Vestibular Dark Cells Research Articles

Mechanism of volume regulation of vestibular dark cells of the gerbil

The volume response of vestibular dark cells of the gerbil to a hyposmotic challenge was investigated. Tissues including dark cells were perfused in preparations in which the perfusate had access to both sides of the epithelium and the height of the dark cell layer was measured as an indicator of its volume. We found that dark cells showed a fast and strong regulatory volume decrease (RVD) and prevented cell swelling in hypotonic media. This mechanism was dependent upon extracellular [K+] and [Cl-]. Ion selectivity of this mechanism was K+ = Rb+ > Cs+ > Na+ = NMDG+ (N-methyl-D-glucamine) for cations and Cl- = SCN- = NO3- > > gluconate- for anions. RVD of dark cells was inhibited by K(+)- channel blockers barium, quinidine and lidocaine, by Cl(-)-channel blockers 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, by an Na(+)-K+ ATPase inhibitor ouabain and by low temperature, but was not inhibited by a loop diuretic bumetanide, by carbonic anhydrase inhibitors acetazolamide and ethoxyzolamide, by a K(+)-channel blocker tetraethylammonium, by a Cl(-)-channel blocker 5-nitro-2 (3-phenylpropylamino)-benzoic acid or by an inhibitor of the Na(+)-H+ exchanger amiloride. These data suggest that the RVD of dark cells occurs via separate K+ and Cl- channels which are different from those active under isosmotic condition, and is presumably activated by a hyposmotic stimulus.

Ion selectivity of volume regulatory mechanisms present during a hypoosmotic challenge in vestibular dark cells

Volume regulation during a hypoosmotic challenge (RVD) in vestibular dark cells from the gerbilline inner ear has previously been shown to depend on the presence of cytosolic K + and Cl −, suggesting that it involves KCl efflux. The aim of the present study was to characterize hypoosmotically-induced KCl transport under conditions where a hypoosmotic challenge causes KCl influx via the pathways normally used for efflux. Net osmolyte movements were monitored as relative changes in cell volume measured as epithelial cell height ( CH). A hypoosmotic challenge (298 to 154 mosM) in the presence of 3.6 or 25 mM K + and loop-diuretics (piretanide or bumetanide) caused an increase in CH by about a factor of 1.2 presumably due to the net effect of primary swelling defined as osmotic dilution of the cytosol and RVD involving KCl efflux. A hypoosmotic challenge in the presence of 79 mM K + and loop-diuretics, however, caused CH to increase by a factor of over 2.4. Presumably, this large increase in CH was due to the sum of primary and secondary swelling. Secondary swelling depended on the presence of extracellular K + and Cl − suggesting that it involved KCl influx followed by water. The ion selectivity of secondary swelling was K + = Rb +>Cs + ⪢ Na + = NMDG + and Cl − = NO 3 − = SCN − ⪢ gluconate −. Secondary swelling was not inhibited by Ba 2+, tetraethylammonium, quinidine, lidocaine, amiloride, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid, 4-acetamido-4′-diisothiocyanatostilbene-2,2′-disulfonic acid, 4,4′-dinitrostilbene-2,2′-disulfonic acid, 5-nitro-2(3-phenylpropylamino)benzoic acid, acetazolamide, or ethoxyzolamide. These data define a profile of the hypoosmotically-induced KCl transport pathways. The ion selectivity and the blocker insensitivity are consistent with the involvement of the apical slowly activating K + channel (I sK or minK channel) and the basolateral 360 pS Cl − channel. The involvement of these channels, however, remains to be demonstrated.

Comparison of ion transport mechanisms between vestibular dark cells and strial marginal cells

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.

The pH-sensitivity of transepithelial K+ transport in vestibular dark cells.

