1 Institut National de la Santé et de la Recherche Médicale U-426 and 2 U-478, Faculté Xavier Bichat, 75870 Paris Cedex 18; and 3 Department of Oto-Rhino-Laryngology, Hôpital Beaujon, AP-HP, 92118 Clichy, France
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ABSTRACT |
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In the cochlea, endolymph is a K-rich and Na-poor fluid. The
purpose of the present study was to check the presence and to assess
the role of epithelial Na channel (ENaC) in this organ. -,
-, and
-ENaC subunit mRNA, and proteins were detected in rat cochlea by
RT-PCR and Western blot.
-ENaC subunit mRNA was localized by in situ
hybridization in both epithelial (stria vascularis, spiral prominence,
spiral limbus) and nonepithelial structures (spiral ligament, spiral
ganglion). The
-ENaC-positive tissues were also positive for
-subunit mRNA (except spiral ganglion) or for
-subunit mRNA
(spiral limbus, spiral ligament, and spiral ganglion), but the signals
of
- and
-subunits were weaker than those observed for
-subunit. In vivo, the endocochlear potential was recorded in guinea
pigs under normoxic and hypoxic conditions after endolymphatic
perfusion of ENaC inhibitors (amiloride, benzamil) dissolved either in
K-rich or Na-rich solutions. ENaC inhibitors altered the endocochlear
potential when Na-rich but not when K-rich solutions were perfused. In
conclusion, ENaC subunits are expressed in epithelial and nonepithelial
cochlear structures. One of its functions is probably to maintain the
low concentration of Na in endolymph.
endolymph; stria vascularis; in situ hybridization; endocochlear potential; hypoxia
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INTRODUCTION |
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IN THE COCHLEA, THE
MEMBRANOUS labyrinth is a complex sensorineural epithelium
composed of sensory hair cells and their supporting cells and of
various nonsensory epithelia, namely, the stria vascularis lined by the
connective tissue of the spiral ligament, the spiral prominence, the
outer sulcus cells, the spiral limbus, and Reissner's membrane (Fig.
1). The basolateral side of the
membranous labyrinth is bathed by perilymph, an extracellular fluid
whose chemical composition is similar to that of blood and
cerebrospinal fluid (35). Apically, the fluid that bathes
the hair bundles of sensory cells is called endolymph and has a unique
composition among mammalian extracellular fluids. It is K rich (170 mM), almost devoid of Na (1 mM), and hyperosmolar (30-40 mM over
blood osmolality) (35). The transepithelial potential
between perilymphatic and endolymphatic compartments is +100 mV,
endolymph positive. Homeostasis of the volume, pressure, and
electrochemical composition of endolymph is necessary to the
mechanoelectrical transduction, i.e., the transformation of sound into
nerve impulse through the apical transduction channels of the sensory
hair cells. Transport systems involved in the homeostasis of K
concentration in endolymph have been mainly elucidated. K is secreted
in endolymph by the marginal cells of the stria vascularis through an
apical IsK/KvLQT1 K channel coupled with basolateral
Na+-K+-ATPase and Na-K-Cl cotransport
(39). It is transported out of endolymph by hair cell
apical transduction channels and probably by outer sulcus cells'
apical nonselective cationic channels (23), and is then
probably recycled toward the stria vascularis through spiral ligament
fibroblasts (34, 37).
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At variance with K flux, the pathway of cochlear transepithelial Na flux remains poorly understood. The Na electrochemical gradient that takes place between endolymph and perilymph suggests that the membranous labyrinth is an Na-reabsorbing epithelium. Koefoed-Johnsen and Ussing (19) showed that Na is reabsorbed through high-resistance epithelia by functional coupling between passive Na diffusion through the apical membrane and active extrusion of intracellular Na by basolateral Na+,K+-ATPase. In the cochlea, basolateral Na+,K+-ATPase has been detected in several epithelial cells such as stria vascularis marginal cells (35) and outer sulcus cells (27). The apical transport system responsible for Na reabsorption from endolymph remains unknown. A good candidate would be the epithelial Na channel, ENaC. Indeed, this channel has been shown to participate to transepithelial Na transport in many epithelia such as kidney, colon, lung, sweat glands, and salivary glands (2, 13, 15).
