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Despite well-documented neurotoxic and ototoxic properties, styrene remains commonly used in industry. Its effects on the cochlea have been extensively studied in animals, and epidemiological and animal evidence indicates an impact on balance. However, its influence on the peripheral vestibular receptor has yet to be investigated. Here, we assessed the vestibulotoxicity of styrene using an in vitro model, consisting of three-dimensional cultured newborn rat utricles filled with a high‑potassium (K + ) endolymph-like fluid, called "cysts". K + entry in the cyst ("influx") and its exit ("efflux") are controlled by secretory cells and hair cells, respectively. The vestibular epithelium's functionality is thus linked to K + concentration, measured using a microelectrode. Known inhibitors of K + efflux and influx validated the model. Cysts were subsequently exposed to styrene (0.25; 0.5; 0.75 and 1 mM) for 2 h or 72 h. The decrease in K + concentration measured after both exposure durations was dose-dependent, and significant from 0.75 mM styrene. Vacuoles were visible in the cytoplasm of epithelial cells from 0.5 mM after 2 h and from 0.25 mM after 72 h. The results presented here are the first evidence that styrene may deregulate K + homeostasis in the endolymphatic space, thereby altering the functionality of the vestibular receptor., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Specific pharmacological blockade of KCNQ (Kv7) channels with XE991 rapidly (within 20 min) and profoundly alters inner ear gravity receptor responses to head motion (Lee et al., 2017). We hypothesized that these effects were attributable to the suppression of K + secretion following blockade of KCNQ1-KCNE1 channels in vestibular dark cells and marginal cells. To test this hypothesis, K + secretion was independently inhibited by blocking the Na + -K + -2Cl - cotransporter (NKCC1, Slc12a2) rather than KCNQ1-KCNE1 channels. Acute blockade of NKCC1 with ethacrynic acid (40 mg/kg) eliminated auditory responses (ABRs) within approximately 70 min of injection, but had no effect on vestibular gravity receptor function (VsEPs) over a period of 2 h in the same animals. These findings show that, vestibular gravity receptors are highly resistant to acute disruption of endolymph secretion unlike the auditory system. Based on this we argue that acute suppression of K + secretion alone does not likely account for the rapid profound effects of XE991 on gravity receptors. Instead the effects of XE991 likely require additional action at KCNQ channels located within the sensory epithelium itself., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Eukaryotic cells exhibit negative resting membrane potential (RMP) owing to the high K(+) permeability of the plasma membrane and the asymmetric [K(+)] between the extracellular and intracellular compartments. However, cochlear fibrocytes, which comprise the basolateral surface of a multilayer epithelial-like tissue, exhibit a RMP of +5 to +12 mV in vivo. This positive RMP is critical for the formation of an endocochlear potential (EP) of +80 mV in a K(+)-rich extracellular fluid, endolymph. The epithelial-like tissue bathes fibrocytes in a regular extracellular fluid, perilymph, and apically faces the endolymph. The EP, which is essential for hearing, represents the potential difference across the tissue. Using in vivo electrophysiological approaches, we describe a potential mechanism underlying the unusual RMP of guinea pig fibrocytes. The RMP was +9.0 ± 3.7 mV when fibrocytes were exposed to an artificial control perilymph (n = 28 cochleae). Perilymphatic perfusion of a solution containing low [Na(+)] (1 mM) markedly hyperpolarized the RMP to -31.1 ± 11.2 mV (n = 10; p < 0.0001 versus the control, Tukey-Kramer test after one-way ANOVA). Accordingly, the EP decreased. Little change in RMP was observed when the cells were treated with a high [K(+)] of 30 mM (+10.4 ± 2.3 mV; n = 7; p = 0.942 versus the control). During the infusion of a low [Cl(-)] solution (2.4 mM), the RMP moderately hyperpolarized to -0.9 ± 3.4 mV (n = 5; p < 0.01 versus the control), although the membranes, if governed by Cl(-) permeability, should be depolarized. These observations imply that the fibrocyte membranes are more permeable to Na(+) than K(+) and Cl(-), and this unique profile and [Na(+)] gradient across the membranes contribute to the positive RMP.

Slc26a4 (Δ/Δ) mice are deaf, develop an enlarged membranous labyrinth, and thereby largely resemble the human phenotype where mutations of SLC26A4 cause an enlarged vestibular aqueduct and sensorineural hearing loss. The enlargement is likely caused by abnormal ion and fluid transport during the time of embryonic development, however, neither the mechanisms of ion transport nor the ionic composition of the luminal fluid during this time of development are known. Here we determine the ionic composition of inner ear fluids at the time at which the enlargement develops and the onset of expression of selected ion transporters. Concentrations of Na(+) and K(+) were measured with double-barreled ion-selective electrodes in the cochlea and the endolymphatic sac of Slc26a4 (Δ/+), which develop normal hearing, and of Slc26a4 (Δ/Δ) mice, which fail to develop hearing. The expression of specific ion transporters was examined by quantitative RT-PCR and immunohistochemistry. High Na(+) (∼141 mM) and low K(+) concentrations (∼11 mM) were found at embryonic day (E) 16.5 in cochlear endolymph of Slc26a4 (Δ/+) and Slc26a4 (Δ/Δ) mice. Shortly before birth the K(+) concentration began to rise. Immediately after birth (postnatal day 0), the Na(+) and K(+) concentrations in cochlear endolymph were each ∼80 mM. In Slc26a4 (Δ/Δ) mice, the rise in the K(+) concentration occurred with a ∼3 day delay. K(+) concentrations were also found to be low (∼15 mM) in the embryonic endolymphatic sac. The onset of expression of the K(+) channel KCNQ1 and the Na(+)/2Cl(-)/K(+) cotransporter SLC12A2 occurred in the cochlea at E19.5 in Slc26a4 (Δ/+) and Slc26a4 (Δ/Δ) mice. These data demonstrate that endolymph, at the time at which the enlargement develops, is a Na(+)-rich fluid, which transitions into a K(+)-rich fluid before birth. The data suggest that the endolymphatic enlargement caused by a loss of Slc26a4 is a consequence of disrupted Na(+) transport.

