Transepithelial voltage and resistance of vestibular dark cell epithelium from the gerbil ampulla

doi:10.1016/0378-5955(94)90287-9

Hearing Research

Volume 73, Issue 1, February 1994, Pages 101-108

https://doi.org/10.1016/0378-5955(94)90287-9 Get rights and content

Abstract

Transepithelial voltage (V_t) and resistance (R_t) were measured across the dark cell epithelium of the gerbil ampulla using a micro Ussing chamber of improved design in order to test the view that the histologically similar epithelia in the utricle and in the ampullae exhibit similar electrophysiologic functions. V_t was found to be 8.0 ± 0.3 mV and R_t was 11.6 ±0.4 ohm-cm² (N= 179) when both sides of the tissue were perfused with symmetric perilymph-like solution. The equivalent short circuit current (I_sc = V_t/R_t) was 712±18 μA/cm² (N = 179). I_sc was reduced from 638 ± 60 to 48 ± 16 μA/cm² (N = 14) by basolateral perfusion of 10⁻³ M ouabain and from 538 ± 27 to 27 ± 4 μA/cm² (N = 15) by basolateral perfusion of 5 · 10⁻⁵ M bumetanide. Basolateral K⁺ steps (Na⁺ substitution) from 3.6 to 25 mM increased V_t from 6.5 ± 0.5 to 12.2 ± 0.6 mV and reduced R_t from 9.7 ± 0.7 to 7.4 ± 0.5 ohm-cm² (N = 43). Apical K⁺ steps from 3.6 to 25, to 100 mM or to 145 mM led to a decrease in both V_t and R_t. The steady state V_t during apical perfusion of 145 mM K⁺ was near zero. Upon return to 3.6 mM K⁺, V_t transiently overshot its original level. Apical Cl⁻ steps from 150 to 50 mM (gluconate substitution) monophasically decreased V_t from 5.9 ± 0.7 to 4.1 ± 0.8 mV (N = 15) and increased R_t from 9.6 ± 1.3 to 12.0 ± 1.5 ohm-cm² (N= 14). Apical perfusion of 5 · 10⁻⁴ M DIDS increased V_t from 7.1 ± 0.9 to a peak value of 11.9 ± 1.7 mV (N = 6) and decreased R_t from 10.2 ± 2.1 to 9.0 ± 1.7 ohm-cm² at the time of the peak V_t. The present results are qualitatively similar to data obtained in the utricle, suggesting a functional similarity between the dark cell epithelium in these two regions of the vestibular labyrinth. Further, the data support the hypothesis that the vestibular dark cells contribute a lumen-positive voltage only when the K⁺ concentration of endolymph falls below the level normally found in vivo.

