1 Medical Research Council Group in Lung Development and Divisions of 2 Respiratory and 3 Neonatal Research, Hospital for Sick Children Research Institute and Department of Paediatrics, University of Toronto, Toronto, Ontario, Canada M5G 1X8; and 4 Hospital for Children and Adolescents, University of Helsinki, 00290 Helsinki, Finland
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ABSTRACT |
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Cultured rat fetal distal lung epithelial cells
(FDLEs), when switched from fetal (3%) to postnatal (21%)
O2 concentrations, have increased
epithelial Na+ channel (ENaC) mRNA
levels and amiloride-sensitive Na+
transport [O. Pitkänen, A. K. Tanswell, G. Downey, and H. O'Brodovich. Am. J. Physiol. 270 (Lung Cell. Mol.
Physiol. 14): L1060-L1066, 1996]. The
mechanisms by which O2 mediates
these effects are unknown. After isolation, FDLEs were kept at 3%
O2 overnight, then switched to
21% O2 (3-21%
O2 group) or maintained at 3%
O2 (3-3%
O2 group) for 48 h. The
amiloride-sensitive short-circuit current
(Isc) in the
3-21% O2 group was double
that in the 3-3% O2 group.
Amiloride-sensitive Isc could not be
induced by medium conditioned by 21%
O2-exposed FDLEs but was reversed
by returning the cells to 3% O2.
Neither the cyclooxygenase inhibitor ibuprofen, liposome-encapsulated catalase, nor hydroperoxide scavengers (U-74389G or Trolox) blocked the
O2-induced amiloride-sensitive
Isc. In contrast,
the cell-permeable superoxide scavenger
tetramethylpiperidine-N-oxyl (TEMPO)
eliminated the O2-induced
increases in amiloride-sensitive
Isc and ENaC mRNA levels. The switch from 3 to 21%
O2 induced the transcription factor nuclear factor-B, which could also be blocked by TEMPO. We
conclude that 1) the
O2-induced increase in
amiloride-sensitive Isc is reversible
and 2) the
O2-induced increase in
amiloride-sensitive Isc and ENaC mRNA
levels is associated with activation of nuclear factor-
B and may be
mediated, at least in part, by superoxide.
epithelial sodium channel; nuclear factor-B; amiloride; type II
epithelium; tetramethylpiperidine-N-oxyl; reactive
oxygen species
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INTRODUCTION |
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BEFORE BIRTH, THE FETAL LUNG is filled with liquid that
has been secreted by its epithelium. This liquid secretion results from
the active transport of Cl
from the interstitium to the alveolar lumen, a phenomenon that is
necessary for the normal development of the lung (1). At birth,
however, fluid secretion must cease, and the lung liquid must be
cleared before effective gas exchange can take place. The major
component of this liquid clearance results from active epithelial
Na+ transport from the lumen to
the interstitium (5, 25-27). Indeed, a transgenic
mouse deficient in the amiloride-sensitive epithelial Na+ channel (ENaC) fails to clear
its fetal lung liquid and dies shortly after birth (17).
How the lung converts from a fluid-secreting to an absorbing organ is
incompletely understood and has been the subject of many studies. There
is general agreement that increases in the circulating levels of
vasoactive compounds such as catecholamines (5) and arginine
vasopressin (30) can initiate this conversion in mature lungs in utero.
However, as soon as the levels of these vasoactive agents return to
basal values, the fetal lung reverts to fluid secretion. The change in
O2 concentration from the fetal (~3%) to the postnatal (21%) environment represents another
potential signal for the perinatal lung. Others (3) have previously
demonstrated that the ability of late-gestation fetal distal lung
explants to form fluid-filled cysts is dependent on
O2 concentration. Our laboratory
(31) has also shown that when primary cultures of fetal distal lung
epithelial cells (FDLEs) were switched from 3 to 21%
O2, there was an increase in the
amount of amiloride-sensitive short-circuit current
(Isc) and mRNA
coding for the -,
-, and
-subunits of ENaC. This effect was
observed within 18 h of incubation of FDLEs in 21%
O2. Although a 48-h incubation led
to a further augmentation in amiloride-sensitive
Isc, an 8-h
incubation had no effect.
