Luminal non-selective cation and outwardly rectifying chloride channels in cultured strial marginal cells from gerbil

Ionic channels located on the luminal side of strial marginal cells (MCs) of gerbil in culture were investigated using the patch-clamp technique. Two types of channels were identified. The most frequently recorded single-channel activity corresponded to a non-selective cation (NSC) channel with a conductance of 23.7 ± 0.2 pS (n = 18) in symmetrical NaCl conditions. The channel was activated by internal Ca²⁺ and inhibited by internal adenine nucleotides and flufenamic acid. Spontaneous activity of NSC channels was found in 16% of the cell-attached patches and with a very high density (9 ± 2 levels/patch, n = 28) in 100% of the excised patches. An outwardly rectifying chloride (ORC) channel was also identified in 14% of the patches but only after excision. The channel exhibited at 0 mV a unit conductance of 26.8 ± 1.3 pS (n = 8) and a strong outward rectification in symmetrical NaCl conditions, and the open probability increased with depolarization. The luminal NSC channel and the ORC channel evidenced in this study might participate in the production of endolymph. Although extrapolation of the presents results to the in vivo situation should be made with caution, this study suggests that culture of strial MCs may be a suitable model for investigation of endolymph physiology.     

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.     

Voltage-dependent K(+) currents in spiral prominence epithelial cells of rat cochlea

It has been suggested that spiral prominence is associated with ion transport, but the characterization of ion channels has not been explored so far. We studied the electrical properties and ion conductances of the spiral prominence epithelial cells (SPECs), which are epithelial cells covering the luminal side of spiral prominence, in the upper turn of neonatal rat cochlea using a whole-cell variant patch clamp technique. The cell capacitance was 16.3+/-2.1 pF (n=33) and the resting membrane potential was -68. 9+/-2.5 mV (n=14) in perilymph-like bath solution. It was found that those SPECs possess a large voltage-activated, outwardly rectifying K(+) current and a small inwardly rectifying K(+) current. The outward K(+) current was activated by depolarizing pulses more positive than -30 mV, and sensitive to tetraethylammonium chloride (20 mM), 4-aminopyridine (10 mM), but not to Ba(2+) (0.5 mM). Tail current analysis revealed that it was primarily K(+)-selective. The time course of activation was well fitted by an exponential function raised to second power. The small inwardly rectifying K(+) current was sensitive to Ba(2+) (0.5 mM), and the Ba(2+)-sensitive current was K(+)-selective. In cell-attached or inside-out patch recordings, no discernible K(+) channel currents were found in the apical membrane of SPECs. Based on these results, we conclude that SPECs have two types of voltage-dependent K(+) currents, which are most likely located in the basolateral membrane.     

F-actin cleavage in apoptotic outer hair cells in chinchilla cochleas exposed to intense noise

Sensory transduction in the cochlea and the vestibular labyrinth depends on the cycling of K+. In the cochlea, endolymphatic K+ flows into the sensory hair cells via the apical transduction channel and is released from the hair cells into perilymph via basolateral K+ channels including KCNQ4. K+ may be taken up by fibrocytes in the spiral ligament and transported from cell to cell via gap junctions into strial intermediate cells. Gap junctions may include GJB2, GJB3 and GJB6. K+ is released from the intermediate cells into the intrastrial space via the KCNJ10 K+ channel that generates the endocochlear potential. From the intrastrial space, K+ is taken up across the basolateral membrane of strial marginal cells via the Na+/2Cl-/K+ cotransporter SLC12A2 and the Na+/K+-ATPase ATP1A1/ATP1B2. Strial marginal cells secrete K+ across the apical membrane into endolymph via the K+ channel KCNQ1/KCNE1, which concludes the cochlear cycle. A similar K+ cycle exists in the vestibular labyrinth. Endolymphatic K+ flows into the sensory hair cells via the apical transduction channel and is released from the hair cells via basolateral K+ channels including KCNQ4. Fibrocytes connected by gap junctions including GJB2 may be involved in delivering K+ to vestibular dark cells. Extracellular K+ is taken up into vestibular dark cells via SLC12A2 and ATP1A1/ATP1B2 and released into endolymph via KCNQ1/KCNE1, which concludes the vestibular cycle. The importance of K+ cycling is underscored by the fact that mutations of KCNQ1, KCNE1, KCNQ4, GJB2, GJB3 and GJB6 lead to deafness in humans and that null mutations of KCNQ1, KCNE1, KCNJ10 and SLC12A2 lead to deafness in mouse models.     

