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

The vestibule is the end organ devoted to sensing of head movements in space. To function properly, its mechano-receptors require the presence of a unique apical extracellular medium, the endolymph. Numerous studies have elucidated the mechanisms involved in the production and homeostasis of this unique medium and the responses of sensory cells to stimulation. However, anatomical constraints have prevented direct and simultaneous studies of their relationships. The aim of this study was the development of an in vitro model that would allow concomitant investigations on maturation and physiological properties of both the hair cells and their endolymphatic compartment. A three-dimensional (3D) culture of newborn rat utricles using an extracellular matrix sustaining 3D cellular growth was developed during 3, 6, or 10 days in vitro (DIV). Using morphological and electrophysiological techniques, we describe the de novo formation of a cyst. It was composed of the sensory epithelium and non-sensory cells-canalar, dark and intermediate cells-that polarized so that their apical surface faced its lumen. During the time of culture, the utricular potential (UP) was steady (-1.1+/-5.0 mV) in oxygenated condition, while in anoxia, the UP significantly decreased to -8.4+/-1.0 mV at 8 DIV. Over the same period, the K+ concentration in the cyst increased up to 86.1+/-33.9 mM (versus 5.6+/-1.5 mM in the bath). These observations indicated that the mechanisms generating the UP and the K-secretory activity were functional at this stage. Concomitantly, the hair cells acquired mature and functional properties: the type 1 and type 2 phenotypes, a mean resting membrane potential of -68.1+/-4.6 mV and typical electrophysiological responses. This preparation provides a powerful means to simultaneous access the hair cells and their endolymphatic compartment, with the possibility to use multi-technical approaches to investigate their interdependent relationships.

Endolymphatic sodium homeostasis by Reissner's membrane.

Cochlear sensory transduction depends on active extrusion of sodium ion (Na(+)) from the luminal fluid, endolymph. Reissner's membrane epithelium forms much of the barrier between cochlear endolymph and perilymph and we hypothesized that Reissner's membrane might be responsible for this function. We found that Reissner's membrane isolated from gerbil produced a short circuit current (I(sc)) directed into the apical side, consistent with cation absorption and/or anion secretion. I(sc) was inhibited by amiloride analogs in the potency sequence benzamil>amiloride>>ethylisopropylamiloride, consistent with Na(+) absorption through an epithelial sodium channel in the apical cell membrane. I(sc) was also inhibited by an inhibitor of Na(+),K(+)-ATPase, ouabain, and by the K(+) channel blockers Ba(2+), 4-aminopyridine and quinine but not tetraethylammonium nor glibenclamide, consistent with the presence of a voltage-activated K(+) channel. Bumetanide, an inhibitor of the Na(+),2Cl(-),K(+)-cotransporter, had no effect on I(sc). Contrary to previous hypotheses, no evidence was found for electrogenic secretion of Cl(-) under control of cAMP since neither forskolin nor genistein affected I(sc) when Na(+) absorption was blocked. These results provide the first direct evidence that Reissner's membrane contributes to normal cochlear function by absorption of Na(+) from endolymph.

Immunolocalization of P2Y4 and P2Y2 purinergic receptors in strial marginal cells and vestibular dark cells.

K+ secretion by strial marginal cell and vestibular dark cell epithelia is regulated by UTP and ATP at both the apical and basolateral membranes, suggesting control by P2Y2 and/or P2Y4 purinergic receptors. Immunolocalization was used to determine the identity and distribution of these putative receptors. Membrane proteins from gerbil brain, gerbil vestibular labyrinth and gerbil stria vascularis were isolated and analyzed by Western blot. P2Y2 antibody stained one band at 42 kDa for each tissue, whereas P2Y4 antibody stained 3 bands on gerbil brain (75, 55 and 36 kDa), one band on gerbil stria vascularis (55 kDa) and two bands on vestibular labyrinth (42 and 56 kDa). All bands were absent when the antibodies were blocked with their respective antigenic peptide. P2Y4 was immunolocalized by fluorescence confocal microscopy to only the apical membrane of strial marginal cells and vestibular dark cells and was similar to apical immunostaining of KCNE1 in the same cells. By contrast, P2Y2 was observed on the basolateral but not the apical membrane of dark cells. Similarly, in the stria vascularis P2Y2 was observed in the basolateral region but not the apical membrane of marginal cells. Additional staining was observed in the spiral ligament underlying the stria vascularis. These findings identify the molecular bases of the regulation of K+ secretion by apical and basolateral UTP in the inner ear.