In the present study, we determined whether the induction of FDLE
Na+ transport by postnatal
O2 was permanent or reversible. In
addition, we investigated possible mechanisms for the increased gene
expression and functional activity of ENaC in FDLEs. This included
experiments to determine whether the change in
O2 concentration resulted in the
release of a stable compound into the cell medium that could induce
amiloride-sensitive
Isc and whether
the increase in amiloride-sensitive Isc could be
prevented by various inhibitors, including blockers of prostaglandin
production and the inducible form of nitric oxide synthase, or by
scavengers of reactive oxygen species (ROS). Because our experiments
demonstrated that the cell-permeable superoxide scavenger
tetramethylpiperidine-N-oxyl (TEMPO)
blocked the postnatal O2-mediated
induction of ENaC mRNA expression, we performed additional experiments
to determine whether the ROS-sensitive transcription factor nuclear
factor (NF)-B (for a review, see Ref. 14) was activated during
exposure of our primary cultures of FDLEs to postnatal
O2 concentrations.
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METHODS |
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Primary cell culture. FDLEs were isolated and cultured as previously described (28). In brief, 20-day-gestation Wistar rats (breeding day = day 0, term = 22 days; Charles River, St. Constant, PQ) were killed with an anesthetic overdose. Fetal lung tissue was dispersed with 0.125% (wt/vol) trypsin, and the resulting cell pellet was further incubated with 0.1% collagenase (wt/vol) to separate associated fibroblasts from epithelial cells. Fibroblasts were separated from epithelial cells with a differential adherence and centrifugation procedure. FDLEs were then seeded at 1 × 106 cells/cm2 onto 0.4-µm pore size Snapwell cell culture inserts (Corning Costar, Cambridge, MA) for Ussing chamber studies and at 0.5 × 106 cells/cm2 onto 75-mm-diameter, 0.4-µm pore size Transwell cell culture inserts (Corning Costar) for subsequent RNA isolation, preparation of conditioned medium (CM), and nuclear extract preparations. All cells were submersion cultured in Dulbecco's modified Eagle's medium (DMEM; 4.5 g/l of glucose with 2 mM L-glutamine and 110 mg/l of sodium pyruvate) supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum (FBS), 100 U/ml of penicillin G sodium, and 100 µg/ml of streptomycin sulfate. All cell culture reagents were purchased from GIBCO BRL (Burlington, ON).
O2 environment and interventions. After being seeded, FDLEs were returned to an incubator containing 3% O2-5% CO2-balance N2 for ~20 h. Then the medium was replaced with fresh medium (containing inhibitors or vehicle alone; see Inhibitors and ROS scavengers). The monolayers were then either transferred to 21% O2 or kept at 3% O2 for approximately the next 48 h.
Reversibility of O2 induction. To test the reversibility of O2-induced Na+ transport, we placed some of the monolayers at 3% O2 after seeding (3-21% group) and some directly into 21% O2 (21-3% O2 group). After remaining at the initial O2 concentration for ~48 h, the 3-21% O2 group was moved to 21% O2 and the 21-3% O2 group was moved to 3% O2. These monolayers stayed at the final O2 concentration for an additional 48 h. A third group remained in 3% O2 throughout the entire 4-day experimental period (3-3% O2 group).
Studies with CM. To determine whether FDLEs exposed to postnatal O2 concentrations released a soluble factor that was capable of stimulating amiloride-sensitive Isc, FDLEs were seeded onto 75-mm Transwell membranes and left in 3% O2 overnight. After the addition of fresh medium, one-half of the monolayers were returned to 3% O2, whereas the other half were transferred to 21% O2. After 48 h, CM was collected from above the monolayers and centrifuged at 200 g for 5 min, and the supernatant was stored at ![]() |
RESULTS |
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Postnatal O2 stimulates
amiloride-sensitive
Na+ transport.
