KGF prevents hyperoxia-induced reduction of active ion transport in alveolar epithelial cells

Zea Borok, Salim Mihyu, Valentino F. J. Fernandes, Xiao-Ling Zhang, Kwang-Jin Kim, and Richard L. Lubman

Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California 90033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We evaluated the effects of acute hyperoxic exposure on alveolar epithelial cell (AEC) active ion transport and on expression of Na+ pump (Na+-K+-ATPase) and rat epithelial Na+ channel subunits. Rat AEC were cultivated in minimal defined serum-free medium (MDSF) on polycarbonate filters. Beginning on day 5, confluent monolayers were exposed to either 95% air-5% CO2 (normoxia) or 95% O2-5% CO2 (hyperoxia) for 48 h. Transepithelial resistance (Rt) and short-circuit current (Isc) were determined before and after exposure. Na+ channel alpha -, beta -, and gamma -subunit and Na+-K+-ATPase alpha 1- and beta 1-subunit mRNA levels were quantified by Northern analysis. Na+ pump alpha 1- and beta 1-subunit protein abundance was quantified by Western blotting. After hyperoxic exposure, Isc across AEC monolayers decreased by ~60% at 48 h relative to monolayers maintained under normoxic conditions. Na+ channel beta -subunit mRNA expression was reduced by hyperoxia, whereas alpha - and gamma -subunit mRNA expression was unchanged. Na+ pump alpha 1-subunit mRNA was unchanged, whereas beta 1-subunit mRNA was decreased ~80% by hyperoxia in parallel with a reduction in beta 1-subunit protein. Because keratinocyte growth factor (KGF) has recently been shown to upregulate AEC active ion transport and expression of Na+-K+-ATPase under normoxic conditions, we assessed the ability of KGF to prevent hyperoxia-induced changes in active ion transport by supplementing medium with KGF (10 ng/ml) from day 2. The presence of KGF prevented the effects of hyperoxia on ion transport (as measured by Isc) relative to normoxic controls. Levels of beta 1 mRNA and protein were relatively preserved in monolayers maintained in MDSF and KGF compared with those cultivated in MDSF alone. These results indicate that AEC net active ion transport is decreased after 48 h of hyperoxia, likely as a result of a decrease in the number of functional Na+ pumps per cell. KGF largely prevents this decrease in active ion transport, at least in part, by preserving Na+ pump expression.

alveolar epithelium; growth factor; keratinocyte growth factor; oxygen toxicity; sodium transport


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ALTHOUGH OXYGEN IS AN important adjunct to therapy in hypoxemic respiratory failure from diverse causes, exposure to high oxygen tension may contribute to acute lung injury as a result of increased production of reactive oxygen metabolites (16). Exposure to high concentrations of oxygen results in a well-defined sequence of pathophysiological changes in the lungs of all mammalian species studied (22, 37). Even before the onset of any structural changes, hyperoxic lung injury is characterized by the accumulation of interstitial edema, followed by an increase in epithelial permeability and alveolar flooding. These changes reflect effects of hyperoxia on both endothelial and epithelial components of the alveolar epithelial barrier.

The intact alveolar epithelium forms a barrier that is highly resistant to the passive flux of solutes and water from the lung interstitium into the alveolar air spaces (29). Alveolar fluid balance is further regulated due to active transepithelial ion transport by alveolar epithelial cells (AEC). Vectorial Na+ transport across the epithelium occurs predominantly via apical Na+ entry into the cells through amiloride-sensitive epithelial Na+ channels (ENaC) and extrusion via basolateral Na+ pumps (Na+-K+-ATPase), leading to net absorption of fluid as a result of the osmotic gradient thus created (9, 15, 18, 19, 38). Together, the barrier and transport properties of the alveolar epithelium are largely responsible for maintaining the relatively fluid-free condition of the alveolar air spaces. Compromise of the alveolar epithelial barrier, or interference with the normal mechanisms of AEC Na+ transport, can contribute to the pathological accumulation of alveolar edema fluid following acute lung injury.

The effects of hyperoxia on transepithelial transport and alveolar fluid clearance have been studied in a variety of experimental models, including whole lung, isolated lung, cultured AEC, and isolated alveolar type II (AT2) cells following in vivo or in vitro exposure (1, 6-8, 17, 21, 23, 27, 33-35, 37, 39, 48, 49, 51). However, the use of different experimental conditions, such as the degree of hyperoxia or the duration of exposure, has resulted in apparently discrepant results among many of these studies. Both increases and decreases in solute and water transport, Na+ pump activity and expression, and Na+ channel expression have been reported as effects of hyperoxia on AEC (6-8, 17, 21, 23, 33, 34, 35, 49, 51). Some of these apparent discrepancies may also be the result of unrecognized differences in the responses of alveolar type I (AT1) and AT2 cells to injury. Although AT1 cells have traditionally been thought to be more susceptible to injury than AT2 cells, technical difficulties inherent in isolating purified functional populations of AT1 cells from adult lung have precluded direct evaluation of potential effects of hyperoxia on AT1 cell function.

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor family, has been shown to ameliorate lung injury from a number of different causes, including bleomycin, radiation, acid instillation, and hyperoxia (13, 20, 31, 36, 40, 44, 46, 47). The beneficial effects of KGF in lung injury have been attributed to increased AT2 cell proliferation, but the possibility of additional cytoprotective effects has not been fully explored. We have recently demonstrated that KGF increases expression of Na+ pumps accompanied by an increase in active ion transport across AEC monolayers grown on permeable supports (2). These findings suggest that upregulation of active transepithelial ion transport could be an additional mechanism that may contribute to the beneficial effects of KGF in hyperoxic lung injury.

