Oxygen modulates Na+ absorption in middle ear epithelium

F. Portier1, T. van den Abbeele1, E. Lecain1, E. Sauvaget1, B. Escoubet2, P. Tran Ba Huy1, and P. Herman1

1 Laboratoire d'Otologie Expérimentale, Faculté Lariboisière-St-Louis and 2 Department of Physiology, Institut National de la Santé et de la Recherche Médicale Unité 426, Faculté Xavier Bichat, Université Paris VII, 75010 Paris, France


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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 alpha -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.

secretory otitis media; ouabain; benzamil; ion transport; transepithelial sodium; short-circuit current; epithelial sodium channel; sodium-potasium-adenosine 5'-triphosphatase


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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 alpha -subunit of the epithelial Na+ channel (alpha -ENaC) was evidenced, which paralleled the decrease of Na+ absorption.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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, 10-6 M hydrocortisone, 10-7 M dexamethasone, 100 U/ml penicillin, 100 µg/ml streptomycin, 15 mM HEPES, and 2 mM L-glutamine.

For electrophysiological measurements, confluent MESV monolayers were trypsinized and plated (105 cells/cm2) onto tissue culture-treated polycarbonate filters (0.4-µm pore, 1 cm2; Snapwell, Costar, Cambridge, MA).

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 (10-6 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 (10-6 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 beta -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). beta -Actin expression was used as an internal standard, since the level of beta -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 alpha -hENaC probe was used (22). Antisense RNA probes were synthesized from the translated region of the alpha -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 beta -actin mRNA was used for standardization. alpha -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.


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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 Omega  · cm2 for control vs. 1,334 ± 141 Omega  · cm2 after 18-h hypoxia; not significant (NS); n = 10].

The Isc decrease paralleled that of Isc,Na: 1) Isc,Na decreased in a time-dependent manner from 1.71 ± 0.23 µA/cm2 in control (n = 12) to 0.64 ± 0.10 µA/cm2 (P < 0.01; n = 8) after 18-h hypoxia, and 2) the Isc and Isc,Na fit curves (1-phase exponential decay, R2 = 0.91 for Isc and R2 = 0.98 for Isc,Na) were parallel (Fig. 1).


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Time-response relationship for effect of hypoxia on short-circuit current (Isc) of MESV monolayers. Isc and fraction of Isc sensitive to apical 10-6 M benzamil (Isc,Na) were measured for normoxic cells [0 h; 150 mmHg PO2 assayed in culture medium (mPO2)] and after 1-, 3-, 6-, 12-, and 18-h hypoxia (32 mmHg mPO2). Recorded values were fitted by a 1-phase exponential decay: R2 = 0.91 for Isc; R2 = 0.98 for Isc,Na (Graph Pad Prism, Graph Pad Software, San Diego, CA). Values are means ± SE. * Significantly different from control (P < 0.05; n = 8-12).

The dose-effect relationship of this process was evaluated. Cells were incubated in various hypoxic conditions. Isc,Na decreased significantly with mPO2: 1.39 ± 0.14 µA/cm2 (n = 4) for control (mPO2 150 mmHg) and 1.45 ± 0.06 (NS; n = 3), 1.10 ± 0.07 (P < 0.05; n = 3), and 0.73 ± 0.07 µA/cm2 (P < 0.01; n = 3) for mPO2 of 100, 50, and 32 mmHg, respectively (Fig. 2).


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 2.   Dose-response relationship for effect of hypoxia on Isc,Na of MESV monolayers. MESV monolayers were incubated for 18 h in normoxic conditions (150 mmHg mPO2) or hypoxic conditions (100, 50, or 32 mmHg mPO2), and benzamil-sensitive Isc,Na (10-6 M benzamil) was subsequently measured. Values are means ± SE of Isc,Na (n = 3-4). * Significantly different from control (P < 0.05; n = 3-4). § Significantly different (P < 0.05; n = 3).

The hypoxia-induced decrease of Isc,Na in MESV cells was partly reversible. In 6- or 18-h hypoxic cells (mPO2 32 mmHg), Isc,Na was not significantly different from control after a 6-h reoxygenation (Fig. 3): 1.85 ± 0.17 µA/cm2 in control (n = 4), 1.15 ± 0.09 µA/cm2 in 6-h hypoxia (P < 0.05; n = 4), 1.47 ± 0.13 µA/cm2 in 6-h hypoxia and reoxygenation (NS; n = 3), 0.53 ± 0.07 µA/cm2 in 18-h hypoxia (P < 0.01; n = 3), and 1.37 ± 0.23 µA/cm2 in 18-h hypoxia and reoxygenation (NS; n = 3).