The purpose of the present study was to detect the presence of -,
-, and
-ENaC subunit mRNA and proteins in the rat cochlea by
RT-PCR and Western blot and to localize ENaC mRNA within the rat
cochlea by in situ hybridization. The function of cochlear ENaC was
investigated in vivo in guinea pigs by assessing the effect of locally
applied ENaC inhibitors, amiloride and benzamil, on the endocochlear potential.
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METHODS |
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The procedures concerning animals reported in the present work were approved by Ministère de l'Agriculture et de la Forêt (approval no. 5521).
RT-PCR
RNA extraction from rat cochlea.
Two Long-Evans rats (Janvier, Le Genest, France) were killed with a
lethal injection of pentobarbital sodium (100 mg/kg ip) and quickly
decapitated. The four temporal bones were rapidly removed, and the
cochlear soft tissues were microdissected in a 4°C PBS [Dulbecco's,
without calcium, magnesium, and sodium bicarbonate, (GIBCO-BRL, Life
Technologies, Gaithersburg, MD)]. Microdissected tissues were pooled
and transferred in 100 µl of lysis buffer (4 M guanidium thiocyanate,
25 mM sodium citrate, pH 7.0, 0.5% N-laurylsarcosine, 0.1 M
-mercaptoethanol) and mechanically minced. Total mRNA was extracted
with phenol water and isoamyl alcohol-chloroform (1:24, vol/vol) and
precipitated by isopropanol (8). The pellet was
resuspended in 20 µl of water. Genomic DNA was eliminated by adding 4 µl of 5× transcription buffer (GIBCO-BRL), 0.5 µl (10 U/µl) of
RNase-free DNase I (Amersham Pharmacia Biotech, Piscataway, NJ), and 1 µl of RNasine (40 U/µl; Promega, Madison, WI). This solution was
heated for 60 min at 37°C. An ethanol precipitation was then
performed, and the pellet was dried and resuspended in 20 µl of water.
Reverse transcription of RNA extracted from rat cochlea. cDNA was synthetized by reverse transcription of RNA in solution (2 µl RNA solution extracted from rat cochlea) with 400 U Moloney murine leukemia virus RT (200 U/µl; GIBCO-BRL) in a buffer containing (in mM) 250 Tris · HCl, pH 8.3, 275 KCl, 15 MgCl2, and 1 D-nucleotide triphosphate (final volume: 40 µl). To verify the possibility of genomic DNA amplification during the PCR reaction, RT negative controls were performed by omitting the RT in the solution.
PCR.
The main characteristics of the primers used to amplify the cDNA of the
-,
-, and
-ENaC subunits are specified in Table 1.
- and
-ENaC primers, but not
-primers, amplified cDNA fragments whose corresponding genomic DNA
encompassed at least one intron. The cDNA segments were amplified from
10 µl of the RT product or of the RT-negative control in a PCR buffer
(in mM: 20 Tris · HCl, 50 KCl, 1.5 MgCl2) and in
the presence of 0.2 mM of dNTP and 1 U of Taq polymerase at
5 U/µl (GIBCO-BRL). Each PCR cycle comprised denaturation at 94°C
for 30 s, annealing during 30 s at variable temperatures
(Table 1), and elongation at 72°C for 1 min. Forty cycles were used.
The reaction was stopped by a temperature decrease to 4°C. A 10-µl
sample of the PCR product was run on a 1% agarose gel with ethidium
bromide. A 100-bp molecular weight (MW) ladder permitted the
verification of the length of the amplified fragment. The whole
procedure was repeated in three series of two animals.
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In Situ Hybridization
The temporal bones of Long-Evans rats were rapidly removed (see above) and immersed in a solution of 4% paraformaldehyde (PFA) in PBS (0.1 M, pH 7.4). The cochleas were dissected in PFA, and the round and oval windows were opened to facilitate PFA entry in the cochlea. Cochleas were fixed in PFA for 24 h, decalcified during 21 days in 8% EDTA, and paraffin embedded. In situ hybridization was performed on 7-µm sections (Leitz) with radiolabeled riboprobes. To prepare riboprobes, part of the 3'-untranslated region to theWestern Blot
Western blot was performed on 14 cochlear soft tissues microdissected from 7 Long-Evans rats. Cochlear proteins homogenates were extracted from a 200-µl solution containing (in mM) 50 Tris · HCl, pH 7.5, 150 NaCl, 1% Triton X-100, and 5 EDTA.SDS-PAGE was performed with solubilized membranes (15-63 µg of proteins) and prestained MW markers (Sigma), on 7% polyacrylamide minigels (Mini PROTEAN II, Bio-Rad, Hercules, CA). Proteins were subsequently transferred from the gels electrophoretically to nitrocellulose membranes (Mini Trans-Blot Module, Bio-Rad). Transfer efficiency was checked by Ponceau red staining of the nitrocellulose membranes.
