Inner ear defects induced by null mutation of the isk gene.

The isk gene is expressed in many tissues. Pharmacological evidence from the inner ear suggests that isk mediates potassium secretion into the endolymph. To examine the consequences of IsK null mutation on inner ear function, and to produce a system useful for examining the role(s) IsK plays elsewhere, we have produced a mouse strain that carries a disrupted isk locus. Knockout mice exhibit classic shaker/waltzer behavior. Hair cells degenerate, but those of different inner ear organs degenerate at different times. Functionally, we show that in mice lacking isk, the strial marginal cells and the vestibular dark cells of the inner ear are unable to generate an equivalent short circuit current in vitro, indicating a lack of transepithelial potassium secretion.

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- = NO3- = SCN- >> gluconate-. Secondary swelling was not inhibited by Ba2+, 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 (IsK or minK channel) and the basolateral 360 pS Cl- channel. The involvement of these channels, however, remains to be demonstrated.

Progressive irreversible hearing loss is caused by stria vascularis degeneration in an Slc26a4-insufficient mouse model of large vestibular aqueduct syndrome.

Hearing loss of patients with enlargement of the vestibular aqueduct (EVA) can fluctuate or progress, with overall downward progression. The most common detectable cause of EVA is mutations of SLC26A4. We previously described a transgenic Slc26a4-insufficient mouse model of EVA in which Slc26a4 expression is controlled by doxycycline administration. Mice that received doxycycline from conception until embryonic day 17.5 (DE17.5; doxycycline discontinued at embryonic day 17.5) had fluctuating hearing loss between 1 and 6 months of age with an overall downward progression after 6 months of age. In this study, we characterized the cochlear functional and structural changes underlying irreversible hearing loss in DE17.5 mice at 12 months of age. The endocochlear potential was decreased and inversely correlated with auditory brainstem response thresholds. The stria vascularis was thickened and edematous in ears with less severe hearing loss, and thinned and atrophic in ears with more severe hearing loss. There were pathologic changes in marginal cell morphology and gene expression that were not observed at 3 months. We conclude that strial dysfunction and degeneration are the primary causes of irreversible progressive hearing loss in our Slc26a4-insufficient mouse model of EVA. This model of primary strial atrophy may be used to explore the mechanisms of progressive hearing loss due to strial dysfunction.

How do loop diuretics act?

In the thick ascending limb of the loop of Henle, NaCl reabsorption is mediated by a Na+/2Cl-/K+ cotransport system, present in the luminal membrane of this nephron segment. Loop diuretics such as furosemide (frusemide), piretanide, bumetanide and torasemide bind reversibly to this carrier protein, thus reducing or abolishing NaCl reabsorption. This leads to a decrease in interstitial hypertonicity and thus to a reduced water reabsorption. In nephron segments other than the thick ascending limb, loop diuretics have no quantitative importance with respect to their saluretic and diuretic activities. Loop diuretics also reduce Ca++ and Mg++ reabsorption in the thick ascending limb in a way which is still not clear. Furthermore, these drugs increase the urinary K+ excretion by enhancing distal tubular K+ secretion and reducing K+ reabsorption in the loop of Henle. Finally, by reduction of active NaCl transport, loop diuretics drastically reduce the substrate requirement and oxygen dependence of the thick ascending limb cells. This renders these cells, which are characterised by high transport rates and only limited substrate reserves, less vulnerable in acute renal failure.

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.

Aminoglycoside antibiotics inhibit maxi-K+ channel in single isolated cochlear efferent nerve terminals.

Patch clamp recordings were obtained from isolated cochlear efferent nerve terminals. The effect of aminoglycoside antibiotics on single maxi-K+ channels was determined. At positive voltages (cytosol with respect to extracellular side), neomycin, streptomycin, and kanamycin significantly reduced the single channel current amplitude of the maxi-K+ channel from the cytosolic side. The IC50 for neomycin was 9.10(-4) M from the cytosolic side and >> 10(-3) M from the extracellular side. Streptomycin and kanamycin were less potent. No significant difference in inhibition of the single channel current amplitude by 2.5.10(-4) M cytosolic neomycin was observed between 7.10(-4) M and 10(-6) M free cytosolic Ca2+. Neomycin had no significant effect on the open probability of the maxi-K+ channel either from the cytosolic or from the extracellular side. These findings demonstrate that the maxi-K+ channel in cochlear efferent nerve terminals can be a site of action for aminoglycoside antibiotics.