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 3.   Reversibility of hypoxia effect on Isc,Na. Isc,Na was measured in normoxic cells (150 mmHg mPO2; open bars), in 6- or 18-h hypoxic cells (32 mmHg mPO2; hatched bars), and in 6-h reoxygenated cells (solid bars). Values are means ± SE of Isc,Na (n = 3-4). * Significantly different from control (P < 0.05; n = 3-4). § Significantly different (P < 0.05; n = 3).

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.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Intracellular ATP content

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 (10-6 M) in the apical bath. Our results show that benzamil decreased ouabain-sensitive Rb+ influx in normoxic cells (91.71 ± 8.67 pg 86Rb+/filter; P < 0.01; n = 3). However, hypoxic and benzamil effects on ouabain-sensitive Rb+ influx were not additive, as shown in Fig. 4. In hypoxic cells, benzamil did not further decrease ouabain-sensitive Rb+ influx (47.73 ± 10.16 pg 86Rb+/filter with benzamil vs. 38.61 ± 1.06 pg 86Rb+/filter without benzamil; NS; n = 3).


View larger version (9K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of hypoxia on ouabain-sensitive 86Rb+ uptake (OsRb). Na+-K+-ATPase activity was determined as the 5 mM ouabain-sensitive 86Rb+ uptake in normoxic or 18-h hypoxic monolayers (32 mmHg mPO2) with or without benzamil (Bz; 10-6M) in apical bath during incubation period. * Significantly different from control (P < 0.05; n = 3).

Effect of hypoxia on alpha -ENaC subunit mRNA levels. RNase protection assays were performed to evaluate the expression of alpha -ENaC subunit mRNA transcripts in normoxic and hypoxic MESV cells. beta -Actin expression was used as an internal standard, since the level of beta -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 alpha -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 alpha -ENaC mRNA transcripts was not different from control (116.3 ± 12.46 AU; NS; n = 5-8; Fig. 5).


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5.   Modulation by hypoxia of expression of epithelial Na+ channel alpha -subunit (alpha -ENaC) mRNA transcripts. RNase protection assay was performed on cell lysis from normoxic (150 mmHg mPO2; CTL) and hypoxic (32 mmHg mPO2; Hypoxia) MESV monolayers. Latter were incubated for 18 h under hypoxic conditions, eventually followed by a 6-h reoxygenation (Reox.). Antisense RNA probe was synthesized from translated region of human alpha -ENaC. beta -Actin was used for standardization. * Significantly different from control (P < 0.05; n = 5-8). § Significantly different (P < 0.05; n = 8).


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

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).

In numerous cell types, decrease of PO2 induces irreversible damage (6, 13). However, hypoxia was also shown to induce specific nontoxic effects, such as modulation of ion transport in lung epithelial cells (31, 32) or cardiomyocytes (14). In MESV cells, the decrease in Na+ absorption was not related to irreversible cellular damage: 1) ultrastructural study did not reveal cellular modifications or damage, 2) continuous measurement of RT attested for the absence of leak in the monolayers, and 3) the hypoxia-induced effects on Isc,Na were reversible after reoxygenation.

Hypoxia promotes a decrease of the expression of transcripts encoding alpha -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 alpha -subunit (bp 1036-1259) of the hENaC (alpha -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 alpha -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 alpha -ENaC mRNA transcripts was not related to irreversible cellular damage, since 1) beta -actin mRNA transcripts were not modified by hypoxia and 2) after an 18-h hypoxia, reoxygenation allowed parallel Isc,Na and alpha -ENaC mRNA recovery. Hypoxia-induced decrease in Na+ absorption might also have been related to downregulation of beta - and gamma -subunits. However, it should be stated that the alpha -subunit of the Na+ channel exhibits, when expressed in oocytes, all the characteristics of the highly selective channel, whereas beta - and gamma -subunits only allow maximal activity of active Na+ channels (7, 36).

