1 Departments of Pediatrics, 2 Anesthesiology, and 3 Biostatistics, University of Alabama at Birmingham, Birmingham Alabama 35226
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ABSTRACT |
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Regulation of active Na+
transport across fetal distal lung epithelial cells (FDLE) by
corticosterone (CST), corticotropin-releasing hormone (CRH), and oxygen
tension may be crucial for postnatal adaptation. FDLE isolated from
19-day rat fetuses (term: 22 days) were grown on permeable supports to
confluent monolayers (duration 3 days) in 2.5, 5, 12, or 20%
O2 with 5% CO2-balance N2 and
mounted in Ussing chambers for measurement of short-circuit currents
(Isc). FDLE monolayers grown in 20%
O2 had significantly higher levels of total
Isc and of their amiloride-sensitive
(Iamil) and ouabain-sensitive (Iouab) components than hypoxic cells. Values
(µA/cm2 ± SE) for 2.5-5% O2 and
20% O2 were, respectively, Isc
5.3 ± 0.2 vs. 8.4 ± 0.3 (P < 0.001),
Iamil 3.4 ± 0.2 vs. 4.3 ± 0.2 (P < 0.01), and Iouab 3.4 ± 0.6 vs. 9.1 ± 0.6 (P < 0.001). Addition of
CST but not CRH to the culture medium at any O2
concentration increased Iamil. FDLE cells grown
at 5% O2 expressed significantly lower levels of -,
-, and
-epithelial Na+ channel (ENaC), and of the
1-Na+-K+-ATPase, as determined
by Western blotting. We conclude that higher O2
concentrations increased total vectorial Na+ transport, and
the function of Na+-K+-ATPase and apical
amiloride-sensitive Na+ conductance, whereas CST only
increased ENaC function.
Ussing chamber; short-circuit current; sodium-potassium-adenosinetriphosphatase; epithelial sodium channel; oxygen; corticosterone; corticotropin-releasing hormone
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INTRODUCTION |
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SHORTLY AFTER BIRTH,
active reabsorption of Na+ across the alveolar epithelium
creates an osmotic gradient favoring the reabsorption of fetal lung
fluid. Na+ enters the apical membranes of alveolar
epithelial cells through amiloride-sensitive epithelial Na+
channels (ENaC) and is extruded across the basolateral membranes by the
ouabain-sensitive Na+-K+-ATPase
(50). The importance of active Na+ transport
in fetal fluid reabsorption was clearly demonstrated by the work of
Hummler et al. (23), who showed that newborn mice lacking
the (pore-forming)-subunit of the amiloride-sensitive channel
(
-ENaC) failed to clear their lung fluid and died within 40 h
from respiratory failure. Additional studies have shown that active
Na+ transport plays an important part in decreasing
alveolar fluid, thus optimizing gas exchange, in both the neonatal and
adult lung (44, 45).
Active alveolar epithelial Na+ transport may be compromised
in a number of pathological situations, including premature birth and
respiratory distress syndrome (4, 7). During lung
inflammation, reactive oxygen and nitrogen species, generated by
epithelial and inflammatory cells (17, 22, 53), may damage
apical and basolateral Na+ transporters. In addition,
volutrauma during mechanical ventilation (12) was found to
decrease lung liquid clearance (36). Both O2
toxicity and volutrauma contribute to the pathogenesis of respiratory distress syndrome and bronchopulmonary dysplasia. For these reasons, enhancing Na+ reabsorption across the newborn and adult
alveolar epithelium may be of significant clinical benefit. In previous
studies, we have shown that treatment of fetal distal lung epithelial
(FDLE) cells with an adenoviral vector encoding for the
1-subunit of the Na+-K+-ATPase
increased vectorial Na+ transport (62).
