Oxygen induction of epithelial Na+ transport
requires heme proteins
Bijan
Rafii1,
Chris
Coutinho1,
Gail
Otulakowski1, and
Hugh
O'Brodovich1,2
1 Medical Research Council Group in Lung
Development, Program in Lung Biology Research, Hospital for Sick
Children Research Institute, and 2 Department of
Pediatrics, University of Toronto, Toronto, Ontario, Canada M5G 1X8
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ABSTRACT |
Fetal distal lung epithelial (FDLE) cells
exposed to a postnatal O2 concentration of 21% have higher
epithelial Na+ channel (ENaC) mRNA levels and
Na+ transport relative to FDLE cells grown in a fetal
O2 concentration of 3%. To investigate the mechanism of
this process, FDLE monolayers were initially cultured in 3%
O2, and then some were switched to a 21% O2
environment. Incubation of FDLE cells with the iron chelator
deferoxamine, CoCl2, NiCl2, or an
inhibitor of heme synthesis prevented or diminished the O2
induction of amiloride-sensitive short-circuit current in FDLE
cells. Similarly, defer- oxamine and cobalt prevented
O2-induced ENaC mRNA expression. Exposure of FDLE cells
grown under hypoxic conditions to carbon monoxide increased both ENaC
mRNA expression and amiloride-sensitive short-circuit current.
We therefore concluded that induction of ENaC mRNA expression and
amiloride-sensitive Na+ transport in FDLE cells by a
physiological increase in O2 concentration seen at birth
requires iron and heme proteins.
epithelial sodium channel; alveolar epithelium
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INTRODUCTION |
BEFORE BIRTH, THE FETAL LUNG is filled with fluid. This
fluid is critical for normal development of the lung and is secreted by
the fetal lung epithelium through a process driven by active secretion
of Cl
from the interstitium of the lung into the
lumen of the air space, with subsequent movement of water down its
osmotic gradient. After birth, however, reversal of this phenomenon
occurs: fluid is cleared from the lumen of alveoli through a process
driven by absorption of Na+ from the apical side of alveoli
into the interstitium of the lung and subsequent vectorial movement of
water down its osmotic gradient.
Discovery of the epithelial Na+ channel (ENaC) (5, 6, 16)
has shed new light on the mechanism of Na+ transport.
Although the stoichiometry of the ENaC is still controversial (7, 9),
it is generally believed to consist of the three subunits,
,
,
and
(6). The
-subunit is considered essential for the proper
function of the channel in the lung because the mouse lacking this
subunit fails to clear its fetal lung liquid and dies within the first
40 h after birth (14). Although mice lacking the
- and
-subunits
of ENaC have near-normal lung clearance, they die shortly after
birth because of an abnormal electrolyte balance (3, 18, 22),
likely due to the inability of the kidneys to maintain electrolyte homeostasis.
Although there is some information available, the actual mechanism
involved in the dramatic conversion of the lung from a fluid-secreting
to a fluid-absorbing organ is poorly understood. For example, it is
known that an increase in the circulating levels of adrenaline in the
fetal lamb (4) can rapidly and reversibly switch the fetal lamb from
fluid secretion to fluid absorption. However, this is a reversible
phenomenon, and because the increase in the level of this hormone
declines shortly after birth, it is uncertain whether this signal
is sufficient to permanently convert the lung to
Na+ absorption. Fetal (20, 23) and adult (17, 21) distal lung epithelia have enhanced Na+ transport and higher mRNA
levels of all three subunits of ENaC in a 21% O2 relative
to a 3% O2 environment, a phenomenon that is reversible
and is likely mediated, at least in part, by reactive oxygen species
(23). Therefore, O2 may be an important molecular switch to
"turn on" lung epithelial Na+ absorption at birth and
maintain it at sufficiently high levels thereafter.
