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RECEPTORS AND SIGNAL TRANSDUCTION

Gastric type H⁺,K⁺-ATPase in the cochlear lateral wall is critically involved in formation of the endocochlear potential

¹Division of Molecular and Cellular Pharmacology, Department of Pharmacology, and ²Department of Otolaryngology, Graduate School of Medicine, Osaka University, Suita, Osaka; and ³Department of Otolaryngology, Kyoto Prefectural University of Medicine, Kyoto, Japan

Submitted 13 May 2006 ; accepted in final form 25 June 2006

	ABSTRACT

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Cochlear endolymph has a highly positive potential of approximately +80 mV known as the endocochlear potential (EP). The EP is essential for hearing and is maintained by K⁺ circulation from perilymph to endolymph through the cochlear lateral wall. Various K⁺ transport apparatuses such as the Na⁺,K⁺-ATPase, the Na⁺-K⁺-2Cl^– cotransporter, and the K⁺ channels Kir4.1 and KCNQ1/KCNE1 are expressed in the lateral wall and are known to play indispensable roles in cochlear K⁺ circulation. The gastric type of the H⁺,K⁺-ATPase was also shown to be expressed in the cochlear lateral wall (Lecain E, Robert JC, Thomas A, and Tran Ba Huy P. Hear Res 149: 147–154, 2000), but its functional role has not been well studied. In this study we examined the precise localization of H⁺,K⁺-ATPase in the cochlea and its involvement in formation of EP. RT-PCR analysis showed that the cochlea expressed mRNAs of gastric ₁-, but not colonic ₂-, and -subunits of H⁺,K⁺-ATPase. Immunolabeling of an antibody specific to the ₁ subunit was detected in type II, IV, and V fibrocytes distributed in the spiral ligament of the lateral wall and in the spiral limbus. Strong immunoreactivity was also found in the stria vascularis. Immunoelectron microscopic examination exhibited that the H⁺,K⁺-ATPase was localized exclusively at the basolateral site of strial marginal cells. Application of Sch-28080, a specific inhibitor of gastric H⁺,K⁺-ATPase, to the spiral ligament as well as to the stria vascularis caused prominent reduction of EP. These results may imply that the H⁺,K⁺-ATPase in the cochlear lateral wall is crucial for K⁺ circulation and thus plays a critical role in generation of EP.

hydrogen, potassium-adenosine triphosphatase; stria vascularis; spiral ligament

It is assumed that K⁺ circulation from perilymph to endolymph through the cochlear lateral wall is essential to establish the high K⁺ concentration ([K⁺]) in endolymph and EP (90). The lateral wall is divided into two components, 1) the spiral ligament, a connective tissue comprising several types of fibrocytes, and 2) the stria vascularis, an epithelial tissue containing marginal, intermediate, and basal cells. K⁺ secreted from the hair cells is transported to the endolymphatic space via the epithelium on the basilar membrane, the spiral ligament, and the stria vascularis. Whereas most of this K⁺ transport pathway is connected with gap junctions (10, 13, 35, 79), the steps between the hair cells and the epithelium, between the epithelium and the spiral ligament, and between the basal cells and the marginal cells in the stria vascularis are not. At each step, K⁺ should be secreted from the former and absorbed by the latter. Finally, the marginal cells in the stria vascularis unidirectionally carry K⁺ from the intrastrial space to the endolymphatic space, the scala media (37, 81, 83). Several K⁺ transport apparatuses, including Na⁺,K⁺-ATPase, Na⁺-K⁺-2Cl^– cotransporter (NKCC1), and K⁺ channels (KCNQ1/KCNE1 and Kir4.1), have been identified expressed in the spiral ligament and/or stria vascularis. They are thought to play pivotal roles in cochlear K⁺ circulation, because application of specific inhibitors for each apparatus reduced EP (24, 43, 47, 48) and targeted ablation of the genes for NKCC1 and the K⁺ channels collapsed scala media (54, 81, 84). The K⁺ channel Kir5.1, expressed in the fibrocytes in the lower part of the ligament, may negatively regulate K⁺ circulation and may be important for precise control of K⁺ flow (23).

It was recently reported that the gastric type H⁺,K⁺-ATPase is expressed in the cochlear lateral wall (49), but its details have not been studied. In this study, we have examined the precise cellular and subcellular localization of the H⁺,K⁺-ATPase in the cochlea and its functional role in EP formation. Using RT-PCR analysis, we found the expression of gastric ₁- and accessory -subunits, but not of the colonic ₂-subunit, in cochlea. The ₁-subunit was specifically localized in particular types of fibrocytes in the lateral wall and the limbus, as well as at the basolateral site of marginal cells. Both perilymphatic and vascular perfusion of Sch-28080, a specific inhibitor of the gastric H⁺,K⁺-ATPase, suppressed EP. The unique distribution pattern and the pharmacological observations strongly suggest that the gastric-type H⁺,K⁺-ATPase plays a crucial role in cochlear K⁺ circulation and thus in generation of the EP.

