Claudins in the tight junctions of stria vascularis marginal cells

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Abstract

In the mammalian cochlea, tight junctional strands are visible on freeze fracture images of marginal cells and other inner ear epithelia. The molecular composition of the strial tight junctions is, however, largely unknown. We investigated the expression of integral tight junction-proteins, claudin-1 to -4, and occludin, in stria vascularis of the guinea-pig cochlea, as compared to kidney. Western blot analysis revealed a strong expression of claudin-4 and occludin in strial tissue, and confocal immunofluorescence microscopy demonstrated their presence in the tight junctions of the marginal cells. In addition, a moderate level of claudin-3 and claudin-1 was detected and both were located in the marginal tight junctions. Claudins-1, -3, and -4 are characteristic of epithelia with low paracellular permeability and claudin-4 is known to restrict the passage of cations through epithelial tight junctions. In the marginal cells, these claudins appear to be responsible for the separation of the potassium-rich endolymph from the sodium-rich intrastrial fluid. In contrast, Western blot analysis and confocal microscopy demonstrated that the marginal cell epithelium does not contain claudin-2, which forms a cation-selective pore in tight junctions. Its absence indicates a cation-tight paracellular pathway in the marginal cells.

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Materials and methods

Tissue preparation. Healthy, pigmented, adult guinea pigs of both sexes with intact Preyer’s reflex were anesthetized and subsequently killed by inhalation of 100% CO2. The cochleae were opened and the bony walls were micromechanically removed as described previously in detail [14]. The stria vascularis of each cochlear turn was transferred to a cell-tak (BD Biosciences, Bedford, USA) coated coverslip in a culture dish (35 mm diameter; Nalge Nunc International, Denmark). With the culture dish on

Tight junction protein expression

Immunoblot analyses were performed for the tight junction-proteins claudin-1 to -4 and occludin. Crude membrane fractions were used because these proteins are integral membrane proteins forming tight junction strands. All claudins were detected as a band at ∼23 kDa (Fig. 1B). The protein expression of the kidney sample served for reference (100%) since all of the claudins studied are expressed in different nephron segments as well [16].

Western blot analysis (Figs. 1A and B) and subsequent

Discussion

In the cochlea, the barrier function of epithelial tight junctions is essential for sensory transduction and the compartmentalization of the fluid-filled spaces of the cochlea. Thus, mutation of the tight junction-protein claudin-14 can lead to hereditary deafness DFNB29 [12]. There are, however, only few reports on integral tight junction proteins of the mammalian inner ear. In cultured epithelial cells from semicircular canals (without receptor organs), the intracellular tight

Acknowledgements

We thank Anja Fromm for the excellent technical assistance. The work was supported by the Deutsche Forschungsgemeinschaft (DFG) and by Funds of the Medical Faculty, Free University of Berlin.

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