Rie Nishioka

Expressions of Aquaporin-2, Vasopressin Type 2 Receptor, Transient Receptor Potential Channel Vanilloid (TRPV)1, and TRPV4 in the Human Endolymphatic Sac†

Objective: To localize aquaporin (AQP)2, vasopressin type 2 receptor (V2‐R), and transient receptor potential channel vanilloid subfamily 1, 4 (TRPV1, TRPV4) in the human endolymphatic sac (ES).

Hormonal Aspects of Ménière’s Disease on the Basis of Clinical and Experimental Studies

Conclusions: Endolymph homeostasis is thought to be mediated by the vasopressin-aquaporin-2 (VP-AQP2) system in the inner ear. Endolymphatic hydrops, the morphological characteristics of Ménière’s disease (MD), seems to reflect the malregulation of the VP-AQP2 system in inner ear fluid. The elevation of plasma vasopressin (p-VP) level, which is often observed in MD and its related diseases, might be one of the causative factors underlying these diseases. Purpose of Review: Review of the role of the VP-AQP2 system in the inner ear fluid homeostasis and in the formation and development of endolymphatic hydrops. Recent Clinical and Experimental Findings: A clinical survey has revealed that the p-VP level is often elevated in MD and its related diseases and that the increase in the p-VP level was closely linked to vertigo attacks in MD. Experimental studies have revealed that proteins and mRNAs of aquaporin-2 and vasopressin type 2 receptor were expressed in the stria vascularis of the cochlea and the epithelium of the endolymphatic sac, and that the volume of the endolymphatic compartment was mediated by the activity of the VP-AQP2 system in the inner ear.

Expression of aquaporin1, 3, and 4, NKCC1, and NKCC2 in the human endolymphatic sac

OBJECTIVE To locate aquaporin (AQP) 1, 3, and 4, Na-K-2Cl cotransporter (NKCC) 1 and 2 in the human endolymphatic sac (ES). METHODS A sample of human ES was harvested during the removal of vestibular schwannoma via the translabyrinthine approach. The sample was immediately fixed in 4% paraformaldehyde and embedded in OCT compound. Immunohistochemistry was performed with AQP1, 3, and 4, NKCC1, and NKCC2 polyclonal antibodies. RESULTS AQP1, AQP3, and NKCC2 were strongly expressed in the epithelial layer of the ES. AQP4 and NKCC1 were weakly expressed in the epithelial layer of the ES. CONCLUSIONS As it is impossible to perform quantitative analysis based on the fluorescence intensity of each immunoreactivity, we have presented the existence of AQP1, 3, and 4, NKCC1, and NKCC2 in the ES. The expression of NKCC1 and 2 indicated that the ES may have both secretory and adsorptive functions to maintain the homeostasis of endolymph.

Expression of aquaporins and vasopressin type 2 receptor in the stria vascularis of the cochlea

Recently, considerable evidence has been accumulated to support the novel view that water homeostasis in the inner ear is regulated via the vasopressin-aquaporin 2 (VP-AQP2) system in the same fashion as in the kidney. Indeed, multiple subtypes of AQPs including AQP-2 are reported to be expressed in the cochlea. However, the mechanism that underlies VP-AQP-2 mediated water homeostasis remains to be elucidated. In the present study, the localizations of AQP-1, -2, -3, -4, -5, -7, -8, -9, and vasopressin type 2 receptor (V2-R) in the stria vascularis (SV) were molecular biologically and immunohistochemically examined to evaluate the role of the AQP water channel system in water homeostasis of the SV. A RT-PCR study revealed that AQPs and V2-R mRNA are expressed in the cochlea. As for their immunohistochemical localization, the AQP-2 protein is expressed on the basal side of the basal cells of the SV, and proteins of AQP-3 and V2-R are expressed on the apical side of the basal cells. AQP-7 and -9 proteins are expressed on the apical side of marginal cells. AQP-4, -5, and -8 protein expressions could not be detected in the lateral wall of the cochlea. From the present results, water flux in the SV is thought to be regulated at the level of the basal cells by vasopressin. Furthermore, such a distribution of AQP-2, -3, and V2-R suggests that VP-AQP-2 mediated water transport might work actively in the basal cells from perilymph towards endolymph containing AQP-1, -7 and -9.

