Evaluating the Expression and Function of Carbonic Anhydrase IV in Bovine Corneal Endothelial Cells

Abstract

Abstract: : Purpose: Previous functional studies have revealed that membrane–associated carbonic anhydrase IV (CA IV) is expressed in bovine corneal endothelial cells (BCEC). However, the molecular study of CA IV has not been reported in BCEC. In this study, we further tested the molecular expression, distribution and function of CA IV in BCEC. Methods: RT–PCR and sequencing were performed to identify the mRNA expression of CA IV in cultured BCEC. In addition, immunoblot analysis was used to further demonstrate the protein expression of CA IV. Immunofluorescence and confocal microscopy determined the distribution of CA IV in BCEC. Intracellular pH was measured with the pH sensitive fluorescent dye BCECF. This was used to measure apical CO₂ flux–induced intracellular pH changes following inhibition of CA IV activity using extracellular carbonic anhydrase inhibitors (CAIs) or siRNA knockdown of CA IV. Results: RT–PCR showed a single band at the predicted size. Sequencing analysis further confirmed the identity of CA IV. Immunoblot analysis showed a positive band at ∼40 kD, which is consistent with other studies. Immunofluorescence and confocal microscopy demonstrated that CA IV in BCEC is localized at the apical membrane. Transfection of 30 nM siRNA specific for CA IV reduced CA IV protein expression by more than 90%. 10 µM benzolamide significantly decreased apical CO₂ efflux–induced intracellular pH changes by 48 ± 3 % relative to the control (n=15). Conclusion: Membrane–associated carbonic anhydrase IV is functionally expressed in the apical membrane of BCEC. CO₂ diffusion may contribute to apical HCO₃^– flux through the action of carbonic anhydrase IV, thereby increasing HCO₃^– efflux from the apical membrane.

Keywords: ion transporters • PH regulation/protons • cornea: endothelium