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., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Partial labyrinthectomy can result in maintenance of hearing under certain circumstances, and the mechanism of the hearing impairment caused by labyrinthectomy is unclear. We hypothesized that disruption of the membranous labyrinth results in electrical leakage and electrolyte imbalance. This study investigated the change in cochlear function by measurement of endocochlear potential (EP) and potassium concentration ([K(+)]) caused by vestibular labyrinth destruction in the acute phase. Hartley guinea pigs underwent lateral semicircular canal (LSCC) transection with suctioning of the perilymph, ampullectomy, or destruction of the LSCC, superior SCC, and lateral part of the vestibule. The EP and [K(+)] were monitored using double-barreled ion-selective microelectrodes in the second turn of cochlea. The EP showed little to mild change after LSCC transectioning or ampullectomy, but declined variously and drastically after vestibulotomy. The EP did not recover but [K(+)] partially recovered after vestibulotomy. Disturbance of the mechanism of cochlear function caused by vestibular labyrinth destruction may involve reduction in the [K(+)] concentration in the endolymph., (2009 Elsevier B.V. All rights reserved.)

A total of 4668 patients from four university hospitals, all representing cases with inner ear tumours or other inner ear disorders were evaluated. No primary cancer or melanoma could be detected. These results are in compliance with publications of other institutions discussing their own results in detecting inner ear pathologies. This finding is unusual and leads to the assumption that the epithelial cells of the auditory inner ear may be protected from malignant transformations. The aim of this study is to investigate the reasons for this supposed anticarcinogenic privilege of the auditory inner ear. The auditory inner ear has some unique features in the organism by reversing the relationship between intra- and extracellular ion concentrations of potassium and sodium in the endolymph. Furthermore it has a physiologically extremely high membrane potential and an unusual transmembrane and extracellular circulation of potassium ions. How these aspects contrast with the corresponding features at the carcinoma site, will be investigated. This comparison will be further deepened by reviewing the published actions of antitumoral substances in connection with these issues. On the basis of this comparative study one might speculate that the auditory inner ear is privileged by an anticarcinogenic principle and that the specific features of membrane potential, certain potassium channels and the unique transmembrane as well as extracellular potassium movements may play a decisive role in it.

Cochlear marginal cells and vestibular dark cells transport potassium into the inner ear endolymph, a potassium-rich fluid, the homeostasis of which is essential for hearing and balance. We have formulated an integrated mathematical model of ion transport across these epithelia that incorporates the biophysical properties of the major ion transporters and channels located in the apical and basolateral membranes of the constituent cells. The model is constructed for both open- and short-circuit situations to test the extremes of functional capacity of the epithelium and predicts the steady-state voltages, ion concentrations, and transepithelial currents as a function of various transporter and channel densities. We validate the model by establishing that the cells are capable of vectorial ion transport consistent with several experimental measurements. The model indicates that cochlear marginal cells do not make a significant direct contribution to the endocochlear potential and illustrates how changes to the activity of specific transport proteins lead to reduced K(+) flux across the marginal and dark cell layers. In particular, we investigate the mechanisms of loop diuretic ototoxicity and diseases with hearing loss in which K(+) and Cl(-) transport are compromised, such as Jervell and Lange-Nielsen syndrome and Bartter syndrome, type IV, respectively. Such simulations demonstrate the utility of compartmental modeling in investigating the role of ion homeostasis in inner ear physiology and pathology.

The endolymph fills the lumen of the inner ear membranous labyrinth. Its ionic composition is unique in vertebrates as an extracellular fluid for its high-K(+)/low-Na(+) concentration. The endolymph is actively secreted by specialized cells located in the vestibular and cochlear epithelia. We have investigated the early phases of endolymph secretion by measuring the endolymphatic K(+) concentration in the chicken vestibular system during pre-hatching development. Measurements were done by inserting K(+)-selective microelectrodes in chicken embryo ampullae dissected at different developmental stages from embryonic day 9 up to embryonic day 21 (day of hatching). We found that the K(+) concentration is low (<10mM/L) up to embryonic day 11, afterward it increases steeply to reach a plateau level of about 140 mM/L at embryonic day 19--21. We have developed a short-term in vitro model of endolymph secretion by culturing vestibular ampullae dissected from embryonic day 11 chicken embryos for a few days. The preparation reproduced a double compartment system where the luminal K(+) concentration increased along with the days of culturing. This model could be important for (1) investigating the development of cellular mechanisms contributing to endolymph homeostasis and (2) testing compounds that influence those mechanisms.