	MATERIALS AND METHODS

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Animals. The experimental protocol of this study was carefully reviewed and approved by the Animal Research Committee of Osaka University Medical School. All the experiments were carried out under the supervision of the committee and in accordance with Guidelines for Animal Experiments of Osaka University and the Japanese Animal Protection and Management Law. The animals were fed and allowed water freely until they were used for the experiments.

RT-PCR. Adult male C57 Black6 mice (8–10 wk old) were deeply anesthetized with pentobarbital sodium (50 mg/kg). Total RNA was extracted from their whole cochlea, kidney, and distal colon with TRIzol reagent in accordance with the manufacturer's protocol (Invitrogen, Carlsbad, CA). DNase I was added to each 5 µg of RNA, and the mixture was incubated for 15 min at room temperature. After incubation, the mixture was treated with EDTA for 10 min at 65°C and then with proteinase K for 10 min at 65°C. RNA was purified with phenol and chloroform and then precipitated with ethanol. The RNA obtained was used to synthesize cDNA with oligo(dT) primers, one-twentieth of which was used for one PCR reaction. The DNA amplification was performed in a final volume of 30 µl. PCR cycling conditions were 94°C for 2 min, followed by 30 cycles of 94°C for 15 s, 57°C for 30 s, and 72°C for 45 s, with a final step of 72°C for 7 min. Amplified product (15 µl) was separated using electrophoresis in a 2% agarose gel and visualized with ethidium bromide. We analyzed transcripts of ₁-, ₂-, and -subunits of mouse H⁺,K⁺-ATPase by primer pairs as following: the upstream primer 1F (5'-CTTCAGGAACAAGATCCTGGTGA-3') and the downstream primer 1R (5'-GAAGGATAGATTCCCTCCAATGG-3') for ₁, 2F (5'-CTTTGTTGCCATCATGGTCC-3') and 2R (5'-GTATGCTTCACACAGTTTTC-3') for ₂, and 3F (5'-CTTCAACAACCCCCATGACCC-3') and 3R (5'-AGGACGGGCAAATGATCACAG-3') for .

Immunohistochemistry. All of the cochlear samples for immunohistochemistry were prepared as described previously (24). Viable dominant spotting (W^v/W^v) mice were purchased from SLC (Hamamatsu, Japan). Briefly, deeply anesthetized mice were perfused from their left ventricle with 4% paraformaldehyde-0.1 M sodium phosphate, pH 7.4, and cochleas were isolated. The cochleas were decalcified in EDTA solution (0.12% EDTA in PBS, pH 7.4) for 5 days.

An affinity-purified rabbit polyclonal antibody against gastric H⁺,K⁺-ATPase ₁-subunit was kindly provided by Dr. Noriaki Takeguchi (Toyama Medical and Pharmaceutical University) (2). The immunohistochemistry was performed as reported previously (26, 32). The cochlear cryosections (12 µm) were incubated with anti-₁-subunit antibody (1:2,000 dilution) and fluorescein isothiocyanate (FITC)-labeled anti-rabbit antibody. Nuclei were detected using anti-heterochromatin 1 (HP1) antibody (Chemicon, Temecula, CA) with Texas red (TXR)-labeled secondary antibody. The samples were examined under a confocal microscope (LSM 510; Carl Zeiss, Jena, Germany). We analyzed the expression of the ₁-subunit in cochlea of adult male rats and guinea pigs using the same procedure.

Isolation of cells from stria vascularis and cellular immunolabeling. The temporal bones of anesthetized mice were removed, and the stria vascularis was dissociated. Tissue strips of stria vascularis were incubated for 30 min at 25°C in a normal Tyrode solution (136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.53 mM MgCl₂, 5.5 mM glucose, and 5.0 mM HEPES-NaOH buffer, pH 7.4) containing 0.2% trypsin and then disrupted mechanically with pipetting (82).

Anti-barttin antibody, which was raised in chicken and affinity purified, was kindly provided by Drs. Shinichi Uchida and Sei Sasaki (Tokyo Medical and Dental University). Immunolabeling was performed using the same protocol as the slice immunohistochemistry described in Immunohistochemistry. The isolated cells were treated with anti-barttin (1:100 dilution), anti-₁-subunit (1:2,000 dilution), and anti-Kir4.1 (1:2,000 dilution) antibodies and then visualized with FITC- or TXR-labeled secondary antibodies (see Fig. 3).

View larger version (96K): [in this window] [in a new window]   Fig. 1. RT-PCR analysis of H+,K+-ATPase subunits in mouse cochlea. Sets of primers specific for 1-, 2-, and -subunits of H+,K+-ATPase were used for PCR reactions as indicated. Total RNAs obtained from kidney, colon, and cochlea were treated with (lanes 1 and 3) or without (lanes 2 and 4) RT and then amplified with the specific primers. PCR products were stained with ethidium bromide.

