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Light cupula is an emerging concept accounting for positional nystagmus. It can be diagnosed when persistent geotropic direction-changing positional nystagmus (PG DCPN) is observed in a head-roll test. Although hypotheses explaining light cupula phenomenon such as "light debris", "lighter cupula", and "heavier endolymph" have been proposed, the mechanism underlying light cupula has not been clearly elucidated yet. In the present study, we proposed a new hypothesis accounting for light cupula, i.e., density difference between perilymph and endolymph could elicit characteristic PG DCPN in a head-roll test. We also discussed the mechanism how membranous canal containing endolymph became buoyant within the perilymphatic space under constant influence of gravity when the density of perilymph was higher than that of endolymph.

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This review is focused on the unusual composition of the endolymph of the inner ear and its function in mechanoelectrical transduction. The role of K(+) and Ca(2+) in excitatory influx, the very low Na(+), Ca(2+) and Mg(2+) concentrations of endolymph, stereocilia structure of hair cells and some proteins involved in mechanosensory signal transduction with emphasis on auditory receptors are presented and analyzed in more details. An alternative hypothetical model of ciliary structure and endolymph with a 'normal' composition is discussed. It is concluded that the unique endolymph cation content is more than an energy saving mechanism that avoids disturbing circulatory vibrations to achieve a much better mechanosensory resolution. It is the only possible way to fulfil the requirements for a precise ciliary mechanoelectrical transduction in conditions where pressure events with quite diverse amplitudes and duration are transformed into adequate hair cell membrane depolarizations, which are regulated by a sensitive Ca(2+)-dependent feedback tuning.

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OBJECTIVE: To examine whether the changes in endolymphatic ion concentrations were involved in hair cells degeneration in claudin-14 knockout (KO) mice (Cldn14-/-), we measured the endocochlear potential (EP) along with concentrations of K+, Na+, H+, or Ca2+ ([K]e, [Na]e, pHe, [Ca]e) in Cldn14-/-, in which hair cells were selectively damaged, and compared with measurements in wild type mice (Wt). METHODS: We used the Cldn14-/- from 3 weeks of age, in which the auditory brain responses (ABR) was severely diminished. Using double-barreled ion-selective microelectrodes, we measured [K]e, [Na]e, pHe, and [Ca]e in both Wt and Cldn14-/- at 8-10 weeks of age. RESULTS: (1) In Wt, the EP was +92mV. [K]e, [Na]e, pHe, and [Ca]e were 169mM, ∼1.0mM, 7.50, and 395nM, respectively. In the Cldn14-/-, the EP was +96mV. [K]e, [Na]e, pHe, and [Ca]e were 167mM, ∼1.0mM, 7.73, and 179nM, respectively. No significant differences in the above values were observed between Wt and Cldn14-/-. (2) A significant linear correlation between EP and [Ca]e (R=0.93) was observed for both Wt and Cldn14-/-, but no correlation was observed between EP and K+, Na+, or H+. CONCLUSION: These findings suggest that (1) the changes in endolymphatic ion concentrations might not be involved in hair cells degeneration in Cldn14-/-, (2) [Ca]e might be regulated by EP in both Wt and Cldn14-/-.

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In the cochlea, K(+) is essential for mechano-electrical transduction. Here, we explore cochlear structure and function in mice lacking K(+) channels of the two-pore domain family. A profound deafness associated with a decrease in endocochlear potential is found in adult Kcnk5(-/-) mice. Hearing occurs around postnatal day 19 (P19), and completely disappears 2 days later. At P19, Kcnk5(-/-) mice have a normal endolymphatic [K(+)] but a partly lowered endocochlear potential. Using Lac-Z as a gene reporter, KCNK5 is mainly found in outer sulcus Claudius', Boettcher's and root cells. Low levels of expression are also seen in the spiral ganglion, Reissner's membrane and stria vascularis. Essential channels (KCNJ10 and KCNQ1) contributing to K(+) secretion in stria vascularis have normal expression in Kcnk5(-/-) mice. Thus, KCNK5 channels are indispensable for the maintenance of hearing. Among several plausible mechanisms, we emphasize their role in K(+) recycling along the outer sulcus lateral route.