Effects of epinephrine on ouabain-sensitive, K(+) -dependent P-nitrophenylphosphatase activity in strial marginal cells of guinea pigs

In strial marginal cells, Na+/K(+)-ATPase activity is abundant, and contributes to maintain the characteristic electrolyte composition of the cochlear endolymph. In the present study, to clarify the relationship between epinephrine and strial Na+/K(+)-ATPase activity, the ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K(+)-NPPase) activity of strial marginal cells was investigated with a cerium-based method in normal guinea pigs and guinea pigs treated with reserpine, epinephrine, and reserpine plus epinephrine. In our previous study, K(+)-NPPase activity had almost completely decreased 3 to 20 days after reserpine administration. In the present study, at 10 days after reserpinization and following repeated epinephrine treatment, enzyme activity was detectable. These results suggest that exogenous epinephrine was able to restore strial K(+)-NPPase activity in the reserpine-treated animals, and that epinephrine might increase strial Na+/K(+)-ATPase activity.     

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.     

KCNQ1/KCNE1 K+ channel and P2Y4 receptor are co-expressed from the time of birth in the apical membrane of rat strial marginal cells

CONCLUSION: KCNQ1/KCNE1 K(+) channels and P2Y(4) receptors are expressed in the apical membrane of rat strial marginal cells from postnatal day 1 (P1) and maintained throughout development. OBJECTIVES: The purpose of the present study was to investigate the developmental expression of KCNQ1/KCNE1 K(+) channel and of P2Y(4), which is an important metabotropic regulator of KCNQ1/KCNE1 K(+) channel in strial marginal cells. MATERIALS AND METHODS: Sprague-Dawley rats at different stages of development (P1, P3, P5, P7, P14, and P21) were studied. The spiral ligament with the stria vascularis was detached from the cartilaginous or bony cochlea and prepared for a voltage-sensitive vibrating probe and immunohistochemistry. RESULTS: Chromanol 293B, a blocker of KCNQ1/KCNE1 K(+) channel, inhibited short-circuit currents (I ( sc )) from P1 to P21. Similarly, I ( sc ) were found to be decreased by uridine 5'-triphosphate at all ages. The antagonist profiles indicated that the apical P2Y receptor is P2Y(4) subtype. KCNQ1, KCNE1, and P2Y(4) were immunolocalized in the apical region of stria vascularis at P1.     

Recording of mechano-electrical transduction currents by a nystatin perforated-patch method

Mechano-electrical transduction (MET) currents in isolated cochlear hair cells of chicks were recorded by use of a nystatin perforated-patch method. The membrane of a cell-attached patch was permeabilized by nystatin in the patch pipette, thus providing electrical continuity between the pipette and the cytoplasm of the cell without loss of cytoplasmic compounds. The current-voltage relationship was linear for the inward-going MET current at negative membrane potentials, but outward currents were reduced at positive membrane potentials, evidence of inward-going rectification. Elevation of the intracellular concentration of calcium at positive membrane potential, mediated via a voltage-dependent Ca2+ channel, may suppress the outward-going MET current by acting from within the cell.     

Reactive blue 2, an antagonist of rat P2Y4, increases K+ secretion in rat cochlea strial marginal cells

Extracellular ATP decreases K+ secretion in strial marginal cells via apical P2Y4 receptors. We investigated the effect of reactive blue 2 (RB-2), an antagonist of rat P2Y4, on rat strial marginal cells using a voltage-sensitive vibrating probe. The application of RB-2 increased K+ secretion in a dose-dependent manner, and this increase was characterized as a peak followed by a partial relaxation to a steady-state. Moreover, this response was similar to that caused by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Suramin had no similar effect, except at high concentration. Thus, we tested the effects of these chemicals on P2Y4 receptors in strial marginal cells. Both RB-2 and DIDS had antagonistic activities at P2Y4, and the antagonist potency at P2Y4 paralleled the potency of K+ secretion. Interestingly, 2'- and 3'-O-(4-benzoyl-benzoyl)adenosine 5'-triphosphate (BzATP) exhibited an agonistic effect at P2Y4 receptor, which was blocked by RB-2, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Based on these results, we speculate that direct and/or indirect inhibitory mechanisms between P2Y4 and KENQ1/KCNE1 K+ channels exist in strial marginal cell.     

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.     

