Low-salt diet increases mRNA expression of aldosterone-regulated transporters in the intermediate portion of the endolymphatic sac.

The endolymphatic sac is a small sac-shaped organ at the end of the membranous labyrinth of the inner ear. The endolymphatic sac absorbs the endolymph, in which the ion balance is crucial for inner ear homeostasis. Of the three sections of the endolymphatic sac, the intermediate portion is the center of endolymph absorption, particularly sodium transport, and is thought to be regulated by aldosterone. Disorders of the endolymphatic sac may cause an excess of endolymph (endolymphatic hydrops), a histological observation in Meniere's disease. A low-salt diet is an effective treatment for Meniere's disease, and is based on the assumption that the absorption of endolymph in the endolymphatic sac abates endolymphatic hydrops through a physiological increase in aldosterone level. However, the molecular basis of endolymph absorption in each portion of the endolymphatic sac is largely unknown because of difficulties in gene expression analysis, resulting from its small size and intricate structure. The present study combined reverse transcription-quantitative polymerase chain reaction and laser capture microdissection techniques to analyze the difference of gene expression of the aldosterone-controlled epithelial Na+ channel, thiazide-sensitive Na+-Cl- cotransporter, and Na+, K+-ATPase genes in the three individual portions of the endolymphatic sac in a rat model. A low-salt diet increased the expression of aldosterone-controlled ion transporters, particularly in the intermediate portion of the endolymphatic sac. Our findings will contribute to the understanding of the physiological function of the endolymphatic sac and the pathophysiology of Meniere's disease.

Phosphatidylinositol 4,5-bisphosphate is localized in the plasma membrane outer leaflet and regulates cell adhesion and motility.

Phospholipids are distributed asymmetrically in the plasma membrane (PM) of mammalian cells. Phosphatidylinositol (PI) and its phosphorylated forms are primarily located in the inner leaflet of the PM. Among them, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a well-known substrate for phospholipase C (PLC) or phosphoinositide-3 kinase, and is also a regulator for the actin cytoskeleton or ion channels. Although functions of PI(4,5)P2 in the inner leaflet are well characterized, those in the outer leaflet are poorly understood. Here, PI(4,5)P2 was detected in the cell surface of non-permeabilized cells by anti-PI(4,5)P2 antibodies and the pleckstrin-homology (PH) domain of PLCδ1 that specifically binds PI(4,5)P2. Cell surface PI(4,5)P2 signal was universally detected in various cell lines and freshly isolated mouse bone marrow cells and showed a punctate pattern in a cholesterol, sphingomyelin, and actin polymerization-dependent manner. Furthermore, blocking cell surface PI(4,5)P2 by the addition of anti-PI(4,5)P2 antibody or the PH domain of PLCδ1 inhibited cell attachment, spreading, and migration. Taken together, these results indicate a unique localization of PI(4,5)P2 in the outer leaflet that may have a crucial role in cell attachment, spreading, and migration.

Tricellulin Expression and its Deletion Effects in the Endolymphatic Sac.

OBJECTIVES: Tricellulin is a tight junction (TJ)-forming protein that participates in the sealing function of tricellular TJs. Tricellulin-knockout (Tric-/-) mice show progressive hearing loss with degeneration of hair cells in the cochlea without physiological or physical disorders. In the present study, we investigated the tricellulin expression and its deletion effects in the endolymphatic sac (ES) using Tric-/- mice. MATERIALS AND METHODS: The ES epithelia from wild-type (WT) mice were laser-microdissected, and RT-PCR was performed. The ES sections from Tric-/- and WT mice were immunostained with an anti-tricellulin antibody. Hematoxylin and eosin staining was performed for morphological examination. The inner ear of Tric-/- mice was perfused with biotinylation reagents, and the ES sections were observed for tracer permeability assay after applying streptavidin-Alexa Fluor 488 conjugate. RESULTS: The tricellulin expression was confirmed by RT-PCR and by immunohistochemistry in the WT ES. The ES in Tric-/- mice showed normal morphology and revealed no biotin leakage from the lumen. CONCLUSION: The ES in Tric-/- mice showed no changes in morphology or disruption in macromolecular barrier function. The effects of solute leakages in the ES of Tric-/- mice may be very limited and compensatable, or that the ES epithelia may have other sealing system covering the lack of tricellulin.

A fungal Argonaute interferes with RNA interference.