To detect -,
-, and
-ENaC subunits, rabbit polyclonal
antibodies, whose specificity had been previously tested
(9), were used. For generation of these antibodies, fusion
proteins from the NH2-terminal end of rat
-ENaC (amino
acids E10-F77), from the COOH-terminal end of rat
-ENaC (amino
acids G559-E636), and from the COOH-terminal end of rat
-ENaC (amino
acids A570-L650) were used (9, 10). Nitrocellulose
membranes were blocked for 45 min at ambient temperature in 5% nonfat
milk powder/Tris · HCl-buffered saline (TBS-T, 150 mM NaCl, 10 mM Tris · HCl, pH 8.5, 0.1% Tween 20) and exposed overnight at
4°C to anti-
-,
-, and
-ENaC subunit antibodies diluted
1:1,000 in TBS-T. After washing in TBS-T, the membranes were exposed
for 1 h at room temperature to the second antibody
(peroxidase-labeled anti-rabbit Ig antibody, Amersham Life Sciences)
diluted 1:7,500 in TBS-T. After washing in TBS-T, antigen-antibody
complexes were detected by autoradiography by using enhanced
chemiluminescence (ECL Western blotting analysis system, Amersham).
In Vivo Experiments
Adult male pigmented guinea pigs (300-400 g body wt, Elevage d'Ardenay, France) were anesthetized with intramuscular injection of 2 ml/kg body wt of a mixture of ketamine (50 mg/ml, Panpharma, Fougères, France) and xylazine (2%, Rompun, Bayer, Leverkusen, Germany; 2:1, vol/vol). A heating table maintained the temperature between 36.5 and 37.5°C. The animal's head was maintained in a ventral position by a head holder.The bulla was opened through a lateral approach, and the cochlea was exposed. Glass micropipettes were bevelled to a tip diameter of 10-15 µm. The micropipette was inserted in a straight holder containing an Ag-AgCl pellet and endowed with a perfusion port (PC-S3 30° holder, Phymep, Paris, France). The holder was connected to an electrometer amplifier (604 differential electrometer, Keithley Instruments, Cleveland, OH). The perfusion port was coupled to a syringe with pump (341A, Sage Instrument, Cambridge, MA) with a polyethylene tube (external diameter: 0.96 mm; internal diameter: 0.58 mm; Polylabo, Strasbourg, France). The micropipette, holder, and polyethylene tube were filled with Na-rich (in mM: 125 NaCl, 25 NaHCO3, 25 mannitol) or K-rich (in mM: 126 KCl, 1 NaCl, 25 KHCO3, 25 mannitol) solutions. Immediately before injection, solutions were warmed to 37°C and gassed with 95% O2 and 5% CO2 so that their final pH was within the physiological range (7.35-7.45). An Ag-AgCl reference electrode was placed on the neck muscles. The micropipette was handled with a micromanipulator under stereomicroscopic observation. The tip of the micropipette was moved to the perilymph of the scala tympani through the round window and then to the endolymph of the scala media of the first cochlear turn through the basilar membrane (Fig. 1). The zero potential was determined in the perilymph of the scala tympani. Endocochlear potential of the first cochlear turn was recorded before, and 30 s, 1 min, 2 min, 3 min, and 15 min after the beginning of endolymphatic perfusion (100 nl/min for 3 min).
Normoxia experiments.
During these experiments, animals breathed spontaneously. Five
experimental series of perfusions of the scala media were performed: 1) K-rich solution (n = 11); 2)
K-rich solution with 104 M amiloride (Sigma, St. Louis,
MO) (n = 11); 3) Na-rich solution (n = 7); 4) Na-rich solution with
10
4 M amiloride (n = 5); and
5) Na-rich solution with 10
6 M benzamil
(n = 4; Fluka, Buchs, Switzerland).