View larger version (109K): [in this window] [in a new window]   Fig. 2. Expression and distribution of H+,K+-ATPase 1-subunit in mouse cochlea. Mouse cochlear sections were treated with an antibody specific to the 1-subunit, and the immunoreactivity was visualized using FITC-conjugated secondary antibody. Images were obtained with a confocal microscope. A: low-magnification image. The 1-subunit (green) was expressed in stria vascularis (StV), the lower portion of spiral ligament containing types II and IV fibrocytes, the suprastrial zone (SSZ) comprising type V fibrocytes, and spiral limbus (Lim). Moderate labeling also was detected in spiral ganglions (SG). Note that no signal was detectable in outer sulcus cells (OSC) or in the region of type I and III fibrocytes (arrowheads). SV, scala vestibuli; SM, scala media; ST, scala tympani; TM, tectrial membrane. Roman numerals indicate fibrocyte types. B–D: higher magnification images from different regions of the cochlea. In the lower part of the ligament (B), OSC and endothelial cells of blood vessels (V) were found to be free from 1 labeling, indicating that the subunit was certainly expressed in the fibrocytes. The outline of the OSC is indicated by the white line (B). In stria vascularis, 1 immunoreactivity was observed in the middle part, but at neither the apical side of marginal cells (arrows) nor in the basal cell (BC) layer (C, arrowheads). Moderate labeling also was visible in the cytosol of ganglion neurons (GC) but not in satellite cells (SC) (D). These sections also were stained with anti-heterochromatin 1 antibody (red) to visualize the nuclei (B–D) (25).

View larger version (40K): [in this window] [in a new window]   Fig. 3. Cellular distribution of the H+,K+-ATPase 1-subunit. Cells were dissociated from stria vascularis of C57 Black6 mice, and immunolabeling was performed as described in MATERIALS AND METHODS. A: a marginal cell (MC). Double immunolabeling was performed with anti-1-subunit of H+,K+-ATPase and anti-barttin antibodies. The positive signals were visualized using Texas R (TXR; red)- and FITC (green)-conjugated secondary antibodies, respectively. A MC expressing barttin (green) (15) was 1 immunopositive. The transmission illuminated images (Trans) show that the MC has a flat face on one side (arrows) and infoldings on the other side (arrowheads), which correspond to its apical and basolateral membranes, respectively (72). B: an intermediate cell (IC). Note that the cell is back-illuminated because of abundant pigments. Double labeling of 1-subunit and barttin (top) and the single labeling of Kir4.1 (bottom) were performed with their specific antibodies. The intermediate cell was free from both 1 and barttin immunoreactivity. Anti-Kir4.1 antibody (red) intensely labeled the cell, which ensures that it is an IC.

Measurement of EP. Albino guinea pigs weighing 250–300 g were treated with pentobarbital sodium (50 mg/kg im) and vecuronium bromide (1.0 mg/kg im) and artificially ventilated with room air. The cochlea was exposed using a ventrolateral approach. We inserted a glass microelectrode filled with 150 mM KCl solution into the scala media of the second turn through the spiral ligament. EP was measured with an electrometer amplifier (FD223; WPI, Sarasota, FL). An Ag-AgCl wire placed in the thigh muscle served as reference. The recording system was zeroed with the electrode in the perilymph of spiral ligament before insertion into the scala media. Vascular perfusion of the stria vascularis was performed at a rate of 1.5 ml/min until the reaction became a plateau (8–10 min) through a polyethylene tube located in the vertebral artery at the same side of the cochlea. Perilymphatic perfusion of the scala tympani was performed at a rate of 10 µl/min through a perfusion pipette connected to a syringe pump (UltraMicroPump II; WPI). The tip of the perfusion pipette was inserted into a small fenestration that was made in the bony wall of the scala tympani in the basal turn. The outlet for the perfusate was made at the top of the first turn. The artificial perilymph contained 126.0 mM NaCl, 5.0 mM KCl, 1.2 mM CaCl₂, 1.0 mM MgCl₂, 24 mM NaHCO₃, 0.5 mM NaH₂PO₄, 4.0 mM glucose, and 5 mM HEPES-NaOH buffer (34). Omeprazole (AstraZeneca, Osaka, Japan) was dissolved in control normal Tyrode solution or control artificial perilymph. The solution containing omeprazole, which is strongly alkaline, was adjusted to neutrality before its perfusion. Sch-28080 (Sigma, St. Louis, MO) was dissolved in methanol and stored at –20°C. Just before application, Sch-28080 was diluted in the control solutions, which also contained PUREBRIGHT MB-37 (10 mg/ml; Nippon Yushi, Tokyo, Japan). PUREBRIGHT MB-37 is a polymer-type solubilizer that is reported to be of low toxicity (44). The final concentration of methanol was <2%. The perfusion of either control Tyrode or artificial perilymph containing 2% methanol and 10 mg/ml PUREBRIGHT had no effect on EP (data not shown).