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Transplantation of neural stem cells (NSCs) into the cochlea to replace irreversibly damaged sensory epithelia is a potentially valuable remedy for hearing loss. Several mammalian stem cell lines are being successfully transplanted into, or migrated to, the endolymph (EL) fluids environment of the cochlea. However, the survival rate of transplanted cells is relatively low. This study focused on the effect of altering the potassium (K(+)) concentration of artificial EL on cell survival and apoptosis of olfactory bulb neural precursor cells (OB NPCs) in vitro. OB NPCs were prepared and placed in media for 24h, supplemented either with artificial EL, or artificial EL-like solutions of different K(+) concentrations. Survival, apoptotic features and ultrastructural changes in the cells are noted. Artificial EL-like solutions, especially with K(+) concentrations of 50mM or more, resulted in a series of necrotic or apoptotic events. Lower K(+) concentrations (30mM) decreased apoptosis and necrosis, improving the survival rate of cultured NPCs. Thus, it is conceivable that the external K(+) concentration in EL is a key environmental factor to regulate the survival of exogenous stem cells.

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OBJECTIVE: Endothelin has many biological activities, including regulation of the functions of the cochlear lateral wall. The present study aimed to analyse the expression of endothelin receptors in the cochlear lateral wall, and to investigate the significance of such receptors in maintaining the homeostatic environment of the cochlea. METHODS: The cochleae of healthy guinea pigs were fixed, decalcified, embedded in paraffin and serially sectioned. Expression of the endothelin receptor subunits A and B in the cochlear lateral wall was examined using an immunohistochemical technique. RESULTS: Different degrees of endothelin receptor subunit A and endothelin receptor subunit B like activity were found distributed in the cells of the cochlear lateral wall. CONCLUSION: These findings support the theory that endothelin, via its receptors, plays an important role in maintaining the homeostatic environment of the cochlea.

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OBJECTIVE: To investigate the relationship between endolymphatic hydrops and perilymphatic potassium. METHODS: 20 pigmented guinea pigs were used: 10 for scala vestibuli study and 10 for scala tympani study. Acute endolymphatic hydrops was produced by microinjection of an artificial endolymph into the scala media. Injections were performed in the second turn at rates up to 500 nl/min for a period of 10 min. The injection volume was up to 5 microl. Endocochlear potential (EP) was monitored during injections. Simultaneous with the injections, the potassium concentrations in scala vestibuli (K(SV)) or tympani (K(ST)) perilymph were measured with ion-sensitive double-barreled microelectrodes sealed into in the scalae in the 3rd turn with cyanoacrylate glue. RESULTS: For endolymphatic injections of

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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.

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The fractionation of metals from water to otolith is an area of research that has received relatively limited attention, especially in freshwater systems. The objectives of the present research were to study the metal partitioning between otolith and endolymph of two freshwater species: Lake trout (Salvelinus namaycush), and burbot (Lota lota). We also included the chemical analyses of water and blood from fish of the same species collected in the same area but during different years. These results provide insight regarding the partition of metals between water and fish. This is one of the first studies to provide a range of trace metal concentrations for endolymph and the growing otolith (both aragonite and vaterite) and to directly measure otolith-endolymph partition coefficients for freshwater fish. The trace elements (Mg, Sr, and Ba) most often used as otolith elemental tracers were the ones with the lowest uptake from water to blood. We found that endolymph and whole blood had similar metal concentrations, with Mg and Fe being the only elements enriched in whole blood. Results showed few significant differences in trace metal content between wild lake trout and burbot endolymph (except for K, Mg, and Ba), but significant differences existed between their aragonitic otoliths. These results suggest two different crystallization processes in these species or the presence of different proteins (and/or organic matrices) that would selectively influence elemental incorporation in the otoliths.