Hypoxia experiments.
Animals were tracheotomized and artificially ventilated with a Harvard
volumetric ventilator for small rodents. Tidal volume and ventilation
frequency were 2 ml and 60 beats per min, respectively. One minute
after the beginning of the 3-min endolymphatic perfusion, a 3-min
hypoxia was induced by ventilating the animals with pure N2. Evolution of the endocochlear potential was compared in
two experimental conditions of K-rich solution perfusion in absence (n = 6) or presence of 106 M
benzamil (n = 6).
Statistics
Results are expressed as means ± SE. Statistical significance between experimental groups was assessed by Student's paired or unpaired t-test, with P < 0.05 considered significant. ![]() |
RESULTS |
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RT-PCR
mRNA encoding
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In Situ Hybridization
Negative controls with sense probes showed quite a high background signal because of the long exposition in the photoemulsion (6 wk) (Fig. 3). With
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Western Blot
The three ENaC subunit proteins were detected by Western blot in cochlear soft tissues with similar MW to the ones previously reported in mouse and rat kidney cortical collecting duct cell lines (Fig. 4) (3, 9).
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In Vivo Experiments
Initial endocochlear potential and mechanical effect of
endolymphatic perfusion.
Initial endocochlear potential values of the different experimental
groups were not statistically different (one-way ANOVA, P = 0.15). Nevertheless, these values were about +8 mV
higher before K-rich solution perfusion (+84 ± 2.7 mV,
n = 11, and +83 ± 3.8 mV, n = 11, in control and amiloride groups, respectively; Fig.
5) than before Na-rich solution perfusion
(+75 ± 2.9 mV, n = 7, +73 ± 4.9 mV,
n = 5, and +80 ± 2.3 mV, n = 4, in control, amiloride, and benzamil groups, respectively; Fig.
6) or before K-rich solution perfusion in
artificially ventilated animals (+74 ± 5.0 mV, n = 6, and +75 ± 3.5 mV, n = 6, in control and
benzamil groups, respectively; Fig. 7).
Values obtained before K-rich solution perfusion under normoxic
conditions were similar to the ones usually reported at the first turn
of guinea pig cochlea (35). In the two other groups, the
relatively low endocochlear potential values could have resulted from a
small Na leak out of the microelectrode into endolymph before the
beginning of the Na-rich perfusion, and from a small hypoxia- and/or
stress-related hormone release related to tracheotomy and artificial
ventilation before K-rich solution perfusion under hypoxic conditions.
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Lack of effect of ENaC inhibitors under normoxic conditions. When K-rich solution was perfused, endocochlear potential was not altered at 15 min compared with the beginning of the experiment (Fig. 5) (P > 0.05, paired Student's t-test). Amiloride did not modify the evolution of the EP under this experimental condition (Fig. 5).
When Na-rich solution was perfused, the endocochlear potential value progressively fell after the end of the perfusion, reaching 34% ± 3.9 (n = 7) of its initial value 15 min after the beginning of the perfusion. Amiloride and benzamil, in Na-rich solution, rapidly increased the endocochlear potential as soon as 30 s, and up to 3 min, and 15 min after the beginning of the perfusion, respectively (Fig. 6). This rapid effect suggests that ENaC was functionally active at the apical membrane of epithelial cells facing endolymph.Effect of ENaC inhibitors under hypoxic conditions. As previously reported (5), hypoxia induced a rapid endocochlear potential decrease that was maximal at the end of hypoxia, i.e., 4 min after the beginning of the experiment (Fig. 7). At that time, endocochlear potential was 15% ± 6.0 (n = 6) of the initial value (endocochlear potential at 1 min: +79 ± 6.2 mV; endocochlear potential at 4 min: +10 ± 4.5 mV, n = 6). The addition of benzamil in the perfused K-rich solution did not modify the endocochlear potential during hypoxia (19% ± 7.0 of the initial value at 4 min, n = 6, Fig. 7).