Cochlear endolymph contains 150 mm K+ and has a highly positive potential of approximately +80 mV. The specialized ionic composition and high potential in endolymph are essential for hearing and maintained by circulation of K+ from perilymph to endolymph through the cochlear lateral wall. Various types of K+ channel such as Kir4.1 and KCNQ1/KCNE1 are expressed in stria vascularis of the lateral wall and play essential roles in K+ circulation. In this study, we examined a distribution of another K+ channel, Kir5.1, and found it specifically expressed in the spiral ligament of the cochlear lateral wall. Specific immunoreactivity for Kir5.1 was detected in type II, IV and V fibrocytes of the ligament and spiral limbus, all of which are directly involved in K+ circulation. Kir5.1 was not found in either type I or III fibrocytes. Although Kir5.1 assembles with Kir4.1 to form a functional Kir channel in renal epithelia and retinal Müller cells, double-immunolabelling revealed that they were expressed in distinct regions in the cochlea lateral wall, i.e. Kir4.1 only in stria vascularis vs. Kir5.1 in spiral ligament. During development, the expression of Kir5.1 subunits started significantly later than Kir4.1 and was correlated with the 'rapid' phase of the elevation of endocochlear potential (EP). Kir5.1 and Kir4.1 channel-subunits may therefore play distinct functional roles in K+ circulation in the cochlear lateral wall.

The endolymphatic sac (ES) is believed to be the locus for endolymph volume regulation in the inner ear. It has recently been shown that induced endolymph volume changes in the cochlea result in anatomical changes in the ES, suggesting that function of the sac varies according to endolymph volume status. In the present study we have recorded luminal concentrations of K(+) and Na(+) from the ES and the endolymphatic sac potential (ESP) during cochlear endolymph volume changes. ES recordings were made by an extradural approach, thereby preserving normal cerebrospinal fluid resting pressure. Cochlear endolymph volume changes were generated by performing injections or withdrawals through a pipette inserted into endolymph by a round window approach. The pre-treatment concentrations of K(+) and Na(+) in the ES were found to be 8.4 mM (S.D. 3.3, n=8) and 128. 6 mM (S.D. 18.4, n=10) respectively, and the mean ESP was 14.4 mV (S. D. 5.2, n=18). Endolymphatic injections were found to produce a sustained increase in the K(+) content of the ES by an average of 19. 9 mM and to decrease Na(+) by 30.7 mM measured 50 min after the start of injection. The time for K(+) increase to occur was found to correlate with the injected volume, with larger injected volumes producing a more rapid increase. Endolymphatic withdrawals were found to induce a slow decline in endolymphatic K(+) by an average of 3.4 mM measured at 50 min after withdrawal, although no significant change of Na(+) was detected. Volume-induced ESP changes were highly variable. Injections produced a small increase in the mean ESP and withdrawals produced a small decrease but neither change was statistically significant and some animals showed potential changes in the opposite direction. These data show that a change in cochlear endolymph volume status results in a physiologic response of the ES which is sustained for a considerable period. If the ES plays a part in the restoration of normal endolymph volume, this process appears to proceed slowly, based on the prolonged time courses of ionic changes observed.

The present study was designed to clarify the chronological developmental process of monovalent ions (Na(+), K(+), Cl(-)) in the endolymph of the mouse in relation to the development of the endocochlear potential (EP). The EP and ionic concentrations were measured simultaneously with the ion-sensitive double-barreled microelectrodes from the scala media of the basal turn. The EP increased abruptly 7 days after birth (DAB) and reached approximately 80 mV 14 DAB. In the earliest postnatal days, the endolymphatic Na(+) concentration was significantly higher than that in adult mice, however, the K(+) and the Cl(-) concentrations were lower. The concentrations of all the monovalent ions in endolymph reached adult levels at 7 DAB when the EP was still under 20 mV. These data strongly suggest the presence of a different mechanism between the production of monovalent ions, especially of high K(+) in the endolymph and that of EP., (Copyright 2000 S. Karger AG, Basel)

DFN3, an X chromosome-linked nonsyndromic mixed deafness, is caused by mutations in the BRN-4 gene, which encodes a POU transcription factor. Brn-4-deficient mice were created and found to exhibit profound deafness. No gross morphological changes were observed in the conductive ossicles or cochlea, although there was a dramatic reduction in endocochlear potential. Electron microscopy revealed severe ultrastructural alterations in cochlear spiral ligament fibrocytes. The findings suggest that these fibrocytes, which are mesenchymal in origin and for which a role in potassium ion homeostasis has been postulated, may play a critical role in auditory function.

The stria vascularis in the cochlea generates the endocochlear potential (EP) and secretes K+-rich endolymph; both are indispensable for normal sound transduction by hair cells. K+ conductance in the intermediate cell, one of the several types of cells constituting the stria vascularis, was investigated by the whole-cell patch-clamp technique. Inwardly-rectifying K+ (Kir) currents were the major currents observed. The currents were inhibited dose-dependently by Ba2+, quinine, verapamil and Cs+, but not by tetraethylammonium (20 mM), 4-aminopyridine (5 mM) or Cd2+ (1 mM). The similarity between the effect of inhibitors on Kir currents and on the EP (Takeuchi et al., Hearing Res., 101 (1996) 181-185) suggests a direct contribution of the Kir conductance to the generation of the EP.

