Cation transport in the ampulla of the semicircular canal and in the endolymphatic sac

Summary We examined the effects of anoxia and ethacrynic acid on the endolymphatic potential and cation activity in the superior ampulla of the guinea pig, using double-barrelled ion-exchanger microelectrodes. In normal guinea pigs the ampullar endolymphatic potential was +3.9±1.2 mV (n=32), the Cl^– activity 130±4.6 mM (n=9), and the Na⁺ activity 18.4±4.4 mM (n=20). After anoxia, the ampullar DC potential decreased rapidly and reversed its polarity within 5 min. It then decreased gradually for 60 min and increased afterwards to approximately zero. K⁺ activity decreased gradually after a latency of 10 min, whereas Na⁺ activity increased. During the gradual decrease of a negative ampullar endolymphatic potential, an increase in Na⁺ activity was observed. Thirty minutes after the intravenous injection of ethacrynic acid (100 mg/kg), the potential began to decrease, changed to a negative polarity, and approached a maximum negative level 100 min after the injection. The decrease in K⁺ activity corresponded to the reduction of potential whereas Na⁺ activity remained unchanged. The DC potential of the endolymphatic sac in normal guinea pigs was + 14.7±5.1 mV (n=17). The Na⁺ concentration was 103.3±14.7 mM (n=14) and the K⁺ concentration was 11.6 ±0.8 mM (n=4). After anoxia, the DC potential decreased rapidly and approached 0 mV within 8 min. No negative potential could be observed. The Na⁺ concentration began to increase 2 min after anoxia and reached the extracellular Na⁺ concentration about 30 min later. No significant effect of intravenous administration of ethacrynic acid (100 mg/kg) on DC potential and Na + concentration could be observed. The results suggest the presence of a different ion transport system in the endolymphatic sac from that of the cochlea and the ampullae of the semicircular canals.     

速尿对壶腹嵴内电位及Ca^2＋活度的影响

为广探讨速尿试验改善梅尼埃病前庭功能的可能机理，应用钙离子选择性微电极观察速尿对正常及实验性膜迷路积水豚鼠的前庭壶腹嵴内电位（AEP）及其内淋巴Ca2＋活度的影响。结果表明，实验性膜迷路积水动物AEP显著下降（P＜0．001），Ca2 活度明显升高（P＜0．01）。经静注10mg／kg速尿约6min的潜伏期后，AEP评始下降，40min达最低，为3．04±0．53mV，但与注射前比较地显著性差异。Ca2 活度于60min上升至最高，为（710±1．32）×10－4M，80min开始回落。结果支持小剂量速尿对实验膜迷路积水前庭有直接作用。并就Ca2＋在速尿试验作用机理中的意义进行讨论。     

Endolymph formation in the inner ear of pigeons

The endolymphatic space of pigeons was studied by using double-barrelled electrodes with a potassium liquid ion exchanger. The K+ activity of the endolymph was 155 mM in the cochlea and 133 mM in the ampulla, respectively. Positive DC potential in the cochlea (+14.5 mV) was much lower than in guinea pigs (+80 mV) whereas in the ampulla of pigeons the DC potential (+7.4 mV) was 2 times higher than that of guinea pigs (+3.9 mV). General application of ethacrynic acid in pigeons induced a weak change in DC potential and no typical intercellular edema in the cochlea and ampulla. Local application of ethacrynic acid and ouabain in the cochlea and ampulla of pigeons induced a negative DC potential of between -30 and -40 mV. This negative DC potential was higher than the anoxia-induced negative potential. Short hypoxia during a drug-induced DC potential resulted in a decrease in DC potential above the diffusion potential. Below the diffusion potential additional hypoxia increased the DC potential independent of the cause of intoxication.     

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.     

Adenylate cyclase modulation of endocochlear potential during suppression of strial Na(+)-K+ ATPase.