Effects of lithium on endolymph homeostasis and experimentally induced endolymphatic hydrops

There is evidence to suggest that water homeostasis in the inner ear is regulated via the vasopressin (VP)-aquaporin 2 (AQP2) system in the same fashion as in the kidney. The VP-AQP2 system in the kidney is well known to be inhibited by lithium, resulting in polyuria due to a decrease in reabsorption of water in the collecting duct of the kidney. Therefore, lithium is also likely to inhibit the VP-AQP2 system in the inner ear, and consequently exert some influence on inner ear fluid homeostasis. In this study, we investigated the effects of lithium on AQP2 expression in the rat inner ear, and on the cochlear fluid volume in hydropic ears of guinea pigs. A quantitative PCR study revealed that lithium reduced AQP2 mRNA expression in the cochlea and endolymphatic sac. Lithium application also decreased the immunoreactivity of AQP2 in the cochlea and endolymphatic sac. In a morphological study, lithium intake significantly reduced endolymphatic hydrops dose-dependently. These results indicate that lithium acts on the VP-AQP2 system in the inner ear, consequently producing a dehydratic effect on the endolymphatic compartment.

Decompression effects of erythritol on endolymphatic hydrops.

OBJECTIVE The effect of the uptake of erythritol with and without the addition of pectin on fecal condition, p-OSM and p-AVP levels, and the endolymphatic volume was investigated to consider the possibility that erythritol is applicable as a therapeutic agent for Menieres disease. MATERIALS AND METHODS Two experiments were performed using 100 female Hartley guinea pigs. Experiment 1 was designed to morphologically investigate the influence of the uptake of erythritol with or without the addition of pectin on the endolymphatic volume. Experiment 2 was designed to investigate changes in p-OSM and p-AVP levels after the uptake of erythritol with or without the addition of pectin. RESULTS (1) Endolymphatic hydrops significantly decreased after the uptake of a mixture of erythritol and pectin, but did not decrease after the uptake of pectin or erythritol (p<0.001). (2) The fecal condition was muddy in all animals with the uptake of erythritol alone, but muddy or very soft feces were not observed in animals with a mixture of pectin and erythritol. p-AVP and p-OSM levels were significantly elevated in animals with the uptake of erythritol alone or a mixture of erythritol and pectin. Notably, the increase in p-AVP and p-OSM levels was significantly more evident in animals with the uptake of erythritol alone (one-way ANOVA, p<0.001). CONCLUSIONS The addition of pectin almost completely suppressed erythritol-induced diarrhea. Consequently, the secondary elevation of p-AVP and p-OSM due to diarrhea was also reduced. The uptake of a mixture of erythritol and pectin markedly decompressed endolymphatic hydrops, although the uptake of erythritol alone did not. The difference of the decompression effect between animal groups with the uptake of erythritol alone and a mixture of erythritol and pectin seemed to be attributable to the difference of p-AVP levels due to diarrheal state.

A comparison of dehydration effects of V2-antagonist (OPC-31260) on the inner ear between systemic and round window applications