Consistent with a previous report by Pitkänen et al.
(31), when we switched the FDLE monolayers from 3 to 21%
O2, the amiloride-sensitive Isc increased
from 1.3 ± 0.1 to 2.5 ± 0.1 µA/cm2, baseline
Isc increased
from 2.7 ± 0.1 to 4.0 ± 0.2 µA/cm2, and baseline
R increased from 1,245 ± 66 to
1,687 ± 99 · cm2
(P < 0.05 for all changes;
n = 50 monolayers). There was no
change in amiloride-insensitive
Isc (1.4 ± 0.1 vs 1.5 ± 0.1 µA/cm2;
P > 0.05).
O2-induced increase in amiloride-sensitive Isc is reversible. In this series of experiments, the amiloride-sensitive Isc increased by an average of 60% after the FDLEs were switched from 3 to 21% O2 (Fig. 1). When FDLEs were switched from 21 to 3% O2 (21-3% O2 group), the amiloride-sensitive Isc declined to the same level as the 3-3% O2 group (P > 0.05 between the 2 groups; n = 3).
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O2-induced FDLEs do not release a stable soluble factor capable of inducing amiloride-sensitive Isc. Figure 2 illustrates that CM derived from FDLEs maintained in 21% O2 did not induce amiloride-sensitive Isc in FDLEs that were kept at 3% O2 compared with the group that received the noninduced CM. Furthermore, the groups that received CM had amiloride-sensitive Isc values similar to those of O2-matched control groups receiving fresh medium, indicating that the process of CM preparation did not deprive the cell culture medium of factors that may have been essential for the ability of the cell to sustain ion transport.
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Postnatal O2 concentration activates the
nuclear transcription factor NF-B.
Because the transcription factors NF-
B and AP-1 are known to be
redox sensitive (22) and NF-
B is known to be activated by ROS (35),
we tested nuclear extracts from FDLEs grown under 3 or 21%
O2 using electrophoretic mobility shift assays with
radiolabeled oligonucleotides corresponding to consensus binding sites
for these two transcription factors. Three hours after FDLEs were switched from 3 to 21% O2, there
was increased binding activity to an NF-
B consensus oligonucleotide
(Fig.
5A).
This activation was prevented when FDLEs were incubated with 1 mM TEMPO
during the O2 induction period. In
contrast, AP-1-binding activity was not induced under these conditions
(Fig. 5B).
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DISCUSSION |
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This study demonstrated that a change from a fetal (3%) to a postnatal
(21%) O2 concentration induces a
reversible increase in amiloride-sensitive
Isc of FDLEs. We
demonstrated that this increase in
Na+ transport was associated with
an increase in the levels of mRNAs that code for the subunits of ENaC.
This induction of Na+ transport
could not be reproduced with CM from FDLEs that had been exposed to
postnatal O2, and it was not
prevented by pharmacological blockers of cyclooxygenase (ibuprofen) or
NO synthase (L-NAME). The
induction of FDLE Na+ transport is
due, at least in part, to an alteration in intracellular ROS,
specifically superoxide, because the superoxide scavenger TEMPO
prevented both the O2 induction of
FDLE Na+ transport and increases
in the level of ENaC mRNA. Our experiments also show that this
perinatal physiological change in
O2 concentration is associated
with activation of the redox-sensitive transcription factor NF-B and
that this activation of NF-
B could also be prevented with TEMPO.
Together these results suggest a possible mechanism by which an
increase in O2 concentration and a
concomitant change in the intracellular redox state of FDLE at birth
may result in increased Na+
transport in the distal regions of the lung.
