Biochemical properties and immunohistochemical localization of carbonic anhydrase in the sacculus of the inner ear in the salmon Oncorhynchus masou

Carbonic anhydrase (CA) in the inner ear sacculus was examined by activity assay, Western blotting and immunohistochemistry to determine its role in otolith calcification. An immunoreactive protein with a molecular mass of approximately 28 kDa was detected by Western blotting. The CO2 hydration activity in the cytosol fraction of the sacculus was 5.4 units/mg protein, while little or no activity was detected in the nuclear and mitochondrial fractions. The enzyme activity was highly inhibited by acetazolamide. The concentration of 50% inhibition was 8.16 nM and the inhibition constant of the activity was 8.25 nM. Transitional and squamous epithelial cells of the sacculus were immunopositive with an anti-CA II antibody, but sensory epithelial cells and mitochondria-rich cells in the transitional epithelium were not. These results suggest that transitional epithelial cells other than mitochondria-rich cells and squamous epithelial cells play an important role in otolith calcification by supplying bicarbonate to otoliths and/or by eliminating protons from endolymph.