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PubMed1998

Cytochemical and patch-clamp studies of calcium influx through voltage-dependent Ca2+ channels in vestibular supporting cells of guinea pigs.

To clarify whether or not vestibular supporting cells have voltage-dependent Ca2+ channels, cytochemical and patch-clamp studies were performed using cells isolated from the ampullae of the semicircular canal of the guinea pig. Image analysis used fura-2 as a Ca(2+)-sensitive fluorescence dye and showed that the intracellular Ca2+ concentration ([Ca2+]i) increased with bath application of high (150 mM)K+, but was unaffected by 80 mM K+. The increase in [Ca2+]i induced by high K+ was completely blocked by 1 microM nifedipine as an L-type Ca2+ channel antagonist. In the patch-clamp whole-cell recording of the isolated supporting cells, the voltage-dependent inward current was induced by a depolarizing pulse lasting 2 s in a high (50 mM) Ca2+ and tetraethylammonium-containing external solution replaced by choline chloride and a Cs(+)-containing internal solution. The inward current was obtained when the membrane was depolarized to -50 mV and maximum current was observed at -10 to +10 mV. This inward current was completely blocked by 1 microM nifedipine. These findings strongly suggest that voltage-dependent Ca2+ channels exist in the vestibular supporting cells and regulate Ca2+ concentration in the vestibular endolymph.

Animals↗
PubMed1994

Changes of ampulla pressure in the semicircular canal of pigeons by caloric stimulation.

Still now several hypotheses about the mechanisms of the caloric nystagmus have been in conclusive. In this study we confirmed the convection effect and the volume change effect of the endolymph in horizontal semicircular canal following the caloric stimulation using pigeons (Columba livia). Although the direction of the caloric nystagmus depended on the head position and the stimulus site of calorization, the caloric nystagmus disappeared after plugging of horizontal semicircular canal. On the other hand, the ampulla pressure increased by cold calorization and decreased by hot calorization and these pressure changes had no relation to the head position. These results show that the main role of the mechanisms of the caloric nystagmus under 1G is the convection effect but the volume change effect may act on the caloric nystagmus not only under 1G but also under microgravity.

Animals↗
PubMedDate not supplied

The preparation of acetic acid for use in otic drops and its effect on endocochlear potential and pH in inner ear fluid.

The ototoxicity of an otic drop preparation containing 2% acetic acid and 3% propylene glycol (VoSol, Denver Chemical Co., Humacao, PR) was investigated according to measurements of endocochlear potential (EP) and inner ear fluid pH. The application of this preparation to the round window membrane for 30 minutes caused a depression in EP from 80.5 +/- 2.5 mV (mean +/- SD; n = 6) to 11.7 +/- 7.7 mV, and lowered inner ear fluid pH from 7.55 +/- 0.09 to 5.06 +/- 0.19 (n = 6) in perilymph and from 7.52 +/- 0.07 to 5.88 +/- 0.63 (n = 6) in endolymph. Two percent acetic acid produced similar changes after 30 minutes: EP was reduced from 83.0 +/- 2.2 mV to 34.0 +/- 2.9 mV and endolymphatic pH from 7.49 +/- 0.04 to 6.83 +/- 0.21 (n = 4). However, the application of artificial perilymph of pH 4 titrated with HCl induced no significant changes in either EP or endolymphatic pH. We suggest that the mechanisms of ototoxicity in the otic drop preparation are Na+ and K+-ATPase inhibition, and that such inhibition is due to the intracellular acidification of strial cells resulting from the penetration of acetic acid across the cell membrane, and to the direct and synergistic actions of propylene glycol.

Acetates↗
PubMed1986

Some hydrodynamic properties of the posterior canal in the frog labyrinth related to neuronal responses.

Measurements of the posterior-canal radius of curvature (R), the semicircular-duct radius (r) and the cupula radius (rC) were performed in the frog labyrinth. The Steinhausen-van Egmond equation led to an estimate of the canal endolymph flow, cupula deflection and sensory hair bending during constant angular accelerations (0.2-64 degrees/s2) of opposite directions and increasing duration (1.2-12 s). This analysis suggests that the non-linear canal afferent discharge behaviour exhibited by some units may not arise at the presynaptic level but rather postsynaptically, at the encoder site.

