Positive Endocochlear Potential Research Articles

Considering gene therapy to protect from X-linked deafness DFNX2 and associated neurodevelopmental disorders.

Mutations and deletions in the gene or upstream of the gene encoding the POU3F4 transcription factor cause X-linked progressive deafness DFNX2 and additional neurodevelopmental disorders in humans. Hearing loss can be purely sensorineural or mixed, that is, with both conductive and sensorineural components. Affected males show anatomical abnormalities of the inner ear, which are jointly defined as incomplete partition type III. Current approaches to improve hearing and speech skills of DFNX2 patients do not seem to be fully effective. Owing to inner ear malformations, cochlear implantation is surgically difficult and may predispose towards severe complications. Even in cases where implantation is safely performed, hearing and speech outcomes remain highly variable among patients. Mouse models for DFNX2 deafness revealed that sensorineural loss could arise from a dysfunction of spiral ligament fibrocytes in the lateral wall of the cochlea, which leads to reduced endocochlear potential. Highly positive endocochlear potential is critical for sensory hair cell mechanotransduction and hearing. In this context, here, we propose to develop a therapeutic approach in male Pou3f4 -/y mice based on an adeno-associated viral (AAV) vector-mediated gene transfer in cochlear spiral ligament fibrocytes. Among a broad range of AAV vectors, AAV7 was found to show a strong tropism for the spiral ligament. Thus, we suggest that an AAV7-mediated delivery of Pou3f4 complementary DNA in the spiral ligament of Pou3f4 -/y mice could represent an attractive strategy to prevent fibrocyte degeneration and to restore normal cochlear functions and properties, including a positive endocochlear potential, before hearing loss progresses to profound deafness.

Building a Sound Wall in the Cochlea: Development of the Stria Vascularis

The stria vascularis is a specialized epithelial structure of the mammalian cochlea that produces endolymph, the potassium‐rich fluid responsible for the positive endocochlear potential of the cochlear lumen. This positive extracellular potential is the major driving force for proper signal transduction by sensory cells in the ear, and thus normal hearing. The stria vascularis is the “battery” of the auditory organ: it is localized in the lateral wall of the cochlea, and consists of a multilayered highly vascularized epithelium. It is composed of three main cell types with distinct embryonic origins: the marginal cells derived from the otic epithelium , the intermediate cells which are specialized melanocyte‐like cells derived from migratory neural crest cells, and the basal cells that are derived from otic mesenchyme. Mesoderm‐derived blood vessels are interspersed between these cells throughout the stria. Defects in the development of the stria vascularis are part of a number of inheritable deafness syndromes in humans, including Hirschsprung disease and Waardenburg, Jervell and Lange‐Nielsen, Bartter and SeSAME syndromes. Strial degeneration is estimated to cause more than 30% of age related hearing loss or presbycusis, which affects between 25–40% of the population over 65 years and over 60% of the population over 80 years old. The causes of strial atrophy remain poorly understood. Over the past decade, a great effort has been made to understand the development of the sensory cells in the mammalian cochlea with the aim of applying their developmental principles to regenerative therapies. By contrast, very little is known about the developmental potential of the stria vascularis, or even the mechanisms of strial degeneration due to aging. Our focus is to uncover the normal development of the stria, the cellular interactions that lead to its formation, and the potential for regeneration in cases of congenital strial deafness.Support or Funding InformationHearing Health FoundationThe Hartwell FoundationNIDCD R01DC015785This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reduced Membrane Insertion of CLC-K by V33L Barttin Results in Loss of Hearing, but Leaves Kidney Function Intact.

In the mammalian ear, transduction of sound stimuli is initiated by K+ entry through mechano-sensitive channels into inner hair cells. K+ entry is driven by a positive endocochlear potential that is maintained by the marginal cell layer of the stria vascularis. This process requires basolateral K+ import by NKCC1 Na+−2Cl−−K+ co-transporters as well as Cl− efflux through ClC-Ka/barttin or ClC-Kb/barttin channels. Multiple mutations in the gene encoding the obligatory CLC-K subunit barttin, BSND, have been identified in patients with Bartter syndrome type IV. These mutations reduce the endocochlear potential and cause deafness. As CLC-K/barttin channels are also expressed in the kidney, patients with Bartter syndrome IV typically also suffer from salt-wasting hyperuria and electrolyte imbalances. However, there was a single report on a BSND mutation that resulted only in deafness, but not kidney disease. We herein studied the functional consequences of another recently discovered BSND mutation that predicts exchange of valine at position 33 by leucine. We combined whole-cell patch clamp, confocal microscopy and protein biochemistry to analyze how V33L affects distinct functions of barttin. We found that V33L reduced membrane insertion of CLC-K/barttin complexes without altering unitary CLC-K channel function. Our findings support the hypothesis of a common pathophysiology for the selective loss of hearing due to an attenuation of the total chloride conductance in the stria vascularis while providing enough residual function to maintain normal kidney function.