Hypoxia-induced Na+ absorption modulation may not only occur through alpha -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 alpha -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 alpha -hENaC subunit cDNA. We are also indebted to Christine Clerici and Carole Planès for helpful discussion.


    FOOTNOTES

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.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

1.   Al Bazzaz, F. J. Regulation of salt and water transport across airway mucosa. Clin. Chest Med. 7: 259-272, 1986[Medline].

2.   Arnould, T., C. Michiels, I. Alexandre, and J. Remacle. Effects of hypoxia upon intracellular calcium concentration of human endothelial cells. J. Cell. Physiol. 152: 215-221, 1992[Medline].

3.   Blot-Chabaud, M., F. Jaisser, J. P. Bonvalet, and N. Farman. Effect of cell sodium on Na+/K+-ATPase-dependent sodium efflux in cortical collecting tubule of rabbits under different aldosterone status. Biochim. Biophys. Acta 1022: 126-128, 1990[Medline].

4.   Boucher, R. C., M. J. Stutts, M. R. Knowles, L. Cantley, and J. T. Gatzy. Na+ transport rate in cystic fibrosis respiratory epithelia. Abnormal basal rate and response to adenylate cyclase activation. J. Clin. Invest. 78: 1245-1252, 1986[Medline].

5.   Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

6.   Bunn, H. F., and R. O. Poyton. Oxygen sensing and molecular adaptation to hypoxia. Physiol. Rev. 76: 839-885, 1996[Abstract/Free Full Text].

7.   Canessa, C. M., L. Schild, G. Buell, B. Thorons, and B. C. Rossier. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367: 463-467, 1994[Medline].

8.   Clerici, C., S. Couette, A. Loiseau, P. Herman, and C. Amiel. Evidence for Na-K-Cl cotransport in alveolar epithelial cells: effect of phorbol ester and osmotic stress. J. Membr. Biol. 147: 295-304, 1995[Medline].

9.   De Serres, L. M., M. R. Van Scott, H. C. Pillsbury, and J. Prazma. Bioelectric properties of gerbil middle ear epithelia. Arch. Otolaryngol. Head Neck Surg. 117: 416-421, 1991[Medline].

10.   Doctor, R. B., R. Bacallao, and L. J. Mandel. Method for recovering ATP content and mitochondrial function after chemical anoxia in renal cell cultures. Am. J. Physiol. 266 (Cell Physiol. 35): C1803-C1811, 1994[Abstract/Free Full Text].

11.   Doyle, W. J., J. T. Seroky, and C. M. Alper. Gas exchange across the middle ear mucosa in monkeys. Arch. Otolaryngol. Head Neck Surg. 121: 887-892, 1995[Medline].

12.   Furukawa, M., K. Ikeda, M. Yamaya, T. Oshima, H. Sasaki, and M. Takasaka. Effects of extracellular ATP on ion transport and [Ca2+]i in Mongolian gerbil middle ear epithelium. Am. J. Physiol. 272 (Cell Physiol. 41): C827-C836, 1997[Abstract/Free Full Text].

13.   Graven, K. K., L. H. Zimmerman, E. W. Dickson, G. L. Weinhouse, and H. W. Farber. Endothelial cell hypoxia associated proteins are cell- and stress- specific. J. Cell. Physiol. 157: 544-554, 1993[Medline].

14.   Grinwald, P. M. Sodium pump failure in hypoxia and reoxygenation. J. Mol. Cell. Cardiol. 24: 1393-1398, 1992[Medline].

15.   Grontved, A., A. Moller, and L. Jorgensen. Studies on gas tension in the normal middle ear. Acta Otolaryngol. (Stockh.) 109: 271-277, 1990[Medline].

16.   Hanley, M. J. Isolated nephron segments in a rabbit model of ischemic acute renal failure. Am. J. Physiol. 239 (Renal Fluid Electrolyte Physiol. 8): F17-F23, 1980[Medline].

17.   Harell, M., H. Moverley, D. Levy, and J. Sadé. Gas composition of the human nose and nasopharyngeal space. Acta Otolaryngol. (Stockh.) 116: 82-84, 1996[Medline].

18.   Hergils, L., and B. Magnuson. Human middle ear gas composition studied by mass spectrometry. Acta Otolaryngol. (Stockh.) 110: 92-99, 1990[Medline].