The pH-sensitivity of transepithelial K+ transport was studied in vitro in isolated vestibular dark cell epithelium from the gerbil ampulla. The cytosolic pH (pHi) was measured microfluorometrically with the pH-sensitive dye 2',7'-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) and the equivalent short-circuit current (Isc), which is a measure for transepithelial K+ secretion, was calculated from measurements of the transepithelial voltage (Vt) and the transepithelial resistance (Rt) in a micro-Ussing chamber. All experiments were conducted in virtually HCO3(-)-free solutions. Under control conditions, pHi was 7.01 +/- 0.04 (n = 18), Vt was 9.1 +/- 0.5 mV, Rt 16.7 +/- 0.09 omega cm2, and Isc was 587 +/- 30 microA/cm2 (n = 49). Addition of 20 mM propionate- caused a biphasic effect involving an initial acidification of pHi, increase in Vt and Isc and decrease in Rt and a subsequent alkalinization of pHi, decrease of Vt and increase of Rt. Removal of propionate- caused a transient effect involving an alkalinization of pHi, a decrease of Vt and Isc and an increase in Rt, pHi in the presence of propionate- exceeded pHi under control conditions. Effects of propionate- on Vt, Rt and Isc were significantly larger when propionate- was applied to the basolateral side rather than to the apical side of the epithelium. The pHi-sensitivity of Isc between pH 6.8 and 7.5 was -1089 microA/(cm2.pH-unit) suggesting that K+ secretion ceases at about pHi 7.6. Acidification of the extracellular pH (pHo) caused an increase of Vt and Isc and a decrease of Rt most likely due to acidification of pHi. Effects were significantly larger when the extracellular acidification was applied to the basolateral side rather than to the apical side of the epithelium. The pHo sensitivity of Isc between pH 7.4 and 6.4 was -155 microA/(cm2.pH unit). These results demonstrate that transepithelial K+ transport is sensitive to pHi and pHo and that vestibular dark cells contain propionate- uptake mechanism. Further, the data suggest that cytosolic acidification activates and that cytosolic alkalinization inactivates the slowly activating K+ channel (IsK) in the apical membrane. Whether the effect of pHi on the IsK channel is a direct or indirect effect remains to be determined.

Melanocytes in the stria vascularis and vestibular labyrinth of the Mongolian gerbil (Meriones unguiculatus).

Inner ear melanocytes are mainly present in the cochlea, vestibular organ, and endolymphatic sac, but their exact biological function has not been determined. In this investigation, we study the pigment cells in the membranous labyrinth of the gerbil. The inner ear melanocytes of M. unguiculatus show an irregular dendritic shape with cytoplasmic processes. These cells are disposed following the distribution of strial marginal and vestibular dark cells that have an important metabolic activity. Gerbil inner ear melanocytes are characterized by the presence of melanosomes, which are homogeneously dense organelles, of variable size and shape, that are surrounded by a membrane. In these cells, the Golgi apparatus plays a important role in melanin synthesis. When melanocytes were incubated in L-DOPA solution, the vesicles and cisterns of the Golgi apparatus exhibited a positive tyrosinase reaction. An interesting observation is the relation between melanocytes and inner ear capillaries. Sometimes, near to sensory vestibular areas, the melanocytes were in contact with Schwann cells and with myelinated fibres of vestibular nerve. The ultrastructural findings of this investigation are consistent with the hypothesis that melanocytes may have functional significance in the inner ear.

Hypo-osmotic challenge stimulates transepithelial K+ secretion and activates apical IsK channel in vestibular dark cells.

Volume regulation of vestibular dark cells from the gerbilline inner ear in response to a hypo-osmotic challenge depends on the presence of cytosolic K+ and Cl-. The present study addresses the questions: (i) whether and by what mechanism K+ is released during volume regulation, (ii) whether the osmolarity of the basolateral medium has an effect on the steady-state rate of transepithelial K+ transport and (iii) whether there is cross-talk between the basolateral membrane responsible for K+ uptake and the apical membrane responsible for K+ release. K+ secretion (JK+,probe) and current density (Isc,probe) were measured with vibrating probes in the vicinity of the apical membrane and the transepithelial potential (Vt) and resistance (Rt) were measured in a micro-Ussing chamber. The equivalent short-circuit current (Isc) was calculated. The current (IIsK), conductance (gIsK) and inactivation time constant (tau IsK) of the IsK channel and the apparent reversal potential of the apical membrane (Vr) were obtained with the cell-attached macropatch technique. Vr was corrected (Vrc) for the membrane voltage (Vm) measured separately with microelectrodes. A hypo-osmotic challenge (294 to 154 mosM by removal of 150 mM mannitol) on the basolateral side of the epithelium increased JK+,probe and Isc,probe by a factor of 2.7 and 1.6. When this hypo-osmotic challenge was applied to both sides of the epithelium Vt and Isc increased from 5 to 14 mV and from 189 to 824 microA/cm2 whereas Rt decreased from 27 to 19 omega-cm2. With 3.6 mM K+ in the pipette IIsK was outwardly directed, tau IsK was 267 msec and the hypo-osmotic challenge caused IIsK and gIsK to increase from 14 to 37 pA and from 292 to 732 pS. Vrc hyperpolarized from -44 to -76 mV. With 150 mM K+ in the pipette IIsK was inwardly directed, tau IsK was 208 msec and the hypo-osmotic challenge caused IIsK and gIsK to increase in magnitude from 0 to -21 pA and from 107 to 1101 pS. Vrc remained unchanged (-2 vs. 1 mV). These data demonstrate that a hypo-osmotic challenge stimulates transepithelial K+ secretion and activates the apical IsK channel. The hypo-osmotically-induced increase in K+ secretion exceeded the estimated amount of K+ release necessary for the maintenance of constant cell volume, suggesting that the rate of basolateral K+ uptake was upregulated in the presence of the hypo-osmotic challenge and that cross-talk exists between the apical membrane and the basolateral membrane.