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Pituitary adenylate cyclase activating polypeptide (PACAP) is widely distributed in ocular tissues, including the lacrimal gland. PACAP has been shown to influence the activity of several exocrine glands, but its effects on the composition of the tear film are not known yet. Similarly, the presence of PACAP has already been shown in the inner ear, but it is not known whether PACAP influences the composition of the endolymph. The aim of the present study was to investigate whether systemic injection of PACAP has any modulatory effects on the protein composition of the tear film and endolymph using chip electrophoresis and mass spectrometry analysis. Tear and endolymph samples were collected from rats and chickens, respectively, at various time points after systemic injection of PACAP. Fluid samples were further processed for chip electrophoretic studies. No difference was found in the protein composition of the endolymph between control and PACAP-treated animals. In contrast, tear samples showed a marked difference after PACAP treatment. Proteins in the molecular range 50-70 kDa, which showed a different chip electropherogram profile in every PACAP-treated sample, were further analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. PACAP treatment induced a repression in certain keratins, while others were induced after PACAP injection. Furthermore, PACAP treatment decreased aldehyde dehydrogenase expression. The present study provides a base for further studies on the in vivo effects of PACAP on the composition of tear film. These investigations may have important clinical relevance because of the noninvasive sample collection, the correlation between tear proteins and ocular diseases, and the possible presence of biomarkers for both ophthalmological and systemic pathological conditions.

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OBJECTIVES: We determined the chemical composition of the human endolymphatic sac luminal fluid in a patient with Mondini dysplasia. METHODS: Four milliliters of endolymphatic sac luminal fluid was sampled in a 41-year-old patient who presented with bilateral Mondini dysplasia and underwent auditory brain stem implantation. Ion and protein concentrations, as well as the osmolarity of the fluid, were analyzed by means of biochemical automated procedures. RESULTS: The sodium, potassium, and protein concentrations and the osmolarity of the sample were 87 mmol/L, 24 mmol/ L, 33.5 g/L, and 258 mOsm/L, respectively. CONCLUSIONS: The composition of the endolymphatic sac luminal fluid found in our patient was similar to that determined in guinea pigs under physiological conditions, and very different from the usual composition of the cochleovestibular endolymph. Contamination of the cochleovestibular endolymphatic compartment by this sodium-rich, potassium-poor, and hypo-osmotic fluid might impair cochleovestibular function by altering the chemical composition of the cochleovestibular endolymph.

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The concentrations of free Na+, K+, Ca(+, and Cl(-)in endolymph and perilymph from the inner ear of the oyster toadfish, Opsanus tau, were measured in vivo using double-barreled ion-selective electrodes. Perilymph concentrations were similar to those measured in other species, while endolymph concentrations were similar to those measured previously in elasmobranch fish, though significantly different from concentrations reported in mammals. Perilymph concentrations (mean +/- std. dev.) were as follows: Na+, 129 mmol l(-1) +/- 20; K+, 4.96 mmol l(-1) +/- 2.67; Ca2+, 1.83 mmol l(-1) +/- 0.27; and Cl(-), 171 mmol l(-1) +/- 20. Saccular endolymph concentrations were Na+, 166 mmol l(-1) +/- 22; K+, 51.4 mmol l(-1) +/- 16.7; Ca2+, 2.88 mmol l(-1) +/- 0.27; and Cl(-), 170 mmol l(-1) +/- 12; and semicircular canal (utricular vestibule) endolymph concentrations were Na+, 122 mmol l(-1) +/- 15; K+, 47.7 mmol l(-1) +/- 13.2; Ca2+, 1.78 mmol l(-1) +/- 0.48; Cl(-), 176 mmol l(-1) +/- 27. The relatively high concentrations of Ca2+ and Na+ in the endolymph may have significant implications for the physiological function of the mechanoelectrical transduction channels in the vestibular hair cells of fish compared to those of their mammalian counterparts.

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The outer sulcus cells are epithelial cells covering the luminal side of spiral sulcus of cochlea. It has been suggested that outer sulcus cells contribute to cation absorption from the lumen of the cochlea. We investigated the electrical properties and the effects of endothelin-1 (ET-1) on the outward potassium currents in mouse outer sulcus cells using a whole-cell patch clamp technique. The cell capacitance was 3.16+/-0.66 pF (n =35) and the resting membrane potential was -98.4+/-1.6 mV (n=6) in extracellular fluid bath solution. The outward K+ currents were activated by depolarizing pulses more positive than -60 mV, and was sensitive to TEA (10 mM). Tail current analysis revealed that it was primarily K+ selective. Application of ET-1 caused a decrease of outward potassium currents within seconds, whereas treatment with BQ123, a competitive inhibitor of the ET type-A receptor, counteracted the inhibitory effect of ET-1. These results suggest that ET-1 inhibits outward potassium currents through the activation of ET type-A receptor. ET-1 may play an important role in maintaining the ionic homeostasis of endolymph.