In addition to their well-known effects on surfactant production, prenatally administered glucocorticoids may also improve lung function in premature infants by stimulating increased pulmonary Na+ absorption (24, 63). Mineralocorticoids and glucocorticoids have been shown to increase both electrogenic Na+ absorption and fluid clearance by enhancing transcription of both ENaC and Na+-K+-ATPase genes (5, 60). Increased amiloride-sensitive short-circuit current (Isc) was found after exposure of fetal alveolar cells to glucocorticoids (11), although it is unclear whether this effect was the result of upregulation of ENaC, Na+-K+-ATPase, or both. The effect of corticosteroids on the oubain-sensitive current (Iouab), as an indicator of Na+-K+-ATPase activity has not yet been measured. Furthermore, there is significant evidence that both levels of expression of Na+ transporters (ENaC and Na+-K+-ATPase) and levels of Na+ transport across alveolar epithelial cells are dependent on O2 tension (2, 3, 19, 29, 37, 48, 52, 54, 55, 67). Moreover, the interactions between corticosteroids and the increased O2 concentration, to which the alveoli are exposed after birth, remain undefined.
Corticotropin-releasing hormone (CRH) is a hypothalamic and placental hormone that controls adrenal glucocorticoid production through the release of ACTH from the hypophysis. Importantly, CRH is also abundant in other organs (39) and thought to control the timing of birth (47). Therefore, CRH may also be involved in lung maturation independent of its stimulatory effect on ACTH and corticosterone (CST) release. Direct effects of CRH on fetal alveolar cells are completely unknown.
After the considerations outlined above, we hypothesized that Na+ transport through rat fetal alveolar cells is 1) higher in cells grown at ambient air O2 tension than in cells grown at reduced O2 tension, 2) increased by CST, the major glucocorticoid in the rat, and 3) modulated by CRH. We investigated these hypotheses by measuring Isc across alveolar cell monolayers in Ussing chambers. Using selective antagonists, we further examined whether current changes were attributable to changes in function of ENaC, Na+-K+-ATPase, or both. We also measured ENaC and Na+-K+-ATPase subunit protein expression by Western blotting.
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METHODS |
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FDLE cell isolation and culture. The procedure for isolating FDLE cells has been described previously (27). In brief, lungs of 19- to 20-day gestation fetal rats (term = 22 days) were digested in a solution containing 0.125% trypsin and 0.4 mg/ml DNase in Eagle's minimal essential medium (MEM) for 10 min. Digestion was stopped by the addition of MEM containing 10% FBS. Cells were collected by centrifugation and resuspended in 15 ml of MEM containing 0.1% collagenase and DNase. This solution was incubated for 15 min at 37°C. Collagenase activity was then neutralized by the addition of 15 ml of MEM containing 10% FBS. The cells were plated two times for 1 h to remove contaminating fibroblasts. The supernatant contained epithelial cells with >95% purity (27). Cells were counted and seeded on permeable Transwell culture inserts (Corning, Corning, NY) with 0.4-µm pore size. For Ussing chamber experiments, cells were seeded on Transwell no. 3413 (surface area 0.33 cm2) at a density of 5 × 105 cells/insert. For Western blotting, cells were seeded on Transwell no. 3412 (surface area 4.5 cm2) at a density of 2 × 106 cells/insert. Immediately after seeding, the cells were placed in atmospheres containing 2.5, 5, 12, or 20% O2, which resulted in atmospheric PO2 values of 19, 38, 91, and 152 mmHg, respectively, and 5% CO2-balance N2. The lower O2 concentrations were selected to resemble fetal PO2 values, which vary between 16 and 25 mmHg (1, 61). The same O2 concentration was maintained throughout the culture period for each condition. To promote optimal attachment to filters, cells were cultured initially overnight in MEM with 10% FBS and 1% penicillin/streptomycin. On the 2nd and 3rd day, these media were replaced by serum-free complete medium (Cellgro; Mediatech, Herndon, VA) supplemented with 1 µM CST (C2505; Sigma, St. Louis, MO), 3 nM CRH (C3042; Sigma), or both, as indicated. Cells subjected to the different experimental conditions were always age-matched and derived from the same litter.
Measurement of bioelectric properties of FDLE cells.
All experiments were performed on the 4th day of culture, ~72 h after
seeding. Monolayers grown on type 3413 filters were used in Ussing
chamber experiments when their transepithelial resistance exceeded 0.2 k · cm2. The chambers were filled
with a solution containing (in mM) 145 Na+, 5 K+, 1.2 Ca2+, 1.2 Mg2+, 125 Cl
, 25 HCO
-free solutions (all
Cl
replaced by gluconate) to avoid interference of
Cl
secretion with the current measurements.
Measurement of transport protein expression.