DIDS increases K+ secretion through an IsK channel in apical membrane of vestibular dark cell epithelium of gerbil.

Vestibular dark cell epithelium secretes K+ via IsK channels in the apical membrane. The previous observation that disulfonic stilbenes increased the equivalent short circuit current (Isc) suggested that these agents might be useful investigative tools in this tissue. The present experiments were conducted to determine if the increase in Isc was associated with an increase in K+ flux and if the effect was directly on the IsK channel or indirectly via a cytosolic intermediary. Measurements of transepithelial K+ flux with the K(+)-selective vibrating probe and of changes in net cellular solute flux by measurements of epithelial cell height showed that 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) increased K+ flux by a factor of 1.96 +/- 0.71 and caused net solute efflux. The apical membrane was partitioned with a macropatch pipette and DIDS was applied either to the membrane outside the pipette, inside the pipette or to the entire apical membrane. DIDS inside the pipette increased the current across the patch, the membrane conductance, the slowly-inactivating (IsK) component of the membrane current and shifted the reversal voltage toward the equilibrium potential for K+. DIDS outside the patch decreased the patch current and conductance, consistent with shunting of current away from the membrane patch. These findings strongly support the notion that DIDS increases K+ secretion through IsK channels in the apical membrane of vestibular dark cell epithelium by acting directly on the channels or on a tightly colocalized membrane component.

Ion transport mechanisms responsible for K + secretion and the transepithelial voltage across marginal cells of stria vascularis in vitro

It has long been accepted that marginal cells of stria vascularis are involved in the generation of the endocochlear potential and the secretion of K +. The present study was designed to provide evidence for this hypothesis and for a cell model proposed to explain K + secretion and the generation of the endocochlear potential. Stria vascularis from the cochlea of the gerbil was isolated and mounted into a micro-Ussing chamber such that the apical and basolateral membrane of marginal cells could be perfused independently. In this preparation, the transepithelial voltage ( V t ) and resistance ( R t ) were measured across marginal cells and the resulting equivalent short circuit current ( I sc ) was calculated ( I sc = V t R t ). Further, K + secretion ( J K +, probe ) was measured with a K +-selective vibrating probe in the vicinity of the apical membrane. In the absence of extrinsic chemical driving forces, when both sides of the marginal cell epithelium were bathed with a perilymph-like solution, V t was 8 mV (apical side positive), R t was 10 ohm-cm 2 and I sc was 850 μA/cm 2 ( N = 27). J K +,probe was outwardly directed from the apical membrane and reversibly inhibited by basolateral bumetanide, a blocker of the Na + /Cl − /K + cotransporter. On the basolateral but not apical side, ouabain and bumetanide each caused a decline of V t and an increase of R t suggesting the presence of the Na,K-ATPase and the Na + /Cl − /K + cotransporter in the basolateral membrane. The responses to [Cl −] steps demonstrated a significant Cl − conductance in the basolateral membrane and a small Cl − conductance in the paracellular pathway or the apical membrane. The responses to [Na +] steps demonstrated no significant Na + conductance in the basolateral membrane and a small Na + or nonselective cation conductance in the apical membrane or paracellular pathway. The responses to [K +] steps demonstrated a large K + conductance in the apical membrane. Apical application of 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS) and basolateral elevation of K + caused an increase in V t and a decrease in R t consistent with stimulation of the apical K + conductance. Similar observations have been made in vestibular dark cells, which suggest that strial marginal cells and vestibular dark cells are homologous and transport ions by the same pathways. Taken together, these observations are incompatible with a model for the generation of the endocochlear potential which ascribes the entire potential to the strial marginal cells [Offner et al. (1987) Hear. Res. 29, 117–124]. However, the data are compatible with the notion that marginal cells contribute not more than a few mV to the endocochlear potential as suggested by Salt et al. [Laryngoscope 97, 984–991, 1987].