The unique electrical properties in an extracellular fluid of the mammalian cochlea; their functional roles, homeostatic processes, and pathological significance.

The cochlea of the mammalian inner ear contains an endolymph that exhibits an endocochlear potential (EP) of +80 mV with a [K+] of 150 mM. This unusual extracellular solution is maintained by the cochlear lateral wall, a double-layered epithelial-like tissue. Acoustic stimuli allow endolymphatic K+ to enter sensory hair cells and excite them. The positive EP accelerates this K+ influx, thereby sensitizing hearing. K+ exits from hair cells and circulates back to the lateral wall, which unidirectionally transports K+ to the endolymph. In vivo electrophysiological assays demonstrated that the EP stems primarily from two K+ diffusion potentials yielded by [K+] gradients between intracellular and extracellular compartments in the lateral wall. Such gradients seem to be controlled by ion channels and transporters expressed in particular membrane domains of the two layers. Analyses of human deafness genes and genetically modified mice suggested the contribution of these channels and transporters to EP and hearing. A computational model, which reconstitutes unidirectional K+ transport by incorporating channels and transporters in the lateral wall and connects this transport to hair cell transcellular K+ fluxes, simulates the circulation current flowing between the endolymph and the perilymph. In this model, modulation of the circulation current profile accounts for the processes leading to EP loss under pathological conditions. This article not only summarizes the unique physiological and molecular mechanisms underlying homeostasis of the EP and their pathological relevance but also describes the interplay between EP and circulation current.

Properties of ATP-gated ion channels assembled from P2X2 subunits in mouse cochlear Reissner's membrane epithelial cells.

In the cochlea, Reissner's membrane separates the scala media endolymphatic compartment that sustains the positive endocochlear potential and ion composition necessary for sound transduction, from the scala vestibuli perilymphatic compartment. It is known that with sustained elevated sound levels, adenosine 5'-triphosphate (ATP) is released into the endolymph and ATP-gated ion channels on the epithelial cells lining the endolymphatic compartment shunt the electrochemical driving force, contributing to protective purinergic hearing adaptation. This study characterises the properties of epithelial cell P2X(2)-type ATP-activated membrane conductance in the mouse Reissner's membrane, which forms a substantial fraction of the scale media surface. The cells were found to express two isoforms (a and b) of the P2X(2) subunit arising from alternative splicing of the messenger RNA (mRNA) transcript that could contribute to the trimeric subunit assembly. The ATP-activated conductance demonstrated both immediate and delayed desensitisation consistent with incorporation of the combination of P2X(2) subunit isoforms. Activation by the ATP analogue 2meSATP had equipotency to ATP, whereas α,β-meATP and adenosine 5'-diphosphate (ADP) were ineffective. Positive allosteric modulation of the P2X(2) channels by protons was profound. This native conductance was blocked by the P2X(2)-selective blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and the conductance was absent in these cells isolated from mice null for the P2rX2 gene encoding the P2X(2) receptor subunit. The activation and desensitisation properties of the Reissner's membrane epithelial cell ATP-gated P2X(2) channels likely contribute to the sensitivity and kinetics of purinergic control of the electrochemical driving force for sound transduction invoked by noise exposure.

Pannexin1 channels dominate ATP release in the cochlea ensuring endocochlear potential and auditory receptor potential generation and hearing.