	RESULTS

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Expression of gastric type H⁺,K⁺-ATPase in mouse cochlea. The H⁺,K⁺-ATPase is classified into two types: gastric and colonic (nongastric). Two types of the -subunit of H⁺,K⁺-ATPase, i.e., ₁ and ₂, have been identified. The functional H⁺,K⁺-ATPases are combinations of a catalytic -subunit and an accessory -subunit (14, 70). The assembly of the ₁- and -subunits of the H⁺,K⁺-ATPase constitutes the gastric type proton pump (2, 63). On the other hand, the ₂-subunit of H⁺,K⁺-ATPase assembles with the ₁-subunit of Na⁺,K⁺-ATPase in native tissues such as epithelia of colon, kidney, and prostate and forms nongastric type of proton pump, although the ₂-subunit can associate with the -subunit of H⁺,K⁺-ATPase in vitro (8, 9, 45, 59, 60). To examine which subunits were expressed in cochlea, we performed RT-PCR analyses of total RNAs obtained from mouse whole cochlea, kidney, and colon with the primers specific for each subunit (Fig. 1).

Each set of the primers amplified a clear band when the total RNA of kidney or colon was used as a template (Fig. 1, lane 1). Using the same primers, we found that cochlea clearly expressed the mRNAs of ₁- and -subunits (lane 3). On the other hand, the ₂ transcript was not detected in cochlear RNA (lane 3). The band of ₂ was not visible even when the PCR products were stained with SYBR Green I, which is 25 times more sensitive than ethidium bromide to detect DNA (data not shown). All of the fragments depicted in Fig. 1 are specific, because PCR amplification of total RNA untreated with reverse transcriptase gave no signals (lanes 2 and 4). These results indicate that it is the gastric-type H⁺,K⁺-ATPase and not the colonic type H⁺,K⁺-ATPase that is predominantly expressed in mouse cochlea.

Distribution of H⁺,K⁺-ATPase in mouse cochlea. We next performed immunolabeling of cochlear cryosections with a specific antibody against the carboxy-terminal end of the ₁-subunit of gastric H⁺,K⁺-ATPase (2) and analyzed it on a confocal microscope (Fig. 2). The efficacy of this antibody was proven by specific and intense labeling of parietal cells in stomach (Supplemental Fig. 1),¹ as has been previously reported with other antibodies against ₁- or -subunits of gastric H⁺,K⁺-ATPase (16, 20).

We first examined the labeling in mouse cochlea at low magnification. The antibody clearly labeled specific components of cochlear lateral wall (Fig. 2A, green). A moderate signal was detected in spiral ganglions as well (Fig. 2A). In the lateral wall, we observed strong ₁ immunolabeling in the lower part of spiral ligament, where type II and IV fibrocytes dominate, and also in stria vascularis, as reported previously (49). In addition, we detected clear labeling in the suprastrial zone and spiral limbus, where type V and another type of fibrocyte are respectively distributed (Fig. 2A). The regions with types I and III fibrocytes were free from labeling (Fig. 2A). A similar expression pattern for the -subunit was also observed in cochleae of rats and guinea pigs (Supplemental Fig. 2). All of the reactions were specific, because no immunolabeling was detected when the antibody preincubated with excess immunized oligopeptide was used (data not shown). There was a faint signal in the tectrial membrane, which often shows nonspecific reactions (12, 64).

We then examined the distribution of the ₁-subunit in spiral ligament and stria vascularis at a higher magnification. In the ligament, careful observation identified the "dot"-like labeling around ovoid or round nuclei that is associated with fibrocytes (Fig. 2B) (71). Labeling was not detected in either endothelial cells around blood vessels or outer sulcus cells (Fig. 2B). We therefore concluded that the ₁-subunit was expressed specifically in the fibrocytes of this region, i.e., types II and IV.

In stria vascularis, a strong signal was diffusely detected in its middle part, where basolateral membranes of marginal cells and apical membranes of intermediate cells are localized (Fig. 2C). No labeling was visible at either the apical site of marginal cells (arrows) or in the basal cell layer (arrowheads). As in the spiral ligament, we could not detect any ₁ immunoreactivity in endothelial cells in stria vascularis (Fig. 2C). These results strongly suggest that the ₁-subunit is localized at either the basolateral membrane of marginal cells or the apical site of intermediate cells. The resolution of confocal microscopic analysis was, however, insufficient to precisely determine the localization of the ₁-subunit, because the basolateral membrane of marginal cells and the apical membrane of intermediate cells are associated in close vicinity of 150–200 Å and are prominently invaginated (28, 72). In spiral ganglions, we detected moderate labeling of the ₁-subunit in the neurons but not in satellite cells (Fig. 2D).

The RT-PCR study revealed that the cochlea expressed mRNA of the -subunit of the H⁺,K⁺-ATPase (see Fig. 1). However, we could not detect any immunolabeling with commercially available anti--subunit antibodies. This may be due to either very low protein expression level of the subunit in cochlea or loss of its antigenicity during the decalcification treatment (see MATERIALS AND METHODS).