Basolateral K+ conductance establishes driving force for cation absorption by outer sulcus epithelial cells.

Outer sulcus epithelial cells were recently found to actively reabsorb cations from the cochlear luminal fluid, endolymph, via nonselective cation channels in the apical membrane. Here we determined the transport properties of the basolateral membrane with the whole-cell patch clamp technique; the apical membrane contributed insignificantly to the recordings. Outer sulcus epithelial cells exhibited both outward and inward currents and had a resting membrane potential of -90.4 +/- 0.7 mV (n = 78), close to the Nernst potential for K+ (-95 mV). The reversal potential depolarized by 54 mV for a tenfold increase in extracellular K+ concentration with a K+/Na+ permeability ratio of 36. The most frequently observed K+ current was voltage independent over a broad range of membrane potentials. The current was reduced by extracellular barium (10-5 to 10-3 m), amiloride (0.5 mm), quinine (1 mm), lidocaine (5 mm) and ouabain (1 mm). On the other hand, TEA (20 mm), charybdotoxin (100 nm), apamin (100 nm), glibenclamide (10 microm), 4-aminopyridine (1 mm) and gadolinium (1 mm) had no significant effect. These data suggest that the large K+ conductance, in concert with the Na+,K+-ATPase, of the basolateral membrane of outer sulcus cells provides the driving force for cation entry across the apical membrane, thereby energizing vectorial cation absorption by this epithelium and contributing to the homeostasis of endolymph.

P2X2 receptor mediates stimulation of parasensory cation absorption by cochlear outer sulcus cells and vestibular transitional cells.

Cochlear outer sulcus cells (OSC) and vestibular transitional cells (VTC) are part of the parasensory epithelium in the inner ear and are located in homologous positions between the sensory hair cells and the cation secretory epithelial cells in the cochlea and the vestibular labyrinth. OSC are known to sustain a reabsorptive transepithelial current and to contain an immunoreactivity for P2X(2) purinergic receptors. This study addresses whether OSC and VTC share functional similarities and extends this hypothesis to the question of whether both cell types contain functional P2X(2) receptors. The current density (I(sc)) was recorded with the vibrating probe technique and was found to be similar in VTC and OSC. Both gadolinium and flufenamic acid reduced I(sc) in VTC, as reported previously for OSC. I(sc) was stimulated by extracellular ATP but not by selective agonists of P2Y receptors. Purinergic receptor agonists increased I(sc) with a potency order of ATP > 2'- and 3'-O-(4-benzoyl-benzoyl)adenosine 5'-triphosphate alpha,beta-methyleneadenosine 5'-triphosphate in both OSC and VTC. In the presence of suramin (100 micrometer) or gadolinium (100 micrometer), the responses of ATP were inhibited significantly in both OSC and VTC. This pharmacological profile is consistent with that of the P2X(2) receptor. These results demonstrate that VTC participate in vestibular parasensory cation absorption and that both OSC and VTC regulate their parasensory cation flux via P2X(2) receptors, which would regulate the endolymphatic concentration of the current-carrying ion species in auditory and vestibular transduction.

Immunolocalization of ClC-K chloride channel in strial marginal cells and vestibular dark cells.