Forskolin, an adenylate cyclase activator, produces a reversible elevation of the endocochlear potential (EP) (Doi et al., 1990a). To determine whether strial Na(+)-K+ ATPase activity is essential for the forskolin-dependent EP elevation, we examined, by means of K(+)-selective microelectrodes, the effects of forskolin on the EP and the endolymphatic K+ activity ([K+]) while strial Na(+)-K+ ATPase was suppressed by ouabain. Perilymphatic perfusion with ouabain (10(-3) M) decreased the EP from 78.5 +/- 2.4 mV to -27.6 +/- 2.4 mV (N = 8) at 37.9 +/- 3.7 min after the start of perfusion and decreased the [K+] from 138.7 +/- 5.4 mM to 103.7 +/- 3.7 mM (N = 3). Successive perfusion with forskolin (2 x 10(-4) M) with ouabain (10(-3) M) increased the EP by 15.1 +/- 1.5 mV (N = 8) but did not influence the [K+] decrease from 101 +/- 3.6 mM to 95 +/- 1.3 mM (N = 3). Forskolin (2 x 10(-4) M) with ouabain (10(-3) M) without a preceding ouabain perfusion decreased the EP from 76.2 +/- 2.3 mV to -12.9 +/- 1.8 mV (N = 6) at 65.3 +/- 2.1 min after the start of perfusion. These results indicate that adenylate cyclase can modulate the EP in the absence of strial Na(+)-K+ ATPase activity and that adenylate cyclase activation can attenuate the EP drop induced by strial Na(+)-K+ ATPase suppression.     

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

The ampullar endolymphatic potential (AEP) was studied in the guinea pig during ischemia and asphyxia and following systemic application of ethacrynic acid. In addition the specialized and nonspecialized portions of the ampullar wall were analyzed for ATP and P-creatine at different conditions of metabolic interference. Under control conditions the AEP amounted to + 4.6 +/- 1.2 mV. In both types of hypoxia the decline of the AEP proceeded on a much slower time scale than that of the cochlear endolymphatic potential (CEP), and the maximum negativity reached was considerably less. Quantitative analysis of both types of ampullar wall tissue indicated a much slower decline in hypoxia of ATP levels than in the stria vascularis. Changes in P-creatine levels were considerably more rapid. The AEP became reduced and changed polarity also by intoxication with ethacrynic acid (EA), but higher dosages (above 70 mg/kg) were necessary than for effects upon the CEP and much longer time periods were required for attainment of maximum negativity. The maximum negativity of the AEP was significantly greater at a dosage of 100 mg/kg of EA than during ischemia. At the point of maximum depression of the AEP P-creatine levels in both types of ampullar tissue were unchanged, but ATP levels were significantly reduced in the specialized portions of ampullar wall.     

Ionic activities of the inner ear fluid and ionic permeabilities of the cochlear duct in endolymphatic hydrops of the guinea pig

Ionic activities (K+, Na+, and Cl-) of the perilymph and endolymph of the basal turn were measured using ion-selective microelectrodes in experimentally induced endolymphatic hydrops of the guinea pig. Three months following the obstruction of the endolymphatic duct and sac, the endocochlear potential (EP) of hydroptic ears was measured at 59.7 +/- 9.6 mV (N = 12) which was significantly lower than the EP of the contralateral control ears (84.4 +/- 2.8 mV, N = 12). A paired t-test (P greater than 0.05) showed no significant differences of ion concentrations of the inner ear fluid between the hydroptic and contralateral ears. Ion permeabilities of the cochlear duct following anoxia were calculated according to the Nernst-Planck equation. Comparing hydroptic and normal ears following anoxia, a statistically significant decrease was observed in the permeability coefficients for K+. Similarly, K+ conductance was significantly lower in the hydroptic ears than in the normal ears. Total conductance of the cochlear duct, defined as the sum of each ion conductance, was 0.560 siemens in the normal ears and 0.217 siemens in the hydroptic ears. On the basis of the Goldman-Hodgkin-Katz equation, preexisting negative EP in the normal state was calculated to be -24.5 mV in normal ears and -21.4 mV in hydroptic ears. Therefore, the positive component of the EP was 108.9 mV in normal ears and 81.1 mV in hydroptic ears. These findings suggest that the pathophysiology of hydrops involves changes in K+ permeability and the inhibition of the electrogenic transport processes.     

Membrane potential measurement in isolated outer hair cells of the guinea pig cochlea using conventional microelectrodes.