Osmotic water permeability of capillaries from the isolated spiral ligament: new in-vitro techniques for the study of vascular permeability and diameter.

Perilymph is separated from blood by a barrier called the blood-labyrinth or blood-perilymph barrier in analogy to the blood-brain or blood-cerebrospinal fluid barrier. These barriers consist mainly of vascular endothelial cells. To characterize the blood-labyrinth barrier we developed in vitro techniques for the quantitative determination of the osmotic water permeability and for the determination of changes in the diameter of isolated inner ear capillaries. Both techniques rely on measurement of the velocity of marker red cells trapped in the lumen of capillaries. The velocity of marker red cells is a measure for the capillary permeability when a water flux across the capillary wall is induced by an osmotic gradient or a measure for a change in the capillary diameter. With these techniques the osmotic water permeability coefficient (Pf) and the pH sensitivity of isolated capillaries from the spiral ligament of the inner ear was determined. Pf at 23 degrees C was (1.49 +/- 0.17) 10(-3) cm/s at pH 7.4 and (1.61 +/- 0.23) 10(-3) cm/s at pH 6.8 (n = 12: mean +/- SEM: n = number of tissues). Pf at 37 degrees C was (2.26 +/- 0.23) 10(-3) cm/s at pH 7.4 and (2.35 +/- 0.17) 10(-3) cm/s at pH 6.8 (n = 13). No change in capillary diameter was observed when the pH of the interstitial fluid was lowered from pH 7.4 to 6.8. These data demonstrate that Pf and the capillary diameter of spiral ligament capillaries are pH independent and suggest that water crosses the blood-labyrinth barrier via an aqueous pathway. Further, these data suggest that the relatively low Pf is another characteristic shared by the blood-labyrinth and the blood-brain barrier.

Maxi-K+ channel in single isolated cochlear efferent nerve terminals.

Patch clamp recordings were obtained from isolated cochlear efferent nerve terminals. Channel activity was found in 85% of membrane patches, was present in on-terminal and excised patches and was characterized to originate from a maxi-K+ channel. An average of 2.0 +/- 0.1 (N = 33) maxi-K+ channels were found per active patch. In symmetrical solutions, the current-voltage relationship was linear and the single-channel conductance was 221 +/- 5 pS (N = 22). The open probability of the maxi-K+ channel increased with depolarization of the membrane potential and with an increasing free Ca2+ concentration on the cytosolic side. The open probability was insensitive to changes in the free Ca2+ concentration on the extracellular side. TEA (20 mM) and charybdotoxin (10(-7) M) decreased the open probability to nearly zero from the extracellular side but had no effect from the cytosolic side. The high incidence with which this channel was found suggests that the maxi-K+ channel is physiologically relevant which might include protection against overstimulation of the efferent synapse.

Vestibular dark cells contain the Na+/H+ exchanger NHE-1 in the basolateral membrane.

