in vitro model for transport studies of alveolar epithelial barrier. Pharm Res 18: 253–255, 2001.[CrossRef][ISI][Medline]
  24. Kim KJ, Cheek JM, and Crandall ED. Contribution of active Na+ and Cl fluxes to net ion transport by alveolar epithelium. Respir Physiol 85: 245–256, 1991.[CrossRef][ISI][Medline]
  25. Liley HG, Ertsey R, Gonzales LW, Odom MW, Hawgood S, Dobbs LG, and Ballard PL. Synthesis of surfactant components by cultured type II cells from human lung. Biochim Biophys Acta 961: 86–95, 1988.[ISI][Medline]
  26. Marunaka Y, Niisato N, and Ito Y. Beta agonist regulation of sodium transport in fetal lung epithelium: roles of cell volume, cytosolic chloride and protein tyrosine kinase. J Korean Med Sci 15 Suppl: S42–S43, 2000.[ISI][Medline]
  27. Marunaka Y, Niisato N, O'Brodovich H, and Eaton DC. Regulation of an amiloride-sensitive Na+-permeable channel by a beta2-adrenergic agonist, cytosolic Ca2+ and Cl in fetal rat alveolar epithelium. J Physiol 515: 669–683, 1999.[Abstract/Free Full Text]
  28. Mason RJ, Williams MC, Widdicombe JH, Sanders MJ, Misfeldt DS, and Berry LC Jr. Transepithelial transport by pulmonary alveolar type II cells in primary culture. Proc Natl Acad Sci USA 79: 6033–6037, 1982.[Abstract]
  29. Matalon S. Mechanisms and regulation of ion transport in adult mammalian alveolar type II pneumocytes. Am J Physiol Cell Physiol 261: C727–C738, 1991.[Abstract/Free Full Text]
  30. Matalon S and O'Brodovich H. Sodium channels in alveolar epithelial cells: molecular characterization, biophysical properties, and physiological significance. Annu Rev Physiol 61: 627–661, 1999.[CrossRef][ISI][Medline]
  31. Matthay MA, Folkesson HG, and Clerici C. Lung epithelial fluid transport and the resolution of pulmonary edema. Physiol Rev 82: 569–600, 2002.[Abstract/Free Full Text]
  32. Matthay MA, Folkesson HG, and Verkman AS. Salt and water transport across alveolar and distal airway epithelia in the adult lung. Am J Physiol Lung Cell Mol Physiol 270: L487–L503, 1996.[Abstract/Free Full Text]
  33. Modelska K, Pittet JF, Folkesson HG, Courtney Broaddus V, and Matthay MA. Acid-induced lung injury. Protective effect of anti-interleukin-8 pretreatment on alveolar epithelial barrier function in rabbits. Am J Respir Crit Care Med 160: 1450–1456, 1999.[Abstract/Free Full Text]
  34. O'Grady SM and Lee SY. Chloride and potassium channel function in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 284: L689–L700, 2003.[Abstract/Free Full Text]
  35. Planes C, Blot-Chabaud M, Matthay MA, Couette S, Uchida T, and Clerici C. Hypoxia and beta 2-agonists regulate cell surface expression of the epithelial sodium channel in native alveolar epithelial cells. J Biol Chem 277: 47318–47324, 2002.[Abstract/Free Full Text]
  36. Ridge KM, Olivera WG, Saldias F, Azzam Z, Horowitz S, Rutschman DH, Dumasius V, Factor P, and Sznajder JI. Alveolar type 1 cells express the alpha2 Na,K-ATPase, which contributes to lung liquid clearance. Circ Res 92: 453–460, 2003.[Abstract/Free Full Text]
  37. Ridge KM, Rutschman DH, Factor P, Katz AI, Bertorello AM, and Sznajder JL. Differential expression of Na-K-ATPase isoforms in rat alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 273: L246–L255, 1997.[Abstract/Free Full Text]
  38. Sakuma T, Folkesson HG, Suzuki S, Okaniwa G, Fujimura S, and Matthay MA. Beta-adrenergic agonist stimulated alveolar fluid clearance in ex vivo human and rat lungs. Am J Respir Crit Care Med 155: 506–512, 1997.[Abstract]
  39. Sakuma T, Okaniwa G, Nakada T, Nishimura T, Fujimura S, and Matthay MA. Alveolar fluid clearance in the resected human lung. Am J Respir Crit Care Med 150: 305–310, 1994.[Abstract]
  40. Sakuma T, Pittet JF, Jayr C, and Matthay MA. Alveolar liquid and protein clearance in the absence of blood flow or ventilation in sheep. J Appl Physiol 74: 176–185, 1993.[Abstract]
  41. Sartori C, Fang X, McGraw DW, Koch P, Snider ME, Folkesson HG, and Matthay MA. Selected contribution: long-term effects of {beta}2-adrenergic receptor stimulation on alveolar fluid clearance in mice. J Appl Physiol 93: 1875–1880, 2002.[Abstract/Free Full Text]
  42. Saumon G and Basset G. Electrolyte and fluid transport across the mature alveolar epithelium. J Appl Physiol 74: 1–15, 1993.[Abstract]
  43. Smith JJ and Welsh MJ. Fluid and electrolyte transport by cultured human airway epithelia. J Clin Invest 91: 1590–1597, 1993.[ISI][Medline]
  44. Sznajder JI, Olivera WG, Ridge KM, and Rutschman DH. Mechanisms of lung liquid clearance during hyperoxia in isolated rat lungs. Am J Respir Crit Care Med 151: 1519–1525, 1995.[Abstract]
  45. Uyekubo SN, Fischer H, Maminishkis A, Illek B, Miller SS, and Widdicombe JH. cAMP-dependent absorption of chloride across airway epithelium. Am J Physiol Lung Cell Mol Physiol 275: L1219–L1227, 1998.[Abstract/Free Full Text]
  46. Van Os CH, Wiedner G, and Wright EM. Volume flows across gallbladder epithelium induced by small hydrostatic and osmotic gradients. J Membr Biol 49: 1–20, 1979.[ISI][Medline]