1 Laboratoire d'Otologie
Expérimentale, The physiology of the middle ear is primarily concerned with
keeping the cavities air filled and fluid free to allow transmission of
the sound vibrations from the eardrum to the inner ear. Middle ear
epithelial cells are thought to play a key role in this process, since
they actively transport Na+ and
water. The PO2 of the middle ear
cavities varies from 44 to 54 mmHg in healthy human ears but may be
lower in the course of secretory otitis media. The effect of chronic
hypoxia on ion transport was investigated on a middle ear cell line
using the short-circuit current technique. Chronic hypoxia reversibly decreased the rate of Na+
absorption across the MESV cell line. Although a decrease in cellular
ATP content was observed, the decrease of
Na+ absorption seemed related to a
primary modulation of apical Na+
entry. As revealed by RNase protection assay, the decrease in the rate
of apical Na+ entry strictly
paralleled the decrease in the expression of transcripts encoding the
secretory otitis media; ouabain; benzamil; ion transport; transepithelial sodium; short-circuit current; epithelial sodium
channel; sodium-potasium-adenosine 5'-triphosphatase
THE PHYSIOLOGY OF THE middle ear is primarily concerned
with keeping the cavities air filled and fluid free, to allow
transmission of the sound vibrations from the eardrum to the inner ear.
Middle ear epithelial cells are thought to play a key role in this
process. They eliminate mucus from the tympanic cavity by means of
apical cilia (1) and also actively transport
Na+ and water to clear any fluid
present in excess (9, 20).
The perfusion of the mucosa is thought to be poor, since high
PCO2 and low
PO2 have been reported in the middle
ear cavities (18). Therefore, as far as oxygen is concerned, epithelial
cells may rely not only on the capillary bed but also on the gas
mixture of the middle ear cavities. This gas mixture has long been
thought to originate from boluses of exhaled air, since the middle ear
communicates with the pharynx through the eustachian tube. This would
likely allow the PO2 to reach 120 mmHg (17). However, several recent works have reported that the middle
ear gas composition differs dramatically from that of atmospheric air
and is similar to the composition of mixed venous blood (11, 26, 35).
This might be related to the very short openings of the eustachian
tube. The PO2 varies from 44 mmHg
(18) to 54 mmHg (15) in healthy human ears but may be lower in the
course of secretory otitis media, from 31 to 51 mmHg (24). The middle
ear cells are thus putatively exposed to a minor hypoxia.
Hypoxia is central to many pathophysiological disorders. Most mammalian
cells are very sensitive to the decrease in
PO2, which may lead to irreversible
cellular damage (13). However, a certain respiratory epithelium was
shown to be quite resistant to prolonged hypoxia, with a reversible
modulation of the rate of ion transport (33). Because the middle ear
epithelium is also of respiratory type, the evaluation of middle ear
epithelial cell function under hypoxia represents an important issue.
In this work, we report that chronic hypoxia reversibly decreased the
rate of Na+ absorption across
middle ear epithelial cells. Although a decrease in cellular ATP
content was observed, the decrease of
Na+ absorption seemed related to a
primary modulation of apical Na+
entry. A decrease in the expression of transcripts encoding the Cell culture.
Techniques have been described elsewhere (23). The MESV cell line was
previously established from Mongolian gerbil
(Meriones unguiculatus) middle ear epithelial
primary culture by infection with simian virus 40 (20). Briefly, MESV
cells were regularly subcultured in a humidified 5%
CO2 incubator at 37°C, in a
medium composed of DMEM-medium 199 (1:1 vol/vol) containing 5% FCS, 10 ng/ml epidermal growth factor, 5 µg/ml transferrin, 2 nM
triiodothyronine, 5 µg/ml insulin,
10
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-subunit of the epithelial Na+
channel. This effect of oxygen on
Na+ absorption might account for
1) the presence of fluid in the middle ear in the course of secretory otitis media and
2) the beneficial effect of the
ventilation tube in treating otitis media that allows the
PO2 to rise and restores the fluid clearance.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-subunit of the epithelial Na+
channel (
-ENaC) was evidenced, which paralleled the decrease of
Na+ absorption.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
6 M hydrocortisone,
10
7 M dexamethasone, 100 U/ml penicillin, 100 µg/ml streptomycin, 15 mM HEPES, and 2 mM
L-glutamine.