Vestibular dark cells and strial marginal cells transport K+ by similar mechanisms. We have shown that K+ transport in vestibular dark cells is sensitive to the cytosolic pH (pHi) (Wangemann et al. (1995a): J. Membrane Biol. 147: 255-262). The present study addresses pharmacologically the questions whether vestibular dark cells and strial marginal cells from the gerbil contain a Na+/H+ exchanger (NHE) and in which membrane, apical or basolateral, NHE is located. Further, the study addresses the question which NHE subtype is present in vestibular dark cells. pHi was measured micro-fluorometrically with the pH-sensitive dye 2',7'-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF), cell volume which is a measure of the net balance between ion influx and efflux was monitored as cell height (CH) and the equivalent short circuit current (Isc) which is a measure of transepithelial K+ secretion was calculated from measurements of the transepithelial voltage (Vt) and the transepithelial resistance (Rt). Changes in pHi were induced by 20 or 40 mM propionate. In the presence of propionate a transient acidification of pHi was observed in vestibular dark cells as well as a subsequent alkalinization to pHi values exceeding those under control conditions. The alkalinization of pHi in the presence of propionate was inhibited by the NHE blockers amiloride and EIPA. Propionate-induced swelling of vestibular dark cells was inhibited by amiloride. The NHE blocker amiloride caused in vestibular dark cells an acidification of pHi and a decrease in CH. Amiloride caused in both vestibular dark cells and strial marginal cells a transient stimulation of Isc when added to the basolateral side but not added to the apical side. Similar effects and pHi were observed in vestibular dark cells with the amiloride analog ethyl-isopropyl-amiloride (EIPA) and similar effects on Isc were observed with EIPA and the NHE blocker HOE694 when applied to the basolateral side of vestibular dark cell epithelium. The IC50 for these basolateral effects of EIPA, HOE694 and amiloride on Isc in vestibular dark cells were 2 x 10(-7) M, 8 x 10(-7) M and 4 x 10(-5) M. These observation suggest that vestibular dark cells and strial marginal cells contain NHE in their basolateral membrane, that K+ transport in strial marginal cells in pHi sensitive similar to K+ transport in vestibular dark cells and that NHE in vestibular dark cells consists of the subtype NHE-1.

K(+)-induced stimulation of K+ secretion involves activation of the IsK channel in vestibular dark cells.

Vestibular dark cells in the inner ear secrete K+ from perilymph containing 4 mM K+ to endolymph containing 145 mM K+. Sensory transduction causes K+ to flow from endolymph to perilymph, thus threatening the homeostasis of the perilymphatic K+ concentration which is crucial for maintaining sensory transduction since the basolateral membranes of the sensory cells and adjacent neuronal elements need to be protected from K(+)-induced depolarization. The present study addresses the questions (1) whether increases in the perilymphatic K+ concentration by as little as 1 mM are sufficient to stimulate KCl uptake across the basolateral membrane of vestibular dark cells, (2) whether K(+)-induced stimulation of KCl uptake causes stimulation of the IsK channel in the apical membrane, and (3) whether the rate of transepithelial K+ secretion depends on the perilymphatic (basolateral) K+ concentration when the apical side of the epithelium is bathed with a solution containing 145 mM K+, as in vivo. Uptake of KCl was monitored by measuring cell height as an indicator for cell volume. The current (IIsK), conductance (gIsK) and inactivation time constant (tau IsK) of the IsK channel as well as the apparent reversal potential of the apical membrane (Vr) were obtained with the cell-attached macro-patch technique. Vr was corrected for the membrane voltage previously measured with microelectrodes. The rate of transepithelial K+ secretion JK was obtained as equivalent short circuit current from measurements of the transepithelial voltage (Vt) and resistance (Rt) measured in the micro-Ussing chamber. Cell height of vestibular dark cells was 7.2 microns (average). Elevations of the extracellular K+ concentration from 3.5 to 4.5 mM caused cell swelling with an initial rate of cell height change of 11 nm/s. With 3.6 mM K+ in the pipette IIsK was outwardly directed and elevation of the extracellular K+ concentration from 3.6 to 25 mM caused an increase of IIsK from 12 to 65 pA, gIsK from 152 to 950 pS and tau IsK from 278 to 583 ms as well as a hyperpolarization of Vr from -50 to -60 mV. With 150 mM K+ in the pipette IIsK was inwardly directed and the elevation of the extracellular K+ concentration caused an increase of IIsK from -1 to -143 pA, gIsK from 141 to 1833 pS and tau IsK from 248 to 729 ms. Vr remained within +/- 10 mV from zero. JK was 4.8 nmol x cm-2 x s-1 when the both the apical side and the basolateral side of the epithelium were perfused with a solution containing 3.5 mM K+. Elevation of the basolateral K+ concentration by 1 mM caused JK to increase by 1.1 nmol x cm-2 x s-1 or 23%. When the basolateral side of the epithelium was perfused with a solution containing 3.5 mM K+ and the apical side with a solution containing 145 mM K+, as in vivo, JK was 0.8 nmol x cm-2 x s-1 and elevation of the basolateral K+ concentration by 1 mM caused JK to increase by 0.8 nmol x cm-2 x s-1 or 100%. These data suggest that physiologically relevant increases in the perilymphatic K+ concentration increase JK by increasing KCl uptake across the basolateral membrane and activation of K+ release via the IsK channel in the apical membrane. Thus, the data demonstrate that vestibular dark cells adjust the rate of K+ secretion into endolymph according to the perilymphatic K+ concentration.

