Expression and localization of Na(+)-HCO(3)(-) cotransporter in bovine corneal endothelium.
Functional studies support the presence of the Na(+)-HCO(3)(-) cotransporter (NBC) in corneal endothelium and possibly corneal epithelium; however, molecular 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 approximately 130 kDa for freshly isolated and cultured 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 epithelial 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 product 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 basolateral membrane in corneal endothelial cells. Furthermore, Na(+)-dependent HCO(3)(-) fluxes and HCO(3)(-)-dependent cotransport with Na(+) were elicited only from the basolateral side of corneal endothelial cells. Therefore, we conclude that pNBC is present in the basolateral membrane of both fresh and cultured bovine corneal endothelium and weakly expressed in the corneal epithelium.
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School of Optometry, Indiana University, Bloomington, Indiana 47401, USA.
Bonanno
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Jelamskii
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D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000166:cytology / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D007425:Intracellular Membranes / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D027982:Sodium-Bicarbonate Symporters; D014018:Tissue Distribution
D002352:Carrier Proteins; D027982:Sodium-Bicarbonate Symporters