Immunohistochemical localization of the epithelial sodium channel in the rat inner ear

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Abstract

Endolymph in membranous labyrinth is a K+-rich and Na+-poor fluid, and perilymph is conversely Na+-rich and K+-poor. Electrolyte transport between endolymph and perilymph is important for regulation of volume and osmotic pressure of the labyrinth. The epithelial sodium channel (ENaC) is a good candidate protein for Na+ transport in the tight epithelia, which has been well demonstrated in other tissues such as kidney, colon and lung. The purpose of the present study was to investigate the cellular localization of ENaC subunits in the rat inner ear immunohistochemically with the specific polyclonal rabbit antibodies against the rat α-, β- and γ-ENaC. All three subunits of ENaC were extensively labeled in the cochlea including the stria vascularis, spiral ligament, organ of Corti, spiral limbus, Reissner's membrane and spiral ganglion, and in the vestibule including the sensory epithelia and stroma cells of the macula utriculi, macula sacculi and ampullary crest. In conclusion, our results suggest that functional ENaC in the labyrinth may work in concert with other Na+ and K+ transport molecules to regulate endolymph and to maintain homeostasis in the inner ear.

Introduction

The membranous labyrinth in the temporal bone is filled with endolymph that is a K+-rich and Na+-poor fluid. In the cochlear endolymph, the K+ concentration is 160 mM and Na+ concentration is 1.5 mM, and in the vestibular endolymph, the K+ concentration is 150 mM and Na+ concentration is 9 mM (Bosher and Warrant, 1971; Wangemann and Schacht, 1996). This unique ion composition of endolymph is essential for the function of the sensory hair cells in the cochlea and vestibule, and for generating a high positive endocochlear potential (EP) (Salt et al., 1987; Wangemann, 1995).

Though the mechanism underlying potassium transport in labyrinth is still controversial, Na,K-ATPase and Na–K–Cl co-transporter, the most important transport molecules involved in K+ recycling, have been demonstrated to be widely distributed in the cochlea and vestibule. These molecules may play important roles in establishing the K+ gradients between perilymph and endolymph, and in generating the EP (Pitovski and Kerr, 2002; Crouch et al., 1997; ten Cate et al., 1994; Fina and Ryan, 1994). On the other hand, the mechanism underlying sodium transport in the inner ear remains incompletely understood, though it is known that sodium can be pumped against electrochemical gradient out of the basolateral membrane of the marginal cells of the stria vascularis and the dark cells of vestibule by Na,K-ATPase.

In recent years, more and more researchers have focused on the amiloride-sensitive epithelial sodium channel (ENaC) in an attempt to elucidate the transport mechanism of Na+ in the tight epithelia (Kellenberger and Schild, 2002; Alvarez de la Rosa et al., 2000). It has been found that ENaC may play critical roles in (re)absorbing Na+ from apical plasma membrane in variant tissues and cells such as kidney (Hager et al., 2001), colon (Epple et al., 2000) and lung (Talbot et al., 1999). ENaC allows passive apical entry of Na+ into cell, then Na+ is actively transported out of the cell by basolateral Na,K-ATPase. It has also been demonstrated that ENaC is regulated by hormones such as glucocorticoid, mineralocorticoid aldosterone, vasopressin and other factors (Garty and Palmer, 1997). However, no study has been reported about the expression patterns of ENaC subunit proteins in the inner ear to date.

The present study reports for the first time the localization of α, β and γ subunits of ENaC in the adult rat inner ear by immunohistochemistry. The cellular distribution of different subunits of ENaC was examined by immunolabeling with specific antibodies. Our results showed that all three subunits of ENaC, α, β and γ, were extensively expressed in the rat inner ear, which suggests that they may work in combination to regulate endolymph and to maintain homeostasis in the inner ear.

Section snippets

Animals and tissue preparation

Eight adult Wistar rats aged 4–8 months were used. The procedures concerning animals reported in this study were approved by the Animal Care and Use Committee at the Third Military Medical University. After being anesthetized intraperitoneally with pentobarbital sodium (40 mg/kg body weight), rats were perfused, after clamping the descending vessels, via cardiac puncture with 200 ml 0.9% saline, followed by 4% paraformaldheyde at pH 7.3 till the neck became stiff. Following decapitation, the

Results

Our results showed that all three subunits of ENaC, α-, β- and γ-, were widely distributed in the labyrinth. In the cochlea (Fig. 1), immunolabeling of α-, β- and γ-ENaC was found extensively in the stria vascularis, spiral ligament, spiral limbus, Reissner's membrane and spiral ganglion. However, the labeling in the limbus differed for all three forms. The α subunit was labeled diffusely through the limbus, the β subunit was very little in the limbus and the γ subunit labeled the interdental

Discussion

ENaC, belonging to the degenerin/ENaC gene superfamily, is composed of three homologous subunits, α-, β- and γ-ENaC, with a stoichiometry of α2βγ or α3β3γ3 (Alvarez de la Rosa et al., 2000). In several tight epithelial such as kidney, colon and lung, electrogenic entry of Na+ from the lumen into the cells is mediated by the ENaC located in the apical plasma membrane, which represents the rate-limiting step for Na+ absorption. At the basolateral membrane, Na+ is pumped out of the cell by

Acknowledgments

The authors thank Dr. Pascal Barbry for kindly providing the polyclonal antibodies against α-, β- and γ-ENaC. We also thank Mrs. Jin-Qing Yang and Miss Juan Du for their invaluable assistance with the pathological techniques.

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