Endolymphatic Sac Epithelial Cells Research Articles

Localization of melatonin and its receptors (melatonin 1a and 1b receptors) in the mouse inner ear

Background: In the inner ear, evidence has been gathered indicating that melatonin plays important roles in inner ear physiology and pathophysiology. However, no attempt has been made previously to investigate the localization or expression of melatonin and its receptors in the whole inner ear.Aims/objectives: To analyze the presence of melatonin and its receptors in the normal mouse inner ear.Material and methods: C57BL6/J mice were used in this study. The localizations of melatonin, MT1a and MT1b in the inner ear, i.e. cochlea, vestibular end organs, vestibular ganglion and endolymphatic sac (ES), were studied by immunohistochemistry.Results: The organ of Corti, spiral ganglion, vestibular ganglion, vestibular sensory cells, vestibular dark and transitional cells, and ES epithelial cells showed an immunofluorescence reaction to melatonin, MT1a and MT1b.Conclusion and significance: The present findings show that melatonin and its receptors (MT1a and MT1b) are present in the inner ear, thus supporting the hypothesis that melatonin plays a physiological role in the inner ear.

Immunolocalization of calcium sensing and transport proteins in the murine endolymphatic sac indicates calciostatic functions within the inner ear

An exceptionally low calcium (Ca2+) concentration in the inner ear endolymph ([Ca2+]endolymph) is crucial for proper auditory and vestibular function. The endolymphatic sac (ES) is believed to critically contribute to the maintenance of this low [Ca2+]endolymph. Here, we investigated the immunohistochemical localization of proteins that are presumably involved in the sensing and transport of extracellular Ca2+ in the murine ES epithelium. Light microscopic and fluorescence immunolabeling in paraffin-embedded murine ES tissue sections (male C57BL/6 mice, 6–8 weeks old) demonstrated the presence of the calcium-sensing receptor CaSR, transient receptor potential cation channel subtypes TRPV5 and TRPV6, sarco/endoplasmic reticulum Ca2+-ATPases SERCA1 and SERCA2, Na+/Ca2+ exchanger NCX2, and plasma membrane Ca2+ ATPases PMCA1 and PMCA4 in ES epithelial cells. These proteins exhibited (i) membranous (apical or basolateral) or cytoplasmic localization patterns, (ii) a proximal-to-distal labeling gradient within the ES, and (iii) different distribution patterns among ES epithelial cell types (mitochondria-rich cells (MRCs) and ribosome-rich cells (RRCs)). Notably, in the inner ear membranous labyrinth, CaSR was exclusively localized in MRCs, suggesting a unique role of the ES epithelium in CaSR-mediated sensing and control of [Ca2+]endolymph. Structural loss of the distal ES, which is consistently observed in Meniere’s disease, may therefore critically disturb [Ca2+]endolymph and contribute to the pathogenesis of Meniere’s disease.

Gastric-type H+,K+-ATPase in mouse vestibular end organs

Conclusion: Gastric type H+,K+-ATPase in the vestibular end organs may be of importance for K+ circulation and may also be related to pH regulation in vestibular end organs and endolymphatic sac.Objective: To analyze the expression of gastric-type H+,K+-ATPase in normal mouse vestibular end organs.Methods: 8 weeks old CBA/J mice were used in this study. The presence of gastric-type H+,K+-ATPase α and β in the vestibular end organs, viz. utricle, saccule, ampulla, vestibular ganglion, and endolymphatic sac, was investigated using immunohistochemistry.Results: In the vestibular end organs, H+,K+-ATPase α and β were almost identical. H+,K+-ATPase was expressed in sensory cells, the basolateral surface of dark cells, fibrocytes, in vestibular ganglion cells, and in the upper region of the endolymphatic sac epithelial cells.