Pannexin1 (Panx1) is a gap junction gene in vertebrates whose proteins mainly function as non-junctional channels on the cell surface. Panx1 channels can release ATP under physiological conditions and play critical roles in many physiological and pathological processes. Here, we report that Panx1 deficiency can reduce ATP release and endocochlear potential (EP) generation in the cochlea inducing hearing loss. Panx1 extensively expresses in the cochlea, including the cochlear lateral wall. We found that deletion of Panx1 in the cochlear lateral wall almost abolished ATP release under physiological conditions. Positive EP is a driving force for current through hair cells to produce auditory receptor potential. EP generation requires ATP. In the Panx1 deficient mice, EP and auditory receptor potential as measured by cochlear microphonics (CM) were significantly reduced. However, no apparent hair cell loss was detected. Moreover, defect of connexin hemichannels by deletion of connexin26 (Cx26) and Cx30, which are predominant connexin isoforms in the cochlea, did not reduce ATP release under physiological conditions. These data demonstrate that Panx1 channels dominate ATP release in the cochlea ensuring EP and auditory receptor potential generation and hearing. Panx1 deficiency can reduce ATP release and EP generation causing hearing loss.

Low channel conductances in capillary endothelial cells of guinea pig cochlea contribute to the blood‐labyrinth‐barrier

The blood‐labyrinth‐barrier at the strial vascularis (SV) capillaries separates big physical (>100 mV) and chemical gradients between the intrastrial space and blood stream while maintains necessary metabolites exchanges. The mechanisms required to fulfill these functions remain poorly understood. Using whole‐cell and single channel recording techniques, we explored the channel mechanisms in the capillary endothelial cells (ECs) and pericytes (PCs) in acutely isolated SV capillary segments and in fully dispersed state. We found that 1) with physiological solutions in‐and‐out, SV ECs in segments had a membrane potential ~−30 mV, a Cinput ~28 pF and a Rinput ~ 510 MΩ (~10 pF and ~2 GΩ from isolated single cells). The I/V curve of ECs mostly exhibited a mild outward rectification at voltages positive to −40 mV, was slightly flattened by 4‐AP, TEA, and NFA, but not significantly changed by Ba2+, La3+, nitrendipine, apamin, glipizide or 20 mM K+. 2) In contrast, PCs had a membrane potential ~−50 mV, a Cinput ~85 pF and a Rinput ~ 306 MΩ (~10 pF and ~450 MΩ for isolated single cells), and an I/V relation with prominent inward rectification at potentials <−60 mV. The rectification was enhanced by [K]o elevation and abolished by 0.1 mM Ba2+. 3) Both cell types showed 18β‐glycyrrhetinic acid‐sensitive electro‐coupling. 4) Cellattached and outside‐out patch recordings from ECs occasionally detected activities of ~50 pS K+‐channels with very low open probability. We conclude that SV ECs normally express little active channels, resulting in high Rinput thus to economically preserve the highly positive endocochlear potential, whereas SV PCs appear as a regulatory element. Supported by NIH grant R01 DC004716 (ZGJ).

How is the highly positive endocochlear potential formed? The specific architecture of the stria vascularis and the roles of the ion-transport apparatus

Cochlear endolymph, an extracellular solution containing 150 mM K(+), exhibits a positive potential of +80 mV. This is called the endocochlear potential (EP) and is essential for audition. The mechanism responsible for formation of the EP has been an enigma for the half century since its first measurement. A key element is the stria vascularis, which displays a characteristic tissue structure and expresses multiple ion-transport apparatus. The stria comprises two epithelial layers: a layer of marginal cells and one composed of intermediate and basal cells. Between the two layers lies an extracellular space termed the intrastrial space (IS), which is thus surrounded by the apical membranes of intermediate cells and the basolateral membranes of marginal cells. The fluid in the IS exhibits a low concentration of K(+) and a positive potential similar to the EP. We have demonstrated that the IS is electrically isolated from the neighboring extracellular fluids, perilymph, and endolymph, which allows the IS to sustain its positive potential. This IS potential is generated by K(+) diffusion across the apical membranes of intermediate cells, where inwardly rectifying Kir4.1 channels are localized. The low K(+) concentration in the IS, which is mandatory for the large K(+)-diffusion potential, is maintained by Na(+),K(+)-ATPases and Na(+),K(+),2Cl(-)-cotransporters expressed at the basolateral membranes of marginal cells. An additional K(+)-diffusion potential formed by KCNQ1/KCNE1-K(+) channels at the apical membranes of marginal cells also contributes to the EP. Therefore, the EP depends on an electrically isolated space and two K(+)-diffusion potentials in the stria vascularis.