The process of O2 induction of ENaC mRNA expression in
fetal lung distal epithelial (FDLE) cells would require an
O2-sensing cellular repertoire. A putative heme
O2 sensor has been implicated in mediating the induction of
erythropoietin (Epo) synthesis and vascular endothelial growth factor
mRNA expression by hypoxia in a liver cell line (11, 12). The function
of this sensor is dependent on extracellular iron levels and can be
modulated by iron chelators, selected divalent transition metals such
as Co2+ and Ni2+, and carbon monoxide (CO).
Although it is an increase rather than a decrease in O2
concentration that stimulates ENaC mRNA expression and Na+
transport in FDLE cells, we speculated that we may obtain insight into
the mechanism of O2 induction using these agents and approaches.
In this study, we show that iron, but not the iron binding proteins
contained within serum, is required for the induction of
amiloride-sensitive Na+ transport and ENaC mRNA expression
when FDLE cells are exposed to 21% O2. Three series of
experiments suggested that this process is mediated through or
dependent on heme-containing protein(s). First, when FDLE cells were
cultured in the presence of Co2+, a transition metal that
displaces iron from the protoporphyrin moiety of heme proteins and
locks the heme molecule in its deoxygenated conformation (30), 21%
O2 could no longer induce ENaC mRNA expression and
amiloride-sensitive Na+ transport in these monolayers.
Second, when FDLE cells were incubated in the presence of CO, a gas
that binds to the heme molecule and locks it in its oxygenated
conformation (11), there was an induction of ENaC mRNA expression and
Na+ transport, even though we maintained the FDLE cells in
a 3% O2 environment. Third, incubation of FDLE cells with
dioxoheptanoic acid (DHA), an inhibitor of heme synthesis (29),
attenuated the induction of FDLE cell Na+ transport by a
21% O2 environment.
 |
METHODS |
Primary cell culture. FDLE cells were isolated and cultured as
previously described (19). In brief, 20-day-gestation Wistar rats
(breeding day = day 0, term 22 days; Charles River, St.
Constant, PQ) were killed with an ether overdose. Fetal lung tissue was dispersed with 0.125% trypsin, and the resulting cell pellet was further incubated with 0.1% collagenase to separate associated fibroblasts from epithelial cells. Dissociated fibroblasts were separated from epithelial cells with a differential adherence and
centrifugation procedure. FDLE cells 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. All cells were
submersion cultured for a total of 3 days after being seeded in
Dulbecco's modified Eagle's medium (4.5 g/l of glucose with 2 mM
L-glutamine and 110 mg/l of sodium pyruvate) supplemented with 10% fetal bovine serum (Cansera, Rexdale, ON), 100 U/ml of penicillin G sodium, and 100 µg/ml of streptomycin sulfate. All cell
culture reagents were purchased from GIBCO BRL (Life Technologies, Burlington, ON).
O2 environment and interventions. After being
seeded, the FDLE cells were returned to an incubator containing 3%
O2-5% CO2-balance N2. After 24 h,
the medium was replaced with fresh medium that contained various
agents. Immediately thereafter, the monolayers were either transferred
to 21% O2 or kept in 3% O2 for the next 48 h.
For the CO experiments, the FDLE cells were placed in a hypoxic chamber
(Fisher Scientific, Unionville, ON), purged with a gas mixture
containing 3% O2, 5% CO2, 10% CO, and
balance N2 and then tightly sealed. The chamber was then
placed within the 3% O2 incubator. The concentration of
these gases was analyzed by the manufacturer (Praxair, Oshawa, ON). The
O2 concentration in the hypoxic chamber was monitored
during the 48-h experimental period with a Miniox I O2
analyzer (MSA Medical Products, Pittsburgh, PA). After the 48-h
induction period, the monolayers were used for either Ussing chamber
studies or Northern analyses. Deferoxamine, CoCl2,
NiCl2, ZnCl2, ferrous ammonium sulfate, and DHA
were purchased from Sigma-Aldrich Canada (Mississauga, ON).