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

The K+ secretory epithelium of the vestibular labyrinth (dark cells) was impaled with glass microelectrodes in order to test the hypothesis that it contains a large Cl- conductance. In the first series of experiments, the short-circuited epithelium was perfused on both sides by a solution containing 150 mmol/l Cl-. The membrane voltage (PD) was -18 +/- 1 mV (N = 101), showed a Gaussian distribution, and the estimated input resistance of the cell (R 'cell') was 17 +/- 3 M omega. The PD responded to 10(-4) mol/l ouabain with a depolarization, suggesting the presence of a (Na(+) + K+)-ATPase. The PD responses to Cl- steps yielded an apparent transference number tCl = 0.34 +/- 0.03 (N = 65) and those to K+ steps yielded a tK = 0.16 +/- 0.01 (N = 48). In the second series of experiments, cells presumed to be Cl(-)-depleted were impaled in Cl(-)-free solutions. The distribution of the PD was not Gaussian; PDs as negative as -90 mV were observed. Cells with a highly negative PD also had a high R 'cell'. With the addition of Cl- the PD collapsed to -19 +/- 1 mV and R collapsed to 16 +/- 3 M omega (N = 145) which are not significantly different from values obtained in the first series of experiments when cells were impaled in a solution containing 150 mmol/l Cl-. Alternating the bath perfusate between Cl(-)-free and Cl(-)-containing solutions led to large PD transients.(ABSTRACT TRUNCATED AT 250 WORDS)

The isolated in vitro perfused spiral modiolar artery: pressure dependence of vasoconstriction.

We developed a new technique, the isolated in vitro perfused spiral modiolar artery, which allowed the continuous measurement of the vascular diameter and control of the intravascular pressure. An isolated section of the spiral modiolar artery from the gerbil was perfused on one end with a set of concentric pipettes and occluded on the other end in order to apply a defined intravascular pressure in the range between 10 and 230 cm H2O. The preparation was continuously superfused with a NaCl solution. The diameter of the spiral modiolar artery in NaCl solution displayed little dependence on the applied intravascular pressure. The diameter was 73 +/- 10 microm (n = 5) at 10 cm H2O and increased with pressure to 85 +/- 7 microm (n = 5) at the highest applied pressure (220 or 230 cm H2O). Elevation of the K+ concentration from 3.6 to 150 mM in the superfusate caused a transient vasoconstriction. The amplitude of the K+-induced vasoconstriction depended strongly on the applied intravascular pressure. At 10 cm H2O the amplitude was maximal and the outer diameter decreased transiently by 49 +/- 9% (from 73 +/- 10 to 38 +/- 9 microm; n = 5). The amplitude of K -induced vasoconstriction was nearly maximal at pressures lower than 30 cm H2O, declined at higher pressures, and was not significantly different from zero at pressures larger than 100 cm H2O. These observations in conjunction with an estimation of the intravascular pressures in vivo suggest that cochlear blood flow can be regulated on two levels: (1) cochlear blood flow can be regulated by controlling the vascular diameter of the spiral modiolar artery (intracochlear blood flow regulation) and (2) intracochlear blood flow regulation can be modulated by altering the perfusion pressure which is controlled by the vasculature upstream of the cochlea.

Endothelin-A receptors mediate vasoconstriction of capillaries in the spiral ligament.