Ca2+ regulation of endocochlear potential in marginal cells

We examined the effect of the cytosolic Ca(2+) concentration ([Ca(2+)](c)) in marginal cells on the asphyxia- or furosemide-induced decrease in the endocochlear potential (EP) by perfusing the endolymph with or without a Ca(2+) chelator or inhibitors of Ca(2+)-permeable channels or Ca(2+)-pump during transient asphyxia or intravenous administration of furosemide. We obtained the following results. (1) Endolymphatic administration of SKF96365 (an inhibitor of TRPC and L-type Ca(2+) channels) or EGTA-acetoxymethyl ester (EGTA-AM) significantly inhibited both the transient asphyxia-induced decrease in EP (TAID) and the furosemide-induced decrease in EP (FUID). (2) Endolymphatic perfusion with nifedipine significantly inhibited the TAID but not the FUID. (3) The recovery from the FUID was significantly suppressed by perfusing the endolymph with EGTA-AM, nifedipine, or SKF96365. (4) Endolymphatic administration of thapsigargin inhibited both the FUID and TAID. (5) The recovery rate from the FUID was much slower than that from the TAID, indicating that furosemide may inhibit the Ca(2+)-pump. (6) A strong reaction in immunohistochemical staining for TRPC channels was observed in the luminal and basolateral membranes of marginal cells. (7) A positive staining reaction for the gamma subunit of epithelial Na(+) channels was observed in the luminal and basolateral membranes of marginal cells. (8) Positive EP was diminished toward 0 mV by the endolymphatic perfusion with 10 muM amiloride or 10 muM phenamil. Taken together, these findings suggest that [Ca(2+)](c) regulated by endoplasmic Ca(2+)-pump and Ca(2+)-permeable channels in marginal cells may regulate the positive EP, which is partly produced by the diffusion potential of Na(+) across the basolateral membrane in marginal cells.

Cellular characterization of Connexin26 and Connnexin30 expression in the cochlear lateral wall

Gap junctions in the cochlear lateral wall, which consists of the stria vascularis (SV) and spiral ligament (SPL), are important for generating a positive endocochlear potential and high potassium concentration in the endolymph. In this study, the cellular expression of connexin 26 (Cx26) and Cx30 in the cochlear lateral wall of rats and guinea pigs was examined by immunofluorescent staining and confocal microscopy. Co-labeling for Kir4.1 revealed that the stria intermediate cells had extensive labeling for Cx26 and Cx30 with a leaf-like distribution. Cx26 and Cx30 also co-distributed hexagonally around the basal cells. However, no labeling was observed in the marginal cells. In the SPL, punctate Cx26 and Cx30 labeling was distributed along vertical lines orthogonal to the cochlear longitudinal direction. Intense labeling for Cx26 and Cx30 was found in type II fibrocytes in the spiral prominence and central region, but Cx26 labeling was absent in the middle region just beneath the SV, where only Cx30 labeling was observed. Outer sulcus (OS) cells and their root processes also exhibited intense labeling for Cx26 and Cx30. Neither Cx26 nor Cx30 was immunopositive in the hyaline region beneath the OS, in the subcentral region (type IV fibrocytes), or in the tension (type III) fibrocytes beneath the bone. Cx26 and Cx30 labeling was also absent in the lateral wall blood vessels. Thus, Cx26 and Cx30 have distinct cell-specific distributions in the SV and SPL, suggesting that they can form different pathways for transporting ions/nutrients in the cochlear lateral wall.

Conservation of hearing by simultaneous mutation of Na,K-ATPase and NKCC1.

Although drug-induced and age-related hearing losses are frequent otologic problems affecting millions of people, their underlying mechanisms remain uncertain. The inner ear is exclusively endowed with a positive endocochlear potential (EP) that serves as the main driving force for the generation of receptor potential in hair cells to confer hearing. Deterioration of EP leads to hearing loss or deafness. The generation of EP relies on the activity of many ion transporters to establish active potassium (K(+)) cycling within the inner ear, including K(+) channels, the Na-K-2Cl co-transporter (NKCC1), and the alpha(1) and alpha(2) isoforms of Na,K-ATPase. We show that heterozygous deletion of either NKCC1, alpha(1)-Na,K-ATPase, or alpha(2)-Na,K-ATPase independently results in progressive, age-dependent hearing loss with minimal alteration in cochlear morphology. Double heterozygote deletion of NKCC1 with alpha(1)-Na,K-ATPase also shows a progressive, though delayed, age-dependent hearing loss. Remarkably, double heterozygote deletion of NKCC1 with alpha(2)-Na,K-ATPase demonstrates a striking preservation of hearing threshold both initially and with age. Measurements of the EP confirm the anticipated drop in potential for genotypes that demonstrate age-dependent hearing loss. The EP generated by the NKCC1 + alpha(2)-Na,K-ATPase double heterozygote, however, is maintained at a level comparable to that of the control condition, suggesting a potential advantage in this combination of ion transporter modification. These observations provide insight into the detailed mechanisms of EP generation, and results of combination-knockout experiments may have important implications in the future treatment of drug-induced and age-related hearing losses.