Cellular and subcellular localization of H⁺,K⁺-ATPase in stria vascularis of mouse cochlea. We attempted to more precisely determine the localization of the ₁-subunit in stria vascularis at either the basolateral membrane of marginal cells or the apical membrane of intermediate cells. For this purpose, we first examined the viable dominant spotting (W^v/W^v) deaf mouse mutant, which is reported to lack intermediate cells in the stria vascularis (6, 76). As observed in the wild-type mouse (see Fig. 2), we still detected clear ₁ immunoreactivity in the stria vascularis of this mutant mouse (Supplemental Fig. 3, A and B), whereas the immunoreactivity of Kir4.1, which locates in intermediate cells, disappeared completely (Supplemental Fig. 3C). This observation suggests that the H⁺,K⁺-ATPase is expressed in marginal cells of W^v/W^v mouse.

We next prepared the cryosections of wild-type mouse cochlea again, and, in the stria vascularis, compared the localization of the ₁-subunit with that of either barttin, a constituent of a Cl^– channel selectively expressed at the basolateral membrane of marginal cells but not in intermediate cells (15), or Kir4.1, a marker protein for intermediate cells, using a double-immunolabeling technique. However, as expected, even at a high magnification under confocal microscopic analysis, the resolution of merged images was not enough to conclude which membrane expressed the H⁺,K⁺-ATPase (data not shown). This must be due to the fact that the basolateral membrane of marginal cells and the apical membrane of intermediate cells have abundant infoldings and are tightly associated together in very close vicinity, as mentioned above. Therefore, we isolated marginal and intermediate cells from stria vascularis of wild-type mice and examined the expression of the ₁-subunit of the H⁺,K⁺-ATPase in each cell type (Fig. 3).

Morphologically, marginal cells can be identified with their highly convoluted and infolded basolateral membrane (72). We also visualized barttin immunoreactivity as another hallmark for marginal cells (15). The barttin-immunopositive cells (Fig. 3A, green) possessed a highly convoluted and infolded basolateral membrane (arrowheads), ensuring that they were marginal cells. These cells were labeled with the anti-₁-subunit antibody (Fig. 3A, red). Notably, labeling was detected at the basolateral side (arrowheads) but not at the apical membrane (arrows), of the marginal cells (Fig. 3A). Intermediate cells are a type of melanocytes (27, 82). Thus isolated intermediated cells could be identified with the melanin pigments. The cells with black pigments were devoid of either ₁ or barttin immunoreactivity (Fig. 3B, top), although they were clearly labeled with anti-Kir4.1 antibody (Fig. 3B, bottom, red) (1, 23). These results suggest that the H⁺,K⁺-ATPase is expressed in marginal but not in intermediate cells.

Finally, we conducted an immunoelectron microscopic examination on mouse stria vascularis (Fig. 4). Because it is often difficult in colored animals like C57 Black6 mice to distinguish the positive signal (black) of immunoreactivity from the pigmented inclusion bodies of intermediate cells, we examined the cochlea of albino ddY mice for this experiment. Marginal cells project their abundant basolateral processes to endothelial cells and pericytes of blood vessels (Fig. 4A). We detected prominent labeling of anti-₁ antibody at the invaginated basolateral membrane of marginal cells (Fig. 4, A and B, asterisks). Little signal was visible on the apical site of these cells (Fig. 4A, arrows). Endothelial cells and pericytes were free from staining (Fig. 4B). We also carefully examined intermediate cells and their surroundings. The ₁-immunopositive infoldings of basolateral membrane of marginal cells (Fig. 4C, asterisks) tightly wrapped the soma and the processes of intermediate cells. The highly magnified images confirmed that the immunoreactivity was detected at the basolateral site of marginal cells (Fig. 4, D and E, asterisks) but not either on the somatic membrane (Fig. 4D, arrowheads) or at the projections (Fig. 4E, arrowheads) of intermediate cells. Accordingly, we conclude that the ₁-subunit of gastric H⁺,K⁺-ATPase is localized specifically at the basolateral membrane of marginal cells in stria vascularis.

View larger version (159K): [in this window] [in a new window]   Fig. 4. Subcellular localization of the H+,K+-ATPase 1-subunit in stria vascularis. This immunoelectron microscopic analysis was performed in stria vascularis of an albino ddY mouse with the antibody against the 1-subunit. A: an area where a MC is closely associated with a blood vessel (BV). The inset in A is shown enlarged in B. Immunoreactivity was specifically detected at the invaginated basolateral membrane (A and B, asterisks) but not at the apical membrane (A, arrows) of the MC. Vascular endothelial cells (EC) and pericytes (PC) were free from staining (B). C: an area where the basolateral membranes of MCs twine around an IC. The insets in C are shown as higher magnification images in D and E. Arrowheads highlight the membrane of the IC. Whereas clear immunoreactivity was visible at the infoldings of basolateral membrane of MCs (asterisks), no positive signal was detected in the cell body or the processes of the IC.