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DISCUSSION |
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The purpose of the present study was to check the presence and to
assess the role of ENaC in the cochlea. ENaC -,
-, and
-subunits were detected in rat cochlea both at the mRNA (RT-PCR) and
protein (Western blot) levels. ENaC mRNA was localized by in situ
hybridization in several epithelial (stria vascularis, spiral
prominence, spiral limbus) and nonepithelial (spiral ligament, spiral
ganglion) structures of rat cochlea. This Na channel may be involved in
the maintenance of the Na chemical gradient between Na-poor endolymph
(1 mM) and Na-rich perilymph (140 mM). Thus the in vivo function of
this channel likely is to contribute to Na transport out of endolymph
to maintain a very low endolymphatic Na concentration.
Presence of ENaC in Epithelial and Nonepithelial Rat Cochlear Tissues
ENaC mRNA was localized by in situ hybridization in several epithelial and nonepithelial tissues. The labeled epithelial structures, stria vascularis, spiral prominence, and spiral limbus, border endolymph. In these epithelia, ENaC is probably involved in Na transepithelial vectorial transport resulting in Na extrusion from endolymph.ENaC mRNA was also localized in two nonepithelial cochlear tissues, the spiral ligament, and the spiral ganglion. This channel has been found in several nonepithelial cells such as keratinocytes (6), lymphocytes (28), eye endothelial cells, fibroblasts, and neurons (photoreceptors, bipolar cells, ganglia) (26). The functions of ENaC in nonepithelial cells probably depend on the cell type and remain to be elucidated. In spiral ligament fibrocytes, ENaC may be more specifically involved in vectorial ion transports. This structure has been proposed to be involved in the recycling of K from the organ of Corti to the stria vascularis (4, 32, 34). As a matter of fact, these cells are endowed with tight junctions and with several Na and K transport systems (Na-K-Cl cotransport, Na+,K+-ATPase, Na+/H+ exchanger). ENaC might regulate the K recycling pathway by modifying the resting potential and intracellular Na concentration of spiral ligament fibrocytes.
The observation of positive signals for ENaC subunits mRNA in the cochlear spiral ganglion is in accordance with recent works in which ENaC was detected in nervous structures such as eye photoreceptors, bipolar cells, and ganglia (26). The role of ENaC in these structures is unclear. As reported for Phe-Met-Arg-Phe-amide-activated Na channel, another member of the degenerin/ENaC superfamily (22), ENaC may be functionally coupled to neurotransmission processes.
Concerning the stochiometry of ENaC subunits, the presence of the three
subunits of ENaC in the rat cochlea was attested to at both the mRNA
and protein levels by RT-PCR and Western blot, respectively. In situ
hybridization clearly showed that the -subunit mRNA was expressed in
several epithelial and nonepithelial structures of the cochlea.
However, because of the low signal intensity obtained with
- and
-subunit mRNA-specific probes in the present study, further work is
needed to assess the exact distribution of
- or
-subunits of ENaC
in the cochlea. Additional information concerning the topography of
ENaC-subunits could be brought at the protein level by
immunohistochemistry. Note that this does not preclude the existence of
a functional Na channel in those tissues because the expression of
-subunit in Xenopus laevis oocytes was sufficient to give
rise to an amiloride-sensitive Na current, and the addition of
- or
-subunits increased the activity of the Na channel (7).