Small RNA (sRNA)-mediated gene silencing phenomena, exemplified by RNA interference (RNAi), require a unique class of proteins called Argonautes (AGOs). An AGO protein typically forms a protein-sRNA complex that contributes to gene silencing using the loaded sRNA as a specificity determinant. Here, we show that MoAGO2, one of the three AGO genes in the fungus Pyricularia oryzae (Magnaporthe oryzae) interferes with RNAi. Gene knockout (KO) studies revealed that MoAGO1 and MoAGO3 additively or redundantly played roles in hairpin RNA- and retrotransposon (MAGGY)-triggered RNAi while, surprisingly, the KO mutants of MoAGO2 (Δmoago2) showed elevated levels of gene silencing. Consistently, transcript levels of MAGGY and mycoviruses were drastically reduced in Δmoago2, supporting the idea that MoAGO2 impeded RNAi against the parasitic elements. Deep sequencing analysis revealed that repeat- and mycovirus-derived small interfering RNAs were mainly associated with MoAGO2 and MoAGO3, and their populations were very similar based on their size distribution patterns and positional base preference. Site-directed mutagenesis studies indicated that sRNA binding but not slicer activity of MoAGO2 was essential for the ability to diminish the efficacy of RNAi. Overall, these results suggest a possible interplay between distinct sRNA-mediated gene regulation pathways through a competition for sRNA.

The difference in endolymphatic hydrostatic pressure elevation induced by isoproterenol between the ampulla and the cochlea.

OBJECTIVE: The purpose of the study was to investigate the difference in the responses of endolymphatic hydrostatic pressure to isoproterenol, ß-adrenergic receptor agonist, between pars superior and pars inferior. METHODS: The hydrostatic pressure of endolymph and perilymph and endolymphatic potential in the ampulla and the cochlea during the intravenous administration of isoproterenol were recorded using a servo-null system in guinea pigs. RESULTS: The hydrostatic pressure of endolymph and perilymph in the ampulla and cochlea was similar in magnitude. Isoproterenol significantly increased hydrostatic pressure of ampullar and cochlear endolymph and perilymph with no change in the ampullar endolymphatic potential and endocochlear potential, respectively. The isoproterenol-induced maximum change of endolymphatic hydrostatic pressure in ampulla was significantly (p<0.01) smaller than that in the cochlea. In ears with an obstructed endolymphatic sac, the action of isoproterenol on endolymphatic hydrostatic pressure in the ampulla disappeared like that in the cochlea. CONCLUSION: Isoproterenol elevates endolymphatic hydrostatic pressure in different manner between the vestibule and the cochlea.

Ion transport its regulation in the endolymphatic sac: suggestions for clinical aspects of Meniere's disease.

Ion transport and its regulation in the endolymphatic sac (ES) are reviewed on the basis of recent lines of evidence. The morphological and physiological findings demonstrate that epithelial cells in the intermediate portion of the ES are more functional in ion transport than those in the other portions. Several ion channels, ion transporters, ion exchangers, and so on have been reported to be present in epithelial cells of ES intermediate portion. An imaging study has shown that mitochondria-rich cells in the ES intermediate portion have a higher activity of Na+, K+-ATPase and a higher Na+ permeability than other type of cells, implying that molecules related to Na+ transport, such as epithelial sodium channel (ENaC), Na+-K+-2Cl- cotransporter 2 (NKCC2) and thiazide-sensitive Na+-Cl- cotransporter (NCC), may be present in mitochondria-rich cells. Accumulated lines of evidence suggests that Na+ transport is most important in the ES, and that mitochondria-rich cells play crucial roles in Na+ transport in the ES. Several lines of evidence support the hypothesis that aldosterone may regulate Na+ transport in ES, resulting in endolymph volume regulation. The presence of molecules related to acid/base transport, such as H+-ATPase, Na+-H+ exchanger (NHE), pendrin (SLC26A4), Cl--HCO3- exchanger (SLC4A2), and carbonic anhydrase in ES epithelial cells, suggests that acid/base transport is another important one in the ES. Recent basic and clinical studies suggest that aldosterone may be involved in the effect of salt-reduced diet treatment in Meniere's disease.

Deletion of Tricellulin Causes Progressive Hearing Loss Associated with Degeneration of Cochlear Hair Cells.

Tricellulin (also known as MARVELD2) is considered as a central component of tricellular tight junctions and is distributed among various epithelial tissues. Although mutations in the gene encoding tricellulin are known to cause deafness in humans (DFNB49) and mice, the influence of its systemic deletion in vivo remains unknown. When we generated tricellulin-knockout mice (Tric(-/-)), we found an early-onset rapidly progressive hearing loss associated with the degeneration of hair cells (HCs); however, their body size and overall appearance were normal. Tric(-/-) mice did not show any morphological change pertaining to other organs such as the gastrointestinal tract, liver, kidney, thyroid gland and heart. The endocochlear potential (EP) was normal in Tric(-/-) mice, suggesting that the tight junction barrier is maintained in the stria vascularis, where EP is generated. The degeneration of HCs, which occurred after the maturation of EP, was prevented in the culture medium with an ion concentration similar to that of the perilymph. These data demonstrate the specific requirement of tricellulin for maintaining ion homeostasis around cochlear HCs to ensure their survival. The Tric(-/-) mouse provides a new model for understanding the distinct roles of tricellulin in different epithelial systems as well as in the pathogenesis of DFNB49.