Measurement of FDLE monolayer bioelectric properties. The
bioelectric properties of the FDLE monolayers were determined as previously described (19) with modified Ussing chambers (World Precision Instruments, Sarasota, FL) while the cells were bathed in
37°C Hank's balanced salt solution (GIBCO BRL) supplemented with
1.8 g/l of sodium bicarbonate and equilibrated with a 5% CO2-balance air gas mixture. The FDLE monolayers were
maintained under open-circuit conditions, and their short-circuit
current (Isc) was determined every 10 min with a
voltage-current clamp (Physiologic Instruments, San Diego, CA) until
stabilized (~20 min). The amiloride-sensitive Isc
was determined by the addition of 0.1 mM amiloride (in DMSO with a
final dilution of 1:1,000; Sigma-Aldrich) to the apical side of
monolayers. The transepithelial resistance (R) was calculated
by dividing the transepithelial potential difference by the
Isc.
Northern analyses. RNA was extracted from FDLE monolayers with
4 ml of TRIzol Reagent (GIBCO BRL) according to the manufacturer's instructions. The final pellet was dissolved in water treated with
dimethyl pyrocarbonate (Sigma-Aldrich), and then 20 µg of total RNA
were size-fractionated on a 1% agarose-1× MOPS-2% formaldehyde gel. RNA was subsequently transferred to Hybond N+ nylon
membranes (Amersham, Oakville, ON). The blots were then ultraviolet
cross-linked and hybridized with 32P random-primed rat
-ENaC (
-rENaC),
-rENaC, and
-rENaC cDNA fragments [bp
74-403 for
-rENaC, bp 2025-2401 for
-rENaC, and bp
2161-2520 for
-rENaC (6)] in ExpressHyb solution
(Clontech, Palo Alto, CA) following the manufacturer's instructions.
After a wash in 0.1× sodium citrate-sodium chloride plus 0.1%
sodium dodecyl sulfate at 50°C for 1 h, the blots were exposed to
autoradiography film at
80°C. Autoradiographic bands were
quantified with an Agfa (Duoscan) scanner and the National Institutes
of Health Scion Image version 1.6 quantitation program. The mRNA levels
were normalized to 18S rRNA content by hybridizing the blots with a
full-length mouse 18S rRNA 32P random-primed cDNA probe
(American Type Culture Collection, Manassas, VA).
Data analysis. We used the INSTAT version 3.0 statistical
program (GraphPad, San Diego, CA) to perform ANOVA followed by Tukey's post hoc test to examine significant differences between experimental groups. Probability (P) values of <0.05 were considered to be significant. All data are expressed as means ± SE, and unless otherwise specified, comparisons were made against the data
corresponding to the 3% O2 untreated control groups. Each
n represents a single monolayer from one primary culture of
FDLE cells. There were a minimum of three primary culture preparations
used for each experimental group.
 |
RESULTS |
Iron, but not serum iron binding proteins, is required for
O2 induction of FDLE cell Na+ transport. To
study the iron dependency of O2-induced Na+
transport in FDLE cells, we incubated FDLE monolayers in 21% O2 with various concentrations of the iron chelator
deferoxamine for 48 h. Deferoxamine prevented the induction of
amiloride-sensitive Isc by 21% O2 in a
dose-dependent manner (IC50 ~3 µM; Fig.
1). As Rafii et al. (23) have previously
described, amiloride- insensitive Isc was not
affected by switching FDLE cells from a 3 to a 21% O2
environment. In a separate set of experiments, incubation of FDLE cells
with 10 µM deferoxamine for the 48-h O2 induction period prevented the induction of amiloride-sensitive Isc
by 21% O2 (Fig. 2).
Neutralizing the effect of deferoxamine by coincubation of the
deferoxamine-treated FDLE cells with excess iron (20 µM ferrous ammonium sulfate) restored the ability of FDLE cells to be induced by
O2 (Fig. 2). Deferoxamine treatment had no effect on the
amiloride-sensitive Isc of monolayers incubated in
3% O2 (Fig. 2). Treatment of the cells with deferoxamine
did not lead to a lower R in the monolayers, indicating that
the effect of deferoxamine was not due to a nonspecific toxic effect on
these cells (Table 1). Baseline
Isc in deferoxamine-treated cells, however, was
significantly lower than that in the 21% O2 control group
(P < 0.05; Table 1).