In this study, we evaluated the effects of hyperoxia (95% for 48 h) on AEC active ion transport and expression of Na+-K+-ATPase and Na+ channels in AEC monolayers and the ability of KGF to modulate hyperoxia-induced changes in AEC ion transport. We demonstrate that acute exposure to hyperoxia decreases AEC active ion transport, likely due to decreased expression of Na+-K+-ATPase beta 1-subunits. KGF abrogates the inhibitory effects of hyperoxia on Isc while preserving Na+ pump beta 1-subunit expression.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell isolation and preparation of rat AEC monolayers. AT2 cells were isolated from adult male Sprague-Dawley rats by disaggregation with elastase (2.0-2.5 U/ml; Worthington Biochemical, Freehold, NJ), followed by differential adherence on IgG-coated bacteriologic plates (14). The enriched AT2 cells were resuspended in a minimal defined serum-free medium (MDSF) consisting of DMEM and Ham's F-12 nutrient mixture in a 1:1 ratio (DMEM/F12; Sigma Chemical, St. Louis, MO), supplemented with 1.25 mg/ml BSA (Collaborative Research, Bedford, MA), 10 mM HEPES, 0.1 mM nonessential amino acids, 2.0 mM glutamine, 100 U/ml sodium penicillin G, and 100 µg/ml streptomycin (3). Cells were seeded onto tissue culture-treated polycarbonate (Nuclepore) filter cups (Transwell, Corning Costar, Cambridge, MA) at a density of 1.0 × 106 cells/cm2. Cultures were maintained in a humidified 5% CO2 incubator at 37°C. Media were changed, thereby removing nonadherent cells, on the second day after plating. From day 2, the monolayers were incubated in either MDSF or MDSF supplemented with KGF (10 ng/ml). The dose of KGF used was based on previous studies demonstrating a half-maximal effective dose for the effect of KGF on Isc of 0.5 ng/ml (2). AT2 cell purity (>= 90%) was assessed by staining freshly isolated cells for lamellar bodies with tannic acid (32). Cell viability (>90%) was measured by trypan blue dye exclusion. Cell number in the monolayers was evaluated by counting cell nuclei as previously described (5).

Measurement of monolayer bioelectric properties. Transepithelial resistance (Rt; kOmega · cm2) and spontaneous potential difference (SPD; mV) were measured using a rapid-screening device (Millicell-ERS; Millipore, Bedford, MA). Equivalent short-circuit current (Isc; µA/cm2) was calculated from the relationship Isc = SPD/Rt as previously described (2, 4, 9, 12). Comparison of Isc data obtained using a Millicell device with Isc data obtained from Ussing chamber studies previously showed these measurements to be equivalent in this system (25). Rt and Isc were determined before the hyperoxic exposure period (day 5) and 48 h thereafter (day 7).

Experimental design and hyperoxic exposure. To evaluate the effects of hyperoxia on AEC active ion transport, confluent AEC monolayers maintained in MDSF with or without KGF were exposed to either 95% air-5% CO2 ("normoxia") or 95% O2-5% CO2 ("hyperoxia") for 48 h in humidified, sealed exposure chambers beginning on day 5. Analysis of gas samples from the hyperoxia exposure chambers on day 7 confirmed that O2 concentration was maintained at >= 95% for the 48-h period. RNA and protein were harvested from AEC in MDSF with or without KGF before exposure on day 5 and from normoxia- or hyperoxia-exposed monolayers on day 7.

RNA isolation and Northern analysis. Total RNA was isolated by the acid phenol-guanidinium-chloroform method (10). Equal amounts of RNA (5-10 µg) were denatured with formaldehyde, size fractionated by agarose gel electrophoresis under denaturing conditions, and transferred to nylon membranes. RNA was immobilized by ultraviolet cross-linking (Hybond N+, Amersham Life Science, Cleveland, OH). Blots were prehybridized for 2 h at 65°C in 1 M sodium phosphate buffer (pH 7), 7% SDS, and 1% BSA. Hybridization was performed for 16 h at 65°C in the same buffer. Blots were probed with isoform-specific cDNA probes for the alpha 1 and beta 1 isoforms of Na+-K+-ATPase (E. Benz, Johns Hopkins University), and the alpha -, beta -, and gamma -subunits of the rat ENaC (rENaC; C. Canessa, Yale University and B. Rossier, Université de Lausanne). Probes were labeled with [alpha -32P]dCTP (Amersham) by the random primer method using a commercially available kit (Boehringer Mannheim, Indianapolis, IN). Blots were washed at high stringency [0.5× SSC (75 mM NaCl, 7.5 mM sodium citrate, pH 7.0) with 0.1% SDS at 55°C] and visualized by autoradiography. Differences in RNA loading were normalized using a 24-mer oligonucleotide probe for 18S rRNA end labeled with [gamma -33P]ATP. Expression of Na+ channel and Na+ pump subunit mRNA was quantified by densitometry.

Western analysis. SDS-PAGE was performed using the buffer system of Laemmli (26), and immunoblotting was performed using procedures modified from Towbin et al. (42). For detection of alpha 1-subunits of Na+-K+-ATPase, AEC monolayers were solubilized directly into 2% SDS sample buffer. Equal amounts of cell protein in sample buffer were resolved by SDS-PAGE under reducing conditions and electrophoretically blotted onto Immobilon-P (Millipore, Marlborough, MA). The blotted sheets were blocked for 2 h with 5% nonfat dry milk in Tris-buffered saline (TBS; 20 mM Tris, 500 mM NaCl, pH 7.5), and then incubated with primary antibody (Ab) overnight for detection of Na+ pump subunits by immunoblot. The monoclonal anti-alpha 1 Ab 6H (24) (M. Caplan, Yale University) was used for detection of Na+ pump alpha 1-subunits. Blots were washed with TBS with 0.5% Tween 20 and incubated with horseradish peroxidase-linked goat anti-mouse IgG conjugates for 1 h, and antigen-Ab complexes were visualized by enhanced chemiluminescence (ECL, Amersham, Arlington Heights, IL). Relative intensity of Na+ pump alpha 1-subunit protein bands was quantified by densitometry and compared after calibration with standard dilutions of control cell lysate. Protein concentrations were determined using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA), with BSA as a standard.

For detection of the beta 1-subunits, AEC monolayers were first immunoprecipitated with the monoclonal anti-Na+ pump beta -subunit Ab IEC 1/48 (A. Quaroni, Cornell University) (30), deglycosylated, and processed for SDS-PAGE as previously described (50). The polyclonal anti-beta 1-subunit Ab FP (A. McDonough, University of Southern California) was used for detection of beta -subunits precipitated by Ab IEC 1/48, since the latter recognizes only the undenatured beta -subunit and is not useful for immunoblotting. Blots were incubated with horseradish peroxidase-linked goat anti-rabbit IgG conjugates for 1 h, and antigen-Ab complexes were visualized by chemiluminescence.