Prox1 expression in the endolymphatic sac revealed by whole-mount fluorescent imaging of Prox1-GFP transgenic mice

This study describes a technical breakthrough in endolymphatic sac research, made possible by the use of the recently generated Prox1-GFP transgenic mouse model. Whole-mount imaging techniques through the decalcified temporal bone and three-dimensional observations of Prox1-GFP mouse tissue revealed the positive labeling of the endolymphatic sac in adult stage, and allowed, for the first time, the GFP-based identification of endolymphatic sac epithelial cells. Prox1 expression was observed in all parts of the endolymphatic sac epithelia. In intermediate portion of the endolymphatic sac, mitochondria-rich cells did not express Prox1, although ribosome-rich cells showed strong GFP labeling. The anatomical relationship between the endolymphatic sac and the surrounding vasculature was directly observed. In the endolymphatic sac, expression of Prox1 may suggest progenitor cell-like pluripotency or developmental similarity to systemic lymphatic vessels in other organs. This whole-mount imaging technique of the endolymphatic sac can be combined with other conventional histological, sectioning, and labeling techniques and will be very useful for future endolymphatic sac research.

Localization of aquaporins in the mouse vestibular end organs

Conclusion: We found that aquaporins (AQPs) in the fluid transporting cells, such as vestibular dark cells and endolymphatic sac epithelial cells, seem to be of importance in fluid transport in the inner ear, while those in the sensory and ganglion cells may play a functional role in sensory cell transduction. Objective: Expression of AQPs (0–12) was analyzed in normal mouse vestibular end organs. Methods: CBA/J mice were used in this study. Localization of AQPs 0–12 in the vestibular end organs and endolymphatic sac was investigated by immunohistochemistry. Results: The AQPs were found abundantly distributed in many structures in the vestibular end organs, i.e. vestibular sensory and supporting cells, vestibular dark cells, vestibular ganglion cells, and the endolymphatic sac.

Oxygenated fixation demonstrates novel and improved ultrastructural features of the human endolymphatic sac

The purpose of the present study is to describe in detail the ultrastructure of the human endolymphatic sac using a new and improved method of fixation as well as a refined surgical approach in obtaining specimens. Transmission electron microscopy of the human endolymphatic sac, employing an oxygenated fixative. Eighteen tissue samples of the human endolymphatic sac were obtained during surgery for vestibular schwannoma using the translabyrinthine approach. The specimens were fixed in 2% glutaraldehyde in an oxygenated fluorocarbon blood substitute vehicle before preparation by routine methods for transmission electron microscopy. We focused on the epithelial cell layer, subepithelial tissue, intraluminal content, and vascular tissue in both the intra- and extraosseous part of the endolymphatic sac. We observed well-defined endolymphatic sac epithelial cell lining in all 18 specimens. In general, we found very well-preserved specimens with well-defined intracellular structures. In contrast to the results in former studies, a minimum of fixation artifacts was observed in the present study. Three different cell types were observed in the intraosseous part of the sac: mitochondria-rich cells, ribosome-rich cells, and nonclassifiable cells. A fourth cell type was found in the extraosseous part. Novel ultrastructural features of the epithelial lining and the subepithelial layer are described and discussed. The results in the present study indicate an improvement in obtaining human tissue with optimal fixation for ultrastructural analysis and provide several novel morphologic observations. The potential functions of the endolymphatic sac are discussed with reference to former studies.

Expression of Anion Exchangers in Cultured Human Endolymphatic Sac Epithelia

Pendrin acts as a Cl-/HCO3- exchanger and is responsible for endolymphatic fluid volume and pH homeostasis in human endolymphatic sac epithelial cells. The endolymphatic sac (ES) is part of the membranous labyrinth in the inner ear that plays an important role in maintaining homeostasis of the endolymphatic fluid system. However, the exact mechanism of fluid volume and pH regulation is not fully understood yet. We aimed to demonstrate the expression of various anion exchangers (AEs), including pendrin, in cultured human endolymphatic sac epithelial (HESE) cells. Endolymphatic sac specimens were harvested during acoustic neuroma surgery (n = 24) using the translabyrinthine approach and then subcultured with high epidermal growth factor (EGF) (25 ng/ml) media and differentiated using low-EGF (0.5 ng/ml) media. The cultured cells were classified according to the morphology on TEM. The Cl-/HCO3- exchanger activity was assessed by pHi measurement using pH sensitive dye 2', 7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF/AM). We performed reverse transcriptase-polymerase chain reaction and immunohistochemical staining for AEs. We determined that 7.3 ± 6.7% of cells differentiated into mitochodria-rich cells and 50.2 ± 15.1 of cells differentiated into ribosome-rich cells. bAE3, AE4, SLC26A4, SLC26A6, and SLC26A11 were also expressed in cultured HESE cells. The cultured cells had Cl-/HCO3- and Cl-/formate exchange activity on the luminal membrane, which is sensitive to anion channel inhibitors (DIDS 500 μM). Furthermore, we showed that pendrin (SLC26A4) was expressed in cultured HESE cell membranes. Our results suggest that AEs, including pendrin, are expressed in epithelia of ES and may have role in maintaining ionic homeostasis, and the HESE culture system are useful for uncovering the functional role of ES epithelial cells.