The purpose of this study was to determine whether endothelin-1 (ET-1), endothelin-2 (ET-2) or endothelin-3 (ET-3) alter the vascular diameter of capillaries in the spiral ligament. Changes in vascular tone were measured in capillaries from the isolated spiral ligament in vitro. Capillaries were occluded on one end and opened on the other end. Red blood cells trapped in the capillaries served as markers for a luminal volume defined by the red cell itself, the capillary wall and the occluder. Movement of the red cell toward the open end was taken as evidence for vasoconstriction and movement of the red cell toward the occluder was taken as evidence for vasodilation. The inner diameter of the capillaries was 7.0 microm and decreased maximally by a factor of 0.8 in response to ET-1 and ET-2 (both 10(-8) M). Vasoconstriction induced by ET-1 and ET-2 was concentration-dependent in the range between 10(-12) and 10(-8) M whereas ET-3 (10(-8) M) had no effect. The EC50s for ET-1 and ET-2 were 1.2 x 10(-10) M and 1.4 x 10(-9) M, respectively. Thus, the potency order was ET-1 > ET-2 >> ET-3. Vasoconstriction induced by ET-1 and ET-2 was completely inhibited by the competitive antagonist 10(-6) M BQ-123 (cyclic D-Asp-L-Pro-D-Val-L-Leu-D-Trp). Vasoconstriction induced by ET-1 or ET-2 continued for more than 1 min after removal of agonist from the perfusate. Rapid vasodilation of capillaries preconstricted by ET-1 was observed in response to 10(-3) M sodium nitroprusside. Sodium nitroprusside, however, had no significant effect on the vascular diameter of resting capillaries. These results demonstrate that capillaries in the spiral ligament can constrict and the endothelin-mediated vasoconstriction occurs via ET(A) receptors.

The Na+/H+ exchanger in transitional cells of the inner ear.

The cytosolic pH (pHi) of transitional cells from the ampulla of the gerbil was measured micro-fluorometrically with the pH-sensitive dye 2',7'-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) to assess the possible contribution of a Na+/H+ exchanger to the regulation of pHi. All experiments were conducted in virtually HCO3(-)-free solutions. Under control conditions, pHi was 7.19 and addition of 10(-5) M ethylisopropylamiloride (EIPA), a blocker of Na+/H+ exchange, caused a small but significant acidification of pHi. A transient exposure to 21.4 mM NH4Cl caused a rapid cytosolic alkalinization followed by a brisk acidification and prompt recovery of pHi to its control value. The cytosolic buffer capacity (Bi) was determined in the absence of Na+ from changes in pHi which were elicited by [NH4+] steps. Bi was 4.7 mM/pH at pHi 7.19 and varied with pHi. The initial net proton flux JH, representative of Na+/H+ exchange activity, was calculated from the product of the initial rate of alkalinization after an NH4(+)-prepulse and Bi at the corresponding pHi. JH was dependent on the extracellular Na+ with an apparent Km of 64 mM, sensitive to the cytosolic [H+] with an apparent Km of 2.7 * 10(-7) M (i.e. pH 6.6), and inhibited by EIPA with an IC50 of 5 * 10(-7) M. These data suggest that transitional cells contain in the basolateral membrane a Na+/H+ exchanger of the amiloride-sensitive subtype.

Two types of chloride channel in the basolateral membrane of vestibular dark cells.

Transepithelial and cell membrane potential measurements have suggested that the basolateral membrane of gerbil vestibular dark cells contains Cl- conductive pathways. We used the patch clamp technique to search this membrane for Cl- conductive channels which could account for the macroscopic observations. Two types of Cl- channel were found in both cell-attached and excised membrane patches. One type was found with an incidence of 19% and had a single-channel conductance of 95 +/- 1 pS (N = 20) in symmetrical Cl- solutions. The other type was found with an incidence of 3% and had a large single-channel conductance of 360 +/- 11 pS (N = 12) in symmetrical Cl- solutions (LC-type Cl- channel). Both types of Cl- channel had linear current-voltage relations and at least 2 substates. In asymmetrical Cl- solutions (gluconate substitution) the current-voltage relations fit the Goldman-Hodgkin-Katz current equation for Cl-. Neither channel was blocked by Zn2+, NPPB, DIDS, DNDS or quinine. The 95 pS channel exhibited a spontaneous 'rundown' of its activity within 1 to 10 min after being excised. This rundown was not reversed by the catalytic subunit of protein kinase A. Channel activity was not dependent on the presence of cytosolic Ca2+ nor markedly altered by variations in cytosolic pH between 6.5 and 8.0. The two Cl- channels were distinguished by the membrane voltage ranges in which they were active and by their anion selectivity. The open probability of the 95 pS channel was insensitive to voltage and the anions NO3-, I- and Br- were only half as permeable as Cl-. By contrast, the LC-type Cl- channel was mostly active between about +/- 30 mV and equally permeable to NO3-, I-, Br- and Cl-. The 95 pS Cl- channel may account for the observed transepithelial and intracellular voltage responses to Cl- concentration steps and provide the path for the recirculation of Cl- across the basolateral membrane. The LC-type Cl- channel shows the same lack of anion discrimination as the anion pathway activated during hyposmotic challenge.