Materials. Human recombinant KGF was obtained from R&D Systems (Minneapolis, MN). Cell culture media and all other chemicals were purchased from Sigma and were of the highest commercial quality available.

Statistical analysis. Results are expressed as means ± SE. Significance (P < 0.05) of differences in mean cell number, Rt, Isc, and densitometric measurements of Na+ channel mRNA and Na+ pump subunit mRNA and protein expression were determined either by Student's t-test or by ANOVA (when multiple pairwise comparisons were required).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Cell number. Monolayer cell number was determined in MDSF with or without KGF before and after hyperoxic exposure. There were no significant differences in cell number on day 5 between monolayers maintained in MDSF with or without KGF (Fig. 1A). Cell number was 39% greater in MDSF plus KGF than in MDSF plus +O2 on day 7 but was not significantly different among other conditions (Fig. 1B).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of keratinocyte growth factor (KGF) and/or hyperoxia on alveolar epithelial cell (AEC) monolayer cell number. A: cell number was not different on day 5 for monolayers maintained in minimal defined serum-free medium (MDSF) + KGF vs. those cultivated in MDSF alone (n = 3). B: cell number on day 7 was greater for MDSF + KGF than MDSF + hyperoxia (O2), but not different among all other conditions. * Significantly different from MDSF + O2 (n = 3).

Bioelectric properties. AEC cultivated on polycarbonate filters in MDSF form functional monolayers that actively transport Na+, with Rt ~3.0 kOmega · cm2 and Isc ~2.5 µA/cm2 on day 5. As illustrated in Fig. 2A, Rt is not significantly reduced for monolayers grown in MDSF in hyperoxia relative to normoxia. Although Rt is reduced in KGF-exposed monolayers in both normoxia and hyperoxia relative to those grown in MDSF, there is no difference in Rt for monolayers grown in KGF under either normoxic or hyperoxic conditions. Isc for hyperoxia-exposed monolayers maintained in MDSF is 53% less than that for corresponding normoxia-exposed controls at 48 h, whereas monolayers cultivated in MDSF plus KGF demonstrate an 87% increase in transepithelial active ion transport compared with monolayers in MDSF. As shown in Fig. 2B, treatment with KGF prevents the hyperoxia-induced decrease in Isc.


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 2.   Effects of KGF and/or hyperoxia on AEC monolayer bioelectric properties. A: transepithelial resistance (Rt). Exposure of monolayers grown in MDSF to hyperoxia for 48 h results in no significant change in Rt relative to normoxia-exposed controls on day 7. Rt is reduced in monolayers maintained in MDSF + KGF with or without O2 relative to monolayers grown in MDSF in normoxia, although there is no difference between monolayers maintained in MDSF + KGF in normoxia vs. hyperoxia. * Significantly different from MDSF in normoxia (n = 10). B: short-circuit current (Isc). AEC monolayers grown in MDSF exposed to hyperoxia for 48 h show a decrease in Isc compared with normoxia-exposed controls on day 7, consistent with a reduction in active transepithelial ion transport. Isc is significantly higher in normoxia-exposed AEC monolayers maintained in MDSF + KGF than in monolayers maintained in MDSF. Isc for KGF-treated monolayers cultivated under hyperoxic conditions was not different from that of monolayers grown in MDSF in normoxia. * Significantly different from all other conditions. ** Significantly different from other conditions except for MDSF in normoxia (n = 10).

rENaC subunit mRNA expression. As shown in the representative Northern blot in Fig. 3A, AEC monolayers express mRNA for the alpha -, beta -, and gamma -subunits of rENaC on day 7 in culture in MDSF. mRNA levels for the alpha - and gamma -subunits show no significant reduction after hyperoxic exposure for 48 h, whereas mRNA for beta -subunit rENaC is reduced by 30%. Monolayers maintained in MDSF + KGF under normoxic conditions demonstrate decreased levels of Na+ channel alpha -, beta -, and gamma -subunit mRNA on day 7 relative to MDSF. Hyperoxia does not significantly increase Na+ channel mRNA levels in MDSF + KGF monolayers, with levels remaining significantly below those for monolayers grown in MDSF (Fig. 3, A and B).



View larger version (57K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of KGF and/or hyperoxia on Na+ channel mRNA. A: as shown in this representative Northern blot, mRNA levels for alpha - and gamma -subunits of rat epithelial Na+ channel (rENaC) are unchanged on day 7 in MDSF following O2 exposure for 48 h relative to controls maintained in normoxia, whereas an ~30% reduction occurs in beta -subunit rENaC mRNA levels. Monolayers treated with KGF with or without hyperoxia show decreased mRNA levels for all rENaC subunit levels relative to monolayers cultivated in MDSF and normoxia. RNA loading was similar among all conditions, as indicated by equivalence of signal following hybridization with an 18S rRNA oligonucleotide probe. B: average relative densitometric values of hyperoxia- and/or KGF-treated monolayers as a fraction of normoxic controls grown in MDSF. * Significantly different from MDSF in normoxia (n = 3).

Na+-K+-ATPase alpha 1- and beta 1-subunit expression. AEC cultivated for 5 days in MDSF express abundant mRNA for the alpha 1- and beta 1-subunit isoforms of Na+-K+-ATPase, with levels of these isoforms remaining constant through day 7 in normoxia-exposed monolayers (data not shown). After exposure to hyperoxia for 48 h, Na+ pump alpha 1-subunit mRNA levels remain unchanged relative to those in normoxia-exposed monolayers. There is a decline in levels of Na+ pump beta 1-subunit mRNA to ~20% compared with normoxia-exposed cells at 48 h. Abundant expression of both Na+ pump alpha 1- and beta 1-subunit protein in AEC monolayers is also observed on day 5 in MDSF, as we have previously shown (12). Abundance of Na+ pump alpha 1-subunit protein does not decrease significantly in monolayers following exposure to hyperoxia. Na+ pump beta 1-subunit protein, quantified using the relative size of the endoglycosidase-dependent band, decreases to ~35% of that observed in normoxic controls (see Fig. 5, A and B).