Membrane potential of the isolated outer hair cells (OHCs) from the guinea pig cochlea was measured using conventional microelectrodes filled with 200 mM KCl. The resting membrane potential during superfusion with the standard physiological saline solution containing 3.5 mM K+ was -47.3 +/- 1.4 mV (N = 72), which was higher than those previously reported for isolated OHCs studied by using microelectrodes. Addition of ouabain (10(-5)-10(-3) M), the specific Na+, K+ ATPase inhibitor, depolarized the cell slowly and progressively, indicating the presence of low but definite Na+, K+ ATPase activity in the plasma membrane of OHCs. The magnitude of membrane potential was mainly dependent on the extracellular K+ concentration ([K+]O). A ten-fold increase of [K+]O depolarized the membrane potential by 49.6 +/- 1.0 mV (N = 58). A decrease of [Na+]O to one tenth of the control hyperpolarized the membrane potential by about 2 mV. Decreasing extracellular Cl- from 131.3 mM to 27.5 mM did not cause a significant change in the membrane potential. Using the Goldman-Hodgkin-Katz equation, assuming a negligible contribution of Cl- to the membrane potential and total monovalent cat ion concentration of the cytosol similar to the extracellular fluid, we calculated the permeability ratio of K+ versus Na+ to 131 +/- 19 and intracellular K+ concentration to 33.3 +/- 1.9 mM.     

Calcium transport in the endolymphatic sac

Ca++ activity and DC potential were measured in vivo in the endolymphatic sac (ES) of guinea pigs by means of double-barrelled ion-sensitive microelectrodes. We found a positive DC potential of 14 mV and a Ca++ activity of 4.7 X 10(-4) M. Anoxia induced a decrease in the DC potential and an increase in Ca++ activity; however, no negative DC potential was measured during permanent anoxia. The Ca++ activity measured was in agreement with the Ca++ value calculated with the Nernst equation, assuming a positive DC potential. On the basis of these data, it was suggested that the Ca++ in the ES is in electrochemical equilibrium with the surrounding fluid and no active Ca++ transport is necessary in the ES of guinea pigs.     

The ionic and electric environment in the endolymphatic sac of the chinchilla: relevance to the longitudinal flow

The ionic composition of the endolymph in the endolymphatic sac (ES) of the chinchilla was measured using double-barreled ion-selective micro-electrodes. The DC potential of the ES was 9.3 +/- 1.8 mV (N = 18). The K+, Na+, and Cl- concentrations of the ES were 13.3 +/- 4.7 mM (N = 6), 129.0 +/- 8.8 mM (N = 6), and 124.3 +/- 16.6 mM (N = 6), respectively. In light of the chemical potentials of the cochlear endolymph previously reported [Ikeda and Morizono (1989), Hear. Res., 39, 279-286] the pressure gradient of the endolymph between the cochlea and ES was calculated to be 71.5 mmHg at 38 degrees C. The contribution of the osmotic and hydrostatic pressure gradients of the endolymph to the longitudinal flow is discussed.     

Effect of kanamycin sulfate on the endocochlear dc potential of guinea pigs.

The magnitudes of the endocochlear dc potential (EP) during adequate ventilation and during five minutes of anoxia were recorded in control guinea pigs and guinea pigs administered various dosages of kanamycin sulfate. The magnitudes of the EP after five minutes of anoxia were -44.6 +/- 5.9 mV in the controls and +26.6 +/- 9.9 mV in the guinea pigs that received kanamycin for seven or ten days. This change occurred after five or six days of administration of kanamycin. However, there was no significant change in the magnitude ot the EP with adequate ventilation with kanamycin intoxication. There is a significant correlation between the magnitude of the negative EP and the maximum output of the cochlear microphonics (r = -.770, P less than .001). These results suggest that the EP may not be the mathematical summation of the positive electrogenic potential and the negative diffusion potential. The mechanism for generating the negative EP during anoxia may have some relationship to hair cell integrity.     

Electrophysiological measurements of the stria vascularis potentials in vivo

Glass microelectrodes were introduced into the stria vascularis (SV) to measure the DC potential profile of the SV in vivo from its apical as well as basal side. The K+ concentration gradient in the lateral cochlear wall was measured by means of double barrel K+-selective microelectrodes. A positive potential 10.4 +/- 6.3 mV higher than the endocochlear potential (EP) was found in the SV. In noise-exposed animals the positive potential found in the SV was 14.6 +/- 7.2 mV higher than the EP. K+ concentration observed during penetration into the SV was in the range of 50-100 mmol/l. Simultaneous measurement of the DC positive potential in the SV and EP showed a nearly parallel time course during anoxia with a tendency to increase the difference between the SV potential and the EP. The difference reached approximately 30 mV 20 min after the beginning of anoxia. It may be assumed that the electrogenic pump localized at the basolateral membrane of the marginal cell (MC) is a source of the positive potential inside the MC and is hence a source of the potential drop across the luminal side of the MC membrane.     