Presence of Adrenergic Receptors in Rat Endolymphatic Sac Epithelial Cells

Intravenous application of catecholamines produces a depression in the endolymphatic sac direct current potential (ESP) and increases endolymphatic pressure via the β-adrenergic receptor (AR) in guinea pigs, suggesting that catecholamines play a role in the endolymphatic system. However, the localization of ARs in the endolymphatic sac (ES) is still undetermined. The presence of ARs in the rat ES was investigated by reverse transcriptase-polymerase chain reaction using laser capture microdissection (LCM) and immunohistochemical analysis. Expression of α(1A)-, α(1B)-, α(2A)-, α(2B)-, β(1)-, β(2)- and β(3)-ARs was observed in LCM samples of ES epithelia. Immunohistochemical analysis using specific antibodies showed immunofluorescence of β(2)- and β(3)-ARs in epithelial cells of the ES intermediate portion, and no specific staining results were obtained for α(1)-, α(2A)-, α(2B)- and β(1)-ARs. The presence of β(2)-AR with no clear immunostaining of β(1)-AR in ES epithelial cells is in accordance with previous electrophysiological and pharmacological results, which suggests that β(2)-AR mediates the action of catecholamines on the ESP. The presence of β(3)-AR in the ES epithelial cells and its absence in the stria vascularis implies that β(3)-AR plays a specific role in the ES.

Localization of aquaporins 1, 2, and 3 and vasopressin type 2 receptor in the mouse inner ear

Conclusion: It is suggested that aquaporins (AQPs) 1, 2, and 3, and vasopressin type 2 receptors (V2Rs) in the fluid transporting cells, such as stria vascularis, vestibular dark and transitional cells, and endolymphatic sac epithelial cells, have an important role in fluid transport in the inner ear, while those in the sensory and ganglion cells may play a functional role in the sensory cell transduction system. Objective: To analyze expression of AQP1, AQP2, and AQP3 as well as V2Rs in the normal mouse inner ear. Methods: CBA/J mice were used in this study. Localization of AQP1, AQP2, AQP3, and V2Rs in the inner ear, i.e. cochlea, vestibular end organs, and endolymphatic sac, was investigated by immunohistochemistry. Results: The results show that AQP1, AQP2, AQP3, and V2Rs are abundantly distributed in many inner ear structures, i.e. stria vascularis, inner and outer hair cells, spiral ganglion cells, vestibular sensory and ganglion cells, vestibular dark and transitional cells, and the endolymphatic sac.

The Detailed Localization Pattern of Na+/K+/2Cl− Cotransporter Type 2 and Its Related Ion Transport System in the Rat Endolymphatic Sac

The endolymphatic sac (ES) is a part of the membranous labyrinth. ES is believed to perform endolymph absorption, which is dependent on several ion transporters, including Na(+)/K(+)/2Cl(-) cotransporter type 2 (NKCC-2) and Na(+)/K(+)-ATPase. NKCC-2 is typically recognized as a kidney-specific ion transporter expressed in the apical membrane of the absorptive epithelium. NKCC-2 expression has been confirmed only in the rat and human ES other than the kidney, but the detailed localization features of NKCC-2 have not been investigated in the ES. Thus, we evaluated the specific site expressing NKCC-2 by immunohistochemical assessment. NKCC-2 expression was most frequently seen in the intermediate portion of the ES, where NKCC-2 is believed to play an important role in endolymph absorption. In addition, NKCC-2 expression was also observed on the apical membranes of ES epithelial cells, and Na(+)/K(+)-ATPase coexpression was observed on the basolateral membranes of ES epithelial cells. These results suggest that NKCC-2 performs an important role in endolymph absorption and that NKCC-2 in apical membranes and Na(+)/K(+)-ATPase in basolateral membranes work coordinately in the ES in a manner similar to that in renal tubules.