Hypoxic exposure. To achieve hypoxic exposure, culture dishes were placed in a humidified airtight incubator with inflow and outflow valves, and the desired hypoxic gas mixture (0% O2-5% CO2-95% N2, 5% O2-5% CO2-90% N2, or 10% O2-5% CO2-85% N2) was delivered at a constant flow rate for 20 min. The airtight incubator was kept at 37°C for 1, 3, 6, 12, 18, or 24 h, while control normoxic cells were placed in a 21% O2-5% CO2-74% N2 humidified incubator for the same period of time. In our conditions, values of PO2 assayed in the culture medium (mPO2) were 32, 50, 100, and 150 mmHg after 18-h incubation with 0, 5, and 10% O2 (hypoxia) and 21% O2 (normoxia), respectively.
Morphology. To investigate the morphological impact of hypoxia exposure on MESV cells, cells were incubated 18 h in normoxia or hypoxia. Afterwards, cells were rinsed with cacodylate buffer (0.1 M) and fixed overnight with 2.5% glutaraldehyde. Specimens were then washed in buffer, postfixed with 1% OsO4, dehydrated, and embedded in Epon. The sections were counterstained with uranyl acetate and further processed for transmission electron microscopy (EM 410, Philips, Eindhoven, The Netherlands).
Determination of cellular protein content. The method for the quantification of the protein content in MESV cells utilized the principle of protein-dye binding (5). BSA was used as standard. Results are expressed in micrograms of protein per well or per filter.
Bioelectric measurements.
Cells were used 5 days after seeding. Filters were mounted into
micro-Ussing chambers perfused with medium. The perfusion medium was
lifted with the gas in which the cells were preincubated (0, 5, 10, or
21% O2). Chambers were
connected to a voltage-current clamp device (DVC1000, World Precision
Instruments, New Haven, UK). Short-circuit conditions were maintained
throughout the experiment, and the short-circuit current
(Isc) was
continuously recorded on a pen chart recorder (Servotrace, Sefram,
Paris, France). Every 30 s, voltage was clamped at 1 mV, so that the
transepithelial resistance
(RT) could be
determined by Ohm's law. Two currents were studied:
1)
Isc across MESV
monolayers and 2)
Isc,Na, the fraction of Isc
sensitive to benzamil (106
M), a highly selective apical Na+
channel inhibitor (36).
Determination of intracellular ATP content.
Intracellular ATP content was determined with luciferase assay
according to Doctor et al. (10). Cells were cultured on six-well plates
for 4 days and then incubated for 18 h in normoxia or 1, 3, 6, or 18 h
in hypoxia. The cells were washed, and ice-cold 3% (3 N) perchloric
acid was added. After 3 min, the monolayer was scraped from the dish
and the resulting mixture was centrifuged (10 min at 2,000 rpm). The
pellets were resuspended in 0.1 N NaOH for protein determination. The
supernatant was neutralized with KOH and stored at 80°C
until measurement was performed. ATP content of the supernatant was
measured in a luminometer (Hewlett-Packard Picolite luminometer,
Packard Instrument) using an ATP determination kit (Calbiochem).
Standard curves of log photons vs. log ATP were linear over the range
from 10
8 to
10
4 M ATP. Results were
expressed in micromoles of ATP per milligram of protein. Each data
point is the mean of three measurements.
Measurement of the ouabain-sensitive
86Rb+
uptake.
The ouabain-sensitive Rb+ influx,
determined by the difference between
86Rb+
uptake with and without ouabain, was used as an indicator of Na+-K+-ATPase
activity (8). MESV cells were incubated for 18 h in normoxia or
hypoxia, with or without benzamil in the apical bath (106 M). Uptake
measurements were performed at 37°C in a solution derived from
Eagle's essential medium containing (in mM) 120 NaCl, 5 RbCl, 1 MgSO4, 0.15 Na2HPO4,
0.2 NaH2PO4,
4 NaHCO3, 1 CaCl2, 5 glucose, and 20 HEPES (pH
7.4). The osmolality was adjusted with mannitol to 350 mosmol/kg. Cells
were preincubated in the presence of ouabain (5 mM) or vehicle, and
then uptake was performed for 5 min with
86Rb+
(2 µCi/ml) in the basal compartment. Uptake solutions used on hypoxic
cells were preequilibrated with the hypoxic gas to avoid reoxygenation
during the uptake procedure. Uptake was stopped by washing the cells
three times with ice-cold buffer. Radioactivity was then extracted by
Triton X-100 (1%) and counted in a scintillation counter. The protein
content of each filter was determined, and results were expressed as
picograms of
86Rb+
per filter.