Cells grown on the large Transwell 3412 filters were placed on ice and
washed with ice-cold PBS solution. Filters were overlaid with 100 µl
of lysis buffer and incubated on ice for 30 min. The composition of the
lysis buffer was 50 mM Tris (pH 7.4), 50 mM NaCl, 10 mM
MgCl2, 2 mM EGTA, 0.2% IgePal, 100 µM of the proteinase inhibitors leupeptin, antipain,
N-p-tosyl-L-lysine chloromethyl ketone, and N-p-tosyl-L-phenylalanine
chloromethyl ketone, and 1 mM phenylmethylsulfonyl fluoride. After 30 min, filters were excised from their holders and transferred to
Eppendorf tubes, carefully collecting all fluid that had flown off.
Filters grown under identical conditions were pooled with their fluid
in a single tube for each condition. The tubes were sonicated for
30 s in ice water and then centrifuged at 500 g and
4°C for 10 min to accumulate the fluid at the bottom of the tubes.
The almost dry filters were discarded, leaving the protein-rich lysate.
Protein content was measured using a standard protein assay (BCA, no. 23223; Pierce, Rockford, IL). Proteins were denaturated by 10 min of
incubation with 10% 2-mercaptoethanol at 95°C or, for
Na+-K+-ATPase, at 37°C for 20 min and
separated by SDS-PAGE through a 10% gel. Proteins were then
transferred to a prewetted polyvinylidene difluoride membrane. Blots
were probed by overnight incubation at 4°C with rabbit antibodies
against
-ENaC,
-ENaC (Alpha Diagnostics, San Antonio, TX),
-ENaC (gift from the laboratory of Douglas C. Eaton, Emory
University, Atlanta, GA),
1-Na+-K+-ATPase (Research
Diagnostics, Flanders, NJ), or
1-Na+-K+-ATPase (gift from the
laboratory of Phillip Factor, Northwestern University, Chicago, IL),
all diluted 1:1,000 in 50 mM Tris, 0.9% NaCl, and 0.1% Tween 20 (TBST) containing 3% nonfat dry milk. Blots were washed in TBST, and
bound primary antibody was detected by incubation for 1 h at room
temperature with a goat anti-rabbit polyclonal antibody, conjugated to
horseradish peroxidase (diluted 1:10,000 in TBST-3% milk). Blots were
washed again, and horseradish peroxidase activity was detected by
enhanced chemiluminescence (ECL; Amersham, Piscataway, NJ). Band
intensity was measured by densitometry using FluorChem software on an
Alpha Innotech Imager. In some experiments, primary antibodies were
preincubated with blocking peptides (derived from the same sources as
each antibody; concentration of peptide to antibody 10:1) for 30 min
before probing of blots.
Statistical analysis. Significant differences among group means and interactions were determined by three-way ANOVA and Tukey's post hoc test, using SAS software (SAS Institute, Cary, NC). P < 0.05 was considered significant.
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RESULTS |
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Electrophysiological studies.
All FDLE cells used in the electrophysiological studies were obtained
from 11 different cell isolations. The mean value for transepithelial
resistance for all monolayers was 0.55 ± 0.28 k · cm2 (mean ± SD,
n = 250).
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Protein expression.
FDLE cells used for Western blotting were obtained from four different
cell isolations. All three subunits of ENaC were detected in the cell
lysates. The -,
-, and
-subunits formed bands at 150, 130, and
160 kDa, respectively (Fig.
6A). An additional
-ENaC band of ~100 kDa molecular mass was detected only in cells cultured in hypoxia in the presence of CST. Blots in which binding of primary antibody was inhibited by the presence of blocking peptides showed no
immunoreactivity, thus confirming specificity of the
- and
-antibodies (data not shown). Expression of all three subunits was
reduced significantly only by culturing cells in the presence of
hypoxia and CST (Fig. 6B, P < 0.01, n = 5 individual blots analyzed for each subunit). In
the absence of CST, O2 tension did not have a significant
influence on ENaC subunit expression, and, in the presence of room air,
CST did not influence ENaC expression (Fig. 6B).