Monolayers maintained in MDSF + KGF demonstrate increased levels of both Na+ pump alpha 1- and beta 1-subunit mRNA on day 5 relative to MDSF (data not shown). Levels of Na+ pump alpha 1-subunit mRNA remain increased in the presence of KGF following exposure to hyperoxia relative to both normoxia- and hyperoxia-exposed cells in MDSF on day 7, whereas the reduction in beta 1-subunit mRNA observed after exposure to hyperoxia in MDSF on day 7 is partially prevented (Fig. 4, A and B). Monolayers treated with KGF and grown through day 7 in normoxia have increased abundance of Na+ pump alpha 1-subunit protein relative to those grown in MDSF alone in either normoxia or hyperoxia. Monolayers cultivated with KGF under normoxic conditions show large increases in Na+ pump beta 1-subunit protein abundance compared with those grown in MDSF alone in either normoxia or hyperoxia. Monolayers cultivated with KGF under hyperoxic conditions show an increase in Na+ pump beta 1-subunit protein abundance that is smaller than that for monolayers grown in KGF in normoxia but is still greater than that observed for those grown in MDSF alone in either normoxia or hyperoxia (Fig. 5, A and B).



View larger version (76K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of KGF and/or hyperoxia on Na+-K+-ATPase alpha 1- and beta 1-subunit mRNA. A: as shown in this representative Northern blot, alpha 1-subunit levels are unchanged, whereas beta 1-subunit levels are decreased in MDSF following hyperoxic exposure. In the presence of KGF, alpha 1-subunit of Na+-K+-ATPase is increased relative to both normoxia- and hyperoxia-exposed monolayers in MDSF. In addition, KGF pretreatment partially blocks hyperoxia-induced reduction in Na+ pump beta 1-subunit mRNA seen in MDSF. B: average relative densitometric values of Na+ pump subunit mRNA levels in hyperoxia- and/or KGF-treated monolayers as a fraction of normoxic controls grown in MDSF. * Significantly different from MDSF in normoxia. ** Significantly different from MDSF with or without O2 (n = 7).




View larger version (34K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of KGF and/or hyperoxia on Na+-K+-ATPase alpha 1- and beta 1-subunit protein expression. A: these representative Western blots demonstrate that beta 1-subunit protein abundance is decreased after 48 h of hyperoxic exposure in MDSF, whereas there is no significant change in alpha 1-subunit protein abundance. In the presence of KGF, the decrease in beta 1-subunit protein following hyperoxia is prevented. B: average relative densitometric values of Na+ pump subunit protein abundance in hyperoxia- and/or KGF-treated monolayers as a fraction of normoxic controls grown in MDSF. * Significantly different from all other conditions. ** Significantly greater than MDSF with or without O2 (n = 3 for alpha 1-subunit; n = 4 for beta 1-subunit).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we demonstrate that exposure of AEC monolayers (in MDSF) to 95% O2 for 48 h (hyperoxia) results in a decrease in transepithelial active ion transport. This decrease is accompanied by reduced levels of Na+-K+-ATPase beta 1-subunit mRNA and protein, which decline in parallel with the observed decrease in Isc. Expression of mRNA and protein for the alpha 1-subunit of Na+-K+-ATPase remains unchanged following hyperoxia. Expression of mRNA for the alpha - and gamma -subunits of rENaC is also unchanged by hyperoxia, and that of the beta -subunit is only modestly diminished. Because effects on Isc must depend on changes in either Na+ channels, Na+ pumps, or both, these data suggest that the hyperoxia-induced decrease in active ion transport across AEC monolayers is mediated primarily via effects on Na+ pump abundance that are driven by a decrease in expression of the Na+ pump beta 1-subunit. Despite reduced expression of all three rENaC subunits in AEC monolayers in MDSF plus KGF, hyperoxia-induced decreases in active transepithelial ion transport and Na+ pump beta 1-subunit mRNA and protein expression are partially prevented by KGF. KGF also effects increases in Na+ pump alpha 1-subunit mRNA and protein expression under both normoxic and hyperoxic conditions. These data imply that KGF ameliorates the hyperoxia-induced reduction of active ion transport across AEC monolayers by its effects on Na+ pumps. KGF alters Na+ pump abundance under hyperoxic conditions by a relative increase in alpha 1-subunit mRNA and protein levels, a relative preservation of beta 1-subunit mRNA and protein levels, or both mechanisms.

Several lines of evidence support the hypothesis that the hyperoxia-induced impairment of transepithelial transport across AEC monolayers described herein and the ameliorative effects of KGF on this process both occur primarily via effects on Na+ pump subunit mRNA and protein levels. As shown in Fig. 2A, hyperoxia did not cause significant reductions in Rt (an index of monolayer and tight junction integrity) relative to normoxia-exposed controls. AEC monolayers remain electrically "tight" (Rt > 1,000 Omega  · cm2) following hyperoxia, implying continued low paracellular permeability. Although significant loss of alveolar epithelial barrier function through cell loss or impaired tight junctions could occur with more severe hyperoxic injury, these findings make it unlikely that either the large (~50%) hyperoxia-induced decrements in Isc (an index of active ion transport) under the conditions of our experiments or their relative preservation in the presence of KGF results from effects on passive solute and water permeability (Fig. 2B). The effects of hyperoxia and KGF specifically do not appear to be mediated by a change in cell number per monolayer, as illustrated in Fig. 1, A and B.

Hyperoxia reduces Isc across AEC monolayers (MDSF), whereas Isc remains higher across hyperoxia-exposed AEC monolayers treated with KGF (MDSF plus KGF) relative to either normoxia-exposed or hyperoxia-exposed monolayers maintained in MDSF (Fig. 2B). These KGF-sensitive effects of hyperoxia on Isc reflect alterations in active transepithelial Na+ transport across AEC monolayers. As we have previously demonstrated, Isc is almost entirely due to Na+ reabsorption across AEC monolayers (9, 25). Furthermore, KGF does not alter the fraction of Isc across AEC monolayers that is amiloride or ouabain sensitive (2), consistent with the assertion that neither the ion species actively transported nor the specific ion transport pathways they traverse are changed by this growth factor. This does not preclude, however, a complex effect of both stimuli on the function and/or abundance of amiloride-sensitive Na+ channels (the major Na+ entry pathway), Na+ pumps (the major Na+ exit pathway), or both.