Na+Homeostasis in the Inner Ear

homeostasis in the inner ear is important to maintain normal hearing and balance. transport in the inner ear is reported to be occurred in non-sensory epithelium of inner ear which forms a barrier between endolymphatic space and perilymphatic space. Functionally identified and constitutively active absorption sites in the inner ear are Reissner's membrane, outer sulcus cells, vestibular transitional cells, saccular nonsensory epithelial cells, and endolymphatic sac epithelial cells. transport in these epithelial cells is mediated by apically located epithelial channels (ENaC), nonselective cation channels and basolaterally located , -ATPase. absorption is increased by glucocorticoid through glucocorticoid receptor or ATP through purinergic receptors depending on cell types.

Functional and Molecular Expression of Epithelial Sodium Channels in Cultured Human Endolymphatic Sac Epithelial Cells

Epithelial sodium channels are expressed in cultured human endolymphatic sac (ES) epithelial (HESE) cells and epithelial sodium channel (ENaC) expression is suppressed by interleukin 1beta. The ES is part of the membranous labyrinth in the inner ear that plays an important role in maintaining homeostasis of the endolymphatic fluid system. However, the exact mechanism of fluid volume regulation is not yet known. The ES specimens were harvested during acoustic neuroma surgery (n = 13) using the translabyrinthine approach and were subcultured with high-epidermal growth factor (25 ng/mL) media. The serially passaged HESE cells differentiated into a monolayer of confluent cells and some of the cultured cells had features of mitochondria-rich cells. Reverse transcription-polymerase chain reaction revealed that ENaC subunits are expressed in the cultured HESE cells. We also confirmed the presence of an ENaC-dependent short-circuit current in the cultured HESE cells. Interestingly, ENaC mRNA expression and ENaC-dependent current decreased after treatment with interleukin 1beta (10 nmol/L for 24 h). These findings suggest that ENaC plays an important role in fluid absorption in the human ES and that its function may be altered during inflammatory conditions.

Interferon‐γ suppresses Na+–H+ exchanger in cultured human endolymphatic sac epithelial cells

Adequate regulation of endolymphatic pH is essential for maintaining inner ear function. The Na(+)-H(+) exchanger (NHE) is a major determinant of intracellular pH (pH(i)), and facilitates Na(+) and fluid absorption in various epithelia. We determined the functional and molecular expression of NHEs in cultured human endolymphatic sac (ES) epithelial cells and examined the effect of IFN-gamma on NHE function. Serial cultures of human ES epithelial cells were generated from tissue samples. The molecular expression of NHE1, -2, and -3 isoforms was determined by real-time RT-PCR. The functional activity of NHE isoforms was measured microfluorometrically using a pH-sensitive fluorescent dye, 2',7'-bis(carbonylethyl)-5(6)-carboxyfluorescein (BCECF), and a NHE-inhibitor, 3-methylsulfonyl-4-piperidinobenzoyl guanidine methanesulfonate (HOE694). NHE1, -2, and -3 mRNAs were expressed in human ES epithelial cells. Functional activity of NHE1 and -2 was confirmed in the luminal membrane of ES epithelial cells by sequentially suppressing Na(+)-dependent pH(i) recovery from intracellular acidification using different concentrations of HOE694. Treatment with IFN-gamma (50 nM for 24 h) suppressed mRNA expression of NHE1 and -2. IFN-gamma also suppressed functional activity of both NHE1 and -2 in the luminal membrane of ES epithelial cells. This study shows that NHEs are expressed in cultured human ES epithelial cells and that treatment with IFN-gamma suppresses the expression and functional activity of NHE1 and -2.