Role of ENaC in the Regulation of Na Endolymph Concentration
The Na electrochemical gradient that exists between Na-poor endolymph and Na-rich perilymph in physiological conditions indicates that active Na absorption from endolymph is achieved by epithelia lining these compartments. The observation of ENaC mRNA in some of these epithelia, namely the stria vascularis, spiral prominence, and spiral limbus, suggests that ENaC probably contributes to this active Na resorption. Other apical Na transport systems such as nonselective cationic channels in outer sulcus cells (23), apical transduction channels in hair cells (25), or NHE3 isoform of the Na+/H+ exchanger in marginal cells (4) may also contribute to Na extrusion from endolymph. The lack of effect of amiloride (present study, Fig. 5; 33) and benzamil (Fig. 7) on the endocochlear potential in K-rich endolymph suggests that ENaC activity is not a major determinant of the endocochlear potential. When the scala media was perfused with a Na-rich solution, ENaC activity became, as expected, a determinant of the endocochlear potential, as shown by the endocochlear potential increase induced by the luminal application of amiloride and benzamil (Fig. 6). This result confirms that ENaC is located in the apical membrane of epithelia facing endolymph, and is consistent with previous observations (12, 38). Indeed, recording of short circuit current in stria vascularis marginal cell line mounted into Ussing chamber showed an amiloride-sensitive transepithelial Na flux (38). Similarly, a transepithelial Na flux inhibited by amiloride has also been observed in in vitro preparations of frog semicircular canal ampulla (12), an inner ear structure in which epithelial transport systems are quite similar to those observed in the cochlea (39).It must be emphasized that the effects of amiloride and benzamil in
Na-rich solutions cannot be due to the inhibition of hair cell
nonselective cationic transduction channels (31), because no endocochlear potential increase was observed when amiloride was
administered in K-rich solutions (present study, 33). Neither did the
endocochlear potential increase result from the inhibition of apical
Na+/H+ exchanger, because this electroneutral
antiporter is not inhibited by benzamil at the low concentration used
in the present study (final concentration < 106 M)
(18). Thus the endocochlear potential increase induced by amiloride and benzamil during Na-rich perfusions presumably resulted from the inhibition of Na reabsorption from endolymph through apical ENaC.
Physiologically, endolymph is a Na-rich fluid during the fetal period. During the ultimate phase of inner ear functional maturation, i.e., at the end of gestation for the guinea pig, or during the first week of life in mice and rats, endolymph becomes a Na-poor and K-rich fluid (1, 30). ENaC could be involved in the clearance of endolymphatic Na during this shift in the composition of endolymph. This role of ENaC in the inner ear can be parallelled with the role of ENaC in lung maturation, during which ENaC participates in the clearance of the fluid that fills newborn airways (16). When the K-rich and Na-poor composition of endolymph is settled, the function of ENaC remains necessary to clear the Na that reaches the endolymphatic compartment by paracellular passive leak.
In pathophysiology, an increase in endolymphatic Na concentration has been described during anoxia, from 3-6 to 32 mM, after 2- and 32-min anoxia, respectively (5). In the present study, administration of benzamil in the scala media did not alter the evolution of the endocochlear potential during and after a 3-min period of hypoxia (Fig. 6). This may be due to the fact that the modification of Na concentration (approximately or equal to +6 mM) induced by this short period of hypoxia was insufficient to significantly influence the endocochlear potential. Endolymphatic Na concentration may also be increased during endolymphatic hydrops, which is an increase in endolymph volume. Endolymphatic hydrops constitutes the anatomic substratum of Menière's disease, a syndrome characterized by attacks of vertigo, sensorineural hearing loss, and tinnitus. Attacks of vertigo during this pathology have been ascribed to the sudden disruption of distended Reissner's membrane (25) with subsequent contamination of endolymph by Na. In this pathology, ENaC may participate in the clearance of Na out of endolymph.
In conclusion, all three ENaC subunit mRNAs and proteins were detected in rat cochlea. ENaC mRNA was localized by in situ hybridization in both epithelial and nonepithelial cochlear structures. The rapid endocochlear potential increase induced by endolymphatic perfusion of ENaC inhibitors, amiloride and benzamil, dissolved in Na-rich solution, indicates that ENaC is functionally active in the apical membrane of epithelia facing endolymph, stria vascularis, spiral prominence, and spiral limbus. This strongly suggests that ENaC transport activity contributes to maintenance of a very low Na concentration in endolymph.
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ACKNOWLEDGEMENTS |
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The authors would like to thank Marcel Blot-Chabaud for kind encouragement and helpful advice. They are grateful to Maurice Bichara for critically reading the manuscript and for constructive comments.
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FOOTNOTES |
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This work was supported by grants from Faculté Xavier Bichat, Université Paris 7, Institut National de la Santé et de la Recherche Médicale, and Association de Recherches Physiologiques.
Address for reprint requests and other correspondence: V. Couloigner, INSERM - Unité de recherche U426, UFR Xavier Bichat, 16, rue Henri Huchard BP416, 75870 Paris Cedex 18 France (E-mail: Vincent.Couloigner{at}wanadoo.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12 May 2000; accepted in final form 20 September 2000.
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