K+ effluxed from outer hair cells and their nerves is thought to flow laterally to strial marginal cells for recycling into scala media. Observations reported here provide evidence that K+ effluxed from inner hair cells and inner radial nerves travels medially through border cells, inner sulcus cells (ISCs), limbal fibrocytes and interdental cells (IDCs) for return to endolymph. Morphologic features of ISCs in the medial route resembled those of Hensen and Claudius cells in the lateral indicating an ion transport role for ISCs like that of Hensen and Claudius cells. Na,K-ATPase in plasmalemma of IDCs testified to their capacity to resorb and transport K+ through their known gap junctions. IDCs were differentiated into three subgroups. The most lateral IDCs formed short and long columns. Long columns contacted the medialmost ISC inferiorly and the undersurface of the tectorial membrane superiorly providing thereby a potential transcellular route for K+ transit from ISCs to endolymph. Short columns faced inner sulcus below and tectorial membrane above and accordingly possessed cells with opposite polarity at the bottom and top of the column. Short columns thus appeared situated to resorb electrolytes from limbal stroma for release into inner sulcus and beneath tectorial membrane at opposite ends of the column. The central IDCs were positioned for resorbing and transporting K+ effluxing from the Na,K-ATPase-rich stellate fibrocytes which spread toward the IDCs from near the inner sulcus. The most medial IDCs lined cuplike invaginations near the attachment of Reissner's membrane and lay apposed to light fibrocytes located between supralimbal fibrocytes and the medial IDCs. Content of Na,K-ATPase and position in the K+ transport route likened the limbal stellate fibrocytes to the spiral ligament type II fibrocytes and supralimbal fibrocytes to suprastrial fibrocytes in the lateral wall. From content of creatine kinase and position in the transport path, limbal light fibrocytes appeared analogous to spiral ligament type I fibrocytes. The additional finding that limbal fibrocytes showed unchanged or upregulated Na,K-ATPase immunoreactivity in aged gerbils with strial atrophy provided further evidence for an independent medial transport route and for the survival of inner hair cells in presbyacusis.

Objective: To investigate the effect of acoustic overstimulation on concentrations of cations in cochlear endolymph and analyze the relationship between the ionic changes in endolymph and the hearing loss., Methods: The endocochlear potentials (EP), K+, Na+ and Ca2+ concentration in cochlear endolymph were examined in vivo for normal and 167 +/- 2 dB SPL impulse noise exposed groups of guinea pig bored on time course by means of double-barreled ion-selective microelectrodes. Brain stem auditory evoked potential (BAEP) was used to evaluate the auditory function. Ca(2+)-ATPase activity was demonstrated cytochemically in the lateral cochlear wall as induced by Ando et al with slight modification., Results: The K+ and Ca2+ concentration exhibited significant changes in 8-hour groups (P < 0.05 for K+ and P < 0.01 for Ca2+). Then the K+ concentration was eventually resumed to the initial levels in accordance with the EP recovery in 7 days, while during the same period Ca2+ concentration was always significantly higher than that in control group (P < 0.01). The BAEP threshold shifts were correlated well with changes in ionic concentrations, especially Ca2+ (P < 0.001), in the endolymph induced by acoustic trauma. Although the normal positive EP was observed 7 days after noise exposure, the function of the vascular stria was not completely restored as revealed by the fact that the Ca(2+)-ATPase was diffused to the apical membrane surface., Conclusions: Endolymph compartment intrinsic mechanism for maintaining ionic composition is seriously deteriorated after high impulse noise stimulation. The changes of the unique environment of endolymph may play an important role in the mechanism of sensorineural hearing loss induced by acoustic trauma.

Membrane currents in marginal cells cultured from rat stria vascularis were studied using the whole-cell patch-clamp technique. Two types of voltage-dependent whole-cell currents were observed in the voltage range from -150 mV to +50 mV: an outwardly rectifying current and an inwardly rectifying current. The outwardly rectifying current, which was activated by depolarizing pulses more positive than -30 mV, was sensitive to TEA (20 mM) and relatively not to Ba2+ (0.5 mM). Tail current analysis revealed that the outward currents were primarily K+-selective. The conductance of the current was half-maximal at 0.5 mV and a substantial portion of current was not inactivated by the depolarizing prepulses from -30 mV to +20 mV. The inwardly rectifying current with rapid exponential activation was observed with hyperpolarizing voltage pulses. The zero-current potential of this current was dependent on extracellular K+ concentration. In contrast to the outwardly rectifying current, this current was blocked by extracellular application of Ba2+, not by TEA. The conductance of this current increased with the increase in external K+ concentration. Our data suggest that marginal cells cultured from rat stria vascularis express at least two types of voltage-dependent K+ currents which may serve as K+ secretory pathways into endolymph.

Localization of the immunoreactivity in the lateral wall of the rabbit cochlear duct was examined using a post-embedding immunogold method with a polyclonal antiserum raised against the rabbit parotid Na-K-Cl cotransporter. In the stria vascularis, the labeling was significant on the basolateral membrane infolding of marginal cells, whereas no labeling was seen on the luminal membrane of these cells. Immunoreactivity was also detected on the cell membranes of various other cells. These include fibrocytes of the spiral ligament and the spiral prominence, and vascular endothelial cells in the stria vascularis and the spiral ligament. In contrast, virtually no gold particles were seen on the membrane of intermediate cells, basal cells of the stria vascularis, the epithelial cells of the spiral prominence, or Reissner's membrane. Our result on the localization of the Na-K-Cl cotransporter in marginal cells is consistent with electrophysiological studies (Wangemann et al. (1995) Hear. Res. 84, 19-29). Our result on fibrocytes is discussed in relation to K+ circulation into endolymph from perilymph (Schulte and Steel (1994) Hear. Res. 78, 65-76).