Endocochlear potential and endolymphatic K+ changes induced by gap junction blockers

OBJECTIVE: To examine the effects of gap junction blockers on the endocochlear potential (EP) and endolymphatic potassium concentration ([K(+)](e)). MATERIAL AND METHODS: The EP and [K(+)](e) were monitored using double-barreled ion-selective microelectrodes in the second turn of the guinea pig cochlea during perilymphatic perfusion. RESULTS: When the perilymphatic scalae of the cochlea were perfused with artificial perilymph containing 10 mM n-heptanol the EP was decreased by -8.8+/-1.4 mV (n=10), and this was accompanied by a decline in the [K(+)](e) of -6.7+/-2.1 mM (n=6). Perilymphatic application of 10 mM hexanol also produced declines in both the EP and [K(+)](e). In control studies, perilymphatic perfusion with 10 mM ethanol showed no remarkable changes in either the EP or [K(+)](e). Anoxia during perfusion with heptanol resulted in the generation of a negative EP, similar to the situation in controls. CONCLUSIONS: A decline in the EP together with a lowering of [K(+)](e) induced by long-chain n-alkanols, which act as gap junction blockers, may be explained by an interruption in potassium ion transport related to a gap junction dysfunction.     

The Ca2+ activity of cochlear endolymph of the guinea pig and the effect of inhibitors

The Ca2+ concentrations in cochlear perilymph and endolymph of the guinea pig were measured with double-barreled Ca2+-selective microelectrodes and showed 1.76 +/- 0.74 X 10(-3) M and 2.20 +/- 0.19 X 10(-5) M, respectively. The electrochemical potential gradient for Ca2+ between perilymph and endolymph was 23.2 mV and the existence of an active transport mechanism from the former to the latter was suggested. Vanadate given perilymphatically decreased the Ca2+ concentration in endolymph with a slight elevation of the endocochlear potential and was suspected of blocking the active transport. The Ca2+ concentration in endolymph was abruptly increased by anoxia or the intravenous administration of 60 mg/kg furosemide and was slightly increased by the intravenous administration of 30 mg/kg furosemide or 100 mg/kg acetazolamide. The endolymphatic pH measured with pH-microelectrodes under various conditions indicates that the mechanism of increase in the Ca2+ concentration is attributed not to the liberation of Ca2+ from the surrounding tissues caused by a fall in pH but to the increased influx of Ca2+ from perilymph due to the depression of the endocochlear potential.     

Magnesium ion activity in the mammalian endolymph measured with ion-selective microelectrodes

The free Mg++ concentration in endolymph was measured with Mg++-selective microelectrodes based on the neutral ligand ETH 1117. The property of Mg++ microelectrodes was obtained from calibration solutions, containing various Mg++ concentrations with the background electrolytes resembling endolymph. The range between 10 and 0.1 mM Mg++ concentrations changed the potentials of Mg++ microelectrodes by 14.4 +/- 3.0 mV. The endocochlear potential and the Mg++ concentration in the endolymph were 82.0 +/- 5.0 mV and 0.77 +/- 0.29 mM in the guinea pig, and 84.4 +/- 4.9 mV and 1.12 +/- 0.24 mM in the chinchilla, respectively. These results are discussed in the light of the dependence of Na+, K+-ATPase and its interaction with Ca++.     

The effect of kanamycin on experimentally induced endolymphatic hydrops

Summary In guinea pigs with endolymphatic hydrops 6 months after operation the DC potential had decreased from +80 mV to +50 mV, while the sodium activity had increased. The twofold increase in Na⁺ activity could explain the increased degree of endolymphatic hydrops. The K⁺ activity and Cl^– activity were unchanged. However, endolymphatic hydrops could be produced in kanamycin-induced deaf guinea pigs after obliteration of the endolymphatic sac and duct. The physiological data on DC potential, K⁺, and Na⁺ activity were similar to those for non-treated animals. When the animals were treated with kanamycin 6 months after the development of endolymphatic hydrops, there was no effect on the morphological and functional changes compared to controls. The data suggest that regulation of the inner ear fluid occurs independent of an intact auditory system. The effect of aminoglycoside treatment in Ménière's disease cannot be explained by an effect on endolymphatic hydrops formation.     

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.     