Ultrastructure of the vestibular dark cell area in patients with acoustic neurinoma.

The human vestibular dark cell (DC) areas of the utricle and ampulla of the lateral semicircular canal were investigated ultrastructurally in 7 patients with acoustic neurinoma. Two types of DCs mainly constituted the epithelial cells of the DC area. One type of DC had basolateral infoldings that were closely interwoven with the melanocyte processes, and the pinocytotic vesicles were frequently found within the basolateral infoldings and the melanocyte processes. The other type of DC had basolateral infolsings stacked upon each other. The former type of DC was more frequently found than the latter type in the DC area adjacent to the sensory epithelium and in the mid portion of the DC area. However, the latter type of DC was more predominant in the DC area distant from the sensory epithelium. These findings suggest that the activity of DCs is modulated by the melanocytes and that the former type of DC has a more active role in ion and/or fluid transport than the latter type of DC. Thus, the DC area near the sensory epithelium might be more actively engaged in the production and regulation of vestibular endolymph than that distant from the sensory epithelium.

Otoconia on the vestibular dark cells of the ampullar areas.

Morphological and metabolic differences in the otoconia on vestibular dark cells of the ampullar area before and after streptomycin treatment were studied by scanning electron microscopy and X-ray microanalyser. Young adult pigmented guinea pigs were used in this study. In the control group, distribution of the otoconia showed a regular pattern. In the SM group the otoconia decreased in numbers and the regularity of the distribution pattern seemed to disappear. Calcium content of the otoconia in SM group also decreased. From these findings, it was concluded that SM might affect not only the sensory and supporting cells but also the vestibular dark cells.

Expression of mRNAs Encoding α and β Subunit Isoforms of Na,K-ATPase in the Vestibular Labyrinth and Endolymphatic Sac of the Rat

The distribution of mRNAs coding for three different isoforms of the α and two of the β subunit of Na,K-ATPase was studied in the rat vestibular system using in situ mRNA hybridization. The dark cells of the utricular macula and of the ampullae of the semicircular canals expressed high levels of mRNA encoding the α1 and β2 isoforms of the Na,K-ATPase, a composition that in the cochlea has been uniquely found in the stria vascularis. However, in the dark cells it was coupled with a weak expression of β1. The sensory epithelia of the vestibular system showed α1 and β1 expression at much higher levels than in the cochlear sensory epithelium. Weak expression limited to the α1, β1, and β2 isoforms was observed in the endolymphatic sac, contrasting previous cytochemical results which suggested extensive Na,K-ATPase activity to the sac. The results support the widely held hypothesis that the vestibular dark cells play a role similar to that of the stria vascularis in endolymph production. They indicate that the ion transport requirements of the vestibular sensory epithelia may be different than those in the cochlea. They also suggest that the endolymphatic sac may not be a major site of inner ear ion exchange.

Slowly activating voltage-dependent K+ conductance is apical pathway for K+ secretion in vestibular dark cells

Dark cell epithelium secretes K+ into the lumen of the vestibular labyrinth by a previously unidentified apical transport mechanism. Previous single-channel patch-clamp studies demonstrated nonselective cation channels and maxi-K+ channels in the apical membrane, but in too low a density to account for transepithelial K+ transport. In this report, we demonstrated with the cell-attached macro-patch-clamp technique an outward apical membrane current at 0-mV pipette voltage, which was stimulated by elevating bath K+ concentration from 3.6 to 25 mM and inhibited by 10 microM bumetanide, similar to their known effects on transepithelial short-circuit current and K+ secretion. Furthermore, the patch current was activated over several seconds by a sustained depolarization and deactivated over several hundred milliseconds by a hyperpolarization. Current-voltage relationships from tail currents were obtained with either NaCl or KCl in the pipette. Depolarization from -40 to +40 mV led to an increased conductance by a factor of 7.3 +/- 1.7 (n = 7) and 19.2 +/- 7.6 (n = 6) for NaCl and KCl, respectively, and to a reversal voltage near the presumed equilibrium potential for K+. The results demonstrate that dark cell K+ secretion occurs via K(+)-selective channels with characteristics similar to those associated with the IsK protein.