We first perfused the drug into perilymph to examine the role of the H⁺,K⁺-ATPase in the cochlear spiral ligament (Fig. 5). Sch-28080 decreased EP in a dose-dependent fashion (Fig. 5D). Sch-28080 (300 µM) dramatically decreased EP from +81.3 to +34.0 mV in 15 min in the example shown in Fig. 5A. Upon washout of the blocker, EP gradually returned to the initial level, indicating that its inhibitory effect is reversible (Fig. 5A). Sch-28080 (1 mM) suppressed EP more strongly, and the potential reached a negative value of –9.2 mV (Fig. 5B). These results suggest that the H⁺,K⁺-ATPase in spiral ligament is involved in formation of EP. On the other hand, perfusion of the perilymph with a high concentration of omeprazole (5 mM), an irreversible inhibitor of gastric H⁺,K⁺-ATPase that is active only in an acidic environment (<pH 5) (51, 89), had little effect on EP (Fig. 5C; n = 4). This may suggest that the extracellular fluid surrounding the strial H⁺,K⁺-ATPase is not acidic enough to activate the drug.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Effects of perilymphatic perfusion of blockers of gastric H+,K+-ATPase on endocochlear potential (EP). The EP of guinea pigs was measured with perilymphatic (A–C) perfusion of blockers (Sch-28080 or omeprazole) specific for gastric H+,K+-ATPase. The blockers were applied for the periods indicated by horizontal bars, as described in MATERIALS AND METHODS. Sch, Sch-28080. D: dose-dependent reduction of EP by Sch-28080. Error bars represent SE.

View larger version (21K): [in this window] [in a new window]   Fig. 6. Effects of vascular perfusion of blockers of gastric H+,K+-ATPase on EP. The EP of guinea pigs was measured with vascular (A and B) perfusion of the blockers Sch-28080 and omeprazole. The blockers were applied for the periods indicated by horizontal bars, as described in MATERIALS AND METHODS. C: effects of Sch-28080 on EP under different conditions. Error bars represent SE.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Specificity of Sch-28080 in inhibition of EP Sch-28080 is an inhibitor specific to gastric H⁺,K⁺-ATPase. In in vitro experiments using the vesicles isolated from stomach, gastric glands, and the human embryonic kidney (HEK) cells exogenously expressing ₁- and -subunits of the gastric H⁺,K⁺-ATPase, it was reported that the IC₅₀ value of the drug to inhibit the ATPase is 0.2–5.2 µM and that 100 µM of Sch-28080 is enough for complete inhibition (3, 85, 88). It was shown that at this concentration, the drug did not affect the activity of other transporters such as Na⁺,K⁺-ATPase and NKCC1 (4, 5, 7, 50, 68, 85, 92). In this study, we needed to perfuse 1 or 3 mM of Sch-28080 to the perilymph or the vertebral artery, respectively, to achieve complete suppression of EP (Figs. 5 and 6). It is therefore possible that the application of such high concentrations of Sch-28080 affected ion transport apparatuses other than H⁺,K⁺-ATPase and thus suppressed EP. Measurement of the actual concentration of Sch-28080 at the sites of action in cochlear lateral wall may be needed to reach the final conclusion whether the drug action is specific to inhibition of the H⁺,K⁺-ATPase. At present, however, it is technically very difficult to achieve this task, because the volume of the extracellular solution surrounded with invaginated basolateral membrane of marginal cells and that of the perilymph around infolded processes of the fibrocytes are very small (28, 71–73).

Nevertheless, on the basis of the relationship between the in vitro properties of other inhibitors and their effects on EP, we can roughly estimate the concentration of Sch-28080 in the lateral wall of living animals. For example, it was shown that total suppression of EP by inhibiting NKCC1 at the basolateral site of marginal cells or in the fibrocytes of the ligament required 1 mM of furosemide when applied through vertebral artery or perilymph (42, 47, 69), whereas 100 µM of furosemide was enough to completely inhibit the NKCC1 itself with the IC₅₀ value of 2.5–3.0 µM (66, 77). Thus the drugs in the perfusates are expected to be diluted to 1/10 at the site of action in cochlea. Therefore, it would be the case that the actual concentration of Sch-28080 reaching the marginal cells in the stria or the fibrocytes in the ligament may be on the order of 100–300 µM. Because it was also shown in many previous studies that Sch-28080 at the concentration of 100 µM selectively blocked gastric H⁺,K⁺-ATPase and did not significantly affect other apparatuses such as Na⁺,K⁺-ATPase and NKCC1 (4, 5, 7, 50, 68, 85, 92), EP reduction by Sch-28080 observed in this study would be mainly attributable to suppression of the H⁺,K⁺-ATPase.