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DISCUSSION |
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Although FDLE cells are generally considered to be secretory
rather than absorptive in vivo, we found the majority of their Isc to be amiloride sensitive and insensitive to
removal of Cl from the bath. This phenotype is indicative
of Na+ absorption. It is possible that the cells change
their properties during the time in cell culture, which must be
considered when interpreting our results. Within this generalization,
O2 and CST exposure during culture had marked effects on
Isc.
The amiloride-sensitive component of Isc was
~70% of total Isc, which was slightly higher
than in a previous study (35). Furthermore, herein we
observed that removal of Cl had little effect on
Isc while previously we reported that ~40% of
Isc was the result of secretion of anions
(Cl
and HCO
Compared with lower O2 concentrations, room air (20% O2) profoundly increased baseline Isc and its amiloride-sensitive and ouabain-sensitive components in intact monolayers. Likewise, experiments with permeabilized monolayers showed an increase in both amilmax and ouabmax after exposure to 20% O2. This indicates that both ENaC and Na+-K+-ATPase activities are dependent on O2 and upregulated when O2 concentration is increased to 20%.
The differences in total Isc and amiloride-sensitive and ouabain-sensitive Isc were not quite reflected in the measured protein expression. For example, we found that the combination of hypoxia and CST treatment resulted in a marked downregulation of ENaC subunit expression, which is in accordance with the lower Isc found in these monolayers. However, hypoxia without CST treatment induced a similar reduction in Isc but without reducing ENaC subunit protein expression. This may be explained by two facts. First, protein expression was evaluated in whole cell lysates, which contain both cytoplasmic and membrane-associated proteins. It is therefore impossible to determine how much functional ENaC or Na+-K+-ATPase protein was actually inserted in the membranes and in functional conditions. Western blot analysis of preparations of isolated apical and basolateral membranes would be necessary to clarify this point, but such samples are technically very difficult to obtain from cells grown on permeable supports. Second, multiple intracellular regulatory systems, such as protein kinases, modulate the functional activity of ion transport proteins. Therefore, even when isolated cell membranes are used, measurement of expressed protein may not reflect actual activity. Thus our data also show that measurement of protein expression is no substitute for actual measurement of ion transport.
The molecular masses of ENaC - and
-subunits were somewhat higher
than those found by others in adult alveolar type II cells (26). However, we confirmed specificity of the antibodies
by repeating the binding in the presence of blocking peptides, which blocked binding completely. Furthermore, older studies have also found
higher molecular masses, i.e., between 135 and 150 kDa, depending on
cell type and culture conditions (20, 42, 43, 51, 66). We
speculate that differences in glycosylation, as well as the length of
the actual protein molecule between species and cell lines, may be
responsible for different molecular masses of the isolated proteins.
Such differences may even be modulated by culture conditions, as shown
by the lower molecular mass band that appeared in the
-ENaC blots
when cells were cultured in hypoxia and in the presence of CST.
Few previous studies have addressed the effect of O2 on Na+ transport. Hyperoxia resulted in increased expression and activity of ENaC and Na+-K+-ATPase (29, 48, 52, 67). A similar dependence was described in the lower PO2 range, in accordance with our results. FDLE cell monolayers responded with increased amiloride- and ouabain-sensitive Isc after atmospheric PO2 was increased from 23 to 100 mmHg (3, 55). In another study using FDLE cells, increased ENaC expression, total current, and Iamil were found after raising the PO2 from 23 to 160 and 380 mmHg (54), but Na+- K+-ATPase activity was not assessed in this study. Furthermore, the expression of ENaC and Na+-K+- ATPase subunits was increased with increasing PO2 in A549 cells (65) and alveolar cells from adult rats (18, 37, 38). Live animals kept in hypoxia showed similar effects (65). We chose to test several hypoxic O2 concentrations because the real PO2 at the cellular level was likely to be lower than the O2 concentration in the atmosphere around the cell culture. PO2 depends on the balance between diffusion of O2 through the culture media and the metabolic rate of the cells (59, 64). The latter of these parameters was unknown, and the testing of several PO2 values in the range of 19-152 mmHg therefore increased the likelihood that some culture conditions resemble fetal conditions, which are characterized by PO2 values between 16 mmHg (tissue) and 20-25 mmHg (arterial; see Refs. 1 and 61). It turned out that results were similar after cell culture in 2.5, 5, or 12% O2, whereas only cells grown at 20% O2 had distinctively higher currents, suggesting that PO2 needs to exceed a certain threshold to affect ion transport. Therefore, cells grown with 12% O2 appeared to add little information and were not included in all experiments. During the actual measurements in Ussing chambers, hypoxia was not continued because this would have been technically difficult, and the duration of measurements was much shorter than the 4 h that is the minimum exposure needed to alter epithelial properties (54).