Acceleration↗
PubMed1978

The effects of ethacrynic acid upon the potassium concentration in guinea pig cochlear fluids.

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.

Animals↗
PubMed1983

Holes in the reticular lamina after noise exposure: implication for continuing damage in the organ of Corti.

Three different histological techniques (scanning electron microscopy, phase contrast microscopy and light microscopy) were used to examine the organ of Corti after a damaging noise exposure. In the region of maximal injury, all outer hair cells were missing 1-2 h after the exposure had ended. Degenerated hair cells are eventually replaced by phalangeal scars. However, in these short-term recovery ears, a series of holes which were the exact size and shape of the missing outer hair cell apices were found in the reticular lamina. These holes may provide a communication route between the endolymphatic space and the fluid spaces of the organ of Corti for a period of time following a damaging noise exposure. The noise-related degeneration of supporting cells, nerve fibers and even some sensory cells may be secondary to contamination of the fluid spaces with potassium-rich endolymph.

Animals↗
PubMed1988

Potassium ion conductance of the cochlear partition: differences between the chinchilla and guinea pig.

The time courses of the endocochlear potential (EP) and the K+ concentrations in the inner ear fluid under permanent anoxia were observed in the chinchilla and guinea pig using K+-selective microelectrodes. The EP following 30 min of anoxia in the chinchilla (-10.9 +/- 2.2 mV) showed a significantly less negative value than that of the guinea pig (-25.7 +/- 2.6 mV). The K+ concentration in endolymph induced by anoxia decreased less in the chinchilla than in the guinea pig. The average K+ conductance of the cochlear partition 10-30 min after anoxia in the guinea pig (0.1703 +/- 0.0792 S) was approximately 7.9 times that of the chinchilla (0.0216 +/- 0.0042 S), which is thought to be responsible for the difference of the anoxic EP between the two species.

Animals↗
PubMed1991

Pilocarpine elicits the interdental cell secretory activity of the inner ear.

Subcutaneous injection of pilocarpine in guinea pigs resulted in the following ultrastructural changes: 1) the apical cavities of the interdental cells were filled with a substance indistinguishable from the overlying amorphous layer of the TM; 2) a great number of spherical structures appeared over the limbal portion of the tectorial membrane. In TEM photomicrographs these structures displayed the same appearance as the amorphous layer of the TM and were usually continuous to it; 3) the number of holes that decorate the upper surface of the limbal portion of the TM was dramatically increased and it was found that they connect the endolymphatic space to the apical cavities of the interdental cells; 4) there was an increase in the number of the small extracellular vesicles found in the clear spaces of the tectorial membrane. These facts suggest that pilocarpine stimulates the secretion of the interdental cells, confirming the existence of the secretory processes previously described (Prieto et al., 1990). These findings can be related to the turnover of the TM in the adult animal and, perhaps, to the secretion of some organic compound to the endolymph. We postulate that the actions of pilocarpine on the interdental cells are most probably mediated by the activation of muscarinic acetylcholine receptors in these cells.

Animals↗
PubMed1991

Old theme and new reflections: hearing impairment associated with endolymphatic hydrops.