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Fig. 1.
Deferoxamine inhibited fetal lung distal epithelial (FDLE) cell
Na+ transport in a dose-dependent manner. FDLE monolayers
were incubated with indicated concentrations of deferoxamine in 21%
O2. After 48 h, amiloride-sensitive short-circuit current
(Isc) for these monolayers was measured
(IC50 ~3 µM; n = 4-19 monolayers/group).
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Fig. 2.
O2 induction of Na+ transport in FDLE cells
required iron. FDLE monolayers were initially cultured in 3%
O2 for 24 h (control group), and then some monolayers were
treated with (+) and without ( ) 10 µM deferoxamine (Def) or 10 µM Def plus 20 µM ferrous ammonium sulfate (Fe). Subsequently,
monolayers were either left in 3% O2 or switched to 21%
O2. After 48 h, amiloride-sensitive Isc
for these monolayers was measured (n = 12-28
monolayers/group). * P < 0.05 compared with 3%
O2 control group.
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To investigate whether iron-containing or iron binding proteins within
serum are necessary for the O2 induction of Na+
transport in FDLE cells, we conducted our O2 induction
experiments in the presence and absence of serum. As in monolayers
incubated in the presence of serum, FDLE cells exposed to 21%
O2 in serum-free medium had a higher amiloride-sensitive
Isc than those kept in a 3% O2
concentration (Fig. 3). The monolayers
incubated under serum-free conditions, however, had a significantly
lower R than those of the serum-treated 21% O2
control group (P < 0.05), although the baseline
Isc in these monolayers was comparable to that of the serum-treated control cells (Table 1).

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Fig. 3.
O2 induction of Na+ transport in FDLE cells was
independent of serum proteins. FDLE monolayers were initially cultured
in DMEM plus 10% fetal bovine serum (FBS) in 3% O2 for 24 h, and then medium was changed to either DMEM or DMEM plus 10% FBS and
cells were maintained at either 3 or 21% O2. After 48 h,
amiloride-sensitive Isc for these monolayers was
measured (n = 9-12 monolayers/group). * P < 0.05 compared with 3% O2 control group.
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Effect of CoCl2 and other transition metals on
O2 induction of FDLE cell Na+ transport.
Transition metals with a low binding affinity for O2, such
as Co2+, are known to displace iron in the protoporphyrin
ring of heme proteins and thereby chemically mimic the hypoxic
conditions in cells. To study the effect of Co2+ on
amiloride-sensitive Na+ transport, we incubated FDLE
monolayers with various concentrations of CoCl2 for 48 h in
21% O2. Co2+ inhibited amiloride-sensitive
Isc in a dose-dependent manner (IC50
~100 µM; Fig. 4). In a separate set of
experiments, we incubated FDLE monolayers with 100 µM
CoCl2, NiCl2, or ZnCl2 for the 48-h duration of the O2 induction period. Co2+
prevented 21% O2 induction of amiloride-sensitive
Isc (Fig. 5). FDLE
cells incubated with Ni2+, another divalent transition
metal with similar hypoxia-inducing properties as Co2+ (11,
26), similarly failed to respond to induction by 21% O2
(P < 0.05 vs. 3% O2 control group; n = 12 monolayers/group; data not shown). However, monolayers incubated
with Zn2+, a transition metal shown not to displace iron in
heme proteins (27), did not block 21% O2-induced
amiloride-sensitive Isc (Fig. 5). This latter
finding suggests that the effect of Co2+ is not due
to a nonspecific effect of transition metals. Although treatment of the monolayers with Co2+ led to lower
baseline Isc, R was not affected
by this treatment (Table 1).