We tested the reversibility of O2 induction of amiloride-sensitive Isc by returning FDLE monolayers to 3% O2 after an initial incubation in 21% O2. After the switch back to 3% O2, amiloride-sensitive Na+ transport in this group declined to the same level as that of the group that had remained in 3% O2 throughout the study. This observation is in agreement with recent studies that have shown that Na+ uptake (21, 32) and mRNA expression for all three ENaC subunits (32) decline in adult rat type II epithelial cells when these cells were switched from 21% to hypoxic O2 concentrations.
Any increase in O2 concentration leads to an increase in intracellular ROS levels due to an increased rate of respiration. Because an increased O2 concentration, ROS, and hydroperoxides are known to stimulate prostaglandin synthesis and secretion (9, 16, 38, 39) and prostaglandins alter Na+ transport by otic epithelium (16), we treated FDLEs with ibuprofen before O2 induction. Ibuprofen, at concentrations known to inhibit cyclooxygenase (38), did not prevent 21% O2 induction of amiloride-sensitive Isc, thus making cyclooxygenase-derived prostaglandins unlikely mediators in the process of 21% O2-induced Na+ absorption.
It is believed that lung epithelium may regulate its biological character by secreting autocrine factors (7). To determine whether changing FDLEs from a 3% to a 21% O2 environment produced factors that could stimulate amiloride-sensitive Isc, we incubated FDLE in 3 or 21% O2-derived CM. Medium derived from FDLEs grown in 21% O2 was ineffective in stimulating amiloride-sensitive Isc in FDLEs cultured in 3% O2. Because prostaglandins are secreted molecules, these experiments further enforced the notion that prostaglandins do not mediate 21% O2-induced Na+ transport. We cannot, however, rule out the possibility that an unstable factor was secreted by FDLEs under the 21% O2 conditions because such factors would not be detected with our experimental protocol.
NO is an essential mediator in many cellular processes. It can play a role as both an oxidant and an antioxidant molecule (12). The mechanism by which NO acts varies from cell to cell, but, in general, it acts through a cGMP pathway (24). Atrial naturetic peptide acts via cGMP to regulate the 25-pS nonselective cation channel in kidney epithelium (20). Although a study (27) from our laboratory has shown that a brief (<1-h) exposure to atrial naturetic peptide is ineffective in regulating amiloride-sensitive Isc in FDLEs, Compeau et al. (10) also observed that activated macrophages reduce amiloride-sensitive Isc in FDLEs by an NO-dependent mechanism. To test the possible role NO may play in O2-induced Na+ transport in FDLEs, we incubated FDLE monolayers with an inhibitor of the inducible form of NO synthase (L-NAME) for the duration of the O2 induction protocol. L-NAME was unable to block the O2-induced increase in amiloride-sensitive Isc. Therefore, we do not believe 21% O2 induction of amiloride-sensitive Isc is mediated by NO.
There has been an increasing recognition of the role of ROS in signal transduction and gene regulation (reviewed in Ref. 19). Our results demonstrated that the superoxide scavenger TEMPO inhibited the 21% O2-induced amiloride-sensitive Na+ transport and ENaC mRNA expression. We chose TEMPO for this study because of its high cellular permeability and its lack of hydroperoxide scavenging activity (8, 34; Rafii, Tanswell, and O'Brodovich, unpublished observations). The SOD-catalase mimic EUK-8, at a concentration that was much lower than we used for TEMPO, also diminished the O2-induced amiloride-sensitive Isc to the 3% O2 level. On the other hand, neither catalase nor the cell-permeable hydroperoxide scavengers U-74389G and Trolox were able to block O2 induction of amiloride-sensitive Isc. NAC, a substrate source for the synthesis of glutathione (cofactor for the glutathione peroxidase that detoxifies H2O2), was equally ineffective, indicating that the change in the overall intracellular redox balance was not a factor in inducing amiloride-sensitive Isc on the switch of FDLEs from 3 to 21% O2. SOD, either alone or when encapsulated with pH-sensitive liposomes, did not block O2-induced amiloride-sensitive Isc. The failure of SOD to block O2-induced amiloride-sensitive Isc may have been due to its instability or its lack of uptake by FDLEs under these culture conditions for the 48-h O2 induction period. Unlike the small and diffusible TEMPO, large proteins such as SOD may also have stearic limitations in reaching relevant sites of superoxide production. Indeed, exogenous SOD has been shown to be an ineffective antioxidant in other whole cell systems (8, 23).