The cause(s) of the hearing impairment associated with Menière's disease are not understood but are undoubtedly associated with the inner ear endolymphatic hydrops. Two major hypotheses have been proposed and widely received: endolymphatic overpressure followed by leaky membranes and subsequently the mixing of high K+ endolymph with perilymph. Our recent data on an experimental model of endolymphatic hydrops have provided grounds for renewed reflections on the pathology. Indeed our data might be interpreted without involving either of the above hypotheses and suggests that the symptoms of Menière's disease might be accounted for by a flow of perilymph from scala vestibuli towards scala tympani with the mixing of the two perilymphs which are similar but not identical in composition. The higher K+ concentration arriving from the scala vestibuli into the scala tympani at the apex of the cochlea via the helicotrema is likely to be toxic to hair cell and auditory nerve fiber function. The mixing of the two perilymphs could result in deterioration of low frequency sensitivity, provoke low frequency tinnitus and in the long term cause spiral ganglion cell degeneration at the apex of the cochlea. The feeling of fullness in the ear might be the result of the decreasing perilymph volume in the scala vestibuli which could give rise to inner ear conductive losses. The patency of the cochlear aqueduct might play a role in determining the high risk group of individuals likely to manifest the symptoms of endolymphatic hydrops.

Ear↗
PubMed1993

Effect of lymph composition on an in vitro preparation of the alligator lizard cochlea.

The effects of different artificial lymphs on the cochlear duct of the alligator lizard were studied in an in vitro preparation. The duct was dissected and cemented to the glass floor of a chamber that had been filled with an artificial lymph. The vestibular membrane was removed and latex beads (1-5 microns in diameter) were allowed to settle on the endolymphatic surface of the duct. During perfusion with an artificial lymph solution, the positions of beads were measured and video images of the duct were obtained. Artificial lymphs were isosmotic and included artificial endolymph (AE), artificial perilymph (AP), Leibovitz's L-15 culture medium, an AE solution whose calcium concentration was the same as that of AP, and AE and AP solutions in which gluconate was substituted for chloride ions. Results obtained in AE were consistently different from those in other lymphs. The displacements of beads, the projected area of the papilla, the occurrence of blebs, and direct observation of cells in the duct all indicated that the tissue swelled in AE (with or without 2 mmol/l Ca) but showed no consistent shrinking or swelling in any of the other artificial lymphs. Thus for the solutions we used, the presence of both potassium and chloride was required to elicit the swelling response to isosmotic artificial lymphs. There were some regional differences in the swelling response: the swelling of the endolymphatic surface of the tissue in a direction orthogonal to the basilar membrane surface was smaller on the free-standing region of the basilar papilla than either on the tectorial membrane or on the hyaline epithelial cells. The preparation was osmotically stable in AP and in both AE and AP solutions in which gluconate was substituted for chloride ions. After exposure to these solutions for as much as 300 min, the preparation showed no gross signs of deterioration visible with the light microscope, and continued to exhibit a highly specific osmotic response to the composition of the bathing medium.

Animals↗
PubMed1995

Role of endolymphatic anion transport in forskolin-induced Cl- activity increase of scala media.

To determine the role of anion transport in the forskolin-induced Cl- increase of scala media (SM), effects of forskolin on the EP (endocochlear potential) and Cl- activity (ACl) in SM were examined with double-barrelled Cl(-)-selective microelectrodes. The experiments were carried out on guinea pig cochleae, using a few anion transport inhibitors: IAA-94 for a Cl- channel blocker, bumetanide (BU) for an Na+/K+/2Cl- cotransport blocker, and SITS and DIDS for Cl-/HCO3- exchange blockers. The application of forskolin (200 microM) into scala vestibuli (SV) caused a 20 mEq increase of endolymphatic ACl and a 15 mV elevation of EP, and IAA-94 with forskolin completely abolished these responses. Although each application of BU, SITS or DIDS did not completely suppress EP elevation, the concurrent application of these inhibitors completely suppressed EP with endolymphatic ACl increase. The results indicate the involvement of Cl- channels, Na+/K+/2Cl- cotransport and Cl-/HCO3- exchange in forskolin-induced increase of ACl and EP. The role of adenylate cyclase activation and Cl- transport in endolymph homeostasis was discussed.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1994

Mineralocorticoid type I receptor in the rat cochlea: mRNA identification by polymerase chain reaction (PCR) and in situ hybridization.