RNase protection assay.
RNase protection assay was performed directly on cell lysis as
described earlier (22). MESV monolayers were incubated for 18 h in
normoxia, hypoxia, or hypoxia followed by reoxygenation. Total RNA
equivalent of 10 cells or yeast tRNA (Boehringer) was hybridized with 5 × 105 counts/min (cpm) for
human ENaC (hENaC) and 5 × 104 cpm for -actin or
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) radiolabeled probes in
80% formamide, 40 mM PIPES (pH 7.4), 400 mM NaCl, and 1 mM EDTA at
50°C overnight, and RNase digestion (40 µg/ml RNase A and 2 µg/ml T1; Boehringer) was performed at 30°C for 60 min. Digestion
by proteinase K (125 µg/ml; Boehringer) was then done at 37°C for
30 min. After phenol extraction and ethanol precipitation, protected
fragments were separated by urea-PAGE. Gels were fixed with 10%
CH3COOH and vacuum dried before
exposure to Kodak X-OMAT AR 5 films or quantification with an Instant
Imager (Packard Instrument).
-Actin expression was used as an
internal standard, since the level of
-actin mRNA was not
significantly modified by hypoxia, whereas the level of GAPDH mRNA was
significantly increased after 18 h of hypoxic exposure and could not be
used as a reliable standard. Results were expressed as the ratio of expression of the mRNA of interest to actin mRNA, in arbitrary units (AU).
cRNA probes.
As previously described, the -hENaC probe was used (22). Antisense
RNA probes were synthesized from the translated region of the
-subunit (bp 1036-1259) of hENaC. The cRNA synthesis (Promega kit) was done using
[32P]UTP (Amersham; sp
act >15 TBq/mmol). The
[32P]cRNA probe was
307 bp, and the protected fragment was ~110 bp. Rat
-actin mRNA
was used for standardization.
-hENaC subunit cDNA was a gift from
Richard Boucher (Chapel Hill, NC).
Reagents. All chemicals were purchased from Sigma (St. Louis, MO). Tracers were all provided by Amersham (Amersham, UK). Culture media and reagents were from GIBCO BRL (Cergy-Pontoise, France). Plasticware was from Costar (Cambridge, MA). Gas mixtures were from Carboxyque and CFPO.
Statistical analysis. Results are expressed as means ± SE of n separate experiments. Three values were averaged for measurement of Na+-K+-ATPase activity and intracellular ATP content. Comparisons of means were performed by using one- or two-way ANOVA (as appropriate) followed by Fisher's least significant difference test or Bonferroni's test for comparison with control. Differences were considered significant at P < 0.05.
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RESULTS |
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Morphology. Dome formation is a constant feature of MESV cells when grown on nonporous supports. Surprisingly, incubation for 18 h in hypoxic conditions dramatically prevented dome formation.
Transmission electron microscopy did not reveal any morphological differences, such as cellular hypertrophy, increase in cell height, or amplification of basolateral areas, between cells grown in normoxia and 18-h hypoxia. Neither cilia nor secretory granules could be observed in either condition.Effect of hypoxia on
Isc.
Incubation of cell monolayers in a hypoxic medium
(mPO2 32 mmHg) induced a
time-dependent decrease of
Isc from 3.21 ± 0.57 µA/cm2 in normoxia
(control; n = 12) to 1.28 ± 0.22 µA/cm2
(P < 0.01;
n = 8) after 18-h hypoxia. The
RT was not
modified by hypoxia [1,623 ± 154 · cm2 for
control vs. 1,334 ± 141
· cm2 after
18-h hypoxia; not significant (NS); n = 10].
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Determination of intracellular ATP content.
The inhibitory effect of hypoxia on
Na+ absorption could be related to
a metabolic effect, such as decrease of the ATP cellular content.