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 (Vt) and resistance (Rt) were measured across marginal cells and the resulting equivalent short circuit current (Isc) was calculated (Isc = Vt/Rt). Further, K+ secretion (JK+,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, Vt was 8 mV (apical side positive), Rt was 10 ohm-cm2 and Isc was 850 microA/cm2 (N = 27). JK+,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, oubain and bumetanide each caused a decline of Vt and an increase of Rt 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 Vt and a decrease in Rt 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].(ABSTRACT TRUNCATED AT 400 WORDS)

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

Transepithelial voltage (Vt) and resistance (Rt) 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. Vt was found to be 8.0 +/- 0.3 mV and Rt 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 (Isc = Vt/Rt) was 712 +/- 18 microA/cm2 (N = 179). Isc was reduced from 638 +/- 60 to 48 +/- 16 microA/cm2 (N = 14) by basolateral perfusion of 10(-3) M ouabain and from 538 +/- 27 to 27 +/- 4 microA/cm2 (N = 15) by basolateral perfusion of 5 x 10(-5) M bumetanide. Basolateral K+ steps (Na+ substitution) from 3.6 to 25 mM increased Vt from 6.5 +/- 0.5 to 12.2 +/- 0.6 mV and reduced Rt 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 Vt and Rt. The steady state Vt during apical perfusion of 145 mM K+ was near zero. Upon return to 3.6 mM K+, Vt transiently overshot its original level. Apical Cl- steps from 150 to 50 mM (gluconate substitution) monophasically decreased Vt from 5.9 +/- 0.7 to 4.1 +/- 0.8 mV (N = 15) and increased Rt from 9.6 +/- 1.3 to 12.0 +/- 1.5 ohm-cm2 (N = 14).(ABSTRACT TRUNCATED AT 250 WORDS)

Functional evidence for a monocarboxylate transporter (MCT) in strial marginal cells and molecular evidence for MCT1 and MCT2 in stria vascularis.

The transport of lactate, pyruvate and other monocarboxylates across plasma membranes of metabolically active cells such as strial marginal cells (SMC) may be important under aerobic conditions as well as under ischemic and hypoxic conditions. This study addresses the question whether SMC from the gerbil contain a membrane transport mechanism for monocarboxylates. The type of transporter was identified in functional studies by monitoring uptake of monocarboxylates into SMC through measurement of the cytosolic pH (pHi) with the pH-sensitive dye 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Further, subtypes of the functionally identified transporter which are present in stria vascularis were identified as transcripts by cloning and sequencing the reverse-transcription polymerase chain reaction (RT-PCR) products. All functional experiments were performed under nominally HCO3--free conditions. The monocarboxylates acetate and pyruvate (each 20 mM) induced an acidification of pHi. In contrast, the dicarboxylate malonate (20 mM) had no significant effect on pHi. Alpha-cyano-4-hydroxycinnamate (CHC; 5 mM), a blocker of H+/monocarboxylate cotransporter (MCT), reduced reversibly the acidification induced by 5 mM pyruvate. In contrast, 1 microM DIDS, a blocker of band-3 protein, had no significant effect on the acidification induced by 20 mM acetate. The presence of the transcripts for each of the MCT subtypes, MCT1 and MCT2, was determined by RT-PCR of stria vascularis from gerbil. RT-PCR performed with primers for the MCT1 and MCT2 subtypes on total RNA from stria vascularis revealed PCR products of the predicted sizes. Sequence analysis confirmed that amplified MCT1 and MCT2 cDNA fragments encoded a nucleotide sequence of MCT1 and MCT2, respectively. These observations suggest that SMC contain a MCT and that stria vascularis contains RNA for the subtypes MCT1 and MCT2 subtypes.