Localization of megalin in rat vestibular dark cells and endolymphatic sac epithelial cells

Conclusion: Megalin immunoreactivity was observed in kidney proximal tubule cells, vestibular dark cells, and epithelial cells of the endolymphatic sac. Endocytic mechanisms appear to differ between the endolymphatic sac and proximal tubule cells. We speculate that megalin is secreted by a certain type of cell into the endolymphatic space, and is then absorbed from the endolymphatic space by another type of cell to maintain endolymphatic sac homeostasis. Objectives: We previously detected megalin immunoreactivity in the rat cochlear duct. Megalin may be involved in endocytosis in the vestibular organ and endolymphatic sac. To examine this possibility, we extended our immunocytochemical investigation to the rat inner ear cells with special attention to vestibular dark cells and endolymphatic sac. Materials and methods: We observed immunoreactivity of megalin under light and electron microscopy. The primary antibody was rabbit polyclonal antibody that had been raised against rat immunoaffinity-purified megalin. Results: The luminal membrane and subapical area of dark cells in the semicircular canal were immunolabeled. The stainable substance in the endolymphatic space was strongly stained. The cytoplasm of epithelial cells was also stained in various patterns.

Large Na+ influx and high Na+, K+–ATPase activity in mitochondria-rich epithelial cells of the inner ear endolymphatic sac

Fluid in the mammalian endolymphatic sac (ES) is connected to the endolymph in the cochlea and the vestibule. Since the dominant ion in the ES is Na(+), it has been postulated that Na(+) transport is essential for regulating the endolymph pressure. This study focused on the cellular mechanism of Na(+) transport in ES epithelial cells. To evaluate the Na(+) transport capability of the ES epithelial cells, changes in intracellular Na(+) concentration ([Na(+)](i)) of individual ES cells were measured with sodium-binding benzofurzan isophthalate in a freshly dissected ES sheet and in dissociated ES cells in response to either the K(+)-free or ouabain-containing solution. Analysis of the [Na(+)](i) changes by the Na(+) load and mitochondrial staining with rhodamine 123 showed that the ES cells were classified into two groups; one exhibited an intensive [Na(+)](i) increase, higher Na(+), K(+)-ATPase activity, and intensive mitochondrial staining (mitochondria-rich cells), and the other exhibited a moderate [Na(+)](i) increase, lower Na(+), K(+)-ATPase activity, and moderate mitochondrial staining (filament-rich cells). These results suggest that mitochondria-rich ES epithelial cells (ca. 30% of ES cells) endowed with high Na(+) permeability and Na(+), K(+)-ATPase activity potentially contribute to the transport of Na(+) outside of the endolymphatic sac.

Soluble Megalin Is Accumulated in the Lumen of the Rat Endolymphatic Sac

The endolymphatic sac (ES) is believed to play an important role in maintaining homeostasis in the inner ear by the absorption and endocytosis of endolymph. Megalin is a 600-kDa multiligand endocytic receptor expressed in certain types of absorptive epithelia including kidney proximal tubules. We analyzed the immunoreactivity for megalin in rat ES by immunofluorescence, immunogold electron microscopy, and immunoblotting. With immunostaining, the luminal substances of the ES were strongly stained for megalin. Megalin was also localized in luminal macrophage-like cells and both types of epithelial cell (mitochondria-rich cells and ribosome-rich cells). In these cells, the megalin was localized in the lumen of endosomes, but was not membrane associated. This localization pattern indicates that the megalin in these cells is not a membrane receptor, but merely one of the constituents that are endocytosed from the lumen of the ES. Immunoblotting indicated that the megalin in the ES is a 210-kDa molecule lacking a cytoplasmic domain. This suggests that the megalin in the ES may be a soluble form, different from the 600-kDa membrane-bound receptor expressed in kidneys. Taken together, it is likely that the megalin in the ES lumen is a soluble component and may be endocytosed by the ES epithelial cells. Furthermore, we found that the tectorial membrane, an acellular structure in the cochlea, gave a strong megalin immunoreaction. Since the cochlea is connected to the ES, the megalin may be transported alone or with the components of the tectorial membrane from the cochlea to the ES lumen through longitudinal flow.

Co-expression of pendrin, vacuolar H+-ATPase alpha4-subunit and carbonic anhydrase II in epithelial cells of the murine endolymphatic sac.