人鼻粘膜上皮细胞Na^＋通道的初步研究

明确人鼻粘膜上皮细胞Ｎａ＾＋通道的特性，为研究Ｎａ＾＋通道在鼻粘膜病理性改变及治疗中的作用奠定理论基础。方法利用膜片钳技术在经无血清气－液面培养的鼻源性鼾症患者手术切除下鼻甲标本的鼻上皮细胞进行Ｎａ＾＋通道基本特性研究。     

Positive endocochlear potential: mechanism of production by marginal cells of stria vascularis

The positive endocochlear potential (EP+) and high K+ concentration of the endolymph in the scala media of the mammalian cochlea are unusual. They have long been assumed to be due to a putative K-pump in the luminal membrane of the marginal cells of the stria vascularis, which were believed to have a negative internal potential. We show that the cell potential is more positive than the EP+, and that the ion pump is conventional Na,K-ATPase, probably in the basolateral membrane. The latter was determined from experiments in which the ionic environment of the strial cells was controlled by perfusion of the perilymphatic space of the cochlea, in the absence of vascular circulation. While the usual EP+ was maintained by normal perfusate, replacement of Na+ by choline resulted in a negative EP, showing that Na,K-ATPase is necessary for the production of EP+. Elimination of K+ as well as Na+ from the perfusate did not change the value of the negative EP, showing that no K-ATPase is involved.     

Ultracytochemical study of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity in the inner ear of the squirrel monkey

The localization of ouabain-sensitive, K+-dependent p-nitrophenylphosphatase (K+-NPPase) activity of the Na+, K+-ATPase complex was studied ultracytochemically in the squirrel monkey inner ear. In the stria vascularis the reaction products showing K+-NPPase activity were limited to the cytoplasmic side of the plasmalemmal infoldings of the marginal cells. In the spiral prominence, a weak reaction was also found on the cytoplasmic process of the stromal cell, while no or little reaction was detected on the spiral prominence epithelium. In the dark cells of vestibular labyrinth the reaction products were observed on the basolateral interdigitation of the plasmalemma. In contrast, no reaction was observed on the apical cell surface. K+-NPPase activity was most intense in the strial marginal cell, followed by the dark cell of the ampulla and the utricle. The present results revealed that the dark cells in the vestibular labyrinth are involved in endolymph homeostasis.     

Immunoreactivity of endothelins and endothelin receptor in the stria vascularis of the mouse cochlea

Immunoreactivities of endothelin-1, endothelin-3, endothelin receptor type A, and Na,K-ATPase were investigated in the stria vascularis of adult male WBB6F1 +/+ mice and in that of W/Wv mutants lacking strial intermediate cells. In the +/+ mice, electron microscopic immunoreactivity for the endothelins was seen on the rough endoplasmic reticulum, Golgi apparatus, cytoplasmic vesicles and lysosomes exclusively in the strial intermediate cells by the postembedment method. Immunoreactive endothelin receptor A was localized along the plasma membrane of strial marginal cells of both wild and mutant types although the immunoreactivity of the latter was much less than that of the former by the preembedment method. These findings suggest that the endothelins, which are produced in the strial intermediate cells, may play a role in the maintenance of the stria vascularis function in the +/+ mice. Since the plasma membrane of the marginal cells of the W/Wv mice, which do not generate a high positive endocochlear potential, also showed immunoreactivity for Na,K-ATPase, it seems likely that the endothelins are involved in the activation of sodium pump of the strial marginal cells by mediation of endothelin receptor A. In addition, the role of lysosomes in the crinophagy of the endothelins in the strial intermediate cells is proposed in the +/+ mice.     

Cellular localization of rat Isk protein in the stria vascularis by immunohistochemical observation.

A novel rat membrane protein, termed Isk protein, that exhibits a voltage-dependent potassium channel activity was first reported through molecular cloning combined with an electrophysiological assay (Takumi et al., 1988). In the present study, we made an attempt to identify the cellular localization of the rat Isk protein in the stria vascularis using two types of antibodies that specifically react with the distinct parts of the rat Isk protein. Immunohistochemical analysis showed that the rat Isk protein was present only on the endolymphatic surface of the marginal cell. The possibility that the Isk protein is involved in potassium permeation in the luminal membrane of the marginal cell will be also discussed.     