Effectively, hypoxia significantly affected the cellular ATP content in
MESV cells beyond 3 h of hypoxia (Table 1). However, a
partial recovery of cellular ATP content was observed after 18-h
hypoxia.
|
Measurement of ouabain-sensitive 86Rb+ uptake. Incubation for 18 h in hypoxia dramatically decreased the ouabain-sensitive 86Rb+ uptake from 164.99 ± 2.63 pg 86Rb+/filter for normoxic cells to 38.61 ± 1.06 pg 86Rb+/filter for hypoxic cells (P < 0.001; n = 3).
To evaluate the target of hypoxia in our system, either apical or basal, we measured ouabain-sensitive Rb+ influx with or without benzamil (10
|
Effect of hypoxia on -ENaC subunit mRNA levels.
RNase protection assays were performed to evaluate the expression of
-ENaC subunit mRNA transcripts in normoxic and hypoxic MESV cells.
-Actin expression was used as an internal standard, since the level
of
-actin mRNA was not significantly modified by hypoxia (11.73 ± 0.96 cpm for control cells vs. 9.95 ± 1.77 cpm for hypoxic
cells; NS; n = 7-8). Exposure of
MESV cells to hypoxia for 18 h induced a 50% decrease in the
expression of
-ENaC mRNA (67.5 ± 7.7 vs. 135.3 ± 13.25 AU
for normoxic cells; P < 0.05;
n = 5-8). This decrease
paralleled the decrease of
Isc,Na (63%)
observed in the
Isc assays. When
hypoxic cells were allowed to recover in 150 mmHg
O2, the level of
-ENaC mRNA
transcripts was not different from control (116.3 ± 12.46 AU; NS;
n = 5-8; Fig.
5).
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DISCUSSION |
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This work reports a reversible hypoxia-induced decrease of Na+ absorption in middle ear epithelial cells. This modulation was likely related to a reversible decrease in the expression of the apical Na+ channel mRNA rather than to metabolic effects of hypoxia.
Hypoxia decreases the rate of
Na+ absorption
in the middle ear epithelium.
Ion transport activity of the middle ear epithelium has been previously
studied in primary cultured cells (12, 19) and in the MESV cell line
(20) by the Isc
technique. In these cells, most of the electrogenic ion transport is
related to an active absorption of
Na+ from the luminal to the basal
compartment, occurring through apical amiloride-sensitive
Na+ channels. This process is
thought to drive a water flow and to contribute to the maintenance of
air-filled and fluid-free cavities. The
Na+ absorption was herein
evaluated as
Isc,Na (22).
Chronic hypoxia induced a time-dependent decrease of
Isc in MESV cells
(~60% after 18 h of exposure). This hypoxia-induced decrease
paralleled the decrease in Na+
absorption, as evaluated by
Isc,Na (~63%
after 18 h of exposure; see Fig. 1). The fraction of
Isc insensitive
to benzamil may be related to an apical
Cl channel (12, 21).
Because of an unfavorable electrochemical gradient, this
Cl
channel seems to be
active only when Na+ entry is
blocked (12), as in the case of human nasal epithelial cells (4).
Hypoxia promotes a decrease of the expression of transcripts
encoding -ENaC.