Distribution of immunoreactive alpha- and beta-subunit isoforms of Na,K-ATPase in the gerbil inner ear.

Biochemical and histochemical studies have demonstrated abundant Na,K-ATPase in the inner ear and provided new information concerning the ion transport capacities of specialized cell types. To extend these earlier observations, we immunostained inner ears from adult gerbils with antibodies specific for the three known alpha- and the two known beta-isoforms of Na,K-ATPase. Different inner ear cell types contained specific and distinct combinations of alpha- and beta-subunit isoforms. Strial marginal cells and vestibular dark cells expressed the alpha 1- and beta 2-isoforms, whereas other positive epithelial cells expressed alpha 1 in combination with beta 1. Ganglion neurons and their peripheral processes showed positive immunostaining for the alpha 3- and beta 1-subunit isoforms. Subpopulations of fibrocytes in the spiral prominence, suprastrial and supralimbal regions, and vestibular system expressed either the alpha 1- or alpha 2-isoform, or both. The differential expression of Na,K-ATPase subunit isoforms presumably reflects different K+ and Na+ transport capacities among inner ear cell types which, working in concert, serve to generate and maintain the unique ionic and electrical environment in the mammalian inner ear.

Expression of α and β subunit isoforms of Na,K-ATPase in the mouse inner ear and changes with mutations at the W v or Sl d loci

Mice homozygous for mutations at the viable dominant spotting (W v) and Steel-dickie (Sl d) loci exhibit a similar phenotype which includes deafness. The auditory dysfunction derives from failure of the stria vascularis to develop normally and to generate a high positive endocochlear potential (EP). Because strial function is driven by Na,K-ATPase its expression was investigated in inner ears of W v W v and Sl d Sl d mice and their wild-type littermates by immunostaining with antisera against four of the enzyme's subunit isoforms. Wild-type mice from two different genetic backgrounds showed an identical distribution of subunit isoforms among inner ear transport cells. Several epithelial cell types coexpressed the α1 and β1 subunits. Vestibular dark cells showed no reactivity for β1 but expressed abundant β2, whereas, strial marginal cells stained strongly for both β isoforms. The only qualitative difference between mutant and wild-type mice was the absence of β1 subunit in marginal cells of the mutant's stria. However, it is unlikely that this difference accounts for failure of mutants to generate a high EP because the β1 subunit is not present in the stria vascularis of either rats or gerbils with normal EP values. Strong immunostaining for Na,K-ATPase in lateral wall fibrocytes of normal mice along with diminished immunoreactivity in the mutants supports the concept that these strategically located transport fibrocytes actively resorb K + leaked across Reissner's membrane into scala vestibuli or effluxed from hair cells and nerves into scala tympani. It is further speculated that the resorbed K + normally is siphoned down its concentration gradient into the intrastrial space through gap junctions between fibrocytes and strial basal and intermediate cells where it is recycled back to endolymph via marginal cells. Thus, failure of mutants to generate a positive EP could be explained by the absence of intermediate cells which may form the final link in the conduit for moving K + from perilymph to the intrastrial compartment.

Potassium secretion by vestibular dark cell epithelium demonstrated by vibrating probe

Detection of motion and position by the vestibular labyrinth depends on the accumulation of potassium within a central compartment of the inner ear as a source of energy to drive the transduction process. Much circumstantial evidence points to the vestibular dark cell (VDC) epithelium as being responsible for concentrating K+ within the lumen. We have used the vibrating probe technique to directly observe voltage and ion gradients produced by this tissue to put this assumption on a solid experimental footing. Relative current density (Isc,probe) over the apical membrane of VDC epithelium was measured with the vibrating voltage-sensitive probe, and this technique was validated by performing maneuvers known to either stimulate or inhibit the transepithelial equivalent short circuit current. Basolateral bumetanide (5 x 10(-5) M) and ouabain (1 x 10(-3) M) caused a decrease in Isc,probe by 55 +/- 6% and 39 +/- 3%, respectively while raising the basolateral K+ concentration from 4 to 25 mM caused an increase by 35 +/- 8%. A K+ gradient directed toward the apical membrane was detected with the vibrating K(+)-selective electrode, demonstrating that, indeed, the VDC epithelium secretes K+ under control conditions. This secretion was inhibited by bumetanide (by 94 +/- 7%) and ouabain (by 52 +/- 8%). The results substantiate the supposition that dark cells produce a K+ flux and qualitatively support the correlation between this flux and the transepithelial current.