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Three-dimensional cultured ampullae from rats as a screening tool for vestibulotoxicity: Proof of concept using styrene
2023, Toxicology
Numerous ototoxic drugs, such as some antibiotics and chemotherapeutics, are both cochleotoxic and vestibulotoxic (causing hearing loss and vestibular disorders). However, the impact of some industrial cochleotoxic compounds on the vestibular receptor, if any, remains unknown. As in vivo studies are long and expensive, there is considerable need for predictive and cost-effective in vitro models to test ototoxicity. Here, we present an organotypic model of cultured ampullae harvested from rat neonates. When cultured in a gelatinous matrix, ampulla explants form an enclosed compartment that progressively fills with a high-potassium (K⁺) endolymph-like fluid. Morphological analyses confirmed the presence of a number of cell types, sensory epithelium, secretory cells, and canalar cells. Treatments with inhibitors of potassium transporters demonstrated that the potassium homeostasis mechanisms were functional. To assess the potential of this model to reveal the toxic effects of chemicals, explants were exposed for either 2 or 72 h to styrene at a range of concentrations (0.5–1 mM). In the 2-h exposure condition, K⁺ concentration was significantly reduced, but ATP levels remained stable, and no histological damage was visible. After 72 h exposure, variations in K⁺ concentration were associated with histological damage and decreased ATP levels. This in vitro 3D neonatal rat ampulla model therefore represents a reliable and rapid means to assess the toxic properties of industrial compounds on this vestibular tissue, and can be used to investigate the specific underlying mechanisms.
Acute blockade of inner ear marginal and dark cell K<sup>+</sup> secretion: Effects on gravity receptor function
2018, Hearing Research
Citation Excerpt :
One can argue that the NKCC1 cotransporter in vestibular dark cells is less sensitive to EA than that in marginal cells thus requiring higher doses to achieve the effect. This is not the case however, since the cotransporters are the same and it has been shown that K+ secretion rates in both vestibular dark cells and stria marginal cells are equally suppressed by loop diuretics (Wangemann and Marcus, 1990; Marcus et al., 1987, 1994; Marcus and Marcus, 1987; Wangemann et al., 1995; Wangemann, 1995). Thus, differential sensitivity of the NKCC1 cotransporter to EA is not a likely explanation of the current findings.
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.
Ion flow in stria vascularis and the production and regulation of cochlear endolymph and the endolymphatic potential
2011, Hearing Research
Citation Excerpt :
The stria’s need for many cell types is emphasized by the lack of these cell layers in the vestibular system, with its much lower stimulus frequencies and different constraints. The utricle exchanges Na+ and K+ with only a single layer of MC-like ‘dark cells’ (Marcus and Marcus, 1987; Marcus and Shipley, 1994; Marcus et al., 1994; Wangemann and Marcus, 1990, 1992) without BC or IC support (producing endolymph with much less electrogenic current and so with a lower positive endolymph potential). The saccule has neither a stria nor dark cells, but parasitises both the cochlea and utricle to have any endolymph at all, and an endolymphatic potential that is only slightly more positive than the K+ diffusion potential (Sellick and Johnstone, 1975).
This paper reviews some of what is known about ion transport through the cells of the mammalian stria vascularis, and discusses how the endolymph and endocochlear potential in scala media are produced by the stria’s main cell types. It discusses the role of each cells’ ion transport proteins from an engineering perspective, and the advantages and disadvantages in using the different transport proteins in the different cells to perform their different roles. To aid this discussion, the use of spreadsheet analysis in the modelling of ion transport in single cells and homogenous epithelia is outlined, including the current-voltage (IV) characteristics of the three main categories of transport proteins (pores, ports and pumps), and the constraint equations that apply under various conditions (the voltage or ionic steady states in the open- and closed-circuit conditions). Also discussed are the circulation of K⁺ within the cochlea, and the chloride, salt and water balance of scala media and stria vascularis, and what transport processes may be required to maintain such a balance.
EphB2 and ephrin-B2 regulate the ionic homeostasis of vestibular endolymph
2007, Hearing Research
The ability to transport cations and anions across epithelia is critical for the regulation of pH, ionic homeostasis, and volume of extracellular fluids. Although the transporters and channels that facilitate ion and water movement across cell membranes are well known, the molecular mechanisms and signal transduction events that regulate these activities remain poorly understood. The Eph family of receptor tyrosine kinases and their membrane-anchored ephrin ligands are well known to transduce bidirectional signals that control axon guidance and other cell migration/adhesion events during development. However, these molecules are also expressed in non-motile epithelial cells, including EphB2 in K⁺-secreting vestibular dark cells and ephrin-B2 in the adjacent transitional cells of the inner ear. Consistent with these expression patterns, mice with cytoplasmic domain mutations that interfere with EphB2 forward signaling or ephrin-B2 reverse signaling exhibit a hyperactive circling (waltzing) locomotion associated with a decreased amount of endolymph fluid that normally fills the vestibular labyrinth. Endolymph is unusual as an extracellular fluid in that it is normally high in K⁺ and low in Na⁺. Direct measurement of this fluid in live animals revealed significant decreases in K⁺ concentration and endolymphatic potential in both EphB2 and ephrin-B2 mutant mice. Our findings provide evidence that bidirectional signaling mediated by B-subclass Ephs and ephrins controls the production and ionic homeostasis of endolymph fluid and thereby provide the first evidence that these molecules can control the activities of mature epithelial cells.
Higher serum aldosterone correlates with lower hearing thresholds: A possible protective hormone against presbycusis
2005, Hearing Research
Aldosterone hormone is a mineralocorticoid secreted by adrenal gland cortex and controls serum sodium (Na⁺) and potassium (K⁺) levels. Aldosterone has a stimulatory effect on expression of sodium–potassium ATPase (Na, K-ATPase) and sodium–potassium–chloride cotransporter (NKCC) in cell membranes. In the present investigation, the relation between serum aldosterone levels and age-related hearing loss (presbycusis) and the correlation between these levels versus the degree of presbycusis in humans were examined. Serum aldosterone concentrations were compared between normal hearing and presbycusic groups. Pure-tone audiometry, transient evoked otoacoustic emissions (TEOAE), hearing in noise test (HINT) and gap detection were tested for each subject and compared to the serum aldosterone levels. A highly significant difference between groups in serum aldosterone concentrations was found (p = 0.0003, t = 3.95, df = 45). Highly significant correlations between pure-tone thresholds in both right and left ears, and HINT scores versus serum aldosterone levels were also discovered. On the contrary, no significant correlations were seen in the case of TEOAEs and gap detection. We conclude that aldosterone hormone may have a protective effect on hearing in old age. This effect is more peripheral than central, appearing to affect inner hair cells more than outer hair cells.
Three-dimensional culture of newborn rat utricle using an extracellular matrix promotes formation of a cyst
2005, Neuroscience
The vestibule is the end organ devoted to sensing of head movements in space. To function properly, its mechano-receptors require the presence of a unique apical extracellular medium, the endolymph. Numerous studies have elucidated the mechanisms involved in the production and homeostasis of this unique medium and the responses of sensory cells to stimulation. However, anatomical constraints have prevented direct and simultaneous studies of their relationships. The aim of this study was the development of an in vitro model that would allow concomitant investigations on maturation and physiological properties of both the hair cells and their endolymphatic compartment.
A three-dimensional (3D) culture of newborn rat utricles using an extracellular matrix sustaining 3D cellular growth was developed during 3, 6, or 10 days in vitro (DIV). Using morphological and electrophysiological techniques, we describe the de novo formation of a cyst. It was composed of the sensory epithelium and non-sensory cells—canalar, dark and intermediate cells—that polarized so that their apical surface faced its lumen. During the time of culture, the utricular potential (UP) was steady (−1.1±5.0 mV) in oxygenated condition, while in anoxia, the UP significantly decreased to −8.4±1.0 mV at 8 DIV. Over the same period, the K⁺ concentration in the cyst increased up to 86.1±33.9 mM (versus 5.6±1.5 mM in the bath). These observations indicated that the mechanisms generating the UP and the K-secretory activity were functional at this stage. Concomitantly, the hair cells acquired mature and functional properties: the type 1 and type 2 phenotypes, a mean resting membrane potential of −68.1±4.6 mV and typical electrophysiological responses. This preparation provides a powerful means to simultaneous access the hair cells and their endolymphatic compartment, with the possibility to use multi-technical approaches to investigate their interdependent relationships.