Secretion of K(+) into endolymph depends on a particular constellation of ion transport proteins in the apical and basolateral membranes of strial marginal cells and vestibular dark cells. One fundamental component is the large chloride conductance of the basolateral membrane, which recycles chloride taken up by the Na(+)-K(+)-Cl(-) cotransporter in the same membrane. Evidence has been reported recently that supports ClC-K, a channel subunit previously thought to be specific to the kidney, as being the molecular entity underlying this conductance. We have isolated protein from the gerbil kidney, stria vascularis and vestibular labyrinth and found by Western blot analysis a 60 kDa band, a 48 kDa band and 54 and 70 kDa bands, respectively, specifically labeled by ClC-K antibody. Subsequent immunohistochemical observations of the inner ear tissues with a confocal microscope on fluorescently labeled tissue sections showed the staining to be restricted to the basolateral region of strial marginal cells and vestibular dark cells. The cochlear staining was distinct from the distribution of the Kir4.1 (KCNJ10) K(+) channel, known to be present only in strial intermediate cells. These findings support the contention that ClC-K is an important component of the basolateral Cl(-) conductance that participates in K(+) secretion by these epithelia.

Estrogen acutely inhibits ion transport by isolated stria vascularis.

We investigated the nongenomic effects of female sex steroid hormones on the short circuit current (I(sc,probe)) across gerbil stria vascularis using the voltage-sensitive vibrating probe. The strial marginal cell epithelial layer produces I(sc,probe) by secreting K+ via I(Ks) channels in the apical membrane. Application of 17beta-estradiol (E2) caused a decrease of I(sc,probe) in a dose-dependent manner (10 nM-10 microM) within seconds. Tamoxifen, a competitive inhibitor of the intracellular estrogen receptor, did not change the inhibitory effect of E2. Activation of I(Ks) channels by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid in the presence and absence of E2 was used to test the mechanism of action. The results were consistent with a direct inhibitory effect of E2 on the I(Ks) channels. By contrast, progesterone caused a transient increase of I(sc,probe). These results suggest that E2 decreases secretion of K+ by inhibition of I(Ks) channels via a nongenomic mechanism at concentrations near those occurring under some physiologic conditions while progesterone caused only transient effects on I(sc,probe).

Apical P2Y4 purinergic receptor controls K+ secretion by vestibular dark cell epithelium.

It was previously shown that K+ secretion by vestibular dark cell epithelium is under control of G protein-coupled receptors of the P2Y family in the apical membrane that are activated by both purine and uridine nucleotides (P2Y2, P2Y4, or P2Y6). The present study was conducted to determine the subtype of purinergic receptor and to test whether these receptors undergo desensitization. The transepithelial short-circuit current represents electrogenic K+ secretion and was found to be reduced by UTP, ATP, and diadenosine tetraphosphate, but not UDP. Neither pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 30 microM) nor suramin (100 microM) inhibited the effect of UTP. The potencies of the agonists were consistent with rodent P2Y4 and P2Y2, but not P2Y6, receptors. The ineffectiveness of suramin was consistent with P2Y4, but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in vestibular labyrinth. Sustained exposure to ATP or UTP for 15 min caused a constant depression of short-circuit current with no apparent desensitization. The results support the conclusion that regulation of K+ secretion across vestibular dark cell epithelium occurs by P2Y4 receptors without desensitization of the response.

Nonselective cation and BK channels in apical membrane of outer sulcus epithelial cells.