The PO2 variations have been shown to
affect ion transport function. Depending on the model, hypoxia
modulates apical ionic channels (31, 32) and/or basolateral
Na+-K+-ATPase
activity (16, 29, 32). To characterize the target of hypoxia in our
system, the
Na+-K+-ATPase
activity was measured by the ouabain-sensitive
86Rb+
uptake, with or without a benzamil-induced apical
Na+ entry inhibition. We observed
that hypoxia induced a 75% decrease in
Rb+ uptake, i.e., in
Na+-K+-ATPase
activity. This could have been related to an effect of hypoxia either
on apical Na+ entry or on basal
extrusion through the pump. If the only target of hypoxia were the
basal pump, one would expect the addition of benzamil, which blocks the
apical Na+ entry, to reduce the
intracellular Na+ concentration
and, as a result, the activity of the basal pump. In that case, hypoxia
combined with benzamil should yield a lower Na+-K+-ATPase
activity than hypoxia alone, which was not the case. This result
suggests that the actual target of hypoxia was the apical Na+ entry, which does not preclude
a simultaneous effect on the basal pump. Furthermore, hypoxia
significantly decreased
Isc,Na after 12 h, a delay compatible with a transcriptional effect. For these reasons,
the hypoxia-induced modulation of the expression of transcripts encoding ENaC was measured. The ribonuclease protection assay was
performed with a probe synthesized from the translated region of the
-subunit (bp 1036-1259) of the hENaC (
-ENaC). Although some
interspecies differences have restrained the size of the protected
fragment, examination of rat and human sequences reveals that the
translated region used is highly maintained, with a 92% homology, the
longest identical sequence being 144 bp long (22). Hypoxia decreased by
50% the expression of
-ENaC subunit transcripts. Although
posttranscriptional events might affect the effective production of
Na+ channels, this result
paralleled the hypoxia-induced decrease of the transepithelial
Na+ absorption observed in
functional assays
(Isc,Na decreased
by 60%). The decrease of
-ENaC mRNA transcripts was not related to
irreversible cellular damage, since
1)
-actin mRNA transcripts were
not modified by hypoxia and 2) after
an 18-h hypoxia, reoxygenation allowed parallel
Isc,Na and
-ENaC mRNA recovery. Hypoxia-induced decrease in
Na+ absorption might also have
been related to downregulation of
- and
-subunits. However, it
should be stated that the
-subunit of the
Na+ channel exhibits, when
expressed in oocytes, all the characteristics of the highly selective
channel, whereas
- and
-subunits only allow maximal activity of
active Na+ channels (7, 36).
Hypoxia-induced
Na+ absorption
modulation may not only occur through -ENaC mRNA
transcript modulation.
It has been shown in other tight epithelia that the rate-limiting step
for transepithelial Na+
reabsorption is Na+ entry (34),
whereas
Na+-K+-ATPase
adapts its activity to maintain a low intracellular
Na+ concentration (3). The data we
present strongly support a downregulation of
-ENaC mRNA. However,
translation or posttranslation hypoxia-induced regulation cannot be
excluded because 1) neither the
Na+ channels produced nor those
actually present on the apical cellular membrane were quantified, and
2) hypoxia might modulate ion
transport function because of cytosolic factors such as intracellular
Ca2+ increase (2, 32) or
intracellular ATP decrease (28). Actually, a significant decrease in
cellular ATP content was observed in hypoxic conditions in MESV cells.
A 70% decrease after a 6-h incubation was followed by a partial
recovery for a longer incubation time (49% at 18 h), which may be
related to an activation of anaerobic metabolism. This decrease may
contribute to the effect of hypoxia on
Na+ absorption. However,
the Michaelis constant of the
Na+-K+-ATPase
for ATP is very low, so that only drastic depletion can affect the
Na+-K+-ATPase
activity (25). Last, although the
Na+-K+-ATPase
activity decrease seemed to be related to a decrease of apical
Na+ entry and apico-basal
coupling, a transcriptional modulation cannot be excluded.
Pathophysiological incidence of the epithelial effect of hypoxia-reoxygenation. Some in vivo experiments (31, 36) have indicated that PCO2 may be high in the middle ear cavities (~60 mmHg). Because of the high diffusion rate coefficient of CO2, this fact suggests that the perfusion of the mucosa is poor and that cellular metabolism may rely on the O2 content of the middle ear cavities. This oxygen content, measured as the middle ear PO2, is physiologically near 50 mmHg (15, 18). In pathological conditions, the middle ear PO2 may further decrease because of eustachian tube dysfunction or vascular modifications, although available data are scarce and controversial (19, 24).
Our data demonstrate that hypoxia dramatically downregulates the epithelial Na+ absorption and thus the osmotically induced water flux from the apical to the basolateral side in the middle ear epithelium. As far as these in vitro experimental data can be extrapolated to the in vivo situation, the oxygen-induced modulation of Na+ absorption might account for 1) fluid excess in the course of secretory otitis media and 2) the beneficial effect of the ventilation tube in treating otitis media because of a reoxygenation-induced improvement in fluid clearance from the middle ear cavities. ![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Richard Boucher for kindly providing the
-hENaC subunit cDNA. We are also indebted to Christine Clerici and
Carole Planès for helpful discussion.
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FOOTNOTES |
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This work was supported by grants from Fondation pour la Recherche Médicale (France).
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: F. Portier, Hopital Lariboisiere, Service ORL 2, Rue Ambroise Paré, 75010 Paris, France.
Received 4 March 1998; accepted in final form 21 October 1998.
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