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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.

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Endolymph, the extracellular solution in cochlea, contains 150 mM K(+) and exhibits a potential of approximately +80 mV relative to neighboring extracellular spaces. This unique situation, essential for hearing, is maintained by K(+) circulation from perilymph to endolymph through the cochlear lateral wall. Recent studies have identified ion-transport molecules involved in the K(+) circulation and their pathophysiological relevance.

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The aim of the present work was to examine the modifications of the organic composition of fish endolymph under environmental conditions (day-night cycle, starvation and Cl2-stress) known to modify otolith growth. Endolymph electrophoretic patterns were compared. An antibody raised against the trout otolith organic matrix allowed examining the variations of organic matrix precursors in the endolymph under the above conditions. Western blot analysis showed bands around 60-80 kDa. A 50% decrease of immunolabelling was observed during the night whereas increases were seen after starvation (factor 3) or stress (factor 2) suggesting that these variations could be related to the organic matrix deposit. A factor retarding in vitro CaCO3 crystallization (FRC) was shown to co-precipitate with endolymph proteins and its apparent molecular mass (determined by measuring the activity after electro elution of gel electrophoresis) was estimated around 20 kDa. The FRC activity was stable during day-night cycle whereas it decreased by 70% and nearly 100% under starvation and stress respectively. These results suggest that the FRC, although retarding in vitro crystallization, plays a major role in the process of otolith calcification and that the decreases measured after starvation and stress are responsible for the decreases of the otolith growth. The variations of these two parameters (precursors and FRC) could contribute for the changes in the microstructure of the otolith.

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BACKGROUND: Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-). METHODS: Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry. RESULTS: Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2. CONCLUSIONS: These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome.

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The endolymphatic sac is a non-sensory organ of the inner ear. It is connected to the endolymphatic compartment that is filled with endolymph, a potassium-rich fluid that bathes the apical side of inner ear sensory cells. The main functions ascribed to the endolymphatic sac are the regulation of the volume and pressure of endolymph, the immune response of the inner ear, and the elimination of endolymphatic waste products by phagocytosis. Functional alteration of these functions, leading to deficient endolymph homeostasis and/or altered inner ear immune response, may participate to the pathophysiology of Ménière's disease, an inner ear pathology that causes episodes of vertigo, sensorineural hearing loss and tinnitus, and is characterized by an increase in volume of the cochleo-vestibular endolymph (endolymphatic hydrops).

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The soluble organic matrix (OM) of various biominerals (red coral skeleton, oyster shell, sea urchin test, turbot otolith, chicken eggshell) was extracted after demineralization with acetic acid. The protein content of the OM varies strongly from 0.02 to 1.6 microg/mg biomineral whereas proteoglycans present less variations (from 0.7 to 1.4 microg/mg biomineral). Electrophoresis of biominerals OM shows differences in their protein pattern although several bands are present in all matrices. OM of all biominerals shows carbonic anhydrase activity but no activity was detectable in the endolymph. OM of all biominerals also displays an anticalcifying activity. After separation of the OM extracts by chloroform-methanol, 80% of the anticalcifying activity was found in the methanol phase except in the urchin test. After OM precipitation with trichloracetic acid, 70% of the activities was found in the supernatants. Partial biochemical characterization suggests that the anticalcifying factor is a polyanionic and water-soluble molecule, which could be proteoglycans. The endolymph surrounding the otolith also displays an anticalcifying activity although its inhibitous activity was 50 times lower than that of the otolith OM. However, the anticalcifying activity of the endolymph is assumed by a proteic structure (80% activity precipitated with TCA treatment). Our results suggest that both carbonic anhydrase and anticalcifying activities are widespread and play a significant role in the regulation of biomineral formation. Results are discussed in relation to the calcification process that takes place at the fluid-mineral interface.

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