The outer sulcus epithelium was recently shown to absorb cations from the lumen of the gerbil cochlea. Patch clamp recordings of excised apical membrane were made to investigate ion channels that participate in this reabsorptive flux. Three types of channel were observed: (i) a nonselective cation (NSC) channel, (ii) a BK (large conductance, maxi K or K(Ca)) channel and (iii) a small K(+) channel which could not be fully characterized. The NSC channel found in excised insideout patch recordings displayed a linear current-voltage (I-V) relationship (27 pS) and was equally conductive for Na(+) and K(+), but not permeable to Cl(-) or N-methyl-d-glucamine. Channel activity required the presence of Ca(2+) at the cytosolic face, but was detected at Ca(2+) concentrations as low as 10(-7) m (open probability (P(o)) = 0.11 +/- 0.03, n = 8). Gadolinium decreased P(o) of the NSC channel from both the external and cytosolic side (IC(50) approximately 0.6 microm). NSC currents were decreased by amiloride (10 microm - 1 mm) and flufenamic acid (0.1 mm). The BK channel was also frequently (38%) observed in excised patches. In symmetrical 150 mm KCl conditions, the I-V relationship was linear with a conductance of 268 pS. The Goldman-Hodgkin-Katz equation for current carried solely by K(+) could be fitted to the I-V relationship in asymmetrical K(+) and Na(+) solutions. The channel was impermeable to Cl(-) and N-methyl-d-glucamine. P(o) of the BK channel increased with depolarization of the membrane potential and with increasing cytosolic Ca(2+). TEA (20 mm), charybdotoxin (100 nm) and Ba(2+) (1 mm) but not amiloride (1 mm) reduced P(o) from the extracellular side. In contrast, external flufenamic acid (100 microm) increased P(o) and this effect was inhibited by charybdotoxin (100 nm). Flufenamic acid inhibited the inward short-circuit current measured by the vibrating probe and caused a transient outward current. We conclude that the NSC channel is Ca(2+) activated, voltage-insensitive and involved in both constitutive K(+) and Na(+) reabsorption from endolymph while the BK channel might participate in the K(+) pathway under stimulated conditions that produce an elevated intracellular Ca(2+) or depolarized membrane potential.

K+ and Na+ absorption by outer sulcus epithelial cells.

Transduction of sound into nerve impulses by hair cells depends on modulation of a current carried primarily by K+ into the cell across apical transduction channels that are permeable to cations. The cochlear function thus depends on active secretion of K+ accompanied by absorption of Na+ by epithelial cells enclosing the cochlear duct. The para-sensory cells which participate in the absorption of Na+ (down to the uniquely low level of 1 mM) were previously unidentified and the existence of a para-sensory pathway which actively absorbs K+ was previously unknown. A relative short circuit current (Isc,probe, measured as the extracellular current density with a vibrating electrode) was directed into the apical side of the outer sulcus epithelium, decreased by ouabain (1 mM), an inhibitor of Na+, K(+)-ATPase, and found to depend on bath Na+ and K+ but on neither Ca2+ nor Cl-. Isc,probe was shown to be an active current by its sensitivity to ouabain. On-cell patch clamp recordings of the apical membrane of outer sulcus cells displayed a channel activity, which carried inward currents under conditions identical to those used to measure Isc,probe. Both Isc,probe and non-selective cation channels (27.4+/-0.6 ps, n = 22) in excised outside-out patches from the apical membrane were inhibited by Gd3+ (1 mM). Ics,prob was also inhibited by 5 mM lidocaine, 1 mM quinine and 500 microM amiloride but not by 10 microM amiloride. These results demonstrate that outer sulcus epithelial cells contribute to the homeostasis of endolymph by actively absorbing Na+ and K+. An entry pathway in the apical membrane was shown to be through non-selective cation channels that were sensitive to Gd3+.

Divalent cations inhibit IsK/KvLQT1 channels in excised membrane patches of strial marginal cells.