Previous work has shown that the endocochlear potential (EP) decreases with age in the gerbil. Concomitant with the EP decrease is an age-related loss of activity of Na,K-ATPase in the lateral wall and stria vascularis. We hypothesized that the EP decrease is associated with a similar decrease in the endolymphatic potassium concentration [Ke+]. This hypothesis was tested using double-barrelled, K(+)-selective electrodes introduced into scala media through the round window in young and quiet-aged gerbils. Results show that the means (+/- S.D.) of the [Ke+] in young and aged gerbils were not significantly different (178.2 +/- 14.2 mM and 171.2 +/- 34.4 mM, respectively), although the intersubject variability was much greater in the aged animals than in the young. These values of [Ke+] are slightly higher than those found for other mammals and may reflect the higher plasma osmolarity found in the gerbil. The concentration of perilymphatic potassium [Kp+] in scala tympani at the round window was also similar for the young and aged groups (3.57 +/- 1.17 mM and 4.18 +/- 2.03 mM, respectively). On the other hand, mean EP values in the young and aged gerbils were 92.0 +/- 5.7 mV and 64.8 +/- 15.8 mV, respectively and were statistically different (P < 0.001). Overall, EP and [Ke+] showed little correlation (R2 = 0.23), except that when [Ke+] fell below 150 mM, the EP was always less than 60 mV. An analysis of the chemical potential for Ke+ with respect to Kp+ shows that it was similar for young and aged gerbils (overall mean of 103.1 +/- 13.7 mV) and remained constant with respect to the EP, in spite of an overall electrochemical potential of Ke+ that varied from 120 to 210 mV. Thus, the system maintains Ke+ homeostasis at the expense of the EP, even when the EP is on the verge of collapse.

This study evaluated the electrochemical potentials and potassium concentration (Ck+) profiles in the perilymph, endolymph, marginal cells, and spiral ligament of adrenalectomized rats in which endogenous corticosteroids had been removed. Electrochemical potentials recorded at the four cochlear sites were not affected by adrenalectomy (ADX). Ck+ was greater in the endolymph of the ADX animals as compared to control animals. Additionally, there was an increase of Ck+ in the marginal cells, perilymph, and spiral ligament tissues of the ADX animals as compared to control animals, although the observed increases were not statistically significant. In a previous study (Ma et al., 1995a), it was found that potassium levels in the blood plasma of ADX animals were higher than those identified in normal rats; thus, ADX may have a systemic effect on Ck+ that is detectable in both tissues and fluids within the cochlea. Even though Ck+ was elevated within the cochlea in the ADX model, the functional response of the inner ear, as assessed electrophysiologically, was not altered.

The effects of removal of endogenous corticosteroids via bilateral adrenalectomy in combination with noise exposure (30 min at 100 dB) were determined by recording compound action potential (CAP) and endocochlear potentials (EP), and by measuring potassium concentrations (K+e) within the endolymph. Thirty-eight Long-Evans rats were divided into groups according to experimental treatments: adrenalectomy (ADX) or non-ADX and noise exposure or non-noise exposure. CAP thresholds, EP and K+e values were subjected to repeated-measures analysis of variance with group and time as factors classifying the measurements. Noise exposure resulted in significant elevations of CAP thresholds in both the ADX and non-ADX animals, but had no effect on either EP or endolymphatic K+e. Recovery was noted during all post-exposure measurement periods and was significantly faster for ADX animals. EP and K+e did not change during or after noise exposure. ADX animals showed a non-significant reduction of EP and a statistically significant increase of K+e during all measurement periods as compared to non-ADX animals.

Morphologic similarities between strial marginal cells and vestibular dark cells have long been recognized and it has long been accepted that both of these cell types are involved in the secretion of K+ into endolymph. Functional similarities of these two epithelia however, were considered unlikely as long as strial marginal cells were assumed to generate the endocochlear potential which has no equivalent in the vestibular labyrinth. The recently introduced concept that strial marginal cells transport K+ but that the mechanism for the generation of the endocochlear potential is located in another cell type provided the basis to hypothesize that ion transport mechanisms and their regulation are similar in vestibular dark and strial marginal cells. The present review compiles evidence in support of this hypothesis.

In vitro, the frog semicircular canal secretes an endolymph-like fluid, i.e. a K-rich, positively polarized fluid. This electrogenic K secretion involved basolateral Na+, K(+)-ATPase and Na-K-Cl co-transporter and a luminal protein possessing sulfhydryl groups blocked by N-ethylmaleimide. Streptomycin, an ototoxic antibiotic, is known to block the non-specific mechano-dependent channels in the sensory cells of the ampulla of the semicircular canal. The aim of the present study was to investigate the possible effect of streptomycin on the K fluxes in the ampulla of the semicircular canal. The posterior frog semicircular canal was isolated and the lumen was filled with perilymph-like solution containing or not containing 0.5 mM streptomycin. The luminal K concentration and the transepithelial potential were measured and the unidirectional K fluxes calculated. The K influxes (into the lumen, pmoles/min/mm2) were 114 +/- 25.9 and 111 +/- 3.2 (mean +/- SE, n = 3) in the absence and presence of streptomycin, respectively. The transepithelial potential was not altered (4.0 +/- 1.08 mV versus 3.4 +/- 1.03 mV, n = 3). When ouabain (10(-3)M) was added to the basolateral solution together with luminal streptomycin, no further alteration occurred as compared with the effect of ouabain alone. These results suggest that in these conditions, the sensory organ does not have a major role in the endolymphatic K secretion in the ampulla of the frog semicircular canal.