Effect of Norepinephrine on Ouabain-sensitive,K+-dependent p-Nitrophenylphosphatase Activity in Strial Marginal Cells of the Cochlea in Normal and Reserpinized Guinea Pigs

On the basolateral infoldings of the strial marginal cells in the cochlea, Na-K ATPase activity is abundant. To clarify the humoral control by norepinephrine, K-NPPase activity of strial marginal cells in the cochlea was investigated in normal, reserpine, norepinephrine (NE), reserpine plus NE-treated guinea pigs using a cerium-based method. K-NPPase activity was almost completely decreased 3-20 days after reserpine administration. At 10 days after reserpinization and following NE repeated treatment, enzyme activity was detectable. These results suggested that norepinephrine might restore and regulate strial K-NPPase activity.     

Potassium recycling pathways in the human cochlea

OBJECTIVES/HYPOTHESIS: Potential pathways for recycling potassium (K+) used in the maintenance of inner ear electrochemical gradients have been elucidated in animal models. However, little is known about K+ transport in the human cochlea. This study was designed to characterize putative K+ recycling pathways in the human ear and to determine whether observations from animal models can be extrapolated to humans. STUDY DESIGN: A prospective laboratory study using an immunohistochemical approach to analyze the distribution of key ion transport mediators in the human cochlea. METHODS: Human temporal bones were fixed in situ within 1 to 6 hours of death and subsequently harvested at autopsy. Decalcification was accomplished with the aid of microwaving. Immunohistochemical staining was then performed to define the presence and cell type-specific distribution of Na,K-ATPase, sodium-potassium-chloride cotransporter (NKCC), and carbonic anhydrase (CA) in the inner ear. RESULTS: Staining patterns visualized in the human cochlea closely paralleled those seen in other species. Anti-Na,K-ATPase stained strongly the basolateral plasma membrane of strial marginal cells and nerve endings underlying hair cells. This antibody also localized Na,K-ATPase to type II, type IV, and type V fibrocytes in the spiral ligament and in limbal fibrocytes. NKCC was present in the basolateral membrane of strial marginal cells as well as in type II, type V, and limbal fibrocytes. Immunoreactive carbonic anhydrase was present in type I and type III fibrocytes and in epithelial cells lining Reissner's membrane and the spiral prominence. CONCLUSIONS: The distribution of several major ion transport proteins in the human cochlea is similar but not identical to that described in various rodent models. These results support the presence of a complex system for recycling and regulating K+ homeostasis in the human cochlea, similar to that described in other mammalian species.     

An electronmicroscopic study of microtubules in the development of marginal cells of the mouse stria vascularis

This study provides an ultrastructural evaluation of cytoplasmic microtubules during the maturation of marginal cells of the mouse stria vascularis. Postnatal marginal cells are cuboidal in structure and over a period of days develop the numerous cellular processes typical of transporting epithelia. Immediately after birth, marginal cells contain numerous microtubules randomly oriented throughout the cytoplasm. Golgi bodies and vesicles are also abundant. The initiation of cellular extension is characterized by the presence of sheet-like extensions of plasma membrane about areas of the cell periphery. Subsequently, large organelle-containing processes form, whose plasmalemma appear pleated due to the presence of the above mentioned sheet-like extensions. Typically, these processes contain microtubules oriented parallel to the direction of their extension. Within these processes, microtubules are closely associated with organelles, such as mitochondria, and may function to displace and stratify these organelles within the processes. The number and length of microtubules increase as the processes grow larger. In the adult mouse, the strial marginal cell processes are attenuated and contain almost exclusively microtubules and mitochondria. The membrane pleats unfold, apparently to provide plasma membrane for cellular extension. The data strongly implicate microtubules in strial development. Furthermore, it is suggested that the depolymerization of microtubules in the adult may underlie strial atrophy.     

Ultrastructure of cultured marginal cells of the guinea pig cochlea.

Explants of stria vascularis and spiral ligament of guinea pig cochlea were kept in primary culture. On the explant, proliferating marginal cells advanced by 15 microns/day, suggesting that in vivo defects of the strial epithelium can be covered by new marginal cells. The marginal cells growing in the cell culture dish had a diameter of 12.8 +/- 0.7 microns and formed an epithelial monolayer. Adjacent cells were connected by desmosomes and tight junctions. The cells were uniformly polarized. The apical membrane had small invaginations and numerous microvillus-like extensions, and the convoluted lateral membrane interdigitated with adjacent cells. The basal infoldings were smaller in cultured cells than in vivo; mitochondria were dispersed in the entire cytoplasm rather than concentrated in basolateral infoldings. The basal membrane infoldings of cultured marginal cells did not interdigitate with underlying fibroblast-like cells. Marginal cells were separated from underlying fibroblast-like cells by a fluid-filled space which was sometimes enlarged, leading to the formation of "domes" in the otherwise planar epithelium.