The IsK/KvLQT1 K+ channel in the apical membrane of strial marginal cells and vestibular dark cells is an essential ion transport pathway for the secretion of K+ into the endolymph of the inner ear. Study of this control point has been impeded by rundown of channel activity upon excision into commonly used cytosolic solutions. This paper describes conditions under which patches of apical membrane of strial marginal cells and vestibular dark cells from gerbil containing this channel can be excised, retaining its characteristic voltage dependence, kinetic properties, ion permeability sequence and pharmacological sensitivity, similar to those found during on-cell and perforated-patch whole cell recordings (Shen et al., Audit. Neurosci. 3 (1997) 215-230). Those excised-patch conditions include removal of Mg2+ from the cytosolic solution and use of a K+-rich pipette electrolyte. The inhibition of channel activity by Mg2+ was found to be a general feature of divalent cations; the channel was also inhibited by Ca2+, Ba2+ and Sr2+. The concentrations causing 50% inhibition of IsK/KvLQT1 channel current were 7 x 10(-5) M, 6 x 10(-6) M, 3 x 10(-4) M and 7 x 10(-5) M, respectively. It was also found that a chemical cross-linking agent, 3,3'-dithio-bis(sulfosuccinimidyl propionate) (DTSSP), which was previously shown to persistently activate IsK/KvLQTI channels expressed in Xenopus oocytes, maintained in excised patches channel activity which retained voltage dependence and pharmacological sensitivity. These data demonstrate that (1) the channel complex is inhibited by Ca2+, Mg2+ and other divalent cations, (2) the activation by Ca2+ observed previously in whole-cell preparations was due to action via other cellular pathways. These findings must be taken into account when considering the action of receptors which alter the cytosolic Ca2+ activity.

Protein kinase C mediates P2U purinergic receptor inhibition of K+ channel in apical membrane of strial marginal cells.

Strial marginal cells (SMC) electrogenically secrete K+ via slowly activating K+ (I[sK]) channels, consisting of I(sK) regulatory and KvLQT1 channel subunits, and the associated short circuit current (I[sc]) is inhibited by agonists of the apical P2U receptor [Liu et al., Audit. Neurosci. 2 (1995) 331-340]. Measurements of relative K+ flux (JK) with a self-referencing K+-selective probe demonstrated a decrease in JK after apical perfusion of 100 microM ATP. On-cell macro patch recordings from the apical membrane of gerbil SMC showed a decrease of the I(sK) channel current (I[IsK]) by 88 +/- 8% during pipette perfusion of 100 microM ATP. The magnitude of the decrease of L(sc) by ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (20 nM) decreased I(IsK) (gerbil: by 62 +/- 10%; rat: by 72 +/- 6%) in perforated-patch whole-cell recordings while the inactive analog, 4alphaPMA, had no effect. By contrast, elevation of cytosolic [Ca2+] by A23187 increased the whole-cell I(IsK). The expression of the isk gene transcript was confirmed and the serine responsible for the species-specific response to PKC was found to be present in the gerbil I(sK) sequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the I(sK) channel of SMC in response to activation of the apical P2U receptor and predict that the secretion of endolymph in the human cochlea may be controlled by PKC in the same way as in our animal model.

Ototoxic effect of erythromycin on cochlear potentials in the guinea pig.

The mechanism of hearing loss due to the administration of intravenous erythromycin was investigated in the albino guinea pig, and it was found for the first time that this drug causes cochlear dysfunction. The endocochlear potential (EP) and the cochlear microphonics (CM) recorded at the first cochlear turn transiently decreased when erythromycin was administered intravenously at dosages of 100 and 150 mg/kg. The averaged maximum decrease in EP was 16 mV (n = 5) and 33 mV (n = 5) for 100 and 150 mg/kg, respectively. The maximum decrease in the CM was about 25% when the EP reached its lowest value with the injection of 150 mg/kg. A complete recovery of the EP and CM ensured within 20 minutes after each erythromycin dose. The perilymphatic perfusion of 3 mmol/L of erythromycin decreased the EP and CM; however, in contrast to the intravenous administration, the decrease of the CM was nearly complete and both the EP and CM were irreversible. Hearing loss due to intravenously administered erythromycin could likely be attributle to the transient dysfunction of the stria vascularis, although concomitant dysfunction of the central auditory pathway cannot be excluded.

cAMP increases apical IsK channel current and K+ secretion in vestibular dark cells.