Expression of mineralocorticoid type I receptor (MR) gene in the rat cochlea was determined using molecular biological techniques. We synthesized complementary DNA (cDNA) from rat cochlear total RNA and then amplified MR cDNA fragments by polymerase chain reaction (PCR). The amplified cDNA fragments were subcloned into an expression vector and the nucleotide sequence was analyzed to confirm the expression of mRNA encoding MR in the cochlea. We then synthesized digoxigenin-labeled riboprobes with this cloned DNA template and examined the localization of MR mRNA in the cochlea by in situ hybridization. The amino acid sequence of MR cDNA expressed in the cochlea was identical to that of the MR first cloned in the rat hippocampus. In situ hybridization showed the expression of MR mRNA in marginal cells of the stria vascularis, suggesting that aldosterone may regulate microhomeostasis of the endolymph, presumably by modulating Na, K-ATPase activity. Intense MR signal was also identified in spiral ganglion cells, the function of which remains to be determined.

Aldosterone↗
PubMed1994

The osmotic response of the isolated tectorial membrane of the chick to isosmotic solutions: effect of Na+, K+, and Ca2+ concentration.

Changes in the size, shape, and structure of the isolated tectorial membrane of the chick were measured in response to isosmotic changes in the ionic composition of the perfusion solution. Substitution of artificial perilymph (AP) for artificial endolymph (AE) caused a small (approximately 15%), slow (time constants tau approximately 12 min) shrinkage of the thickness of the tectorial membrane that was largely reversed on return to AE. Substitution of AP for AE alters not only the predominate cation (from K+ to Na+) but also the Ca2+ concentration (from < 7 mumol/l to 2 mmol/l). Additional experiments were performed to separate effects of each of these changes. When a high-Na+, low-Ca2+ solution was substituted for a high-K+, low-Ca2+ solution (AE), the tectorial membrane swelled significantly, often to more than twice its original thickness (the largest swelling was 337%), with a slow time course (tau approximately 23 min). Addition of the Ca2+ to either high-K+ or high-Na+ solutions caused rapid shrinkage of the tectorial membrane (tau approximately 2-3 min). Addition of the Ca2+ chelator EGTA caused rapid swelling (tau approximately 4 min). Large osmotic responses were only partially reversible and caused long-lasting changes. For example, long-duration solution changes that produced large, rapid osmotic responses early in an experiment tended to produce smaller and slower responses later in the experiment. In contrast, the small osmotic responses to short-duration solution changes were repeatable for tens of hours. Changes in ionic composition of the bath affected not only the thickness of the tectorial membrane but also its other dimensions. Responses were not generally isotropic; both the size and shape of the tectorial membrane generally changed. Consistent changes in microstructure accompanied the osmotic changes.

Animals↗
PubMed1994

Modulation of cyclic AMP production by strial marginal cells from gerbil in culture.

To further investigate the role of marginal cells (MCs) in the secretion of endolymph and because of the limitations encountered in investigating these cells in vivo, we used primary cultures of MCs derived from explants of gerbil stria vascularis and investigated modulation of the adenylate cyclase-cyclic AMP system. After 10 days on type I collagen coated plastic dishes, a confluent monolayer of epithelial-like cells was obtained which exhibited the morphologic and immunohistochemical features of the native marginal cells. The cyclic AMP (cAMP) content was determined at 37 degrees C, after 5 min of incubation with various agents, in the presence of a specific inhibitor of type III cAMP-dependent phosphodiesterase, RO 20-1724. The adenylate cyclase-cAMP system was associated with beta 2-adrenergic receptors. The cAMP content was increased by isoproterenol (23-fold), a beta-agonist, but not by octopamine, an alpha-agonist, and the affinity for ICI 118.551, a specific beta 2-antagonist, was greater than for CGP 20712A, a specific beta 1-antagonist (Kd: 0.03 x 10(-6) M and 15 x 10(-6) M respectively). The cAMP content was maximally increased by prostaglandin E2 > beta 2-adrenergic agonist >> vasopressine type 2 receptor agonist (26-, 23-, and 3-fold the basal cAMP content, respectively). The present study demonstrates that cultured marginal cells retain some of their in vivo properties including a modulated enzymatic cAMP system. This culture model should allow further in-depth investigation of the function of marginal cells.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

The osmotic response of the isolated, unfixed mouse tectorial membrane to isosmotic solutions: effect of Na+, K+, and Ca2+ concentration.