Together, these data suggest that superoxide anion and not H2O2 (the product of dismutated superoxide) is the ROS mediating the 21% O2 induction of amiloride-sensitive Na+ transport in FDLEs. These results are in contrast with other studies suggesting H2O2 as the ROS capable of modulating ion transport. Catalase, and not SOD, inhibits Na+ current in gerbil ear epithelium (16) and Na+-K+-ATPase activity in adult lung type II epithelial cells (15) that have been stimulated with the xanthine/xanthine oxidase ROS-generating system. However, these studies were conducted at ROS concentrations that may have been higher than those predicted to be generated by exposing FDLEs to postnatal 21% O2.
Our study shows that the change in O2 concentration was associated with increased levels of mRNA coding for ENaC. Although we did not directly assess transcription, it is likely that there was increased synthesis of ENaC mRNA. Others (11) have described O2-responsive elements on the human glutathione peroxidase promoter. In addition to the potential for an O2-responsive element in the ENaC promoter, it is also possible that there are other genes in the FDLEs that are O2 responsive and alter ENaC expression through their expressed proteins or metabolic by-products.
The transcription factors NF-B and AP-1 are known to be sensitive to
the intracellular redox state and ROS (14, 22). Both NF-
B and AP-1
are key players in the regulation of gene expression by cellular redox
state (14, 22, 36) and the promoter for
-rENaC contains an NF-
B
consensus binding element (GGGGAGTTCC at
519 to
510) and
a 12-O-tetradecanoylphorbol 13-acetate (CTAGTCA at
284 to
278) response element (29). In the
present study, we have provided preliminary data indicating that
NF-
B is activated by a postnatal 21%
O2 concentration and that this NF-
B activation can be blocked by the superoxide scavenger TEMPO. This is in agreement with another study (33) that has shown that
NF-
B is activated in tumor cells on posthypoxic reoxygenation. Because catalase and hydroperoxide scavengers were ineffective in
blocking O2-induced
amiloride-sensitive
Isc, it is likely
that superoxide acts directly and not through its dismutated by-product H2O2.
In conclusion, our experiments have demonstrated that the physiological increase in O2 concentration at the time of birth increases distal lung epithelial Na+ transport. This increase is a reversible phenomenon and is likely mediated by an increase in ROS that cause increased gene expression of ENaC. Because FDLEs (28) and human distal lung epithelial cells (2) have similar bioelectric properties, these findings may be relevant to the normal transition from fetal to postnatal life and in the recovery of patients with pulmonary edema.
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ACKNOWLEDGEMENTS |
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We thank Eukarion for providing EUK-8 and Dr. Susan Doctrow for helpful comments.
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FOOTNOTES |
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This research was supported by the Medical Research Council Group in Lung Development.
A. K. Tanswell is the Hospital for Sick Children Women's Auxiliary and University of Toronto Chair in Neonatology. O. Pitkänen was supported by a grant from the Finnish Cultural Foundation.
Preliminary results of this study were presented in abstract form (Am. J. Respir. Crit. Care Med. 155: A648, 1997).
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: H. O'Brodovich, Hospital for Sick Children, Univ. of Toronto, 555 Univ. Ave., Toronto, Ontario, Canada M5G 1X8.
Received 22 January 1998; accepted in final form 12 June 1998.
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