FXYD6 Is a Novel Regulator of Na,K-ATPase Expressed in the Inner Ear

The exquisite sensitivity of the cochlea, which mediates the transduction of sound waves into nerve impulses, depends on the endolymph ionic composition and the endocochlear potential. A key protein in the maintenance of the electrochemical composition of the endolymph is the Na,K-ATPase. In this study, we have looked for the presence in the rat inner ear of members of the FXYD protein family, recently identified as tissue-specific modulators of Na,K-ATPase. Only FXYD6 is detected at the protein level. FXYD6 is expressed in various epithelial cells bordering the endolymph space and in the auditory neurons. FXYD6 co-localizes with Na,K-ATPase in the stria vascularis and can be co-immunoprecipitated with Na,K-ATPase. After expression in Xenopus oocytes, FXYD6 associates with Na,K-ATPase alpha1-beta1 and alpha1-beta2 isozymes, which are preferentially expressed in different regions of the inner ear and also with gastric and non-gastric H,K-ATPases. The apparent K(+) and Na(+) affinities of alpha1-beta1 and alpha1-beta2 isozymes are different. Association of FXYD6 with Na,K-ATPase alpha1-beta1 isozymes slightly decreases their apparent K(+) affinity and significantly decreases their apparent Na(+) affinity. On the other hand, association with alpha1-beta2 isozymes increases their apparent K(+) and Na(+) affinity. The effects of FXYD6 on the apparent Na(+) affinity of Na,K-ATPase and the voltage dependence of its K(+) effect are distinct from other FXYD proteins. In conclusion, this study defines the last FXYD protein of unknown function as a modulator of Na,K-ATPase. Among FXYD protein, FXYD6 is unique in its expression in the inner ear, suggesting a role in endolymph composition.

Endolymphatic Perfusion with EGTA-Acetoxymethyl Ester Inhibits Asphyxia- and Furosemide-Induced Decrease in Endocochlear Potential in Guinea Pigs

We examined the effect of the Ca(2+) concentration in the endolymph ([Ca](e)) or in the endolymphatic surface cells ([Ca](i)) on the endocochlear potential (EP) by using an endolymphatic or perilymphatic perfusion technique, respectively. (i) A large increase in [Ca](e) up to approximately 10(-3) M with a fall in the EP was induced by transient asphyxia ( approximately 2 min) or by the intravenous administration of furosemide (60 mg/kg), and a significant correlation was obtained between the EP and p[Ca](e) (= -log [Ca](e), r = 0.998). (ii) Perfusion of the endolymph with 10 mM EGTA for 5 min neither produced any significant change in the EP nor altered the asphyxia-induced change in EP (DeltaEP(asp)), suggesting that neither [Ca](e) nor the Ca(2+) concentration gradient across the stria vascularis contributed directly to the generation of the EP in the condition of low [Ca](e). In contrast, endolymphatic perfusion with high Ca(2+) (more than 10 mM) produced a decrease in EP and a significant correlation was obtained between the EP and the Ca(2+) concentration of perfusion solution (r = 0.982), suggesting that Ca(2+) permeability may exist across the stria vascularis. (iii) The administration of a Ca(2+) chelator, EGTA-acetoxymethyl ester (AM, 0.3 mM), to the endolymph, which produced a gradual increase in EP, suppressed significantly, by 60-80%, DeltaEP(asp) or furosemide-induced changes in EP. In contrast, perilymphatic administration of 0.5 mM EGTA-AM caused no significant suppression of the DeltaEP(asp). These findings suggest that [Ca](i) plays an important role in generating/maintaining a large positive EP.

Compartmentalization established by claudin-11-based tight junctions in stria vascularis is required for hearing through generation of endocochlear potential.

