Mechanisms regulating ion transport in the secretory epithelia of the inner ear : an experimental study

Sammanfattning: The apical portions of the cells in the sensory end organs for audition and equilibrium border a fluid called endolymph. Endolymph is the only extracellular fluid in the body which is characterized by an intracellular-like ion composition. The unique ion composition of the endolymph is supposed to be a prerequisite for making possible impulse transduction in the sensory cells of the cochlea. The molecular mechanisms responsible for the production and the regulation of endolymph composition remain largely unknown. The secretory epithelia in the cochlea, the stria vascularis (SV), and the epithelial cells of the endolymphatic sac are believed to be involved in these mechanisms. In this study fresh tissue preparations of the SV and cultured cells from the SV and the endolymphatic sac were established. Marginal cells and intermediate cells from the SV and epithelial cells from the endolymphatic sac, besides fibroblasts, could be identified in the cell cultures. The cultured cells showed morphological characteristics similar to those occuring in vivo. All cell types, identified in the cell cultures, responded with a rapid and reversible increase in cytoplasmic free Ca2+ concentration, [Ca2+]i, when stimulated with ATP. This effect was not directly coupled to influx of Ca2+ from the extracellular space, which indicates that it probably reflects inositol triphosphate-mediated release from the endoplasmatic reticulum. When stimulation with different K+ concentrations was performed, no change in [Ca2+]i could be seen. This indicates that the cultured cells from the SV and the endolymphatic sac lack voltagedependent Ca2+ -channels activated by an increase in extracellular K+. Part of the ototoxic effects of gentamicin and ethacrynic acid are supposed to be due to disturbances in the function of the SV. By adding gentamicin and ethacrynic acid to the cultured SV cells and a clonal insulin-secreting cell line (HIT), the direct toxic effects on these secretory cells were investigated. The morphological changes seen in the cultured cells after exposure to gentamicin and ethacrynic acid, were similar to those changes previously described to occur in vivo. When gentamicin was added to the cultured cells, no change in [Ca2+]i could be seen. Accordingly, the SV cells and HIT cells reacted with a rapid and reversible increase in [Ca2+]i when stimulated with ATP and K+, respectively. However, after exposure to ethacrynic acid, no increase in [Ca2+]i could be seen when the cells were stimulated with ATP (SV) or K+ (HIT cells). This inability to react with an increase in [Ca2+]i may constitute part of the pathophysiology of ethacrynic acid toxicity. Despite the low Ca2+ concentration of the endolymph, Ca2+ has to be actively transported into the endolymphatic space. The mechanisms responsible for this active transport are not known. The presence of plasma membrane-bound Ca2+-ATPase (PMCA ATPase) was investigated and a method where different dissected inner ear tissues were used for immunoblotting were established. PMCA ATPase was present in all the endolymph-producing secretory epithelia of the inner ear, indicating that PMCA ATPase plays a role in the regulation of the Ca2+ concentration of the endolymph. The importance of Na+, K+-ATPase for the development and maintenance of the endocochlear potential has been studied extensively. Na+, K+-ATPase is present in abundance in the SV. In this study the presence of PKC isoforms [alpha], [delta] and [zeta] CaM kinase II, PP-1 isoforms [alpha] and [gamma] and I-1 in the SV was shown by using immunoblotting. These intracellular components have been proposed to participate in the hormonal short-term regulation of Na+, K+-ATPase activity in the kidney. The presence of these intracellular components of phosphorylation/dephosphorylation system in the SV suggests that hormonal short-term regulation of Na+, K+-ATPase activity also is possible in the SV.