Our current data indicate that hyperoxia negatively influences Na+ transport across AEC monolayers. As shown in Fig. 3, A and B, hyperoxia has no significant effect on mRNA levels for either alpha  or gamma  rENaC subunits and only a modest decrease in the level of beta  rENaC subunit mRNA, suggesting that the effect of hyperoxia on ENaC Na+ channels is not the primary cause of reduced active Na+ transport. We cannot exclude the possibility that exposure to hyperoxia alters ENaC subunit abundance posttranscriptionally, due in part to the current lack of availability of antibodies suitable for quantifying rENaC subunits. It is also possible that other types of Na+ channels are upregulated or that oxidation of Na+ channels directly alters their function [as others have reported (49)], although changes in rENaC subunit abundance alone seem an unlikely explanation for the observed hyperoxia-induced reduction in active ion transport across AEC monolayers.

Conversely, hyperoxia appears to induce a large decrease in Na+ pump subunit abundance in AEC monolayers. The Na+ pump is a heterodimeric protein consisting of alpha - (catalytic) and beta - (regulatory) subunits, of which the alpha 1 and beta 1 isoforms are predominantly expressed in alveolar epithelium (2, 12, 50). As shown in Fig. 5, A and B, the relative abundance of the beta 1-subunit protein declines by 80% following 48 h of hyperoxic exposure. If beta -subunits are rate limiting for the formation of heterodimers under the conditions of these experiments, as has been described in other cell types and conditions (28), hyperoxia likely causes a reduction in the total number of Na+ pumps present in the cells. Although measurements of plasma membrane Na+ pump vs. intracellular pools would be useful to confirm the loss of basolateral heterodimers, the reduction in Isc suggests that the decrease in total Na+ pumps reflects a loss of functional pumps at the plasma membrane. This decrease in Na+ pump beta -subunits is associated with a reduction in beta -subunit mRNA level, consistent with a possible role of transcriptional regulation of Na+ pump expression due to hyperoxia as has previously been suggested (45).

The ameliorative effect of KGF on hyperoxia-induced reductions in active Na+ transport does not appear to be related to effects on rENaC expression. KGF decreases mRNA levels of all three units of this Na+ channel while increasing Isc across normoxia-exposed AEC monolayers (2). rENaC alpha -, beta -, and gamma -subunit mRNA levels are diminished in KGF-treated hyperoxia-exposed monolayers compared with those grown in MDSF in normoxic conditions, and rENaC beta - and gamma -subunit mRNA levels are lower in KGF-treated and hyperoxia-exposed monolayers than in those grown in MDSF under hyperoxic conditions as well. Although the same caveats may apply for the effects of KGF on Na+ channel expression and function as those cited above for hyperoxia, data on rENaC mRNA levels suggest that changes in the abundance of the subunits of this type of Na+ channel cannot account for the effects of KGF on Isc.

KGF prevents the hyperoxia-induced decrease in Na+ pump beta -subunit abundance, and presumably in total Na+ pumps, thereby increasing Isc across hyperoxia-treated monolayers. As illustrated in Fig. 5, A and B, the decrease in Na+ pump beta 1-subunit protein abundance relative to AEC monolayers grown in MDSF under normoxic conditions does not occur in hyperoxia-exposed AEC monolayers in MDSF plus KGF. This effect occurs in parallel with an increase in Na+ pump alpha 1-subunit mRNA level relative to both normoxia- and hyperoxia-exposed monolayers and a partial block of the hyperoxia-induced reduction in the beta -subunit mRNA level (Fig. 4, A and B). These data suggest two possibly independent actions of KGF in AEC subjected to hyperoxia, namely, an increase in Na+ pump alpha -subunit mRNA levels as previously described in normoxia-exposed monolayers (2) and a protective effect on the hyperoxia-induced reduction in Na+ pump beta -subunit mRNA levels. Taken together, these data suggest that hyperoxia reduces Isc across AEC monolayers in association with decreased Na+ pump subunit expression. KGF ameliorates both the decrease in Isc and decrease in Na+ pumps via multiple, probably independent, regulatory effects on Na+ pump subunit expression.

The effects of hyperoxia on lung Na+ transport and fluid balance have been studied in a variety of rat models. In subacute models (e.g., 85% O2 for 7 days followed by a variable recovery period), hyperoxia has been reported to augment amiloride-sensitive Na+ flux and water clearance (39), increase expression of Na+ channel mRNA or protein (21, 49), and increase Na+ pump activity and subunit protein (23). These findings suggest that, under conditions of subacute injury, the alveolar epithelium is able to adapt by upregulating Na+ transport pathways in a compensatory fashion to enhance alveolar fluid clearance. Studies on the effects of shorter exposures (48-64 h) to higher concentrations of O2 (97-100%) have yielded less consistent results. For example, Nici et al. (33) found that lung mRNA levels for Na+-K+-ATPase alpha 1- and beta 1-subunits increased in animals exposed to 97% O2 for 60 h compared with normoxic controls, and Garat et al. (17) demonstrated that alveolar fluid clearance mechanisms in anesthetized rats are relatively preserved following exposure to 100% O2 for 40 h. Zheng et al. (51) recently showed no change in active Na+ transport or D-glucose flux in lungs isolated from animals exposed to >95% O2 for 48 or 60 h, whereas Olivera et al. (34) demonstrated a decrease in active Na+ transport and lung liquid clearance in lungs isolated from rats exposed to 100% O2 for 64 h, with Na+-K+-ATPase activity reduced in parallel fashion in AT2 cells isolated from these animals. The divergent results among these studies remain puzzling but may indicate that acute exposure to hyperoxia may result in either alveolar edema or enhanced fluid clearance, depending on some thus far unidentified specific conditions or factors present at the time of exposure.

In the current study, exposure of AEC monolayers to 95% O2 for 48 h in vitro results in a large decrease in active ion transport and Na+ pump and beta 1-subunit protein abundance but little change in Na+ channel expression and no change in Rt (i.e., no change in monolayer permeability). Our data therefore support the concept that diminished AEC active ion transport contributes to the development of alveolar edema resulting from acute hyperoxic lung injury primarily, if not exclusively, due to downregulation of Na+ pump expression. Although it is not yet possible to reconcile the results of all of the studies cited above with those of our current study, it is likely that some of the dissimilarities can be accounted for by the use of different experimental model systems. For example, previous studies on effects of hyperoxia on AEC transport have focused almost exclusively on Na+-K+-ATPase activity and expression in AT2 cells isolated following in vivo exposure or exposed to increased levels of O2 immediately after isolation. AEC cultivated on plastic or other inflexible substrata, as in the current study, gradually acquire the phenotypic characteristics of AT1 cells via a process of transdifferentiation. The marked downregulation of active ion transport pathways observed under these conditions may therefore reflect an increased susceptibility of AT1 cells to oxidant damage, whose effects are not compensated for by less marked effects on AT2 cells or by AT2 cell proliferation in vitro.