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Fig. 4.
Cobalt inhibited FDLE cell Na+ transport in a
dose-dependent manner. FDLE monolayers were incubated with indicated
concentrations of CoCl2 in 21% O2. After 48 h,
amiloride-sensitive Isc for these monolayers was
measured (IC50 ~100 µM; n = 4-16
monolayers/group).
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Fig. 5.
Cobalt but not zinc inhibited O2-induced Na+
transport in FDLE cells. FDLE monolayers were initially cultured in 3%
O2 for 24 h, and then some monolayers were incubated with
100 µM of either CoCl2 or ZnCl2.
Subsequently, monolayers were either left in 3% O2 or
switched to 21% O2. After 48 h, amiloride-sensitive
Isc for these monolayers was measured (n = 8-24 monolayers/group). * P < 0.05 compared with 3%
O2 control group.
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Effect of iron chelation and Co2+ on ENaC mRNA
expression. To test whether the effects of iron chelation and
Co2+on Na+-channel activity correlated with
changes in the ENaC mRNA levels, we performed Northern analyses
on several FDLE monolayers exposed to either deferoxamine or
Co2+. The
- and
-ENaC mRNA levels changed in
parallel with the changes in FDLE amiloride-sensitive
Isc (Fig.
6). Deferoxamine blocked O2-induced
- and
-ENaC mRNA expression. The effect of
deferoxamine on
-ENaC mRNA levels was reversed by coincubation of
deferoxamine-treated cells with ferrous ammonium sulfate. As with
deferoxamine, Co2+ was able to abrogate
O2-induced
- and
-ENaC mRNA expression. The changes
in
-ENaC mRNA levels (data not shown) in response to deferoxamine,
iron, and Co2+ showed a statistically insignificant pattern
similar to that for
- and
-ENaC (Fig. 6).



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Fig. 6.
Def and cobalt abrogated O2-induced - and -RNA
expression in FDLE cells. FDLE monolayers were initially cultured in
3% O2 for 24 h, and then some monolayers were incubated
with either 10 µM Def, 10 µM Def plus 20 µM Fe, or 100 µM
CoCl2. Subsequently, monolayers were either left in 3%
O2 or switched to 21% O2. After 48 h, total
RNA from these monolayers was extracted for Northern analyses.
A: representative blots of -, -, and -epithelial
Na+ channels (ENaCs) and 18S rRNA (18S). B and
C: densitometric analyses of multiple blots of - and
-ENaCs, respectively, normalized first to 18S rRNA and then to
corresponding 21% O2 group (n = 2-4
monolayers/group; each monolayer was prepared from a separate harvest
of FDLE cells). * P < 0.05 compared with 3%
O2 control group.
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CO induces amiloride-sensitive Na+ transport and
ENaC mRNA expression. Our hypotheses of heme involvement in
O2 induction of ENaC would predict that CO, a molecule that
mimics binding of O2 to heme proteins, might stimulate ENaC
mRNA expression and Na+ transport in FDLE cells.
We incubated FDLE monolayers in a 3% O2-10% CO gas
mixture and measured amiloride-sensitive Isc and steady-state mRNA levels for
-,
-,and
-ENaC after 48 h. As predicted, CO induced amiloride-sensitive
Isc with respect to the 3% O2 control
group (Fig. 7) even though the monolayers
were incubated in 3% O2. Both baseline
Isc and R for the CO-treated group were
comparable to those for the 21% O2 untreated control group
(Table 1). Similarly, CO increased the level of
- and
-mRNA
expression compared with that in the 3% O2 control group (P < 0.05; Fig. 8,
A-C). Levels of
-ENaC mRNA were unresponsive to
induction by CO (Fig. 8, A and D).

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Fig. 7.
Carbon monoxide (CO) induced Na+ transport in FDLE cells.
FDLE monolayers were initially cultured in 3% O2 for 24 h,
and then some monolayers were incubated in 10% CO plus 3%
O2 while others were incubated in either only 3% or only
21% O2. After 48 h, amiloride-sensitive
Isc was measured (n = 12 monolayers/group).