V2-antagonist (OPC-31260 (OPC)) application to the scala tympani reduced endolymphatic hydrops. In the present study, we investigated whether systemic administration or local infusion via the round window (RW application) of OPC would be more suitable for clinical use. In Experiment 1, the increase ratios of the cross-sectional area of the scala media of experimentally induced endolymphatic hydrops were quantitatively assessed among four groups of non-OPC application, RW application of xanthan gum, systemic application of OPC and RW application of OPC. In Experiment 2, the effects of systemic and RW applications of OPC on plasma vasopressin (p-VP) concentrations and plasma osmolality (p-OSM) were investigated. In Experiment 3, endocochlear DC potential (EP) was measured in guinea pigs with the RW application of OPC. Electron microscopic observations of the stria vascularis and the hair cells were also made. Both systemic and RW applications of OPC significantly reduced endolymphatic hydrops. However, systemic application resulted in the distension of the Reissners membrane in the non-operated ear, which seemed to be caused by elevated p-VP levels resulting from the systemic application of OPC. In contrast, RW application of OPC produced no apparent toxic effects in the inner ear, as indicated electrophysiological or morphological changes. Thus, drug delivery via the round window is more useful for the clinical application of OPC for medical decompression.

Expression and immunolocalization of aquaporin-6 (Aqp6) in the rat inner ear

Conclusion. Since aquaporin-6 (Aqp6) protein was located in the membrane of intracellular vesicles of the stria vascularis, endolymphatic sac, and vestibule, Aqp6 might be involved in some distinct physiological function of acid–base metabolism and water balance in endolymphatic fluid homeostasis. However, its lack of expression on the plasma membrane indicates that Aqp6 does not have a direct role in water flux via the plasma membrane. Objective. To evaluate the expression and immunolocalization of Aqp6 in the rat inner ear. Materials and methods. Wistar rats were used. Aqp6 mRNA expression in the rat inner ear was investigated in the vestibulum as well as in the cochlea and endolymphatic sac using the reverse transcription-polymerase chain reaction (RT-PCR) method, and detailed immunolocalization of Aqp6 in the rat inner ear was investigated using immunohistochemical methods including immunofluorescence microscopy and immunoelectron microscopy. Results. We obtained novel data showing that not just Aqp6 mRNA but also Aqp6 protein is expressed in the cochlea, endolymphatic sac, and vestibule. Immunoelectron microscopic studies revealed that the immunolabelled gold was diffusely seen in the intracellular area of the stria vascularis, endolymphatic sac, and vestibule, but never in the plasma membranes.

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

Abstract 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.

Presence and regulation of epithelial sodium channels in the marginal cells of stria vascularis

Conclusion. This study indicates that epithelial Na+-selective channels (ENaC) recycle Na+ via clathrin-mediated endocytosis in the marginal cells of the stria vascularis and that clathrin-independent endocytosis appeared to be modulated by the amount of Na+ transported. These results suggest the presence of ENaC in the luminal membrane of marginal cells and that ENaC are an efficient pathway for the uptake of Na+ from the endolymph. Objective. The ENaC found in many transporting epithelia play a key role in the regulation of salts and water homeostasis, cellular pH, cell volume, and cell function. Both biochemical and physiological approaches have been used to identify, characterize, and quantify this important channel, but its location in the marginal cells of the stria vascularis has not been fully clarified. The aim of this study was to determine the localization and regulation of ENaC. Materials and methods. Forty healthy female guinea pigs were used: 20 for the control experiment, 10 for the amiloride experiment, and 10 for the aldosterone experiment. We perfused cationized ferritin (CF) and microperoxidase (MPO) as tracers for clathrin-mediated and clathrin-independent endocytosis, respectively, into the cochlear duct. After 30 min of endolymphatic perfusion, the tissues were fixed and CF- and MPO-loaded endosomes within the marginal cell were observed by transmission electron microscopy. The numbers of CF- and MPO-loaded endosomes were compared between the three groups. Results. In the amiloride group, the numbers of CF- and MPO-loaded endosomes decreased in comparison with the control. In the aldosterone group, the numbers of CF- and MPO-loaded endosomes decreased and increased, respectively. Recently, it has been reported that ENaC are endocytosed via clathrin-mediated endosomes and aldosterone decreases the rate of endocytosis of ENaC. In this study, the results of the aldosterone experiment were consistent with those of recent studies.