We studied AEC monolayers grown in serum-free medium on permeable supports to enable us to evaluate direct effects of hyperoxia on alveolar epithelial Na+ transport pathways in the absence or presence of interactions with mesenchyme-derived growth factors that may be induced by inflammatory stimuli in vivo under some conditions. Our current data, which show a marked ameliorative effect of KGF on hyperoxic downregulation of Na+ pump expression and transepithelial transport, further support the concept that such growth factors can profoundly influence AEC transport properties (2, 9). KGF, a member of the fibroblast growth factor family, is an epithelial mitogen that acts as a paracrine mediator of mesenchymal-epithelial interactions in a variety of tissues, including the skin, liver, gastrointestinal tract, and alveolar epithelium. The target specificity of KGF is determined by expression of the KGF receptor, whose expression is restricted to epithelial cells. KGF has been shown to ameliorate lung injury from a number of different causes, including bleomycin, radiation, acid instillation, and hyperoxia (13, 20, 31, 36, 40, 44, 46, 47). The beneficial effects of KGF in lung injury have been attributed to an increase in cell proliferation, but recent studies suggest an additional role for KGF in upregulating alveolar fluid clearance. For example, intratracheal administration of KGF to rats exposed to hyperoxia results in improved survival accompanied by decreased accumulation of pleural effusions and intra-alveolar exudates, suggesting an effect of KGF on alveolar fluid clearance mechanisms (20). Consistent with these observations in hyperoxia-induced lung injury, similar protective effects of KGF have been observed in a rat model of pulmonary edema induced by alpha -naphthylthiourea, in which pretreatment of animals with KGF upregulates active alveolar epithelial ion transport and significantly attenuates the development of lung leak (31).

We have also recently demonstrated that treatment of AEC monolayers with KGF results in a sustained increase in transepithelial active ion transport accompanied by an increased Na+ pump abundance (2). In the present study, we similarly demonstrate that KGF induces an increase in AEC active ion transport under normoxic conditions, reflected by a >80% increase in Isc at day 7. As in the previous study, this KGF-induced upregulation of alveolar epithelial Na+ transport appears to be driven by an increase in Na+ pump alpha 1-subunit mRNA levels, with no effect seen on beta 1-subunit mRNA. In contrast, hyperoxia appears to inhibit AEC active ion transport via effects on Na+ pump expression that are driven largely by changes in beta 1-subunit mRNA, since levels of the alpha 1-subunit are unchanged. After exposure to hyperoxia for 48 h, levels of alpha 1 mRNA in monolayers pretreated with KGF are increased relative to those observed for monolayers grown in MDSF under normoxic conditions. In addition, the reduction of levels of beta 1 mRNA that are observed following hyperoxia in MDSF is largely prevented by KGF. Taken together, these data suggest that hyperoxia and KGF exert their effects on AEC transport and Na+ pump expression by distinct mechanisms related to differential effects on cellular expression of the Na+ pump alpha - and beta -subunit genes. Further studies will be required to distinguish whether these effects are transcriptionally and/or posttranscriptionally mediated, as well as to determine the signal transduction pathways relevant for each stimulus.

In the current study, AEC monolayers were treated with KGF from days 2 through 5 in culture before exposure to hyperoxia. This period of pretreatment was chosen to allow full expression of KGF effects on AEC transport properties to develop before the monolayers were subjected to hyperoxic injury (2). Previous studies showing a reduction in lung injury from acid aspiration, radiation, bleomycin, and peroxide due to KGF either only attempted or were only able to show ameliorative effects of pretreatment (13, 20, 31, 36, 40, 44, 46, 47). For prevention of some forms of lung injury, particularly those that occur unpredictably, pretreatment with KGF might not be possible or practical. Because we did not attempt concurrent treatment or posttreatment with KGF following hyperoxic exposure, the potential for protective effects of KGF given during or after the initiation of lung injury remains undetermined. Nonetheless, when early treatment for hyperoxic exposure can be instituted in the clinical situations in which it most commonly occurs, such as mechanical ventilation of patients with respiratory failure using high inspired oxygen concentrations, a role for KGF may potentially exist.

In summary, we demonstrate that hyperoxia reduces active ion transport across AEC monolayers accompanied by a decrease in Na+-K+-ATPase beta 1-subunit mRNA and protein. KGF partially prevents the hyperoxia-induced reduction in active Na+ transport and Na+ pump expression, accompanied by increased Na+ pump alpha 1-subunit expression and by a relative preservation of Na+-K+-ATPase beta 1-subunit expression. We conclude that hyperoxia downregulates AEC transport and Na+ pump expression in vitro, an effect ameliorated by pretreatment with KGF. These results suggest a possible role for KGF in the prevention and treatment of alveolar edema resulting from hyperoxia.


    ACKNOWLEDGEMENTS

We thank Dr. Edward D. Crandall for numerous helpful discussions and review of the manuscript. We also note with appreciation the expert technical support of Monica Flores, Martha Jean Foster, and Susie Parra.


    FOOTNOTES

This work was supported in part by the American Lung Association, the American Heart Association (Greater Los Angeles Affiliate), National Heart, Lung, and Blood Institute (NHLBI) Clinical Investigator Development Award HL-02836, NHLBI Grants HL-03609, HL-38578, HL-38621, HL-38658, and HL-51928, and the Hastings Foundation.

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

Address for reprint requests and other correspondence: Z. Borok, Div. of Pulmonary and Critical Care Medicine, GNH 11900, University of Southern California, 2025 Zonal Ave., Los Angeles, CA 90033 (E-mail: zborok{at}hsc.usc.edu).

Received 18 August 1998; accepted in final form 10 March, 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Allen, C. B., X. L. Guo, and C. W. White. Changes in pulmonary expression of hexokinase and glucose transporter mRNAs in rats adapted to hyperoxia. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L320-L329, 1998[Abstract/Free Full Text].

2.   Borok, Z., S. I. Danto, L. L. Dimen, X.-L. Zhang, and R. L. Lubman. Na+-K+-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L149-L158, 1998[Abstract/Free Full Text].