* P < 0.05 compared with 3% O2
control group.
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Fig. 8.
CO induced - and - but not -ENaC mRNA expression. FDLE
monolayers were initially cultured in 3% O2 for 24 h, and
then some monolayers were incubated in 10% CO plus 3% O2
while others were incubated in either only 3% or only 21%
O2. After 48 h, total RNA was extracted for Northern
analyses. A: representative blot from 3 different experiments.
B-D: densitometric analyses of multiple blots of
-, -, and -ENaC, respectively, normalized first to 18S rRNA
and then to corresponding 21% O2 group (n = 7 monolayers/group; each monolayer was prepared from a separate harvest
of FDLE cells). * P < 0.05 compared with 3%
O2 control group.
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Inhibition of heme synthesis attenuates O2 induction of
Na+ transport in FDLE cells. To provide further
evidence supporting the role of a heme protein in the O2
induction of Na+ transport in FDLE cells, we incubated FDLE
monolayers in the presence of the heme synthesis inhibitor DHA (2 mM)
throughout the 48-h period in which they were exposed to 21%
O2. DHA attenuated the ability of 21% O2 to
induce Na+ transport (Fig.
9). The treatment of cells with DHA
led to a lower baseline Isc but not to a lower
R (Table1), indicating that its effect was not
due to a nonspecific toxic property of this compound.

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Fig. 9.
Heme synthesis inhibitor dioxoheptanoic acid (DHA) attenuated
O2-induced Na+ transport in FDLE cells. FDLE
monolayers were initially cultured in 3% O2 for 24 h, and
then some monolayers were incubated with 2 mM DHA. Subsequently,
monolayers were either left in 3% O2 or switched to 21%
O2. After 48 h, amiloride-sensitive Isc
in these monolayers were measured (n = 23-24
monolayers/group). * P < 0.05 compared with 3%
O2 control group. # P < 0.05 compared with 21% O2 control group.
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 |
DISCUSSION |
In this study, we have shown that O2-induced ENaC mRNA
levels and amiloride-sensitive Na+ transport is dependent
on the presence of adequate amounts of iron and that it is inhibited by
micromolar concentrations of Co2+. Both
- and
-ENaC
mRNA expression and Na+ transport in FDLE cells can be
induced by CO, whereas O2-induced Na+ transport
can be attenuated by an inhibitor of heme synthesis. Together these
results point to involvement of a heme-containing molecule in the
O2 induction of ENaC mRNA expression and Na+
transport in FDLE cells.
Because tissue culture serum contains iron-containing proteins, we
evaluated the relevance of these proteins by carrying out O2 induction of FDLE cells in the presence and absence of
serum. Na+ transport in monolayers cultured in the absence
of serum was induced by O2 similar to that in cells
cultured in the presence of serum. However, serum-free culture of FDLE
monolayers significantly decreased their R values, suggesting
that factors within serum may be important for the integrity of tight
junctions. These experiments provide evidence that the phenomenon of
O2 induction does not require iron-containing proteins or
other factors within serum.
