Identification and cloning of the Na/HCO(3-) cotransporter (NBC) in human corneal endothelium.
Fluid secretion by the corneal endothelium is associated with the net flux of HCO(3)(-) from basolateral (stromal) to apical (anterior chamber) sides of the tissue. In this study we asked if Na(+)/HCO(3)(-) cotransporter (NBC-1) protein expression and functional activity are present in freshly isolated human corneal endothelium. Immunoblot analysis using a polyclonal antibody to NBC-1 showed a single band at approximately 130 kDa. Indirect immunofluorescence indicated that NBC-1 is expressed on the basolateral, but not apical side of human corneal endothelium. RT-PCR was used to determine whether the kidney or pancreatic isoform of NBC-1 is expressed. Using the specific primers for pNBC and kNBC isoforms, RT-PCR showed that only pNBC could be detected in human corneal endothelium. The product was cloned and confirmed by sequencing. Full-length NBC-1 was also cloned from human corneal endothelium. This clone (hcNBC) is 100% identical to the longer, more common form of NBC [pNBC; 1079 amino acids (aa); 122 kDa in human heart, pancreas and prostate]. To test for functional activity of NBC-1, freshly isolated endothelium was loaded with the pH sensitive fluorescent dye BCECF and HCO(3)(-) fluxes were measured. HCO(3)(-) fluxes were Na(+)-dependent, electrogenic and H(2)-DIDS sensitive. We conclude that the long isoform of the sodium bicarbonate cotransporter (pNBC-1) is expressed on the basolateral side of fresh human corneal endothelium (hcNBC). The shorter form, kNBC, could not be detected. As in bovine corneal endothelium, hcNBC is instrumental in loading HCO(3)(-) into endothelial cells from the basolateral membrane.
Sun
Xing Cai
XC
School of Optometry, Indiana University, 800 E. Atwater Ave. Bloomington, IN 47401, USA. jbonanno@indiana.edu
Bonanno
Joseph A
JA
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004728:Endothelium, Corneal / Q000378:metabolism*; D005136:Eye Proteins / Q000032:analysis*; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D006801:Humans; D007536:Isomerism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D027982:Sodium-Bicarbonate Symporters / Q000032:analysis*
D005136:Eye Proteins; C498898:SLC4A4 protein, human; D027982:Sodium-Bicarbonate Symporters