Will Rogers Institute Pulmonary Research Center, Division of Pulmonary and Critical Care Medicine, University of Southern California, Los Angeles, California 90033
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ABSTRACT |
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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 -,
-, and
-subunit and
Na+-K+-ATPase
1- and
1-subunit mRNA levels were
quantified by Northern analysis.
Na+ pump
1- and
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
-subunit mRNA
expression was reduced by hyperoxia, whereas
- and
-subunit mRNA
expression was unchanged. Na+ pump
1-subunit mRNA was unchanged,
whereas
1-subunit mRNA was decreased ~80% by hyperoxia in parallel with a reduction in
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
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
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INTRODUCTION |
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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
1-subunits. KGF abrogates the
inhibitory effects of hyperoxia on
Isc while
preserving Na+ pump
1-subunit expression.
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METHODS |
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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;
k · 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
1 and
1 isoforms of
Na+-K+-ATPase
(E. Benz, Johns Hopkins University), and the
-,
-, and
-subunits of the rat ENaC (rENaC; C. Canessa, Yale University and B. Rossier, Université de Lausanne). Probes were labeled with
[
-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
[
-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
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-
1 Ab 6H
(24) (M. Caplan, Yale University) was used for detection of
Na+ pump
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
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.
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).
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RESULTS |
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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).
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Bioelectric properties.
AEC cultivated on polycarbonate filters in MDSF form functional
monolayers that actively transport
Na+, with
Rt ~3.0
k · 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.
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rENaC subunit mRNA expression.
As shown in the representative Northern blot in Fig.
3A, AEC
monolayers express mRNA for the -,
-, and
-subunits of rENaC on day 7 in culture in MDSF. mRNA
levels for the
- and
-subunits show no significant reduction
after hyperoxic exposure for 48 h, whereas mRNA for
-subunit rENaC
is reduced by 30%. Monolayers maintained in MDSF + KGF under normoxic
conditions demonstrate decreased levels of
Na+ channel
-,
-, and
-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).
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Na+-K+-ATPase
1- and
1-subunit expression.
AEC cultivated for 5 days in MDSF express abundant mRNA for the
1- and
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
1-subunit mRNA levels remain
unchanged relative to those in normoxia-exposed monolayers. There is a
decline in levels of Na+ pump
1-subunit mRNA to ~20%
compared with normoxia-exposed cells at 48 h. Abundant expression of
both Na+ pump
1- and
1-subunit protein in AEC
monolayers is also observed on day 5 in MDSF, as we have previously shown (12). Abundance of
Na+ pump
1-subunit protein does not
decrease significantly in monolayers following exposure to hyperoxia.
Na+ pump
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).
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DISCUSSION |
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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
1-subunit mRNA and protein,
which decline in parallel with the observed decrease in
Isc. Expression
of mRNA and protein for the
1-subunit of
Na+-K+-ATPase
remains unchanged following hyperoxia. Expression of mRNA for the
-
and
-subunits of rENaC is also unchanged by hyperoxia, and that of
the
-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
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
1-subunit mRNA and protein
expression are partially prevented by KGF. KGF also effects increases
in Na+ pump
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
1-subunit mRNA and protein
levels, a relative preservation of
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 · 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 or
rENaC subunits and only a modest
decrease in the level of
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 - (catalytic) and
-
(regulatory) subunits, of which the
1 and
1 isoforms are predominantly
expressed in alveolar epithelium (2, 12, 50). As shown in Fig. 5,
A and
B, the relative abundance of the
1-subunit protein declines by
80% following 48 h of hyperoxic exposure. If
-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
-subunits is
associated with a reduction in
-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 -,
-, and
-subunit
mRNA levels are diminished in KGF-treated hyperoxia-exposed monolayers
compared with those grown in MDSF in normoxic conditions, and rENaC
- and
-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 -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
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
1-subunit mRNA level relative
to both normoxia- and hyperoxia-exposed monolayers and a partial block
of the hyperoxia-induced reduction in the
-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
-subunit
mRNA levels as previously described in normoxia-exposed monolayers (2)
and a protective effect on the hyperoxia-induced reduction in
Na+ pump
-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
1- and
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
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
-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
1-subunit mRNA levels, with no
effect seen on
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
1-subunit mRNA, since levels of
the
1-subunit are unchanged.
After exposure to hyperoxia for 48 h, levels of
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
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
- and
-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
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
1-subunit expression and by a
relative preservation of Na+-K+-ATPase
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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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
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
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
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
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
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
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 -,
-, and
-subunit mRNAs of epithelial Na+ channel along rat respiratory tract.
Am. J. Physiol.
272 (Cell Physiol. 41):
C131-C141,
1997
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
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
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
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
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
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
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 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
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
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
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
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
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
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
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
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 -subunit in alveolar epithelial cells.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L85-L94,
1997
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