Electrochemical profiles for monovalent ions in the stria vascularis: cellular model of ion transport mechanisms

The electrochemical profiles for K+, Na+, and Cl- ions in tissues of the lateral wall of the cochlea in the chinchilla were measured using an ion-selective microelectrode. Based upon the changes of the d.c. resting potential and ion composition, five distinct compartments were identified in the lateral wall. The first compartment, corresponding to the spiral ligament, showed an ionic composition similar to perilymph. The second, corresponding to the basal cell layer of the stria vascularis, showed a characteristic recording of the ion-sensitive barrel: a spike-like change of the ion concentration (K+: 100.1 +/- 16.8 mM, Na+: 33.9 +/- 20.2 mM, Cl-: 70.2 +/- 9.4 mM). The third compartment, corresponding to the extracellular space of the stria vascularis, showed a higher d.c. potential (60.4 +/- 9.5 mV) than that of the second region, with a low K+ concentration (22.1 +/- 14.9 mM) and a high Na+ (78.3 +/- 5.7 mV) than the fifth compartment, corresponding to the scala media (78.1 +/- 4.1 mV), and K+ and Na+ concentrations similar to those of endolymph, while the Cl- concentration in the fourth compartment (117.6 +/- 21.5 mM) was lower than that of endolymph (143.3 +/- 13.9 mM). A thermodynamic study of electrochemical potential gradients suggests the possibility that the Na-K pump and Na-K-2Cl cotransport exist at the basolateral membrane of the marginal cells.     

Effects of heavy metal ions in endocochlear DC potential and cochlear microphonics in the guinea pig--the influence of calcium on the cochlea]

Endocochlear DC potential (EP) and cochlear microphonics (CM) in the guinea pig under the influence of the divalent heavy metal cations of manganese, nickel, cobalt and cadmium, and the trivalent cation of lanthanum were investigated. The area from the scala tympani to the scala vestibuli was perfused with control and test solutions. CM decreased gradually to 50-80%, but EP showed no change after perfusion with a solution containing 1 mM of metal ions. At a concentration of 10 mM, EP decreased from 80 mV to 10-20 mV and CM decreased to 15-55%. At 100 mM, EP increased by about 10 mV at the beginning of perfusion, remained steady for 1 min, and then rapidly decreased to 0-10 mV. Meanwhile, CM continued to decrease, finally sustaining a 10-56% reduction. The decrease in EP and CM were irreversible, and perfusion of the area with the standard solution for 20 min had no effect. The osmolarity of the artificial perilymph containing 100 mM of metal ions was twice as high as that of the normal physiological solution. The effects of osmolarity, however, were excluded because perfusion with an artificial perilymphatic solution made hypertonic by either NaCl or sucrose changed neither EP nor CM. The application of 100 mM of metal ions topically to the round window membrane caused no change in EP. The alkali metal ions are known to inhibit inward Ca2+ current. Therefore, the present results suggest that Ca2+ ions play a role in maintaining EP generation in the stria vascularis and CM generation in the organ of Corti.     

Arguments against a mediating role of the adenylate cyclase--cyclic AMP system in the ototoxic action of loop diuretics

Previous studies in our laboratory have indicated that adenylate cyclase of the stria vascularis is strongly inhibited in vitro by ethacrynic acid and furosemide. In order to test whether the in vitro effects upon the enzyme are also present under in vivo conditions, ethacrynic acid was perfused perilymphatically for 15 min and 20 min at a concentration of 10(-3) M. Cyclic AMP of the stria vascularis was reduced by 27% and 34%, respectively, but ATP also declined significantly, suggesting unspecific effects. When ethacrynic acid was applied intravenously at a dosage of 50 mg/kg, and the endolymphatic potential allowed to decline to -10mV, no significant changes in cyclic AMP and ATP were seen. The absence of effects upon cyclic AMP in the early stage of systemic intoxication with ethacrynic acid is strong evidence against a mediating role of adenylate cyclase in the ototoxic action of ethacrynic acid. When a bolus of 3 x 10(-2) M furosemide was applied intra-arterially the endolymphatic potential declined at the exceedingly rapid rate of about 10 mV/sec, strongly suggesting that the action of the drug takes place in the vicinity of the capillaries of the stria vascularis. In view of the proposition that adenylate cyclase appears to be located primarily at eh luminal aspect of the stria vascularis, this constitutes further evidence against a role of the enzyme in the mediation of the specific ototoxic effects of loop diuretics. Other recent evidence against a mediatory role of the adenylate cyclase--cyclic AMP system is discussed.