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References (24)

Transepithelial cation movements in gerbil utricles

Am. J. Otolaryngol.

Transepithelial electrical responses to Cl⁻ of nonsensory region of gerbil utricle

Biochim. Biophys. Acta

Sidedness of action of loop diuretics and ouabain on nonsensory cells of utricle: A micro Ussing chamber for inner ear tissues

Hear. Res.

Calcium gradients in inner ear endolymph

Am. J. Otolaryngol.

The membrane potential of vestibular dark cells is controlled by a large Cl⁻ conductance

Hear. Res.

Production of endolymph in the semicircular canal of the frog Rana Esculenta

J. Physiol.

Stilbenes stimulate T84 Cl⁻ secretion by elevating Ca²⁺

Am. J. Physiol.

Stilbene disulfonate blockade of colonie secretory Cl⁻ channels in planar lipid bilayers

Am. J. Physiol.

Membrane proteins related to anion permeability of human red blood cells. I. Localization of disulphonic stilbene binding sites in proteins involved in permeation

J. Membrane Biol.

Distribution, structure, and function of dark cells in the vestibular labyrinth

Ann. Otol.

The cochlear potentials. II. The nature of the cochlear endolymphatic resting potential

Pflügers Arch.

Effects of anoxia and ethacrynic acid upon ampullar endolymphatic potential and upon high energy phoshates in ampullar wall

Laryngoscope

Cited by (47)

Three-dimensional cultured ampullae from rats as a screening tool for vestibulotoxicity: Proof of concept using styrene

Acute blockade of inner ear marginal and dark cell K<sup>+</sup> secretion: Effects on gravity receptor function

Ion flow in stria vascularis and the production and regulation of cochlear endolymph and the endolymphatic potential

EphB2 and ephrin-B2 regulate the ionic homeostasis of vestibular endolymph

Higher serum aldosterone correlates with lower hearing thresholds: A possible protective hormone against presbycusis

Three-dimensional culture of newborn rat utricle using an extracellular matrix promotes formation of a cyst

Transepithelial voltage and resistance of vestibular dark cell epithelium from the gerbil ampulla

Abstract

Am. J. Otolaryngol.

Biochim. Biophys. Acta

Hear. Res.

Am. J. Otolaryngol.

Hear. Res.

Production of endolymph in the semicircular canal of the frog Rana Esculenta

J. Physiol.

Stilbenes stimulate T84 Cl− secretion by elevating Ca2+

Am. J. Physiol.

Stilbene disulfonate blockade of colonie secretory Cl− channels in planar lipid bilayers

Am. J. Physiol.

Membrane proteins related to anion permeability of human red blood cells. I. Localization of disulphonic stilbene binding sites in proteins involved in permeation

J. Membrane Biol.

Distribution, structure, and function of dark cells in the vestibular labyrinth

Ann. Otol.

The cochlear potentials. II. The nature of the cochlear endolymphatic resting potential

Pflügers Arch.

Effects of anoxia and ethacrynic acid upon ampullar endolymphatic potential and upon high energy phoshates in ampullar wall

Laryngoscope

Stilbenes stimulate T84 Cl⁻ secretion by elevating Ca²⁺

Stilbene disulfonate blockade of colonie secretory Cl⁻ channels in planar lipid bilayers