Putative role of gastric H⁺,K⁺-ATPase in cochlear stria vascularis. The cochlear stria vascularis is known playing essential roles in generation of EP (Fig. 7A) (83, 90). Salt et al. (65) found a unique space sandwiched between basolateral membrane of marginal cells and apical membrane of intermediate or basal cells in stria vascularis. This so-called intrastrial space (IS) is filled with an unusual extracellular fluid that contains low [K⁺] ([K⁺]_IS) of 1–2 mM and exhibits a highly positive potential of approximately +100 mV with reference to the body fluid (Fig. 7B) (31, 65). IS is electrically isolated with two tight-junction shields: the marginal cell layer and the basal cell layer (19, 33, 39, 40, 78). Intermediate and basal cells are connected through gap junctions not only to each other but also to fibrocytes, endothelial cells, and pericytes (38, 80). Thus all of these cells form an electrical syncytium, which is called the connective tissue gap-junction network (Fig. 7B) (38, 80). The membrane potential (E_m) of the syncytium relative to the perilymph is –5–0 mV (31, 55, 65). The apical membrane of intermediate cells is highly K⁺ permeable because of abundant expression of Kir4.1 (1, 23). Takeuchi et al. (81) found that the resting membrane potential of isolated intermediate cells was almost identical to the equilibrium potential for K⁺. They also examined the effects of Ba²⁺ ([Ba²⁺]) in bath solution containing low [K⁺], similar to [K⁺]_IS, on the E_m of the intermediate cells. The dependence of membrane potential changes on [Ba²⁺] was quite similar to the dependence of EP decline on [Ba²⁺] that was perfused into the artery (81). The E_m of intermediate cells may therefore directly reflect the high potential of IS and this potential as a source of EP. It is also strongly suggested that Kir4.1 is responsible for formation of the potential difference across the apical membrane of intermediate cell. This idea is also supported by the observation that Kir4.1-null mice completely lost EP (54). If Kir4.1 is the key player for establishment of the high potential of IS, the low [K⁺]_IS (1–2 mM) must be constantly maintained to form the potential difference (65, 81, 91). The NKCC1 and Na⁺,K⁺-ATPase that are expressed at the basolateral membrane of marginal cells are responsible for maintaining low [K⁺]_IS (Fig. 7B), and vascular perfusion of respective inhibitor largely reduces EP (46, 48). It is therefore possible that the H⁺,K⁺-ATPase localizing at the basolateral membrane of marginal cells also participates in maintenance of low [K⁺]_IS alongside NKCC1 and Na⁺,K⁺-ATPase.

View larger version (48K): [in this window] [in a new window]   Fig. 7. Schematic model of cochlear K+ circulation and formation of EP in the lateral wall. A: K+ exiting from the hair cells is circulated to the type II and IV fibrocytes in the spiral ligament (SL) through the epithelial tissue gap-junction network, which is composed of ECs on the basilar membrane and OSC in the ligament. K+ is taken up by types II and IV fibrocytes in the SL and then transported to the StV via the connective tissue gap-junction network, comprising the fibrocytes, basal cells, and ICs (see detail in B). MCs secrete K+ across their apical membrane to maintain the endolymph. The concentration of K+ ([K+]) and the potential of each cochlear fluid are indicated. B: ion transport molecules expressed in the StV and the SL that participate in cochlear K+ circulation. The potential and [K+] in each compartment are indicated. The involvement of H+,K+-ATPase in formation of EP and cochlear K+ circulation is described in text. FC, fibrocytes; BC, basal cells; IS, intrastrial space; TJ, tight junction. Pink circles, Na+,K+-ATPase; blue circles, Na+,K+,2Cl– cotransporter (NKCC1); yellow cylinders, Cl– channel.

However, this may not be enough to explain why strong inhibition of any one of the H⁺,K⁺-ATPase, the NKCC1, and the Na⁺,K⁺-ATPase expressed in stria vascularis caused complete suppression of EP (Fig. 6) (46, 47). One possibility might be that, at the basolateral site of marginal cells, the three apparatuses are functionally coupled to form a complex of machinery for effective K⁺ transport, and that disruption of any one of them would significantly impair K⁺ transport function of the complex. Further studies are needed to clarify this possibility.

Putative role of gastric H⁺,K⁺-ATPase in cochlear spiral ligament. It is assumed that K⁺ circulation from perilymph to endolymph through cochlear lateral wall is essential for maintaining primarily endolymphatic high [K⁺] and thus EP. Two functional components are considered to be critically involved in the K⁺ circulation. They are the epithelial tissue and connective tissue gap junction networks (Fig. 7A). The former is composed of the supporting cells beneath the hair cells, the epithelial cells on basilar membrane, and the outer sulcus cells, whereas the latter comprises the fibrocytes in spiral ligament and some cells in stria vascularis, as stated above. K⁺ released from the hair cells seems to be absorbed by the supporting cells and then transported to the stria vascularis via the two gap junction networks. There is no gap-junctional connection between the two networks. The type II and V fibrocytes contacting with the outer sulcus cells should therefore take up K⁺ by an active process for the K⁺ circulation. The NKCC1 and the Na⁺,K⁺-ATPase in the fibrocytes may contribute to this process (37). Perilymphatic perfusion of K⁺-free solution, which may remove the extracellular K⁺ around the NKCC1 and the Na⁺,K⁺-ATPase and thus cause them to dysfunction, and that of the inhibitors for these K⁺ uptake apparatuses dramatically reduce EP (52). This highlights a possible involvement of polarized K⁺ transport via the fibrocytes in EP formation. In the present study we have shown that these particular fibrocytes also express the gastric type of the H⁺,K⁺-ATPase (Fig. 2, A and B) and that a specific blocker for this pump, Sch-28080, applied to perilymph, caused suppression of EP (Fig. 5, A and B). Therefore, it may be reasonable to suggest that the H⁺,K⁺-ATPase in the fibrocytes is also involved in K⁺ transport in the ligament and thus generation of EP. The H⁺,K⁺-ATPase would accelerate cochlear K⁺ circulation by taking up K⁺ in exchange to H⁺ from perilymph alongside of the NKCC1 and the Na⁺,K⁺-ATPase (Fig. 7B). This also may occur in fibrocytes in the suprastrial zone of the spiral ligament and in those of the spiral limbus, which are thought to be another pathway of K⁺ transport from endolymph to perilymph (90).