Claudins are cell adhesion molecules working at tight junctions (TJs) that are directly involved in compartmentalization in multicellular organisms. The cochlea includes a rather peculiar compartment filled with endolymph. This compartment is characterized by high K+ concentration (approximately 150 mM) and a positive endocochlear potential (approximately 90 mV; EP), both indispensable conditions for cochlear hair cells to transduce acoustic stimuli to electrical signals. These conditions are thought to be generated by the stria vascularis, which is adjacent to the endolymph compartment. The stria vascularis itself constitutes an isolated compartment delineated by two epithelial barriers, marginal and basal cell layers. Because TJs of basal cells are primarily composed of claudin-11, claudin-11-deficient (Cld11-/-) mice were generated with an expectation that the compartmentalization in stria vascularis in these mice would be affected. Auditory brainstem response measurements revealed that Cld11-/- mice suffered from deafness; although no obvious gross morphological malformations were detected in Cld11-/- cochlea, freeze-fracture replica electron microscopy showed that TJs disappeared from basal cells of the stria vascularis. In good agreement with this, tracer experiments showed that the basal cell barrier was destroyed without affecting the marginal cell barrier. Importantly, in the endolymph compartment of Cld11-/- cochlea, the K+ concentration was maintained around the normal level (approximately 150 mM), whereas the EP was suppressed down to approximately 30 mV. These findings indicated that the establishment of the stria vascularis compartment, especially the basal cell barrier, is indispensable for hearing ability through the generation/maintenance of EP but not of a high K+ concentration in the endolymph.

Immunological identification of an inward rectifier K+ channel (Kir4.1) in the intermediate cell (melanocyte) of the cochlear stria vascularis of gerbils and rats.

The cochlear stria vascularis produces the positive endocochlear potential (EP) and the endolymph. Both the EP and the endolymph are essential for the physiological function of hair cells. The intermediate cell is one of several cell types constituting the stria vascularis. It is known that inward rectifier K+ channels can play a constitutive role in the determination of the resting membrane potential. Localization of a member of the inward rectifier K+ channel family, Kir4.1, in the stria vascularis of gerbils and rats was investigated by immunological methods. A polyclonal antibody specific to the C-terminus of the rat Kir4.1 channel was raised in rabbits. Immunostaining of dissociated cells revealed that the Kir4.1 channel was localized to the intermediate cell, but not to the epithelial marginal cell. Subcellular localization of the Kir4.1 channel to the plasma membrane of the intermediate cell was confirmed by immunoelectron microscopy. Immunostaining of whole-tissue preparations revealed a network-like structure composed of intermediate cells. It seems likely that the Kir4.1 channel mediates the inwardly rectifying K+ current in the intermediate cell as shown previously by electrophysiological methods, and that this channel plays key roles in the production of the EP and K+ transport in the stria vascularis.

Potassium channel ether à go-go mRNA expression in the spiral ligament of the rat

Identification of the K + transporters located in the lateral wall of the cochlea is essential for a better understanding of the mechanisms by which a positive endocochlear potential and a high K + concentration are achieved in endolymph. In this study, we have determined the distribution of the K + channel rat ether à go-go (eag) mRNA in the cochlea. After reverse transcription of adult rat cochlear tissues, cDNA was amplified with primers specific to eag channel. The eag mRNA was localized in cochlear tissues by in situ hybridization using specific oligonucleotide probes tailed with digoxigenin conjugated UTP. Eag mRNA was detected in the organ of Corti but mainly in the fibrocytes of the spiral ligament but not in spiral prominence or in stria vascularis. The expression pattern of rat eag transcript in spiral ligament is complementary to the Na +,K +-ATPase distribution in the cochlear lateral wall. The localization of eag mRNA suggests that eag potassium channel may be produced in the corresponding cells. Considering the importance of the K + gradient in the cochlea, the result reported here suggests that eag channel may play a role in the control of K + fluxes in the spiral ligament.