Adenosine 3',5'-cyclic monophosphate (cAMP) is known to stimulate exogenous IsK channel current in the Xenopus oocyte expression system. The present study was performed to determine whether elevation of cytosolic cAMP in a native mammalian epithelium known to secrete K+ through endogenously expressed IsK channels would stimulate K+ secretion through these channels. The equivalent short circuit current (Isc) across vestibular dark cell epithelium in gerbil was measured in a micro-Ussing chamber and the apical membrane current (IsK) and conductance (gIsK) of IsK channels was recorded with both the on-cell macro-patch and nystatin-perforated whole-cell patch-clamp techniques. It has previously been shown that Isc can be accounted for by transepithelial K+ secretion and that the apical IsK channels constitute a significant pathway for K+ secretion. The identification of the voltage-dependent whole-cell currents in vestibular dark cells was strengthened by the finding that a potent blocker of IsK channels, chromanol 293B, strongly reduced IIsK from 646 +/- 200 to 154 +/- 22 pA (71%) and gIsK from 7.5 +/- 2.6 to 2.8 +/- 0.4 nS (53%). Cytoplasmic cAMP was elevated by applying dibutyryl cyclic AMP (dbcAMP), or the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-20-1724. dbcAMP (1 mM) increased Isc and IIsK from 410 +/- 38 to 534 +/- 40 microA/cm2 and from 4.3 +/- 0.8 to 11.4 +/- 2.2 pA, respectively. IBMX (1 mM) caused transient increases of Isc from 415 +/- 30 to 469 +/- 38 microA/cm2 and Ro-20-1724 (0.1 mM) from 565 +/- 43 to 773 +/- 58 microA/cm2. IBMX increased IIsK from 5.5 +/- 1.5 to 16.9 +/- 5.8 pA in on-cell experiments and from 191 +/- 31 to 426 +/- 53 pA in whole-cell experiments. The leak conductance due to all non-IsK channel sources did not change during dbcAMP and IBMX while 293B in the presence of dbcAMP reduced IIsK by 84% and gIsK by 62%, similar to unstimulated conditions. These results demonstrate that the cAMP pathway is constitutively active in vestibular dark cells and that the cAMP pathway stimulates transepithelial K+ secretion by increasing IsK channel current rather than by altering another transport pathway.

cAMP increases K+ secretion via activation of apical IsK/KvLQT1 channels in strial marginal cells.

In the cochlea, K+ is secreted by electrodiffusion across the apical membrane of strial marginal cells via the IsK/KvLQT1 ('IsK') channel. This channel complex has been reported to be activated in other systems by adenosine 3',5'-cyclic monophosphate (cAMP). Since several reports had suggested that cAMP is a second messenger in the cochlea, the effect of the cAMP pathway on transepithelial K+ secretion by strial marginal cells of the gerbil was studied. Both the transepithelial current (Isc) and K+ flux (JK) across strial marginal cell epithelium were measured; Isc in a micro-Ussing chamber and JK as the gradient of K+ concentration near the apical membrane. The apical membrane current (IIsK) and conductance (gIsK) of IsK channels were recorded with the on-cell macro-patch and the nystatin-perforated whole-cell patch clamp techniques. It has previously been shown that the apical IsK channel constitutes the primary pathway for K+ secretion. Cytoplasmic cAMP was elevated by applying dibutyryl cyclic-AMP (dbcAMP) or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) at 37 degrees C. dbcAMP (1 mM) increased Isc by 51 +/- 4% and IIsK in on-cell and whole-cell recordings increased by 214 +/- 63% and 390 +/- 61% above the control value, respectively. IBMX (1 mM) caused transient increases of Isc by 53 +/- 3% and IIsK in on-cell recordings by 177 +/- 75% above the control value. The leak conductance due to all non-IsK channel sources did not change in the presence of dbcAMP or IBMX. dbcAMP (1 mM at 24 degrees C) increased JK by 53 +/- 16% and Isc by 18 +/- 4%. IBMX (1 mM at 24 degrees C) had no effect, suggesting reduced activity of adenylate cyclase at this temperature. Our results demonstrate that the cAMP pathway is constitutively active in strial marginal cells and that the cAMP pathway stimulates transepithelial K+ secretion by increasing IsK channel current rather than by altering another transport pathway.

