Expression and localization of Na+-HCO3- cotransporter in bovine corneal endothelium

Functional studies support the presence of the Na+-HCO3- cotransporter (NBC ) in corneal endothelium and possibly corneal epithelium; however, molecula r identification and membrane localization have not been reported. To test whether NBC is expressed in bovine cornea, Western blotting was performed, which showed a single band at similar to 130 kDa for freshly isolated and c ultured endothelial cells, but no band for epithelium. Two isoforms of NBC have recently been cloned in kidney (kNBC) and pancreas (pNBC). RT-PCR was run using cultured and fresh bovine corneal endothelial and fresh corneal e pithelial total RNA and specific primers for kNBC and pNBC. RT-PCR analysis for pNBC was positive in endothelium and weak in epithelium. The RT-PCR pr oduct was subcloned and confirmed as pNBC by sequencing. No specific bands for kNBC were obtained from corneal cells. Indirect immunofluorescence and confocal microscopy indicated that NBC locates predominantly to the basolat eral membrane in corneal endothelial cells. Furthermore, Na+-dependent HCO3 - fluxes and HCO3--dependent cotransport with Na+ were elicited only from t he basolateral side of corneal endothelial cells. Therefore, we conclude th at pNBC is present in the basolateral membrane of both fresh and cultured b ovine corneal endothelium and weakly expressed in the corneal epithelium.