Using 110 male albino guinea pigs, and applying electrophysiological methods, we investigated the effects of 4 kHz tone at moderate sound pressure level on the inner ear at different exposure times (6, 12, 24 and 48 h). Longer than 12 h exposure to 4 kHz tone had a considerable effect on the hearing, inducing a decrease in the maximum output voltage of cochlear microphonics and an increase in the K+ concentration in the endolymph.

Using 97 male albino guinea pigs and applying electrophysiological methods, the effects of a 6 kHz tone were investigated at a moderate sound pressure level to the inner ear during a 24-h exposure time. Following exposure to the 6 kHz tone at 90 dB, cochleas showed decreased maximum output voltage of cochlear microphonics (CM) and action potential (AP). In the endolymph, K+ ion and Na+ ion concentrations remained unchanged during 40 min anoxia and 90 dB tone exposure. At 80 dB sound exposure decreases in maximum output voltage of CM and AP and decreases in the absolute value of EP could not be detected. Endolymph K(+)-ion Na(+)-ion concentrations were also unchanged. These findings indicate that diffusion potentials are induced at the same time as decreases of maximum output voltage in CM induced by exposure to sound at 90 dB.

The endocochlear potential (EP), and the concentration of K+, Na+ and Cl- were measured simultaneously in endolymph of guinea pigs. The EP was 85.6 +/- 0.8 mV in normal guinea pigs, 90.7 +/- 0.8 mV in the kanamycin-treated animals, and 91.6 +/- 1.2 mV in those treated with nitrogen mustard-N-oxide (NMNO). Thirty minutes after the onset of anoxia, the EP (negative EP) was -29.3 +/- 1.0 mV in the normal group, -0.2 +/- 1.0 mV in the kanamycin-treated group, and -1.9 +/- 1.3 mV in the NMNO-treated group. The permeability coefficients of K+ (Pk), Na+ (Pna) and Cl- (Pcl) across the endolymph-perilymph barrier during the period of 20-30 min after the onset of anoxia in the normal group were (341.6 +/- 38.2) x 10(-9) cm3 sec-1, (53.0 +/- 8.1) x 10(-9) cm3 sec-1 and (111.8 +/- 27.2) x 10(-9) cm3 sec-1, respectively. Pk was decreased in the kanamycin- and NMNO-treated groups. Pna did not differ between the normal and treated groups. Pcl was increased in the kanamycin- and NMNO-treated groups. The K+:Na+:Cl- permeability ratio was 1:0.16:0.32 in the normal group, 1:1.12:11.6 in the kanamycin-treated group, and 1:0.44:5.60 in the NMNO-treated group. The results indicate that the lack of development of a negative EP in the kanamycin- and NMNO-treated guinea pigs was attributable to the increased Pcl and the decreased Pk across the endolymph-perilymph barrier, probably the organ of Corti, during anoxia.

The endolymphatic potential is assumed to result from active K transport into the endolymphatic compartment and passive K diffusion in the opposite direction. However, in several in vivo experiments, changes in the endolymphatic potential differed from those in the endolymphatic K concentration. Moreover, in in vitro experiments, a negative endolymphatic potential was observed in the presence of ouabain without K gradient between the two compartments. These observations suggest that the coupling between the K transport and the genesis of the endolymphatic potential is not tight. Several factors may separately influence the endolymphatic potential and the K transport such as the acid-base equilibrium, the integrity of Reissner's membrane, the hormonal status, and the Na transport.

1. The mechanisms of K+ secretion into endolymph were studied on a preparation of isolated semicircular canal with different pharmacological inhibitors. Three periods of 5 or 30 min were performed, the first as control, the second in the presence of the drugs added to the apical or the basolateral bathing solution, and the third as recovery. Apical fluid was sampled at the beginning and the end of each period, transepithelial potential was recorded, Na+, K+, and Cl- concentrations, and K+ efflux, with 86Rb+ as a tracer, were measured and K+ fluxes were calculated. 2. When both sides of the epithelium were bathed with perilymph-like solution, the epithelium absorbed Na+, secreted K+, and generated a lumen positive potential. 3. The ATPases inhibitors, ouabain (10(-5) and 10(-3) M) and N-ethylmaleimide (10(-4) and 10(-3) M) inhibited the electrogenic K+ secretion when added to the basolateral fluid. N-ethylmaleimide (10(-3) M) applied to the apical fluid during a 5 min period decreased the K+ influx by 43% and the transepithelial potential by 66%. Other ATPase inhibitors, harmaline (10(-3) M), omeprazole (10(-4) M), vanadate (10(-4) M and 10(-3) M), N,N'-dicyclohexylcarbodiimide (DCC, 10(-5) M), 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl, 5 x 10(-6) M and 5 x 10(-5) M), and bafilomycin (10(-7) M) did not affect the K+ transport nor the transepithelial potential when they were added to the apical fluid. 4. The Na(+)-K(+)-Cl- co-transporter inhibitor, bumetanide, decreased both the transepithelial potential and the K+ transport when added to the basolateral solution but not to the apical one. At 10(-6) M, bumetanide maximally decreased the K+ influx by about 60%. 5. K+ channel blockers, quinine (10(-4) M), TEA (5 x 10(-3) M), added to the apical solution and barium (2 x 10(-3) M) added to either the apical or the basolateral solutions, did not affect the K+ transport and the transepithelial potential. 6. The carbonic anhydrase inhibitor acetazolamide (10(-3) M) added to both apical and basolateral solutions did not affect the K+ transport and the transepithelial potential. 7. It is concluded that, in the ampulla of the semicircular canal, a basolateral Na(+)-K(+)-Cl- co-transporter energized by the Na+, K(+)-ATPase was involved for 60% in the K+ secretion into endolymph. The electrogenic K+ transport would partly depend on a N-ethylmaleimide-sensitive protein possibly located at the apical plasma membrane or intracellularly.