Interestingly, exposure to 1 µM of the glucocorticoid CST did not
change baseline Isc in our experiments and
brought only an increase in Iamil but not in the
Iouab. Likewise, the
Iamil in experiments involving permeabilization
of the basolateral membrane (amilmax) showed a marked
increase after exposure to CST in culture. In contrast,
ouabmax was even slightly reduced after CST exposure. Thus
CST appears to upregulate only the function of ENaC and not the
Na+-K+-ATPase if used at physiological
concentrations. This is further supported by the observation that, in
contrast to normal Cl-based solution, baseline
Isc was increased after CST exposure if measured
in Cl
-free conditions (Fig. 2). In normal
Cl
solution, Cl
secretion serves as a
potential additional pathway for ion movement and may have contributed
to the total Isc to a varying extent, compensating for changes in ENaC activity. In Cl
-free
solution, Na+ absorption remains the only source of
current. Therefore, changes in Na+ channel activity
elicited by CST may only become apparent in Cl
-free
solution, whereas, in normal solution, total current may be more
dependent on the Na+-K+-ATPase activity,
which drives both Na+ absorption and Cl
secretion.
Corticosteroids have been found to increase amiloride-sensitive
Na+ transport and Na+ channel mRNA
transcription in pulmonary epithelia (1, 10, 25).
Furthermore, pretreatment with hydrocortisone enhanced a
terbutaline-induced, amiloride-sensitive potential increase across
cultured fetal lung buds (33) and a terbutaline-induced increase of amiloride-sensitive Isc across rat
FDLE monolayers (11). Prenatal administration of
dexamethasone to pregnant rats increased fetal expression of the
-ENaC subunit but not the
- and
-subunits (60).
In a human cancer cell line (A549 cells), dexamethasone treatment
mainly increased expression of the
- and
-subunits of ENaC, which
profoundly changed the biophysical properties of the channels formed
(34). However, most previous studies used dexamethasone, a
much more potent corticosteroid than CST, and it was given in
pharmacological doses to malignant cell lines. Our results presented
here demonstrate distinctly different actions of a physiological
steroid used in physiological concentrations on native cells (6,
8, 9, 21).
Very limited data regarding the effect of corticosteroids on lung
epithelial Na+-K+-ATPase are available.
Prenatal administration of dexamethasone to pregnant rats did not
change fetal expression of the 1- and
1-subunits of the Na+-K+-ATPase
(60), although no measurement of
Iouab was performed in that study. Our study
provides the first data on Na+-K+-ATPase
function after corticosteroid treatment of lung epithelia and supports
the notion that corticosteroids do not increase
Na+-K+-ATPase expression and activity in fetal
lungs. We also show that the action of corticosteroids in physiological
doses is dependent on ambient O2 concentration.
CRH levels in normal fetal rats have not been reported. In umbilical venous blood of normal human fetuses, CRH levels were between 15 and 63 pmol/l (13, 49, 56, 57). However, it is possible that fetal concentrations are much higher under pathological conditions, which are associated with increased maternal CRH levels (14-16, 56). We used 3 nM CRH in this study, which is ~50 times higher than in normal human fetuses and comparable to the highest concentrations achieved in humans at birth (13).