19.   Hergils, L., and B. Magnuson. Middle ear gas composition in pathologic conditions: mass spectrometry in otitis media with effusion and atelectasis. Ann. Otol. Rhinol. Laryngol. 106: 743-745, 1997[Medline].

20.   Herman, P., R. Cassingena, G. Friedlander, P. Soler, A. Grodet, P. Tran Ba Huy, and C. Amiel. Middle ear cell line that maintains vectorial electrolyte transport. J. Cell. Physiol. 154: 615-622, 1993[Medline].

21.   Herman, P., G. Friedlander, P. Tran Ba Huy, and C. Amiel. Ion transport by primary cultures of Mongolian gerbil middle ear epithelium. Am. J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F373-F380, 1992[Abstract/Free Full Text].

22.   Herman, P., C. T. Tan, T. Van Den Abbeele, B. Escoubet, G. Friedlander, and P. Tran Ba Huy. Glucocorticosteroids increase sodium transport in middle ear epithelium. Am. J. Physiol. 272 (Cell Physiol. 41): C184-C190, 1997[Abstract/Free Full Text].

23.   Herman, P., T. Y. Tu, A. Loiseau, C. Clerici, R. Cassingena, A. Grodet, G. Friedlander, C. Amiel, and P. Tran Ba Huy. Oxygen metabolites modulate sodium transport in gerbil middle ear epithelium: involvment of PGE2. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L390-L399, 1995[Abstract/Free Full Text].

24.   Ingelstedt, S., B. Jonson, and H. Rundcrantz. Gas tension and pH in middle ear effusion. Ann Otol. 84: 198-202, 1975.

25.   Jorgensen, P. L. Sodium and potassium ion pump in kidney tubules. Physiol. Rev. 60: 864-917, 1980[Free Full Text].

26.   Levy, D., M. Herman, M. Luntz, and J. Sadé. Direct demonstration of gas diffusion into the middle ear. Acta Otolaryngol. (Stockh.) 115: 276-278, 1995[Medline].

27.   Luntz, M., D. Levy, J. Sadé, and M. Herman. Relationship between the gas composition of the middle ear and the venous blood at steady state. Laryngoscope 105: 510-512, 1995[Medline].

28.   Mandel, L. J. Primary active sodium transport, oxygen consumption and ATP: coupling and regulation. Kidney Int. 29: 3-9, 1986[Medline].

29.   Mason, J., F. Beck, A Dörge, R. Rick, and K. Thurau. Intracellular electrolyte composition following renal ischemia. Kidney Int. 20: 61-70, 1981[Medline].

30.   Ostfeld, E. J., and A. Silberberg. Gas composition and pressure in the middle ear: a model for the physiological steady state. Laryngoscope 101: 297-304, 1991[Medline].

31.   Pitkänen, O., A. K. Tanswell, G. Downey, and H. Obrodovich. Increased PO2 alters the bioelectric properties of fetal distal lung epithelium. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L1060-L1066, 1996[Abstract/Free Full Text].

32.   Planès, C., B. Escoubet, M. Blot-Chabaud, and C. Clerici. Hypoxia reduces expression and activity of sodium channels sensitive to amiloride in cultivated pneumocytes II (Abstract). Arch. Physiol. Biochem. 104: D86, 1996.

33.   Planès, C., B. Escoubet, M. Blot-Chabaud, G. Friedlander, N. Farman, and C. Clerici. Hypoxia downregulates expression and activity of epithelial sodium channels in rat alveolar epithelial cells. Am. J. Respir. Cell Mol. Biol. 17: 508-518, 1997[Abstract/Free Full Text].

34.   Rossier, B. C., C. M. Canessa, L. Schild, and J. D. Horisberger. Epithelial sodium channels. Curr. Opin. Nephrol. Hypertens. 3: 487-496, 1994[Medline].

35.   Sadé, J., M. Luntz, and D. Levy. Middle ear gas composition and middle ear aeration. Ann. Otol. Rhinol. Laryngol. 104: 369-373, 1995[Medline].

36.   Voilley, N., E. Lingueglia, G. Champigny, M. G. Mattéi, R. Waldmann, M. Lazdunski, and P. Barbry. The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning. Proc. Natl. Acad. Sci. USA 91: 247-251, 1994[Abstract].


Am J Physiol Cell Physiol 276(2):C312-C317
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society