Cystic fibrosis transmembrane conductance regulator in the endolymphatic sac of the rat.

OBJECTIVE: Na(+) and Cl(-) are dominant ions in the endolymphatic fluid in the endolymphatic sac and are important for volume regulation in the endolymphatic sac. An epithelial sodium channel (ENaC) and other Na(+) transporters have been identified in the endolymphatic sac epithelia, and they are involved in the regulation of endolymph. Although the presence of Cl(-) channels in the endolymphatic sac epithelia has been speculated, no Cl(-) channels have been identified. In this study, we confirmed the expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the endolymphatic sac by reverse transcriptase polymerase chain reaction (RT-PCR) and by immunohistochemical staining. METHODS: Pure mRNA from endolymphatic sac epithelia was prepared using laser capture microdissection (LCM) and examined using RT-PCR. Localization of CFTR and ENaC in the endolymphatic sac was examined using immunohistochemistry. RESULTS: mRNA of the CFTR was expressed in the endolymphatic sac. Immunohistochemical analysis showed the expression of the CFTR on apical side of the endolymphatic sac epithelia and co-localization with the ENaC. CONCLUSION: RT-PCR and immunohistochemistry were used to identify the expression of CFTR in the endolymphatic sac epithelia, which gives us a clue for understanding Cl(-) transport in the endolymphatic sac. These results suggest a pathway for Cl(-), possibly through interaction with the ENaC, which may regulate the endolymph in the endolymphatic sac.

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 ß-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)-, ß(1)-, ß(2)- and ß(3)-ARs was observed in LCM samples of ES epithelia. Immunohistochemical analysis using specific antibodies showed immunofluorescence of ß(2)- and ß(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 ß(1)-ARs. The presence of ß(2)-AR with no clear immunostaining of ß(1)-AR in ES epithelial cells is in accordance with previous electrophysiological and pharmacological results, which suggests that ß(2)-AR mediates the action of catecholamines on the ESP. The presence of ß(3)-AR in the ES epithelial cells and its absence in the stria vascularis implies that ß(3)-AR plays a specific role in the ES.

Presence of FXYD6 in the endolymphatic sac epithelia.

A homeostasis of the electrochemical properties and volume of the endolymph in the inner ear is essential for hearing and equilibrium sensing and is maintained by ion-transport across an epithelial tissue, the endolymphatic sac. One of the key proteins in the maintenance is Na(+), K(+)-ATPase. Although we previously found that the Na(+), K(+)-ATPase in the sac plays a pivotal role in the control of the endolymphatic volume, the mechanism remains unclear. Therefore, in this study, we examined the expression of FXYD6, a functional modulator of the Na(+), K(+)-ATPase, in the epithelial cells of the endolymphatic sac using various approaches. Laser capture microdissection RT-PCR was used to identify FXYD6 mRNA in the endolymphatic sac. Immunolabeling with the specific antibody showed that FXYD6 was predominantly expressed in the intermediate portion of the endolymphatic sac, and it was colocalized with the Na(+), K(+)-ATPase. Because the Na(+), K(+)-ATPase in this region is known to exhibit a high level of activity, an interaction of FXYD6 with this transporter may be critically involved in the regulation of the characteristics of the endolymph.

The mRNA of claudins is expressed in the endolymphatic sac epithelia.

OBJECTIVE: Claudins are a family of membrane proteins which localize to tight junctions (TJs). Recent studies have shown that claudins can form pores for ions in the TJs and regulate the permeability of epithelial paracellular ion transport. The endolymphatic sac (ES) is a part of the inner ear, absorbing the endolymphatic fluid. ES dysfunction may result in endolymphatic hydrops. In this study, we focused on the paracellular transport and examined claudin mRNA expression in the ES epithelia. MATERIALS AND METHODS: Total RNA was isolated from whole ES epithelia of rats by laser capture microdissection. RT-PCR was used to evaluate the expression of claudins. The expression of each claudin mRNA in the epithelial cells of rat ES was confirmed by in situ hybridization. RESULTS: RT-PCR indicated the expression of cldn2, cldn4, cldn6, cldn7, cldn9, cldn11, cldn12, and cldn14. The expression of these claudin mRNAs in the epithelial cells of rat ES was confirmed by in situ hybridization. CONCLUSION: We demonstrated mRNA expression of multiple claudins in the rat ES epithelia. These results in the ES epithelia were consistent with a role of claudins in paracellular ion transport.