3.   Borok, Z., S. I. Danto, S. M. Zabski, and E. D. Crandall. Defined medium for primary culture de novo of adult rat alveolar epithelial cells. In Vitro Cell. Dev. Biol. 30A: 99-104, 1994.

4.   Borok, Z., A. Hami, S. I. Danto, R. L. Lubman, K.-J. Kim, and E. D. Crandall. Effects of EGF on alveolar epithelial junctional permeability and active sodium transport. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L559-L565, 1996[Abstract/Free Full Text].

5.   Borok, Z., A. Hami, S. I. Danto, S. M. Zabski, and E. D. Crandall. Rat serum inhibits progression of alveolar epithelial cells toward the type I cell phenotype in vitro. Am. J. Respir. Cell Mol. Biol. 12: 50-55, 1995[Abstract].

6.   Carter, E. P., S. E. Duvick, C. H. Wendt, J. Dunitz, L. Nici, O. D. Wangensteen, and D. H. Ingbar. Hyperoxia increases active alveolar Na+ resorption in vivo and type II cell Na,K-ATPase in vitro. Chest 105: 75S-78S, 1994[Medline].

7.   Carter, E. P., O. D. Wangensteen, J. Dunitz, and D. H. Ingbar. Hyperoxic effects on alveolar sodium resorption and lung Na-K-ATPase. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L1191-L1202, 1997[Medline].

8.   Carter, E. P., O. D. Wangensteen, S. M. O'Grady, and D. H. Ingbar. Effects of hyperoxia on type II cell Na-K-ATPase function and expression. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L542-L551, 1997[Abstract/Free Full Text].

9.   Cheek, J. M., K.-J. Kim, and E. D. Crandall. Tight monolayers of rat alveolar epithelial cells: bioelectric properties and active sodium transport. Am. J. Physiol. 256 (Cell Physiol. 25): C688-C693, 1989[Abstract/Free Full Text].

10.   Chomczynski, P., and N. Sacchi. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

11.   Clerici, C. Sodium transport in alveolar epithelial cells: modulation by O2 tension. Kidney Int. 53, Suppl. 65: S79-S83, 1998.

12.   Danto, S. I., Z. Borok, X.-L. Zhang, M. Z. Lopez, P. Patel, E. D. Crandall, and R. L. Lubman. Mechanisms of EGF-induced stimulation of sodium reabsorption by alveolar epithelial cells. Am. J. Physiol. 275 (Cell Physiol. 44): C82-C92, 1998[Abstract/Free Full Text].

13.   Deterding, R. R., A. M. Havill, T. Yano, S. C. Middleton, C. R. Jacoby, J. M. Shannon, W. S. Simonet, and R. J. Mason. Prevention of bleomycin-induced lung injury in rats by keratinocyte growth factor. Proc. Assoc. Am. Physicians 109: 254-268, 1997[Medline].

14.   Dobbs, L. G., R. Gonzalez, and M. C. Williams. An improved method for isolating type II cells in high yield and purity. Am. Rev. Respir. Dis. 134: 141-145, 1986[Medline].

15.   Farman, N., C. R. Talbot, R. Boucher, M. Fay, C. M. Canessa, B. Rossier, and J. P. Bonvalet. Non-coordinated expression of alpha -, beta -, and gamma -subunit mRNAs of epithelial Na+ channel along rat respiratory tract. Am. J. Physiol. 272 (Cell Physiol. 41): C131-C141, 1997[Abstract/Free Full Text].

16.   Folz, R. J., C. A. Piantadosi, and J. D. Crapo. Oxygen toxicity. In: The Lung: Scientific Foundations, edited by R. G. Crystal, J. B. West, E. R. Weibel, and P. J. Barnes. Philadelphia, PA: Lippincott-Raven, 1997, p. 2713-2723.

17.   Garat, C., M. Meignan, M. A. Matthay, D. F. Luo, and C. Jayr. Alveolar epithelial fluid clearance mechanisms are intact after moderate hyperoxic lung injury in rats. Chest 111: 1381-1388, 1997[Abstract/Free Full Text].

18.   Goodman, B. E., and E. D. Crandall. Dome formation in primary cultured monolayers of alveolar epithelial cells. Am. J. Physiol. 243 (Cell Physiol. 12): C96-C100, 1982[Abstract/Free Full Text].

19.   Goodman, B. E., K. J. Kim, and E. D. Crandall. Evidence for active sodium transport across alveolar epithelium of isolated rat lung. J. Appl. Physiol. 62: 2460-2477, 1987[Abstract/Free Full Text].

20.   Guery, B. P., C. M. Mason, E. P. Dobard, G. Beaucaire, W. R. Summer, and S. Nelson. Keratinocyte growth factor increases transalveolar sodium reabsorption in normal and injured rat lungs. Am. J. Respir. Crit. Care Med. 155: 1777-1784, 1997[Abstract].

21.   Haskell, J. F., G. Yue, D. J. Benos, and S. Matalon. Upregulation of sodium conductive pathways in alveolar type II cells in sublethal hyperoxia. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L30-L37, 1994[Abstract/Free Full Text].

22.   Jackson, R. M. Molecular, pharmacologic, and clinical aspects of oxygen-induced lung injury. Clin. Chest Med. 11: 73-87, 1990[Medline].

23.   Johnson, C. R., Y. Guo, E. S. Helton, S. Matalon, and R. M. Jackson. Modulation of rat lung Na+,K+-ATPase gene expression by hyperoxia. Exp. Lung Res. 24: 173-188, 1998[Medline].

24.   Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. Monoclonal antibody to Na,K-ATPase: immunocytochemical localization along nephron segments. Kidney Int. 28: 899-913, 1985[Medline].

25.   Kim, K.-J., J. M. Cheek, and E. D. Crandall. Contribution of active Na+ and Cl- fluxes to net ion transport by alveolar epithelium. Respir. Physiol. 85: 245-256, 1991[Medline].

26.   Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

27.   Lasnier, J. M., O. D. Wangensteen, L. S. Schmitz, C. R. Gross, and D. H. Ingbar. Terbutaline stimulates alveolar fluid resorption in hyperoxic lung injury. J. Appl. Physiol. 81: 1723-1729, 1996[Abstract/Free Full Text].