The endolymph in the endolymphatic sac (ES) is acidic (pH 6.6-7). Maintaining this acidic lumen is believed to be important for the normal function of the ES. The acid-base regulation mechanisms of the ES are unknown. Here we investigated the expression patterns of acid-base regulators, including vacuolar (v)H+-ATPase (proton pump), carbonic anhydrase (CA) II, and pendrin in the murine ES epithelium by immunohistochemistry (IHC) and compared their expression patterns by double immunostaining. We found that pendrin and vH+-ATPase were co-localized in the apical membrane of a specific type of ES epithelial cell. Pendrin- and vH+-ATPase-positive cells also expressed cytoplasmic CA II. Co-expression of pendrin, vH+-ATPase, and CA II in the same subgroup of ES cells suggests that this specific type of ES cell is responsible for the acid-base balance processes in the ES and pendrin, vH+-ATPase, and CA II are involved in these processes.

Calcium-sensitive nonselective cation channel identified in the epithelial cells isolated from the endolymphatic sac of guinea pigs.

We identified a Ca2+-sensitive cation channel in acutely dissociated epithelial cells from the endolymphatic sac (ES) of guinea pigs using the patch-clamp technique. Single-channel recordings showed that the cation channel had a conductance of 24.0 +/- 1.3 pS (n = 8) in our standard solution. The relative ionic permeability of the channel was in the order K+ = Na+ > Ca2+ >> Cl-. This channel was weakly voltage-dependent but was strongly activated by Ca2+ on the cytosolic side at a concentration of around 1 mm in inside-out excised patches. With cell-attached patches, however, the channel was activated by much lower Ca2+ concentrations. Treatment of the cells, under cell-attached configuration, with ionomycin (10 microm), carbonyl cyanide 3-chlorophenylhydrazone (CCCP, 20 microm), or ATP (1 mm), which increased intracellular Ca2+ concentration ([Ca2+]i), activated the channel at an estimated [Ca2+]i from 0.6 microm to 10 microm. It is suggested that some activators of the channel were deteriorated or washed out during the formation of excised patches. Based on this Ca2+ sensitivity, we speculated that the channel contributes to the regulation of ionic balance and volume of the ES by absorbing Na+ under certain pathological conditions that will increase [Ca2+]i. This is the first report of single-channel recordings in endolymphatic sac epithelial cells.

Evidence for the presence of a Na+-H+ exchanger in the endolymphatic sac epithelium of guinea-pigs.

The intracellular pH (pHi) of epithelial cells from the endolymphatic sac (ES) of the guinea-pig was measured microfluorometrically with the pH-sensitive fluorescent dye, 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to examine the presence of a Na+-H+ exchanger (NHE) in the ES epithelial cells. pHi recovery from acid loading with an NH4+-prepulse in a nominally HCO3-free solution was dependent on extracellular Na+ ([Na+]o) and was inhibited by amiloride and its analogue ethylisopropylamiloride (EIPA), suggesting that a decreased pHi induced by an acute acid load may be equilibrated by a NHE. In the steady-state, amiloride had no effect on pHi, indicating that the NHE activity is low at the resting pHi. However, the intracellular acidification induced by the removal of [Na+]o was inhibited by the simultaneous application of amiloride. H+-efflux rate (JH, mean activity of NHE), which was calculated as the product of the recovery rate (dpHi/dt) from the acid loading and the intrinsic buffering capacity (betai) at the corresponding pHi, was decreased as pHi was increased. The concentration/response curve for the inhibition of initial JH by EIPA revealed an apparent 50% inhibitory constant (Ki ) of 0.85 microM. Kinetic analysis of initial JH as a function of [Na+]o revealed a Michaelis-Menten constant (Km) of 24.14 mM for Na+-dependent H+ efflux. The results indicate that NHE in the ES epithelium belongs to an amiloride-sensitive subtype.

Calcium Mobilization in Isolated Epithelial Cells of the Endolymphatic Sac

Endolymphatic sac epithelial cells were isolated from the endolymphatic sac of the guinea pig by enzymatic and mechanical dissociation. The intracellular free calcium ion concentrations ([Ca²⁺]i) of the isolated cells were determined using the Ca²⁺-sensitive dye fura-2. The isolated cells were classified into two types, i.e. light and dark cells. In the resting state, [Ca²⁺]i in the cells was variable in both types of cells. In the presence of 200 µM ATP, there was a rapid rise in [Ca²⁺]i. These findings suggest that endolymphatic sac epithelial cells may have receptor-mediated Ca channels which may play an important role for a nerve-mediated local feedback system of the endolymphatic sac to regulate homeostasis of endolymph volume, pressure and electrolyte balance.