Specific RNA collection from the rat endolymphatic sac by laser-capture microdissection (LCM): LCM of a very small organ surrounded by bony tissues.

Laser-capture microdissection (LCM) is an excellent tool to selectively obtain target tissue or cells. The endolymphatic sac (ES) is part of the inner ear, and a large part of the ES is located in the temporal bone. The rat ES is conventionally harvested using stereomicroscopy. In this method, contamination is unavoidable because of its size and location; therefore, additional checks, such as in situ hybridization, are necessary to confirm the cellular localization, and quantitative analysis is difficult in the ES. We have shown a selective epithelial tissue method using LCM to obtain RNA without contamination from ES epithelial tissue.

Measurement of nasal resistance by rhinomanometry in 892 Japanese elementary school children.

OBJECTIVE: The normal value of nasal resistance in adults has been reported (0.25 Pa/cm³/s), but that in children has not. In this study, we measured nasal resistance in Japanese school children by employing rhinomanometry. METHODS: An otolaryngologist examined 939 Japanese school children with regard to the presence or absence of nasal diseases and tonsil size. Nasal resistance was measured by rhinomanometry employing the active anterior method in 892 children. A questionnaire concerning the condition during sleep, such as the presence or absence of snoring and sleep apnea syndrome, was performed. RESULTS: The mean nasal resistance was 0.43 ± 0.50 Pa/cm³/s: 0.46 ± 0.65 and 0.39 ± 0.22 Pa/cm³/s in boys and girls, respectively. Of the 892 children, Grade 3 and 4 tonsil hypertrophy was noted in 84 (9%), but the presence of tonsil hypertrophy did not influence nasal resistance. Nasal diseases were noted in 335 children (38%) and the nasal condition was normal (the normal group) in 557 (62%). Nasal resistance was 0.56 ± 0.75 Pa/cm³/s in the nasal disease group and 0.36 ± 0.21 Pa/cm³/s in the normal group, showing that the resistance was significantly higher in the nasal disease group. The resistance tended to decrease as the school grade increased. In the normal group, 290 children (33%) experienced no problem regarding the upper airway, such as snoring and sleep apnea syndrome, based on a questionnaire, and nasal resistance was 0.35 ± 0.17 Pa/cm³/s. CONCLUSION: This normal nasal resistance value may be adopted for the objective evaluation of nasal obstruction and effects of treatment in pediatric nasal diseases.

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.

Expression and localization of 11beta-hydroxysteroid dehydrogenase (11betaHSD) in the rat endolymphatic sac.

CONCLUSIONS: 11beta-Hydroxysteroid dehydrogenase type 2 (11bHSD-2) enables aldosterone to bind to mineralocorticoid receptors (MRs) selectively by converting cortisol (corticosterone) into inactive metabolites. Its expression in the endolymphatic sac (ES) suggests that aldosterone may selectively act on the ES through its binding to MRs by the action of 11betaHSD-2, and supports the notion that ES is an aldosterone target organ. We propose that 11betaHSD-2 is a dominant isoform of 11betaHSDs in the ES, and the ES (especially the intermediate portion of the ES) may be the main aldosterone target in the inner ear. OBJECTIVE: The purpose of this study was to examine 11bHSD isoform expression in the rat inner ear, mainly 11betaHSD-2 in the ES. MATERIALS AND METHODS: In the ES and whole cochlea, 11betaHSDs were examined by RT-PCR using highly specific ES RNA by laser capture microdissection. In addition, 11betaHSD-2 localization in the rat ES was determined by immunohistochemistry. RESULTS: RT-PCR demonstrated 11betaHSD-2 expressed in the rat ES. In addition, its localization was observed mainly in the intermediate portion and a faint immune positive signal was observed in other parts of the ES. In contrast, 11bHSD-1 was undetectable in the ES by RT-PCR. Both types of 11betaHSDs were expressed in rat cochlea.

A new approach for selective rat endolymphatic sac epithelium collection to obtain pure specific RNA.

The endolymphatic sac (ES) is an organ that is located in the temporal bone. Its anatomical location makes ES tissue collection without any contamination very difficult, and sometimes accurate molecular analyses of the ES are prevented due to this matter. In the present study, a new selective ES epithelial tissue collection method was attempted using laser capture microdissection to obtain pure ES RNA without any contamination. The validity of this method was demonstrated by RT-PCR with three specific primer pairs against osteocalcin, calponin H1, and NKCC2, which are specific proteins in bone, smooth muscle, and kidney/ES cells, respectively. From the RT-PCR results, the high specificity and sufficient sensitivity of the new method was indicated. It is considered that the new method is optimal for ES collection without contamination and it will be able to contribute to future analyses of the ES.