Expression Of The Epithelial Sodium Channel And Aquaporin,and Regulation Of The Epithelial Sodium Channel By Aldosterone In The Inner Ear

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. It has been well demonstrated that endolymph is produced by the stria vascularis and vestibular dark cells, and reabsorbed by endolymphatic sac. 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. Endolymphatic hydrops caused by disequilibrium of endolymph production and absorption has generally been established to be the pathological base of Meniere's disease. To date the mechanism underlying sodium and water transport of endolymph in the inner ear remains incompletely understood though the mechanism underlying potassium transport in labyrinth has been investigated in numerous reports.It has been well known that the epithelial sodium channel(ENaC) in tight epithelia such as kidney, colon and lung is a rate-limiting molecule for Na~+ absorption, which is regulated by SGK1(serum and glucocorticoid inducible kinasel) and Nedd4(Neural precursor cell-expressed, developmentally downregulated gene), and that aquaporin is the key molecule for water transport in epithelia. The present study investigates the expression of ENaC and AQPs and the regulation of ENaC by aldosterone in the inner ear as the following. Firstly, the celluar localizations of ENaC, SGK1 and Nedd4 isoforms in the inner ear of rat and guinea pig were detected immunohistochemically with special polyclonal antibodies. The expressions of mRNAs encodig aENaC and SGK1 were studied by in situ hybridization with digoxin-labelled cDNA probes as well. Secondly, auditory threshold and ultra-struture of the stria vascularis were compared between the normal rats, adrenalectomized (ADX) rats and ADX rats injected intraperitoneally with aldosterone. Additonaly, regulation of aENaC and SGK1 by aldosterone was studied by Western blot and RT-PCR with theADX animals. Thirdly, expression patterns of AQPl, 2, 3 and 4 in the inner ear of rat and guinea pig were investigated by immunohistochemstry with special polyclonal antibodies. The following is our results:1. In the cochlea of guinea pig, the immunolabels of a-, 0-, y-ENaC and SGK1 were positive in all subregions excluding the stria vascularis, with the exception that aENaC was negative in the spiral ganglion. Nevertheless, Nedd4 isoforms were detected in all parts. In the endolymphatic sac, ENaC, SGK1, and Nedd4 were all detected in the epithelial cells and subepithelial tissuses. The transcripts of aENaC and SGK1 were found in the stria vascularis, spiral limbus, lower part of spiral ligament and endolymphatic sac.2. In the rat cochlea, the a-, 0- and y-ENaC, SGK1 and Nedd4 isoforms proteins were extensively distributed in subregions. The expression pattern of aENaC mRNA was similar to that of aENaC protein. The positive label of SGK1 mRNA was detected in cochlea excluding the spiral ganglion and Reissner's membrane.3. No obvious differences were found in auditory threshold and ultrastucture of the stria vascularis between the normal rats and ADX rats.4. RT-PCR analysis showed that there existed significant differences in the expression of aENaC mRNA between the normal rats, ADX rats and ADX rats injected intraperito-neally with aldosterone.5. Western blot analysis showed that there were significant differences in the expression of SGK1 protein between the normal rats, ADX rats and ADX rats injected intraperi-toneally with aldosterone.6. Immunohistochemical examination showed that in the cochlea of rat and guinea pig, AQPl, 3 and 4 were widely distributed in various locations in the similar patterns except that AQP3 in the stria vascularis was lightly weaker than AQPl and AQP4. AQP2 was labeled only in the Reissner's membrane. In the endolymphatic sac of guinea pig, AQP1, AQP3 and AQP4 were strongly expressed in the epithelial cells and subepithelial cells similarly with the exception that AQP3 was lightly weaker than AQPl and AQP4. No AQP2 immunoreactivity was detected in the endolymphatic sac.Our results suggest that:I. ENaC, SGK1 and Nedd4 expressed with different patterns in the inner ear may form a functional chain to regulate the sodium metabolism in endolymph.2. Though no changes of auditary function and ultrastructure of the stria vascularis occour in the absence of circulating adrenal hormones, aldosterone may regulate the expression of SGKl in the early phase and regulate that of ENaC in the late phase in labyrinth. As a result, sodium metabolism in endolymph may be modulated by aldosterone.3. Different members of the AQP family expressed with different patterns in the labyrinth may work in concert to regulate water mebabolism in endolymph and to maintain homeostasis in the inner ear.