Endolymphatic potential is assumed to result from active K transport into the endolymphatic compartment and passive K diffusion in the opposite direction. However, in several in vivo experiments, changes in endolymphatic potential differed from that in endolymphatic K concentration. Moreover, in in vitro experiments, a negative endolymphatic potential was observed in presence of ouabain without K gradient between the two compartments. These observations suggest that the coupling between the K transport and the genesis of the endolymphatic potential is not tight. Several factors may influence separately the endolymphatic potential and the K transport such as the acid-base equilibrium, the integrity of the Reissner's membrane, the hormonal status and the Na transport.

The potassium concentration was measured in the perilymphatic and endolymphatic spaces of the chick's cochlea using ion-specific microelectrodes. The mean values in post-hatching chicks were 8.1 and 161 mM/l, respectively and are thus the same as in mammals. The high potassium concentration is reached at the latest at stage E42, and thus before the tegmentum vasculosum is fully developed. The endocochlear potential of chicks was maximally + 13 mV and thus somewhat below the published values for birds. Positive potentials in the endolymphatic space of 5 mV or more were already present in animals of age E41 and E42 and the developmental process is complete at the latest one day after hatching.

The inner ear epithelium secretes potassium (K+) and absorbs sodium (Na+). The authors' experiments utilized, for the first time, isolation of the nonsensory from the sensory regions of the utricle in the vestibular labyrinth of the gerbil by means of injecting insulating seals. It was found that the isolated nonsensory region accumulated rubidium (Rb+) (as a marker for K+) in the endolymph to a level of eight times (41 mmol/l) that in the incubation medium (5 mmol/l) over a 90-minute period. However, Na+ rose during this period, suggesting that cells excluded from this preparation (sensory regions) may be involved in Na+ reabsorption. Further observations of the time course of changes in the luminal concentrations of cations suggested that the endpoints observed at 90 minutes represented a new steady state in which both Na+ and K+ concentrations were higher and lower, respectively, than under control conditions (by about 40 mmol/l each), but they were still each displaced from electrochemical equilibrium. This implies that the processes of Na+ absorption and/or K+ secretion may be shared in some way between the two regions. This finding is unexpected, based on previous models of endolymph production, in which ionic regulation has been assigned to nonsensory regions alone.

The mammalian inner ear is located deep within the temporal bone. The organ of Corti, the delicate sensory system for sound, is surrounded by two fluid systems; the potassium-rich endolymph and the sodium-rich perilymph. The pathogenesis of inner ear deafness is thought to be largely due to an imbalance of potassium and sodium ions in the inner ear fluids. Dynamic changes in K+ in the endolymph and perilymph were studied in the guinea-pig following cetrimide (cetrimonium bromide, a powerful cationic detergent which shows ototoxicity) applications on the round window membrane, intramuscular injection of potassium bromate (bread whitener, known to cause renal damage and permanent deafness in animals and man). Maximum fall in K+ concentration in the endolymoh (mM/min) and maximum K+ conductance (mM/min/mV) were 3.54 +/- 1.65 and 0.036 +/- 0.02 in cetrimide, and 1.85 +/- 0.35 and 0.021 +/- 0.009 in potassium bromate, respectively. In view of these findings, the influence of the active transport mechanism to K+ concentrations are discussed in comparison with dynamic changes in endolymph K+ induced by asphyxia and ethacrynic acid.

Healthy guinea pigs were exposed to broadband noise at 115 dBA for 7 days. When perfusion of the scala vestibuli was carried with artificial perilymph containing 43K, the 43K concentrations in the perilymph of the scala tympani were very low in both control and noise-exposed guinea pigs. The transport rate constant for K+ was computed by utilizing compartmental analysis. The results indicate that exposure to noise at 115 dBA for 7 days does not alter the permeability to K+ of Reissner's membrane or the lateral wall of the cochlear duct but does decrease K+ conductance of the organ of Corti.

The results obtained from tracer kinetics, measured by Konishi et al. (1978) and June (1979), are combined to construct a two-compartment model of the guinea-pig cochlea. The potassium fluxes are calculated using a mathematical two-compartment theory. For the basal turn, one finds a circulating flux between the endolymphatic and perilymphatic spaces of 190 pMol/min and 1 mm cochlea, whereas a minor value of 100 pmol/min/mm is calculated for the flux from the perilymphatic scales out of the cochlea.

Endolymph of the scala media (SM) and perilymph of the scala vestibuli (SV) and scala tympani (ST) were collected from the basal turn of anesthetized guinea pigs before and after intravenous administration of glycerol (3 g/kg). Sound-evoked responses were recorded during the test periods. Blood, CSF, and perilymph of the ST were also collected continuously after the injection. The osmolalities and chloride concentrations of the collected samples were determined. In another experiment, the continuous changes of potassium and chloride concentrations in endolymph and perilymph of the ST before and after the injection were measured by ion-selective electrodes. The osmolalities in CSF and perilymph lagged behind the increase in serum osmolality. The osmolalities in endolymph and perilymph increased gradually after the injection, reached maximum values after 90 minutes, and then decreased. The changes in chloride and potassium concentrations in endolymph and perilymph had similar tendencies. But the increases in chloride concentrations in perilymph of the SV and ST were much less than that in endolymph. We propose that most of the osmolality increase in perilymph is due to glycerol or other osmotically active substances and that the osmolality increase in endolymph is due to water shift.