Cell volume control in vestibular dark cells during and after a hyposmotic challenge

Cell height was measured as an index of volume in a preparation of vestibular dark cells in which the perfusate had access to both sides of the epithelium. In response to a hyposmotic challenge induced by removal of 75 mM NaCl, cell height increased to 107%; however, cell width did not increase. Significantly larger increases in cell height were observed in the absence of Cl- or K+ or in the presence of ouabain, lidocaine, barium, or quinidine, at 7 degrees C, or after fixation with glutaraldehyde. However, no significantly different swelling was observed during a hyposmotic challenge in the absence of Na+ or in the presence of bumetanide or ethoxyzolamide. Subsequent return to control osmolarity caused a regulatory volume increase that was dependent on Na+, Cl-, and K+, inhibited by bumetanide, ouabain, or 7 degrees C, however not inhibited by ethoxyzolamide, barium, quinidine, or lidocaine. The data suggest that cell volume control during the hyposmotic challenge involved a mechanism dependent on cytosolic KCl and the Na(+)-K(+)-ATPase and that the Na(+)-Cl(-)-K+ cotransporter was involved in regulatory volume increase.

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

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-cm 2 ( 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/cm 2 ( N = 179). I sc was reduced from 638 ± 60 to 48 ± 16 μA/cm 2 ( N = 14) by basolateral perfusion of 10 −3 M ouabain and from 538 ± 27 to 27 ± 4 μA/cm 2 ( 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-cm 2 ( 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-cm 2 ( 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-cm 2 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.

Otoconia on Vestibular Dark Cells in the Ampullary Area

Morphological and metabolic differences in the otoconia on vestibular dark cells of the ampullary area before and after streptomycin treatment were studied by scanning electron microscope and X-ray microanalyzer.Young adult pigmented guinea pigs were used in this study. In the control group, distribution of the otoconia showed a regular pattern. In the SM group, the otoconia were decreased and their distribution pattern was irregular. The calcium content of the otoconia was also decreased in the SM group. These findings indicate that SM might affect not only the sensory and supporting cells but also the vestibular dark cells.

Application of non-radioisotopic in situ hybridization to the inner ear--expression of osteopontin

Osteopontin (OPN) is considered to be a non-collagenous bone matrix protein which is involved in the ossification process. However, OPN has recently been observed in ectopic sites such as the kidney and nervous tissues. In the present study, expression of OPN mRNA was examined in the rat inner ear by non-radioisotopic in situ hybridization. Signals of OPN mRNA were observed in the marginal cells of the stria vascularis, spiral ganglions, vestibular sensory hair cells and vestibular dark cells. OPN protein was detected only in otoliths by immunohistochemistry. The reasons for the presence of OPN mRNA in the cochlea and dark cells of the vestibulum were unclear. On the other hand, findings of the sacculus and utriculus suggest that OPN is one of the protein components of rat otoliths and that vestibular sensory hair cells are involved in the production of otoliths.

Effect of gentamycin on vestibular dark cells and melanocytes: an ultrastructural and cytochemical study.

Vestibular dark cells areas in gentamycin (GM)-intoxicated guinea pigs were examined ultrastructurally and cytochemically. After GM treatment the dark cells represented cytoplasmic vacuoles, wide intercellular space, less osmiophilic cytoplasm and irregularly shaped basal portions. Cytochemically, Na+,K(+)-ATPase activity was demonstrated on the basolateral folded plasma membranes of the dark cells similarly in both GM-intoxicated and control guinea pigs. These findings revealed that the dark cells still possessed a function of regulating the ionic composition of the endolymph, although they showed various structural changes. On the other hand, the melanocytes extended their cytoplasmic processes into the basement membrane and the intercellular space of the dark cells. Melanosomes markedly increased in number dose-dependently by GM treatment. The significance and the function of the melanocytes in the dark-cell areas are discussed.