Immunoreactivity of endothelins and endothelin receptor in the stria vascularis of the mouse cochlea

Immunoreactivities of endothelin-1, endothelin-3, endothelin receptor type A, and Na,K-ATPase were investigated in the stria vascularis of adult male WBB6F 1 +/+ mice and in that of W/W v mutants lacking strial intermediate cells. In the +/+ mice, electron microscopic immunoreactivity for the endothelins was seen on the rough endoplasmic reticulum, Golgi apparatus, cytoplasmic vesicles and lysosomes exclusively in the strial intermediate cells by the postembedment method. Immunoreactive endothelin receptor A was localized along the plasma membrane of strial marginal cells of both wild and mutant types although the immunoreactivity of the latter was much less than that of the former by the preembedment method. These findings suggest that the endothelins, which are produced in the strial intermediate cells, may play a role in the maintenance of the stria vascularis function in the +/+ mice. Since the plasma membrane of the marginal cells of the W/W v mice, which do not generate a high positive endocochlear potential, also showed immunoreactivity for Na,K-ATPase, it seems likely that the endothelins are involved in the activation of sodium pump of the strial marginal cells by mediation of endothelin receptor A. In addition, the role of lysosomes in the crinophagy of the endothelins in the strial intermediate cells is proposed in the +/+ mice.

Dye-coupling of melanocytes with endothelial cells and pericytes in the cochlea of gerbils.

Intercellular connections via gap junctions in the stria vascularis, which constitutes the lateral wall of the cochlear duct, were investigated by the Lucifer yellow microinjection method with the aid of a confocal laser microscope. The dye injected into an intermediate cell (melanocyte) diffused into capillary endothelial cells and pericytes as well as other intermediate cells, basal cells, and fibrocytes in the spiral ligament; whereas the dye injected into a marginal cell (epithelial cell) was confined to the injected cell. The observation of dye-coupling between intermediate cells and endothelial cells and pericytes makes likely the possibility that these cells work together to play a role in the specific function of the stria vascularis (i.e., production of the positive endocochlear potential and the endolymph) and adds endothelial cells and pericytes to the current "two-cell model" of the stria vascularis.

Effect of artificial endolymph injection into the cochlear duct on the endocochlear potential

We investigated the effect of acute endolymphatic hydrops on the positive endocochlear potential (+EP) and negative endocochlear potential (−EP). The +EP was measured in guinea pigs during injection (without outlet) and perfusion (with outlet) of artificial endolymph into the cochlear duct. The −EP was measured during anoxia after the injection or the perfusion had finished. Injection of artificial endolymph produced a slight transient increase in the +EP, and a significant decrease in the magnitude of the −EP. Chronic endolymphatic hydrops produces both +EP and −EP decrease. The +EP decrease in chronic endolymphatic hydrops may cause the chronic change of the inner ear. The +EP increase in acute endolymphatic hydrops may be caused by a shift of the basilar membrane. However, the mechanism of the `transient' +EP increase is not clear. The −EP decrease was not observed in animals whose cochlear duct was perfused with artificial endolymph. Therefore, the artificial endolymph itself did not cause the decrease in magnitude of the −EP. Dysfunction of the hair cells is a possible explanation for the −EP decrease but the mechanism of such a decrease is not clear in the present study. However, the results of this study support the notion that small increases in endolymphatic pressure below the resolution of recent measurements ( DeMott and Salt, 1997) can lead directly to a reduction of the −EP during hydrops. The animal model described here can eliminate the chronic effect of hydrops, therefore, this model is useful for investigations into the effect of hydrops itself on the inner ear and the mechanism of hearing loss in Ménière's disease.

An ATP-Dependent Inwardly Rectifying Potassium Channel, KAB-2 (Kir4.1), in Cochlear Stria Vascularis of Inner Ear: Its Specific Subcellular Localization and Correlation with the Formation of Endocochlear Potential

Cochlear endolymph has a highly positive potential of approximately +80 mV. This so-called endocochlear potential (EP) is essential for hearing. Although pivotal roles of K+ channels in the formation of EP have been suggested, the types and distribution of K+ channels in cochlea have not been characterized. Because EP was depressed by vascular perfusion of Ba2+, an inhibitor of inwardly rectifying K+ (Kir) channels, but not by either 4-aminopyridine or tetraethylammonium, we examined the expression of Kir channel subunits in cochlear stria vascularis, the tissue that is supposed to play the central role in the generation of positive EP. Of 11 members of the Kir channel family examined with reverse transcription-PCR, we could detect only expression of KAB-2 (Kir4.1) mRNA in stria vascularis. KAB-2 immunoreactivity was specifically localized at the basolateral membrane of marginal cells but not in either basal or intermediate cells. Developmental expression of KAB-2 in marginal cells paralleled formation of EP. Furthermore, deaf mutant mice (viable dominant spotting; WV/WV) expressed no KAB-2 in their marginal cells. These results suggest that KAB-2 in marginal cells may be critically involved in the generation of positive EP.