A number of findings indicated that CRH, a hypothalamic hormone that controls adrenal glucocorticoid production through the release of ACTH from the hypophysis, may have additional effects independent of CST, which may affect maturation of fetal tissues and thus alveolar Na+ transport. It is well known that postnatal glucocorticoid administration, no matter how early, is not nearly as effective as prenatal administration in improving lung function of preterm infants (58). After showing that fetal growth and lung function after premature birth in lambs was more strongly influenced if glucocorticoids were given to the ewe rather than directly to the fetus, Jobe et al. (28) speculated that part of the glucocorticoid effect may be mediated through another, as yet unidentified, factor. CRH was a potential candidate for this factor for several reasons. First, it is made not only in the hypothalamus but also in a number of other tissues, including the placenta and the lung (39), but the biological reason for this production has never been elucidated. Second, CRH shares homology to some intestinal signaling peptides. Third, CRH is thought to control the timing of birth (40). CRH levels rise steadily throughout pregnancy. High levels early in pregnancy have been associated with premature birth, whereas low levels are associated with postterm delivery (40, 46, 47). Fourth, there is a positive feedback between fetal glucocorticoids and CRH levels, with glucocorticoids stimulating placental release of CRH (30, 31, 41). Finally, increased levels of CRH have been observed in pathological situations, such as pregnancy-induced hypertension (14-16, 56). One could speculate that either increased prenatal levels of CRH or withdrawal of placental CRH at birth might enhance maturational responses in alveolar cells.
Contrary to our hypothesis, exposure to CRH during culture did not significantly alter Na+ transport. The small trends observed after exposure to CRH may become statistically significant with a higher number of experiments; however, differences that small would not be physiologically meaningful. It is unlikely that our CRH concentration was too low. In preliminary experiments using culture media containing serum, a 10-fold higher CRH concentration (30 nM) did not make any difference either. However, our study does not exclude that CRH may have other nonelectrophysiological effects on fetal alveolar cells, such as increasing surfactant production and maturation, which were beyond the scope of this study. Finally, CRH may not have the same effects in rats as it may have in sheep or humans, where most of the research regarding its influence on the timing of birth was done. In vivo, CRH will also have indirect effects through CST release from the adrenals.
Despite permeabilization, which effectively removed the apical barrier for Na+ entry, ouabmax was not much higher than the Iouab in intact monolayers. However, in intact monolayers, the K+ that is exchanged for Na+ by the basolateral Na+-K+-ATPase leaves the cell through basolateral K+ channels. Therefore, K+ remains on the basolateral side and does not contribute to net charge movement, which is being measured. With a permeabilized apical membrane, K+ is likely to leave the cell through the apical membrane in the negatively charged apical compartment, thus diminishing the measured net charge movement. Given the stochiometry of the Na+-K+-ATPase (3 Na+ for 2 K+), one can therefore assume that the true extrusion rate of Na+ is three times higher than the measured ouabmax. It is very unlikely, but possible, that a small amount of apically instilled amphotericin B may have gained access to the basolateral membrane. The pores that would then form in the basolateral membrane would have decreased measurable Na+-K+-ATPase activity. In this case, our measurements of the ouabmax would have underestimated the true Na+-K+-ATPase activity, but this does not invalidate our conclusions. On the other hand, when permeabilizing the basolateral membrane in the presence of an apical-to-basolateral Na+ gradient, any amphotericin B that gets to the apical membrane would open further pathways for the movement of Na+ down the concentration gradient and thus lead to an overestimation of apical membrane Na+ permeability. Such pores, however, are insensitive to amiloride, and possible confounding effects can thus be eliminated by evaluating only the Iamil.
In conclusion, our data indicate that ENaC function in rat FDLE cells is increased by exposure to increased O2 concentration or corticosteroids. In contrast, the Na+-K+-ATPase function is increased by exposure to increased O2 concentration but not by corticosteroids. CRH did not have detectable electrophysiological effects on FDLE cells.
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ACKNOWLEDGEMENTS |
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We thank T. Bamberg, G. Davis, C. Myles, and J. Ware for excellent technical assistance, Drs. C. J. Venglarik and Ahmed Lazrak for many helpful comments and suggestions, and Dr. W. A. Carlo for reviewing the manuscript.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants HL-31197, HL-51173, P30 DK-54781, and HL-48129. I. C. Davis is a Parker B. Francis Families Fellow in Pulmonary Research.
Present address: U. H. Thome, University Children's Hospital, 89070 Ulm, Germany.
Address for reprint requests and other correspondence: S. Matalon, Univ. of Alabama at Birmingham, 901 19th St. South, BMR II, Rm. 223, Birmingham, AL 35233-1924 (E-mail: sadis.matalon{at}ccc.uab.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published November 15, 2002;10.1152/ajplung.00218.2002
Received 8 July 2002; accepted in final form 18 October 2002.
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