Future questions and direction. In summary, we have shown that the gastric type of H⁺,K⁺-ATPase is abundantly expressed in cochlear stria vascularis and spiral ligament and may be critically involved in EP formation, probably through control of the K⁺ circulation in the lateral wall. Several issues, however, remain to be addressed for comprehensive understanding of the roles of the H⁺,K⁺-ATPase in auditory function.

The H⁺,K⁺-ATPase extrudes H⁺ in exchange to K⁺ from intracellular milieu to extracellular fluid. Thus this pump also may be involved in the pH control of cochlear fluids. Whereas Sch-28080 perfused to the lateral wall largely suppressed EP, vascular or perilymphatic perfusion of omeprazole, another specific inhibitor of gastric type H⁺,K⁺-ATPase, did not significantly affect EP (Figs. 5 and 6). This observation may suggest that the extracellular solution in IS or in the ligament may not be acidic enough, because omeprazole is activated only in a strong acidic environment of pH <5 (51, 89). Actually, it has been reported that pH of perilymph is 7.8–8.0 (56). This may be due to the effective neutralization of the protons by HCO₃^– secreted via Cl^–/HCO₃^– exchangers expressed in the outer sulcus cells neighboring the fibrocytes (75). Although the pH value of IS solution has not yet been measured, the following observations in other tissues may support the idea that the solution would not be strongly acidic. For example, in the prostate of mice, the secretions are mildly acidic with a pH value of 6.4. The slight acidification is, however, caused by the colonic H⁺,K⁺-ATPase expressed in the prostate epithelia, because the secretions are alkalinized to pH 7 when the ₂-subunit-gene is ablated (60). In kidney, although gastric type H⁺,K⁺-ATPase is shown expressed in its epithelia and involved in the control of proton transport in vitro with omeprazole (17, 41), in vivo application of the inhibitor to healthy human subjects did not affect their urinary pH (29, 58). Accordingly, differently from the gastric mucosa, the primary role of H⁺,K⁺-ATPase in these organs, and probably also in cochlea, would be the absorption of K⁺ from the extracellular fluid and the pH control of intracellular milieu rather than extracellular fluid.

However, there remains a possibility that abnormality in the proton production by the H⁺,K⁺-ATPase could be linked with some pathological conditions of inner ear. Indeed, venous injection of sodium bicarbonate solution is an effective therapy for vertigo and dizziness induced by different defects in the inner ear, including Méniere's disease (21). Thus the relevance of gastric type H⁺,K⁺-ATPase in various auditory diseases should be examined. It may therefore be of interest to examine whether the H⁺,K⁺-ATPase-null mice (61, 62, 74) exhibit hearing disorder or whether there are hereditary or sporadic mutations of the gene encoding the ATPase in deaf patients. Further studies on the subjects described above are needed to clarify the physiological and pathological roles of the H⁺,K⁺-ATPase in cochlea.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by the following research grants and funding: Grant-in-Aid for Scientific Research on Priority Areas (2) 12144207 (to Y. Kurachi), Grant-in-Aid for Scientific Research on Priority Areas 17081012 (to H. Hibino), Grant-in-Aid for Young Scientists (A) 17689012 (to H. Hibino), and Japan-France Integrated Action Program (SAKURA) (to Y. Kurachi) from the Ministry of Education, Science, Sports and Culture of Japan, and the Uehara Memorial Foundation (to Y. Kurachi), Kanae Foundation for Life & Socio-Medical Science (to H. Hibino), Inamori Foundation (to H. Hibino), and Yamanouchi Foundation for Research on Metabolic Disorders (to H. Hibino).

	ACKNOWLEDGMENTS

 
We thank Dr. Ian Findlay (Université de Tours, Tours, France) for critically reading this manuscript.

	FOOTNOTES

 

Address for reprint requests and other correspondence: Y. Kurachi, Div. of Molecular and Cellular Pharmacology, Graduate School of Medicine, Osaka Univ., 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan (e-mail: ykurachi{at}pharma2.med.osaka-u.ac.jp)

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.

¹ The online version of this article contains supplemental data.

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