28.   Lescale-Matys, L., C. B. Hensley, R. Crinkovic-Markovic, D. S. Putnam, and A. A. McDonough. Low K increases Na+-K+-ATPase in LLC-PK1/Cl4 cells by differentially increasing beta, not alpha, subunit mRNA. J. Biol. Chem. 265: 17935-17940, 1990[Abstract/Free Full Text].

29.   Lubman, R. L., K.-J. Kim, and E. D. Crandall. Alveolar epithelial barrier properties. In: The Lung: Scientific Foundations, edited by R. G. Crystal, J. B. West, E. R. Weibel, and P. J. Barnes. Philadelphia, PA: Lippincott-Raven, 1997, p. 585-601.

30.   Marxer, A., B. Stieger, A. Quaroni, M. Kashgarian, and H.-P. Hauri. Na+,K+-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta  subunit. J. Cell Biol. 109: 1057-1068, 1989[Abstract].

31.   Mason, C. M., B. P. Guery, W. R. Summer, and S. Nelson. Keratinocyte growth factor attenuates lung leak induced by alpha-naphthylthiourea in rats. Crit. Care Med. 24: 925-931, 1996[Medline].

32.   Mason, R. J., S. R. Walker, B. A. Shields, and J. E. Henson. Identification of rat alveolar type II epithelial cells with a tannic acid and polychrome stain. Am. Rev. Respir. Dis. 131: 786-788, 1985[Medline].

33.   Nici, L., R. Dowin, M. Gilmore-Hebert, J. D. Jamieson, and D. H. Ingbar. Upregulation of rat lung Na-K-ATPase during hyperoxic injury. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L307-L314, 1991[Abstract/Free Full Text].

34.   Olivera, W. G., K. M. Ridge, and J. I. Sznajder. Lung liquid clearance and Na,K-ATPase during acute hyperoxia and recovery in rats. Am. J. Respir. Crit. Care Med. 152: 1229-1234, 1995[Abstract].

35.   Olivera, W., K. Ridge, L. D. Wood, and J. I. Sznajder. Active sodium transport and alveolar epithelial Na-K-ATPase increase during subacute hyperoxia in rats. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L577-L584, 1994[Abstract/Free Full Text].

36.   Panos, R. J., P. M. Bak, W. S. Simonet, J. S. Rubin, and L. J. Smith. Intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality of rats. J. Clin. Invest. 96: 2026-2033, 1995[Medline].

37.   Royston, B. D., N. R. Webster, and J. F. Nunn. Time course of changes in lung permeability and edema in the rat exposed to 100% oxygen. J. Appl. Physiol. 69: 1532-1537, 1990[Abstract/Free Full Text].

38.   Russo, R. M., R. L. Lubman, and E. D. Crandall. Evidence for amiloride-sensitive sodium channels in alveolar epithelial cells. Am. J. Physiol. 262 (Lung Cell. Mol. Physiol. 6): L405-L411, 1992[Abstract/Free Full Text].

39.   Sznajder, J. I., W. G. Olivera, K. M. Ridge, and D. H. Rutschman. Mechanisms of lung liquid clearance during hyperoxia in isolated rat lungs. Am. J. Respir. Crit. Care Med. 151: 1519-1525, 1995[Abstract].

40.   Takeoka, M., W. F. Ward, H. Pollack, D. W. Kamp, and R. J. Panos. KGF facilitates repair of radiation-induced DNA damage in alveolar epithelial cells. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L1174-L1180, 1997[Abstract/Free Full Text].

41.   Toshiyuki, Y., R. R. Deterding, W. S. Simonet, J. M. Shannon, and R. J. Mason. Keratinocyte growth factor reduces lung damage due to acid instillation in rats. Am. J. Respir. Cell Mol. Biol. 15: 433-443, 1996[Abstract].

42.   Towbin, H. H., T. Staehelin, and J. Gordon. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc. Natl. Acad. Sci. USA 76: 43-50, 1979.

43.   Ulrich, T. R., E. S. Yi, K. Longmuir, S. Yin, R. Blitz, C. F. Morris, R. M. Housley, and G. F. Pierce. Keratinocyte growth factor is a growth factor for type II pneumocytes in vivo. J. Clin. Invest. 93: 1298-1306, 1994[Medline].

44.   Waters, C. M., U. Savla, and R. J. Panos. KGF prevents hydrogen peroxide-induced increases in airway epithelial cell permeability. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L681-L699, 1997[Abstract/Free Full Text].

45.   Wendt, C. H., H. Towle, R. Sharma, S. Duvick, K. Kawakami, G. Gick, and D. H. Ingbar. Regulation of Na-K-ATPase gene expression by hyperoxia in MDCK cells. Am. J. Physiol. 274 (Cell Physiol. 43): C356-C364, 1998[Abstract/Free Full Text].

46.   Yano, T., R. R. Deterding, W. S. Simonet, J. M. Shannon, and R. J. Mason. Keratinocyte growth factor reduces lung damage due to acid instillation in rats. Am. J. Respir. Cell Mol. Biol. 15: 433-442, 1996[Abstract].

47.   Yi, E. S., S. T. Williams, H. Lee, D. M. Malicki, E. M. Chin, S. Yin, J. Tarpley, and T. R. Ulich. Keratinocyte growth factor ameliorates radiation- and bleomycin-induced lung injury and mortality. Am. J. Pathol. 149: 1963-1970, 1996[Abstract].

48.   Yue, G., and S. Matalon. Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L407-L412, 1997[Abstract/Free Full Text].

49.   Yue, G., W. J. Russell, D. J. Benos, R. M. Jackson, M. A. Olman, and S. Matalon. Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats. Proc. Natl. Acad. Sci. USA 92: 8418-8422, 1995[Abstract].

50.   Zhang, X.-L., S. I. Danto, Z. Borok, J. T. Eber, P. Martín-Vasallo, and R. L. Lubman. Identification of Na+-K+-ATPase beta -subunit in alveolar epithelial cells. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 16): L85-L94, 1997[Abstract/Free Full Text].

51.   Zheng, L. P., R. S. Du, and B. E. Goodman. Effects of acute hyperoxic exposure on solute fluxes across the blood-gas barrier in rat lungs. J. Appl. Physiol. 82: 240-247, 1997[Abstract/Free Full Text].


Am J Physiol Cell Physiol 276(6):C1352-C1360
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society