Other investigators have used divalent transition metals to study the
mechanisms whereby changes in O2 concentration alter cellular physiological functions. It is well known that
Co2+and Ni2+ can displace iron in the
protoporphyrin ring of heme molecules (26, 30). When these compounds
replace iron within the heme group, there is a reduction in the
affinity of the heme molecules for O2 (26, 30). Therefore,
Co2+- and Ni2+-containing heme molecules are
locked in their deoxygenated conformation. Using this information,
others (11) have demonstrated that Co2+ can mimic the
hypoxic induction of the glycoprotein Epo by liver cells and that CO, a
molecule that binds to heme proteins and locks it in the oxygenated
conformation, can, in turn, abrogate this induction. The current
working hypothesis on hypoxic induction for Epo is that when liver
cells are exposed to hypoxia or Co2+, there is a
concomitant activation of the transcription factor hypoxia-inducible
factor-1 (HIF-1) (25). HIF-1 binds to an enhancer in the gene encoding
Epo, resulting in increased transcription. In contrast to the effect of
hypoxia on Epo, hypoxia downregulates FDLE cell Na+
transport and ENaC mRNA levels. Thus if HIF-1 is involved in our
system, one must speculate that hypoxic induction of HIF-1 increases
the expression of a protein that is capable of suppressing ENaC
expression. To gain more insight into this issue, we also incubated
some monolayers with deferoxamine in hypoxic O2
concentrations. Under 3% O2, deferoxamine had no effect on
Na+ transport. Because deferoxamine inhibits
Na+ transport in 21% but not in 3% O2, we
suggest that the phenomenon of O2 regulation of ENaC likely
involves activation under a 21% O2 rather than suppression
under a 3% O2 environment.
CO binds to heme proteins in a manner analogous to O2
binding to hemoglobin and locks the molecule in its oxygenated
conformation (11). Our present observation that
- and
-ENaC mRNA
expression and Na+ transport were induced in monolayers
incubated with CO under hypoxic conditions supports our hypothesis that
a heme-containing protein is involved in O2 signaling
in FDLE cells. In contrast to
- and
-ENaC,
-ENaC
mRNA expression was neither suppressed by Co2+ nor induced
by CO. We, therefore, speculate that the mechanism and pathways of
induction of
-ENaC by O2 may be different from those of
either
- or
-ENaC. We also performed additional experiments to
further investigate the potential role of heme proteins in O2 induction. In those studies, O2 induction
was done in the presence of DHA, an inhibitor of aminolevulinate
dehydratase (29), an enzyme in the heme synthetic pathway. DHA
significantly reduced O2 induction of FDLE cells, further
supporting a role for heme proteins in O2 induction of
Na+ transport in FDLE cells. Inability of DHA to completely
inhibit O2-induced Na+ transport could be
attributed to its inability to completely inhibit intracellular heme
synthesis (24).
To date, heme-mediated activation of gene transcription in response to
O2 has been conclusively demonstrated only in the
FixL/FixR system of the nitrogen-fixing bacterium Rhizobium
meliloti (10). Some previously identified heme proteins, however,
have been proposed as potential candidates for this O2
sensor in eukaryotic systems. These include cytochrome P-450
(8) and NADPH oxidase (1, 13, 31), which under normoxic conditions bind
and convert O2 to superoxide, and NO-inducible guanylate
cyclase (28).
Our results do not rule out the possibility that there is also a
nonheme O2 sensor similar to that described in neurons (15) or an iron-sulfur cluster-type molecule such as aconitase that produces
reactive oxygen species in response to changes in environmental O2 concentration. Rather, our results suggest that heme
proteins play an essential role in the pathway(s) leading to increased Na+ transport by FDLE cells when they are exposed to
postnatal O2 concentrations.
In conclusion, our experiments have demonstrated that induction of
Na+ transport and ENaC mRNA expression in FDLE cells by
O2 requires iron and heme-containing protein. Further
studies are required to determine whether the heme protein-dependent
increase in ENaC mRNA levels occurs through increasing the stability or
inducing the synthesis of the mRNA message under higher postnatal
oxygen concentration. Because FDLE (19) and human distal lung
epithelial cells (2) have similar bioelectric properties, these
findings may be relevant to the normal transition of the lung from
fetal to postnatal life and in the recovery of patients with pulmonary edema.
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ACKNOWLEDGEMENTS |
This research was supported by a Medical Research Council (Canada)
Group Grant in Lung Development.
 |
FOOTNOTES |
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: H. O'Brodovich,
Hospital for Sick Children, 555 University Ave., Toronto, Ontario,
Canada M5G 1X8 (E-mail: hugh.obrodovich{at}sickkids.on.ca).
Received 1 July 1999; accepted in final form 13 September 1999.
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