Changes in the size, shape, and structure of the isolated tectorial membrane (TM) of the mouse were measured in response to isosmotic changes in the ionic composition of the bathing solution. Substitution of artificial perilymph (AP) for artificial endolymph (AE) caused a small (approximately 1%) shrinkage of the TM's thickness. This substitution alters not only the predominate cation (from K+ to Na+) but also the Ca2+ concentration (from 20 mumol/l to 2 mmol/l). When the predominate cation was changed from K+ to Na+, while holding Ca2+ concentration constant, results depended on Ca2+ concentration: there was a small (approximately 1%) swelling for 20 mumol/l Ca2+, larger (approximately 14%) swelling for lower (< 7 mumol/l) concentrations of Ca2+, and little response for 2 mmol/l Ca2+ or for solutions containing the Ca2+ chelator EGTA. Addition of Ca2+ while holding the predominate cation constant caused shrinkage of the TM; both removal of Ca2+ and addition of the Ca2+ chelator EGTA caused swelling. Swelling responses were largely reversible if the magnitude of the swelling was small. Responses greater than a few percent were only partially reversible and caused long-lasting changes. Changes in ionic composition of the bath affected not only the thickness of the TM but also its other dimensions. Solution changes that increase TM thickness tend to cause radial shearing motions of the surfaces of the TM, which are accompanied by small decreases in width. Little change in length was observed. Although the responses were non-isotropic, increases in thickness were highly correlated with increases in volume. Swelling of the TM was also accompanied by a reduction in prominence of its radially oriented fibrillar structure. These results for the isolated TM of the mouse are qualitatively similar to those obtained previously for the isolated chick TM (Freeman et al., 1994) but different from those obtained for the in vitro mouse TM (Kronester-Frei, 1979a).

Acoustic Stimulation↗
PubMed1995

Extracellular adenosine 5'-triphosphate (ATP) in the endolymphatic compartment influences cochlear function.

There is strong evidence for the presence of P2 purinoceptors on cochlear tissues, but the role of extracellular ATP in cochlear function is still unclear. Our previous studies have determined the presence of ATP in the cochlear fluids and indicated that the purinoceptors are substantially localized to the tissues lining the endolymphatic compartment. This implies that extracellular ATP may have an humoral role confined to the endolymphatic space. In order to study the influence of extracellular ATP in the endolymphatic space, a series of studies were undertaken in which ATP (10 microM to 10 mM) in artificial endolymph (EL) (test solution: 2-12.5 nl) was injected into the scala media and the effect on the cochlear microphonic (CM) and endocochlear potential (EP) evaluated. A double-barrelled pipette, with one barrel containing the test solution and the other artificial EL (control solution) was inserted into scala media of the third turn of the guinea-pig cochlea. A known volume (2-12.5 nl) of test or control solution was then pressure-injected into the space. ATP had a significant dose-dependent suppressive effect on both EP and CM with a threshold of approximately 2 x 10(-14) mol; the response was readily reversible, also in a dose-dependent fashion. Artificial EL of the same volume had no effect on EP and CM. The ATP effect on EP was blocked by the P2 purinoceptor antagonists suramin and reactive blue 2 (RB2). Neither adenosine (2 x 10(-13) to 2 x 10(-11) mol) nor suramin or RB2 on their own had any effect on EP and CM. This study provides the first evidence for an effect of extracellular ATP in the endolymphatic compartment on cochlear function which is mediated via P2 purinoceptors. This provides supporting evidence for an humoral role for extracellular ATP in the modulation of cochlear function.

Adenosine↗
PubMed1995

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.

Adenosine Triphosphate↗
PubMed1995

Combined effects of adrenalectomy and noise exposure on compound action potentials, endocochlear potentials and endolymphatic potassium concentrations.

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.

Acoustic Stimulation↗