P2U purinergic receptor inhibits apical IsK/KvLQT1 channel via protein kinase C in vestibular dark cells.

Vestibular dark cells (VDC) are known to electrogenically secrete K+ via slowly activating K+ (IsK) channels, consisting of IsK regulatory and KvLQT1 channel subunits, and the associated short-circuit current (Isc) is inhibited by agonists of the apical P2U (P2Y2) receptor (J. Liu, K. Kozakura, and D. C. Marcus. Audit. Neurosci. 2: 331-340, 1995). Measurements of relative K+ flux (JK) with a self-referencing K(+)-selective probe demonstrated a decrease in JK after apical perfusion of 100 microM ATP. On-cell macropatch recordings from gerbil VDC showed a decrease of the IsK channel current (IIsK) by 83 +/- 7% during pipette perfusion of 10 microM ATP. The magnitude of the decrease of Isc by ATP was diminished in the presence of inhibitors of phospholipase C (PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of PKC by phorbol 12-myristate 13-acetate (PMA, 20 nM) decreased IIsK by 79 +/- 3% in perforated-patch whole cell recordings, whereas the inactive analog, 4 alpha-PMA, had no effect. In contrast, elevation of cytosolic Ca2+ concentration by A-23187 increased the whole cell IIsK. The expression of the isk gene transcript was confirmed, and the serine responsible for the species-specific response to PKC was found to be present in the gerbil IsK sequence. These data provide evidence consistent with a direct effect of the PKC branch of the PLC pathway on the IsK channel of VDC in response to activation of the apical P2U receptor and predict that the secretion of endolymph in the human vestibular system may be controlled by PKC in the same way as in our animal model.

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.

Vibrating probes: new technology for investigation of endolymph homeostasis.

It is well known that the function of the cochlear and vestibular labyrinth depends on the high concentration of potassium (K+) in the luminal fluid, endolymph. Homeostasis of endolymphatic ion composition has been attributed to the stria vascularis and vestibular dark cells but with little prior experimental basis. The extremely small domain of each epithelial cell type bounding the endolymphatic space has precluded study of ion fluxes from these cells. The voltage-sensitive and (+)-selective vibrating probes were adapted recently for the demonstration of electrogenic K+ secretion and its regulation by stria vascularis and vestibular dark cell epithelium. The isolated stria vascularis and vestibular dark cell epithelium are known to produce a transepithelial current directed toward the endolymphatic side and this current has been shown to be sensitive to bumetanide, an inhibitor of the Na(+)-Cl(-)-K+ cotransporter. The vibrating probes were used to demonstrate that this current is carried by K+ and that the K+ flux is also sensitive to bumetanide. Several other agents and maneuvers which alter the transepithelial current (e.g. apical DIDS and basolateral hypotonic challenge) were found to produce similar changes in the K+ flux. The technique holds the promise of discovery of the contribution to the homeostasis of endolymph of other cell types in the inner ear.

Inhibitory effect of erythromycin on ion transport by stria vascularis and vestibular dark cells.

A previous study showed that systemic administration of erythromycin caused a reversible decline in the endocochlear and cochlear microphonic potentials. Those data were thought to suggest that erythromycin caused hearing loss by interference with ion transport processes in the stria vascularis. The present study was undertaken to test this hypothesis by measuring the effects of erythromycin perfused on either the apical or basolateral side on the transepithelial short circuit current (Isc), a measure of the K+ secretion rate. Isc was measured from preparations of strial marginal cells and the homologous vestibular dark cells in vitro with a micro-Ussing chamber. Erythromycin was found to have no effect when perfused on the apical side but to cause a reversible decrease in Isc when perfused on the basolateral side for both epithelia. These data are consistent with the notion that at least one ototoxic effect of erythromycin is the inhibition of K+ secretion 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.