Ca(2+)-activated nonselective cation, maxi K+ and Cl- channels in apical membrane of marginal cells of stria vascularis.

Patch clamp recordings on the apical membrane of marginal cells of the stria vascularis of the gerbil were made in the cell-attached and excised configuration. Marginal cells are thought to secrete K+ into and absorb Na+ from endolymph. Four types of channel were identified; the most frequently observed channel was a small, nonselective cation channel which was highly similar to that found in the apical membrane of vestibular dark cells (Marcus et al., (1992) Am. J. Physiol. 262, C1423-C1429). The small nonselective cation channel was equally conductive (26.7 +/- 0.3 pS; N = 49) for K+, Na+, Rb+, Li+ and Cs+, 1.6 times more permeable to NH4+, but not permeable to Cl-, Ca2+, Ba2+ or N-methyl-D-glucamine. This channel yielded linear current-voltage relations which passed nearly through the origin (intercept: -2.2 +/- 0.4 mV, N = 49) when conductive monovalent cations were present on both sides of the membrane in equal concentrations. Channel activity required the presence of Ca2+ at the cytosolic face but not the extracellular (endolymphatic) face; there was essentially no activity for cytosolic Ca2+ less than or equal to 10(-7) M Ca2+ and full activity for greater than or equal to 10(-5) M. Cell-attached recordings had a conductance of 28.6 +/- 2.2 pS (N = 6) and a reversal voltage of -2.2 +/- 5.2 mV (N = 3) which was interpreted to reflect the intracellular potential of marginal cells under the present conditions. The three other types of channel were a Cl- channel (approximately 50 pS; N = 2), a maxi-K+ channel (approximately 230 pS; N = 1), and another large channel, probably cation nonselective (approximately 170 pS; N = 1). The 27 pS nonselective cation channel may be involved in K+ secretion and Na+ absorption under stimulated conditions which produce an elevated intracellular Ca2+; however, consideration of the apparent channel density in relation to the total transepithelial K+ flux suggests that these channels are not sufficient to account for K+ secretion.

Ca2+-dependence and nifedipine-sensitivity of vascular tone and contractility in the isolated superfused spiral modiolar artery in vitro.

The regulation of the vascular diameter of the spiral modiolar artery may play a major role in the regulation of cochlear blood flow and tissue oxygenation since the spiral modiolar artery provides the main blood supply to the cochlea. The goal of the present study was to determine whether vascular tone and contractility of the spiral modiolar artery depend on the presence of extracellular Ca2+ and involves nifedipine-sensitive Ca2+ channels. The spiral modiolar artery was isolated and superfused in vitro and the diameter was measured continuously by video microscopy. Isolated segments of the spiral modiolar artery had an outer diameter of 61 +/- 3 microm (n = 59) and displayed vasomotion characterized by 5-15 clearly distinguishable constrictions per min. Removal of Ca2+ from the superfusion medium caused a reversible relaxation and cessation of vasomotion and was used to determine the magnitude of basal vascular tone. The basal vascular tone consisted of a sustained reduction of the vascular diameter to 95.1 +/- 0.3% (n = 51) of the maximal diameter in Ca2+-free medium. Nifedipine reduced the basal vascular tone with an IC50 of (1.1 +/- 0.3) x 10(-9)) M although 22% of the basal vascular tone was insensitive to nifedipine. Elevation of the K+ concentration from 3.6 to 150 mM caused a transient vasoconstriction which was dependent on the presence of extracellular Ca2+. Nifedipine fully inhibited K+-induced vasoconstriction with an IC50 of (2.0 +/- 0.7) x 10(-9) M. Norepinephrine (10(-4) M) caused a transient vasoconstriction and an increase of vasomotion at branch points of the spiral modiolar artery. Norepinephrine-induced vasoconstriction was fully inhibited in the absence of Ca2+ and partially inhibited by 10(-7) M nifedipine. These observations suggest that the spiral modiolar artery contains voltage-dependent nifedipine-sensitive Ca2+ channels which are involved in the maintenance of basal vascular tone as well as in the mediation of K+- and norepinephrine-induced contractility. Further, the data suggest that cytosolic Ca2+ stores, if present in the spiral modiolar artery, are of limited capacity compared to other vessels.