The endocochlear potential (EP), potassium concentration in the endolymph (Ke+) and in the perilymph (Kp+) were measured in guinea-pigs during anoxia of different duration. Specific K+ double-barrel microelectrodes with liquid ion exchanger were used. The resting K+ concentration in the endolymph was 146.8 +/- 9.2 mM and in the perilymph 3.2 +/- 0.5 mM. The following time course of events was observed in the cochlea during anoxia: 40-50 s after the arrest of ventilation the K+ concentration decreased by 0.1-0.2 mM in the scala vestibuli, which was time related to a rapid fall of EP to negative values. Perilymphatic K+ started to increase in both scalae with a latency of 2-2.5 min, reaching a concentration of about 14 mM 60 min after the arrest of ventilation. The endolymphatic K+ began to decrease after a latency of 2.5-3 min, and 60 min after the arrest of ventilation an 80% concentration (average 112 mM K+) was reached as compared to the initial value. From the comparison of K+ concentration changes with the experimental values of the negative EP, it may be assumed that the negative EP is mainly generated by the K+ gradient between the perilymph and endolymph.

The endocochlear potential and potassium concentrations in endolymph and perilymph were simultaneously measured in the basal turn of the guinea pig cochlea with double-barreled K+ selective electrodes. The K+ conductance and K+ permeability coefficient of the endolymph-perilymph barrier were calculated from the rate of change of endolymph K+ concentration relative to the K+ electrochemical potential difference recorded during permanent anoxia. When anoxia was induced in guinea pigs treated with kanamycin, the rate of decline of the electrochemical potential difference for K+ between the endolymph and perilymph was reduced when compared to normal guinea pigs. In guinea pigs exposed to broad band noise at 115 dBA for periods from 11-15 days, the rate of decline of the electrochemical potential difference for K+ across the endolymph-perilymph barrier was reduced but not to the extent found in guinea pigs treated with kanamycin. The K+ conductance and K+ permeability coefficient of the endolymph-perilymph barrier showed substantial decreases in noise exposed and kanamycin treated guinea pigs, as compared to normal guinea pigs. The magnitude of decrease of K+ permeability of the endolymph-perilymph barrier by noise or kanamycin was correlated with suppression of the maximum output of the cochlear microphonic.

The kinetics of radioactive potassium, chloride, and water entry into endolymph, perilymph, and cerebrospinal fluid were studied after intravenous administration of tracers in anesthetized and nephrectomized rats. Samples of cochlear endolymph, perilymph of scala vestibuli, perilymph of scala tympani, and cisternal cerebrospinal fluid were obtained. The data showed: 1) a rapid turnover of water in endolymph, perilymph, and cerebrospinal fluid, since 3H2O equilibrated with plasma in a few minutes; 2) a slow entry of 42K and 36Cl in perilymph, since 36Cl equilibrated with plasma after 2 h and 42K did not at 6 h; 3) an extremely slow entry of 42K and 36Cl in endolymph, since no equilibrium with plasma was obtained within the 5 h of the experiments. The comparison of the compartmental analysis of our data with the results of other studies using perilymphatic perfusion of tracers indicated that perilymph rather than plasma may be considered as the precursor of endolymph.

A survey of experimental evidence showing the influence of an increase in potassium concentration in the perilymph on cochlear and vestibular nerve branches is discussed. Informations have indicated that a concentration lower than 20--30 mM produced an increase in action potential frequency in nerve preparations and that a low potassium concentration in animal experiments produced an ipsilateral nystagmus. Increasing the concentration of potassium in the endolymph to values not over the normal endolymph concentration, blocked the conduction of action potentials in the vestibular nerve branches resulting in a reversible contralateral nystagmus. From these results it may be concluded that all objective symptoms of a Ménière attack can be experimentally reproduced by one single factor: the potassium concentration in the perilymph. The known ruptures in the distended walls in a hydrops, with a diffusion of endolymph potassium into the perilymph surrounding the nerve branches to the vestibular and cochlear areas have consequently been assumed to produce the basis for Ménière attacks.

After i.v. injection of 50 mg/kg ethacrynic acid (EA), potassium concentration in the endolymph (Ke+) measured with K+-specific microelectrodes decreases by 10 mM at the most and endocochlear potential falls to negative values. Potassium concentration in the perilymph (Kp+) generally does not change, but sometimes a transient decrease in Kp+ level of about 0.5 mM was observed, presumably due to the electrogenic effect of the time-related decrease of the endocochlear potential. When anoxia is induced approximately 120 min after EA administration Ke+ slowly decreases. The decrease in Ke+ 50 min after the arrest of ventilation is smaller when compared with the Ke+ anoxic decrease without preceding EA administration. The endocochlear potential, which falls to negative values during anoxia after EA administration, does not return to the zero level as in the case when only anoxia is applied. Similarly, during anoxia, which follows EA administration, the perilymphatic Ke+ concentration increases more slowly than in the case when only anoxia is introduced. It is assumed from the results that EA abolishes activity of the positive electrogenic K+ pump and reduces the passive permeability of the walls of the cochlear duct to the potassium ions.